IL102495A - Universal standard reagents, method of preparing same and use thereof - Google Patents

Universal standard reagents, method of preparing same and use thereof

Info

Publication number
IL102495A
IL102495A IL102495A IL10249592A IL102495A IL 102495 A IL102495 A IL 102495A IL 102495 A IL102495 A IL 102495A IL 10249592 A IL10249592 A IL 10249592A IL 102495 A IL102495 A IL 102495A
Authority
IL
Israel
Prior art keywords
compound
reactive
reagent
reaction
measured
Prior art date
Application number
IL102495A
Other versions
IL102495A0 (en
Original Assignee
Patchornik Avraham
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
Application filed by Patchornik Avraham filed Critical Patchornik Avraham
Priority to IL102495A priority Critical patent/IL102495A/en
Publication of IL102495A0 publication Critical patent/IL102495A0/en
Priority to PCT/US1993/006980 priority patent/WO1994001771A1/en
Priority to JP6503596A priority patent/JPH08505220A/en
Priority to US08/362,519 priority patent/US5576216A/en
Priority to AU47844/93A priority patent/AU4784493A/en
Priority to EP93918367A priority patent/EP0650595A4/en
Publication of IL102495A publication Critical patent/IL102495A/en

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
    • C07D207/02—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D207/30—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having two double bonds between ring members or between ring members and non-ring members
    • C07D207/34—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having two double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D207/36—Oxygen or sulfur atoms
    • C07D207/40—2,5-Pyrrolidine-diones
    • C07D207/404—2,5-Pyrrolidine-diones with only hydrogen atoms or radicals containing only hydrogen and carbon atoms directly attached to other ring carbon atoms, e.g. succinimide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C211/00—Compounds containing amino groups bound to a carbon skeleton
    • C07C211/01—Compounds containing amino groups bound to a carbon skeleton having amino groups bound to acyclic carbon atoms
    • C07C211/02—Compounds containing amino groups bound to a carbon skeleton having amino groups bound to acyclic carbon atoms of an acyclic saturated carbon skeleton
    • C07C211/09—Diamines
    • C07C211/10—Diaminoethanes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C211/00—Compounds containing amino groups bound to a carbon skeleton
    • C07C211/43—Compounds containing amino groups bound to a carbon skeleton having amino groups bound to carbon atoms of six-membered aromatic rings of the carbon skeleton
    • C07C211/44—Compounds containing amino groups bound to a carbon skeleton having amino groups bound to carbon atoms of six-membered aromatic rings of the carbon skeleton having amino groups bound to only one six-membered aromatic ring
    • C07C211/52—Compounds containing amino groups bound to a carbon skeleton having amino groups bound to carbon atoms of six-membered aromatic rings of the carbon skeleton having amino groups bound to only one six-membered aromatic ring the carbon skeleton being further substituted by halogen atoms or by nitro or nitroso groups
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C215/00—Compounds containing amino and hydroxy groups bound to the same carbon skeleton
    • C07C215/02—Compounds containing amino and hydroxy groups bound to the same carbon skeleton having hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton
    • C07C215/04—Compounds containing amino and hydroxy groups bound to the same carbon skeleton having hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being saturated
    • C07C215/06—Compounds containing amino and hydroxy groups bound to the same carbon skeleton having hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being saturated and acyclic
    • C07C215/16—Compounds containing amino and hydroxy groups bound to the same carbon skeleton having hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being saturated and acyclic the nitrogen atom of the amino group being further bound to carbon atoms of six-membered aromatic rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C223/00—Compounds containing amino and —CHO groups bound to the same carbon skeleton
    • C07C223/02—Compounds containing amino and —CHO groups bound to the same carbon skeleton having amino groups bound to acyclic carbon atoms of the carbon skeleton
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C229/00—Compounds containing amino and carboxyl groups bound to the same carbon skeleton
    • C07C229/02—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton
    • C07C229/04—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated
    • C07C229/06—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having only one amino and one carboxyl group bound to the carbon skeleton
    • C07C229/18—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having only one amino and one carboxyl group bound to the carbon skeleton the nitrogen atom of the amino group being further bound to carbon atoms of six-membered aromatic rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C233/00—Carboxylic acid amides
    • C07C233/01—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms
    • C07C233/34—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms having the nitrogen atom of at least one of the carboxamide groups bound to a carbon atom of a hydrocarbon radical substituted by amino groups
    • C07C233/35—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms having the nitrogen atom of at least one of the carboxamide groups bound to a carbon atom of a hydrocarbon radical substituted by amino groups with the substituted hydrocarbon radical bound to the nitrogen atom of the carboxamide group by an acyclic carbon atom
    • C07C233/36—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms having the nitrogen atom of at least one of the carboxamide groups bound to a carbon atom of a hydrocarbon radical substituted by amino groups with the substituted hydrocarbon radical bound to the nitrogen atom of the carboxamide group by an acyclic carbon atom having the carbon atom of the carboxamide group bound to a hydrogen atom or to a carbon atom of an acyclic saturated carbon skeleton
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C233/00—Carboxylic acid amides
    • C07C233/64—Carboxylic acid amides having carbon atoms of carboxamide groups bound to carbon atoms of six-membered aromatic rings
    • C07C233/77—Carboxylic acid amides having carbon atoms of carboxamide groups bound to carbon atoms of six-membered aromatic rings having the nitrogen atom of at least one of the carboxamide groups bound to a carbon atom of a hydrocarbon radical substituted by amino groups
    • C07C233/78—Carboxylic acid amides having carbon atoms of carboxamide groups bound to carbon atoms of six-membered aromatic rings having the nitrogen atom of at least one of the carboxamide groups bound to a carbon atom of a hydrocarbon radical substituted by amino groups with the substituted hydrocarbon radical bound to the nitrogen atom of the carboxamide group by an acyclic carbon atom
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C243/00—Compounds containing chains of nitrogen atoms singly-bound to each other, e.g. hydrazines, triazanes
    • C07C243/24—Hydrazines having nitrogen atoms of hydrazine groups acylated by carboxylic acids
    • C07C243/26—Hydrazines having nitrogen atoms of hydrazine groups acylated by carboxylic acids with acylating carboxyl groups bound to hydrogen atoms or to acyclic carbon atoms
    • C07C243/34—Hydrazines having nitrogen atoms of hydrazine groups acylated by carboxylic acids with acylating carboxyl groups bound to hydrogen atoms or to acyclic carbon atoms to carbon atoms of a carbon skeleton further substituted by nitrogen atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C271/00—Derivatives of carbamic acids, i.e. compounds containing any of the groups, the nitrogen atom not being part of nitro or nitroso groups
    • C07C271/06—Esters of carbamic acids
    • C07C271/08—Esters of carbamic acids having oxygen atoms of carbamate groups bound to acyclic carbon atoms
    • C07C271/10—Esters of carbamic acids having oxygen atoms of carbamate groups bound to acyclic carbon atoms with the nitrogen atoms of the carbamate groups bound to hydrogen atoms or to acyclic carbon atoms
    • C07C271/22—Esters of carbamic acids having oxygen atoms of carbamate groups bound to acyclic carbon atoms with the nitrogen atoms of the carbamate groups bound to hydrogen atoms or to acyclic carbon atoms to carbon atoms of hydrocarbon radicals substituted by carboxyl groups
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C311/00—Amides of sulfonic acids, i.e. compounds having singly-bound oxygen atoms of sulfo groups replaced by nitrogen atoms, not being part of nitro or nitroso groups
    • C07C311/30—Sulfonamides, the carbon skeleton of the acid part being further substituted by singly-bound nitrogen atoms, not being part of nitro or nitroso groups
    • C07C311/37—Sulfonamides, the carbon skeleton of the acid part being further substituted by singly-bound nitrogen atoms, not being part of nitro or nitroso groups having the sulfur atom of at least one of the sulfonamide groups bound to a carbon atom of a six-membered aromatic ring
    • C07C311/38—Sulfonamides, the carbon skeleton of the acid part being further substituted by singly-bound nitrogen atoms, not being part of nitro or nitroso groups having the sulfur atom of at least one of the sulfonamide groups bound to a carbon atom of a six-membered aromatic ring having sulfur atoms of sulfonamide groups and amino groups bound to carbon atoms of six-membered rings of the same carbon skeleton
    • C07C311/39—Sulfonamides, the carbon skeleton of the acid part being further substituted by singly-bound nitrogen atoms, not being part of nitro or nitroso groups having the sulfur atom of at least one of the sulfonamide groups bound to a carbon atom of a six-membered aromatic ring having sulfur atoms of sulfonamide groups and amino groups bound to carbon atoms of six-membered rings of the same carbon skeleton having the nitrogen atom of at least one of the sulfonamide groups bound to hydrogen atoms or to an acyclic carbon atom
    • C07C311/41—Sulfonamides, the carbon skeleton of the acid part being further substituted by singly-bound nitrogen atoms, not being part of nitro or nitroso groups having the sulfur atom of at least one of the sulfonamide groups bound to a carbon atom of a six-membered aromatic ring having sulfur atoms of sulfonamide groups and amino groups bound to carbon atoms of six-membered rings of the same carbon skeleton having the nitrogen atom of at least one of the sulfonamide groups bound to hydrogen atoms or to an acyclic carbon atom to an acyclic carbon atom of a hydrocarbon radical substituted by nitrogen atoms, not being part of nitro or nitroso groups
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C323/00—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups
    • C07C323/50—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and carboxyl groups bound to the same carbon skeleton
    • C07C323/51—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and carboxyl groups bound to the same carbon skeleton having the sulfur atoms of the thio groups bound to acyclic carbon atoms of the carbon skeleton
    • C07C323/57—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and carboxyl groups bound to the same carbon skeleton having the sulfur atoms of the thio groups bound to acyclic carbon atoms of the carbon skeleton the carbon skeleton being further substituted by nitrogen atoms, not being part of nitro or nitroso groups
    • C07C323/58—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and carboxyl groups bound to the same carbon skeleton having the sulfur atoms of the thio groups bound to acyclic carbon atoms of the carbon skeleton the carbon skeleton being further substituted by nitrogen atoms, not being part of nitro or nitroso groups with amino groups bound to the carbon skeleton
    • C07C323/59—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and carboxyl groups bound to the same carbon skeleton having the sulfur atoms of the thio groups bound to acyclic carbon atoms of the carbon skeleton the carbon skeleton being further substituted by nitrogen atoms, not being part of nitro or nitroso groups with amino groups bound to the carbon skeleton with acylated amino groups bound to the carbon skeleton
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D295/00—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms
    • C07D295/04—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms
    • C07D295/06—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms substituted by halogen atoms or nitro radicals
    • C07D295/073—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms substituted by halogen atoms or nitro radicals with the ring nitrogen atoms and the substituents separated by carbocyclic rings or by carbon chains interrupted by carbocyclic rings
    • 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
    • G01N33/52—Use of compounds or compositions for colorimetric, spectrophotometric or fluorometric investigation, e.g. use of reagent paper and including single- and multilayer analytical elements
    • 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

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Hematology (AREA)
  • Immunology (AREA)
  • Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Urology & Nephrology (AREA)
  • Molecular Biology (AREA)
  • Microbiology (AREA)
  • Cell Biology (AREA)
  • Biotechnology (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)
  • Investigating Or Analyzing Non-Biological Materials By The Use Of Chemical Means (AREA)
  • Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)

Description

UNIVERSAL STANDARD REAGENTS, METHOD OF PREPARING AND USE THEREOF UNIVERSAL STANDARD REAGENTS, METHOD OF PREPARING SAME AND USE THEREOF The present invention relates to a new class of chemical reagents for quantitatively measuring compounds containing reactive functional groups. The invention further relates to methods of preparing such reagents and using them. Specifically, the invention relates to a new class of chemical reagents which can be used as universal standards for quantatively measuring any compound which can form a derivative with the reagents when the compound is present in mixtures and together with homologues. More specifically, the reagents of this invention are organic chemicals which have specific moieties that can be measured quantatively, colorometrically, fluoroscopically, or by other spectral means such as UV, IR, NMR and ESR. Some of these reagents are new chemical compounds.
It is thus an object of this invention to provide a new class of analytical reagents which can serve as universal standards for visual spectroscopic analysis of different compounds, without requiring comparison with a standard for each compound measured.
Another object of the present invention is to provide analytical methods for measuring compounds quantitatively, with reagents whose molar ε and that of their derivatives is substantially constant.
Yet another object of the invention is to provide a method of quantitatively measuring homologous compounds with a single standard reagent without requiring external standards . 2 102,495/ 3 A further object of the invention is to provide some new chemical compounds suitable for use as universal standard colorimetric or spectrometric analytical reagents.
Until now, the common methods for quantitatively measuring chemical compounds having functional groups were based on the reaction of these compounds via their functional groups with a reagent, usually a chromophoric or fluorescent reagent, and measuring the derivative spectrometrically by comparing the absorption of this derivative with that of a known external standard of such a derivative compound. For example, the determination of amino acids with fluoro-2,4-dinitrobenzene (FDNB), as described by Sanger, Biochem. J., Vol. 45, p. 563 (1984), is carried out by converting the amino acids into their dinitrophenyl derivatives. The actual quantification of each amino acid is arrived at by comparing the UV absorption of the corresponding dinitrophenyl derivative with that of standard amino acid dinitrophenyl products, e.g., DNP-Gly, DNP-Ala, etc. The external standard, however, is different for each amino acid measured. Similarly, amino acids can be analyzed quantitatively fluorometrically by reacting them with a fluorescent reagent such as Dansyl chloride, Dabsyl chloride or O-phthalaldehyde to form the fluorescent derivative. The actual quantification again requires a specific external standard for each amino acid.
The present invention provides a method for the quantitative determination of an organic compound designated herein by A, said compound A having quantitatively reactable functional groups, said method consisting essentially of quantitatively reacting compound A, in a sample containing compound A, with a reagent comprising a compound of the general formula I: 3 102,495/ 3 Q-B-f (I) wherein: Q stands for an organic moiety which can be measured quantitatively, visually by color, spectroscopically, or fluorometrically; B represents a non-reactive organic bridging unit linking the moiety Q to a reactive functional group said bridging unit including non-reactive CH2- CH2 linkages and being of sufficient length or size to prevent any possible interaction of Q that might alter its spectroscopic properties even upon derivatization; and is a reactive group which can react with a compound to form covalently bonded derivatives, according to the reaction n(Q-B-f) + A→Q-B-f-A + (n-l)Q-B-f wherein: Q, B and / are as defined above, and n equals 1 or more; separating any excess reagent (n-\)Q-B-f; and measuring the absorption of the reaction product Q-B- f - A, wherein the molar absorbtivity ε of said reaction product Q-B-f -A and said reagent Q-B-f are substantially constant and wherein the molar concentration of the reaction product Q-B- f - A is equal to the measured absorption divided by ε, whereby division of said measured absorption by ε provides the value of the molar concentration of the reaction product, thus eliminating the requirement for comparing the result with an external standard. 4 102,495/ 3 The invention further provides a method for studying the reaction rates of a given functional group present in different types of compounds, comprising reacting a known mixture of compounds having the same functional group with less than stoichiometric amounts of a reagent comprising a compound of the general formula I: Q- B - f (I) wherein: Q stands for an organic moiety which can be measured quantitatively, visually by color, spectroscopically, or fluorometrically; B represents a non-reactive organic bridging unit linking the moiety Q to a reactive functional group said bridging unit including non-reactive CH2- CH2 linkages and being of sufficient length or size to prevent any possible interaction of Q that might alter its spectroscopic properties even upon derivatization; and is a reactive group which can react with a compound to form covalently bonded derivatives; separating the derivative reaction products; and measuring the absorption of each derivative product using the reagent as a universal standard, wherein the molar absorbtivity ε of each said derivative product is substantially the same, whereby division of said measured absorption of each said derivative product by the same value ε provides the value of the molar concentration of the derivative product and the reaction rates of the functional groups present in each of the different compounds are proportional to the molar concentration of each of the derivative products. 5 102,495/4 The invention still further provides a method for the quantitative determination of the quantity of each of a plurality of components of a mixture, each of said components being designated herein as Ai, A2 . . .Am, m being a whole number and each component carrying the same quantitatively reactable functional group , said method comprising quantitatively reacting said mixture of components A\, A2 . . .Am with an excess of a reagent comprising a compound of the general formula I: Q - B - f (I) wherein: Q stands for an organic moiety which can be measured quantitatively, visually by color, spectroscopically, or fiuorometrically; B represents a non-reactive organic bridging unit linking the moiety Q to a reactive functional group said bridging unit including non-reactive CH2- CH2 linkages and being of sufficient length or size to prevent any possible interaction of Q that might alter its spectroscopic properties even upon derivatization; and is a reactive group which can react with a compound to form covalently bonded derivatives; according to the reaction n(Q - B - f) + A → Q - B - f - A + {n - \)Q - B - f wherein: n is greater than the sum of A A2 . . .Am, and separating said reaction products by chromatographic methods known per se. 5A 102,495/ 1 Generally, the compounds to be analyzed and measured are reacted with an excess reagent Q - B - f and the product is isolated from excess reagent by known methods of separation such as chromatography, or by adding excess of another reactive compound A ' similar to A in its chemistry, but carrying a functional group that can help in separating the excess reagent Q - B - f from the reaction mixture. Such compounds A ' may be polymers with functional groups similar to those of A, which, upon reaction with excess reagent, form separable solids. A ' could also contain functional groups such as -SO3H or -N-, which would make the reaction product with Q- B - f water-soluble or separable. The product is then measured visually by color or spectroscopically. The molar concentration of the reagent and derivative will have the same ε, irrespective of which compound A being determined in the reaction is quantitative.
Some representative moieties for Q are shown in Table 1 : - 6 - 102495/2 'TABLE 1 102495/2 - 7 - TABLE 1 contd.
Structural formula Name H3C dabsylamido- dabsyl-N, H3C dabsyl- dabsyloxy- fluoren-9-ylirethyloxycarl-onyl- ( FMOC ) - 8 - 102495/2 TABLE 1 contd.
Name Structural formula fluoren-9-ylmethyloxycarbamido- fluoren-9-ylmethyloxycarbonato- - 9 - 102495/2 Some representative bridging units B are: (CnHIn) - where n = 2 to 18; (C^H^Z)^ where m = 2 to 3, Z = NH, O, S and p = 1 to 18; where n and m are as defined above.
Some representative reactive functional groups f that can be reacted with compounds A to form spectroscopically measurable derivatives not requiring any external standard are listed in Table 2. 102495/2 - 10 TABLE 2 f-grouD Suitable for measuring the Following A Groups 1) -NH2 acylating, sulphonating or phosphorylating reagents, reactive esters, aldehydes, mixed anhyarides, carbon dioxide, alkylating epoxides, acrylates, β· lactams 2) -NH-R1 as for 1), except for aldehydes 3) -NHR1«2 alkylating reagents (CH3X, X=Halogen) β ->) - R1 R2 R3 ionic reagents in aqueous solution (ion exchange reaction) 5) -CO0H CH2N2, ArCHNj, ROH or RSH (DCC coupling method) 6) -CO-OL -OL=leaving group such as Nucleophiles, such as -NH2, NHR, -MHR1R2, ROH, RSH, CNe, N39, etc. OBT, Q-SUC, nitro- phenol etc. 7) -CO NH NH2 aldehydes, saccharides S) -NH C0CH,X (X=Halogen) RSH 9) -NHCOCH2CH2CH2SH alkylating reagents such as a Iky I halogens, activated double bond (Michael addition) as in acrylates 10) -CH2-CH=CH2 halogens and H-hal 11) -CH2-hydroxy quinoline Zn2*, CU**, Au, Ca** EDTA, hydroxamates 12) -CHO S02/H20, ROH, RSH, CN®, proteins with or without help of NaBCNH-j 13) -SO3H amine salts, basic inorganic salts, i.e. Na, K, Ca 14) -S02NH HH2 R CHO, 8r2, Cl2, I2 (RC0)20 and acylating reagents; aldehydes 15) -CH2 a model for aromatic Friedel Craft reactions 16) COOCH3 kinetics of ester hydrolysis, hydrazinolysis, ami nono lysis 17) -CH2S3Hal selective for indoles and derivatives 18) OH as models for sugars in the formation of glycosidic bonds 102495/2 For example, in the analytical reagent D PNH( CH2 ) 2NH2 , where DNP = 2, 4-dinitrophenyl, Q = 2 , 4-dinitrophenylamine (DNP-NH-) , B = -(CH2)2- and f = -NH2. We measured various acyl compounds with this reagent. By mixing excess DNPNH(CH2)2NH2 with an unknown acylating (RCO"**) compound, DNP-NH( CH2 ) 2NHCOR is formed. After removal of excess reagent, the new derivative compound can be measured spectrometrically, its measured value being proportional to the amount of RCO*. The acylating compound can encompass a wide variety of acylating groups such as RCO*, CH3C0-, CeHsCO-, t-BuOCO-, ArS02-, etc.
Similarly, the analytical reagent DNPNH( CH2) 2COOSu, (Su = succinimide) , where Q = DNPNH- , B = -(CH2)2- and f = COOSu, was successfully reacted with a wide variety of compounds carrying a primary or secondary amine function such as (C2HS)2NH, CeHsNH , H2NNH2 , CeHsCH2NH2, HO-(CH2) 3NH2, HS(CH2)2NH2, etc., to give derivatives that can be determined spectroscopically without external standards. Using excess succinimide active esters as reagent gives quantitative conversion of the amines to amides .
In addition to using the moved reagents of this invention for the quantitative determination of compounds, it is possible to determine the relative reactivities of two given compounds A and A2 by reacting an excess of each of Aa. and A2 with the same analytical reagent Q-B-f . The ratio of the amounts of their respective products Q-B-f-Ax and Q-B-f-A2 is a measure for their relative reactive. For example, when 10 equivalents of acetic anhydride (A = CH3CO) and 10 equivalents of benzoic anhydride (A2 = CeHsCO) are reacted with 1 equivalent of DNP-NH(CH2)2 NH2, the ratio in which their products, DNP-NH(CH2) aNHCOCH3 and DNPNH(CH2)2 NHCOCeHB, respectively, are found in the - 12 - 102495/2 resulting reaction mixture is a measure for the reactivity of acetic anhydride and benzoic anhydride as acylating agents .
The reagents of this invention are also useful for measuring reactive gases. Thus, carbon dioxide can be measured in a very convenient way by reacting it with a toluene solution of DNP-NH( CH2 ) 2NH2 , as follows: + - DNP - NH ( CH2 ) 2NH2 + C02 — > DNPNH ( CH2 ) 2NH3OCONH ( CH2 ) 2NHDNP (yellow, insoluble in water) (yellow, water soluble) The latter compound is a colored salt and can be extracted with . water and measured spectrometrically. Remnants of the original reagent will dissolve in toluene.
With this class of reagents it is possible to follow reactions involving colored chromophores by visual examination with very small quantities (nanomoles).
In accordance with this invention, not only soluble material or gases can be analysed with the novel universal standard reagents, but it is also possible to determine quantitatively heterogeneous matrices such as found in polymers and polymeric reagents, which can be covalently bound to the reagents.
Thus, e.g., when an insoluble, but swellable polymer P has a reactive group A attached to it, the reactive polymer can be represented by the formula (g)-a-A. a is the linking group between the insoluble polymer matrix (P) and the group A and is covalently bound to © and stable to any cleavage during the chemical manipulation. This polymer, when reacted with excess of a reagent Q-B-f, forms reaction 102495/2 products which are (1) insoluble polymer - (P) -a itself; and (2) Q-B-f-A, where Q,'B, f and A are as defined above After removing the excess reagent Q-B-f, and the polymer ^P) -a- from the reaction mixture, the amount of A originally bound to a give weight of polymer® -a-A can b calculated by measuring spectrometrically the amount of Q E-f-A formed. The value found for Q-B-f-A is then proportional to -the original amount of A attached to the polymer of (¾ -a-A.
More specifically, the amount of reactive RC0+ groups bound to a polymer can be determined as shown by the equation: + {n-l)NH2(CH-)2NKDtf? + RCONH ( CH3 ) 2NHDNP wherein n is a whole integer.
Alternatively, the reactive polymer may be of the kind represented by(^ -A, where A is covalently bound to the polymeric matrix. Here, too, excess reagent Q-B-f is used to react with the polymer to give a high yield of reaction to form(^ -A-f-B-Q and the excess reagent is washed out. In this case, additional chemical cleavage is necessary to release the Q group to be measured. Special, easily hydrolyzable or cleavable groups are used. To mention a few: ester (-C0O-), amide (-C0 H-), carbamide (- 0C0 H-) and S-S bonds are quite often used.
In cases where the reactive polymer mentioned above, (?) -a-A, comprises an active ester group, the the a - 14 - 102495/2 (α = CN~, Ν3~, benzhydroxythiazole [ΒΤΟΗ], imidazole, etc.), forms an unstable compound RCOa in solution. To measure the quantity of RCOa an excess of DNPNH( CH2 ) 2NH2 is added to the filtrate of the reaction mixture, and a stable compound RCOHN{CH2)2NHDNP is formed and measured easily as described. This allows the measurement of polymeric-bound active reagent and soluble RCOa.
While the invention will now be described in connection with certain preferred embodiments in the following examples so that aspects thereof may be more fully understood and appreciated, it is not intended to limit the invention to these particular embodiments. On the contrary, it is intended to cover all alternatives, modifications and equivalents as may be included within the scope of the invention as defined by the appended claims. Thus, the following examples which include preferred embodiments will serve to illustrate the practice of this invention, it being understood that the particulars shown are by way of example and for purposes of illustrative discussion of preferred embodiments of the present invention only and are presented in the cause of providing what is believed to be the most useful and readily understood description of formulation procedures as well as of the principles and conceptual aspects of the invention. - 15 - 1Q2495/2 Determination of Functional Groups on Solid Support Example 1 Equal aliquots (5 x 10"AM) of a solution of 2 , 4-dinitropheny1 amino ethylenediamine (DNP H( CH2 ) 2NH2 ) in CH2C12 were reacted with equal volumes 25 x 10~*M of a solution of the following compounds in CH2C12: 1. acetyl chloride 2. acetic anhydride 3. benzoyl chloride 4. tosyl chloride 5. trimethylsilyl chloride 6. oxalyl chloride 7. thionyl chloride To each reaction mixture a five-fold excess of triethyl amine, with respect to the chloride, was added to prevent the formation of the HC1 salts of the intended products (except for reaction mixture 2 , where it cannot be formed) . The formation of the expected products was confirmed by TLC analysis of a second series of equal reaction mixtures.
It was shown that in all cases 1-7 the UV-absarption measured at = 480A was the same as for the original reagent. This shows that the different acyl groups attached to DNPNH(CH2)2NH2 have no influence on the absorption spectrum of the chromophore, because of the ethylene bridging unit. It also shows that DNPNH(CH2)2NH2 is a sensitive reagent for quantifying various acylating reagents when used in excess and the excess is removed by flash chromatography after completion of the reaction. HC1 salts have a slightly different ε value because of an altered chromophore as - 16 - 102495/2 compared to that in 2,4 . dinitrophenyl amino ethylene diamine.
For separating the excess DNPNH( CHa ) 2NH2 reagent from the acylated product, a quite simple and effective procedure was found, using a separation column as follows: A column having a diameter of about 4 mm and length of 2-4 cm was filled with a mixture comprising 90% silica and 10% polymeric sulfuric acid ion exchange resin. The acylated reaction mixture was poured onto the column. The acylated DNPNH( CH2 ) 2NHa was eluted with 0.2 to 2 ml of solvent such as methylene chloride or ethyl acetate, optionally containing up to 30% methyl, ethyl or isopropyl alcohol. The excess DNPNH( CH2) aNH2 remained on the column. This separation method is good for detecting quantities as low as 1 mm.
Example 2 Equal aliquots (5.3 x 10""*M) of a " solution of DNP-NH(CHa)aCOOSu in DMSO/CH2Cl2 were reacted with equal aliquots (25 x 10~*M) of solutions of the following amines in DMS0/CH2C12: 1. ( C2HB ) 2NH ' 2. C6HSCH2NH2 3. HaNNHa 4. CeHsNH2 5. HS-CHa-CHaNHaHCl 6. H03S-CH2-CHa-NH2-HCl The DNP-NH(CH2)a COOSu was added in approximately two-fold excess. In reaction mixtures 5 and 6, excess triethyalinine was added. The reaction mixtures were heated (60°C) for one - 17 - 102495/2 hour. TLC tests (on a second equal set of reaction mixtures) showed the formation of new amide products. When the reaction mixtures were measured at =348OA, the UV-absorption of the products formed was similar to that of the original Q-B-f reagent. This shows that the ethylene bridging unit prevents any influence by the different amides on the DNPNH-chromophore group. Moreover, the experiment shows that DNP-NH(CH2)2COOSu can be used successfully as an analytical reagent for quantifying various amines , when applied in excess and the excess is removed after the completion of the reaction.
Example 3 Equal aliquots 'CI.4 x 10~3M) of a solution of dansyl ethylenediamine in CH2C12 were reacted with equal aliquots (7 x 10~3M) of a solution of the following compounds in CH2C12: 1. acetic anhydride 2. benzoyl chloride 3. tosyl chloride Compounds 2 and 3 were added in approximately five-fold excess. One equivalent triethyl amine with respect to tosyl. chloride was added as well.
The formation of the expected dansyl ethylene diamine derivatives was confirmed by a TLC check of a second set of similar reaction mixtures. When the reaction mixtures were diluted to an arbitraily chosen, but exactly known, volume, and measured fluorimetrically at »3400A, it was found that the fluorescence of the rection mixtures was equal to that of an equimolar dansyl ethylene amine solution. This implies that acyl groups connected to dansyl ethylene - 18 - 102495/2 diamine via the ethylene bridging unit do not influence the fluorescent proeprties of the dansyl group itself. Moreover, it shows that dansyl ethylene diamine, like DNPNH( CH2) 2NH2 , can serve as an analytical reagent for quantifying various acylating compounds, when reacted in excess and the excess is removed (by flash chromatography) after completion of the reaction.
Synthesis of Reagents Example 4 DNP-NH(CH2)2NH2 90.0 g of chloro-2,4-dinitrobenzene (0.44 M) was added portionwise to a cooled (0°C stirred) solution of 179 g ethylenediamine (200 ml, 2.98 M) in 400 ml methanol. The solid reactant gradually dissolved in the reaction mixture. After completion of the addition, the stirring continued for another 4 hours at 0°C.
Workup A precipitate formed and was identified as DNP-NH-CH2-CH2-NH-DNP side product. It was filtered off, washed repeatedly with water and dried. Yield: 2.0 g. To the filtrate was added ice water, and the resulting precipitate was filtered and repeatedly washed with cold water. The crude product was dried. Yield: 52.0 g (52%). A further purification was made by dissolving 10 g of the crude product in 400 ml boiling 1.0 NHC1. The solution was cooled and more cold water was added. It was then extracted with ethyl acetate until TLC showed that the last extraction was clean. The aqueous fraction was made alkaline with concentrated ammonia, cooled to 0eC and the resulting - 19 - 102495/2 precipitate filtered off . It was washed with water several times and dried. This treatment afforded 6.5 g very pure, yellow needles.
Example 5 DNP- H( CH2 ) aNHCOONH(CH2) 2NHDNP Part of the crude DNPNH(CH2) 2NH3 prepared was dissolved in methanol and a current of carbon dioxide was led through the clear solution. A yellow, spongy precipitate formed. After 20 minutes the influx of carbon dioxide was stopped, and the precipitate filtered off, washed with cold methanol and dried.
Example 6 DNP~NH(CH2)eNH2 10 g fluorodinitrobenzene (FDNB, 53.5 mM) was added portionwise to a stirred solution of 17.4 g hexamethylene-diamine (150 mM) in 500 ml chloroform. The FDNB reacted quickly, and yellow precipitate was immediately formed.
Workup After stirring for 4 hours, the precipitate formed was filtered off and the resulting filtrate was washed with 1.5 1 of diethyl ether. Yield: 10.1 g (66% ) tic on silica gel in CH2C12. Rf « 0.77. - 20 - 102495/2 Example 7 23.0 g piperazine (267 mM) and 10.0 g chlorodinitrobenzene (CDNB) (49.4 mM) were mixed (with stirring) in 150 ml of methanol at room temperature . The reactants went into solution within 10 minutes, but then a new product started to precipitate. The reaction mixture was stirred for a total of 4 hours.
Workup The precipitate was filtered off, the filtrate evaporated to half its original volume. About 240 ml CH2C12 was added, whereupon the organic layer was washed 8 times with water. The presence of piperazine in the washings was checked with ninhydrine. Then the organic layer was extracted 6 times with HC1 4 N solution; it became almost colorless. The aqueous layer in turn was made alkaline with a 25% Ni OH solution and the product was washed back with CH2C12. The CH2C12 layer was dried over MgSO*, filtered and evaporated till dry. The resulting orange syrup crystallized totally in about 3 weeks. Yield: 24.2 g. - 21 - 102495/2 Example 8 DNPNH( CH2) 3OH 10.1 g chlorodinitrobenzene (CDNB, 50 mM) was added at once to a cooled stirred (0eC) solution of 11.3 g 3-aminopropanol (1.4 ml, 150 mM) in 50 ml of methanol. The CDNB went very slowly into solution; there was still some left after 1 hour. After 2 hours, the reaction mixture was brought to a boil and kept at a reflux for a few minutes. After cooling off, a yellow precipitate formed, which was filtered off, washed with a little cold methanol and sucked dry. This afforded 11.5 g of crude material (95%). Further purification could be obtained by recrystallization from hot methanol.
Example 9 DNP-NH(CH2)2COOH This synthesis was conducted in three steps: a) HCl-H2N{CH3)2COOCH3: 17.8 g B-alanine (H2N(CH2) 2COOH, 200 mM) was partly dissolved in 200 ml dry methanol and cooled to -20°C. 20 ml S0C12 (275 mM) was added in 40 minutes, during which the cooling remained in effect. The acid went slowly into solution. Shortly after completion of the addition, the cooling was removed, allowing the reaction mixture to reach room temperature. It was stirred for 16 additional hours to remove S02. 102495/2 Workup The solution was evaporated to dryness, affording white hygroscopic crystals. They were dried in vacuo. Yield: 26.8 g white crystals (96%), mp: 99-101eC. b) DNP-NH( CH2) 2COOCH3 A solution of 20.26 g CD B (100 mM) in 50 ml THF was added dropwise to a mixture of 13.96 g HC1 H2N(CH2)2COOCH3 (100 mM) and 28.0 ml triethylamine (200 mM) in 300 ml THF at room temperature. The addition lasted 2 hours and 15 minutes, while stirring vigorously. Thereafter, the stirring continued for 40 hours.
Workup The reaction mixture was filtered off , and the resulting filtrate evaporated to dryness. The yield was 23.9 g (89%), • it was an orange yellow amorphous powder. c) DNPNH(CH2)2COOH 15.0 g (55.7 mM) of DNPNH(CH2)aCOOCH3 was dissolved (with periodical warming to about 40eC of the reaction mixture) in 200 ml of a 0.5 N NaOH solution (1.8 eq NaOH) . The starting material dissolved slowly; the mixture was stirred for a total of 24 hours.
Workup The solution was filtered after warming again to 50°C. The pH was brought to about 2 with concentrated hydrochloric acid. The flaky precipitate formed was filtered off and - 23 - 102495/2 washed with ice water, and dried in vacuo. Yield: 11.13 g of a yellow, amorphous powder (78%) .
Example 10 DNP H( CH2) 2COONSuc 9.29 g dicyclohexyl' carbodiimide (DCC, 45 mM; 1.5 eq) was added at once to a cooled (0eC) solution of 7.65 g DNPNH(CH2)2COOH (30 mM) and 3.45 g N-hydroxy succinimide (3 mM) in 150 ml dry THF. The cooling remained in effect for about 2 hours, then the reaction mixture was allowed to slowly reach room temperature. Stirring continued for 72 hours .
Workup The precipitate formed was filtered off and the filtrate evaporated to dryness. The resulting residue was recrystallized from hot iso-propanol. This afforded 6.32 g orange-brown crystals (57%).
Example 11 DNP-NH( CH2) 2CONHNH2 A solution of 6.4 ml hydrazine (200 mM) in 35 ml iso-propanol was dropped during 10 minutes into a solution of 10.76 g DNPNH( CH2) 2COOCH3 (40 mM) in 135 ml dry THF at room temperature. After completion of the addition, the mixture was brought to a boil and refluxed for 3 hours. - 24 - 102495/2 Workup The reaction mixture was filtered off over a paper filter and the filtrate left for crystallization overnight (first fraction) . The concentrated mother liquor afforded a second fraction. Yield: 6.75 g (63%), fine orange-brown needles.
Example 12 DNPNH( CHa) 2NHCOCH3 A solution of 0.785 g of acetylchloride (0.71 ml, 10 mM) in 25 g dry THF was dropped slowly into a stirred, cooled (0eC) solution of 2.26 g DNPNH( CH2 ) 2NH2 (10 mM) and 1.6 ml triethyl amine (12 mM) in 50 ml dry THF. After completion of the addition, the cooling remained in effect for 30 minutes, and it was then removed to allow the reaction mixture to reach room temperature.
Workup The reaction mixture (rm) was evaporated to dryness. The residue was washed with small volumes of cold water over a glass filter. The resulting filter cake was dried in vacuo. Yield: 1.9 g.
Example 13 DNPNH( CH2) 2NHCOCeH5 A solution of 1.40 g benzoylchloride (1.16 ml, 10 mM) in 25 ml dry THF was added dropwise to a stirred, cooled (0eC) solution of 2.26 g DNPNH(CHa)NH2 (10 TOM) and 1.67 ml triethyl amine (12 mM) in 50 ml dry THF. After' the end of the addition, the cooling remained in effect for 1 hour and 102495/2 - 25 - it was then taken away to allow the reaction mixture to slowly reach room temperature.
Workup The workup was done as in Examples 9 and 11. Yield: 2.2 g.
Example 14 DNPNH( CH2) 2NHS02C6H4CH3 A solution of 1.92 g toluenesulphonyl chloride (tosyl chloride, 10 mM) in 30 ml dry THF was added dropwise to a stirred, cooled (0eC) solution of 2.26 g DNPHH( CH2 ) 2NH2 (10 mM) and 1.67 ml triethyl amine (12 mM) in 50 ml dry THF.
Cooling was maintained for 2 hours, then it was removed. Stirring at room temperature continued for 20 hours.
Workup The workup was done as in Examples 9 and 10. Yield: 2.5 g.
Example 15 DNPNH(CH2)2CeHs 2.03 g chloro-2 , 4-dinitrobenzene was added portionwise to a stirred, cooled (0eC) solution of 6.1 g phenethylamine (6.3 ml, 50 mM) in 100 ml methanol. After all CNDB had reacted away (4 hours) the ice bath was removed. 102495/2 - 26 - Workup To the reaction was added ice water, and the pH made acidic with concentrated hydrochloric acid. The precipitate formed was filtered off and wahsed several times with cold water over a glass filter. The filter cake was dried in vacuo. Yield: 2.5 g.
Example 16 DNPNHCH2-CH=CH2 3.05 g chloro-2, 4-dinitrobenzene (15 mM) was added portionwise to a cold, stirred solution of 8.57 g allylamine (11.25 ml, 150 mM) in 100 ml of methanol. Stiring continued at 0eC for 4 hours and then for 18 additional hours at room temperature .
• Worku The addition of ice water to the reaction mixture caused a precipitate. The pH was made acidic with concentrated hydrochloric acid, whereupon the precipitate was filtered off and washed with several volumes of cold water over a glass filter. The product obtained was dried in vacuo. Yield: 4.5 g.
Example 17 DNPNH-CH2CH(OMe)3 and DNPNHCH2CH0 2.03 g chloro-2, -dinitrbbenzene (10 mM) was added portionwise to a cold (O'C), stirred solution of 5.26 g aminoacetaldehyde dimethyl acetal (5.45 ml; 50 M) in 50 ml methanol. The stirring continued for 4 hours at 0°c and 20 - 27 - 102495/2 additional hours at room temperature. Then the solution was made acidic with 0.5 N HCl solution, and more cold water was added.
Workup The precipitate . formed was filtered off and washed repeatedly with cold water, until the washings had a netural pH. The produce was then dried in vacuo. Yield: 2.4 g.
Example 18 FMOC-L-Amino Acid-DNP Derivatives General Procedure: The FMOC-L-amino acid DNP derivatives were all prepared from the N-protected FMOC-L-amino acids and DNPNH( CH2) 2NH2 or DNPNH( CH2) aNHCOONH( CH2) aNHDNP, which can be regarded as a "DNP dimer" and thus as 2 euqivalents of DNP-NH(CH2)2NH2 by the DCC coupling method. l-Hydroxybenzotriazole (BTOH) was used as a catalyst and DMF as a solvent. A typical synthesis of this type was carried out as follows: 5 mM of the FMOC-L-amino acid was dissolved together with 5 mM 1-hydroxybenzotriazole in 15-50 g of dry DMF, whereupon the solution was cooled to between -10 to 15eC. DCC was added in excess, for most reactions 7.5 to 9.0 mM (50-80% excess). Cooling remained in effect for about 1 hour, and then the mixtures were allowed to. react overnight. The workup was usually done by first adding 1-2 ml 1 N HCl solution to the reaction mixture for destroying excess of DCC, except for FMOC-L-amino acids protected by acid sensitive groups, such as S-acetamidomethyl-L-cystein, TRT-L-histidine, MTR-L-Arginine , TRT-L-cysteine , - 28 - 102495/2 PMC-L-Arginine , S-t-butyl-L-cysteine . Then the DCU formed was filtered off over a glass filter and it was washed with DMF until all yellow products had been washed out. The filtrate was made alkaline with a 5% NaHC03 solution and ice water added. The precipitate was washed repeatedly with cold water (usually over a Buchner filter) and dried in vacuo. In most cases the products obtained were already clean, but furthre purification could be achieved by recrystallisation from various solvents of mixtures thereof. The yields of the compounds ranged from 36-93%. The synthetic data of 20 different FMOC-L-amino acid DNP derivatives are compiled in Table 3.
TABLB 3 F value in Ethylacetate : Recrysta Compound Yield (%) Ethanol = 0:1 mp (0°C) mi FMOC-L-Valine -DNP 48 0.64 214.5-216 FMOC-L-Phenylalanine-DNP · 81 0.62 151-154 CH3 OH : DM FMOC-L- Leucine-DNP 36 0.63 172-174 CH3OH:DM FMOC-L-Glycine-DNP 70 0.44 148-151 CH3OH:CH FMOC-L- Isoleucine -DNP 40 0.62 212-213.5 CH3 OH : DM FMOC- e-BOC-L- Lysine -DNP 80 0.57 169-171 CH3 OH : DM FMOC-L-Asparaginyl-DNP 91 0.50 191-193 CH3 OH : DM FMOC-L-Methionine-DNP 82 0.60 194-197 CH3 : DMF FMOC-L-Aspartic acid 71 0.64 133-136 CH3 OH : DM FMOC-L-Proline-DNP 78 0.37 142-144 CH3 OH : DM FMOC-L- ryptophan -DNP 93 0.61 178.5-180 CH3 OH : DM FMOC-L-Glutamic acid- 77 0.63 187.5 -1B9 CH3 OH : DM -5-t-butylester-DNP FMOC-O-t-butyl-L-Serine-DNP 81 0.63 151-153 CH3 OH : DM FMOC-O-t-butyl-L-Tyrosine-DNP 71 0.64 182-184 CH3 OH : DM FMOC-S-Acetamidomethyl- 60 0.68 180-183 CH3 OH : DM -L-Cysteine FMOC-trityl-im-L-Histidine-DNP 0.40 99-105 CH3OH:DM FMOC-Mtr-L-Arginine-DNP 70 0.68 122-126 CH3 OH : DM FMOC-trityl-S-L-Cysteine-DNP 81 0.47 119-122 Hexane : FMOC-Pmc-L-Arginine-DNP 42 0.47 CH3 OH : DM FMOC-S-t-butyl-L-Cysteine- 85 0.68 165-167 EtOAc:Et -DNP - 30 - 102495/2 Example 19 Dansyl-S02NH( CH2 ) 2NH2 A solution of 134.3 mg dansyl chloride (0.5 mM) in 3 ml dry CH2C12 was slowly dropped (with a Pasteur pivette) into a cooled (0eC) 0.6 g ethylene diamine (0.67 ml, 10 mM) in 5 ml dry Ca2Cl2. During the addition and therefafter, the solution of dansyl chloride and the reaction mixture was shielded from light. The reaction mixture was stirred for 45 minutes at 0°C, and another 15 minutes after cooling was taken away.
Workup The reaction mixture was evaporated to dryness in vacuo, then taken up in 50 ml CH2C12. The solution was washed four times with 0.6 N Rose. The organic layer was dried over MgS04. This was filtered off and the solvent was removed. Yield: 40 mg (27%), yellow oil.
Example 20 / \ Dansyl-S02- NH A solution of 135 mg dansyl chloride (0.5 mM) in 3 ml dry CH2C12 was dropped slowly into an ice cooled solution of 0.61 g piperazine (10 mM) in 15 ml dry CH2C12. The dansyl chloride solution was shielded from light during the - 31 - 102495/2 addition and ensuing reaction. The reaction mixture was stirred for 2 hours at 0eC, and overnight at room temperature.
Workup The reaction mixture -was diluted with CHaCla to about 60 ml, washed with 0.6 N KOH (4 x 10 ml), dried over MgSO*, filtered, and evaporated to dryness. Yield: 150 mg (94%), yellowish, fluorescent oil which crystallized upon standing.
Example 21 It is possible, by using the reagents of this invention, to learn about the reaction path and side reactions of given reactions. For example, thyroxin peptides were reacted with t-butyloxycarbonyl anhydride ( (t-BOC) aO) part of the thyroxin was blocked with the t-BOC group to form the t-BOC-0 phenyl derivative. When subjected to TLC on D P NH(CHa)aNHa impregnated plates, the product and t-BOC DNPNH derivative was detected. By adding ammonia the t-BOC group was removed completely from the thyroxin and the pure thyroxin recovered.
This TLC with reagent impregnated along the base line can be used for (a) qualitative or quantitative detection or determination of functional groups, and (b) determining the reactivity of certain functional groups. - 22 - 102495/2 Example 22 The Use of DNPNH( CHa) 3OH in Sugar Transformations 1-Halo activated glucose tetra acetate, when reacted with DNPNH(CH2) 3OH, yielded B- and a-glycosidic products in ratios of 96.4% B to 3.6% a, based on UV determination at 248 n . When fi and a products were separated and analyzed, their max and EM were identical, as expected.
The B-glucoside tetra acetate of glucose was hydrolyzed with a variety of hydroxylated or alkoxylated reagents, such as OH*, 0-Me, O*prop. In all cases hydrolysis takes place, but surprisingly enough, the amounts of tri- and di-acetate derivatives were found in small quantities, but monoacetate was already observed, even at the beginning of the reaction.
The use of the reagents of the present invention enabled the prediction of the existence of self-catalytic reactions in the sugar acetate molecule itself.
Example 23 Triglyceride Analysis Upon refluxing a fraction of 1 mg of a tri-glyceride with DNPNH( CH2) 3NHNH2 in toluene, cleavage of the glyceride esters occurred and the corresponding hydrazxdes were formed and analyzed quantitatively using H.P.L.C. separation. 102495/2 - 33 - Example 24 The Use of Chiral Amines in the Determination of Chiral Amino Acids When excess of HC1 salt of -L-Val CONH(CHa) 2NHD P HCl was coupled to CbzDL-PheC02H using conventional peptide chemistry (DCC + OH Sue), the two diasterearners L-L and D-L of Cbz-Phe-Val-CO-NH(CH)2DNP were separated by H.P.L.C., and their quantities were 1:1.
It will be evident to those skilled in the art that the invention is not limited to the details of the foregoing illustrative examples and that the present invention may be embodied in other specific forms without departing from the essential attributes thereof, and it is therefore desired that the present embodiments and example be considered in all respects as illustrative and not restrictive, reference being made to the appended claims, rather than to the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.

Claims (7)

34 102,495/ 4 WHAT IS CLAIMED IS:
1. A method for the quantitative determination of an organic compound designated herein by A, said compound A having quantitatively reactable functional groups, said method consisting essentially of: quantitatively reacting compound A, in a sample containing compound A, with a reagent comprising a compound of the general formula I: Q-B-f (I) wherein: Q stands for an organic moiety which can be measured quantitatively, visually by color, spectroscopically, or fluorometrically; B represents a non-reactive organic bridging unit linking the moiety Q to a reactive functional group/ said bridging unit including non-reactive CH2- CH2 linkages and being of sufficient length or size to prevent any possible interaction of Q that might alter its spectroscopic properties even upon derivatization; and /is a reactive group which can react with a compound to form covalently bonded derivatives, according to the reaction n(Q-B-f) + A→Q-B-f-A + (n-\)Q-B-f wherein: Q, B and / are as defined above, and n equals 1 or more; separating any excess reagent (n - \)Q -B-f; and 35 102,495/ 3 measuring the absorption of the reaction product Q- B - f - A , wherein the molar absorbtivity ε of said reaction product Q- B - f - A and said reagent Q - B - f are substantially constant and wherein the molar concentration of the reaction product Q- B - f - A is equal to the measured absorption divided by ε, whereby division of said measured absorption by ε provides the value of the molar concentration of the reaction product, thus eliminating the requirement for comparing the result with an external standard.
2. A method for the quantitative determination of an organic compound as claimed in claim 1, wherein said molar absorption is measured spectroscopically.
3. A method for the quantitative determination of an organic compound as claimed in claim 1, wherein said molar absorptiion is measured visually.
4. A method as claimed in claim 1, wherein A is an amino acid.
5. The method as claimed in claim 4 for determining mixtures of amino acids, comprising: reacting said mixture of amino acids with a reagent as claimed in claim 1 ; separating the amino acid reaction products; and measuring said products spectroscopically with said reagent serving as a universal standard for all the amino acid reaction products. 36 102,495/4
6. A method according to claim 1 for the quantitative determination of the quantity of each of a plurality of components of a mixture, each of said components being designated herein as Ab A2 . . .Am, m being a whole number and each component carrying the same quantitatively reactable functional group , said method comprising: quantitatively reacting said mixture of components A1? A2 . . .Am with an excess of a reagent comprising a compound of the general formula I: Q - B - f (I) wherein: Q stands for an organic moiety which can be measured quantitatively, visually by color, spectroscopically, or fluorometrically; B represents a non-reactive organic bridging unit linking the moiety Q to a reactive functional group said bridging unit including non-reactive CH - C¾ linkages and being of sufficient length or size to prevent any possible interaction of Q that might alter its spectroscopic properties even upon derivatization; and is a reactive group which can react with a compound to form covaiently bonded derivatives; according to the reaction n(Q - B - f) + A → Q- B - f - A + (n - \)Q - B - f wherein: n is greater than the sum of A\, A2 . . Am, and separating said reaction products by chromotographic methods known per se. 37 102,495/3
7. A method for studying the reaction rates of a given functional group present in different types of compounds, comprising: reacting a known mixture of compounds having the same functional group with less than stoichiometric amounts of a reagent comprising a compound of the general formula I: Q - B - f (I) wherein: Q stands for an organic moiety which can be measured fluorometrically; B represents a non-reactive organic bridging unit linking the moiety Q to a reactive functional group said bridging unit including non-reactive CH2- CH2 linkages and being of sufficient length or size to prevent any possible interaction of Q that might alter its fluorometric properties even upon derivatization; and is a reactive group which can react with a compound to form covalently bonded derivatives; separating the derivative reaction products; and measuring the fluorescence of each derivative product using the reagent as a universal standard, wherein the molar absorptivity ε of each said derivative product is substantially the same, whereby division of said measured fluorescence of each said derivative product by the same value ε provides the value of the molar concentration of the derivative product and the reaction rates of the functional groups present in each of the different compounds are proportional to the molar concentration of each of the derivative products. for the Applicant: WOLFF, BREGMAN AND GOLLER
IL102495A 1992-07-14 1992-07-14 Universal standard reagents, method of preparing same and use thereof IL102495A (en)

Priority Applications (6)

Application Number Priority Date Filing Date Title
IL102495A IL102495A (en) 1992-07-14 1992-07-14 Universal standard reagents, method of preparing same and use thereof
PCT/US1993/006980 WO1994001771A1 (en) 1992-07-14 1993-07-14 Universal standard reagents, method of preparing same and use thereof
JP6503596A JPH08505220A (en) 1992-07-14 1993-07-14 Universal standard reagent and its preparation and use
US08/362,519 US5576216A (en) 1992-07-14 1993-07-14 Universal standard reagents, method of preparing same and use thereof
AU47844/93A AU4784493A (en) 1992-07-14 1993-07-14 Universal standard reagents, method of preparing same and use thereof
EP93918367A EP0650595A4 (en) 1992-07-14 1993-07-14 Universal standard reagents, method of preparing same and use thereof.

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
IL102495A IL102495A (en) 1992-07-14 1992-07-14 Universal standard reagents, method of preparing same and use thereof

Publications (2)

Publication Number Publication Date
IL102495A0 IL102495A0 (en) 1993-01-14
IL102495A true IL102495A (en) 1998-06-15

Family

ID=11063819

Family Applications (1)

Application Number Title Priority Date Filing Date
IL102495A IL102495A (en) 1992-07-14 1992-07-14 Universal standard reagents, method of preparing same and use thereof

Country Status (5)

Country Link
EP (1) EP0650595A4 (en)
JP (1) JPH08505220A (en)
AU (1) AU4784493A (en)
IL (1) IL102495A (en)
WO (1) WO1994001771A1 (en)

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996018899A1 (en) * 1994-12-16 1996-06-20 President And Fellows Of Harvard College Method for detecting a phosphorylated amino acid in an intact protein
GB9903548D0 (en) * 1999-02-16 1999-04-07 Novartis Ag Organic compounds
WO2005000795A2 (en) * 2003-06-10 2005-01-06 Smithkline Beecham Corporation Aniline derivatived androgen-, glucocorticoid-, mineralcorticoid- and progesterone- receptor modulators

Family Cites Families (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US2434564A (en) * 1943-06-08 1948-01-13 Rohm & Haas Substituted nitro aromatic amines as insecticides
US2636032A (en) * 1949-04-12 1953-04-21 Abbott Lab N, n'-disubstituted piperazines and process of preparing same
US3978045A (en) * 1973-08-13 1976-08-31 Mitsubishi Chemical Industries Ltd. N2 -dansyl-L-arginine derivatives, and the pharmaceutically acceptable acid addition salts thereof
US4148791A (en) * 1975-07-04 1979-04-10 Sandoz Ltd. Phenyl-azo-phenyl compounds having at least one hydroxy or oxo substituent on a sulfinyl or sulfamoyl group in the para position of the diazo component radical
DE3266967D1 (en) * 1982-01-05 1985-11-21 Int Inst Cellular Molecul Path Method of immunoassay
JPH0634013B2 (en) * 1985-03-25 1994-05-02 栄治 石川 Highly sensitive immunoassay
JPH0792455B2 (en) * 1986-07-04 1995-10-09 株式会社ミドリ十字 Aqueous solvent for immunological test
US4914040A (en) * 1988-03-03 1990-04-03 Boehringer Mannheim Gmbh Reagent and method for determination of a polyvalent substance using an immunoaggregate

Also Published As

Publication number Publication date
EP0650595A1 (en) 1995-05-03
AU4784493A (en) 1994-01-31
IL102495A0 (en) 1993-01-14
EP0650595A4 (en) 1996-08-21
JPH08505220A (en) 1996-06-04
WO1994001771A1 (en) 1994-01-20

Similar Documents

Publication Publication Date Title
Kim et al. “BOP” as a reagent for mild and efficient preparation of esters
Bernatowicz et al. 1H-Pyrazole-1-carboxamidine hydrochloride an attractive reagent for guanylation of amines and its application to peptide synthesis
EP2688867B1 (en) Heterobifunctional esters for use in labeling target molecules
EP2252584B1 (en) Compounds and methods for rapid labeling of n-glycans
US9389183B2 (en) Fluorogenic hydrazine-substituted compounds
US5304645A (en) Resorufin derivatives
CA2660300C (en) Sugar chain-capturing substance and use thereof
AU2007221425A1 (en) Mass tags for quantitative analyses
US5576216A (en) Universal standard reagents, method of preparing same and use thereof
Nimura et al. Activated carbamate reagent as derivatizing agent for amino compounds in high-performance liquid chromatography
IL102495A (en) Universal standard reagents, method of preparing same and use thereof
Haugland Covalent fluorescent probes
US10793518B2 (en) Hydrazinyl and aminooxy compounds and their methods of use
EP2157088A1 (en) Indicyanine dyes and the derivatives thereof for analysing biological micromolecules
Games A novel acridone derivative for the fluorescence tagging and mass spectrometric sequencing of peptides
US9448212B2 (en) Method for the detection of sulphur containing phosphorylating agents
US5097040A (en) Benzotriazole derivatives and reagents for determining carboxylic acids thereof
Byun et al. Preparation of polymer-bound pyrazolone active esters for combinatorial chemistry
JP2012528119A (en) Cross-linking reagent for intermolecular interaction analysis
US5151517A (en) Derivatives of tetrahydro-2,3,6,7,1h,5h,11h-(1)benzopyrano(6,7,8,i,j)quinolizinone-11 usable as markers for organic compounds, particularly biological compounds with a view to their detection by chemiluminescence or fluorescence
Smith et al. Molecular complex evaluation. A simultaneous assay of binding using substrate mixtures
US4647671A (en) Cleavable cross-linking reagent
Soundararajan et al. Synthesis and binding of new polyfluorinated aryl azides to. alpha.-chymotrypsin. New reagents for photoaffinity labeling
Cory et al. [9] Design of exo affinity labeling reagents
Zubair et al. Saccharin

Legal Events

Date Code Title Description
FF Patent granted
KB Patent renewed
KB Patent renewed
MM9K Patent not in force due to non-payment of renewal fees