DE10320351A1 - Production of a coated support by stepwise synthesis of a receptor comprising a linker and probe comprises monitoring the synthesis by incorporating one or more labeled synthesis units into the linker and the probe - Google Patents

Abstract

Producing a coated support, comprising preparing a support having reactive surface groups and building up a functionalized surface on the support by stepwise synthesis of a receptor comprising a linker and probe comprises monitoring the synthesis by incorporating one or more labeled synthesis units into the linker and the probe, is new. An independent claim is also included for diphenylmethane derivatives of formula (XI) and triphenylmethane derivatives of formula (XII): [Image] M : polycyclic aryl or heteroaryl; A 1, A 2H, O, OR, NHR or NRR; R : 1-20C hydrocarbyl; C* : a functional group; M' : a label; and Y : a bond or a spacer with a chain length of up to 20 C atoms and optionally one or more heteroatoms.

Description

The The present invention relates to a method for validating the Synthesis of arrays, especially biopolymers, by stepwise Structure from protected and marked synthesis building blocks.

To the current state of the art, the receptors (probes) of an array through covalent or non-covalent interaction immobilized or synthesized in situ on the solid phase. The building blocks chemical polymerization processes are generally referred to as "synthons". The functional groups of a synthon allow a targeted chemical reaction with suitable other functional groups on a second synth. As a rule, these are reactive centers masked by so-called protecting groups, which are in a suitable chemical environment can be removed, creating a control of the synthesis process possible will be implemented, since only those functional groups that do not wear a protective group. Dependent Protective groups in general can depend on the particular synthesis strategy through a change chemical or physicochemical environmental parameters, such as the redox potential, the pH value or the temperature, but also through the entry of electromagnetic energy, e.g. by irradiation with light of a certain wavelength become.

For the sequence specific Synthesis of oligomers, such as oligonucleotides, oligonucleotide derivatives, peptides or carbohydrates made up of different, but finally many Monomer units are built, the sequential introduced by Merryfield Polymerization by condensation on a solid phase has proven itself (R. B. Merryfield (1963), J. Am. Chem. Soc. 85: 2149-2154; R. B. Merryfield (1965) Science 150: 179-185). The Merryfield synthesis starts on a carrier-bound functional group that can carry a protecting group and itself can be activated by removing them. This will couple one in solution supplied, next Monomers allowed that after polymerization through condensation in turn as a starting point for a further polymerization step is available. The successive Repetition of activation and subsequent polymerization by Condensation leads finally to Synthesis of the desired Oligomers.

The products of a conventional solid-phase-supported (bio) polymer synthesis are separated from the solid phase by cleaving a predetermined breaking point, for example by hydrolysis of an oxalate or succinate, and are therefore known analysis methods such as NMR, HPLC, CE, MS, phosphor imager etc., accessible. By combining a cleavable spacer amidite (predetermined breaking point = ester, sulfones, etc.) and a fluorescent branched phosphoramidite, the strategy described above can also be applied to arrays in the sense of a modular approach. The cleavage products are 3'-fluorescent labeled. In this way one can draw conclusions about the coupling efficiency [ US 6,238,862 B1, J. Burmeister, A. Azzawi, G. v. Kiedrowski, Tethedron Lett. 1995, 3667-68]. The same result is achieved when a trifunctional molecule is used which provides functionality for polymer synthesis, one for labeling and one for anchoring to the solid phase. The third functionality also has an unstable bond [ US 5,290,925 ].

A another possibility for quality control is additional to condense cleavable phosphate group-labeled phosphoramidites at the 5 'ends. The signal from the reporter group then becomes quality control the synthesis. After splitting off the reporter group, 5'-phosphorylated ones are obtained Probes [Beier, WO 00/15837].

The Process for the manufacture of finished chemical surfaces, which is described in WO 01/36086, has functional groups as its task in situ, i.e. during of the functionalization process and the later application process customized a surface to provide specific chemical or physicochemical properties.

Other Methods use "starburst" dendrimers around the Increase the number of reaction sites, which it finally to an increase the density of functional groups on the surface and indirectly to an improvement in the detection signal (M. Beier, J. D. Hoheisel (1999) Nucleic Acids Res. 27: 970-1977) comes or better signal-to-noise ratios can be achieved (Niemeyer et al. (2000), Chembiochem. 2: 685-694).

By using so-called inverse synthons, these are nucleosides that 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 00/61594; WO 01/55451; MCPirrung, Org. Lett. 2001, 3.8, 1105-1108].

For an in situ array synthesis is a site-specific deprotection of individuals Protection groups absolutely necessary. A possibility for spatially resolved synthesis uses the process of photolithography and should use the example of DNA synthesis explained : In the light-controlled synthesis of nucleic acid chips find photolabile nucleoside derivatives. Here takes place the chain structure of the nucleic acid fragments usually using phosphoramidite synthons instead. The building blocks each carry a temporary Photo protection group that can be removed by exposure to light. The synthesis principle sees a cyclic sequence of condensation and deprotection steps (by light). The efficiency with Such a light-controlled synthesis can take place in Essentially due to the photolabile protective groups used, in particular by the efficiency with which it is removed in the irradiation step can be certainly.

at the photo protection groups previously used for light-controlled synthesis it is usually the NVOC protective groups (S.P.A. Fodor et al., Science 251 (1991), 767 ff.), MeNPOC (A.C. Pease et al., Proc. Natl. Acad. Sci. 91 (1994), 5022 ff.), DMBOC (M.C. Pirrung, J. Chem. 60 (1995), 1116 ff.) and NPPOC (A. Hassan et al., Tetrahedron 53 (1997), 4247 ff.). Other known photolabile protective groups in the nucleoside or Nucleotide chemistry is o-nitrobenzyl groups and their derivatives (cf. e.g. Pillai, Org. Photochem. 9: 225 (1987); Walker et al., J. Am. Soc. 110: 7170 (1988). As further photolabile protective groups the 2- (o-nitrophenyl) ethyl group (Pfleiderer et al., In: "Biosphosphates and their Analogues - Synthesis, Structure, Metabolism and Activity ", ELSEVIER Science Publishers B.V. Amsterdam (1987), 133 ff.) And derivatives thereof (WO 97/4434, WO 96/18634, WO 02/20150) proposed.

The currently for the light-controlled synthesis of nucleic acids used photolabile Protective groups (e.g. NVOC, MeNPOC, NPPOC) generally stand out due to a comparatively low absorption coefficient the wavelength from the light. Irradiation of the photolabile nucleoside derivatives usually takes place with Hg high pressure lamps at a wavelength of 365 nm. The low absorption coefficient of the photolabile protecting group used at this wavelength with the result that only a very small proportion of the irradiated Light to excite the molecules can be used. Furthermore, it concerns the used photolabile protecting groups mostly around colorless derivatives. this in turn has the consequence that during synthesis not possible is to use simple spectroscopic methods to detect whether the photolabile protective group is still on the nucleoside derivative or already partially or completely has been abstracted by the entry of light. It can be thus it is difficult or impossible to follow the process of abstraction.

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 ₁ R ₂ -OC (O) X, where Ar is a condensed aromatic, for example pyrene and R ₁ R ₂ substituted aromatics can. 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, Helv. Chim Acta 2001, 84, 3735-3740]. The fluorescence signals of the coumarin can be used for quality control.

An alternative to photolithography is DNA synthesis, which is based on tandem protective groups. This type of protective group is obtained by combining two protective groups established for nucleotide chemistry, e.g. B. 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). The cleavage of the colored trityl cation of the two-stage protective groups, which has a much higher absorption coefficient than the cleavage products in other photoprotection processes based on benzyl or nitrobenzyl, further opens up the possibility of direct online process control. This leads to an improvement in quality control for biochips.

The effort to influence the properties of the trityl protecting group has a long tradition [A. Taunton-Rigby et al., J. Org. Chem. 1972, 37, 956-964]. Some working groups have increased the hydrophobic character for purification using RP chromatography [R. Ramage et al. Tetrahedron Lett. 34 (1993) 7133; H. Seliger et al., Angew. Chem. 1981, 93, 709], others have varied the exocyclic groups so that the cleavage conditions are reversed (base-labile trityle) [M. Sekine et al., Bull. Chem. Soc. Jpn. 1985, 58, 336; M. Sekine et al., J. Am. Soc. 1986, 108, 4581] or can be split off under particularly gentle conditions, for example hydrazinolysis of 4- (9-fluorenenylmethoxyoxycarbonyl) oxy- or amino-4 ', 4''- dimethoxytriyl [E. Happ et al., Nucleosides and Nucleotides 1988, 7, 813-816.]. The fluorine-labeled trityl compounds serve strictly as a protective group with unusual lability properties.

Furthermore, 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, 5157].

The 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.

at the array-based Polymer synthesis of sometimes complex substance libraries one usually faces the problem that only the synthesis is carried out and then experienced afterwards whether the synthesis was successful. To make matters worse added to the surfaces that arrays created in situ can be difficult to access. This can quality control through standard surface analysis methods significantly affect. Because of economical reasons would it be therefore desirable to have a process that provides an early indication of quality, still while processing.

A The object underlying the present invention was a method for producing solid-phase arrays develop, which an improved quality control over the Prior art methods allowed.

• (a) Bereitstellen eines Trägers, der an seiner Oberfläche reaktive Gruppen aufweist,
• (b) Aufbauen einer funktionalisierten Oberfläche auf dem Träger durch schrittweise Synthese eines Rezeptors, umfassend ein Linker- und ein Sondenelement aus Synthonbausteinen,

dadurch gekennzeichnet, dass der Syntheseprozess (i) durch Einführung von einem oder mehreren Synthonbausteinen mit mindestens einer nachweisbaren Markierungsgruppe in das Linkerelement und (ii) durch Einführung von einem oder mehreren Synthonbausteinen mit mindestens einer nachweisbaren Markierungsgruppe in das Sondenelement des Rezeptors überwacht wird.This object is achieved by a method for producing a coated carrier, comprising the steps:

• (a) providing a support which has reactive groups on its surface,
• (b) building a functionalized surface on the support by stepwise synthesis of a receptor, comprising a linker and a probe element from synthon building blocks,

characterized in that the synthesis process is monitored (i) by introducing one or more synthon building blocks with at least one detectable labeling group into the linker element and (ii) by introducing one or more synthon building blocks with at least one detectable labeling group into the probe element of the receptor.

object the present invention, a method in which the quality of the array surface is checked is preferred but is not affected, and one or more or all of the following Steps of biopolymer synthesis can be followed online, i.e. to increase efficiency can one or more steps, e.g. the first, the nth, the 2nth, penultimate or / and last etc. step in which linker and probe synthesis are checked.

The Synthesis process according to the invention includes the introduction of one or more synthon building blocks with at least one detectable Marker group in the linker element and the introduction of one or more synthon building blocks with at least one detectable Marker group in the probe element of the receptor. It can for example when building the linker and / or the probe element a single marked synthon building block or several marked Syntonic blocks, e.g. two, three, four etc. marked syntonic blocks, be used. If necessary, all synthon building blocks for the synthesis of the linker element and / or the probe element at least one detectable marker group contain. In a preferred embodiment of the method according to the invention the first and third steps in building the linker element or / and the probe element with a labeled synthon building block carried out. Steps without using labeled synthon building blocks can be found on performed in a conventional manner become.

In addition to the functionalization density, which correlates directly with the later probe density, a uniform distribution of the functional groups is of crucial importance. For the assessment of the Homogeneity can be a variety of labels in the synthesis of 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 derivatives from combinatorial chemistry. The biopolymers can be synthesized in any direction, for example in the 5 '→ 3' or / and in the 3 '→ 5' direction for nucleic acids. Accordingly, 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 which can be integrated into a system for the synthesis of arrays or biochip carriers according to WO 00/13018 are particularly preferred.

at the inventive method can Synthon building blocks are used that have several detectable side by side Marker groups included. So can introduce to the linker element marker groups are used next to the marker groups for introduction are detectable in the probe element.

The Synthesis of the linker element and synthesis of the probe element according to the present The invention preferably comprises several steps, the respective elements can be constructed from several synthon building blocks. The linker element becomes one or more non-functional Synthon building blocks that are different from the functional ones Synthon blocks for are the probe element. Preferred examples of linker synthons are alkyl radicals, Oligoethylene glycol residues or combinations of alkyl and aryl residues.

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. Furthermore, R and R 'can be alkyl, aryl, alkenyl and / or allyl radicals and / or other useful organic radicals.

To completed linker synthesis, the coupling of probes takes place as well through step-by-step synthesis from synthesis building blocks depending on the used Synthesis strategy, e.g. Peptide, oligonucleotide or carbohydrate synthesis on the solid phase or by site-specific and / or non-site-specific Complete probes can be immobilized. Particularly preferred Building blocks for the oligonucleotide synthesis are phosphoramidites.

The carrier can basically arbitrarily selected 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, semi-metals, Have metal oxides or plastic. Are particularly preferred the microparticles disclosed in PCT / EP 99/06315 and those in PCT / EP 99/06316 and PCT / EP 99/06317 disclosed carriers with planar or with microchannels (cross section e.g. 10–1000 μm) Surfaces. Reference is expressly made to the disclosure of the named documents taken.

The Synthesis of the receptor molecules can on the entire surface of the carrier or take place at selected reaction sites depending on the location.

at 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 to the probe element uses detectable labeling groups. If necessary, you can all Synthon building blocks for the synthesis of the linker element or the probe element at least contain a detectable marker group.

The Detectable marker groups can be split off during or / and after the synthesis of the linker element or the probe element. Different marker groups can be at different times be split off from the proceedings.

The following types of connection are preferably used in the process relevant to the invention. When checking the surface for installation in the linker element, connections from the following group I (types I – VII) can be used:
Type I: P ^m -C *
Type II: MLP ^m -C *
Type III: P ^m -LV (L'-H-L '' - M) -L '''- C *
Type IV: P ^{m '} P ^m -LV (L'-H-L''- M) -L''' - C *
Type V: MLP ^{m '} P ^m -LV (L'-H-L''- M) -L''' - C *
Type VI: P ^{m '} P ^m -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 ^{m '} is a protective group orthogonal to P ^m , which can carry a label m' for detection. The protective group P ^{m '} can be split off selectively under conditions in which the protective group P ^{m is} stable. For example, 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 marks, that can be used for detection. M, m and optionally m 'detectable independently.

she can possibly also independently be removable, e.g. for the compounds of types III, IV or VI. The markings are expedient also compatible with synthetic chemistry.

L to L '' 'mean any spacer, e.g. organic Groups, such as alkylene groups, optionally heteroatoms, such as O, N, P and S.

H means a fissile group, e.g. an ester, disulfide, sulfone or diol group.

V means a trifunctional molecule / atom, such as a nucleoside, Trihydroxyalkyl or dihydroxyaminoalkyl.

C * means a functional group or a functionalized support surface, in particular a linker module.

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.

Of the compounds of group I are compounds of types I and / and II particularly preferred.

When checking the probe for installation in the probe element, compounds of the following group II (types I, II and VI – X) can be used:
Type I: P ^m -C *
Type II: MLP ^m -C *
Type VI: P ^{m '} P ^m -C *
Type VII: MLP ^{m '} P ^m -C *
Type VIII: P ^m -LH-L'-C *
Type IX: P ^{m '} Pm-LH-L'C *
Type X: MLP ^{m '} P ^m -LH-L''- 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 (m') for detection. The protective group P ^{m '} can be split off selectively under conditions in which the protective group P ^{m is} stable. For example, 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. M, m and optionally m 'can be detected independently. They can also be split off independently. The markings are expediently also compatible with synthetic chemistry.

L to L '' mean any Spacers, e.g. organic groups, such as alkylene groups, optionally Heteroatoms such as O, N, P and S can contain.

H means a fissile group, e.g. an ester, disulfide, sulfone or diol group.

C * means a probe module, e.g. 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.

Of the compounds of group II are particularly types I and / or II prefers.

Around the inventive method to explain the individual types should be based on general examples from phosphoramidite chemistry and later on specific embodiments are illustrated. The connections described are marked that based on the emission of their one to three fluorescent labels, and partly based on their absorption, a quality assessment possible is. Let of course the executions also transferred to other synthesis strategies and / or labeling groups.

A preferred embodiment is as follows: First a labeled synthon is condensed onto the functionality of the support surface. After the fluorescence determination the markings are either at the next deprotection step, in the sense of a step-by-step biopolymer synthesis, split off, or on End of synthesis removed so that it can detect the detection signal Don't falsify hybridization can.

in the In the case of trityl-containing protective groups or marked photo-protective groups comes a second analysis, the spectroscopic analysis of the colored Trityl cations or the now colored (compared to the cleavage products conventional photo protection groups) or possibly fluorescent Cleavage products of the photo-deprotection process, for carrying. The data obtained are compared with an evaluation criterion and further processed after a positive assessment.

The types of compounds, for example phosphoramidites III-V, 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, for example two fluorescent dyes or fluorescence in combination with radioactive labeling, wherein m and M, which are part of the P ^{m '} P ^m , P ^m and MLP ^m' 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.

If the starting groups of the P ^{m '} P ^m , P ^m and MLP ^m' P ^m cleavage products, which enable spectroscopic online process control, are incorporated into nucleoside phosphoramidites, the compounds that can be used for a probe quality control are obtained.

The following preferred examples of the individual types of connections are possible:

The Conventional quality control methods for polymer synthesis (CE, HPLC, MS etc.) can essentially be taken into account the three-dimensional structure of arrays and in particular by Use of molecules of type VIII – X transferred to the products from the array synthesis, provided that the functionalization process of the surface is sufficiently high Functionalization density delivers.

worin M eine polycyclische Aryl- oder Heteroarylgruppe ist, A₁ und A₂ jeweils unabhängig ausgewählt sind aus H, O, OR, NHR oder NR₂, worin R eine C₁-C₂₀-Kohlenwasserstoffgruppe ist, die gegebenenfalls ein oder mehrere Heteroatome tragen kann, z.B. eine Alkyl-, Aryl-, Aralkyl- oder Alkarylgruppe und C* eine funktionelle Gruppe, z.B. ein Linker- oder Sondensynthonbaustein, ist. Die Gruppe M ist vorzugsweise eine Aryl- oder Heteroarylgruppe mit mindestens 3 oder 4 kondensierten Ringen, z.B. eine Pyrengruppe. Weiterhin ist M vorzugsweise eine fluoreszierende Markierungsgruppe, z. B. ein Coumarin, ein Pyren, Cy5, Cy3, ein Rhodamin oder ein fluoreszierendes Nanopartikel. Gegebenenfalls kann M über einen Spacer mit dem Grundgerüst verbunden sein.The invention further relates to compounds of the general structure (XI):

wherein M is a polycyclic aryl or heteroaryl group, A ₁ and A _{2 are} each independently selected from H, O, OR, NHR or NR ₂ , wherein R is a C ₁ -C ₂₀ hydrocarbon group which may optionally carry one or more heteroatoms can, 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. Furthermore, M is preferably a fluorescent labeling group, e.g. B. 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 A ₁ and A ₂ is preferably NHR or NR ₂ . At least one of A ₁ and A ₂ is particularly preferably a dialkylamino group, the alkyl radicals having 1 to 20 C atoms. Compounds (XI), in which A ₁ or / and A _{2 is} an NHR or NR ₂ group, surprisingly show a higher hydrolysis stability and thus better releasability and a higher color intensity.

worin M' eine Markierungsgruppe ist, Y eine Bindung oder ein Spacer mit einer Kettenlänge von bis zu 20 C-Atomen und gegebenenfalls einen oder mehreren Heteroatomen ist, A₁ und A₂ jeweils unabhängig ausgewählt sind aus H, O, OR, NHR oder NR₂, worin R eine C₁-C₂₀-Kohlenwasserstoffgruppe ist, die gegebenenfalls ein oder mehrere Heteroatome tragen kann, z.B. eine Alkyl-, Aryl-, Aralkyl- oder Alkarylgruppe und C* eine funktionelle Gruppe, z.B. ein Linker- oder Sondensynthonbaustein, ist. Vorzugsweise ist M' eine fluoreszierende Markierungsgruppe, z.B. eine Coumaringruppe ... .The invention further relates to compounds of the general structure (XII):

wherein 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 ₁ and A _{2 are} each independently selected from H, O, OR, NHR or NR ₂ , wherein R is a C ₁ -C ₂₀ hydrocarbon group which can 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 ₁ and A ₂ is NHR or NR ₂ , particularly preferably A ₁ or / and A _{2 is} a dialkylamino group, where an alkyl radical can contain up to 20 carbon atoms.

at Y is preferably a non-photolabile compound (XII) Structure, i.e. the group M 'can not split off from the basic structure of the connection by exposure become.

The Compounds (XI) and (XII) represent preferred examples of syntonic building blocks for use in a method as previously described.

An exemplary embodiment of a photolabile protective group, which can be used, for example, as group P ^m in type I compounds, is shown in 1 shown. It is an intramolecular triplet-sensitized o-nitrophenylethyl photoprotecting group that bears a pyrene residue. Other suitable groups of this type are, for example, in DE 102 60 592.0 described.

2 shows compounds of the type MLP ^m -C *, which can be used, for example, for compounds of type II. Pm is an optionally substituted trityl group to which a label M is optionally coupled via a linker.

The 3 to 12 show examples of compounds of type III (P ^m -LV (L'-H-L '' - 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.

13 shows a compound of type VI (P ^{m '} P ^m -C *), which is a fluorescent two-stage protective group.

In 14 a compound of type VII (MLP ^{m '} P ^m -C *) is shown, 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. There are also photoactivatable NppoC groups (P ^{m '} ) on the trityl group.

15 shows a compound of the 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).

Claims (31)

A method for producing a coated carrier, comprising the steps: - providing a carrier which has reactive groups on its surface, - building up a functionalized surface on the carrier by stepwise synthesis of a receptor, comprising a linker and a probe element made of synthon building blocks, characterized in that the synthesis process is monitored (i) by introducing one or more synthon building blocks with at least one detectable labeling group into the linker element and (ii) by introducing one or more synthon building blocks with at least one detectable labeling group into the probe element of the receptor. A method according to claim 1, characterized in that one uses synthon building blocks that have a labeling group contain. A method according to claim 1 or 2, characterized in that that one uses synthon building blocks that can detect several side by side Marker groups included. Method according to one of claims 1 to 3, characterized in that that marker groups to introduce into the linker element next to marker groups for an introduction to the probe element is detectable. Method according to one of claims 1 to 4, characterized in that that one, two, three or all Synthon building blocks for the synthesis of the linker element at least one detectable labeling group contain. Method according to one of claims 1 to 5, characterized in that that one, two, three or all Synthon building blocks for the synthesis of the probe element at least one detectable labeling group contain. Method according to one of the preceding claims, characterized characterized that the synthesis process is monitored online. Method according to one of the preceding claims, characterized characterized in that the probe elements of the receptor are selected from nucleic acids (in any synthesis direction, such as DNA or RNA, nucleic acid analogues, such as PNA or LNA, carbohydrates, peptides, derivatives of combinatorial chemistry and combinations thereof. Method according to one of the preceding claims, characterized characterized that monitoring includes an optical measurement. A method according to claim 9, characterized in that the optical measurement is a determination of absorption, emission, Diffraction, scattering or ellipsometry. Method according to one of the preceding claims, characterized characterized that monitoring a measurement of radioactivity includes. Method according to one of the preceding claims, characterized characterized that monitoring includes a measurement of plasmon resonance. Method according to one of the preceding claims, characterized characterized that monitoring includes an electronic measurement. A method according to claim 13, characterized in that electronic measurement is a determination of electron diffraction or electrical signals. Method according to one of the preceding claims, characterized characterized that you can build the carrier in an integrated Performs synthesis / analysis device. Method according to one of the preceding claims, characterized in that at least used a trifunctional synthon building block to build the linker and / or the probe. Method according to one of the preceding claims, characterized in that at least one synthon building block for incorporation into the linker element selected from one of the compounds (types I to VII) is used: type I: P ^m -C * type II: MLP ^m -C * Type III: P ^m -LV (L'-H-L '' - M) -L '''- C * Type IV: P ^m' P ^m -LV (L'-H-L '' - M) - L '''- C * Type V: MLP ^m' P ^m -LV (L'-H-L '' - M) -L '''- C * Type VI: P ^m' P ^m -C * Type VII : MLP ^{m '} P ^m -C * where P ^{m is} a protective group P which can carry a label m for detection, the protective group and label being compatible with synthetic chemistry, P ^{m' is} a protective group P orthogonal to P ^m , which is a Label m 'can be used for detection, where M denotes a label that can be used for detection, where m, M and optionally m' can be detected independently, and where the labels are compatible with synthetic chemistry, L-L "" any spacer mean, for example organic groups, H is a cleavable group, V is a trifunctional onelles molecule / atom and C * means a functional group or a functionalized support surface. A method according to claim 17, characterized in that at least one of the compounds of types (I) and / and (II) used. Method according to claim 17 or 18, characterized in that that C * means a linker block. Method according to one of the preceding claims, characterized in that at least one synthon building block for installation in the probe element selected from one of the compounds of types (I, II or VI to X) is used: Type I: P ^m -C * Type II: MLP ^m -C * Type VI: P ^{m '} P ^m -C Type VII: MLP ^m' P ^m -C * Type VIII: P ^m -LH-L'-C * Type IX: P ^{m '} P ^m -LH- L'C * Type X: MLP ^{m '} P ^m -LH-L''- C * where P ^{m represents} a protective group P which can carry a label m for detection, the protective group and label being compatible with synthetic chemistry, P ^{m 'is} a protective group P orthogonal to P ^m , which can carry a label m' for detection, the protective group and the label being compatible with synthetic chemistry, M denotes a label which can be used for detection, where m, M and optionally m 'are detectable independently, and the labels are compatible with synthetic chemistry, L-L''mean any spacer, H is a cleavable group and d C * means a probe module. A method according to claim 20, characterized in that at least one of the compounds of types (I) and / and (II) used. Compounds of the general structure (XI):

wherein M is a polycyclic aryl or heteroaryl group, A ₁ and A _{2 are} each independently selected from H, O, OR, NHR or NR ₂ , wherein R is a C ₁ -C ₂₀ hydrocarbon group which may optionally carry one or more heteroatoms can, for example an alkyl, aryl, aralkyl or alkaryl group and C * is a functional group. Compounds according to claim 22, characterized in that M is an aryl or heteroaryl group with at least 3 or 4 is condensed rings. Compounds according to claim 23, characterized in that M is a pyrene group, ... Compounds according to any one of claims 22 to 24, characterized in that at least one of A ₁ and A _{2 is} NHR or NR ₂ . Compounds of the general structure (XII):

wherein 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 ₁ and A _{2 are} each independently selected from H, O, OR, NHR or NR ₂ , wherein R is a C ₁ -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. Compounds according to claim 26, characterized in that M 'is a fluorescent Marker group is. Compounds according to claim 26 or 27, characterized in that M 'Coumarin, Is fluorescein, pyrene, Cy5, Cy3, rhodamine or a nanoparticle. Compounds according to any one of claims 26 to 28, characterized in that at least one of A ₁ and A _{2 is} NHR or NR ₂ . Compounds according to any one of claims 26 to 29, characterized in that Y is a non-photolabile structure includes. Use of compounds according to one of claims 22 to 30 as synthon building blocks in a method according to any one of claims 1 to 21st