CN102250238A - Method for synthesizing swainsonine antigen - Google Patents

Abstract

The invention discloses a method for synthesizing a swainsonine antigen, comprising the following steps of: firstly performing a reaction between halo alkyl aryl fatty acid and alcohol to generate halo alkyl aryl fatty acid ester, followed by a reaction between halo alkyl aryl fatty acid ester and swainsonine to generate a quaternary ammonium salt with a structure of aromatic nucleus, performing a reaction between the quaternary ammonium salt and sodium hydroxide for deesterification to generate a sodium salt, adjusting the pH value by the use of hydrochloric acid to generate SW-halo alkyl aryl fatty acid; condensing SW-halo alkyl aryl fatty acid with bovine serum albumin (BSA) to produce SW-BSA, sieving through a glucan gel column chromatography for desalination, freeze drying, determining the proportion of BSA and SW, and adding an immunoadjuvant for emulsification to obtain the SW immune antigen. The antigen is white and oily with the effective content being 5mg/mL and the effective immune period being 12 months. The effective protection rate reaches more than 90%.

Description

The antigenic method of synthesized swainsonine

Technical field

The present invention relates to the antigenic method of a kind of biological chemistry synthesized swainsonine, belong to technical fields such as organic chemistry, toxicology and immunology.

Background technology

(swainsonine SW) is main poisonous alkaloid component in the loco weed to trihydroxyoctahydroindolizidine, is that domestic animals such as horse, ox, sheep large quantities of major causes of being poisoned to death occur after because of the loco weed that searches for food.The crude protein content of leguminous plants loco weed is up to 11%～12%, and is suitable with clover, and the potentiality that are used as good forage grass are arranged, and simultaneously, has vital role in preventing and fixing sand.Studies show that in recent years, SW has significant anti-cancer activity, for further investigation and the application of loco weed provides more wide prospect.Therefore, how to solve correct effectively preventing domestic animal and poison and the contradiction that makes full use of, become the focus of extensive concern day by day.Verified, by preparation SW-BSA antigen immune goat, goat time of poisoning behind the loco weed of searching for food is significantly postponed, demonstrate its application prospect aspect the poisoning of control domestic animal, but used preparation method's program is loaded down with trivial details, the reaction process detection difficult, productive rate is low, the antigen cost is too high, and immunogenicity of antigens is not fully up to expectations, has limited its further research application.

Research SW mechanism of poisoning and urgent needs such as animal body intracellular metabolite kinetics and anticancer mechanism are set up a kind of SW immunochemistry and are detected novel method, thereby have also proposed new requirement for the antigenic preparation of SW obtains high antibody of tiring.

Summary of the invention

Technical problem to be solved by this invention is at the deficiencies in the prior art, provides a kind of synthesized swainsonine antigenic method.By new technological line, heuristic routine is simple, process is easy to control and monitoring, and antigenicity is good, the synthetic method of high specificity, for its further research use and lay the foundation.

The present invention adopts following technical scheme:

The antigenic method of a kind of synthesized swainsonine, at first brine alkyl aryl fatty acid and alcohol reaction are generated brine alkyl aryl fatty acid ester, generate the quaternary ammonium salt that has aromatic ring structure with the trihydroxyoctahydroindolizidine reaction, take off ester with the sodium hydroxide reaction again and generate sodium salt, hydrochloric acid is regulated pH, generates SW-brine alkyl aryl fatty acid; Last become SW-BSA with bovine serum albumin (BSA) condensation, the desalination of mistake dextrane gel column chromatography, the wherein ratio of BSA and SW is measured in lyophilize, and immunological adjuvant emulsification obtains the SW immunizing antigen.

The antigenic method of synthesized swainsonine according to claim 1 is characterized in that, concrete preparation is carried out according to the following steps:

The first step, compound 1 synthetic:

Get the 60mL anhydrous methanol, the ice bath cooling is stirred simultaneously and is dropwise added about 40ml Acetyl Chloride 98Min. down, then adds 0.1～10g to chloromethyl benzoic acid, and stirring is spent the night.Remove solvent under reduced pressure, residue carries out silica gel column chromatography to be separated, and chloroform is an eluent, and substep is collected, must be to the chloromethyl benzoic acid methyl esters;

With 1.71～171mg (0.1～10mmol) SW and 2 times of amounts the chloromethyl benzoic acid methyl esters is dissolved in the 10ml dry acetone, add the NaI of catalytic amount again; 80 ℃ of heating in water bath back flow reaction, TLC follows the tracks of the detection reaction process, and about 24h afterreaction is complete.Steam and remove acetone, residue carries out silica gel column chromatography, uses chloroform and chloroform-methanol (1: 1) wash-out successively, collects chloroform-methanol wash-out part, gets white solid after steaming desolventizes, and promptly compound 1;

Synthesizing of the second step compound 2:

Compound 1 is dissolved in 6ml methyl alcohol, adds 6mL 6mol/L NaOH solution, stirring reaction 2h; Transfer pH 5-6 with 6mol/L HCl; 70 ℃ remove solvent under reduced pressure, add the 5ml anhydrous methanol in residue, and stirred for several minute is filtered, and filter residue is used the 5ml methanol wash again, collects and merging filtrate and washings, and steaming desolventizes; Residue carries out silica gel column chromatography, methanol-eluted fractions, and substep is collected; TLC detects, and collects target compound, merges target tube, removes solvent under reduced pressure, gets white solid compound 2;

The 3rd step, compound 3 synthetic:

Its schedule of operation DCC or EDC method is routinely carried out.

0.1～10mmol compound 2 and 0.005～0.5mmol BSA are dissolved in 4ml MES solution, add EDC 100mg, stirred overnight at room temperature; Cross sephadex LH-20 post with reaction solution next day, the deionized water wash-out, and sulphosalicylic acid detects, and collects protein part, and the dialysis tubing of packing into concentrates postlyophilization, gets white powder target compound---compound 3;

The 4th step, the antigenic preparation of SW:

Take by weighing compound 3, i.e. SW-BSA lyophilized powder, add physiological saline solution after, add the equivalent white-oil adjuvant, fully emulsified, be SW antigen.

The synthetic route that this invention relates to is simple, and reaction system is stable, can detect in real time reaction process by Ultraviolet Detector, also can adopt ultraviolet spectral analysis to measure the mol ratio of SW and BSA on the antigen simultaneously.Antigen is white oily, effective content 5mg/mL, and immune validity period 12 months, Effective Vate of Protection reaches more than 90%.This antigen can be used for preparing anti-SW monoclonal antibody and research with the height anti-SW antibody of tiring, and also can be used for animal immune, the poisoning of avoiding animal to cause because of the loco weed that searches for food.Antibody horizontal after the animal via immunity can be by detecting as the indirect elisa method of envelope antigen with SW-OVA.

Description of drawings

Fig. 1 is the mass spectrum (MS) of SW-chloromethyl benzoic acid methyl esters;

Fig. 2 is a SW-chloromethyl benzoic acid methyl esters ¹H NMR;

Fig. 3 is a SW-chloromethyl benzoic acid methyl esters ¹³C NMR;

Fig. 4 is the mass spectrum (MS) of key intermediate species trihydroxyoctahydroindolizidine-chloromethyl benzoic acid;

Fig. 5 is key intermediate species trihydroxyoctahydroindolizidine-chloromethyl benzoic acid ¹H NMR;

Fig. 6 is key intermediate species trihydroxyoctahydroindolizidine-chloromethyl benzoic acid ¹³C NMR;

Fig. 7 is the mass spectrum (MS) of chlorination N-(4-carbamyl phenmethyl) trihydroxyoctahydroindolizidine ammonium;

Fig. 8 is chlorination N-(4-carbamyl phenmethyl) trihydroxyoctahydroindolizidine ammonium ¹H NMR;

Fig. 9 is chlorination N-(4-carbamyl phenmethyl) trihydroxyoctahydroindolizidine ammonium ¹³C NMR;

Figure 10 compound 1 and compound 2 tomographic maps, wherein B is the SW-chloromethyl benzoic acid; A is a SW-chloromethyl benzoic acid methyl esters.

Figure 11 is SW-BSA and acid amides UV scanning result; BSA, SW-BSA concentration is 0.5 * 10 ^-5Mol/L, amide concentration is 8.77 * 10 ^-5Mol/L.

Embodiment

For clearer explanation the present invention, the spy provides example, and the present invention is described in further detail.

Synthesizing of embodiment 1 artificial antigen

Undertaken by following technological line: at first brine alkyl aryl fatty acid and alcohol reaction are generated brine alkyl aryl fatty acid ester, the latter generates the bitter horse toxin quaternary ammonium salt that has aromatic ring structure with the trihydroxyoctahydroindolizidine reaction again.Products therefrom again with sodium hydroxide generation ester hydrolysis reaction, generate SW-aryl fatty acid binding substances, last and bovine serum albumin (BSA) or ovalbumin (OVA) are condensed into the SW-BSA binding substances (SW artificial antigen) that contains the aromatic ring bridge.

Wherein, X=Cl, Br, I, Y=O (CH ₂) _m, (CH ₂) _m, Z=CH ₂, CH ₂CO ₂, n, m=0,1,2 ...

The first step, compound 1 synthetic

Reaction formula:

Schedule of operation is:

Get the 60mL anhydrous methanol, the ice bath cooling is stirred simultaneously and is dropwise added about 40ml Acetyl Chloride 98Min. down, then adds 0.1～10g to chloromethyl benzoic acid, and stirring is spent the night.Remove solvent under reduced pressure, residue carries out silica gel column chromatography to be separated, and chloroform is an eluent, and substep is collected.TLC detects (developping agent: CHCl ₃-MeOH 5: 1 V/V), merges each pipe of target product place, remove under reduced pressure behind the solvent water white transparency oily thing, be the chloromethyl benzoic acid methyl esters.

With 1.71～171mg (0.1～10mmol) SW and 2 times of amounts the chloromethyl benzoic acid methyl esters is dissolved in the 10ml dry acetone, add an amount of NaI again.80 ℃ of heating in water bath back flow reaction, TLC follows the tracks of the detection reaction process, and about 24h afterreaction is complete.Steam and remove acetone, residue carries out silica gel column chromatography, uses chloroform and chloroform-methanol (1: 1) wash-out successively, collects chloroform-methanol wash-out part, gets white solid after steaming desolventizes, and promptly compound 1.Carrying out structure by Spectrum Analysis identifies.

Synthesizing of the second step compound 2

Reaction formula:

Schedule of operation:

Compound 1 is dissolved in 6ml methyl alcohol, adds 6mL 6mol/L NaOH solution, stirring reaction 2h.Transfer pH 5-6 with 6mol/L HCl.70 ℃ remove solvent under reduced pressure, add the 5ml anhydrous methanol in residue, and stirred for several minute is filtered, and filter residue is used the 5ml methanol wash again, collects and merging filtrate and washings, and steaming desolventizes.Residue carries out silica gel column chromatography, methanol-eluted fractions, and substep is collected.TLC detects, and collects target compound, merges target tube, removes solvent under reduced pressure, gets white solid compound 2.Carrying out structure by Spectrum Analysis identifies.

The 3rd step, compound 3 synthetic

Reaction formula:

Schedule of operation:

0.1～10mmol compound 2 and 0.005～0.5mmol BSA are dissolved in 4ml MES solution, add EDC 100mg, stirred overnight at room temperature.Cross sephadex LH-20 post with reaction solution next day, the deionized water wash-out, and sulphosalicylic acid detects, and collects protein part, and the dialysis tubing of packing into concentrates postlyophilization, gets white powder target compound---compound 3.

The 4th step, the calculating of SW-BSA combination rate

Respectively the BSA and the compound 3 (SW-BSA) of accurate weighing are dissolved in distilled water and constant volume to same concentration, carry out UV scanning respectively, measure the maximum absorption wavelength (λ max) of the two and at the maximum light absorption value (A) of maximum absorption wave strong point.

Accurate weighing compound 1, add excessive methanol ammonia room temperature reaction 2h after, concentrate drying is chlorination N-to carbamyl phenmethyl trihydroxyoctahydroindolizidine, is compound 4.With distilled water the solution that it is mixed with proper concn is carried out UV scanning, measure the maximum light absorption value of maximum absorption wave strong point, calculate its molar extinction coefficient (ε) than law according to bright.Mensuration is calculated as follows SW and BSA mol ratio in the binding substances 3.

$\frac{{\mathrm{SW}}_{\mathrm{mol}}}{{\mathrm{BSA}}_{\mathrm{mol}}}=\frac{A_{\mathrm{SW}-\mathrm{BSA}}-A_{\mathrm{BSA}}}{\mathrm{\ϵ}\×\left[\mathrm{BSA}\right]}$

The 5th step, the antigenic preparation of SW

Schedule of operation: take by weighing compound 3, i.e. SW-BSA lyophilized powder, add physiological saline solution after, add equivalent freund's adjuvant or incomplete Freund's adjuvant, with the abundant mixing emulsification of micro-vortex mixer, as immunizing antigen, be used for the laboratory animal immunity, detect prepared immunogenicity of antigens or preparation antibody.

The test of embodiment 2 animal immunes

1. antigen prepd

Take by weighing the SW-BSA lyophilized powder, add physiological saline solution after, add equivalent freund's adjuvant or incomplete Freund's adjuvant, with the abundant mixing emulsification of micro-vortex mixer, as immunizing antigen.

2. animal immune

Get 6 of mouse, wherein use the SW-BSA immunity for 5, remaining 1 injection PBS in contrast.2 week immunity are 1 time at interval, each 3 subcutaneous injection immunogens of every nape part 0.2mL (100 μ g).Initial immunity Freund's complete adjuvant antigen, after this immunity Freund's incomplete adjuvant antigen.After the 3rd 1 week of immunity, every mouse is taken a blood sample from the eye socket venous plexus, separation of serum, and indirect elisa method is measured antibody titer, treats that ratio P/N＞2 of gaging hole and negative hole OD value are judged to the positive.

Other gets 4 rabbit, wherein uses the SW-BSA immunity for 3, and 1 injection PBS is as negative control.Just exempt from equivalent mixed liquor with SW-BSA and Freund's complete adjuvant, 500 μ g/ only, the branch subcutaneous injection, the two full Freund's complete adjuvant antigens of exempting to too many or too much for use, dosage and method are same just to be exempted from.Two exempt from 2 weeks of back, use indirect ELISA and agar diffusion test respectively and carry out antibody test.

3. the detection of antigen-specific

Use the agar bed board of 1%NaCl solution preparation 1.5%, rabbit anteserum after the immunity and contrast rabbit anteserum carried out agar diffusion test, detect immunize rabbit serum respectively with SW-BSA, SW-OVA, SW, the reactivity of BSA and OVA is established simultaneously in the immune serum and to be added SW and detect its sealing process.

4. animal immune test-results

Mouse and rabbit through 2 immunity after, the blood sampling separation of serum, with SW-OVA as the envelope antigen coated elisa plate, indirect ELISA detects antibody titer, and the result shows that the anti-SW antibody of mice immunized and rabbit anteserum is all positive, tire and can reach 1: 2000 (table 1, table 2).

4 immunity backs of table 1 mice serum SW antibody test result

2 week of table 2 immunity back rabbit anteserum SW antibody test result

Agar diffusion test detects immunizing rabbit serum result and shows, immune serum and SW-BSA, and precipitation line all appears in BSA between the SW-OVA, and and SW, no visible precipitate line between the OVA does not all have the visible precipitate line between above-mentioned each antigen and the negative serum.Be used for the agar diffusion test discovery after using SW and immune serum effect, itself and SW-BSA, BSA, the precipitation line between the SW-OVA significantly weakens.Show no antigen intercrossing between OVA and the BSA, contain special antibody in the immune serum at SW.

Embodiment 3

Present embodiment provides detected result and data among the embodiment 1.

1.1 compound 1 (SW-chloromethyl benzoic acid methyl esters) is referring to figs. 1 to Fig. 3;

Behind compound 1 purifying is buff powder, has extremely strong water-absorbent, under the 254nm on TLC garnet, the SW color reaction positive (blue spot) is with reference to figure 10A.

Analyzing its molecular weight through spectroscopic measurement is 322, through comparing with document, proves that the synthetic product structure conforms to fully with notional result, and its POP analytical results is as follows:

ESI?MS?m/z?322(M ⁺)： ¹³C?NMR(125MHz，CD ₃OD)：δ167.6(C-10)，134.6(C-1′)，134.3(C-3′)，133.3(C-4′)，131.2(C-2′)，77.4(C-δa)，71.6(C-2)，69.4(C-1)，68.8(C-3)，65.3(C-8)，65.1(C-9)，59.2(C-5)，52.9(OCH ₃)，28.3(C-7)，16.6(C-6)。 ¹H?NMR(400MHz，CD ₃OD)：δ8.13(d，J＝8.8Hz，2H，H-3′)，7.78(d，J＝8.8Hz，2H，H-2′)，3.93(s，3H，OCH ₃)，5.15(d，J＝13.2Hz，1H，H-9a)，4.72(d，J＝13.2Hz，1H，H-9b)，3.18(dd，J＝3.6，12.8Hz，1H，H-5a)，3.39(d，J＝12.8Hz，1H，H-5b)，2.47(m，1H，H-6a)，2.35(m，1H，H-6b)，1.91(d，J＝13.2Hz，1H，H-7a)，1.78(d，J＝14.4Hz，1H，H-7b)，3.85(d，J＝4.0Hz，2H，H-3a)，3.80(dd，J＝2.4，14.4Hz，1H，H-8a)，4.37(m，1H，H-8)，4.46(m，1H，H-2)，4.50(m，1H，H-1)。

1.2 compound 2 (trihydroxyoctahydroindolizidine-chloromethyl benzoic acid), with reference to figure 4 to Fig. 6;

Compound 2 usefulness TLC detect, and developping agent is a chloroform: acetone (5: 3), under the 254nm on TLC garnet, its Rf value is 0.118, the SW color reaction positive.With reference to figure 10B.

Analyzing its molecular weight through spectroscopic measurement is 308, consistent with expection.Through comparing with document, prove that the synthetic product structure is correct, its POP analytical results is as follows:

ESI?MS?m/z?308[M ⁺]. ¹³C?NMR(125Hz，acetone-d ₆+CD ₃OD)：δ134.0(C-2’)，131.3(C-3’)，100.8(C-1’，C-4’)，77.2(C-8a)，71.6(C-2)，69.3(C-1)，68.7(C-3)，65.4(C-8)，65.0(C-9)，59.0(C-5)，28.2(C-7)，16.5(C-6). ¹H?NMR(400MHz，acetone-d ₆+CD ₃OD)：δ8.13(d，J＝7.42Hz，2H，H-3’)，7.78(d，J＝7.68Hz，2H，H-2’)，5.19(d，J＝13.2Hz，1H，H-9a)，4.78(d，J＝13.2Hz，1H，H-9b)，4.63(t，J＝6.0Hz，1H，H-1)，4.51(brs，1H，H-2)，4.44(m，1H，H-8)，4.10(dd，J＝8.0，12.4Hz，1H，H-8a)，4.02(d，J＝5.6Hz，1H，H-3a)，3.47(d，J＝12.8Hz，1H，H-3b)，3.24(m，2H，H-5)，2.49(m，1H，H-6a)，2.33(m，1H，H-6b)，1.95(d，J＝13.2Hz，H-7a)，1.81(m，1H，H-7b)。

1.3 compound chlorination N-is to carbamyl phenmethyl trihydroxyoctahydroindolizidine (with reference to figure 7-Fig. 9)

Its structure of spectroscopic measurement analytical proof is correct, and its POP analytical results is as follows:

ESI?MS?m/z?307[M ⁺]， ¹³C?NMR(125MHz，acetone-d ₆)：δ171.2(C-10)，136.7(C-1′)，134.2(C-2′)，133.2(C-4′)，129.9(C-3′)，78.5(C-8a)，71.7(C-2)，70.0(C-1)，68.9(C-3)，65.4(C-8)，65.2(C-9)，57.9(C-5)，28.1(C-7)，16.9(C-6)。 ¹H?NMR(400MHz，acetone-d ₆)：δ8.01(d，J＝8.0Hz，2H，H-3′)，7.82(d，J＝8.0Hz，2H，H-2′)，5.20(d，J＝13.2Hz，1H，H-9a)，4.84(d，J＝13.2Hz，1H，H-9b)，4.63(t，J＝6.0Hz，IH，H-1)，4.53(m，1H，H-2)，4.50(m，1H，H-8)，4.17(dd，J＝8.0，12.4Hz，1H，H-8a)，3.47(d，J＝12.8Hz，1H，H-3b)，3.24(m，2H，H-5)，2.49(m，1H，H-6a)，2.33(m，1H，H-6b)，1.90(d，J＝13.2Hz，H-7a)，1.80(6rd，J＝14.4Hz，H-7b)。

1.4 compound 3 (SW-BSA)

Needle crystal is white in color after the synthetic SW-BSA freeze-drying, pressing BSA calculates, productive rate is 95%, with reference to Figure 11, according to UV scanning, under same concentration and the same terms, the maximum absorption wavelength of BSA is 275.0nm, the maximum absorption wavelength of SW-BSA is 268.0nm, and obvious displacement takes place maximum wavelength, and SW-BSA coupling success is described.After measured, the coupling ratio of SW and BSA is 20: 1.

Should be understood that, for those of ordinary skills, can be improved according to the above description or conversion, and all these improvement and conversion all should belong to the protection domain of claims of the present invention.

Claims (2)

1. antigenic method of synthesized swainsonine, it is characterized in that, at first brine alkyl aryl fatty acid and alcohol reaction are generated brine alkyl aryl fatty acid ester, generate the quaternary ammonium salt that has aromatic ring structure with the trihydroxyoctahydroindolizidine reaction, take off ester with the sodium hydroxide reaction again and generate sodium salt, hydrochloric acid is regulated pH, generates SW-brine alkyl aryl fatty acid; Last become SW-BSA with bovine serum albumin (BSA) condensation, the desalination of mistake dextrane gel column chromatography, the wherein ratio of BSA and SW is measured in lyophilize, and immunological adjuvant emulsification obtains the SW immunizing antigen.

2. the antigenic method of synthesized swainsonine according to claim 1 is characterized in that, concrete preparation is carried out according to the following steps:

The first step, compound 1 synthetic

Get the 60mL anhydrous methanol, the ice bath cooling is stirred simultaneously and is dropwise added about 40ml Acetyl Chloride 98Min. down, then adds 0.1～10g to chloromethyl benzoic acid, and stirring is spent the night.Remove solvent under reduced pressure, residue carries out silica gel column chromatography to be separated, and chloroform is an eluent, and substep is collected, must be to the chloromethyl benzoic acid methyl esters;

With 1.71～171mg (0.1～10mmol) SW and 2 times of amounts the chloromethyl benzoic acid methyl esters is dissolved in the 10ml dry acetone, add the NaI of catalytic amount again; 80 ℃ of heating in water bath back flow reaction, TLC follows the tracks of the detection reaction process, and about 24h afterreaction is complete.Steam and remove acetone, residue carries out silica gel column chromatography, uses chloroform and chloroform-methanol (1: 1) wash-out successively, collects chloroform-methanol wash-out part, gets white solid after steaming desolventizes, and promptly compound 1;

Synthesizing of the second step compound 2:

Compound 1 is dissolved in 6ml methyl alcohol, adds 6mL 6mol/L NaOH solution, stirring reaction 2h; Transfer pH 5-6 with 6mol/L HCl; 70 ℃ remove solvent under reduced pressure, add the 5ml anhydrous methanol in residue, and stirred for several minute is filtered, and filter residue is used the 5ml methanol wash again, collects and merging filtrate and washings, and steaming desolventizes; Residue carries out silica gel column chromatography, methanol-eluted fractions, and substep is collected; TLC detects, and collects target compound, merges target tube, removes solvent under reduced pressure, gets white solid compound 2;

The 3rd step, compound 3 synthetic:

Its schedule of operation DCC or EDC method is routinely carried out.

0.1～10mmol compound 2 and 0.005～0.5mmol BSA are dissolved in 4ml MES solution, add EDC 100mg, stirred overnight at room temperature; Cross sephadex LH-20 post with reaction solution next day, the deionized water wash-out, and sulphosalicylic acid detects, and collects protein part, and the dialysis tubing of packing into concentrates postlyophilization, gets white powder target compound---compound 3;

The 4th step, the antigenic preparation of SW

Take by weighing compound 3, i.e. SW-BSA lyophilized powder, add physiological saline solution after, add the equivalent white-oil adjuvant, fully emulsified, be SW antigen.

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