WO2013169640A1

WO2013169640A1 - Diaminobenzoic or ortho-phenylene diamine conjugated peptides as functional saccharide tagging reagents

Info

Publication number: WO2013169640A1
Application number: PCT/US2013/039677
Authority: WO
Inventors: Wen-Bin Yang
Original assignee: Academia Sinica; Liang, Chi-Ming
Priority date: 2012-05-05
Filing date: 2013-05-06
Publication date: 2013-11-14
Also published as: TW201408690A

Abstract

Novel designs of tagged saccharides useful in analytical and diagnostic applications are disclosed. Saccharide reagents disclosed mclude monosaccharide, oligosaccharide or polysaccharide subanit 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

OIAMI OBE ZOIC OR ORTH O-F HENYLE E DIAMINE CONJUGATED PEPTIDES AS .FUNCTIONAL SACCHARIDE TAGGING REAGENTS

INVENTOR:

Wen-Bin YANG

TECHNICAL FIELD OF THE INVENTION

flMMll This invention relates to the field of carbohydrate analysis, In particular, the invention relates to the field of labeling polysaccharides with detectable tags for analytical and diagnostic applications. More particularly, the invention relates to ortho-substiiuted, diami no- aromatic compounds conjugated with peptides to form saccharide tagging reagents.

BACKGROUND OF THE INVENTIO

ffM)02| Saccharides play essentia! biological function, for instance, -, A^y-glycans in glycoproteins, 0-/ ~antigen.s in HpopoSysaccharides (LPS). However, saccharides are difficult to separate and measure in nature due to lack of chromophobes and low ionization ability in MS. Thus, saccharides are undetectable by conventional photometric methods (e.g. UV/fluorescence in .liquid chromatography) or MS analysis.

f0O<)3| Development, of labeled saccharide is an important topic in. analytical chemistry. Although some methods have been developed for saccharide labeling, these approaches suffer from some drawbacks. An example is the tagging of free saccharides by reductive arai.nation with amino-phospholipids or with neutral or acidic fluorescent reagents. (Hase S. et ah, J. Biochem. 95 : 197-203 (1996)). Although this approach is useful and practical in. some applications, fractionation of saccharides tagged by these methods has certain limitations. (Price NPJ et al, Anal Chem, 2010, 82, 2893; Pabst M. et al„ Anal Biochem. 2009, 384, 263; Tripathi, R. P„ et al. Current Org. Chem. 2008, 12, 1093; Bigge i.C, et al, Anaf.

Biochem. 1995, 230, 229; Harvey, D.J. J Chmmatogr, B 201 1 , 879, 1 196).

jiiO j Development of labeled saccharide is an important topic in analytical chemistry.

Although some methods have been developed for saccharide labeling, these approaches suffer from some drawbacks. Some examples are the tagging of free saccharides by reductive animation with amino-phosphol ipids or with neutral or acidic fluorescent reagents. (Lin, C et al, J, Org. Chem, 2008, 73, 3848-3853; Lin, C et al, Rapid Cammm Mats Spectrom. 2 1.0, 24, 85-94; Lin, C et al., Molecules 2010, 1 5, 1340-1353; Lin, C et al. Molecules 201 1 , 5.6, 652-664; Chang, Y. L et al., J. Mass Sp trom. 201 1 , 46, 247-255; Hase S et ah, J. Biochem. 95: 197-203 ( 1996)). AS -52 S P01

|0 05| Conjugation (covalent coupling) of polysaccharides to proteins or peptides has been used. Coupling polysaccharide fragments to proteins or peptides through their reducing end by direct reductive animation leads to poor incorporation of the saccharides.

{0006] Thus, there is a Song-felt need for a tagging agent for purposes of tagging and labeling saccharides which allows further applications of the tagged, functionally labeled saccharides.

SUMMARY OF THE INVENTION

|0007j The presen t disclosure is based on a number of newly designed tagged saccharides useful in analytical and diagnostic application,

jO OSj Accordingly, in one aspect the present disclosure relates to modified glycosides having the formula Y-X wherein Y represents a monosaccharide, oligosaccharide or polysaccharide subunits, in which the subunk is linked in a linear or branch chain by gSyeoskiie linkages, and wherein X represents a tagging moiety comprising an ortho- diaminobertzoic( D AB)-peptide.

0 91 Disclosed herein are 3_}4-diaiTsinobenzoic(DAB)-peptide and the 3,4- diammobenwtc(DAB)-peptide conjugated functio molecules used as taggers to tag a saccharide at a reducing end of the saccharide to comprise a behzimidazoie unit (saccharide- Bl -peptide or saccharide-BlM-peptide conjugated function molecules). Tagged saccharide can be generated by coupling an ort¾o-diamlnobenzoic(DAB>peptide tagging agent.

|00t0] In some embodiments, the o /^o-diaminobenzoic-pe tide tagging agent, is selected from the group consisting of S3AB---6His, DAB~3His, DAB-Lys >iotin)₍ DAB-L s(FITC), DAB-l.ys~resin,

00111 some embodiments, the DAB-peptide is generated by condensing a peptide or a functional label-conjugated peptide with Λ-Boc-diaminobenzoic acid by solid phase peptide synthesis or other chemical process.

j^'0012^'j 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, iyxose, allose. altrose, gufose, rhamn se, arabinose, fticose, N- aeetyigaiactosamine, glucuronic acid, ga!acturonic acid Globo FL GD2, GD3, G I. a, (30 Sb, GTlb, GTIa, Gb3, Gb5, SSEA oligosaccharides, Fucosyl GM L GM2, GM3, blood group an.fige.nsf A, B, O, H), Forssman antigens, Lewis a, Lewis b, Lewis X, Sialyl Lewis X, Lewis Y, lactose based O-giycans, N-acety!glucosamme core structures, sialyifactose, sial fated AS -52 S P01 oligosaccharides, sulphated oligosaccharides, phosphated oligosaccharides, manno- oligosaceharides, eello-oligosaeeriarides, xylo-ojigosaceharides, chittvoligosaccharides, raalto-oUgosac-charides and polysaccharides,

{0013] In some embodiments, the tagging moiety further comprises a functional label, wherein the functional label is a detectable label suitable for photometric analysis.

|0014] In some embodiments, the detectable label is selected from the group consisting of BODIFY Dyes, Cascade Blue Dyes, Coumarin, Fluorescein (FiTC/FAM), Haptens,

Lissamme Rhodamine B Dyes, NBD, Oregon Green Dyes, Texas Red Dyes, bimane azide, Marina Blue, Pacific Blue, Rhodamine 6G Dyes, Rhodamine Green Dyes, Rhodamine ed Dyes, Tetramethylrhodamine, DNP, Digoxigenin, biotm, avidin, streptavidin, protein, luciferin, an anti-dye antibody_* 5-(and 6)-cartraxyflooreseei«, 5- or 6-carboxyfluorescein, 6- (tluorescein)-5'(and 6)-earhoxamido hexanoic acid, fluorescein isothiocyanate, rhodamine, tetramethylrhodamine, Cy2,.Cy3, CyS, A CA, FerCP, R-phycoerythriw (RPE)

ailophycoeryihrin ( APC), Texas Red, Princeton Red, Green fluorescent protein (GFP) coated CdSe nanoerystallites, DNP, biotin, digoxiginin, horse radish peroxidase (HRP), alkaline phosphatase (AP), fJ-galactosidase (GAL), gIucose^»6^»phosphate dehydrogenase, B-N- aeetvlglucosamimsdase. fi-e^'lucuronidase, invertase. Xanthine Oxidase, glucose oxidase, peptides, antibodies, and linkers.

|0015] In some embodiments, the functional label is a detectable label suitable for mass spectrometric (MS) analysis.

{0016] In some embodiments, the functional label-conjugated peptide comprises a detectable label selected from fluorescent labels, enzyme labels, radioisotopes, chemimminescent labels, blolumineseent labels, polymers, .metal particles, antibodies and dyes.

|00!7] In some embodiments, the functional label-conjugated peptide is selected from Lys- biothi, Lys-FiTC, Lys-streptavidin, Lys-D P, Lys-resio, Lys~solid supporters or Lys- antibody.

{0018] In some embodiments, the modified glycoside, Y-X, is coupled to a solid support, wherein the solid support is selected from a resin, a bead, a planar support, a glass slide, a polycarbonate slide, an aluminum slide, a nanoparticle, a chromatography medium, a magnetic particle and a metal.

16019] Another aspect of the present disclosure relates to a. kit for photometric analysis of saccharides, the kit comprising a composition mentioned above. AS -52 S PO!

{0020} Another aspect of the present disclosure relates to a kit for chromatographic separation of saccharides, the kit comprising a composition, mentioned above.

{0021 } Another aspect of the present disclosure relates to a kit for mass specirometric analysis of saccharides, the kit comprising a composition mentioned above,

{0022] Another aspect of the present disclosure relates to a method for preparing a tagged saccharide of the formula: Y-X, wherein Y represents a monosaccharide, oligosaccharide or polysaccharide subunit, and wherein X represents a tagging moiety comprising a 2,3- dtaminobenzoic (DAB)-peptide, the method comprising the steps: (I ) providing an ortho- DAB-pcptide tagging reagent; and (2) coupling the DAB-peptkle tagging reagent to a saccharide, in the presence of iodine as catalyst and an acidic solvent.

| 023j i some examples, the method mentioned above further comprises a step by coupling the tagged saccharide to a solid support.

{0024] In some embodiments, the solid support is selected from a resin, a bead, a planar support, a glass slide, a polycarbonate slide, a nanop&rtick, a chromatography medium, a magnetic particle and a metal.

|0025] Another aspect of the present disclosure relates to a method, for preparing an DAB- peptide taggin reagent, the method comprising the steps (I ) providing N-Boc-3,4- dlami.nobenzo.ic acid (N~Boc-DAB); (2) providing a peptide optionally conjugated to a functional label; and (3) conjugating the A^?-13oc-3,4-diamino enzoic acid to the peptide or peptide-eonjugated functional molecules by means of a peptide synthesizer or other chemical process,

|ftt)26] In some embodiments, the functional molecule label, comprises a detectable label selected irom fluorescent labels, enzyme labels, radioisotopes, ehemiluminescent labels, bioluminescent labels, polymers, metal particles, resins, slides, plates, antibodies, and dyes.

|0027| In some embodiments, the functional molecule-conjugated peptide is selected from Lys-biothi, !,ys~FlTC, L-ys-sirepiavidin, Lys-D P, Lys-antibody or other amino acid conjugates,

{0028] Another aspec t of the present disclosure relates to a method f or chroma tographic isolation of saccharides, the method comprising the steps^* (I ) tagging the saccharide with an DAB-pepti.de tagging reagent, wherein the peptide is conjugated to a detectable label; and (.2) subjecting a composition comprising the saccharide to chromatography. AS -52 S PO! fO029j Another aspect of the present disclosure relates to a method for enhancing ass intensity of a saccharide during mass spectrometry (MS), the method comprising the steps: ( !) tagging the saccharide with an DAB-peptide tagging reagent, wherein the peptide is conjugated to a functional label; and (2) analyzing a composition comprising the saccharide by mass spectrometry, wherein the mass intensity of the tagged saccharide is enhanced.

BRIEF DESCRIPTION OF THE DRAWINGS

j003(fj The following drawings of schemes and figures form part of the present specification and are included to further demonstrate certain aspects of the present disclosure, the inventions of which can be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.. The patent or application file contains at least one drawing executed in color. Copies of this paten or patent application publication with color dra ing(s) will be provided by the Office upon request and payment of the necessar fee.

§0031] Figure 1 ; Schematic view of conjugation of saccharide -to tagging agents, DAB- peptides and DAB-peptide-Rs, achieved by iodine catalytic oxidation condensation of saccharide and DAB^••■peptides or DAB -peptkie~Rs.

fiM)32] Figure 2; Schematic view of preparation of DAB-peptide tagging reagents and DAB- peptide conjugated resin or other bio-molecules tagging reagents for saccharide tagging, fO033) Figure 3: Applications of saccharide-DAB-peptides in g!yeomies,

100341 Figure 4: The MS of 3,4-diamino.benzoic-(His).? tagger {C₂s%<?N _{f f} (¾; 564.0 Da) measured by MALDI-TOF MS.

0035j Figure 5: The MS of 3,4-diaminoberizoie~(His)₆ tagger (C^Hso acOe; 975.2 Da) measured by MALDl-TOF MS.

10036] Figure 6: The molecular weight of condensed maltohexose-DAB obtained

(C«i½N₂Q_w a; 1 1453 Da) by MALDl-TOF MS.

ffl03?] Figure 7: The molecular weight of condensed maltotetraose-DAB-(His)<s obtained (C¾;_f.H^ ₂o02*; 1613.1 Da) by MALDl-TOF MS.

|0038J Figure 8: The molecular weight of condensed nialtohexose-DAB-(llis).; obtained (Qi B^N l sO^Na 1 556 J Da); 1556. i Da) by MALDl-TOF MS. AS -52 S P01

I0 39J Figure 9: The molecular weight of condensed maKohexose-DA 8~(His)6 obtained (C _mNz(jOnl 1945.3 Da) by MALDI-TOF MS.

.DETAILED DESCRIPTION OF THE INVENTION |Θ04Θ) in the following detailed description of embodiments of the present disclosure, reference is made to the accompanying drawings in which like references indicate similar elements, and in which is shown, by way of illustration specific embodiments in. which the present disclosure may be practiced. These embodiments are described in sufficient detail to enable those skilled in the art to practice the present disclosure, and it is to be understood that other embodiments may be utilized and thai logical, structural, functional, and other changes may be made without departing from the scope of the present disclosure. The following detailed description is, therefore, not to be taken in a limiting sense.

f004J | Disclosed herein are modified glycosides, the composition comprising ( I ) a monosaccharide, oligosaccharide or polysaccharide summit, the summit is linked in a linear or branch chain b glyeosidie linkages, and (2) a tagging moiety.

|(M 42J The tagging moiety comprises an ortho-substitoted, diamino-aromatic compound. In different embodiments of the invention the ortho-sub$tituted,diamino-aromatic compound is 1,2-diaminobenzene (also known as *^*/&o-phenylenediamine ; OPD), 3.4-diaminobenzoic acid, or 2,3-diaminopyridine. in a preferred aspect, the tagging moiety comprises 3,4- diaminobenzoic acid peptide (DAB-peptide) conjugate.

10043} The term "polysaccharide" and "oligosaccharide" as used herein are used in their broadest sense to refer to saccharides comprising a plurality of repeating units, including, but not limited to saccharides having from 2 to over 2,000 repeating units. Typically, as accepted in. the art and as used herein, the term "polysaccharide" refers to a saccharide having from, about 10 to about 2,000 or more repea ing units. As accepted in the art and as used herein, the term "Oligosaccharide" refers to a saccharide having from, about 2 to about if) repeating units. The repeating unit of a polysaccharide may be a single monosaccharide molecule or may be a piiirality saccharide (ex. mixed saccharide types). In certain embodiments, the repeating unit is 2 or more monosaccharide .molecules. In accordance with the method of the invention, fragments of polysaccharides or oligosaccharides from di ffering types and/or strains of bacteria may be chemically joined or synthetically synthesized to form a single

polysaccharide or oligosaccharide chain comprising multiple epitopes from the ^'multiple types and/or strains of bacteria from which the fragments were originally derived and/or identified; AS -52 S P01 accordingly, the composition of the repeating unit(s) of the -polysaccharide or oligosaccharide of the invention need not he constant over the entire saccharide chain. The polysaccharides or oligosaccharides encompassed by the methods of the invention comprise one or more amino sugars, i n certain embodiments, said one or more amino sugar is a component of the repeating unit of the polysaccharide or oligosaccharide. In. other embodiments, the said one or more amino sugar is not. a component of the repeating unit of the polysaccharide or oligosaccharide.

|0044| The tagging .moiety can be made by any conventional methods, e.g., standard methods of solid phase peptide synthesis or chemical synthesis well known to any one of ordinary skill in the art. For example, histidme, lysine, and functional molecule-conjugated peptides (e.g. biotin, fluorescent dyes, proteins etc.) can be condensed with N-Boc-2₅3-diammobenzoic acid (DAB) though amid bond formation by peptide synthesis.

flM)4S| The term "peptide" used herein re fers to a polymer composed of one or more amino acid monomers and is shorter than a protein. Preferably, each of the cancer-targeting peptides described herein includes up to 50 (e.g., up to 10 or 20} amino acids. In some examples, the cancer-targeting peptides each contain 4*20 amino acid .residues (e.g., 4- 0, - 1.0, 6- 1 5, or 6^» 20 amino acid residues). These peptides can contain naturally-occurring amino acid residues, or modified amino acids, hi one example, either the jV-termmus or the Otermmus of a cancer- targeting peptide Is modified, e.g., containing an ~N¾ group at the C-ter inos,

|0046] The pepiides ma be sy thesized by soiid phase chemistry techniques following the procedures described by Steward et al. in Solid Phase Peptide Sy nthesis, 2nd Ed., Pierce Chemical Company, Rockford, ill., ( 1 84) using a Rainin PTl Symphony synthesizer. For solid phase peptide synthesis, techniques may be found in Stewart et al, in "Solid Phase Peptide Synthesis", W. I-L Freeman Co. (San Francisco), .196.3 and Meienhofer, Hormonal Proteins and Peptides, 1973, For classical solution sy nthesis, see for example Schroder et al. in "The Peptides", volume I , Acacemic Press (New York), in general, such methods comprise the sequential addition of one or more amino acids or suitably protected amino acids to a growing peptide chain on a polymer. Normally, either the amino or earboxy! grou of the first amino acid is protected by a suitable protecting group. The protected and/or derivatized amino acid is then either attached to an inert solid support or utilized in solution by adding the next amino acid in the sequence having the complimentary (amino or carboxyS) group suitably protected and under conditions suitable for forming the amide linkage. The AS -52 S PO! protecting group is then removed from this .newly added amino acid residue and the next amino acid (suitabl protected) is added, and so forth.

|0O47] The lagging agents. DAB-peptides, described herein can. be used as detectable labels and are capable of for labeling saccharides at reducing end to form saccharide-DAB- peptides, thereby facilitating saccharide purification and analysis, in addition, DAB-peptides conjugated saccharides can increase ionization ability of saccharides, thereby improving sensitivity in MS analyses,

ΙΘΘ48] In some aspects the 3,4-dianitnoben¾oic acid (DAB) itself can be converted fo a detectable moiety when it is enxroaticaliy oxidized by horse radish peroxidase and hydrogen peroxide or urea peroxide to yield a fiuorophore -

100 ] Conjugation of saccharide to tagging agents, DA B-pepiides and .DAB-pepiide-Rs, can be achieved by iodine catalytic oxidation condensation of saccharide and DAB-peptides or DAB-peptide-Rs as shown in Figure 1.

fCHISdJ In some embodiments, saeelutride-DAB-peptides described herein comprise saccharide-DAB-b!iss, saccha.ride-~DAB~6Srlis~resin_t $ac«haride~DAB~3Flis,, saeeharide- DAB-3His-resin, saccharide-DAB-Lys-biotin, saccharide-DAB-Lys-FlTC, saccharide- DAB-61 ·! is, saccharide— D AB-3 His, saechar ide— D A B- Lys-biotia, saceharide-D B-Lys- resin,

[0051] Any of saccharide~DAB~peptides can be further conjugated with (attached to) a detectable label or immobilized t solid support such as resin, nanoparticle, plate, chip etc. for saccharide bind assay, image and other applications.

f(H)52] As used herein, "conjugation", "conjugated^'' or "attached" means two entities are associated. The association between the two entities can be either direct or via a linker, such as a polymer linker. Conjugated or attached can include covalent or noneovaient bonding as well as other farms of association, such as entrapment, e.g., of one entity on or within the other, or of either or both entities on or within a third entity, such as a micelle.

|0053] In one aspect, the peptide in saccbaride-DAB-peptide is attached to a detectable label, which is a compound that allows recognition, either directly or indirectly, the peptide conjugated to it such that the saccharide can be detected, measured, and/or qualified.

Examples of such "detectable labels" are intended to include, but are not limited to, fluorescent labels, chemi luminescent labels, colon metric labels, enzymatic markers, AS -52 S P01 radioactive isotopes, and affinity tags such as biotfn. Such labels can be conjugated to the peptide, directly or indirectly, by conventional methods.

|0054| Detection, of the present labeling saccharide is performed using methods and reagents well known to those skilled in the art. A preferred method of detection of the invention is through the use of fluorescence. Fluorescence can be visualised with a variety of imaging techniques, including ordinary light or fluorescence microscopy, con focal laser-scanning microscopy, and flow cytometry.

|0 55) In another example, saccharide- DAB-peptides comprise saecharscle--DAB--(His)6 and saccharide- DA -(flis).i bound to N.i column, thereby facilitating separation and purification of saccharides through immobilized metal affinity chromatogra hy (I AC). .In addition, saccharide~DAB~-peptide~resin comprise saccharide-DAB-CHis^-resin and saccharide- DAB--{His)H'es n thereby facilitating separation and purification of saccharides washed, filtered or cenirifuged.

i(M> ] Preparation of DAB-peptide tagging reagents and DAB-pepiide conjugated resin or other bio-molecules tagging reagents for saccharide tagging is shown in Figure 2.

|ΘΘ57) Comparison with other saccharide tagging methods, the saccbarlde-DAB~peptides have a number of advantages in saccharide detection. Applications of saccharide-DAB- peptides in giyeomics are shown in Figure 3.

M58] The present disclosure also provides a. rapid method for purification, identification and derivation of saccharides and without destroyed glycans function in glycomic studies, even when a tiny amount (less than Ϊ pmol) of saccharide is present in the sample,

|0059] The terms used in this specification generally have their ordinary meanings in the art, within the context of the invention, and in the specific context where each term is used. Certain terms that are used to describe the invention are discussed below, or elsewhere in the specification, to provide additional guidance to the practitioner regarding the description, of the invention. For convenience, certain terms may be highlighted, for example using italics and/or quotation marks. The use of highlighting has no influence on the scope and meaning of a term; the scope and meaning of a term is the same, in the same context, whether or not it is highlighted. It will be appreciated that same thing can be said in more than one way.

Consequently, alternative language and synonyms may be used for any one or more of the terms discussed herein, nor is any special significance to be placed upon whether or not a term is elaborated or discussed herein. Synonyms for certain terms are provided. A recital, of AS -52 S PO! one or .more synonyms does .not exclude the use of other synonyms. The use of examples anywhere in this specification including examples of any terms discussed herein is illustrative only, and in no way limits the scope and meaning of the invention or of any exempli t ed term. Likewise, the invention Is not limited to various embodiments given in this

specification..

|W)68] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains, in. the case of conflict, the present document, includin definitions will control..

EXAMPLES

|0M1J Without intent to limit the scope of the invention, exemplary instruments, apparatus, methods and their related results according to the embodiments of the present invention are given beiow. Note that titles or subtitles may be used in the examples for convenience of a reader, which in no way should limit the scope of the invention. Moreover, certain theories are proposed and disclosed herein; however, in no way they, whether they are right or wrong, should limit the scope of the invention so long as the invention is practiced according to the invention without regard for an particular theory or scheme of action.

AS -52 S PO!

Example 2: Synthesis of A^'~Boc-3_^

{0062] A^r~Boc {N'i ri-buiox ' carbonyl) protected 3,4-d.iaraiiiobeozoic acid precursor (3,4- DAB) was prepared b reacting 3,4Hiiamioohenzotc acid (I -52g, 10 romol) and di-terh butoxy diearbonate (( -Boej-OASSg, 30 mnioi) with triethyteminc ( Et_?; 7.0 mi, 60 rnmol) in chloroform (CHC¾, 200 mt) at room temperature (25 ^1>C) stirring for overnight (24 h). The resuKiag solution was extracted with water, dried with a^SO^. to give A^r-Boc-3,4~ diammobeozoic acid (2<0g, 79%) as a brown powder. ^!H MR (600 MHz, d-McOH) ·¾ 7.96 is, ! i:L Ari l), 7,75 (dd, .1 ii J ~ 8.4, 1 ,9 Bz, Aril), 7.60 (d, i I I J~ 8.4, 1 .9 Hz. Aril), 4.80 ibrs, 2H, NH), 1.52 (_s, 18H, CM₃). ^,3C NMR (150 MHz, d-MeOH) 173.2, 156.4, 155.5,

135.7, 133.6, 130.3, 127.9, 127.8, 123.6, ! .7, 8 L6, 28.8, 28.7. Mass f N-Boe~DAB

(Cn¾ ;?O_ft; 352,4 Da) was measured.

Example 3: Synthesis of 3.4-diaroi.«oben¾oiC'(ffi$^'h tagger

|6063] The Λ'-Boc (N-terf-butoxy carbonyl) protected 3,4-diaminobenzo!C precursor (N-Boe- DAB), which was obtained by block amino group on DAB using d er?-b«ty1 diearbonate (t- B0C2O), was added 3 histidine units by solid phase peptide synthesis (AB1433A Peptide Synthesizer) and followed hydrolysis to gi ve 3,4-dtaminober!Zoic~(His)j tagger (DAB-His- His-His). The MS of 3,4-diaminobenzoic-(His)s tagger (CssH N j sOj; 564.0 Da) was measured ^'by MALDl- OP MS as shown in Figure 4.

Example 4; Synthesis of 3,4-dianiinobcnzoic-(Hts)j-resm tagger

|0064| The A~Boe (A ^rf-butox carbonyl) protected 3,4-dianiinobenzoic precursor (7V~Boc- DAB). which was obtained by block amino group on DAB using dwert-butyi dicarbonate (i- AS -52 S P03.

Boc), was added 3 histidme units by solid phase peptide synthesis with TentaGal resin to give 3,4~diaminobenzoic-(His) 5-re¾}n tagger (DA8~His~.His-His-resm)»

Example S: Synthesis of 3.4-diai¾>nob R2oic-{^'His^ tagger

ft)M5] The ,Υ-Boc {N-fcn'-butox carbonyl) protected 3,4-diaminobenzoic precursor (N-Boe- DAB), which was obtained by block amino group n DAB using d eri-baty! dicarbonate {>- Boc). was added 6 histidme units by solid phase peptide synthesis (ABI 433A. Peptide Synthesizer) and followed hydrolysis to give 3,4-diaminobenzoic-{His)(, tagger (DAB-F!ls- His-His-His-His-Kis). The MS of 3,4-diaminobenzoic-(His)_¾j tagger (CcsBjoNaA; 975.2 Da) was measured by MALDS~T0F MS as shown in Figure 5.

Example 6: Synthesis of 3,4>diammobenzoie~(H is restn tagger

|(H½6) The N-Boc {A-ierf-butoxy carbonyl) protected 3 ,4-d iarainobenzoic precursor (Λ-Boe- DAB), which was obtained by block amino group on DAB using di- r/-buty.! dicarbonate (t* Boc), was added 6 histidme units by solid phase peptide synthesis with TentaGal resin to give 3,4~diaminoben oic-(l:iis)6-resjn ta.gger (DA8-His-His-HiS-His-Hi$-Hf^'is-resin).

Example 7: Synthesis of 3.4'diam>nobenzoie-Lys~(Biotm tagger

Θ067} The A/~Boc (A¾'r/»bnioxy carbonyl) protected 3,4-diammobe«zol:c precursor (Λ-Boc- DAB), which was obtained by block amino group on DAB using di-½r?-butyl dicarbonate (Y- Boc). was added lysine-(Biotin) unit by solid phase peptide synthesis (ABI 433A Peptide Synthesizer) and followed hydrolysis to give 3,4-diaminobenzoic-lysine»-(Biotin) tagger (DAB-Lys-(Siotin), The MS of 3,4-diaminobenzoic->lysine-(Biotin) tagger (506.3 Da) was measured by A1.DI-TOF MS. AS -52 S PO!

Example 8: Synthesis qimaltohesose---PAB.

|Θ068] The native maltdhcxose^' (611 or 06; 1.0 mg) was iaggec! with 3_t4^«diamtn0 eazoic acid (DAB; LO mg) in presence of catalytic amount of iodine at acetic acid solution to form the maitohexose-DAB product The molecular weight of condensed maltohexose-DAB was obtained

1 145.3 Da by MALDl-TOF MS as shown in Figure 6,

|0O69j The native roalioietraose (04; 1.0 mg) was tagged with 3,4«diaminobenzoic~(His)6

(DAB-{i'Iis)<i; 1.0 mg) in presence of catalytic amouiit of iodine at acetic acid solution to form the nialtotetniose~DAB-(li is)(, product. The molecular weight of condensed

maltotetraose-DAB~( is)6 was obtained

1613.1 Da) by MALDl-TOF MS as shown in Figure 7,

Example 10: Sy rsthesi s mal tohexose- P A B-f H is h

[6078] The native rnaltohexose (06; 1.0 mg) was tagged with 3,4-dkminobenxoic~{His)3 (OAB-(His>5; 1 ,0 mg) in presence of catalytic amount of iodine at acetic acid solution to form the- maltohexo$e-DAB-(Hi$)s product. The molecular weight of condensed

maltohexose>DAB~(H.is)j was obtained (C^H^NI _sO_i5Na; 1556.1 Da) by MALPJ-TOF MS as shown in Figure 8,

E^n¾ je J. j : Sy uthes ^; is o f mg1 se ? B (Hj.s)fe

HO ft_c ¾^'^HO ¾'5= ^¾ ^ O^^HO^ AS -52 S P01 0 71 | The native maitohexose (G6; 1.0 rag) was tagged with 3_<4-dia.ininobe.nzoic---(ilis)6 CDAB-(His}₍₁; 1.0 nig) in presence of catalytic amount of iodine at acetic acid solution to form the maiiohexose-DAB™(His)_tl product. The molecular weight of condensed raa!tohexose-DAiHHts)* was obtained (C-^HwNa ^; 1945,3 Da) by MALDKiOF MS as shown in Figure 9.

|0072J Although specific embodiments of the invention have been described herein for purposes of illustration, various modifications may be made without deviating from the spirit and scope of the invention. Accordingly, the invention is not limited except as by the appended claims.

0 73} While the invention has been described in conjunction with the deiaiied description thereof the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the appended claims.

f.0074] The patent and scientific literature referred to herein establishes the knowledge that i available to those with skill in the art. All United. States patents and published, or unpublished. United States patent applications cited herein are incorporated by reference. Ail published foreign _.patents and patent applications cited herein are hereby incorporated by reference. All other published references, documents, manuscripts and scientific literature cited herein are hereb incorporated by reference.

Claims

AS -52 S PO!

We claim: ί . A composition comprising a modified glycoside having the formula:

Y-X,

wherein Y represents a monosaccharide, oligosaccharide or -polysaccharide subunit* in which the monosaccharides comprising the oligosaccharide or polysaccharide subunU are linked together in a linear or branch chain by giyeosklic linkages, and

wherein X represents a tagging moiety comprising an ortho~substituted,diamino~ aromatic compound.

2. The composition of claim 1 wherein the monosaccharide subunii, or the

oligosaccharide or polysaccharide suhunit, are the same or different and are selected from the group consisting of glucose, galactose, fructose, ribulose, rnanoose, ribose, arabinose, xylose, lyxose, aSJose, altrose, gulose, rhamnose, arabinose. fucose, -aeetyigaiactosamitie, glucuronic acid, galacturonie acid Globo If, GDI, GD3_:, GO l a, GQI b, G^*flh, Cili a, Gb3, Gb5, SSEA oligosaccharides, Fucosyi OM I , QM2, GM3, blood group antigens{A, B, Q, H), Forssman antigens, Lewis a, Lewis b, Lewis X, Sialyl Lewis X. Lewis Y. lactose based O- glyeans, N-acetylglucosamine core structures, siaiyl-laetose, sialylated oligosaccharides, sulphated oligosaccharides, phosphated oligosaccharides, manno-oiigosaecharides, cello- oligosaccharides, xykvoligosaeriharides, ehtto-oltgosaccharides, malto-oiigosaecharides and polysaccharides.,

3. The composition of claim 1 wherein the tagging moiety further comprises a functional label.

4. The composition of claim 3 wherein the functional label is a detectable label suitable for photometric analysis.

5. The composition of claim 4 wherein the detectable label is selected from the group consisting of BODJPY Dyes, Cascade Blue Dyes, Coumarin, Fluorescein (FITCV^'FAM), Haptens, Lissamine Rhodamine B Dyes, NBD, Oregon Green Dyes, Texas Red Dyes, bimane azide, Marina Blue. Pacific Blue, Rhodamine 6G Dyes. Rhodamine Green. Dyes, Rhodamine Red Dyes, Tetramethylrhodamine, DNP, Digoxigenin, biotin, avidin, streptavidm, protein, !ucilerin, an anti-dye antibody, 5~(and 6)-cafbox il uorescein, 5- or 6-carboxy fluorescein, 6- AS -52 S P03.

(i1uorescein}-5~(an.d 6)-carboxamido hexanoie acid, fluorescein isothipcyanate, rhodamtne, ieiraraethyirhodamine, C 2, Cy3. CyS, AMCA, PetCP, R-phycoerythrin (RPB)

alloph eoerythrin (APC), Texas Red, Princeton Red, Green fluorescent protein (GFP) coated CdSe nanocrysiallites- DNP, biotin, digoxiginin, horse radish peroxidase (HRP), alkaline phosphatase (AP), β-gatactosidase (GAL), gi eose-6-phosphaie dehydrogenase, S-N- aceiyiglucosamimidase. β-glucuromdase, mveriase. Xanthine Oxidase, glucose oxidase, peptides, antibodies, and linkers.

6 , The composition of claim 3 wherein the functional label is a detectable label suitable for mass spectroraetric (MS) analysts.

7. The composition, of claim. .1 wherein the tagging moiety is at least one of a ortho- phenyienediamme (OPD), an or/Ao-diaminobetizoic acid (DAB), a OAB-pepti.de or a DAB- peptide-conjugated functional molecu ie,

8. The composition of claim 7 wherein the D B-peptkle comprises a. benzimidazole unit.

9. The composition of claim 7 wherein the.DAB-peptide tagged saccharide is generated by a method comprising:

coupling a DAB, or DAB-peptide, or DAB-peptide conjugated tagging agent to a reducing end on a saccharide.

IG\ The composition of claim 9 wherein the DAB-peptide tagging agent is selected from the group consisting of DAB-CHfcfr, DAB~ His)j, DAB-Lys-b.iotin, DA.B~Lys-F.iTC, DAB peptkle-resin and DAB-peptide-eonjugated functional molecule.

1 1 . The composi tion of claim 9 wherein the DAB-peptide is generated by condensing a peptide or a functional label-conjugated peptide with Λ'-Boc-diaminobenzoie acid (A'-Boc- DAB) by solid phase peptide synthesis or other chemical process.

12. The composition of claim 1.1 wherein the peptide comprises His, Lys or other amino acids.

13. The composition of claim 1 1 wherein the functional label-conjugated peptide comprises a detectable label selected from fluorescent labels, enzyme labels, radioisotopes, AS -52 S PO! chemiluminesceni labels, bioiumineseent labels, polymers, metal particles, linkers, antibodies, and dyes.

14. The composition of claim 1 .5 wherein the functional label-conjugated peptide is selected from Lys-bsotio, Lys-FITC, Lys-sireptavklio, Lys-DNP or Lys-antibody,

15. The composition of claim i wherein the modified glycoside, Y-X, is coupled to a solid support,

16. The composition of claim 15, wherein the solid support is selected from, a resin, a bead, a planar support, a glass slide, a polycarbonate slide, a nanopafticie, a chromatography medium, a magnetic particle and a metal.

17. The composition of claim 15, wherein the solid support is coupled to the peptide residue on the modified glycoside, Y -X.

! 8. A kit for photometric analysis- of saccharides, the kit comprising a composition according to any of claims 1 - 1 7.

19. A kit for chromatographic separation of saccharides, the kit comprising a composition according to any of claims 1 -17.

20. A kit for mass spectrometry analysis of saccharides, the kit comprising a composition according to any of claims 1-17.

21. A method for preparin a tagged saccharide of the formula: Y -X, wherein Y represents a monosaccharide, oligosaccharide or polysaccharide subunit, and wherein X represents a tagging moiety comprising a 3,4~diaminohen'«>tc (DAB)-peptide, the method comprising:

providing an DAB-peptide tagging reagent;

coupling the DAB, or DAB-peptide, or DAB-peptide conjugated functional molecule tagging reagents to a saccharide, in the presence of iodine as catalyst and an acidic solvent; and

isolating the tagged, saccharide of the formula: Y -X.

2.2. The method of claim 21 , further comprising:

coupling the tagged saccharide to a solid support. AS -52 S PO!

23. The method of claim 22, wherein the solid support is selected from a resin, a bead, a planar support, a glass slide, a polycarbonate sl ide, a nanoparticle, a chromatography medium, a magnetic particle, a linker and a meial.

24. A method for preparing an DAB-peptide tagging reagent the method comprising: providing N-8oe~3,4~diammotaenzoic acid (N-Boe-DAB);

providing a peptide optionally conjugated to a functional label;

conjugating the A'-Boc-DAB to Che peptide,

25. The method of claim 24, wherein, the .JV-BOC-DAB is conjugated to the peptide by a peptide synthesizer or a chemical process.

26. The method of claim 24, wherein the functional label comprises a detectable label selected from fluorescent labels, enzyme labels, radioisotopes, chemilumiuescetit labels, bio!uminesoent labels, polymers, metal particles, linkers, antibodies, and dyes.

27. The method of claim 24, wherein, the functional label-conjugated peptide is selected from Lys-btotm, Lys- ITC, Lys-streptavkiin, Lys-DNP, Lys-antibody or other amino acids.

28. A method for chromatographic isola tion of saccharides, the method comprising:

tagging the saccharide with a DAB-peptide tagging reagent, wherein the peptide is conjugated to a detectable label; and

subjecting a composition comprising the saccharide to chromatography.

29. The method of claim 28, wherein, the saceharlde-D AB, sacclmnde-D AB-pepiide and functional molecule labeled saccharide-DAB-conjugated peptide comprises an imidazole ring.

30. A method for enhancing mass intensit of a saccharide during mass spectrometry (MS), the method comprising;

tagging the saccharide with a DAB-peptide tagging reagent, wherein the peptide is conjugated to a functional label; and

analyzin a composition comprising the saccharide by mass spectrometry, wherein a mass intensity signal of the tagged saccharide is enhanced. AS -52 S P03.

31 . The method of claim 30, wherein the sacchaiide-DAB, saecha:ride-DAB-peptide and functional molecule labeled saccharidc-DAB-conjugated peptide comprises an imidazole ring.