WO2017132177A1 - Rapide marquage par fluorescence de glycanes et d'autres biomolécules par des signaux activés par spectrométrie de masse - Google Patents

Rapide marquage par fluorescence de glycanes et d'autres biomolécules par des signaux activés par spectrométrie de masse Download PDF

Info

Publication number
WO2017132177A1
WO2017132177A1 PCT/US2017/014790 US2017014790W WO2017132177A1 WO 2017132177 A1 WO2017132177 A1 WO 2017132177A1 US 2017014790 W US2017014790 W US 2017014790W WO 2017132177 A1 WO2017132177 A1 WO 2017132177A1
Authority
WO
WIPO (PCT)
Prior art keywords
glycan
acid
compound
glycans
compounds
Prior art date
Application number
PCT/US2017/014790
Other languages
English (en)
Inventor
Darryl W. Brousmiche
Matthew A. LAUBER
Original Assignee
Waters Technologies Corporation
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from US15/005,619 external-priority patent/US11352325B2/en
Application filed by Waters Technologies Corporation filed Critical Waters Technologies Corporation
Publication of WO2017132177A1 publication Critical patent/WO2017132177A1/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D207/00—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom
    • C07D207/46—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with hetero atoms directly attached to the ring nitrogen atom
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
    • C07D401/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings
    • C07D401/12—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings linked by a chain containing hetero atoms as chain links
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2400/00—Assays, e.g. immunoassays or enzyme assays, involving carbohydrates
    • G01N2400/10—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • G01N2400/38—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence, e.g. gluco- or galactomannans, e.g. Konjac gum, Locust bean gum, Guar gum
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2560/00—Chemical aspects of mass spectrometric analysis of biological material

Definitions

  • Fluorescent labeling of N-glycans is beneficial to detecting glycans because it improves both sensitivity and selectivity of the detection as well as the chromatographic behavior of glycans.
  • Amino acid analysis is a fundamental process in protein research and is of particular importance to clinical chemists and pharmaceutical manufacturers when glycosylation profiling of proteins must be monitored to ensure consistency of a therapeutic product.
  • the functional group of the compound can only be estimated.
  • Mass spectrometry is then required to identify the specific compound.
  • MS is used to determine what the molecular makeup is.
  • MS active compounds useful in rapid fluorescence tagging of glycans such as N -I inked glycans and other bio-molecules including, but not limited to, proteins, peptides and amino acids.
  • These MS active, fluorescent compounds have three functional components: (a) a tertiary amino group or other MS active atom; (b) a highly fluorescent moiety, and (c) a functional group that rapidly reacts with amines, such as an isocynanate or succidimidylcarbamate.
  • the reactive functional group provides rapid tagging of desired biomolecules, and the fluorescent moiety provides for a strong fluorescent signal.
  • the tertiary amino group substituent provides a strong MS signal.
  • there is no specific connectivity between the rapidly reacting functional group, the fluorescent moiety or the tertian,' amino group as any group can be attached to the other.
  • the invention relates to compounds of the various formulas described herein.
  • Each compound can act as a reagent for rapid fluorescence tagging of biomolecules and enhanced MS signaling.
  • the MS active, rapid fluorescence tagging compounds can be of the structural Formula I:
  • ⁇ a is selected from ester, amide, amine, ether, urea, carbamate, carbonate, thiol, thiourea. thiocarbamate, alky! or carbonyl;
  • the S active, rapid fluorescence tagging compounds can also be of the staictural Formula
  • R a is selected from ester, amide, amine, ether, urea, carbamate, carbonate, thiol, thiourea, thiocarbamate, a!ky! or carbonyl;
  • the compounds of the formulas described herein may have optica! centers and therefore may occur in different enantiomeric and disastereomeric configurations.
  • the invention described herein includes all enantiomers, diastereoniers and other stereoi somers of such compounds of each formula, as well as racemic compounds and racemic mixtures and other mixtures of stereoisomers thereof.
  • FIG. I shows the fluorescence and MS detection of the glycans GOT, GIF, and G2F released from 0.8 ⁇ Herceptin IgG labeled with 2,5-dioxopyrrolidin-l-yl (4-((2- (diethylamino)ethyl)carbamoyl)phenyl)carbamate.
  • FIG. 2 shows the fluorescence and MS detection of the giycan G2FBS2 released from 0.8 ⁇ Herceptin IgG labeled with 2,5-dioxopyrrolidin-l-yl (4-((2- (diethylamino)ethyl)carbamoyl)phenyl)carbamate.
  • Novel compounds useful in the rapid fluorescence tagging of glycans such as N-l inked glycans and other bio-molecules including, but not limited to, proteins, peptides and amino acids and with enhanced MS signaling are provided herein. These compounds are used to analyze glycans and/or other biomolecules in a sample. To analyze the biomolecule, the molecule is rapidly labeled with a compound described herein and then subjected to liquid chromatography, mass spectrometry, and fluorescence detection.
  • alkoxy refers to an alkyl ether radical, wherein the term alkyl is as defined below.
  • suitable alkyl ether radicals include methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, iso-butoxy, sec-butoxy, tert-butoxy, and the like.
  • alkyl refers to a straight-chain or branched-chain alkyl radical containing from 1 to and including 20, preferably 1 to 10, and more preferably I to 6, carbon atoms. Alkyl groups may be optionally substituted as defined herein without changing or effecting the fluorescent or mass spec properties of the molecule.
  • alkyl radicals include methyl, ethyl, n-propyl, isopropyl, n-butyi, isobutyi, sec-butyl, tert- butyl, pentyl, iso-amyl, hexyl, octy! , noyl and the like.
  • alkylene refers to a saturated aliphatic group derived from a straight or branched chain saturated hydrocarbon attached at two or more positions, such as methylene i C ' l i - !
  • alkyiammo may be a mono- or dialkyiated groups (also referred to "dialkylamino”) such as, for example, N-m ethyl amino, N-ethylamino, N,N- dimethyiamino, ⁇ , ⁇ -ethylmethylamino and the like and combination, refers to— -NRR', wherein R is independently selected from the group consisting of hydrogen and alkyl, and R' is alkyl, any of which may themselves be optionally substituted and the dialkyamino group can further comprise a spacer (sometimes referred to as a linker or linker group).
  • a molecular spacer or simply a "spacer” in chemistry is any flexible part of a molecule that provides a connection between two other parts of a molecule
  • parent molecular moiety means and includes phenyl, quinoline, coumarin or rhodamine.
  • amino refers to -NR , wherein R and R are independently selected from the group consisting of hydrogen, alkyl, acyi, heteroalkyl, aryl, cycloalkyl, heteroaryl, and heterocycloalkyl, any of which may themselves be optional ly substituted.
  • aryl as used herein, alone or in combination, means a carbocyclic aromatic system containing one, two or three rings wherein such rings may be attached together in a pendent manner or may be fused.
  • aryl embraces aromatic radicals such as benzyl, phenyl, naphthy!, anthracenyl, phenanthryl, indanyl, indenyl, annulenyl, azulenyl, tetrahydronaphthyl, and biphenyl.
  • benzo and "benz,” as used herein, alone or in combination, refer to the divalent radical derived from benzene. Examples include benzothiophene and benzimidazole.
  • carbamate refers to an ester of carbamic acid (- HCOO-) which may be attached to the parent molecular moiety from either the nitrogen or acid end, and which may be optionally substituted as defined herein.
  • O-carbamyl refers to a -OC(0) RR', group-with R and R' as defined herein.
  • N-carbamyP as used herein, alone or in combination, refers to a ROC(0)NR'- group, with R and R' as defined herein.
  • carbonyl when alone includes formyl [-C(0)H] and in combination is a -C(O)- group.
  • Carboxy refers to -C(0)OH or the corresponding “carboxylate” anion, such as is in a carboxylic acid salt.
  • An "O-carboxy” group refers to a RC(0)0- group, where R is as defined herein.
  • a “C-carboxy” group refers to a -C(0)OR groups where R is as defined herein.
  • cycioalkyl refers to a carbocyclic substituent obtained by removing a hydrogen from a saturated carbocyclic molecule and having three to fourteen carbon atoms. In one embodimeni, a cycioalkyl substituent has three to ten carbon atoms. Examples of cycioalkyl include cyclopropyl, cyclobutyl, cyciopentyl and cyciohexyl .
  • halo or halogen, as used herein, alone or in combination, refers to fluorine, chlorine, bromine, or iodine.
  • haloalkoxy refers to a haloalkyl group attached to the parent molecular moiety through an oxygen atom.
  • haloalkyl refers to an alky! radical having the meaning as defined above wherein one or more hydrogens are replaced with a halogen. Specifically embraced are monohaloalkyl , dihaloalkyl and polyhaloalkyl radical s.
  • a monohaloalkyl radical for one example, may have an iodo, bromo, chloro or fluoro atom within the radical.
  • Dihalo and polyhaloalkyl radicals may have two or more of the same halo atoms or a combination of different halo radicals.
  • haloalkyl radical s examples include fiuoromethyl, difluoromethyl, trifluoromethyl, chloromethyl, dichioromethyl, trichloromethyl, pentafluoroethyl, heptafluoropropyS , difluorochloromethyl, dichlorofluoromethyl , difluoroethyl, difluoropropyl, dichloroethyl and dichioropropyl.
  • Haloalkylene refers to a haloalkyl group attached at two or more positions. Examples include fluorom ethylene (-CFH-), difluoromethyiene (-CF?, --), chloromethylene (-CHC1-) and the like.
  • heteroalkyl refers to a stable straight or branched chain, or cyclic hydrocarbon radical, or combinations thereof, fully saturated or containing from 1 to 3 degrees of unsaturation, consisting of the stated number of carbon atoms and from one to three heteroatoms selected from the group consisting of O, N, and S, and wherein the nitrogen and sulfur atoms may optionally be oxidized and the nitrogen heteroatom may optionally be quaternized.
  • the heteroatom(s) O, N and S may be placed at any interior position of the heteroalkyl group. Up to two heteroatoms may be consecutive, such as, for example, -CH 2 -NH-OCH 3 .
  • heterocycloalkyl and, interchangeably, “heterocycle,” as used herein, alone or in combination, each refer to a saturated, partially unsaturated, or fully unsaturated monocyclic, bicyclic, or tricyclic heterocyclic radical containing at least one, preferably 1 to 4, and more preferably 1 to 2 heteroatoms as ring members, wherein each said heteroatom may be independently selected from the group consisting of nitrogen, oxygen, and sulfur, and wherein there are preferably 3 to 8 ring members in each ring, more preferably 3 to 7 ring members in each ring, and most preferably 5 to 6 ring members in each ring.
  • Heterocycloalkyl and “heterocycle” are intended to include sulfones, sulfoxides, N-oxides of tertiary nitrogen ring members, and carbocyciic fused and benzo fused ring systems; additionally, both terms also include systems where a heterocycle ring is fused to an aryl group, as defined herein, or an additional heterocycle group.
  • Heterocycle groups of the invention are exemplified by aziridinyl, azetidinyl, 1,3-benzodioxolyl, dihydroisoindolyl, dihydroisoquinolinyl, dihydrocinnolinyl, dihydrobenzodioxinyl, dihydro[l,3]oxazolo[4,5-b]pyridinyl, benzothiazolyl, dihydroindolyl, dihy-dropyridinyl, 1,3-dioxanyl, 1 ,4-dioxanyl, 1 ,3-dioxolanyl, isoindolinyl, morpholinyl, piperazinyl, pyrrolidinyl, tetrahydropyridinyl, piperidinyl, thiomorpholinyl, and the like.
  • the heterocycle groups may be optionally substituted unless specifically prohibited.
  • the term "optionally substituted” means the anteceding group may be substituted or unsubstituted.
  • the substituents of an "optionally substituted” group may include, without limitation, one or more substituents independently selected from the following groups or a particular designated set of groups, alone or in combination: lower alkyl, lower alkenvl, lower alkynyl, lower alkanoyl, lower heteroalkyl, lower heterocycloalkyl, lower haloalkyi, lower haioalkenyl, lower haloalkynyl, lower perhaloaikyl, lower perhaloalkoxy, lower cycloalkyl, phenyl, aryl, aryloxy, lower alkoxy, lower ha!oalkoxy, oxo, lower acyloxy, carbonyl, carboxyl, lower aikyiearbonyl, lower carboxyester, lower carboxamido, cyano, hydrogen, halogen, hydroxy, amino, lower alky
  • An optionally substituted group may be unsubstituted (e.g., -CH 2 CH 3 ), fully substituted (e.g., -CF 2 CF 3 ), monosubstituted (e.g., -CH 2 CH 2 F) or substituted at a level anywhere in-between fully substituted and monosubstituted (e.g., -CH 2 CF 3 ).
  • substituents are recited without qualification as to substitution, both substituted and unsubstituted forms are encompassed.
  • substituent is qualified as "substituted," the substituted form is specifically intended.
  • different sets of optional substituents to a particular moiety may be defined as needed; in these cases, the optional substitution will be as defined, often immediately following the phrase, "optionall substituted with.”
  • OCN OCN
  • SCN SCN
  • aryi, heterocycle, R, etc. occur more than one time in a formula or generic structure, its definition at each occurrence is independent of the definition at every other occurrence.
  • certain groups may be attached to a parent molecular moiety or may occupy a position in a chain of elements from either end as written.
  • an unsymmetrical group such as -C(0)N(R)- may be attached to the parent molecular moiety at either the carbon or the nitrogen.
  • bond refers to a covalent linkage between two atoms, or two moieties when the atoms joined by the bond are considered to be part of larger substructure.
  • a bond may be single, double, or triple unless otherwise specified.
  • a dashed line between two atoms in a drawing of a molecule indicates that an additional bond may be present or absent at that position.
  • a hydrogen bond is an electromagnetic attractive interaction between polar molecules, where hydrogen is bonded to an electronegative atom such as nitrogen or oxygen.
  • the hydrogen bond represents a strong dipole-dipole attraction. These hydrogen-bond attractions can occur between molecules (intermolecular) or within different parts of a single molecule (intramolecular).
  • the electronegative atom is considered a hydrogen bond acceptor, whether it is bonded to a hydrogen atom or not. While many possible hydrogen bonds can exist between two compounds, an exemplary' " bonding of a compound with another could take one of many forms, including, but not limited to, for example the following:
  • Individual stereoisomers of compounds can be prepared synthetically from commercially available starting materials which contain chiral centers or by preparation of mixtures of enantiomeric products followed by separation such as conversion to a mixture of diastereomers followed by separation or reciystallization, chromatographic techniques, direct separation of enantiomers on chiral chromatographic columns, or any other appropriate method known in the art.
  • Starting compounds of particular stereochemistry are either commercially available or can be made and resolved by techniques known in the art.
  • the compounds of the present invention may exist as geometric isomers.
  • the present invention includes ail cis, trans, syn, anti,
  • compounds may exist as tautomers; all tautomeric isomers are provided by this invention.
  • the compounds of the present invention can exist in unsoivated as well as solvated forms with pharmaceutically acceptable solvents such as water, ethanol, and the like. In general, the solvated forms are considered equivalent to the unsoivated forms for the purposes of the present invention.
  • the compounds described herein can also be in the form of a salt or solvate, in particular acid addition salts.
  • a salt for example, in acid-base neutralization, an acid and a base react to form water and a salt. Basically, to react together, there must be the transfer of protons between acids and bases.
  • different acids can produce different ions. For example, an Arrhenius acid produces hydronium ions when it dissociates in water.
  • a Bronsted-Lowry acid is a proton donor that donates hydrogen ions to the base.
  • proton acceptors and proton donors are the basis for the reaction and are referred to sometimes as a conjugate base or a conjugate acid.
  • a conjugate pair refers to acids and bases with common features, where there is an equal loss/gain of protons between the pairs.
  • M l is the conjugate acid to the base XI L because NH 3 gains a hydrogen ion to form H ⁇ as H 2 0 donates a hydrogen ion to form OH " , the conjugate base.
  • a Lewis acid accepts an electron pair and a Lewis base donates an electron pair donor. Accordingly, the proton H + can be an electron pair acceptor.
  • a compound can be both, a Lewis acid and a Lewis base, depending on the reaction.
  • methyl iodide can behave as both, a Lewi s acid and a Lewis base, where the methyl group is donated to form a salt.
  • acids which can be employed to form a salt of any of the compounds provided herein include inorganic acids and organic acids as well known to those skilled in the art such as, but not limited to, N-hydroxysuccinimide, hydrochloric, hydrofluoric, hydroiodic, hydrobromic, sulfuric, hydrosulfuric, thiosulfuric, hydrocyanic, phosphoric, phosphorous, hydrochlorous, chlorous, nitrous, nitric, chloric, perchloric, sulfurous, oxalic, maleic, succinic, and citric. Salts may also be formed by coordination of the compounds with an alkali metal or alkaline earth ion.
  • acids can form a salt including, but not limited to, 1 - hydroxy-2-naphthoic acid, 2,2-dichioroacetic acid, 2-hydroxyethanesulfonic acid, 2-oxoglutaric acid, 4-acetamidobenzoic acid, 4-aminosalicylic acid, acetic acid, adipic acid, ascorbic acid (L), aspartic acid (L), benzenesulfonie acid, benzoic acid, camphoric acid (+), camphor- 10-sulfonic a cid (+), capric acid (decanoic acid), caproic acid (hexanoic acid), capryiic acid (octanoic acid), carbonic acid, cinnamic acid, citric acid, cyclamic acid, dodecylsulfuric acid, ethane- 1,2- di sulfonic acid, ethanesulfonic acid, ethanesulfonic acid, formic acid, fuma
  • the counterion can be the conjugate base formed after reacting a compound or groups of compounds with an acid. In other words, counterion holds the opposite charge to that of the compound or compounds it is associated with.
  • the counterion represents the anionic part of the salt.
  • counterions of a salt compound described herein can include, but are not limited to, any of the following common anions and oxoanions: N-hydroxysuccinimidyl, hydride (FT), fluoride (F “ ), chloride (CF), bromide (Br " ), iodide (F), oxide (O 2” ), hydroxide (OIF), peroxide (O?.
  • hypochlorite ClOf
  • chlorite C10 2 "
  • chlorate C10 3 ⁇ X perchlorate
  • C10 4 ⁇ sulfite SO3 2"
  • sulfate S ( V " )
  • hydrogen sulfate H ( V)
  • thiosulfate S2O3 2"
  • nitrite N0 2 "
  • nitrate NO3 '
  • phosphite PO3 2”
  • phosphate P0 4 3”
  • (mono)hydrogen phosphate HPO 4 2”
  • di hydrogen phosphate I ⁇ 2 ⁇
  • carbonate C0 3 2'
  • hydrogen carbonate HCO 3 "
  • oxalate C 2 0 4 2”
  • cyanate NCO "
  • isocyanate OCN "
  • thiocyanate SCN "
  • chromate Cr0 4 2”
  • dichromate Cr 2 0 7
  • the M S active, rapid fluorescence tagging compounds can also be of the Formula ⁇ :
  • R 2a is selected from ester, amide, amine, ether, urea, carbamate, carbonate, thiol, thiourea, thiocarbamate, alkyl or carbonyl;
  • the M S active, rapid fluorescence tagging compounds can be of the Formula IV: wherein
  • R 1 is C or :
  • R ja is selected from ester, amide, amine, ether, urea, carbamate, carbonate, thiol, thiourea, thiocarbamate, alky! or carbonyl;
  • the MS active, rapid fluorescence tagging compounds can be of the Formula V:
  • R 1 is NH; NR where R is a!kyi, or O,
  • R 3a is selected from ester, amide, amine, ether, urea, carbamate, carbonate, thiol, thiourea, thiocarbamate, alk l or carbonyl,
  • R ⁇ a is selected from ester, amide, amine, ether, urea, carbamate, carbonate, thiol, thiourea, thi
  • R a R 3b , R JC ; R 4 " and R 4b are independently selected from H, optionally substituted alkyl; x 0 1 2.
  • the MS active, rapid fluorescence tagging compounds can be of the Formula VII:
  • ester selected from ester, amide, amine, ether, urea, carbamate, carbonate, thiol, thiourea, ocarbamate, alkvl or carbony
  • the M S active, rapid fluorescence tagging compounds can also be of the Formula VI i wherei
  • R " is selected from ester, amide, amine, ether, urea, carbamate, carbonate, thiol, thiourea, thiocarbamate, alky! or carbonyl;
  • the MS active, ra id fluorescence tagging compounds can also be of the Formula IX:
  • R " is selected from ester, amide, amine, ether, urea, carbamate, carbonate, thiol, thiourea, thiocarbamate, alkyl or carbonyl;
  • the MS active, rapid fluorescence tagging compounds can also be of the Formula
  • R 2d is selected from ester, amide, amine, ether, urea, carbamate, carbonate, thiol, thiourea, thiocarbamate, alkyl or carbonyl;
  • the organic layer was separated and the aqueous layer was extracted with 2 mL of dichloromethane.
  • the organic phases were combined, dried, and then evaporated to dryness to provide the crude material. This was subjected to standard organic chemistry purification techniques to provide the desired materia! C in > 95% purity.
  • procainamide A was added to 47 g of dry acetonitrile in a dry 100 mL Erienmeyer flask equipped with a stir bar, and allowed to dissolve.
  • 3.2 g of ⁇ , ⁇ -disuccinimidylcarbonate (DSC) B was dissolved in 417 g of dry acetonitri le, and the system was purged with N 2 .
  • the solution of procainamide was then transferred to the dropping funnel, and added dropwise to the DSC solution over the course of 1 hour. The solution was then allowed to stir for 4 hours.
  • N-linked and O-linked glycans are common giycans from recombinant biotherapeutic proteins, N-glycans being the more prominent.
  • N-linked glycans are attached to asparagines via an N-acetylglucosamine ("GlcNAc") residue in an Asn-Xxx-(Ser, Thr) motif where Xxx can be any amino acid except proline.
  • GlcNAc N-acetylglucosamine
  • O-linked glycans are attached to either Serine or Threnine. relinked glycans can be removed from the glycoprotein chemically or enzymatically. Analytical methods of analyzing N-linked glycans have become considerably sophisticated.
  • CE-, HPAEC- PAD, HILIC-LC/FLR, RPLC/MS, MALDI-MS are the most common analytical instrumentations.
  • Liquid chromatography ( "' ! .( " ) separation with fluorescence detection is widely used in the pharmaceutical industry for the characterization of enzymatically/chemically released gl can, typically tagged with a fluorescent dye at the reducing end of a glycan, Kalyan R. Anumula & Shirish T.
  • the sample preparation step can be very time consuming as it requires enzymatic digestion on the protein to release N-linked glycans followed by fluorescence tagging reaction.
  • the derivatization with a fluorescence moiety accomplished by reductive animation can require up to 4 hours.
  • Derivatization using the aromatic amine, 2-aminobenzamide (2AB) is the most established method and requires this reductive amination.
  • the 2AB tag improves the MS sensitivity compared to the non-labeled glycan and is fluorescently active.
  • N-linked glycans using novel chemical reagents. These tags are designed to enhance the analyte mass spectrometry response. This same chemical tag maybe used for amino acid and peptide labeling.
  • the reaction mechanism may be the same for all three types of molecules, whereby the derivatization occurs at the amine moiety. Amino acid analysis, peptide mapping and glycan profiling are each an integral part of the overall biotherapeutic protein characterization. Therefore, it is advantageous to have rapid universal fluorescent derivatization methods which improve detection of the MS instrumentation.
  • New molecules also referred herein to as "reagents" specific for N-linked glycans amino acids and peptides, are provided for enhanced MS detection and rapid fluorescence tagging of glycans and other biomolecules with enhanced MS signals.
  • the reaction times necessary to carry out the tagging process is measured in seconds, rather than minutes or hours.
  • the described molecules are useful in a wide variety of processes that rely on giycan and amino acid/peptide analysis for essential information of a product, process, or protein. As such, the molecules described herein may be used in processes such as protein characterization, cell culture monitoring, synthetic peptide manufacturing, and food analysis.
  • the reagents provided herein can consist of three functional components a) a tertiary amino group or other MS active atom, b) a highly fluorescent moiety, and c) a functional group that rapidly reacts with amines (such as an isocyanate, or succidimidylcarbamate).
  • Other reagents can consist of two functional components a) a tertiary amino group or other MS active atom, and b) a functional group that rapidly reacts with amines (such as an isocyanate, or succidimidylcarbamate).
  • the components serve the following purposes: (1) the amino group or MS active group gives good MS signal; (2) the fluorescent moiety provides a good fluorescence signal; and (3) the reactive functional group gives rapid tagging of desired biomolecules.
  • the enhanced MS signal observed upon utilization of a 2AB tagging reagent is a function of the amino or amine group present in the system following tagging.
  • rapid tagging agents contain no amino functionality following tagging of the desired biomolecule. Rather, these compounds have functionalities which do not provide the same electron density for mass spectrometry applications (e.g., urea or carbamate) and tie up the electron density resulting in low MS signal.
  • Biomolecules are organic compounds that are involved in the maintenance and metabolic processes of living organisms. Many disease conditions are due to impaired amino acid metabolism (e.g. phenylketonuria). As noted above, a biomolecule can also be a therapeutic agent such as peptide based pharmaceuticals that have been used to treat the disease. Glycans, amino acids, peptides and proteins are closely monitored during protein drug development and production. Many biomolecules can be detected by tagging them with a fluorescent label. The resulting conjugate or complex will show fluorescence, thereby facilitating their detection. There is recent movement, in the industry towards using MS for detection and quantitation of biomolecules. Fluorescent detection is still widely used for its sensitivity and quantitative analysis. Therefore, the combination of liquid chromatography, mass spectrometry and fluorescent detection is an analytical platform that can be used for a comprehensive protein analysis. Notwithstanding, there is no single technique that is capable of providing a complete structural analysis of N-linked glycans.
  • Mammalian and plant glycoproteins are biomolecules that commonly contain any one or more three types of constituent glycans, oligo- and polysaccharides. Glycans are important for protein folding and any alteration thereof may eliminate or alter activity. Often an immune response is triggered by an unrecognized glycan. Of the three types of glycans, analytical methods of analyzing N-linked glycans have become considerably sophisticated.
  • N-linked glycans are attached to asparagines via an N-acetylglucosamine (GlcNAc) residue in an Asn-Xxx-(Ser, Thr) motif where Xxx can be any amino acid except proline.
  • the N-glycan can be removed from the glycoprotein with hydrazine whether manually or with the aid of automated hydrazinolysis equipment.
  • the reagent cleaves peptide bonds between the N-linked glycan and asparagines to produce the glycan biomolecule.
  • enzymes are available for releasing N-glycans.
  • N-glycosidase F PNGase-F
  • PNGase-F N-glycosidase F
  • a commonly used enzyme cleaves the intact glycan as the glycosylamine leaving aspartic acid in place of the asparagine at the N-linked site of the protein.
  • Harvey, D.J. Identification of Protein-Bound Carbohydrates by Mass Spectrometry, 1 PROTEOMICS 311 at 31 1-312, 317 (2001), incorporated herein by reference.
  • a commonly used tactic to maximize the sensitivity of an assay is to convert the compound of interest into a derivative that exhibits a better response for the particular detection method being utilized.
  • the selection of a derivatizing agent is a critical choice in the development of an analytical procedure. The derivatizing agent affects the ultimate sensitivity and accuracy of the analysis by maximizing the sensitivity, yield and stability of the derivatized molecules.
  • the following determinations must be performed separately: (1 ) the glycosylated sites; (2) the glycosylated site occupancy; (3) the structure and amount of each glycan at each site: and (4) the number of glycoforms. Id. at 3 12, incorporated herein by reference.
  • MS can provide the answers to each of these steps. Hence the need for enhanced MS signals.
  • structural determination of the glycan is complicated.
  • the monosaccharide unit, the anomericity and ring size of each monosaccharide, the monosaccharide sequence and ring conformation together with identification of other groups must be determined. With the exception of ring conformation, MS can be used directly or indirectly to make these determinations using MALDI and/or ESI as the preferred MS technique. Id. at 3 13-3 16, incorporated herein by reference.
  • Reducing- Terminal Derivatization incorporated herein by reference.
  • Reductive amination while producing an MS active compound, is a very slow process and can take four (4) hours to tag the reagent to the compound.
  • Reducing-terminal derivatives may also be prepared by reactions other than reductive amination. Id. at 3 19, incorporated herein by reference.
  • glycans are not readily detectable due to the absence of a strong chromophore or fluorophore.
  • Free glycans released from glycoproteins enzymatically or chemically can be analyzed directly via MALDI MS or ESI MS/MS directly without any chemical tagging. Ying Qing Yu et al., A Rapid Sample Preparation Method for Mass Spectrometric Characterization of N-linked Glycans, 19 RAPID COMM. MASS SPECTROMETRY 233 1 (2005). This label-free approach is suitable for qualitative analysis for glycans.
  • Reductive amination while producing an MS active compound, is a very slow process and can take up to four hours to complete.
  • aromatic amine compounds that are used for reductive amination for glycans, most of them giving a low to moderate MS response.
  • procainamide can be used to enhance glycan MS response.
  • procainamide labeling procedure is similar to other commonly used reductive amination reagents, and therefore, it still takes a half day for the labeling step.
  • phanquinones and benzooxadiazoles are nitrogen containing fluorophores that can be used as pre-column derivatization agents. These compounds are devoid of intrinsic fluorescence. However, on conjugation with amino acids, they form the corresponding fluorescent conjugates.
  • the present compounds are particularly useful for derivatizing glycans and also amino acids and proteins because they react quickly with the molecules and form a stable, highly fluorescent MS derivative.
  • the general methodology for an analysis of a glycan or amino acid using the compounds of the subject invention consists of three closely related processes: (1) formation of derivatives in the sample; (2) separation of the derivatives; and (3) detection of the separated derivatives.
  • the first step is generally performed by reacting a mixture with one of the present reagents to yield a distinct compound. These derivatives provide a fluorescent signal which can then be detected in the detection stage of the analysis.
  • the separation step is based upon the differences in the chemical structure of the derivatives.
  • the derivatized amino acids differ from each other in the same way that the chemical structures of the precursor amino acids differ.
  • the derivatives must be separated so that the detector signal can be correctly related to the concentration of each derivative.
  • the derivatized amino acids can be separated and detected by chromatography, e.g., by high performance liquid chromatography (HPLC) or capillary zone electrophoresis (CZE). HPLC is particularly useful for this purpose. These technologies are well suited for this purpose because they are selective and can be used with very small samples. It is also possible to carry out the separation step by separating the amino acids prior to their derivatization.
  • the detection step is generally carried out using either an absorbance or fluorescence detector. As each derivative is eluted from the chromatographic column after separation, its presence and quantity is detected by a mass spectrometer and/or by the aborbance or emission of light. The sensitivity of the assay depends upon the strength of the signal produced.
  • reverse phase HPLC can be also used to analyze the peptide digests.
  • a given peptide digest there may be from 20 to 150 different peptides, each of which must be resolved and quantified.
  • the available sample is very small.
  • the analyst may be determining the structure of a protein that is isolated from an organism or one that has been synthesized by recombinant DNA technologies. Typically, nanomole quantities of a protein digest are studied. Due to the scarcity and cost of many proteins, it is very desirable to use as small a sample as possible.
  • N-linked glycans were released from a glycoprotein (Herceptin) using PNGase F prior to labeling with 2,5-dioxopyrrolidin-l-yl (4-((2-(diethylamino)ethyl)carbamoyl)phenyl)carbamate.
  • the 2,5- dioxopyrrolidin-l-yl (4-((2-(diethylamino)ethyl)carbamoyl)phenyl)carbamate was solubilized in water free acetonitrile to a final concentration of 45 ⁇ ⁇ .
  • 100 ⁇ of 2,5-dioxopyrrolidin-l-yl (4- ((2-(diethylamino)ethyl)carbamoyl)phenyl)carbamate solution was added to the released glycan sample and left at room temperature for 5 minutes, during which time the labeling reaction finished.
  • the labeled sample was lyophilized using a speed vac.
  • the lyophilized sample was reconstituted in 60% acetonitrile/water solution prior to chromatographic separation using FflLIC LC method. As shown in FIGS. I and 2, the samples were analyzed using fluorescence and MS detection.
  • Absorbance detection is generally used in protein mapping work. Two different detection processes which are often used for this purpose are: a) detection at 210-215 nm using a single wavelength detector; and b) broadband spectral detection using a photodiode array (PDA) detector.
  • PDA photodiode array
  • all peptides absorb at that wavelength, thus the user can ensure that all peptides eluted from the column are detected.
  • One difficulty with this technique is that a wide variety of compounds absorb in this region of the spectrum, and extreme care must be taken to ensure that all reagents, eluents, glassware, etc. are scrupulously clean to ensure that the observed signal is solely from the peptides.
  • the PDA detector collects the spectra of the eluent at specific time intervals (e.g. a spectrum between 200 and 350 nm is collected every second). This provides more information than a single wavelength and thus can assist in distinguishing between peptides which may elute with similar retention times.
  • Peptide mapping often involves the qualitative and quantitative analysis of trace levels of peptides in the digested protein.
  • the identification and quantitation of peptides in complex mixtures in the present method is effected by a three stage process: a) tagging the peptides of interest with the heterocyclic aromatic carbamates or other reactive groups, which exhibit a stronger absorbance or fluorescence signal than the original compound; b) separating the derivatized samples; and c) detecting the derivatized peptides by absorbance or fluorescence techniques.
  • the separation conditions for a derivative are frequently drastically different from the separation of the starting compounds. Likewise, the efficiency of a separation has a serious impact on the detection process.
  • heterocyclic aromatic carbamates and/or similar reactive groups provides a mapping method adaptable for use with nanogram quantities of protein. Further, the methods described herein provide a means for enhancing the sensitivity of known methodologies for detecting peptides in biological samples, such as tissue, urine, blood and saliva.
  • the analytical procedure must provide accurate quantitation of each component present in a complex mixture. To accomplish this, it is necessary to resolve the components of interest, not only from each other, but from components generated by the derivatization procedure. Quantitative conversion of all underivatized glycans and amino acids, including secondary amino acids, to single products is highly desirable, and facilitates good quantitation.
  • Detection selectivity is another advantageous feature for amino acid derivatives. Underivatized amino acids ail absorb weakly in the low UV (200-220 nm) range, but detection at such wavelengths is subject to interference by many compounds present in sample mixtures or chromatographic mobile phases. Derivatization with reagents absorbing at approximately 254 nm provides a measure of selectivity, but any aromatic organic compounds, frequently present in biological samples, can interfere at this wavelength. Reagents that enable detection via fluorescence, electrochemical response or visible-range absorbance would be desirable for superior detection selectivity.
  • OPA o-phthalaldehyde
  • mercaptan The o-phthalaldehyde (OPA)/mercaptan method.
  • OPA o-phthalaldehyde
  • the formation of the derivatives is rapid.
  • a significant difficulty with this method is the adduct is fairly unstable, and must be prepared very shortly before the detection step.
  • An additional problem is that this reagent will not form a derivative with secondary amino acids.
  • FMOC 9-fluorenylmethylchloroformate
  • PITC phenylisothiocyanate method
  • the dansyl chloride method provides stable derivatives with a minimum detectability in the order of about 1 .5 pmol. It is able to detect secondary amines and cysteine, but it results in multiple derivatives.
  • Fluorescent succinimidocarbamates have been used as derivatizing agents for amines, amino acids, peptides, phosphates and other classes of compounds.
  • succinimidocarbamate reagent When the succinimidocarbamate reagent is used to tag a compound with a fluorescent group, a detection limit of about 1 pmol can be achieved.
  • These reagents are used in conjunction with modern separation techniques such as high performance liquid chromatography, thin layer chromatography or capi llary electrophoresis.
  • Succinimidyl activated carbamates have been prepared by reacting carbocyclic aromatic amines with di-(N-succinimidyl) carbonate. Takeda et al ., 24 TETRAHEDRON LETT,, 4569 (1983).
  • Waters AccQTag method is a precolumn derivatization technique for peptide and protein hydrolysate amino acids.
  • the AccQTag methodology is based on a derivatizing reagent developed specifically for amino acid analysis.
  • Waters AccQFluor reagent (6-aminoquinolyl-N-hydrozysuccinimidyl carbamate, or ACQ) is an N-hydroxysuccinimide-activated heterocyclic carbamate, a known class of amine-derivatizing compounds. See, EP0533200 Bl .
  • This reagent converts both primary and secondary amino acids to stable, fluorescent derivatives and hydrolyz.es to yield 6-aminoquinoline, a non-interfering byproduct.
  • the AccQFluor reagent reacts rapidly with primary and secondary amino acids to yield highly stable ureas that fluoresce strongly at 395nm, The resulting derivatives are stable at room temperature for up to one (!) week.
  • Waters AccQTag Amino Acid Analysis method is the derivatizing reagent and a simple, pre-column derivatization protocol.
  • Waters AccQFluor Reagent is a highly reactive compound, 6-aminoquinolyl-N-hydroxysuccinimidyl carbarmate (AQC), which forms stable derivatives with primary and secondary amino acids in a matter of seconds. The derivatives are easily separated by reversed phase HPLC using Waters AccQTag Amino Acid Analysis System in less than 35 minutes.
  • AMQ aminoquinoline
  • the derivatization protocol - adding reagent to and heating a properly buffered sample - is simple and straight forward.
  • the amino acid derivatives can be injected directly without further sample preparation. Common buffer salts and detergents have little effect on reaction yield or on the reproducibility of results.
  • Another example of derivatization chemistry for HPLC analysis of a broad range of samples is the InstantAB 1M kit from Prozyme which is used to tag N-linked glycans, for example, the Glyko® InstantABTM kit (available from Prozyme, Inc., Hayward, California).
  • InstantAB IM provides rapid tagging and strong fluorescence but produces a weak MS signal , in fact, the MS signal of this molecule is significantly reduced when compared to the standard 2- AB reagent.
  • molecules that contain a tertiary amine, a fluorescent moiety and a reactive functional group may simply contain the tertiary amine and reactive functional groups. All molecules, however, undergo rapid functionalization. Through the use of a tertiary amine, all of the molecules are MS active (active in mass spectrometry), while others may also be fluorescent.
  • the current state of the art utilizes tagging molecules that either 1) react very slowly and give good MS/fluorescence signals or 2) react quickly and have good fluorescence signal, but have poor MS signal.
  • the lack of MS signal in the currently rapid reacting molecules is believed to arise from the lack of an electron rich amine - any nitrogen present loses electron density as part of urea or carbamate functionality.
  • the present molecules can have heterocyclic aromatic groups that exhibit a higher fluorescence quantum yield that that of carbocyclic aromatics used as tags. Nimura et al Anal. Chem. 58, 2372 (1986). This increase in the fluorescence quantum yield of the tag provides an increase in the sensitivity of the tagged amine.
  • the emission maximum of an amine compound derivatized with the reactive group is at a significantly different wavelength than the emission maximum of the free heterocyclic amine. The wavelength shift has very significant implications for fluorescence detection of tagged amines. Furthermore, since the observed fluorescence is predominantly from the derivative, background noise is eliminated or reduced and a more sensitive assay obtained.
  • the molecules provided herein do not provide a work-around for proper sample preparation. To obtain high quality mass spectra, the condition of the sample is of critical importance. Compounds other than the anaiyte will generally have an adverse effect on ion yield and must be removed. Indeed, while small amounts of sodium are essential for ionization by MALDI, carbohydrates are particularly susceptible to the effects of salts. Moreover, many carbohydrates occur as mixtures. Therefore it is important to ensure that isolation and purification techniques do not cause fractionation of the sample with a loss of quantitative information. Exemplar ⁇ '- is sialic acids which often are lost from glycoproteins when pH is too low or sample temperature too high.
  • N-linked glycans were released from 0.8 ⁇ ig of Herceptin using standard PNGase F protocols prior to labeling with 2,5-dioxopyrrolidin-l-yl (4-((2-)
  • the labeled sample was then lyophilized using a speed vac and reconstituted in 60% acetonitrile/water solution prior to chromatographic separation using a HILIC LC method and analysis fluorescence and MS detection, as shown in FIGS. 1 and 2.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Urology & Nephrology (AREA)
  • Biomedical Technology (AREA)
  • Hematology (AREA)
  • Immunology (AREA)
  • Molecular Biology (AREA)
  • Cell Biology (AREA)
  • Microbiology (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Biotechnology (AREA)
  • Plural Heterocyclic Compounds (AREA)

Abstract

L'invention concerne des réactifs comprenant des molécules fluorescentes activables par spectrométrie de masse (SM) et ayant une fonctionnalité activée pour réagir avec des amines utiles dans le marquage de biomolécules, telles que des N-glycanes, et leurs utilisations.
PCT/US2017/014790 2016-01-25 2017-01-24 Rapide marquage par fluorescence de glycanes et d'autres biomolécules par des signaux activés par spectrométrie de masse WO2017132177A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US15/005,619 US11352325B2 (en) 2011-09-28 2016-01-25 Rapid fluorescence tagging of glycans and other biomolecules with enhanced MS signals
US15/005,619 2016-01-25

Publications (1)

Publication Number Publication Date
WO2017132177A1 true WO2017132177A1 (fr) 2017-08-03

Family

ID=59398777

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2017/014790 WO2017132177A1 (fr) 2016-01-25 2017-01-24 Rapide marquage par fluorescence de glycanes et d'autres biomolécules par des signaux activés par spectrométrie de masse

Country Status (1)

Country Link
WO (1) WO2017132177A1 (fr)

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009100155A1 (fr) * 2008-02-04 2009-08-13 Prozyme, Inc. Compositions et procédés pour le marquage rapide de n-glycanes
WO2013049622A1 (fr) * 2011-09-28 2013-04-04 Waters Technologies Corporation Rapide marquage par fluorescence de glycanes et autres biomolécules ayant des signaux ms accrus
US20140350263A1 (en) * 2011-09-28 2014-11-27 Waters Technologies Corporation Rapid fluorescence tagging of glycans and other biomolecules with enhanced ms signals

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009100155A1 (fr) * 2008-02-04 2009-08-13 Prozyme, Inc. Compositions et procédés pour le marquage rapide de n-glycanes
WO2013049622A1 (fr) * 2011-09-28 2013-04-04 Waters Technologies Corporation Rapide marquage par fluorescence de glycanes et autres biomolécules ayant des signaux ms accrus
US20140350263A1 (en) * 2011-09-28 2014-11-27 Waters Technologies Corporation Rapid fluorescence tagging of glycans and other biomolecules with enhanced ms signals

Similar Documents

Publication Publication Date Title
US11448652B2 (en) Rapid fluorescence tagging of glycans and other biomolecules with enhanced MS signals
EP3309549B1 (fr) Rapide marquage par fluorescence de glycanes et autres biomolécules ayant des signaux ms accrus
US11352325B2 (en) Rapid fluorescence tagging of glycans and other biomolecules with enhanced MS signals
EP2990401A1 (fr) Marquage par fluorescence rapide des glycanes et d'autres biomolécules avec des signaux de ms améliorés
JP2013506115A (ja) 還元剤として２−ピコリンボランを使用する炭水化物の還元的アミノ化及び分析
US20210223253A1 (en) Methods for labeling amine-containing compounds
US20220381788A1 (en) Methods of enhancing ms detection of tagged glycans
Zou et al. Enhanced transglycosylation activity of an Endo-F3 mutant by ligand-directed localization
WO2017132177A1 (fr) Rapide marquage par fluorescence de glycanes et d'autres biomolécules par des signaux activés par spectrométrie de masse
US11061023B2 (en) Fluorescence tagging of glycans and other biomolecules through reductive amination for enhanced MS signals
EP3744708B1 (fr) Calibrant pour l'étalonnage de chromatographie liquide de n-glycans étiquetés

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 17744787

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 17744787

Country of ref document: EP

Kind code of ref document: A1