DYNAMIC COMBINATORIAL LIBRARY BASED ON PSEUDOPEPTIDES AND ITS USE FOR THE DETECTION OF CYSTEINE AND OTHER BIOTHIOLS

Abstract

The present invention relates to a dynamic chemical network that mimics the transfer of information and ultimately produces a chemical response, for example a readable signal. More specifically, the invention discloses a dynamic system able to selectively sense a biologically-relevant analyte, such as cysteine, in its reduced or its oxidized form (cystine) in aqueous media and in a biofluid (such as urine) for diagnostic application. Due to this property, said dynamic system is useful for the detection of cysteine or its derivatives in biological fluids such as urine.

Claims

1. A mixture of molecules comprising at least a compound as defined by formula (I): ##STR00016## or an isomer or a salt thereof, wherein: Ra and Rb are independently selected from H, —CONRR′, —COOR, —NRR′, —OH, —C(NH)NH.sub.2, —CH(OH)CH.sub.3, aryl-(C.sub.6-C.sub.10) or heteroaryl-(C.sub.6-C.sub.10); R and R′ are independently selected from H or alkyl-(C.sub.1-C.sub.3); m is an integer selected from 0, 1, 2, 3 or 4; n is an integer selected from 1 or 2; at least a compound of formula (II): ##STR00017## or an isomer or a salt thereof, wherein: n′ is an integer selected from 1 or 2; and at least a compound of formula (III): ##STR00018## or an isomer or a salt thereof, wherein: Rc represents an aromatic chromophore; Y is selected from NHCO or CONH; Z is selected from H, —NH.sub.2, —OH or —COOH; p is an integer selected from 0, 1, 2 or 3.

2. The mixture of molecules according to claim 1, which also comprises at least a compound of formula (IV): ##STR00019## or an isomer or a salt thereof, wherein: Rd is selected from H or S(CH.sub.2)qCH(NH.sub.2)COOH; q is an integer selected from 1, 2 or 3.

3. The mixture of molecules according to claim 1, wherein m is 1 and n is 1.

4. The mixture of molecules according to claim 1, wherein Ra is CONH.sub.2.

5. The mixture of molecules according to claim 1, wherein Rb is CONH.sub.2.

6. The mixture of molecules according to claim 1, wherein n′ is 1.

7. The mixture of molecules according to claim 1, wherein Rc is selected from the following chromophores: ##STR00020##

8. The mixture of molecules according to claim 1, wherein Y is —NHCO.

9. The mixture of molecules according to claim 1, wherein Z is —NH.sub.2.

10. The mixture of molecules according to claim 1, wherein p is 1.

11. The mixture of molecules according to claim 1, wherein Rd is H.

12. The mixture of molecules according to claim 1, wherein q is 1.

13. A dynamic combinatorial library comprising the mixture of molecules of claim 1.

14. An in vitro method for the detection of a compound of formula (IV): ##STR00021## or an isomer or a salt thereof, wherein: Rd is selected from H or S(CH.sub.2)qCH(NH.sub.2)COOH; q is an integer selected from 1, 2 or 3 in a sample comprising the following steps: a) mixing at least a compound of formula (I) with at least a compound of formula (II) and at least a compound of formula (III) according to claim 1; b) contacting the sample comprising the compound of formula (IV) with the mixture of step (a); c) measuring the fluorescence emission of the mixture obtained in step (b); d) detecting a significant deviation from a standard; and e) assigning the sample to the group of samples comprising a compound of formula (IV) when a significant difference has been detected in stage (d).

15. The method according to claim 14, wherein the molecule of formula (IV) is selected from cysteine or cystine.

16. A kit for the detection of a compound of formula (IV) in a sample of ##STR00022## or an isomer or a salt thereof, wherein: Rd is selected from H or S(CH.sub.2)qCH(NH.sub.2)COOH; q is an integer selected from 1, 2 or 3 comprising a mixture of compounds of formula (I), (II) and (III) as described in claim 1.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0070] FIG. 1: Shows the fluorescence emission spectra of the dynamic library formed by Ia, IIa and IIIa (sensor) (25% DMSO in aqueous bis-tris buffer at pH 6.5) and under increasing concentrations of Cys. [Cys]=No L-Cys (dotted line), 0.025 mM, 0.05 mM, 0.1 mM, 0.25 mM, 0.5 mM, 1 mM, 2.5 mM.

[0071] FIG. 2: (A) Shows the fluorescence emission spectra of the library formed by Ia, IIa and IIIa alone (black line) and in the presence of 0.5 mM Cys (Δ), 0.25 mM cystine (.circle-solid.), 0.5 mM N-acetyl-Cys (∘) and 0.5 mM GSH (.box-tangle-solidup.). (B) Shows the plot of the excimer/monomer ratio for the different biothiols. (C) and (D) show the sensing of Cys (0-1.0 mM) and CySS (0-0.5 mM)) respectively, in the presence of the three basic amino acids (Lys, Orn and Arg) at non-pathological upper limit concentrations found in urine. (E) and (F) show, as in (C) and (D), but at pathological concentrations in urine samples (5 mM Lys, 1.4 mM Orn and 1.2 mM Arg). [Cys] or [CySS]=No L-Cys (dotted line), 0.05 mM, 0.1 mM, 0.25 mM, 0.5 mM. (G) Shows, as in (C) but in a buffer containing (Lys, Orn, Arg, Asn, Gly, Ala, His, Asp, β-Ala, Ser, Tyr and Met). [Cys]=No L-Cys (dotted line), 0.05 mM, 0.1 mM, 0.25 mM, 0.5 mM, 1 mM.

[0072] FIG. 3: (A) Shows the selected normalized fluorescence spectra of urine (dotted line), sensor alone (solid black line), sensor+urine (∘) and after increasing concentrations of added Cys (0.25, 0.5, 1 mM (grey) to 2.5 mM (.circle-solid.)). (B) Shows the scatter plot of the excimer/monomer emission of the library alone (Blank), for different urine (U) samples of healthy volunteers and for the urine samples plus additional Cys.

EXAMPLES

1. Synthesis of the Compounds of the Mixture of Molecules of the Invention

[0073] A) Compounds of formula (I)

[0074] Step 1: To a solution of protected amino acid 1 (PG1 is Fmoc and PG2 is Boc or tBu) (7.11 mmol) in dry DMF (15 mL), HOBt (1.25 g, 9.27 mmol) and DCC (2.23 g, 10.8 mmol) were added under nitrogen atmosphere. The resulting mixture was cooled down to 0° C. and then a solution of m-phenylenediamine (334 mg, 3.09 mmol) in dry DMF (10 mL) was added via cannula. The mixture was stirred at room temperature for 60 hours, filtered, and the filtrate was diluted with DCM, washed with saturated aqueous NaHCO3 and saturated aqueous NaCl, dried over MgSO4 and concentrated under reduced pressure. The residue was purified by flash chromatography using AcOEt/hexane as eluents to yield compounds (i).

[0075] Step 2: Compounds (i) (0.5 mmol) were dissolved in 4.0 mL of 20% piperidine in dry DMF. The mixture was allowed to react for 4 hours and then diethyl ether was added over the reaction mixture and the product was filtered off and washed with diethyl ether to obtain compounds (ii) which were used without further purification.

[0076] Step 3: Tritylsulfanyl acetic acid (501 mg, 1.50 mmol) was dissolved in dry DMF (20 mL) and EDC.HCl (312 mg, 1.63 mmol), HOBt (228 mg, 1.69 mmol) and DIPEA (1.6 mL, 4.59 mmol) were added over the solution. The reaction mixture was cooled down to 0° C. and (ii) (0.715 mmol) was added over the mixture. The mixture was stirred at room temperature for 48 hours and then diluted with DCM, washed with saturated aqueous NaHCO3 and saturated aqueous NaCl, and dried under reduced pressure. The crude product was purified by flash chromatography using AcOEt/hexane as eluents to give compounds (iii).

[0077] Step 4: To a solution of (iii) (0.16 mmol) in DCM (1.0 mL), TFA (8.5 mL), TIS (332 μL, 1.28 mmol) and EDT (160 μL, 1.91 mmol) were added rapidly and under stirring. The reaction mixture was stirred at room temperature for 2 hours, after which the solvents were partially evaporated using a N.sub.2 flow. Diethyl ether was added over the reaction mixture and the product was filtered off and washed with diethyl ether. The product was purified by reversed-phase flash chromatography using a mixture of MeCN+0.07% (v/v) TFA and H2O+0.1% (v/v) TFA as mobile phase. Compounds (I) were obtained after lyophilisation of the mobile phase.

[0078] (2S,2′S)-N1,N1′-(1,3-phenylene)bis(2-(2-mercaptoacetamido)succinamide) (Ia) (Asn): 1H NMR (400 MHz, DMSO-d6): δ=9.97 (s, 2H), 8.34 (d, 2H), 7.93 (t, 1 H), 7.36 (s, 2H), 7.29 (dd, 2H), 7.24-7.15 (m, 1H), 6.91 (s, 2H), 4.67 (q, 2H), 3.17 (d, 4H), 2.73 (t, 2H), 2.62-2.41 (m, 4H) ppm. HRMS (ESI+) calcd. for C.sub.18H.sub.24N.sub.6O.sub.6S.sub.2 [M+H]+ (m/z): 485.1277, found: 485.1279.

[0079] (2S,2′S)-N1,N1′-(1,3-phenylene)bis(2-(2-mercaptoacetamido)pentanediamide) (Ib) (Gln): 1H NMR (400 MHz, DMSO-d6): δ=10.09 (s, 2H), 8.30 (d, J=7.7 Hz, 2H), 7.96 (t, 1H), 7.35-7.27 (m, 4H), 7.26-7.20 (m, 1H), 6.77 (s, 2H), 4.39 (td, 2H), 3.25-3.12 (m, 4H), 2.75 (t, 2H), 2.22-2.04 (m, 4H), 1.99-1.88 (m, 2H), 1.88-1.76 (m, 2H) ppm. HRMS (ESI+) calcd. for C.sub.20H.sub.28N.sub.6O.sub.6S.sub.2 [M+H]+ (m/z): 513.1590, found: 513.1592.

[0080] (2S,2′S)-N,N′-(1,3-phenylene)bis(3-hydroxy-2-(2-mercaptoacetamido)propanamide) (Ic) Ser: 1H NMR (400 MHz, MeOD-d4): δ=7.92 (t, 1 H), 7.37-7.30 (m, 2H), 7.29-7.23 (m, 1H), 4.56 (t, 2H), 3.93-3.82 (m, 4H), 3.28 (s, 4H) ppm. HRMS (ESI+) calcd. for C.sub.16H.sub.22N.sub.4O.sub.6S.sub.2 [M+H]+ (m/z): 431.1059, found: 431.1054.

[0081] (2S,2′S)-N,N′-(1,3-phenylene)bis(3-hydroxy-2-(2-mercaptoacetamido)butanamide) (Id) (Thr): 1H NMR (400 MHz, MeOD-d4): δ=9.80 (s, 2H), 8.18 (d, 2H), 7.95-7.89 (m, 1H), 7.36-7.30 (m, 2H), 7.29-7.23 (m, 1H), 4.48-4.42 (m, 2H), 4.24 (qd, 2H), 3.35-3.29 (m, 4H), 1.24 (d, 6H) ppm. HRMS (ESI+) calcd. for C.sub.18H.sub.26N.sub.4O.sub.6S.sub.2 [M+H]+ m/z): 459.1372, found: 459.1371.

[0082] (2S,2′S)-N,N′-(1,3-phenylene)bis(3-(4-hydroxyphenyl)-2-(2-mercaptoacetamido)propanamide) (Ie) (Tyr): 1H NMR (400 MHz, DMSO-d6): δ=10.11 (s, 2H), 9.17 (br s, 2H), 8.34 (d, 2H), 7.88 (t, 1H), 7.32-7.25 (m, 2H), 7.25-7.18 (m, 1H), 7.06 (d, 4H), 6.64 (d, 4H), 4.58 (td, 2H), 3.12 (d, 4H), 2.92 (dd, 2H), 2.75 (dd, 2H), 2.63 (t, 2H) ppm. HRMS (ESI+) calcd. for C.sub.28H.sub.30N.sub.4O.sub.6S.sub.2 [M+H]+(m/z): 583.1685. found: 583.1696.

[0083] (2S,2′S)-N,N′-(1,3-phenylene)bis(3-(1H-indol-3-yl)-2-(2-mercaptoacetamido)propanamide) (If) (Trp): 1H NMR (400 MHz, DMSO-d6): δ=10.82 (d, 2H), 10.17 (s, 2H), 8.37 (d, 2H), 7.93 (s, 1H), 7.64 (d, 2H), 7.35-7.27 (m, 4H), 7.24-7.18 (m, 1H), 7.16 (d, 2H), 7.05 (ddd, 2H), 6.97 (ddd, 2H), 4.72 (td, 2H), 3.23-3.11 (m, 6H), 3.02 (dd, 2H), 2.65 (t, 2H) ppm. HRMS (ESI+) calcd. for C.sub.32H.sub.32N.sub.6O.sub.4S.sub.2 [M+H]+ (m/z): 629.2004, found: 629.2003.

[0084] (3S,3′S)-4,4′-(1,3-phenylenebis(azanediyl))bis(3-(2-mercaptoacetamido)-4-oxobutanoic acid) (Ig) (Asp): 1H NMR (400 MHz, MeOD-d4): δ=7.85 (t, 1H), 7.35-7.29 (m, 2H), 7.28-7.22 (m, 1H), 4.90-4.81 (m, 2H), 3.24 (s, 4H), 2.91 (dd, 2H), 2.78 (dd, 2H)ppm. HRMS (ESI+) calcd. for C18H22N4O8S2 [M+H].sup.+ (m/z): 487.0957, found: 487.0956.

[0085] (4S,4′S)-5,5′-(1,3-phenylenebis(azanediyI))bis(4-(2-mercaptoacetamido)-5-oxopentanoic acid) (Ih) Glu: 1H NMR (400 MHz, MeOD-d4): δ=7.90 (t, 1H), 7.35-7.30 (m, 2H), 7.29-7.23 (m, 1H), 4.53 (dd, 2H), 3.24 (s, 4H), 2.45 (t, 4H), 2.25-2.12 (m, 2H), 2.08-1.96 (m, 2H) ppm. HRMS (ESI+) calcd. for C.sub.20H.sub.26N.sub.4O.sub.8S.sub.2 [m+H].sup.+ (m/z): 515.1270, found: 515.1271.

[0086] (2S,2′S)-N,N′-(1,3-phenylene)bis(6-amino-2-(2-mercaptoacetamido)hexanamide) (Ii) (Lys): 1H NMR (400 MHz, MeOD-d4): δ=8.01-7.96 (m, 1H), 7.35-7.19 (m, 3H), 4.49 (dd, 2H), 3.24 (s, 4H), 2.93 (t, 4H), 2.01-1.87 (m, 2H), 1.86-1.63 (m, 6H), 1.62-1.39 (m, 4H) ppm. HRMS (ESI+) calcd. for C.sub.22H.sub.36N.sub.6O.sub.4S.sub.2 [M+H].sup.+ (m/z): 513.2318, found: 513.2319.

[0087] (2S,2′S)-N,N′-(1,2-phenylene)bis(5-amino)pentanamide) (Ij) (Orn(L 1H NMR (400 MHz, MeOD-d4): δ=8.02-7.97 (m, 1H), 7.34-7.18 (m, 3H), 4.54 (dd, 2H), 3.25 (s, 4H), 3.07-2.90 (m, 4H), 2.05-1.89 (m, 2H), 1.88-1.68 (m, 6H) ppm. HRMS (ESI+) calcd. for C.sub.20H.sub.32N.sub.6O.sub.4S.sub.2 [M+H].sup.+ (m/z): 485.2005, found: 485.2007.

[0088] (2S,2′S)-N,N′-(1,3-phenylene)bis(5-guanidino-2-(2-mercaptoacetamido)pentanamide) (Ik) Arg: 1H NMR (400 MHz, MeOD-d4): δ=7.99 (s, 1H), 7.33-7.20 (m, 3H), 4.52 (dd, 2H), 3.29-3.11 (m, 8H), 2.00-1.87 (m, 2H), 1.86-1.57 (m, 6H) ppm. HRMS (ESL.sup.+) calcd. for C.sub.22H.sub.36N.sub.10O.sub.4S.sub.2 [M+H].sup.+ (m/z): 569.2441, found: 569.2435.

B) Compounds of formula (II)

[0089] Compounds of formula (II) can be synthesised as previously described in K. R. West, K. D. Bake and S. Otto, Org. Lett., 2005, 7, 2615-2618.

[0090] 2,2′,2″-(benzenetricarbonyltris(azanediyl))tris(3-mercaptopropanoic acid) (IIa) 1H-NMR (500 MHz, DMSO-d6): δ 9.04 (d, 1H), 8.52 (s, 1H), 4.58 (m, 1H) 3.03 (m, 1H), 2.93 (m, 1H), 2.61 (t, 1H) ppm. HRMS: Calc. for C.sub.18H.sub.21N.sub.3O.sub.9S.sub.3 [M+H].sup.+ 520.0518, found 520.0526.

C) Compounds of Formula (III)

[0091] Compounds of formula (III) can be synthesized as follows:

Synthesis of Compound IIIa

[0092] ##STR00011##

[0093] (R)-2-amino-N-(pyren-1-yl)-3-(tritylthio)propanamide (3a): Boc-Cys(Trt)-OH (2.134 g, 4.60 mmol) was dissolved in dry DMF (6 mL) and then HBTU (2.094 g, 5.52 mmol) and HOBt (0.746 g, 5.52 mmol) were added. The reaction mixture was stirred during 2 min and after 1-aminopyrene (200 mg, 0.92 mmol) was added. The solution was stirred at room temperature under an inert atmosphere of Ar for 5 hours (TLC monitoring). The mixture was diluted with DCM, washed with saturated aqueous NaHCO.sub.3 and saturated aqueous NaCl, dried over MgSO4 and concentrated under reduced pressure. The crude product was purified by flash chromatography using EtOAc/hexane/toluene as eluent (from 20/60/20 to 40/40/20) to give 311 mg (51% yield) of 3a as a light pink solid.

[0094] .sup.1H NMR (400 MHz, CDCl.sub.3-d): δ=8.90 (s, 1H, NHCOC*H), 8.46 (d, 1H, CH.sub.AT), 8.19-8.11 (m, 3H, CH.sub.Ar), 8.09-7.96 (m, 4H, CH.sub.Ar)), 8.00 (s, 1 H, CH.sub.Ar), 7.52 (dd, 6H, CH.sub.Ar), 7.33 (dd, 6H, CH.sub.Ar), 7.26 (d, 3H, CH.sub.Ar), 4.98 (d, 1H, C*HNHCO), 4.24-4.11 (m, 1H, C*H), 2.89 (ddd, 2H, C*HCH.sub.2), 1.53 (s, 9H, CH.sub.3) ppm. HRMS (ESI.sup.−) calcd. for C.sub.43H.sub.38N.sub.2O.sub.3S [M−H].sup.− (m/z): 661.2575, found: 661.2525.

[0095] (R)-2-amino-3-mercapto-N-(pyren-1-yl)ppropanamide (IIIa): To a solution of 3a (280 mg, 0.42 mmol) in DCM (1.5 mL), 1 mL of trifluoroacetic acid (TFA), 1,2-Ethanedithiol (EDT, 0.64 mL, 7.61 mmol) and triisopropylsilane (TIS, 1.30 mL, 6.34 mmol) were added rapidly and under stirring. The reaction mixture was stirred at room temperature for 2 hours, after which the solvents were partially evaporated using a N2 flow. Diethyl ether was added over the reaction mixture and the product was filtered off and washed with diethyl ether. The product was purified by reversed-phase flash chromatography using a mixture of MeCN+0.07% (v/v) TFA and H2O+0.1% (v/v) TFA as mobile phase (gradient: from 0% to 10% MeCN in H2O). After lyophilization 75 mg (55% yield) of IIIa.1TFA were obtained as a white solid.

[0096] .sup.1H NMR (400 MHz, MeOH-d.sub.4): δ=8.29-8.17 (m, 6H, H.sub.5, H.sub.6, H.sub.8, H.sub.10, H.sub.12, H.sub.13), 8.11 (q, 2H, H.sub.2, H.sub.3), 8.05 (t, 1H, H.sub.9), 4.48 (dd, 1H, C*HCH.sub.2), 3.38-3.24 (dd, 2H, C*HCH.sub.2) ppm. HRMS (ESI.sup.+) calcd. for C.sub.19H.sub.16N.sub.2OS [M+H].sup.+ (m/z): 321.1061, found: 321.1041.

Synthesis of Compound IIIb

[0097] ##STR00012##

[0098] (R)-tert-butyl (1((5-aminonaphthalen-1-yl)amino)-1-oxo-3-(tritylthio)propan-2-yl)carbamate (3b). To a solution of Boc-Cys(Trt)-OH (1 g, 2.15 mmol) in dry DMF (10 mL) and EDC.HCl (0.723 g, 3.77 mmol), HOBt (0.597 g, 4.42 mmol) and DIPEA (1.32 mL, 9.7 mmol) were added. The reaction mixture was stirred during 2 min and 1,5-diaminonaphthalene (170 mg, 1.08 mmol) was added over the mixture. The solution was stirred at room temperature under an inert atmosphere of Ar for 48 hours, and the formation of the product was followed by TLC. The mixture was diluted with DCM, washed with saturated aqueous NaHCO3 and saturated aqueous NaCl, and dried under reduced pressure. The crude product was purified by flash chromatography using EtOAc/Hexane as eluent (from 30% to 50% EtOAc) to give 356 mg (55% yield) of 3b as a dark pink solid.

[0099] .sup.1H NMR (400 MHz, CDCl.sub.3-d): δ=8.51 (s, 1H, CONH), 7.99 (d, 1H, CH.sub.Ar), 7.62 (d, J=8.5 Hz, 1H, CH.sub.Ar), 7.46 (d, 5H), 7.39 (t, 1H), 7.32-7.19 (m, 12H, CH.sub.Ar, CONH), 6.80-6.73 (m, 2H, CH.sub.Ar), 4.90 (d, 1H, C*HCH.sub.2), 2.80 (ddd, 2H, CH.sub.2STrt), 1.46 (s, CH.sub.3) ppm HRMS (ESI.sup.+) calcd. for C.sub.37H.sub.37N.sub.3S [M+H].sup.+ (m/z): 604.2634, found: 604.2637.

[0100] (R)-2-amino-N-(5-aminonaphthalen-1-yl)-3-mercaptopropanamide (IIIb). To a solution of 3b (200 mg, 0.191 mmol) in DCM (1 mL), 1 mL of trifluoroacetic acid (TFA), 1,2-Ethanedithiol (EDT, 0.3 mL, 3.44 mmol) and triisopropylsilane (TIS, 0.6 mL, 2.29 mmol) were added rapidly and under stirring. The reaction mixture was stirred at room temperature for 2 hours, after which the solvents were partially evaporated using a N2 flow. Diethyl ether was added over the reaction mixture and the product was filtered off and washed with diethyl ether. The product was purified by reversed-phase flash chromatography using a mixture of MeCN+0.07% (v/v) TFA and H2O0.1% (v/v) TFA as mobile phase (gradient: from 0% to 10% MeCN in H2O). After lyophilisation 77 mg (83% yield) of IIIb.2TFA were obtained as a white solid.

[0101] .sup.1H NMR (400 MHz, MeOH-d.sub.4): δ=7.92 (d, 2H, CH.sub.Ar), 7.75 (d1H, CH.sub.Ar), 7.61 (t, 1H, CH.sub.Ar), 7.50 (t, 1H, CH.sub.Ar), 7.36 (d, 1H, CH.sub.Ar), 4.38 (t, 1H, C*HCH.sub.2), 3.30-3.12 (dd, 2H, CH.sub.2SH) ppm. HRMS (ESI.sup.+) calcd. for C.sub.13H.sub.15N.sub.3OS [M+H].sup.+ (m/z): 262.1014, found: 262.1001.

Synthesis of Compound IIIc

[0102] ##STR00013##

[0103] N-(5-aminonaphthalen-1-yl)-2-(tritylthio)acetamide (3c). Tritylsulfanyl acetic acid (500 mg, 1.50 mmol) was dissolved in dry DMF (6 mL) and EDC.HCl (383 mg, 2 mmol), HOBt (304 mg, 2.25 mmol) and DIPEA (0.87 mL, 5 mmol) were added over the solution. The reaction mixture was stirred during 2 min and 1,5-diaminonaphthalene (158 mg, 1 mmol) was added over the mixture. The solution was stirred at room temperature under an inert atmosphere of Ar for 48 hours, and the formation of the product was followed by TLC. The mixture was diluted with DCM, washed with saturated aqueous NaHCO3 and saturated aqueous NaCl, and dried under reduced pressure. The crude product was purified by flash chromatography using EtOAc/Hexane as eluent (from 30% to 50% EtOAc) to give 287 mg (61% yield) of 3c as a pink solid.

[0104] .sup.1H NMR (400 MHz, CDCl3-d): δ=8.44 (s, 1H, CH2CONH), 7.83 (d, 1H, CH.sub.Ar), 7.61 (d, 1H, CH.sub.Ar), 7.48 (d, 6H, CH.sub.Ar), 7.37 (t, 1H, CH.sub.Ar), 7.30-7.23 (m, 7H, CH.sub.Ar), 7.18 (t, 3H, CH.sub.Ar), 7.06 (d, 1 H, CH.sub.Ar), 6.78 (d, 1H, CH.sub.Ar), 3.46 (s, 2H, CH.sub.2CONH) ppm. HRMS (ESI.sup.+) calcd. for C.sub.31H.sub.26N.sub.2OS [M+H].sup.+ (m/z): 475.1844, found: 475.1815.

[0105] N-(5-aminonaphthalen-1-yl)-2-mercaptoacetamide (IIIc). To a solution of 3c (166 mg, 0.35 mmol) in DCM (1 mL), 1 mL of trifluoroacetic acid (TFA), 1,2-Ethanedithiol (EDT, 0.53 mL, 6.30 mmol) and triisopropylsilane (TIS, 1.07 mL, 5.24 mmol) were added rapidly and under stirring. The reaction mixture was stirred at room temperature for 2 hours, after which the solvents were partially evaporated using a N2 flow. Diethyl ether was added over the reaction mixture and the product was filtered off and washed with diethyl ether. The product was purified by reversed-phase flash chromatography using a mixture of MeCN+0.07% (v/v) TFA and H2O+0.1% (v/v) TFA as mobile phase (gradient: from 0% to 8% MeCN in H2O). After lyophilisation 60 mg (51% yield) of IIIc-1TFA were obtained as a white solid.

[0106] .sup.1H NMR (400 MHz, MeOH-d.sub.4): δ=7.96 (d, 1H, CH.sub.Ar), 7.88 (d, 1H, CH.sub.Ar), 7.74 (d, 1 H, CH.sub.Ar), 7.63 (t, 1H, CH.sub.Ar), 7.53 (t, 1H, CH.sub.Ar), 7.41 (d, 1H, CH.sub.Ar), 3.48 (s, 2H, CH.sub.2SH) ppm. HRMS (ESI.sup.+) calcd. for C.sub.12H.sub.12N.sub.2OS [M+H].sup.+ (m/z): 233.3035, found: 233.3015.

Synthesis of Compound IIId

[0107] ##STR00014##

[0108] N-(5-aminonaphthalen-1-yl)-3-(tritylthio)propanamide (3d). To a solution of 3-(Tritylsulfanyl)propanoic acid (500 mg, 1.43 mmol) in dry DMF (10 mL), EDC.HCl (383 mg, 2 mmol), HOBt (304 mg, 2.25 mmol) and DIPEA (0.87 mL, 5 mmol) were added. The reaction mixture was stirred during 2 min and 1,5-diaminonaphthalene (158 mg, 1 mmol) was added over the mixture. The solution was stirred at room temperature under an inert atmosphere of Ar for 48 hours, and the formation of the product was followed by TLC. The mixture was diluted with DCM, washed with saturated aqueous NaHCO3 and saturated aqueous NaCl, and dried under reduced pressure. The crude product was purified by flash chromatography using EtOAc/Hexane as eluent (from 30% to 50% EtOAc) to give 301 mg (62% yield) of 3d as a dark pink solid.

[0109] .sup.1H NMR (400 MHz, DMSO-d): δ=9.70 (s, 1H, CONH), 7.90 (d, 1 H, CH.sub.Ar), 7.54 (d, 1H, CH.sub.Ar), 7.38-7.32 (m, 12H, CH.sub.Ar), 7.30-7.17 (m, 6H, CH.sub.Ar), 6.69 (dd, 1 H, CH.sub.Ar), 5.73 (s, 2H, NH.sub.2), 2.56 (t, 2H, COCH.sub.2CH.sub.2), 2.39 (t, 2H, CH.sub.2CH.sub.2STrt) ppm. HRMS (ESI.sup.+) calcd. for C.sub.32H.sub.28N2OS [M+H].sup.+ (m/z): 489.2001, found: 489.2003.

[0110] N-(5-aminonaphthalen-1-yl)-3-mercaptopropanamide (IIId). To a solution of 3d (240 mg, 0.49 mmol) in DCM (1 mL), 1 mL of trifluoroacetic acid (TFA), 1,2-Ethanedithiol (EDT, 0.74 mL, 8.84 mmol) and triisobutylsilane (TIS, 1.52 mL, 5.89 mmol) were added rapidly and under stirring. The reaction mixture was stirred at room temperature for 2 hours, after which the solvents were partially evaporated using a N2 flow. Diethyl ether was added over the reaction mixture and the product was filtered off and washed with diethyl ether. The product was purified by reversed-phase flash chromatography using a mixture of MeCN+0.07% (v/v) TFA and H2O+0.1% (v/v) TFA as mobile phase (gradient: from 0% to 10% MeCN in H2O). After lyophilisation 107 mg (61% yield) of IIId.1TFA were obtained as a white solid.

[0111] .sup.1H NMR (400 MHz, MeOH-d.sub.4): 67 =8.01 (d, 1H, CH.sub.Ar), 7.87 (d, 1H, CH.sub.Ar), 7.69 (d, 1H, CH.sub.Ar), 7.63 (t, 1H, CH.sub.Ar), 7.52 (t, 1H, CH.sub.Ar), 7.43 (d, 1H, CH.sub.Ar), 3.28 (dt, 2H, COCH.sub.2CH.sub.2), 2.86 (d, 2H, CH.sub.2CH.sub.2SH) ppm. HRMS (ESI.sup.+) calcd. for C.sub.13H.sub.14N.sub.2OS [M+H]+ (m/z): 233.3035, found: 233.3015.

Synthesis of Compound IIIe

[0112] ##STR00015##

[0113] (R)-tert-butyl 2-(8,10-dihydropyrene-1-carboxamido)-3-(tritylthio)propanoate (3e). To a solution of pyrene carboxylic acid (250 mg, 1.06 mmol) in dry DMF (6 mL) EDC.HCl (681 mg, 3.55 mmol), HOBt (562 mg, 4.16 mmol) and DIPEA (1.25 mL, 9.13 mmol) were added. The reaction mixture was stirred during 2 min and H-Cys(Trt)-OtBu (468 mg, 1.12 mmol) was then added. The solution was stirred at room temperature under an inert atmosphere of Ar for 48 hours, and the formation of the product was followed by TLC. The mixture was diluted with DCM, washed with saturated aqueous NaHCO3 and saturated aqueous NaCl, and concentrated by distillation under reduced pressure. The crude product was purified by flash chromatography using EtOAc/Hexane as eluent (from 10% to 30% EtOAc to give 470 mg (77% yield) of 5e as a yellow solid.

[0114] .sup.1H NMR (400 MHz, CDCl-d.sub.6) δ=8.72 (d, 1H, CONHC*H), 8.32-8.08 (m, 8H, CH.sub.Ar), 7.47 (dd, 6H, CH.sub.Ar), 7.32-7.15 (m, 9H, CH.sub.Ar), 6.64 (d, 1H, CH.sub.Ar), 4.98 (dt, 1H, C*H), 2.96-2.81 13.04 (s, 1H, OH), 8.99 (d1H, CONH), 8.60 (d, 1H, CH.sub.4), (m, 2H, C*HCH.sub.2), 1.58 (s, 9H, CH.sub.3) ppm. HRMS (ESL) calcd. for C43H37NO3S [M+H]+(m/z): 647.8330, found: 647.8310.

[0115] (R)-2-(8,10-dihydropyrene-1-carboxamido)-3-mercaptopropanoic acid (IIIe). To a solution of 5e (215 mg, 0.33 mmol) in DCM (1.5 mL), 1 mL of trifluoroacetic acid (TFA), 1,2-Ethanedithiol (EDT, 0.5 mL, 5.97 mmol) and triisobutylsilane (TIS, 1.30 mL, 4.98 mmol) were added rapidly and under stirring. The reaction mixture was stirred at room temperature for 2 hours, after which the solvents were partially evaporated using a N.sub.2 flow. Diethyl ether was added over the reaction mixture and the product was filtered off and washed with diethyl ether. The product was purified by reversed-phase flash chromatography using a mixture of MeCN+0.07% (v/v) TFA and H2O+0.1% (v/v) TFA as mobile phase (gradient: from 0% to 10% MeCN in H2O). After lyophilisation 67 mg (58% yield) of 3e were obtained as a white solid.

[0116] .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ=13.04 (s, 1H, OH), 8.99 (d1H, CONH), 8.60 (d, 1H, CH.sub.Ar), 8.36 (dd. 3H, CH.sub.Ar), 8.30-8.22 (m, 3H, CH.sub.Ar), 8.16 (d, 1H, CH.sub.Ar), 8.13 (t, 1H, CH.sub.Ar), 4.71 (td, 1H, C*H), 3.14-2.89 (m, 2H.sub.2SH). 2.70 (s, 1SH) ppm. HRMS (ESI.sup.+) calcd. for C.sub.20H.sub.15NO.sub.3S [M+H].sup.+ (m/z): 350.0851, found: 350.0839.

2. General Procedure for the Preparation of DCL Sensors and Fluorescence Analysis of the DCLs

a) General Procedure for the Preparation of the DCL Sensors:

[0117] A 66.7 mM BIS-TRIS methane buffer was prepared by dissolving 1.39 g of the free amine in 100 mL of milli-Q water and adjusting the pH of the solution to 6.5 by the addition of HCl (aq). Individual concentrated stock solutions for the different building blocks (BBs) were prepared in DMSO. Mixture stock solutions containing the necessary BBs for the generation of the libraries (I, II and III) were prepared from these individual solutions. The reaction mixtures were then prepared by dilution of the stock solutions ensuring no differences in concentration between the reaction mixtures of the same batch.

[0118] Unless otherwise specified, the DCL sensors were prepared in the reaction mixtures at final concentrations of 0.1 mM for the bi- and tripodal BBs (I and II) and 0.05 mM of monopodal BB III, in a final 50 mM BIS-TRIS methane aqueous buffer (pH 6.5) with 25% DMSO.

[0119] The samples were prepared by adding 15 μL of the stock mixture in DMSO to 45 μL of the 66.7 mM buffered solution, to a final volume of 60 μL.

[0120] According to this general procedure, the following dynamic library sensors composed of a mixture of compounds were prepared:

Ia, IIa, IIIa

Ia, IIa, IIIb

Ia, IIa, IIIc

Ia, IIa, IIId

Ia, IIa, IIIe

b) General Procedure for the Fluorescence Analysis of the DCLs:

[0121] Once the oxidation of the free thiols was complete (measured by HPLC or L-MS) the reaction mixtures were analyzed by fluorescence spectroscopy.

[0122] For the DCLs performed at 0.05 mM of III, the fluorescence samples were prepared by diluting the reaction mixture to a final volume of 2060 μL with a solution of 50% 100 mM BIS-TRIS aqueous buffer and 50% DMSO, reaching a final theoretical concentration of 1.5 μM for III. The mixtures containing a different concentration of III were diluted to reach a final concentration of 1.5 μM. Therefore, for measurement purposes the samples were diluted 1:34 with H.sub.2O/DMSO (1:1 (v:v)) prior to the data acquisition. After studying the effect of the DMSO in the sensing system it was found that using a buffered solution at 50 mM with 50% DMSO to perform the measurements was optimal to avoid precipitation/aggregation processes and bleaching of the excimer signal.

[0123] After determining the different fluorescence spectra of all the DCLs, DCL formed by compounds Ia, IIa and IIIa was selected for further studies.

3. Evaluation of the Sensivity and Selectivity of the Method of the Invention

[0124] The compound IIIa presents suitable fluorescence spectra of its reduced and oxidized ([IIIa].sub.2) forms. Thus, [IIIa].sub.2 presents an excimer emission band at about 500 nm that is not present in the monomer, which is characterized by a low-wavelength band with fine structure (c.a. 350-450 nm). A library containing Ia and IIa (0.1 mM each), and IIIa (0.05 mM) minimizes the formation of [IIIa].sub.2 homodimer as read by LC-MS and fluorescence spectroscopy, with almost no excimer emission (FIG. 1, dotted line). When cysteine (IVa) is added to the reaction media the excimer band increases (FIG. 1, grey lines to black) due to the formation of [IIIa].sub.2.

[0125] The emission at 501 nm is detected after adding a Cys concentration as low as 50 μM and increasing the concentration of Cys (IVa) results in an increase of the fluorescent response (FIG. 2). This can be ratiometrically read by analyzing the excimer (501 nm) over monomer (385 nm) emission relationship. Thus, taking into account this ratio, the response to the presence of Cys goes from 1.4 (50 μM) to 3.8 (1 mM) times the blank. Considering that the normal presence of Cys in urine is ˜35 μM and the occurrence of stone-producing cystinuria starts from approximately 0.8 mM, thus this method fits within these values.

[0126] Thanks to the dynamic nature of the sensing system based on disulfide formation and exchange, presence of Cystine (CySS or IVb) behaves similar to Cys (FIG. 2A, .circle-solid. and Δ, respectively).

[0127] This ability, allows this dynamic sensor detecting cysteine in its reduced or oxidized forms in aqueous media with no need for an extra preparation step.

Selectivity

[0128] Selectivity in Front of Other Thiols such as GSH or N-acetylcysteine

[0129] The system of the invention, preferably that formed by Ia, IIa and IIIa, gives almost no response to the presence of other biologically relevant cysteine derivatives like GSH (FIG. 2A, .box-tangle-solidup.) or N-acetylcysteine (FIG. 2A, ∘), overcoming the possible incidence of false-positives.

[0130] The selectivity for IVa and IVb is illustrated in FIG. 2B by the excimer/monomer ratio plot.

Cross Reactivity

[0131] Additionally, the behavior of the dynamic sensor system in the presence of the amino acids that can be typically found in urine was also tested, either at normal or pathological concentrations.

[0132] It was started with the basic amino acids (Lys, Orn, Arg), because they also show high values in cystinuria patients. The dynamic sensor (Ia, IIa and IIIa) is able to detect IVa and IVb (FIGS. 2C and D) in the presence of these basic amino acids at concentrations in the upper limit of the expected in regular urine samples. More importantly, the sensor also provides a clear readout of IVa (Cys) in the presence of pathological concentrations of these basic amino acids (FIGS. 2E and 2F).

[0133] This highlights the robustness of the network sensor, which is able to detect the much lower concentrations of the thiol analytes in the presence of potentially competing high concentrations of basic amino acids.

[0134] Likewise, the sensor responded very efficiently to minute concentrations of both Cys and cystine in an extremely competitive media containing most of the amino acids that can be found in urine (Lys, Orn, Arg, Asn, Gly, Ala, His, Asp, β-Ala, Ser, Tyr, Met) at their physiological concentrations (FIG. 2G).

4. Measuring of Cystine Levels in Urine Samples

[0135] Once evaluated the sensitivity and the selectivity of the method, the dynamic sensing system was tested in real samples with urine from healthy volunteers.

General Procedure for the Preparation of the DCLs in Human Urine:

[0136] 300 mM BIS-TRIS methane buffer was prepared as explained above.

[0137] Individual stocks of the building blocks (BBs) Ia, IIa and IIIa in DMSO were prepared. The DCLs were prepared in the reaction mixtures at final concentrations of 0.1 mM for Ia and IIa and 0.05 mM of IIIa in a final 50 mM BIS-TRIS methane aqueous buffer urine solution (pH 6.5) with 25% DMSO.

[0138] The samples were prepared by adding 15 μL of the stock mixture to 45 μL of the 300 mM buffered urine solution (containing 35 μL of urine sample and 10 μL of 300 mM buffered solution), to a final volume of 60 μL. The system was spiked with different concentrations of Cys (IVa).

General Procedure for the Fluorescence Analysis of the DCLs:

[0139] Once the oxidation of the free thiols was complete, each reaction mixture was analyzed by fluorescence spectroscopy.

[0140] The fluorescence samples were prepared by diluting the reaction mixture to a final volume of 2060 μL with a solution of 50% 100 mM BIS-TRIS aqueous buffer and 50% DMSO, reaching a final theorical concentration of 1.5 μM for IIIa. Therefore, for measurement purposes the samples were diluted 1:34 with 1:1 H.sub.2O/DMSO (v:v) prior to the data acquisition.

[0141] Eleven samples of different volunteers were tested, and three extra measures of the combination of two samples for avoiding cross response were tested too (Table 1). The fluorescence spectra of the urine without added sensor were measured to confirm that no other metabolites in this fluid could interfere with the analysis. The positive response of the sensor to the naturally excreted cysteine was also measured.

TABLE-US-00001 TABLE 1 Selected real samples of urine from healthy volunteers used for test the sensing system described. Sample's Volunteer Number Age Gender 1 29 F 2 24 F 3 34 F 4 38 F 5 29 F 6 30 M 7 23 M 8 62 F 9 65 M 10 34 F 11 27 F 12 (1 + 6) 29 + 30 F + M 13 (2 + 7) 24 + 23 F + M 14 (8 + 9) 62 + 65 F + M

[0142] The fluorescence spectra of the urine without added sensor was carried out to confirm that no other metabolites in this fluid could interfere with the analysis (FIG. 3A, dotted line). The positive response of the sensor to the naturally excreted cysteine in the urine samples was also measured (solid black line with empty circles in FIG. 3A and U samples in FIG. 3B). Moreover, the addition of cysteine (IVa) into the sample produced the increase of the band at 501 nm, with a detection range that goes from normally occurring Cys in urine (U in FIG. 3B) to pathological concentrations (U+1.0, 2.5 mM, grey). Thus, we could also sense abnormal concentrations of cysteine that would not yet cause calculi (U+0.25, 0.5 mM, black).