GB2412657A

GB2412657A - Fluorophore-labelled solid supports

Info

Publication number: GB2412657A
Application number: GB0506480A
Authority: GB
Inventors: Silva Amilra Prasanna De; Sheenagh Marie Weir
Original assignee: Queens University of Belfast
Current assignee: Queens University of Belfast
Priority date: 2004-03-31
Filing date: 2005-03-31
Publication date: 2005-10-05
Also published as: GB0407212D0; GB2412657A8; GB0506480D0

Abstract

A labelled solid support comprising a solid support and at least one fluorophore label is described wherein the fluorophore label operates as a logic gate in the presence of an ionic species. A library of labelled solid supports is also described wherein the solid support comprises a chemical entity bound thereto and each label corresponds to a single chemical entity. The library comprises at least 10<3> solid supports.

Description

24 1 2657 1 "Labelled Solid Support comprising Fluorophore Label" 3 The

present invention relates to a tagging system for 4 combinatorial chemistry and screening assays.

6 There is a real desire in the pharmaceutical and chemical 7 industries to have the ability to manufacture or screen 8 thousands of candidate compounds. Methodologies, termed 9 "combinatorial chemistry", have been developed whereby numerous compounds can be produced or screened 11 simultaneously. Only those few compounds which indicate 12 a positive result (ie. one shown to have the desired 13 characteristics) are then isolated and identified for 14 further investigation.

16 One common way of enhancing the handling, presentation 17 and/or subsequent isolation of candidate compounds is to 18 attach each compound to an individual inert bead, which 19 also contains a label. The label is desirably unique to the particular chemical entity bound to that bead and 21 enables subsequent identification. At present there are 22 severe limitations on the accuracy with which the labels 1 themselves can be resolved from one another and 2 consequently multiple copies (eg. twenty copies) of each 3 chemical entity, each labelled identically, is included 4 to avoid identification errors. Moreover there is a limitation on the number of distinct labels available 6 which reduces the numbers of different compounds which 7 can be manufactured and/or screened at any one time.

9 Additionally the label chosen needs to be non-destructive to the entity labelled.

12 There is considerable interest in the use of "one bead, 13 one compound" combinatorial chemistry where only a single 14 bead has the desired activity. For this approach to be successful, it is essential to have the ability to 16 reliably determine the identity of the 0.1-lnmol of 17 sample bound to the single bead of interest.

19 In Nature 1993 Vol 364 p42, we described the ability to use a blue fluorescent compound as an AND logic gate; the 21 labels fluoresced only in the presence of both Ha+ and H+ 22 ions.

24 Likewise, in J. Am. Chem. Soc. 2000 Vol 122, p3965 we described the use of a further fluorescent compound which 26 could be used as an AND gate (fluorescence being 27 dependent upon the presence of both Ca2+ and H+ ions) and 28 a related light-absorbing compound which acted as a XOR 29 gate (transmittance occurred only in the presence of either Ca2+ or H+ ions, but not in the presence of both) 31 and the use of these compounds for numerical operations 32 was discussed.

1 We have now found that such fluorescent compounds can be 2 attached to beads (such as the inert beads commonly used 3 for high through-put assays) without adversely affecting 4 their unique ability to fluoresce in the presence of specific combinations of ions. Based upon this 6 observation, it has been recognized that such 7 fluorophores could be used as labels for identification 8 of unique beads. Moreover, our experiments have 9 demonstrated that a high degree of resolution of the TO differently labelled beads can be achieved, enabling 11 significant reduction in the need for duplication of 12 samples and enabling a higher number of different samples 13 to be screened in a single assay, thereby increasing the 14 efficiency of the assay.

16 In one aspect, the present invention provides a labelled 17 solid support comprising a solid support with one or more 18 fluorophore labels attached thereto, wherein the 19 fluorophore label operates as a logic gate in the presence of a pre- determined ionic species or mixture of 21 ionic species.

23 Preferably the labelled solid support comprises one or 24 two fluorophore labels.

26 The fluorophore label itself comprises a fluorophore 27 covalently bound (optionally via a spacer) to a receptor 28 able to bind an ionic species, such that the fluorescence 29 of the fluorophore is altered upon binding of the ionic species to the receptor.

1 In the compounds described fluoresence may be achieved by 2 photoinduced electron transfer (PET) or Internal Charge 3 Transfer (ICT).

In PET an electron is transferred from the highest 6 occupied molecular orbital of a donor in its ground state 7 to the highest occupied molecular orbital of an acceptor 3 in its excited state. In the compounds described PET is 9 arranged by coupling a receptor (donor moiety) to a 1O fluorophore (acceptor moiety) via a 6-spacer. The 11 presence of the spacer means that the fluorophore and 12 receptor are spatially distinct and any orbital 13 interactions between these portions of the sensor are 14 minimized. The fluorophore moiety is the site of both excitation and emission whereas the receptor moiety is 16 responsible for complexing to the guest ionic species.

17 Figure 1 shows a schematic representation of these 18 compounds.

In ICT, the fluorophore label has electron donors and 21 acceptors linked by a conjugated bridge so forming a 22 single delocalised unit. The electron donor pushes 23 electron density into the -system whilst the electron 24 acceptor pulls electrons from it. A more integrated structure, generally lacking a spacer, is required for a 26 molecule to achieve ICT. A schematic representation is 27 shown in Figure 2.

29 The fluorophore and receptor are chosen so that an electron transfer can occur from the receptor to the 31 fluorophore upon excitation which quenches the excited 32 state of the fluorophore and leads to a change in the 33 fluorescence. The excited state energy must be 1 sufficient to oxidise the receptor and reduce the 2 fluorophore. The introduction of a guest ionic species 3 which can bind to the receptor alters the oxidation 4 potential of the receptor and so changes the conditions at which PET occurs.

7 The labelled solid support may comprise more than one 8 fluorophore, and where multiple fluorophores are present 9 each fluorophore may behave according to the same or different logic operations. Suitably each fluorophore 11 behaves according to the AND, PASS-1, NOT, NAND, OR, NOR, 12 XOR, XNOR, INHIBIT, ENABLED OR, or YES logic operation.

14 Preferably, a first fluorophore of a labelled solid support behaves according to YES logic operation, and a 16 second fluorophore of a labelled solid support behaves 17 according to a PASS-1 logic operation.

19 Suitably the fluorophore is anthracene, pyrene, 9 cyanoanthracene, 6amino-3-benzimidazobenz-isoquinolin-3 21 one or many others which can be a part of "fluorophore 22 spacer-receptor" PET systems and "fluorophore-receptor'' 23 ICT systems.

Suitably the solid support has a It to 5% loading, 26 preferably a 0.1%, 0.25%, 0.5% or 1% loading.

28 Preferably two fluorophore labels are attached to the 29 solid support. Advantageously each fluorophore label behaves according to a different logic operation and/or 31 makes a different fluorescence intensity contribution to 32 the combination. Suitably the fluorophore labels are 2 33 [(9'-anthryl-methyl)(methyl)-amino]acetic acid and 3-(9- 1 anthryl) propanoic acid. Alternatively the fluorophore 2 labels are 2-[methyl(1'-pyrenylmethyl)amino]acetic acid 3 and 4-(1-pyrenyl)butyric acid. These are only two 4 examples of several pairs which can be profitably examined, other examples of suitable fluorophore label 6 pairs include anthracene and perylene and anthracene and 7 4-amino-1-8-naphthalimide. There is particular benefit 8 to using such double fluorophore labelled solid supports 9 because this hugely increases the number of distinguishable encodings that are possible for the 11 beads.

13 Suitably the fluorophores make a different fluorescence 14 intensity contribution to the combination. Each fluorophore may emit fluorescence at a different 16 intensity.

18 The spacer molecule is suitably -CH2-, but other lower 19 alkylene groups (e.g. -CH2CH2- or -CH2CH2CH2-) are also

suitable.

22 The solid support may be any suitable support surface, 23 but for convenience of handling is preferably a bead or 24 the like. Generally the solid support will be chemically robust and will be chosen for its inert behaviour during 26 the reactions to which the bound molecules will be 27 exposed. Suitable materials include inert polymers with 28 some hydrophilicity, such as polystyrene-based Tentagel, 29 silica or glass. Optionally the polymer may be derivatized with a functional group or linker.

32 In a further aspect, the present invention provides a 33 device for tracking a library member of a combinatorial 1 library, the device comprising a support adapted for 2 linking to a library member or a precursor thereof, 3 wherein said support is attached to a fluorophore label 4 able to operate as a logic gate in the presence of a mixture of ionic species.

7 The combinatorial library may consist of 103 to 106 or 8 more different members. It would be advantageous to 9 screen a library of 106 to 108 different members, but samples of 102 to 103 members may be tested at any one 11 time.

13 The library members may be monomers or oligomers and may 14 be used to screen for activity with or to identify peptides, nucleic acids, carbohydrates or the like.

16 Optionally, the library members may be screened for the 17 ability to bind to, activate, repress or mimic enzymes, 13 receptors, antibodies, hormones, growth factors and the 19 like.

21 Optionally, the combinatorial library may be subjected to 22 chemical transformation steps prior to such screening and 23 the fluorophore label discussed above would provide 24 "birth-to-death" identification of the associated member.

26 The present invention also provides a labelled library or 27 random oligomers or other members, where each member is 28 linked to a solid support itself attached to a 29 fluorophore label able to operate as a unique logic gate such that the identity of the member can be reliably 31 ascertained.

1 The present invention also provides a method for 2 deciphering the content of a combinatorial library, said 3 method comprising labelling each member of the library 4 with a unique fluorophore label able to act as a logic gate in the presence of predetermined ionic species or 6 mixture of ionic species, and detecting fluorescence 7 under different ionic conditions. We note that all the 8 experiments described here use protons (H+) as the ionic 9 species to demonstrate the principle. However, other ionic species such as Ca2+ and Na+ can be used with 11 similar success given our previous success with logic 12 gates driven by such species. The availability of 13 multiple ionic species as inputs raises further the 14 number of distinguishable logic gates that can be used for encoding.

17 In a further aspect, the present invention provides a 18 method for identifying an isolated member of a 19 combinatorial library wherein each member is attached to a predetermined unique fluorophore label, the method 21 comprising measuring the fluorescence of the fluorophore 22 label in the presence of different ionic species or 23 mixture of species, and thereby determining the identity 24 of the unique fluorophore label and hence the identity of the member attached thereto.

27 The present invention will now be further described with 28 reference to the following non-limiting examples and 29 figures in which: 31 Figure 1 shows a schematic representation of PET 32 transfer.

1 Figure 2 shows a schematic representation of ICT 2 transfer.

4 Figure 3 shows a family of absorption spectra as a function of pH of the model compound (82) which mimics 6 the surface of a Tentagel-S-NH2 bead grafted with 2-(9' 7 anthyrlmethyl)(methyl)amino]acetic acid. The pH values 8 in order of increasing absorbance at 370nm are 11.2, 9 10.6, 9.9, 9.5, 8.8, 8.4, 7.4, 7.0, 6.5, 6.1, 5.4, 4.8, 4.3, 3.9, 3.5, 3.0 and 2.8.

12 Figure 4 shows a family of fluorescence emission spectra 13 as a function of pH of the model compound (82) which 14 mimics the surface of a Tentagel-S-NH2 bead grafted with 2-(9'-anthyrlmethyl(methyl)amino]acetic acid. The 16 excitation wavelength is 368nm. The pH values in order 17 of increasing intensity at 421nm are 10.6, 9.6, 8.5, 7.5, 18 7.0, 6.6, 6.2, 5.7, 5.4, 5.2, 4.9, 4.6, 4.3, 4.0, 3.6, 19 3.3, 3.0 and 2.8.

21 Figure 5 shows an emission-pH profile (at 421nm) for the 22 model compound (82) which mimics the surface of a 23 Tentagel-S-NH2 bead grafted with 2-(9' 24 anthyrlmethyl(methyl)amino]acetic acid 82.

26 Figure 6 shows an emission-pH profile (at 421nm) of Nl 27 benzyl-2-[(9'-anthrylmethyl)(methyl)amino]acetamide (83).

29 Figure 7 shows a family of fluorescence emission spectra as a function of pH of 2-[(9' 31 anthrylmethyl)(methyl)amino]acetic acid grafted onto 32 Tentagel-S-NH2 beads (88) at 2.5% loading. The pH 33 volumes in order of increasing intensity at 423nm are 1 10.3, 9.1, 8.0, 7.1, 6.3, 5.7, 5.1, 4.8, 4.5, 4.1, 3.8, 2 3.6 and 3.2.

4 Figure 8 shows the emission-pH profile (at 423nm) of 2 [9'-anthrylmethyl)(methyl)amino]acetic acid grafted onto 6 Tentagel-S-NH2 beads (88).

8 Figure 9 shows a family of fluorescence emission spectra 9 as a function of pH of protected 2-[9' anthrylmethyl)(methyl)amino] acetic acid (89) (2.5%). The 11 excitation wavelength is 368nm. The pH values in order 12 of increasing intensity at 423nm are 10.3, 8.0, 7.3, 6.6, 13 6.1, 5.8, 5.5, 5.1, 4.9, 4.5, 4.1, 3.8, 3.5, and 3.3.

Figure 10 shows an emission-pH profile (at 423nm) of 2 16 [9'-anthrylmethyl)(methyl)amino]acetic acid (89).

18 Figure 11 shows a family of fluorescence emission spectra 19 as a function of pH of Tentagel-S-NH2 beads having 9 Bromomethyl-10cyanoanthracene grafted at 1% (55). The 21 ordinate is fluorescence intensity. The excitation 22 wavelength is 390nm. The pH values in order of 23 increasing intensity at 45Onm are 9.5, 6.8, 6.1, 5.4, 24 4.8, 4.3, 3.9, 3.5, 3.0, 2.7, 2.5 and 2.3.

26 Figure 12 shows an emission-pH profile (at 450nm) of 27 Tentagel-S-NH2 beads having 9-Bromomethyl-10 28 cyanoanthracene grafted at 1% (55).

Figure 13 shows a family of fluorescence emission spectra 31 as a function of pH of 1-(9 -anthrylmethyl)-4 32 piperidinecarboxylic acid (93). The excitation 33 wavelength is 368nm. The pH values in order of 1 increasing intensity at 422nm are 10.7, 9.5, 9.0, 8.0, 2 7.5, 7.1, 6.6, 6.4, 5.8, 5.4, 5.2, 4.9, 4.5, 4.1, 3.9, 3 3.6 and 3.3.

Figure 14 shows an emission-pH profile (at 422nm) for 1 6 (9 -anthrylmethyl)-4-piperidinecarboxylic acid (93).

8 Figure 15 shows a family of absorption spectra as a 9 function of pH of Nl-(2''-methoxyethyl)-2[(methyl)(1' pyrenylmethyl) amino]acetamide (98). The pH values in 11 order of increasing absorbance at 326nm are 3.0, 3.4, 12 3.8, 4.2, 4.6, 5.2, 6.1, 6.9, 7.5, 8.5, 9.2, 9.8 and 13 10.5.

Figure 16 shows an absorbance-pH profile (at 326nm) of 16 Nl-(2''-methoxyethyl)-2 [(methyl) (l' 17 pyrenylmethyl)amino]acetamide (98).

19 Figure 17 shows a family of fluorescence emission spectra as a function of pH of Nl-(2''-methoxyethyl)-2 21 [(methyl)(l'-pyrenylmethyl)amino]acetamide (98). The 22 excitation wavelength is 326nm. The pH values in order 23 of increasing intensity at 377nm are 10.3, 9.3, 8.7, 7.3, 24 6.5, 6.2, 5.8, 5.5, 5.2, 4.8, 4.1, 3.7, 3.4 and 3.1.

26 Figure 18 shows an emission-pH profile (at 377nm) of Nl 27 (2''-methoxyethyl)-2[(methyl)(1' 28 pyrenylmethyl)amino]acetamide (98).

Figure 19 shows a family of fluorescence emission spectra 31 as a function of pH of 3-(9 -anthryl)propanoic acid 32 (105). The excitation wavelength is 368nm. The pH 1 values in order of increasing intensity at 421nm are 2 10.9, 9.6, 8.4, 7.3, 6.4, 5.4, 3.8 and 3.0.

4 Figure 20 shows an emission-pH profile (at 421nm) of 3 (9 -anthryl)propanoic acid (105).

7 Figure 21 shows a family of fluorescence emission spectra 8 as a function of pH of 2-[methyl(1'-pyrenylmethyl)amino] 9 acetic acid (99). The excitation wavelength is 326nm.

The pH values in order of increasing intensity at 378nm 11 are 10.4, 9.0, 8.1, 7.3, 6.6, 6.0, 5.6, 5.1, 4.8, 4.5, 12 4.1, 3.8, 3.6 and 3.3.

14 Figure 22 shows an emission-pH profile (at 378nm) for 2 [methyl(1'-pyrenylmethyl)amino] acetic acid (99).

17 Figure 23 shows absorption spectra upon pH variation of 18 Nl-[2-methoxyethyl)-3-(9-anthryl)propanamide (104).

19 The pH values in order of increasing absorbance at 368nm are 2.8, 3.3, 3.7, 4.2, 5.0, 6.0, 6.7, 7.9, 8.5, 9.5, 21 10.0 and 11.2.

23 Figure 24 shows an absorbance-pH profile (at 368nm) of 24 Nl-[2-methoxyethyl)-3-(9-anthryl)propanamide (104).

26 Figure 25 shows a family of fluorescence emission spectra 27 as a function of pH of Nl-[2''-methoxyethyl)-3-(9- 28 anthryl)propanamide (104). The excitation wavelength is 29 368nm. The pH values in order of increasing intensity at 421nm are 11.0, 9.9, 9.0, 8.3, 7.7, 7.0, 6.2, 5.2, 4.2, 31 3.7, 3.2 and 2.6.

1 Figure 26 shows an emission-pH profile (at 421nm) for N1- 2 [2''-methoxyethyl)-3-(9'-anthryl)propanamide (104).

4 Figure 27 shows a family of absorption spectra as a function of pH for Nl-(2''-methoxyethyl)-4-(1'-pyrenyl) 6 butanamide (106). The pH values in order of increasing 7 absorbance at 326nm are 10.1, 9.3, 8.4, 6.7, 5.8, 5.0, 8 4.3, 3.7 and 3.1.

Figure 28 shows an absorbance-pH profile (at 326nm) for 11 Nl-(2''-methoxyethyl)-4-(1'-pyrenyl) butanamide (106).

13 Figure 29 shows a family of fluorescence emission spectra 14 as a function of pH of Nl-(2-methoxyethyl)-4-(l,- pyrenyl) butanamide (106). The excitation wavelength is 16 326nm. The pH values in order of increasing intensity at 17 377nm are 10.3, 9.5, 8.5, 7.2, 6.2, 5.5, 4.9, 4.5, 4.1, 18 3.8 and 3.3.

Figure 30 shows an emission-pH profile (at 377nm) for Nl 21 (2''-methoxyethyl)-4-(1'-pyrenyl) butanamide (106).

23 Figure 31 shows a family of fluorescence emission spectra 24 as a function of pH of Tentagel-S-NH2 beads (107) with 2.5 loading of 1-pyrenebutyric acid. The excitation 26 wavelength is 326nm. The pH values in order of 27 increasing value at 379nm are 3.1, 3.5, 3.8, 4.2, 4.7, 28 5.7, 6.4, 7.2, 8.0 and 10.4.

Figure 32 shows an emission-pH profile (at 379nm) for 31 Tentagel-S-NH2 beads (107) with 2.5% loading of 1 32 pyrenebutyric acid.

1 Figure 33 shows a family of fluorescence emission spectra 2 as a function of pH of double labelled Tentagel-S-NH2 3 beads (108) with 2-[9'-anthryl-methyl)(methyl)-amino] 4 acetic acid and 3-(9'-anthryl) propanoic acid. The excitation wavelength is 368nm. The pH values in order 6 of increasing intensity at 421nm are 10.5, 9.5, 8.2, 7.3, 7 5.9, 5.1, 4.8, 4.5, 4.0, 3.5, 3.2 and 2.9.

9 Figure 34 shows an emission-pH profile (421nm) for double labelled Tentagel-S-NH2 beads (108) with 2-[9'-anthryl 11 methyl)(methyl)-amino] acetic acid and 3-(9'-anthryl) 12 propanoic acid.

14 Figure 35 shows a family of fluorescence emission spectra as a function of pH of double labelled Tentagel-S-NH2 16 beads (109) with loading 2-(9' 17 anthrylmethyl)(methyl)amino]acetic acid at 2.5% loading 18 and 3-(9'-anthryl)propanoic acid at 5% loading. The 19 excitation wavelength is 268nm. The pH values in order of increasing intensity at 421nm are 10.7, 9.1, 8.0, 7.0, 21 6.4, 5.0, 4.5, 4.1, 3.5, 3.0 and 2.8.

23 Figure 36 shows an emission-pH profile (at 421nm) for 24 double labelled Tentagel-S-NH2 beads (109) with loading 2-(9'-anthrylmethyl)(methyl)amino]acetic acid at 2.5% 26 loading and 3-(9'-anthryl)propanoic acid at 5% loading.

28 Figure 37 shows a family of fluorescence emission spectra 29 of double labelled Tentagel-S-NH2 beads (110) with 2 [(9'-anthryl-methyl)(methyl)-amino]acetic acid at 5\ 31 loading and 3-(9'-anthryl)propanoic acid at 2.5% loading 32 as a function of pH. The excitation wavelength is 368nm.

33 The pH values in order of increasing intensity at 421nm 1 are 10.5, 10.2, 9.1, 8.2, 7.0, 6.3, 5.7, 4.9, 3.9, 3.4 2 and 3.2.

4 Figure 38 shows an emission-pH profile (at 421nm) for double labelled Tentagel-S-NH2 beads (110) with 2-[(9- 6 anthryl-methyl)(methyl)-amino]acetic acid at 5% loading 7 and 3-(9'-anthryl)propanoic acid at 2.5% loading.

9 Figure 39 shows a family of fluorescence emission spectra as a function of pH of double labelling Tentagel-S-NH2 11 beads (111) with 2-[methyl(1'-pyrenylmethyl)amino] and 1 12 pyrenebutyric acid. The excitation wavelength is 326nm.

13 The pH values in order of increasing intensity at 379nm 14 are 10.5, 9.0, 8.0, 6.9, 6.1, 5.5, 5.0, 4.5, 4.1, 3.8, 3.5 and 3.3.

17 Figure 40 shows an emission-pH profile (at 379nm) for 18 double labelling Tentagel-S-NH2 beads (111) with 2 19 [methyl(l'-pyrenylmethyl)amino] and 1-pyrenebutyric acid.

21 Figure 41 shows a family of fluorescence emission spectra 22 as a function of pH of double labelling Tentagel-S-NH2 23 beads (111) with 2-[methyl(1'-pyrenylmethyl)amino] and 1 24 pyrenebutyric acid. The excitation wavelength is 326nm.

The pH values in order of increasing intensity at 379nm 26 are 10.5, 9.3, 8.2, 7.5, 6.7, 5.7, 5.0, 4.5, 4.2, 3.9 and 27 3.4.

29 Figure 42 shows an emission-pH profile (at 379nm) for double labelling Tentagel-S-NH2 beads (111) with 2 31 [methyl(1'-pyrenylmethyl)amino] and 1-pyrenebutyric acid.

1 Figure 43 shows a family of fluorescence emission spectra 2 as a function of pH of 3-(9'-anthryl)-2-(2n 3 pyridyl)propanoic acid grafted onto Tentagel-S-NH2 beads 4 with 2.5% loading (119). The excitation wavelength is 370nm. The pH values in order of increasing intensity at 6 420nm are 0.5, 1.0, 2.0, 2.5, 3.0, 4.1, 4.7, 5.4, 6.5, 7 8.2 and 10.4.

9 Figure 44 shows an emission-pH profile (at 420nm) of 3 (9'-anthryl)-2-(2"-pyridyl)propanoic acid grafted onto 11 Tentagel-S-NH2 beads with 2.5% loading (119).

13 Figure 45 shows a family of absorption spectra as a 14 function of pH of Nl-(2'''-methoxyethyl)-3-(9'-anthryl) 2-(2''-pyridyl)propanamide (118). The pH values in order 16 of increasing absorbance at 368nm are 2.1, 3.0, 3.5, 4.0, 17 4.7, 5.0, 5.4, 6.3, 5.8, 7.3, 8.0, 8.9 and 11.2.

19 Figure 46 shows an absorbance-pH profile (at 368nm) of Nl-(2'''-methoxyethyl)-3-(9'-anthryl)-2-(2- 21 pyridyl)propanamide (118).

23 Figure 47 shows a family of fluorescence emission spectra 24 as a function of pH of Nl-(2'''-methoxyethyl)-3-(9anthryl)-2-(2''-pyridyl)propanamide (118). The 26 excitation wavelength is 370nm. The pH values in order 27 of increasing intensity at 416nm are 2.8, 3.1, 3.4, 3.6, 28 4.0, 4.2, 5.0, 5.2, 5.5, 6.0, 7.2, 7.8 and 11.2.

Figure 48 shows an emission-pH profile (at 416nm) of Nl 31 (2'''-methoxyethyl)-3-(9'-anthryl)-2-(2'' 32 pyridyl)propanamide (118).

1 Figure 49 shows a family of fluorescence emission spectra 2 of 3-[(9'-anthrylmethyl)(2" pyridylmethyl)amino] 3 propanoic acid grafted onto Tentagel-S-NH2 beads with 4 2.5% loading (131) as a function of pH. The excitation wavelength is 370nm.

7 Figure 50 shows an emission-pH profile (424nm) of 3-[(9- 8 anthrylmethyl)(2" pyridylmethyl)amino]propanoic acid 9 grafted onto Tentagel-S-NH2 beads (131) with 2.5% loading.

12 Figure 51 shows a family of absorption spectra as a 13 function of pH of Nl-(2'''-methoxyethyl)-3-[(9' 14 anthrylmethyl)(2''-pyridylmethyl)amino] propanamide (129). The pH values in order of increasing absorbance 16 at 367nm are 3.1, 3.4, 3.5, 3.8, 4.1, 4.4, 4.8, 5.1, 5.4, 17 5.9, 6.4, 6.6,6.8, 7.0, 7.4, 7.8, 8.0, 8.4, 8.8, 9.3 and 18 10.1.

Figure 52 shows an absorbance-pH profile (at 367nm) of 21 Nn-(2'''-methoxyethyl)-3-[(9'-anthrylmethyl)(2'' 22 pyridylmethyl)amino] propanamide (129).

24 Figure 53 shows a family of fluorescence emission spectra as a function of pH of Nl-(2'''-methoxyethyl)-3-[(9- 26 anthrylmethyl)(2''-pyridylmethyl)amino] propanamide 27 (129). The excitation wavelength is 370nm.

29 Figure 54 shows an emission-pH profile (421nm) of N1 (2'''-methoxyethyl)-3-[(9'-anthrylmethyl)(2'' 31 pyridylmethyl)amino] propanamide (129).

1 Figure 55 shows a family of fluorescence emission spectra 2 as a function of pH of Tentagel-SNH2 beads (112) double 3 labelled with 2-[(9'-anthrylmethyl)(methyl)amino]acetic 4 acid (79) and 2-[(methyl)(1'-pyrenylmethyl)amino]acetic acid (94). The excitation wavelength is 368nm. The pH 6 values in order of increasing intensity at 422nm are 7 10.8, 9.0, 8.1, 6.9, 5.9, 5.6, 5.1, 4.8, 4.4, 4.1, 3.9, 8 3.5 and 303.

Figure 56 shows an emission-pH profile (at 422nm) of 11 Tentagel-SNH2 beads (112) double labelled with 2-[(9' 12 anthrylmethyl)(methyl)amino]acetic acid (79) and 2 13 [(methyl)(l'pyrenylmethyl)amino]acetic acid (94). The 14 excitation wavelength is 368nm.

16 Figure 57 shows a family of fluorescence emission spectra 17 as a function of pH of Tentagel-S-NH2 beads (112) double 18 labelled with 2-[(9'-anthrylmethyl)(methyl)amino]acetic 19 acid (79) and 2-[(methyl)(l'pyrenylmethyl)amino]acetic acid (94). The excitation wavelength is 326nm. The pH 21 values in order of increasing intensity at 379nm are 22 10.5, 8.0, 7.1, 6.3, 5.7, 5.2, 4.9, 4.6, 4.1, 3.8 and 23 3.60 Figure 58 shows an emission-pH profile (at 379nm) for 26 Tentagel-S-NH2 beads (112) double labelled with 2-[(9' 27 anthrylmethyl)(methyl)amino]acetic acid (79) and 2 28 [(methyl)(l'pyrenylmethyl)amino]acetic acid (94). The 29 excitation wavelength is 326nm.

31 Figure 59 shows a family of fluorescence emission spectra 32 as a function of pH of Tentagel-S-NH2 beads (62) grafted 33 onto 4-Bromomethyl-7-methoxy coumarin (1% loading). The 1 ordinate is fluorescence intensity. The excitation 2 wavelength is 332nm. The pH values in order of 3 increasing intensity at 409nm are 10.5, 9.6, 8.2, 7.1, 4 6.4, 5.9, 5.1, 4.2, 3.4, 2.7 and 2.3.

6 Figure 60 shows an emission-pH profile (at 409nm) for 7 Tentagel-S-NH2 beads (62) grafted onto 4-Bromomethyl-7 8 methoxy coumarin (1% loading).

Figure 61 shows a family of fluorescence emission spectra 11 as a function of pH of 6-chloro-3-benzimedozobes 12 isoquinolin-3-one grafted onto Tentagel-S-NH2 beads with 13 1% loading (70). The ordinate is fluorescence intensity.

14 The excitation wavelength is 476nm. The pH values in order of increasing intensity at 511nm are 9.0, 8.3, 6.8, 16 6.3, 5.4, 4.7, 4.2, 3.6, 3.0, 2.5, 2.2 and 2.1.

18 Figure 62 shows an emission-pH profile (at 511nm) of 6 19 chloro-3-benzimedozobes-isoquinolin-3-one grafted onto Tentagel-S-NH2 beads with 1% loading (70).

22 Figure 63 shows an emission-pH profile for 6-chloro-3 23 benzimedozobes-isoquinolin-3-one grafted onto Tentagel-S 24 NH2 beads with 1% loading (70). The excitation wavelength is 514nm.

27 Figure 64 shows absorption spectra as a function of pH of 28 3-[(2'-Methoxyethyl)amino]-7H 29 benzo[de]benzo[4,5]imidazo[2,1-a]isoquinolin-7-one (68).

The pH values in order of increasing absorbance at 468nm 31 are 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 3.0, 3.2, 3.5, 3.8, 32 4.2, 4.7, 5.5, 6.2, 7.2 and 8.9.

1 Figure 65 shows an absorbance-pH profile (at 468nm) of 3 2 [(2'-Methoxyethyl)amino]-7H 3 benzo[de]benzo[4,5]imidazo[2,1-a]isoquinolin-7-one (68).

Figure 66 shows a family of fluorescence emission spectra 6 of 3-[(2'-Methoxyethyl)amino]-7H 7 benzo[de]benzo[4,5]imidazo[2,1-a]isoquinolin-7-one (68) 8 as a function of pH. The ordinate is fluorescence 9 intensity. The excitation wavelength is 476nm. The pH values in order of increasing intensity at 510nm are 1.9, 11 2.1, 2.3, 2.4, 2.5, 2.8, 3.1, 3.6, 4.2,5.0, 5.3, 5.7, 6.1 12 and 7.3.

14 Figure 67 shows an emission-pH profile (510nm) of 3-t(2' Methoxyethyl) amino]-7H-benzo[de]benzo[4,5]imidazo[2,1 16 a]isoquinolin-7-one (68).

18 Figure 68 shows a family of fluorescence emission spectra 19 as a function of pH of 3-[(2'-Methoxyethyl)amino]-7H benzo[de]benzo[4, 5]imidazo[2,1-a]isoquinolin-7-one (68).

21 The ordinate is fluorescence intensity. The excitation 22 wavelength is 514nm. The pH values in order of 23 increasing intensity at 559nm are 7.3, 6.4, 6.1, 5.5, 24 5.1, 4.6, 4.0, 3.7, 3.4, 3.0, 2.7, 2.5, 2.3 and 2.2.

26 Figure 69 shows an emission-pH profile (at 559nm) of 3 27 [(2'-Methoxyethyl)amino]-7H 28 benzo[de]benzo[4,5]imidazo[2,1-a]isoquinolin-7-one (68).

Figure 70 shows an electronic representation of the PASS 31 1 Logic Operation. 1 Figure 71 shows an electronic representation of the 2 parallel logic

operation of PASS l:YES(1:1).

4 Figure 72 shows an electronic representation of the parallel logic operation of PASS 1:YES(2:1).

7 Figure 73 shows an electronic representation of the 8 parallel logic operation of PASS 1:YES(1:2).

Figure 74 shows an electronic representation of the 11 parallel operation for Tentagel-SNH2 beads (112) double 12 labelled with 2-[(9'-anthrylmethyl)(methyl)amino]acetic 13 acid (79) and 2-[(methyl)(1'-pyrenylmethyl)amino]acetic 14 acid (94).

16 Figure 75 shows a family of fluorescence emission spectra 17 as a function of pH of double labelled Tentagel-S-NH2 18 beads (113) with 2-[(methyl)(1' l9 pyrenylmethyl)amino]acetic acid (94) and 3-(9' anthrylmethyl)propanoic acid (100). The excitation 21 wavelength is 368nm. The pH values in order of 22 increasing intensity at 419nm are 10.7, 9.0, 8.1, 7.2, 23 6.0, 5.0, 4.5, 4.0, 3.7 and 3.2.

Figure 76 shows an emission-pH profile (at 419nm) of 26 double labelled Tentagel-S-NH2 beads (113) with 2 27 [(methyl)(1'-pyrenylmethyl)amino]acetic acid (94) and 3 28 (9'-anthrylmethyl)propanoic acid (100).

Figure 77 shows an electronic representation of the 31 parallel logic operation for double labelled Tentagel-S 32 NH2 beads (113) with 2-[(methyl)(1' 33 pyrenylmethyl)amino]acetic acid.

1 Figure 78 shows a family of fluorescence emission spectra 2 as a function of pH of double labelled Tentagel-S-NH2 3 beads (113) with 2-[(methyl)(1' 4 pyrenylmethyl)amino]acetic acid (94) and 3-(9' anthrylmethyl)propanoic acid (100). The excitation 6 wavelength is 326nm. The pH values in order of 7 increasing intensity at 379nm are 10.6, 9.1, 8.0, 7.2, 8 6.2, 5.6, 5.2, 4.9, 4.5, 4.1, 3.8, 3.5 and 3.3.

Figure 79 shows an emission-pH profile (at 379nm) of 11 double labelled Tentagel-S-NH2 beads (113) with 2 12 [(methyl)(1'-pyrenylmethyl)amino]acetic acid (94) and 3 13 (9'-anthrylmethyl)propanoic acid (100). The excitation 14 wavelength is 326nm.

16 Examples

18 Example 1: Method for fluorescence measurements on 19 derivatised beads 21 Our recent tests with our simple fluorescence equipment 22 are successful with 5 beads or more. Better equipment 23 which is available at a higher price will easily allow 24 measurements of similar accuracy to be performed on single beads.

27 The spectrometer was equipped with two optical fibres, 28 one guiding the light beam from the excitation slit to 29 the sample and back to the emission slit respectively. A few beads (at least 5) were placed in the holder. A 31 buffer solution was then added dropwise onto the beads.

32 The beads were washed ten times with this buffer. At 33 this point it should be noted that extreme care has to be 1 taken in order not to move the bead holder. All 2 measurements must be carried out with the beads in a 3 fixed position. The holder was designed to allow this as 4 the beads naturally fell into the centre. After the beads settled the wavelength band at which there was 6 maximum emission intensity was recorded. The beads were 7 then agitated in the holder and an intensity integrated 8 and averaged over one minute at the maximum emission 9 wavelength was recorded. This was repeated 10 times.

The results of these integrated intensities showed that 11 there is good reproducibility of the fluorescence signal 12 from the beads. After these measurements were completed 13 the buffer solution was removed. Subsequent measurements 14 were carried out in the same way only using different buffer solutions.

1 Example 2: Synthesis of 2-[(9'-anthrylmethyl)(methyl) 2 amino]acetic acid (79) PPh3 3 Br OH Br 80 CH3NHCH2CO2C2H5.HCI K2CO3 CH2CI2 MeOH/H2O/THF NaOH

IN N 0 0

OH O.: 4 79 81 6 Using the format described by Miller (Copeland, G.T., 7 Miller, S.J., J. Am. Chem. Soc; 1999, 121, 4306), the 8 first prefabricated logic gate 79 was designed. To 9 prepare the logic gate, 9-(bromomethyl)anthracene, 80, triphenylphosphine (2.68 g, O.OlOmol) was dissolved in 11 acetonitrile (20ml, anhydrous) and bromine (0.49ml, 12 0.0096mol) was added dropwise. 9-Anthrylmethanol (approx.

13 O.lg, 0.48mmol) was added and allowed to stir for 5 min. 14 The remainder of 9-anthrylmethanol (1.9 g, O.OO9mol) was then added. The reaction mixture was allowed to stir for 16 a further 1 hour and 30 mine. The yellow precipitate was 17 collected and recrystallized using chloroform producing 18 yellow crystals (Yield: 82 %).

1 1H NMR (CDC13)300 MHz .51(s, 1H, Ar-_), 8.31(d, 2H, 2 Ar-_, J=8 Hz), 8.05(d, 2H, Ar-_, 3 J=8 Hz), 7.65(t, 2H, Ar-_, J=8 4 Hz), 7.51(t, 2H, Ar-_, J=8 Hz), 5.56(s, 2H, Ar-C_ 2) 7 13C NMR(CDCl3)75 MHz 627.0, 123.5, 125.4, 126.8, 8 129.2, 129.3, 131.6.

irvmax(KBr): 2361, 1621, 1446, 1257, 1196, 11 729 cml.

13 m/z(%), (EI): 270(M+, 23), 191(100).

To prepare ethyl-2-[(9'-anthrylmethyl)(methyl)amino] 16 acetate, 81, starting from 80 (0.13g, 0.49mmol), 17 sarcosine ethylester hydrochloride (0.075g, 0.49mmol) and 18 potassium carbonate (3g, anhydrous) were refluxed in 19 CH2C12 (lOml) overnight. Potassium carbonate was then filtered off and washed with CH2C12 The solvent was 21 evaporated off producing a yellow oil, 81 (Yield: 86.7%).

22 Found: 307.157229 23 Required for C20H21NO2 307.155632 1H NMR (CDC13)500 MHz 68.41(d, H, Ar-_, J=9 Hz), 26 8.31(s, 1H, Ar-_), 7.88(d, 2H, 27 Ar-_, J=8 Hz), 7.43(t, 2H, Ar-_, 28 J=8 Hz), 7.35(t, 2H, Ar-_, ]=7 29 Hz), 4.60(s, 2H, Ar-CH2), 4.10(q, 2H, OC_ 2, 7 Hz), 3.30(s, 31 2H, NC_ 2), 2.38(s, 3H, NC_3), 32 1.17(t, 3H, CH2C_ 3, J=3 Hz).

1 l3C NMR(CDCl3)125 MHz 613.2, 40.8, 51.2, 56.4, 59.3, 2 122. 4, 123. 8, 124. 2, 124.6, 3 125.9, 127. 7, 128.2, 128. 7, 4 170.3.

6 irvmax(KBr): 3052, 2980, 2375, 1733, 1624, 7 1447, 1188, 1031, 886, 732, 533 8 cm1.

m/z (%), (ES): 330 (M+Na+, 20), 308 (M+H+, 100), 11 309 (45) 13 To prepare 2- [ (9' -anthrylmethyl) (methyl) amino] acetic 14 acid, 79, 81 (9Omg, 0.3mmol) was dissolved in THF (lOml).

A solution of NaOH (0.24g, 6mmol) in MeOH:H2O (10 ml:2ml) 16 was added. The solution was left to stir overnight. The 17 solvent was then evaporated off. The yellow solid was 18 dissolved in H2O and neutralised with acetic acid 19 producing a yellow precipitate, which was filtered off (Yield: 75%, m.p. > 300. 0 C) . 22 U.V. 351nm (MeOH:H2O, 1:1)= 6200 dm3mollcm 2 3 U. V. 3 69nm ( MeOH: H2O, 1:1) = 8 2 0 0 dm3mo 1 1cm 24 U.V. 389nm (MeOH:H2O, 1:1) = 6900 dm3mol1cm 26 Found: 279.125929 2 7 Requi red f or Cl3Hl7NO2 2 7 9.12 7 0 2 9 28 1H NMR (MeOH-d6) 300 MHz 68.69 (d, 2H, Ar-_, J=9 Hz), 29 3.63 (s, 1H, Ar-_), 7.63 (t, 2H, Ar-H, J=8 Hz), 7.49 (t, 2H, Ar-H, 31 J=8 Hz), 5.34 (s, 2H, Ar-CH2), 1 3.88(s, 2H, NC_ 2), 2.70(s, 3H, 2 NCH3) 4 13C NMR(MeOH-d6)75 MHz 633.0, 43.0, 55.0, 63.0, 127.0, 129.0, 131.0, 132.0, 135.0.

7 irvmax(3r): 1625, 3433, 1111, 1396cm 9 m/z(%), (ES): 303(M+Na++H+, 22), 302(M+Na+, 100), 191(20).

12 Example 3: 2-[(9'-anthrylmethyl)(methyl)amino]acetic acid 13 (79) grafted onto Tentagel-S-NHz beads The product 79 was then reacted with 2-methoxyethylamine 16 in the presence of DCC and HOBT used as coupling and 17 activating reagents respectively to give the model 18 compound 82 i.e. a molecule which would mimic the surface 19 of a Tentagel-S-NH2 bead grafted with 79.

o'^'_'N H 21 82 22 W-Visible spectroscopic investigations of 82 24 Absorption spectra were obtained for 82 in MeOH:H2O (50:50, v/v) using aliquots of H3PO4(aq) and NaOH(aq) to 26 vary pH. These spectra can be seen in Figure 3.

1 The absorption spectra seen in Figure 3 show the 2 characteristic anthracene signature. If the spacer 3 component in the molecule is minimizing interaction 4 between the anthracene fluorophore and the amine receptor then according to the PET sensor design, minimal change 6 in absorption spectra would result upon guest 7 complexation. However it is clear from Figure 3 that 8 upon protonation there is a red-shift of 4 nm in the 9 absorption spectra. This can be explained by the fact that the substituted carbon in 82 has a strong electron 11 withdrawing group attached. Internal Charge Transfer 12 (ICT) excited states are set up. The system is now more 13 delocalized, therefore a red-shift is observed.

Figure 3 yields six isosbestic points, 340, 350, 359, 16 368, 378, and 388nm. By using 368nm as an excitation 17 wavelength, fluorescence spectra of 82 were recorded 18 using the same conditions as were used for the absorption 19 measurement (Figure 4). Figure 5 shows the emission-pH profile (at 421nm).

22 This profile clearly shows a switching behaviour with the 23 'on' state at low pH values and the 'off' state at high 24 pH values due to the reversible binding to the receptor preventing PET. The resulting pKa is 5.5, the same value 26 which was observed with the absorption experiments. This 27 result shows that 82 is behaving according to the YES 28 logic operation.

Although the molecule demonstrates an efficient 'off' 31 state of fluorescence with increasing pH values, the 'on' 32 state of fluorescence deviates from what is expected. It 33 appears from Figure 5 that there is a second mode of 1 switching which prevents observation of a plateau. This 2 was investigated by changing the structure of the model 3 compound to 83 which showed the ideal fluorescence 4 behaviour (not shown).

6 Preparation of Nl-benzyl-2-[(9'-anthrylmethyl) 7 (methyl)amino]acetamide (83) 9 Instead of reacting 79 with 2-methoxyethylamine to give 82, benzylamine was used to produce Nl-benzyl-2 11 [9'anthyrlmethyl)(methyl)amino]acetamide 83.

12 83 was excited at 368nm and the change in emission 13 against pH at 421nm can be seen in Figure 6. o g4:N 83

17 79 (0.07 g, 0.25mmol), DCC (0.062g, 0.30mmol) and HOBT 18 (0.038 g, 0.25mmol) were dissolved in CH2C12.

19 Benzylamine (0.028 ml, 0.25mmol) was added and stirred at 0 C for 1 hour. The reaction mixture was then stirred 21 overnight at room temperature. The suspension was 22 filtered and the filtrate evaporated. The residue was 23 partitioned between EtOAc (15 ml) and 5 NaHCO3 (10 ml).

24 The organic layer was washed with NaHCO3 (2x10 ml) and saturated sodium chloride (2x10 ml). It was then dried 26 and the solvent evaporated to producing a yellow oil 27 (Yield: 63).

1 1H NMR (CDCl3)300 MHz 68.35(s, 1H, Ar-_), 8.23(d, 2H, 2 Ar-H, J=8 Hz), 7.92(d, 2H, Ar-H, 3 J=9 Hz), 7.34(m, 2H, Ar-_), 4 7.15(m, 3H, Ar-_), 6.84(m, 2H, Ar-H), 4.49(s, 2H, Ar-CH2), 6 4.09(s, 2H, Ar-_), 3.06(s, 2H, 7 NCH2), 2.40(s, 3H, NC_3).

9 13C NMR(CDCl3)125 MHz 641.5, 43.0, 51.4, 52.3, 123.0, 123.9, 124.9, 125.2, 126.1, 11 126.2, 127.1, 127.8, 128.0, 12 130.1, 132.5, 133.1, 137.0, 13 138.8, 169.7.

irVmax(KBr): 3435, 2929, 2361, 2119, 1651, 16 1262, 1034, 732 cm1.

18 Tentagel-S-NH2beads were treated with 79 by stirring for 19 three hours in DMF with DIC and HOBT. They were then washed successively with DMF, DMF:MeOH and MeOH to remove 21 any physisorbed switch molecule. A set of beads 88 was 22 prepared with 2.5% loading of 79. Higher loadings were 23 also tested. The synthetic scheme can be seen below. 1 0

too of NH2 - DMF/ HOBT/ 9^ooNJb 1 88 3 Fluorescence emission studies of 88 Fluorescence spectroscopic measurements were performed on 6 the beads by washing them with MeOH:H2O (50:50, v/v) 7 solutions and adjusting the pH of the solution with 1M 8 H3PO4 (aq) and lM NaOH (aq), or more dilute solutions if 9 necessary of various pH values.

11 These measurements (Figure 7 and 8) show that a YES gate 12 has been successfully prefabricated and covalently bonded 13 via an amide bond onto Tentagel-S-NH2 bead, 88 where 'on 14 off' switching is observed when protons are the inputs.

The YES truth table is shown below.

16 IN OUT 17 (Hi) (Flue)

O O 1 1

1 Example 4: 2-[9'-anthrylmethyl)(methyl)amino]acetic acid 2 (79) grafting onto Tentagel-S-NHz beads having all free 3 amine groups protected (89) Beads 88 were added to a 50 ml round bottom flask with 6 DMF (20 ml) and stirred for 10 minutes. FMOC (0.067 g, 7 0.225mmol), HOBT (0.34 g, 2.25mmol) and DIC (2.99 ml, 8 19.2mmol) were then added and the reaction vessel was 9 agitated for 3 hours. The beads were washed sequentially with DMF (x2), DMF:MeOH (x2) and MeOH (x2). This 11 procedure was repeated to ensure the reaction of all free 12 amines producing beads 89.

14 One of the reasons suggested for the lowering of the phi value of the switch molecule while anchored onto the 16 bead, was that the initial protonation of the free amines 17 on the beads delayed the protonation of the fluorophore 18 appended receptor, thus lowering the ply value. The 19 beads 88 which were already grafted with 2.5% of 79 were treated exhaustively with FMOC in DMF with DIC and HOBT.

21 The beads 89 were washed with DMF, DMF:MeOH and MeOH.

22 The ninhydrin test was used to ensure all free amines had 23 reacted. Fluorescence spectroscopic properties were then 24 examined.

26 Fluorescence emission studies of 89 28 Fluorescence measurements were carried out using similar 29 conditions and procedures used with previously examined beads. The excitation wavelength was 368nm.

31 Fluorescence spectra are seen in Figure 9 and the 32 emission-pH profile (at 422nm) is presented in Figure 10.

1 Spectral position and shape seen in Figure 9 is the same 2 as that observed in for the unprotected bead. However 3 the pH profile has a few obvious differences. Firstly, 4 the pKa value has increased to 5.5 which is the same value as was obtained for the model compound 82. This 6 confirms the belief that the free amines on the bead are 7 affecting the pKa value of the switch when on the polymer 8 bead. The pH profile also shows the expected 'switching 9 off' action at high pH values although it is not as efficient as that observed with the unprotected beads 88.

11 This reduction of efficiency is because PET is slowed 12 somewhat by the lower polarity imposed by the FMOC groups 13 with their fluorenyl units.

Example 5: 9-Bromomethyl-10-cyanoanthracene (48) grafted 16 onto aminomethyl polystyrene with 10% loading (49) Br NH2 + Toluene as ON 48 49 18 Aminomethyl polystyrene beads (2g) were suspended in 19 toluene (lOml) containing triethylamine (3701). After 30 minutes stirring a solution of 48 (0.05g, 0.18mmol) 21 dissolved in toluene (50ml) was added over 45 minutes.

22 This reaction mixture was refluxed for 2 nights using a 23 Dean-Stark apparatus. The beads were then washed 24 refluxing in acetonitrile overnight followed by successive washings in toluene, THF, THF: MeOH (50:50, 26 v/v) and MeOH.

1 Several different solvents were tried in the 2 derivatisation of the beads and it was found that 3 provided a solvent which will swell the beads is used 4 during synthesis and washing, derivatising the polymer beads with the fluorescent PET switch can be achieved 6 successfully.

8 Example 6: Tentagel-S-NH2 beads having 9-Bromomethyl-10 9 cyanoanthracene (48) grafted at 1% (55) 11 A set of beads 55 was prepared with 1% of grafted amino 12 groups loaded on to the probe molecule 48. Higher 13 loadings were also tested. pH titrations (in MeOH:H2O, 14 50:50 v/v) of this set of beads resulted in the spectra shown in Figure 11 The corresponding emission-pH 16 profiles are presented in Figure 12.

18 Figure 11 shows the expected cyanoanthracene spectral 19 shape as expected, the (0-0) band is relatively intense at 1% loading due to less self-absorption by the local 21 high concentration of fluorophore. Figure 11, shows 22 similar spectral properties to the model compound 51 i.e. 23 it is behaving close to the ideal situation.

The emission-pH profile shows that efficient switching is 26 occurring. Fluorescence intensity is quenched with 27 increasing pH value due to an electron transfer 28 originating from the free amine to the excited 29 fluorophore.

31 The pH profile shows that the pea values for the bead is 32 approximately 5.0. This value is lower than that 33 obtained for the model compound 51 (where the value is 1 6.8) and switching is observed over a broader range (3.5 2 pH values). This can be explained using the same points 3 discussed concerning derivatized aminomethyl polystyrene 4 i.e. hydrophobicity, gradual protonation of fluorophore appended receptors and initial protonation of free amines 6 on the bead making further protonation of fluorophore 7 appended receptors more difficult.

9 Although the derivatised Tentagel-S-NH2 beads 55 are still not behaving exactly like the model compound 51 it 11 is noted that they are closer to ideal behaviour than 12 that obtained with the derivatised aminomethyl 13 polystyrene beads, demonstrating that switching 14 efficiency is increased in more hydrophilic environments.

1 Example 7: 1-(9 -anthrylmethyl)-4-piperidinecarboxylic 2 acid (90) grafted onto Tentagel-S-NH2 beads (93) o

OH

(\o of/\ NH2 DMF/ - HOBT/ DIC O/-\O N 3 93 Fluorescence emission studies of 93 7 Fluorescence measurements were performed on the beads 8 (93) by washing them with MeOH:HO solutions of varying 9 pH value. The same excitation wavelength used for the model compound 92 (368nm) was employed. The fluorescence 11 spectra are seen in Figure 13 and the emission-pH profile 12 (at 420nm) is presented in Figure 14.

14 The pKa value for this polymer-bound switch is 6.3 and switching is occurring over 2.5 pH units. This value is 16 lower than that obtained with the model compound 92 but 17 can be explained using arguments discussed earlier. The 18 most important point is that this pKa value is over 2 pH l9 units higher than that obtained for the beads grafted with 79 at the same loading, hence making the two sets of 1 beads easily distinguishable by measuring fluorescence 2 intensity at different pH values.

4 Example 8: Preparation of Ethyl 2-(methyl)(1' pyrenylmethyl) amino] acetate (97) 7 1-(Bromomethyl)pyrene (0.07g, 0.25mol), sarcosine 8 ethylester hydrochloride (0.04g, 0.25mol), and potassium 9 carbonate (2.5g, anhydrous) were dissolved in CH2C12 and refluxed overnight. The potassium carbonate was filtered 11 of f and the solvent was evaporated of f under reduced 12 pressure yielding a yellow oil (Yield: 72.3%) . 14 lH NMR (CDCl3) 300 MHz 68.49 (d, 1H, Ar-_, J=6 Hz), 7.97-8.05 (m, 4H, Ar-_), 7.83 16 7.89 (m, 4H, Ar-_), 4.24 (s, 2H, 17 Ar-C_ 2), 4.08 (q, 2H, CH3C_ 2, J=4 18 Hz), 3.24 (s, 2H, N-C_ 2), 2.34 (s, 19 3H, N-C_3), 1.16 (t, 3H, CH2C_ 3, J=4 Hz).

22 13C NMR(CDC13)75 MHz 613.0, 41.0, 57.0, 58.0, 59.0, 23 112.8, 113.0, 118.0, 121.0, 24 122.0, 123.0, 123.7, 124.0, 125.0, 126.3, 126.4, 128.0, 26 129 0, 130.0, 130.2, 131.0, 27 170.0.

29 irVmax(KBr): 3041, 2980, 2797, 2360, 1920, 1738, 1457, 1184, 1045, 846, 31 710 cml.

1 m/z(), (CI): 215(75), 332(M+H+, 100), 2 546(17).

4 Example 9: Preparation of 2-[(Methyl)(1'-pyrenylmethyl) amino]acetic acid (94) 7 97 (0.24 g, 0.72mmol) was dissolved in THE (10 ml). NaOH 8 pellets (0.58 g, 0.014mol) were dissolved in H2O:MeOH (10 9 ml:2 ml) and added to the THE solution. The solution was stirred overnight at which point the solvent was 11 evaporated off yielding a yellow solid. This was 12 dissolved in H2O and the solution was neutralized using 13 glacial acetic acid. A yellow precipitate formed which 14 was filtered and washed with water (Yield: 74%, m.p. 187.0-189.0 C).

17 U.V. 327nm, (MeOH:H2O, 1:1)= 30000 dm3mollcm 18 U.V. 352nm, (MeOH:H2O, 1:1)= 44000 dm3mollcm 1H NMR (MeOH-d6)300 MHz 68.73 (d, 1H, Ar-H, J=9 Hz), 21 8.18-8.30(m, 4H, Ar-_), 8.00 22 8.16 (m, 4H, Ar-H), 4.95(s, 2H, 23 Ar-C_2), 4.75(br s, 1H, N-_), 24 3.86(s, 2H, N-CH2, J=4 Hz), 2.95(s, 3H, N-C_3).

27 13C NMR(MeOH-d6)75 MHz 340.0, 57.0, 59.0, 122.0, 123.0, 28 124.0, 124.4, 125.0, 125.5, 29 125.7, 126.2, 127.0, 128.0, 129.0, 129.8, 130.4, 130.5, 31 131.0, 133.0, 169.0. \

1 irvmax(KBr): 3367, 3038, 2361, 1589, 1560, 2 1438, 1414, 1315, 849 cm1.

3 m/z(%), (CI): 215(10), 361(M+, 100), 362(22).

Example 10: Preparation of N1-(2''-methoxyethyl)-2 6 [(methyl)(1'-pyrenylmethyl)amino]acetamide (98) 1 a

NH 0 9 98

11 2-[methyl(1'-pyreneylmethyl)amino]acetic acid (94) 12 (0.064g, 0.21mmol), DCC (0.052g, 0.25mmol), and HOBT 13 (0.032g, 0.21mmol) were dissolved in CH2C12 at 0 C. 2 14 Methoxyethylamine (0.016g, 0.21mmol) was added at 0 C and stirred for 1 hour and then at room temperature 16 overnight. The suspension was filtered and the filtrate 17 evaporated. The resulting yellow oil was dissolved in 18 EtOAc and washed with NaHCO3 (x2) and saturated NaCl.

19 The solvent was evaporated off producing a yellow oil (Yield: 61%).

22 U.V. 314nm, (MeOH:H2O, 1:1)= 13000 dm3mol1cm 23 U.V. 327nm (MeOH:H2O, 1:1)= 30000 dm3mol1cm 24 U.V. 343nm (MeOH:H2O, 1:1)= 43000 dm3mol1cm 26 1H NMR (CDCl3)300 MHz 68.46 (d, 1H, Ar-H, ]=9 Hz), 27 8.15-8.22 (m, 4H, Ar-H), 8.02 \ 1 8.13 (m, 4H, Ar-H), 7.93(br s, 2 1H, N-H), 4.28(s, 2H, Ar-C 2), 3 3.26(t, 2H, NH-CH2, J=5 Hz), 4 3.20(t, 2H, O-CH2, J=6 Hz), 3.17(s, 3H, N-CH2CO), 3.15(s, 6 3H, O-CH3), 2.45(s, 3H, N-CH3).

8 13C NMR(MeOH-d6)75 MHz 25.7, 34.4, 38.9, 44.2, 56.1, 9 71.3, 123.8, 124.9, 125.1, 125.4, 125.6, 125.7, 126.4, 11 127.5, 127.7, 127.8, 128.6, 12 130.2, 131.1, 131.5, 131.6, 13 131.8, 171.2.

i.r. (vmax) her: 3375, 3042, 2929, 2117, 1678, 16 1518, 1124, 847, 711 cml.

18 The spectral properties of 98 were investigated in 19 MeOH:H2O (50:50, v/v) using aliquots of NaOH(aq) and HCl/H3PO4(aq) to vary pH values. The absorption spectra 21 are shown in Figure 15. An absorbance-pH profile can be 22 seen in Figure 16.

24 In Figure 15 the expected distinct vibrational fine structure of pyrene is observed with maximum absorbance 26 at 342, 326 and 313nm. These are red-shifted upon 27 protonation by lnm. This was also observed for the 28 anthracene amino acid model compound, 82. Anthracene and 29 pyrene show delocalisation in the excited state typical of aromatic hydrocarbon fluorophores. However when 31 protonated, the amine substituent distorts the system.

32 The substituted carbon acts as though it has a strong 1 electron-withdrawing group attached. The system behaves 2 to a significant extent like an ICT system. The - 3 electrons in the system are therefore more delocalized 4 which is shown as a small but significant red-shift in the absorption spectra. The loss of the aromatic 6 hydrocarbon -* fluorophore is also evidenced by some 7 reduction of band resolution in favourable cases.

9 Figure 16 shows a pKa value for this molecule of 6.0.

This is a slightly higher pKa than was observed for 82 11 (pKa 5.5) which can be explained with reference to steric 12 inhibition of salvation. When 98 is protonated the 13 charge is easily dispersed due to ease of salvation.

14 However when 82 is protonated steric hindrance from neighbouring peri hydrogens allows less salvation.

16 Therefore charge dispersion is difficult thus lowering 17 the pKa value.

19 Fluorescence Emission Studies of 98 21 Different emission colours were investigated. 326nm (0,1 22 band) was chosen as the excitation wavelength because if 23 there are any anthracene fluorophores in the sample being 24 tested, excitation at this wavelength is where the least interference from anthracene emission is found. This will 26 be an issue when multiple labelling begins. The 27 fluorescence emission spectra obtained, using the same 28 conditions as for the absorption spectra, can be seen in 29 Figure 17. We note that pH-induced absorbance changes of 98 are small at this excitation wavelength. Figure 18 31 shows the emission-pH profile obtained when emission at 32 377nm was plotted against pH. \

1 The pKa value for 98 is 6.0 which is in agreement with 2 the result found from the absorption spectral study.

3 Figure 18 shows that 98 is performing the YES logic 4 operation where fluorescence is high in the presence of protons and low in the absence of protons.

7 This experimental result demonstrates the use of tags 79 8 and 94 for identification of beads whilst attached 9 individually or together. Not only will the colour identify beads but the emission spectral shape observed 11 will also confirm which label is present. Shape can be 12 furthered defined in such structured spectra in terms of 13 relative vibrational band heights (de Silva, A.P., 14 Gunaratne, H.Q.N., Gunnlaugeson, T., Huxley, A.J.M., McCoy, C.P., Rademacher, J.T., Rice, T.E., Chem. Rev., 16 1997, 97, 1515).

1 Example 11: Preparation of 3-(9 -anthryl)propanoic acid 2 (98) grafted onto Tentagel-S-NH2 beads with 10% loading 3 (105)

OOH

: DMF/ HOBT/

DIC

(\0 O/\NH2 0 O N 4 105 Tentagel-S-NH2 beads (2 g, 0.45mmol/g), 100 (0. 0226g, 6 0.09mmol), HOBT (0.014g, 0.9mmol) and DIC (1.2ml, 7 7.68mmol) were stirred in DMF (20ml) for 3 hours. The 8 beads were then washed sequentially in DMF, DMF:MeOH and 9 MeOH.

11 pH titrations were performed on the beads by washing them 12 with different buffer solutions and exciting them at 13 368nm. Figure 19 shows the fluorescence spectra obtained 14 and the emission-pH profile is shown by Figure 20.

16 Figure 20 shows, as expected, that no switching is 17 occurring with varying pH value. 100, whilst grafted 18 onto the polymer beads, to give 105 is performing the 19 PASS 1 logic operation.

IN OUT

3 (H) (Flue) 4 0 1 1 1 8 Example 12: 2-[methyl(1'-pyrenylmethyl)amino] acetic acid 9 (94) grafted onto Tentagel-S-NH2 beads with 2.5% loading (99)

O

(34\0 0/ 3;\NH2 K" J(OH DMF/

O O N go

12 Tentagel-S-NH2 beads (0.5g) were swollen in DMF (20ml) 13 for 10 minutes. DIC (0.075ml, 0.48mmol), HOST (0.0086g, 14 0.056mol) and 94 (0.0017g, 0.0056mol) was added.

16 The reaction mixture was allowed to stir for 3 hours.

17 The beads were then collected by filtering and were 18 washed successively with DMF (x2), DMF:MeOH (x2) and MeOH 19 (x2).

1 The beads 99 were washed with various solutions of 2 MeOH:H2O (50:50, v/v) with different pH values and 3 excited at 326nm. The resulting fluorescence spectra are 4 displayed in Figure 21. Plotting emission (at 378nm) against pH results in the emission-pH profile shown in 6 Figure 22.

8 Figure 22 displays the same spectral shape as was 9 observed with 98. From the emission-pH profile we see that effective switching is observed. The beads are 11 performing the YES logic operation. The pea value is 5.0 12 which is lower than the value obtained for the model 13 compound 98 and the switch is occurring over 2.5 pH 14 units.

16 These results show that by choosing a particular 17 fluorophore, exciting at a known wavelength and observing 18 the emission over a specific range, different emission 19 colours will be potentially easily distinguished. This therefore demonstrates the possibility of this method for 21 coding.

23 Example 13: Preparation of Nq-[2''-methoxyethyl)-3-(99 24 anthryl)propanamide (104)

ON OOH

28 104 100 1 100 (0.084 g, 0.34mmol) was refluxed in 2 2 methoxyethylamine (excess) for 12 hours. Excess amine was 3 removed by distillation under vacuum. The resulting 4 brown tar was dissolved in CH2C12 and washed with NaHCO3.

The organic layer was then dried over sodium sulphate and 6 the solvent removed under vacuum. The tar was then 7 purified using a column (eluent, 9:1, ether:hexane) 8 (Yield: 50%).

1H NMR (CDC13)300 MHz 68.37(s, 1H, Ar-_), 8.30(d, 2H, 11 Ar-_, J=9 Hz), 8.02 (d, 2H, Ar-H, 12 J=83 Hz), 7.53 (t, 2H, Ar-_, J=8 13 Hz), 7.47(t, 2H, Ar-_, J=7 Hz) , 14 5.60 (br s, 1H, NH), 4.00 (t, 2H, Ar-C_ 2, J=8 Hz), 3.34 (t, 2H, 16 OCH2, J=5 Hz), 3.26 (t, 2H, NC_ 2, 17 J=5 Hz), 3.21 (s, 3H, OCH3) , 18 2.66 (t, 2H, Ar-CH2C_ 2, J=7 Hz) . 19 13C NMR(CDCl3)75 MHz 625.0, 26.0, 35.0, 56.0, 57.0, 124.0, 125.0, 126.0, 128.0, 21 129.0, 130.0, 131.0, 132.0, 22 140.0.

24 irvmax(KiBr): 3445, 2943, 2361, 1639, 1558, 1457, 1123, 729cml.

27 The spectral properties of 104 were investigated in 28 MeOH:H2O (50:50, v/v) using aliquots of NaOH(aq) and 29 H3PO4 (aq) to vary pH values. The absorption spectra obtained are shown by Figure 23. Maximum wavelengths can 31 be seen at 349, 368 and 387nm. Figure 24 shows a plot of 32 absorbance, at 368nm, against pH. 1 104 is a monosubstituted anthracene molecule just as in 2 82. The

expected anthracene spectra are observed and the 3 main bands are in similar positions as those seen with 4 82. It is noticeable from these figures that only small changes in absorbance intensity is observed with 6 increasing pH value. The band positions remain constant.

8 Fluorescence emission studies of 104 Fluorescence emission studies were then performed using 11 the same conditions as outlined for the absorption 12 experiment. The excitation wavelength chosen was 368nm.

13 This is the same wavelength as was chosen to excite 14 molecule 82. Fluorescence spectra are shown in Figure 25 and the emission (at 421nm)-pH profile is in Figure 26.

17 Figure 26 clearly shows that no switching behaviour is 18 observed with pH. 104 is performing the PASS 1 logic 19 operation (See Figure 70).

21 Example 14: Preparation of Nn-(2-methoxyethyl)-4-(l' 22 pyrenyl) butanamide (106)

OH NO O\

95 106 1 95 (0.5 g, 0.0017mol), DCC (0.42 g, 0.002Imol) and HOBT 2 (0.26 g, 0.0017mol) were dissolved in CH2C12 at 0 C. 2 3 Methoxyethylamine (0.15 ml, 0.0017mol) was added at 0 C 4 and stirred for 1 hour and then at room temperature overnight. The suspension was filtered and the filtrate 6 evaporated. The resulting yellow oil was dissolved in 7 EtOAc and washed with NaHCO3 (X 2) and saturated NaCl.

8 The solvent was evaporated producing a yellow solid 9 (Yield: 71\, m.p. 123.0-124.0 C).

11 U.V. 325nm (MeOH:H2O, 1:1)= 27000 dm3mol1cm 12 U.V. 341nm, (MeOH:H2O, 1:1)= 41000 dm3mol1cml 13 1H NMR (CDC13)300 MHz 68.33(d, 1H, Ar-_, J=7 Hz), 14 8.15-8.18(m, 4H, Ar-_), 8.02 8.06 (m, 3H, Ar-H), 7.98 (d, 1H, 16 Ar-_, J=8 Hz), 5.70(br s, 1H, N 17 H), 3.37-3.48 (m, 4H, O-CH2C_2), 18 3.33(s, 3H, O-C_3), 2.30-2.21 19 (m, 6H, Ar-(C 2)3).

13C NMR(MeOH-d6)75 MHz 632.7, 39.2, 36.0, 39.2, 58.7, 21 71.2, 123.0, 124.7, 124.8, 22 124.9, 125.0, 125.1, 126.0, 23 126.7, 127.4, 128.0, 129.0, 24 130.0, 131.0, 131.4, 136.0, 172.6.

27 irvmax(KlBr): 3446, 3289, 2927, 2361, 1647, 28 1557, 1458, 1125, 840, 582 cm1.

m/z(), (ES): 368(M+Na+, 30), 346(M+H+, 5), 31 282 (100). \

1 The spectral properties of 106 were studied by observing 2 the change in absorption spectra with varying pH value.

3 These measurements were carried out in MeOH:H2O (50:50, 4 v/v) using aliquots of NaOH(aq) and H3PO4(aq) to vary pH.

* The absorption spectra are shown in Figure 27 and the 6 absorbance-pH profile is seen in Figure 28.

8 Figure 27 shows the same spectral signature as 98, i.e. 9 three distinct vibrational bands at 341, 326, and 312 nm which is expected as 106 is a mono-substituted pyrene 11 compound just like 98. The absorbance maximum is at the 12 0-0 band (341nm), again because of the pyrene 0-0 band's 13 dependence on solvent polarity. The pH profile in Figure 14 28 shows very little change in absorbance with pH.

Fluorescence measurements were then performed on 106 16 using the same conditions as outlined for the absorption 17 experiment, resulting in spectra shown in Figure 29. The 18 excitation wavelength used was 326nm. The emission (at 19 377nm)-pH profile can be seen in Figure 30.

1 Example 15: Preparation of 95 grafted onto Tentagel-S-NH2 2 beads with 2.5% loading (107) o

POOH

(40 0/\ NH2 os' DMF/ W I HOBT/ DIC 0 O N 3 107 Tentagel-S-NH2 beads (0.5g) were swollen in DMF (20ml) 6 for 10 minutes. DIC (0.075ml, 0.48mmol), HOBT (0.0086 g, 7 0.056mmol) and 95 (0.0016g, 0.0056mmol) were added. The 8 reaction mixture was allowed to stir for 3 hours. The 9 beads were then collected by filtering and were washed successively with DMF(x2), DMF:MeOH (x2) and MeOH (x2).

12 The beads 107 were washed with solutions of different pH 13 values and excited at 326nm. The fluorescence spectra 14 obtained are shown in Figure 31 and the emission-pH profile (at 422nm) is presented in Figure 32.

17 These figures demonstrate that 107 is working effectively 18 as a PASS 1 logic gate with fluorescence remaining high 19 at both high and low pH values just as in the model compound 106.

1 Example 16: Preparation of double labeled Tentagel- S-NH2 2 beads (108) with 2-[(9'-anthryl-methyl)(methyl)-amino] 3 acetic acid (79) and 3-(9'-anthryl) propanoic acid (100)

KNIN 4 108

6 Tentagel-S-NH2 beads (0.5g) were swollen in DMF (20ml) 7 for 10 minutes. DIC (0.15ml, 0.96mmol), HOBT (0.017g, 8 0.112mmol) and 79 (0.0031g, 0.0112mmol) were added. The 9 reaction mixture was allowed to stir for 3 hours. The beads were then collected by filtering and washed with 11 DMF(x2). This set of beads were then stirred in DMF 12 (20ml) with DIC (0.15ml, o.g6mmol), HOBT (0.017g, 13 0.112mmol) and 100 (0.0028g, 0.0112mmol). The beads were 14 then collected by filtering and were washed successively with DMF (x2), DMF:MeOH (x2) and MeOH (x2). Beads with 16 different ratios of 79 and 100 were prepared using this 17 procedure.

19 Fluorescence measurements were performed on the beads in MeOH:H2O (50:50, v/v) using NaOH(aq) and H3PO4(aq) to vary 21 the pH value. The excitation wavelength used was 368nm 22 as this is where the individually labelled beads were 23 excited. Fluorescence spectra are shown in Figure 33 and 24 the emission-pH profile is presented in Figure 34. A small wavelength shift may be noticed in Figure 33 which 1 is due to the slightly different emission spectral 2 signatures of the fluorophores in the two different logic 3 gates. Such a small deviation from ideal behaviour is 4 not unexpected due to the short spacer within the YES gate.

7 The relatively low intensity of the 0-0 band at 403nm, 8 seen in Figure 34 is similar to that observed for the 9 individually labelled beads. This is due to the relatively high total fluorophore loading, 10%. It is 11 clear from these figures that in the fully protonated 12 state, fluorescence intensity is reaching a maximum.

13 With increasing pH values the fluorescence is quenched 14 but only reaching half of the initial fluorescence intensity. Fluorescence is still observed at this level 16 even at pH 10.5. This result shows that two logic 17 operations are occurring in parallel. When only 100 is 18 grafted onto the bead the fluorescence intensity remains 19 high at both low and high pH values i.e. PASS 1 logic.

When 79 is grafted onto the bead alone, fluorescence 21 intensity reduces to near zero at high pH values i.e. YES 22 logic. With beads 108, the two logic operations are 23 combining together with equal weighting causing the ratio 24 of fluorescence at low pH values to that of high pH values to be close to 2:1, shown in Table 1. This result 26 shows that energy migration is not critical in basic 27 conditions. The apparent pKa value 4.4 is close to the 28 expected value of 4.8. The electronic representation of 29 the logic situation is shown in Figure 71. The symbol is a reminder that a summed output from the two gates is 31 observed.

Table 1

Input Output (PASS 1) Output (YES) Total Output 3 _ e men Fluorescence Fluorescence 4 This experiment shows that combining logics in controlled concentrations is easily accomplished. Therefore beads 6 which display a total fluorescence output with ratio 3:2 7 at low to high pH values should be easily obtained by 8 grafting the bead with 2.5 of the YES switch 79 and 5% 9 of the PASS 1 switch 100, to form double labelled Tentagel-S-NH2 beads (109).

12 A set of beads were prepared with the abovementioned 13 concentrations using procedures described previously.

14 Fluorescence measurements were performed on the beads 109 by exciting at 368nm at chosen pH values. Fluorescence 16 spectra displayed in Figure 35 were obtained. The 17 emission-pH profile is shown in Figure 36.

19 Figures 35 and 36 show that in the fully protonated state the fluorescence intensity is a maximum, even though the 21 plateau is not fully defined in this case. With 22 increasing pH values fluorescence is being quenched but 23 only by close to 1/3 of its initial value. Therefore the 24 ratio of fluorescence intensity at low pH values to that at high pH values is nearly 3:2, as shown in the logic 26 Table below (Table 2). The pea value is 3.5 which 27 deviates somewhat from the expected value of 4.8 in this 28 instance. The electronic representation for the combined 29 logic operation is shown in Figure 72.

1 Table 2

Input Output (PASS Output (YES) Total Output (H) 1) Fluorescence Fluorescence Fluorescence

_

4 The final combination of this type to be examined was where Tentagel-SNH2 beads were grafted with 5% YES 6 switch 79 and 2.5% PASS 1 switch 100, to form double 7 labelled Tentagel-S-NH2 beads (110). Fluorescence 8 measurements were performed on these beads 110 using 9 conditions discussed previously resulting in the spectra displayed in Figure 37. The emission-pH profile is 11 presented in Figure 38.

13 From this set of results it is clear that fluorescent 14 switches 79 and 100 are again operating in parallel whilst grafted onto the polymer bead together. This gives 16 a ratio of close to 3:1 of fluorescence intensity at low 17 pH values to that of high pH values, shown in the logic 18 table below (Table 3). The electronic representation for 19 the combined logic situation is shown in Figure 73.

21 Table 3

Input Output (PASS Output (YES) Total Output (H) 1) Fluorescence Fluorescence Fluorescence 1 A summary of all the combinations of fluorescent logic 2 switches 79 and 100 grafted onto Tentagel-S-NH2beads, 3 which were studied can be seen in the logic table below

4 (Table 4).

6 Table 4

Predicted Experimental ratio of ratio of YES PASS 1 fluorescence fluorescence (% loading) (% loading) intensity at intensity at Low pH:High pH Low pH:High 1 (5 %) 1 (5 %) 2:1 2.3:1 2 (5 %) 1 (2.5 %) 3 1 - 3.5:1 1 (2.5 %) 2 (5 %) 3:2 3.1:2 1 (2.5 %) 1:1 1 15:1 0 1 (2.5 %) 1:1 0.95:1 9 Summary of all combinations of 79 and 100 on Tentagel-S NH2 beads studied.

12 This set of experiments show that Tentagel-S-NH2 beads 13 can be labelled with different ratios of the fluorescent 14 logic gates 79 and 100 and can be easily distinguished by comparing their fluorescence intensity at both low and 16 high pH values. As can be seen in the logic table above 17 the experimental ratio is close to the predicted ratio.

19 Only five different ratios of switch molecules grafted onto the bead have been examined but the close comparison 21 of experimental ratios with predicted ratios suggests 22 that various different ratios will also be 1 distinguishable. Therefore a near infinite variety of 2 distinguishable ratios can be identified provided the 3 experimental error of the reading instrument for 4 intensity ratios is borne in mind. It can therefore be said that this method of coding will be very valuable.

7 The fluorescence quantum yields of the two gates can 8 alter slightly due to polarity effects on the excited 9 state of the YES gate with some ICT character as well as shielding from oxygen quenching. Hydrophobicity control 11 of location of fluorescent sensors has been observed 12 before. Another more trivial contributor to small 13 discrepancies is that the two gates have slightly 14 different spectral signatures. The chosen monitoring wavelength will then naturally have a small bias for one 16 gate or the other (except at the isoemissive point).

18 Example 17: Preparation of double labelling Tentagel-S 19 NH2 beads (111) with 2-[methyl(1'-pyrenylmethyl)amino] (94) and 1-pyrenebutyric acid (95) o /NN 22 (111) 24 Tentagel-S-NH2 beads (0.5g) were swollen in DMF (20ml) for 10 minutes. DIC (0.075ml, 0.48mmol), HOBT (0.0086g, 26 0.056mol) and 94 (0.0017 g, 0.0056mol) were added. The 1 reaction mixture was allowed to stir for 3 hours. The 2 beads were then collected by filtering and washed with 3 DMF (x2). This set of beads were then stirred in DMF (20 4 ml) containing DIC (0.075 ml, 0.48mmol), HOBT (0.0086 g, 0.056mmol) and 95 (0.0016g, 0.0056mmol). The reaction 6 mixture was allowed to stir for 3 hours. The beads were 7 then collected by filtering and were washed successively 8 with DMF(X2), DMF:MeOH (x2) and MeOH (x2). Other double 9 labelled beads were prepared using a similar procedure.

11 Fluorescence measurements were performed on the beads in 12 MeOH:H2O (50:50, v/v) using NaOH(aq) and H3PO4(aq) to vary 13 the pH value. The excitation wavelength used was 326nm 14 as this is where the individually labelled beads were excited. Figure 39 shows the fluorescence spectra 16 obtained and Figure 40 shows the emission-pH profile.

17 These figures show that just like the situation where 18 Tentagel-S-NH2 beads were grafted with different 19 concentrations of switch molecules containing anthracene fluorophores and the ratio of fluorescence at high and 21 low pH values was predictable the same can be done for a 22 bead labelled with different logic gates with pyrene as 23 the fluorophore. Figure 40 clearly shows that with 24 increasing pH value fluorescence intensity is being quenched until virtually half its initial value. This is 26 what would have been expected as both logic gates are 27 grafted onto the bead with equal concentrations. The 28 logic table shown below (Table 5) shows the resulting 29 ideal ratio of fluorescence intensity obtained at high and low pH value.

1 Table 5

Input Output (PASS 1) Output (YES) Total Output I (H Fluorescence Fluorescence Fluorescence 3 1 1 1 1 2 4 An example where the switch molecules 94 (YES) and 95 (PASS 1) have been grafted onto the Tentagel-S-NH2 beads 6 with unequal loadings has also been examined. In this 7 case 5% of 95 and 2. 5 of 94 were grafted onto the 8 Tentagel-S-NH2 beads. Fluorescence spectra are displayed 9 in Figure 41 and the emission-pH profile is shown in Figure 42.

12 Figure 42 shows a ratio of virtually 3:2 of fluorescence 13 intensity at low to high pH value. This could have been 14 predicted due to the presence of twice as much of the PASS 1 logic gate as the YES logic gate.

17 Table 6

InputOutput (PASS Output (YES) Total Output (H)1) Fluorescence Fluorescence L:FluoreM Hence Combined logic for parallel PASS 1: YES (2:1) with pyrene 21 f1uorophores. 22 -

23 It is important to discuss at this stage that when logic 24 gates with similar fluorophores are operating on the bead 1 in parallel their total fluorescence outputs are summed 2 and treated according to many-valued logic. The outputs 3 are no longer binary numbers. Conventional computers are 4 unable to cope with many-valued logic. The main reasons are; 6 (i) data is transmitted between points within or 7 outside the processsor so noise induced corruption is a 8 problem, 9 (ii) while a computer is processing information a series of operations are occurring. Within each 11 operation errors may occur. These errors propagate.

13 Binary form is therefore the safest for computers to 14 avoid data corruption. However our molecular computational elements are designed to encode or identify 16 numbers of large populations. Multi-valued logic output 17 is not a problem in this application since computation is 18 a single step with no transmission along noisy lines.

Example 18: Preparation of Ethyl 3-(9'-anthryl)-2-(2- 21 pyridyl)propanoate (117)

N

23 117 24 Ethyl-2-pyridyl acetate (0.19ml, 1.22mmol) was dissolved in anhydrous THE (15ml) and cooled down to -78 C using 26 dry ice and acetone. This solution was allowed to stir \ 1 for 15 minutes. Sodium bis (trimethyl-silyl)amide, in 2M 2 THF (3.37ml, 6.74mmol) was added dropwise and the 3 solution allowed to stir at -78 C for 1 hour. 9 4 bromomethylanthracene (0.27g, lmmol) dissolved in anhydrous THF (lOml) was added dropwise using a cannula.

6 This was allowed to stir overnight. NH4Claq, (lOml, 7 saturated) was added slowly to quench the reaction. The 8 organic layer was separated, washed with 1M HCl and 9 NaHCO3 (saturated) and the solvent removed yielding a yellow oil (Yield: 53%).

12 lH NMR (CDCl3)300 MHz 68.62 (d, TH, Ar-H, J=5 Hz), 13 8.30(s, 1H, Ar-H), 8.13(d, 2H, 14 Ar-_, J=9 Hz), 7.93(d, 2H, Ar-_, J=8 Hz), 7.83(m, 5H, Ar-_), 16 7.06(t, 1H, Ar-_, J=6 Hz), 17 6.75(d, 1H, Ar-_, J=8 Hz), 18 4.50(t, 1H, Ar-C_, J=10 Hz), 19 4.27(q, 2H, OCH2CH3, J=9 Hz), 4.03(m, 2H, Ar-C_2CH), 0.98(t, 21 3H, OCH2CH3, J=7 Hz).

23 l3C NMR(CDCl3)125 MHz 614.3, 30.6, 55.2, 61.5, 122.6, 24 123.9, 124.9, 125.2, 126.0, 127.0, 129.5, 130.5, 131.6, 26 131.8, 136.8, 149.9, 158.6, 27 173.1.

29 irvmax(KBr): 3435, 3050, 2361, 1584, 1388, 726 cm1.

1 m/z(%), (ES): 356(M+H+, 100), 378(M+Na+, 10), 2 711(5), 733(15).

4 Example 19: Preparation of 3-(9'-Anthryl)-2-(2''-pyridyl) propanoic acid (115) 7 117 (0.12g, 0.0014mol) was dissolved in THF (lOml). NaOH 8 (0.26g, 0.0067mol) was dissolved in H2O:MeOH (lOml:2ml) 9 and added to the THE solution. The solution was stirred overnight at which point the solvent was evaporated off 11 yielding a yellow solid. The solid was dissolved in H2O 12 and the solution was neutralized using glacial acetic 13 acid. The resulting yellow precipitate was filtered and 14 washed with water (Yield: 73%).

16 1H NMR (CDC13)300 MHz 68.54 (d, 1H, Ar-H, J=5 Hz), 17 8.35(s, 1H, Ar-H), 7.95(m, 4H, 18 Ar-_), 7.48(m, 4H, Ar-H), 19 7.08(t, 1H, Ar-_, J=6 Hz), 6.97(t, 1H, Ar-H, J=7 Hz), 21 5.75(d, 1H, Ar-_, J=8 Hz), 22 4.50(m, 1H, Ar-C_CH2), 4.22(m, 23 2H, Ar-CH2CH).

13C NMR(CDC13)75 MHz 634.7, 52.8, 123.2, 123.9, 26 125.2, 126.0, 126.5, 127.6, 27 129.6, 130.5, 131.6, 132.0, 28 138.0, 147.0, 157.2, 174.1.

irVmax(KBr): 3434, 3051, 2925, 2362, 1943, 31 1718, 1591, 1158, 88, 733 cml.

1 m/z(%), (ES): 328(M+H+, 100), 350(M+Na+, 48), 2 655(10), 677(20).

4 Example 20: 3-(9'-Anthryl)-2-(2''-pyridyl)propanoic acid (115) grafted onto Tentagel-S-NH2 beads with 2.5% loading 6 (119) q (\0 0/\ NH + gal DMF/ HOBT/ :\0 OWN

H

8 Tentagel-S-NH2 beads (0.5g) were swollen in DMF (20ml) 9 for 10 minutes. DIC (0.075ml, 0.48mmol), HOBT (0.0086g, 0.056mmol) and 115 (0.0021g, 0.0056mmol) were added. The 11 reaction mixture was allowed to stir for 3 hours. The 12 beads were then collected by filtering and were washed 13 successively with DMF (x2), DMF:MeOH (x2) and MeOH (x2).

Fluorescence measurements were performed on 119 by 16 washing the beads with MeOH:H2O (50:50, v/v) solutions of 1 chosen pH values where NaOH(aq) and H3PO4(aq) were used 2 to vary the pH value. The excitation wavelength used was 3 370nm, the same wavelength as was used for 118. The 4 fluorescence spectra obtained are shown in Figure 43 and the emission- pH profile is displayed in Figure 44.

6 These results show that fluorescence is switched off at 7 low pa values and fluorescence enhancement is observed 8 with increasing pH value. This shows that the NOT logic 9 behaviour is still detected when 115 is grafted onto Tentagel-S-NH2 beads. The pKa value is 2.2, as obtained 11 from figure 44. This value is lower than the value of 12 4.0 obtained for the model compound 118. This shift in 13 ply value has also been observed for the other switch 14 molecules, which have been studied, whilst grafted onto Tentagel-S-NH2 beads and is explained by the hydrophobic 16 environment of the switch molecules and gradual 17 protonation of fluorophore-appended receptors. However 18 these results do show that the NOT logic gate can be 19 synthesized and successfully grafted onto Tentagel-S-NH2 beads.

IN OUT

(H) (Flue) 0 1 26 1 0 29 Example 21: Preparation of Nl-(2'''-methoxyethyl)-3-(9- anthryl)-2-(2''-pyridyl)propanamide (118) 32 115 (0.03g, 0.4mmol), DCC (0.023g, 0.1mmol), and HOBT 33 (0.014g, 0.01mmol) were dissolved in CH2C12 at 0 C. 2 1 Methoxyethylamine (0.OOlml, 0.08mmol) was added at 0 C 2 and stirred for 1 hour and then at room temperature 3 overnight. The suspension was filtered and the filtrate 4 evaporated. The resulting yellow oil was dissolved in EtOAc and washed with NaHCO3 (x2) and saturated NaCl.

6 The solvent was evaporated producing a yellow oil 7 (Yield: 73%).

8 1H NMR (CDCl3) 300 MHz 68.55 (d, 1H, Ar-_, J=5 Hz), 9 8.28(s, 1H, Ar-_), 7.91(m, 4H, Ar-H), 7.37 (m, 4H, Ar-_), 11 7.22 (t, 1H, Ar-_, J=4 Hz), 12 7.03 (t, 1H, Ar-H, J=6 Hz), 13 6.66 (d, 1H, Ar-H, J=8 Hz), 14 4.47 (m, 1H, Ar-CHCH2), 4.14 (m, 2H, Ar-CHC_ 2), 3.29 (m, 7H, 16 C_ 2 CH2 OC_3) 18 13C NMR(CDCl3)125 MHz 833.3, 34.0, 34.9, 58.7, 71.1, 19 122.1, 124.3, 124.6, 124.8, 126.6, 127.2, 129.0, 130.1, 21 131.3, 134.1, 136.5, 149.0, 22 158.7, 172.3.

24 irvmax(KBr): 3328, 2930, 2116, 1663, 1570, 1126, 908, 732 cm1.

27 The spectral properties of 118 were investigated in a 28 MeOH:H2O (50:50, v/v) solution using aliquots of NaOH(aq) 29 and HCl (aq) to vary pH. Figure 45 displays the influence of pH on the absorption spectra. The absorbance-pH 31 profile is shown by Figure 46. \

1 Figure 45 displays a typical anthracene signature with 2 the main band positions in basic solution at 350, 368 and 3 388nm. Upon protonation these bands are red-shifted by 4 lnm. This small shift which, is not expected according to the principle of PET sensor design, can be attributed 6 to the formation of an ICT excited state of the 7 fluorophore due to a perturbation across the dimethylene 8 spacer by the charge on the pyridinium unit. Figure 46 9 shows that the pea value for 118 is 4.0. It also shows that only small changes in absorption intensity are 11 observed with varying pH value.

13 Fluorescence measurements were then performed on 118 14 using the same conditions as outlined for the absorption spectroscopic experiments. The excitation wavelength was 16 370nm. The fluorescence spectra obtained are presented 17 in Figure 47. The emission-pH profile is shown in Figure 18 48 displaying a ply value of 4.0 for 118. This is 19 clearly the NOT logic operation.

21 Example 22: Preparation or Ethyl 3-[(2' 22 pyridylmethyl)amino]propanoate (127) 26 N 29 127 Ethyl-3- bromopropionate (0.56g, 0.003mol) was dissolved 31 in toluene (30ml). 2-aminomethylpyridine (1.58ml, 32 0.015mol) was added and allowed to stir overnight. The 33 solution was filtered and extracted with 0.1M NaHCO3. \

1 The organic phase was dried over Na2SO4 and the solvent 2 removed yielding a clear oil (Yield: 86).

4 1H NMR (CDCl3)300 MHz 88.57 (d, 1H, Ar-_, J=6 Hz), 7.62 (t, 1H, Ar-_, J=8 Hz), 6 7.31 (d, 1H, Ar-_, J=8 Hz), 7 7.13(t, 1H, Ar-_, J=6 Hz), 8 4.12(q, 2H, OC_2CH3, J=7 Hz), 9 3.92(s, 2H, Ar-C_ 2), 2.93 (t, 2H, NCH2C_2, J=6 Hz), 2.54 (t, 2H, 11 NC_2CH2, J=7 Hz), 1.24 (t, 3H, 12 OCH2C_3, J=7 Hz).

14 13C NMR(CDC13)75 MHz 614.3, 32.7, 45.0, 55.0, 60.0, 122.0, 128.0, 137.0, 149.0, 16 156.0, 172.0.

18 irvmax(KBr): 3398, 2982, 1729, 1593, 1444, 19 1183, 763 cml.

21 Example 23: Preparation of Ethyl 3-[(9' 22 anthrylmethyl)(2''-pyridylmethyl)amino]propanoate (128)

N

26 N. O 28 O. / 1 127 (0.26g, 0.0012mol), 9-bromomethylanthracene (0.34g, 2 0.0012mol) and K2CO3 (2g, anhydrous) were refluxed 3 overnight in CH2C12 (3Oml). The solution was allowed to 4 cool, K2CO3 was filtered off and the solvent was removed giving a yellow oil. After loading onto a column of 6 flash silica the product, a yellow solid, was eluted with 7 a ether: hexane mixture in the ratio 90:10 v/v (Yield: 8 52%, m.p. 111.1-111.3 C).

9 1H NMR (CDCl3)300 MHz 68.46(m, 4H, Ar-_), 7.97(d, 2H, Ar-H, J=7 Hz), 7. 50(m, 5H, Ar 11 H), 7.24(d, 1H, Ar-_, J=7 Hz), 12 7.06(m, 1H, Ar-H), 4.63(s, 2H, 13 Ar-CH2), 3.86(q, 2H, OCH2, J=7 14 Hz), 3.81(s, 2H, Ar(py)-CH2), 3.01(t, 2H, NCH2C_ 2, J=7 Hz), 16 2.58(t, 2H, NCH2CH2, J=7 Hz), 17 1.00(t, 3H, OCH2C_ 3, J=7 Hz).

18 13C NMR(CDCl3)75 MHz 814.4, 33.4, 50.9, 51.4, 60.2, 19 60.7, 122.2, 123.8, 125.2, 125.6, 125.7, 128.1, 129.4, 21 130.0, 131.7, 131.8, 136.5, 22 148.8, 160.4, 172.8.

23 irvmax(KBr): 3437, 2921, 1732, 1589, 1320, 24 1181, 1027, 758, 733 cm1.

m/z(%), (ES): 421(M+Na+, 20), 399(M+H+, 100).

27 Example 24: Preparation of 3-[(9'-Anthrylmethyl)(2', 28 pyridylmethyl)amino]propanoic acid (120) 128 (0.55g, 0.0014mol) was dissolved in THF (20ml). NaOH 31 (0.55g, 0.014mol) was dissolved in H2O:MeOH (20ml:4ml) 32 and added to the THF solution. The solution was stirred \ 1 overnight at which point the solvent was evaporated off 2 yielding a yellow solid. The solid was dissolved in H2O 3 and the solution was neutralised using glacial acetic 4 acid. The resulting yellow precipitate was filtered and washed with water (Yield: 78%, m.p. 170.1-170.4 C).

NH2CH2CI2 PNo + Br O K2CO3:gN CH2C12 rB, K2CO3, N NaOH N gO OH THFlMeoH/ 128 7 1H NMR (CDCl3)300 MHz 68.47(d, 1H, Ar-H), 8.39(s, 1H, 8 Ar-H), 8.19(d, 2H, Ar-_, J=8 9 Hz), 7.96(d, 2H, Ar-_, J=8 Hz), 7.48(m, 5H, Ar-_), 7.14(m, 1H, 11 Ar-H), 6.88(d, 1H, Ar-_, J=8 12 Hz), 4.77(s, 2H, Ar-CH2), 13 3.96(s, 2H, -Ar(py)-C_ 2) 14 3.09(t, 2H, NCH2C_ 2, J=6 Hz), 2.55(t, 2H, NCH2CH2, J=6 Hz).

1 13C NMR(CDCl3)125 MHz 632.1, 50.3, 51.1, 58.3, 122.5, 2 123.4, 124.1, 125.0, 126.4, 3 127.0, 128.6, 129.2, 131.3, 4 131.4, 136.8, 14.7, 156.9, 173.7.

6 irvmax(KBr): 3436, 1591, 1384, 1189, 776, 7 735, 602 cm1.

9 m/z(%), (ES): 371(M+H+, 100), 339(95).

11 Example 25: 3-[(9'-Anthrylmethyl)(2'' 12 pyridylmthyl)amino]propanoic acid (120) grafted onto 13 Tentagel-S-NH2 beads with 2.5% loading (131) W\0 O/\NH2 +

ON O

l HOBT/ (in\ H Tentagel-S-NH2 beads (0.5g) were swollen in DMF (20ml) 16 for 10 minutes. DIC (0.075ml, 0.48mmol), HOBT (0.0086g, 17 0.056mmol) and 120 (0.0018g, 0.0056mmol) were added. j

1 The reaction mixture was allowed to stir for 3 hours.

2 The beads were then collected by filtering and were 3 washed successively with DMF (x2), DMF:MeOH (x2) and MeOH 4 (x2).

6 Fluorescence measurements were performed on the beads by 7 washing them with solutions of MeOH:H2O (50:50, v/v) of 8 chosen pH values. The excitation wavelength used was 9 370nm. Figure 49 presents the fluorescence spectra obtained and the emission-pH profile is shown in Figure 11 50.

13 Figure 50 shows a bell-shape curve. Although not ideal, 14 fluorescence is switching 'off' at both high and low pH value which shows that the 'off-on-off' switching action 16 is occurring when 120 is grafted onto Tentagel-S-NH2 17 beads. A more detailed discussion and the logic table 18 are presented under Example 26. The pKa values are 4.6 19 and 0.5 (from calculated pH values). This shift in pKa value i.e. compared to that of the model compound 129 21 (see next example), is in agreement with previously 22 discovered results. However it is visible from Figure 50 23 that the quenching at both high and low pH values is 24 incomplete. This is again probably due to the lower polarity of the switch microenvironment when it is 26 connected to the polymer beads. Also, as incomplete 27 switching was observed in the model compound 129 this 28 effect is also expected when the tag is attached onto the 29 polymer beads.

1 Example 26: Preparation of Nq-(2'''-methoxyethyl)-3-[(9- 2 anthrylmethyl)(2''-pyridylmethyl)amino] propanamide (129) N rO

O 129

7 120 (0.148g, 0.4mmol), DCC (0.099g, 0.48mmol), and HOBT 8 (0.06g, 0.15mmol) were dissolved in CH2C12 at 0 C. 2 9 Methoxyethylamine (0.032ml, 0.36mmol) was added at 0 C and stirred for 1 hour and then at room temperature 11 overnight. The suspension was filtered and the filtrate 12 evaporated. The resulting yellow oil was dissolved in 13 EtOAc and washed with NaHCO3 (x2) and saturated NaCl.

14 The solvent was evaporated producing a yellow oil (Yield: 711).

17 1H NMR (CDC13)500 MHz 68.57(d, 1H, Ar-H, J=5 Hz), 18 8.38(s, 1H, Ar-H), 8.19(d, 2H, 19 Ar-H, J=9 Hz), 7.97(d, 2H, Ar-H, J=10 Hz), 7.63(m, 1H, Ar-_), 21 7.44(m, 4H, Ar-H), 7.26(d, 1H, 22 Ar-H, J=8 Hz), 7.19(m, 1H, Ar 23 H), 4.60(s, 2H, Ar-CH2), 3.93(s, 24 2H, Ar(py)-C_ 2), 3.23(s, 3H, OCH3), 3.02(t, 2H, NCH2C_2C, J=6 26 Hz), 2.87(t, 2H, NCH2CH2O, J=6 27 Hz), 2.84(t, 2H, NCH2CH2O, J=6 i 1 Hz), 2.38(t, 2H, NC_2CH2C, J=6 2 Hz).

4 13C NMR(CDCl3)75 MHz 632.6, 34.9, 50.3, 51.0, 58.5, 60.4, 70.9, 121.9, 123.7, 124.4, 6 124.9, 126.1, 127.2, 128.0, 7 130.0, 132.2, 132.4, 137.0, 8 150.0, 159.0, 172.1.

irvmax(KBr): 3421, 2930, 2117, 1653, 1558, 11 1448, 1122, 889, 734 cml.

13 m/z(%), (CI): 192(5), 428(M+H+, 100), 429(27). The spectral properties of 129 were investigated in a 16 MeOH:H2O (50:50,

v/v) solution using aliquots of NaOH(aq) 17 and HCl(aq) to vary pH which was measured directly using 18 a glass electrode. Figure 51 shows the absorption 19 spectra obtained and the absorbance-pH profile is shown by Figure 52.

22 Figure 51 displays a series of anthracene absorption 23 spectra with maximum absorbance at 349, 367 and 387 nm in 24 neutral solution. This figure also shows that upon protonation a red- shift of 4nm is occurring. This is 26 explained by the fact that the substituted carbon, in 27 129, acts as though it has a strong electron withdrawing 28 group attached when it is protonated. Internal Charge 29 Transfer (ICT) excited states are therefore set up. The system is now more delocalized, therefore a red-shift is 31 observed. It also visible that upon protonation the band 32 shape is less distinct and the absorbance intensity is 33 decreased by a significant amount in comparison to the 1 other mono-substituted anthracene compounds studied.

2 This may be due to - stacking induced by hydrogen 3 bonding of the pyridine nitrogen to a water molecule as 4 shown by 130. This produces rigidification in 129 by the formation of a seven membered ring and allows the 6 electron-accepting pyridinium moiety to approach the 7 electron-rich anthracene unit resulting in charge 8 transfer. This may also cause the red-shift which was 9 observed upon protonation. Figure 52 shows a pKa value of 6.5 for the tertiary amine within 129. The pKa value 11 of the pyridine moiety is not measurable in this 12 experiment in this pH range. NN//H

TO 'H'''o 130 17 The fluorescence properties of 129 were then examined by 18 using the same procedure as outlined for the absorption 19 spectroscopic experiments. The excitation wavelength was 370nm which is an isosbestic point as obtained from 21 Figure 51. The fluorescence spectra obtained are shown 22 in Figure 53. The emission-pH profile is shown by Figure 23 54.

From Figure 54 it is visible that two switching modes are 26 apparent where pKa values are 6.3 and 2.8. Fluorescence 27 is quenched at low and high pH values whilst fluorescence 28 is only observed at medium pH values. At high pH values \ 1 (>5.5) excitation of the anthracene fluorophore causes a 2 single electron transfer from the tertiary amine group 3 (receptor) to the fluorophore causing fluorescence 4 quenching. At medium pH values (3.0-5.5) the amine group (receptor) is protonated and the pyridine group 6 (receptor) is not. The electron transfer from the amine 7 group is therefore prevented so fluorescence is observed.

8 At low pH (<3.0) values the amine group (receptor) and 9 the pyridine group (receptor) are both occupied by protons. As previously discussed the pyridinium unit is 11 a powerful electron-accepting species and protonation 12 causes a one-electron reduction of the receptor, by a 13 second electron transfer from the photoexcited 14 fluorophore, which is easier due to the electrostatic attraction of the incoming electron to the protonated 16 receptor. Fluorescence is therefore quenched by this PET 17 process leading to another 'off' state.

19 It is noticeable from Figure 54 that at high pH values a complete quenching effect is not observed. While part of 21 this could be due to a lower polarity of the 22 microenvironment, the bulk of this effect is likely due 23 to the difficulty of salvation of the crowded amine 24 center. However by modifying 129 through increasing the distance between the amine and the pyridyl and carbonyl 2 6 group to decrease crowding of the anthracene more 27 efficient quenching may be observed. However for the 28 purpose of comparison with other compounds this mono 29 substituted anthracene compound was examined.

31 It can be said that molecule 129 has an 'off-on-off, 32 switching action and gives rise to rapid visual 33 indication of a concentration window of target species 1 which can be tuned due to the simplicity of the PET 2 design. This 'off-on-off' switching action cannot be 3 described by a specific binary logic type but it does 4 have similarities with tunnel diodes in electronics (Ryder, J.D., Engineering Electronics, 2nd ed; McGraw 6 Hill, New York, 1967). The same phenomenon has been 7 recently observed in the field of molecular electronics, 8 though the term 'negative differential resistance' has 9 been coined to describe it. These results can be summarized in the logic table (Table 7) shown below.

12 Table 7

-

IN OUT

(H) (Fluorescence) 0 (pH>5.5) 1 (pH=3.0-5.5) 2 (pH<3.0) Example 27: Synthesis of Tentagel-S-NH2 beads (112) with 16 2-(9'-anthrylmethyl)(methyl)amino]acetic acid (79) and 2 17 [(methyl)(l'pyrenylmethyl)amino]acetic acid (94) 19 Tentagel-S-NH2 beads were reacted with 94 in DMF with HOST and DIC. The beads were successively washed with 21 DMF, DMF:MeOH and MeOH. These beads were then treated 22 with 79 using the same procedure producing double 23 labelled 112. | I

2.5 % 2.5 loading loading 3 Fluorescence emission studier of 112 The influence of pH on fluorescence of 112 was 6 investigated by washing the beads with MeOH:H2O (50:50, 7 v/v) solutions of chosen pH value and exciting initially 8 at 368nm which is where the anthracene YES switch 9 molecule is usually excited. Importantly, the pyrene fluorophore has negligible absorption at this wavelength 11 as seen for instance in Figure 15. The fluorescence 12 spectra obtained are presented in Figure 55 and the 13 emission-pH profile is shown in Figure 56.

Figure 55 shows the typical anthracene signature with 16 maximum fluorescence peaks at 401, 422 and 446nm. From 17 Figure 56 it can be observed that the switch molecule is 18 still performing the YES logic operation where 19 fluorescence is quenched with increasing pH value. The pKa value is 5. 0 which is similar to that obtained for 21 the individually labelled bead. These results show that 22 by choosing the correct excitation wavelength the 23 behaviour of the YES switch molecule with the anthracene 1 fluorophore can be observed alone. The truth table 2 (Table 8) is shown below and the corresponding electronic 3 symbol is also shown in Figure 74. Of course, only the 4 blue fluorescence arm is detected in this experiment so far.

7 Table 8

l Input (H+) Output (YES) Output (YES) Blue fluorescence Violet 1 O I = Truth table for 112 where the excitation wavelength is 11 368 am.

13 Further fluorescence measurements were then performed on 14 112 using the same procedure as before only changing the excitation wavelength to 326nm. This is the wavelength 16 where beads grafted with the pyrene YES switch molecule 17 alone were excited. Of course, some excitation of the 18 anthracene fluorophore cannot be avoided. In fact the 19 wavelength of 326nm achieves perhaps the best selectivity of exciting pyrene fluorophores in the presence of 21 anthracene. The fluorescence spectra obtained are shown 22 in Figure 57.

24 The fluorescence spectra of compound 112 (Figure 57) displays fluorescence maxima at 379, 400, 421 and 446nm.

26 Fluorescence peaks for a monosubstituted pyrene molecule 27 are commonly observed at 379, 400 and 421 nm, however in 28 this case the bands at 400 and 421nm have unusually high \ 1 intensity. As was found in the previous set of 2 measurements anthracene emits with bands around 400, 421 3 and 446nm. It can therefore be said that the increase of 4 bands at 400, 421 and 446nm is due to the emission from anthracene molecules which are grafted onto this bead and 6 which will be slightly excited at 326nm. However, if 7 fluorescence is monitored at 379nm only pyrene emission 8 is seen. An emission-pH profile where emission at 379nm 9 is plotted against pH is shown in Figure 58.

11 From this profile it is clear that if fluorescence is 12 observed at 379nm the pyrene logic gate 94 is performing 13 the YES logic operation with high fluorescence intensity 14 in the presence of protons. The corresponding truth table (Table 9) is shown below.

17 Table 9

Input (H+) Output (YES) Output (YES) Blue fluorescence Violet t f 1 oars cenc e | Truth table for 112 where the excitation wavelength is 21 326nm.

23 The initial concern with this experiment was the 24 possibility of highly efficient EET since pyrene's emission 0-0 band and anthracene's absorption band 26 overlap, especially in the region of their 0-0 27 transitions. However it can be quickly concluded that 28 100% efficient EET is not observed as this would have 1 resulted in no pyrene excitation being observed. This is 2 due to the large dimension of the bead (0.lmm diameter) 3 which should minimise EET i f we assume that the gates are 4 uniformly distributed. The fluorescence from a donor and EET from a donor to an acceptor compete equally at donor- 6 acceptor separations of about 5nm (the forster distance).

7 The EET rate falls off with the sixth power of separation 8 distance (Birkes, J.B. Photophysics of Aromatic 9 Molecules, Wiley, 1970). Nevertheless, some small contribution of EET cannot be ruled out though it is 11 certainly not large enough to jeopardize the successful 12 use of polymer beads carrying two (or more) logic gates 13 with different fluorophores. The conclusion that EET is 14 not large is also borne out by analyzing excitation spectra.

17 Example 28: 4-Bromomethyl-7-methoxy coumarin (56) grafted 18 onto Tentagel-S-NHz beads (62) 4-Bromomethyl-7-methoxy coumarin, 56 is commercially 21 available (fluorescent reagent for HPLC determination of 22 carboxylic acids Dunges, W., Anal. Chem; 1977, 49, 442; 23 Lam, S., Grushka, E., J. Chromatogr., 1978, 168, 297) and 24 was easily grafted onto Tentagel-S-NH2 beads by refluxing in toluene overnight. These beads were then washed 26 successively in THF, THF:MeOH (50:50, v/v) and MeOH. A 27 set of beads was prepared (62) where the tag loading was 28 1. The reaction scheme is shown below.

(+0 0/\NH2 /\0 O:/\N - o 2 Fluorescence emission studies of 62 4 Fluorescence spectroscopic measurements were performed on the beads in MeOH:H2O (50:50, v/v) solutions of chosen pH 6 value using aliquots of NaOH(aq) and H3PO4(aq) to vary pH 7 which was measured directly using a glass electrode. The 8 excitation wavelength was 332nm (de Silva, A. P., 9 Gunaratne, H. Q. N., Lynch, P. L. M., Patty, A. J., Spence, G. L., J. Chem. Soc. Perkin. Trans 2, 1993, 11 1611). Fluorescence emission spectra obtained can be 12 seen in Figure 59. Emission-pH profiles are shown by 13 Figure 60.

Figure 59 shows a broad spectrum typical of a coumarin 16 fluorophore with a maximum fluorescence intensity 17 wavelength at 410 nm. Efficient switching can be 18 observed in Figure 60 where quenching of fluorescence is 19 obtained with increasing pH values as is expected for these molecular PET-based switches. The beads 62 1 implement the YES logic operation where fluorescence 2 (output) is only observed in the presence of an input 3 (protons). The pKa value in 62 is 5.5 which is close to 4 the ideal situation (7.1) (see de Silva, A.P., Gunaratne, H. Q. N., Lynch, P. L. M., Patty, A. J., Spence, G. L., 6 J. Chem. Soc. Perkin. Trans 2, 1993, 1611) and switching 7 is occurring over 3 pH units.

9 Molecular switch design

IN Not

ICI 11 67

13 Derivatives of 67 can be used as optical sensors and have 14 been used as dyes for flaw detection in solids (de Silva, A. P., Gunaratne, H. Q. N., Lynch, P. L. M; Patty, A. J., 16 Spence, G. L.; J. Chem. Soc. Perkin. Trans 2, 1993, 17 1611). It communicates with wavelengths in the visible 18 range. The molecular design adopted here is based on an 19 integrated 'fluorophore-receptor' system. Unlike the PET signalling system no spacer is present which preserves 21 the modularity of the system i.e. prevents interaction 22 between the fluorophore and receptor. This is therefore 23 a more complex signalling system and is more difficult to 24 predict. When 67 id treated with an amine the product that is formed has an internal charge transfer (ICT) 26 excited state. This is due to the electronic push-pull 27 system created by the 6-amino donor and the electron 1 accepting carbonyl and imine groups. Therefore when 67 2 is reacted with Tentagel-S-NH2 beads a system which has 3 an ICT excited state is obtained.

Example 29: 6-Chloro-3-benzimedozobes-isoquinolin-3-one 6 (67) grafted onto Tentagel-S-NH2 beads with 1% loading 7 (70) 9 67 was grafted onto the beads with a 1% loading by refluxing in THF overnight producing beads 70 Tentagel-S 11 NH2 beads (0.5g) were suspended in a solution of THF 12 (lOml) containing triethylamine (92.51). After stirring 13 for 10 minutes a solution of 67 (0.0007g, 0.0022mmol) in 14 THF was added which was then stirred for 4 hours and refluxed for 4 nights. The resulting beads were than 16 washed successfully in THF, THF:MeOH (50:50 v/v) and 17 MeOH.

(woo o/\ NH2 Ot,N,pN Cl

H CIAO

IN No

1 Synthetic scheme for 70.

3 Fluorescence properties of 70 were examined by washing 4 the beads with various solutions in MeOH:H2O (50:50, v/v) of differing pH values and exciting at 476nm. The 6 fluorescence emission spectra obtained can be seen in 7 Figure 61. The emission-pH profile at 511nm is shown by 8 Figure 62.

Figure 61 shows that the pKa value is 2.5 and switching 11 is occurring over 2 pH units, similar to the model 12 compound 68. The Tentagel-S-NH2 beads which have been 13 previously grafted with other molecules such as 48 and 56 14 gave a pKa value much lower than the model compound pKa value. This was explained in terms of hydrophobicity of 1 the amine receptor's microenvironment which causes 2 protonation difficulties, thus lowering the pKa value.

3 Another problem was that the free amine groups were most 4 probable for initial protonation due to their exposed nature compared to the amine near the fluorophore.

6 Protonation of the fluorophore-appended receptors is 7 delayed therefore the pKa value is reduced. However the 8 pKa value 2.5, shown by the beads 70 is the same as that 9 shown by the model compound 68 (see next example). When 67 is grafted onto the bead the receptor unit is at a far 11 enough distance so that it is not buried in the bead 12 environment. Protonation can occur almost immediately.

13 This leads to a pKa value similar to that obtained in 68.

Further fluorescence spectral measurements were performed 16 on these beads using the same conditions as previously, 17 only the excitation wavelength was altered to 468nm. The 18 emission (at 559nm)-pH profile can be seen in Figure 63.

The plot, shows a general switch action indicative of YES 21 logic. These measurements show that the beads 70 behave 22 similarly to the model compound 68 (see next example) 23 i.e. they can perform two logic operations by choosing 24 different excitation wavelengths.

26 Example 30: Synthesis of 3-[(2'-Methoxyethyl)amino]-7H 27 benzo[de]benzot4,5]imidazot2,1-a]isoquinolin-7-one (68) 29 67 (0.3 g, l.Ommol) was refluxed overnight with excess 2 methoxyethylamine (which mimics the surface of a grafted 31 Tentagel-S-NH2 bead) creating the integrated fluorophore 32 receptor system 68, by the SN2 nucleophilic attack of the 33 nitrogen lone pair of 2-methoxyethylamine on the chloro 1 substituted carbon of 67. The resulting product was 2 distilled under vacuum to remove excess amine. A red tar 3 remained which was dissolved in CH2C1 and washed with 4 saturated NaHCO3 solution. The CH2C12 layer was dried over sodium sulfate and solvent was removed under reduced 6 pressure producing a red solid. After loading onto a 7 column of flash silica and washing with CH2C12, the 8 product was eluted with a CH2Cl2:MeOH mixture in the 9 ratio 95:5 v/v (Yield: 75%, m.p. 133.0-134.0 C).

11 1H NMR (CDC13)300 MHz 68.84 (d, 1H, Ar-_, J=7 Hz), 12 8.73(d, 1H, Ar-H, J=ll Hz), 13 8.56(d, 1H, Ar-H, J=8 Hz), 14 8.29(d, 1H, Ar-H, J=7 Hz), 7.79(m, 2H, Ar-_), 7.42(m, 2H, 16 Ar-H), 6.83(d, 1H, Ar-H, J=8 17 Hz), 5.61(br s, 1H, -NH), 18 3.81(t, 2H, CH2O, J=5 Hz), 19 3.60(m, 2H, CH2CH2O), 3.53(s, 1H, OCH3) 22 13C NMR(CDCl3)125 MHz 643.1, 59.0, 70.1, 104.7, 116.0, 23 119.0, 120.0, 121.0, 123.7, 24 124.7, 124.8, 125.5, 127.2, 127.3, 130.2, 131.6, 135.0.

27 irvmax(KBr): 3437, 2924, 2375, 1652, 1558, 28 1456, 1103, 762 cml.

1 W-Visible spectroscopic investigations of 68 3 The spectral properties of 68 were investigated in a 4 MeOH:H2O (50:50, v/v) solution using aliquots of NaOH(aq) and HCl(aq) to vary pH. Figure 64 displays the influence 6 of pH on the absorption spectra. The absorbance-pH 7 profile is shown by Figure 65.

9 This investigation showed that larger changes in absorbance are observed with 68 when compared with 11 molecule compound 51 (molecule based on the PET 12 signalling system). This is expected due to the 13 insulating properties of the spacer module in the latter.

14 A blue-shift is observed with increasing pH values where the band at 475nm is shifted to 468nm. Also with 16 increasing pH values the absorbance intensity of the band 17 at 468nm is decreased. A pKa value of 2.5 is obtained 18 from the absorbance-pH profile in Figure 65.

Fluorescence emission studies of 68 22 An isosbestic point is observed in Figure 64 at 476nm.

23 This is used as the excitation wavelength for 24 fluorescence studies of 68 which was examined in a MeOH:H2O (50:50 v/v) solution using aliquots of NaOH(aq) 26 and HCl(aq) to vary the pH value. Fluorescence spectra 27 shown in Figure 65 were obtained. Figure 67 shows the 28 emission-pH profile at 510nm.

Figure 65 displays a broad emission band with maximum 31 fluorescence observed at 510nm. Figure 67 shows that 68 32 is behaving as a pH sensor with a pKa value of 2.5 but is 33 showing opposite switching action than that observed with 1 the aminomethylanthracene system (model compound 51) i.e. 2 the green fluorescence is switched off as the pH value is 3 decreased. 68 possesses an electronic push-pull system 4 which generates ICT excited states. At low pH values delocalisation is greater within 68 due to the 6 protonation of the acceptor imine nitrogen and the ICT 7 nature is more extensive than at high pH values.

8 Therefore at low pH values longer wavelength orange 9 emission is seen, whereas the green fluorescence dominates at high pH. However, the quantum efficiency of 11 the orange emission is rather small. This explains the 12 result shown by the fluorescence spectra i.e. at high pH 13 values fluorescence intensity in the green region is high 14 and decreases with decreasing pH.

16 It can therefore be said that 68 can be used to perform 17 the NOT logic operation.

19 An observation from the absorption spectra in Figure 64 was that at 514 nm as pH is decreased the absorbance 21 increases. A solution of 68 was excited at this 22 wavelength at chosen pH values. The series of 23 fluorescence spectra obtained can be seen in Figure 68.

By exciting at 514nm the intensity of fluorescence in the 26 orange region is now high at low pH and the intensity at 27 high pH values is low. The pH profile in Figure 69 shows 28 that with increasing pH value fluorescence at 559nm is 29 being reduced. These experiments therefore show that 68 can be reconfigured as a YES logic gate by changing the 31 excitation wavelength.

1 An important observation from these measurements is that 2 the intensity of fluorescence in the protonated form when 3 excited at 514nm (orange emission) is much lower than the 4 intensity observed when 68, unprotonated, is excited at 476nm (green emission). This can be explained in terms 6 of hydrogen bonding, where the proton bonded to the 7 electronegative heteroatom N. can hydrogen bond 8 efficiently with protic solvents as shown in 69. A 9 fluorescence deactivation pathway is opened decreasing the fluorescence quantum yield.

O. N. N-Hit '' to HN: 13 An important feature of 68 is therefore that it can 14 perform two different types of logic operations by angling the excitation to a particular wavelength.

17 Example 31: Synthesis of double labelled Tentagel-S-NH2 18 beads (113) with 2-[methyl(1'-pyrenylmethyl)amino]acetic 19 acid (94) and 3-(9'-anthrylmethyl)propanoic acid (100).

21 Tentagel-S-NH2 beads were reacted with 100 in DMF with 22 HOBT and DIC. The beads were successively washed with 23 DMF, DMF:MeOH and MeOH. These beads were then treated 1 with 94 using the same procedure used for 112 producing 2 double labelled beads 113.

,>.NW:(N,N 2.5% 2.5% 6 loading 113 loading 8 Fluorescence emission studies of 113 Fluorescence measurements were performed on 113 by 11 washing the beads with MeOH:H2O (50:50, v/v) solutions of 12 chosen pH value and exciting initially at 368 nm which is 13 where the beads grafted with the PASS 1 switch molecule 14 were excited. We note again that the pyrene fluorophore is unaffected by radiation of this wavelength. The 16 fluorescence spectra obtained are presented in Figure 75 17 and the emission-pH profile is shown in Figure 76.

19 Figure 75 which displays a typical anthracene fluorescence signature shows that emission from the 21 anthracene fluorophore alone is observed. Figure 76 22 clearly shows that the anthracene type tag molecule is 23 performing the PASS 1 logic operation which is what is 24 expected i.e. fluorescence does not vary with pH value.

The truth table concerning the blue fluorescence is shown 1 in Table 9 and the electronic symbol for the whole system 2 is in Figure 77.

Input (H+) Output (PASS 1) Output (YES) Blue fluorescence Violet fluorescence 1 1 -- Table 9 Truth tableFor 113 where the excitation 6 waver ength i s 3 68 am.

8 Fluorescence measurements were then performed on 113 9 using the same conditions as discussed previously only the excitation wavelength was 326 nm. This is where the 11 pyrene fluorophore is normally excited and where maximum 12 selectivity is achieved against exciting anthracene 13 fluorophores. Fluorescence spectra obtained are seen in 14 Figure 78 and the emission-pH profile is presented in Figure 79.

17 Figure 78 shows similar band positions as were obtained 18 when 112 was excited at 326 nm i.e. 379, 398, 419 and 446 19 nm. Emission at bands 398, 419 and 446 nm have contributions due to some interference from the excited 21 anthracene fluorophore. If Figure 78 is studied closely 22 it can be seen that with increasing pH value fluorescence 23 at 398, 419 and 446 nm is not completely quenched. This 24 is evidence for the excitation of the anthracene fluorophore which is present as part of a PASS 1 logic 26 gate and as discussed previously the fluorescence of this 27 molecule is not expected to be quenched with increasing 28 pH values. This residual is also an indication of the 1 anthracene emission contribution during experiments with 2 326 nm excitation aimed at pyrene-based logic gates.

3 However if emission is observed at 379 nm pyrene emission 4 alone is seen. The emission-pH profile in Figure 79 shows that by observing at this wavelength the expected 6 behaviour of the pyrene YES logic gate is observed where 7 fluorescence is quenched in the absence of protons. The 8 pKa value is 5.0 which is the same value obtained when 9 similar measurements were performed on Tentagel-S-NH2 beads which were grafted with the pyrene YES switch 11 alone. The truth table is shown below.

Input (H+) Output (PASS l) Output (YES) Blue fluorescence Violet O f: 14 Table 10 Truth table for 113 where the excitation wavelength is 326 am.

16 We can conclude from analyzing excitation spectra that 17 EET is not large enough to endanger the validity of the 18 encoding.

Fluorescent switch molecules have been produced in situ 21 by covalent attachment onto polymer beads forming 22 'fluorophore-spacer-receptor' PET systems or the 23 behaviorally more complex integrated 'fluorophore 24 receptor' system which has ICT excited states. The influence of tag loading was examined and efficient 26 switching behaviour was observed under the correct 27 conditions. Multiple labelling of Tentagel-S-NH2 beads 28 was also successful where emission and excitation signals 1 from both fluorophores can be distinguished and observed 2 at chosen wavelengths.

Claims

1 Claims 3 1. A labelled solid support comprising a solid 4 support and at

least one fluorophore label wherein the fluorophore label operates as a 6 logic gate in the presence of an ionic 7 species.

9 2. A labelled solid support as claimed in Claim 1 wherein each fluorophore behaves according 11 to the AND, PASS-1, NOT, NAND, OR, NOR, XOR, 12 XNOR, INHIBIT, ENABLED OR, or YES logic 13 operation.

3. A labelled solid support as claimed in either 16 one of Claims 1 and 2 comprising more than 17 one fluorophore wherein each fluorophore 18 behaves according to the same logic 19 operation.

21 4. A labelled solid support as claimed in Claim 22 3 comprising two fluorophores wherein both 23 fluorophores behave according to a YES logic 24 operation.

26 5. A labelled solid support as claimed in either 27 one of Claims 1 and 2 comprising more than 28 one fluorophore wherein each fluorophore 29 behaves according to a different logic operation.

1 6. A labelled solid support as claimed in Claim 2 5 comprising a first fluorophore and a second 3 fluorophore wherein the first fluorophore 4 behaves according to a YES logic operation, and the second fluorophore behaves according 6 to a PASS-1 logic operation.

8 7. A labelled solid support as claimed in any 9 preceding claim wherein the fluorophore label comprises a fluorophore covalently bound to a 11 receptor said receptor being able to bind an 12 ionic species such that upon binding of the 13 ionic species to the receptor the 14 fluorescence of the fluorophore is altered.

16 8. A labelled solid support as claimed in Claim 17 7 wherein the fluorophore is covalently bound 18 to a receptor via a spacer molecule.

9. A labelled solid support as claimed in Claim 21 8 wherein the spacer is -CH2-, -CH2CH2-, 22 -CH2CH2CH2-.

24 10. A library of labelled solid supports as claimed in any preceding claim said solid 26 support comprising a chemical entity bound 27 thereto wherein the library comprises at 28 least 103 solid supports and each label 29 corresponds to a single chemical entity.

31 11. A method of identifying a chemical entity 32 bound to a labelled solid support as claimed 1 in any one of Claims 1 to 9 from a library of 2 labelled solid supports wherein each label 3 corresponds to a single chemical entity 4 comprising the steps of: preparing a key of which label corresponds 6 to which chemical entity; 7 sequentially exposing said labelled solid 8 support to an ionic species at predetermined 9 concentrations and to a mixture of said ionic species at predetermined 11 concentrations; and 12 observing the conditions required to produce 13 fluorescence of said label, thereby 14 identifying said label.