CN111057131A

CN111057131A - Bacitracin and analysis method of components thereof

Info

Publication number: CN111057131A
Application number: CN201911285218.5A
Authority: CN
Inventors: 张含智; 刘浩; 赵敬丹; 秦峰; 闻宏亮; 裘亚
Original assignee: SHANGHAI INSTITUTE FOR FOOD AND DRUG CONTROL
Current assignee: SHANGHAI INSTITUTE FOR FOOD AND DRUG CONTROL
Priority date: 2019-12-13
Filing date: 2019-12-13
Publication date: 2020-04-24

Abstract

The invention discloses a novel Bacitracin (Bacitracin) component and a high performance liquid chromatography-mass spectrometry (HPLC-MS) analysis method thereof. The component is verified to be bacitracin A of double methylene and vinyl or bacitracin Y of double methylene and ethyl by HPLC-MS and secondary mass spectrum analysis, and the novel component is named bacitracin HZ (bacitracin HZ). The present invention reveals the presence of novel non-protein amino acids such as 2-amino-3-vinyl-5-hexenoic acid, 2-amino-4-vinyl-5-hexenoic acid, 3-vinyl cysteine, 2-vinyl cysteine. The bacitracin HZ has antibacterial activity or toxicity different from that of common bacitracin components, has potential application value, and the structure confirmation of the compounds also provides research basis for strictly controlling the quality of bacitracin.

Description

Bacitracin and analysis method of components thereof

Technical Field

The invention relates to the field of pharmaceutical analytical chemistry, in particular to a bacitracin and an analysis method of components of the bacitracin.

Background

Bacitracin (structural formula shown in figure 1 and table 1) is a multicomponent antimicrobial peptide produced by bacillus subtilis and bacillus licheniformis, can strongly inhibit gram-positive bacteria, and is mainly used for treating penicillin-resistant staphylococcus infection. Bacitracin has a special bacteriostatic mechanism, can interfere the synthesis of bacterial cell walls, and simultaneously inhibits the regeneration of phospholipid receptors in the synthesis of peptidoglycan, so that the bacitracin has the characteristics of wide antibacterial spectrum, difficult generation of drug resistance and the like. Zinc ions are added during the pharmaceutical process to enhance the activity.

TABLE 1R of bacitracin structural formulae A and B_nExpression (2)

As the bacillus peptide is a fermentation product and has more bacillus peptide components, the components such as the bacillus peptide A, B, C, F and the like are included in the United states Pharmacopeia 40, 19 components or related substances are included in the European Pharmacopeia 9.6, and other unknown components are also included. High performance liquid chromatography (dipotassium hydrogen phosphate/acetonitrile/methanol system) adopted in United states Pharmacopeia 40 edition and European Pharmacopeia 9.6 edition selects a non-volatile mobile phase system, is not suitable for direct mass spectrometry, and cannot realize on-line determination of the structure of an unknown component.

Disclosure of Invention

Aiming at the problems in the prior art, the invention discloses a bacitracin and an analysis method of components thereof, which is a high performance liquid chromatography-mass spectrometry (HPLC-MS) analysis method. The bacitracin is verified to be bismethyleneated and vinylated bacitracin A or bismethyleneated and ethylated bacitracin Y and named bacitracin HZ (bacitracin HZ) by primary mass spectrometry and secondary mass spectrometry.

In order to achieve the above object, the technical solution provided by the present invention comprises:

in a first aspect, the present invention provides a novel bacitracin, designated bacitracin HZ. The bacitracin is composed of bismethylenated and vinylated bacitracin A or bismethylenated and ethylated bacitracin Y.

Further, the constituent units of the methylated and vinylated bacitracin A include isoleucine at position 1, cysteine at position 2, leucine at position 3, glutamic acid at position 4, isoleucine at position 5, lysine at position 6, ornithine at position 7, isoleucine at position 8, phenylalanine at position 9, histidine at position 10, aspartic acid at position 11, asparagine at position 12, the isoleucine at position 1 and the cysteine at position 2 form a thiazoline ring, and the amino acids at positions 6 to 12 form a cyclic peptide 7. the bismethylated and vinylated bacitracin A refers to β '-methyl and gamma' -methyl of the isoleucine at position 1 of the procymitracin A are methylated, and the β "position of the cysteine at position 2 is vinylated, and has the following structure:

or β ' -methyl and gamma ' -methyl at isoleucine position 1 of the protopanoxapeptide A are methylated, and α ' position at cysteine position 2 is vinylated, and the structure is as follows:

further, the constituent units of the bismethyleneated and ethylated bacitracin Y include isoleucine at position 1, cysteine at position 2, leucine at position 3, glutamic acid at position 4, isoleucine at position 5, lysine at position 6, ornithine at position 7, isoleucine at position 8, phenylalanine at position 9, histidine at position 10, aspartic acid at position 11, asparagine at position 12, isoleucine at position 1 and cysteine at position 2 form a thiazole ring, and amino acids at positions 6 to 12 form a peptide 7, wherein the bismethyleneated and ethylated bacitracin Y refers to β '-methyl and γ' -methyl of isoleucine at position 1 of procymitracin Y being methylated, and β "of cysteine at position 2 being ethylated, and has the following structure:

further, the configuration of the bismethyleneated and vinylated bacitracin a, β' and α ", β" -carbon is either the R configuration or the S configuration.

Further, the constituent amino acids of bismethyleneated and vinylated bacitracin a are in L-form or D-form.

Further, the configuration of the β' -carbon of the bismethyleneated and ethylated bacitracin Y is R configuration or S configuration.

Further, the constituent amino acids of the bismethyleneated and ethylated bacitracin Y are in L-form or D-form.

Further, isoleucine at

position

1, 5 or 8 of bacitracin HZ may be converted into leucine, and the resulting composition after the conversion may be bismethylenated or may not be bismethylenated.

Further, the chemical structural formula of substituting isoleucine at position 1 of the bismethyleneated and vinylated bacitracin a with leucine and the chemical structural formula of substituting isoleucine at position 1 of the bismethyleneated and ethylated bacitracin Y with leucine are respectively as follows:

in a second aspect, the present invention provides novel unnatural protein amino acids found in bacitracin HZ: 2-amino-3-vinyl-5-hexenoic acid A, 2-amino-4-vinyl-5-hexenoic acid B, 3-vinyl cysteine C, 2-vinyl cysteine D, the structural formula is as follows:

further, bacitracin includes oxidation products, epimerization products, hydrolysis products, sulfation products, sulfonation products, etc. of bacitracin HZ, or other bacitracin products.

A third aspect of the invention is to provide a bacitracin drug substance or a bacitracin zinc drug substance or a pharmaceutical combination comprising bacitracin HZ.

The fourth aspect of the present invention provides a pharmaceutical preparation comprising the above-mentioned crude drug or pharmaceutical composition, which is an injection, an ointment or an eye ointment.

The fifth aspect of the present invention is to provide a method for obtaining components of bacitracin and structures thereof as a high performance liquid chromatography-mass spectrometry (HPLC-MS) analysis method.

Further, the MS ionization method may be selected from electrospray ionization, atmospheric pressure ionization, and the like, and is preferably electrospray ionization.

Further, the mass analyzer of the MS may select a quadrupole time-of-flight mass spectrum, a linear or three-dimensional ion hydrazine mass spectrum, a triple quadrupole mass spectrum, an orbital ion hydrazine mass spectrum, or the like, preferably a quadrupole time-of-flight mass spectrum.

Further, bacitracin HZ and other bacitracin components can be structurally judged by information of primary mass spectrum and secondary mass spectrum.

Further, the MS and MS/MS scanning ranges from 50 to 2000 amu.

Further, the secondary mass collision energy setting range is 10-30eV, preferably 20eV, 30 eV.

Further, the molecular weight of bacitracin HZ was determined to be 736.8908([ M + 2H)]²⁺) The characteristic ions of the secondary mass spectrum are m/z 604.32, 491.23, 382.14, 362.19, 334.19, 253.10, 249.11 and 110.07.

Compared with the prior art, the invention has the following beneficial effects:

(1) the invention discovers for the first time that bacitracin HZ, namely the bacitracin A subjected to bismethylene and vinylation or the bacitracin Y subjected to bismethylene and ethylation, may have antibacterial activity or toxicity different from common bacitracin components, and has potential application value, and the structure confirmation of the compound also provides a research basis for strictly controlling the quality of bacitracin;

(2) the invention identifies the structure of the bismethyleneated and vinylated bacitracin A or the bismethyleneated and ethylated bacitracin Y for the first time through MS and MS/MS data;

(3) the present invention reveals the presence of novel non-natural protein amino acids, such as 2-amino-3-vinyl-5-hexenoic acid, 2-amino-4-vinyl-5-hexenoic acid, 3-vinyl cysteine, 2-vinyl cysteine;

(4) the HPLC-MS/MS method can be used for directly analyzing the structures of other components in bacitracin, namely, a primary mass spectrum is comprehensively adopted to obtain molecular weight, secondary mass spectrum fragment ions and amino acid residue ions to estimate the component structures, and the analysis strategy can also be popularized to the structure analysis of other polypeptide antibiotics.

Drawings

FIG. 1 is a general structural formula of a peptide component of Bacillus in the background of the invention;

FIG. 2 is a mass spectrometric total ion flow diagram of a bacitracin test article in an embodiment of the present invention;

FIG. 3 is a first order mass spectrum of bacitracin HZ in one embodiment of the present invention;

FIG. 4 is a secondary mass spectrum of bacitracin HZ in one embodiment of the present invention;

FIG. 5 is a graph showing the cleavage pattern of bacitracin A in one embodiment of the present invention;

FIG. 6 shows bacitracin HZ: the chemical structural formula of the bismethyleneated and vinylated bacitracin A;

FIG. 7 shows bacitracin HZ: chemical structural formula of bismethyleneated and ethylated bacitracin Y;

FIG. 8 shows bacitracin HZ: chemical structural formula of bismethyleneated and vinylated bacitracin A-I (isoleucine substituted by leucine at position 1);

FIG. 9 illustrates bacitracin HZ: the chemical structural formula of dimethylenated and ethylated bacitracin Y-I (isoleucine substituted by leucine at position 1);

FIG. 10 shows the chemical structure of amino acids of a novel unnatural protein according to one embodiment of the invention;

wherein, A: 2-amino-3-vinyl-5-hexenoic acid, B: 2-amino-4-vinyl-5-hexenoic acid, C: 3-vinyl cysteine, D: 2-vinyl cysteine.

Detailed Description

The present invention will be described in detail and specifically with reference to the following examples and drawings so as to provide a better understanding of the invention, but the following examples do not limit the scope of the invention.

The bacitracin related in the invention takes bacitracin 'United states Pharmacopeia' as a concrete implementation case, but can also be applied to other bacitracin reference substances and preparations.

Example 1

This example provides a novel bacitracin, designated bacitracin HZ. The bacitracin is composed of bismethylenated and vinylated bacitracin A or bismethylenated and ethylated bacitracin Y. Wherein, the constituent unit of the methylated and vinylated bacitracin A comprises 1-position isoleucine, 2-position cysteine, 3-position leucine, 4-position glutamic acid, 5-position isoleucine, 6-position lysine, 7-position ornithine, 8-position isoleucine, 9-position phenylalanine, 10-position histidine, 11-position aspartic acid and 12-position asparagine, the 1-position isoleucine and 2-position cysteine form thiazoline ring, and 6-12 amino acids form cyclic 7 peptide. The constituent unit of the bismethylenated and ethylated bacitracin Y comprises isoleucine at position 1, cysteine at position 2, leucine at position 3, glutamic acid at position 4, isoleucine at position 5, lysine at position 6, ornithine at position 7, isoleucine at position 8, phenylalanine at position 9, histidine at position 10, aspartic acid at position 11 and asparagine at position 12, wherein the isoleucine at position 1 and the cysteine at position 2 form a thiazole ring, and the amino acids at positions 6 to 12 form a cyclic peptide 7.

Bacillin A, which is bismethylenated and vinylated, means that β '-methyl and γ' -methyl at isoleucine position 1 are methylenated and β "position at cysteine position 2 is vinylated, and its structure is shown in (I), or β '-methyl and γ' -methyl at isoleucine position 1 are methylenated and α" position at cysteine position 2 is vinylated, and its structure is shown in (II), bacillin Y, which is bismethylenated and ethylated, means that β '-methyl and γ' -methyl at isoleucine position 1 and β "position at cysteine position 2 are methylenated, and its structure is shown in (III).

The configuration of β 'and α', β '-carbon in the bismethyleneated and vinylated bacitracin A is R configuration or S configuration, the constituent amino acid is L type or D type, the configuration of β' -carbon of the bismethyleneated and ethylated bacitracin Y is R configuration or S configuration, and the constituent amino acid is L type or D type.

Isoleucine at

position

1, 5 or 8 of bacitracin HZ may be converted to leucine, and the resulting composition may or may not be bismethylenated. Wherein, the chemical structural formula A-I that isoleucine at position 1 of the dimethylenated and vinylated bacitracin A is substituted by leucine and the chemical structural formula Y-I that isoleucine at position 1 of the dimethylenated and ethylated bacitracin Y is substituted by leucine are respectively as follows:

example 2

This example provides the use of bacitracin HZ in medicine. A bacitracin bulk drug or a pharmaceutical composition comprising bacitracin HZ and a pharmaceutical preparation comprising the same.

The pharmaceutical preparation comprises bacitracin for injection, bacitracin ointment and bacitracin eye ointment.

Wherein the specification of the injection is 1000 units per milliliter (dissolved in isotonic sodium chloride injection);

the specification of the ointment is 1 g: 500 units, 5g:4000 units;

the specification of the eye ointment is 1 g: 500 units, 2g:1000 units.

Verification experiment

An ammonium formate-acetonitrile separation system (Chinese pharmacy journal, 2018, 53(23):2041-2046 and Chinese invention patent: 201811467424.3) is developed in the early stage of the research, has a good separation effect on various components of bacitracin, and can be directly used for mass spectrometry. The structure of the bacitracin HZ is analyzed by adopting a mass spectrometry method of an ammonium formate-acetonitrile separation system in the verification experiment.

Instrument and reagent

Agilent model 1290 high performance liquid chromatograph-6550 QTOF-MS (Agilent Technologies, USA).

Bacitracin United states Pharmacopeia control (batch No.: R048 CO).

Second, experimental conditions

The invention adopts a liquid phase method in Chinese invention patent (Zhangzhi, etc., a high performance liquid chromatography method for analyzing bacitracin component, Chinese invention patent 201811467424.3), namely, 50mmol/L ammonium formate solution (pH value is adjusted to 4.0 by formic acid) is taken as a mobile phase A, acetonitrile is taken as a mobile phase B, elution is carried out according to the linear gradient condition in the following table 2, and the flow rate is 1 ml/min; the column temperature is 30 ℃; the sample injection amount is 4 mul; the detection wavelength was 254 nm. ESI-Q/TOF-MS is used as a detector, a primary mass spectrum and a secondary mass spectrum are collected in a positive ion scanning mode, the collection range is m/z 50-1700, and the collision energy of the secondary mass spectrum is 10-30 eV.

TABLE 2 Linear elution conditions for mobile phase A and mobile phase B

Third, bacitracin reference sample solution preparation and analysis

Precisely weighing bacitracin 20mg as reference substance in United states Pharmacopeia, placing into 10ml measuring flask, adding mobile phase A-mobile phase B (77:23, V: V) to dissolve and quantitatively dilute to scale, shaking up, and the concentration is 2 mg/ml. The total ion flow chart is shown in figure 2 and the elution positions of bacitracin A and bacitracin HZ are shown in figure 3 by HPLC-ESI-Q/TOF-MS analysis (the primary mass spectrum of bacitracin HZ is shown in figure 4).

Structure analysis of bacitracin A

The structure analysis process is described by taking bacitracin A as an example, and the excimer ion peak [ M + H ] of bacitracin A]⁺Has an m/z of 1422.76. Bacitracin A readily adds two H's in solution or during electrospray⁺And mainly with [ M +2H ]]²⁺(m/z, 711.89) is present. With [ M +2H ]]²⁺As the parent ion, the secondary mass spectrogram of bacitracin A is obtained by adjusting the fragmentation voltage, and the cracking rule is shown in FIG. 5. When bacitracin A is cracked from a linear chain end (N end), firstly removing isoleucine decarboxylation positive ions (a1 ions, m/z, 86.10) to obtain x11 ions (m/z, 1337.67), then removing thiazoline cyclic acyl to obtain y10 ions, then removing leucyl, glutamyl and isoleucyl to obtain y9, y8 and y7 ions, and finally obtaining the cyclic heptapeptide; the above peptides all volatilize the amino group on asparagine. Corresponding b ions such as b5, b4, b3 and b2, and also b ions may lose acyl groups (e.g., b3 ion loses acyl groups to obtain fragment ion m/z 284.18) or a1 ion obtains fragment peaks at the lower molecular weight. Cyclic peptides have low abundance of characteristic ions, two cleavage pathways, one of which is through the process of loss of phenylalanyl and isoleucyl groups (with loss of fragment ion m/z 627.29), aspartyl amino (fragment ion m/z 513.25) and aspartyl (fragment ion m/z 398.22), the last fragment ion (m/z, 243.18) comprising only lysine and ornithine; the second is the loss of ornithinyl and lysyl, aspartyl amino, aspartyl to give characteristic fragments. At the low end, it can be observed that the amino acid residues constituting bacitracin A, such as fragment ions (m/z, 86.10) after acyl group loss of leucyl or isoleucyl group, glutamyl residues (m/z, 102.06), ornithiyl residues (m/z, 115.09 and 87.09), phenylalanyl residues (m/z, 120.08), histidyl residues (m/z, 138.07 and 110.07), aspartic acid residues (m/z, 88.04), asparagine residues (m/z, 87.06) and lysyl amine residues (m/z, 129.10), and detailed data of each amino acid residue and the above fragment ion information can reduce the sequence of bacitracin A, and can also provide reference for structure derivation of other components.

Structure analysis of bacitracin HZ

The M/z of bacitracin HZ is 736.89([ M + 2H)]²⁺) Compared with bacitracin A, the molecular weight is increased by 50, and the secondary mass spectrum fragment ion ratio of bacitracin A shows that characteristic ions m/z 491.23, 362.19, 334.19 and 249.11 have 50 increased molecular weights compared with corresponding ions 441.22, 312.17, 284.18 and 199.09 of bacitracin A, which indicates that linear peptide segments are changed, the characteristic ion a1 has m/z of 110.07 increased by 24 compared with a1 of bacitracin A, which predicts that β '-methyl and gamma' -methyl of isoleucine at position 1 are methylated, the fragment ion 253.09 is 26 larger than the corresponding ion 227.09 of bacitracin A, and combines m/z 249.11 and 110.07, so that the modified site is judged to be on the thiazoline ring, and the vinyl is predicted to be generated at position β 'or position α' of cysteine at position 2, namely, the dimethylene and vinyl bacitracin A are shown in the structural formula in figure 6.

It is also possible to convert the thiazoline ring into the thiazoline ring, and the β' position is ethylated, i.e., bismethylenated and ethylated bacitracin Y, see the structural formula in FIG. 7.

The isoleucine at position 1 may also be converted to leucine, i.e., the two methyl groups at γ' are methylated, and the bacitracin HZ structure may also be as shown in FIG. 8 (i.e., bismethylenated and vinylated bacitracin A-I), FIG. 9 (i.e., bismethylenated and ethylated bacitracin Y-I).

Four novel unnatural protein amino acids, such as 2-amino-3-vinyl-5-hexenoic acid (A), 2-amino-4-vinyl-5-hexenoic acid (B), 3-vinyl cysteine (C), 2-vinyl cysteine (D), have the structural formula shown in FIG. 10, wherein the chiral of α -carbon is not identified, and R or S configuration is possible, wherein A or B corresponds to the 1-position amino acid of bacitracin, and C or D corresponds to the 2-position amino acid of bacitracin.

In summary, the present invention has found a novel bacitracin fraction and its structure can be analyzed by HPLC-MS and MS/MS methods, which are named bacitracin HZ, i.e., bismethyleneated and vinylated bacitracin A and bismethyleneated and ethylated bacitracin Y. In addition, the present invention reveals the presence of novel non-natural protein amino acids, such as 2-amino-3-vinyl-5-hexenoic acid, 2-amino-4-vinyl-5-hexenoic acid, 3-vinyl cysteine, 2-vinyl cysteine. The novel multi-double bonded component may have different antibacterial activities and biological activities, and has potential application prospects.

The embodiments of the present invention have been described in detail, but the embodiments are merely examples, and the present invention is not limited to the embodiments described above. Any equivalent modifications and substitutions to those skilled in the art are also within the scope of the present invention. Accordingly, equivalent changes and modifications made without departing from the spirit and scope of the present invention should be covered by the present invention.

Claims

1. Bacitracin which is a bismethyleneated and vinylated bacitracin a (formula i or formula ii) or a bismethyleneated and ethylated bacitracin Y (formula iii):

2. the bacitracin of claim 1 wherein the isoleucine at position 1, 5 or 8 of said bacitracin is converted to leucine and the resulting composition after conversion is either bismethylenated or not bismethylenated;

wherein, the chemical structural formula A-I that isoleucine at position 1 of the dimethylenated and vinylated bacitracin A is substituted by leucine and the chemical structural formula Y-I that isoleucine at position 1 of the dimethylenated and ethylated bacitracin Y is substituted by leucine are respectively as follows:

3. the bacitracin of claim 2 wherein said bacitracin presents novel non-protein amino acids: 2-amino-3-vinyl-5-hexenoic acid A, 2-amino-4-vinyl-5-hexenoic acid B, 3-vinyl cysteine C, 2-vinyl cysteine D, the structural formula is as follows:

4. a novel bacitracin according to claim 1 wherein said bacitracin comprises an oxidation product, epimerization product, hydrolysis product, sulfation product or sulfonation product of said bacitracin.

5. A drug substance or pharmaceutical composition comprising the bacitracin of any one of claims 1-4.

6. A pharmaceutical formulation comprising a drug substance or pharmaceutical composition according to claim 5, wherein the formulation comprises an injection, an ointment or an eye ointment.

7. An analytical method for obtaining the bacitracin of claim 1 and its structure characterized in that said method is high performance liquid chromatography-mass spectrometry.

8. The method of claim 7, wherein the MS mass analyzer is selected from the group consisting of quadrupole time of flight mass spectrometry, linear or three-dimensional ion hydrazine mass spectrometry, triple quadrupole mass spectrometry, orbital ion hydrazine mass spectrometry, and the like.

9. The method of claim 7, wherein the MS ionization is selected from electrospray ionization, atmospheric pressure ionization, and the like.

10. The assay of claim 7, wherein the MS and MS/MS scan ranges from 50 to 2000 amu.