阅读说明：本技术 用于使用封闭的2-aa进行聚糖分析的方法和试剂盒 (Methods and kits for glycan analysis using blocked 2-AA ) 是由 S·弗拉森科 F·T·哈克 N·A·格雷罗 于 2019-01-31 设计创作，主要内容包括：本发明提供在标记聚糖以供分析的程序中使用以叔丁氧羰基(“Boc”)基团封闭的邻氨基苯甲酸(2-AA)的方法,以及提供2-AA-Boc和用于使封闭的2-AA解封闭的酸、用于聚糖标记和分析的试剂盒。(The present invention provides methods of using anthranilic acid (2-AA) blocked with a tert-butyloxycarbonyl ("Boc") group in a procedure to label glycans for analysis, as well as provides 2-AA-Boc and an acid for deblocking the blocked 2-AA, kits for glycan labeling and analysis.)

1. A method of labeling glycans with anthranilic acid ("2-AA"), the method comprising: (a) obtaining 2-AA ("2-AA-Boc") conjugated with a tert-butyloxycarbonyl ("Boc") group,

(b) removing said Boc group by incubating said 2-AA-Boc with an acid for a time and at a temperature sufficient to remove said Boc group from said 2-AA-Boc, thereby obtaining 2-AA, an

(c) Incubating a solution containing the 2-AA and the glycan under conditions that allow the 2-AA to label the glycan, thereby labeling the glycan with 2-AA.

2. The process of claim 1, wherein the acid is trifluoroacetic acid, difluoroacetic acid, or hydrochloric acid.

3. The method of claim 2, wherein the acid is difluoroacetic acid ("DFA").

4. The method of claim 1, wherein the temperature sufficient to remove the Boc group from the 2-AA-Boc is 50-90 ℃.

5. The method of claim 4, wherein the temperature sufficient to remove the Boc group from the 2-AA-Boc is about 70 ℃.

6. The method of claim 1, wherein the time sufficient to remove the Boc group from the 2-AA-Boc is from about 1 hour to overnight.

7. The method of claim 1, wherein the acid incubated with the 2-AA-Boc is solvent-free.

8. The method of claim 1, wherein the acid incubated with the 2-AA-Boc is in an aprotic, organic solvent.

9. The method of claim 8, wherein the aprotic, organic solvent is dimethyl sulfoxide ("DMSO"), dimethylformamide ("DMF"), or N-methylformamide.

10. The method of claim 9, wherein the aprotic, organic solvent is dimethyl sulfoxide.

11. The method of claim 1, wherein the acid is present at a concentration of 1M to 6M.

12. The method of claim 11, wherein the acid is DFA and is present at a concentration of about 4M.

13. The process of claim 1, wherein the acid is in an aqueous solution and the process further comprises a step (b') between steps (b) and (c) of drying the 2-AA to remove the aqueous solution.

14. The method of claim 1, wherein the incubating of step (c) is at about 50-90 ℃.

15. The method of claim 1, wherein the incubating of step (c) is at about 65 ℃.

16. The method of claim 1, wherein the solution of step (c) further comprises a reducing agent.

17. The method of claim 15, wherein the reducing agent is sodium cyanoborohydride or picoline borane.

18. A kit for labeling glycans with anthranilic acid ("2-AA") by reductive amination, the kit comprising: (a) a vessel containing a 2-AA ("2-AA-Boc") conjugated with a tert-butyloxycarbonyl ("Boc") group, and (b) a vessel containing an acid suitable for removing said Boc group from said 2-AA-Boc in a concentration sufficient to remove said Boc group from said 2-AA-Boc.

19. The kit of claim 18, wherein the acid suitable for removing the Boc group from the 2-AA-Boc is suitable for removing the Boc group under anhydrous conditions.

20. The kit of claim 19, wherein the acid is trifluoroacetic acid, difluoroacetic acid, or hydrochloric acid.

21. The kit of claim 20, wherein the acid is difluoroacetic acid.

22. The kit of claim 21, wherein the difluoroacetic acid is solvent-free.

23. The kit of claim 18, wherein the acid is in an aprotic, organic solvent.

24. The kit of claim 23, wherein the aprotic, organic solvent is dimethyl sulfoxide ("DMSO"), dimethylformamide ("DMF"), or N-methylformamide.

25. The kit of claim 23, wherein the aprotic, organic solvent is DMSO.

26. The kit of claim 23, wherein the acid is present in the aprotic, organic solvent at a concentration of 1M to 6M.

27. The kit of claim 23, wherein the acid is present in the aprotic, organic solvent at a concentration of about 4M.

28. The kit of claim 18, wherein the amount of 2-AA-Boc is 5-10 mg.

29. The kit of claim 18, further comprising a reducing agent.

30. The kit of claim 29, wherein the reducing agent is sodium cyanoborohydride.

31. The kit of claim 29, wherein the reducing agent is picoline borane.

32. The kit of claim 29, further wherein the reducing agent is dissolved in an organic solvent.

33. The kit of claim 32, further wherein the solvent is DMSO.

Background

Determining glycan (glycan) characteristics of biological samples has been of importance in many cases. For example, it is desirable to determine glycan characteristics of glycoproteins, such as antibody therapeutics, to ensure consistent biological properties. Furthermore, the production of such therapeutic agents requires monitoring of the glycosylation profile in order to prevent changes in fermentation conditions that may adversely affect the yield or properties of the agent.

A variety of labels have been developed on which glycans can be attached for detection by analysis, for example mass spectrometry analysis of the labelled glycans by observing their fluorescence. The presence of N-glycans on glycoproteins is often analyzed by: the PNGase F enzyme releases the N-glycans from the glycoproteins and the resulting glycosylamines are labeled with reagents such as those taught in co-owned U.S. patent nos. 8,124,792 and 8,445,292. Another way to label glycans is to reductively aminate them using fluorescent dyes such as 2-aminobenzamide ("2-AB") or anthranilic acid ("2-AA"). See, for example, Bigge et al, "Nonsective and Efficient Fluorescent Labeling of Glycansusing 2-Amino Benzamide and Anthranilic Acid," anal. biochem.230: 229-. The labeled glycans are then typically separated, for example, by high performance liquid chromatography ("HPLC") or capillary electrophoresis, and then provided to an analytical device, such as a fluorescence detector.

Although 2-AB is still widely used to label glycans by reductive amination, it is becoming more difficult to use 2-AA for these purposes due to limitations imposed by the Controlled substrates Act 21u.s.c. § 801 and the like, at least in the united states. Section 802(34) of the act lists anthranilic acid, its esters, and its salts as "list 1 chemicals" for use in the preparation of controlled substances. List 1 chemicals are subject to various requirements regarding record keeping, distribution and shipping. 2-AA is believed to be slightly more sensitive than 2-AB for labeled glycans. It would be desirable if there were a convenient way to provide 2-AA in a form suitable for glycan analysis, but complying with Drug Administration ("DEA") regulations regarding anthranilic acid, its esters and salts thereof.

There remains a need for methods and kits that make 2-AA useful for reductive amination and labeling of glycans, while still complying with DEA regulations that control distribution and transport of 2-AA, its esters, and salts thereof. Surprisingly, the present invention has been found to meet these and other needs.

Summary of The Invention

In some embodiments, the present invention provides methods of labeling glycans with anthranilic acid ("2-AA"). In some embodiments, the method comprises: obtaining 2-AA ("2-AA-Boc") conjugated with a tert-butyloxycarbonyl ("Boc") group, removing the Boc group by incubating the 2-AA-Boc with an acid for a time and at a temperature sufficient to remove the Boc group from the 2-AA-Boc, thereby obtaining 2-AA, and incubating a solution containing the 2-AA and a glycan under conditions that allow the 2-AA to label the glycan, thereby labeling the glycan with the 2-AA. In some embodiments, the acid is trifluoroacetic acid, difluoroacetic acid, or hydrochloric acid. In some embodiments, the acid is difluoroacetic acid ("DFA"). In some embodiments, the temperature sufficient to remove the Boc group from the 2-AA-Boc is from 50 to 90 ℃. In some embodiments, the temperature sufficient to remove the Boc group from the 2-AA-Boc is about 70 ℃. In some embodiments, the time sufficient to remove the Boc group from the 2-AA-Boc is from about 1 hour to overnight. In some embodiments, the acid incubated with the 2-AA-Boc is solvent-free (neat). In some embodiments, the acid incubated with the 2-AA-Boc is in an aprotic, organic solvent. In some embodiments, the aprotic, organic solvent is dimethyl sulfoxide ("DMSO"), dimethylformamide ("DMF"), or N-methylformamide. In some embodiments, the aprotic, organic solvent is dimethyl sulfoxide. In some embodiments, the acid is present at a concentration of 1M to 6M. In some embodiments, the acid is DFA and is present at a concentration of about 4M. In some embodiments, the acid is in an aqueous solution, and the method further comprises drying the 2-AA after incubating the acid with the 2-AA-Boc to remove the aqueous solution prior to incubating the 2-AA with glycans. In some embodiments, the incubation of the 2-AA with glycans is at about 50-90 ℃. In some embodiments, the incubation of the 2-AA with the glycan is at about 65 ℃. In some embodiments, the solution of 2-AA and the glycan further comprise a reducing agent. In some embodiments, the reducing agent is sodium cyanoborohydride or picoline borane.

In a second group of embodiments, the present invention provides kits for labeling glycans with anthranilic acid ("2-AA") by reductive amination. In some embodiments, the kit comprises: a container comprising a 2-AA ("2-AA-Boc") conjugated with a tert-butyloxycarbonyl ("Boc") group, and a container comprising an acid suitable for removing said Boc group from said 2-AA-Boc, said acid being present in a concentration sufficient to remove said Boc group from said 2-AA-Boc. In some embodiments, the acid suitable for removing the Boc group from the 2-AA-Boc is suitable for removing the Boc group under anhydrous conditions. In some embodiments, the acid is trifluoroacetic acid, difluoroacetic acid, or hydrochloric acid. In some embodiments, the acid is difluoroacetic acid. In some embodiments, the difluoroacetic acid is solvent-free. In some embodiments, the acid is in an aprotic, organic solvent. In some embodiments, the aprotic, organic solvent is dimethyl sulfoxide ("DMSO"), dimethylformamide ("DMF"), or N-methylformamide. In some embodiments, the aprotic, organic solvent is DMSO. In some embodiments, the acid is present in the aprotic, organic solvent at a concentration of 1M to 6M. In some embodiments, the acid is present in the aprotic, organic solvent at a concentration of about 4M. In some embodiments, the amount of 2-AA-Boc is 5-10 mg. In some embodiments, the kit further comprises a reducing agent. In some embodiments, the reducing agent is sodium cyanoborohydride. In some embodiments, the reducing agent is picoline borane. In some embodiments, the reducing agent is dissolved in an organic solvent. In some embodiments, the solvent is DMSO.

Detailed Description

Reductive amination is a common method of labeling glycans for analysis. Two fluorophores, anthranilic acid (2-aminobenzoic acid, also known as "2-AA"), and 2-aminobenzamide, also known as "2-AB", are widely used to label glycans by reductive amination due to the stability and sensitivity of attachment to the glycans. According to the Sigma-Aldrich website, the sensitivity of 2-AA marker glycans was slightly higher than 2-AB. Unfortunately, as described in the background of the invention, the U.S. drug administration ("DEA") imposes restrictions and record keeping requirements on the use of anthranilic acid, its esters and its salts, since they can also be used to synthesize the controlled substance, hypnone. These limitations make it more difficult to use 2-AA for the legitimate use of glycan labeling and analysis in the united states.

Surprisingly, the present invention provides kits and methods for labeling glycans for use in assays that allow for the transport and use of derivatives of 2-AA that are not anthranilic acid, nor esters or salts of anthranilic acid, but that only require slight modifications to the standard workflow for labeling glycans can allow for the introduction and use of the derivatives for labeling glycans.

The kits and methods of the invention employ a 2-AA having an amino group protected by a tert-butyloxycarbonyl ("Boc") group. The use of Boc groups to protect amino groups in organic synthesis during peptide synthesis is well known. Typically, a Boc protecting group is added, the desired synthetic steps are performed, and the protecting group is removed by incubating the compound bearing the Boc group with an acid. Depending on the acid, the incubation can be carried out at ambient temperature, but is preferably carried out at a temperature of 50-90 ℃. (for ease of reference, a 2-AA having a Boc group attached to the amino moiety of the 2-AA molecule is sometimes referred to herein as a "2-AA-Boc", also referred to as a "blocked 2-AA" or a "protected 2-AA". The removal of the Boc group from the 2-AA-Boc is sometimes referred to as a "deprotected" 2-AA or a "deblocked" 2-AA.

Although the use of protection and deprotection of amino groups in peptide synthesis is well known, it has not been commonly used in labeling and analysis of glycans, nor has 2-AA been used specifically in glycan labeling, as blocking and unblocking of 2-AA increases the steps and time of the labeling workflow, in stark contrast to the general desire of practitioners to reduce the number of steps and the time to perform an analysis. Thus, blocking the amino group on the 2-AA or using Boc protected 2-AA forms is counterintuitive. However, here conjugation of 2-AA to Boc yielded compounds that were not 2-AA or esters or salts of 2-AA, and thus were not considered list 1 chemicals. Furthermore, the deprotection step results in 2-AA being present in a mixture with the acid. This combination of acid and 2-AA can be used to label glycans by reductive amination.

Addition and removal of Boc protecting groups

the addition and removal of t-Boc protecting groups has been practiced for decades. See, for example, Lundt et al, "Removal Soft-butyl and t-butyl protective groups with trifluoracetic acid," chem. biol. & Drug Design,12(5): 258-; atherton et al, J.chem.Soc., chem.Commun.,13:537-539 (1978); hemmasi and Bayer, Int J Pept Protein Res.9(1):63-70 (1977); schnolzer et al, "In situ catalysis In Boc-chemistry localized peptide synthesis. Rapid, high yield analysis of differential sequences," Int JPept Protein Res. 1992Sep-Oct; 40(3-4) 180-93; and Han et al, "Fast, effective and active detection of tert-butyl-butylcarbonyl (Boc) group using HCl/dioxane (4m)," J.peptide Res.,58, 338-. The addition and removal of Boc protecting Groups is generally taught, for example, in Green and Wuts, "Protective Groups in Organic Synthesis, Wiley-Interscience, NY (1999), pages 518, 525 and 736, 739.

Boc-protected 2-AA is available from, for example, Sigma Aldrich Corp. (st. louis, MO, which company lists as "2- (Boc-amino) benzoic acid", and Bachem (Bachem Americas, inc., Torrance, CA, catalognumber a-3240.) alternatively, Boc groups can be added to compounds by known techniques, such as 2-AA. for example, vickipedia, Boc protecting groups can be added using di-tert-butyl dicarbonate in the presence of a base such as sodium bicarbonate under "aqueous solution conditions. The protected compound was heated at 65 ℃ with aqueous hydrochloric acid and toluene, and the protected compound was dissolved in 50/50 mixture of dichloromethane and trifluoroacetic acid ("TFA").

The Schnolzer reference cited above teaches a method for the rapid removal of Boc protecting groups from amino acids using 100% TFA. As noted above, deprotection schemes typically require the use of strong acids such as 3M hydrochloric acid or trifluoroacetic acid.

Some deprotection schemes known in the art use acids dissolved in aqueous solution to remove Boc groups. Aqueous solutions may be used in the methods and kits of the invention (the acid dissolved in the aqueous solution is sometimes referred to herein as "aqueous acid"). In these methods, a "drying" step is used to remove water after the 2-AA is deprotected, since the subsequent reductive amination of the glycan must be performed under anhydrous conditions. In a preferred embodiment, the acid is solvent-free or dissolved in an aprotic organic solvent (such as dimethyl sulfoxide ("DMSO"), dimethylformamide ("DMF"), or N-methylformamide) that is compatible with reductive amination so as to eliminate the need to remove the 2-AA after deblocking before labeling the glycan of interest. Tetrahydrofuran may be used but is less preferred because it is volatile. In some preferred embodiments, the organic solvent is DMSO. The mixture of the selected acid and organic solvent is sometimes referred to herein as an "acid solution".

The use of a solvent-free acid or an acid dissolved in an organic solvent may eliminate the "drying" (dry down) step between deblocking of the 2-AA and subsequent reductive amination of the glycan with the deblocked 2-AA and is therefore preferred. As mentioned above, a solvent-free acid may be used. If an organic solvent is used, the acid is preferably used at a concentration of 1M to 6M, more preferably about 3-5M, even more preferably about 4M, where "about" means. + -. 0.25M herein.

In a preferred embodiment, the 2-AA-Boc is deprotected by incubating the 2-AA-Boc with an acid, aqueous acid or acidic solution for a time and at a temperature sufficient to remove the Boc group from the blocked 2-AA. If the 2-AA-Boc is deprotected in aqueous acid, then a "drying" step can be used to leave the deprotected 2-AA in an anhydrous state, ready for use in labeling glycans.

In some embodiments, the deblocking of the 2-AA and labeling of the glycans with the 2-AA are performed in the same vessel (a so-called "one-pot" procedure). This avoids multiple transfers of the reactant into another vessel, each transfer resulting in the loss of some of the glycans to be analyzed. For example, 2-AA-Boc can be mixed with an acid, aqueous acid, or acidic solution in a vial, and then the vial is placed in a heating block for a desired period of time, from one hour to overnight. The vial is typically heated to 50-90 ℃. In some preferred embodiments, the vial is heated to about 70 ℃, wherein "about" in the case of temperature means ± 5 ℃.

If the 2-AA-Boc has been deprotected using aqueous acid, it is usually subsequently dried, then the organic acid is added, and the glycan to be labelled is added. In a preferred embodiment, in which the 2-AA-Boc has been deprotected by incubation with an acid under anhydrous conditions (such as with an acid in an anhydrous acid or aprotic organic solvent) for a time and temperature sufficient to remove the Boc group, the glycan sample can be added directly to the anhydrous solution, labeled with deblocked 2-AA by reductive amination. For example, as shown in scheme 1, 2-AA-Boc can be deblocked by incubating 2-AA-Boc in DMSO with an acid (e.g., TFA) overnight at 70 ℃:

scheme 1

As the skilled person will appreciate, other combinations of incubation times and temperatures for removal of Boc groups from protected amino acids using specific acids are known in the art and can be readily adapted for the purpose of Boc group removal from 2-AA-Boc. Since difluoroacetic acid (DFA) is not as strong as the TFA acid used in deprotected Boc groups commonly used in peptide synthesis, if DFA is used, the incubation time or temperature or both of deblocked 2-AA should be increased appropriately to reflect the weaker strength of DFA compared to the protocol using TFA as the acid. Any particular time and temperature can be readily tested with any particular acid at any particular concentration in any particular solvent to determine if it results in complete deblocking of the 2-AA-Boc. Since both 2-AA and 2-AA-Boc are fluorescent, complete deprotection can be easily tested for the resulting product by, for example, performing HPLC, examining the fluorescence of the eluted product and determining the relative amounts of 2-AA and 2-AA-Boc.

The reducing agent is preferably present throughout the reductive amination of the labeled glycan. Since the use of reducing agents in such labeling is well known in the art, it is only briefly described herein. Reducing agents suitable for reductive amination of glycans include sodium cyanoborohydride and picoline borane. The reducing agent is typically in an organic solvent, such as DMSO or Tetrahydrofuran (THF), but in certain work-flows, an acid or acid solution may be added to the reducing agent while it is in solid form, with the reducing agent then being dissolved in the acid or acid solution. The reducing agent is typically added in a ratio of 1:1, 1:2, 1:3, or 1:4 in the solution containing the reducing agent and the solution containing 2-AA and the acid or the acid solution. The concentration of reducing agent in the final mixture is typically 0.5 to 2M.

The unblocked 2-AA can be used in the normal workflow for reductive amination of the glycan of interest. 2-AA is typically used for labeling of glycans at a concentration of 0.1M to 2M, more preferably at a concentration of 0.3M to 1M, more preferably at a concentration of about 0.3M to about 0.6M, most preferably at a concentration of 0.4M, wherein "about" means. + -. 0.05M herein.

Reagent kit

The present invention provides a kit comprising 2-AA-Boc and a reagent for deblocking the 2-AA-Boc. For example, the kit may provide one or more containers containing an amount of 2-AA-Boc suitable for a selected number of glycan assays.

For example, a multi-well plate, such as a 96-well plate, can be used to label glycans with 2-AA. Conveniently, the amount of 2-AA-Boc in the container can be selected such that upon deblocking, an amount of 2-AA is produced sufficient to label the glycan sample in the wells of a 96-well plate. The amount of 2-AA used to label glycans is quite small, with 40mg of 2-AA generally being sufficient to label glycans in all wells of a 96-well plate. The kit further comprises one or more containers containing an acid, aqueous acid or aqueous acid solvent solution selected for deblocking the 2-AA-Boc. Conveniently, the container holding the 2-AA-Boc, the container holding the acid, aqueous acid or acidic solvent solution, or both containers are of such dimensions that once the 2-AA-Boc has been mixed with the acid or acid-containing solution, the container holding the resulting mixture can be placed in a heated block and incubated at a selected temperature to remove the Boc group, resulting in a deblocked 2-AA.

Containers for labeled deblocked 2-AA typically contain 5-30mg of about 0.4M 2-AA solution. Containers containing the same amount and concentration of 2-AA-Boc, once deblocked, will yield an equivalent amount of 2-AA for labeling.

In a preferred embodiment, the kit is not shipped or dispensed with a container already mixed with the 2-AA-Boc and the acid, as this will result in a mixture containing some deblocked 2-AA and acid during dispensing and shipping. It is contemplated that the 2-AA-Boc and the acid will be mixed together only at the site where the labeling and analysis of the glycan sample is to be performed, and will typically be mixed just prior to the reductive amination of the glycan sample.

The kit may further comprise a reducing agent useful for reductive amination of 2-AA. For example, the kit may include sodium cyanoborohydride or picoline borane as the reducing agent. The reducing agent is added to the acid or acid-containing solution prior to incubating the acid or acidic solution with the 2-AA-Boc.

The kit preferably further comprises instructions for using the included reagents to provide deblocked 2-AA, the instructions comprising time and temperature conditions for deblocking the 2-AA-Boc using the provided acid or acidic solution.

Analysis of glycans labeled with 2-AA

Once deprotection of the 2-AA-Boc has occurred, it can be determined by standard techniques such as Abo et al, "Determination of monoacscholaris derivitized with 2-aminobenzoic Acid by Bacillus subtilis electrophoresis," Methods Mol biol.2013; 984:45-50, Rustoghi et al, "Analysis of N-acetylaminosources by CE: a synergistic differentiation study," electrophosphoresis.2009Aug; 30(15) 2632-9, or Jiang et al, Anal Chim acta.2017Apr 15; 962:32-40, using the resulting deblocked 2-AA to label the glycan of interest. Conveniently, the glycan(s) to be analyzed may be added to a receiving vessel containing the now deblocked 2-AA and subjected to reductive amination to label the glycan(s) according to standard, well known procedures, such as those described in the references cited above. Conversely, the now deblocked 2-AA can be pipetted, poured, or otherwise transferred into a separate vessel containing the glycan to be labeled, and reductive amination of the glycan can be performed in the separate vessel. Optionally, a washing step may be performed to remove any excess label. Common washing methods for labeled glycans with 2-AA by reductive amination include hydrophilic interaction liquid chromatography or "HILIC", or the use of graphitized carbon. The labeled glycans can then be analyzed by standard methods, such as those in the references cited above. For example, the labeled glycans can be pipetted or eluted from the receiving vessel, separated by high performance liquid chromatography or capillary electrophoresis, and the presence of the labeled glycans detected by observing fluorescence of the separated, labeled glycans.

Examples