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Fluorescent Chemosensors for Organic Compounds based on Cu(II) Complex

Fluorescent Chemosensors for Organic Compounds based on Cu(II) Complex
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대학원 화학·나노과학과
이화여자대학교 대학원
본 연구에서는 생리학적 pH의 수용액에서 옥살레이트(oxalate)를 효과적이고 선택적으로 감지(sensing)할 수 있도록 naphthalene의 1번, 8번 위치에 1-methyl-1,4,7-triazonane이 결합되어 있는 리간드(L)와 두 개의 Cu(II)가 결합된 화합물인 [Cu2(L)]4+를 디자인, 합성하였으며 이들의 결합 평형 상수(binding equilibrium constant)와 다양한 음이온에 대한 선택성(selectivity)을 형광 데이터(fluorescent data)로 확인하였다. 옥살레이트는 금속과 결합할 수 있는 네 개의 산소 원자를 가지고 있어 [Cu2(L)]4+화합물과 매우 쉽고 강하게 1:1로 결합하고, 이는 옥살레이트와 [Cu2(L)]4+의 결정 구조로 확인 할 수 있다. [Cu2(L)]4+와 옥살레이트의 결합 평형상수는 ‘형광 앙상블적 접근(fluorescence ensemble approach)’을 통해 측정할 수 있었고 이 과정에서 지시약은 이오신 Y(eosin Y)를 사용하였다. 형광의 세기가 매우 강한 이오신 Y (1.0×10-6M)의 경우, 이의 100배 당량에 해당하는 [Cu2(L)]4+ (1.0×10-4M)와 결합하게 되면 본연의 형광 세기(fluorescent intensity)를 거의 잃게 된다. 이러한 결합물에 옥살레이트 이온을 첨가하면 옥살레이트와 이오신 Y 사이에 치환 반응이 일어나게 되고, [Cu2(L)]4+에서 떨어져 나온 이오신 Y는 본연의 형광을 다시 회복되는 것을 확인 할 수 있었다. 이러한 지시약 치환법(indicator displacement method)을 통하여 옥살레이트와 [Cu2(L)]4+의 결합 평형상수는 (1.3 ± 0.1)×105 M-1으로 계산되었다. 옥살레이트의 선택성을 확인하기 위해서, 옥살레이트와 같이 4개의 산소 원자를 가지는 말로네이트, 석시네이트, 그리고 글루타레이트를 가지고 동일한 방법으로 실험을 진행하였다. 그 결과 옥살레이트의 결합 평형 상수는 말로네이트에 비해4배, 석시네이트에 비해 50배 그리고 글루타레이트에 비해 200배의 높은 값으로 계산되었다. 이러한 결과로 pH=7.0의 수용액에서 [Cu2(L)]4+가 옥살레이트를 매우 단단하고 가역적이며 선택적으로 감지할 수 있음을 증명하였다.;In the development of chemosensors, one important feature is the mechanism through which the receptor signals the binding of the analyte. Many analytes of interest do not incorporate their own chromophore or fluorophore, and development of a sensor that could detect these without chemical modification is preferred. A colorimetric or fluorescent indicator needs to be associated with the receptor, and the most common way is to covalently attach the indicator. Upon binding the analyte, a change is induced in the sensor to indicate that binding has occurred. This can be seen in Scheme 1, where upon addition of an analyte the chromophore is “switched on” giving a signal change. A common example of an indicator is one used to detect changes in pH such as phenolphthalein. Another commonly used signaling technique involves PET. This generally entails a fluorophore- spacer-receptor system, where the fluorescence can be “switched” on or off upon binding. A popular example of this utilizes the fluorophore, anthracene, attached to an amine built into the binding site. In the absence of an analyte, the lone pair of the amine quenches the fluorescence of the fluorophore through PET. Chemosensors generally comprise a metal-ion recognition site and a signal transduction domain. Cyclam and many of its derivatives are well-known for their unique ionophoric properties toward some representative transition metal ions. We have tried to develop a new fluoroionophore by utilizing the excellent molecular recognition ability of cyclam by appending the efficient signaling fluorophore of anthracene or pyrene functions in ‘fluorophore-spacerreceptor’mode. This paper reports that the pronouncedly selective ON-OFF type fluorescence quenching behavior of The Ligand (L)-Cu(II) complex toward carbonyl groups. Reaction of 1,4,8,11-tetraazacyclotetradecane (cyclam) with 0.5 equiv of 2-bromo-N-(4,6-dihydropyren-1-yl)acetamide(1) (K2CO3, CH3CN) afforded desired mono-functionalized cyclam derivative (2) (yield based on (1) = 82%). Because the cyclam and various related derivatives are known to have strong affinity toward Cu2+, Ligand(L) is combined with Cu2+ in no time at all. In aqueous HEPES buffered solution, the fluorescence intensity of The Ligand (L)-Cu(II) complex observed at 384 nm. Interesting observation is that the The Ligand (L)-Cu(II) complex showed a selective and efficient quenching effect toward carbonyl group guests in aqueous buffered solution. For example, treatment of the ionophore solution with 1200 equiv of nicotinamide’s derivatives, 3-pyridinylaldehyde, benzamide, thymine and barbiturate resulted in almost complete quenching of the pyrene fluorescence in 0.05M HEPES solution. Another interesting observation is the fluorescence sameness with pyridine, 3-picolylamine and nicotine in aqueous buffered solution. That might be useful for the design of selective molecular switching system utilizing the selective ON-OFF type responses of carbonyl group.;Considerable efforts have been devoted to the selective sensing of anions because of the important roles that they play in various chemical and biological processes. Fluorescent sensing of anions have become particularly attractive due to its simplicity and low detection limit. Sensing of oxalate is useful in food chemistry and in clinical analysis. The level of oxalate in urine is an indicator of calcium oxalate kidney stones. Current methods for oxalate detection such as colorimetry, liquid and gas chromatography, and capillary electrophoresis often require sample pre-treatment, and expensive equipments. During the last decade, many artificial sensors for dicarboxylates have been developed. However, very few examples of effective fluorescent sensors for oxalate have been reported to date. In general, it is challenging to develop receptors that bind tightly, reversibly and selectively to small molecules in water for sensing purposes. In case of oxalate, there are four oxygen atoms that can coordinate to metal ions. We reasoned that a couple of well positioned metal complexes could cooperatively bind to all four oxygens of oxalate tightly, reversibly and selectively over other dicarboxylates such as malonate, succinate and glutarate. Here we report a dinuclear metal complex ([Cu2(L)Cl4]) that can be used for fluorescent detection of oxalate in water at physicological pH by chemosensing ensemble approach. The ligand (L) and dinuclear Cu(II) complex [Cu2(L)]4+ were prepared according to the method previously described. The crystal structure of oxalate shows that the two copper in the structure are octahedral with oxalate bridging the two metal ions. One of the two chlorides has been replaced with a solvent water molecule. It is evident from the structure that oxalate fits nicely to the receptor forming a 1:1 complex. In order to measure the equilibrium constant for binding of oxalate to the dinuclear metal complex, chemosensing ensemble approach with eosine Y was used. Upon addition of [Cu2(L)]4+ to a solution of eosine Y (1.0 M) buffered at pH = 7.0 (50 mM HEPES), the fluorescence intensity of eosine Y (E-Y) sharply decreased and resulted in complete quenching of the emission above 150 equiv of [Cu2(L)]4+. Nonlinear least-squares fitting of the titration profiles indicated formation of a 1:1 complex with binding constant Ks = (5.6 ± 0.2) x 104 M-1. The receptor-eosine pair was titrated by the indicator displacement method with some representative dicarboxylate anions: oxalate, malonate, succinate, and glutarate. In a typical experiment, increasing amounts of oxalate was added to a chemosensing ensemble solution containing E-Y (1.0×10-6 M) and [Cu2(L)]4+ (1.0×10-4 M) in a buffered solution at pH 7.0 (50 mM HEPES). A revival of the indicator fluorescence was observed upon addition of oxalate. Various fluorescent data show the increase in fluorescence of this chemosensing ensemble solution with increase in oxalate concentration. This result indicates the successful competitive binding of the oxalate ion and displacement of the indicator from the receptor. The binding constant for oxalate anion was measured to be Ks = (1.3 ± 0.1) ×105 M-1 by fitting the data with a competitive binding equilibria model. The above method gave binding constants Ks = (3.1 ± 0.2) × 104 M-1, (2.1 ± 0.3) × 103 M-1, and (6.9 ± 1.2) × 102 M-1 for malonate, succinate, and glutarate respectively. The receptor binds oxalate about 4-fold, 50-fold, and 200-fold more tightly than malonate, succinate, and glutarate respectively. The equilibrium constant for binding of acetate to the receptor is too small to be measured accurately by the above method (Ks < 102 M-1). If the value of the binding constant of a dicarboxylate like oxalate is greater than the square of the binding of acetate, it could be reasoned that there is cooperativity for dicarboxylate binding. The dinuclear copper complex represents a relatively simple receptor that binds tightly and selectively to oxalate over other dicarboxylates (malonate, succinate, glutarate). The two metal complexes in the crystal structure appear to be ideally positioned for binding oxalate with an inter-metal distance of about 5.2 Å. The parallel orientation of the two metal complexes is suitable for coordinating the four oxygen atoms of oxalate. Based on inspection of the crystal structure, it is likely that less steric and ring strain are introduced upon binding of oxalate to the receptor than when other dicarboxylates are bound to the receptor. Indeed, molecular mechanics and DFT computations show that the computed trend for binding of the four dicarboxylates to the receptor is in agreement with the experimental trend obtained by the fluorescence ensemble approach (oxalate > malonate > succinate > glutarate). In summary, a dinuclear copper complex that binds tightly and selectively to oxalate over other dicarboxylates (malonate, succinate, glutarate) is reported. We developed a highly sensitive and selective fluorescence assay for sensing oxalate in water at neutral pH based on the receptor. Crystal structure of oxalate bound to the receptor together with molecular mechanics and DFT computations provide insights into the tight and selective binding of the anion by the receptor.;본 연구에서는 사이클램(cyclam)에 1-아미노파이렌(1-aminopyrene)이 치환된 리간드(ligand, L)와 구리(Ⅱ) 금속 이온의 결합으로 이루어진 The Ligand (L) - Cu(II) complex [Cu(L)]2+를 디자인하고 합성하였다. 합성 과정은 세 단계의 과정으로 비교적 쉽고 간단한 합성법으로 구성되어 있다. 합성된 [Cu(L)]2+의 경우 게스트(Guest) 분자와 결합을 이룰 수 있는 구리(II) 이온과 형광적 변화를 나타낼 수 있는 파이렌(pyrene)을 동시에 가지고 있어 형광센서로서의 구조적 요건을 충분히 가지고 할 수 있다. 리간드에 구리 이온의 농도를 다르게 하여 적정을 해 본 결과 두 물질의 비율이 1:1일 때 나타내는 형광 세기에서 큰 변화가 없는 것으로 나타났다. 또한 [Cu(L)]2+의 질량 분석 결과 분자량이 458.2g/mol로 측정되어 본 화합물의 합성이 바르게 이루어졌음을 증명하였다 [Cu(L)]2+의 선택성을 알아보기 위하여, 0.05M HEPES 완충용액에서 [Cu(L)]2+의 형광을 측정한 뒤 다양한 게스트 화합물을 첨가해 주었다. 니코틴아마이드(nicotinamide)와 이것의 이성질체들, 니코틴 산(nicotinic acid), 벤자마이드(benzamide), 3-피리디닐알데하이드(3-pyridinylaldehyde), 티민(thymine), 바비추레이트(barbiturate)가 게스트 화합물로 들어갔을 때에는 형광의 변화가 눈에 띄게 나타났지만 피리딘(pyridine), 3-피콜릴아민(3-picolylamine), 니코틴(nicotine)이 게스트로 들어 갔을 때에는 형광의 변화가 보이지 않았다. 형광의 변화를 보인 게스트 화합물의 특징은 공통적으로 카보닐기(carbonyl group)를 가지고 있다는 것임으로 [Cu(L)]2+는 카보닐기에 대한 선택성이 있는 화학센서로써 활용 가능하다는 것을 증명하였다.
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