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EP0650595A4 - Reactifs standard universels, procede de preparation et utilisation de ces reactifs. - Google Patents

Reactifs standard universels, procede de preparation et utilisation de ces reactifs.

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Publication number
EP0650595A4
EP0650595A4 EP93918367A EP93918367A EP0650595A4 EP 0650595 A4 EP0650595 A4 EP 0650595A4 EP 93918367 A EP93918367 A EP 93918367A EP 93918367 A EP93918367 A EP 93918367A EP 0650595 A4 EP0650595 A4 EP 0650595A4
Authority
EP
European Patent Office
Prior art keywords
reagent
compound
compounds
reactive
reaction
Prior art date
Legal status (The legal status 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 status listed.)
Withdrawn
Application number
EP93918367A
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German (de)
English (en)
Other versions
EP0650595A1 (fr
Inventor
Avraham Patchornik
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
PATCHORNIK ZIPORA
Original Assignee
PATCHORNIK ZIPORA
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Publication of EP0650595A1 publication Critical patent/EP0650595A1/fr
Publication of EP0650595A4 publication Critical patent/EP0650595A4/fr
Withdrawn legal-status Critical Current

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    • 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

Definitions

  • 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.
  • 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.
  • 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.
  • Another object of the present invention is to provide analytical methods for measuring compounds quantitatively, with reagents whose molar ⁇ 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.
  • a further object of the invention is to provide some new chemical compounds suitable for use as universal standard colorimetric or spectrometric analytical reagents.
  • 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 is different for each amino acid measured.
  • amino acids can be analysed quantitatively fluorometrically by reacting them with a fluorescent reagent such as dansyl chloride, Dabsyl chloride or O-pthalaldehyde to form the fluorescent derivative.
  • the actual quantification again requires a specific external standard for each amino acid.
  • the present invention provides a new universal standard chemical reagent for quantitative visual and spectrometric analysis of compounds having reactive functional groups, including mixtures and homologs of said compounds, said 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, spectro ⁇ scopically or fluorometrically; B represents a non-reactive organic bridging unit linking the moiety Q to a reactive functional group f, said bridging unit being of sufficient length or size to prevent any possible interaction of Q that might alter its spectroscopic properties even upon derivatization; and f is a reactive group which can react with a compound to form covalently bonded derivatives.
  • said bridging unit B is of sufficient length and/or size to prevent any possible spacial interaction of Q that might alter its spectroscopic properties even upon derivatization. This is particularly relevant for fluoroscopic analysis where interference is possible even from a distance.
  • f is any reactive group which can react with a compound to form covalently bonded derivatives having the formula Q-B-f-A.
  • Such a reaction can be represented as follows:
  • n(Q-B-f) + A > Q-B-f-A + (n-l)Q-B-f
  • A represents any analysable compound containing a reactive functional group that will form a covalent bond with the reactive group f.
  • the invention also provides a method for the quantitative determination of an organic compound designated herein by A, said compound A having a reactive functional group, said method comprising reacting said compound A with the reagent of the present invention, according to the reaction: n(Q-B-f ) + A > Q-B-f-A + ( n-l )Q-B-f
  • Q, B, and f are as defined hereinabove and n equals 1 or more; separating any excess reagent (n-1)Q-B-f; and measuring the molar absorption ⁇ of the reaction product Q-B-f-A which will be substantially the same as that of the reagent Q-B-f, thus eliminating the requirement for comparing the measured result with an external standard.
  • said molar absorption is measured spectroscopically or visually.
  • Said method is especially applicable for the determination of amino acids, and the invention also provides a method for determining mixtures of amino acids, comprising reacting said mixture of amino acids with a reagent according to the present invention; 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.
  • a method for studying reaction rates of functional groups 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 of the present invention; separating the derivative reaction products; spectroscopically or fluorometrically measuring the molar concentration of each derivative using the reagent as a universal standard, the reaction rates of the functional groups present in each of the different compounds being proportional to the molar concentration of each of the compounds in the reaction products.
  • said reaction products are preferably chromotographically separated.
  • the compounds to be analysed 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 -SO 3 H 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 ⁇ irrespective of which compound A is being determined if the reaction is quantitative.
  • n and are as defined above.
  • DNP-NH(CH 2 ) 2 NHCOR an unknown acylating (RCO"* " ) compound.
  • RCO acylating
  • 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 "1" , CH 3 CO-, C 6 H 5 CO-, t-BuOCO-, ArS0 2 -, etc.
  • the reagents of this invention are also useful for measuring reactive gases.
  • carbon dioxide can be measured in a very convenient way by reacting it with a toluene solution of DNP-NH(CH 2 ) 2 NH 2 , as follows:
  • 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.
  • the reactive polymer 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.
  • ⁇ is the linking group between the insoluble polymer matrix(P)and the group A and is covalently bound to (g) and stable to any cleavage during the chemical manipulation.
  • This polymer when reacted with excess of a reagent Q-B-f, forms reaction - 13 -
  • the amount of reactive RCO " * ⁇ groups bound to a polymer can be determined as shown by the equation:
  • n is a whole integer.
  • the reactive polymer may be of the kind represented by (p) -A, where A is covalently bound to the polymeric matrix.
  • excess reagent Q-B-f is used to react with the polymer to give a high yield of reaction to form(p) -A-f-B-Q and the excess reagent is washed out.
  • 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 (-COO-), amide (-CONH-), carbamide (-OCONH-) and S-S bonds are quite often used.
  • (g)- ⁇ -A comprises an active ester group
  • To measure the quantity of RCO ⁇ an excess of DNPNH(CH 2 ) 2 NH 2 is added to the filtrate of the reaction mixture, and a stable compound RCOHN(CH 2 ) 2 NHDNP is formed and measured easily as described. This allows the measurement of polymeric-bound active reagent and soluble RCO ⁇ .
  • 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(CH 2 ) 2 NH 2 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(CH 2 ) 2 NH 2 remained on the column. This separation method is good for detecting quantities as low as 1 mm.
  • Equal aliquots (5.3 x 10 ⁇ *M) of a solution of DNP-NH(CH 2 ) 2 COOSu in DMS0/CH 2 C1 2 were reacted with equal aliquots (25 x 10 ⁇ *M) of solutions of the following amines in DMS0/CH 2 C1 2 :
  • the DNP-NH(CH 2 ) 2 COOSu was added in approximately two-fold excess. In reaction mixtures 5 and 6, excess triethyalmine was added. The reaction mixtures were heated (60°C) for one 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 «3480A, 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(CH 2 ) 2 COOSu 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.
  • dansyl ethylene diamine like DNPNH(CH 2 ) 2 NH 2 , 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.
  • 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.
  • the FMOC-L-amino acid DNP derivatives were all prepared from the N-protected FMOC-L-amino acids and DNPNH(CH 2 ) 2 NH 2 or DNPHH(CH 2 ) 2 NHCOONH(CH 2 ) 2 NHDNP, which can be regarded as a "DNP dimer” and thus as 2 euqivalents of DNP-NH(CH 2 ) 2 NH 2 by the DCC coupling method.
  • 1-H ⁇ drox ⁇ benzotriazole (BTOH) was used as a catalyst and DMF as a solvent.
  • a typical synthesis of this type was carried out as follows:
  • 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, PMC-L-Arginine, S-t-butyl-L-c ⁇ steine. 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% NaHC0 3 solution and ice water added.
  • reaction mixture was diluted with CH 2 C1 2 to about 60 ml, washed with 0.6 N KOH (4 x 10 ml), dried over MgS0 4 , filtered, and evaporated to dryness. Yield: 150 mg (94%), yellowish, fluorescent oil which crystallized upon standing.
  • thyroxin peptides were reacted with t-butyloxycarbonyl anhydride ((t-BOC) 2 0); part of the thyroxin was blocked with the t-BOC group to form the t-BOC-0 phenyl derivative.
  • t-BOC t-butyloxycarbonyl anhydride
  • the product and t-BOC DNPNH derivative was detected.
  • 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.
  • the B-glucoside tetra acetate of glucose was hydrolyzed with a variety of hydroxylated or alkoxylated reagents, such as OH e , O e Me, O e 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.

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Abstract

L'invention se rapporte à un réactif chimique standard universel servant à l'analyse quantitative par moyen visuel et par spectrométrie de composés contenant des groupes fonctionnels réactifs, y compris des mélanges et des homologues des ces composés. Le réactif chimique decrit comprend un composé représenté par la formule générale: Q-B-f, dans laquelle: Q représente une fraction organique qui peut être mesurée quantitativement, par un moyen visuel grâce à la couleur, par spectroscopie ou par fluorométrie; B représente une unité organique non réactive à fonction de passerelle, qui relie la fraction Q à un groupe fonctionnel réactif f, cette unité à fonction de passerelle ayant une longueur ou une grandeur suffisante pour empêcher toute interaction éventuelle de l'élément Q, qui pourrait altérer ses propriétés spectroscopiques même en cas de dérivation; et f représente un groupe réactif qui peut réagir avec un composé pour former des dérivés liés par covalence.
EP93918367A 1992-07-14 1993-07-14 Reactifs standard universels, procede de preparation et utilisation de ces reactifs. Withdrawn EP0650595A4 (fr)

Applications Claiming Priority (3)

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
IL10249592 1992-07-14
PCT/US1993/006980 WO1994001771A1 (fr) 1992-07-14 1993-07-14 Reactifs standard universels, procede de preparation et utilisation de ces reactifs

Publications (2)

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EP0650595A1 EP0650595A1 (fr) 1995-05-03
EP0650595A4 true EP0650595A4 (fr) 1996-08-21

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EP93918367A Withdrawn EP0650595A4 (fr) 1992-07-14 1993-07-14 Reactifs standard universels, procede de preparation et utilisation de ces reactifs.

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EP (1) EP0650595A4 (fr)
JP (1) JPH08505220A (fr)
AU (1) AU4784493A (fr)
IL (1) IL102495A (fr)
WO (1) WO1994001771A1 (fr)

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WO1996018899A1 (fr) * 1994-12-16 1996-06-20 President And Fellows Of Harvard College Procede de detection d'un acide amine phosphoryle dans une proteine intacte
GB9903548D0 (en) * 1999-02-16 1999-04-07 Novartis Ag Organic compounds
WO2005000795A2 (fr) * 2003-06-10 2005-01-06 Smithkline Beecham Corporation Composes chimiques
CN119320571A (zh) * 2024-10-15 2025-01-17 河南晶鑫生物科技有限公司 一种造纸用环保型染料及其制备方法

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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 (ja) * 1985-03-25 1994-05-02 栄治 石川 高感度免疫測定法
JPH0792455B2 (ja) * 1986-07-04 1995-10-09 株式会社ミドリ十字 免疫学的試験用水性溶媒
US4914040A (en) * 1988-03-03 1990-04-03 Boehringer Mannheim Gmbh Reagent and method for determination of a polyvalent substance using an immunoaggregate

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Also Published As

Publication number Publication date
AU4784493A (en) 1994-01-31
JPH08505220A (ja) 1996-06-04
WO1994001771A1 (fr) 1994-01-20
IL102495A0 (en) 1993-01-14
IL102495A (en) 1998-06-15
EP0650595A1 (fr) 1995-05-03

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