METHOD

Abstract: A method of monitoring reaction progress on solid-supported substrates using derivatisation and HPLC.

Description: Method 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. There is a great deal of interest in the use of combinatorial chemistry and variomer (parallel) synthesis, in particular on solid-phase supports, to generate libraries of compounds for testing in biological assays to discover new leads for drug discovery. US Patent 5,324,483 discloses apparatus for multiple simultaneous synthesis on a solid-phase support followed by cleavage to yield the compounds of interest. REM resin represents one of the most synthetically flexible solid-phase support systems, and compounds with a high degree of diversity or purity are obtained (Brown A R et al J. Am. Chem. Soc. (119) 3288-3295 (1997) and Ouyang X et al J. Org. Chem. (63) 1027-1032 (1998)). 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. For example, mass spectrometry (MS), although a useful technique for the analysis of combinatorial libraries, is prone to the formation of adducts and degradation of the analyte which complicates interpretation of the data. 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. Chu and Reich have described the use of nitrophenyl isothiocyanate- O-trityl (NPIT) for the quantitative or qualitative analysis of amines on solid support (Biorganic and Medicinal Chemistry Letters 5 (10) 1053-1058 (1995). Shah et al have described the use of Ellman's reagent [5,5 -dithio bis(l- nitrobenzoic acid)(DTNB)] for the quantification of cysteine residues bound to a polystyrene matrix via a glycine linker molecule (Analytical Proceedings Including Analytical Communications, April 1995, 32, 149-153). There still remains, however, the need for rapid and reliable methods using conventional techniques which can be used to monitor the progress of reactions during the preparation of compounds on solid phase supports. High performance liquid chromatography (HPLC) is a widespread analytical technique in the analysis of organic compounds and the most common form of detection in HPLC is ultraviolet (UV) detection. This method of 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. It has now been found that the derivatisation of a compound bound to a resin with a chromophore and the subsequent cleavage of the chromophore-derivatised compound from the resin, provides a reproducible and user friendly method for the quantitative detection of the compound. In particular when combined with an appropriate HPLC system the method provides for the quantitative detection of the individual components of a mixture of compounds, including for example the unreacted substrate and reaction product or products of a chemical reaction. Accordingly, 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: Thus when the compound is an amine the labelling moiety can be any moiety which provides a detectable labelled derivative, for example an arylisocyanate, such as phenylisocyanate. Alternatively, when the labelling moiety is phenylisocyanate, the compound may be any compound which provides a detectable labelled derivative, for example an amine such as β-alanine. A preferred compound is an amine, for example β-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. An example of a UV chromophone is phenylisocyanate (PIC). It will be appreciated that 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. For example the labelled derivative produced by β-alanine and PIC is a urea. It is considered that the use of phenylisocyanate in the method described above is a novel use. Hence, in an alternative aspect, 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 Protecting Groups Kocienski P J, Georg Thieme Verlag Stuttgart, New York. Although 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. Furthermore, 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. When a mixture of compounds is present, the labelling moiety enabling derivatisation is chosen so as to be capable of derivatising each of the compounds in the mixture. When a mixture of compounds is present, 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). Suitably, 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 In one preferred aspect the method comprises: (a) derivatising a sample of the bound compound with PIC (b) cleaving the derivatised compound from the solid support, (c) determining the amount of derivatised compound by HPLC using UV detection. In more detail, a reaction aliquot containing the reaction substrate immobilised on the resin support is suspended in tetrahydrofuran (THF), and n-propylamine and phenylisocyanate added. The solvent is then removed under reduced pressure and the residue suspended in trifluoroacetic acid (TFA). 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. In a further aspect, 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. The invention will now be described by the following example which is not intended to limit the invention in any way: Example The reaction of several aromatic aldehydes with resin-bound β-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 β-alanine present. The PIC method avoids the overestimation of the amount of unreacted β-alanine present which results from the production of additional β-alanine by TFA hydrolysis of the imines (Scheme 2). Solid-supported imines were obtained by exposure of the polymer-bound β-alanine (Ramage R and Stewart A S J J. Chem. Soc. Perkin Trans. (1) 1947-1950 (1993)) to a twofold molar excess of the aromatic aldehydes in a trimethyl orthoformate suspension for eighteen hours (Look G C et al Tetrahedron Lett. (36) 2937-2940 (1995)). For the PIC derivatisation, the solid resin support (about 3mg) was suspended in tetrahydrofuran (THF) (1ml), and n-propylamine (10 microlitres of a 2.6M solution in THF) and phenylisocyanate (100 microlitres of an 88M solution in THF) added. After about one hour, the solvent was removed under reduced pressure and the residue suspended in 95% trifluoroacetic acid (TFA) for about one hour. The excess reagents were removed by evaporation and the residue dispersed in methanol (1 ml). The mixture was filtered and the filtrate analysed by re versed-phase HPLC. For MS analysis, the components were cleaved from the resin with TFA using standard procedures. , ■JC N' Scheme 1 Scheme 2 HPLC analysis was performed under the following conditions: Instrument: Pump: SHIMADZU LC-10AS Autoinjector: SHIMADZU SIL-9A Detector: SHIMADZU SPD-M10A Column: 150mm x 4.6mm i.d. ULTRACARB ODS30 5micron Mobile phase: A: Deionised water:acetonitrile:trifluoroacetic acid 900:100:0.5 v/v B: Deionised water:acetonitrile:trifluoroacetic acid 100:900:0.5 v/v Gradient elution 20% B (5mins.) to 80% B (lOmins.) over 20 mins. (Linear programme: gradient) Reequilibrate at 20%B for 10 mins. Analysis time: 35mins. Flow rate: 1 ml min- 1 Detector: UV at 220nm MS analysis was performed under the following conditions: Instrument: FINNIGAN TSQ700 Ionisation mode: Electrospray Sample introduction: Direct loop injection Flow rate: 0.2ml min- 1 in a 1 : 1 v/v mixture of acetonitrile and water Data acquisition: Selective Ion Monitoring for β-alanine at m/z 90 [M+H]+ and m z 131 [M+H+MeCN]+ The results are shown in Table 1 and clearly demonstrate the superior and consistent results from the PIC method over the MS method (Entries 2, 3, 4, 7, and 8), taking into account the overestimation of the β-alanine by imine hydrolysis. Three imines (entries 1, 5, and 6) proved to be resistant to TFA hydrolysis and hence good agreement between the results from PIC method and MS were obtained. Nevertheless, due to the unpredictable behaviour of imines towards TFA, the PIC method is the only reliable procedure to assess and moreover quantify the progress of this reaction. Table 1. HPLC (PIC) and MS Results For Reactions On β-Alanine Resin

Claims: Claims 1. 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. 2. A method according to claim 1 in which the the labelling moiety is phenylisocyanate (PIC). 3. A method according to claim 1 in which the compound bound to a solid support is β-alanine. 4. A method according to any one of claims 1 to 3 which method comprises: (a) derivatising a sample of the bound compound with PIC, (b) cleaving the derivatised compound from the solid support, (c) determining the amount of derivatised compound by HPLC using UV detection.

Inventors: ARTICO MARCO, MENA RENZO, ZANONI GIUSEPPE, ARTICO, MARCO, MENA, RENZO, ZANONI, GIUSEPPE
Applicants: ARTICO MARCO, MENA RENZO, SMITHKLINE BEECHAM SPA, ZANONI GIUSEPPE, ARTICO, MARCO, MENA, RENZO, SMITHKLINE BEECHAM S.P.A., ZANONI, GIUSEPPE
Application year: 1999
Main classification: G01N31
IPC codes: G01N21, G01N30, G01N31, G01N33