Patent Application
20230184751
The present invention relates to a system and method for carbohydrate analysis in a flow chemistry manner. The present invention at least features continuous reaction of glycan hydrolysis and saccharide labeling which is helpful to improve the existing approaches in glycan structural analysis.
Carbohydrate analysis is essential for using glycans in biological research, clinical analysis, and biotechnological production.1 The primary structure of a glycan is defined not only by the constituent monosaccharides, but also by their linkages and branching. Often the nature and position of nonglycan substituents such as aglycan, esters (e.g., acetate, sulphate and phosphate) need to be determined. Finally, methods for solving the three-dimensional structures of glycans are also needed.
Various approaches for analyses of glycan structure have been reported.2-5 Structural and compositional analysis of glycans often require hydrolysis to release the monosaccharides, for which acidic hydrolysis is most typically used. Many hydrolysis protocols for glycan hydrolysis have been reported.6-11 Monosaccharide analyses are usually by liquid chromatography (LC), mass spectrometry (MS), nuclear magnetic resonance (NMR) or any combination of the three techniques. Moreover, the released monosaccharides can be derivatized to facilitate the detection and quantification by LC analysis.12,13 Suitable derivatization also aids in improving ionization efficiency for MS analysis. Capillary electrophoresis mass spectrometry (CE-MS),14-16 LC-MS17-19 and NMR20,21 can be used to determine the structures of complex glycans and substantial to extend over NADA tagging.22 We have previously explored a method using naphthalene-2,3-diamine (NADA) for derivatization of aldoses and α-ketoacid type saccharides (e.g., sialic acid) to their corresponding naphthimidazole (NAIM) and quinoxalinone (QXO) derivatives (
A reducing sugar may exist in the cyclic form as the α and β anomers, which sometimes obscure 1H-NMR signals. The sugar-NAIM derivative eliminates this obstacle in NMR analysis.25 We have previously shown that NAIM derivatization provides a simple method for quantitative NMR analysis of monosaccharides and disaccharides, including arabinose (Ara), xylose (Xyl), rhamnose (Rha), glucose (Glc), mannose (Man), galactose (Gal), N-acetylgalactosamine (GalNAc), glucuronic acid (GlcUA), maltose (Mal) and lactose (Lac).25 The NAIM derivative of each saccharide shows a single characteristic vinyl H-2 proton at a distinct position to facilitate the quantitative analysis. This NAIM method is especially useful for identification and quantification of multiple kinds of glycans for their compositional analysis. In addition, the sugar-NAIM carries a hydrophobic naphthimidazole group that can enhance ionization in MS detection.26 The UV and fluorescence active NAIM modifier can also assist in the LC analysis. The limit of detection for sugar-NAIM compounds can possibly reach sub-micromolar range on using a fluorescence detector. Furthermore, the D-/L-enantiomeric pairs of sugar-NAIM compounds derived from common monosaccharides, including ribose (Rib), Ara, Xyl, Rha, fucose (Fuc), Glc, Man, Gal, N-acetylgalactosamine (GalNAc), GlcUA and galacturonic acid (GalUA), are resolved on an uncoated fused-silica capillary using sulfated-α-cyclodextrin as the chiral selector.27
The present invention provides a new technology for carbohydrate analysis in a flow chemistry manner. In particular, the present invention features combining glycan degradation and saccharide derivatization in a flow system, optionally along with the use of detection means, for example, chromatography, MS and NMR techniques, leading to rapid carbohydrate compositional analysis.
In one aspect, the present invention provides a method for analyzing a glycan molecule, comprising the steps of:
In another aspect, the present invention provides a flow chemistry system for analyzing a glycan molecule, comprising
In a further aspect, the prevent invention provides an apparatus for analyzing a glycan molecule, which comprises
The details of one or more embodiments of the invention are set forth in the description below. Other features or advantages of the present invention will be apparent from the following detailed description of several embodiments, and also from the appending claims.
The foregoing summary, as well as the following detailed description of the invention, will be better understood when read in conjunction with the appended drawings. For the purpose of illustrating the invention, there are shown in the drawings embodiments which are presently preferred. It should be understood, however, that the invention is not limited to the precise arrangements and instrumentalities shown.
Unless defined otherwise, all technical and scientific terms used herein have the same meanings as is commonly understood by one of skill in the art to which this invention belongs.
As used herein, the articles “a” and “an” refer to one or more than one (i.e., at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element.
The term “comprise” or “comprising” is generally used in the sense of include/including which means permitting the presence of one or more features, ingredients or components. The term “comprise” or “comprising” encompasses the term “consists” or “consisting of.”
As used herein, “around”, “about” or “approximately” can generally mean within 20 percent, particularly within 10 percent, and more particularly within 5 percent of a given value or range. Numerical quantities given herein are approximate, meaning that the term “around”, “about” or “approximately” can be inferred if not expressly indicated.
As used herein, the term “glycan” refers to an oligosaccharide or a polysaccharide. An oligosaccharide is a saccharide polymer composed of a relatively small number (e.g. two to ten) of monosaccharides, while a polysaccharide is a saccharide polymer composed of a relatively large number (e.g. more than ten) of monosaccharides. The term “monosaccharides” as used herein refers to the simplest form of carbohydrates. Examples of monosaccharides include but are not limited to glucose, fructose, galactose, xylose, mannose, fucose, rhamnose and ribose.
As used herein, the term “hydrolysis” with respect to a carbohydrate refers to the breakdown of a glycan molecule to monosaccharides, partially or completely. It can be carried out by an acidic hydrolysis process or enzymatic hydrolysis process.
As used herein, the term “sugar derivatization reaction” refers to a reaction to modify sugars for the purpose of their structural or functional analysis. Typically, most of the labels used for the derivatization of carbohydrates possess a chromophor or a flurophor which provides a sensitive detection of these analytes by means of spectroscopic methods, for example. In particular, a method using naphthalene-2,3-diamine (NADA) has been reported for derivatization of saccharides to their corresponding naphthimidazole (NAIM) derivatives. The NAIM derivative of a saccharide shows a single characteristic vinyl H-2 proton at a distinct position to facilitate the quantitative analysis and useful for identification and quantification of multiple kinds of glycans for their compositional analysis. Further, the sugar-NAIM derivatives carry a hydrophobic naphthimidazole group that can enhance ionization in MS detection, and also the UV and fluorescence active NAIM modifier can also assist in the LC analysis.
As used herein, the term “flow chemistry” refers to a process where a chemical reaction is run in a continuously flowing stream rather than in batch production. Typically, pumps move fluid into a tube, and where tubes join one another, the fluids contact one another. The use of a microreactor greatly facilitates the NAIM derivatization, resulting in a shorter reaction time and improved yield.28 Flow chemistry systems for multiple-step synthesis of many other bioactive compounds and natural products29-32 enhance the yield as well as contribute to safety.
According to the present invention, a new technology for carbohydrate analysis is provided which features combining the reactions of glycan hydrolysis and saccharide derivatization in a flow chemistry, leading to improvement of carbohydrate analysis.
In particular, the present invention provides a method for carbohydrate analysis, which comprises the steps of:
In certain embodiments, the glucan molecule to be analyzed comprises oligosaccharides (e.g. di, tri, tetra saccharides) and/or polysaccharides.
In certain embodiments, the detectable label for the sugar derivation reaction is a naphthimidazole molecule.
In certain embodiments, the sugar derivatives are analyzed by nuclear magnetic resonance spectroscopy (NMR), liquid chromatography (LC), gas chromatography (GC), mass spectrometry (MS) and any combination thereof.
In certain embodiments, the glycan hydrolysis reaction is performed in a hydrolysis unit, the sugar derivation reaction is performed in a derivatization unit, and the hydrolysis unit is connected to the derivatization unit via connective tubing to provide a continuous flow path where the glycan hydrolysate flows from the hydrolysis unit into the derivatization unit for the sugar derivatization reaction.
In certain embodiments, the monosaccharides include ribose (Rib), arabinose (Ara), xylose (Xyl), rhamnose (Rha), fucose (Fuc), glucose (Glc), mannose (Man), galactose (Gal), N-acetylgalactosamine (GalNAc), glucuronic acid (GlcUA), and/or galacturonic acid (GalUA).
In certain embodiments, the hydrolysis reaction is effected by acidic hydrolysis or enzymatic hydrolysis.
In certain embodiments, the acidic hydrolysis is carried out under pH 1-5 at a temperature in the range of 60° C. to 150° C. for 5 to 120 minutes.
In certain embodiments, the enzymatic hydrolysis is performed with one or more enzymes selected from the group consisting of amylase, glucanase, cellulase, galactosidase, neuraminidase, glycosyltransferase, sialyltransferase, and any combinations thereof.
The present invention also provides a flow chemistry system for carbohydrate analysis, which comprises
The present invention further provides an apparatus for performing a method for carbohydrate analysis as described herein. Specifically, the apparatus of the present invention comprises a flow chemistry system as described herein in combination with an analytical system adapted for interaction with the flow chemistry system for the measurement of one or more characteristics of the sugar derivatives, and a data processing system comprising sugar database and a means for comparing the one or more characteristics of the sugar derivatives measured by the analytical system with the sugar database to determine the composition and sugar sequence of the glycan molecule.
In certain embodiments, the hydrolysis unit includes a first reservoir A containing a solution of the glycan molecule, a first reservoir B containing an acidic solution, a hydrolysis reactor and a first collection valve, connected with connective tubing and configured to enable the solution of the glycan molecule and the acidic solution to flow into the hydrolysis reactor where the hydrolysis reaction is performed and the resultant glycan hydrolysate flows into the derivatization unit when the first collection valve is in an open position. See
In certain embodiments, the derivatization unit includes a second reservoir A containing the fluorescent label, a second reservoir B containing the glycan hydrolysate, a mixer, a derivatization reactor and a second collection valve, connected with connective tubing and configured to enable the fluorescent label and the glycan hydrolysate to flow into the mixer to form a mixture of the fluorescent label and the glycan hydrolysate, and the mixture to flow into the derivatization reactor to produce the fluorescent labelled sugar derivatives. See
In certain embodiments, the measurement for one or more characteristics of the sugar derivatives is performed by nuclear magnetic resonance spectroscopy (NMR), liquid chromatography (LC), gas chromatography (GC), mass spectrometry (MS) and any combination thereof.
The present invention is further illustrated by the following examples, which are provided for the purpose of demonstration rather than limitation. Those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.
This study demonstrates the utilization of flow chemistry system for continuous glycan hydrolysis and saccharide labeling to assist with the existing methods in glycan structural analysis. Acidic hydrolysis of glycans could be accelerated in a flow system. Aldoses and α-ketoacid type saccharides were effectively labeled with naphthalene-2,3-diamine (NADA) at 60° C. for 10 min to form the fluorescent naphthimidazole (NAIM) and quinoxalinone (QXO) derivatives, respectively. The NADA-labeled derivatives improved the structural determination and composition analysis for their parent saccharides by using matrix-assisted laser desorption ionization time-of-flight mass spectrometer (MALDI-TOF-MS), liquid chromatography mass spectrometer (LC-MS) and nuclear magnetic resonance (NMR). Furthermore, this protocol was applied to determine the SA-Gal-Glc sequence of GM3-sugar out of 6 possible permutations.
Iodine, glacial acetic acid, NADA, HCl, and D2O were purchased from Merck & Co., Inc. (Darmstadt, Germany). 2,5-Dihydroxybenzoic acid (2,5-DHB), glucose, maltose, maltotriose, lactose and other monosaccharides were purchased from Sigma-Aldrich (St. Louis, Miss., US). GM3-sugar was purchased from Dextra Laboratories Ltd. (Reading, UK). Maltotetraose was purchased from Supelco Analytical (Mainz, Germany). All chemicals and solvents were of analytical grade and used without further purification. The NAIM labeling kit used in this study was a gift from Sugarlighter Co., Inc. (New Taipei City, Taiwan).25
The procedure follows the published method.22 A mixture of monosaccharide (2.0 mg, 11 μmol), naphthalene-2,3-diamine (2.0 mg, 13 μmol) and iodine (2.0 mg, 8 μmol) in glacial acetic acid (1.0 mL) was stirred at room temperature. The labeling reaction was completed in 3 h as indicated by thin-layer chromatography (TLC). The mixture was concentrated by rotary evaporation under reduced pressure to give the sugar-NAIM derivative. Other sugars were also derivatized in this fashion. Alternatively, sugar-NAIM derivatives were prepared by using a NAIM labeling kit (Sugarlighter Co.).25
A Vapourtec flow reactor E-series with V-3 peristaltic pumps (Vapourtec Ltd., Bury St Edmunds, Suffolk, UK) was used for flow chemistry. Our setup is shown in
The procedure for preparing sugar-NAIM in a flow chemistry system was modified from the batch preparation method.22,25 The flow-diagram of NAIM tagging process in Vapourtec easy-MedChem flow chemistry system is shown in
The diagram of glycan hydrolysis set-up in a flow chemistry system is shown in
The stock solutions of saccharides (1.2×10−3 to 5×10−3 M) were prepared in dd-H2O containing 0.1% formic acid and 50% CH3CN. The stock solutions of matrix 2,5-DHB (10 mg/mL, 6.5×10−2 M) and NaCl (1.7×10−2 M) were prepared in dd-H2O containing 0.1% formic acid/CH3CN (1:1 v/v). The sample for MALDI-MS measurement was typically prepared by combining 10 μL of saccharide stock solution with 10 μL of matrix stock solution and 5 μL of NaCl solution to give a final volume of 25 μL in an Eppendorf tube. Then, 2 μL of this sample solution was applied to the sample plate by a dried-droplet method (i.e., placing a droplet of the sample solution on a mass spectrometer's sample stage and drying the droplet at room temperature),24 instead of a vacuum drying process. Samples of saccharide-NAIM derivatives were similarly prepared for MALDI-MS determination. The mass spectrometer used to acquire the spectra was a Voyager Elite Applied Biosystems (Foster City, Calif., US). The accelerating voltage was set at 20 kV in either positive or negative ion mode. Typically, spectra were obtained by accumulating 800-1000 laser shots for quantification. Laser energy per pulse was calibrated with a laser power meter (PEM 101, Laser Technik, Berlin, Germany) so that laser fluence could be precisely measured. The delay extraction time was adjusted from 10 to 500 ns. The grid voltage was set at 95% of the accelerating voltage; the guide wire voltage was 0.2% of the accelerating voltage. The laser beam diameter was measured as ˜100 μm on the sample target. The laser fluence was in the range of 50-300 mJ/cm2. The flight tube pressure inside the vacuum was always kept between 10−7 and 10−6 torr.
Velos Pro dual-pressure linear ion trap MS from Thermo Fisher Scientific (San Jose, Calif., US) was used for linear trap quadrupole Fourier transform mass spectrometry (LTQ-FTMS). The saccharide sample was similarly prepared as described above and subjected to LC-MS analysis. In brief, a sample solution was prepared by dissolving the saccharide (or sugar-NAIM derivative) in dd-H2O (0.5 mL) containing 0.1% formic acid. The sample solution (5 μL) was then injected into a Xbridge C18 column (1.0 mm i.d.×15.0 cm, 3.5 μm particle size, and 130 Å pore size). The flow rate was set 0.05 mL/min, the gradient elution was applied (0-20 min, 2-98% CAN/H2O) and a UV detector was used for the LTQ-FTMS analysis.
1H-NMR spectra were recorded on a Bruker AV600 MHz NMR spectrometer (GmbH, Rheinstetten, Germany). This is a two-channel system equipped with a 5 mm DCI dual cryoprobe for high sensitivity 1H/13C observation. The sugar-NAIM sample was dissolved in D2O solution containing (CH3)2SO (0.03-0.1%) as an internal standard. Quantification of sugars was based on the integral areas of the characteristic proton signals. For example, the area of H-2 in individual hexose-NAIM derivative was compared with that of (CH3)2SO (integral region from δ 2.792 to 2.727 ppm for six protons of the two methyl groups). The acquisition parameters were equipped with a high-performance actively shielded standard bore 14.09 Tesla superconducting magnet. 1H-NMR acquisition parameters: 90° pulse, P1=9.95 μs, PL1=−0.8 dB; relaxation delay D1=2 sec; number of acquisition aq=1.9530824 (s); type of baseline correction: quad; window function: EM; LB=0.5 Hz; software for spectral processing and regression analysis: TopSpin 3.0.
We have previously prepared a series of sugar-NAIM derivatives in batch-wise manner by treating aldoses with NADA and iodine in a flask with magnetic stirring.22 This reaction usually completed in 3-6 h at room temperature. The reaction time was reduced to 1-2 h on using a NAIM labeling kit through the enhanced concentration of NADA and iodine.25 Using a flow chemistry system further improved the labeling reaction (
Taking D-glucose as an example, the formation of Glc-NAIM derivative was ˜20% for 5 min at 25° C. in a flow system, and essentially completed at 20 min (
We first investigated the acidic hydrolysis of di-, tri- and tetrasaccharides in a flow chemistry system. Maltose (1.0 mg/mL) was treated with 4 M HCl at 80° C. for 10 min in a flow system to cause partial hydrolysis (˜65%) according to the MALDI-TOF-MS analysis of the product mixture (
We then investigated the degradation of a disaccharide that contained two different monosaccharide components in the flow chemistry system. Common saccharides (e.g., Glc, Man and Gal) are hardly distinguished by MS when they have the same molecular weight. High performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD) is often used for direct separation and detection of the saccharide components by elution with strong base (NaOH).33-36 In comparison, the conventional reversed-phase high-pressure liquid chromatograph (HPLC) is more easily accessed to separate the proper derivatives of sugar components, such as sugar-NAIM compounds.22,37 In addition, HPLC can be linked with MS for analysis of oligosaccharides with prior derivatization.37,38 For glycan compositional analysis, those monosaccharides obtained from glycan hydrolysis were recycled into the flow system to generate the sugar-NAIM derivatives, even at low sample loads. The prepared sugar-NAIM derivatives were concentrated by rotary evaporation under reduced pressure, and analyzed by LC-MS without further purification.
Taking lactose as an example, the glucose and galactose components were obtained by hydrolysis in a flow chemistry system. After NADA labeling, the Glc-NAIM and Gal-NAIM derivatives were analyzed by LC-MS. The residue was separable on a C18 capillary column and identified by linear trap quadrupole Fourier transform mass spectrometry (LTQ-FTMS) (
GM3 is a common glycosphingolipid in tissues. The carbohydrate portion (GM3-sugar) is a trisaccharide SA(2α,3)Gal(1β,4)Glc comprising sialic acid, galactose and glucose. In this study, GM3-sugar (5.0 mg, 8.0 μmol) was hydrolyzed with 4 M HCl at 120° C. for 10 min in a flow system, and the hydrolysate was analyzed by MALDI-TOF-MS (
In addition to composition analysis, the lysate of GM3-sugar was concentrated and labeled with NADA in a flow chemistry system to obtain the corresponding NAIM and QXO derivatives (
We further combined glycan hydrolysis and NADA tagging in a continuous flow system to simplify the procedure for preparation of sugar NAIM (or QXO) derivatives. An additional peristaltic pump reactor was installed to the Vapourtec E-series Flow Chemistry System (
Automated polymer-supported synthesis of oligosaccharides is rapidly progressing.39-41 Immobilization of complex glycan onto a polymer or solid surfaces can be advanced to structural analysis with the assistance of flow chemistry system. We have previously demonstrated that an arginine-tagged phenylenediamine can successfully catch tetrasialic acid.24 We thus propose to modify the surface of polymer (or solid) with ortho-phenylenediamine moieties as depicted in
We have previously demonstrated the use of α-amylase, endo-β-1,3-glucanase and cellulose for specific digestion of maltohexaose, laminarihexaose and cellohexaose, respectively (
In this study, we demonstrated that glycan hydrolysis and saccharide tagging were accelerated in a flow chemistry system. Aldoses and α-ketoacid type saccharide components were mixed with NADA and iodine at 60° C. for 10 min to form the light-absorbing sugar-NAIM and sugar-QXO derivatives. This new method improved the structural determination, compositional analysis and possibly sequencing of the parent glycan by using a combination of LC, MS and NMR techniques. For example, the hetero trisaccharide GM3-sugar was hydrolyzed in 4 M HCl at 120° C. for 10 min and NADA labeled in a flow system. Since the product mixture was found to contain Glc-NAIM, Gal-NAIM, Lac-NAIM and SA-QXO by MALDI-TOF-MS, LC-MS and 1H-NMR analyses, the results concluded that GM3-sugar has a sequence of SA-Gal-Glc out of 6 possible permutations. As demonstrated in this study, application of flow chemistry system for continuous glycan hydrolysis and NADA labeling can assist the existing methods in glycan sequencing. At this moment, we still use micromolar amount of glycan sample; however, one should be able to conduct this experimental protocol with smaller amounts of glycans when advanced instruments are available. For complete glycan sequencing, one must elucidate the linkage position and anomeric configuration in each monosaccharide component. This is still a challenging task, even though many hurdles have been overcome by using chemical, biological and instrumental methods in concert.2,3,9,17
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This application claims the benefit of U.S. provisional application No. 63/025,184, filed May 15, 2020 under 35 U.S.C. § 119, the entire content of which is incorporated herein by reference.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2021/032420 | 5/14/2021 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63025184 | May 2020 | US |
