EP1144994A2 - Verfahren zur markierung von an festträger gebundenen verbindungen - Google Patents

Verfahren zur markierung von an festträger gebundenen verbindungen

Info

Publication number
EP1144994A2
EP1144994A2 EP99944295A EP99944295A EP1144994A2 EP 1144994 A2 EP1144994 A2 EP 1144994A2 EP 99944295 A EP99944295 A EP 99944295A EP 99944295 A EP99944295 A EP 99944295A EP 1144994 A2 EP1144994 A2 EP 1144994A2
Authority
EP
European Patent Office
Prior art keywords
compound
solid support
detection
labelling
solid
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Withdrawn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
EP99944295A
Other languages
English (en)
French (fr)
Other versions
EP1144994A3 (de
Inventor
Marco Artico
Renzo Mena
Giuseppe Zanoni
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.)
GlaxoSmithKline SpA
Original Assignee
SmithKline Beecham SpA
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 SmithKline Beecham SpA filed Critical SmithKline Beecham SpA
Publication of EP1144994A2 publication Critical patent/EP1144994A2/de
Publication of EP1144994A3 publication Critical patent/EP1144994A3/de
Withdrawn legal-status Critical Current

Links

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N31/00—Investigating or analysing non-biological materials by the use of the chemical methods specified in the subgroup; Apparatus specially adapted for such methods
    • G01N31/22—Investigating or analysing non-biological materials by the use of the chemical methods specified in the subgroup; Apparatus specially adapted for such methods using chemical indicators
    • 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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids

Definitions

  • This invention relates to a method for detecting a compound which is reversibly bound to a solid support and in particular to a method for monitoring the progress of a chemical reaction carried out on a solid support.
  • a disadvantage of solid-phase chemistry is that it is often difficult to apply common analytical techniques, such as chromatography or spectroscopy, in a conventional manner to monitor the progress of a reaction step.
  • MS mass spectrometry
  • Solid-phase NMR has been used for reaction monitoring, but this is a specialised technique and may require complex apparatus (Brown A R et al J. Am. Chem. Soc. (119) 3288-3295 (1997)).
  • Spectrophotometric methods have also been applied to combinational libraries e.g.
  • NPIT nitrophenyl isothiocyanate- O-trityl
  • High performance liquid chromatography is a widespread analytical technique in the analysis of organic compounds and the most common form of detection in HPLC is ultraviolet (UV) detection.
  • UV detection relies on the presence of a chromophore in the component of interest which absorbs ultraviolet radiation. This absorbance may be used to quantify the amount of the component present.
  • a disadvantage of UV detection is that compounds which do not possess a UV chromophore will not be detected.
  • the invention provides a method for detecting a compound which is reversibly bound to a solid support, which method comprises derivatising said compound with a labelling moiety and cleaving the labelled derivative produced from the solid support, thereby enabling detection of the labelled derivative.
  • a suitable compound is a product or a substrate in a chemical reaction such as a solid phase combinatorial chemical reaction.
  • the compound and labelling moiety are limited only in that they must be capable of providing a labelled derivative:
  • the labelling moiety can be any moiety which provides a detectable labelled derivative, for example an arylisocyanate, such as phenylisocyanate.
  • the labelling moiety is phenylisocyanate
  • the compound may be any compound which provides a detectable labelled derivative, for example an amine such as ⁇ -alanine.
  • a preferred compound is an amine, for example ⁇ -alanine.
  • Suitable solid supports include: Wang resin, Merrifield resin, Rink acid resin, Sieber amide resin, and hydroxyethyl-photolinker AM resin.
  • the labelling moiety is suitably a chromophore, especially a UV chromophore which therefore provides a labelled derivative detectable via UV detection methods.
  • UV chromophone phenylisocyanate (PIC).
  • the labelled derivative is any derivative formed by the compound and the labelling moiety which is thereafter detectable because of the presence of the labelling moiety or any part thereof in the labelled derivative.
  • the labelled derivative produced by ⁇ -alanine and PIC is a urea.
  • the use of phenylisocyanate in the method described above is a novel use.
  • the invention provides phenylisocyanate for use as a UV chromophore, in particular for use in the derivatisation of a compound reversibly bound to a solid support which after cleavage of said labelled derivative enables the detection of the labelled derivative formed by UV detection methods.
  • Cleaving the derivatised compound from the solid support is carried out using standard methodology appropriate to the nature of the compound and the labelled derivative. For example by using trifluoroacetic acid hydrolysis. Other methods are those disclosed in Protective Groups in Organic Synthesis (2nd Edition) Green T W and Wuts P G M, Wiley-Interscience, New York ( 1991 ) and
  • UV detection is the preferred detection method of the invention, it is considered that any detection method may be used which is capable of detecting the labelled derivative e.g. MS (ESI or APcI) or NMR.
  • the method of the invention can be used to detect a single discrete compound or the components of a mixture of compounds.
  • One common application of the detection of the separate components of a mixture of compounds is to monitor the progress of a chemical reaction, such as a solid-phase chemical reaction, when one or more products may be produced and unreacted substrate may also be present.
  • the labelling moiety enabling derivatisation is chosen so as to be capable of derivatising each of the compounds in the mixture.
  • the analytical method is chosen so as to be capable of separating the mixture of labelled derivatives, thereby enabling detection of each of the labelled derivatives, for example high pressure liquid chromatography (HPLC).
  • HPLC high pressure liquid chromatography
  • the method of the invention is used to quantitatively detect the amount of a compound, including a compound being a component of a mixture of compounds
  • the method comprises:
  • reaction substrate immobilised on the resin support is suspended in tetrahydrofuran (THF), and n-propylamine and phenylisocyanate added.
  • THF tetrahydrofuran
  • TFA trifluoroacetic acid
  • the excess reagents are removed by evaporation and the residue dispersed in methanol.
  • the mixture is filtered and the filtrate analysed by reversed-phase HPLC. This method can be used to quantify the components present, after suitable calibration against appropriate standards. Methods of calibration of HPLC systems are well-known to those skilled in the art.
  • the method may be used for the quantification of a suitable functional group on a solid phase support, for instance to estimate the loading achieved of a resin in a preliminary reaction step.
  • Example 2 The reaction of several aromatic aldehydes with resin-bound ⁇ -alanine to produce a series of imines was monitored by HPLC using the PIC derivatisation method (Scheme 1) and MS to determine the amount of unreacted ⁇ -alanine present.
  • the PIC method avoids the overestimation of the amount of unreacted ⁇ -alanine present which results from the production of additional ⁇ -alanine by TFA hydrolysis of the imines (Scheme 2).
  • Solid-supported imines were obtained by exposure of the polymer-bound ⁇ -alanine (Ramage R and Stewart A S J J. Chem. Soc. Perkin Trans.
  • HPLC analysis was performed under the following conditions:

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Immunology (AREA)
  • Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Chemical & Material Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Hematology (AREA)
  • Pathology (AREA)
  • Physics & Mathematics (AREA)
  • Urology & Nephrology (AREA)
  • Biomedical Technology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Cell Biology (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Biophysics (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
  • Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
  • Investigating Or Analyzing Non-Biological Materials By The Use Of Chemical Means (AREA)
  • Crystals, And After-Treatments Of Crystals (AREA)
  • Plural Heterocyclic Compounds (AREA)
EP99944295A 1998-07-21 1999-07-20 Verfahren zur markierung von an festträger gebundenen verbindungen Withdrawn EP1144994A3 (de)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
ITMI981684 1998-07-21
IT98MI001684A ITMI981684A1 (it) 1998-07-21 1998-07-21 Metodo di monitoraggio dell'avanzamento della reazione di substrati su supporto solido
PCT/EP1999/005336 WO2000005578A2 (en) 1998-07-21 1999-07-20 Method for labelling solid support bound compounds

Publications (2)

Publication Number Publication Date
EP1144994A2 true EP1144994A2 (de) 2001-10-17
EP1144994A3 EP1144994A3 (de) 2002-09-11

Family

ID=11380484

Family Applications (1)

Application Number Title Priority Date Filing Date
EP99944295A Withdrawn EP1144994A3 (de) 1998-07-21 1999-07-20 Verfahren zur markierung von an festträger gebundenen verbindungen

Country Status (5)

Country Link
EP (1) EP1144994A3 (de)
JP (1) JP2002521664A (de)
CA (1) CA2338205A1 (de)
IT (1) ITMI981684A1 (de)
WO (1) WO2000005578A2 (de)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2855183B1 (fr) 2003-05-23 2005-08-12 Agronomique Inst Nat Rech Procede de clonage du rat par transfert nucleaire

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO0005578A3 *

Also Published As

Publication number Publication date
WO2000005578A2 (en) 2000-02-03
JP2002521664A (ja) 2002-07-16
CA2338205A1 (en) 2000-02-03
WO2000005578A3 (en) 2001-10-11
ITMI981684A1 (it) 2000-01-21
ITMI981684A0 (it) 1998-07-21
EP1144994A3 (de) 2002-09-11

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