CN105622748B - A kind of C16H25NO2 detection antigen and preparation method thereof - Google Patents
A kind of C16H25NO2 detection antigen and preparation method thereof Download PDFInfo
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- CN105622748B CN105622748B CN201610079188.2A CN201610079188A CN105622748B CN 105622748 B CN105622748 B CN 105622748B CN 201610079188 A CN201610079188 A CN 201610079188A CN 105622748 B CN105622748 B CN 105622748B
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Abstract
The present invention relates to a kind of C16H25NO2s to detect antigen, and general structure is as follows:Wherein, m=1,2,3,4,5.And the preparation method of C16H25NO2 detection antigen, first by O demethyls on C16H25NO2 phenyl ring, then draws carboxyl on the position O that O- removes first C16H25NO2 by bromobutyrate, then slough ethyl ester using C16H25NO2 as raw material, is coupled by carboxyl and high molecular weight protein.Material used in synthesis and preparation process of the invention and catalyst are common product, therefore more practicability, suitable for industrial production and can generate economic value；The present invention does not use chloroform, and toxicity is lower, has more safety.
Description
Technical field
The present invention relates to technical field of immunoassay；It particularly relates to chemical synthesis process and synthesizes since C16H25NO2 go
First C16H25NO2 and its derivative, then the preparation method that C16H25NO2 detects antigen is made with high molecular weight protein coupling.
Background technique
Following background technique is used to help reader and understands the present invention, and is not construed as the prior art.
C16H25NO2 (Tramadol) is a kind of non-opium central analgesics, suitable to μ-opioid recdptor affinity
In the affinity of 1/6000 pair of κ and δ receptor of morphine be only the 1/25 of μ receptor.Tramadol hydrochloride is ground by company, German Glan Thailand
System exploitation was listed in Germany first in 1977, had been obtained extensive in many country's listings including China at present
Clinical application.C16H25NO2 Orally-administrable, drop rectum with drug, intramuscular injection or vein are slowly injected, or are intravenously dripped after dilution
Note.C16H25NO2 mainly acts on central nervous system, and over administration can generate dependence, to the effect of human body similar to morphine and Hai Luo
Cause.C16H25NO2 can be used for treating medium to serious pain.Some researches show that C16H25NO2 is to norepinephrine and serum tension
The effect of prime system system and the effect for mitigating pain, can mitigate the pain of depression and anxiety disorder.
As a kind of currently the only non-opium central analgesics, due to its analgesic effect it is powerful and additive low so
Clinically it is used widely.The common treatment for various postoperative analgesias, pain caused by cancer, pain of childbirth etc., with what is studied it
Deeply, in terms of cold-resistant war, cough-relieving and some other treatment, C16H25NO2 has played great effect.
But with being widely used for C16H25NO2, the problem of C16H25NO2 habituation, causes to pay close attention to.According to the relevant information, normal person
If taken 200 milligrams of C16H25NO2 daily, pharmacological dependence can be about generated after half a year, and if taking 300 to 400 milligrams (6 daily
To 8 medicines) it is even more, it can be addicted in a short time.2008, tramadol hydrochloride is carried out control by China.
Therefore the exploitation for carrying out related reagent of C16H25NO2 Misuse detection and products thereof just becomes needs.
Summary of the invention
The present invention connects crosslinking arm again and with immunogene using C16H25NO2 as Material synthesis haptens, from the position O on phenyl ring
Property protein coupling, the comlete antigen with reactionogenicity and immunogenicity is obtained with this；This antigen is applied to inspection
It surveys in card and carries out the detection whether C16H25NO2 is abused.In order to obtain the specific antibody for being directed to haptens, crosslinking arm is generally wanted
Follow two principles: the structure for having complicated of 1. trying not generally is preferred with hydrocarbon chain, can avoid generating anti-friendship so as far as possible
The antibody of joint arm；2. have certain length, generally 1-10 atom, it preferably 1-6, in this way can be sudden and violent by haptens
It is exposed at the appearance of protein, rather than is hidden by protein.Therefore the crosslinking agent of the immunizing antigen selection of synthesis is usually bromine
Ethyl acetate, bromobutyrate or bromocaproic acid ethyl ester etc., then reacted by de- ethyl ester with the amino on protein, crosslinking arm is
The simple hydrocarbon chain of structure, length are 4-8 atom.
Specifically, a kind of C16H25NO2 provided by the invention detects antigen, and general structure is as follows:
Wherein, m=1,2,3,4,5
In some preferred embodiments, high molecular weight protein be bovine serum albumin(BSA) (BSA), bovineγ-globulin (BGG) or
Chicken egg white (OVA).
On the other hand, the preparation method of C16H25NO2 detection antigen provided by the invention, specifically: using C16H25NO2 as raw material,
First by O demethyls on C16H25NO2 phenyl ring, carboxyl is then drawn on the position O that O- removes first C16H25NO2 by bromobutyrate,
Ethyl ester is sloughed again, is coupled by carboxyl and protein；Its reaction equation is as follows:
Reaction equation one:
Reaction equation two:
Wherein, product 1: removing first C16H25NO2,
Product 2:4- (3- (- ((dimethylamino) methyl)-hydroxy-cyclohexyl) phenoxy group) ethyl butyrate,
Product 3:4- (3- (- ((dimethylamino) methyl)-hydroxy-cyclohexyl) phenoxy group) butyric acid,
Product 4: C16H25NO2 antigen.
Wherein, reaction equation is first is that obtain the synthesis process of product 3 by C16H25NO2 Material synthesis；Second reaction equation is product 3
It is coupled the process of high molecular weight protein.
The present invention also provides the preparation methods of specific C16H25NO2 detection antigen, and its step are as follows:
(1) under protection of argon gas, C16H25NO2 and methionine are dissolved in methanesulfonic acid, reaction 48-96h is stirred at room temperature；Instead
It should be dried, filtered after the completion with being extracted with ethyl acetate after NaOH tune PH to 11-12, Rotary Evaporators, which are drawn, to be done, and column chromatography mentions
It is pure, obtain product 1；Wherein, C16H25NO2 and methionine molar ratio are 1:2~1:6；
(2) product 1 is dissolved in acetonitrile, brominated alkanes ester and potassium carbonate is added, flowed back 12~16 hours；After reaction
Filtering, dry, Rotary Evaporators, which are drawn, to be done, and obtains product 2；Wherein, product 1 and brominated alkanes ester molar ratio are 1:1.2~1.5；
(3) product 2 is dissolved in methanol, 4N NaOH is added, is stirred at room temperature 8-16H, reaction be added after terminating it is positive from
Sub- resin agitating filters after detecting PH < 7, and Rotary Evaporators, which are drawn, to be done, and column chromatographic purifying obtains product 3；Wherein, methanol and 4N NaOH
Volume ratio is 5:1；
(4) by product 3,1- ethyl-(3- dimethylaminopropyl) carbodiimide hydrochloride (EDC), N- hydroxysuccinimidyl acyl
Imines (NHS) is dissolved in N,N-Dimethylformamide (DMF), and 8H is stirred at room temperature.Then it instills in protein solution, is stirred overnight
Obtain product 4；Wherein, product 3, DEC, NHS ratio are 1:1.2~1.5:1.2~1.5.
Beneficial effect
The invention has the following advantages:
1, material and catalyst used in synthesis and preparation process of the invention are common product, therefore more practical
Property, suitable for industrial production and economic value can be generated；
2, the present invention does not use chloroform, and toxicity is lower, has more safety.
Detailed description of the invention
Fig. 1 is the general structure that C16H25NO2 detects antigen；
Fig. 2 is the structural schematic diagram of colloidal-gold detecting-card；
Fig. 3 is colour atla schematic diagram；
Fig. 4 is C16H25NO2 antigen for small molecule testing result picture；
Fig. 5 is the accelerated stability result picture of antigen.
Specific embodiment
The present invention is described in detail with reference to the accompanying drawing.
Antigen is detected by Material synthesis of C16H25NO2, specific practice is that C16H25NO2 is Material synthesis haptens, from phenyl ring
O position connect crosslinking arm and be coupled again with the protein with immunogenicity, obtained with this with reactionogenicity and immunogenicity
Comlete antigen.
Embodiment 1: the preparation of C16H25NO2 detection antigen
1. intermediate product (product 3): 4- (3- (- ((dimethylamino) methyl)-hydroxy-cyclohexyl) phenoxy group) butyric acid
Preparation
Step 1: under protection of argon gas, C16H25NO2 100mg and methionine 150mg are dissolved in methanesulfonic acid 3.5ml, room
Temperature is stirred to react 72h；TLC (methylene chloride/methanol=3/1) monitors reaction process, raw material point Rf=0.7, product point Rf=
0.8；Three times with the extraction of 20ml ethyl acetate merged organic phase with 4N NaOH tune PH to 12, dried, filtered with Na2SO4, revolved
Turn evaporimeter and draw dry, column Chromatographic purification, obtains product 1 (removing first C16H25NO2) meter 68mg.Yield 81.8%；
13CNMR(DMSO)δ:158.564,145.233,131.187,118.365,114.219,112.365,70.552,
58.089,48.223,46.586,41.858,37.126,28.023,24.835,22.635
ESI m/z=250 (M+1)
Step 2: 68mg product 1 is dissolved in 10ml acetonitrile, it is added 4- bromobutyrate 64mg, potassium carbonate 225mg, 80 DEG C
It is heated to reflux 12 hours.TLC (methylene chloride/methanol=3/1) monitoring reaction, product point Rf=0.9.It filters after reaction,
It is dried, filtered with Na2SO4, Rotary Evaporators, which are drawn, to be done, and (4- (3- (- ((dimethylamino) the methyl)-hydroxyl of 84mg product 2 is obtained
Cyclohexyl) phenoxy group) ethyl butyrate) yield 85.3%；
13CNMR(DMSO)δ:173.614,158.564,145.233,131.181,118.365,114.219,
112.365,70.552,67.143,62.584,57.721,48.565,46.586,41.858,37.126,31.431,
28.023,25.617,24.835,22.635,15.005
ESI m/z=364 (M+1)
Step 3: taking 84mg product 2 in the round-bottomed flask of 50mL, it is dissolved in 5ml methanol, is then added 1ml4N's
Reaction is stirred at room temperature overnight in NaOH；TLC (methylene chloride/methanol=5/1) monitors reaction process.After reaction is basic, it is added
Resin cation is gently mixed, and after standing 4 hours, PH < 7, filtering, Rotary Evaporators, which are drawn, to be done, and column chromatographic purifying obtains, and 59mg is produced
Object 3 (4- (3- (- ((dimethylamino) methyl)-hydroxy-cyclohexyl) phenoxy group) butyric acid), yield 93.5%；
13CNMR(DMSO)δ:173.882,158.345,144.961,131.853,117.672,113.856,
112.015,70.132,68.584,57.231,49.155,46.225,42.010,37.105,30.984,28.655,
25.332,24.252,22.335,
ESI m/z=336 (M+1)
The preparation that intermediate product 2. (product 3) coupling carrier albumen forms C16H25NO2 detection antigen
Step 1: 60mg product 3 is taken to be dissolved in 2ml DMF, concentration 30mg/ml.Configure the PBS-BGG albumen of 10mg/ml.
Step 2: 41mg EDC is added in 2ml × 30mg/ml product 3,8H is stirred at room temperature in 25mg NHS.
Step 3: taking the PBS-BGG protein solution 27ml of step 1, the mixed liquor that step 2 obtains then is instilled, room temperature is anti-
It should stay overnight to obtain final product 4, i.e. C16H25NO2 antigen, such as Fig. 1.PBS dialyses 3 days, and measuring concentration is 5.72mg/ml.
Embodiment 2: application of the antigen in colloidal gold colloidal gold detection test paper strip
Diameter is prepared in the colloidal gold of 20-40nm with four chlorauride of reduction of sodium citrate, then by colloid gold label to anti-
The antibody of C16H25NO2 indicates the concentration of gold labeling antibody with the light absorption value (OD) of λ max to upper；
Gold labeling antibody is loaded on golden mark machine, the gold labeling antibody of certain OD value is equably sprayed into polyester film (gold-labelled pad
202) on, 37 degree of baking ovens is put into after spray is good and dry 8h；It is cut into suitable size, is put into the aluminium foil bag equipped with drying machine, room temperature is deposited
It puts spare；
By C16H25NO2 antigen diluent to suitable concentration, with T line position corresponding on film machine point to nitrocellulose filter 203
206, corresponding 207 position of C line is plus corresponding antigen (for whether detecting the detection card equally on nitrocellulose filter 203
Effectively), 37 degree of drying 12h；
Sample pad 201--- glass fibre passes through the processing of buffer and surface-active, and 37 degree of drying 10h are spare；
By sample pad 201, gold-labelled pad 202, nitrocellulose filter 203, blotting paper 204, kilocalorie 205 is assembled into colloidal gold inspection
Paper card 200 is tested, sees Fig. 2；
The small molecule solution for preparing respective concentration, is added in sample pad 201, result is read after 5min；
Requirement of the product to the Cutoff value of C16H25NO2 (Tramadol) is 100ng/ml, i.e., -50% is 50ng/ml ,+
50% is 150ng/ml.
Wherein:
Remarks 1: all small molecules to be measured are dissolved in negative urine；
Remarks 2: standard color card: the ratio color range of standard color card is between 0-10, i.e. G1-G10, color from scratch, face
Color depth is incremented by successively, as shown in Figure 5.
Remarks 3: examination criteria: the detection T line 206 that will test test strips is compared with standard color card:
Line depth indicates negative when being greater than G8；
In the case of -50%cutoff, line depth ﹥ G4 is qualification；
Line depth ﹤ G3.5 is qualification in the case of+50%cutoff
Table one: detection sensitivity result of the antigen to C16H25NO2 small molecule (see Fig. 3)
Table two: assembled detection (is blocked 55 DEG C to dry 72 hours) result (see Fig. 4) by the accelerated stability of antigen
Table three: the cross reaction value of analog
Analog concentration to be measured when the line intensity of test strips detection analog reaches critical value G3.5
From table one, table two and table three: C16H25NO2 detection antigen is applied to progress C16H25NO2 detection in test strips and meets
Examination criteria, and thermal stability is good, analog cross reaction is small, and therefore, the detection antigen is qualified.
Claims (4)
1. a kind of C16H25NO2 detects antigen, which is characterized in that its general structure is as follows:
Wherein, m=1,2,3,4,5.
2. C16H25NO2 according to claim 1 detects antigen, which is characterized in that high molecular weight protein is bovine serum albumin(BSA)
(BSA), bovineγ-globulin (BGG) or chicken egg white (OVA).
3. a kind of preparation method of C16H25NO2 detection antigen as claimed in claim 1 or 2, which is characterized in that be with C16H25NO2
Then raw material is drawn on the position O that O- removes first C16H25NO2 first by O demethyls on C16H25NO2 phenyl ring by bromobutyrate
Carboxyl, then ethyl ester is sloughed, it is coupled by carboxyl and high molecular weight protein；Its reaction equation is as follows:
Reaction equation one:
Reaction equation two:
Wherein, product 1: removing first C16H25NO2,
Product 2:4- (3- (- ((dimethylamino) methyl)-hydroxy-cyclohexyl) phenoxy group) ethyl butyrate,
Product 3:4- (3- (- ((dimethylamino) methyl)-hydroxy-cyclohexyl) phenoxy group) butyric acid,
Product 4: C16H25NO2 antigen.
4. preparation method according to claim 3, which is characterized in that specific preparation process is as follows:
(1) under protection of argon gas, C16H25NO2 and methionine are dissolved in methanesulfonic acid, reaction 48-96h is stirred at room temperature；It has reacted
It is extracted with ethyl acetate, dries, filters after Cheng Houyong NaOH tune PH to 11-12, Rotary Evaporators, which are drawn, to be done, and column Chromatographic purification obtains
To product 1；Wherein, C16H25NO2 and methionine molar ratio are 1:2~1:6；
(2) product 1 is dissolved in acetonitrile, brominated alkanes ester and potassium carbonate is added, flowed back 12~16 hours；It crosses after reaction
Filter, dry, Rotary Evaporators, which are drawn, to be done, and obtains product 2；Wherein, product 1 and brominated alkanes ester molar ratio are 1:1.2~1.5；
(3) product 2 is dissolved in methanol, 4N NaOH is added, 8-16H is stirred at room temperature, cation tree is added in reaction after terminating
Rouge stirring, is filtered after detecting PH < 7, and Rotary Evaporators, which are drawn, to be done, and column chromatographic purifying obtains product 3；Wherein, methanol and 4N NaOH volume
Than for 5:1；
(4) by product 3,1- ethyl-(3- dimethylaminopropyl) carbodiimide hydrochloride (EDC), n-hydroxysuccinimide
(NHS) it is dissolved in N,N-Dimethylformamide (DMF), 8H is stirred at room temperature.Then it instills in protein solution, is stirred overnight to obtain
Product 4；Wherein, product 3, DEC, NHS ratio are 1:1.2~1.5:1.2~1.5.
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