ES2308932B1 - NEW FLUORESCENT CHEMICAL SENSORS FOR THE DETECTION OF CITRATE AND CITRUS ACID. - Google Patents
NEW FLUORESCENT CHEMICAL SENSORS FOR THE DETECTION OF CITRATE AND CITRUS ACID. Download PDFInfo
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- ES2308932B1 ES2308932B1 ES200701456A ES200701456A ES2308932B1 ES 2308932 B1 ES2308932 B1 ES 2308932B1 ES 200701456 A ES200701456 A ES 200701456A ES 200701456 A ES200701456 A ES 200701456A ES 2308932 B1 ES2308932 B1 ES 2308932B1
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- compound
- side chain
- phenylalanine
- radical derived
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- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 title claims abstract description 67
- KRKNYBCHXYNGOX-UHFFFAOYSA-K Citrate Chemical compound [O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O KRKNYBCHXYNGOX-UHFFFAOYSA-K 0.000 title claims abstract description 59
- 238000001514 detection method Methods 0.000 title abstract description 12
- 239000000126 substance Substances 0.000 title abstract description 7
- 150000001875 compounds Chemical class 0.000 claims abstract description 75
- 150000001413 amino acids Chemical class 0.000 claims abstract description 8
- 125000004169 (C1-C6) alkyl group Chemical group 0.000 claims abstract description 5
- 229910052783 alkali metal Inorganic materials 0.000 claims abstract description 4
- 150000001340 alkali metals Chemical class 0.000 claims abstract description 4
- 229910052757 nitrogen Inorganic materials 0.000 claims abstract description 4
- 125000004191 (C1-C6) alkoxy group Chemical group 0.000 claims abstract description 3
- 125000003118 aryl group Chemical group 0.000 claims abstract description 3
- 125000004435 hydrogen atom Chemical group [H]* 0.000 claims abstract description 3
- 229910052760 oxygen Inorganic materials 0.000 claims abstract description 3
- 125000001424 substituent group Chemical group 0.000 claims abstract description 3
- 229910052717 sulfur Inorganic materials 0.000 claims abstract description 3
- 238000000034 method Methods 0.000 claims description 45
- COLNVLDHVKWLRT-QMMMGPOBSA-N L-phenylalanine Chemical compound OC(=O)[C@@H](N)CC1=CC=CC=C1 COLNVLDHVKWLRT-QMMMGPOBSA-N 0.000 claims description 30
- COLNVLDHVKWLRT-UHFFFAOYSA-N phenylalanine Natural products OC(=O)C(N)CC1=CC=CC=C1 COLNVLDHVKWLRT-UHFFFAOYSA-N 0.000 claims description 30
- UFWIBTONFRDIAS-UHFFFAOYSA-N Naphthalene Chemical compound C1=CC=CC2=CC=CC=C21 UFWIBTONFRDIAS-UHFFFAOYSA-N 0.000 claims description 20
- 239000003446 ligand Substances 0.000 claims description 14
- 238000011088 calibration curve Methods 0.000 claims description 11
- 238000002360 preparation method Methods 0.000 claims description 10
- 238000006243 chemical reaction Methods 0.000 claims description 9
- 125000004122 cyclic group Chemical group 0.000 claims description 9
- 239000000203 mixture Substances 0.000 claims description 9
- MWPLVEDNUUSJAV-UHFFFAOYSA-N anthracene Chemical compound C1=CC=CC2=CC3=CC=CC=C3C=C21 MWPLVEDNUUSJAV-UHFFFAOYSA-N 0.000 claims description 7
- 125000001500 prolyl group Chemical group [H]N1C([H])(C(=O)[*])C([H])([H])C([H])([H])C1([H])[H] 0.000 claims description 7
- 125000006239 protecting group Chemical group 0.000 claims description 7
- 230000003213 activating effect Effects 0.000 claims description 5
- 239000003795 chemical substances by application Substances 0.000 claims description 5
- QOSSAOTZNIDXMA-UHFFFAOYSA-N Dicylcohexylcarbodiimide Chemical compound C1CCCCC1N=C=NC1CCCCC1 QOSSAOTZNIDXMA-UHFFFAOYSA-N 0.000 claims description 4
- KZSNJWFQEVHDMF-BYPYZUCNSA-N L-valine Chemical compound CC(C)[C@H](N)C(O)=O KZSNJWFQEVHDMF-BYPYZUCNSA-N 0.000 claims description 4
- SMWDFEZZVXVKRB-UHFFFAOYSA-N Quinoline Chemical compound N1=CC=CC2=CC=CC=C21 SMWDFEZZVXVKRB-UHFFFAOYSA-N 0.000 claims description 4
- KZSNJWFQEVHDMF-UHFFFAOYSA-N Valine Natural products CC(C)C(N)C(O)=O KZSNJWFQEVHDMF-UHFFFAOYSA-N 0.000 claims description 4
- DZBUGLKDJFMEHC-UHFFFAOYSA-N acridine Chemical compound C1=CC=CC2=CC3=CC=CC=C3N=C21 DZBUGLKDJFMEHC-UHFFFAOYSA-N 0.000 claims description 4
- 238000005259 measurement Methods 0.000 claims description 4
- 230000008569 process Effects 0.000 claims description 4
- BBEAQIROQSPTKN-UHFFFAOYSA-N pyrene Chemical compound C1=CC=C2C=CC3=CC=CC4=CC=C1C2=C43 BBEAQIROQSPTKN-UHFFFAOYSA-N 0.000 claims description 4
- 150000003839 salts Chemical class 0.000 claims description 4
- 239000004474 valine Substances 0.000 claims description 4
- JPVYNHNXODAKFH-UHFFFAOYSA-N Cu2+ Chemical class [Cu+2] JPVYNHNXODAKFH-UHFFFAOYSA-N 0.000 claims description 3
- NQTADLQHYWFPDB-UHFFFAOYSA-N N-Hydroxysuccinimide Chemical compound ON1C(=O)CCC1=O NQTADLQHYWFPDB-UHFFFAOYSA-N 0.000 claims description 3
- DGEZNRSVGBDHLK-UHFFFAOYSA-N [1,10]phenanthroline Chemical compound C1=CN=C2C3=NC=CC=C3C=CC2=C1 DGEZNRSVGBDHLK-UHFFFAOYSA-N 0.000 claims description 3
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 claims description 3
- 150000001879 copper Chemical class 0.000 claims description 3
- HGHJYCKSBMCGRK-UHFFFAOYSA-N 2,7-dimethylacridine-3,6-diamine Chemical compound CC1=C(N)C=C2N=C(C=C(C(C)=C3)N)C3=CC2=C1 HGHJYCKSBMCGRK-UHFFFAOYSA-N 0.000 claims description 2
- 150000001412 amines Chemical class 0.000 claims description 2
- 239000002585 base Substances 0.000 claims description 2
- GVEPBJHOBDJJJI-UHFFFAOYSA-N fluoranthrene Natural products C1=CC(C2=CC=CC=C22)=C3C2=CC=CC3=C1 GVEPBJHOBDJJJI-UHFFFAOYSA-N 0.000 claims description 2
- 229910021592 Copper(II) chloride Inorganic materials 0.000 claims 1
- 125000005577 anthracene group Chemical group 0.000 claims 1
- ORTQZVOHEJQUHG-UHFFFAOYSA-L copper(II) chloride Chemical group Cl[Cu]Cl ORTQZVOHEJQUHG-UHFFFAOYSA-L 0.000 claims 1
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 18
- 229960005190 phenylalanine Drugs 0.000 description 15
- 239000000243 solution Substances 0.000 description 14
- 239000002253 acid Substances 0.000 description 9
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 9
- 150000007942 carboxylates Chemical class 0.000 description 8
- 238000010521 absorption reaction Methods 0.000 description 7
- 239000010949 copper Substances 0.000 description 7
- 230000035945 sensitivity Effects 0.000 description 7
- 108010036824 Citrate (pro-3S)-lyase Proteins 0.000 description 6
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 6
- KWYUFKZDYYNOTN-UHFFFAOYSA-M Potassium hydroxide Chemical compound [OH-].[K+] KWYUFKZDYYNOTN-UHFFFAOYSA-M 0.000 description 6
- 229940024606 amino acid Drugs 0.000 description 5
- 238000004458 analytical method Methods 0.000 description 5
- 239000003814 drug Substances 0.000 description 5
- 239000000047 product Substances 0.000 description 5
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 4
- 238000005481 NMR spectroscopy Methods 0.000 description 4
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 4
- 230000008901 benefit Effects 0.000 description 4
- 238000006073 displacement reaction Methods 0.000 description 4
- 235000013305 food Nutrition 0.000 description 4
- 239000002904 solvent Substances 0.000 description 4
- VZCYOOQTPOCHFL-UHFFFAOYSA-N trans-butenedioic acid Natural products OC(=O)C=CC(O)=O VZCYOOQTPOCHFL-UHFFFAOYSA-N 0.000 description 4
- 108090000790 Enzymes Proteins 0.000 description 3
- 102000004190 Enzymes Human genes 0.000 description 3
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 3
- 208000000913 Kidney Calculi Diseases 0.000 description 3
- 206010029148 Nephrolithiasis Diseases 0.000 description 3
- 230000004913 activation Effects 0.000 description 3
- 229910052802 copper Inorganic materials 0.000 description 3
- -1 copper halide Chemical class 0.000 description 3
- 238000003745 diagnosis Methods 0.000 description 3
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 3
- 238000002330 electrospray ionisation mass spectrometry Methods 0.000 description 3
- 230000001575 pathological effect Effects 0.000 description 3
- 238000007430 reference method Methods 0.000 description 3
- 238000010992 reflux Methods 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 239000007787 solid Substances 0.000 description 3
- QTBSBXVTEAMEQO-UHFFFAOYSA-M Acetate Chemical compound CC([O-])=O QTBSBXVTEAMEQO-UHFFFAOYSA-M 0.000 description 2
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- FEWJPZIEWOKRBE-JCYAYHJZSA-N Dextrotartaric acid Chemical compound OC(=O)[C@H](O)[C@@H](O)C(O)=O FEWJPZIEWOKRBE-JCYAYHJZSA-N 0.000 description 2
- VZCYOOQTPOCHFL-OWOJBTEDSA-N Fumaric acid Chemical compound OC(=O)\C=C\C(O)=O VZCYOOQTPOCHFL-OWOJBTEDSA-N 0.000 description 2
- JVTAAEKCZFNVCJ-UHFFFAOYSA-M Lactate Chemical compound CC(O)C([O-])=O JVTAAEKCZFNVCJ-UHFFFAOYSA-M 0.000 description 2
- OFOBLEOULBTSOW-UHFFFAOYSA-L Malonate Chemical compound [O-]C(=O)CC([O-])=O OFOBLEOULBTSOW-UHFFFAOYSA-L 0.000 description 2
- ONIBWKKTOPOVIA-UHFFFAOYSA-N Proline Natural products OC(=O)C1CCCN1 ONIBWKKTOPOVIA-UHFFFAOYSA-N 0.000 description 2
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 2
- 230000002159 abnormal effect Effects 0.000 description 2
- 238000002835 absorbance Methods 0.000 description 2
- WPYMKLBDIGXBTP-UHFFFAOYSA-N benzoic acid Chemical compound OC(=O)C1=CC=CC=C1 WPYMKLBDIGXBTP-UHFFFAOYSA-N 0.000 description 2
- 125000001584 benzyloxycarbonyl group Chemical group C(=O)(OCC1=CC=CC=C1)* 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 125000002915 carbonyl group Chemical group [*:2]C([*:1])=O 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 150000001860 citric acid derivatives Chemical class 0.000 description 2
- 238000011156 evaluation Methods 0.000 description 2
- 238000009472 formulation Methods 0.000 description 2
- JFCQEDHGNNZCLN-UHFFFAOYSA-N glutaric acid Chemical compound OC(=O)CCCC(O)=O JFCQEDHGNNZCLN-UHFFFAOYSA-N 0.000 description 2
- 150000004820 halides Chemical group 0.000 description 2
- 229940049920 malate Drugs 0.000 description 2
- VZCYOOQTPOCHFL-UPHRSURJSA-N maleic acid Chemical compound OC(=O)\C=C/C(O)=O VZCYOOQTPOCHFL-UPHRSURJSA-N 0.000 description 2
- BJEPYKJPYRNKOW-UHFFFAOYSA-N malic acid Chemical compound OC(=O)C(O)CC(O)=O BJEPYKJPYRNKOW-UHFFFAOYSA-N 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 230000007246 mechanism Effects 0.000 description 2
- 230000004060 metabolic process Effects 0.000 description 2
- UYWQUFXKFGHYNT-UHFFFAOYSA-N phenylmethyl ester of formic acid Natural products O=COCC1=CC=CC=C1 UYWQUFXKFGHYNT-UHFFFAOYSA-N 0.000 description 2
- XNGIFLGASWRNHJ-UHFFFAOYSA-L phthalate(2-) Chemical compound [O-]C(=O)C1=CC=CC=C1C([O-])=O XNGIFLGASWRNHJ-UHFFFAOYSA-L 0.000 description 2
- 208000023958 prostate neoplasm Diseases 0.000 description 2
- 238000011002 quantification Methods 0.000 description 2
- 239000011734 sodium Substances 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- 239000001509 sodium citrate Substances 0.000 description 2
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 description 2
- 238000001228 spectrum Methods 0.000 description 2
- KDYFGRWQOYBRFD-UHFFFAOYSA-L succinate(2-) Chemical compound [O-]C(=O)CCC([O-])=O KDYFGRWQOYBRFD-UHFFFAOYSA-L 0.000 description 2
- 229940095064 tartrate Drugs 0.000 description 2
- RRONHWAVOYADJL-HNNXBMFYSA-N (2s)-3-phenyl-2-(phenylmethoxycarbonylamino)propanoic acid Chemical compound C([C@@H](C(=O)O)NC(=O)OCC=1C=CC=CC=1)C1=CC=CC=C1 RRONHWAVOYADJL-HNNXBMFYSA-N 0.000 description 1
- MXZANEWAFZMPKW-UHFFFAOYSA-O 1h-imidazol-3-ium-3-amine Chemical compound NN1C=C[NH+]=C1 MXZANEWAFZMPKW-UHFFFAOYSA-O 0.000 description 1
- IYGWEQDZEJYALU-UHFFFAOYSA-N 2-amino-N-anthracen-1-yl-3-phenylpropanamide Chemical compound C=1C=CC2=CC3=CC=CC=C3C=C2C=1NC(=O)C(N)CC1=CC=CC=C1 IYGWEQDZEJYALU-UHFFFAOYSA-N 0.000 description 1
- KXDHJXZQYSOELW-UHFFFAOYSA-M Carbamate Chemical compound NC([O-])=O KXDHJXZQYSOELW-UHFFFAOYSA-M 0.000 description 1
- 241000207199 Citrus Species 0.000 description 1
- RYGMFSIKBFXOCR-UHFFFAOYSA-N Copper Chemical compound [Cu] RYGMFSIKBFXOCR-UHFFFAOYSA-N 0.000 description 1
- 229910021591 Copper(I) chloride Inorganic materials 0.000 description 1
- 238000005033 Fourier transform infrared spectroscopy Methods 0.000 description 1
- 238000001157 Fourier transform infrared spectrum Methods 0.000 description 1
- 206010060862 Prostate cancer Diseases 0.000 description 1
- 208000000236 Prostatic Neoplasms Diseases 0.000 description 1
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 150000007513 acids Chemical class 0.000 description 1
- 239000004480 active ingredient Substances 0.000 description 1
- 238000007792 addition Methods 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 125000006242 amine protecting group Chemical group 0.000 description 1
- 125000003277 amino group Chemical group 0.000 description 1
- 239000012491 analyte Substances 0.000 description 1
- YUENFNPLGJCNRB-UHFFFAOYSA-N anthracen-1-amine Chemical compound C1=CC=C2C=C3C(N)=CC=CC3=CC2=C1 YUENFNPLGJCNRB-UHFFFAOYSA-N 0.000 description 1
- 239000003146 anticoagulant agent Substances 0.000 description 1
- 229940127219 anticoagulant drug Drugs 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 125000001797 benzyl group Chemical group [H]C1=C([H])C([H])=C(C([H])=C1[H])C([H])([H])* 0.000 description 1
- 235000013361 beverage Nutrition 0.000 description 1
- 238000012742 biochemical analysis Methods 0.000 description 1
- 239000004202 carbamide Substances 0.000 description 1
- 150000004649 carbonic acid derivatives Chemical class 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 235000020971 citrus fruits Nutrition 0.000 description 1
- 230000015271 coagulation Effects 0.000 description 1
- 238000005345 coagulation Methods 0.000 description 1
- 238000010668 complexation reaction Methods 0.000 description 1
- 150000004699 copper complex Chemical class 0.000 description 1
- OXBLHERUFWYNTN-UHFFFAOYSA-M copper(I) chloride Chemical compound [Cu]Cl OXBLHERUFWYNTN-UHFFFAOYSA-M 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 239000012738 dissolution medium Substances 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 235000013399 edible fruits Nutrition 0.000 description 1
- 238000000921 elemental analysis Methods 0.000 description 1
- 238000004993 emission spectroscopy Methods 0.000 description 1
- 150000002148 esters Chemical class 0.000 description 1
- 230000005284 excitation Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- 239000000706 filtrate Substances 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 238000012921 fluorescence analysis Methods 0.000 description 1
- 238000001506 fluorescence spectroscopy Methods 0.000 description 1
- 238000002189 fluorescence spectrum Methods 0.000 description 1
- 239000007789 gas Substances 0.000 description 1
- 238000004817 gas chromatography Methods 0.000 description 1
- 150000004677 hydrates Chemical class 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-M hydroxide Chemical compound [OH-] XLYOFNOQVPJJNP-UHFFFAOYSA-M 0.000 description 1
- 150000007529 inorganic bases Chemical class 0.000 description 1
- 238000004811 liquid chromatography Methods 0.000 description 1
- 238000004949 mass spectrometry Methods 0.000 description 1
- 239000002207 metabolite Substances 0.000 description 1
- 229910052751 metal Inorganic materials 0.000 description 1
- 239000002184 metal Substances 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 150000002902 organometallic compounds Chemical class 0.000 description 1
- 125000002524 organometallic group Chemical group 0.000 description 1
- 239000000825 pharmaceutical preparation Substances 0.000 description 1
- 229920005646 polycarboxylate Polymers 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 239000003755 preservative agent Substances 0.000 description 1
- 230000002335 preservative effect Effects 0.000 description 1
- BDERNNFJNOPAEC-UHFFFAOYSA-N propan-1-ol Chemical compound CCCO BDERNNFJNOPAEC-UHFFFAOYSA-N 0.000 description 1
- 238000003908 quality control method Methods 0.000 description 1
- 239000011541 reaction mixture Substances 0.000 description 1
- 238000001953 recrystallisation Methods 0.000 description 1
- 235000021067 refined food Nutrition 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 238000009666 routine test Methods 0.000 description 1
- 235000017557 sodium bicarbonate Nutrition 0.000 description 1
- 229910000030 sodium bicarbonate Inorganic materials 0.000 description 1
- 238000004611 spectroscopical analysis Methods 0.000 description 1
- 239000003381 stabilizer Substances 0.000 description 1
- 239000007858 starting material Substances 0.000 description 1
- JDVPQXZIJDEHAN-UHFFFAOYSA-M succinamate Chemical compound NC(=O)CCC([O-])=O JDVPQXZIJDEHAN-UHFFFAOYSA-M 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 230000001131 transforming effect Effects 0.000 description 1
- 238000000870 ultraviolet spectroscopy Methods 0.000 description 1
- 238000002371 ultraviolet--visible spectrum Methods 0.000 description 1
- 210000002700 urine Anatomy 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
- 229910052724 xenon Inorganic materials 0.000 description 1
- FHNFHKCVQCLJFQ-UHFFFAOYSA-N xenon atom Chemical compound [Xe] FHNFHKCVQCLJFQ-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C237/00—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups
- C07C237/02—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups having the carbon atoms of the carboxamide groups bound to acyclic carbon atoms of the carbon skeleton
- C07C237/04—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups having the carbon atoms of the carboxamide groups bound to acyclic carbon atoms of the carbon skeleton the carbon skeleton being acyclic and saturated
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
- C07F1/00—Compounds containing elements of Groups 1 or 11 of the Periodic Table
- C07F1/08—Copper compounds
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/76—Chemiluminescence; Bioluminescence
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C2603/00—Systems containing at least three condensed rings
- C07C2603/02—Ortho- or ortho- and peri-condensed systems
- C07C2603/04—Ortho- or ortho- and peri-condensed systems containing three rings
- C07C2603/22—Ortho- or ortho- and peri-condensed systems containing three rings containing only six-membered rings
- C07C2603/24—Anthracenes; Hydrogenated anthracenes
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
- G01N2021/7769—Measurement method of reaction-produced change in sensor
- G01N2021/7786—Fluorescence
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- Plasma & Fusion (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Analytical Chemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Engineering & Computer Science (AREA)
- Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
Abstract
Nuevos sensores químicos fluorescentes para la detección de citrato y ácido cítrico.New fluorescent chemical sensors for citrate and citric acid detection.
Los compuestos de fórmula (I), donde R_{1} es un radical de una cadena lateral de un aminoácido natural; y R_{2} es un C-radical derivado de uno de los sistemas de anillo conocidos de 1-5 anillos; siendo los anillos aromáticos, aislados o parcialmente/totalmente fusionados y teniendo 5-6 miembros; siendo cada miembro independientemente seleccionado entre C, CH, N, NH, O y S; estando uno o más de los átomos de hidrógeno de estos miembros opcionalmente sustituido por sustituyentes seleccionados entre (C_{1}-C_{6})-alquilo y (C_{1}-C_{6})-alcoxilo; NH_{2}; F, Cl, Br; COOM donde M es un metal alcalino o alcalinotérreo; y OH; n es 0 ó 1; m es un entero entre 1 y 2; son sensores fluorescentes selectivos de citrato/ácido cítrico que proporcionan un límite de detección mejorado.The compounds of formula (I), where R 1 is a radical of a side chain of a natural amino acid; and R2 it is a C-radical derived from one of the systems of known ring of 1-5 rings; being the rings aromatic, isolated or partially / fully fused and having 5-6 members; being each member independently selected from C, CH, N, NH, O and S; being one or more of the hydrogen atoms of these members optionally replaced by substituents selected from (C 1 -C 6) - alkyl and (C 1 -C 6) -alkoxy; NH2; F, Cl, Br; COOM where M is an alkali metal or alkaline earth; and OH; n is 0 or 1; m is an integer between 1 and 2; They are selective citrate / citric acid fluorescent sensors that They provide an improved detection limit.
Description
Nuevos sensores químicos fluorescentes para la detección de citrato y ácido cítrico.New fluorescent chemical sensors for citrate and citric acid detection.
La presente invención se refiere al campo del análisis químico y bioquímico. Más particularmente la presente invención se refiere a nuevos compuestos organometálicos y a su uso como sensores químicos.The present invention relates to the field of chemical and biochemical analysis. More particularly the present invention refers to new organometallic compounds and their use as chemical sensors
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Entre las técnicas empleadas para el análisis de sustancias en disolución se encuentran la espectrometría de masas, frecuentemente acoplada a la cromatografía de gases y de líquidos, y técnicas espectrofotométricas, principalmente de absorción UV-Vis. Más recientemente, se han desarrollado métodos de análisis basados en técnicas mucho más sensibles como es la espectroscopia de fluorescencia o de emisión, del tipo de estado estacionario y resuelta en el tiempo. Estas técnicas basadas en fluorescencia han resultado ser herramientas potentes para la investigación y el análisis en sectores tales como biomedicina, farmacia, medioambiente o la industria de la alimentación.Among the techniques used for the analysis of substances in solution are mass spectrometry, frequently coupled to gas and liquid chromatography, and spectrophotometric techniques, mainly absorption UV-Vis. More recently, they have developed analysis methods based on much more sensitive techniques such as fluorescence or emission spectroscopy, of the type of state stationary and resolved in time. These techniques based on fluorescence have turned out to be powerful tools for the research and analysis in sectors such as biomedicine, pharmacy, environment or the food industry.
El análisis de fluorescencia puede proporcionar información de la estructura, movilidad, tamaño macromolecular, distancias o movimientos conformacionales de las moléculas enlazadas a los fluoróforos, gracias a que el proceso de emisión de fluorescencia se da en una escala de tiempo comprendida entre nanosegundos y milisegundos, dependiendo del sistema de fluorescencia utilizado.Fluorescence analysis can provide structure information, mobility, macromolecular size, distances or conformational movements of molecules linked to fluorophores, thanks to the process of emission of fluorescence occurs on a time scale between nanoseconds and milliseconds, depending on the system of fluorescence used.
Para la utilización de dichas técnicas de fluorescencia se han desarrollado un gran número de sensores fluorescentes o quimiosensores fluorogénicos, capaces de detectar y cuantificar, en muy bajas concentraciones, diferentes analitos (cfr. R. Martínez-Máñez et al., Chem. Rev. 2003, vol. 103, pp. 4419-4476; J. F. Callan et al., Tetrahedron, 2005, vol. 61, pp. 8551-8588; A. P. de Silva et al., Chem. Rev. 1997, vol. 97, pp.1515-1566; y P.D. Beer et al., Anqew. Chem. Int. Ed. 2001, vol. 40, pp. 486-516).For the use of these fluorescence techniques, a large number of fluorescent sensors or fluorogenic chemosensors have been developed, capable of detecting and quantifying, at very low concentrations, different analytes (cf. R. Martínez-Máñez et al., Chem. Rev. 2003, vol. 103, pp. 4419-4476; JF Callan et al., Tetrahedron , 2005, vol. 61, pp. 8551-8588; AP de Silva et al., Chem. Rev. 1997, vol. 97, pp .1515-1566; and PD Beer et al., Anqew. Chem. Int. Ed. 2001, vol. 40, pp. 486-516).
Entre los analitos de mayor interés se encuentran los carboxilatos y los policarboxilatos, más particularmente el citrato/ácido cítrico, dado el importante papel que juega en el metabolismo de los seres vivos y su utilidad como componente de numerosos preparados farmacéuticos, alimentos, bebidas y diversos productos industriales.Among the analytes of greatest interest are find carboxylates and polycarboxylates, more particularly citrate / citric acid, given the important role which plays in the metabolism of living beings and their usefulness as component of numerous pharmaceutical preparations, food, Beverages and various industrial products.
El citrato/ácido cítrico se utiliza como conservante y acidulante y es necesaria su determinación en frutas y en los productos de alimentación procesados industrialmente; en la industria farmacéutica, donde el citrato/ácido cítrico se utiliza como estabilizante de gran variedad de formulaciones farmacéuticas, como contra-anión para la preparación de principios activos, y en forma de ácido se utiliza en formulaciones efervescentes en combinación con carbonatos y bicarbonatos.Citrate / citric acid is used as preservative and acidulant and its determination in fruits is necessary and in industrially processed food products; in the pharmaceutical industry, where citrate / citric acid is used as a stabilizer of a variety of formulations pharmaceuticals, as counter-anion for preparation of active ingredients, and in the form of acid is used in effervescent formulations in combination with carbonates and baking soda
Dada la importancia del citrato/ácido cítrico en el metabolismo de los seres vivos, su determinación es una importante cuestión en investigación biomédica o bioquímica y en medicina clínica. La determinación del ácido cítrico y de sus metabolitos relacionados es de gran utilidad en la investigación del funcionamiento celular y en la detección de niveles patológicos de dichos analitos como elemento de diagnóstico. La determinación del citrato/ácido cítrico, se utiliza para el diagnóstico de diversos desordenes y condiciones patológicas relacionadas con concentraciones anómalas de ácido cítrico/citrato, por ejemplo para la detección de procesos cancerosos como tumores de próstata o para la evaluación de la probabilidad de sufrir cálculos renales, entre otros. Otros usos del citrato en medicina incluyen su utilización como anticoagulante, tanto en experimentos de coagulación como en la conservación de los bancos de sangre.Given the importance of citrate / citric acid in the metabolism of living beings, its determination is a important issue in biomedical or biochemical research and in clinical medicine The determination of citric acid and its Related metabolites is very useful in research of cellular functioning and in the detection of pathological levels of said analytes as a diagnostic element. The determination of citrate / citric acid, is used for the diagnosis of various disorders and pathological conditions related to abnormal concentrations of citric acid / citrate, for example for the detection of cancerous processes such as prostate tumors or for the evaluation of the probability of suffering kidney stones, between others. Other uses of citrate in medicine include its use as an anticoagulant, both in coagulation experiments and in the conservation of blood banks.
La determinación de ácido cítrico en medicina se lleva a cabo mediante resonancia magnética nuclear (en el caso del cáncer de próstata) o mediante el método de la citrato liasa (en el caso de los cálculos renales). Adicionalmente, el método conocido de la citrato liasa implica varios pasos, siendo el más largo la reacción entre las enzimas y el ácido cítrico presente en la muestra. Generalmente se ha de esperar de 15 a 20 minutos antes de medir.The determination of citric acid in medicine is carried out by nuclear magnetic resonance (in the case of prostate cancer) or by the citrate lyase method (in the case of kidney stones). Additionally, the known method of citrate lyase involves several steps, the longest being the reaction between enzymes and citric acid present in the sample. Generally, wait 15-20 minutes before to size.
En el estado de la técnica se conocen una variedad de sensores fluorescentes para la determinación de carboxilatos, generalmente complejos organometálicos que incluyen grupos fluorescentes o potencialmente fluorescentes. Una aproximación se basa en el diseño de sensores que presentan una especificidad geométrica con el analito. Así, L. A. Cabell et al. describen un sensor fluorescente que consiste en Cu (II) coordinado a un ligando 1,10-fenantrolina que está unido a un receptor bis(aminoimidazolio) (cfr. J.Chem. Soc., Perkin Trans. 2, 2001, pp. 315-323). C. Schmuck et al. describen un receptor guanidiniocarbonil pirrol tricatiónico (cfr. J. Am. Chem. Soc. 2005, vol.127, pp. 3373-3379). También L. Fabrizzi et al. describen receptores trifurcados que contienen tres subunidades de ciclama-cobre (II) útiles como sensores para carboxilatos, en particular para citratos (cfr. Org. Lett. 2005, vol 7, pp. 2603-2606). Desafortunadamente, se trata de sensores estructuralmente complejos que implican procedimientos de preparación largos y difíciles de llevar a cabo a escala industrial y con rendimientos globales no superiores al 10% en la mayoría de los casos, lo que implica un aumento del coste del sensor. Además, el mecanismo de actuación de dichos sensores se basa en una "reacción de desplazamiento" de un colorante o indicador previamente unido al receptor, el cual al ser desplazado señaliza la presencia del citrato. Dicho mecanismo es complejo (a: complejación del indicador por el receptor y b: desplazamiento por el citrato) y por tanto susceptible a sufrir interferencias.A variety of fluorescent sensors for the determination of carboxylates, generally organometallic complexes including fluorescent or potentially fluorescent groups, are known in the state of the art. An approach is based on the design of sensors that have a geometric specificity with the analyte. Thus, LA Cabell et al . describe a fluorescent sensor consisting of Cu (II) coordinated to a 1,10-phenanthroline ligand that is bound to a bis (aminoimidazolium) receptor (cf. J.Chem. Soc., Perkin Trans. 2 , 2001, pp. 315 -323). C. Schmuck et al. describe a tricationic guanidiniumcarbonyl pyrrole receptor (cf. J. Am. Chem. Soc. 2005, vol.127, pp. 3373-3379). Also L. Fabrizzi et al . describe trifurcated receptors containing three cyclo-copper (II) subunits useful as carboxylate sensors, in particular for citrates (cf. Org. Lett. 2005, vol 7, pp. 2603-2606). Unfortunately, these are structurally complex sensors that involve long and difficult preparation procedures to be carried out on an industrial scale and with overall yields not exceeding 10% in most cases, which implies an increase in the cost of the sensor. In addition, the actuation mechanism of said sensors is based on a "displacement reaction" of a dye or indicator previously attached to the receptor, which when displaced signals the presence of citrate. This mechanism is complex (a: complexation of the indicator by the receptor and b: displacement by the citrate) and therefore susceptible to interference.
El método actualmente de referencia para la determinación de citrato y ácido cítrico emplea la citrato liasa como enzima que desencadena una serie de reacciones medibles por absorción (cfr. European Standard EN 1137, dec. 1994). Este método de referencia no es adecuado para las aplicaciones en que se necesita una sensibilidad y selectividad altas.The currently reference method for citrate and citric acid determination employs citrate lyase as an enzyme that triggers a series of measurable reactions by absorption (cf. European Standard EN 1137, dec. 1994). This method reference is not suitable for applications in which It needs high sensitivity and selectivity.
Así, de lo que se conoce en el estado de la técnica, se deduce que sería deseable encontrar nuevos sensores de citratos que sean selectivos, con un límite de detección bajo.Thus, of what is known in the state of the technique, it follows that it would be desirable to find new sensors citrates that are selective, with a low detection limit.
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Los inventores han encontrado un grupo de nuevos compuestos de formula (I) que pueden actuar como sensores selectivos de citrato y que proporcionan un límite de detección mejorado. Adicionalmente presentan la ventaja de que su procedimiento de preparación es breve y sencillo y procede con rendimientos globales altos.The inventors have found a group of new compounds of formula (I) that can act as sensors citrate selective and that provide a limit of detection improved. Additionally they have the advantage that their Preparation procedure is short and simple and proceeds with high overall yields
Así en un primer aspecto de la presente invención se refiere a los compuestos de fórmula (I),So in a first aspect of the present invention refers to the compounds of formula (I),
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donde: R_{1} es un radical de una cadena lateral de un aminoácido natural; y R_{2} es un C-radical derivado de uno de los sistemas de anillo conocidos de 1-5 anillos; siendo los anillos aromáticos, aislados o parcialmente/totalmente fusionados y teniendo 5-6 miembros; siendo cada miembro independientemente seleccionado entre C, CH, N, NH, O y S; estando uno o más de los átomos de hidrógeno de estos miembros opcionalmente sustituido por sustituyentes seleccionados entre el grupo formado por (C_{1}-C_{6})-alquilo y (C_{1}-C_{6})-alcoxilo; NH_{2}; F, Cl, Br; COOM donde M es un metal alcalino o alcalinotérreo; y OH; n es 0 ó 1; m es un entero entre 1 y 2; con la condición de que cuando R_{1} es una cadena lateral de la prolina, m es 1, y R_{1} junto al C al que está unido y el NH forman conjuntamente un ciclo de 5 miembros.where: R_ {1} is a radical of a side chain of a natural amino acid; and R2 is a C-radical derived from one of the ring systems known of 1-5 rings; being the rings aromatic, isolated or partially / fully fused and having 5-6 members; being each member independently selected from C, CH, N, NH, O and S; being one or more of the hydrogen atoms of these members optionally substituted by substituents selected from the group formed by (C 1 -C 6) - alkyl and (C 1 -C 6) -alkoxy; NH2; F, Cl, Br; COOM where M is an alkali metal or alkaline earth; and OH; n is 0 or 1; m is an integer between 1 and 2; with the condition that when R_ {1} is a side chain of the proline, m is 1, and R1 next to the C to which it is attached and the NH together they form a cycle of 5 members.
En la presente invención por cadena lateral de un aminoácido natural se entiende la cadena en la posición a respecto al grupo amino y al grupo carboxilo de un aminoácido existente en la naturaleza. En la siguiente tabla se muestra el nombre, la abreviatura y la cadena lateral R_{1} de los aminoácidos naturales más importantes:In the present invention by side chain of a natural amino acid means the chain at position a with respect to the amino group and the carboxyl group of an amino acid existing in nature. The following table shows the name, abbreviation and side chain R_ {1} of the most important natural amino acids:
En el caso de la prolina, la cadena lateral R_{1} junto al C al que está unida y el NH forman conjuntamente un ciclo de 5 miembros.In the case of proline, the side chain R1 next to the C to which it is attached and the NH together a cycle of 5 members.
En una realización preferida de la invención, el compuesto de fórmula (I) es aquél donde R_{1} es la cadena lateral de un aminoácido seleccionado entre fenilalanina y valina.In a preferred embodiment of the invention, the compound of formula (I) is that where R_ {1} is the side chain of an amino acid selected from phenylalanine and valine.
En otra realización preferida, el compuesto de fórmula (I) es aquél donde R_{2} es un C-radical derivado de un sistema de anillo de 2 a 4 anillos. En otra realización todavía más preferida, el sistema de anillo se selecciona entre naftaleno, antraceno, acridina, pireno, 2,4,6-trifenil-pirilio, quinolina, 1,10-fenantrolina, estando el sistema de anillo opcionalmente sustituido por (C_{1}-C_{6})-alquilo o NH_{2}. En otra realización todavía más preferida, el sistema de anillo es el antraceno.In another preferred embodiment, the compound of formula (I) is one where R2 is a C-radical derived from a ring system of 2 to 4 rings. In other even more preferred embodiment, the ring system is select between naphthalene, anthracene, acridine, pyrene, 2,4,6-triphenyl-pyrilium, quinoline, 1,10-phenanthroline, the ring system being optionally substituted by (C 1 -C 6) - alkyl or NH2. In another even more preferred embodiment, the system of Ring is the anthracene.
Los compuestos más preferidos son los de la siguiente lista:The most preferred compounds are those of the following list:
(a) compuesto (I) con R_{1}= cadena lateral de fenilalanina; R_{2}= C-radical derivado de 1-antraceno; m= 2; y n=0;(a) compound (I) with R1 = side chain of phenylalanine; R2 = C-radical derived from 1-anthracene; m = 2; and n = 0;
(b) compuesto (I) con R_{1}= cadena lateral de valina; R_{2}= C-radical derivado de 1-antraceno; m=2; y n=0;(b) compound (I) with R1 = side chain of valine R2 = C-radical derived from 1-anthracene; m = 2; and n = 0;
(c) compuesto (I) con R_{1}= cadena lateral de fenilalanina; R_{2}= C-radical derivado de 1-naftaleno; m=2; y n=0;(c) compound (I) with R1 = side chain of phenylalanine; R2 = C-radical derived from 1-naphthalene; m = 2; and n = 0;
(d) compuesto (I) con R_{1}= cadena lateral de fenilalanina; R_{2}= C-radical derivado de 2-naftaleno; m=2; y n=0;(d) compound (I) with R1 = side chain of phenylalanine; R2 = C-radical derived from 2-naphthalene; m = 2; and n = 0;
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(e) compuesto (I) con R_{1}=cadena lateral de fenilalanina; R_{2}= C-radical derivado de 1-naftaleno; m=2; y n=1;(e) compound (I) with R1 = side chain of phenylalanine; R2 = C-radical derived from 1-naphthalene; m = 2; and n = 1;
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(f) compuesto (I) con R_{1}=cadena lateral de fenilalanina; R_{2}= C-radical derivado de 2-naftaleno; m=2; y n=1;(f) compound (I) with R1 = side chain of phenylalanine; R2 = C-radical derived from 2-naphthalene; m = 2; and n = 1;
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(g) compuesto (I) con R_{1}=cadena lateral de fenilalanina; R_{2}= C-radical derivado de 2-antraceno; m=2; y n=0(g) compound (I) with R1 = side chain of phenylalanine; R2 = C-radical derived from 2-anthracene; m = 2; and n = 0
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(h) compuesto (I) con R_{1}=cadena lateral de fenilalanina; R_{2}= C-radical derivado de 1-antraceno; m=2; y n=1(h) compound (I) with R1 = side chain of phenylalanine; R2 = C-radical derived from 1-anthracene; m = 2; and n = 1
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(i) compuesto (I) con R_{1}=cadena lateral de fenilalanina; R_{2}= C-radical derivado de 2-antraceno; m=2; y n=1(i) compound (I) with R1 = side chain of phenylalanine; R2 = C-radical derived from 2-anthracene; m = 2; and n = 1
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(j) compuesto (I) con R_{1}=cadena lateral de fenilalanina; R_{2}= C-radical derivado de 10-antraceno; m=2; y n=1(j) compound (I) with R1 = side chain of phenylalanine; R2 = C-radical derived from 10-anthracene; m = 2; and n = 1
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(k) compuesto (I) con R_{1}=cadena lateral de fenilalanina; R_{2}= C-radical derivado de 1-pireno; m=2; y n=1(k) compound (I) with R1 = side chain of phenylalanine; R2 = C-radical derived from 1-pyrene; m = 2; and n = 1
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(l) compuesto (I) con R_{1}=cadena lateral de fenilalanina; R_{2}= C-radical derivado de 1-pireno; m=2; y n=0(l) compound (I) with R1 = side chain of phenylalanine; R2 = C-radical derived from 1-pyrene; m = 2; and n = 0
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(m) compuesto (I) con R_{1}=cadena lateral de fenilalanina; R_{2}= C-radical derivado de 2,4,6-trifenil-pirilio; m=2; y n=0(m) compound (I) with R1 = side chain of phenylalanine; R2 = C-radical derived from 2,4,6-triphenyl-pyrilium; m = 2; Y n = 0
(n) compuesto (I) con R_{1}=cadena lateral de fenilalanina; R_{2}= C-radical derivado de 2,4,6-trifenil-pirilio; m=2; y n=0(n) compound (I) with R1 = side chain of phenylalanine; R2 = C-radical derived from 2,4,6-triphenyl-pyrilium; m = 2; Y n = 0
(o) compuesto (I) con R_{1}=cadena lateral de fenilalanina; R_{2}= C-radical derivado de 3,6-diamino-2,7-dimetilacridina; m=2; y n=0(o) compound (I) with R1 = side chain of phenylalanine; R2 = C-radical derived from 3,6-diamino-2,7-dimethylacridine; m = 2; and n = 0
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Compuestos de fórmula (I) todavía más preferidos son el compuesto de fórmula (a) y el compuesto de fórmula (b).Compounds of formula (I) still more preferred they are the compound of formula (a) and the compound of formula (b).
Tal como se ha mencionado anteriormente, los compuestos de la presente invención presentan la ventaja de que pueden prepararse mediante procedimientos de preparación sencillos con pocos pasos sintéticos y con rendimientos globales altos. Esto conlleva una disminución del coste de dichos compuestos gracias a un gasto inferior de reactivos, disolventes y mano de obra, así como una mayor facilidad para su fabricación a nivel industrial.As mentioned above, the Compounds of the present invention have the advantage that can be prepared by simple preparation procedures with few synthetic steps and high overall yields. This entails a decrease in the cost of said compounds thanks to a lower cost of reagents, solvents and labor as well as a greater facility for manufacturing at the level industrial.
Así, un aspecto adicional de la presente invención es proporcionar un procedimiento de preparación del complejo de Cu (II) de fórmula (I) que comprende hacer reaccionar al menos dos equivalentes de un ligando de fórmula (II) o una sal del mismo,Thus, an additional aspect of the present invention is to provide a method of preparing the Cu (II) complex of formula (I) comprising reacting at least two equivalents of a ligand of formula (II) or a salt of the same,
donde R_{1}, R_{2}, m, y n tienen los valores definidos en la fórmula (I) incluyendo que cuando R_{1} es una cadena lateral de la prolina, m es 1, y R_{1} junto al C al que está unido y el NH forman conjuntamente un ciclo de 5 miembros, con una sal de cobre (II) o un hidrato de la misma. Más preferiblemente, la sal de cobre es un haluro de cobre. Todavía más preferiblemente, el haluro de cobre es CuCl_{2} o uno de sus hidratos.where R_ {1}, R2, m, and n have the values defined in formula (I) including that when R_ {1} is a proline side chain, m is 1, and R1 next to the C to which it is attached and the NH together a 5-member cycle, with a copper (II) salt or a hydrate of the same. More preferably, the copper salt is a halide of copper. Even more preferably, the copper halide is CuCl_2 or one of its hydrates
Generalmente, los complejos metálicos de formula (I) se preparan con dos equivalente del ligando (L) o de una sal del mismo y un equivalente de sal de cobre. Normalmente, la reacción se lleva a cabo en presencia de un disolvente apropiado como por ejemplo agua, metanol, o mezclas de los mismos y en presencia de una base inorgánica, por ejemplo, un hidróxido de metal alcalino tal como hidróxido potásico, y a una temperatura comprendida entre 0°C y la temperatura de reflujo del disolvente utilizado.Generally, the metal complexes of formula (I) are prepared with two equivalent of the ligand (L) or a salt thereof and an equivalent of copper salt. Normally the reaction is carried out in the presence of an appropriate solvent such as water, methanol, or mixtures thereof and in presence of an inorganic base, for example, a hydroxide of alkali metal such as potassium hydroxide, and at a temperature between 0 ° C and solvent reflux temperature used.
Las condiciones más adecuadas para llevar a cabo el procedimiento varían en función de los parámetros considerados por el experto en la materia, como por ejemplo los productos de partida, la temperatura y similares. Se ajustarán en cada caso para conseguir la máxima cantidad de complejo. Estas condiciones podrán ser fácilmente determinadas por dicho experto en la materia mediante pruebas rutinarias, y con la ayuda de las enseñanzas de los ejemplos presentes en esta memoria.The most suitable conditions to carry out the procedure vary depending on the parameters considered by the person skilled in the art, such as the products of heading, temperature and the like. They will be adjusted in each case to Get the maximum amount of complex. These conditions may be easily determined by said expert in the field through routine tests, and with the help of the teachings of the examples present in this report.
El compuesto de partida de fórmula (II) puede prepararse mediante un procedimiento que comprende las etapas de:The starting compound of formula (II) can be prepared by a procedure comprising the steps from:
(a) activar el grupo carboxilo del compuesto de fórmula (V) por reacción con un agente activante;(a) activate the carboxyl group of the compound of formula (V) by reaction with an activating agent;
donde p es un grupo protector de aminas, z es 0 ó 1, y R_{1} tiene la misma definición que en la reivindicación 1; con la condición de que cuando R_{1} es una cadena lateral de la prolina, Z es 0, y R_{1} junto al C al que está unido y el N forman conjuntamente un ciclo de 5 miembros;where p is a protective group of amines, z is 0 or 1, and R_ {1} has the same definition as in the claim 1; with the proviso that when R_ {1} is a proline side chain, Z is 0, and R1 next to the C to which is joined and the N together form a cycle of 5 members;
(b) hacer reaccionar el compuesto obtenido en la etapa (a) con un compuesto de fórmula H_{2}N-(CH_{2})_{n}-R_{2} para dar el compuesto de fórmula (III), donde p, Z, y R_{1}, tienen la misma definición que en la fórmula (V), con la condición de que cuando R_{1} es una cadena lateral de la prolina, Z es 0, y R_{1} junto al C al que está unido y el N forman conjuntamente un ciclo de 5 miembros; y R_{2} y n tienen los mismos valores definidos para el compuesto de fórmula (I); y(b) react the compound obtained in the step (a) with a compound of formula H 2 N- (CH 2) n -R 2 to give the compound of formula (III), where p, Z, and R1, have the same definition than in formula (V), with the proviso that when R_ {1} is a proline side chain, Z is 0, and R_ {1} together to the C to which it is attached and the N together form a cycle of 5 members; and R2 and n have the same values defined for the compound of formula (I); Y
(c) desproteger el compuesto de fórmula (III).(c) deprotect the compound of formula (III).
El procedimiento se ilustra en el siguiente Esquema 1:The procedure is illustrated in the following Scheme 1:
Esquema 1Scheme one
La activación del ácido se lleva a cabo con un agente activante que lo modifica para convertirlo en un centro reactivo. La activación del grupo ácido se lleva a cabo mediante cualquiera de los procedimientos conocidos en el estado de la técnica (cfr. M. B. Smith, J. March, March's Advanced Organic Chemistry, John Wiley & Sons, Inc., (2007)), por ejemplo transformando el grupo ácido en haluro de ácido, éster, carbamato, succinamato, o en cualquier otro grupo apropiado conocido para la activación del ácido.Acid activation is carried out with an activating agent that modifies it to become a reactive center. The activation of the acid group is carried out by any of the procedures known in the state of the art (cf. MB Smith, J. March, March's Advanced Organic Chemistry , John Wiley & Sons, Inc., (2007)), by example by transforming the acid group into acid halide, ester, carbamate, succinamate, or any other appropriate group known for acid activation.
Preferiblemente el agente activante es una mezcla de N-hidroxisuccinimida y diciclohexilcarbodiimida.Preferably the activating agent is a mixture of N-hydroxysuccinimide and dicyclohexylcarbodiimide.
Un grupo protector de amina adecuado se
selecciona de aquellos conocidos en la técnica. Preferiblemente, el
grupo protector es benciloxicarbonilo, pero para el propósito de la
invención se pueden utilizar otros grupos protectores. Asimismo, el
grupo protector se puede introducir y eliminar por métodos conocidos
en la técnica (cfr. Protective Groups in Organic Synthesis,
Wiley-Interscience, (1999)). Las condiciones
específicas de reacción dependen del grupo protector
utilizado. Por ejemplo, cuando se utiliza el benciloxicarbonilo,
este se puede desproteger en condiciones ácidas.A suitable amine protecting group is selected from those known in the art. Preferably, the protecting group is benzyloxycarbonyl, but for the purpose of the invention other protecting groups can be used. Likewise, the protecting group can be introduced and removed by methods known in the art (cf. Protective Groups in Organic Synthesis , Wiley-Interscience, (1999)). Specific reaction conditions depend on the protective group
used. For example, when benzyloxycarbonyl is used, it can be deprotected under acidic conditions.
En el caso de que de la etapa (c) resulte una sal del compuesto de fórmula (II), ésta puede utilizarse directamente para la preparación del complejo de cobre, o bien convertirla primero en el compuesto libre.In the event that stage (c) results in a salt of the compound of formula (II), this can be used directly for the preparation of the copper complex, or turn it into the free compound first.
Aunque todos los nuevos compuestos definidos por la formula (I) incluyen un radical fluorescente o potencialmente fluorescente, los complejos de Cu (II) de la invención sólo presentan fluorescencia cuando se ponen en contacto con el anión citrato, lo que les permite que puedan ser utilizados como sensores. Se ha observado que además son altamente selectivos frente a citrato, ya que las soluciones de los sensores de la presente invención con otros carboxilatos diferentes al citrato, por ejemplo acetato, malato, glutarato, malonato, succinato, tartrato, lactato, benzoato, ftalato, fumarato o maleato, no presentan una fluorescencia significativa. La selectividad se basa en la capacidad del citrato de liberar el ligando presente en el complejo de fórmula (I).Although all new compounds defined by formula (I) include a fluorescent or potentially radical fluorescent, the Cu (II) complexes of the invention only they show fluorescence when they contact the anion citrate, which allows them to be used as sensors It has been observed that they are also highly selective against to citrate, since the sensor solutions of the present invention with other carboxylates other than citrate, for example acetate, malate, glutarate, malonate, succinate, tartrate, lactate, benzoate, phthalate, fumarate or maleate, do not have a significant fluorescence. The selectivity is based on the citrate's ability to release the ligand present in the complex of formula (I).
Así, un aspecto adicional de la presente invención es el uso del complejo de Cu (II) de fórmula (I) como sensores fluorescentes de citrato/ácido cítrico. El uso de los sensores de fórmula (I) como sensores químicos para la detección de citrato/ácido cítrico proporciona una selectividad frente a otros carboxilatos y tricarboxilatos mejorada, además de un límite de detección de citrato muy favorable, ya que su rango de sensibilidad y de cuantificación se sitúa en concentraciones de orden micromolar (10^{-6} M).Thus, an additional aspect of the present invention is the use of the Cu (II) complex of formula (I) as fluorescent citrate / citric acid sensors. The use of sensors of formula (I) as chemical sensors for the detection of citrate / citric acid provides a selectivity against others enhanced carboxylates and tricarboxylates, in addition to a limit of very favorable citrate detection, since its sensitivity range and quantification is placed in micromolar order concentrations (10-6 M).
Dicha selectividad y sensibilidad mejoradas se deben a un incremento de la intensidad de fluorescencia de hasta el 1500% en las soluciones de los sensores de la presente invención al ponerse en contacto con el ión citrato. Este dato conlleva que, en presencia de citrato, la señal se multiplica por un factor de 15 en comparación con la señal medida en una solución de sensores en ausencia de citrato.Such improved selectivity and sensitivity are due to an increase in fluorescence intensity of up to 1500% in the solutions of the sensors of the present invention to Get in touch with the citrate ion. This data implies that, in presence of citrate, the signal is multiplied by a factor of 15 in comparison with the measured signal in a sensor solution in absence of citrate
Un aspecto adicional de la presente invención se refiere al procedimiento para la determinación de citrato/ácido cítrico que comprende las etapas de:A further aspect of the present invention is refers to the procedure for citrate / acid determination Citrus comprising the stages of:
(a) preparar una curva de calibrado a partir de soluciones de citrato de concentraciones conocidas;(a) prepare a calibration curve from citrate solutions of known concentrations;
(b) poner en contacto la muestra a analizar con el complejo de Cu (II) de formula (I) a un pH mayor o igual a 7,5; y(b) contact the sample to be analyzed with the Cu (II) complex of formula (I) at a pH greater than or equal to 7.5; Y
(c) medir la fluorescencia de la muestra y determinar el contenido de citrato usando la curva de calibrado.(c) measure the fluorescence of the sample and determine the citrate content using the curve of calibrated.
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La preparación de la curva de calibrado puede realizarse mediante métodos conocidos en el estado de la técnica. Así, la curva de calibrado puede prepararse a partir de soluciones de citrato de diferentes concentraciones conocidas, las cuales se ponen en contacto con el sensor de la presente invención y posteriormente se mide la intensidad de fluorescencia de cada una de ellas con un espectrofluorímetro. Mediante las diversas técnicas de regresión conocidas se relacionan los datos de concentración con la intensidad de señal y se construye un algoritmo útil para la determinación de la concentración de citrato en función de la intensidad de la fluorescencia de la muestra. Más concretamente el intervalo de la curva de calibrado que se utiliza para la determinación es aquél en el que la relación entre la intensidad de señal fluorescente y la concentración es lineal. En este caso, para el ajuste de los datos se utiliza la siguiente expresión:The preparation of the calibration curve can be carried out by methods known in the state of the art. Thus, the calibration curve can be prepared from solutions of citrate of different known concentrations, which are contact the sensor of the present invention and subsequently the fluorescence intensity of each one is measured of them with a spectrofluorimeter. Through the various techniques known regression data concentration data are related to signal strength and a useful algorithm is constructed for the determination of citrate concentration based on the fluorescence intensity of the sample. More specifically the interval of the calibration curve that is used for the determination is one in which the relationship between the intensity of fluorescent signal and the concentration is linear. In this case, for The data setting uses the following expression:
I_{F} = a + b\cdotcI_ {F} = a + b \ cdotc
donde:where:
I_{F} es la intensidad de fluorescenciaI_ {is the fluorescence intensity
c es la concentración de citratoc is the citrate concentration
a y b son la ordenada en el origen y la pendiente, respectivamente, de la recta resultante del ajuste de los datos.a and b are the ordinate at the origin and the slope, respectively, of the straight line resulting from the adjustment of the data.
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Para la determinación de citrato/ácido cítrico de una muestra problema se pone en contacto la solución de sensor con la muestra problema, opcionalmente tratada y preparada previamente para poder ser analizada, y se mide la intensidad de fluorescencia emitida. El resultado se obtiene mediante el algoritmo previamente establecido.For the determination of citrate / citric acid of a sample problem the sensor solution is contacted with the sample problem, optionally treated and prepared previously to be analyzed, and the intensity of emitted fluorescence. The result is obtained by previously established algorithm.
En el caso de que el pH de la muestra sea inferior a aproximadamente 7,5, se trata con una cantidad adecuada para llevarla a un pH mayor o igual a aproximadamente 7,5 para llevar a cabo la medición.In case the pH of the sample is less than about 7.5, it is treated with an adequate amount to bring it to a pH greater than or equal to approximately 7.5 for Carry out the measurement.
Los sensores de la presente invención pueden utilizarse en diversos sectores. Dichos sectores incluyen la industria de alimentación; la industria farmacéutica; biomedicina, bioquímica y medicina clínica.The sensors of the present invention can be used in various sectors These sectors include the food industry; the pharmaceutical industry; biomedicine, Biochemistry and clinical medicine.
Los complejos de la invención proporcionan la sensibilidad y selectividad apropiadas para, mediante la determinación del citrato/ácido cítrico, el diagnóstico de diversos desordenes y condiciones patológicas relacionadas con concentraciones anómalas de ácido cítrico/citrato, por ejemplo para la detección de procesos cancerosos como tumores de próstata o para la evaluación de la probabilidad de sufrir cálculos renales, entre otros. A diferencia de los métodos conocidos en el estado de la técnica, el método de la presente invención proporciona un análisis mejorado en su sensibilidad, rapidez y sencillez. Por ejemplo, la sensibilidad del método de la presente invención es entre cien y mil veces mayor que el método de la citrato liasa, que es el método estándar para la determinación de citrato y ácido cítrico de la Unión Europea (cfr. European Standard EN 1137, dec. 1994).The complexes of the invention provide the appropriate sensitivity and selectivity for, by citrate / citric acid determination, the diagnosis of various disorders and pathological conditions related to abnormal concentrations of citric acid / citrate, for example for the detection of cancerous processes such as prostate tumors or for the evaluation of the probability of suffering kidney stones, between others. Unlike the known methods in the state of the technique, the method of the present invention provides an analysis Improved sensitivity, speed and simplicity. For example, the sensitivity of the method of the present invention is between one hundred and thousand times greater than the citrate lyase method, which is the method standard for the determination of citrate and citric acid of the European Union (cf. European Standard EN 1137, dec. 1994).
A diferencia del método de la citrato Iiasa que implica varios pasos y generalmente se ha de esperar de 15 a 20 minutos antes de medir, en el caso de los complejos de la presente invención, la reacción ocurre prácticamente de manera instantánea, o al menos durante el tiempo que se tarda en mezclar sensor y la muestra problema (1-2 segundos). La ventaja de la rapidez del método de la presente invención es de gran importancia a la hora de desarrollar sistemas automáticos de medición en tiempo real, como por ejemplo los requeridos en a) control de calidad alimentario (con objeto de detectar a tiempo posibles fallos en la calidad de fabricación de un producto), o b) análisis médicos urgentes para el diagnóstico rápido de enfermedades ligadas a la presencia de citrato o ácido cítrico en fluidos, como por ejemplo orina o sangre, o en tejidos.Unlike the Iiase citrate method that It involves several steps and generally you have to wait from 15 to 20 minutes before measuring, in the case of the complexes of the present invention, the reaction occurs virtually instantaneously, or at least for the time it takes to mix sensor and the shows problem (1-2 seconds). The advantage of the rapidity of the method of the present invention is of great importance to when developing automatic time measurement systems real, such as those required in a) quality control food (in order to detect possible failures in time product manufacturing quality), or b) medical analysis urgent for the rapid diagnosis of diseases linked to presence of citrate or citric acid in fluids, such as urine or blood, or in tissues.
A lo largo de la descripción y las reivindicaciones la palabra "comprende" y sus variantes no pretenden excluir otras características técnicas, aditivos, componentes o pasos. Para los expertos en la materia, otros objetos, ventajas y características de la invención se desprenderán en parte de la descripción y en parte de la práctica de la invención. Los siguientes ejemplos y dibujos se proporcionan a modo de ilustración, y no se pretende que sean limitativos de la presente invención.Throughout the description and the claims the word "comprises" and its variants not they intend to exclude other technical characteristics, additives, components or steps. For those skilled in the art, other objects, advantages and features of the invention will be partly detached of the description and in part of the practice of the invention. The following examples and drawings are provided by way of illustration, and are not intended to be limiting of the present invention.
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Fig.1: muestra las curvas de absorción y fluorescencia del ligando libre y del complejo. Las curvas a y b son curvas de absorción, la curva b corresponde al ligando libre de formula (II) (con R_{1}= CH_{2}C_{5}H_{6}, R_{2}= 1-antraceno, m= 2, n= 0) y la curva a corresponde al correspondiente complejo de fórmula (I); y las curvas c y d son las curvas de fluorescencia, la curva c corresponde a dicho complejo y la curva d corresponde a su respectivo ligando libre; donde A representa la absorbancia, I la intensidad de la emisión de fluorescencia y L la longitud de onda en nm.Fig. 1: shows the absorption curves and fluorescence of the free ligand and the complex. The curves a and b they are absorption curves, curve b corresponds to the free ligand of formula (II) (with R 1 = CH 2 C 5 H 6, R 2 = 1-anthracene, m = 2, n = 0) and the curve a corresponds to the corresponding complex of formula (I); and the curves c and d are the fluorescence curves, the curve c corresponds to said complex and curve d corresponds to their respective free ligand; where A represents the absorbance, I the intensity of the emission of fluorescence and L the wavelength in nm.
Fig.2: muestra las diferentes curvas de emisión del complejo en función de la concentración de citrato, donde I representa la intensidad de la emisión de fluorescencia, C la concentración de citrato y L la longitud de onda en nm.Fig. 2: shows the different emission curves of the complex depending on the citrate concentration, where I represents the intensity of the fluorescence emission, C the citrate concentration and L wavelength in nm.
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Fig.3: muestra la curva de calibrado de la intensidad de fluorescencia a 418 nm frente a concentración de citrato según el método de la presente invención (línea sólida) en comparación con los resultados de la medición mediante el método de la citrato-liasa (línea discontinua), donde A representa la absorbancia, I la intensidad de la emisión de fluorescencia y C la concentración de citrato en mol\cdotl^{-1}.Fig. 3: shows the calibration curve of the fluorescence intensity at 418 nm versus concentration of citrate according to the method of the present invention (solid line) in comparison with the measurement results by the method of citrate lyase (dashed line), where A represents the absorbance, I the intensity of the emission of fluorescence and C the citrate concentration in mol • -1.
Fig.4: muestra la curva de calibrado en la zona de respuesta lineal (0,1-10 \muM), con un coeficiente de correlación de R=0,99792.Fig. 4: shows the calibration curve in the area linear response (0.1-10 µM), with a correlation coefficient of R = 0.99792.
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(a) En un matraz de 500 ml se introdujeron (25,8 g, 86,0 mmol) de benciloxicarbonil-L-fenilalanina junto con 10,2 g (86,0 mmol) de N-hidroxisuccinimida. La mezcla se disolvió en 250 ml de THF seco y se enfrió a 0°C. Gota a gota, se adicionó una disolución de 20,50 g (98,36 mmoles) de diciclohexilcarbodiimida (DCC) en 100 ml de THE seco, y la mezcla de reacción se mantuvo durante un periodo de 5 h a una temperatura entre 0-5°C. Trascurrido este tiempo, la urea formada se separó por filtración y el filtrado se evaporó a presión reducida. El crudo de reacción se purificó mediante recristalización en 2-propanol (rendimiento: 86% de compuesto obtenido de fórmula (IV) con p= BzO, R_{1}=CH_{2}C_{5}H_{6}). ^{1}H RMN (300 MHz, CDCl_{3}) \delta 0,96 (t, 3H), 1,59 (m, 2H, j=14,2, 6,6 Hz), 3,20 (dd, 2H, j=14,2, 5,6 Hz), 5,00-5,13 (m, 3H), 5,39 (d, 1H, j=8,6 Hz), 7,24-7,38 (m, 10H); IR (KBr) 3297, 1814, 1785, 1747, 1679, 1541 cm^{-1}. ESI-MS m/z=419,1 (M+Na^{+}). Análisis Elemental calculado (C_{21}H_{20}N_{2}O_{6}): C 63,6; H 5,1; N 7,1; Encontrado: C 63,5; H 5,5; N 7,4.(a) In a 500 ml flask were introduced (25.8 g, 86.0 mmol) of benzyloxycarbonyl-L-phenylalanine together with 10.2 g (86.0 mmol) of N-hydroxysuccinimide. The mixture was dissolved in 250 ml of dry THF and cooled to 0 ° C. Drop by drop, a solution of 20.50 g (98.36 mmol) of dicyclohexylcarbodiimide (DCC) in 100 ml of THE dry, and the reaction mixture was maintained over a period of 5 h at a temperature between 0-5 ° C After this time, the urea formed is filtered off and the filtrate was evaporated under reduced pressure. The reaction crude was purified by recrystallization from 2-propanol (yield: 86% of compound obtained of formula (IV) with p = BzO, R 1 = CH 2 C 5 H 6). 1 H NMR (300 MHz, CDCl 3) δ 0.96 (t, 3H), 1.59 (m, 2H, j = 14.2, 6.6 Hz), 3.20 (dd, 2H, j = 14.2, 5.6 Hz), 5.00-5.13 (m, 3H), 5.39 (d, 1H, j = 8.6 Hz), 7.24-7.38 (m, 10H); IR (KBr) 3297, 1814, 1785, 1747, 1679, 1541 cm -1. ESI-MS m / z = 419.1 (M + Na +). Elemental Analysis Calculated (C 21 H 20 N 2 O 6): C 63.6; H 5.1; N 7.1; Found: C 63.5; H 5.5; N 7.4.
(b) El compuesto obtenido en la etapa (a) (5,0 g, 12,6 mmol) se disolvió en THE anhidro (40 mL) y a esa disolución se le añadió otra de 1-aminoantraceno (2,7 g, 12,6 mmol) en THE (10 ml). Tras reflujo de 18 h bajo atmósfera de nitrógeno se filtró y evaporó a presión reducida. El sólido obtenido se lavó con agua básica, agua neutra y 2-propanol caliente. El producto se secó en estufa a presión reducida (temp. 60-70°C) durante 24 h para dar el compuesto de fórmula (III) con n=0, p= BzO, R_{2}=1-antraceno, R_{1}=CH_{2}C_{5}H_{6} (rendimiento: 70%). ^{1}H RMN (500 MHz, DMSO) \delta 3,04 (dd, 1H, j=9,7 Hz, j'=12,7 Hz), 3,22 (dd, 1H, j=5,5 Hz, j'=13,4 Hz), 4,75 (m, 1H), 5,06 (s, 2H), 7,25 (m, 1H), 7,33 (m, 1H), 7,43 (d, 6H, j=7,1 Hz), 7,50 (t, 2H, j=7,8 Hz), 7,54 (dd, 2H, j=2,9 Hz, j'=6,4 Hz), 7,61 (d, 1H, j=7,0 Hz), 7,80 (d, 1H, j=8,0 Hz), 7,95 (d, 1H, j=8,5 Hz), 8,00 (m, 1H), 8,53 (s, 1H), 8,60 (s, 1H), 10,20 (s,1H); IR (KBr) 3269, 1684, 1653, 1531 cm^{-1}. ESI-MS m/z=497,2 (M+Na^{+}), 513,3 (M+K^{+}).(b) The compound obtained in step (a) (5.0 g, 12.6 mmol) was dissolved in the anhydrous (40 mL) and at that solution another 1-aminoanthracene (2.7 g, 12.6 was added mmol) in THE (10 ml). After reflux of 18 h under atmosphere of Nitrogen was filtered and evaporated under reduced pressure. Solid obtained was washed with basic water, neutral water and 2-hot propanol. The product was dried in an oven under reduced pressure (temp. 60-70 ° C) for 24 h to give the compound of formula (III) with n = 0, p = BzO, R 2 = 1-anthracene, R 1 = CH 2 C 5 H 6 (yield: 70%). 1 H NMR (500 MHz, DMSO) δ 3.04 (dd, 1H, j = 9.7 Hz, j '= 12.7 Hz), 3.22 (dd, 1H, j = 5.5 Hz, j' = 13.4 Hz), 4.75 (m, 1H), 5.06 (s, 2H), 7.25 (m, 1H), 7.33 (m, 1H), 7.43 (d, 6H, j = 7.1 Hz), 7.50 (t, 2H, j = 7.8 Hz), 7.54 (dd, 2H, j = 2.9 Hz, j '= 6.4 Hz), 7.61 (d, 1H, j = 7.0 Hz), 7.80 (d, 1H, j = 8.0 Hz), 7.95 (d, 1H, j = 8.5 Hz), 8.00 (m, 1H), 8.53 (s, 1H), 8.60 (s, 1H), 10.20 (s, 1 H); IR (KBr) 3269, 1684, 1653, 1531 cm -1. ESI-MS m / z = 497.2 (M + Na +), 513.3 (M + K +).
(c) A 3,0 g (6,3 mmol) del compuesto obtenido en la etapa (b) se añadieron 14 ml de HBr/AcOH (33%) y la mezcla se agitó durante 50 minutos. Se añadió éter dietílico lo que condujo a la aparición de un precipitado verde. Se filtró y lavó con éter dietílico. El sólido obtenido se secó a vacío y a 60-70°C durante 24 h para dar el bromohidrato del compuesto de fórmula (II) (rendimiento: 97%). ^{1}H RMN (500 MHz, DMSO) \delta 3,28 (m, 1H), 3,40 (bs, 1H), 4,55 (s, 1 H), 7,33 (m, 1 H), 7,40 (m, 4H), 7,51 (t, 1 H, j=7,8 Hz), 7,57 (m, 2H), 7,61 (d, 1H, j=7,1 Hz), 7,98 (d, 1H, j=8,5 Hz), 8,00 (d, 1H, j=8,0 Hz), 8,10 (d, 1H, j=8,0 Hz), 8,27 (s, 1H), 8,50 (bs, 3H), 8,61 (s, 1H), 10,45 (s, 1H); IR (KBr) 3425 3028 1665 1549 cm^{-1}. ESI-MS m/z=341,3 (M^{+}-Br^{-}).(c) At 3.0 g (6.3 mmol) of the compound obtained in step (b) 14 ml of HBr / AcOH (33%) was added and the mixture was stirred for 50 minutes. Diethyl ether was added which led to the appearance of a green precipitate. It was filtered and washed with ether diethyl The solid obtained was dried under vacuum and at 60-70 ° C for 24 h to give the bromohydrate of the compound of formula (II) (yield: 97%). 1 H NMR (500 MHz, DMSO) δ 3.28 (m, 1H), 3.40 (bs, 1H), 4.55 (s, 1 H), 7.33 (m, 1 H), 7.40 (m, 4H), 7.51 (t, 1 H, j = 7.8 Hz), 7.57 (m, 2H), 7.61 (d, 1H, j = 7.1 Hz), 7.98 (d, 1H, j = 8.5 Hz), 8.00 (d, 1H, j = 8.0 Hz), 8.10 (d, 1H, j = 8.0 Hz), 8.27 (s, 1H), 8.50 (bs, 3H), 8.61 (s, 1H), 10.45 (s, 1 H); IR (KBr) 3425 3028 1665 1549 cm -1. ESI-MS m / z = 341.3 (M + - Br -).
(d) El compuesto del título se preparó disolviendo 0,427 mmol del compuesto obtenido en la etapa (c) y 0,214 mmol de CuCl_{2}\cdot2H_{2}O en MeOH. Tras la mezcla de dichas disoluciones no hay cambio de color apreciable. Se añadieron 0,854 mmol de KOH a la disolución y se observó un oscurecimiento de la mezcla final tras disolverse la base. Se puso la mezcla a reflujo 1 h y al cabo de dicho tiempo se evaporó el disolvente y se lavó con agua. Tras secado en estufa a vacío (65°C) durante 20 h se obtuvo un sólido verde oscuro con un rendimiento del 51%. El complejo obtenido se caracterizó mediante espectroscopia infrarroja, espectrofotometría de absorción y fluorescencia. El espectro FT-IR del compuesto del título preparado mediante el procedimiento de la invención muestra una banda de vibración de carbonilo en 1591 cm^{-1}. El espectro de UV-Vis (agua:metanol, 3:7 v/v, pH=8) del complejo muestra máximos de absorción en las longitudes de onda de 350, 367 y 384 nm. El espectro de emisión de fluorescencia del compuesto (agua:metanol, 3:7 v/v, pH=8) con una longitud de onda de excitación de 370 nm, muestra un máximo de emisión a 418 nm.(d) The title compound was prepared dissolving 0.427 mmol of the compound obtained in step (c) and 0.214 mmol CuCl 2 • 2 O 2 in MeOH. After mixing such solutions there is no noticeable color change. They were added 0.854 mmol of KOH to the solution and a darkening of the final mixture after dissolving the base. The mixture was set to reflux for 1 h and after that time the solvent was evaporated and washed with water. After drying in a vacuum oven (65 ° C) for 20 hours, obtained a dark green solid with a yield of 51%. He complex obtained was characterized by spectroscopy infrared, absorption spectrophotometry and fluorescence. He FT-IR spectrum of the title compound prepared by the method of the invention shows a band of carbonyl vibration in 1591 cm -1. The spectrum of UV-Vis (water: methanol, 3: 7 v / v, pH = 8) of the complex shows maximum absorption at wavelengths of 350, 367 and 384 nm. The fluorescence emission spectrum of the compound (water: methanol, 3: 7 v / v, pH = 8) with a wavelength of 370 nm excitation, shows a maximum emission at 418 nm.
La comparación entre los espectros de FT-IR de ligando libre (compuesto de la etapa (c)) y del complejo del título obtenido en la etapa (d) muestra un gran desplazamiento de la banda de vibración de carbonilo, desde 1663 cm^{-1} en el ligando hasta 1591 cm^{-1} en el complejo. Una comparación entre los espectros de UV-Vis (en agua:metanol, 3:7 v/v, pH 8) de ligando libre y del complejo permite apreciar tanto desplazamientos de la banda de absorción del ligando a 320-400 nm como disminución de la intensidad de emisión en el complejo respecto al ligando libre (cfr. Fig. 1).The comparison between the spectra of Free ligand FT-IR (compound of step (c)) and of the title complex obtained in step (d) shows a large displacement of the carbonyl vibration band, since 1663 cm -1 in the ligand up to 1591 cm -1 in the complex. A comparison between UV-Vis spectra (in water: methanol, 3: 7 v / v, pH 8) of free ligand and complex allows to appreciate both displacements of the absorption band of the ligand at 320-400 nm as a decrease in emission intensity in the complex with respect to the free ligand (cf. Fig. 1).
La curva de calibrado se obtiene utilizando 30 ml de una disolución no fluorescente del complejo (concentración del complejo= 2x10^{-5} M, NaCl 0,15 M, metanol:agua 7:3 v/v, a pH=7,9), a la que se le añaden alícuotas de entre 5 y 200 microlitros de una disolución de citrato sódico (concentración de citrato = 1,5x10^{-3} M, NaCl 0,15M, agua) y ajustando el pH a 7,9 tras cada una de las adiciones. De este modo se aumenta la concentración de citrato en la disolución desde 0 a 100 micromolar. Debido a ello se produce un incremento de la emisión a 418 nm (cfr. Fig. 2), que permite relacionarla señal de emisión fluorescente con la concentración de citrato presente en el medio de disolución (cfr. Fig. 4). Para la medida de la emisión de fluorescencia se empleó un espectrofotómetro Spex Fluorolog 3 equipado con una lámpara de xenón de 450 W, excitando a 370 nm y registrando la emisión entre 380 y 550 nm. En concreto, de la curva de calibrado se utiliza el rango de concentración 0,1 – 10 micromolar, en el cual la relación entre la intensidad de señal fluorescente y la concentración de señal es lineal. Se estima un límite de detección de aproximadamente 1 micromolar de citrato. En la Fig. 3 se ha representado en escala logarítmica la intensidad de emisión de fluorescencia en función de la concentración de citrato presente en el medio (trazo más grueso).The calibration curve is obtained using 30 ml of a non-fluorescent solution of the complex (concentration of the complex = 2x10-5 M, 0.15 M NaCl, methanol: water 7: 3 v / v, a pH = 7.9), to which aliquots of between 5 and 200 are added microliters of a sodium citrate solution (concentration of citrate = 1.5x10-3M, 0.15M NaCl, water) and adjusting the pH to 7.9 after each of the additions. This increases the concentration of citrate in the solution from 0 to 100 micromolar. Because of this there is an increase in the emission at 418 nm (cfr. Fig. 2), which allows the fluorescent emission signal to be related to the concentration of citrate present in the dissolution medium (cf. Fig. 4). To measure the fluorescence emission, used a Spex Fluorolog 3 spectrophotometer equipped with a 450 W xenon lamp, exciting at 370 nm and recording the emission between 380 and 550 nm. Specifically, the calibration curve the concentration range 0.1 - 10 micromolar is used, in the which the relationship between the fluorescent signal intensity and the Signal concentration is linear. A detection limit is estimated of approximately 1 micromolar citrate. In Fig. 3 it has been represented in logarithmic scale the emission intensity of fluorescence depending on the concentration of citrate present in the middle (thicker stroke).
En la Fig. 3 se comparan dichos resultados con los que resultan de aplicar el método actualmente de referencia, trazo más fino y punteado, para la determinación de citrato y ácido cítrico, método que emplea la citrato liasa como enzima que desencadena una serie de reacciones medibles por absorción (cfr. European Standard EN 1137, dec. 1994).In Fig. 3 these results are compared with those resulting from applying the currently reference method, finest and dotted line, for the determination of citrate and acid citric, a method that uses citrate lyase as an enzyme that triggers a series of measurable reactions by absorption (cf. European Standard EN 1137, dec. 1994).
La sensibilidad de la determinación usando el complejo de fórmula (I) frente a citrato es más de cien veces superior que utilizando el método de referencia.The sensitivity of the determination using the complex of formula (I) against citrate is more than one hundred times higher than using the reference method.
b) Determinación de citrato: la determinación de citrato se llevó a cabo midiendo por triplicado la fluorescencia de cinco muestras de concentración conocida de citrato (por pesada de citrato sódico y disolución en un volumen dado). Se ha utilizado la curva de calibrado de la Fig.3 para determinar la concentración de cada muestra y evaluar la precisión del método.b) Citrate determination: citrate determination was carried out by measuring in triplicate the fluorescence of five samples of known citrate concentration (by weighing sodium citrate and dissolving in a given volume). The calibration curve of Fig. 3 has been used to determine the concentration of each sample and evaluate the accuracy of the method.
Los resultados se han incluido en la Tabla 2.The results have been included in the Table 2.
El ensayo descrito demuestra la utilidad del complejo de fórmula (I) con R_{1}=cadena lateral de fenilalanina para la cuantificación del anión citrato mediante fluorescencia en el rango micromolar en disoluciones acuosas.The described trial demonstrates the usefulness of complex of formula (I) with R1 = phenylalanine side chain for the quantification of citrate anion by fluorescence in the micromolar range in aqueous solutions.
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El complejo arriba mencionado es sensible selectivamente frente a citrato y no frente a otros carboxilatos biológicamente relevantes, utilizando idéntica metodología. Así, se ensayaron los ácidos siguientes (en forma de carboxilatos) en las mismas condiciones arriba indicadas sin apreciarse un cambio importante de la fluorescencia: acetato, malato, glutarato, malonato, succinato, tartrato, lactato, benzoato, ftalato, fumarato y maleato. Las variaciones de intensidad de fluorescencia en presencia de dichos carboxilatos fueron inferiores al 10% de la variación de intensidad experimentada en presencia de citrato.The above mentioned complex is sensitive selectively against citrate and not against other carboxylates biologically relevant, using identical methodology. Thus tested the following acids (in the form of carboxylates) in the same conditions indicated above without appreciating a change important fluorescence: acetate, malate, glutarate, malonate, succinate, tartrate, lactate, benzoate, phthalate, fumarate and maleate. Fluorescence intensity variations in presence of said carboxylates were less than 10% of the Intensity variation experienced in the presence of citrate.
Claims (12)
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BURGUETE, M.I. et al. "{}Nickel complexes from a-amino amides as efficient catalysts for the enantioselective Et2Zn addition to benzaldehyde"{}. Tetrahedron Letters, 2003, Volumen 4, Número 36, páginas 6891-6894. Ver página 6892, esquema 2, tabla 1. * |
FABBRIZZI, L. et al. "{}Metal-Containing Trifurcate Receptor that Recognizes and Senses Citrate in Water"{}. Organic Letters, 2005, Volumen 7, Número 13, páginas 2603-2606. * |
KOMORITA, T. et al. "{}Metal complexes with Amino Acid Amides. III. Geometrical Structures and Electronic Spectra of Bis(a-amino- acid-amidato)palladium (II), -nickel (II) and -copper (II)."{} Bulletin of the American Society of Japan, 1971, Volumen 44, páginas 3353-3363. Ver página 3355, columna 2, apartado 10. * |
KRÄMER, R. "{}Fluorescent Chemosensors for Cu2+ Ions: Fast, Selective and Highly Sensitive"{}. Angewandte Chemie International Edition, 1998, Volumen 37, Número 6, páginas 772-773. Ver esquema 1. * |
KRONECK, P.M.H. et al. "{}Model Studies on the Coordination of Copper In Biological Systems"{}. European Journal of Biochemistry, 1980, Volumen 109, Número 2, páginas 603-612. Ver página 606, tabla 1; página 604, ecuación 1; página 607, figura 1. * |
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