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WO1992009568A1 - Reactif de marquage par fluorescence et applications - Google Patents

Reactif de marquage par fluorescence et applications Download PDF

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Publication number
WO1992009568A1
WO1992009568A1 PCT/AU1991/000560 AU9100560W WO9209568A1 WO 1992009568 A1 WO1992009568 A1 WO 1992009568A1 AU 9100560 W AU9100560 W AU 9100560W WO 9209568 A1 WO9209568 A1 WO 9209568A1
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Prior art keywords
hydrazine
fmoc
derivative
organic molecule
tricyclic compound
Prior art date
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PCT/AU1991/000560
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English (en)
Inventor
Milton Thomas William Hearn
Renen Zhang
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Monash University
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Monash University
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Priority to AU90548/91A priority Critical patent/AU9054891A/en
Publication of WO1992009568A1 publication Critical patent/WO1992009568A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
    • C07H15/18—Acyclic radicals, substituted by carbocyclic rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C281/00—Derivatives of carbonic acid containing functional groups covered by groups C07C269/00 - C07C279/00 in which at least one nitrogen atom of these functional groups is further bound to another nitrogen atom not being part of a nitro or nitroso group
    • C07C281/02—Compounds containing any of the groups, e.g. carbazates
    • 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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals
    • G01N33/533—Production of labelled immunochemicals with fluorescent label
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C2603/00—Systems containing at least three condensed rings
    • C07C2603/02—Ortho- or ortho- and peri-condensed systems
    • C07C2603/04—Ortho- or ortho- and peri-condensed systems containing three rings
    • C07C2603/06—Ortho- or ortho- and peri-condensed systems containing three rings containing at least one ring with less than six ring members
    • C07C2603/10—Ortho- or ortho- and peri-condensed systems containing three rings containing at least one ring with less than six ring members containing five-membered rings
    • C07C2603/12—Ortho- or ortho- and peri-condensed systems containing three rings containing at least one ring with less than six ring members containing five-membered rings only one five-membered ring
    • C07C2603/18—Fluorenes; Hydrogenated fluorenes

Definitions

  • the present invention relates generally to a new derivatisation reagent useful in the quantitative determination of organic molecules containing an aldehyde and/or keto group, such as sugars, by reversed phase HPLC.
  • compositional analysis of the oligosaccharide moieties of glycoconjugates is of fundamental importance in structural studies on these compounds. Over the past decade, several methods have been developed for the analysis of saccharides in the mid-picomole to low nanomole range. Historically, gas chromatography methods have been used to improve sensitivity(l) with derivatised sugars. In HPLC methods, post-column derivatization has been employed for analysis of saccharides and a number of fluorescent reagents have been examined(2-5). Most recently, anion exchange chromatography with amperometric detection has been used for the analysis of underivatised carbohydrates(6).
  • one aspect of the present invention is directed to a fluorescent labelling reagent capable of reacting with an aldehyde or keto group of an organic molecule which reagent comprises a tricyclic compound having a hydrazine moiety depending from the central ring thereof.
  • the present invention is particularly applicable to sugars.
  • the tricyclic compound is a derivative of fluorenylmethyl and more preferably 9-fluorenylmethyl.
  • the fluorescent labelling reagent is 9-fluorenyloxycarbonyl hydrazine having the structural formula (I):
  • Fmoc-hydrazine CH 2 OCONH-NH 2 Fmoc-hydrazine
  • Fmoc-hydrazine this compound will be referred to as "Fmoc-hydrazine”.
  • reference herein to "Fmoc-hydrazine” is not to be construed as a limitation to the ambit of the present invention which extends to the tricyclic- hydrazine compounds stated above. Accordingly, reference to “Fmoc- hydrazine” or “9-fluorenyloxycarbonyl hydrazine” herein is considered to encompass all fluorescent labelling reagents contemplated by the present invention including any derivatives or functional analogues of Fmoc-hydrazine.
  • substituents contemplated herein include, alkyl (e.g. methyl, ethyl), substituted alkyl, alkenyl, substituted alkenyl, acyl, dienyl, arylalkyl, arylalkenyl, aryl, substituted aryl, heterocyclic, substituted heterocyclic, cycloalkyl, substituted cycloalkyl, halo (e.g.
  • alkyl, substituted alkyl, alkenyl and substituted alkenyl and the like is meant to encompass straight and branched molecules, lower (Cj - C_) and higher (more than Cg) derivatives.
  • substituted includes all the substituents setforth above.
  • the reagent Fmoc-hydrazine of the present invention is convenientiy synthesized by the reaction of hydrazine hydrate with 9-fluorenylmethyl chloroformate (Fmoc-Cl) dissolved in acetonitrile or dichloromethane. It should be noted, however, that the present invention is not limited to Fmoc- hydrazine prepared only by this method and variations in conditions and starting materials may be made without departing from the scope of the present invention.
  • Fmoc-Cl may be substituted by any of the groups listed above and, hence, form the corresponding substituted Fmoc- hydrazine the only requirement being that the resulting substituted Fmoc- hydrazine be able to derivatise sugars as herein described
  • the present invention contemplates Fmoc-hydrazine and any functional biochemical and/or chemical derivatives thereof.
  • Fmoc-hydrazine is meant to include above-mentioned substituted derivatives.
  • the present invention also provides Fmoc-hydrazine either in solid of liquid form packaged for sale in a suitable container. There is no absolute requirement that the Fmoc-hydrazine be pure or even substantially pure. Suitable Fmoc-hydrazine can be obtained by reacting Fmoc-Cl and hydrazine without further purification.
  • Fmoc-hydrazine provides an improved means for derivatising organic aldehydes and ketones such as sugars.
  • the present invention contemplates a method for derivatising an organic aldehyde and/or ketone prior to reversed phase HPLC which method comprises contacting said organic aldehyde and/or ketone with a fluorescence labelling effective amount of a tricyclic compound having a hydrazine moiety depending from a central ring of said tricyclic compound for a time and under conditions sufficient for said organic aldehyde or ketonesugar to be derivatised.
  • the tricyclic compound is a derivative of fluorenylmethyl and more preferably 9-fluorenylmethyl.
  • the fluorescent labelling reagent is Fmoc-hydrazine including its derivatives as outlined above and the organic molecule is a sugar.
  • sucrose any polyhydroxylated aldehyde or ketone and includes trioses, tetroses, pentoses, hexoses and septoses as well as higher sugars.
  • the term “sugar” also includes derivatives thereof including a sugar in combination with other molecules (i.e. glycoconjugates) such as in glycoproteins, lipopolysaccharides and the like.
  • “Sugar” and “saccharide” are used herein interchangeably and include disaccharide, trisaccharide and polysaccharide. The present invention is not limited to any particular stereo isomer of a sugar.
  • phosphosugars may also be used provided that the resulting derivatives allow a reducing sugar to be used.
  • the method of the present invention therefore, provides a highly sensitive and reliable method for the analysis of sugars at the sub-picomole to low picomole level.
  • the present invention extends to the determination of organic molecules containing an aldehyde and/or keto group such as glycoconjugates, aliphatic and/or aromatic aldehydes and/or ketones, and ketoacid and/or analogues thereof. For reasons of brevity, all such molecules are encompassed by the term "sugar".
  • the principle product of the derivatisation procedure is a sugar-hydrazone derivative and generally a sugar-tricyclic compound-hydrazone derivative.
  • the product is a sugar-Fmoc-hydrazone derivative.
  • the present invention therefore, provides sugar-Fmoc-hydrazone derivatives including any substituted Fmoc-hydrazone and/or sugar derivatives as outlined above.
  • the sugar-Fmoc-hydrazone derivative is packaged for sale in solid or liquid form in a suitable container. Such derivatives are useful, for example, as standards in reversed phase HPLC analysis of sugars.
  • the present invention also provides a kit useful for derivatising sugars and/or in the quantitative determination of sugars comprising a first compartment adapted to containing Fmoc-hydrazine and a second compartment adapted to contain one or more sugar-Fmoc-hydrazone derivatives for use as a standard.
  • the kit may contain additional compartments adapted to contain or receive one or more sugars for labelling or the sugar(s) to be labelled may be added directly to the Fmoc-hydrazine compartment. Further compartments may also be provided to contain appropriate buffers, diluents and/or other reagents required in the derivatisation process.
  • the kit contemplated herein would most conveniently be used in conjunction with a suitable HPLC facility and additional apparatus may also be required such as a water bath.
  • Figure 1 is a diagrammatic representation showing the synthesis of Fmoc- hydrazine and its reaction with a carbonyl compound.
  • Figure 2 is a graphical representation of a chromatogram of Fmoc-hydrazine unpurified after synthesis, chromatographic conditions: column Zorbax ODS 150x4.6 mm, mobile phase 30% acetonitrile 0.08M acetic acid, isocratic elution flow rate l.Oml/min.
  • Figure 3 is a graphical representation showing the effect of excitation wavelength and emission wavelength on fluorescence intensity, O Mannose; x xylose.
  • Figure 4 is a graphical representation showing the effects of molecular ratio of monosaccharide to Fmoc-hydrazine on derivatisation reaction at 65°C for 120 min.
  • Figure 5 is a graphical representation showing the effect of concentration of acetic acid on derivatisation of Fmoc-hydrazine with sugars. O Mannose; x xylose.
  • Figure 6 is a graphical representation depicting the effect of reaction time of derivatisation of Fmoc-hydrazine with sugars. 1. Mannose; 2. Ribose; 3. Xylose; 4. Glucose; 5. Galactose; 6. Fructose; 7. Maltose. Reactions at 65°C.
  • Figure 7 is a graphical representation showing (A) the chromatogram of sugar Fmoc-hydrazones, sample injected after derivatization. Chromatographic conditions: column Zorbax ODS 150 x 4.6mm; mobile phase A 20% acetonitrile 0.08M acetic acid, B 50% acetonitrile 0.08M acetic acid gradient elution for 30 min flow rate 1.0 ml/min. 1. Mannose, 2. Fructose, 3. Xylose. (B) The chromatogram of sugar Fmoc-hydrazones, stored for two weeks at 4°C (conditions as for (A)).
  • Figure 8 is a graphical representation showing RP-HPLC of sugar Fmoc- hydrazones. The separation was achieved by gradient elution. Conditions: column Zorbax C - 8 150 x 4.6 mm mobile phase A 27-5% acetonitrile 0.08 M acetic acid, B 30% acetonitrile 0.08 M acetic acid gradient elution for 30 min flow rate 1.0 ml/min. 1. Lactose, 2. Maltose, 3. Mannose, 4. Galactose, 5. Fructose, 6. Ribose, 7. Xylose.
  • Figure 9 is a graphical representation of calibration graphs for reducing sugars.
  • Man mannose; Rib, ribose; Xyl, xylose; Lac, lactose; Fru, Fructose.
  • Figure 10 is a graphical representation of an analysis of constituent monosaccharides of calf serum fetuin (A) and ovalbumin (B). Sample amount injected, 7 ng each as protein. Solid and dotted lines represent the results obtained for the hydrolysates for 5h and 8h at 100°C, respectively.
  • A peak.l containing mannose 0.7 and 0.94 pmol; peak 2 containing galactose 1.06 and 1.46 pmol.
  • peak 1 containing mannose 0.91 and 1.3 pmol; peak 2 containing galactose 0.26 and 0.48 pmol.
  • Eluant 30% acetonitrile in 0.08 M acetic acid. Other chromatographic conditions were the same as those described in Figure 8.
  • Figure 11 is a graphical representation depicting calibration graphs for mannose (Man) and galactose (Gal).
  • Fmoc-Cl(9-fluorenylmethyl chloroformate) and carbohydrates were purchased from Sigma Chemical Co., hydrazine hydrate from BDH Chemicals Australia Pty Ltd, acetic acid (A.R.) from AJAX Chemicals, ethanol from MAY & BAKER Australian Pty Ltd, acetonitrile and methyl alcohol (Chrom AR HPLC) were obtained from Mallinckrodt Australia Pty Ltd. Ovalbumin (chicken egg) and calf serum fetuin were obtained from Sigma Chemical Co.
  • Acid hydrolysis of proteins was carried out as follows. Samples were dissolved with 200 ⁇ l of water in screw cap teflon tubes and 200 ⁇ l of 4M TFA was added. The samples were hydrolysed in a boiling water bath for 5-8h. The tubes were cooled and the samples were dried by nitrogen, dissolved in ethanol and subjected to derivatisation as described above.
  • the apparatus used for HPLC was DuPont Instruments 8800 system (ternary chromatographic pump; series 8800 gradient controller; column oven). Detection of the saccharide derivatives was performed with Perkin-Elmer LS-5 Luminescence Spectrometer. The wavelengths of excitation and emission were 270 and 320 nm, respectively. The separation of monosaccharide derivatives was performed at ambient temperature and at a flow-rate of 1.0 ml/min using DuPont Chromatographic Bioseries C-18, C-8 and Phenyl column (5 ⁇ m, 150 x 4.6 mm).
  • the mobile phase (A) was 27.5% of aqueous acetonitrile containing 0.08 M acetic acid and (B) was 30% of aqueous acetonitrile containing 0.08 M acetic acid. Isocratic elution was 30% of aqueous acetonitrile containing 0.08 M acetic acid.
  • Fmoc-hydrazine was synthesized as described in Example 1. The reversed phase HPLC analysis of the crude product is shown in Figure 2. The new compound was also characterised by its organic elemental analysis and mass spectra. The data of organic elemental analysis are given in Example 1, whilst mass spectroscopy indicated (m/e, relative mass) of 255(molecular ion. MH+). On the basis of these data, the compound synthesized is characterised as 9- fluorenyloxycarbonylhydrazine as depicted in Figure 1.
  • the derivative yield of fructose increased slowly with the increasing mole ratio.
  • the reactivity of sugars with Fmoc-hydrazine is also shown from Figure 4.
  • the effects of the concentration of acetic acid on derivatisation yield were found to affect the rate of hydrazone formation. As shown in Figure 5, the optimum concentration of acetic acid was around 0.5%. When the concentration of acetic acid was reduced to 0.05%, the reaction was nearly stopped; over 1% of acetic acid the recovery for hexose (mannose) was decreased. The use of trichloroacetic acid and trifluoroacetic acid was examined for the derivatisation reaction, but no satisfactory results were obtained.
  • the optimum temperature for hydrazone formation was found to be 65°C, at lower temperature the reaction was slower, while at higher temperatures it was difficult to maintain a constant concentration of acetic acid.
  • the time at which the reaction reached completion at 65°C was different for the various sugars. From Figure 6, it can be seen that the complete derivatization for most of the sugars examined was achieved at around 180 min. When extending the reaction time to 240 min, the recovery of the derivatised sugars remained constant.
  • the elution order of sugar derivatives was different for C-8, C-18 and Phenyl columns under the same elution conditions. Disaccharides were the earliest to elute on all of the columns examined due to their more hydrophilic properties.
  • the elution order of the lactose and maltose derivatives on the alkyl bonded phases was reversed on the phenyl bonded phase, they can not be separated on C-18 bonded phase but were resolved on the C-8 phase.
  • the selectivity of the sugar derivatives examined is better on the C-8 column than on the other two columns. The separation of the sugar derivatives is shown in Figure 8. The effects of mobile phase were also examined.
  • Fluorescent response The fluorescence response of ten sugars, normalised to fucose in terms of peak area, are listed in Table 2.
  • the linearity of the detector response was investigated by injection of progressive dilutions of the sugar Fmoc-hydrazones. As shown in Figure 9, the response was linear over the range of lOpmol - llOpmol. Further studies have shown that this linear response can be extended out to lOfmol.
  • Figures 10 (A) and (B) indicate that under the experimental conditions, using hydrolysis of the sample under temperature conditions of a boiling water bath, the sugar release with 2 M TFA for 5h was not completed. When extending the time of hydrolysis the recovery of sugars was increased. The results also proved that this method can be used for the determination of component monosaccharides of glycoproteins in the sub-picomole to low picomole level.
  • the sugar composition was calculated from the hydrolysis sample and the calibration curve of standard sugars derivatised under the same conditions ( Figure 11).

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Abstract

Réactif de marquage par fluorescence apte à réagir avec les groupes aldéhyde ou céto d'une molécule organique renfermant le premier de ces groupes et/ou le second. Ledit réactif comporte un composé tricyclique possédant une fraction hydrazine suspendue à son cycle central. Dans un mode préféré de réalisation, on a prévu la 9-fluorényloxycarbonyl-hydrazine (Fmoc-hydrazine) ainsi qu'un procédé de dérivation de molécules organiques à l'aide de la Fmoc-hydrazine.
PCT/AU1991/000560 1990-12-03 1991-12-02 Reactif de marquage par fluorescence et applications Ceased WO1992009568A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU90548/91A AU9054891A (en) 1990-12-03 1991-12-02 Fluorescence labelling reagent and use therefor

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AUPK369090 1990-12-03
AUPK3690 1990-12-03

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994004741A1 (fr) * 1992-08-24 1994-03-03 Henkel Kommanditgesellschaft Auf Aktien Procede de lavage
WO1997002277A3 (fr) * 1995-06-30 1997-03-13 Ca Nat Research Council Procede de production de glycoconjugues synthetiques a liaison n

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3586718A (en) * 1965-01-08 1971-06-22 Hoffmann La Roche Dibenzo(a,d)cyclohepta(1,4)dien-5-ones
US3660485A (en) * 1970-10-05 1972-05-02 Searle & Co Fluorene-9-carboxylic acid hydrazides
US3911017A (en) * 1969-04-04 1975-10-07 Sandoz Ag 5-Aminomethyl-5H-dibenzo{8 a,d{9 cycloheptenes
DE2551919A1 (de) * 1974-11-20 1976-08-12 Chinoin Gyogyszer Es Vegyeszet Fungicides mittel und verfahren zur herstellung der wirkstoffe
DE3417413A1 (de) * 1984-05-11 1985-11-14 Dr. Karl Thomae Gmbh, 7950 Biberach Dicarbonsaeure-monohydrazid-derivate, ihre herstellung und ihre verwendung als kopplungskomponenten
US4818704A (en) * 1986-05-16 1989-04-04 Eka Nobel Ab Method for the separation and determination of enantiomeric amine compounds using an optically active agent
EP0328121A1 (fr) * 1988-02-11 1989-08-16 University Of Houston Analogues de mitomycine contenant un groupe hydrazino substitué

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3586718A (en) * 1965-01-08 1971-06-22 Hoffmann La Roche Dibenzo(a,d)cyclohepta(1,4)dien-5-ones
US3911017A (en) * 1969-04-04 1975-10-07 Sandoz Ag 5-Aminomethyl-5H-dibenzo{8 a,d{9 cycloheptenes
US3660485A (en) * 1970-10-05 1972-05-02 Searle & Co Fluorene-9-carboxylic acid hydrazides
DE2551919A1 (de) * 1974-11-20 1976-08-12 Chinoin Gyogyszer Es Vegyeszet Fungicides mittel und verfahren zur herstellung der wirkstoffe
DE3417413A1 (de) * 1984-05-11 1985-11-14 Dr. Karl Thomae Gmbh, 7950 Biberach Dicarbonsaeure-monohydrazid-derivate, ihre herstellung und ihre verwendung als kopplungskomponenten
US4818704A (en) * 1986-05-16 1989-04-04 Eka Nobel Ab Method for the separation and determination of enantiomeric amine compounds using an optically active agent
EP0328121A1 (fr) * 1988-02-11 1989-08-16 University Of Houston Analogues de mitomycine contenant un groupe hydrazino substitué

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
AUST. J. CHEM. 1977, 30, 2255-62, "Cyano esters and malononitriles. IV cyano (FLUORENYL) acetohydrazides and fluorenyl pyrazolones", LATIF, N; MISHRIKY, N.; HAMMAD, M, see formula 2, compounds 2(a)-2(g). *
CHEMICAL ABSTRACTS, Volume 115, No. 5, issued 5 August 1991, (COLUMBUS, OHIO, U.S.A.), R. ZHANG, R. CAO, M.W. HEARN, "Synthesis and application of FMOC-hydrazine for the quantitative determination of saccharides by reversed-phase high-performance liquid chromatography in the low and sub-picomole range", see page 433, column 2, the abstract No. 45378p; & ANAL. BIOCHEM. 1991, 195(1) 160-7(ENG). *
IDEM. *
J. ORG. CHEM., Vol. 37, No. 22, 1972, 3404-3409, "The 9-fluorenyl methoxycarbonyl amino-protecting group", L. CARPINO; G. HAN, see formula 19a page 3406, column 1 page 3407, column 2 page 3409. *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994004741A1 (fr) * 1992-08-24 1994-03-03 Henkel Kommanditgesellschaft Auf Aktien Procede de lavage
US5498546A (en) * 1992-08-24 1996-03-12 Henkel Kommanditgesellschaft Auf Aktien Washing process
WO1997002277A3 (fr) * 1995-06-30 1997-03-13 Ca Nat Research Council Procede de production de glycoconjugues synthetiques a liaison n
US5668272A (en) * 1995-06-30 1997-09-16 National Research Council Of Canada Method for producing synthetic N-linked glycoconjugates

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