WO2004085394A1 - 7-nitroindoline derivatives and their uses - Google Patents

Abstract

7-nitroindoline compounds are disclosed that comprise a triplet sensitising group such as substituted or unsubstituted benzophenone group and can be used to cage effector species. In particular, the inclusion of a triplet sensitising group linked to the 4 or 5 position of a 7-nitroindoline derivative provides compounds which can be photolysed to release the effector species with unexpectedly enhanced photolysis efficiency. The triplet sensitising group may be linked directly to the 7-nitroindoline or via a spacer group. In some examples, the triplet sensitising group and/or the spacer group can be selected to enhance other properties of the caged compound such as its solubility, spectroscopic properties or stability (e.g. stability of the linkage between the nitroindoline moiety and the triplet sensitiser). This can help to improve the performance of the caged compound, especially in aqueous environments containing dissolved oxygen.

Description

7-Nitroindoline Derivatives and Their Uses

Field of Invention

The present invention relates to 7-nitroindoline derivatives and their uses as caged compounds from which effector species such as neurotransmitters and a ino acids are releasable on irradiation with light.

Background of Invention Photorelease of bioeffector species from masked precursors ("caged compounds") by illumination with a brief pulse of near-UV light is an effective means to initiate rapid processes in a biological system.¹ In particular, we have been concerned to devise means for rapid (preferably sub- millisecond) release of neuroactive species upon flash photolysis that could be used to release such compounds within brain slices or related neuronal preparations such as patch-clamped single cells. To this end, we have previously developed particular l-acyl-7-nitroindolines 1 and 2 (shown here in generic form with a general acyl group) that photolyse in aqueous solution according to the outline reactions shown in Scheme l .^{2, 3} The methoxy- substituted compounds of generic structure 2 gave more efficient photorelease (by a 2-3 fold factor) , owing to a combination of higher molar absorbance in the near-UV spectrum and higher quantum yield.³ The specific L- glutamate conjugates 5 and 6 have been used in a range of physiological applications where they successfully mimic the actions of endogenous glutamate .^4~6

The mechanism of the photochemical cleavage of these 1- acyl-7-nitroindolines has been studied and photorelease of the carboxylate that derives from the 1-acyl group was found to have a half time of ~150 ns upon nanosecond time scale flash irradiation in neutral aqueous solution at ambient temperature.⁷

Other caging groups have been introduced in recent years that have had potential for release of neuroactive amino acids by strategies that involve blocking either the amino or carboxylate functional group of the active species . These include the α-carboxy-2-nitrobenzyl (CNB) derivatives of L-glutamate or GABA (compounds 7 and 8),⁸ the brominated 7-hydroxycoumain-4-methyl (BHC) carbamates and esters 9 and 10 respectively⁹ and a brominated hydroxyquinolinylmethyl (BHQ) model compound ll.¹⁰ However, most of these compounds have some drawbacks for use as rapid release agents. The CNB ester-caged species and the BHC ester 10 have relatively poor resistance to spontaneous hydrolysis, so have a tendency to leak the effector species prior to a light flash, with consequent possibilities of receptor desensitisatio . The BHC carbamate 9 is stable to spontaneous hydrolysis and undergoes rapid photocleavage but release of the actual effector is dependent on a subsequent non-photochemical decarboxylation reaction. Previous data¹¹ indicate that this process has a rate constant of 150 s^"1 at pH 7, 21°C, implying that the release rate of the amino acid itself is on a time scale of 4-5 ms, much slower than in normal synaptic functioning. The BHQ system 11 has not been evaluated for its utility in biological systems, but was reported to be more resistant to hydrolysis than the BHC ester. In comparison, the nitroindoline caged compounds have high resistance to hydrolysis.²³

It remains a considerable problem in the art to provide caged compounds which are capable of efficiently releasing bioeffector species and which are chemically robust enough to be used under the conditions found in biological systems, e.g. in aqueous environments in the presence of oxygen.

Summary of the Invention

Broadly, the present invention relates to 7-nitroindoline compounds useful for caging effector species which comprise a triplet sensitising group such as substituted or unsubstituted benzophenone group. The present invention is based on the realisation that the photochemistry for the release of the effector species proceeded via the triplet excited state of the 7- nitroindoline derivatives. Surprisingly, the present inventors have found that the inclusion of a triplet sensitising group linked to the 4 and/or 5 position of a 7-nitroindoline derivative used to cage an effector species provides compounds which can be photolysed to release the effector with unexpectedly enhanced photolysis efficiency. The triplet sensitising group may be linked directly to the 7-nitroindoline or via a spacer group. In particularly preferred embodiments, the triplet sensitising group and/or the spacer group can be selected to enhance other properties of the caged compound such as its solubility, spectroscopic properties or stability (e.g. stability of the linkage between the nitroindoline moiety and the triplet sensitiser) . This can help to improve the performance of the caged compound, especially in aqueous environments containing dissolved oxygen.

Accordingly, in a first aspect, the present invention provides a compound represented by the formula:

wherein:

Ri is an effector species linked to the nitrogen atom at the 1-position of the indoline ring via an acyl linkage or is a group which is capable of linkage to an effector species ;

R₂ and R₃ are selected from hydrogen, a substituted or unsubstituted alkyl group, or R₂ and R₃ together form a substituted or unsubstituted cycloalkyl group;

R₄ is hydrogen, a substituted or unsubstituted alkyl group, a substituted or unsubstituted alkoxy group or a triplet sensitising group; and

R₅ is hydrogen, a substituted or unsubstituted alkyl group, a substituted or unsubstituted phenyl group, a triplet sensitising group, a group represented by(CH₂)_nY or (CH₂) _m0 (CH₂) _nY, where m and n are independently between 1 and 10 and Y is selected from hydrogen, C0₂H or salts thereof, OP0₃ ^2_ or salts thereof, OSO₃ ^"" or salts thereof, or C0₂R_δ , wherein R₆ is an alkyl or substituted alkyl group; wherein at least one of R₄ and R₅ is a triplet sensitising group.

In embodiments of the present invention in which the 1- acyl-7-nitroindolines are used to cage an effector group, preferably the linkage to Ri group is represented by:

wherein X is -C-R, -O-R or -NH-R, wherein R is the remaining part of the effector species. Examples of preferred effector species are described below and include glycine, GABA and glutamate.

According to the present invention, the provision of a triplet sensitising group provides this class of 7- nitroindoline with unexpectedly enhanced photolysis efficiency, as well as other properties that make the compounds of the invention particularly apt for use in high throughput screening applications and patch clamp experiments. Suitable triplet sensitising groups are well known to those skilled in the art. Preferred triplet sensitising groups have triplet energies of at least 4 kcal/mol above that of the triplet energy of the parent compound¹³⁰. In the present case, the 1-acyl nitroindoline has an estimated triplet energy in the region of 60 kcal/mol⁷, and so preferred triplet sensitising groups have triplet energies that are preferably at least 54 kcal/mol. A particularly preferred triplet sensitising group, 4, 4 ' -dialkoxybenzophenone, has a triplet energy of -70 kcal/mol (see Tables 1-1 and 1-2 of reference¹³³) . Furthermore, advantageously, the triplet lifetime of the triplet sensitising group should be sufficiently long that there is time to transfer the energy to the molecule which it is desired to excite. It is also preferred that the efficiency of intersystem crossing is sufficiently high to ensure efficient population of the triplet state of the sensitiser. In this respect, a preferred class of substituted or unsubstituted benzophenones is ideal in this respect, with φisc ~ 1. As the triplet sensitisers of the present invention work use intramolecular energy transfer, it is preferred that, of the incident light that falls on the sample, the majority is absorbed by the sensitiser rather than the nitroindoline moiety, to obtain the triplet state of the sensitiser that then transfers its energy to the nitroindoline system.

The skilled person is well able to balance these factors and, based on the teaching herein, to select suitable triplet sensitising groups for use in accordance with the present invention, e.g. with reference to Tables 1-1 and 1-2 of reference¹³³. One preferred class of triplet sensitising groups are unsubstituted and substituted benzophenones, particularly those having one or more substituents at the 4 or 4,4' positions such as substituted or unsubstituted alkyl, alkoxy, dialkyl or dialkoxy groups. One preferred substituent may be defined as -0- (CH₂) _n ^_0P0₃ ^2_, where n is an integer between 1 and 10, and is preferably where n = 2. Compounds of this sort, such as the exemplified , 4' -dialkoxy derivatives, have strong near-UV absorption. Other types of preferred triplet sensitising groups include, but are not limited to, substituted and unsubstituted anthrones, substituted and unsubstituted xanthones, substituted and unsubstituted carbazoles, substituted and unsubstituted triphenylenes and substituted and unsubstituted heterocyclic analogues of benzophenone such as 3- or 4-benzoylpyridines .

The triplet sensitising group (s) may be linked directly to the 4 and/or 5-position of the nitroindoline ring, or via a linker group, which itself can link to the triplet sensitising group directly or via one of its substituents, if present. The use of a readily formed linker group between the separate parts of the molecule is also advantageous for two reasons, first that it allows so- called convergent synthesis, i.e. complex parts of the overall molecule can be separately assembled and brought together at a late stage of the synthesis. The second reason is that aspects of the synthetic chemistry compatible with one portion of the overall molecule are not necessarily compatible with the other part. Thus, the considerations of suitable protecting groups and reaction conditions are greatly simplified by late-stage assembly of the complete sensitised molecule.

A preferred and exemplified linker group is an amide group having the general formula:

wherein T is the triplet sensitising group, o and p are independently selected from integers from 1 to 10, and the linker group is attached via the 4 and/or 5-position of the nitroindoline. In some embodiments, the amide linker groups may include an oxygen atom between the alkyl groups -(CH₂)_P- and/or -(CH₂)₀- and the nitroindoline and/or the triplet sensitising group. Thus, more generally, the present invention provides a class of linkers which comprise a group represented by - (CH₂) _P-NH-C (=0) - (CH₂) ₀- and more preferably are represented by the general formula -0-(CH₂)_p-NH-C(=0) - (CH₂)_o-0-. In both cases, the nitroindoline and the triplet sensitising groups may be bonded to either end of the linker, i.e. so that the linker group may be in either of the two possible orientations between the nitroindoline and the triplet sensitising group. Preferably, in linkers of all these types, o and p are independently selected from integers which are 1 or 2.

An alternative linker group is represented by the general formula T-O- (CH₂) _o-0-nitroindoline, wherein T is the triplet sensitising group and o is an integer from 1 to 10 and the linker group is attached via the 4 and/or 5- position of the nitroindoline. In the preferred compounds of these types o and p are independently selected from integers which are 1 or 2.

Substituted or unsubstituted amide linkers have the further advantage that they are very stable with respect to hydrolysis. Nevertheless, the particular linkage is not of itself an essential feature of the invention and those skilled in the art can readily design alternatives based on the teaching herein. By way of example, a substituted or unsubstituted alkyl group could also serve as a linker group, e.g. as represented by -(CH₂)₀~_Λ where o is between 1 and 10. Alternative linker group may have one or more methylene groups, a phenylene group, an oligomethylenephenylene, one or more oxygen atoms, etc. The use of amino components within the linker (as opposed to the amide groups) is not preferred as they can lead to the diversion of the photochemistry into non-productive pathways .

The linker group and triplet sensitising group may also be further functionalised to engineer other useful properties into the molecule. By way of example, in the exemplified compounds disclosed herein, a phosphate group is introduced into the triplet sensitising group to improve the aqueous solubility of the compounds. Additionally or alternatively, one or more other solubilising moieties can be included as substituents in any part of the molecule such as a sulfate group, a carboxylate group or a trialkylammonium substituent, or an uncharged solubilising group such as a poly-hydroxy moiety, e.g. a monosaccharide or similar polyol.

Exemplary compounds of the present invention are described in the examples below.

In a further aspect, the present invention provides a method employing a 7-nitroindoline as defined herein which comprises a triplet sensitising group and an effector species, the method comprising irradiating the 7- nitroindoline to cause it to photolyse and release the effector species. Examples of such methods are disclosed below and include patch clamp experiments and high throughput screening methods

Embodiments of the present invention will now be described in more detail by way of example and not limitation with reference to the accompanying figures and schemes.

Brief Description of the Figure

Figure 1. Absorption spectra of the conjugates 12, 32 and 41. As described in General Details, these spectra were calculated by summing the spectra of the benzophenone 19 and the appropriate nitroindoline component. The spectra are not sensitive to a particular aliphatic (amino) acid attached at position 1 of the nitroindoline, so are also appropriate for quantification of conjugates such as 33 (using the appropriate reference spectrum, in this case that of compound 32) . Detailed Description Substituents

Unless otherwise specified, the term "substituted," as used herein, pertains to a parent group which bears one or more substitutents . The term "substituent" is used herein in the conventional sense and refers to a chemical moiety which is covalently attached to, appended to, or if appropriate, fused to, a parent group. A wide variety of substituents are well known in the art, and methods for their formation and introduction into a variety of parent groups are also well known.

Alkyl: The term "alkyl," as used herein, pertains to a monovalent moiety obtained by removing a hydrogen atom from a carbon atom of a hydrocarbon compound having from 1 to 20 carbon atoms, or more preferably 1 to 10 carbon atoms, which may be aliphatic or alicyclic, and which may be saturated, partially unsaturated, or fully unsaturated. Thus, the term "alkyl" includes the sub-classes alkenyl, alkynyl, cycloalkyl, etc., discussed below.

Alkoxy: preferably alkoxy groups are represented by 0(CH₂)n~Y_/ where n and Y are defined above, and may be optionally substituted with one or more of the groups below.

In embodiments of the invention in which a substituted phenyl groups is present, it may include one or more of the substituents set out below. If the phenyl group has less than the full complement of substituents, they may be arranged in any combination. For example, if the phenyl group has a single substituent other than hydrogen, it may be in the 2~ , 3-, or 4-position. Similarly, if the phenyl group has two substituents other than hydrogen, they may be in the 2,3-, 2,4-, 2,5-, 2,6-, 3,4-, or 3, 5-positions . If the phenyl group has three substituents other than hydrogen, they may be in, for example, the 2,3,4-, 2,3,5-, 2,3,6-, 2,4,5-, 2,5,6-, or 3, 4, 5-positions . If the phenyl group has four substituents other than hydrogen, they may be in, for example, the 3,4,5,6-, 2,4,5,6-, 2,3,5,6-, 2,3,4,6-, or 2 , 3, 4 , 5-positions .

In preferred embodiments of the present invention the substituted functional groups as defined herein may be independently selected from: halo; hydroxy; ether (e.g., Cι_₇alkoxy) ; formyl; acyl (e.g., Cι-alkylacyl , C₅-2oarylacyl) ; acylhalide; carboxy; ester; acyloxy; amido; acylamido; thioamido; tetrazolyl; amino; nitro; nitroso; azido; cyano; isocyano; cyanato; isocyanato; thiocyano; isothiocyano; sulfhydryl; thioether (e.g., Cι-₇alkylthio) ; sulfonic acid; sulfonate; sulfone; sulfonyloxy; sulfinyloxy; sulfamino; sulfonamino; sulfinamino; sulfamyl; sulfonamido; phosphoryl groups such as phosphate -O-P(O) (OH) ₂; thiophosphate -O-P (S) (OH) ₂; phosphate esters -O-P(O) (OR' )₂; thiophosphate esters -O-P (S) (OR' ) ₂; phosphonate -0-P(0)OHR'; thiophosphonate -O-P (S) OHR' ; substituted phosphonate -O-P (0) OR' ιR'₂; substituted thiophosphonate -O-P (S) OR' _λR' ₂; -O-P (S) (OH) (SH) ; cyclic phosphate, wherein R' , R'ι and R'₂ are independently hydrogen or Cι_ι₀ unsubstituted or substituted alkyl or aryl; Cι_₇alkyl (including, e.g., unsubstituted Cι-₇alkyl, Cι_₇haloalkyl, Cι-₇hydroxyalkyl, Cι_carboxyalkyl, Cι_₇aminoalkyl, Cs_₂oaryl-C₁_₇alkyl) ; C₃-₂₀heterocyclyl; or C₅-₂oaryl (including, e.g., C₅_₂ocarboaryl, C₅_₂oheteroaryl, Cι_alkyl-C₅-₂oaryl and C₅_₂ohaloaryl) ) .

In one preferred embodiment, the substituent (s) , often referred to herein as R, are independently selected from: -F, -Cl, -Br, and -I;

-OH;

-OMe, -OEt, -O(tBu), and -OCH₂Ph;

-SH; -SMe, -SEt, -S(tBu), and -SCH₂Ph;

-O-P(O) (OH)₂, -O-P(S) (OH)₂,-0-P(0) (OR')₂, -O-P (S ) (OR' ) ₂, -

0-P(0)OHR', -0-P(S)OHR', -O-P (0) OR' _XR' ₂, -O-P ( S) OR' iR' ₂, -

O-P(S) (OH) (SH) and cyclic phosphate, R' , R' _x and R'₂ defined as above; -C(=0)H;

-C(=0)Me, -C(=0)Et, -C(=0) (tBu), and -C(=0)Ph;

-C(=0)OH;

-C(=0)OMe, -C(=0)OEt, and -C (=0) O ( tBu) ;

-C(=0)NH₂, -C(=0)NHMe, -C (=0) NMe₂, and -C(=0)NHEt; -NHC(=0)Me, -NHC(=0)Et, -NHC(=0)Ph, succinimidyl, and maleimidyl;

-NH₂, -NHMe, -NHEt, -NH(iPr), -NH(nPr), -NMe₂, -NEt₂,

-N(iPr)₂, -N(nPr)₂, -N(nBu)₂, and -N (tBu) ₂;

-CN; -N0₂;

-Me, -Et, -nPr, -iPr, -nBu, -tBu;

-CF₃, -CHF₂, -CH₂F, -CC1₃, -CBr₃, -CH₂CH₂F, -CH₂CHF₂, and

-CH₂CF₃;

-OCF₃, -0CHF₂, -OCH₂F, -OCCl₃, -OCBr₃, -OCH₂CH₂F, -0CH₂CHF₂, and -OCH₂CF₃;

-CH₂OH, -CH₂CH₂OH, and -CH (OH) CH₂OH;

-CH₂NH2,-CH₂CH₂NH₂, and -CH₂CH₂NMe₂; and, optionally substituted phenyl.

Unless otherwise specified, included in the above definition of substituents are the well known ionic, salt, solvate, and protected forms of these substituents. For example, a reference to carboxylic acid (-COOH) also includes the anionic (carboxylate) form (-COO^") , a salt or solvate thereof, as well as conventional protected forms. Similarly, a reference to an amino group includes the protonated form (-N⁺HR¹R²) , a salt or solvate of the amino group, for example, a hydrochloride salt, as well as conventional protected forms of an amino group. Similarly, a reference to a hydroxyl group also includes the anionic form (-0^") , a salt or solvate thereof, as well as conventional protected forms .

Effector Species

The Ri group can be an effector species or a group capable of being coupled to or converted into an effector species. The effector species is preferably linked to the indoline via an acyl linkage, which may be derived either from a carboxylate moiety inherently present in the effector or as part of a carbamate or urea linkage between the effector and the indoline, for example:

Depending on particular cases, the linkage to a hydroxyl or amino group on the effector may be performed either before or after the nitration reaction according to the present invention. Where the linkage of effector to indoline is carried out post nitration, it can be accomplished as follows:

phosgene carbamate or urea derivatives as shown above

In cases where the effector species is resistant to the nitration conditions, the non-nitrated indoline can be taken through the same steps to create the acyl linkage to the effector species.

Examples of effector species include labels, drugs, toxins, or carrier or transport molecules. Effector species preferably have a carboxylic acid group which can be coupled to the nitrogen atom at the 1-position on the indoline to form an acyl linked effector species.

Techniques for coupling the photocleavable group to both peptidyl and non-peptidyl coupling partners are well known in the art. In preferred embodiments, the effector species is a biologically active compound such as an amino acid (either L or D-amino acids) , and more particularly neuroactive amino acids such as L-glutamate, GABA and glycine. The procedures described herein can also be readily adapted to linking larger effector groups such as oligopeptides or polypeptides to the photocleavable group. Examples of especially suitable peptides are as follows: thyrotrophin releasing hormone TRH; enkephalins (locally acting endogenous opiates) ; bradykinin; and angiotensin II. Generally, the methods described herein are applicable to any oligopeptides with non-amidated C- termini .

The synthesis of photoreleasable compounds including oligo or polypeptide effector species can be achieved by linking a terminal amino acid (i.e. the C-terminal amino acid) to the photocleavable group and then using polypeptide chain extension techniques to build up the peptide chain stepwise, or by coupling an oligopeptide or polypeptide to the terminal amino acid linked to the photocleavable group. Standard peptide synthesis techniques could also be adapted by linking to the synthesis resin a suitably substituted alkoxy group at position 4. This would give a resin containing the protecting group and C-terminal residue which could be elaborated and eventually cleaved from the resin by standard techniques.

If the photoreleasable compound is synthesized using the scheme described in the examples below with the effector species introduced prior to the nitration reaction, it is preferred that the effector species is stable to the nitration reaction carried out after it is attached to the nitrogen of the indoline ring. In the case of amino acids, this means that the use of amino acids other than tryptophan, tyrosine, cysteine or methionine is preferred.

In other aspects, the present invention provides precursor compounds in which the effector species has not been linked to the photocleavable group (e.g. X is H, COY as defined above, or COC1) and/or in which the nitration reaction has not been carried out (e.g. the substituent at the 7-position is hydrogen) .

After administration, the photoreleasable compound can be activated to release the effector species, conveniently by exposure to a flash of UV light.

Preferred applications of the compounds of the invention are in patch clamp experiments and/or in high throughput screening (HTS) . Patch clamp experiments are a widely used technique in biology that was originally developed to observe ionic current produced when ions flow through ion channels, membrane proteins that regulate the flow of ions across cellular membranes and hence the physiology of cells. This ionic movement creates an electrical current which is tightly regulated by specific signals that cause the ion channels to open and close. The movement of the ions leads to a measurable electrical current that forms the basis of processes such interneuronal and neuromuscular communication. For an introductory review of the technique, see for example Patch Clamping: An Introductory Guide, Molleman, Wiley Europe, 2002.

From its origin, the technique has found many applications including the observation of the function of proteins in lipid bilayers, monitoring the synaptic transmission between neurones in the brain and monitoring changes that occur in cell membranes during secretion. In basic terms, patch clamp experiments employ a pipette or capillary having an opening between about 0.1 and 5μm. A portion of the cell wall of a single cell is sucked into the opening allowing potentials to be applied to and measured across the cell membrane. More recently, patch clamping has been used in assays for the effect of drugs on cells particularly those used to affect ion channels such as sodium or potassium channels.

As the caged compounds of the present invention are compatible with the biological conditions used in such cell based assays and are capable of releasing effector species on irradiation, they are particularly suited in cell based assays such as patch clamp experiments and high throughput screening methods. Thus, the compounds of the invention can be introduced into the vicinity of a cell, e.g. in a patch clamp experiment, and a concentration of the active effector species generated in very short period on irradiation. This enables the effect of the released species to be studies under controlled circumstances. Several reports indicate progress towards high throughput screening in association with patch clamping¹², and neuroactive amino acids and their interactions with specific receptors are targets for therapeutic intervention. The ability to apply a sub-millisecond pulse of neuroactive amino acid to patch clamped cells within a multiple assay format is likely to be an important component of successful assays, avoiding the well-known desensitisation of receptors on neuronal cells that occurs in the prolonged presence of the neuroactive amino acid. In one possible embodiment of such an assay, the array of patch clamped cells would be set up with specific test compounds together with the caged native neuroactive amino acid (such as L-glutamate, GABA or glycine) and the native compound would then be photoreleased by brief illumination of the array.

Experimental General Details ¹H NMR spectra were determined on Varian Unityplus 500 or JEOL FX90Q spectrometers in CDC1₃ solution with TMS as internal reference, unless otherwise specified. Elemental analyses were carried out by MEDAC Ltd., Surrey, U.K. Merck 9385 silica gel was used for flash chromatography . Quantitative amino acid analysis was carried out at the

Department of Biochemistry, University of Cambridge using a Pharmacia AlphaPlus Analyser with ninhydrin detection. Analytical HPLC was performed on a 250 x 4 mm Merck Lichrospher RP8 column at 1.5 mL rnin^"1 flow rate. Preparative HPLC was carried out on a 2 x 30 cm column (Waters Cι₈ packing, Cat. No. 20594) at 2 mL min^"1 flow rate. Detection for analytical and preparative work was at 254 nm. Mobile phases are specified in the sections on particular compounds. Photolysis experiments were performed in a Rayonet RPR-100 photochemical reactor fitted with 16 x 300 nm lamps. Petroleum ether (bp 40-60 °C) was redistilled before use.

Quantitative absorption spectra for the conjugates 12, 32 (also applicable to 33) and 41 were calculated by summing the individual molar absorbance spectra of the benzophenone 19 and the appropriate nitroindoline component. The results are shown in Figure 1 and the calculated absorption coefficients are given in the details of the individual compounds.

4- [ ( t-Butyldimethyl s ilyl ) oxy] -1 -bromobenzene 13

This was adapted from an outline procedure of Angle and Louie.¹⁴ To a solution of 4-bromophenol (10.38 g, 60 mmol) and imidazole (6.13 g, 90 mmol) in dry CH₂C1₂ (150 mL) was added dropwise a solution of tert- butyldimethylsilyl chloride (10.85 g, 72 mmol) in dry CH₂C1₂ (50 mL) , and the mixture was stirred under nitrogen at room temperature for 18 h. The cloudy solution was diluted with CH₂C1₂ (100 mL) and washed successively with 0.5 M aq. HC1, 0.5 aq. NaOH and brine. The organic phase was dried and evaporated to give a colourless liquid which was fractionally distilled under reduced pressure to give 13 as a colourless oil (16.50 g, 96%), bp 76-78°C, 0.25 mm

Hg, (lit.¹⁴ bp 120-125°C, 1 mmHg) ; ¹R NMR: (90 MHz) δ 1 . 32 (2H, d, J = 8 Hz), 6.70 (2H, d, J = 8 Hz), 0.97 (9H, s) and 0.18 (6H, s) .

4- (2-Bromoethoxy) benzaldehyde 14

This preparation was a modification of the method of Reunitz et al.¹⁵ 1, 2-Dibromoethane (188 g, 1 ol) was added in one portion to a solution of 4- hydroxybenzaldehyde (12.21 g, 100 mmol) and KOH (5.61 g, 100 mmol) in a mixture of boiling n-butanol (200 mL) and DMF (50 mL) and the mixture was heated under reflux for 18 h. After cooling to room temperature, the precipitated solid was filtered off, washed with DMF and the filtrate was concentrated under vacuum. The residue was dissolved in Et₂0 (200 mL) and washed with 1 M aq. NaOH and brine, dried and evaporated to give a brown oil (12.51 g) which crystallised on standing at 4°C. Recrystallisation from Et₂0-petroleum ether followed by flash chromatography [EtOAc-petroleum ether (1:9)] of the mother liquor to recover additional product gave 14 as white crystals (8.57 g, 37%) mp 49-50°C (from Et₂0-petroleum ether) (lit.²⁰ mp 52 °C) ; ¹H NMR: (90 MHz) J 9.88 (s, 1H), 7.83 (2H, d, J = 8 Hz), 7.02 (2H, d, J = 8 Hz), 4.37 (2H, t, J = 6 Hz) and 3.66 (2H, t, J = 6 Hz) .

4- (2-Bromoethoxy) - ' - [ (t-butyldimethylsilyl) oxy] benzhydrol

15

A solution of the TBDMS ether 13 (6.32 g, 22 mmol) in dry THF (44 mL) , cooled under N₂ to -78°C, was treated dropwise with t-BuLi (1.7 M solution in pentane; 13.2 mL, 22.5 mmol), keeping the temperature below -76°C. The solution was stirred for 2 h at -78 °C and a solution of 14 (4.58 g, 20 mmol) in dry THF (60 mL) was added dropwise, keeping the same temperature control. The solution was then allowed to warm to 0°C and stirred for a further 2 h, diluted with ether (200 mL) and washed with saturated aq. NaHC0₃ and brine. The organic phase was dried, evaporated and flash chromatographed [petroleum ether, then EtOAc-petroleum ether (1:4)] to give 15 as a pale oil (6.98 g, 80%) which was used in the next step without further purification. ^XH NMR: (90 MHz) δ 7.08-7.36 (4H, m) , 6.68- 6.96 (4H, m) , 5.70 (1H, d, J = 3.6 Hz), 4.24 (2H, t, J = 6 Hz ) , 3 . 58 ( 2H, t , J = 6 Hz ) , 2 . 32 ( 1H, d, J = 3 . 6 Hz ) , 0 . 97 ( 9H , s ) and 0 . 18 ( 6H, s ) .

4 - (2-Bromoethoxy) -4 ' -hydroxybenzophenone 17 A solution of the benzhydrol 15 (6.44 g, 14. 7 mmol) in

CH2CI2 (250 mL) was treated with manganese (IV) oxide (14.7 g) and the mixture was stirred at room temperature for 3.5 h, then filtered through Celite. The filtrate was evaporated to leave the TBDMS ether 16 as a pale oil (6.40 g, 100%) which was used in the next step without further purification. ¹H NMR: (90 MHz) 7.60-7.88 (4H, m) , 6.80- 7.08 (4H, m) , 4.36 (2H, t, J = 6 Hz), 3.66 (2H, t, J = 6 Hz), 1.00 (9H, s) and 0.25 (6H, s) .

A solution of 16 (13.76 g, 32 mmol) in THF (250 mL) was cooled to 0°C and treated with glacial acetic acid (1.92 g, 32 mmol) and 1 M TBAF (32 mL, 32 mmol) for 20 min . The solution was concentrated to ~80 mL, diluted with water (250 mL) and extracted with Et2θ (3 x 120 mL) . The combined organic phases were washed with brine, dried and flash chromatographed [EtOAc-petroleum ether (3:7)] to give 17 as pale crystals (7.57 g, 74%), mp 139-140°C

(EtOAc-petroleum ether), (lit.²¹ 139-142°C).

4- (2-Azidoethoxy) -4 ' -hydroxybenzophenone 18

A mixture of the bromide 17 (5.78 g, 18 mmol) and NaN₃ (3.51 g, 54 mmol) in dry DMF (180 mL) was heated at 100°C for 2.5 h. The solvent was removed under high vacuum and the residue was diluted with EtOAc, washed with water, dried and evaporated. Flash chromatography

[EtOAc-petroleum ether (2:3)] gave 18 as white crystals (4.59 g, 90%), mp 105-106 °C (from EtOAc-petroleum ether); (Found: C, 63.56; H, 4.64; N, 14.73; Calcd. for Cι₅Hι₃N₃0₃: C, 63.60; H, 4.65; N, 14.83%); IR: v_max/cm^_1 3320, 2100, 1625, 1600, 1565, 1320, 1255; ^XH NMR: (500 MHz, CDC1₃ + DMSO- 6) £ 9.80 (1H, br s) , 7.75 (2H, AA'BB', J = 8.8 Hz,), 7.67 (2H, AA'BB', J= 8.8 Hz), 6.99 (2H, AA'BB', J = 8.8 Hz), 6.90 (2H, AA'BB', J= 8.8 Hz), 4.25 (2H, t, J= 4.8 Hz) and 3.66 (2H, t, J = 4.8 Hz).

4- (2-Azidoethoxy) -4 ' - (2-hydroxyethoxy) benzophenone 19 A solution of the phenol 18 (1.19 g, 4.2 mmol) in 2- butanone (80 mL) was mixed with anhydrous K₂C0₃ (1.16 g, 8.4 mmol), Nal (50 mg) and 2-bromoethanol (2.62 g, 21 mmol) and the mixture was heated under reflux. The reaction progress was followed by TLC [EtOAc-petroleum ether (3:2)]. More 2-bromoethanol (2.62 g) and K₂C0₃ (1.16 g) were added at 4 h and 6 h and heating was continued for 7 h total. The solvent was evaporated and the residue was dissolved in water (100 L) and washed with EtOAc (3 x 60 L) . The combined organic phases were washed with brine, dried and evaporated to give 19 (1.07 g, 78%), mp 117-118°C (white flakes from EtOAc-petroleum ether); (Found: C, 62.41; H, 5.23; N, 12.81; Calcd. for Cι₇H₁₇N₃0₄: C, 62.38; H, 5.23; N, 12.83%); UV: Λ^ (EtOH) /nm 221 (£/M^_1cm^_1 22,200), 293 (25,700); _nax [EtOH-25 mM Na phosphate, pH 7.0 (1 : 50) ] /nm 223 /M^cm^"1 15,500), 299 (23,700); IR: v_maχ/cm^_1 3280, 2110, 1635, 1605, 1260; ^XH NMR: (500 MHz) £ 7.77-7.80 (4H, m) , 6.97-7.00 (4H, m) , 4.23 (2H, t, J = 4.8 Hz), 4.17 (2H, t, J = 4.8 Hz), 3.99- 4.03 (2H, m) , 3.65 (2H, t, J = 4.8 Hz) and 2.09-2.15 (1H, m) .

4- (2-Azidoethoxy) -4 ' ~ {2- [di (t- butoxy) phosphoryloxy] ethoxy } benzophenone 20 A solution of 19 (0.72 g, 2.2 mmol) in dry THF (30 L) was treated under nitrogen with lH-tetrazole (0.93 g, 13.2 mmol) and di- t-butyl-N,N-diethylphosphoramidite (93% purity; 1.18 g, 4.4 mmol) and the mixture was stirred at room temperature for 18 h. The solution was cooled to 0°C and treated dropwise with a solution of m-chloroperbenzoic acid (55% peracid; 2.07 g, 6.6 mmol) in CH₂C1₂ (30 mL) . The solution was stirred at 4°C for 1 h, then diluted with CH₂C1₂ (100 mL) and washed with 10 % aq. Na₂S₂0₅. The organic phase was washed with saturated aq. NaHC0₃ and brine, dried and evaporated. Flash chromatography [EtOAc-petroleum ether (7:3)] and trituration with ether gave 20 as white crystals (1.04 g, 91%), mp 53-55°C (from Et₂0-petroleum ether) ; (Found: C, 57.79; H,6.78; N, 8.03; Calcd. for C₂₅H₃₄N₃0₇P: C, 57.80; H, 6.60; N, 8.09%): IR: V_max/cm^"1 2100, 1640, 1600, 1375, 1275, 1250; ^XH NMR: (500 MHz) £7.76-7.81 (4H, m) , 6.95-7.01 (4H, ) , 4.31-4.35 (2H, m) , 4.25-4.29 (2H, ) , 4.23 (2H, t, J = 4.8 Hz), 3.65 (2H, t, J = 4.8 Hz) and 1.50 (18H, s).

l -Acetylindolin-5-yl-acetic acid 24

A solution of methyl l-acetylindoline-5-acetate 23^2a (4.66 g, 20 mmol) in a mixture of MeOH (150 mL) and 2 M aq. NaOH (15 mL, 30 mmol) was stirred at room temperature for 17 h. The solvent was evaporated and the residue was dissolved in water and washed with ether, and the aqueous phase was acidified with cone. HC1. The precipitated solid was filtered, washed with cold water and dried to give 24 as light brown crystals (4.26 g, 97%), mp 216-218°C (from aq. MeCN) ; (Found: C, 65.85; H, 6.01; N, 6.51; Calcd. for

Cι₂H₁₃N0₃: C, 65.74; H, 5.98; N, 6.39%); IR: v_max/cm^_1 1720,

1605, 1580, 1500, 1190; ^:H NMR: (90 MHz, DMSO-ci₆) £ 7.99 (1H, d, J = 8 Hz), 6.88-7.20 (2H, m) , 4.25 (2H, t, J = 8 Hz ) , 3 . 47 ( 2H, s ) , 3 . 12 ( 2H, t, J = 8 Hz ) and 2 . 16 ( 3H, s ) .

l -Acetyl-7-nitroindolin-5-yl-acetic acid 22 The acid 24 (389 mg, 1.77 mmol) was added to a well- stirred suspension of claycop^3b,3c (1.92 g) and acetic anhydride (6 mL) in CC1₄ (12 mL) and the mixture was stirred at room temperature for 18 h. The solid was filtered off, washed with EtOAc (50 L) and the filtrate was washed with brine, dried and evaporated. Flash chromatography [CHCl₃-MeOH (3:2)] afforded 22 as a yellow viscous oil (304 mg, 65%), which was used in the next step without further purification. ^XH NMR: (90 MHz) £7.52 (1H, s), 7.36 (1H, s), 4.20 (2H, t, J = 8 Hz), 3.60 (2H, s), 3.16 (2H, t, J = 8 Hz) and 2.22 (3H, s).

4- { [2- (l -Acetyl- 7-nitroindolin-5-yl) acetamido] ethoxy} -4 ' - [2- (dihydroxyphosphoryloxy) ethoxy] benzophenone 12 A solution of the azide 20 (260 mg, 0.5 mmol) in EtOH (20 mL) was treated with 10% Pd-C (200 mg) and hydrogenated at atmospheric pressure for 30 min. The catalyst was filtered off and washed with EtOH, and the filtrate was evaporated to give the amine 21 (210 mg, 0.42 mmol, 85%) as a viscous oil. ¹H NMR: (90 MHz) £7.60-7.86 (4H, m) , 6.76-7.04 (4H, m) , 3.98-4.40 (6H, m) , 3.12 (2H, t, J = 8

Hz), 2.66 (2H, br s, ) and 1.48 (18H, s,). The crude amine was dissolved in dry MeCN (10 mL) and treated with the acid 22 (200 mg, 0.75 mmol) and 1- (3-dimethylaminopropyl) - 3-ethylcarbodiimide .HC1 (115 mg, 0.6 mmol). The mixture was stirred at room temperature for 18 h, then evaporated and the residue was dissolved in EtOAc and washed successively with 0.5 M aq. HC1, saturated aq. NaHC0₃ and brine, dried and evaporated. Flash chromatography [CHCl₃-MeOH (95:5)] gave the crude product as a yellow gum (184 mg) , which was dissolved in TFA (10 mL) , stirred at room temperature for 1 h and concentrated in vacuo . The residue was dissolved in water (100 mL) and adjusted to pH 7.0 with 1 M aq. NaOH. The solution was washed with ether and analysed by reverse-phase HPLC [mobile phase: 25 mM Na phosphate, pH 6.0-MeOH (5:4)], t_R 5.4 in. The solution was lyophilised, dissolved in 25 mM Na phosphate, pH 6.0 (80 L) and pumped onto the preparative HPLC column. After loading, the column was washed with 25 mM Na phosphate, pH 6.0 for 2 h, then with water for 2 h and finally the product was eluted with water-MeOH (4:1). Fractions containing the product were analysed as above, combined and concentrated in vacuo . The residue was dissolved in water, passed through a 0.2 μm membrane filter and lyophilised. The dried product was dissolved in water (5.4 mL) and quantified by UV spectroscopy [ _nax (H2θ)/nm 300 (fi/ -icrtr¹ 25,500)] to give 12 (Na⁺ salt)

(32.4 mM, 175 μmol, 35% from 20); LRMS (ESI) m/e (M+H)^": Found: 626.3; Calcd. for (C₂₉H₂₈N₃0ιιP + H)^~: 626.2; ¹H NMR: (500 MHz, D₂0, acetone ref.) £7.69 (2H, d, J = 8.7 Hz), 7.57 (2H, d, J= 8.7 Hz), 7.48 (1H, s), 7.35 (1H, s), 7.11, (2H, d, J = 8.7 Hz), 6.77 (2H, d, J = 8.7 Hz), 4.30 (2H, t, J = 4.9 Hz), 4.11-4.15 (4H, m) , 4.03 (2H, t, J = 8 Hz), 3.61 (2H, t, J = 4.9 Hz), 3.53 (2H, s), 2.92 (2H, t, J= 8 Hz) and 2.13 (3H, s).

4-Acetoxy-l -acetylindoline 21

A solution of 4-hydroxyindole 26 (6.66 g, 50 mmol) in acetic acid (250 mL) was treated with NaBH₃CN (9.42 g, 150 mmol) over 0.5 h, keeping the temperature at ~15°C. The mixture was stirred at room temperature for 1 h and water (5 mL) was added and the solvent evaporated. The residue was dissolved in EtOAc (150 mL) and washed with saturated aq. NaHC0₃ and brine, dried and evaporated to give 4- hydroxyindoline as pale crystals (6.76 g. 100%); -"-H NMR: (90 MHz, CDC1₃ + DMSO-de) £ 6.82 (1H, t, J = 8 Hz), 6.20 (1H, d, J = 8 Hz), 6.16 (1H, d, J = 8 Hz) , 3.52 (2H, t, J = 8 Hz) and 2.90 (2H, d, J= 8 Hz). The crude indoline was dissolved in a mixture of acetic acid (50 mL) and acetic anhydride (50 mL) and heated under reflux for 1 h. The solution was diluted with water (10 mL) and the solvents evaporated. The residue was dissolved in EtOAc (150 mL) and washed with saturated aq. NaHC0₃ and brine, dried and evaporated to give 27 as pale crystals (9.01 g, 82%), mp

98-99°C (EtOAc-petroleum ether) ; (Found: C, 65.56; H, 6.07; N, 6.39; Calcd. for C₁₂Hι₃N0₃: C, 65.74; H, 5.98; N, 6.39%); IR: v_max/c ^'1 1755, 1635, 1610, 1215; ^XH NMR: (90 MHz) £ 8.07 (1H, d, J = 8 Hz) , 7.19 (1H, t, J = 8 Hz) ,

6.72 (1H, d, J = 8 Hz), 4.05 (2H, t, J = 8 Hz), 3.03 (2H, t, J = 8 Hz) 2.28 (3H, s) and 2.19 (3H, s) .

1 -Acetyl - 4 -hydroxy indoline 28 A solution of 27 (8.77g, 40 mmol) in MeOH (250 mL) was treated with 2 M aq. NaOH (22 mL, 44 mmol), stirred at room temperature for 0.75 h, diluted with water (100 mL) and concentrated. The residue was acidified to pH 3 with 2 M aq. HC1 and the precipitate was filtered, washed with water and dried under vacuum. The filtrate was extracted with EtOAc and the organic phase was washed with saturated aq. NaHC0₃ and brine, dried and evaporated to give more solid. The combined solids were recrystallised (EtOAc) to give 28 as white crystals (5.75 g, 82%), mp 230-231°C; (Found: C, 67.80; H, 6.26; N, 7.86; Calcd. for CιoHuN0₂ : C, 67.78; H, 6.26; N, 7.90%); IR: v_max/cm^-1 3150, 1630, 1610, 1295; ¹H NMR: (90 MHz, CDC1₃ + DMSO-d₆) 9.10 (1H, br s), 7.57 (1H, d, J = 8 Hz), 6.93 (1H, t, J = 8 Hz), 6.48 ( 1H, d, J = 8 Hz ) , 4 . 05 ( 2H , t , J = 8 Hz ) , 3 . 04 ( 2H, t , J = 8 Hz ) and 2 . 16 ( 3H, s ) .

Methyl (l -acetylindolin-4-yloxy) aceta te 29 A suspension of anhydrous K₂C0₃ (6.64 g, 48 mmol) in acetone (250 mL) was mixed with 28 (5.67 g, 32 mmol) . After 15 min, methyl bromoacetate (7.34 g, 48 mmol) was added and the mixture was heated under reflux for 4 h. The solid was filtered, washed with acetone and the filtrate was evaporated, then re-evaporated from toluene to give 29 as white crystals (7.19 g, 90%), mp 129-131°C

(from EtOAc-petroleum ether); (Found: C, 62.33; H, 6.06; N, 5.54; Calcd. for Cι₃H₁₅N0₄ : C, 62.64; H, 6.07; N, 5.62%); IR: v_max/cm^_1 1770, 1660, 1605, 1440, 1230, 1125; ^XH NMR: (500 MHz) £ 7.88 (1H, d, J = 8 Hz), 7.14 (1H, t, J = 8 Hz), 6.44 (1H, d, J = 8 Hz), 4.66 (2H, s), 4.08 (2H, t, J = 8.5 Hz), 3.79 (3H, s) , 3.20 (2H, t, J = 8.5 Hz) and 2.21 (s, 3H) .

Methyl ( 1 -acety 1- 7 -nitroindol in- 4 -yloxy) aceta te 31 and methyl (l -acetyl -5-nitroindolin-4-yloxy) acetate 30 A solution of 29 (2.49 g, 10 mmol) in a mixture of CC1₄ (80 mL) and acetic anhydride (40 mL) was treated with claycop^3b,3c (6.4 g) and the mixture was stirred at room temperature for 4 h. The solid was filtered and washed with CC1₄ and the filtrate was evaporated. The residue was dissolved in EtOAc and washed with saturated aq. NaHC0₃ and brine, dried and evaporated to give a brown viscous oil. Flash chromatography [EtOAc-petroleum ether (4:1)] afforded two products. The less polar was the 7- nitro isomer 31 as pale yellow crystals (1.76 g, 60%) mp

136-137°C (EtOAc-petroleum ether) ; (Found: C, 53.13; H, 4.82; N, 9.49; Calcd. for C₁₃H₁₄N₂0₆: C, 53.06; H, 4.80; N, 9.52%); UV: ^ (EtOH) /nm 249 (s/M^cπf¹ 19,200), 295 (4,000), 321 (sh) (3,600); _nax [EtOH-25 mM Na phosphate, pH 7.0 (1:25) /nm 247 { ε/M^cm^'1 18,600), 322 (4,400); IR: v_max/cm^_1 1750, 1685, 1615, 1595, 1510, 1460, 1300, 1205, 1110; ¹H NMR: (90 MHz) £ 7.68 (IH, d, J = 9 Hz), 6.45 (IH, d, J= 9 Hz), 4.73 (2H, s), 4.25 (2H, t, J = 8 Hz), 3.80 (3H, s), 3.17 (2H, t, J = 8 Hz) and 2.24 (3H, s) . The more polar product was the 5-nitro isomer 30 as pale yellow needles (0.87 g, 30%) mp 134-135°C (EtOAc-petroleum ether); (Found: C, 53.20; H, 4.83; N, 9.48; Calcd. for

C₁₃Hi₄N₂0₆: C, 53.06; H, 4.80; N, 9.52%); UV: ^_ax (EtOH) /nm 240 (£/M^_1cm^_1 11,900) , 329 (10,200); UV: u_ax [EtOH-25 mM Na phosphate, pH 7.0 (1:25) ] /nm 238 (s/M^cπf¹ 9,500), 342 (10,000); IR: v_max/cm^_1 1765, 1690, 1605, 1585, 1510, 1460, 1315, 1205, 1095; ^XH NMR: (90 MHz) £7.80-8.20 (2H, m) ,

4.65 (2H, s), 4.18 (2H, t, J = 8 Hz), 3.79 (3H, s), 3.33 (2H, t, J = 8 Hz) and 2.26 (3H, s).

(l -Acetyl-7-nitroindolin-4-yloxy) acetic acid 31a A solution of the acetate 31 (0.47 g, 1.6 mmol) in a mixture MeOH (32 mL) and 1 M aq. NaOH (2.4 mL, 2.4 mmol) was stirred at room temperature for 0.5 h and diluted with water. The solvent was evaporated and the residue was acidified to pH 3 with 1 M aq. HC1 and extracted with EtOAc. The combined organic phases were washed with brine, dried and evaporated to give 31a as orange crystals (0.39 g, 87%), mp 206-208°C (from MeOH) ; (Found: C, 51.28; H, 4.30; N, 10.10; Calcd. for Cι₂H₁₂N₂0₆: C, 51.43; H, 4.32; N, 9.99%); IR: v_max/cm^_1 1755, 1645, 1605, 1525, 1455, 1390, 1190, 1105; ^XR NMR: (90 MHz, CDCI3 + DMS0-d₆) £ 7.64 (IH, d, J = 9 Hz), 6.67 (IH, br s), 6.62 (IH, d, J = 9 Hz), 4.73 (2H, s) , 4.26 (2H, t, J = 8 Hz) , 3.16 (2H, t, J = 8 Hz) and 2.23 (3H, s) .

4-{2- [ (l-Acetyl-7 -nitroindolin-4-oxy) acetamido] ethoxy } -4 ' - [2- (dihydroxyphosphoryloxy) ethoxy] benzophenone 32

The azide 20 (324 mg, 0.62 mmol) was hydrogenated and the crude amine (249 mg, 0.5 mmol, 81%) coupled with 31a (207 mg, 0.74 mmol) as described above for 12 and flash chromatographed [CHCl₃-MeOH (96:4)]. The recovered product was dissolved in TFA (10 mL) , stirred at room temperature for 1 h and concentrated in vacuo. The residue was dissolved in water (95 mL) and adjusted to pH 7.0 with 1 M aq. NaOH. The solution was washed with ether and analysed by reverse-phase HPLC [mobile phase: 25 mM Na phosphate, pH 6.0-MeOH (5:4 v/v) ] , t_R 5.6 min. The solution was lyophilised, redissolved in 25 mM Na phosphate, pH 6.0 (120 mL) and pumped onto the preparative HPLC column. The column was washed with 25 mM Na phosphate, pH 6.0 for 2 h, then with water for 2 h and finally eluted with water-MeOH (4:1). Fractions containing the product were analysed as above, combined and concentrated in vacuo. The residue was dissolved in water, passed through a 0.2 μm membrane filter, and lyophilised. The recovered material was dissolved in water (2.5 mL) and quantified by UV spectroscopy to give 32 (Na⁺ salt) (30.5 mM, 76 μmol, 12% from 20); LRMS (ESI) : m/e (M+H)^" Found: 642.4. Calcd. for (C₂₉H₂₈N₃Oi2P + H)^~: 642.1; ^λH NMR: (500 MHz, D2O, acetone ref.) £ 7.70 (2H, d, J = 8.8 Hz) , 7.66 (2H, d, J = 8.8 Hz) , 7.39 (IH, d, J = 9.2 Hz), 7.11 (2H, d, J = 8.8 Hz), 6.89 (2H, d, J = 8.8

Hz), 6.58 (2H, d, J = 9.2 Hz), 4.67 (2H, s) , 4.31 (2H, t, J = 4.9 Hz) , 4.12-4.20 (6H, m) , 3.70 (2H, t, J = 4.9 Hz), 3.05 (2H, t, J = 8 Hz) and 2.20 (3H, s) . Progressive photolysis of 32

A solution of 32 (0.24 mM in 25 mM Na phosphate, pH 7.0) was irradiated in a 1-mm path length cell for increasing times in the range 0-45 s. The extent of photolysis was monitored by UV spectroscopy. Conversion was ~50% after 10 s and no further change was observed after 40 s (Fig. 2). As a control experiment, a solution of l-[4S-(4- amino-4-carboxybutanoyl) ] -4-methoxy-7-nitroindoline 6 (0.20 mM) was irradiated for increasing times up to 180 s under the same conditions. Conversion was ~50% after 45 s and photolysis was complete after 3 min.

Rela tive photolysis efficiencies of 32 and 6

Separate solutions of 32 and 6 (each 0.29 mM in 25 mM Na phosphate, pH 7.0 with 5 mM dithiothreitol) were simultaneously irradiated in 1-mm path length cells . The solutions were analysed by reverse-phase HPLC with mobile phases 25 mM Na phosphate, pH 6.0-MeCN (100:40 v/v) for 32, R 4.6 min and 25 mM Na phosphate, pH 6.0-MeCN (100:25 v/v) for 6, tR 4.4 min. The extent of photolysis of each solution was determined by comparison of peak areas with those of non-irradiated controls and quantification was by measurement of peak heights. After 5 s irradiation, conversions for 32 and 6 were 35% and 8% respectively. A 36% conversion of 6 was reached after 30 s irradiation, indicating that 32 photolysed ~6-fold more efficiently than 6.

.Relative photolysis efficiencies of 12 and 32 Separate solutions of 12 and 32 (each 0.29 mM in 25 mM Na phosphate, pH 7.0 with 5 mM dithiothreitol) were simultaneously irradiated for 5 s in 1-mm path length cells. The solutions were analysed by reverse-phase HPLC [mobile phase 25 mM Na phosphate, pH 6.0-MeCN (100:25 v/v)], ( 4.2 min and 4.6 min for 12 and 32 respectively) . The extent of photolysis of each solution was determined by comparison of peak heights with those of non-irradiated controls. Conversions for 12 and 32 were 33% and 43% respectively, indicating that 12 photolysed ~1.3-fold more efficiently than 32.

Product identification on photolysis of 32

A solution of 32 (3.0 mM in 25 mM Na phosphate, pH 7.0) was lyophilised, re-dissolved in D₂0 and irradiated in an NMR tube for 20 s. The NMR spectrum was recorded and compared with the spectrum of the non-irradiated solution. A new signal observed at 1.91 p. p.m. was identified as acetate ion by a specific increase in its intensity when a solution of sodium acetate in D2O was added.

Methyl { [1 -4S- (4-tert-Butoxycarbonyl) -4- (t- butoxycarbonylamino) butanoyl] indol in- 4 -yloxy } acetate 35 A solution of methyl (l-acetylindolin-4-yloxy) acetate 29 (2.74 g, 11 mmol) in a mixture of MeOH (230 mL) , water (36 mL) and cone. HC1 (18 mL) was refluxed for 4 h. The solution was diluted with water (100 mL) , concentrated to ~200 mL, basified with solid NaHC0₃ and extracted with EtOAc (3 x 100 mL) . The combined organic phases were washed with brine, dried and evaporated to give methyl

(indolin-4-yloxy) acetate 34 (1.77g, 77%) as pale crystals;

^XH NMR (90 MHz) : £ 6.92 (t, J= 7.5 Hz, IH), 6.31 (d, J = 7.5 Hz, IH) , 6.12 (d, J= 7.5 Hz, IH) , 4.62 (s, 2H) , 3.76 (s, 3H) , 3.56 (t, J = 8 Hz, 2H) , 3.05 (t, J= 8 Hz, 2H) . The crude indoline (1.77 g, 8.5 mmol) was dissolved in dry MeCN (80 mL) and treated with N-tert-BOC-L-glutamic acid γ-t-butyl ester (2.88 g, 9.5 mmol) and l-(3- dimethylaminopropyl) -3-ethylcarbodiimide . HC1 (2.301 g, 12 mmol) . The mixture was stirred at room temperature for 18 h, then evaporated and the residue was dissolved in EtOAc and washed successively with 0.5 M aq. HC1, saturated aq. NaHC0₃ and brine, dried and evaporated to give a white foam which after trituration with ether gave 35 as white crystals (3.73 g, 89%), mp 120-122°C (EtOAc-petroleum ether); ^XH NMR (500 MHz): £7.88 (d, J= 8.1 Hz, IH) , 7.13 (t, J = 8.1 Hz, IH) , 6.44 (d, J= 8.1 Hz, IH) , 5.22 (d, J = 7 Hz, IH) , 4.66 (s, 2H) , 4.18-4.26 and 4.10-4.18 (2 x m, rotamers, IH) , 4.057 and 4.062 (2 x t, J= 8.5 Hz), 3.79 (s, 3H) , 3.18 (t, J= 8.5 Hz), 2.44-2.58 (m, 2H) , 2.22-

2.30 (m, IH) , 1.99-2.08 (m, IH) , 1.47(s, 9H) , 1.42 (s, 9H) . Calcd. for C₂₅H₃₆N₂0₈: C, 60.96; H, 7.37; N, 5.68. Found: C, 60.88; H, 7.47; N, 5.59.

Methyl { [ 1 -4S- (4-t-Butoxycarbonyl) -4- (bis-t- butoxycarbonylamino) butanoyl] indolin-4-yloxy} acetate 36 A solution of 35 (3.94 g, 8 mmol) in a mixture of dry CH₂C1₂ (32 mL) and Et3N (48 mL) was treated with di-t- butyl dicarbonate (4.36 g, 20 mmol) and DMAP (98 mg, 0.8 mmol) and the mixture was refluxed under nitrogen for 6 h. The solvents were evaporated and the residue was dissolved in Et₂0 (100 mL) and washed successively with 1 M aq. KHSO4, saturated aq. NaHC0₃ and brine, dried and evaporated to give a viscous oil. Flash chromatography [EtOAc-petroleum ether (2:3)] gave 36 as a pale foam (4.61g, 97%) that was used in the next step without further purification; ^XH NMR (500 MHz): £7.90 (d, J= 8.1 Hz, IH) , 7.12 (t, J= 8.1 Hz, IH) , 6.42 (d, J = 8.1 Hz, IH) , 4.86 (dd, J = 5.2, 9.3 Hz, IH) , 4.66 (s, 2H) , 4.070 and 4.064 (2 x t, J= 8.6 Hz), 3.79 (s, 3H) , 3.18 (t, J= 8.6 Hz), 2.50-2.60 (m, 2H) , 2.42-2.48 (m, IH) , 2.18-2.25 (m, IH) , 1.48 (s, 18H) , 1.46 (s, 9H) . MS (ES⁺) : m/e (M+H) ⁺ Found: 593.4. Cald. for (C₃₀H4₄N₂Oιo + H)⁺ 593.8. Methyl { [1 -4S- (4-t-Butoxycarbonyl) -4- (t- butoxycarbonylamino) butanoyl] -7-nitro-indolin-4- yloxy j acetate 37 and Methyl { [1 -4S- (4-t-Butoxycarbonyl) -4- (t-butoxycarbonylamino) butanoyl] -5-ni troindolin-4- yloxy Jaceta te 38

To a solution of 36 (2.98 g, 5 mmol) in a mixture of CCI4 (40 mL) and acetic anhydride (20 mL) was added claycop (3.2 g) and the mixture was stirred at room temperature for 22 h. The solid was filtered off, washed thoroughly with CHCI3 and the filtrate was washed with saturated aq. NaHC0₃ and brine, dried and evaporated. The residue was dissolved in dry CH₂C1₂ (50 L) and treated with 1 M TFA in CH₂C1₂ (7.5 mL, 7.5 mmol) and stirred at room temperature overnight. The solution was diluted with CH₂C1₂ (100 mL) washed with saturated aq. NaHC0₃ and brine, dried and evaporated to a brown viscous oil. Flash chromatography [EtOAc-petroleum ether (2:3)] gave a pale yellow foam (1.31 g) which after trituration with MeOH gave the 5-isomer 38 as pale crystals (72 mg, 3%), mp 136- 138°C (EtOAc-petroleum ether); H NMR (500 MHz): £ 8.07 (d, J= 9 Hz, IH) , 7.87 (d, J= 9 Hz, IH) , 5.14 (d, J= 6.8 Hz, IH) , 4.66 (s, 2H) , 4.19-4.26 (m, IH) , 416 (t, J = 8 Hz), 3.80 (s, 3H) , 3.35 (t, J = 8 Hz, 2H) , 2.46-2.64 (m, 2H), 2.24-2.33 (m, IH) , 1.96-2.05 (m, IH) , 1.48 (s, 9H) , 1.42 (s, 9H) . Anal. Calcd. for C₂H₃₅N₃O₁₀ : C, 55.86; H,

6.56; N, 7.81. Found: C, 56.18; H, 6.80; N, 7.50. The 7- isomer 37 was isolated from the mother liquor as a pale yellow foam (1.12 g, 42%); ^XH NMR (500 MHz): £ 7.70 (d, J = 9 Hz, IH) , 6.49 (d, J= 9 Hz, IH) , 5.14 (d, J = 6.4 Hz, IH) , 4.73 (s, 2H) , 4.23 (2 x t, J= 8.5 Hz, 2H) , 4.14-4.20 (m, IH) , 3.81 (s, 3H) , 3.18 (t, J = 8.5 Hz), 2.48-2.64 (m, 2H) , 2.22-2.32 (m, IH) , 1.94-2.04 (m, IH) , 1.46 (s, 9H) , 1.44 (s, 9H) . HRMS (MALDI) : m/e (M + Na)+ Calcd for (C₂5H₃₅N₃Oιo + Na) ⁺ : 560.22147. Found: 560.2244.

{ [1-4S- (4-t-Butoxycarbonyl) -4- (bis-t- butoxycarbonylamino) butanoyl] -indolin-4-yloxy} acetic Acid 39

To a solution of the methyl ester 36 (4.35 g, 7.3 mmol) in MeOH (200 mL) was added 1 M aq. NaOH (11 mL, 11 mmol) . After 2.5 h, when consumption of the starting material was confirmed by TLC [EtOAc-petroleum ether-AcOH (9:1:0.1)], the solution was neutralised with 1 M aq. citric acid (11 mL) and concentrated. The residue was diluted with water, acidified to pH 2 with 1 M aq. citric acid and washed with EtOAc. The combined organic phases were washed with brine dried and evaporated to a pale foam which after trituration with EtOH gave 39 as white crystals (3.07g,

72%), mp 65-67°C (Et₂0-petroleum ether); ¹H NMR (500 MHz) : £ 7.91 (d, J = 8.2 Hz, IH) , 7.13 (t, J = 8.2 Hz, IH) , 6.46 (d, J = 8.2 Hz, IH) , 4.86 (dd, J = 4.9, 9.1 Hz, IH) , 4.68 (s, 2H) , 4.08 and 4.06 (2 x t, J = 8.5 Hz, 2H) , 3.16 (t, J = 8.6 Hz, 2H) , 2.50-2.60 (m, 2H) , 2.42-2.50 (m, IH) , 2.16- 2.25 (m, IH) , 1.48 (s, 18H) , 1.46 (s, 9H) . Calcd. for C₂₉H₄₂N₂Oιo + H₂0: C, 58.38; H, 7.40; N, 4.69. Found: C, 58.72; H, 7.40; N, 4.69.

{ [1-4S- (4-t-Butoxycarbonyl) -4- (t- butoxycarbonylamino) butanoyl] -7-nitroindolin-4- yloxyjacetic Acid 40

To a solution of the acid 39 (1.15 g, 2 mmol) in a mixture of CCI4 (50 L) and acetic anhydride (25 mL) was added claycop (3.2 g) and the mixture was stirred at room temperature overnight, when the consumption of the starting material was confirmed by TLC [EtOAc-AcOH (10:0.2)]. The solid was filtered off, washed thoroughly with EtOAc (200 L) and the filtrate was washed with brine, dried and evaporated and re-evaporated from toluene (2 x 50 mL) . The residue was dissolved in dry CH₂C1₂ (50 mL) and treated with 1 M solution of TFA in CH₂C1₂ (3 mL, 3 mmol) and stirred at room temperature overnight. The solution was diluted with CH₂C1₂ (100 mL) washed with brine, dried and evaporated to give a brown viscous oil. Flash chromatography [EtOAc-AcOH (10:0.2)] gave 40 as a pale yellow foam (0.90 g, 86%), contaminated the 5-isomer and used in the next step without further purification.

4- {2- [1 -4S- (4-amino-4-carboxybutanoyl) - 7- (nitroindolin-4- yloxy) acetamido] ethoxy } -4 ' - [2- (dihydroxyphosphoryloxy) ethoxy] benzophenone 33

To a solution of the azide 20 (779 mg, 1.5 mmol) in THF (10.25 mL) containing water (2.25 mmol) was added triphenylphosphine (590 mg, 2.25 mmol) and the mixture was stirred at room temperature under nitrogen for 20 h. TLC [EtOAc-petroleum ether (4:1)] confirmed that all the azide was reduced to the amine. The solvent was evaporated and the residue was dissolved in CHCI3 (30 L) , dried and evaporated to a viscous oil (1.41g) that contained material identical to the amine 21 described above (¹H NMR) , together with triphenylphosphine oxide and unreacted triphenylphosphine. The mixture was dissolved in dry MeCN (50 mL) and treated with the crude acid 40 (785 mg, 1.5 mmol) and 1- (3-dimethylaminopropyl) -3- ethylcarbodiimide .HC1 (401 mg, 2.1 mmol). The mixture was stirred at room temperature under nitrogen for 18 h, then evaporated and the residue was dissolved in EtOAc and washed successively with 0.5 M aq. HC1, saturated aq. NaHC0₃ and brine, dried and evaporated. Flash chromatography [CHCl3~MeOH (95:5)] gave a pale yellow foam (700 mg, 52%) , which was used in the next step without further characterisation. This material (645 mg, 0.71 mmol) was dissolved in TFA (30 mL) , stirred at room temperature for 1 h and concentrated in vacuo . The residue was dissolved in water (180 mL) and adjusted to pH 7.07 with 1 M aq. NaOH. The solution was washed with ether and analysed by reverse-phase HPLC (mobile phase 25 mM Na phosphate, pH 6.0 + 30% MeCN v/v) , R 3.8 min. The solution was lyophilised, dissolved in 25 mM Na phosphate, pH 6.0 buffer (130 mL) and pumped onto the preparative HPLC column. The column was washed with 25 mM Na phosphate, pH 6.0 for 1.5 h and the products were eluted with 25 mM Na phosphate, pH 6.0 +20% MeCN. Fractions containing the first peak were analysed as above, combined and quantified by UV spectroscopy [Λπ,_ax (H2O) /nm 300 { ε/W icm^-1 27,900)] to give 33 (230 μmol) . The solution was concentrated and desalted by re-application to the preparative HPLC column in 25 mM Na phosphate, pH 6.0. The column was first eluted with water for 2 h, then with water + 25% MeOH. Fractions containing the product were analysed, combined and quantified (UV spectroscopy) to give 33 (171 μmol) . The solution was concentrated and the residue dissolved in water, adjusted to pH 8 and stored frozen; ¹E NMR (Na⁺ salt): £ (500 MHz, D2O, acetone ref.) 7.65 (dd, J = 1.8, 8.9 Hz, 2H) , 7.63 (dd, J = 1.8, 8.9 Hz, 2H) , 7.36 (d, J= 9 Hz, IH) , 7.08 (d, J = 8.9 Hz, 2H) , 6.88 (d, J= 8.9 Hz, 2H) , 6.54 (d, J = 9 Hz, IH) , 4.62 (s, 2H), 4.31 (t, J= 4.7 Hz, 2H) , 4.13-4.20 (m, 6H) , 3.76 (t, J = 6.3 Hz, IH) , 3.70 (t, J = 4.7 Hz, 2H) , 3.05 (t, J = 7 . 9 Hz , 2H) , 2 . 69 ( t , J = 7 . 9 Hz , 2H ) , 2 . 08-2 . 15 (m, 2H ) . HRMS (ES⁺) : m/e (M+3H) + Calcd for (C₃₂H₃₃N₄0₁₄P + 3H) ⁺ : 731 . 1960 . Found : 731 . 1951 . Progressive photolysis of 33

A solution of 33 (0.18 mM in 25 mM Na phosphate pH 7.0) was irradiated in a 1-mm path length cell for increasing times in the range of 0-40 s. The extent of photolysis was monitored by UV spectroscopy. Conversion was ~50% after 10 s and no further change was observed after 30 s.

Quantitative photolysis and product analysis for 33 Separate solutions of 33 (0.319 M in 25 M Na phosphate, pH 7.0 containing 5 mM dithiothreitol) were irradiated for varying times (3 or 5 s) in 1-mm path length cells. The solutions were analysed by reverse-phase HPLC (Merck Lichrospher RP8 column; mobile phase 25 mM Na phosphate, pH 6.0 + 30% MeCN v/v, tR 3.5 min) and the extent of photolysis of each solution was determined by comparison of peak areas with those of unphotolysed controls. Aliquots of the photolysed solutions were also subjected to quantitative amino acid analysis . Measured glutamate concentrations were 88-90% of the expected values from the extent of photolysis and were not affected by the concentration of dithiothreitol.

5-Methyl -4-hydroxyindole 43

Following a published method,¹⁹ a solution of 4- hydroxyindole (10.0 g, 75 mmol) in EtOH (50 mL) was mixed with 40% aqueous methylamine (11.0 g, 97.5 mmol) and the mixture was treated dropwise with 37% aqueous formaldehyde (7.0 g, 86 mmol) . The temperature was raised to ~32°C and the mixture was stirred at room temperature for lh. The dark purple solution was diluted with water (200 mL) and washed with CHC1₃ (3 x 100 mL) . The combined organic phases were dried and evaporated to a dark brown viscous oil. Flash chromatography on alumina using EtOAc as eluent gave the product as a brown oil (11.17 g, 78%), which crystallised on standing at 4°C and was used in the next step without further purification. To a solution of this Mannich base (2.13 g, 11.2 mmol) in EtOH (140 mL) was added 5% Pd on alumina (1.6 g) and the mixture was shaken under hydrogen at 5 atm overnight . The catalyst was filtered off, washed with EtOH and the filtrate was evaporated. The residue was flash chromatographed [EtOAc- petroleum ether (1:3)] affording 43 as pale crystals (1.43 g, 87%) , which were used in the next step without further purification.

4 - Ace toxy- 1 -acety -5 -methyl indoline 44

To a solution of 4-hydroxy-5-methylindole 43 (2.80 g, 19 mmol) in acetic acid (90 mL) was added NaBH3CN (3.58 g, 57 mmol) portionwise over 0.5 h, keeping the temperature at ~15°C. The mixture was then stirred at room temperature for 1 h and water (3 mL) was added and the solvent evaporated. The residue was dissolved in EtOAc (50 mL) and washed with saturated aq. NaHC0₃ and brine, dried and evaporated to give 4-hydroxy-5-methylindoline as pale foam

(2.83 g, 100%); H NMR £ (90 MHz) 6.76 (d, J = 8 Hz, IH) , 6.16 (d, J = 8 Hz, IH) , 3.52 (t, J= 8 Hz, 2H) , 2.92 (d, J = 8 Hz, 2H) , 2.14 (s, 3H) . The crude indoline was dissolved in a mixture of acetic acid (20 mL) and acetic anhydride (20 mL) and heated under reflux for 1 h. The resulted dark brown solution was diluted with water (5 mL) and the solvents evaporated. The residue was dissolved in EtOAc (100 mL) and washed with saturated aq. NaHC0₃ and brine, dried and evaporated to give 44 as pale crystals (3.22 g, 73%), mp 103-104°C (EtOAc-petroleum ether) ; ^

NMR: £ (500 MHz) 7.98 (d, J= 8.1 Hz, IH) , 7.06 (d, J = 8.1 Hz, IH) , 4.08 (t, J= 8.5 Hz, 2H) , 3.04 (t, J = 8.5 Hz, 2H) , 2.32 (s, 3H) , 2.20 (s, 3H) , 2.13 (s, 3H) . Calcd for C₁₃Hι₅N0₃: C, 66.94; H, 6.48; N, 6.00. Found: C, 66.51; H, 6.07; N, 5.97.

1-Acetyl -4 -hydroxy-5 -methyl indoline 45 A solution of 44 (3.15 g, 13.5 mmol) in MeOH (95 mL) was treated with 1 M aq. NaOH (14.85 mL, 14.85 mmol), stirred at room temperature for 0.75 h, diluted with water (100 L) and concentrated to remove most of the MeOH. The residue was acidified to pH 3 with 1 M aq. HC1 and the precipitated solid was filtered off, washed with water and dried in a vacuum desiccator. The filtrate was extracted with EtOAc and the organic phase was washed with saturated aq. NaHC0₃ and brine, dried and evaporated to give more solid. The combined solids were recrystallised (MeOH- EtOAc) to give 45 as white crystals (2.06 g, 80%); ¹H NMR:

£ (500 MHz) 8.4 (s, IH) , 7.57 (d, J= 8.1 Hz, IH) , 6.88 (d, J - 8.1 Hz, IH) , 4.05 (t, J = 8.5 Hz, 2H) , 3.13 (t, J = 8.5 Hz, 2H) , 2.91 (s, 3H) , 2.19 (s, 3H) . Calcd for CnH₁₃N0₂: C, 69.09; H, 6.85; N, 7.32. Found: C, 68.93; H, 6.97; N, 7.29.

t-Butyl (l -Acetyl-5-methylindolin-4-yloxy) acetate 46 l-Acetyl-4-hydroxy-5-methylindoline 45 (2.83 g, 16 mmol) was added to a suspension of anhydrous K₂C0₃ (1.24 g, 9 mmol) in acetone (60 mL) and after 15 min t-butyl bromoacetate (2.34 g, 12 mmol) was added and the mixture was heated under reflux for 4 h. The solid was filtered off, washed with acetone and the filtrate evaporated. The residue was dissolved in EtOAc (50 mL) , washed with brine, dried and evaporated to give 46 as white crystals (1.64g, 90%), mp 95-96°C (EtOAc-petroleum ether); ^XH NMR: £ (500 MHz) 7.87 (d, J = 8.1 Hz, IH) , 7.00 (d, J= 8.1 Hz, IH) , 4.38 (s, 2H) , 4.05 (t, J = 8.4 Hz, 2H) , 3.24 (t, J= 8.4 Hz, 2H) , 2.26 (s, 3H) , 2.20 (s, 3H) , 1.50 (s, 9H) . Calcd for Cι₇H₂₃N0₄: C, 66.86; H, 7.59; N, 4.59 Found: C, 67.07; H, 7.64; N, 4.60.

t-Butyl (l-Acetyl-5-methyl-7-nitroindolin~4-yloxy) acetate 47

To a solution of 46 (1.53 g, 5 mmol) in a mixture of CC1₄ (40 mL) and acetic anhydride (20 mL) was added claycop (3.2 g) and the mixture was stirred at room temperature for 4 h. The solid was filtered off, washed with CC1₄ and the filtrate evaporated. The residue was dissolved in

EtOAc and washed with saturated aq. NaHC0₃ and brine, dried and evaporated to give to give 47 as yellow needles (1.15g, 66%), mp 97.5-98.5°C (Et2θ-petroleum ether, with charcoal) ; ^XH NMR: £ (500 MHz) 7.54 (s, IH) , 4.47 (s, 2H) , 4.22 (t, J = 8.0 Hz, 2H) , 3.24 (t, J = 8.0 Hz, 2H) , 2.31 (s, 3H) , 2.24 (s, 3H) , 1.49 (s, 9H) . Calcd for Cι₇H_{22 2}0₆: C, 58.28; H, 6.33; N, 7.99. Found: C, 58.20; H, 6.33; N, 7.96.

(l-Acetyl-5-methyl-7-nitroindolin-4-yloxy) acetic Acid 48 A solution of 47 (1.05 g, 3 mmol) in TFA (10 mL) was stirred at room temperature for 1 h, concentrated and re- evaporated from toluene (2 x 10 mL) to give 48 as light brown crystals (0.66 g, 74%) , mp 188-190°C (EtOAc) ; UV: _na (EtOH)/n 250 (ε/M-¹cm^~1 25,380) , 287 (9,320) shoulder

329 (3,990) ; _nax [EtOH-25 mM Na phosphate, pH 7.0

(1:40) //nm 247 (s M-lcitT¹ 19,670) , 338 (3,570) ; ^lE NMR: £ (500 MHz) 7.45 (s, IH) , 4.57 (s, 2H) , 4.23 (t, J = 8.0 Hz,

2H) , 3.27 (t, J = 8.0 Hz, 2H) , 2.33 (s, 3H) , 2.24 (s, 3H) . Calcd for Cι₃Hι₄N₂0₆: C, 53.06; H, 4.80; N, 9.52. Found: C,

53.16; H, 4.79; N, 9.40. 4- {2- (l -Acetyl-5-methyl-7-nitroindolin-4- yloxy) acetamido] ethoxy } -4 ' - [2- (dihydroxy- phosphoryloxy) ethoxy] benzophenone 41

To a solution of the azide 20 (312 mg, 0.6 mmol) in THF (4.1 mL) containing water (0.9 mmol) was added triphenylphosphine (236 mg, 0.9 mmol) and the mixture was stirred at room temperature under nitrogen for 20 h. The solvent was evaporated and the residue was dissolved in CHCI3 (30 mL) , dried and evaporated to viscous oil. The crude amine 21 was dissolved in dry MeCN (20 mL) and treated with 48 (206 mg, 0.7 mmol) and l-(3- dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (161 mg, 0.84 mmol) . The mixture was stirred at room temperature under nitrogen for 18 h, then evaporated and the residue was dissolved in EtOAc and washed successively with 0.5 M aq. HCl, saturated aq. NaHC0₃ and brine, dried and evaporated. Flash chromatography [CHCI3 -MeOH (95:5)] gave a yellow viscous oil (393 mg, 85%) which was used in the next step without further purification; ¹H NMR: £ (90 MHz) 7.60-7.84 (m, 4H) , 7.32 (s, IH) , 6.88-7.04 (m, 4H) , 4.44 (s, 2H) , 4.04-4.36 (m, 8H) , 3.76-3.98 (m, 2H) , 3.12 (t, J= 8 Hz, 2H) , 2.23 (s, 3H) , 2.18 (s, 3H) , 1.50 (s, 18H) . This material (393 mg, 0.51 mmol) was dissolved in TFA (20 mL) , stirred at room temperature for 1 h and concentrated in vacuo . The residue was dissolved in water (65 mL) and adjusted to pH 7.1 with 1 M aq. NaOH. The solution was washed with ether and analysed by reverse- phase HPLC (mobile phase 25 mM Na phosphate, pH 6.0 + 45% MeCN v/v), t 4.4 min. The solution was lyophilised, dissolved in 25 mM Na phosphate, pH 6.0 buffer (110 mL) and pumped onto the preparative HPLC column. The column was first washed with 25 mM Na phosphate, pH 6.0 for 2 h, then with water for 2 h and then product was eluted with water + 25% MeOH. Fractions containing the product were analysed as above, combined and concentrated in vacuo . The residue was dissolved in water, filtered through a 0.2 μm membrane, lyophilised and the resulting yellow powder was dissolved in water (10.5 mL) , quantified by UV spectroscopy [ nax (H2O) /nm 300 { ε/M^{~ 1}cm^{~ 1} 27,000)], to give 41 (Na⁺salt) (27.5 mM, 289 μmol, 54%); ¹H NMR: £ (500 MHz, D2O, acetone ref.) 7.53 (d, J = 8.5 Hz, 4H) , 7.10 (s, IH) , 6.97 (d, J = 8.5 Hz, 2H) , 6.85 (d, J = 8.5 Hz, 2H) , 4.41 (s, 2H) , 4.21-4.24 (m, 2H) , 4.12-4.19 (m, 4H) , 3.91 (t, J= 7.7 Hz, 2H) , 3.68-3.76 (m, 2H) , 2.92 (t, J= 7.4

Hz, 2H) , 2.10 (s, 3H) , 1.93 (s, 3H) . MS (ES): m/e (M+H) ^~ Calcd for C30H30N3O12P: 656.2. Found: 656.4.

Progressive photolysis of 41 A solution of 41 (0.27 mM in 25 mM Na phosphate pH 7.0) was irradiated in a 1-mm path length cell for increasing times in the range of 0-45 s. The extent of photolysis was monitored by UV spectroscopy. Conversion was -50% after 7 s and no further change was observed after 35 s.

Relative photolysis efficiencies of 32 and 41 Separate solutions of 32 and 41 (each 0.3 mM in 25 mM Na phosphate, pH 7.0 containing 5 mM dithiothreitol) were irradiated in 1-mm path length cells. The solutions were analysed by reverse-phase HPLC (Merck Lichrosphere RP8 column, v/v, flow rate 1.5 mL/min) . For 32, the mobile phase was 25 mM Na phosphate, pH 6.0 + 40% MeCN, t 4.6 min and for 41, the mobile phase was 25 mM Na phosphate, pH 6.0 + 45% MeCN, tR 5.6 min. The extent of photolysis of each solution was determined by comparison of peak areas with those of unphotolysed controls and quantification was by measurement of peak heights. After 5 s irradiation, conversions for 32 and 41 were 39.0% and 45.1% respectively, indicating that 41 photolysed ~1.16- fold more efficiently than 32.

Discussion The present inventors realised that an important consequence of the result that the mechanism for the photolysis of nitroindolines occurred principally via the triplet state was that further improvements might be made to the caging compounds disclosed in the prior art by adding a triplet sensitising group. This was demonstrated by conducting the photolysis in the presence of a water- soluble derivative of benzophenone, 2-benzoylbenzoate, which is a well-known triplet sensitiser. Under anaerobic conditions, photolytic efficiency was greatly enhanced, indicating absorption of light by the sensitiser and intermolecular energy transfer to the nitroindoline⁷. While such processes are well known in photochemistry¹³, the possibility of triplet sensitisation in photolysis of caged compounds as a means to improve photolysis efficiency had not been explored in practice. One reason for this is that oxygen, present in the oxygenated solutions commonly used in biological experiments at ~1 mM concentration is an effective quencher of the long-lived triplet states of typical sensitisers . Therefore, based on the prior art, the expectation was that the triplet state of a sensitiser would be largely quenched before it was able to undergo intermolecular energy transfer to a caged molecule. However it is known that intramolecular energy transfer can occur much more efficiently in the presence of a suitable intramolecular acceptor and we decided to explore whether this strategy could be applied to the nitroindoline photolysis. For simplicity, this was investigated using l-acetyl-7-nitroindolines, i.e. based on compounds 1 and 2 where the R group was CH₃. Our first target was the conjugate 12, in which the benzophenone moiety bears different substituent groups that both enable its linkage to a suitable carboxylate group on the nitroindoline and provide a charged centre to promote aqueous solubility. The synthesis of the sensitiser component 21 is shown in Scheme 2 and its linkage to the nitroindoline 1 (R = CH₃) is shown in Scheme 3. For synthesis of 21, the known compounds 13¹⁴ and 14¹⁵ were coupled via the lithiated derivative of 13, and the product alcohol 15 was oxidised by standard means to the ketone 16. Deprotection of the silyl ether gave the known¹⁵ ketone 17 and displacement of the bromide gave azide 18. Alkylation with bromoethanol gave the azidoalcohol 19 that was converted to the phosphotrieser 20. Finally, hydrogenation of the azido group gave the amine 21 that was suitable for condensation with the nitroindoline moiety (Scheme 3) . There is also an alternative, more satisfactory method for converting the azido group of 20 to the amine 21. This involves treatment with triphenylphosphine in aqueous THF¹⁶ and provides a cleaner synthesis. Details are given in the Experimental section where the synthesis of compound 41 is described.

The carboxylic acid 22 required for the coupling with 21 was obtained by hydrolysis of the ester side chain of 23^a, and subsequent claycop nitration of the resulting acid 24. The nitrated acid 22 was then coupled with the sensitiser 21. This procedure was preferable to direct alkaline hydrolysis of the ester 1 (R = CH₃) as it avoided partial cleavage of the 1-acetyl group from the nitroindoline (see below) . The product was then treated with trifluoroacetic acid to remove the protecting t-butyl groups from the phosphate and the conjugate 12 was purified by preparative reverse-phase HPLC. This material was soluble to at least 30 mM in aqueous media.

Comparative photolysis of 12 and the non-sensitised analogue 25 (available from previous work^a,b and used because of its water solubility) was carried out in a Rayonet RPR100 photochemical reactor fitted with 300-nm lamps. Aqueous solutions of equal concentrations of 12 and 25 at pH 7 were irradiated in separate cuvettes for varying times and the degree of photolysis was estimated by analytical reverse-phase HPLC. After 8 s illumination, the sensitised compound 12 had undergone 40% photolysis while the non-sensitised compound 25 had undergone only 2.2 % photolysis. Thus under these conditions, the sensitised compound was at least 15-fold more photosensitive. It is important to note that no precautions were taken to exclude oxygen from the solutions .

With the success of the first target established, it was desirable to establish whether the methoxy-substituted indoline system 2 would also be susceptible to triplet sensitisation. In order to make available a suitable carboxylate group, the methoxy group was changed to a methoxycarbonylmethyl, as outlined in Scheme 4. Thus 4- hydroxyindole 26 was reduced to the indoline and acetylated to give the diacetyl derivative 27. Selective hydrolysis of the phenolic ester gave monoacetyl compound 28, that was alkylated with methyl bromoacetate to give the indoline 29 with the required ester functionality in place. Nitration with claycop, using the method previously described, ^3b,3c gave a mixture of the 5- and 7- nitroisomers 30 and 31 in approx. 1:2 ratio. These isomers were separated by silica gel chromatography to give 31 as a pure compound. Subsequent hydrolysis of the ester group, followed by coupling with the sensitiser 21 analogously as described above, gave the desired conjugate 32 after TFA deprotection of the phosphate. As for 12, this material was purified by preparative reverse-phase HPLC and, like 12, the compound was soluble to at least 30 mM in aqueous media.

Comparative photolysis of 32 and the non-sensitised compound 6 was performed as for 12 and 25. The results of this experiment showed ~6-fold greater photosensitivity of 32 in comparison to 6. Direct comparison of the two sensitised compounds 12 and 32 showed that photolysis of 32 was approximately 30% more efficient than that of 12, in keeping with the relative efficiencies determined in the other pairwise comparisons. Thus the overall gain in efficiency between compounds of general structure 1 and the final compound 32 is a factor of approx. 20-fold.

Although the gain in efficiency between 12 and 32 was not great, other aspects of the synthetic routes to the two compounds make 32 and derivatives with more complex 1-acyl substituents significantly more accessible. In particular, synthesis of either of the present conjugates requires hydrolysis of an ester present in the side chain. In the route described above for overall construction of 12, this was achieved prior to introduction of the nitro group (i.e. the sequence 23 to 24 to 22 as shown in Scheme 3) . Hydrolysis under the mildest available conditions required approx. 24 hours and, if conducted on the preformed nitroindoline 1 (R = CH₃) would have resulted in significant hydrolysis of the acetyl group from the nitroindoline. Furthermore, in the case of more complex 1-acyl groups that themselves incorporate protecting groups during the synthetic sequence, for example in a protected L-glutamate side chain, significant hydrolysis of those protecting groups would be expected. In contrast, hydrolysis of the ester side chain in compound 31 was complete within 30 min and such mild conditions caused no significant cleavage of the 1-acetyl group.

Synthesis of the L-glutamate conjugate 33 followed the methods outlined above and is depicted in Scheme 5. Thus the amide group of 29 (Scheme 4) was selectively cleaved by methanolic HCl and the indoline 34 was coupled with the γ-carboxylate group of a protected L-glutamate derivative to give 35. It was found necessary further to protect the nitrogen of the amino acid residue as its double BOC derivative¹⁷ to avoid partial nitration during the claycop nitration procedure. This was previously observed as a problem during synthesis of 6 and significantly decreased the yield of the desired product in the nitration step.^3b,3c Nitration of 36 was accompanied by partial cleavage of the newly-introduced second BOC group, but nevertheless proceeded in good yield: subsequent treatment of the crude mixture with 1 M TFA in CH₂C1₂ completed the removal of the second BOC group¹⁸ to leave a mixture of the 7- and 5-nitrated isomers 37 and 38 respectively, that could be separated by flash chromatography. However, attempted alkaline hydrolysis of the methyl ester group in 37 was accompanied by significant cleavage of the 1-acyl substituent. Better results were obtained by reversing the order of steps, i.e. the methyl ester in 36 was first hydrolysed under alkaline conditions and the acid 39 was nitrated with the claycop reagent .^3b'^3c Treatment of this crude product with 1 M TFA in CH₂C1₂ again completed removal of one of the BOC groups . The crude product was principally the 7-nitro isomer 40 shown in Scheme 5 that was not readily separated from a small proportion of its 5-nitro isomer, and the combined materials were coupled with the amine 21 and finally fully deprotected by treatment with neat TFA. Preparative reverse-phase HPLC separated the desired isomer 33 from a trace of the 5- nitro compound.

Photolysis of 33 in aqueous solution proceeded smoothly, as for the related conjugate 32 and quantitative analysis, monitoring disappearance of the starting conjugate by reverse-phase HPLC and formation of the glutamate product by amino acid analysis, indicated, within the precision of the measurements, that formation of glutamate measured was in a 1 : 1 relationship with consumption of the starting material (measured glutamate values were 88-90% of expected concentration) .

Given the efficient photorelease of the L-glutamate product, comparable derivatives of other effectors such as GABA and glycine can readily be envisaged by appropriate modifications .

The successful results with the compounds described above stimulated attempts further to optimise the synthesis and properties of these sensitised conjugates. One problem with the synthetic route is the formation of variable amounts of the unwanted 5-nitro isomer during nitration of 4-alkoxyindolines that have a free 5-position. In previous work in the absence of the intramolecular sensitiser, we had attempted to overcome this by including a 5-methyl substituent³³ but the resulting compound was less photosensitive than compounds such as 6 that lacked this additional substituent. Nevertheless, we considered that further study was warranted and prepared the 5-methyl analogue of 32, starting from known 4-hydroxy-5- methylindole .¹⁹ The synthetic route was identical to that shown in Scheme 4, except for the presence of the 5-methyl substituent and that we made the analogue of 29 as its t- butyl ester. Details are shown in Scheme 6. The eventual conjugate, 41, was -15% more photosensitive than the non- methyl analogue 32. Further developments of the strategy will be to shorten the linker, giving compounds such as 42 or its non-methyl analogue, in the expectation that the intramolecular energy transfer rate will be further enhanced as the donor and acceptor halves of the conjugate are held closer together.

In the work described herein, the incorporation of the triplet sensitiser has been shown to lead to significant enhancement in photolytic efficiency. In further embodiments, the present invention also provides compounds in which the linker between the sensitiser and nitroindoline is shortened, giving compounds such as 42 or its non-methyl analogue. Shortening the linker to hold the donor and acceptor closer together is expected further to enhance the rate of intramolecular energy transfer, so leading to still more efficient photolysis.

References :

The following references are all expressly incorporated by reference .

1. (a) Corrie, J.E.T. ; Trentham, D.R. In Bioorganic Photochemistry; Morrison, H., Ed.; Wiley: New York,

1993; Vol. 2, pp 243-305. (b) Adams, S.R.; Tsien, R.Y. Annu. Rev. Physiol. 1993, 55, 755. (c) Kaplan, J.H. Annu. Rev. Physiol. 1990, 52, 897. (d) Marriott, G., Ed. Methods in Enzymology, Vol. 291. Academic Press: San Diego, 1998. (e) Pelliccioli, A. P.; Wirz, J. Photochem. Photobiol. Sci. 2002, 1, 441.

2. (a) Papageorgiou, G.; Ogden, D.C.; Barth A.; Corrie, J.E.T. J. Am. Chem. Soc. 1999, 121, 6503; (b) Corrie, J.E.T.; Papageorgiou, G. Photoreleasable compounds. WO 00/55133.

3. (a) Papageorgiou, G.; Corrie, J.E.T. Tetrahedron 2000, 56, 8197; (b) Papageorgiou, G.; Corrie, J.E.T. Synth. Commun. 2002, 32, 1571; (c) Corrie, J.E.T.; Papageorgiou, G. Process for producing photocleavable compounds. WO 02/083639.

4. Canepari, M.; Papageorgiou, G.; Corrie, J.E.T.; Watkins, C . ; Ogden, D. J. Physiol. 2001, 533, 765.

5. Canepari, M.; Nelson, L.; Papageorgiou, G.; Corrie, J.E.T.; Ogden, D. J. Neurosci. Methods 2001, 112, 29. 6. Matsuzaki, M. ; Ellis-Davies, G.C.R.; Nemoto, T.;

Miyashita, Y.; lino, M.; Kasai, H. Nat. Neurosci.

2001, 4, 1086. 7. Morrison, J.; Wan, P.; Corrie, J.E.T.; Papageorgiou,

G. Photochem. Photobiol. Sci. 2002, 1, 960. 8. (a) Wieboldt, R.; Gee, K.R.; Νiu, L.; Ramesh, D.;

Carpenter, B.K.; Hess, G.P. Proc. Natl . Acad. Sci.

U.S.A., 1994, 91, 8752; (b) Gee, K.R.; Wieboldt, R.;

Hess, G.P. J. Am. Chem. Soc. 1994, 116, 8366. 9. Furuta, T.; Wang, S.S.H.; Dantzker, J.I.; Dore, T.M.; Bybee, W.J.; Callaway, E.M.; Denk, . ; Tsien, R.Y. Proc . Natl . Acad. Sci . U. S . A. , 1999, 96, 1193.

10. Fedoryak, O.D.; Dore, T.M. Org. Lett . 2002, 4, 3419. 11. Papageorgiou, G.; Corrie, J.E.T. Tetrahedron 1997, 53, 3917.

12. (a) Fertig, N.; Buck, R.H.; Behrends, J.C. Biophys . J. 2002, 82, 3056; (b) Klemic, K.G.; Sigworth, F.J. Biophys . J. , 2003, 84, 135a; (c) Costantin, J.L.; Wittel, A.; Lachnit, W. Biophys . J. , 2003, 84, 295a;

(d) Friis, S.; Krzywkowski, K.M.; Asmild, M.;

Jacobsen, R.B.; Oswald, N.; Schrøeder, R.L.;

Willumsen, N.J. Biophys . J. , 2003, 84, 295a; (e) Ng,

K.; Cutler, . ; Kelly, A.; Weihe, 0.; Velkovska, S.; Warfield, R.; Li, X.; Shetty, S.; Grove, R.; Yang, N.

Biophys . J. , 2003, 84, 296a.

13. (a) Murov, S.L. Handbook of Photochemistry, Marcel Dekker, New York, 1973; (b) Lamola, A.A. in Energy Transfer an Organic Photochemistry, eds. A.A. Lamola and N.J. Turro, Vol. XIV of Techniques in Organic

Chemistry, Interscience, New York, 1969, pp. 17-132; (c) Horspool, W.M. Aspects of Organic Photochemis try, Academic Press, London, 1976.

14. Angle, S.R.; Louie, M.S. J. Org. Chem . 1991, 56, 2853. 15. Ruenitz, P.C.; Bourne, C.S.; Sullivan, K.J.; Moore,

S.A. J. Med . Chem . 1996, 39, 4853.

16. Vaultier, M., Knouzi, N.; Carrie, R. Tetrahedron Lett . 1983, 24 , 763.

17. Bold, G.; Steiner, H.; Moesch, L., Walliser, B. Helv. Chim . Acta 1990, 473, 405.

18. Connell, R.D., Rein, T.; Akermark, B.; Helquist, P. J. Org. Chem . 1988, 53, 3845.

19. Troxler, F.; Bormann, G.; Seemann, F. Helv. Chim . Acta 1968, 51 , 1203. 20. Gatterman, L. Justus Liebigs Ann . Chem . 1907, 357, 313.

21. Katzenellenbogen, J.A.; Tatee, T.; Robertson, D.W. J. Labelled Compd. Radiopharm . 1981, 18, 865.

2 4

Scheme 1. Outline reaction scheme for photolysis of 1- acyl-7-nitroindolines 1 and 2 in aqueous solution .

10

11

Scheme 2. Synthesis of the functionalised sensitiser moiety 21.

Scheme 3. Assembly of conjugate 12.

25

CH,

BrCH₂C0₂Me- K₂C0₃-acetone

31

NaOH-aq. MeOH

31a

Scheme 4 . Synthesis of the methoxycarbonymethoxy- substituted nitroindoline 31 .

+ trace

Scheme 5. Synthesis of the L-glutamate conjugate 33

BrCH₂C0₂Bu^t- K₂C0₃-acetone

_Scheme 6. Synthetic route to conjugate 41

Claims

Claims :

1. A compound represented by the formula:

wherein:

Ri is an effector species linked to the nitrogen atom at the 1-position of the indoline ring via an acyl linkage or is a group which is capable of linkage to an effector species;

R₂ and R3 are selected from hydrogen, a substituted or unsubstituted alkyl group, or R₂ and R₃ together form a substituted or unsubstituted cycloalkyl group;

R is hydrogen, a substituted or unsubstituted alkyl group, a substituted or unsubstituted alkoxy group or a triplet sensitising group; and

R₅ is hydrogen, a substituted or unsubstituted alkyl group, a substituted or unsubstituted phenyl group, a triplet sensitising group, a group represented by(CH₂)_nY; or (CH₂)_mO (CH₂) _nY, where m and n are independently between 1 and 10 and Y is selected from hydrogen, C0H or salts thereof, OP0₃ ^2~ or salts thereof, 0S0₃ ^~ or salts thereof, or C0₂Rε , wherein Re is an alkyl or substituted alkyl group; wherein at least one of R₄ and R₅ is a triplet sensitising group.

2. The compound of claim 1, wherein Ri is represented by -C(=0)-X, and X is -C-R, -O-R or -NH-R, and R is the remaining part of the effector species.

3. The compound of claim 1 or claim 2, wherein the effector species is an amino acid.

4. The compound of any one of claims 1 to 3, wherein 5 the effector species is a neuroactive amino acid.

5. The compound of any one of the preceding claims, wherein the effector species is glycine, GABA or glutamate . lθ^'

6. The compound of any one of the preceding claims, wherein the triplet sensitising group is a linked to the compound via a linker group.

15 7. The compound of any one of the preceding claims, wherein the triplet sensitising group is a substituted or unsubstituted benzophenone, a substituted or unsubstituted anthrone, a substituted or unsubstituted xanthone, a substituted or unsubstituted carbazole, a

20 substituted or unsubstituted triphenylene or a substituted or unsubstituted heterocyclic analogue of benzophenone .

8. The compound of claim 7, wherein the heterocyclic 25 benzophenone analogue is a 3- or 4-benzoylpyridine .

9. The compound of claim 7, wherein the triplet sensitising group is a substituted or unsubstituted benzophenone, optionally having one or more substituents

30 at the 4 or 4,4' position (s) .

10. The compound of claim 9, wherein the benzophenone is substituted with a substituted or unsubstituted alkyl group, a substituted or unsubstituted alkoxy group, a substituted or unsubstituted dialkyl group or a substituted or unsubstituted dialkoxy group.

11. The compound of claim 9 or claim 10, wherein the benzophenone is a 4 , 4' -dialkoxy derivative.

12. The compound of claim 6, wherein the linker group comprises a group represented by the general formula - (CH₂) p-NH-C (=0) - (CH₂) ₀- , wherein o and p are independently selected from integers from 1 to 10, and wherein one end of the linker group is bonded to the 4 and/or 5-position of the nitroindoline and the other end of the linker group is bonded to the triplet sensitising group.

13. The compound of claim 12, wherein the linker group is represented by the general formula -0- (CH₂) _P-NH-C (=0) - (CHz)o-O-.

14. The compound of claim 6, wherein the linker group is represented by the general formula -0- (CH₂) _o-0- , wherein o is an integer from 1 to 10, and wherein one end of the linker group is bonded to the 4 and/or 5-position of the nitroindoline and the other end of the linker group is bonded to the triplet sensitising group.

15. The compound of claim 6, wherein the linker group is a substituted or unsubstituted alkyl group as represented by -(CH₂)₀-, where o is an integer from 1 to 10, and wherein one end of the linker group is bonded to the 4 and/or 5-position of the nitroindoline and the other end of the linker group is bonded to the triplet sensitising group.

16. The compound of any one of claims 12 to 15, wherein in the linker groups o and p is or are independently selected from integers which are 1 or 2.

17. The compound of claim 6, wherein the linker group comprises one or more methylene groups, a phenylene group, an oligomethylenephenylene, or one or more oxygen atoms .

18. The compound of any one of the preceding claims wherein the triplet sensitising group or the linker group includes one or more of a phosphate group, a sulfate group, a carboxylate group, a trialkylammonium substituent, or a poly-hydroxy moiety.

19. The compound of any one of the preceding claims, wherein the triplet sensitising group comprises a substituent represented by -0-(CH₂)_n-Y, where n is an integer between 1 and 10.

20. The compound of claim 19, wherein Y is OP0₃ ^2~.

21. A method which comprises irradiating a compound of any one of the preceding claims to cause it to photolyse and release the effector species.

22. The method of claim 21, wherein the method is a cell based assay.

23. The method of claim 22, wherein the cells are neuronal cells.

24. The method of any one of claims 21 to 23, wherein the photolysis and release steps are used in a patch clamp experiment or in a method of high throughput screening .