WO2004089529A1

WO2004089529A1 - Method for the intramolecular energy transfer for the cleavage of labile functional group from biomolecules and the protected biomolecules

Info

Publication number: WO2004089529A1
Application number: PCT/EP2004/002361
Authority: WO
Inventors: Ulrich Steiner; Dominik WÖLL
Original assignee: Universität Konstanz
Priority date: 2003-04-07
Filing date: 2004-03-08
Publication date: 2004-10-21
Also published as: DE10315772A1

Abstract

The present invention provides a chemical compound and a method for the cleavage of labile functional groups from molecules by the interaction of electromagnetic radiation by the use of the chemical compound. Further, the invention provides a method for the manufacture of DNA chips by spatially addressed light directed nucleotide synthesis on solid substrates. The cleavage of the labile functional group is achieved by intramolecular energy transfer of the excitation energy.

Description

METHOD FOR HE INTRAMOLECULAR ENERGY TRANSFER FOR THE CLEAVAGE OF LABILE FUNCTIONAL GROUP FROM BIOMOLECULES AND THE PROTECTED BIOMOLECULES

The present invention relates to a new chemical compound, as well as to a method of cleaving labile functional groups from molecules by electromagnetic radiation and a method of manufacturing DNA chips by spatially addressed, light controlled nucleotide synthesis on solid substrates.

Methods of cleaving chemical bonds are well-known in chemistry, biochemistry, microbiology and related areas. The cleavage of chemical bonds occurs usually by the following three mechanisms:

Add: tion of thermal energy (thermal dissociation). Add: tion of a radiation energy (photochemical or photolytic dissociation). Add tion of electric energy in a suitable medium (electrolytic dissociation).

The energy required for bond cleavage is different for each chemical bond, even with identical bonding partners and depends especially on the respective chemical environment of each bonding partner.

Easy cleavage of functional groups from molecules plays an important role in many fields of chemistry and biology, like for example in the synthesis of larger chemical units such as in the synthesis of polymers, natural products etc.

In this process, highly reactive groups which may impair the respective intended linking of two molecules or interfere as a result of undesired side reactions are "masked" selectively or protected temporarily and reversibly by a functional protecting group to avoid their participation during the desired linking reaction.

The use of large combinatorial libraries of probe biomolecules immobilized on a substrate as binding partners of target biomolecules provided in a solution is very advantageous in the comparative study of molecular recognition of biomolecules of the same or different structural classes.

The term "biomolecules" refers to compounds of the classes comprising nucleic acids and their derivates, proteins, peptides and carbohydrates. Derivates of nucleic acids include DNA, RNA, LNA, PLA, and chimeras thereof.

Large combinatorial libraries, which use the principle of mutual molecular recognition are primarily of importance for the analysis of nucleic acids. DNA chips, i.e. so-called micro- arrays of spots of DNA or of any selected ohgonucleotide immobilized on glass or polymer substrates, which act as supermultiplex probes for molecular recognition by hybridization (S.P.A. Fodor, Science, 277 (1997) to 393, DNA Sequencing Massively Parallel Genomics) have already been in use in the field of medical research and pharmaceutical research for a long time.

In this field again, DNA chips play an important role in genetic analysis and diagnosis. The so-called spatially addressed, parallel light controlled ohgonucleotide synthesis on solid substrates (see for example S.P.A. Fodor et al., Nature (1993) 364 - 355, Multiplexed Biochemical Arrays with Biological Chips) using photolabile protecting groups, i.e. protecting groups for reactive functionalities of the nucleoside or nucleotide building blocks, which can be selectively cleaved, primarily by use of UV light of a certain wavelength for the protected functionalities to be available again for further reactions, forms the most widely used technique of manufacturing said DNA chips. DNA chips are manufactured by using the above-mentioned technique referred to as photolithography. In this technique, synthesis of the desired ohgonucleotide chains on the substrate is controlled by suitable labile protecting groups which release the bonding site for the next nucleotide upon exposure (primarily using electromagnetic radiation in the UV/VIS range). Until now, these protecting groups have all preferably been photolabile. These photolabile protecting groups can be used to develop a combinatorial strategy by means of spatial, selective exposure to irradiation which produces extremely dense, spatially addressable microarrays of oligonucleotides whose number grows exponentially as the number of synthesis cycles increases. The currently achievable surface area of each element of less than 50 μm² can theoretically accommodate more than 10⁶ probe fields in 1 cm². One method was performed by means of micromirror arrays (S. Singh- Gasson et al., Nature Biotechn. 17 (1999) 974, Maskless fabrication of Light Directed Ohgonucleotide Microarrays using a Digital Micromirror Array), like those used in digital projection technology. This avoids time consuming and expensive fabrication of exposure masks and makes it possible to manufacture DNA chips more rapidly by means of a photolithography.

Currently used photolabile protecting groups still do not yield satisfactory results with respect to the error rate of DNA chips synthesized in this manner (D. J. Lockheart and E. A. Winseler, Nature 405 (2000) 827, Genomics, Gene expression and DNA arrays). The cleavage of protecting groups is not complete enough, as these groups often only exhibit a low capacity for absorbing the UV/VIS wavelength used. Beyond that, partially excited or fully excited protecting groups lead to interfering secondary reactions with undesired reaction products, to the effect, that the bulk of oligonucleotides on the DNA chips cannot be used.

A central point in photolithographic synthesis consists of the use of photolabile protecting groups employed in many chemical variations in organic chemistry and bioorganic chemistry (V.N.R. Pillay, Photolithic Deprotection and Activation of Functional Groups in: Organic Photochemistry, Vol. 9, ed. A. Padwa (Marcel Dekker, New York and Basel, 1987), page 225 and following). The most widely used photolabile protective groups are those based on the 2- nitrobenzyl group (J.E.T. Correy and E.R. Trenton, Caged Nucleotides and Neurotransmitters; in: Biological Applications of Photochemical Switches, in: Bioorganic Photochemistry Series, Vol. 2, ed. Harry Morrison (Whiley Interscience, 1993), page 243 and following).

Until now, the MeNPOC (α-methylnitropiperonyloxycarbonyl) protecting group, which is among the standard protecting groups in DNA chip fabrication, has been preferred among other protecting groups of the 2-nitrobenzyl type in the manufacture of DNA chips, for example, when protecting the terminal 5' OH group during ohgonucleotide synthesis from the 3' to the 5' or from the 5' to the 3' terminus (S.P.A. Fodor et al, Science, 251 (1991), 767, Light Directed, Spatially Adressable Parallel Chemical Synthesis).

The disadvantage of this type of protecting groups lies in the formation of an aromatic nitrosoketone, a very reactive leaving group, after cleavage. This leads to undesired secondary reactions which often cause errors in the nucleotide structure of the resulting ohgonucleotide or polynucleotide. Recently, the DMBOC group (3',5'-dimethoxybenzoinyloxycarbonyl-) has been used as protecting group in polynucleotide synthesis (M.C. Pirrung et al., J. Org. Chem. 63 (1998), 241, Proofing of Photolithographic DNA Syntheses with 3',5'- i dimethoxybenzoinyloxycarbonyl-protected deoxynucleosidephosphoramidites).

Upon cleavage of the DMBOC protecting group, a benzofurane derivative is formed. The benzofurane derivative is fluorescent and can be used for example as an indicator for the progress of the photoreaction. I

Most of the currently known photolabile protecting groups require an irradiation with the 365 nm line of a mercury lamp for several minutes with usual irradiation intensities for a quantitative reaction.

i Beyond that, 2-(2-nitrophenyl)-ethoxycarbonyl compounds, in which the protective groups are cleaved as 2-nitrostyrene derivatives, are known in the manufacture of DNA chips (DE- PS 44 44 996, DE-PS 196 20 170 and US-5,763,599). The cleavage of 2-nitrostyrenes which are generally less reactive also makes these compounds less prone to interfering secondary reactions than the compounds mentioned above, but they still require irradiation at 365 nm.

I

Therefore, the object of the present invention was to provide chemical compounds which enable a loss-free energy transfer, from one "excitation site" to a labile functional group to cleave at least one chemical bond between the labile functional groups and a covalently bonded substrate or molecule, to suppress interfering secondary reactions.

I

This object is solved by providing a chemical compound which comprises the structural motif

S-(L,)_a-B-(L₂)_b-R

I ,wherein S represents a sensitizer synthon, which first excited electronic state is energetically higher than the first excited electronic state of the labile functional group P (also termed as "protecting group synthon"). The presence of conjugated π-systems or conjugated double bonds is especially preferred. It is important, that the sensitizer synthon comprises at least three conjugated double bonds. The double bonds may be either -C=C- double bonds, or double bonds comprising hetero atoms as for example -C=N-, -C=S-, -C=O-, -N=N- or combinations thereof.

In an especially preferred embodiment, the sensitizing synthon comprises the structural motif,

wherein Y = O, S, N, Se or Te, n is 1 or 2, C is a part of an aromatic, heteroaromatic or condensed aromatic or heteroaromatic system, or the structural motif has two aromatic, heteroaromatic or condensed aromatic or heteroaromatic systems and with the proviso that in case that n = 2, then the aromatic, heteroaromatic or condensed aromatic or heteroaromatic system may be the same or different.

Especially preferred are substituted or unsubstituted benzophenone, acridone and xanthone derivatives, according to formula I and II, most preferred are thioxanthone derivatives:

I II

wherein X = O or S, and Y represents O, S, N-R', R' being a monovalent radical like a C| -C alkyl group, C_y-C_y alkoxy or an aryl-group, an ester or an amide group and R represents a substitution pattern at different sites which may be from 0 to 4 substituents which may be the same or different chosen for example from the same class as defined for R'.

The preferred structural motif according to the invention, preferably a motif comprising formula I or II provides an effective intersystem crossing in the excited triplet state, a long triplet lifetime of more than 0.6 microseconds (μs), especially of more than 1 microsecond (μs). Furthermore, the structural motif imparts chemical inertness to the chemical compound in its excited state which is therefore essentially unreactive in this state.

P is a labile, preferably a photolabile functional group (protecting group synthon), selected from the group consisting of

a) compounds of the general formula

wherein R₃ is H, NO , a Ci to C₄ alkyl group, preferably methyl, ethyl, propyl, R₄ is H, NO₂, halogen, C_\ to C alkyl, preferably methyl and ethyl, a substituted or non-substituted aryl group, an acyl, alkoxycarbonyl or acylamino group, R₅ and Re are independently from one or another a H atom, NO₂, halogen, an acyl, alkoxycarbonyl or acylamino group, an alkyl rest of up to 4 C atoms, or a substituted or non-substituted aryl group, or an alkoxide rest with up to 4 C atoms or R₅ and R₆ together form a methylenedioxy group, R₇ and R₈ are independent from one another H, N0₂, halogen, a Ci to C₄ alkyl or alkoxide group, and n represents a number selected from 0, 1, 2, and wherein R₃ to R₈ alone or several together have the meaning of the following group Li, m is 0 or 1 and in the case that m is 1, Z_m is selected from the group consisting of OC(O), OC(S), SO₂, NR'C(O), wherein R' is a Ci to C₆ alkyl, preferably methyl, ethyl or propyl,

b) substituted and unsubstituted, condensed and non-condensed 2-(nitroaryl)ethoxycarbonyl or -thiocarbonyl compounds, substituted and unsubstituted, condensed and non-condensed 2- nitrobenzyl, 2-nitrobenzyloxycarbonyl or thiocarbonyl compounds, substituted and unsubstituted, condensed and non-condensed 2-(nitroheterocycloaryl)ethoxycarbonyl or -thiocarbonyl compounds, and substituted and unsubstituted, condensed and non-condensed 2-(nitroheterocycloalkyl)ethoxy-carbonyl/thiocarbonyl compounds, substituted and unsubstituted 2-nitro-N-methylanilinocarbonyl- or thiocarbonyl derivatives, Li and L₂ are linker groups, which may be the same or different from one another and are selected from the group consisting of OC(O), SC(O), S0₂, SC(S), NHC(O), NR'C(O), wherein R' is a Ci to C₆ alkyl, preferably methyl, ethyl or propyl, and with the proviso that these meanings are not valid for L₂ if m is 1, CR₂, wherein R is selected from H and/or a branched or linear Ci to C₅ substituted or non-substituted alkyl group, wherein in the case of a substituted alkyl group R is selected from the group consisting of OC(O), SC(O), SO₂, SC(S), NHC(O), NR'C(O), wherein R' is a Ci to C₆ alkyl, preferably methyl, ethyl and propyl, or one or more conjugated or non-conjugated C=C double bonds or one or more C≡C triple bonds,

R is a usual reactive functional group, preferably a nucleofuge or electrophilic group, which can react with a suitable substrate N, preferably at a nucleophilic group of the substrate N. R can for example be a halogen atom like CI, Br, I or a derivatized or non-derivatized alcohol, thioalcohol, a carboxyl, thiocarboxyl group. As an alcohol, activated alcohols like for example nitrophenol, hydroxybenzotriazole or pentafluorophenol are preferred. Further, R can be a heteroaromatic or a substituted or a non-substituted azolide, as for example imidazole, triazole, tetrazole, pyridine. R can also form together with L₂ an acetimidate, for example a trichloroacetimidate. Also, R can be a carbonic acid imidazolide, -triazolide, - tetrazolide, pyridinium salt, an ester or ether, activated ester or substituted or non-substituted amino group,

and wherein a and b are a number > 0 and are the same or different from one another.

In another preferred embodiment of the invention, R can also be the substrate N.

The substrate N is for example a nucleoside, an oligo or polynucleotide, an amino acid, an oligopeptide, a carbohydrate and the like. Surprisingly, the chemical compounds according to the invention allow efficient intramolecular energy transfer from a sensitizer synthon to the labile functional group by the combination over a covalent chemical bond. The sensitizer synthon, acts as an "acceptor" for electromagnetic radiation or energy. Thereby, the energy necessary for bond cleavage is delivered without losses to the labile functional group. ^' If the chemical compound according to the invention is linked to a substrate, a selective bond cleavage between the chemical compound according to the invention and the substrate as a predetermined cleavage position between the protecting group synthon and a substrate occurs.

> The term "substrate" as used herein comprises chemical compounds and functional reactive groups on surfaces like hydroxyl, thiol, amino group and the like, more preferably oligonucleotides, proteins, peptides, carbohydrates (sugars) which may form a covalent chemical bond with the compound according to the invention.

) The object of the present invention was further to provide a method wherein the cleavage (which can also be termed as "removal") rate of labile functional groups (that is the reaction time of the cleavage reaction) is considerably decreased and the cleavage reaction is optimized with respect to its yield. A further objective was to minimize the risks of undesired secondary reactions upon cleavage of the labile functional group.

>

This second objective of the present invention is solved by a method for the cleavage of labile functional groups from molecules comprising the following steps:

a) Selection of a sensitizer synthon S and a suitable labile functional group P, whereby ) the first excited electronic state of the sensitizer synthon S is energetically higher than or at the same energy as the first excited electronic state of the labile functional group P.

b) Synthesis of the chemical compound according to the invention by the formation of a

> intramolecular chemical bond between the sensitizer compound S and the labile functional group P.

c) Reaction of the chemical compound according to the invention with a substrate.

) d) Exposure of the reaction product of step c) to electromagnetic irradiation.

The formation of the intramolecular chemical bond in step b) is achieved by usual methods or for example a classical -C-C- bond formation reaction like a Suzuki, Stille or Heck reaction or through ester or amide linkages between sensitizer S and labile functional group P. In the method according to the invention, the sensitizer synthon is excited by electromagnetic irradiation, and transfers the absorbed electromagnetic radiation/energy by a transfer between an excited state of a sensitizer synthon and an excitable state of the functional group intramolecularly to the labile functional group, inducing a cleavage reaction thus removing the labile functional group fast and efficiently.

For the purpose of the present invention it makes no difference, if the method is carried out in solution or on a solid phase, for example on a solid carrier where the molecules containing the labile functional groups are coupled to. Thereby, the method is well suited for a plurality of reactions, for example in the syntheses of oligonucleotides or oligomers or polymers, because especially in the syntheses of polymers and in oligonucleotides, a plurality of undesired secondary reaction occurs like chain or strand breaking, crosslinking, photoaddition reaction of adjacent nucleotide bases and the like.

Depending on the selection of the labile functional groups and the corresponding sensitizer synthon, the respective absorption maximum for electromagnetic radiation is determined. This allows to select the best corresponding wavelength of electromagnetic radiation from the electromagnetic wavelength spectrum.

It is especially preferred that the electromagnetic radiation has a wavelength, which is only in the region of the absorption maximum of the sensitizer synthon. Thereby, only the sensitizer synthon is excited and undesired side reactions do not take place. These side or secondary reactions are induced by the portion of the radiation being absorbed by the substrate of the labile functional group. Further, this preferred embodiment generates a more efficient intramolecular energy transfer from the sensitizer synthon to the labile functional group. Therefore the reaction rate of the cleavage/reaction increases and the yield is considerably improved because undesired secondary reactions do not occur. Furthermore, purification steps due to the separation of the reaction products from undesired side products can be avoided.

It is preferred that the electromagnetic radiation is in the region of the wavelength of UV/VIS radiation (210 - 450 nm). Thereby, the method according to the invention can be used in the manufacture of ohgonucleotide and peptide chips by using common mercury vapor lamps. It is understood, that also other irradiation sources essentially known to an artisan can be used within the context of the present invention.

It is especially preferred, that the first excited singlet state of the sensitizer synthon is equal or lower than the first excited singlet state of the labile functional group. Thereby, the wavelength, and therefore the energy is shifted to a certain area, also termed as a "window" of the electromagnetic spectrum, where the occurrence of secondary reactions, especially in the manufacture of DNA chips can be further minimized.

It is especially preferred, that the triplet-singlet energy gap of the sensitizer synthon is smaller than the triplet-singlet energy gap of a labile functional group. Preferably, the lowest electronic excited state (Ti) of the sensitizer synthon is energetically as high or higher as the lowest electronic excited state (Tj) of the labile functional group. Further, it is preferred that the energy gap of the sensitizer synthon between the state (S|) which corresponds to the light absorption at the longest wavelength and the lowest electronic excited state (Ti) is the same or lower than a corresponding energy gap of the labile functional group. The absorption maximum at the longest wavelength of the sensitizer synthon is at higher wavelength than the absorption maximum at the longest wavelength of the labile functional group. It is preferred that the chemical compound has a high triplet formation quantum yield φr close to the maximum size of 1.

The objective of the present invention is further solved by a method for the manufacture of molecular libraries comprising biomolecules, especially for the manufacture of DNA chips and peptide chips and their analoga and mimetics thereof via spatially addressed light directed synthesis on solid substrates, comprising the following steps:

a) reaction of the un-protected terminal 3' or 5' hyrodroxyl group of a nucleoside and/or nucleotide or nucleic acid analogon or a -COOH or amino group of a suitable peptide arranged on the solid substrate under usual conditions essentially known to an artisan with a chemical compound according to the invention and if necessary purification of the reaction product,

b) selective irradiation of a portion of the reaction product from step a), c) reaction with a nucleoside and/or nucleotide where a free 5' or 3' OH group is protected by a chemical compound according to the invention or with a photolabile group, or reaction with a suitable peptide or with a suitable amino acid,

d) if necessary repetition of steps b) to c).

The application of all of the compounds takes places via usual methods like doctor blades, spraying, jetting, dropping etc. wherein the chemical compound according to the invention can be added in its pure state, in solution, suspension or dispersion.

The selective irradiation in step b) is preferably spatially selective, wherein for example a portion of the surface of the solid substrate is irradiated with electromagnetic radiation, most preferred with radiation in the UV/VIS region.

Thereby, the reaction rate for the cleavage reaction is increased, because the electromagnetic radiation which is absorbed by the sensitizer synthon of the chemical compound according to the invention is transferred intramolecularly from the excited state of the sensitizer synthon to the labile protecting group thereby inducing a selective bond cleavage reaction. The cleavage in the presence of a compound according to the invention takes place much faster (typically 5 to 10 times) at a selected irradiation intensity as with methods in the prior art.

Preferably, the labile functional group is a UV/VIS photolabile group, because suitable radiation wavelength are easily provided. However, also any other groups can be used, which can be irradiated with another electromagnetic radiation of another wavelength, as for example infrared, or radiation with a longer or shorter wavelength.

The method according to the invention is not only suitable for the synthesis of DNA and RNA nucleotides. Also, the synthesis of polynucleotides from nucleic acids analoga, like PNA, LNA or chimera thereof with DNA, RNA or nucleic acid analoga are possible, as well as in solution as on a substrate or chip. It is understood, that the method can also be applied for the synthesis of polypeptides, carbohydrates and other molecules.

The methods according to the invention are especially useful in a automated method. Preferably, such an automated method is designed as a parallel synthesis on a substrate for the manufacture of a nucleotide library, whereby the chemical compounds and the labile protecting group respectively to be used in the method can be deliberately selected.

In a further embodiment of the invention, the present invention comprises a kit, which

> comprises a portion or all reagents and/or adjuvants and/or solvents and/or a working instruction for carrying out one of the methods according to the invention in one spatial unit, whereby the kit comprises at least one or more selected nucleotides, which have preferentially a free 5'-hydroxy function and a protected 3'-hydroxy function or a free 3'-hydroxy function and a protected 5'-hydroxy function. In a further embodiment of the invention, the kit

) comprises suitable peptides and/or amino acid derivatives with protected amino and free carboxyl group or vice versa. These kits allow for the simple realization of the methods according to the invention in solution or, preferably, on substrates.

In still a further embodiment of the invention, the present invention comprises the use of the

> method according to the invention and/or the above-mentioned kits for the manufacture of oligonucleotides or nucleic acid chips, preferably for the automated manufacture of oligonucleotides or nucleic acid chips.

Further advantages and embodiments of the present invention are explained in the description ) and in the figures.

It is understood, that the aforementioned features and the features which will be explained in the following are not only used in the specific combination as mentioned, but also in other combinations or as alone without departing from the scope and the spirit of the present

> invention.

Abbreviations

NPPOC 2-(2-nitrophenyl)propyloxycarbonyl

) MeNPPOC 2-(3, 4-methylendioxy-2-nitrophenyl) propyloxycarbonyl

MeNPOC 2-(3, 4-methylendioxy-2-nitrophenyl) oxycarbonyl

DMBOC dimethoxybenzoinyloxycarbonyl

NPES 2-(2-nitrophenyl)ethylsulfonyl

NPPS 2-(2-nitrophenyl)propylsulfonyl Definitions

The term "nucleotide" as used herein denotes oligonucleotides with at least up to 10 nucleosides, which are linked via 3'-5' as via 5'-3' phosphoric acid ester bonds. The 5 nucleotides according to the invention comprise however also polynucleotides with more than 10 nucleoside building blocks.

The term "synthon" denotes either isolated building blocks or a portion of a chemical compound to be synthesized from several identical or different building blocks or portions. ) An isolated synthon has usually reactive functional groups. The functional groups of a synthon allow for a precise and defined chemical reaction with another functional group from a second synthon.

The invention is explained in the following with the aspect to the following figures and some 5 examples which are not meant to be limiting.

Figure 1 shows in figures la through Id synthetic routes to link a sensitizer and a photolabile protecting group.

) Figure 2 shows in figures 2a and 2b each the reaction of two linked sensitizer/photolabile- protecting-group compounds according to the invention with a substrate.

Figure 3 shows in figure 3a and 3b each a chemical compound according to the invention linked to a substrate. >

Figure 4 shows in Fig. 4a representative examples for the linking site of protecting group synthons to sensitizer synthons (Sens) and in Fig. 4b representative examples of compounds according to the invention making use of the linking sites as shown under Fig. 4a.

)

Figure 5 shows absorption spectra of compounds according to the invention

Figure 6 shows photokinetic decay curves of compounds according to the invention under irradiation at 366 nm in MeOH. Figure 7 shows in Fig. 7a to 7 c biochips made with TS8-T amidite.

Figure 1 shows exemplary syntheses for the linkage of a sensitizer synthon with a photolabile protecting group. The sequence of reaction steps shown in figure la which is familiar to the artisan yields the amino substituted photolabile protecting group (16). Acridone is reacted with ω-halogencarboxyl acids to the respective homologues with a longer alkyl chain. The number of the methylene groups x is an integer between 2 and 10. It is understood, that also suitable substituted ω-halogencarboylic acids, for example α-amino-ω-halogencarboxyl acids and the like can be used within the present invention. The protecting group is coupled to an N-carboxalkyl acridone (11,18) via an esteric bond to yield the linked compound (17). In figure lb, an example is shown that demonstrates how the linkage to the photolabile protecting group is effected through Heck coupling at an aromatic ring of the sensitizer. Figure lc shows how to link acridone and a photolabile 2-nitrobenzyl compound by an oligomethylene chain between the ring nitrogen of acridone and the benzylic carbon of the photolabile protecting group. Reaction step (a) is carried out as described in the literature (Dadabhoy A., Faulkner S., Sammes P.G., J Chem. Soc, Perkin Trans. 2, 2002, 348-357), analogous reactions to reaction step (b) have been successfully performed in our laboratory. Selective reduction of the ester group (c) is done with NaBH in appropriate solvents to yield the linked compound (27) but any other suitable reductive agent may be used as well. An oligomethylene linkage with variable chain length, whereby n is an even number >1, between the acridone nitrogen and the aromatic ring of the 2-nitrobenzylic compound is obtained as shown in figure Id. First, acridone is substituted with a ω-bromo-1-alkene. Hydroboration of the double bond with 9-borobicyclo[3.3.1]nonane (9-BBN) followed by SUZUKI coupling with 2-(5-bromo-2-nitrophenyl) propanol yields the linked compound (28).

Figure 2a shows an exemplary synthesis of a compound according to the invention with different linkers between sensitizers synthon and protecting group synthon and subsequent reaction with a substrate. The variation of the length and the nature of the linker between sensitizer synthon and protecting group synthon allows for fine tuning with respect to energy transfer, conformational stability etc. The respective chlorocarbonic acid ester was obtained by reaction with phosgene or diphosgene. The subsequent coupling of the compound according to the invention to a substrate, for example to the 5' OH group of a 2'- deoxynucleoside, took place via a method essentially known in ohgonucleotide chemistry. The same applies for the coupling in figure 2b (see example 4).

Figure 3 shows in figure 3a and 3b compounds according to the invention on a substrate which have suitable structures ("triade") for the energy transfer upon excitation, for example via UV/VIS irradiation via intramolecular triplet sensitization. The term "triade" denotes within the present invention a chemical unit, comprising a sensitizer synthon, a labile functional group and a substrate.

After excitation of the sensitizer synthon by irradiation of suitable wavelenght, the sensitizer synthon in figure 3a changes via intersystem crossing (ISC) from an excited singlet state in the triplet system and relaxes in the lowest excited triplet state. It is understood, that the same applies for every other protecting group synthon, like benzophenone or thioxanthone derivatives (see figure 3b and example 5). The energy of the triplet state is transferred via triplet triplet energy transfer to the protecting group synthon, whereby the sensitizer synthon and the protecting group synthon are linked by a bridge. After transfer of the energy to the protecting group synthon, the cleavage of the bond between the substrate and the photolabile proctecting group (location C) occurs, so that the substrate can be used selectively for further reactions.

A non-limiting selection of labile reactive groups according to the invention, i.e. so-called "protecting groups", which can be used as a protecting group synthon in the manufacture of the compounds according to the invention are mentioned below. The compounds were synthesized via synthesis methods essentially known to an artisan:

2-(2-chloro-6-nitrophenyl)ethanol, 2-(2-nitrophenyl)propanol, 2-(2-nitrophenyl)ethanol, 2-(4-bromo-2-nitrophenyl)propanol, 2-(5-chloro-2-nitrophenyl)propanol,

2-(5-bromo-2-nitrophenyl)propanol, 2-(5-iodo-2-nitrophenyl)propanol

For further protecting groups or protecting group synthons used within the invention it is referred to figure 4 and the examples below.

It has been found, that sensitizer synthons according to the invention which are suitable for carrying out the methods of the invention have the following non-limiting properties: The sensitizer synthon absorbs preferably at a longer wavelength than the labile protecting group, that is, its first excited singlet (Si) state lies below the first excited singlet (Si) state of the protecting group, i

Further, the sensitizer synthon has at its absorption band at the longest wavelength an absorption coefficient which is as high as possible.

The lowest excited state, that is, the first triplet state (Ti) of the sensitizer synthon lies above ⁾ the lowest excited state, that is the first triplet state (Ti) of the labile protecting group, in the extreme it is energetically similar as the latter. Therefore, the energy gap between the lowest excited singlet (Si) state and the lowest excited triplet (Ti) state of the sensitizer synthon is preferably smaller than of the photolabile protecting group.

> When using nitrophenyl chromophores as portion of the labile protecting groups, the labile protecting group has an energy gap usually in the range of about 130 kJ/mol, so that a plurality of sensitizer synthons according to the invention can be used.

The sensitizer synthon has further a high triplet forming quantum yield φ-r, which contributes ) linearly to the sensitizing efficiency.

Further, the separate sensitizer synthon has a lifetime of the excited state which is as long as possible, in order to provide a high energy transfer efficiency. It has been found, that for a quantitative intermolecular energy transfer with a preferred energy range of the excited state, i a lifetime of more than 0.6 μs is sufficient, especially preferred is a lifetime of more than 1 μs.

The quantum yield φ of the chemical reaction according to one of the methods mentioned before upon cleavage of the chemical compound according to the invention at the labile ) functional group is in specifically preferred embodiments of the invention larger than 0.5.

Non-limiting examples of basic structures of sensitizer synthons, which are useful as synthons for the synthesis of compounds according to the invention are mentioned in the following table 1 : Table 1:

Basic structures of sensitizer synthons

It is understood, that also the substituted derivatives of the compounds mentioned in the table 1 can be used in the context of the present invention, as for example the to C₅ alkyl substituted derivatives, especially the 2 alkyl derivatives, like 2-ethylthioxanthone, 2-and 4 isopropylthioxanthone . Other examples are halogen, ether, ester and amide substituents in all possible positions at the aromatic rings.

The energy (E) of the singlet and triplet state is given in kJ/mol. The absoφtion coefficient ε is given in M^"1 x cm^"1 at the respective wavelength , n stands for a non-polar, p for a polar solvent and b for a solvent of the benzene type. τ means the lifetime of the triplet state in μs and φr means the quantum yield of the intramolecular S|/T| transformation.

Further sensitizer synthons for the manufacture of compounds according to the invention comprise but are not limited to N-methylacridone, 2-ethylthioxanthone, 2-anilino- naphthalene, naphtho-[l,2-c] [l,2,5]-thiadiazole, benzo-[b]-fluorene, 5,7-dimethoxy-3- thionyl-coumarine, 1 ,2-cycloheptandione, 3-acetyl-6-bromo-coumarine, 2-bromo-9-acridone, 4,4'-dibenzylbiphenyl, 2, 6-dithiocoffeine, 1 ,4-dibromonaphthole,

10-phenyl-9-acridone, 2-methyl-5-nitro-imidazole-l-ethanol, l-(2-naphthoyl)-aziridine, 9-(2- naphthoyl)-carbazole, 4,6'-diamino-2-phenyl-benzooxazole, p-thiophenyl, 3-acetyl- phenanthrene, dinaphtho-[l,2-b:2',r-]-thiophene, (E)-piperylene, β-methyl-(E)-styrene, 2- phenyl-benzothiazole, chinoxaline, 9,9'-biphenantryl, naphtho-[l,2-c] [l,2,5]-oxabiazole, phenothiazine, 2-ethoxy-naphthol, 9-phenyl-9-stibafluorene, 9,10-antraquinone, 4,4'- dichlorodiphenyl and their substituted derivatives.

Further sensitizer synthons useful for the puφose of the present invention are for example disclosed in S. L. Murov ,1. Carmichael and G. L. Hug, Handbook of Photochemistry, Marcel Dekker, Inc., New York 1993, the disclosure being incoφorated herein by reference.

Experimental:

The term "usual conditions essentially known to an artisan" as used herein is for example described in US-5,763,599 or DE 44 44 956.

All reactions with moisture sensitive reagents were performed in dried glassware which were cooled down in a stream of nitrogen. All reactions with light sensitive compounds were performed in glassware covered with aluminium foil. NaH (60 % dispersion in mineral oil) was activated by washing with light petroleum ether (3 x 5 mL on 1 g of NaH) prior to use.

Absorption spectra

UV/VIS absoφtion measurements were carried out with a Gary Lambda 18 spectrometer. Laser flash spectroscopy

Irradiation for the laser flash spectroscopy was carried out with a Nd-YAG laser (Spectra Physics Quanta Ray GCR 150, repetition rate 5 Hz, pulse width 4 - 6 ns) which was operated with a tripled frequency (355 nm). The pulse energy was set to 100 mJ, upon use of grey filters the pulse energy was further reduced and adapted to the respective experiment. Laser and detection light beams crossed in a measurement cell under an angle of circa 7°. The detection system consisted of a pulseed Xenon arc lamp (Osram XBO 150) with a power supply built by the laboratory of the University of Konstanz, f/3.4 monochromator (Applied Photophysics), a photomultiplier (Hamamatsu R955) and a CCD camera (Princeton Instruments). The photomultiplier signal was recorded with a LeCroy 9354 A digital oscilloscope and transferred for data analysis to a PC. The data of the CCD camera were analyzed by the program WinSpek/32. A specially designed trigger generator was used to control the cycle time and for the synchronization of the components. The sample was either in a cylindric flow-through cell or in a cuvette made of quartz glass with an optical length of 10 mm. The tubes of the flow-through system are conducted in an exterior tube flushed with nitrogen. The flow rate was such, that at each third laser flash a complete change of the probe in the cell took place.

Irradiation apparatus

The irradiation apparatus consisted of a mercury high pressure lamp (200 W), a heat filter (optical length 5 cm, filled with saturated CuS0₄ solution in water), a collimator lens, an electronically operated shutter, a 366 nm interference filter (Schott) and a cuvette holder for irradiation cuvettes (Hellma QS, 1 cm).

The intensity of the UV lamp was measured daily. As an actinometer system, a methanolic azobenzene solution was used. The photon irradiance of the samples at a wavelength of 366 nm was in the range of 3 • 10^"8 E cm^'V.

The substances were dissolved in the respective solvents (approximately 10 minutes in an ultrasonic bath). With a pipette, 3 ml of the solution were given in a flushable cuvette with a magnetic stirrer. The solutions, which should be oxygen-free were flushed for 15 minutes with nitrogen. Afterwards, the content of the cuvettes were irradiated for times indicated below. Before and after irradiation, an absoφtion spectrum against the respective solvent was recorded. For the puφose of HPLC analysis, the solution was transferred into a micro test tube and put into the autosampler of the HPLC apparatus. The HPLC column was charged with samples of 20 μl each and eluted in a gradient, from water to a water/acetonitrile mixture (1 :1) and further to pure acetonitrile. The analysis of the data detected by an UV diode array was made with the program "HSM manager".

HPLC

The HPLC analyses were carried out with an apparatus from Merck-Hitachi. The apparatus comprised a pump L-7100, an autosampler L-7200, a UV diode array detector L-7450A and an interface L-7000. As a column, a LiChrospher 100 RP-18 (5 μm) from Merck was used. The column was operated by a HSM manager with a Compaq computer.

Esample§

Example 1: Synthetic Route to compound TS4T

1. Synthesis of oNPin-COOMe (2-(2-nitrophenyl)-pent-4-ynoic acid methyl ester)

C₉HgN0₄ C₃H₃Br C₁₂Hι,N0₄ ol. Wl.: 195.17 Mol. Wl.: 118.96 Mol. Wl.: 233.22

The reaction was carried out under nitrogen. o-Nitrophenyl acetic acid methyl ester (3.21 g, 0.0165 mol) was dissolved in 20 ml of THF and propragylbromide (1.3 ml, 2.04 g, 0.0172 mol) was added. The solution was cooled to -80°C and potassium tert-butylate was added under agitation, which led to a deep blue color. The solution was allowed to come to room temperature and stirring was maintained for 12 hours. The blue color lost its intensity and changed to brown violet. At room temperature, water (circa 30 ml) was added to the solution, followed by extraction with diethyl ether (3x), washing with water (lx), drying over MgSO and removal of the solvents. The raw product (3 g) was separated by column chromatography (silica gel, conditioned with petrol ether (PE):ethyl acetate (EE)= 3:1, gradient) into three fractions. 1.84 g of pure product were obtained.

Yield: 1.84 g, 7.89 mmol, 48 % Characterization: orange oil

Η-NMR (400 MHz, CDC1₃): δ 7.98 (dd, ³J=8.2, ⁴J=1.4, IH, H-atom ortho to NO₂), 7.61 (td,

IH, H-atom para to CHR₂), 7.52 (dd, 0J=7.8,

IH, H-atom ortho to CHR₂), 7.47 (td, ³J=7.7, ⁴J=1.5, IH, H-atom para to N0₂), 4.45 (dd, J=8.3; 6.4, IH, Ph- CHR₂), 3.69 (s, 3Η, CH₃), 3.05 (ddd, J=17.0; 6.2; 2.7, 1Η, RCHΗR), 2.85 (ddd, J=17.0; 8.3; 2.7, 1Η, RCΗHR), 1.95 (t, J=2.5, 1Η, CCH).

2. Synthesis of oNPin-OΗ (2-(2-nitrophenyl)-4-pentine-l-ol)

The ester (1.84 g, 0.0789 mmol) obtained in example 1 was dissolved in 25 ml tert-butanol and an excess of sodiumborohydride (525 mg. 0.0139 mol) was added. The suspension while stirring was heated to 80°C and for 4 hours, methanol (3.4 ml) was added dropwise. A gas development was observed. After a further hour of stirring at 80°C, the solvent was allowed to cool down. Water (30 ml) was added, the aqueous phase extracted 3 times with diethylether, the organic phase washed with saturated NH₄C1 solution. The combined organic phases dried over MgS0₄ followed by removal of the solvents. Yield: 1.26 g, 14 mmol, 78 % Characterization: red viscous oil

Η-NMR (250 MHz, CDC1₃): δ 7.79 (dd, IH, H atom ortho to NO₂), 7.61-7.35 (m, 3H, aromatic H atoms), 3.95 (m, 2H, CH₂), 3.60 (quintet, 1Η, Ph-CHR₂), 2.80-2.56 (m, 2Η, CH₂), 1.97 (t, J=2.5, lΗ, CCH). 3. Synthesis of TS4-OH (2-(2-nitrophenyl)-5-(9-oxo-thioxanthene-2-yl)-4-pentine-l-ol)

CnHnNOa C₁₃H₇BrOS Mol. Wt: 205.21 Mol. Wt.: 291.16

The reaction took place under nitrogen. 2-bromothioxanthone (735 mg, 2.52 mmol) was dissolved under gentle heating in 20 ml THF. Cul (32 mg), Pd(PPh₃)₄ (48 mg), triethylamine (5 ml, dry, stored over molecular sieve under N₂) were added to the solution. The ethine (511 mg, 2.49 mmol) dissolved in 5 ml THF were added through a dropping funnel over a period of 2 hours at room temperature to the now dark suspension. Stirring was maintained over night, the solvent was removed, the residue was dissolved in ether and saturated NH₄C1 solution and the organic phase was separated. The aqueous phase was extracted with ether and again the combined organic phases were dried over MgS0₄ and the solvent evaporated. After a separation via column chromatography (silica gel, PE/EE gradient) 318 mg of product were obtained.

Yield: 318 mg, 0.77 mmol, 31 % Characterisation: white foam

Η-NMR (400 MHz, CDCI₃): δ 8.59 (dd, ³J=8.2, ⁴J=1.4, IH, Tx-H ortho to C=0), 8.53 (d, ⁴J=1.7, IH, Tx-H ortho to C=O and CH₂R), 7.82 (d, ³J=8.8, IH, H atom ortho to NO₂), 7.65- 7.39 (rή, 8H, further aromatic H atoms), 4.07 (m, 2H, CH₂), 3.75 (quintet, J=6.8, 1Η, Ph- CHR₂), 3.00 (dd, J=17.0; 6.6, 1Η, RCHΗR), 2.91 (dd, J=17.0; 6.9, 1Η, RCΗHR).

4. Synthesis of TS4-T (5'-O-[2-(nitrophenyl)-5-(9-oxothioxanthene-2-yl)-pent-4- ynyloxycarbonyl]thymidine)

C₂ Hι₇N0₄S C35H29N3O10S Mol. Wt.: 415.46 Mol. Wt.: 683.68

The reaction was carried out under nitrogen. TS4-OH (318 mg, 0.765 mmol) was dissolved in circa 10 ml of dried THF. 3 ml phosgene in toluene solution (phosgene content circa 200 g/1) were added and the solution stirred for 14 hours at room temperature. The solvents were removed and the remaining orange oil was dissolved in 10 ml of dried dichloromethane. Thymidine (200 mg, 0.83 mmol) was dried for 5 hours at high vacuum in a second flask. 10 ml of dried pyridine were added and thymidine was dissolved under gentle heating. The thymidine solution was cooled to -40°C and over a period of 4 hours slowly added to the TS4-OH solution, so that the temperature was maintained between -30°C and 0°C. The solution was allowed to come to room temperature and stirring was maintained for two days. Solvents were removed, an the residue co-evaporated three times with toluene. The product was purified by column chromatography with a PE:EE: MeOH gradient. Yield: 81 mg, 0.118 mmol, 15 % Characterization: white solid

Η-NMR (400 MHz, DMSO): δ 11.27 (s, IH, Thy-NH), 8.46 (dd, ³J=8.1, ⁴J=1.2, 1 Η, Tx-Η ortho to CO), 8.30 (d, ⁴J=1.7, 1Η, Tx-Η ortho to CO und CΗ₂R), 7.91 (d, ³J=8.1, IH, H atom ortho to N0₂), 7.88-7.72 (m, 5H, aromatic H atom), 7.64-7.52 (m, 3H, aromatic H atom), 7.40 (dd, J=4.3; 1.0, IH, aromatic H atom), 6.16 (td, J=6.9; 2.0, IH, HV), 5.40 (broad s, IH, OH), 4.62 (m, 1Η, CΗ-CHΗ-CC), 4.51 (m, 1Η, CΗ-CΗH-CC), 4.32-4.17 (m, 3Η, 2 x H5'+ Hi'), 3.90 (m, 1Η, Hf ), 3.82 (quintett, J=6.8, 1Η, PΗCHR₂), 3.02 (dd, .1=17.3; 6.4, 1Η, RCHΗR), 2.93 (dd, J=17.2; 7.9, 1Η, RCΗHR), 2.10 (m, 2Η, 2 x H ), 1.72 (m, 3H, CH₃) MALDI-MS (Matrix: 2,5-dihydroxybenzoic acid DΗB): M⁺=683.2.

Example 2: Synthetic route to compound TS9T

1. Synthesis of 2-Ηydroxy-° -thioxanthen-9-one

The synthesis was carried out according to the procedures described in Davis E. G., Smiles S., J. Chem. Soc, 1910, 1290, Christopher H., Smiles S., J Chem. Soc, 1911, 2046. A mixture of thiosalicylic acid (3.0 g, 19.46 mmol) and phenol (5.49 g, 58.38 mmol) in concentrated sulfuric acid (60 mL) was stirred at 65 — 70 °C for 4 h. The dark red reaction mixture was cooled to room temperature and poured into ice-water (1.4 L) with stirring. The finely divided precipitate was allowed to settle out, the mother liquor was partially decanted, the precipitate collected by suction and washed with water. For purification the crude product was dissolved it in hot 2 M NaOH (appr. 150 mL), the insoluble impurities was filtered off and the solution was acidified with 2 M HCl to pH 6 to give 2-Hydroxy-°H-thioxanthen-9- one as yellow solid (610 mg, 14 %): m.p. 238—240 °C. R_f 0.72 (CΗ₂C1₂ : MeOH, 9 : 1); R_f 0.62 (petroleum ether : AcOEt, 1 : 1). Η NMR (DMSO-d₆): δ 7.27 (dd, 1 H), 7.55 (dt, 1 H), 7.68 (d, 1 H), 7.23—7.83 (m, 2 H), 7.86 (d, 1 H), 8.45 (dd, 1 H), 10.16 (s, 1 H, OH).

2. Synthesis of 4-Ethyl-3-nitrobenzoie acid

The synthesis was carried out according to the procedure described in Fahim H.A., Fleifel

I A.M., J. Chem. Soc, 1952, 4519. 4-Ethylbenzoic acid (12.0 g, 79.90 mmol) was gradually added over a period of 1.5 h to a stirred fuming nitric acid (16 mL, d 1.52) with such a rate as to keep the reaction temperature below 0 °C (cooling with ice-NaCl bath). The reaction mixture was kept at 0 °C for additional 1 h and then allowed to warm up to room temperature overnight. The reaction mixture was poured into ice-water (700 mL), the precipitated product

! was collected by suction, washed with water until neutral and dried in vacuo over P₂0 to give 4-ethyl-3-nitrobenzoic acid as light yellowish solid (14.36 g, 92 %); m.p. 159 — 160 °C. R_f 0.44 (CH₂C1₂ : MeOH, 9 : 1). Η NMR (CDC1₃): δ 1.33 (t, 3 H, Me), 2.99 (q, 2 H, CH₂), 7.52 (d, 1 H, H-5), 8.24 (dd, 1 H, H-6), 8.59 (d, 1 H, H-2).

) 3. Synthesis of 4-Ethyl-3-nitrobenzoic acid tert-butyl ester

Note: a mechanical stirrer was used.

A stirred solution of 4-ethyl-3-nitrobenzoic acid (4.5 g, 23.1 mmol), 4-(l-pyrrolidino)pyridine (342 mg, 2.3 mmol) and tert-butanol (2.6 g, 35.1 mmol) in dry CH₂C1₂ (70 mL) was treated with NN'-dicyclohexylcarbodiimide (5.23 g, 25.4 mmol) at 0 °C. After 5 min of stirring at 0 °C the cooling bath was removed and stiring of the reaction mixture was continued for 3 h at room temperature. The precipitate was filtered with suction, washed with CH C1₂ and the solvent removed in vacuo. To the oily residue with the precipitate petroleum ether (90 mL) was added and the mixture was passed through a SiO₂ layer (7 g) to remove insoluble dicyclohexylurea.The pad was washed with a mixture petroleum ether- AcOEt (50 : 1, 50 mL). The organic solution was washed with 1 M HCl (2 x 50 mL), 5 % aqueous K CO₃ (1 x 50 mL), dried over MgSO₄. Removal of the solvent in vacuo afforded the title product as a light yellowish oil (5.57 g, 96 %). No further purification was necessary. R_f 0.73 (petroleum ether : AcOEt, 4 : 1). Η NMR (CDC1₃): δ 1.29 (t, 3 H, Me), 1.60 (s, 9 H, t-Bu), 2.95 (q, 2 H, CH₂), 7.42 (d, 1 H, H-5), 8.1 1 (dd, 1 H, H-6), 8.43 (d, 1 H, H-2).

4. Synthesis of 2-(4-fert-Butoι_]

To a stirred solution of 4-ethyl-3-nitrobenzoic acid tert-butyl ester (4.24 g, 16.87 mmol) in dry DMSO (5 mL) paraformaldehyde (763 mg, 25.41 mmol) was added. To this suspension t-

BuOK (70 mg, 0.62 mmol) was added in one portion. The reaction mixture was allowed to stir at room temperature for 1 h and then poured into a saturated aqueous NaCl solution (75 mL). The product was extracted with AcOEt (3 x 40 mL), the combined organic layers were washed with water (2 x 40 mL), dried over MgSO₄ and the solvent was removed in vacuo.

The crude product was purified by flash column chromatography (SiO , 10 — 35 % AcOEt in petroleum ether) to give 2-(4-tert-butoxycarbonyl-2-nitrophenyl)propanol as a yellow oil

(4.75g, 100 %). R_f 0.66 (petroleum ether : AcOEt, 1 : 1). Η NMR (CDC1₃): δ 1.34 (d, 2 FI, Me), 1.59 (s, 9 H, t-Bu), 1.63 (broad s, 1 H, OH), 3.54 (sextet, 1 H, CH), 3.80 (m, 2 H, CH₂), 7.56 (d, 1 H, H-5), 8.15 (dd, 1 H, H-6), 8.30 (d, 1 H, H-2). Η NMR (DMSO-d₆): δ 1.24 (d, 2 H, Me), 1.55 (s, 9 H, t-Bu), 3.27 (m, 1 H, CH), 3.53 (m, 2 H, CH₂), 4.79 (t, 1 H, OH), 7.75 (d, 1 H, H-5), 8.09 (dd, 1 H, H-6), 8.19 (d, 1 H, H-2).

5. Synthesis of 4-[2-(2-Methoxyethoxymethoxy)-l-methylethyl]-3-nitrobenzoic acid tert- butyl ester

To a stirred solution of 2-(4-tert-butoxycarbonyl-2-nitrophenyl)propanol (4.75g, 16.88 mmol) in dry CH C1₂ (15 mL) DIEA (5.6 mL, 32.07 mmol) was added followed by a solution of MEMC1 (2.9 mL, 25.32 mmol) in dry CH₂C1₂ (10 mL). After 4 h of stirring at room temperature a new portion of MEMC1 (0.5 mL, 3.0 mmol) was added and the stirring was prolonged for 17 h. The reaction mixture was diluted with CH₂C1₂ (appr. 60 mL) and the resulting solution was washed with 0.1 M HCl (2 x 30 mL), saturated aqueous NaCO₃ (1 x 30 mL) and water (1 x 30 mL). The organic layer was separated, dried over MgSO and the solvent was removed in vacuo. The crude product was purified by flash chromatography (SiO₂, 10 — 35 % AcOEt in petroleum ether) to give the MEM-protected alcohol 4-[2-(2- methoxyethoxymethoxy)-l-methylethyl]-3-nitrobenzoic acid tert-butyl ester as a yellow oil (5.3 lg, 85 %). R_f 0.39 (petroleum ether : AcOEt, 4 : 1). Η NMR (CDC1₃): δ 1.34 (d, 2 H, Me), 1.60 (s, 9 H, t-Bu), 3.37 (s, 3 H, OMe), 3.46—3.60 (m, 4 H, OCH₂CH₂O), 3.64—3.71 (m, 3 H, CH and OCH₂O), 4.62 (m, 2 H, CHCH?O), 7.56 (d, 1 H, H-5), 8.13 (dd, 1 H, H-6), 8.29 (d, 1 H, H-2). Anal. Calcd for C,₈H₂₇NO₇: C, 58.52; H, 7.37; N, 3.79 %. Found: C, 58.32; H, 7.40; N, 3.98 %.

6. Synthesis of 4-[2-(2-Methoxyethoxymethoxy)-l-methylethyl]-3-nitrobenzoic acid

To a stirred suspension of NaH (2.24 g of 60 % dispersion in mineral oil, 56 mmol) in dry DMF (15 mL) a solution of 4-[2-(2-methoxyethoxymethoxy)-l-methylethyl]-3-nitrobenzoic acid tert-butyl ester (2.59 g, 7.00 mmol) in dry DMF (10 mL) was added. The reaction mixture changed its colour from dark blue to dark brown within a few minutes. The reaction mixture was stirred for 1 h until complete (TLC monitoring, petroleum ether : AcOEt, 1 : 1). The mixture was carefully poured into water (160 mL), the solution was acidified with 3 M HCl to pH 4 — 5 and extracted with ether (2 x 70 mL). The combined organic layers were washed with water (4 x 70 mL), dried over MgSO₄ and the solvent was removed in vacuo. The crude product was purified by flash column chromatography (SiO₂, 25 — 50 % AcOEt in petroleum ether) to give 4-[2-(2-Methoxyethoxymethoxy)-l-methylethyl]-3-nitrobenzoic acid as a yellow oil (2.05 g, 94 %). R_f 0.11 (petroleum ether : AcOEt, 1 : 1). Η NMR (CDC1₃): δ 1.37 (d, 3 H, Me), 3.38 (s, 3 H, OMe), 3.50—3.59 (m, 4 H, OCH₂CH₂O), 3.68—3.74 (m, 3 H, CH and OCH₂O), 4.64 (m, 2 H, CHCH₇O), 7.64 (d, 1 H, H-5), 8.23 (dd, 1 H, H-6), 8.45 (d, 1 H, H-2).

7. Synthesis of 4-[2-(2-methosyethoxymethosy)-l-methylethyl]-3-nitrobensoic acid 9- oxo-9 f-thiosanthene-2-yl ester

Note: For TLC monitoring, a few drops of the reaction mixture were dissolved in water (0.5 mL), acidified with 2 M HCl to pH 4. Then ether (0.5 mL) was added, the mixture well shaken and after separation of the layers the etheral phase was used for TLC. To a stirred solution of the nitrobenzoic acid (300 mg, 0.96 mmol) in dry DMF (9 mL) DMAP (98 mg, 0.79 mmol), 2-hydroxy-9H-thioxanthen-9-one (240 mg, 1.05 mmol) and EDCI (368 mg, 1.92 mmol) were added in that order. The reaction mixture was stirred for 24 h until complete. DMF was removed by co-evaporation with toluene, the oily residue was dissolved in CΗ C1₂ (20 mL) and the organic solution was washed with saturated aqueous NH₄C1 (2 x 15 mL), 5 % aqueous K₂C0₃ (1 x 15 mL) and saturated aqueous NaCl (2 x 15 mL). The organics were separated, dried over MgSO₄ and the solvent was removed in vacuo. Purification by flash column chromatography (SiO₂, 10 — 50 % AcOEt in petroleum ether) afforded the 9-oxo-9H-thioxanthen-2-yl ester as yellow oil (321 mg, 64 %). R_f 0.49 (petroleum ether : AcOEt, 1 : 1). Η NMR (DMSO-d₆): δ 1.40 (d, 3 H, Me), 3.39 (s, 3 H, OMe), 3.49—3.59 (m, 4 H, OCH₂CH₂O), 3.73—3.77 (m, 3 H, CH and OCH₂O), 4.65 (m, 2 H, CHCHO), 7.49—7.57 (m, 2 H, Ar-H), 7.62—7.73 (m, 4 H, Ar-H), 8.37 (dd, 1 H, H-6), 8.45 (d, 1 H, H-l '), 8.58 (d, 1 H, H-2), 8.63 (ddd, 1 H, H-8'). Anal. Calcd for C₂₇H₂₅N0₈S: C, 61.94; H, 4.81; N, 2.68. Found: C, 61.92; H, 4.78; N, 2.70.

8. Synthesis of 4-(2-Hydroxy-l-methylethyl)-3-nitrobenzoic acid 9-0X0-9H- thioxanthene-2-yl ester

To a stirred solution of the MEM-ether (389 mg, 0.74 mmol) in THF (10 mL) cooled to 0 °C 3 M HCl (10 mL) was slowly added. The reaction mixture was gently refluxed for 5 h until complete. The reaction mixture was cooled to room temperature and poured into 0 °C saturated aqueous NaHCO₃ (50 mL). After the evolution of C0₂ had ceased the mixture was extracted with CH C1 (3 x 20 mL), the combined organic extracts were washed with saturated aqueous NaHCO₃ (1 x 20 mL), saturated NaCl (2 x 20 mL), dried over MgS0₄ and the solvent was removed in vacuo. Purification of the crude product by flash column chromatography (Si0 , 20 — 60 % of AcOEt in petroleum ether) followed by crystallisation in ether yielded the deprotected alcohol as yellow crystals (212 mg, 66 %); m.p. 189 — 190 °C. Rr 0.41 (petroleum ether : AcOEt, 1 : 1). Η NMR (DMSO-d₆): δ 1.29 (d, 3 H, Me), 3.55— 3.63 (m, 2 H, CH₂0), 4.89 (t, 1 H, OH), 7.62 (dt, 1 H, Ar-H), 7.77 — 7.93 (m, 4 H, Ar-H), 8.02 (d, 1 H, Ar-H), 8.34—8.40 (m, 2 H, Ar-H), 8.45—8.50 (m, 2 H, Ar-H). Signal of CH overlaps with signal of H₂O. Anal. Calcd for C₂₃H,₇NO₆S: C, 63.44; H, 3.94; N, 3.22. Found: C, 63.30; H, 3.93; N, 3.25.

9. Synthesis of 5-O'-[2-(4-(9-oxo-9fl-thioxanthene-2-yl)carbonyl-2-nitrophenyl) propoxy carbonyl] -thymidine (TS9T)

To a 0 °C stirred solution of trichloromethyl chloroformate (43, 0.40 mmol) in dry THF (2 mL) a solution of alcohol (130 mg, 0.30 mmol) and Et₃N (42 μL, 0.30 mmol) in dry THF (2 mL) was added. The reaction mixture was allowed to stir for 2 h at 0 °C and for 2 h at room temperature until TLC monitoring indicated complete consumption of the alcohol and formation of the chloroformate, R_f 0.62 (CH₂C1₂). The solid was removed by filtration and washed with dry THF. The solvent was removed in vacuo, and the yellow oily residue was dissolved in dry CH₂C1₂ (1.5 mL). In a separate flask thymidine (73 mg, 0.30 mmol) was co- evaporated with dry pyridine (3 x 2 mL) and dissolved in dry pyridine (2 mL). To the stirred solution of thymidine, the solution of the chloroformate was added via a syringe at 0 °C and the reaction mixture was stirred for 18 h at 0 °C. The solvents were removed in vacuo, the residue co-evaporated with EtOH (2 x 3 mL) and then with CH₂C1₂ (3 x 2 ml). Purification of the crude product by flash column chromatography (Si0₂, 0 — 5% of MeOH in CH₂C1₂) followed by crystallisation in ether afforded the photolabile protected thymidine (TS9T) as light yellow solid (123 mg, 58 %); m.p. 162—164 °C. R_f 0.63 (CH₂C1₂ : MeOH, 9 : 1). Η NMR (DMSO-d₆): δ 1.33 (d, 3 H, Me), 1.72 and 1.74 (s, 3 H, Me, diastereomer), 2.11 (m, 2 H, H-2'), 3.61 (m, 1 H, CH), 3.87 (m, 1 H, H-3', diastereomer), 4.15 — 4.44 (m, 5 H, CH₂0, H-4' and H-5⁵), 5.45 (d, 1 H, 3'-OH, diastereomer), 6.17 (t, 1 H, H-l'), 7.41 (m, 1 H, H-Thy), 7.62 (dt, 1 H, Ary-H), 7.77—7.86 (m, 2 H, Ary-H), 7.91 (m, 1 H, Ary-H), 8.01 (m, 2 H, Ary- H), 8.37—8.58 (m, 3 H, Ary-H), 11.33 (s, 1 H, NH). Anal. Calcd for C₃₄H₂₉N₃Oι₂S: N, 5.97. Found: N, 6.16.

Example 3: Preparation of 5'-TS8-dT3'-PA Synthesis of 2-(5,5-Dimethyl-l,3,2-dioxaborinane-2-yl)-9H-thioxanthene-9-one

C₁₃H₇BrOS CIQH₂OB₂0₄ -ιβHι₇B0₃S 291.16 g/mol 225.89 g/mol 324.20 g/mol

In a 100 ml Schlenk flask, 2-bromothioxanthone (694 mg, 2.38 mmol), bis(neopentylglycolato)diboron (577 mg, 2.55 mmol), potassium acetate (730 mg, 7.44 mmol) and PdCl₂(dppf) (two small spatulas) were dissolved under nitrogen atmosphere in 20 ml of DMSO (flushed 30 minutes with nitrogen). The red suspension was stirred for 4 hours at 80°C, whereby the suspension became darker. After standing over night, 80 ml of CH₂C1₂ and 100 ml of water were added and the organic phase were extracted. The aqueous phase was extracted twice with each 30 ml of CH₂C1₂. The combined organic phases were washed with half-saturated NH₄C1 solution and dried over MgS0₄. Evaporation of the solvent yielded the brown oily residue which was purified via column chromatography (silica gel, conditioned with PE:EE=10:1, eluted with PE:EE-gradient). 654 mg (85 %) product were obtained.

Yield: 654 mg, 2.02 mmol, 85 % Characterization: yellowish solid R_f (PE:EE=2:1) = 0.4 Η-NMR (400 MHs, DMSO): δ 8.81 (d, J=0.8 Hz, IH, Tx-H-/), 8.47 (dd, ³J=8.1 Ηz, ⁴J=1.0, 1Η, Tx-H-S), 7.97 (dd, ³J=8.1 Ηz, ⁴J=1.5 Ηz, 1Η, Tx-H-J), 7.83 (d, J=8.1 Ηz, 1 Η, Tx-H-4), 7.79-7.43(m, 2Η, Tx-H_d and Tx-H-5), 7.58 (td, ³J=7.6 Ηz, ⁴J=1.2 Ηz, 1Η, Tx-H- 7), 3.80 (s, 4Η, 2 x,CH₂), 0.97 (s, 6Η, 2 x CH₃).

2. Synthesis of 2-[3-(l-Hydroxyprop-2-yl)-4-nitrophenyl]~9H-thioxanthene-9-one (TS8-OH)

Ci8Hι₇B0₃S C₉H₁₀BrNO₃ C₂₂H₁₇N0₄S 324.20 g/mol 260.08 g/mol 391.44 g/mol In a 250 ml Schlenk flask, 2-(5,5-dimethyl-l,3,2-dioxaborinane-2-yl)-9H-thioxanthene-9-one (1.53 g, 4.72 mmol) and 2-(4-bromo-2-nitrophenyl)propanol (1.35g , 5.19 mmol) were dissolved in 40 ml of TΗF. Water (Millipore, 15 ml) was added and the solution was flushed with nitrogen during 45 minutes to remove oxygen from the solution. Afterwards, 300 mg of solid NaOΗ and PdCl₂(dppf) (120 mg, 0.23 mmol, 5 mol %) were added. The solution was stirred for 8 hours at 65°C, whereby the solution turned slowly dark. Afterwards, 100 ml CΗ C1₂ and 50 ml half-saturated aqueous NH₄C1 were added, the organic phase was extracted and the aqueous phase was extracted three times with CH₂C1₂. The combined organic phases were dried over MgSO , the solvents were evaporated. The residue was suspended in 50 ml MeOH and sonicated for 30 s. The precipitate was filtered off, washed with MeOH and dried in vacuo. Yield: 1.7 g, 4.34 mmol, 92 % Characterization: yellow-orange solid R_f (PE:EE=2:1) = 0.4, R_f(Tol:Ac=4:l) = 0.4

Η-NMR (400 MHz, DMSO): δ 8.74 (d, J=1.95 Hz, IH, Tx-H-/), 8.50 (dd, ³J=8.1 Ηz, ⁴J=1.0 Ηz, 1Η, Tx-H-S), 8.19 (dd, ³J=8.5 Ηz, ⁴J=2.2 Ηz, 1Η, Tx-H-3), 8.02 (d, J=8.5 Ηz, 1Η, Tx-H-4), 7.94 (d, J=8.5 Ηz, 1Η, oNP-H-J), 7.94 (d, J=2.0 Ηz, 1Η, oNF-H-6), 7.89 (d, J=8.1 Ηz, 1Η, Tx-H-J), 7.84 (dd, ³J=8.5 Ηz, ⁴J=2.0 Ηz, 1Η, 6NF-H-4), 7.81 (td, ³J~8.5 Ηz, ⁴J=1.2 Ηz, 1Η, Tx-H-6), 7.62 (td, ³J=8.1 Ηz, ⁴J=1.2 Ηz, 1Η, Tx-H-7), 4.81 (broad, 1Η, OH), 3.65 (m, 2Η, CH₂), 3.33 (m, 1Η, CH), 1.33 (d, 3Η, CHj). MS: (M+Η⁺)=329.0, (M+Na⁺)=41 .0, (M+K⁺)=430.0. Elemental analysis: calculated: C 67.50 %, H 4.38 %, N 3.58 % found: C 66.67 %, H 5.03 %, N 3.65 %

3. Synthesis of 5-0'-[2-(5-(9-oxo-9H-thioxanthene-2-yl)-2-nitrophenyI) propoxycarbonyl] -thymidine (TS8T)

C₂₂H₁₇N0₄S C-₃₃H_2gN₃θι₀S 391.44 g/mol 659.66 g/mol The following reaction steps were performed under a nitrogen atmosphere. TS8-OH (480 mg, 1.23 mmol) was dissolved in 20 ml THF. An excess of phosgene in toluene solution (4 ml, circa 200 g/1) was added and the solution stirred for 5 hours at room temperature. The progress of the reaction was observed via thin layer chromatography. In the solvent CH₂C1₂, the R_f value of the educt is 0.05, the R_f value of the chlorine carbonic acid ester is 0.65. 387 mg of thymidine (1.60 mmol) were placed in a 100 ml Schlenk flask and co-evaporated twice with each 10 ml pyridine. The solvent were removed from the chlorine carbonic acid ester at a vacuum line. The chlorine carbonic acid ester was dissolved in CH C1₂ (circa 30 ml) and poured into a dropping funnel. Thymidine was dissolved in pyridine (circa 15 ml) and the chlorine carbonic acid ester solution was added during 30 minutes to the thymidine solution cooled to -44°C (acetonitrile/dry ice bath). At this temperature, stirring was maintained for 5 hours in the dark. Afterwards, the solution was cooled slowly (over night) to room temperature. The following isolation steps were performed in yellow light. To the reaction solution, 150 ml deionized water and 100 ml CH C1₂ were added. The organic phase was separated and the aqueous phase was extracted twice with CH₂C1 (30 ml each). The combined organic phases were dried over Na₂S0 and the solvents were removed at the rotatory evaporator. To remove the last residues of pyridine, the residue was co-evaporated with toluene three times. CH₂C1 were added to the residue, whereby 248 mg of the product precipitated as a solid. The solid was filtrated and checked for purity by HPLC. Only one peak was observed, so that, the product could be used directly for irradiation experiments. The filtrate contained also product, which could be isolated after column chromatography (silica gel, conditioned with CH₂C1₂, slower CH₂Cl₂:MeOH gradient from 100 % to 97 % CH₂C1₂) (230 mg). The total yield was 478 mg (0.725 mmol, 59 %) of a yellow orange powder.

Characterization: yellow orange powder R_f (CH₂Cl₂:MeOH=20:l) = 0.3, Rf (Tol:EE:MeOH=5:4:l) = 0.3 Η-NMR (400 MHz, DMSO): δ 11.27 (s, IH, NH), 8.78 (d, ⁴J=2.0 Ηz, 1Η, Tx-H-7), 8.51 (d, ³J=8.0 Ηz, 1Η, Tx-H-S), 8.20 (dd, ³J=8.3 Ηz, ⁴J=1.8 Ηz, 1Η, Tx-H-3), 8.08 (s, 1Η, oNP-H-<5), 8.00 (pseudo-t, ³J=8.3 Ηz, 2Η, oNP-H-5 und Tx-H-4), 7.90 (pseudo-d, ³J=8.0 Ηz, 2Η, oNP-H- und Tx-H-5), 7.82 (t, ³J=7.1 Ηz, 1Η, Tx-H-6), 7.63 (t, ³J=7.5 Ηz, 1Η, Tx-H-7), 7.37 (s, 1Η, Thy-H), 6.14 (t, ³J=6.8 Ηz, 1 Η, Ribose-H-7), 5.38 (broad s, 1Η, OH), 4.45 (m, 2Η, oNP-CH(CH₃)-CH₂-O), 4.26-4.15 (m, 3Η, Ribose-H-J und 2 x Ribose-H-5), 3.88 (m, 1Η, Ribose-H-4), 3.63 (m, 1Η, oNP- CH(CH₃)), 2.06 (m, 2H, 2 x Ribose-H-2), 1.68 (s, 3Η, Thy-CH₃), 1.39 (d, ³J=6.8 Ηz, 3Η, oNPCH(CH₃)). elemental analysis: calculated: C 60.08%, Η 4.43%, N 6.37% found: C 58.00%, Η 4.41%, N 6.38%

4 . Synthesis of 5'-TS8-dT3'-Pa

5'-TS8-dT 1.37 g 2.08 mmol 1 Eq DCI 0.12 g 1.04 mmol 0.5 Eq

Bis(diisopropylamino)- 0.94 g 3.12 mmol 1.5 Eq 2-cyanoethoxyphosphane

0.94 g of Bis(diisopropylamine)-2-cyanoethoxyphosphane are added to a suspension of 1.36 g 5'-TS8-dT and 0.12 g of DCI in 50 ml of dichloromethane. After 3 hours conversion is checked by TLC (mobile phase: ethyl acetate). Further 0.94 g of Bis(diisopropylamino)-2- cyanoethoxyphosphane are added to achieve a complete conversion. Two hours later the turbid reaction mixture is filtered over a short column (7.2 g of silica gel, 2 cm column diameter, 7 cm column length, packed with dichloromethane). The column is washed with ethyl acetate. The combined product fractions are concentrated in vacuum and dissolved in approx. 30 ml of ethyl acetate. Under vigorous stirring this solution is dropped slowly in to 800 ml of n-hexane. The precipitate is filtered off, washed with n-hexane, dissolved in dichloromethane, filtered and concentrated in vacuum to give 1.15 g of a yellow foam (yield: 64 % of theory).

³¹P-NMR (300 mHz, DMSO): 149.237 ppm; 149.178 ppm; 149.076 ppm; 148.973 ppm. [S. 42 - 46]

Example 4: Preparation of TS7T 1. Synthesis of 2-(2-nitrophenyl)-4-pentenoic acid methyl ester

To a solution of 2-nitrophenyl acetic adic methyl ester (2.93 g, 15.0 mmol) in 75 mL of MeCN dry K₂CO₃ (17.44 g, 126.15 mmol) and 18-crown-6 (20 mg) were added under a nitrogen atmosphere. To the blue suspension, allyl bromide (3.0 ml, 18.0 mmol) in 15 mL of MeCN were added and the obtained suspension was refluxed for 20 hours. The precipitate was filtered off and washed with MeCN. The solvent was distilled from the filtrate and the obtained oil was dissolved in ether. The organic phase was washed with water, dried over MgSO₄ and the solvent was evaporated under vacuum. Purification was carried out by column chromatography (Si0 , petrolether : EtOAc= 2:1) and yielded a yellowish oil (3.44 g, 93 %). Η NMR (CDC1₃): δ 2.54—2.67 (m, 1 H, H-3), 2.84—2.98 (m, 1 H, H-3), 3.67 (s, 3 H, Me), 4.31 (t, 1 H, H-2), 4.97—5.09 (m, 2 H, H-5), 5.72 (ddt, 1 H, H-4), 7.42 (dt, 1 H, H-4⁵), 7.49— 7.63 (m, 2 H, H-5' and H-6'), 7.89 (dd, 1 H, H-3^*).

2. Synthesis of 2-(2-nitrophenyl)-pent-4-ene-l-ol

To a stirred suspension of 2-(2-nitrophenyl)-4-pentenoic acid methyl ester (1 18 mg, 0.5 mmol) and NaBH₄ (151 mg, 4 mmol) in dry THF (7 mL) MeOH (2 mL) were added slowly over a period of 2 hours under an atmosphere of nitrogen. After the addition of methanol, the solution was stirred for a further hour until the reduction was complete (DC control). Afterwards, MeOH (7 mL ) and water (0.5 mL) were added, the reaction mixture was heavily stirred for 10 minutes and concentrated in vacuo. The residue was dissolved in CH₂C1 (15 mL) and water (15 mL). The organic phase was extracted, washed with water (10 mL) dried over MgSO and the solvent was evaporated in vacuo. Purification was achieved by column chromatography (SiO₂, petrolether : EtO Ac-gradient from 10 % to 50 % EtO Ac) and yielded a yellow oil (84 g, 81 %). Η NMR (CDC1₃): δ 1.67 (broad s, 1 H, OH), 2.36—2.62 (m, 2 H, H-3), 3.48 (m, 1 H, H-2), 3.78—3.93 (m, 2 H, H-l), 4.94—5.05 (m, 2 H, H-5), 5.68 (ddt, 1 H, H-4). 7.36 (dt, 1 H, H-4'), 7.47—7.61 (m, 2 H, H-5' und H-6'), 7.74 (dd, 1 H, H-3'). Elemental analysis calculated for CnH|₃NO₃: calculated N, 6,76 %, found N, 6,70 %.

3. Synthesis of l-[(tert-butyldimethylsiIyl)oxy]-2(2-nitrophenyl)-pent-4-ene

To a solution of 2-(2-nitrophenyl)pent-4-ene-l-ol (800 mg, 3.86 mmol) in dry CH C1₂ (12 mL) imidazole (657 mg, 9.65 mmol) and tert-butyl-dimethyl cholorosilane (1.16 g, 7.72 mmol) were added at 0°C. Afterwards, the solution was stirred for 12 hours at room temperature. A saturated, aqueous NaHCO₃ solution (15 mL) was added, the organic phase was separated and extracted with CH C1₂ (10 mL). The combined organic phases were washed with satured, aqueous NaHC0 solution (15 mL) and dried over MgS0 . After evaporation of the solvent, a raw product was obtained that yielded after column chromatography (Si0₂, petrolether : EtOAc = 8 :1) a bright oil (1.22 g, 98 %)). Η NMR (CDC1₃): δ -0.09 (s, 3 H, Me), -0.06 (s, 3 H, Me), 0.82 (s, 9 H, t-Bu), 2.45 (m, 1 H, H-3), 2.59 (m, 1 H, H-3), 3.43 (m, 1 H, H-2), 3.78 (dd, 2 H, H-l), 4.93—5.04 (m, 2 H, H-5), 5.68 (dm, 1 H, H-4), 7.26—7.35 (m, 1 H, H-4'), 7.49—7.52 (m, 2 H, H-5' and H-6'), 7.72 (dd, 1 H, H- 3')-

4. Synthesis of 2-Iodo-9H-thioxanthene-9-one

2-Iodo-9H-thioxanthen-9-one was synthesized according to methods known in prior art (Schoervaas et al., J. Org. Chem. 1997, 62, 4943) starting from 2-amino-9H-thioxanthen-9- one (Moon et al. J. Ηeterocycl. Chem. 1999, 36, 793). 5. Synthesis of 2-[5-( ter -butyl-dimethysilyI)oxy-4-(2-nitrophenyl)pentyl]-9 /- thioxanthen-9-one

To the solution of l-[(tert-butyldimenthylsilyl)oxy]-2(nitrophenyl)pent-4-ene (450 mg, 1.40 mmol) in dry THF (10 mL) 9-borabicyclo[3.3.1]nonane (9-BBN, 5.60 mL of a 0.5 solution in THF, 2.80 mmol) was added dropwise over a period of 45 minutes under a nitrogen atmosphere. The reaction solution was stirred for 3 hours until the reaction was complete (DC control) and oxygen was removed by 15 minutes flushing with nitrogen. PdCl (dppf) (53 mg, 0.069 mmol), oxygen-free aqueous K PO₄ solution (3 M, 0.88 mL, 2.75 mmol) and oxygen- free DMF (10 mL) were placed under a nitrogen atmosphere in another flask. After 20 minutes of heavy stirring, 2-Iodo-9H-thioxanthen-9-one (465 mg, 1.37 mmol) were added. Afterwards, the borane solution was added via a syringe. The solution was refluxed in the dark for 160 minutes until the reaction was complete (DC control). After cooling down the solution to room temperature, diethyl ether (50 mL) and saturated aqueous NaCl solution (50 mL) were added and extracted. The aqueous phase was once again extracted with diethyl ether, and the combined organic phases were washed with saturated NaΗC0₃ solution (2 x 30 mL) and water (1 x 30 mL) and dried over MgSO₄. The raw product was purified by column chromatography (Si0₂ petrolether : EtOAc-gradient of 3.5 % to 8 % EtOAc). Η NMR (CDC1₃): δ -0.11 (s, 1 H, Me), -0.10 (s, 1 H, Me), 1.34—1.75 (m, 4 H, CH₂), 2.73 (m, 2 H, CH₂), 3.36 (m, 1 H, CH), 3.70 (d, 2 H, CH₂O), 7.31 (dt, 1 H, H-4'), 7.62—7.39 (m, 7 H, Ar- H), 7.68 (dd, 1 H, H-3'), 8.38 (d, 1 H, H-l), 8.62 (dd, 1 H, H-8).

6. Synthesis of 2-[5-hydroxy-4~(2-nitrophenyl)-pentyl]-9H-thioxanthene-9-one

To 2-[5-(tert-butyldimethylsilyl)oxy-4-(2-nitrophenyl)pentyl]-9-H-thioxanthene-9-one (732 mg) in TΗF (12 mL) tetrabutyl ammonium fluoride (1.9 mL, 1.0 M in TΗF) was added under stirring at 0°C. The ice bath was removed and the reaction mixture was stirred for 15 hours, diluted with diethyl ether (50 mL) and washed with with saturated aqueous NΗ₄C1 solution (2 x 30 mL). The aqueous phases were combined and extracted once again with diethyl ether (20 mL). The combined organic phases were washed with saturated NaCl solution (20 mL), dried over MgSO₄ and the solvent was evaporated in vacuo. The product was obtained by column chromatography (SiO₂, Petrolether : EtOAc-gradient of 5 % to 50 % EtoAc) and after crystallization from petrolether as a light yellow solid (303 mg, 53 % for the last two steps starting from 2-[5-(tert-butyldimethylsilyl)oxy-4-(2-nitrophenyl)pentyl]9H-thioxanthene-9- one). Η NMR (DMSO-d₆): δ 1.40—1.70 (m, 4 Η, CΗ₂), 2.70 (broad t, 2 H, Ar-CH₂), 3.52 (m, 1 H, CH), 3.51 (m, 2 H, CH₂O), 4.77 (t, 1 H, OH), 7.42 (dt, 1 H, H-4'), 7.53—7.66 (m, 4 H, Ar-H), 7.29 (m, 4 H, Ar-H), 8.22 (d, 1 H, H-l), 8.46 (dd, 1 H, H-8).

Elemental analysis of C₂ H₂]NO S: calculated C, 68.72 %, H, 5.05 %, N, 3.36 %, found C, 68.64 %, H, 5.11 %, N, 3.40 %.

7. Synthesis of 5'-0-[(2-nitrophenyl)-5-(9-oxo-9 -thioxanthene-2-yl)- pentyloxycarbonyl]-thymidine (TS7T)

5'-O-[4-nitrophenyloxycarbonyl]thymidine (164 mg, 0.40 mmol) was co-evaporated with dry pyridine (4 x 3 mL) and dissolved in dry pyridine ( 5 mL). The previously synthesized alcohol 2-[5-hydroxy-4-(2-nitrophenyl)pentyl]-9H-thioxanthene-9-one (160 mg, 0.31 mmol) was dissolved in dry CΗ₂C1₂ (2 mL) and the solution was added to the prepared pyridine solution. The solution was stirred at room temperature for 48 hours in the dark. The solvents were evaporated in vacuo, and the oily residue was dissolved in CH₂C1 (20 mL) and washed with a 0.1 M HCl solution ( 4 x 10 mL). The combined aqueous phases were washed once again with CH₂C1₂ (1 x 5 mL). The combined organic phases were dried over MgSO₄ and the solvent were evaporated in vacuo. Purification by column chromatography (SiO₂, CH₂C1₂ : MeOH-gradient of 0.5 % to 5 % MeOH) and subsequent crystallization from diethylether yielded TS7T as a light yellow powder (83 mg, 39 %). Melting point: 100 - 102°C. Η NMR (DMSO-d₆): δ 1.37—1.61 (m, 2 H, CH₂), 1.68 and 1.69 (s, 3 H, Me, diastereomer), 1.69— 1.85 (m, 2 H, CH₂), 2.08 (m, 2 H, H-2'), 2.69 (m, 2 H, CH₂), 3.87 (m, 1 H, CH), 4.12—4.31 (m, 4 H, CH₂O and H-5'), 4.38 (m, 1 H, H-3', diastereomer), 5.41 (d, 1 H, 3'-OH, diastereomer), 6.15 (dt, 1 H, H-l'), 7.37 (dd, 1 H, H-Thy), 7.47 (m, 1 H, Ar-H), 7.57 (dt, 2 H, Ar-H), 7.67 (d, 2 H, Ar-H), 7.71—7.85 (m, 4 H, Ar-H), 8.22 (d, 1 H, H-l), 8.45 (dd, 1 H, H- 8), 11.29 (broad s, 1 H, NH). Elemental analysis for C₃₅H₃₃N₃Oι₀S x 1/2 H2O: calculated C, 60.33 %, H, 4.91 %, N, 6.03 %, found C, 60.40 %, H, 5.07 %; N, 5.93 %.

Example 5: Photochemical investigation of compound TS4-T

The following example with TS4-T shows the increase of the reaction rate of a photolabile protecting group, when a sensitizer is linked to the photolabile protecting group.

TS4-T 5'-O-[2-(2-nitrophenyl)-5-(9-oxo-9H-thioxanthene-2-yl)-4-pentinyloxycarbonyl]- thymidine Short time spectroscopy experiments:

The excited triplet state of the thioxanthone moiety of TS4-T was detected by laser flash spectroscopy.

The transient absoφtion spectra have been measured after laser excitation (at 355 nm) of compound TS4-T in oxygen-free methanol. The measured absoφtion maximum at circa 600 nm is characteristic for the triplet state of free thioxanthone. With respect to free thioxanthone, the difference lies in the lifetime of the triplet which is for TS4-T shorter than for free thioxanthone. Decay curves for free thioxanthone (TX, oxygen-free solution) and for TS4-T in oxygen-free and air-saturated solution have been measured at the maximum of the triplet triplet absoφtion of thioxanthone.

The following values for the decay constants of the triplets were obtained:

thioxanthone: ko = 3.9 x 10⁴ s^"1

TS4-T: The decay process is biexponential. The pertinent two rate constants are ki = 3.77 x 10⁶ s^"1 (this decay contributes to 34 %) and i₂ = 0.74 x 10⁶ s¹ (this decay contributes to 66 %)

The lifetime of both triplets was decreased by the influence of oxygen in an oxygen saturated solution to a similar value, corresponding to an effective quenching with &₉[O₂] * 3.4 xlO⁶ s^"1. !

The shorter triplet lifetime of TS4-T compared to free thioxanthone is a proof for the triplet triplet energy transfer in the supramolecular compound according to the invention from the sensitizer synthon on the photolabile 2-(2-nitrophenyl)ethyl chromophore.

) Comparing the average (1.77 x lθV) of the rate constants for the TS4-T triplet decay with the triplet decay constant of free thioxanthone, a value of

*_/iT= 1.73 x lOV is obtained for the rate constant of intramolecular energy transfer.

Photolability:

Upon irradiation with UV light or blue light up to 430 nm, the compound TS4-T is decomposed rapidly. The photochemical decomposition of TS4-T and formation of the deprotected thymidine have been detected via HPLC as a function of exposure time to UV light.

The HPLC diagrams have been recorded in an irradiated methanolic solution of TS4-T after different irradiation times with the light of 366 nm from a mercury high pressure lamp. The peak of the starting material has been observed at a rentention time of 17.6 minutes, the peak of the cleaved thymidine at 7.5 minutes.

Photokinetics:

The photokinetics of the decomposition of the photolabile compound has been quantitatively followed via HPLC and is shown in Figure 6.

Figure 6 shows the photokinetics of the decomposition of TS4-T together with the curves of other exemplary compounds according to the invention upon irradiation at 366 nm in methanol. The concentration of the unreacted compounds is represented as a function of the light dose I₀ x l, where IQ is the photon irradiance and t the irradiation time.

The lines shown in Figure 6 represent fits according to equation (1). For TS4-T, the fitted values of °_em and η~ are 0.40 and 1.5 x 10^'3 M, respectively. The decomposition kinetics observed can be described by the following equation:

(1)

wherein: c is the concentration of the photo reactive compound Co is the starting concentration

I_E,Q the photon irradiance in Einstein per second and cm²

S the illuminated area on the front of the cuvette

V the solution volume in the cuvette d the optical path length of the cuvette

F_pk the photokinetic factor, given by A/(1-10^'A), wherein A is the total absorbance of the solution at a given time Sdir is the molar decadic absoφtion coefficient of the photo reactive chromophore without the linked sensitizer. Its value is here 247 M^"1 cm^"1. φdir the quantum yield of the photoreactive chromophore under direct excitation. ε_se„_s the molar decadic absoφtion coefficient of the free sensitizer. ,_ns the quantum yield of the sensitized photoreaction at a complete energy transfer from the sensitizer to the photolabile protecting group k_qp the rate constant of quenching of the sensitizer triplet by the photoproduct k_tr the rate constant of the intramolecular triplet energy transfer from the sensitizer to the photolabile protecting group

Equation (1) shows, that the partial rates of the direct and intramolecular sensitized photo reaction relate to one another approximately as the products ε_d,_rφd, ^~ 103 M^"'cm^"' and ε<sem Φ _m ~ 980 M^"'cm^"'. This corresponds to an increase in the magnitude of 10 of the reaction rate with TS4-T compared to NPPOC-T without intramolecular sensitizer.

It is of interest to compare the influence on the reaction rate of a free sensitizer with that of an intramolecularly linked sensitizater according to the invention. Using a free sensitizer, its efficiency depends on its concentration. In homogeneous solution, a comparable increase of the reaction rate as with the supramolecular compound is obtained with a free sensitizer concentration of 4 x 10^"5 M. Theoretically, when using infinitely high sensitizer concentration in a 1-cm cuvette, the reaction rate for sensitization with the free sensitizer would become 2.6 times as large as with the linked sensitizer in the supramolecular compound.

On a biochip, there are differences compared to the situation in solution. The photolabile protecting groups are fixed to the chip surface. The excited sensitizer molecules have to diffuse in the case of a separately added sensitizer during their excited lifetime in the layer. Therefore, light absoφtion has only an effect in a very thin solution layer. Upon experiments in air saturated solution with a separate (free) sensitizer, a maximum increase of the factor 2 compared to a "direct" photochemistry has been observed. In the case of a supramolecular compound according to the invention, complex diffusion kinetics is of no importance and the factor 10 of increase in reaction rate observed in homogeneous solution is fully applicable to the situation on the chip.

Air Sensitivity:

Between free thioxanthone as sensitizer and the intramolecularly linked thioxanthone there is a considerable difference with respect to the air sensitivity of the reaction rate.

Photokinetics of NPPOC-T upon sensitization via free thioxanthone and of TS4-T in oxygen- free and in air saturated methanolic solution have been measured.

Whilst the reaction rate in the case of free thioxanthone is increased by a factor of 5 by the influence of air (that is, the quantum yield of sensitization is reduced to the factor 1/5) this increase is reduced in the solution of TS4-T to a factor 3 (that is the quantum yield of sensitization is reduced to 1/3). Therefore, even in the presence of oxygen, the supramolecular. compound TS4-T allows a faster reaction rate than in the case of a unmodified protecting group. This is a great advantage for the processing of DNA chips, because it is no more necessary to remove oxygen from the solution, and secondly, the combination of the sensitizer and oxygen allows the generation of singulet oxygen for the next processing step, that is the oxidation of the phosphite to the phosphate.

Example 6: Investigation of further supramolecular compounds according to the invention.

Five supramolecular protecting group compounds according to the invention and synthesized as described in the foregoing have been studied in further detail:

The bonding of the thioxanthone chromophore of the photolabile 2-nitrophenylethyl fragment occurrs either via linkage to the benzylic C-atom (compounds TS4T, TS6T, TS7T) or via substitution at the phenyl ring (compounds TS8T and TS9T).

The connection via an aliphatic linker at the benzylic C atom is especially advantageous, because of the favourable influence of an alkyl substitutent on the photo-induced H-transfer from the α-C-atom on the nitro group. The alkylation at this CH-acidic position can be achieved via nucleophihc substitution using unsaturated alkyl bromide, allyl bromide or propargyl bromide used in known C-C coupling reactions (HECK, SUZUKI).

An up to now unknown thioxanthone-2-boronic acid ester has been synthesized and reacted in a Suzuki coupling with 2-(5-bromo-2-nitrophenyl)-propanol to obtain compound TS8T where the sensitizer is linked directly to the nitrophenyl ring.

To couple the sensitizer moiety via an ester linkage at the protecting group, the starting material was 4-ethyl-3-nitrobenzoic acid. After protection of the acid group, the ethyl substituent was converted with paraformaldehyde to a 1 -hydroxy-2-propyl substituent. After protection of the alcohol function and deprotection of the carboxyl function, esterification was achieved with 2-hydroxythioxanthone.

The protected thymidines were synthesized for each compound from the free alcohols upon reaction with phosgene or phosgene derivatives and thymidine. The absoφtion spectra of several synthesized supramolecular compounds according to the invention were recorded and are shown in figure 5. Their spectra can be compared to the spectrum of the protecting group compound NPPOC-T. The absoφtion of thioxanthone in the supramolecular compounds is clearly visible. In the wavelength region between 350 and 430 nm, the thioxanthone-specific band contributes to a significant increase in absoφtion. The absoφtion spectra essentially correspond to an independent supeφosition of the absoφtion of the thioxanthone chromophore (in the case of TS4T and TS6T it exhibits an additional band with respect to the unsubstituted thioxanthone in the range of 325 nm due to the conjugated multiple bond) and of the 2-nitrophenyl chromophore. In contrast, the spectrum of TS8T shows a significant change which indicates, that in this compound thioxanthone and the nitrophenyl group form a joint chromophore. However, this chromophore, too, shows the typical photochemical reaction of a 2-nitrobenzyl compound.

All compounds are photolabile. The increase in light sensitivity with respect to the NPPOC compound is shown by the photokinetic curves in Figure 6 representing the decomposition of the starting compound quantified by HPLC as a function of the dose of light applied to the sample. The compounds TS4T, TS7T, and TS8T react fastest, whereby between TS8T and NPPOC-T an increase of a factor more than 10 is obtained. Laser flash spectroscopy measurements of the triplet decay show, that an intramolecular triplet energy transfer took place. The triplet decay time is shorter than in free thioxanthone. The rate of the energy transfer is correlated with a confomiational possibility of the molecules for a good overlap of thioxanthone and nitrobenzene chromophore. All supramolecular compounds have triplet life times at least 10 time shorter than free thioxanthone. The shorter the triplet lifetime as a result of efficient energy transfer the less prone are the triplets to quenching by oxygen. Thus the rates of the photoreactions of compounds TS7T and TS8T which both exhibit triplet lifetimes of less than 200 ns are only little sensitive to oxygen in air. A summary of the photokinetic parameters of new compounds according to the invention is given in Table 2. Table 2 triplet ε (z5/ M^"1cm^"1 ε / M^{' 1} Φ yield of life time at 366 nm at 366 nm at 366 nm thymidine

NPPOC-T 103 250 0,41 90% thioxanthone 22 μs 4300

TS4T 1 ,0 μs 980 3500 0,28 30%

TS6T 2,5 μs 248 3100 0,08 40%

TS7T 0,11 μs 798 3800 0,21 86%

TS8T 0,16 μs 2500 10400 0,24 81 %

TS9T 1 ,6 μs 360 4000 0,09 91%

Example 7: Manufacture of biochips with the compound 5^;-TS-§-T amidite.

Biochips have been manufactured and tested on coupling efficiency according to standard methods on a glass slide by methods in analogy to Fodor et. al Science 1991, 251, p.767 ff. and US Patent No 5,843,655. As nucleoside, the compound 5' TS-8-T amidite was used. PCy3 was used as a marker. 12 coupling steps have been performed, starting from row 1 with the monomer 5'-TS-8-T amidite. Coupling yields for the synthesis cycle of a oligomers were about 90 % with and without solvent (Fig 7a: hexamer, Fig 7b dodecamer and Fig 7C nonamer). Yields for MeNPPOC without solvent are below 70%. The use of compounds according to the invention therefore increases considerably the overall yield. The results are shown in Figs. 7a to 7c.

Irradiation was carried out with (Fig 7a and 7b) and without solvent (DMSO) (Fig 7c). Irradiation times were set to 6 (Fig 7a) and 12 seconds (Fig 7b and Fig 7c). As can be seen from Fig 7a, an irradiation time of 6 seconds is sufficient for complete cleavage of the protecting group. Even in the case without the use of a solvent (Fig 7c), an irradiation time of 12 seconds was sufficient for complete removal of the protecting group. Corresponding values for usual protecting groups like NPPOC or MeNPOC are 70-75 and 60 to 75 seconds respectively. Therefore, the compounds according to the invention increase dramatically the reaction rate of the deprotection reaction.

Claims

Patent claims

1. Chemical compound with the structural motif

S-(L,)_a-P-(L2)b-R

wherein

S is a sensitizer synthon, which first excited electronic state is at the same or a higher energy as the first excited electronic state of the labile functional group P,

P is a labile functional group and selected from the group consisting of

a) compounds of the general formula

wherein R₃ = H, N0₂, halogen, a C| to C₄ alkyl group, preferably methyl, ethyl, propyl, 1^ is H, N0₂, halogen, Cj to C alkyl, preferably methyl, ethyl, a substituted or non-substituted aryl group, an acyl, alkoxycarbonyl or acylamino group, R₅ and R₆ independent from one or another a H atom, NO₂, halogen, an alkyl rest with up 4 C atoms, an alkoxide rest with up to 4 C atoms, an acyl , alkoxycarbonyl or acylamino group, a substituted or non-substituted aryl group, or R₅ and R form together a methylenedioxy group, R₇ and R₈ are independent from one another H, NO₂, halogen, a Ci-to C₄-alkyl or alkoxy group, and n represents a number selected from 0, 1, 2, and R₃ to R₈ have the following meaning of the group Li, m is 0 or 1 and in the case that m is 1, Z is selected from the group consisting of OC(O), OC(S), SO₂, NHC(O), NR'C(O), wherein R' is a C, to C₆ alkyl, preferably methyl, ethyl or propyl, b) and of substituted and non-substituted, condensed and non-condensed 2- (nitroaryl)ethoxycarbonyl or -thiocarbonyl compounds, substituted and non- substituted, condensed and non-condensed 2-nitrobenzyl,

2- nitrobenzyloxycarbonyl or thiocarbonyl compounds, substituted and non- substituted, condensed and non-condensed 2-(nitroheterocycloaryl)ethoxycarbonyl or -thiocarbonyl compounds, and substituted and non-substituted, condensed and non-condensed 2-(nitroheterocycloalkyl)ethoxy-carbonyl/thiocarbonyl compounds, substituted and non-substituted 2-nitro-N-methylanilinocarbonyl- or thiocarbonyl derivatives,

L| and L₂ are linker groups, which are the same or different from one another and are selected from the group consisting of OC(O), SC(O), SO₂, SC(S), NHC(O), NR'C(O), wherein R' is a Ci to C₆ alkyl, preferably methyl, ethyl or propyl, and wherein the above-mentioned meanings L₂ are not applicable, if m is 1 , CR₂, wherein R is selected from H and/or a branched or non-branched Ci to C₅ substituted or non-substituted alkyl group, where in the case that a substituted alkyl group is present, a rest R is selected from the group consisting of OC(O), SC(O), SO₂, SC(S), NHC(O), NR'C(O), wherein R' is a C, to C₆ alkyl, preferably methyl, ethyl and propyl, or one or several conjugated or non-conjugated C=C double bonds or one or several C≡C triple bonds, and

R is a usual reactive functional group, which is capable to react with the suitable substrate N or can be the substrate N,

and a and b are a number > 0 and may be the same or different from one another.

Method for the cleavage of labile functional group from molecule by exposure to electromagnetic irradiation, comprising the following steps:

a) selection of a sensitizer synthon S and of the suitable labile functional group P, wherein the first electronic excited state of the sensitizer synthon S is at the same or higher energy as the first excited electronic state of the selected labile functional group P b) preparation of the chemical compound according to the invention by the formation of an intramolecular chemical bond between the sensitizer synthon S and the labile functional group P

c) reaction of the chemical compound according to the invention with a substrate N

d) exposure to electromagnetic radiation.

3. Method according to claim 2, characterized in that the energy of the electromagnetic radiation is transferred intramolecularly from the sensitizer synthon to the labile functional group.

4. Method according to claim 3, characterized in that the labile functional group and the sensitizer synthon have the same absoφtion maxima for electromagnetic radiation.

5. Method according to claim 2, characterized in that the labile functional group and the sensitizer synthon have different absoφtion maxima for electromagnetic radiation.

6. Method according to one of the preceding claims, characterized in that the electromagnetic radiation has a wavelength, which is in the range of the absoφtion maximum of sensitizer synthon.

7. Method according to claim 6, characterized in that the electromagnetic radiation is in the wavelength range of UV//VIS radiation.

8. Method according to claim 7, characterized in that the labile group is a photolabile group.

9. Method according to one of the preceding claims, characterized in that the lowest electronically excited state (Ti) of the sensitizer synthon is energetically as high or higher than the lowest electronically excited state (Tj) of the labile functional group.

10. Method according to claim 9, characterized in that the energy gap of the sensitizer synthon between light absoφtion of the longest wavelength corresponding to (Si) and of the lowest electronically excited state (Ti) is the same or smaller as the corresponding energy gap of the labile functional group.

11. Method according to one of the preceding claims, characterized in that the absoφtion of the absoφtion band of the longest wavelength of electromagnetic radiation of the sensitizer synthon is longer than 280 nm.

12. Use of the method according to one of the preceding claims for the manufacture of spatially addressed molecular libraries.

13. Method for the manufacture of spatially addressed molecular libraries of biomolecules by spatially addressed light directed synthesis from building blocks of the biomolecules on solid substrates comprising the following steps:

a) reaction of the unprotected terminal 3' or 5' hydroxy group of a nucleoside and/or nucleotides or nucleic acid analogon or the terminal substituted or unsubstituted amino or carboxyl group of a respective peptide ranged on a solid substrate with a chemical compound according to claim 1, which comprises a photolabile protecting group and a sensitizer synthon and purification of the reaction products if necessary,

b) spatially selective irradiation of the surface of the support treated in step a) with electromagnetic radiation in the UV/VIS range,

c) reaction of a nucleoside and/or nucleotide were a free 5' or 3'OH group is protected by a photolabile group or a chemical compound according to claim 1 and/or with a corresponding peptide, which is protected on the amino group or on the carboxy group by a photolabile group or a chemical compound according to claim 1

if necessary repetition of steps b) to c).

14. Method according to claim 13, characterized in that the energy of the electromagnetic radiation is transferred intramolecularly from the sensitizer synthon to the photolabile protecting group.

15. Method according to claim 14, characterized in that step b) is carried out before step c).

16. Method according to claim 13, characterized in that step c) is carried out before step b).

17. Method according to claim 13, characterized in that step c) and step b) are carried out simultaneously.

18. Method according to one of the claims 13 to 17, characterized in that the UV/VIS radiation used in the method has a wavelength, which is in the region of the absoφtion maximum of the sensitizer synthon.

19. Method according to one of the claims 13 to 17, characterized in that the lowest electronically excited state (Ti) of the sensitizer synthon is energetically as high or higher than the lowest electronically excited state (Ti) of the photolabile protecting group.

20. Method according to claim 18, characterized in that the energy gap of the sensitizer synthon between the state (Si) which corresponds to the light absoφtion at the longest wavelength and the state (Ti) which corresponds to the lowest electronically excited state is equal or smaller than the corresponding energy gap of the photolabile protecting group.

21. Method according to claim 19, characterized in that the absoφtion band with the longest wavelength of the electromagnetic radiation of the sensitizer synthon has a wavelength longer than 280 nm.

22. Kit comprising a chemical compound according to claim 1.

23. Kit comprising a portion or all reagents and/or adjuvants and/or solvents and/or working instructions for carrying out a method according to one of the claims 2 to 11 and/or 13 to 20 in one spatial unit.

24. Use of a method according to one of the claims 13 to 20 and/or a kit according to claim 21 and/or 22 for the manufacture of oligonucleotides, polypeptides, or nucleic acid or peptide chips.

24. Use of a method according to one of the claims 2 to 11 and/or a kit according to claim 21 and/or 22 for the manufacture of a spatially addressed molecular libraries and/or of polymers.