EP1596978A2 - Procede pour la constitution validee de jeux d'echantillons - Google Patents

Procede pour la constitution validee de jeux d'echantillons

Info

Publication number
EP1596978A2
EP1596978A2 EP03785930A EP03785930A EP1596978A2 EP 1596978 A2 EP1596978 A2 EP 1596978A2 EP 03785930 A EP03785930 A EP 03785930A EP 03785930 A EP03785930 A EP 03785930A EP 1596978 A2 EP1596978 A2 EP 1596978A2
Authority
EP
European Patent Office
Prior art keywords
group
type
compounds
synthesis
label
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Withdrawn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
EP03785930A
Other languages
German (de)
English (en)
Inventor
Barbro Beijer
Ramon GÜIMIL
Matthias Scheffler
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.)
Febit Holding GmbH
Original Assignee
Febit Biotech GmbH
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
Priority claimed from DE10260591A external-priority patent/DE10260591A1/de
Priority claimed from DE10260592A external-priority patent/DE10260592A1/de
Priority claimed from DE10320351A external-priority patent/DE10320351A1/de
Application filed by Febit Biotech GmbH filed Critical Febit Biotech GmbH
Publication of EP1596978A2 publication Critical patent/EP1596978A2/fr
Withdrawn legal-status Critical Current

Links

Classifications

    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J19/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J19/0046—Sequential or parallel reactions, e.g. for the synthesis of polypeptides or polynucleotides; Apparatus and devices for combinatorial chemistry or for making molecular arrays
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09B—ORGANIC DYES OR CLOSELY-RELATED COMPOUNDS FOR PRODUCING DYES, e.g. PIGMENTS; MORDANTS; LAKES
    • C09B11/00—Diaryl- or thriarylmethane dyes
    • C09B11/04—Diaryl- or thriarylmethane dyes derived from triarylmethanes, i.e. central C-atom is substituted by amino, cyano, alkyl
    • C09B11/06—Hydroxy derivatives of triarylmethanes in which at least one OH group is bound to an aryl nucleus and their ethers or esters
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09B—ORGANIC DYES OR CLOSELY-RELATED COMPOUNDS FOR PRODUCING DYES, e.g. PIGMENTS; MORDANTS; LAKES
    • C09B57/00—Other synthetic dyes of known constitution
    • C09B57/001—Pyrene dyes
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09B—ORGANIC DYES OR CLOSELY-RELATED COMPOUNDS FOR PRODUCING DYES, e.g. PIGMENTS; MORDANTS; LAKES
    • C09B57/00—Other synthetic dyes of known constitution
    • C09B57/02—Coumarine dyes
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/00603—Making arrays on substantially continuous surfaces
    • B01J2219/00605—Making arrays on substantially continuous surfaces the compounds being directly bound or immobilised to solid supports
    • B01J2219/00608—DNA chips
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/00603—Making arrays on substantially continuous surfaces
    • B01J2219/00605—Making arrays on substantially continuous surfaces the compounds being directly bound or immobilised to solid supports
    • B01J2219/00623—Immobilisation or binding
    • B01J2219/00626—Covalent
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/00603—Making arrays on substantially continuous surfaces
    • B01J2219/00605—Making arrays on substantially continuous surfaces the compounds being directly bound or immobilised to solid supports
    • B01J2219/00632—Introduction of reactive groups to the surface
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/00603—Making arrays on substantially continuous surfaces
    • B01J2219/00659—Two-dimensional arrays
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/0068—Means for controlling the apparatus of the process
    • B01J2219/00698—Measurement and control of process parameters
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/0068—Means for controlling the apparatus of the process
    • B01J2219/00702—Processes involving means for analysing and characterising the products
    • B01J2219/00704—Processes involving means for analysing and characterising the products integrated with the reactor apparatus
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00718—Type of compounds synthesised
    • B01J2219/0072—Organic compounds
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00718—Type of compounds synthesised
    • B01J2219/0072—Organic compounds
    • B01J2219/00722—Nucleotides
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00718—Type of compounds synthesised
    • B01J2219/0072—Organic compounds
    • B01J2219/00729—Peptide nucleic acids [PNA]
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

Definitions

  • the present invention relates to a method for validating the synthesis of arrays, in particular of biopolymers, by step-by-step construction from protected and labeled synthesis building blocks.
  • the receptors (probes) of an array are immobilized by covalent or non-covalent interaction or synthesized in situ on the solid phase.
  • the building blocks of a chemical polymerization process are generally referred to as "synthons".
  • the functional groups of one synthon allow a targeted chemical reaction with suitable other functional groups on a second synthon.
  • these reactive centers are masked by so-called protective groups, which can be removed in a suitable chemical environment, which makes it possible to control the synthesis process, since only those functional groups that do not have a protective group are implemented.
  • protective groups can generally be changed by changing chemical or physicochemical environmental parameters, such as the redox potential, the pH or the temperature, but also by introducing electromagnetic energy, e.g. be removed by irradiation with light of a certain wavelength.
  • oligomers such as oligonucleotides, oligonucleotide derivatives, peptides or carbohydrates, which are made up of different but finally many monomer units
  • the sequential polymerization introduced by Merryfield by condensation on a solid phase has proven itself (RB Merryfield (1 963),. At the. Chem. Soc. 85: 2149-21 54; RB Merryfield (1 965) Science 1 50: 1 79-1 85).
  • the Merryfield synthesis begins with a carrier-bound functional group that can carry a protective group and can be activated by its removal.
  • the products of a conventional solid phase-supported (bio) polymer synthesis are broken down by breaking a predetermined breaking point, e.g. by hydrolysis of an oxalate or succinate, separated from the solid phase and are thus the known analytical methods, such as NMR, HPLC, CE, MS, phosphor imager etc. accessible.
  • the cleavage products are 3'-fluorescent labeled. In this way, conclusions can be drawn about the coupling efficiency [US 6,238,862 B1, J.
  • a further possibility for quality control is to condense additional cleavable phosphoramidites labeled with phosphate groups at the 5 'ends. The signal from the reporter group then becomes
  • the process for the production of assembled chemical surfaces which is described in WO 01/36086, has the task of introducing functional groups in situ, that is during the functionalization process, and to provide a surface of specific chemical or physicochemical properties adapted to the later application process.
  • inverse synthons these are nucleosides which have a phosphoramidite on their 5'-hydroxy functions and a protective group on their 3'-hydroxy functions, e.g. DMT or Nppoc, it is possible to carry out chemical oligonucleotide synthesis in the natural 5 ' ⁇ 3' direction.
  • the changed orientation on the array surface opens up another area of application for DNA / RNA microarrays. Among other things, it enables the enzymatic chain extension by polymerases [WO 00/61 594; WO 01/55451; M. C. Pirrung, Org. Lett. 2001, 3.5, 1 105-1 108].
  • the photo-protecting groups hitherto used for light-controlled synthesis are usually the protective groups NVOC (SPA Fodor et al., Science 251 (1 991), 767 ff.), MeNPOC (AC Pease et al., Proc. Natl. Acad. Sei . 91 (1 994), 5022 ff.), DMBOC (MC Pirrung, J. Chem. 60 (1 995), 1 1 16 ff.) And NPPOC (A. Hassan et al., Tetrahedron 53 (1 997), 4247 ff.).
  • NVOC SPA Fodor et al., Science 251 (1 991), 767 ff.
  • MeNPOC AC Pease et al., Proc. Natl. Acad. Sei . 91 (1 994), 5022 ff.
  • DMBOC MC Pirrung, J. Chem. 60 (1 995), 1 1 16 ff.
  • NPPOC A. Hassan et
  • the photolabile protective groups currently used for the light-controlled synthesis of nucleic acids are generally characterized by a comparatively low absorption coefficient at the wavelength of the light radiation.
  • the photolabile nucleoside derivatives are usually irradiated with mercury high-pressure lamps at a wavelength of 365 nm. The lower one
  • the absorption coefficient of the photolabile protective group used at this wavelength means that only a very small proportion of the incident light can be used to excite the molecules.
  • the photolabile protective groups used are mostly colorless derivatives.
  • a further development of the photolithographic process is the use of fluorescent photolabile protective groups.
  • the benzylic photolabile protective groups use molecules of the type ArCR ⁇ -OC ⁇ X, where Ar is a condensed aromatic, e.g. Pyrene and R ⁇ substituted aromatics can be.
  • the fluorescence of the Pymoc group is used to estimate the functionalization density [WO 98/39348].
  • Another approach is the labeling of nitrobenzyl derivatives [C. Muller et al, He / v. Chim Acta 2001, 84, 3735-3740].
  • the fluorescence signals of the coumarin can be used for quality control.
  • DNA synthesis is based on tandem protective groups.
  • This type of protective group is obtained by combining two protective groups established for nucleotide chemistry, for example a photolabile Nppoc (2- [2-nitrophenyl] propyloxycarbonyl chloride) and an acid-labile dimethoxytrityl group, and the derivatives derived therefrom (DE 101 32 025.6, DE 102 60 591 .2 and PCT EP 02/07389).
  • Trityl compounds serve strictly as a protective group with unusual ones
  • the detection limits of the trityl-containing nucleotides and oligonucleotides in HPLC or DC can be reduced by incorporating pyrene into the trityl framework.
  • This compound corresponds to the type: P m -C *, where P m is a protective group P which can carry a label m, for example a fluorescent dye, and C * is a functional group to be protected which corresponds to the 5'-hydroxy group of a nucleoside [JL Fourrey et al. Tetrahedron Lett, 1987, 28, 51 57].
  • Patent US 5,410,068 describes a similar approach to reversibly modify biological compounds for detection, separation and purification. Fluorescent-labeled trityl groups of the type MLPC * are described. M is a label that is used to recognize the molecule, L is a spacer, P is a trityl protecting group and C * is the functional group of a biomolecule.
  • M is a label that is used to recognize the molecule
  • L is a spacer
  • P is a trityl protecting group
  • C * is the functional group of a biomolecule.
  • the problem with the array-supported polymer synthesis of sometimes complex substance libraries is that the synthesis is carried out first and then afterwards one learns whether the synthesis was successful. To make matters worse, the surfaces of the arrays created in situ can sometimes be difficult to access. This can significantly affect quality control using standard surface analysis methods. For economic reasons, it would therefore be desirable to have a process that enables an early quality statement, even during processing.
  • One object underlying the present invention was to develop a method for producing solid-phase-bound arrays, which allows improved quality control compared to the methods of the prior art.
  • the present invention relates to a method in which the quality of the array surface is checked, but preferably not influenced , and one or more or all of the following steps of a biopolymer synthesis can be followed online, ie one or more steps, for example the first, the nth, the 2nth, penultimate or / and last etc. step, can be used to increase the efficiency the linker and probe synthesis are checked.
  • the synthesis process according to the invention comprises the introduction of one or more synthon building blocks with at least one detectable labeling group into the linker element and the introduction of one or more synthon building blocks with at least one detectable labeling group into the probe element of the receptor.
  • a single marked synthon building block or several marked synton building blocks e.g. two, three, four etc. marked syntonic blocks can be used.
  • all synthon building blocks for the synthesis of the linker element and / or the probe element can contain at least one detectable labeling group.
  • the first and third steps in the construction of the linker element and / or the probe element are carried out with a labeled synthon building block. Steps without the use of labeled synthon building blocks can be carried out in a conventional manner.
  • biopolymers e.g. nucleic acids such as DNA or RNA, nucleic acid analogues such as PNA or LNA, oligonucleotides (regardless of the direction of synthesis), saccharides, carbohydrates, peptides, proteins and other mixed forms , but also integrate derivatives from combinatorial chemistry.
  • the biopolymers can be synthesized in any direction, for example in 5 ' ⁇ 3' or / and in 3 ' ⁇ 5' direction for nucleic acids.
  • optical methods such as absorption, emission / light diffraction, light scattering or ellipsometry, but also other methods such as radioactivity, plasmon resonance or electronic methods such as electron diffraction or electrical signals, etc. can be used for the analysis.
  • the analytical methods that can be integrated into a system for the synthesis of arrays or biochip supports according to WO 00/1 301 8 are particularly preferred.
  • synthon building blocks can be used which contain several marker groups which can be detected next to one another.
  • marking groups can be used for introduction into the linker element, which can be detected in addition to the marking groups for introduction into the probe element.
  • the synthesis of the linker element and the synthesis of the probe element according to the present invention preferably each comprise a number of steps, the respective elements being built up from a number of synthon units.
  • the linker element is constructed from one or more non-functional synthon building blocks that are different from the functional synthon building blocks for the probe element.
  • Preferred examples of linker synthons are alkyl radicals, oligoethylene glycol radicals or combinations of alkyl and aryl radicals.
  • the linker molecules synthesized on the carrier contain functional groups which enable the coupling of further linker synthon building blocks or - after the linker synthesis has ended - of probes or probe building blocks.
  • the functional groups of the linker molecules can be selected, for example, from -OR, -NR 2 , -SR, -PO 3 R 2 , -CN, -SCN, -COR ' and -OCOR ' , where RH is a protective group and R 'is H or a protecting group or -OR, -NR 2 or -SR.
  • Farther R and R ' can represent alkyl, aryl, alkenyl and / or allyl radicals and / or other useful organic radicals.
  • oligonucleotide After linker synthesis has been completed, the coupling of probes takes place, which is likewise carried out by step-by-step synthesis from synthesis building blocks, depending on the synthesis strategy used, e.g. Peptide, oligonucleotide or carbohydrate synthesis can be carried out on the solid phase or by site-specific and / or site-specific immobilization of complete probes.
  • Particularly preferred building blocks for oligonucleotide synthesis are phosphoramidites.
  • the carrier can in principle be chosen arbitrarily, e.g. from particles, in particular magnetic particles, microtiter plates and microfluidic carriers (such as fluidic microprocessors) and can have a surface selected from glass, metals, semimetals, metal oxides or plastic.
  • the microparticles disclosed in PCT / EP 99/0631 5 and the supports disclosed in PCT / EP 99/0631 6 and PCT / EP 99/0631 7 with planar surfaces or with microchannels (cross section, for example 10-1000 ⁇ m) are particularly preferred .
  • the synthesis of the receptor molecules can take place on the entire surface of the support or in a site-specific manner at selected reaction sites.
  • Detectable marker groups are used in at least one of the synthon building blocks for introduction into the linker element and at least one of the synthon building blocks for introduction into the probe element.
  • all synthon building blocks for the synthesis of the linker element or the probe element can contain at least one detectable labeling group.
  • Detectable marker groups can be cleaved off during or / and after the synthesis of the linker element or the probe element. Different marker groups can be split off at different times during the process.
  • connection is preferably used in the process relevant to the invention.
  • Compounds of the following group I (types l-VII) can be used in particular for the surface control for the installation in the linker element:
  • Type III P m -LV (L'-HL "-M) -L '" - C *
  • Type IV P m 'P m -LV (L'-HL "-M) -L'" - C *
  • Type V MLP m 'P m -LV (L'-HL "-M) -L'" - C *
  • Type VII MLP m 'P m -C> *
  • P m denotes a protective group P which can carry a label m for detection, the labels M or / and m optionally being linked to P via a linker, and the protective group and labels being compatible with synthetic chemistry.
  • P ' is a protective group orthogonal to P m , which can carry a label m' for detection.
  • M, m and m 'are markings that can be used for detection.
  • the markings are expediently also compatible with synthetic chemistry.
  • L to L '"mean any spacer for example organic groups such as alkylene groups, which may optionally contain heteroatoms such as O, N, P and S.
  • H represents a fissile group, e.g. an ester, disulfide, sulfone or diol group.
  • V represents a trifunctional molecule / atom, such as a nucleoside, trihydroxyalkyl or dihydroxyaminoalkyl.
  • C * means a functional group or a functionalized carrier surface, in particular a linker component.
  • the compounds of types II to VII usually contain several labeling groups, M, m or / and m '. However, those compounds are also recorded which carry only one marker group, ie the protective group P m , for example, does not have to contain a marker group in the compounds, provided the compound contains at least one other marker group, for example M.
  • Type I P m -C *
  • Type II MLP m -C *
  • Type VII -LP m 'P m -C *
  • Type VIII P m -LH-L'-C *
  • Type IX pm m_
  • Type X MLP m 'P m -LHL "-C *
  • P m denotes a protective group (P) which can carry a label (m) for detection, the label M (m) optionally being linked to (P) via a linker, and the protective group and label being compatible with synthetic chemistry.
  • P m ' is a protective group orthogonal to P m , which carries a label (nrT) for detection.
  • P m can be a protective group which can be removed photochemically by exposure and P m 'can be a protective group which can be removed by chemical methods, for example acid or base treatment.
  • M, m and m 'are markings that can be used for detection.
  • the markings are expediently also compatible with synthetic chemistry.
  • L to L "mean any spacer, e.g. organic groups such as alkylene groups, which may optionally contain heteroatoms such as O, N, P and S.
  • H denotes a cleavable group, for example an ester, disulfide, sulfone or diol group.
  • C * means a probe component, for example a nucleotide, a nucleotide analog, an amino acid, an amino acid analog etc.
  • the compounds of types II and VI to X contain one or more labeling groups, ie the protective group P m or / and P m ' need not contain a labeling group if the compound contains at least one other labeling group.
  • a preferred embodiment is as follows: First, a labeled synthon is condensed onto the functionality of the support surface. After the fluorescence determination, the labels are either split off at the next deprotection step, in the sense of a step-by-step biopolymer synthesis, or removed at the end of the synthesis, so that they cannot falsify the detection signal of the hybridization.
  • the compound types are compounds that can make several quality assurance methods accessible at the same time: Homogeneity testing is carried out using the M or M label, which can be detected independently of one another, e.g. two fluorescent dyes or fluorescence in combination with radioactive labeling, wherein m and M, which are part of the p , p m , P m and MLP 'P m groups, are split off before the next coupling step.
  • the labeling groups can, if appropriate, for example in the case of compounds of types III and / or IV, also be removed at a later point in time, for example in the event of the final deblocking. All marking groups are expediently removed until final unblocking, so that their signal cannot interact with the measurement signal on the carrier.
  • Type I P m -C * triplet-sensitized Nppoc
  • Type II MLP m -C * fluorescence-labeled protective group (Coumarin Nppoc)
  • Type VI p ⁇ vpm_p # fluorescence-labeled tandem group (Pyren Nppoc)
  • Type VII MLP m 'P m -C fluorescent labeled tandem group
  • Type VIII P m -LH-L'-C (Nppoc or DMT succinate derivatives)
  • Type IX pm'P m -LH-L'C fluorescent labeled tandem group
  • TypeX ML-P'P m -LHL "-C * fluorescence markers
  • the methods of conventional quality control for polymer synthesis can essentially be transferred to the products from the array synthesis, taking into account the three-dimensional structure of arrays and in particular by using molecules of type VIII-X, provided that the functionalization process of the surface provides a sufficiently high density of functionalization.
  • the invention further relates to compounds of the general structure (XI):
  • M is a polycyclic aryl or heteroaryl group
  • A-, and A 2 are each independently selected from H, O, OR, NHR or NR 2 , wherein R is a C, -C 20 hydrocarbon group, optionally one or more heteroatoms can carry, for example an alkyl, aryl, aralkyl or alkaryl group and C * is a functional group, for example a linker or probe synthon building block.
  • the group M is preferably an aryl or heteroaryl group with at least 3 or 4 fused rings, for example a pyrene group.
  • M is preferably a fluorescent labeling group, for example a coumarin, a pyrene, Cy5, Cy3, a rhodamine or a fluorescent nanoparticle. If necessary, M can be connected to the basic structure via a spacer.
  • At least one of the radicals A and A 2 is NHR or NR 2 .
  • At least one of A. and A 2 is particularly preferably a dialkylamino group, the alkyl radicals having 1 to 20 C atoms.
  • the invention further relates to compounds of the general structure (XII):
  • M ' is a labeling group
  • Y is a bond or a spacer with a chain length of up to 20 C atoms and optionally one or more heteroatoms
  • A, and A 2 are each independently selected from H, O, OR, NHR or NR 2 , wherein R is a C, -C 20 hydrocarbon group which may optionally carry one or more heteroatoms, for example an alkyl, aryl, aralkyl or alkaryl group and C * is a functional group, for example a linker or probe synthon building block
  • M ' is preferably a fluorescent labeling group, for example a coumarin group ....
  • At least one of the radicals A and A 2 is NHR or NR 2 , particularly preferred are A- or / and A 2 is a dialkylamino group, where an alkyl radical can contain up to 20 carbon atoms.
  • Y preferably represents a non-photolabile structure, i.e. the group M 'cannot be split off from the basic structure of the compound by exposure.
  • the compounds (XI) and (XII) represent preferred examples of syntonic building blocks for use in a method as described above.
  • FIG. 1 An exemplary embodiment of a photolabile protective group, which can be used, for example, as group P m in compounds of type I, is shown in FIG. 1. It is an intramolecular triplet-sensitized o-nitrophenylethyl photoprotecting group that bears a pyrene residue.
  • Other suitable groups of this type are described, for example, in DE 1 02 60 592.0.
  • Figure 2 shows connections of the type MLP m -C * , which can be used for connections of type II, for example.
  • Pm is one optionally substituted trityl group to which a label M is optionally coupled via a linker.
  • Figures 3 to 12 show examples of compounds of type III (P m -LV (L'-HL "-M) -L"'- C *) and their synthesis. These are trifunctional molecules, which provide functionality for polymer synthesis, one for a label M and one for anchoring to the solid phase. The second functionality between the label and the trifunctional molecule has a labile bond.
  • the following protective groups or types of protective groups are suitable for P m : DMT, Nppoc, a two-stage, a fluorescent two-stage or a fluorescence-labeled two-stage protective group.
  • Figure 13 shows a compound of type VI (P m 'P m -C * ), which is a fluorescent two-stage protective group.
  • Figure 14 shows a compound of type VII (MLP m, P m -C * ), which is also a fluorescence-labeled two-stage protective group.
  • P m is the trityl group which is linked to a coumarin group (M) via a linker.
  • NppoC groups P '
  • Figure 15 shows a compound of type IX (P m 'P m -LH-L'-C * ). It is a fluorescent two-stage protective group and a spacer which is interrupted by a predetermined breaking point H (OCO-CH 2 -CH 2 -CO-O).

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Prostheses (AREA)

Abstract

L'invention concerne un procédé pour valider la synthèse de jeux d'échantillons, notamment de biopolymères, par synthèse progressive à partir d'unités de synthèse protégées et marquées.
EP03785930A 2002-12-23 2003-12-23 Procede pour la constitution validee de jeux d'echantillons Withdrawn EP1596978A2 (fr)

Applications Claiming Priority (7)

Application Number Priority Date Filing Date Title
DE10260591A DE10260591A1 (de) 2002-12-23 2002-12-23 Photoaktivierbare zweistufige Schutzgruppen für die Synthese von Biopolymeren
DE10260592A DE10260592A1 (de) 2002-12-23 2002-12-23 Intramolekular triplettsensibilisierte o-Nitrophenylethyl-Photoschutzgruppen
DE10260591 2002-12-23
DE10260592 2002-12-23
DE10320351A DE10320351A1 (de) 2003-05-07 2003-05-07 Verfahren zum validierten Aufbau von Arrays
DE10320351 2003-05-07
PCT/EP2003/014826 WO2004058393A2 (fr) 2002-12-23 2003-12-23 Procede pour la constitution validee de jeux d'echantillons

Publications (1)

Publication Number Publication Date
EP1596978A2 true EP1596978A2 (fr) 2005-11-23

Family

ID=32685657

Family Applications (1)

Application Number Title Priority Date Filing Date
EP03785930A Withdrawn EP1596978A2 (fr) 2002-12-23 2003-12-23 Procede pour la constitution validee de jeux d'echantillons

Country Status (3)

Country Link
EP (1) EP1596978A2 (fr)
AU (1) AU2003294947A1 (fr)
WO (1) WO2004058393A2 (fr)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE102005027667A1 (de) * 2005-06-15 2006-12-28 Febit Biotech Gmbh Verfahren zur Qualitätskontrolle funktionalisierter Oberflächen

Family Cites Families (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6147205A (en) * 1995-12-15 2000-11-14 Affymetrix, Inc. Photocleavable protecting groups and methods for their use
EP1117478B1 (fr) * 1998-08-28 2005-06-01 Febit AG Support pour un procede de determination d'analyte et procede de fabrication du support
CA2421732A1 (fr) * 2000-09-11 2002-03-14 Affymetrix, Inc. Groupes de protection photoclivables
EP1330307B8 (fr) * 2000-10-17 2005-11-23 febit biotech GmbH Procede et dispositif pour effectuer une synthese integree et une determination d'une substance a analyser sur un substrat
DE10105079A1 (de) * 2001-02-05 2002-08-08 Febit Ferrarius Biotech Gmbh Fotolabile Schutzgruppen für die Synthese von Biopolymeren
DE10122357A1 (de) * 2001-05-09 2002-11-14 Febit Ferrarius Biotech Gmbh Hybridverfahren zur Herstellung von Trägern für die Analytbestimmung
DE10132025A1 (de) * 2001-07-03 2003-02-06 Febit Ferrarius Biotech Gmbh Zweistufige Schutzgruppen für die Synthese von Biopolymeren

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO2004058393A2 *

Also Published As

Publication number Publication date
AU2003294947A8 (en) 2004-07-22
WO2004058393A3 (fr) 2004-09-10
WO2004058393A2 (fr) 2004-07-15
AU2003294947A1 (en) 2004-07-22

Similar Documents

Publication Publication Date Title
DE69839373T2 (de) Auf die Verwendung von nukleaseresistenten Fragmenten basierende Verfahren und Kits, die für Nukleinsäuretests mit hohem Durchsatz geeignet sind
DE69720041T2 (de) Chemische amplifizierung zur synthese von musterordnungen
DE60222628T2 (de) Herstellung von matrizen aus polynukleotiden
EP1585830A2 (fr) Groupes protecteurs photoactivables subissant un traitement en deux etapes, destines a la synthese de biopolymeres
EP1114186B1 (fr) Procede de controle qualite lors de la constitution de trames oligomeriques
EP1385617B1 (fr) Procedes hybrides permettant de produire des supports con us pour la recherche de substances a analyser
DE19925402C2 (de) Screening von Target-Ligand-Wechselwirkungen
DE10142643A1 (de) Detektion von Wechselwirkungen auf Sonden-Arrays
EP1596978A2 (fr) Procede pour la constitution validee de jeux d'echantillons
DE10320351A1 (de) Verfahren zum validierten Aufbau von Arrays
EP1230015B1 (fr) Procede de production de surfaces chimiques surmoulees
DE10227814A1 (de) Verfahren zur Abspaltung von labilen funtionellen Gruppen aus chemischen Verbindungen
DE10065632A1 (de) Verfahren zum Nachweis von Polynukleotiden
DE102005029811B4 (de) Oligonukleotidanordnungen, Verfahren zu deren Einsatz und deren Verwendung
DE10209203A1 (de) Verfahren zur Abspaltung von labilen funktionellen Gruppen aus chemischen Verbindungen
EP1649045B1 (fr) Incorporation de groupes haptene lors de la production de supports pour la determination de substances a analyser
US20060154253A1 (en) Method for the validated construction of arrays
WO2003040679A2 (fr) Liaison reversible d'un fluorophore sur une surface pour detecter des phenomenes d'association ligat-ligand par extinction de fluorescence
DE10152017A1 (de) Arrays mit Hairpin-Strukturen
DE10315772A1 (de) Chemische Verbindung und Verfahren für den intramolekularen Energietransfer zur Abspaltung von labilen funktionellen Gruppen aus chemischen Verbindungen
DE10156467A1 (de) Träger zur Synthese sowie Verfahren zur Herstellung von Biopolymeren
CH693202A5 (de) Verfahren zur Abspaltung von labilen funktionellen Gruppen aus chemischen Verbindungen.
DE10152147A1 (de) Verfahren zum Aufbringen von Nukleosiden und/oder Nukleotiden auf funktionalisierten Oberflächen sowie Verfahren zur Bestimmung von Kopplungsausbeuten bei der Synthese von Nukleotiden
DE10132025A1 (de) Zweistufige Schutzgruppen für die Synthese von Biopolymeren
DE102005032038A1 (de) Entwicklung einer ortsspezifischen, chemoselektiven und gerichteten photochemischen Mikrostrukturierungstechnik für bio- und materialwissenschaftliche Anwendungen (z.B. zur Herstellung von Mikroarrays)

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 20050721

AK Designated contracting states

Kind code of ref document: A2

Designated state(s): AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IT LI LU MC NL PT RO SE SI SK TR

AX Request for extension of the european patent

Extension state: AL LT LV MK

DAX Request for extension of the european patent (deleted)
RBV Designated contracting states (corrected)

Designated state(s): CH DE FR GB LI

RAP1 Party data changed (applicant data changed or rights of an application transferred)

Owner name: FEBIT HOLDING GMBH

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE APPLICATION IS DEEMED TO BE WITHDRAWN

18D Application deemed to be withdrawn

Effective date: 20120703