CN111848621B

CN111848621B - Lanthanide series cage-shaped compound and preparation method and application thereof

Info

Publication number: CN111848621B
Application number: CN202010604168.9A
Authority: CN
Inventors: 刘涛; 刘元忠
Original assignee: Suzhou Institute of Biomedical Engineering and Technology of CAS
Current assignee: Suzhou Institute of Biomedical Engineering and Technology of CAS
Priority date: 2020-06-29
Filing date: 2020-06-29
Publication date: 2022-05-27
Anticipated expiration: 2040-06-29
Also published as: CN111848621A

Abstract

The invention relates to a lanthanide series cage-shaped compound, a preparation method and application thereof, wherein diamine type cage-shaped terpyridine europium synthesized by the application number CN201610802501 is prepared by controlling the ratio of the diamine type cage-shaped terpyridine europium to a bifunctional coupling agent containing a succinimide group and a sulfhydryl reaction group and reacting at room temperature. The bifunctional coupling agent containing a succinimide group and a thiol-reactive group is selected from SMCC, Sulfo-SMCC, MBS, Sulfo-MBS, SIAB, Sulfo-SIAB, SMPB, Sulfo-SMPB, GMBS, Sulfo-GMBS, SBAP or SIA. The lanthanide cage compound is applied to homogeneous phase time-resolved fluorescence analysis, after the biological molecules are subjected to sulfhydrylation treatment, the lanthanide cage compound directly reacts with a coupling group, and the lanthanide cage compound can be used without further purification, so that the labeling process is greatly simplified.

Description

Lanthanide series cage-shaped compound and preparation method and application thereof

Technical Field

The invention relates to a synthesis technology of a complex, in particular to lanthanide metal (Eu) containing a single coupling group³⁺) A method for synthesizing a cryptate ether complex.

Background

For more than half a century, the mainstream immunoassay technology goes through the stages of radioimmunoassay, enzyme-linked immunoassay, chemiluminescence immunoassay and the like, and is a solid-phase immunoassay technology. The chemiluminescence immunoassay technology is born in the seventies of the last century, the main technology adopted is magnetic particle chemiluminescence, the core hypothesis is that the separation of specific immune complexes and biological matrixes can be realized certainly through magnetic separation, but in clinical practical application, due to the diversity of clinical specimens, cases of magnetic separation failure cannot be avoided, such as 'jump value' phenomenon caused by nonspecific adsorption due to fibrin adhesion, false positive caused by heterophilic antibodies and the like. Magnetic cleaning is a major factor affecting the reliability of the instrument itself. Therefore, the technology of immunoassay without washing has been a research hotspot for nearly two-thirty years.

The time-resolved fluorescence resonance energy transfer immunoassay (TR-FRET) technology was originally developed by a supramolecular rare earth cryptand ether probe discovered by German Bolames (brahms) company based on Jean-Marie Lehn, Nobel chemical prize-winning France, and overcomes the influence of abnormal alignment accuracy of samples such as hemolysis, hyperlipidemia, jaundice and the like in homogeneous detection. Compared with other immunoassay technologies, the technology has many advantages, does not need a solid phase carrier and a solid phase probe, and has the advantages of good precision and accuracy, simple operation, easy automation and miniaturization and the like.

The core of the time-resolved fluorescence resonance energy transfer method is that two fluorescence molecules are called donor and acceptor respectively, wherein the donor can absorb exciting light and emit fluorescence, and the fluorescence can also be transmitted to the acceptor in a fluorescence resonance energy transfer mode and emitted by the acceptor. Specifically, a chelate label of a rare earth element having a cryptic structure is used as a fluorescence donor, and a short-lived fluorescent molecule having a good spectral overlap with the fluorescence donor is used as a fluorescence acceptor, and Fluorescence Resonance Energy Transfer (FRET) occurs between the donor and the acceptor (second fluorescent label) of the cryptic compound of a rare earth element. In fluorescence resonance energy transfer, the lifetime of the acceptor emitted fluorescence approaches that of the donor. Because the donor fluorescence decay period is long, the donor can induce the acceptor to emit fluorescence for a long time, and the fluorescence generated after the acceptor is excited can last for a long time, so that the self-scattered fluorescence with short lifetime can be distinguished through time resolution, and the FRET signal can be easily distinguished from the background of the fluorescence with short lifetime.

The lanthanide series compound has the characteristic of long fluorescence lifetime, and can be used for time-resolved fluorescence detection. In particular, the cage lanthanide complex has good stability and tolerance to various harsh factors because the rare earth ions are positioned in the center of the cage ligand, and can be directly used for detecting complex biological samples such as serum, plasma and the like.

The europium cage-shaped compound formed by terpyridine has high synthesis difficulty and low yield. In particular, compounds for biomarkers ideally have one and only one coupling group, both to achieve molecular labeling and to avoid cross-linking reactions. Since bipyridines generally have symmetric derivatizing groups, compounds generally have two coupling sites, which are not conducive to labeling.

The applicant filed chinese patent application 2016.09.05 for a lanthanide metal cryptate complex, its preparation method and use (application No. CN201610802501, hereinafter referred to as "patent 1"). A molecule having a symmetrical structure (diamine) is described, as shown in formula (I):

the product is in high yield and easy to synthesize, but cannot be used directly for labeling.

The patent filed by the applicant 2015-07-30 is a lanthanide compound and a preparation method and application thereof (CN201510455349, granted). Disclosed are compounds of formula (II):

the synthetic method needs to accurately control the proportion of amination reagents such as ethylenediamine and the like in an amination stage to synthesize an amine structure, realizes single amine group, but cannot be directly used for labeling. Moreover, this synthesis method has a significantly lower yield than the former (product of patent 1) and is not easy to mass-produce. In the preparation process of the compound (II), the addition amount of the ethylenediamine must be strictly controlled during the amination process to prevent excessive conversion into the compound (I), the yield of the crude purified target product is only about 1% from the amination step to the final product yield, and the subsequent conversion into a markable structure further causes loss.

The technical solutions of the above two patents mentioned by the applicant do not have directly couplable groups, and an excessive amount of coupling agent is added after purification to react all primary amino groups, and the primary amino groups are purified by chromatography to be used for labeling, so that the process is complex and time-consuming.

Disclosure of Invention

In order to solve the problems of low synthesis yield, complex probe purification, difficult labeling and the like of the prior lanthanide caged compound, the inventor provides a lanthanide caged compound with a single coupling group, a preparation method and application thereof, and lanthanide metal (europium ion Eu) with a single coupling group is synthesized on the basis of diamino type caged terpyridine europium synthesized by application number CN201610802501 (patent 1)³⁺) The caged complex meets the labeling purpose, is easy to prepare and use, and can be used without using complex chromatographic separation.

In order to achieve the purpose, the invention adopts the following technical scheme:

lanthanide metal (europium ion) cage compounds with a single coupling group have the following structure:

in the structural formula, groups which can be used for coupling comprise maleimide and halohydrocarbon, and the coupling group is connected with one amino group of caged terpyridine europium.

The lanthanide cage compound is prepared by controlling the ratio of caged terpyridine europium containing diprimary amino (intermediate pre K prepared in patent 1) to a bifunctional coupling agent containing succinimide groups and sulfhydryl reaction groups, the preparation of a single coupling group probe is realized by controlling the ratio, the lanthanide cage compound can be used without further purification, and the labeling process is greatly simplified.

The bifunctional coupling agent containing the succinimide group and the sulfhydryl reaction group is a compound containing the succinimide group and a maleimide group or a halogenated hydrocarbon group, and is taken from any one of SMCC, Sulfo-SMCC, MBS, Sulfo-MBS, SIAB, Sulfo-SIAB, SMPB, Sulfo-SMPB, GMBS, Sulfo-GMBS, SBAP and SIA.

In order to avoid generating a plurality of coupling groups by the lanthanide caged compound, the bifunctional coupling agent containing the succinimide group and the sulfhydryl reaction group and the caged terpyridine europium containing the diprimary amino group are mixed according to a molar ratio of 1:1-1:2 and reacted for 30min at room temperature, and time-consuming and complicated purification processes are omitted.

The process scheme is as follows:

the lanthanide cage compound is applied to homogeneous phase time-resolved fluorescence analysis (time-resolved fluorescence resonance energy transfer), and molecular labeling is realized through direct reaction of coupling groups and the lanthanide cage compound after sulfhydrylation treatment of biomolecules.

For antibody labeling, sulfhydryl groups are generated by a reduction method, the sulfhydryl groups of the antibody hinge region can be exposed, the region is close to the antigen binding region of the antibody, and the energy transfer fluorescence pairing distance can be reduced, so that the sensitivity is improved.

The specific detection process is as follows:

the lanthanide caged compounds prepared in the present invention are useful as long-lived fluorescence donors, using short-lived organic small molecules or allophycocyanin as fluorescence acceptors (preferred commercial acceptor molecules include Alexa Fluor647 and Dylight 650.

Constitute the time-resolved fluorescence resonance energy transfer probe pair for clinical detection.

Can be used for nucleic acid detection, immunity detection, enzyme activity detection, etc.

When the probe is used for nucleic acid detection, a neck ring structure can be formed by complementary pairing of nucleic acid, a donor and an acceptor are respectively marked at two ends close to a sequence, and when a target molecule appears, the distance between two fluorescent probes is changed, so that a specific signal can be generated.

For immunoassay, a competition method and a sandwich method can be used. When the sandwich method is adopted to detect the antigen, the two antibodies marked by the donor and the receptor are respectively used for preparing a reagent 1 and a reagent 2, and the two reagents can be packaged separately or used together.

When the two labeled antibodies are combined into one body for use, the reagent package is greatly simplified, and only one reagent is needed for each detection item.

During detection, the labeling reagent and the sample are fully and uniformly mixed, incubated and detected, and no additional washing step is needed.

During detection, the central wavelength of the excitation wavelength is about 320nm, and the central wavelengths of the detection wavelength are about 620nm and 665 nm. After the fluorescence intensities of the two wavelengths are respectively collected, specific signals are calculated through the ratio, and the unknown sample concentration is calculated by utilizing the relationship between the signals and the concentration.

Has the advantages that:

(1) the lanthanide metal (europium ion) cage-shaped compound which is easy to synthesize and has a single coupling group is coupled with biomolecules through active groups capable of reacting with sulfydryl, and can be used for homogeneous phase time-resolved fluorescence immunoassay and the like.

(2) By controlling the proportion of the raw materials, lanthanide metal (europium ion Eu) with a single coupling group is obtained³⁺) The caged complex satisfies the labeling application and can be used without using complex chromatographic separation.

(3) Compared with the compound (II), the amination step does not need to accurately control the content of an aminating agent ethylenediamine, only needs to add excessive ethylenediamine for full amination, and the yield of the compound (I) can reach more than 6 percent and is higher than 1 percent of the compound (II). Easy preparation and high yield.

Drawings

FIG. 1 is a standard curve of the quantitative AFP obtained in example 7.

FIG. 2 is a standard curve of quantitative PCT obtained in example 8.

Detailed Description

The present invention will be described in further detail with reference to specific embodiments.

Example 1:

the intermediate (caged europium terpyridine with a diprimary amino group, named pre K, i.e., compound (I) above) was synthesized according to the synthesis process disclosed in patent 1, and has the following structural formula:

compared with the compound (II), the yield of pre K is improved by 6 times and reaches more than 6 percent.

Example 2: synthesis of lanthanide series cage Compound (Compound K)

The following succinimide group-and maleimide group-or halohydrocarbon group-containing compounds (taken from any one of (SMCC (N-maleimidomethyl) cyclohexane-1-carboxylate succinimide ester, Sulfo-SMCC (Sulfo-SMCC), MBS (m-maleimidobenzoyl-N-hydroxysuccinimide ester), Sulfo-MBS (Sulfo-MBS), SIAB (succinimidyl (4-iodoacetic acid) aminobenzoate), Sulfo-SIAB (Sulfo-SIAB), SMPB (succinimidyl-4- (p-maleimidophenyl) butyrate), Sulfo-SMPB (Sulfo-SMPB), GMBS (N-gamma-maleimidobutyroyl-oxysuccinimide ester), Sulfo-GMBS (Sulfo-GMBS), SBAP (succinimidyl 3- (bromoacetamido) propionate), sia (succinimidyl iodoacetate), thermo fisher) and intermediate pre K prepared in example 1 were mixed in a molar ratio of 1:1 to 1:2 and reacted at room temperature for 30min to obtain compound K, which was used for labeling without purification. The yields are shown in Table 1:

TABLE 1

Compound (I)	A compound: intermediate pre K/molar ratio	Yield of product K
			SMCC	1:1	44％
Sulfo-SMCC	1:1	46％
			MBS	1:1	45％
Sulfo-MBS	1:1	48％
			SIAB	1:1	40％
Sulfo-SIAB	1:1	43％
			SMPB	1:1	49％
Sulfo-SMPB	1:1	52％
			GMBS	1:1	42％
Sulfo-GMBS	1:1	46％
			SBAP	1:1	47％
SIA	1:1	50％

Example 3: preparation of thiolated antibodies

The protein or the macromolecule containing amino group is dissolved in 50mM PBS, 0.15M NaCl, pH 7.2 buffer solution to prepare a solution with the concentration of 1-10 mg/ml. Thiol reducing agent dithiothreitol (10mM, room temperature for 15 minutes) was added to reduce the antibody hinge region to produce thiol groups.

Example 4: k-labeled thiol antibody

The K solution (SMPB-K) prepared in example 2 was added to the thiolated antibody prepared in example 3 in a molar ratio of K to antibody of 1:8, mixed, reacted at 30 ℃ for 90 minutes, and purified to obtain a K-labeled antibody. The antibody concentration can be calculated by measuring the absorbance at a wavelength of 280 nm.

The use concentration of the labeled antibody can be further optimized by an orthogonal test or a chessboard titration method through serial dilution, and the antibody use concentration is about 2nM in the case, and the reagent R1 can be obtained by dilution.

Example 5: receptor-labeled antibody

Alexa Fluor647 (Thermofoisher) is commercially available and receptor labeling is accomplished according to the instructions.

The concentration of the labeled antibody used can be further optimized by serial dilution, either by orthogonal assay or by checkerboard titration, in this case about 20nM, to give reagent R2.

Example 6: detection conditions

The adopted instrument is an instrument with multi-wavelength time-resolved fluorescence and detection functions, such as a multifunctional microplate reader F200 of Dirkan, and a measuring instrument with a full-automatic sample adding function can be adopted for measurement.

Measurement procedure for the following examples: the detection system comprises a reaction reagent of a sample to be detected, R1 and a reaction reagent R2, wherein the two reagents are respectively marked with a fluorescence co-donor (K obtained by the invention) and a fluorescence acceptor, wherein the fluorescence co-donor can emit long-life fluorescence at 620nm, and the fluorescence acceptor can only emit short-life fluorescence, but can also emit long-life fluorescence when receiving the energy of the donor, and only can occur in the fluorescence resonance energy transfer process of forming immune complexes.

The mixed incubation protocol was: AFP item: mixing the 2.5uL sample with each of the 9uL assay reagents R1 and R2, and incubating for 9 minutes; PCT project: the 9.5uL sample and each of the 6.5uL assay reagents R1 and R2 were mixed and incubated for 15 minutes.

Signal measurement: the time-resolved fluorescence at 620nm and 665nm was measured with a time-resolved measurement instrument, respectively, and the ratio was calculated as a measurement signal for calculation.

Generating a calibration curve: the relationship between the measurement signal and the concentration is respectively utilized to establish a calibration curve, and four parameters are adopted to carry out fitting in the range of positive correlation between the concentration and the signal to generate the calibration curve.

Antibody dilution buffer: HEPES 2.38g, KF 4.65g, BSA 0.4g, proclin-3000.1 ml, Tween 200.1 ml, water 200 ml.

Example 7: alpha-fetoprotein (AFP) detection

Using the relationship between the measurement signal value and the concentration, respectively, as shown in table 1: the signal value is the ratio of the fluorescence intensities at 665nm and 620 nm.

TABLE 1

Serial number	Concentration value	Signal value
				1	0.58	146
2	1.17	156
			3	2.34	185
4	9.37	346
			5	37.5	999
6	150	3271
			7	300	5992
8	600	8902
			9	850	9877

From the data in table 1, a calibration curve was generated by fitting four parameters in the range where the concentration and the signal were positively correlated, as shown in fig. 1.

When an unknown sample is detected, the concentration of the unknown sample can be calculated by measuring the signal value of the reaction and substituting the signal value into the calibration curve.

Example 8: procalcitonin (PCT) assay

Using the relationship between the measurement signal and the concentration, respectively, as shown in table 2:

serial number	Concentration value	Signal value
				1	0	131.3
2	0.1	145.5
			3	0.2	157.5
4	0.5	193
			5	2	362.5
6	10	1316
			7	50	6404.3
8	100	9587.5

Using the relationship of the measured signal to the concentration of the concentration, respectively, a calibration curve can be established (fig. 2).

And detecting the signal value of the unknown sample in the range of positive correlation between the concentration and the signal, and calculating the concentration of the unknown sample by using the calibration curve.

The above embodiments do not limit the technical solutions of the present invention in any way, and all technical solutions obtained by means of equivalent replacement or equivalent transformation fall within the protection scope of the present invention.

Claims

1. Lanthanide series cage compounds as described in formulas (III) and (IV), the structures of which are as follows:

2. the process for the preparation of a lanthanide cage compound as described in claim 1, wherein the process scheme is as follows:

the steps of the process are as follows: the europium-doped europium oxide is prepared by reacting at room temperature by controlling the ratio of the europium-doped succinimide group and a bifunctional coupling agent containing succinimide group and sulfhydryl reaction group;

the caged terpyridine europium containing the diprimary amino is a compound shown as a formula (I):

the bifunctional coupling agent containing a succinimide group and a sulfhydryl-reactive group is a compound containing a succinimide group and a maleimide group or a halogenated hydrocarbon group, the compound containing a succinimide group and a maleimide group or a halogenated hydrocarbon group is selected from any one of succinimide 4- (N-maleimidomethyl) cyclohexane-1-carboxy-6-aminocaproic acid, m-maleimidobenzoyl-N-hydroxysuccinimide ester, succinimido (4-iodoacetic acid) aminobenzoate, succinimido-4- (p-maleimidophenyl) butyrate, hydroxysuccinimide acid, succinimido 3- (bromoacetoxy) propionate, N-hydroxysuccinimide iodoacetate.

3. The method of claim 2, wherein the bifunctional coupling agent comprising a succinimide group and a thiol-reactive group and the caged europium terpyridine comprising a diprimary amino group are present in a molar ratio of 1:1 to 1: 2.

4. Use of the lanthanide cage compounds of claim 1 for homogeneous time-resolved fluorescence analysis for non-disease diagnostic and therapeutic uses.

5. The use according to claim 4, wherein the molecular labeling is achieved by direct reaction of a coupling group with a lanthanide cage compound after thiolation of the biomolecule.

6. The use of claim 5, wherein the lanthanide caged compound is used as a long-life fluorescence donor, and the short-life organic small molecule or allophycocyanin is used as a fluorescence acceptor to form a time-resolved fluorescence resonance energy transfer probe pair for detection.

7. The use of claim 6, in nucleic acid detection, wherein complementary nucleic acid pairing is used to form a neck ring structure, the donor and the acceptor are labeled at two ends of the sequence, and when the target molecule appears, the distance between the two fluorescent probes is changed to generate a specific signal; when the reagent is used for immunoassay, a competition method and a sandwich method are utilized, when the antigen is detected by the sandwich method, a donor and an acceptor are respectively used for marking two antibodies to prepare a reagent 1 and a reagent 2, and the two reagents can be independently packaged or can be used together.