TW201408690A - Diaminobenzoic or ortho-phenylene diamine conjugated peptides as functional saccharide tagging reagents - Google Patents

Diaminobenzoic or ortho-phenylene diamine conjugated peptides as functional saccharide tagging reagents Download PDF

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TW201408690A
TW201408690A TW102116113A TW102116113A TW201408690A TW 201408690 A TW201408690 A TW 201408690A TW 102116113 A TW102116113 A TW 102116113A TW 102116113 A TW102116113 A TW 102116113A TW 201408690 A TW201408690 A TW 201408690A
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Wen-Bin Yang
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Abstract

Novel designs of tagged saccharides useful in analytical and diagnostic applications are disclosed. Saccharide reagents disclosed include monosaccharide, oligosaccharide or polysaccharide subunit is coupled at the reducing end to a tagging moiety comprising an ortho-diaminobenzoic(DAB)-peptide. The tagged saccharide comprising detectable or functional groups coupled to the DAB-peptide entity are provided. Methods for use of the tagged saccharides as reagents for chromatography and mass spectrometry are disclosed.

Description

二氨基苯甲酸或鄰苯二胺共軛的胜肽作為功能性的醣類標幟劑 Diaminobenzoic acid or o-phenylenediamine conjugated peptide as a functional saccharide marker

本發明係有關碳水化合物分析法領域。特定言之本發明係有關使用可檢測標幟物標記多醣類之分析與診斷用途領域。更特定言之，本發明係有關一種鄰位經取代之二氨基-芳香系化合物，其與胜肽共軛形成醣類標幟劑。 The invention relates to the field of carbohydrate analysis. In particular, the invention relates to the field of analytical and diagnostic use of labeled polysaccharides using detectable labels. More specifically, the present invention relates to an ortho-substituted diamino-aromatic compound which is conjugated with a peptide to form a saccharide label.

醣類具有必要之生物功能，例如：醣蛋白中之O-,N-聚醣類，脂多醣類(LPS)中之O-/M-抗原。然而，由於醣類缺少發色基團且在MS中之離子化能力低，因此很難自然分離與測定。因此無法採用傳統光測定法(例如：液相層析法中UV/螢光)或MS分析法檢測醣類。 Carbohydrates have the necessary biological functions, such as O-, N-glycans in glycoproteins, O-/M-antigens in lipopolysaccharides (LPS). However, since saccharides lack chromophore groups and have low ionization ability in MS, it is difficult to separate and measure naturally. Therefore, it is not possible to detect sugars by conventional photometry (for example, UV/fluorescence in liquid chromatography) or MS analysis.

帶標記物之醣類之發展係分析化學中重要課題，雖然已發展出一些方法來標記醣類，但此等方法仍有一些缺點。其中一項實例為採用還原性胺化法，使用氨基-磷脂或使用中性或酸性螢光試劑標幟游離醣類。(Hase S.等人之J.Biochem.95：197-203(1996))。雖然此方法適用於某些實際用途，但採用此等方法所標幟醣類之分離法仍受到一些限制。(Price NPJ等人之Anal.Chem.2010,82,2893；Pabst M.等人之Anal.Biochem.2009,384,263；Tripathi,R.P.,et al.Current Org.Chem.2008,12,1093；Bigge J.C.,等人之Anal.Biochem.1995,230,229；Harvey,D.J.J.Chromatogr. B 2011,879,1196)。 The development of labeled sugars is an important topic in analytical chemistry. Although some methods have been developed to label sugars, these methods still have some disadvantages. An example of this is the use of reductive amination, the use of amino-phospholipids or the use of neutral or acidic fluorescent reagents to label free sugars. (Hase S. et al. J. Biochem. 95: 197-203 (1996)). Although this method is suitable for some practical purposes, the separation of sugars by these methods is still subject to some limitations. (Price NPJ et al. Anal. Chem. 2010, 82, 2893; Pabst M. et al. Anal. Biochem. 2009, 384, 263; Tripathi, RP, et al. Current Org. Chem. 2008, 12, 1093; Bigge JC , et al., Anal. Biochem. 1995, 230, 229; Harvey, DJ J Chromatogr. B 2011, 879, 1196).

帶標記物之醣類之發展係分析化學中重要課題，雖然已發展出一些方法來標記醣類，但此等方法仍有一些缺點。其中有些實例為採用還原性胺化法，使用氨基-磷脂或使用中性或酸性螢光試劑標幟游離醣類 (Lin,C等人之J.Org.Chem.2008,73,3848-3853；Lin,C等人之Rapid Commun Mass Spectrom.2010,24,85-94；Lin,C等人之Molecules 2010,15,1340-1353；Lin,C等人之Molecules 2011,16,652-664；Chang,Y.L等人之J.Mass Spectrom.2011,46,247-255；Hase S等人之J.Biochem.95：197-203(1996))。 The development of labeled sugars is an important topic in analytical chemistry. Although some methods have been developed to label sugars, these methods still have some disadvantages. Some of these examples use reductive amination, using amino-phospholipids or using neutral or acidic fluorescent reagents to label free sugars. (Lin, C et al. J. Org. Chem. 2008, 73, 3848-3853; Lin, C et al., Rapid Commun Mass Spectrom. 2010, 24, 85-94; Lin, C et al., Molecules 2010, 15 , 1340-1353; Lin, C et al., Molecules 2011, 16, 652-664; Chang, YL et al., J. Mass Spectrom. 2011, 46, 247-255; Hase S et al. J. Biochem. 95: 197-203 ( 1996)).

過去曾採用多醣類與蛋白質或胜肽之共軛法(共價偶合)。由多醣片段利用其還原端，經由直接還原性胺化法與蛋白質或胜肽偶合時，所納入之醣類少。 Conjugation (covalent coupling) of polysaccharides with proteins or peptides has been used in the past. When a polysaccharide fragment is coupled to a protein or a peptide via a direct reductive amination method using its reducing end, the amount of sugar incorporated is small.

因此，長期需要一種用於標幟及標記醣類之標幟劑，以供進一步應用該經標幟之帶功能標記物之醣類。 Therefore, there is a long-felt need for a marker for labeling and labeling saccharides for further application of the labeled carbohydrate with a functional marker.

本揭示內容係基於適用於分析與診斷用途之許多新穎設計之經標幟醣類。 The present disclosure is based on a number of novel designs of labeled sugars suitable for analytical and diagnostic applications.

因此，在一方面，本發明揭示係有關如式Y-X之經改質糖苷，其中Y代表單醣、寡醣或多醣亞單位，其中該亞單位利用糖苷鏈結體連接成直鏈或分支鏈，且其中X代表包含鄰二氨基苯甲酸(DAB)-胜肽之標幟部份體。 Thus, in one aspect, the invention relates to a modified glycoside of the formula YX, wherein Y represents a monosaccharide, oligosaccharide or polysaccharide subunit, wherein the subunit is linked to a straight or branched chain by means of a glycosidic link, And wherein X represents a part of the body comprising ortho-diaminobenzoic acid (DAB)-peptide.

此處揭示3,4-二氨基苯甲酸(DAB)-胜肽與3,4-二氨基苯甲酸(DAB)-胜肽共軛功能分子，其作為醣類標幟物，供標幟在醣類之還原端，使其包含苯并咪唑單位(醣-BIM-胜肽或醣-BIM-胜肽共軛功能分子)。可由偶合鄰二氨基苯甲酸(DAB)-胜肽標幟劑來產生經標幟之醣類。 Disclosed herein are 3,4-diaminobenzoic acid (DAB)-peptide and 3,4-diaminobenzoic acid (DAB)-peptide conjugated functional molecule, which serve as a sugar label for labeling sugar. The reducing end of the class is such that it contains a benzimidazole unit (sugar-BIM-peptide or sugar-BIM-peptide conjugated functional molecule). The labeled saccharide can be produced by coupling an o-diaminobenzoic acid (DAB)-peptide marker.

在部分具體實施例中，該鄰二氨基苯甲酸-胜肽標幟劑係選自下列各物所組成群中：DAB-6His、DAB-3His、DAB-Lys(生物素)、DAB-Lys(FITC)、DAB-Lys-樹脂。 In some embodiments, the o-diaminobenzoic acid-peptide marker is selected from the group consisting of DAB-6His, DAB-3His, DAB-Lys (biotin), DAB-Lys ( FITC), DAB-Lys-resin.

在部分具體實施例中，該DAB-胜肽係由胜肽或功能性標記物-共軛胜肽與N-Boc-二氨基苯甲酸以固相胜肽合成法或其他化學方法進行縮合法而產生。 In some embodiments, the DAB-peptide is condensed by a peptide or a functional marker-conjugated peptide and N-Boc-diaminobenzoic acid by solid phase peptide synthesis or other chemical methods. produce.

在部分具體實施例中，該醣類亞單位Y係相同或相異，且係選自下列各物所組成群中：葡萄糖、半乳糖、果糖、核酮糖、甘露糖、 核糖、***糖、木糖、來蘇糖、阿洛糖、阿卓糖、古洛糖、鼠李糖、***糖、岩藻糖、N-乙醯基半乳糖胺、葡萄醛酸、半乳糖醛酸、Globo H、GD2、GD3、GD1a、GQ1b、GT1b、GT1a、Gb3、Gb5、SSEA寡醣類、岩藻糖基GM1、GM2、GM3、血型抗原(A、B、O、H)、福斯曼(Forssman)抗原、路易士a(Lewis a)、路易士b(Lewis b)、路易士X(Lewis X)、唾液基路易士X(Sialyl Lewis X)、路易士Y(Lewis Y)、基於乳糖之O-聚醣、N-乙醯基葡糖胺核心結構、唾液基乳糖、唾液基化寡醣類、硫酸化寡醣類、磷酸化寡醣類、甘露-寡醣類、纖維-寡醣類、木-寡醣類、甲殼-寡醣類、麥芽-寡醣類及多醣類。 In some embodiments, the saccharide subunits Y are the same or different and are selected from the group consisting of glucose, galactose, fructose, ribulose, mannose, Ribose, arabinose, xylose, lyxose, allose, altrose, gulose, rhamnose, arabinose, fucose, N-ethyl galactosamine, glucuronic acid, galactose Aldehydic acid, Globo H, GD2, GD3, GD1a, GQ1b, GT1b, GT1a, Gb3, Gb5, SSEA oligosaccharides, fucosyl GM1, GM2, GM3, blood group antigens (A, B, O, H), Fu Forssman antigen, Lewis a, Lewis b, Lewis X, Sialyl Lewis X, Lewis Y, Lactose-based O-glycan, N-acetyl glucosamine core structure, salivary lactose, salivylated oligosaccharide, sulfated oligosaccharide, phosphorylated oligosaccharide, mannose-oligosaccharide, fiber- Oligosaccharides, wood-oligosaccharides, chitin-oligosaccharides, malt-oligosaccharides and polysaccharides.

在部分具體實施例中，該標幟部份體進一步包含功能性標記物，其中該功能性標記物係適合光測定分析法之可檢測標記物。 In some embodiments, the marker moiety further comprises a functional marker, wherein the functional marker is a detectable marker suitable for photometric assays.

在部分具體實施例中，該可檢測標記物係選自下列各物所組成群中：BODIPY染劑、漸層藍(Cascade Blue)染劑、香豆素、螢光素(FITC/FAM)、半抗原(Haptens)、麗絲胺若丹明(Lissamine Rhodamine)B染劑、NBD、奧勒剛綠(Oregon Green)染劑、德州紅(Texas Red)染劑、畢曼疊氮化物(bimane azide)、瑪利納藍(Marina Blue)、太平洋藍(Pacific Blue)、若丹明(Rhodamine)6G染劑、若丹明(Rhodamine)綠色染劑、若丹明(Rhodamine)紅色染劑、四甲基若丹明(Tetramethylrhodamine)、DNP、毛地黃素(Digoxigenin)、生物素、抗生物素蛋白(avidin)、抗生物鏈菌素蛋白、蛋白質、蟲螢光素、抗染劑抗體、5-(與6)-羧基螢光素、5-或6-羧基螢光素、6-(螢光素)-5-(與6)-羧醯胺基己酸、螢光素異硫氰酸酯、若丹明、四甲基若丹明、Cy2、Cy3、Cy5、AMCA、PerCP、R-藻紅素(phycoerythrin)(RPE)、別藻紅素(allophycoerythrin)(APC)、德州紅(Texas Red)、普林斯頓紅(Princeton Red)、塗覆綠色螢光蛋白質(GFP)之CdSe奈米晶粒、DNP、生物素、地高辛(digoxiginin)、辣根過氧化酶(horse radish peroxidase)(HRP)、鹼性磷酸酶(AP)、ß-半乳糖苷酶(GAL)、葡萄糖-6-磷酸脫氫酶、ß-N-乙醯基葡糖胺酶、ß-葡糖醛酸酶、轉化酶、黃嘌呤氧化酶、葡萄糖氧化酶、胜肽、抗體與鏈結體。 In some embodiments, the detectable label is selected from the group consisting of: BODIPY dye, Cascade Blue dye, coumarin, luciferin (FITC/FAM), Haptens, Lissamine Rhodamine B dye, NBD, Oregon Green dye, Texas Red dye, biman azide ), Marina Blue, Pacific Blue, Rhodamine 6G dye, Rhodamine green dye, Rhodamine red dye, Sijia Tetramethylrhodamine, DNP, Digoxigenin, biotin, avidin, avidin, protein, luciferin, anti-drug antibody, 5- (with 6)-carboxyluciferin, 5- or 6-carboxyluciferin, 6-(fluorescein)-5-(and 6)-carboxamidohexanoic acid, luciferin isothiocyanate , rhodamine, tetramethyl rhodamine, Cy2, Cy3, Cy5, AMCA, PerCP, R-phycoerythrin (RPE), allophycoerythrin (APC), Texas Red (Texas Red) ), Princeton Red (Princ Eton Red), CdSe nanocrystals coated with green fluorescent protein (GFP), DNP, biotin, digoxiginin, horseradish peroxidase (HRP), alkaline phosphatase (AP), ß-galactosidase (GAL), glucose-6-phosphate dehydrogenase, ß-N-acetylglucosamine, ß-glucuronidase, invertase, xanthine oxidase , glucose oxidase, peptide, antibody and linker.

在部分具體實施例中，該功能性標記物為適合質譜(MS) 分析法之可檢測標記物。 In some embodiments, the functional marker is suitable for mass spectrometry (MS) A detectable label for the assay.

在部分具體實施例中，該功能性標記物-共軛胜肽包含選自下列之可檢測標記物：螢光標記物、酵素標記物、放射性同位素、化學發光標記物、生物發光標記物、聚合物、金屬粒子、抗體與染劑。 In some embodiments, the functional marker-conjugate peptide comprises a detectable label selected from the group consisting of a fluorescent label, an enzyme label, a radioisotope, a chemiluminescent label, a bioluminescent label, and an polymerization. Materials, metal particles, antibodies and dyes.

在部分具體實施例中，該功能性標記物-共軛胜肽係選自：Lys-生物素、Lys-FITC、Lys-抗生物鏈菌素蛋白、Lys-DNP、Lys-樹脂、Lys-固態擔體或Lys-抗體。 In some embodiments, the functional marker-conjugated peptide is selected from the group consisting of: Lys-biotin, Lys-FITC, Lys-anti-streptoprotein, Lys-DNP, Lys-resin, Lys-solid Carrier or Lys-antibody.

在部分具體實施例中，由經改質糖苷(Y-X)與固態擔體偶合，其中該固態擔體係選自樹脂、珠粒、平面擔體、玻璃載片、聚碳酸酯載片、鋁載片、柰米粒子、層析介質、磁粒與金屬。 In some embodiments, the modified glycoside (YX) is coupled to a solid support, wherein the solid support system is selected from the group consisting of a resin, a bead, a planar support, a glass slide, a polycarbonate slide, and an aluminum slide. , glutinous rice particles, chromatography media, magnetic particles and metals.

本發明另一方面係有關一種醣類之光測定分析法套組，該套組包含上述組成物。 Another aspect of the invention relates to a kit for photometric assays of saccharides comprising the above composition.

本發明另一方面係有關一種醣類之層析分離法套組，該套組包含上述組成物。 Another aspect of the invention relates to a chromatographic separation kit for a saccharide comprising the above composition.

本發明另一方面係有關一種醣類之質譜分析法套組，該套組包含上述組成物。 Another aspect of the invention relates to a mass spectrometry kit for a saccharide comprising the above composition.

本發明另一方面係有關一種製備如式Y-X之經標幟之醣類之方法，其中Y代表單醣、寡醣或多醣亞單位，且其中X代表包含2,3-二氨基苯甲酸(DAB)-胜肽之標幟部份體，該方法包括步驟：(1)提供鄰-DAB-胜肽標幟劑；及(2)由DAB-胜肽標幟劑與醣類在碘(作為觸媒)與酸性溶劑之存在下偶合。 Another aspect of the invention relates to a method of preparing a labeled saccharide of the formula YX, wherein Y represents a monosaccharide, oligosaccharide or polysaccharide subunit, and wherein X represents 2,3-diaminobenzoic acid (DAB) )---------------------------------------------- The medium is coupled with an acidic solvent.

在部分實例中，上述方法進一步包括由經標幟之醣類與固態擔體偶合之步驟。 In some examples, the above method further comprises the step of coupling the labeled sugar to the solid support.

在部分具體實施例中，該固態擔體係選自：樹脂、珠粒、平面擔體、玻璃載片、聚碳酸酯載片、奈米粒子、層析介質、磁粒與金屬。 In some embodiments, the solid support system is selected from the group consisting of resins, beads, planar supports, glass slides, polycarbonate slides, nanoparticles, chromatography media, magnetic particles and metals.

本發明另一方面係有關一種製備DAB-胜肽標幟劑之方法，該方法包括步驟：(1)提供N-Boc-3,4-二氨基苯甲酸(N-Boc-DAB)；(2)提供可視需要與功能性標記物共軛之胜肽；及(3)使N-Boc-3,4-二氨基苯甲酸與胜肽或胜肽-共軛功能性分子利用胜肽合成儀或其他化學方法共 軛。 Another aspect of the invention relates to a method for preparing a DAB-peptide derivative, the method comprising the steps of: (1) providing N-Boc-3,4-diaminobenzoic acid (N-Boc-DAB); (2) Providing a peptide that is conjugated to a functional marker as desired; and (3) using N-Boc-3,4-diaminobenzoic acid with a peptide or peptide-conjugated functional molecule using a peptide synthesizer or Other chemical methods yoke.

在部分具體實施例中，該功能性分子標記物包含選自下列之可檢測標記物：螢光標記物、酵素標記物、放射性同位素、化學發光標記物、生物發光標記物、聚合物、金屬粒子、樹脂、載片、薄片、抗體、與染劑。 In some embodiments, the functional molecular marker comprises a detectable label selected from the group consisting of a fluorescent label, an enzyme label, a radioisotope, a chemiluminescent label, a bioluminescent label, a polymer, a metal particle , resin, slides, flakes, antibodies, and dyes.

在部分具體實施例中，該功能性分子-共軛胜肽係選自：Lys-生物素、Lys-FITC、Lys-抗生物鏈菌素蛋白、Lys-DNP、Lys-抗體或其他胺基酸共軛物。 In some embodiments, the functional molecule-conjugated peptide is selected from the group consisting of: Lys-biotin, Lys-FITC, Lys-anti-streptoprotein, Lys-DNP, Lys-antibody, or other amino acids. Conjugate.

本發明另一方面係有關一種醣類之層析分離方法，該方法包括步驟：(1)使用DAB-胜肽標幟劑標幟醣類，其中該胜肽係與可檢測標記物共軛；及(2)使包含該醣類之組成物進行層析法。 Another aspect of the invention relates to a chromatographic separation method for a saccharide, the method comprising the steps of: (1) using a DAB-peptide marker saccharide, wherein the peptide is conjugated to a detectable label; And (2) subjecting the composition containing the saccharide to chromatography.

本發明另一態樣係有關一種在質譜分析法(MS)期間加強醣類質量強度之方法，該方法包括步驟：(1)使用DAB-胜肽標幟劑標幟醣類，其中該胜肽係與功能性標記物共軛；及(2)採用質譜分析法分析包含該醣類之組成物，其中該經標幟之醣類之質量強度予以提升。 Another aspect of the invention relates to a method of enhancing the mass strength of a saccharide during mass spectrometry (MS), the method comprising the steps of: (1) using a DAB-peptide marker to mark a saccharide, wherein the peptide Conjugating with a functional marker; and (2) analyzing the composition comprising the saccharide by mass spectrometry, wherein the quality of the labeled saccharide is increased.

下列反應圖與圖示形成本說明書之一部份並包括在內，以進一步證實本揭示內容之某些態樣，可參考其中一個或多個圖示並配合本文明確具體實施例之詳細說明更了解本發明。本專利案或申請案包含至少一個彩色圖示。附有彩色圖示之本專利案或申請案之複本可在給付必要費用後向本局要求提供。 The following figures and illustrations form a part of this specification and are included to further clarify certain aspects of the present disclosure, and may refer to one or more of the drawings and the detailed description of the specific embodiments herein. Know the invention. This patent or application contains at least one color representation. A copy of this patent or application with a color graphic may be requested from this Council after payment of the necessary fee.

圖1：醣類與標幟劑(DAB-胜肽及DAB-胜肽-R)共軛之圖解，其係利用碘催化醣類與DAB-胜肽或DAB-胜肽-R之氧化縮合反應達成。 Figure 1: Schematic representation of the conjugation of saccharides with a labeling agent (DAB-peptide and DAB-peptide-R) using iodine-catalyzed oxidative condensation of saccharides with DAB-peptide or DAB-peptide-R Achieved.

圖2：用於標幟醣類之DAB-胜肽標幟劑與DAB-胜肽共軛樹脂或其他生物分子標幟劑之製法圖解。 Figure 2: Diagram of the preparation of DAB-peptide peptides and DAB-peptide conjugated resins or other biomolecular markers for labeling sugars.

圖3：醣-DAB-胜肽於醣質體(glycomic)中之用途。 Figure 3: Use of a sugar-DAB-peptide in a glycoplast.

圖4：由MALDI-TOF MS測定3,4-二氨基苯甲酸-(His)3標幟物之MS(C25H29N11O5；564.0 Da)。 Figure 4: MS (C25H29N11O5; 564.0 Da) of the 3,4-diaminobenzoic acid-(His)3 label was determined by MALDI-TOF MS.

圖5：由MALDI-TOF MS測定3,4-二氨基苯甲酸-(His)6標幟物之MS(C43H50N20O8；975.2 Da)。 Figure 5: MS (C43H50N20O8; 975.2 Da) of the 3,4-diaminobenzoic acid-(His)6 marker was determined by MALDI-TOF MS.

圖6：由MALDI-TOF MS測定縮合之麥芽六醣-DAB之分子量(C43H66N2O32Na；1145.3 Da)。 Figure 6: Determination of the molecular weight of the condensed maltohexaose-DAB (C43H66N2O32Na; 1145.3 Da) as determined by MALDI-TOF MS.

圖7：由MALDI-TOF MS測定縮合之麥芽四醣-DAB-(His)6之分子量(C67H88N20O28；1613.1 Da)。 Figure 7: Determination of the molecular weight of the condensed maltotetraose-DAB-(His)6 (C67H88N20O28; 1613.1 Da) as determined by MALDI-TOF MS.

圖8：由MALDI-TOF MS測定縮合之麥芽六醣-DAB-(His)3之分子量(C61H87N11O35Na；1556.1 Da)。 Figure 8: Determination of the molecular weight of the condensed maltohexaose-DAB-(His)3 (C61H87N11O35Na; 1556.1 Da) by MALDI-TOF MS.

圖9：由MALDI-TOF MS測定縮合之麥芽六醣-DAB-(His)6之分子量(C79H108N20O38；1945.3 Da)。 Figure 9: Determination of the molecular weight of the condensed maltohexaose-DAB-(His)6 (C79H108N20O38; 1945.3 Da) as determined by MALDI-TOF MS.

下列本發明具體實施例之詳細說明中，可參考附圖，其中由類似參考文獻指示類似元素，且其中利用可操作本發明之明確具體實施例說明。此等具體實施例已詳細說明，足使熟悉此相關技術者操作本發明，且咸了解可在不偏離本發明揭示內容下利用其他具體實施例及進行邏輯、結構、功能、及其他方面之變化。因此下列詳細說明並無限制意義。 In the following detailed description of the preferred embodiments of the invention, reference to the claims The specific embodiments have been described in detail, and are intended to be illustrative of the invention . Therefore, the following detailed description is not limiting.

本文揭示經改質之糖苷，該組成物包含(1)單醣、寡醣或多醣亞單位，該等亞單位係利用糖苷鏈結體連接成直鏈或分支鏈，及(2)標幟部份體。 Described herein is a modified glycoside comprising (1) a monosaccharide, oligosaccharide or polysaccharide subunit linked by a glycosidic link to a straight or branched chain, and (2) a part of the label Body.

該標幟部份體包含鄰位經取代之二氨基-芳香系化合物。在本發明不同的具體實施例中，該鄰位經取代之二氨基-芳香系化合物為1,2-二氨基苯(亦稱為鄰-苯二胺；OPD)、3,4-二氨基苯甲酸或2,3-二氨基吡啶。較佳態樣中，該標幟部份體包含3,4-二氨基苯甲酸胜肽(DAB-胜肽)共軛物。 The label moiety comprises an ortho-substituted diamino-aromatic compound. In various embodiments of the invention, the ortho-substituted diamino-aromatic compound is 1,2-diaminobenzene (also known as o-phenylenediamine; OPD), 3,4-diaminobenzene. Formic acid or 2,3-diaminopyridine. In a preferred aspect, the portion of the marker comprises a 3,4-diaminobenzoic acid peptide (DAB-peptide) conjugate.

本文所採用術語「多醣」與「寡醣」之廣義係指包含許多重複單位之醣類，其包括(但不限於)：具有2至超過2,000個重複單位醣類。典型地，此技藝接受且用於本文中之術語「多醣」係指具有約10至約2,000個或更多個重複單位之醣類。此技藝接受且用於本文中之術語「寡醣」係指具有約2至約10個重複單位之醣類。多醣之重複單位可為一個單醣分子 或可為複數個醣類(例如：混合醣類)。在某些具體實施例中，該重複單位為2個或更多個單醣分子。依據本發明方法，來自不同細菌種類與/或菌株之多醣或寡醣片段可採用化學法連接或合成法合成，所形成單一多醣鏈或寡醣鏈即包含來自衍生及/或可辨識該等片段之多種細菌種類及/或菌株之多重抗原決定基；因此，本發明多醣或寡醣重複單位(群)之組成份不一定需要在整個醣鏈中保持一致。本發明方法所涵括之多醣或寡醣包含一個或多個氨基糖。在某些具體實施例中，該一個或多個氨基糖係多醣或寡醣重複單位之組份。在其他具體實施例中，該一個或多個氨基糖不為多醣或寡醣重複單位之組份。 As used herein, the terms "polysaccharide" and "oligosaccharide" are used broadly to refer to a saccharide comprising a plurality of repeating units, including but not limited to: having from 2 to more than 2,000 repeating unit sugars. Typically, the term "polysaccharide" as used herein and as used herein refers to a saccharide having from about 10 to about 2,000 or more repeating units. The term "oligosaccharide" as used in this art and as used herein refers to a saccharide having from about 2 to about 10 repeating units. The repeating unit of the polysaccharide may be a monosaccharide molecule Or it may be a plurality of sugars (for example: mixed sugars). In certain embodiments, the repeating unit is 2 or more monosaccharide molecules. According to the method of the present invention, polysaccharides or oligosaccharide fragments derived from different bacterial species and/or strains may be synthesized by chemical ligation or synthesis, and the single polysaccharide chains or oligosaccharide chains formed may comprise derived and/or identifiable fragments. Multiple antigenic determinants of various bacterial species and/or strains; therefore, the components of the polysaccharide or oligosaccharide repeating unit (group) of the present invention do not necessarily need to be consistent throughout the sugar chain. The polysaccharide or oligosaccharide encompassed by the method of the invention comprises one or more amino sugars. In certain embodiments, the one or more amino sugar polysaccharides or oligosaccharide repeats components of the unit. In other specific embodiments, the one or more amino sugars are not components of a polysaccharide or oligosaccharide repeating unit.

該標幟部份體可依任何習知方法製造，例如：標準固相胜肽合成法或熟悉此相關技術者習知之化學合成法。例如：組胺酸、離胺酸、及功能性分子-共軛胜肽(例如：生物素、螢光染劑、蛋白質，等等)可利用胜肽合成法形成之醯胺鍵，與N-Boc-2,3-二氨基苯甲酸(DAB)縮合。 The portion of the label can be made by any conventional method, such as standard solid phase peptide synthesis or chemical synthesis known to those skilled in the art. For example: histidine, lysine, and functional molecule-conjugate peptides (eg, biotin, fluorescent dyes, proteins, etc.) can be formed by peptide synthesis, and N- Boc-2,3-diaminobenzoic acid (DAB) condensation.

本文所採用術語「胜肽」係指由一個或多個胺基酸單體組成但長度比蛋白質短之聚合物。較佳地，本文所述各靶向癌症之胜肽包括至多50(例如：至多10或20)個胺基酸。有些實例中，該靶向癌症之胜肽分別包含4-20個胺基酸殘基(例如：4-10個、6-10個、6-15個、或6-20個胺基酸殘基)。此等胜肽可包含天然胺基酸殘基，或經改質之胺基酸。一項實例中，該靶向癌症之胜肽之N-端或C-端係經改質，例如：在C-端包含-NH2基團。 The term "peptide" as used herein refers to a polymer composed of one or more amino acid monomers but shorter in length than the protein. Preferably, each of the cancer-targeting peptides described herein comprises up to 50 (eg, up to 10 or 20) amino acids. In some instances, the cancer-targeting peptide comprises 4-20 amino acid residues (eg, 4-10, 6-10, 6-15, or 6-20 amino acid residues, respectively) ). These peptides may comprise a native amino acid residue, or a modified amino acid. In one example, the N-terminus or C-terminus of the peptide that targets the cancer is modified, for example, comprising a -NH2 group at the C-terminus.

胜肽可依據Steward等人於固相胜肽合成法(Solid Phase Peptide Synthesis)，第2版，Pierce Chemical Company,Rockford,Ill.,(1984)中說明之製程，使用Rainin PTI Symphony合成儀，採用固相化學技術合成。有關固相胜肽合成法技術可參見Stewart等人之固相胜肽合成法(Solid Phase Peptide Synthesis),W.H.Freeman Co.(San Francisco),1963與Meienhofer之激素蛋白質與胜肽(Hormonal Proteins and Peptides),1973。有關傳統溶液合成法可參見Schroder等人之胜肽學(The Peptides)，第1冊，Acacemic Press(New York)。一般而言，此等方法包括在聚合物上繼續加長之胜肽鏈上依序添加一或多個胺基酸或經過適當保護之胺基酸。通 常，第一胺基酸上之氨基或羧基均需要使用合適保護基保護。然後由受保護及/或經過衍化之胺基酸附接在惰性固態擔體上，或用在溶液中，在適合形成醯胺鍵之條件下，依序添加下一個具有經過適當保護之互補基團(氨基或羧基)之胺基酸。然後從此新添加之胺基酸殘基上脫除保護基後，再添加下一個胺基酸(適當地保護)，依此類推。 The peptide can be used according to the process described by Steward et al., Solid Phase Peptide Synthesis, 2nd Edition, Pierce Chemical Company, Rockford, Ill., (1984), using a Rainin PTI Symphony synthesizer. Solid phase chemical synthesis. For solid phase peptide synthesis techniques, see Stewart et al., Solid Phase Peptide Synthesis, WHFreeman Co. (San Francisco), 1963 and Meienhofer's Hormone Proteins and Peptides. ), 1973. For a discussion of conventional solution synthesis, see Schroder et al., The Peptides, Vol. 1, Acacemic Press (New York). In general, such methods include the sequential addition of one or more amino acids or suitably protected amino acids to the peptide chain that continues to lengthen on the polymer. through Often, the amino or carboxyl group on the first amino acid needs to be protected with a suitable protecting group. Then, the protected and/or derivatized amino acid is attached to the inert solid support, or used in a solution, and the next suitably protected complementary group is added under conditions suitable for the formation of a guanamine bond. Amino acid of the group (amino or carboxyl group). Then, after removing the protecting group from the newly added amino acid residue, the next amino acid is added (suitably protected), and so on.

本文說明之標幟劑(DAB-胜肽)可用為可檢測標記物，並可標記在醣類之還原端，形成醣-DAB-胜肽，藉以協助醣類之純化與分析。此外，DAB-胜肽共軛醣類可提高醣類之離子化能力，藉以改善其在MS分析法中之敏感性。 The labeling agent (DAB-peptide) described herein can be used as a detectable label and can be labeled on the reducing end of the saccharide to form a sugar-DAB-peptide, thereby assisting in the purification and analysis of the saccharide. In addition, DAB-peptide conjugated saccharides increase the ionization capacity of saccharides, thereby improving their sensitivity in MS assays.

在部分態樣中，當3,4-二氨基苯甲酸(DAB)受到辣根過氧化酶及過氧化氫或尿素過氧化物之酵素性氧化產生螢光團時，其本身即可轉化為可檢測部份體。 In some aspects, when 3,4-diaminobenzoic acid (DAB) is subjected to enzymatic oxidation of horseradish peroxidase and hydrogen peroxide or urea peroxide to produce a fluorophore, it can be converted into a Detect part of the body.

醣與標幟劑(DAB-胜肽及DAB-胜肽-R)之共軛可利用碘催化醣與DAB-胜肽或DAB-胜肽-R之氧化縮合法達成，如圖1所示。 Conjugation of a sugar with a labeling agent (DAB-peptide and DAB-peptide-R) can be achieved by an oxidative condensation method of an iodine-catalyzed sugar with DAB-peptide or DAB-peptide-R, as shown in FIG.

在部分具體實施例中，本文說明之醣-DAB-胜肽包含醣-DAB-6His、醣-DAB-6His-樹脂、醣-DAB-3His、醣-DAB-3His-樹脂、醣-DAB-Lys-生物素、醣-DAB-Lys-FITC、醣-DAB-6His、醣-DAB-3His、醣-DAB-Lys-生物素、醣-DAB-Lys-樹脂。 In some embodiments, the sugar-DAB-peptide described herein comprises a sugar-DAB-6His, a sugar-DAB-6His-resin, a sugar-DAB-3His, a sugar-DAB-3His-resin, a sugar-DAB-Lys. - biotin, sugar-DAB-Lys-FITC, sugar-DAB-6His, sugar-DAB-3His, sugar-DAB-Lys-biotin, sugar-DAB-Lys-resin.

任何醣-DAB-胜肽均可進一步與可檢測標記物共軛(附接)或固定在固態擔體上，如：樹脂、奈米粒子、薄片、晶片，等等，供進行醣類結合性分析法、顯影法與其他用途。 Any sugar-DAB-peptide can be further conjugated (attached) to a detectable label or immobilized on a solid support such as resin, nanoparticle, flakes, wafers, etc. for carbohydrate binding Analytical methods, development methods and other uses.

本文所採用「共軛」、「經共軛」或「附接」意指連接兩種實體。兩種實體之間可直接連接或利用鏈結體連接，如：聚合物鏈結體。共軛或附接法可包括共價或非共價鍵結，及其他連接型式，例如：在其中一個實體上面或裡面捕捉另一個實體，或在第三個實體上面或裡面捕捉其中另一個或兩個實體，如：膠束(micelle)。 As used herein, "conjugate", "conjugated" or "attached" means to connect two entities. The two entities can be directly connected or connected by a link, such as a polymer chain. Conjugation or attachment may include covalent or non-covalent bonding, and other types of connections, such as capturing another entity on or in one of the entities, or capturing one or the other on or in the third entity Two entities, such as micelles.

在一態樣中，醣-DAB-胜肽中之胜肽可附接可檢測標記物，其係一種可直接或間接辨識其所共軛之胜肽之化合物，因此可檢測、測定與/或定性醣類。此等「可檢測標記物」之實例希望包括(但不限於)： 螢光標記物、化學發光標記物、產色標記物、酵素標誌、放射性同位素與親和性標幟物，如：生物素。此等標記物可利用習知方式與胜肽直接或間接共軛。 In one aspect, the peptide in the sugar-DAB-peptide can be attached to a detectable label, which is a compound that directly or indirectly recognizes the peptide to which it is conjugated, and thus can be detected, measured, and/or Qualitative sugars. Examples of such "detectable markers" are intended to include (but are not limited to): Fluorescent markers, chemiluminescent labels, chromogenic markers, enzyme markers, radioisotopes and affinity markers such as biotin. These markers can be directly or indirectly conjugated to the peptide in a conventional manner.

此標記之醣類可採用相關技藝上習知之方法與試劑檢測。本發明較佳檢測法係利用螢光。可採用各種不同顯影技術看到螢光，包括普通光學或螢光顯微鏡、雷射掃描共聚焦顯微鏡、與流式細胞儀。 The labeled sugars can be detected by methods and reagents known in the art. The preferred detection method of the present invention utilizes fluorescence. Fluorescence can be seen using a variety of different development techniques, including general optical or fluorescent microscopes, laser scanning confocal microscopy, and flow cytometry.

在另一項實例中，醣-DAB-胜肽包含與鎳管柱結合之醣-DAB-(His)6與醣-DAB-(His)3，藉以透過固定化金屬親和性層析法(IMAC)促進醣類之分離與純化。此外，醣-DAB-胜肽-樹脂包含醣-DAB-(His)6-樹脂與醣-DAB-(His)3-樹脂，藉以促進經過洗滌、過濾或離心之醣類之分離與純化。 In another example, the sugar-DAB-peptide comprises a sugar-DAB-(His)6 and a sugar-DAB-(His)3 bound to a nickel column, whereby the immobilized metal affinity chromatography (IMAC) is used. Promote the separation and purification of sugars. Further, the sugar-DAB-peptide-resin comprises a sugar-DAB-(His)6-resin and a sugar-DAB-(His)3-resin to promote separation and purification of the washed, filtered or centrifuged sugar.

標幟醣類之DAB-胜肽標幟劑與DAB-胜肽共軛樹脂或其他分子生物標幟劑之製法示於圖2。 The method for preparing DAB-peptide peptide and DAB-peptide conjugated resin or other molecular biological reagents of the standard sugar is shown in Fig. 2.

相較於其他醣類標幟法，醣-DAB-胜肽在檢測醣類上具有許多優點。醣-DAB-胜肽於醣質體(glycomic)中之用途示於圖3。 Compared to other carbohydrate labeling methods, sugar-DAB-peptide has many advantages in detecting sugars. The use of a sugar-DAB-peptide in a glycoform is shown in Figure 3.

本揭示內容亦提供一種在醣質體試驗中檢測及衍化醣類且不需要破壞聚醣功能之快速方法，即使當樣本中醣類含量極低(低於1μmol)。 The present disclosure also provides a rapid method for detecting and derivatizing saccharides in a saccharide assay without the need to destroy the glycan function, even when the saccharide content of the sample is extremely low (less than 1 μmol).

本說明書中，在本發明內容中及使用各術語之明確內容中，其所採用之術語通常具有相關技藝上之一般定義。用於說明本發明之某些術語係如下文或說明書中所討論，以進一步指示操作者與本發明有關之說明。為了方便，可能強調某些術語，例如：利用斜體字與/或引號表示。所強調之術語對該術語之範圍與定義沒有影響；不論是否強調該術語，其在相同內容中之範圍與定義仍保持相同。同樣地咸了解，同一事件可能以另一種方式說明。因此，本文所討論之任何一或多種術語可能採用其他語言與同義字，不論該術語是否在本文中說明或討論，均沒有加諸任何特殊重要性。某些術語會提出同義字。所引用一或多種同義字並未排除其他同義字之用法。本說明書所採用任何實例(包括本文所討論任何術語之實例)均僅供說明，絕未以任何方式限制本發明或任何例舉術語之範圍與定義。 同樣地，本發明未限制本說明書所提出之各種不同具體實施例。 In the present specification, the terms used in the context of the present invention and the use of each term are generally defined in the related art. Certain terms used to describe the invention are discussed below or in the specification to further indicate an operator's description of the invention. For convenience, certain terms may be emphasized, for example, using italics and/or quotation marks. The terminology emphasized has no effect on the scope and definition of the term; whether or not the term is emphasized, its scope and definition remain the same in the same content. Similarly, the same incident may be explained in another way. Thus, any one or more of the terms discussed herein may be in other languages and synonyms, regardless of whether the term is described or discussed herein, without any particular importance. Some terms will be synonymous. The use of one or more synonyms does not exclude the use of other synonyms. The use of any examples, including examples of any terms discussed herein, is for illustrative purposes only and is not intended to limit the scope and definition of the invention or any exemplified terms. As such, the invention is not limited to the various embodiments disclosed herein.

除非另有說明，否則本文所採用所有技術與科學術語均具有熟悉本發明相關技術者習知之相同定義。若有衝突時，以本發明文件(包括其定義)為準。 Unless otherwise stated, all technical and scientific terms used herein have the same definitions as are known to those skilled in the art. In case of conflict, the documents of the invention (including its definition) shall prevail.

實例Instance

在無意限制本發明範圍下，根據本發明具體實施例所例舉儀器、裝置、方法與其相關結果均如下所示。應注意，為了方便讀者，實例中可能採用標題或子標題，但其未以任何方式限制本發明範圍。此外，本文提供並揭示某些理論；然而，不論該等理論是否正確，只要本發明可在不考慮任何特定作用理論或流程下根據本發明進行操作，其絕未以任何方式限制本發明之範圍。 Without limiting the scope of the invention, the instruments, devices, methods and associated results in accordance with the embodiments of the invention are as follows. It should be noted that the title or subtitle may be employed in the examples for the convenience of the reader, but it is not intended to limit the scope of the invention in any way. In addition, certain theories are provided and disclosed herein; however, no matter whether the theory is correct or not, as long as the invention can be practiced in accordance with the present invention without any particular theory of action or flow, it does not limit the scope of the invention in any way. .

實例1：醣類之化學結構Example 1: Chemical Structure of Carbohydrates

實例2：N-Boc-3,4-二氨基苯甲酸(N-Boc-DAB)之合成法Example 2: Synthesis of N-Boc-3,4-diaminobenzoic acid (N-Boc-DAB)

受N-Boc(N-第三丁氧基羰基)保護之3,4-二氨基苯甲酸前體(3,4-DAB)之製法係由3,4-二氨基苯甲酸(1.52g,10mmol)及二碳酸二第三丁氧基酯((t-Boc)2O,6.55g,30mmol)與三乙胺(NEt3；7.0mL,60mmol)於氯仿(CHCl3,200mL)中，於室溫(25℃)下攪拌反應一夜(24h)。使用水萃取所得溶液，經Na2SO4脫水，產生N-Boc-3,4-二氨基苯甲酸(2.0g,79%)之褐色粉末。1H NMR(600MHz,d-MeOH)δ 7.96(s,1H,ArH),7.75(dd,1H,J=8.4,1.9Hz,ArH),7.60(d,1H,J=8.4,1.9Hz,ArH),4.80(brs,2H,NH),1.52(s,18H,CH3).13C NMR(150MHz,d-MeOH)δ 173.2,156.4,155.5,135.7,133.6,130.3,127.9,127.8,123.6,81.7,81.6,28.8,28.7。測定N-Boc-DAB之質量(C17H24N2O6；352.4 Da)。 The 3,4-diaminobenzoic acid precursor (3,4-DAB) protected by N-Boc (N-tert-butoxycarbonyl) is prepared from 3,4-diaminobenzoic acid (1.52 g, 10 mmol). And ditributyl dicarbonate ((t-Boc) 2O, 6.55 g, 30 mmol) and triethylamine (NEt3; 7.0 mL, 60 mmol) in chloroform (CHCl3, 200 mL) at room temperature (25 The reaction was stirred overnight (24 h) under °C. The resulting solution was extracted with water and dried over Na 2 SO 4 to yield a brown powder of N-Boc-3,4-diaminobenzoic acid (2.0 g, 79%). 1H NMR (600MHz, d-MeOH) δ 7.96 (s, 1H, arH), 7.75 (dd, 1H, J = 8.4, 1.9 Hz, ArH), 7.60 (d, 1H, J = 8.4, 1.9 Hz, ArH) , 4.80 (brs, 2H, NH), 1.52 (s, 18H, CH3). 13C NMR (150 MHz, d-MeOH) δ 173.2, 156.4, 155.5, 135.7, 133.6, 130.3, 127.9, 127.8, 123.6, 81.7, 81.6 , 28.8, 28.7. The mass of N-Boc-DAB (C17H24N2O6; 352.4 Da) was determined.

實例3：3,4-二氨基苯甲酸-(His)3標幟物之合成法Example 3: Synthesis of 3,4-diaminobenzoic acid-(His)3 label

採用固相胜肽合成法(ABI 433A胜肽合成儀)在受N-Boc(N-第三丁氧基羰基)保護之3,4-二氨基苯甲酸前體(N-Boc-DAB)(其係使用二碳酸二-第三丁酯(t-Boc2O)封阻DAB上之氨基後製得)上添加3個組胺酸單位後，水解，產生3,4-二氨基苯甲酸-(His)3標幟物(DAB-His-His-His)。採用MALDI-TOF MS測定3,4-二氨基苯甲酸-(His)3標幟物之MS(C25H29N11O5；564.0 Da)，其示於圖4。 The solid phase peptide synthesis method ( ABI 433A peptide synthesizer) was used to protect N-Boc (N-tert-butoxycarbonyl) 3,4-diaminobenzoic acid precursor (N-Boc-DAB) ( It is obtained by adding 3 histidine units to the unit after blocking the amino group on DAB with di-tert-butyl dicarbonate (t-Boc2O), and then hydrolyzing to produce 3,4-diaminobenzoic acid-(His ) 3 marker (DAB-His-His-His). MS (C25H29N11O5; 564.0 Da) of the 3,4-diaminobenzoic acid-(His)3 label was determined by MALDI-TOF MS, which is shown in Fig. 4.

實例4：3,4-二氨基苯甲酸-(His)3-樹脂標幟物之合成法Example 4: Synthesis of 3,4-diaminobenzoic acid-(His)3-resin label

採用固相胜肽合成法，使用TentaGal樹脂，在受N-Boc(N-第三丁氧基羰基)保護之3,4-二氨基苯甲酸前體(N-Boc-DAB)(其係使用二碳酸二-第三丁酯(t-Boc2O)封阻DAB上之氨基後製得)上添加3個組胺酸單位後，水解，產生3,4-二氨基苯甲酸-(His)3-樹脂標幟物(DAB-His-His-His-樹脂)。 Solid phase peptide synthesis, using TentaGal resin, 3,4-diaminobenzoic acid precursor (N-Boc-DAB) protected by N-Boc (N-tert-butoxycarbonyl) After di-tert-butyl dicarbonate (t-Boc2O) blocks the amino group on DAB), after adding 3 histidine units, it is hydrolyzed to produce 3,4-diaminobenzoic acid-(His)3- Resin label (DAB-His-His-His-resin).

實例5：3,4-二氨基苯甲酸-(His)6標幟物之合成法Example 5: Synthesis of 3,4-diaminobenzoic acid-(His)6 label

採用固相胜肽合成法(ABI 433A胜肽合成儀)在受N-Boc(N-第三丁氧基羰基)保護之3,4-二氨基苯甲酸前體(N-Boc-DAB)(其係使用二碳酸二-第三丁酯(t-Boc2O)封阻DAB上之氨基後製得)上添加6個組胺酸單位後，水解，產生3,4-二氨基苯甲酸-(His)6標幟物(DAB-His-His-His-His-His-His)。採用MALDI-TOF MS測定3,4-二氨基苯甲酸-(His)6標幟物之MS(C43H50N20O8；975.2 Da)，其示於圖5。 The solid phase peptide synthesis method ( ABI 433A peptide synthesizer ) was used to protect N-Boc (N-tert-butoxycarbonyl) 3,4-diaminobenzoic acid precursor (N-Boc-DAB) ( It is obtained by adding 6 histidine units to the unit after blocking the amino group on DAB with di-tert-butyl dicarbonate (t-Boc2O), and then hydrolyzing to produce 3,4-diaminobenzoic acid-(His ) 6 markers (DAB-His-His-His-His-His-His). MS (C43H50N20O8; 975.2 Da) of the 3,4-diaminobenzoic acid-(His)6 marker was determined by MALDI-TOF MS, which is shown in Fig. 5.

實例6：3,4-二氨基苯甲酸-(His)6-樹脂標幟物之合成法Example 6: Synthesis of 3,4-diaminobenzoic acid-(His)6-resin label

採用固相胜肽合成法，使用TentaGal樹脂，在受N-Boc(N-第三丁氧基羰基)保護之3,4-二氨基苯甲酸前體(N-Boc-DAB)(其係使用二碳酸二-第三丁酯(t-Boc2O)封阻DAB上之氨基後製得)上添加6個組 胺酸單位後，水解，產生3,4-二氨基苯甲酸-(His)6-樹脂標幟物(DAB-His-His-His-His-His-His-樹脂)。 Solid phase peptide synthesis, using TentaGal resin, 3,4-diaminobenzoic acid precursor (N-Boc-DAB) protected by N-Boc (N-tert-butoxycarbonyl) 6 groups were added to the di-tert-butyl dicarbonate (t-Boc2O) to block the amino group on DAB) After the unit of the amine acid, it is hydrolyzed to produce a 3,4-diaminobenzoic acid-(His)6-resin label (DAB-His-His-His-His-His-His-resin).

實例7：3,4-二氨基苯甲酸-Lys-(生物素)標幟物之合成法Example 7: Synthesis of 3,4-diaminobenzoic acid-Lys-(biotin) label

採用固相胜肽合成法(ABI 433A胜肽合成儀)在受N-Boc(N-第三丁氧基羰基)保護之3,4-二氨基苯甲酸前體(N-Boc-DAB)(其係使用二碳酸二-第三丁酯(t-Boc2O)封阻DAB上之氨基後製得)上添加離胺酸-(生物素)單位後，水解，產生3,4-二氨基苯甲酸-離胺酸-(生物素)標幟物(DAB-Lys-(生物素)。採用MALDI-TOF MS測定3,4-二氨基苯甲酸-離胺酸-(生物素)標幟物之MS(506.3 Da)。 The solid phase peptide synthesis method ( ABI 433A peptide synthesizer) was used to protect N-Boc (N-tert-butoxycarbonyl) 3,4-diaminobenzoic acid precursor (N-Boc-DAB) ( It is prepared by blocking the amino group on DAB by using di-t-butyl dicarbonate (t-Boc2O). After adding lysine-(biotin) unit, it is hydrolyzed to produce 3,4-diaminobenzoic acid. - lysine-(biotin) label (DAB-Lys-(biotin). Determination of MS of 3,4-diaminobenzoic acid-lysine-(biotin) label by MALDI-TOF MS (506.3 Da).

實例8：麥芽糖六醣-DAB之合成法Example 8: Synthesis of maltose hexose-DAB

在觸媒量碘之存在下，於乙酸溶液中，使用3,4-二氨基苯甲酸(DAB；1.0mg)標幟天然麥芽糖六醣(6H或G6；1.0mg)，形成麥芽糖六醣-DAB產物。由MALDI-TOF MS測定縮合之麥芽糖六醣-DAB之分子量(C43H66N2O32Na；1145.3 Da)，其示於圖6。 In the presence of a catalytic amount of iodine, 3,4-diaminobenzoic acid (DAB; 1.0 mg) of natural maltohexaose (6H or G6; 1.0 mg) was used in acetic acid solution to form maltohexose-DAB. product. The molecular weight of the condensed maltohexose-DAB (C43H66N2O32Na; 1145.3 Da) was determined by MALDI-TOF MS, which is shown in Fig. 6.

實例9：麥芽糖四醣-DAB-(His)6之合成法Example 9: Synthesis of maltose tetrasaccharide-DAB-(His)6

在觸媒量碘之存在下，於乙酸溶液中，使用3,4-二氨基苯甲酸-(His)6(DAB-(His)6；1.0mg)標幟天然麥芽糖四醣(G4；1.0mg)，形成麥芽糖四醣-DAB-(His)6產物。由MALDI-TOF MS測定縮合之麥芽糖四醣-DAB-(His)6之分子量(C67H88N20O28；1613.1 Da)，其示於 圖7。 In the presence of catalytic amount of iodine, 3,4-diaminobenzoic acid-(His)6(DAB-(His)6; 1.0 mg) was used in the acetic acid solution to native maltotetraose (G4; 1.0 mg). ), a maltose tetrasaccharide-DAB-(His)6 product is formed. The molecular weight of the condensed maltotetraose-DAB-(His) 6 (C67H88N20O28; 1613.1 Da) was determined by MALDI-TOF MS. Figure 7.

實例10：麥芽糖六醣-DAB-(His)3之合成法Example 10: Synthesis of maltose hexose-DAB-(His)3

在觸媒量碘之存在下，於乙酸溶液中，使用3,4-二氨基苯甲酸-(His)3(DAB-(His)3；1.0mg)標幟天然麥芽糖六醣(G6；1.0mg)，形成麥芽糖六醣-DAB-(His)3產物。由MALDI-TOF MS測定縮合之麥芽糖六醣-DAB-(His)3之分子量(C61H87N11O35Na；1556.1 Da)，其示於圖8。 3,4-diaminobenzoic acid-(His)3(DAB-(His)3; 1.0mg) natural maltohexaose (G6; 1.0mg) in the presence of catalyst iodine in acetic acid solution ), a maltose hexose-DAB-(His) 3 product is formed. The molecular weight of the condensed maltohexose-DAB-(His) 3 (C61H87N11O35Na; 1556.1 Da) was determined by MALDI-TOF MS, which is shown in FIG.

實例11：麥芽糖六醣-DAB-(His)6之合成法Example 11: Synthesis of maltohexose-DAB-(His)6

在觸媒量碘之存在下，於乙酸溶液中，使用3,4-二氨基苯甲酸-(His)6(DAB-(His)6；1.0mg)標幟天然麥芽糖六醣麥芽糖六醣(G6；1.0mg)，形成麥芽糖六醣-DAB-(His)6產物。由MALDI-TOF MS測定縮合之麥芽糖六醣-DAB-(His)之分子量(C79H108N20O38；1945.3 Da)，其示於圖9。 In the presence of catalytic amount of iodine, 3,4-diaminobenzoic acid-(His)6(DAB-(His)6; 1.0 mg) is used in the presence of iodine in the presence of natural maltohexose maltoose hexose (G6). ; 1.0 mg) to form a maltohexaose-DAB-(His)6 product. The molecular weight of the condensed maltohexose-DAB-(His) (C79H108N20O38; 1945.3 Da) was determined by MALDI-TOF MS, which is shown in FIG.

雖然本文所說明本發明之明確具體實施例係供舉例說明，但可在不偏離本發明精神與範圍下進行各種不同修飾。因此除了附錄之請求項外，本發明並不受其限制。 While the invention has been described with respect to the specific embodiments of the present invention, various modifications may be made without departing from the spirit and scope of the invention. Accordingly, the invention is not limited by the scope of the appended claims.

雖然本發明已配合其詳細說明，但上述說明僅供說明，並未限制本發明範圍，本發明範圍僅受附錄之請求項之限制。其他態樣、優點與修飾均在附錄之請求項內。 The present invention has been described in detail with reference to the accompanying drawings. Other aspects, advantages and modifications are included in the appendix.

本文所提及之專利案與科學文獻已建立熟悉此相關技術者可取得之知識。本文所摘錄所有美國專利案與公開或未公開之專利申請案之揭示內容均以引用之方式併入本文中。所有其他已公開參考文獻、手稿與科技文獻之揭示內容已以引用之方式併入本文中。 The patents and scientific literature referred to herein have established knowledge that is available to those skilled in the art. The disclosures of all of the U.S. patents and the disclosures of the entire disclosures are hereby incorporated by reference. The disclosures of all other published references, manuscripts, and scientific literature are incorporated herein by reference.

Claims (31)

一種組成物，其包含如下式之經改質糖苷：Y-X其中Y代表單醣、寡醣或多醣亞單位，其中該等單醣、寡醣或多醣亞單位係利用糖苷鏈結共同連接成直鏈或分支鏈，及其中X代表包含鄰位經取代之二氨基-芳香系化合物之標幟部份體。 A composition comprising a modified glycoside of the formula: YX wherein Y represents a monosaccharide, oligosaccharide or polysaccharide subunit, wherein the monosaccharide, oligosaccharide or polysaccharide subunit is linked together by a glycosidic linkage into a linear chain Or a branched chain, and wherein X represents a partial moiety comprising an ortho-substituted diamino-aromatic compound. 如請求項1之組成物，其中該單醣亞單位或寡醣或多醣亞單位係相同或相異，且係選自由下列各物所組成之群中：葡萄糖、半乳糖、果糖、核酮糖、甘露糖、核糖、***糖、木糖、來蘇糖、阿洛糖、阿卓糖、古洛糖、鼠李糖、***糖、岩藻糖、N-乙醯基半乳糖胺、葡萄醛酸、半乳糖醛酸、Globo H、GD2、GD3、GD1a、GQ1b、GT1b、GT1a、Gb3、Gb5、SSEA寡醣類、岩藻糖基GM1、GM2、GM3、血型抗原(A、B、O、H)、福斯曼(Forssman)抗原、路易士a(Lewis a)、路易士b(Lewis b)、路易士X(Lewis X)、唾液基路易士(Sialyl Lewis X)、路易士Y(Lewis Y)、基於乳糖之O-聚醣、N-乙醯基葡糖胺核心結構、唾液基-乳糖、唾液基化寡醣類、硫酸化寡醣類、磷酸化寡醣類、甘露-寡醣類、纖維-寡醣類、木-寡醣類、甲殼-寡醣類、麥芽-寡醣類與多醣類。 The composition of claim 1, wherein the monosaccharide subunit or oligosaccharide or polysaccharide subunit is the same or different and is selected from the group consisting of glucose, galactose, fructose, ribulose , mannose, ribose, arabinose, xylose, lyxose, allose, altrose, gulose, rhamnose, arabinose, fucose, N-ethyl galactosamine, glucosaldehyde Acid, galacturonic acid, Globo H, GD2, GD3, GD1a, GQ1b, GT1b, GT1a, Gb3, Gb5, SSEA oligosaccharides, fucosyl GM1, GM2, GM3, blood group antigens (A, B, O, H), Forssman antigen, Lewis a, Lewis b, Lewis X, Sialyl Lewis X, Lewis Y (Lewis) Y), lactose-based O-glycan, N-acetyl glucosamine core structure, salivary-lactose, salivylated oligosaccharide, sulfated oligosaccharide, phosphorylated oligosaccharide, mannose-oligosaccharide Classes, fiber-oligosaccharides, wood-oligosaccharides, chitin-oligosaccharides, malt-oligosaccharides and polysaccharides. 如請求項1之組成物，其中該標幟部份體進一步包含功能性標記物。 The composition of claim 1, wherein the label portion further comprises a functional marker. 如請求項3之組成物，其中該功能性標記物為適合光測定分析法之可檢測標記物。 The composition of claim 3, wherein the functional marker is a detectable label suitable for photometric assays. 如請求項4之組成物，其中該可檢測標記物係選自由下列各物所組成之群中：BODIPY染劑、漸層藍(Cascade Blue)染劑、香豆素、螢光 素(FITC/FAM)、半抗原(Haptens)、麗絲胺若丹明(Lissamine Rhodamine)B染劑、NBD、奧勒剛綠(Oregon Green)染劑、德州紅(Texas Red)染劑、畢曼疊氮化物(bimane azide)、瑪利納藍(Marina Blue)、太平洋藍(Pacific Blue)、若丹明(Rhodamine)6G染劑、若丹明(Rhodamine)綠色染劑、若丹明(Rhodamine)紅色染劑、四甲基若丹明、DNP、毛地黃素(Digoxigenin)、生物素、抗生物素蛋白(avidin)、抗生物鏈菌素蛋白、蛋白質、蟲螢光素、抗染劑抗體、5-(與6)-羧基螢光素、5-或6-羧基螢光素、6-(螢光素)-5-(與6)-羧醯胺基己酸、螢光素異硫氰酸酯、若丹明、四甲基若丹明(Tetramethylrhodamine)、Cy2、Cy3、Cy5、AMCA、PerCP、R-藻紅素(RPE)、別藻紅素(APC)、德州紅(Texas Red)、普林斯頓紅(Princeton Red)、塗覆綠色螢光蛋白質(GFP)之CdSe奈米晶粒、DNP、生物素、地高辛(digoxiginin)、辣根過氧化酶(horse radish peroxidase)(HRP)、鹼性磷酸酶(AP)、ß-半乳糖苷酶(GAL)、葡萄糖-6-磷酸脫氫酶、ß-N-乙醯基葡糖胺酶、ß-葡糖醛酸酶、轉化酶、黃嘌呤氧化酶、葡萄糖氧化酶、胜肽、抗體、與鏈結體。 The composition of claim 4, wherein the detectable label is selected from the group consisting of: BODIPY dye, Cascade Blue dye, coumarin, fluorescent (FITC/FAM), hapten (Haptens), Lissamine Rhodamine B dye, NBD, Oregon Green dye, Texas Red dye, Bi Bimane azide, Marina Blue, Pacific Blue, Rhodamine 6G dye, Rhodamine green dye, Rhodamine Red dye, tetramethyl rhodamine, DNP, digoxigenin, biotin, avidin, avidin, protein, luciferin, anti-staining agent Antibody, 5-(and 6)-carboxyluciferin, 5- or 6-carboxyluciferin, 6-(fluorescein)-5-(and 6)-carboxamidohexanoic acid, luciferin Thiocyanate, rhodamine, Tetramethylrhodamine, Cy2, Cy3, Cy5, AMCA, PerCP, R-phycoerythrin (RPE), allophyllin (APC), Texas Red (Texas) Red), Princeton Red, CdSe nanocrystals coated with green fluorescent protein (GFP), DNP, biotin, digoxiginin, horseradish peroxidase (HRP) ), alkaline phosphatase (AP), ß-half Glycosidase (GAL), glucose-6-phosphate dehydrogenase, ß-N-acetylglucosamine, ß-glucuronidase, invertase, xanthine oxidase, glucose oxidase, peptide, Antibodies, and linked bodies. 如請求項3之組成物，其中該功能性標記物為適合質譜(MS)分析法之可檢測標記物。 The composition of claim 3, wherein the functional marker is a detectable label suitable for mass spectrometry (MS) analysis. 如請求項1之組成物，其中該標幟部份體為下列至少一種：鄰-苯二胺(OPD)、鄰二氨基苯甲酸(DAB)、DAB-胜肽或DAB-胜肽-共軛功能性分子。 The composition of claim 1, wherein the label portion is at least one of the following: o-phenylenediamine (OPD), o-diaminobenzoic acid (DAB), DAB-peptide or DAB-peptide-conjugated Functional molecule. 如請求項7之組成物，其中該DAB-胜肽包含苯并咪唑單位。 The composition of claim 7, wherein the DAB-peptide comprises a benzimidazole unit. 如請求項7之組成物，其中該標幟DAB-胜肽之醣類係採用下列方法產生，其包含：由DAB、或DAB-胜肽、或DAB-胜肽共軛標幟劑與醣類還原端偶合。 The composition of claim 7, wherein the saccharide of the DAB-peptide is produced by the following method, comprising: by DAB, or DAB-peptide, or DAB-peptide conjugated reagent and saccharide The reduction end is coupled. 如請求項9之組成物，其中該DAB-胜肽標幟劑係選自下列各物所組成群中：DAB-(His)6、DAB-(His)3、DAB-Lys-生物素、DAB-Lys-FITC、DAB-胜肽-樹脂與DAB-胜肽-共軛功能性分子。 The composition of claim 9, wherein the DAB-peptide marker is selected from the group consisting of DAB-(His) 6 , DAB-(His) 3 , DAB-Lys-biotin, DAB -Lys-FITC, DAB-peptide-resin and DAB-peptide-conjugated functional molecule. 如請求項9之組成物，其中該DAB-胜肽係由胜肽或功能性標記物-共軛胜肽與N-Boc-二氨基苯甲酸(N-Boc-DAB)採用固相胜肽合成法或其他化學方法進行縮合後產生。 The composition of claim 9, wherein the DAB-peptide is synthesized by a solid phase peptide from a peptide or a functional marker-conjugated peptide and N -Boc-diaminobenzoic acid ( N- Boc-DAB). Produced by condensation or other chemical methods. 如請求項11之組成物，其中該胜肽包含His、Lys或其他胺基酸。 The composition of claim 11, wherein the peptide comprises His, Lys or other amino acids. 如請求項11之組成物，其中該功能性標記物-共軛胜肽包含選自下列之可檢測標記物：螢光標記物、酵素標記物、放射性同位素、化學發光標記物、生物發光標記物、聚合物、金屬粒子、鏈結體、抗體、與染劑。 The composition of claim 11, wherein the functional marker-conjugate peptide comprises a detectable label selected from the group consisting of a fluorescent label, an enzyme label, a radioisotope, a chemiluminescent label, and a bioluminescent label. , polymers, metal particles, chains, antibodies, and dyes. 如請求項11之組成物，其中該功能性標記物-共軛胜肽係選自：Lys-生物素、Lys-FITC、Lys-抗生物鏈菌素蛋白、Lys-DNP或Lys-抗體。 The composition of claim 11, wherein the functional marker-conjugated peptide is selected from the group consisting of Lys-biotin, Lys-FITC, Lys-anti-streptoprotein, Lys-DNP or Lys-antibody. 如請求項1之組成物，其中該經改質糖苷Y-X係與固態擔體偶合。 The composition of claim 1, wherein the modified glycoside Y-X is coupled to a solid support. 如請求項15之組成物，其中該固態擔體係選自：樹脂、珠粒、平面擔體、玻璃載片、聚碳酸酯載片、奈米粒子、層析介質、磁粒與金屬。 The composition of claim 15, wherein the solid support system is selected from the group consisting of: a resin, a bead, a planar support, a glass slide, a polycarbonate slide, a nanoparticle, a chromatographic medium, a magnetic particle, and a metal. 如請求項15之組成物，其中該固態擔體係與經改質糖苷Y-X上之胜肽殘基偶合。 The composition of claim 15, wherein the solid support system is coupled to a peptide residue on the modified glycoside Y-X. 一種醣類之光測定分析法套組，該套組包含如請求項1至17中任一項之組成物。 A kit for the determination of light of a saccharide, the kit comprising the composition of any one of claims 1 to 17. 一種醣類之層析分離法套組，該套組包含如請求項1至17中任一項之組成物。 A chromatographic separation kit for a saccharide, the kit comprising the composition of any one of claims 1 to 17. 一種醣類之質譜分析法套組，該套組包含如請求項1至17中任一項之組成物。 A mass spectrometry kit for a saccharide, the kit comprising the composition of any one of claims 1 to 17. 一種製備如式：Y-X之經標幟之醣類之方法，其中Y代表單醣、寡醣或多醣亞單位，及其中X代表包含3,4-二氨基苯甲酸(DAB)-胜肽之標幟部份體，該方法包含：提供DAB-胜肽標幟劑；由DAB、或DAB-胜肽、或DAB-胜肽共軛功能性分子標幟劑與醣類在碘(作為觸媒)及酸性溶劑之存在下偶合；及單離如式：Y-X之經標幟之醣類。 A method for preparing a saccharide of the formula: YX, wherein Y represents a monosaccharide, oligosaccharide or polysaccharide subunit, and wherein X represents a label comprising 3,4-diaminobenzoic acid (DAB)-peptide Part of the body, the method comprises: providing a DAB-peptide derivative; conjugated functional molecular marker by DAB, or DAB-peptide, or DAB-peptide, and saccharide in iodine (as a catalyst) Coupling in the presence of an acidic solvent; and the saccharide of the formula: YX. 如請求項21之方法，其進一步包含：由經標幟之醣類與固態擔體偶合。 The method of claim 21, further comprising: coupling the labeled sugar to the solid support. 如請求項22之方法，其中該固態擔體係選自：樹脂、珠粒、平面擔體、玻璃載片、聚碳酸酯載片、奈米粒子、層析介質、磁粒、鏈結體與金屬。 The method of claim 22, wherein the solid support system is selected from the group consisting of: a resin, a bead, a planar support, a glass slide, a polycarbonate slide, a nanoparticle, a chromatography medium, a magnetic particle, a link, and a metal. . 一種製備DAB-胜肽標幟劑之方法，該方法包含：提供N-Boc-3,4-二氨基苯甲酸(N-Boc-DAB)；提供可視需要與功能性標記物共軛之胜肽；由N-Boc-DAB與胜肽共軛。 A method for preparing a DAB-peptide marker, the method comprising: providing N- Boc-3,4-diaminobenzoic acid ( N- Boc-DAB); providing a peptide that can be conjugated to a functional marker as needed Conjugated with a peptide by N- Boc-DAB. 如請求項24之方法，其中該N-Boc-DAB係利用胜肽合成儀或化學法與胜肽共軛。 The method of claim 24, wherein the N- Boc-DAB is conjugated to the peptide using a peptide synthesizer or chemical method. 如請求項24方法，其中該功能性標記物包含選自下列之可檢測標記物：螢光標記物、酵素標記物、放射性同位素、化學發光標記物、生物發光標記物、聚合物、金屬粒子、鏈結體、抗體、與染劑。 The method of claim 24, wherein the functional marker comprises a detectable label selected from the group consisting of a fluorescent label, an enzyme label, a radioisotope, a chemiluminescent label, a bioluminescent label, a polymer, a metal particle, Chains, antibodies, and dyes. 如請求項24之方法，其中該功能性標記物-共軛胜肽係選自：Lys-生物素、Lys-FITC、Lys-抗生物鏈菌素蛋白、Lys-DNP、Lys-抗體或其他胺基酸。 The method of claim 24, wherein the functional marker-conjugated peptide is selected from the group consisting of: Lys-biotin, Lys-FITC, Lys-anti-streptoprotein, Lys-DNP, Lys-antibody or other amine Base acid. 一種醣類層析分離法，該方法包含：使用DAB-胜肽標幟劑標幟醣類，其中該胜肽係與可檢測標記物共軛；及由包含該醣類之組成物進行層析法。 A method for chromatographic separation of sugars, comprising: using a DAB-peptide marker to mark a saccharide, wherein the peptide is conjugated to a detectable label; and chromatographically consisting of a composition comprising the saccharide law. 如請求項28之方法，其中該醣-DAB、醣-DAB-胜肽與標記功能性分子之醣-DAB-共軛胜肽包含咪唑環。 The method of claim 28, wherein the sugar-DAB, the sugar-DAB-peptide and the sugar-DAB-conjugate peptide of the labeled functional molecule comprise an imidazole ring. 一種在質譜分析法(MS)期間加強醣類強度之方法，該方法包含：使用DAB-胜肽標幟劑標幟醣類，其中由該胜肽與功能性標記物共軛；及採用質譜分析法分析包含該醣類之組成份，其中該經標幟之醣類之質量強度訊號予以提升。 A method of enhancing the strength of a saccharide during mass spectrometry (MS), the method comprising: using a DAB-peptide marker to label a saccharide, wherein the peptide is conjugated to a functional marker; and using mass spectrometry The method analysis comprises a component of the saccharide, wherein the quality intensity signal of the labeled saccharide is increased. 如請求項30之方法，其中該醣-DAB、醣-DAB-胜肽與標記功能性分子之醣-DAB-共軛胜肽包含咪唑環。 The method of claim 30, wherein the sugar-DAB, the sugar-DAB-peptide and the sugar-DAB-conjugate peptide of the labeled functional molecule comprise an imidazole ring.
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