Trimethoprim derivatives, preparation methods thereof, label proteins based on trimethoprim derivatives and applications thereof

Abstract

The present invention provides a trimethoprim derivative, a preparation method thereof, a label protein based on the trimethoprim derivative and an application thereof. The preparation method comprises reacting a compound A#imgabs0# with a substituted amine compound H 2 N‑CH 2 ‑(CH 2 ) n ‑R, wherein n is selected from any natural number of 1 to 5, and R is selected from halogen, and an O-alkylation reaction is performed to obtain compound B#imgabs1# Compound B is subjected to a reductive amination reaction to introduce a benzene ring into compound B to obtain compound C#imgabs2# R2 is an amino protecting group; Compound C is subjected to a coupling reaction with an acrylic acid compound to obtain a trimethoprim derivative TMP3‑NH 2 #imgabs3# Labeling based on this trimethoprim derivative helps to increase the labeling rate.

Description

Technical Field

[0001] The present invention relates to the field of protein labeling, and in particular to a trimethoprim derivative, a preparation method thereof, a label protein based on the trimethoprim derivative, and applications thereof. Background Art

[0002] Protein labeling is one of the most important means of cell labeling. Specific proteins can be labeled by introducing reporter groups through genetic coding. Protein labeling initially relied on fluorescent antibodies, which were used to label proteins through the signal amplification effect of primary antibodies binding to target proteins and fluorescent secondary antibodies. Because it is labeled as endogenous proteins of cells and its ultra-specific staining (nM or even pM level), it has become one of the important means of target detection in the biomedical field. However, antibody labeling can only be performed on fixed cells, and it is still difficult to label living cells. In order to perform imaging and detection at the cellular or even living level, CT and MRI imaging have been developed, and their resolution is at the millimeter level. Photoacoustic imaging takes into account the advantages of imaging depth and resolution, and increases the resolution of deep tissue imaging to the micron or even submicron level. In order to achieve more precise cell positioning and imaging, new protein labeling methods have gradually been developed.

[0003] Early methods of protein labeling used the high reactivity of cysteine ​​or lysine residues to react proteins with excess thiols or amines, such as maleimide or N-hydroxysuccinimide esters. Other methods include tyrosine, glutamic acid, arginine modification or terminal modification. Since Shimomura discovered green fluorescent protein (GFP) in 1962 and Roger Tsien revealed the GFP luminescence mechanism in 1994, protein labeling technology using fluorescent protein tags has gradually developed into one of the most commonly used methods for living cell research. The discovery and application of green fluorescent protein (GFP) and its derivatives have had a great impact on exploring the behavior of proteins in living cells and greatly increased our understanding of biological processes. By constructing fusion proteins encoded by genes and expressing them in cells, dynamic imaging and tracking of living cell proteins can be achieved. However, the size of fluorescent proteins is generally greater than 25kD, and the construction of fusion proteins is likely to affect the structure and function of the target protein.

[0004] In addition to chemical coupling of amino acid modification of proteins and expression of fluorescent protein GFP, protein tag technology is also a good strategy to achieve target protein labeling. That is, the target protein is fused to the tag protein (tag), and then functional molecules such as small molecule dyes that can specifically bind to the tag protein are used to achieve imaging, tracking, and positioning of the target protein.

[0005] There are many kinds of protein tags, and short peptide tags are one of the major categories. Short peptide tags originated from FlAsH developed by Qian Yongjian in 1998. A small fragment of CCXXCC (SEQ ID NO: 2) was introduced into the protein helical region through genetic coding. The introduced fragment is located in the helical region of the original protein, which just makes the four cysteines form a parallelogram, which can specifically bind to the fluorescein analog modified with diarsenic and make it emit green fluorescence. However, the CCXXCC sequence is also expressed in natural proteins, and diarsenic can react with relatively high reducing groups in cells, so the background of this type of tag is high, and arsenic is highly toxic, so it is not widely used at present. The short peptide tags developed later have their own characteristics, but there is still a lack of a tag that combines labeling sensitivity, cytotoxicity and resolution. After coupling fluorescent dyes to nickel, oligomeric His fusion proteins can be labeled, but at present, His tags are more used for protein purification and have not yet been used for super-resolution imaging. Flag-tag is a hydrophilic polypeptide composed of 8 amino acids (DYKDDDDK, SEQ ID NO: 3). Like His-tag, it is mostly used for protein purification, and its cell-level detection still relies on traditional antigen-antibody methods. Others such as HA-tag, c-myc, S1-tag, etc. are mostly used for immune labeling and protein purification.

[0006] With the continuous development of super-resolution imaging technology, super-resolution microscopes that break the traditional optical diffraction limit, such as stimulated emission depletion microscopy (STED), (saturated) structured illumination microscopy ((s)SIM / NL-SIM)), and stochastic optical reconstruction microscopy (STORM), continue to emerge. People have a clearer understanding of multidimensional biological processes such as cells, subcellular organelles, and proteins. The era of super-resolution has arrived. However, new challenges have also emerged. Super-resolution microscopy has more stringent requirements for dyes, requiring them to have high brightness, good photostability, resistance to photobleaching, and low phototoxicity. The brightness of the classic GFP fluorescent protein is one to two orders of magnitude lower than that of small molecule dyes, making it unusable for super-resolution imaging. Protein labeling technology can indirectly link various small molecule dyes with excellent performance and suitable for super-resolution imaging to the target protein, making it widely concerned in the field of imaging and protein labeling. The development of truly easy-to-use protein labeling technology with high labeling efficiency, low background, and little damage to the target protein has become a common call in the field.

[0007] SNAP-tag is the first protein-small molecule tag developed by Kai Johnsson's group in 2003 and has been widely used. The SNAP-tag protein end is human DNA repair protein O 6 -Alkylguanine alkyltransferase (hAGT) can transfer the alkyl group of alkylguanine to its own cysteine. 6 -Benzylguanine (BG) derivatives, the sulfhydryl group on hAGT can attack O 6 The benzyl group of -benzylguanine forms a covalent thioether bond, thereby attaching small molecules to target proteins (e.g. Figure 1 shown).

[0008] SNAP-tag is one of the best commercialized tags. Since acquiring SNAP-tag technology, NEB has launched a series of corresponding substrate dyes and various intermediates, covering a spectrum of 300 to 800 nm, which can stain the cell surface, cytoplasm and nucleus. Later, in order to achieve diversified cell labeling, Kai Johnsson's research group selected O 2 -alkylcytosine derivatives were used as labeling small molecules, and hAGT mutants were obtained through two rounds of error-prone PCR and phage display technology screening, named CLIP-tag. The response of CLIP-tag to BC-fluorescein is about 50 times that of SNAP-tag, and BC derivatives basically do not react with SNAP-tag, thus achieving dual-color simultaneous imaging of cells. Currently, SNAP-tag has been widely used by researchers, while CLIP-tag has not received the same attention, which may be related to the slower actual labeling rate of CLIP-tag (its labeling principle is as follows Figure 1 As shown, wherein BC is benzycytosin, benzylcytosine).

[0009] HaloTag is a protein-small molecule tag developed by Promaga in 2008. It has become the most widely used protein-small molecule tag in the field of super-resolution imaging so far due to its superior labeling efficiency and labeling intensity. HaloTag is based on the dehalogenase (DhaA) of Rhodococcus erythropolis. This type of dehalogenase can bind to chlorinated alkyl chains and remove the chlorine atom from the alkyl chain by nucleophilic substitution. The aspartic acid at position 106 of DhaA forms a covalent bond with the alkyl chain. The histidine at position 272 can hydrolyze the formed covalent bond to form a hydroxyalkyl chain by base catalysis. Mutating the histidine at position 272 to neutral phenylalanine or aspartic acid can prevent hydrolysis and maintain the covalent connection between the protein and the substrate (such as Figure 2 shown).

[0010] Benefiting from Promaga's excellent commercial platform, HaloTag applications have been applied in many areas such as cell imaging, protein purification, protein function and protein interaction. Among them, the most widely used field is super-resolution imaging of living cells. Luke Lavis developed the Janelia Fluor series of dyes, most of which were coupled to HaloTag for testing. The developed dyes can meet the spectral requirements of most HaloTags. Since the spectrum of protein-small molecule tags is adjustable, the HaloTag@Janelia Fluor dye series can cover various spectra and is commercially sold by Promaga.

[0011] Among the above protein tags, HaloTag is a 36kD protein tag, and its molecular weight is about 8kD larger than GFP. Inserting it into the target protein will affect the function of the protein to a certain extent. The molecular weight of SNAP-tag is 19kD, which is more suitable for labeling than HaloTag (36kD). Data show that the fusion protein expression of SNAP-tag is stronger than that of HaloTag, but the small molecule ligand of HaloTag is a chloroalkyl chain, which makes small molecules easier to penetrate the cell membrane, and the labeling rate of HaloTag is indeed higher than that of SNAP-tag. In addition, the tag of SNAP-tag is a mammalian protein, and its cell labeling will produce non-specific staining. It can be said that Halo and SNAP-tag each have their own advantages and disadvantages.

[0012] TMP-tag is a type of protein tag technology developed by Cornish et al. in 2005. TMP (trimethoprim) is a type of antibacterial drug that specifically binds to Escherichia coli dihydrofolate reductase, hinders its folate reduction pathway, and inhibits bacterial DNA synthesis. Figure 3 As shown in the figure, small molecule dyes can be attached to TMP to label the fusion protein encoded by eDHFR. Because the size of eDHFR is only 18.4kD, it is smaller than GFP (28kD), SNAP-tag (19kD) and HaloTag (36kD), and the eDHFR protein is stable and easy to fold. Mouse DHFR has been used as a fusion tag to increase protein expression. When MTX, a substrate of DHFR, is added, the DHFR fusion protein shows a time-dependent increase in expression. The eDHFR-TMP system has been used to induce protein degradation, positron emission tomography (PET), and regulate the activity of nanoantibodies. It can be said that eDHFR-TMP is a widely used biocompatible system. Based on the universality of eDHFR-TMP, it can be considered that TMP-tag has great development prospects.

[0013] Then, to make a covalent version of the TMP dye, an acrylamide bond was modified on the TMP small molecule to react with the thiol group of the cysteine ​​on the eDHFR:L28C mutant to form a covalent link. Figure 4A As shown in the figure, the second generation TMP-tag currently under development shortens the distance between the TMP matrix and acrylamide, shortening the half reaction time to 8 minutes (compared with labeling other labeling molecules, when labeled with biotin, the labeling rate is the fastest, 8 minutes), which basically meets the needs of biological imaging. Due to the lack of rational design and commercial development, although TMP-tag has been used in biological imaging, it has not received the same attention as SNAP-tag and HaloTag.

[0014] In view of this, we are committed to developing the third-generation TMP-tag to further improve the labeling rate and thus make up for the lack of practical tags in the field of living cell super-resolution imaging. Summary of the invention

[0015] The main purpose of the present invention is to provide a trimethoprim derivative, a preparation method thereof, a label protein based on the trimethoprim derivative and an application thereof, so as to solve the problem of low labeling rate of TMP label in the prior art.

[0016] In order to achieve the above object, according to one aspect of the present invention, a method for preparing a trimethoprim derivative is provided, the method comprising: performing an O-alkylation reaction on a compound A and a substituted amine compound to obtain a compound B; performing a reductive amination reaction on the compound B to introduce a benzene ring into the compound B to obtain a compound C; performing a coupling reaction on the compound C and an acrylic acid compound to obtain a trimethoprim derivative TMP3-NH2;

[0017] Wherein, the structural formula of compound A is:

[0018] The structural formula of the substituted amine compound is: H2N-CH2-(CH2) n -R, wherein n is selected from any natural number from 1 to 5, and R is selected from halogen;

[0019] The structural formula of compound B is:

[0020] The acrylic compound is acrylic acid or acryloyl chloride;

[0021] The structural formula of compound C is: R2 is an amino protecting group;

[0022] The structural formula of trimethoprim derivative TMP3-NH2 is:

[0023]

[0024] Furthermore, compound A is subjected to an O-alkylation reaction with a substituted amine compound to obtain compound B, which comprises: conducting an O-alkylation reaction of compound A with a substituted amine compound having an amino protecting group to obtain compound D; and performing a deamination protecting group reaction on compound D to obtain compound B; preferably, the amino protecting group is selected from any one or more of Boc, Cbz, Fmoc, Alloc, Teoc, Pht, Tos, Tfa, Trt, Dmb, PMB and Bn; and preferably, the deamination protecting group reaction is performed under hydrochloric acid or ethyl acetate conditions.

[0025] Further, the compound B is subjected to a reductive amination reaction to introduce a benzene ring into the compound B to obtain the compound C, which comprises: reacting the compound B with an aldehyde compound containing a benzene ring to generate an imine compound; reducing the imine compound to a secondary amine compound under the action of a reducing agent, wherein the secondary amine compound is the compound C; preferably, the aldehyde compound containing a benzene ring is A compound having a substituent arranged at any substitutable position on the benzene ring, the substituent being selected from halogen or C2-C6 alkyl; preferably, the reducing agent is selected from any one of the following: NaBH3CN or NaBH4; preferably, the imine compound is reduced to a secondary amine compound in an alcohol solution of NaBH3CN at a temperature of 20-30°C, preferably 23-26°C; more preferably, the alcohol solution is selected from any one of the following: methanol solution, ethanol, n-propanol or isopropanol.

[0026] Furthermore, compound C is subjected to a coupling reaction with an acrylic compound to obtain a trimethoprim derivative TMP3-NH2, including: under the action of a coupling agent, compound C is subjected to a coupling reaction with an acrylic compound to obtain an intermediate 5; intermediate 5 is subjected to a deamination protecting group reaction to obtain a trimethoprim derivative TMP3-NH2; preferably, the coupling agent is selected from any one of the following: 4-(4,6-dimethoxy-1,3,5-triazine-2-yl)-4-methylmorpholinium-4-ium chloride; preferably, the deamination protecting group reaction is carried out under hydrochloric acid and ethyl acetate conditions.

[0027] Furthermore, the structural formula of the substituted amine compound is H2N-(CH2) n -R, n is selected from any natural number of 2 to 3, and R is selected from bromine; preferably, compound A is obtained by removing a methyl group from trimethylbenzylidine; preferably, trimethylbenzylidine is placed at a temperature of 95 to 105°C, preferably 98-102°C, in hydrobromic acid with a volume concentration of 30 to 50%, preferably 35-45%, for demethylation to obtain compound A; preferably, the duration of the demethylation reaction is 30 to 60 min, more preferably 40 to 50 min.

[0028] According to the second aspect of the present application, a trimethoprim derivative TMP3-NH2 prepared by the above preparation method is provided.

[0029] According to the third aspect of the present application, a label protein based on a trimethoprim derivative is provided, wherein the trimethoprim derivative label protein is obtained by reacting a trimethoprim derivative TMP3-NH2 prepared by any of the above preparation methods with a labeling molecule through the amino group at the terminal; preferably, the labeling molecule is selected from any one of the following: biotin, rhodamine dyes or cyanine dyes; preferably, the labeling molecule is selected from any one of the following: biotin, TMR, SiR, Cy5, Map555 or JF549.

[0030] According to a fourth aspect of the present application, a protein label complex is provided, comprising a target protein and a label coupled to the target protein, wherein the label is selected from the above-mentioned label protein based on trimethoprim derivatives.

[0031] Furthermore, the target protein is an eDHFR fusion protein. Preferably, the eDHFR fusion protein is formed by fusing eDHFR to the N-terminus or C-terminus of the target protein, or by fusing eDHFR into the loop domain region of the target protein; preferably, the eDHFR fusion protein is formed by fusing eDHFR to the N-terminus or C-terminus of any of the following target proteins: a nuclear protein, an endoplasmic reticulum protein, a cytoskeletal protein, an extracellular membrane protein or a mitochondrial inner membrane protein; preferably, the nuclear protein is selected from H2B or NLS; preferably, the endoplasmic reticulum protein is selected from Sec61b; preferably, the cytoskeletal protein is selected from F- actin or tubulin; preferably, the extracellular membrane protein is selected from Tomm20; preferably, the mitochondrial inner membrane protein is selected from adenosine triphosphate synthase membrane subunit E or cytochrome C oxidase subunit 8A; preferably, the eDHFR fusion protein is formed by fusion of eDHFR into the ring domain of firefly luciferase, and more preferably, the ring domain of firefly luciferase is selected from any one of the following: T74 / N75, Y178 / D179, F331 / H332, V384 / N385, R437 / L438, S504 / Q505 or T508 / A509.

[0032] According to the fifth aspect of the present application, a method for labeling a target protein is provided, the labeling method comprising: forming an eDHFR fusion protein with the target protein and eDHFR; and specifically binding the above-mentioned label protein based on trimethoprim derivatives to eDHFR to form an eDHFR fusion protein with a trimethoprim derivative protein label.

[0033] According to the sixth aspect of the present application, a method for imaging a target protein is provided, the imaging method comprising: placing cells containing the target protein under an imaging device for imaging observation, wherein the target protein is a protein labeled according to the above-mentioned labeling method; preferably, the imaging device is a confocal microscope, a structured light illumination microscope, a random optical reconstruction microscope and / or a stimulated emission depletion microscope; preferably, the cells are living cells.

[0034] According to the seventh aspect of the present application, there is provided the use of the above-mentioned trimethoprim derivative-based label protein in protein-related research.

[0035] Furthermore, protein-related research includes any one or more of protein purification, screening of candidate small molecule drugs binding to proteins, protein interactions, protein imaging, localization and tracking; preferably, protein imaging includes multicolor imaging with other imaging labels, and other imaging labels include Halo labels and / or SNAP labels.

[0036] By applying the technical solution of the present invention, the distance between the TMP parent nucleus and the reactive group acrylamide bond for covalent labeling is further shortened from 7 atoms to 3 atoms by using substituted amine compounds for O-alkylation reaction, thereby making the distance between acrylamide and the sulfhydryl group of eDHFR closer, thereby achieving faster covalent labeling. In addition, the introduction of a rigid benzene ring as a connecting molecule between the TMP parent and the reporter group prevents the connected benzylamine from attacking acrylamide itself, shortens the synthesis path, reduces the difficulty of synthesis, and makes the molecule more concise as a whole. The small molecule TMP3-NH2 of the third-generation TMP-Tag synthesized by the above-mentioned synthesis method has an NH2 at its end that facilitates the connection of various dyes or other functionalized small molecules. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] The drawings constituting a part of the present application are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the drawings:

[0038] Figure 1 A schematic diagram of the labeling principle of SNAP-tag and CLIP-tag in the prior art is shown.

[0039] Figure 2 A schematic diagram of the labeling principle of HaloTag in the prior art is shown.

[0040] Figure 3 The figure shows a schematic diagram of the second generation TMP-tag labeling principle in the prior art.

[0041] Figure 4A A schematic diagram of the labeling principle of TMP-tag in the prior art is shown.

[0042] Figure 4B A schematic diagram of the labeling principle of the third-generation TMP-tag provided in the present application is shown.

[0043] Figure 5a The covalent labeling conditions of TMP3-Biotin and eDHFR:L28C protein mutant provided by the present application at different reaction times are shown.

[0044] Figure 5b The graph shows the change in fluorescence intensity of rhodamine dye (TMR, SiR) and Cy5 covalently labeled eDHFR:L28C with TMP3 of the present application as a function of labeling time.

[0045] Figure 5c The change in fluorescence intensity of eDHFR:L28C covalently labeled with TMP3-Cy5 over time is shown;

[0046] Figure 5d The covalent labeling kinetic parameters t of biotin, TMR, JF549 and SiR when labeling eDHFR:L28C by TMP3 of the present application are shown. 1 / 2 (mark half the time required).

[0047] Figure 6a and Figure 6b The imaging effect of covalent TMP-tag on living cells is shown; wherein, Figure 6a The molecular structure of TMP3-Map555 and its subcellular organelle structure labeled Confocol are shown; Figure 6b The molecular structure of TMP3-SiR and its labeled subcellular organelle structure are shown; Figure 6a and Figure 6b The scale bar is 20 μm.

[0048] Figure 7a and Figure 7b The fluorescence intensity of HeLa cells transiently transfected with NLS-sfGFP-SNAP-Halo-eDHFR:L28C labeled with three tags, Halo, SNAP and TMP3, monitored at different time points under a high-content fluorescence microscope; Figure 7a Shown is the time when the fluorescence intensity of cells labeled with different tags reaches half saturation; Figure 7b Shown are the saturated brightness of the three tag markers.

[0049] Figure 8a and Figure 8b The subcellular localization of three tags, Halo, TMP3 and SNAP, in HeLa cells is shown. Figure 8a It shows that both TMP and Halo tag are cytoplasmic localized, while SNAP is partially nuclear localized; Figure 8b The figure shows the localization of fusion proteins formed by directly fusion of three tags to the C-terminus of target proteins (mitochondrial inner membrane proteins: MTS positive control, ATP5ME and Cox8A) in subcellular structures.

[0050] Figure 9a A schematic diagram of the three-dimensional structure of firefly luciferase is shown.

[0051] Figure 9b The difference in luciferase activity when eDHFR is inserted into different loops of firefly luciferase is shown.

[0052] Fig.10a The figure shows the comparison of fluorescence intensity of three tags, TMP3, Halo and SNAP, inserted into sfGFP.

[0053] Fig.10b The figure shows the expression comparison of fusion proteins of three tags, TMP3, Halo and SNAP, inserted into sfGFP.

[0054] Fig.11a The schematic diagram shows the structure of TMP3 inserted into the N23 or L24 site in two different ways when an eDHFR is fused to eDHFR.

[0055] Fig.11b The labeling kinetic curves of fluorescence intensity at different labeling time points are shown, in which TMP3-TMR is inserted into the N23 or L24 site in two different ways for labeling;

[0056] Fig.12a Schematic representation of the structure of eDHFR:L28C inserted into mammalian dopamine receptor D1 is shown.

[0057] Figure 12b Shown is normal membrane localization after TMP3-SiR staining in HeLa cells.

[0058] Fig.13a and Fig.13b The figures show the images of HeLa cells transiently expressing Lifeact-eDHFR:L28C and Tomm20-Halo after staining with TMP3-SiR and Halo-TMR under confocal microscopy and STED microscopy, respectively.

[0059] Fig.13c The figure shows the multicolor labeling and imaging of HeLa cells transiently expressing three marker proteins using three dyes, where TMP3-MaP555 labels F-actin through Lifeact-eDHFR:L28C, Halo-SiR labels Tomm20, and H2B is labeled with SNAP-fluorescein. DETAILED DESCRIPTION

[0060] It should be noted that, in the absence of conflict, the embodiments and features in the embodiments of the present application can be combined with each other. The present invention will be described in detail below in conjunction with the embodiments.

[0061] As mentioned in the background technology, the TMP labeling rate in the prior art is low. In order to further improve the labeling rate, the present application improves the synthesis route of TMP compounds and provides a series of protection schemes of the present application based on the improved TMP compounds.

[0062] In a first typical embodiment of the present application, a method for preparing a trimethoprim derivative is provided, the method comprising: performing an O-alkylation reaction on a compound A and a substituted amine compound to obtain a compound B; performing a reductive amination reaction on the compound B to introduce a benzene ring into the compound B to obtain a compound C; performing a coupling reaction on the compound C and an acrylic acid compound to obtain a trimethoprim derivative TMP3-NH2;

[0063] Wherein, the structural formula of compound A is: The structural formula of the substituted amine compound is: H2N-(CH2) n -R, wherein n is selected from any natural number from 2 to 6, and R is selected from halogen; the structural formula of compound B is: The acrylic compound is acrylic acid or acryloyl chloride; the structural formula of compound C is: The structural formula of trimethoprim derivative TMP3-NH2 is:

[0064]

[0065] The synthesis method of TMP compounds provided in the above-mentioned embodiments provided in the present application further shortens the distance between the TMP nucleus and the acrylamide bond of the reactive group for covalent labeling from 7 atoms to 3 atoms, so that the distance between acrylamide and the thiol group of eDHFR is closer, thereby achieving faster covalent labeling. In addition, the partially redundant flexible PEG chain used to connect dyes (it should be noted that the dyes in this application refer to marker molecules that can be used for imaging, including known dye molecules, such as rhodamine or cyanine dyes; or other marker molecules with fluorescence, such as biotin) in the second-generation TMP is removed, and a rigid benzene ring is introduced as a linker between the TMP parent and the reporter group to prevent the benzylamine connected from attacking acrylamide itself, and also shortens the synthesis path and reduces the difficulty of synthesis, and the molecule as a whole is more concise. The above-mentioned synthesis method synthesizes the small molecule TMP3-NH2 used for the third-generation TMP-Tag, and the NH2 at its end is convenient for connecting various dyes or other functionalized small molecules.

[0066] In a preferred embodiment, compound A is subjected to an O-alkylation reaction with a substituted amine compound to obtain compound B, which comprises: conducting an O-alkylation reaction of compound A with a substituted amine compound having an amino protecting group to obtain compound D; and performing a deamination protecting group reaction on compound D to obtain compound B; preferably, the amino protecting group is selected from any one or more of Boc, Cbz, Fmoc, Alloc, Teoc, Pht, Tos, Tfa, Trt, Dmb, PMB and Bn; and preferably, the deamination protecting group reaction is performed under hydrochloric acid and ethyl acetate conditions.

[0067] In the above preferred embodiment, by selecting a substituted amine compound (such as Br-CH2CH2-NHBoc) for O-alkylation reaction, compared with other compounds in the prior art, such as Br-(CH2)4-COOEt (such as in US2006 / 0211007A1), the distance between the TMP mother core and the subsequent acrylamide bond used for covalent connection is shortened from 7 atoms to 3 atoms, making the distance between the sulfhydryl group of acrylamide and eDHFR closer, thereby achieving faster covalent labeling.

[0068] In a preferred embodiment, the method of subjecting compound B to a reductive amination reaction to introduce a benzene ring into compound B to obtain compound C comprises: reacting compound B with an aldehyde compound containing a benzene ring to generate an imine compound; reducing the imine compound to a secondary amine compound under the action of a reducing agent, wherein the secondary amine compound is compound C; preferably, the aldehyde compound containing a benzene ring is selected from Or a compound having a substituent at any substitutable position on its benzene ring, wherein the specific substituent is selected from halogen (for example, bromine, chlorine or fluorine) or C2-C6 alkyl (for example, methyl, ethyl, n-propyl or isopropyl, etc.), and the substituted position can be at the ortho and / or meta position of the imino group; preferably, the reducing agent is selected from any one of the following: NaBH3CN or NaBH4; preferably, the imine compound is reduced to a secondary amine compound in an alcohol solution of NaBH3CN at a temperature of 20-30°C, preferably 23-26°C; more preferably, the alcohol solution is selected from any one of the following: methanol solution, ethanol, n-propanol or isopropanol.

[0069] In the above preferred embodiment, a rigid benzene ring is introduced as a linker between the TMP matrix and the reporter group (i.e., the dye connected by reaction with -NH2), thereby preventing the connected benzylamine from attacking acrylamide itself. At the same time, the synthesis path is shortened and the difficulty of synthesis is reduced, and the overall molecule is simpler.

[0070] In a preferred embodiment, coupling reaction of compound C with an acrylic compound to obtain a trimethoprim derivative TMP3-NH2 comprises: coupling reaction of compound C with an acrylic compound under the action of a coupling agent to obtain an intermediate 5; deaminating the intermediate 5 to obtain the trimethoprim derivative TMP3-NH2; preferably, the coupling agent is selected from any one of the following: 4-(4,6-dimethoxy-1,3,5-triazine-2-yl)-4-methylmorpholinium-4-ium chloride; preferably, the deaminating protective group reaction is carried out under hydrochloric acid or ethyl acetate conditions.

[0071] In a preferred embodiment, compound A is prepared by the following method: removing a methyl group from trimethylbenzylidine to obtain compound A; preferably, placing trimethylbenzylidine in a temperature of 95-105° C., preferably 98-102° C., in a hydrobromic acid with a volume concentration of 30-50%, preferably 35-45%, for demethylation reaction to obtain compound A; preferably, the demethylation reaction time is 30-60 min, more preferably 40-50 min. Under the above conditions, methyl removal at a specific position can be achieved.

[0072] In a second typical embodiment of the present application, a trimethoprim derivative TMP3-NH2 prepared by any of the above preparation methods is provided. Figure 4B As shown, the distance between the TMP core and the acrylamide bond of the reactive group used for covalent labeling in the trimethoprim derivative TMP3-NH2 is 3 atoms, and the distance between acrylamide and the thiol group of eDHFR is closer, which can achieve faster covalent labeling.

[0073] In a third typical embodiment of the present application, a label protein based on a trimethoprim derivative is provided. The label protein based on a trimethoprim derivative is obtained by reacting the trimethoprim derivative TMP3-NH2 prepared by the above preparation method with a labeling molecule through the terminal amino group reaction.

[0074] Among them, the labeling molecule varies according to the research or use purpose, and the labeling molecule is selected from any one of the following: biotin, rhodamine dyes or cyanine dyes; preferably, the labeling molecule is selected from any one of the following: biotin, TMR, SiR, Cy5, JF549, Map555.

[0075] In a fourth typical embodiment of the present application, a protein label complex is provided, comprising a target protein and a label coupled to the target protein, wherein the label is selected from the above-mentioned label protein based on trimethoprim derivatives. The protein label complex containing the above-mentioned label protein based on trimethoprim derivatives can be imaged, tracked and / or located under an ultra-high resolution microscope, thereby realizing imaging observation of living cells.

[0076] In a preferred embodiment, the target protein is an eDHFR fusion protein. More preferably, the eDHFR fusion protein is formed by fusing eDHFR to the N-terminus or C-terminus of the target protein, or by inserting eDHFR into the loop domain region of the target protein; further preferably, the eDHFR fusion protein is formed by fusing eDHFR to the N-terminus or C-terminus of any of the following target proteins: a nuclear protein, an endoplasmic reticulum protein, a cytoskeletal protein, an extracellular membrane protein or a mitochondrial inner membrane protein; preferably, the nuclear protein is selected from H2B or NLS; preferably, the endoplasmic reticulum protein is selected from Sec61b; preferably, the cytoskeletal protein Preferably, the extracellular membrane protein is selected from F-actin or tubulin; preferably, the extracellular membrane protein is selected from Tomm20; preferably, the mitochondrial inner membrane protein is selected from adenosine triphosphate synthase membrane subunit E or cytochrome C oxidase subunit 8A; preferably, the eDHFR fusion protein is formed by fusion of eDHFR into the ring domain of firefly luciferase, and more preferably, the ring domain of firefly luciferase is selected from any one of the following: T74 / N75, Y178 / D179, F331 / H332, V384 / N385, R437 / L438, S504 / Q505 or T508 / A509.

[0077] The examples of the present application demonstrate that the eDHFR fusion protein labeled with the tag protein of the present application can be imaged, tracked and / or positioned under ultra-high resolution microscopy equipment, thereby enabling observation of subcellular structures of living cells.

[0078] In a fifth typical embodiment of the present application, a method for labeling a target protein is provided, the labeling method comprising: forming an eDHFR fusion protein with the target protein and eDHFR; and forming an eDHFR fusion protein with a trimethoprim tag by specifically binding the above-mentioned label protein based on a trimethoprim derivative to eDHFR. The specific steps for forming the fusion protein can refer to the existing fusion protein expression method, and are not specifically limited in the present application.

[0079] In a sixth typical embodiment of the present application, a method for imaging a target protein is provided, the imaging method comprising: placing cells containing the target protein under an imaging device for imaging observation, wherein the target protein is a protein labeled according to the above-mentioned labeling method; preferably, the imaging device is a confocal microscope, a structured light illumination microscope, a random optical reconstruction microscope and / or a stimulated emission depletion microscope; preferably, the cells are living cells.

[0080] In the seventh typical embodiment of the present application, any of the above-mentioned label proteins based on trimethoprim derivatives is provided for use in protein-related research. Specifically, protein-related research includes any one or more of protein purification, screening of candidate small molecule drugs bound to proteins, protein interactions, protein imaging, positioning and tracking. The above-mentioned various applications are application scenarios for known protein labels. The improved TMP label protein of the present application has the advantage of a faster labeling rate relative to the existing TMP label protein. Therefore, in the field of labeling imaging, positioning and tracking, it is more suitable for use in the field of ultra-microscopic resolution imaging (such as imaging under structured light illumination microscopy, random optical reconstruction microscopy and / or stimulated emission depletion microscopy). The above-mentioned protein imaging includes multicolor imaging with other imaging labels, and other imaging labels include Halo labels and / or SNAP labels.

[0081] The application of the tag protein based on trimethoprim derivatives in the present application in protein purification, screening of candidate small molecule drugs (herein chemical small molecule drugs) bound to proteins, protein interactions, etc. is exemplified in combination with the following specific scenarios:

[0082] For protein purification: The target protein (abbreviated as POI in this application, i.e. Protein of Interest, and the meaning of other positions is the same as here) is fused with the eDHFR:L28C protein and covalently bound to the resin containing the immobilized acrylamide ligand. The 6×His-TEV protease can cut the target protein from the resin, while the eDHFR:L28C protein remains on the resin. After passing through the nickel column, the TEV protease and POI can be separated and purified, and finally the pure target protein without tags and TEV is obtained. Compared with traditional affinity tags (such as His6Tag, GST and MBP), this method has strong specificity and can obtain high recovery rate and high purity target protein.

[0083] For drug screening: fix Histag-eDHFR:L28C on multiple nickel affinity chromatography glass beads through His tag, add different small molecule drugs modified with TMP tag ligand for screening, realize covalent connection between drug and TMP tag, then add target protein solution, mix evenly at room temperature, and wash away POI with PBS. Add sample buffer to nickel affinity chromatography glass beads, and use SDS-PAGE to detect which small molecule drugs can bind His tag-eDHFR:L28C to POI, so as to realize screening of inhibitors of target protein in multiple compounds. The cost is relatively low and the cycle is fast.

[0084] For protein interaction detection (BRET): Protein interaction regulates most biological processes - from short-term interactions in signal transduction to large multi-protein complex assembly, it is now a hot topic of research. At present, protein interaction detection methods are divided into two categories. One is non-cellular detection. The detection results lack verification at the cellular level and lack persuasiveness. Therefore, cellular detection is still needed for verification. The bioluminescence resonance energy transfer (BRET) method is one of the very few methods that can detect protein interactions at the living cell level. BRET is when two protein molecules interact, one protein is fused to express the donor luciferase protein, and the emission spectrum generated by the enzymatic reaction overlaps with the excitation spectrum of the acceptor fluorescent group labeled by another protein, inducing the acceptor molecule to emit fluorescence. Similar to FRET, BRET significantly weakens as the distance increases. The interaction between proteins can be indirectly detected by detecting the BRETratio. In the experiment, cells were allowed to express POI A fused with luciferase protein and POI B fused with eDHFR:L28C protein at the same time. Luciferase was used as an energy donor and the fluorescent group labeled with TMP Tag protein was used as an energy acceptor to detect the BRET ratio signal and explore the interaction between the two target proteins. This method does not require excitation light, has low background, and causes less damage to cells, and can detect protein interactions at the living cell level.

[0085] Below we will further illustrate the beneficial effects of the present application with reference to specific embodiments.

[0086] Example 1

[0087] The synthesis path is as follows:

[0088]

[0089] First, a methyl group of trimethylbenzylidine is removed under high temperature conditions of hydrobromic acid to obtain compound 1. Compound 1 is subjected to O-alkylation reaction with 2-bromoethylamine and then the amino group is deprotected with hydrochloric acid to obtain compound molecule 3. The benzene ring is then introduced through a reductive amination reaction. Compound 3 first reacts with aldehyde to form an imine, and then under mild conditions, the reducing agent NaBH3CN reduces it to secondary amine 4. Acrylic acid and secondary amine 4 are coupled under the action of a highly efficient coupling agent 4-(4,6-dimethoxy-1,3,5-triazine-2-yl)-4-methylmorpholinium-4-ium chloride (DMTMM) to obtain the key intermediate 5. Finally, the terminal amino group is removed to obtain the third-generation TMP molecule: TMP3-NH2. TMP3 and dye can obtain TMP3-dyes by reacting the terminal amino group.

[0090] The NMR data and mass spectrometry data of the final product are as follows:

[0091] The NMR data are:

[0092]

[0093] 1 H NMR (400MHz, Methanol-d4) δ7.50(d,J=8.0Hz,1H),7.44(d,J=8.3Hz,1H),7.37(dd,J=10.5,8.0Hz, 2H),7.28–7.22(m,1H),6.99(dd,J=16.8,10.6Hz,1H),6.76(dd,J=16.7,10.5Hz,0H),6.58(d,J=2. 0Hz,2H),6.30(ddd,J=16.8,7.3,2.0Hz,1H),5.76(ddd,J=10.5,7.5,2.0Hz,1H),5.03(s,1H),4.13 (d, J=9.2Hz, 3H), 4.08 (t, J=5.2Hz, 1H), 3.80 (d, J=4.5Hz, 6H), 3.74 (q, J=5.6Hz, 2H), 3.68 (s, 2H).

[0094] 13 C NMR(101MHz,Methanol-d4)δ168.12,167.84,164.92,154.74,153.48,139.00,135.78,132.73,132.10,12 9.26,128.94,128.53,128.25,127.89,127.20,109.67,105.73,71.52,71.32,55.27,52.18,42.67,32.58.

[0095] The mass spectrometry data are:

[0096] MS(m / z):calculated for C 26 H 32 N6O4[M+H] + 493.25, found 493.3.

[0097] Example 2

[0098] The reaction steps are similar to those of Example 1, except that compound 1 is subjected to O-alkylation reaction with 3-bromopropylamine and then the amino group is deprotected with hydrochloric acid, thereby obtaining the following carbon chain-extended TMP3'-NH2 as the final product.

[0099]

[0100] The NMR data and mass spectrometry data of the final product are as follows:

[0101] The NMR data are:

[0102]

[0103] 1 H NMR(400MHz,Chloroform-d)δ7.69(d,J=7.4Hz,2H),7.21(dd,J=21.8,6.9Hz,4H), 6.81(dd,J=16.7,10.4Hz,1H), 6.58(dd,J=16.7,10.3Hz,1H), 6.42(dd,J=16.6,2. 1Hz,2H),6.37(d,J=4.7Hz,4H),5.71(d,J=2.1Hz,1H),4.36–4.27(m,2H),4.21(t, J=5.1Hz,2H),4.05(t,J=6.1Hz,2H),3.79(d,J=8.5Hz,6H),3.67(d,J=7.5Hz,3H).

[0104] The mass spectrometry data are:

[0105] MS(m / z):calculated for C 27 H 34 N6O4[M+H] + 507.26, found 507.3.

[0106] It should be noted that the third generation TMP3-NH2 molecule provided in Example 1 above effectively improves the time for TMP3-dye and eDHFR to reach half of the labeling, and the half labeling time of TMP-biotin in vitro is shortened from 8 minutes to about 3 minutes, and finally the in vitro labeling is completed instantly. In terms of molecular design, the distance between the TMP mother nucleus and the acrylamide bond of the reactive group used for covalent labeling is further shortened from 7 atoms to 3 atoms, so that the distance between the acrylamide and eDHFR sulfhydryl groups is closer, thereby achieving faster covalent labeling.

[0107] On the other hand, we removed the redundant flexible PEG chain used to connect the dye in the second generation and introduced a rigid benzene ring as a linker between the TMP matrix and the reporter group to prevent the connected benzylamine from attacking acrylamide itself. This also shortened the synthesis path and reduced the difficulty of synthesis, making the overall molecule simpler.

[0108] In summary, we synthesized the small molecule TMP3-NH2 for the third-generation TMP-Tag. Based on the common characteristics of the first-generation and second-generation TMP molecules that can connect to various dyes or other functional small molecules, it can be seen that the NH2 at the end of the third-generation TMP-NH2 of the present application can also connect to various dyes or other functional small molecules.

[0109] In addition, in Example 2, we also synthesized TMP3'-NH2, which has one more carbon than the TMP3-NH2 linker. It shows that the synthesis strategy of the present application is compatible with linkers of different carbon chain lengths. According to the characteristics of the second-generation TMP molecule, the atomic distance between the TMP parent and acrylamide is shortened, which helps to shorten the time required for the labeling reaction. In theory, if the atomic distance between the TMP parent and acrylamide is shorter than 7 atoms, the time required for half labeling will be shortened accordingly. In the following examples, the time required for half labeling of TMP-Biotin, which has the fastest labeling rate among the second-generation TMP molecules, is 8 minutes as a reference object, and the time required for half labeling of the third-generation TMP3-Biotin molecule of the present application is compared.

[0110] Example 3

[0111] We tested the in vitro labeling reaction of TMP3-biotin and eDHFR:L28C protein mutant. We found that Figure 5a (The marker molecule is TMP3-biotin), Figure 5b , Figure 5c and Figure 5d As shown in the figure, after 1 hour of reaction, it can be seen that the binding strength of TMP3 is stronger than that of the second generation TMP. For the labeling reaction of TMP3-biotin, the time required for half of the reaction is shortened from 8 minutes to about 3 minutes.

[0112] In addition, commonly used rhodamine dyes (TMR, SiR), Cy5 and JF549 can also effectively label eDHFR:L28C through TMP3. Gel fluorescence saturation experiments show (see Figure 5b and 5c ), TMP3-rhodamine labels eDHFR:L28C slightly faster than TMP3-Cy5, but the covalent labeling of these fluorophores 1 / 2 All in about 10 minutes. Figure 5d It can be seen from the labeling kinetics that after the third-generation TMP3-NH2 is coupled with fluorescent dyes or other labeling molecules, the time required for labeling half of the time is also the shortest for labeling biotin.

[0113] Example 4

[0114] The dye Map555 and SiR with good cell permeability developed by Kai Johnsson's group were linked to TMP3-NH2 and tested on HeLa cells. Figure 6a and Figure 6b As shown, cell imaging experiments showed that TMP3-Map555 could specifically bind to the nuclear protein (H2B), endoplasmic reticulum protein (Sec61b), and cytoskeletal protein F-actin (Lifeact) coupled to eDHFR:L28C, showing good cellular localization ( Figure 6a The molecular structure of TMP3-Map555 and its subcellular organelle structure labeled with Confocol are shown). Afterwards, TMP3 coupled with SiR also showed high-brightness and high-specificity staining, and more precise cell structures could be displayed in STED super-resolution imaging ( Figure 6b The molecular structure of TMP3-SiR and its labeled subcellular organelle structure are shown, scale bar: 20 μm). These experiments all indicate that TMP3 can highly specifically label subcellular structures of living cells.

[0115] Example 5

[0116] We compared the ability of three Halo SNAP TMP tags to enter cells. HeLa cells transiently transfected with NLS-sfGFP-SNAP-Halo-eDHFR:L28C were labeled with different Tag-SiRs and the fluorescence intensity was monitored at different time points using a high-content fluorescence microscope. A curve was drawn with time as the horizontal axis and the fluorescence ratio of SiR to sfGFP as the vertical axis. The time to half saturation can reflect the speed at which different tags enter cells, such as Figure 7a As shown, Halo-SiR takes about 5 minutes, TMP3-SiR takes about 20 minutes, and SNAP-SiR takes the slowest, about 2 hours. Figure 7b As shown in the figure, the saturation brightness of the three tags, Halo-SiR is slightly brighter than TMP3-SiR, and TMP3-SiR is slightly brighter than SNAP-SiR. However, the difference in saturation brightness is not as obvious as the difference in cell entry rate. Based on this result, when selecting tags for live cell imaging, the recommended priority level is: HaloTag>TMP3-Tag>SNAP-Tag.

[0117] Example 6

[0118] This example evaluates the compatibility of protein tags as fusion proteins. HaloTag, TMP-tag and SNAP-Tag were transiently expressed under the CMV promoter in HeLa cells. Figure 8aAs shown, after staining, TMP-Tag and HaloTag were both cytoplasmic localized, while SNAP-Tag was partially nuclear localized. Next, we fused these three tags with selected mitochondrial proteins. Two mitochondrial inner membrane proteins, adenosine triphosphate synthase membrane subunit E (ATP5ME) and cytochrome C oxidase subunit 8A (Cox8A), were selected, and mitochondrial targeting sequence (MTS) fusion tag proteins were used as positive controls.

[0119] For comparison purposes, the tag protein was directly fused to the C-terminus of the target protein. The localization of the fusion protein was detected after staining. TMP-Tag and SNAP-Tag fusion proteins were cleanly localized to mitochondria. However, ATP5ME-Halo and Cox8A-Halo were mislocalized to the endoplasmic reticulum (ER) or cytoplasm (e.g. Figure 8b This indicates that the larger Halo Tag (36KD) may affect the target protein structure and function compared with the smaller tags DHFR (18KD) and SNAP-Tag (19KD).

[0120] Example 7

[0121] In addition to the traditional method of fusing tags to the N-terminus or C-terminus of the target protein, modern protein engineering is increasingly focusing on the exploration of protein loop domains. Since loop fusion is more likely to affect structure and function, inserting tags into the loop region of the target protein has become a new challenge for protein tagging technology. As a promising loop fusion tag, the biggest advantage of eDHFR is that the distance between NC is short and eDHFR is The NC distances of HaloTag and SNAP-Tag are and We therefore tested labeling of the loop regions of various proteins with TMP3.

[0122] First, we tried to insert eDHFR into different loops of firefly luciferase. The three-dimensional structure of firefly luciferase is shown in Figure 2. Figure 9a As shown, 10 flexible amino acids (specifically GGGGSGGGGS in this embodiment, SEQ ID NO: 1) are inserted into the N-terminus and C-terminus of eDHFR as connectors.

[0123] Assayed in E. coli lysates, luciferase activity was inhibited in all loop fusion constructs but not in the terminal fusions (e.g. Figure 9bThe results show that inserting the tag into the loop region of the target protein does affect the function of the protein. Among the loop fusion positions, T508 / X509 luc508-DHFR showed the highest luminescence rate.

[0124] Using this site, the loop tag was then replaced with eDHFR:L28C, HaloTag, and SNAP-Tag. Although the interface is in principle sufficient to bridge the gaps between the N / C of all tags, eDHFR:L28C inserted at position 508 showed the highest luciferase activity, confirming that protein tags with a small N / C distance are indeed more suitable for loop insertion (e.g. Figure 9b shown).

[0125] Example 8

[0126] We also compared the insertion of TMP-Tag, HaloTag, and SNAP-Tag into sfGFP. After constructing the plasmid and expressing it in E. coli, we detected that the expression of sfGFP-(eDHFR:L28C) strains was about 50% after IPTG induction, and the expression of SNAP and Halo inserted into sfGFP was less than 10% (e.g. Fig.10a eDHFR:L28C inserted in the sfGFP loop can be labeled with TMP3-TMR. Insertion of eDHFR, SNAP, and Halo in the sfGFP loop does not affect the fluorescent properties of the protein, but the fusion of eDHFR shows a higher expression level (as shown in Fig.10b shown).

[0127] Example 9

[0128] An eDHFR was fused to eDHFR to explore the labeling dynamics of TMP3 on the inserted and inserted proteins. The insertion site was selected at N23 / L24, according to Fig.11a The fluorescence intensity at different labeling time points was measured in vitro using TMP3-TMR, and the labeling kinetic curve was drawn (e.g. Fig.11b As shown in Figure 2 ). It can be seen that the labeling time of eDHFR inserted into eDHFR:L28C was about 10 minutes, which was similar to that of eDHFR:L28C. However, the labeling of eDHFR:L28C inserted into eDHFR showed reduced reactivity (t 1 / 2 ~30 minutes). Considering that L28C is very close to the insertion site of N23 / L24, the results indicate that the structure of eDHFR:L28C as a loop tag is intact, and that an intact loop tag is a functional protein.

[0129] Example 10

[0130] Insert eDHFR:L28C into the membrane protein mammalian dopamine receptor D1 (DRD1) (eg Fig.12a In HeLa cells, TMP3-SiR staining showed normal membrane localization (as shown in Figure 12b shown).

[0131] Embodiment 11

[0132] TMP3-tag, HaloTag and SNAP-Tag are not only complementary protein tagging methods, they can also be used orthogonally in high-resolution microscopy to achieve multicolor imaging. For the first time, we imaged Tomm20 and F-actin simultaneously. HeLa cells transiently expressing Lifeact eDHFR:L28C and Tomm20-Halo were stained with TMP3-SiR and Halo-TMR (e.g. Fig.13a As shown). Confocal microscopy shows the structure of fibers and mitochondrial outer membrane. STED microscopy gives higher resolution images. We then tested dual-color labeling of mitochondria using two smaller tags: TMP3 and SNAP. Tomm20-eDHFR:L28C was labeled with TMP3-SiR, and ATP5ME-SNAP was labeled with SNAP-Map555. Fig.13b As shown, under STED microscopy, after 1 h of staining with 400 nM dye, the mitochondrial outer membrane protein Tomm20 showed the mitochondrial outer membrane structure, while ATP5ME was located in the inner membrane and matrix.

[0133] Example 12

[0134] HeLa cells transiently expressing three marker proteins were labeled with three dyes. Fig.13c As shown, TMP3-MaP555 was labeled to F-actin by Lifeact-eDHFR:L28C, Tomm20 was labeled by Halo-SiR, and H2B was labeled with SNAP-fluorescein. We achieved labeling and imaging of up to three protein targets using orthogonal self-labeling tags. The third generation of covalent TMP tags, together with HaloTag and SNAP-Tag, is expected to become a chemical biology tool for multi-protein analysis.

[0135] From the above description, it can be seen that the above embodiments of the present invention achieve the following technical effects:

[0136] 1) Faster labeling rate; 2) Simplified molecular synthesis; 3) Overall performance is better than the currently widely used SNAP-tag; 4) N / C is short and close, making it a better choice for protein loop region fusion; 5) Can be used in conjunction with the current HaloTag and SNAP tag to perform multi-color imaging of multiple cellular proteins.

[0137] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention. Sequence Listing <110> Beijing University <120> Trimethoprim derivatives, preparation methods thereof, label proteins based on trimethoprim derivatives and applications thereof <130> PN166650BJDXYL <160> 3 <170> SIPOSequenceListing 1.0 <210> 1 <211> 10 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> (1)..(10) <223> Flexible amino acid linker <400> 1 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1 5 10 <210> 2 <211> 6 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> (1)..(6) <223> FqV <220> <221> UNSURE <222> (3) <223> The 'Xaa' at location 3 stands for Gln, Arg, Pro, or Leu. <220> <221> UNSURE <222> (4)..(4) <223> The 'Xaa' at position 4 represents Gln, Arg, Pro, or Leu. <400> 2 Cys Cys Xaa Xaa Cys Cys 1 5 <210> 3 <211> 8 <212> PRT <213> Artificial Sequence <220> <221> PALMITATE <222> (1)..(8) <223> Flag-tag <400> 3 Asp Tyr Lys Asp Asp Asp Asp Lys 1 5

Claims

1. A method for preparing a trimethoprim derivative, characterized in that: The preparation method comprises: Compound A is subjected to O-alkylation reaction with a substituted amine compound to obtain compound B; Performing a reductive amination reaction on the compound B to introduce a benzene ring into the compound B to obtain a compound C; The compound C is coupled with an acrylic compound to obtain a trimethoprim derivative TMP3-NH2; Wherein, the structural formula of the compound A is: ; The structural formula of the substituted amine compound is: H2N-CH2-(CH2) n -R, wherein n is selected from any natural number from 1 to 5, and R is selected from halogen; The structural formula of the compound B is: ; The acrylic compound is acrylic acid or acryloyl chloride; The structural formula of the compound C is: , R2 is an amino protecting group; The structural formula of the trimethoprim derivative TMP3-NH2 is: 。 2. The preparation method according to claim 1, characterized in that: Compound A is subjected to O-alkylation reaction with a substituted amine compound to obtain compound B, including: The compound A is subjected to an O-alkylation reaction with a substituted amine compound having an amino protecting group to obtain a compound D; The compound D is subjected to a deamino protecting group reaction to obtain the compound B.

3. The preparation method according to claim 2, characterized in that: The amino protecting group is selected from any one or more of Boc, Cbz, Fmoc, Alloc, Teoc, Pht, Tos, Tfa, Trt, Dmb, PMB and Bn.

4. The preparation method according to claim 2, characterized in that: The deamino protecting group reaction is carried out under hydrochloric acid and ethyl acetate conditions.

5. The preparation method according to claim 1, characterized in that: The compound B is subjected to a reductive amination reaction to introduce a benzene ring into the compound B to obtain a compound C, which comprises: reacting the compound B with the aldehyde compound containing the benzene ring to generate an imine compound; Under the action of a reducing agent, the imine compound is reduced to a secondary amine compound, and the secondary amine compound is the compound C.

6. The preparation method according to claim 5, characterized in that: The aldehyde compound containing the benzene ring is .

7. The preparation method according to claim 5, characterized in that: The reducing agent is selected from any one of the following: NaBH3CN or NaBH4.

8. The preparation method according to claim 5, characterized in that: The imine compound is reduced to a secondary amine compound in an alcohol solution of NaBH3CN at a temperature of 20-30°C.

9. The preparation method according to claim 8, characterized in that: The imine compound is reduced to a secondary amine compound in an alcohol solution of NaBH3CN at a temperature of 23-26°C.

10. The preparation method according to claim 9, characterized in that: The alcohol solution is selected from any one of the following: methanol solution, ethanol, n-propanol or isopropanol.

11. The preparation method according to claim 1, characterized in that: The compound C is coupled with the acrylic compound to obtain the trimethoprim derivative TMP3-NH2, which includes: Under the action of a coupling agent, the compound C is coupled with the acrylic compound to obtain an intermediate 5; The intermediate 5 is subjected to a deamino protecting group reaction to obtain the trimethoprim derivative TMP3-NH2.

12. The preparation method according to claim 11, characterized in that: The coupling agent is selected from any one of the following: 4-(4,6-dimethoxy-1,3,5-triazine-2-yl)-4-methylmorpholinium-4-ium chloride.

13. The preparation method according to claim 11, characterized in that: The deamino protecting group reaction is carried out under hydrochloric acid and ethyl acetate conditions.

14. The preparation method according to any one of claims 1 to 13, characterized in that: The structural formula of the substituted amine compound is H2N-(CH2): n -R, n is selected from any natural number between 2 and 3, and R is selected from bromine.

15. The preparation method according to claim 14, characterized in that: The compound A is obtained by removing a methyl group from trimethoxybenzylidine.

16. The preparation method according to claim 14, characterized in that: The trimethoxybenzylidine is placed in a hydrobromic acid solution with a volume concentration of 30-50% at a temperature of 95-105° C. to undergo a demethylation reaction to obtain the compound A.

17. The preparation method according to claim 14, characterized in that: The trimethoxybenzylidine is placed in a solution of hydrobromic acid with a volume concentration of 30-50% at a temperature of 98-102° C. to undergo a demethylation reaction to obtain the compound A.

18. The preparation method according to claim 14, characterized in that: The trimethoxybenzylidine is placed in a hydrobromic acid solution with a volume concentration of 35-45% at a temperature of 95-105° C. for demethylation reaction to obtain the compound A.

19. The preparation method according to claim 14, characterized in that: The trimethoxybenzylidine is placed in a hydrobromic acid having a volume concentration of 35-45% at a temperature of 98-102° C. for demethylation reaction to obtain the compound A.

20. The preparation method according to claim 14, characterized in that: The duration of the demethylation reaction is 30 to 60 minutes.

21. The preparation method according to claim 14, characterized in that: The duration of the demethylation reaction is 40 to 50 minutes.

22. The trimethoprim derivative TMP3-NH2 prepared by the preparation method according to any one of claims 1 to 21.

23. A protein tag based on trimethoprim derivatives, characterized in that: The trimethoprim derivative protein tag is obtained by reacting the trimethoprim derivative TMP3-NH2 prepared by the preparation method of any one of claims 1 to 21 with a labeling molecule through the terminal amino group reaction.

24. The protein tag according to claim 23, characterized in that The labeling molecule is selected from any one of the following: biotin, rhodamine dyes or cyanine dyes.

25. The protein tag according to claim 23, characterized in that The labeling molecule is selected from any one of the following: biotin, TMR, SiR, Cy5, Map555 or JF549.

26. A protein-label complex, comprising a target protein and a label coupled to the target protein, characterized in that: The tag is selected from the protein tag based on trimethoprim derivatives according to any one of claims 23 to 25, and the target protein is eDHFR fusion protein.

27. The protein tag complex according to claim 26, characterized in that The eDHFR fusion protein is formed by fusing eDHFR to the N-terminus or C-terminus of the target protein, or by inserting eDHFR into the loop domain region of the target protein.

28. The protein tag complex according to claim 26, characterized in that The eDHFR fusion protein is formed by fusing eDHFR with the N-terminus or C-terminus of any of the following target proteins: nuclear protein, endoplasmic reticulum protein, cytoskeleton protein, extracellular membrane protein or mitochondrial inner membrane protein.

29. The protein tag complex according to claim 28, characterized in that The cell nuclear protein is selected from H2B or nuclear localization sequence NLS.

30. The protein tag complex according to claim 28, characterized in that The endoplasmic reticulum protein is selected from Sec61b.

31. The protein tag complex according to claim 28, characterized in that The cytoskeletal protein is selected from F-actin or tubulin.

32. The protein tag complex according to claim 28, characterized in that The extracellular membrane protein is selected from Tomm20.

33. The protein tag complex according to claim 28, characterized in that The mitochondrial inner membrane protein is selected from adenosine triphosphate synthase membrane subunit E or cytochrome C oxidase subunit 8A.

34. The protein tag complex according to claim 28, characterized in that The eDHFR fusion protein is formed by inserting eDHFR into the loop domain of firefly luciferase.

35. The protein tag complex according to claim 34, characterized in that The ring domain region of the firefly luciferase is selected from any one of the following: T74 / N75, Y178 / D179, F331 / H332, V384 / N385, R437 / L438, S504 / Q505 or T508 / A509.

36. A method for labeling a target protein, characterized in that: The marking method comprises: The target protein and eDHFR form an eDHFR fusion protein; The protein tag based on the trimethoprim derivative according to any one of claims 23 to 25 is specifically combined with eDHFR to form an eDHFR fusion protein with the trimethoprim derivative protein tag.

37. A method for imaging a target protein for non-medical purposes, characterized in that: The imaging method comprises: placing cells containing the target protein under an imaging device for imaging observation, wherein the target protein is a protein labeled according to the labeling method according to claim 36.

38. The imaging method according to claim 37, characterized in that The imaging device is a confocal microscope, a structured light illumination microscope, a random optical reconstruction microscope and / or a stimulated emission depletion microscope.

39. The imaging method according to claim 37, characterized in that The cells are living cells.

40. Use of a trimethoprim derivative-based protein tag according to any one of claims 23 to 25 in protein-related research, wherein the protein-related research comprises any one or more of protein purification, screening of candidate small molecule drugs binding to proteins, protein interactions, protein imaging, localization, and tracking.

41. The use according to claim 40, The protein imaging includes multi-color imaging with other imaging labels, and the other imaging labels include Halo labels and / or SNAP labels.

Images