Fluorescent Chemosensors for Monitoring Potassium in Blood and

Chapter 11

Downloaded by NANYANG TECHNOLOGICAL UNIV on August 26, 2015 | http://pubs.acs.org Publication Date: October 20, 1993 | doi: 10.1021/bk-1993-0538.ch011

Fluorescent Chemosensors for Monitoring Potassium in Blood and across Biological Membranes Divakar Masilamani and Mariann E . Lucas Biotechnology Department, Allied-Signal Inc., Morristown, NJ 07962-1021

A r a t i o n a l approach t o developing a fluorescent chemosensor for potassium (K ) i s presented. In t h i s approach, J - M . Lehn's [222] cryptand, which s e l e c t i v e l y binds K , i s covalently attached t o coumarin at positions 6 and 7. In this hybrid system, coumarin plays the r o l e of a transducer t r a n s l a t i n g the free energy of supramolecular i n t e r a c t i o n between the cryptand and K into measurable enhancement of its fluorescence. By immobilizing this system on an o p t i c a l f i b e r , the continuous monitoring of K i n patients undergoing open-heart surgery can be achieved. In methanol, the hybrid system behaves as a reagent and can be used i n automated microfluorometers to assay potassium i n the range of 0-6 mM in the presence of 500-3000 fold excess of sodium (Na ) with 99% accuracy. The fluorescent chemosensor also serves as a t o o l for studying rates of transport of K across b i o l o g i c a l membranes. A mechanism based on photo-induced electron transfer explains the observed fluorescence enhancement caused by K binding. +

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I n t h i s p a p e r , we r e p o r t o u r s u c c e s s i n d e v e l o p i n g a p r a c t i c a l f l u o r e s c e n t c h e m o s e n s o r f o r K* (1) . This sensor was s p e c i f i c a l l y d e s i g n e d f o r a t t a c h m e n t t o a n o p t i c a l f i b e r f o r t h e c o n t i n u o u s s e n s i n g o f K* i n t h e e x t r a c o r p o r e a l blood o f p a t i e n t s undergoing open-heart surgery. At the p r e s e n t t i m e , K* i n b l o o d i s m o n i t o r e d t h r o u g h a b a t c h process. B l o o d samples a r e withdrawn p e r i o d i c a l l y from p a t i e n t s and sent f o r a n a l y s i s by conventional techniques s u c h a s a t o m i c a b s o r p t i o n s p e c t r o m e t r y (2) . There i s a

0097-6156/93/0538-0162$06.25A) © 1993 American Chemical Society In Fluorescent Chemosensors for Ion and Molecule Recognition; Czarnik, A.; ACS Symposium Series; American Chemical Society: Washington, DC, 1993.


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Potassium in Blood and across Membranes

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lag-time b e t w e e n s a m p l i n g and r e p o r t i n g o f t h e results which c a n be c r i t i c a l . A s u d d e n s u r g e i n t h e l e v e l o f K* ( a b o v e 6 mM) may b e a n i n d i c a t i o n t h a t t h e p a t i e n t i s g o i n g into "shock". The lag-time c a n be avoided through continuous monitoring. Our f i b e r o p t i c s - b a s e d s e n s o r w i l l provide t h i s technology. A rational approach t o developing a fluorescent chemosensor requires an understanding of the subtle d i f f e r e n c e s between c l a s s i c a l r e a g e n t s and s e n s o r s (3). The c r i t i c a l thermodynamic f a c t o r t h a t d i s t i n g u i s h e s t h e s e n s o r f r o m t h e r e a g e n t c a n a l s o b e u s e d t o c o n v e r t one t o the other. The t h e r m o d y n a m i c c h a r a c t e r i s t i c s o f s e n s o r s c l e a r l y i n d i c a t e a n e e d f o r e f f i c i e n t t r a n s d u c e r s (4) t h a t t r a n s l a t e t h e weak c h e m i c a l f r e e e n e r g y o f s e n s o r - a n a l y t e interactions i n t o measurable changes i n t h e i r physical properties. We c h o s e f l u o r o p h o r e s f o r t h i s p u r p o s e n o t o n l y b e c a u s e t h e y a r e h i g h l y s e n s i t i v e (5) t o e l e c t r o n i c perturbations, but are also ideal for remote sensing through o p t i c a l f i b e r s (6) • It i s important that the design of our sensor takes i n t o account the c o n d i t i o n s t h a t prevail in the extracorporeal blood. The relative concentrations of other a l k a l i metal i o n s , t h e i r diameters and s o l v a t i o n e n e r g i e s , e t c . w i l l d e f i n e t h e selectivity l i m i t s t h a t a r e r e q u i r e d o f t h e s e n s o r f o r K*. T h e d e s i g n o f f l u o r e s c e n t c h e m o s e n s o r s f o r K* i s s i m p l e in its logic. I t c o m b i n e s known i o n o p h o r e s t h a t a r e h i g h l y s e l e c t i v e i n b i n d i n g K* w i t h a f l u o r e s c e n t g r o u p . T h e two u n i t s a r e c o m b i n e d i n s u c h a way t h a t t h e s u p r a m o l e c u l a r i n t e r a c t i o n s o f K* t h a t c a u s e t h e e l e c t r o n i c p e r t u r b a t i o n s o f t h e l i g a t i n g atoms o f t h e i o n o p h o r e w i l l b e t r a n s m i t t e d t o t h e f l u o r e s c i n g chromophore t h r o u g h c o n j u g a t e d d o u b l e bonds. The c h a l l e n g e i s n o t i n d e s i g n i n g t h e hybrid sensors. It is in making t h e right choice of the components t h a t c o n s t i t u t e the h y b r i d . Here chemical intuition plays a significant role. Once the d e c i s i o n was made on the fluorescent s i g n a l i n g group, several hybrid molecules incorporating the signal group in several K*-selective ionophores were d e s i g n e d and s y n t h e s i z e d . The b e s t c a n d i d a t e s e n s o r f o r K* was identified. The synthesis represents the most f r u s t r a t i n g and t i m e - c o n s u m i n g p a r t o f o u r r e s e a r c h . This a s p e c t w i l l n o t be d i s c u s s e d h e r e . However, an o u t l i n e o f our s y n t h e s i s i s p r e s e n t e d as Appendix I a t t h e end o f t h e c h a p t e r a n d d e t a i l s a r e p r o v i d e d i n two U . S . P a t e n t s (1) .

Fluorescent Sensors and Reagents I n d e v e l o p i n g a f l u o r e s c e n t s e n s o r f o r K*, we n e e d to understand the subtle differences between a classical r e a g e n t and a s e n s o r ( 3 . 4 ) . H i s t o r i c a l l y , r e a g e n t s were d e s i g n e d t o consume a n d transform analytes. Reagents, t h e r e f o r e , are not reusable. They a r e s u i t e d f o r b a t c h a n a l y s i s i n which t h e t r a n s f o r m e d a n a l y t e i s e a s i e r t o a n a l y z e and q u a n t i t a t e . There i s a t i m e - l a g between s a m p l i n g and r e p o r t i n g o f t h e result. T h i s a p p r o a c h i s e x p e n s i v e i n terms o f t i m e , m a t e r i a l and labor.

In Fluorescent Chemosensors for Ion and Molecule Recognition; Czarnik, A.; ACS Symposium Series; American Chemical Society: Washington, DC, 1993.


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FLUORESCENT CHEMOSENSORS FOR ION AND MOLECULE RECOGNITION

Sensors on the other hand, are devices that continuously monitor analytes (4.6). S e n s o r s do not consume a n a l y t e s . T h e y i n t e r a c t w i t h them i n a r e v e r s i b l e f a s h i o n . R e v e r s i b i l i t y i m p l i e s lower e q u i l i b r i u m constants for interactions between the s e n s o r and t h e analyte. Consequently, the free energy changes for these i n t e r a c t i o n s are a l s o s m a l l . Sensors, therefore, need efficient transducers that amplify t h e i r weak c h e m i c a l interaction energies into strong measurable signals (4.6.71. We c h o s e f l u o r o p h o r e s a s t r a n s d u c e r s f o r o u r K* sensors. There are s e v e r a l advantages t o t h i s approach. The measurement o f f l u o r e s c e n c e i s a n e x t r e m e l y s e n s i t i v e t e c h n i q u e (5). A number o f e f f i c i e n t f l u o r e s c e n t s y s t e m s s u c h a s d y e s , f u s e d a r o m a t i c s , e t c . w i t h quantum y i e l d above 0.9 are readily available. More importantly, f l u o r e s c e n t s e n s o r s c a n b e i m m o b i l i z e d on o p t i c a l f i b e r s i n r e m o t e in v i v o m o n i t o r i n g (6) o f K* i n t h e b l o o d o f p a t i e n t s undergoing open-heart surgery. T h e r e i s , h o w e v e r , a d i s a d v a n t a g e w h i c h i s common t o a l l sensors. S e n s o r s o b e y mass l a w s i n c e t h e e q u i l i b r i u m constants of t h e i r interaction with analytes a r e much lower. As a consequence, a p l o t of the sensor s i g n a l v e r s u s l o g o f c o n c e n t r a t i o n o f a n a l y t e i s " S " shaped and not l i n e a r (61. T h i s implies that the concentration of analyte ( i n o u r c a s e K*) must f a l l w i t h i n t h e " d y n a m i c r a n g e " o f t h e " S " c u r v e (6) . S h i f t i n g the "dynamic range" w i l l r e q u i r e a f u n d a m e n t a l c h a n g e i n t h e way t h e s e n s o r i s constructed. T h e r e i s a l s o a n a d v a n t a g e r e s u l t i n g f r o m t h e mass l a w effect. Lower c o n c e n t r a t i o n o f a g i v e n s e n s o r c a n s c a n l a r g e c o n c e n t r a t i o n ranges of the a n a l y t e . T h i s advantage, along with those discussed e a r l i e r , supports our c o n c l u s i o n t h a t sensors i n general are economical to use. They a r e n o t consumed a n d a r e n e e d e d i n s m a l l a m o u n t s . They r e s p o n d i n s t a n t a n e o u s l y t o changes i n a n a l y t e c o n c e n t r a t i o n . In addition, fluorescent sensors are suited for remote s e n s i n g , m i n i a t u r i z a t i o n and automation (6).

Ions i n Human Blood In designing a fluorescent sensor for the in vivo m o n i t o r i n g o f K*, i t i s i m p o r t a n t t o t a k e i n t o a c c o u n t t h e nature, c o m p o s i t i o n and t h e e n v i r o n m e n t s u r r o u n d i n g t h e i o n s t h a t a r e p r e s e n t i n human b l o o d . T a b l e I p r o v i d e s t h e

Table I. Ions in Blood Serum Ions Na K Ca HC0 +

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+ 2

3

cr Li

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Normal Range (mmol/L) 135-148 3.5-5.3 4.5-5.5 23-30 103 0-2.0

pH Range 7.35 - 7.45


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Table II. Ionic Diameters and Free Energy of Hydration Ionic Diameter (A) 1.20 1.90 2.66 2.12

Cation Li Na K Ca +

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+ 2

-AG° (Kcal/mol.)(25°) 122.0 98.5 80.5 379.0

e s s e n t i a l data ( S a a r i , L . A . , Instrumentation Lab I n c . , p e r s o n a l communication, 1984.) The medium i s aqueous (near n e u t r a l pH). Na* i s present i n 35-40 f o l d excess compared t o K*. Because of i t s abundance and i t s s i m i l a r chemical b e h a v i o r , Na* w i l l compete with K* f o r the sensor. In the face o f such a competition, K* can be analyzed r e l i a b l y only i f the sensor shows a s e l e c t i v i t y of the order of 500 f o r K* over Na*. T h i s i s a formidable t a s k . Even though C a i s present i n amounts comparable t o K*, the doubly-charged metal i o n i s easy t o d i s c r i m i n a t e against. G r e l l et a l . (8) have shown t h a t d i v a l e n t calcium i s complexed b e t t e r by a n i o n i c c a r r i e r s r a t h e r than the n e u t r a l ones. Overcoming the l a r g e f r e e energy of h y d r a t i o n (91 (Table II) of a l k a l i and a l k a l i n e e a r t h metal ions i n aqueous medium i s a l s o a c h a l l e n g i n g t a s k . The sensor designed must compete with water f o r the designated i o n . F o r t u n a t e l y , the h y d r a t i o n energy f o r K* i s much s m a l l e r than t h a t f o r Na* because of i t s l a r g e r s i z e . Y e t , i s l a r g e enough t o compete w i t h the sensor f o r K*. Since our f l u o r e s c e n t sensor w i l l be immobilized on an o p t i c a l f i b e r , i t i s important t o evaluate the o v e r a l l performance of such a d e v i c e . In g e n e r a l , o p t i c a l f i b e r technology has given o p t i c a l sensors an edge over other conventional t e c h n o l o g i e s such as e l e c t r o c h e m i c a l probes. O p t i c a l sensors are not subject t o e l e c t r i c a l i n t e r f e r e n c e s and do not r e q u i r e a r e f e r e n c e . The b i o c o m p a t i b i l i t y of o p t i c a l f i b e r s f o r An v i v o a p p l i c a t i o n s has been w e l l e s t a b l i s h e d (6). As d i s c u s s e d e a r l i e r , these d e v i c e s are amenable t o m i n i a t u r i z a t i o n (6). However, there are a l s o disadvantages. Ambient l i g h t often i n t e r f e r e s . Photob l e a c h i n g o f the sensor molecule may a f f e c t i t s long-term s t a b i l i t y (6). 2+

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Design of Fluorescent Chemosensors for K

As o u t l i n e d e a r l i e r , our f l u o r e s c e n t sensors f a r K* are h y b r i d systems made by a t t a c h i n g ionophores (of h i g h s e l e c t i v i t y f o r K*) t o e f f i c i e n t f l u o r e s c i n g groups. In d e s i g n i n g these h y b r i d systems, i t i s important not t o compromise e i t h e r the s e l e c t i v i t y of the ionophore o r the e f f i c i e n c y o f the f l u o r e s c i n g f u n c t i o n . For t h i s reason, we decided not t o use natural antibiotics such as v a l i n o m y c i n i n our h y b r i d systems. Valinomycin shows an u n u s u a l l y h i g h s e l e c t i v i t y f o r K* (9.10). However, the



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s e l e c t i v i t y o f t h i s a n t i b i o t i c i s c o n t r o l l e d b y t h e way i t f o l d s t o accommodate K* ( 9 ) . Conformational f a c t o r s are therefore important. Any e x t e r n a l attachment to this a n t i b i o t i c w i l l a f f e c t t h e f o l d i n g mechanism t h u s a f f e c t i n g its selectivity. On t h e o t h e r h a n d , s y n t h e t i c i o n o p h o r e s a r e b e t t e r suited for attachment to fluorophores. Their ion s e l e c t i v i t i e s are related to the s i z e of t h e i r cavities (9.101 w h i c h i s u s u a l l y u n a f f e c t e d b y a t t a c h m e n t s t o o t h e r molecules. Further, c o n f o r m a t i o n i s n o t an o v e r r i d i n g factor. The f u s i o n o f a f l u o r e s c i n g group t o s y n t h e t i c ionophores will not significantly change their selectivities. T a b l e III summarizes t h e e f f i c i e n c i e s o f selected c r o w n e t h e r s f o r b i n d i n g Na* and K* ( 1 1 1 . E f f i c i e n c i e s are m e a s u r e d i n t e r m s o f l o g o f a s s o c i a t i o n c o n s t a n t s (Ka) in methanol and w a t e r . The c o r r e l a t i o n between t h e d i a m e t e r o f t h e a l k a l i i o n s and t h e c a v i t y d i a m e t e r o f t h e crown e t h e r s i s e v i d e n t from t h e enhanced a s s o c i a t i o n c o n s t a n t s . F o r e x a m p l e , 1 8 - c r o w n - 6 , ( c a v i t y d i a m e t e r 2 . 6 - 3 . 2 A) b i n d s K* ( d i a m e t e r 2 . 6 6 À) more s e l e c t i v e l y t h a n Na* ( d i a m e t e r 1.9 A) . On t h e o t h e r h a n d , 1 5 - c r o w n - 5 ( c a v i t y d i a m e t e r 1 . 7 - 2 . 2 A) i s more s e l e c t i v e f o r Na* t h a n K*. The c a s e o f 2 1 - c r o w n 7 is interesting. G o k e l e t a l . (121 h a v e shown t h a t t h e c a v i t y o f t h i s i o n o p h o r e ( d i a m e t e r 3 . 4 - 4 . 3 À) i s t o o l a r g e f o r Λ b u t l a r g e r s t i l l f o r Na*. A s a r e s u l t , i t i s more s e l e c t i v e f o r K* t h a n 1 8 - c r o w n - 6 . The d i f f e r e n c e i n l o g Ka v a l u e s i n m e t h a n o l b e t w e e n K* a n d Na* f o r 2 1 - c r o w n - 7 is larger (1.9) compared t o 18-crown-6, for which the d i f f e r e n c e i s 1.78. However, 21-crown-7 i s l e s s s e n s i t i v e t h a n 1 8 - c r o w n - 6 i n b i n d i n g K* ( l o g Ka o f 4 . 3 5 v e r s u s 6 . 1 ) . I n w a t e r , l o g Ka v a l u e s a r e 2-4 o r d e r s o f m a g n i t u d e smaller. B e c a u s e o f t h e h i g h s o l v a t i o n e n e r g i e s o f K* a n d Na* w i t h w a t e r , i t i s d i f f i c u l t t o e x t r a c t them i n t o t h e ionophore. T h e l o w e r l o g Ka v a l u e s i m p l y t h a t t h e i o n s a r e exchanged f a s t e r i n water than i n methanol. Consequently, t h e i o n o p h o r e s w i l l b e h a v e more a s s e n s o r s i n w a t e r t h a n i n methanol. Cryptands are b a s k e t - l i k e b i c y c l i c ionophores i n which three strands of polyethers are t i e d together by two n i t r o g e n atoms. They p r o v i d e t h r e e - d i m e n s i o n a l s p a c e s f o r b i n d i n g m e t a l i o n s (11.13.141 They a r e s e v e r a l o r d e r s o f m a g n i t u d e more s e l e c t i v e t h a n c r o w n e t h e r s in binding a l k a l i metal i o n s . T a b l e IV p r o v i d e s d a t a o n d i m e n s i o n s a n d l o g Ka v a l u e s f o r K*, Na* a n d L i * i n w a t e r f o r [222] [221] a n d [211] cryptands. F o r t h e [222] cryptand, the d i f f e r e n c e i n t h e l o g Ka v a l u e b e t w e e n K* a n d Na* i n w a t e r i s 2.54. F o r 1 8 - c r o w n - 6 , t h e d i f f e r e n c e (Table III) is 1.83. Even though the i o n - r e c o g n i z i n g c a p a b i l i t i e s o f the crown e t h e r s and c r y p t a n d s a r e w e l l e s t a b l i s h e d , t h e y a r e s t i l l "passive" sensors. The c o n v e r s i o n o f t h e s e " p a s s i v e i o n o p h o r e s " i n t o e f f i c i e n t f l u o r e s c e n t s e n s o r s depends on t h e c h o i c e o f f l u o r o g e n i c g r o u p a n d t h e way i t i s a t t a c h e d to these ionophores.





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S e l e c t i n g t h e f l u o r o g e n i c g r o u p was c h a l l e n g i n g . We i n i t i a t e d our quest with naphthalene. 2,3-Naphtho-18c r o w n - 6 was s y n t h e s i z e d a n d e v a l u a t e d . This particular i o n o p h o r e h a s b e e n shown b y P e d e r s e n (15) a n d Cram e t a l . (16) (through the extraction technique) to be highly s e l e c t i v e f o r K* o v e r N a * . H o w e v e r , i t was i n s o l u b l e i n water. In d i o x a n e , a s l i g h t quenching o f i t s f l u o r e s c e n c e was o b s e r v e d o n a d d i n g KI and N a l . K* was more s e l e c t i v e t h a n Na*. The p o o r r e s p o n s e o f t h e f l u o r e s c e n c e o f t h i s i o n o p h o r e t o m e t a l i o n - b i n d i n g i s p r o b a b l y due t o t h e weak d i p o l e generated i n i t s excited s t a t e . The c h a r g e s t e n d t o be d e l o c a l i z e d i n t h e n ap h t h al en e s y s t e m s . We n e e d a s y s t e m i n w h i c h t h e e x c i t e d s t a t e d i p o l e moment i s s t r o n g and l o c a l i z e d . We l o o k e d f o r a s y s t e m w i t h h e t e r o a t o m s . C o u m a r i n i s one s u c h s y s t e m ( 1 7 . 1 8 ) . The f l u o r e s c e n c e quantum y i e l d i s o f t h e o r d e r o f 0 . 9 f o r c o u m a r i n a s w e l l a s i t s 7 - h y d r o x y a n d 6 , 7 - d i h y d r o x y compounds ( 1 9 ) . The S t o k e s s h i f t i s c l o s e t o 100 nm. E v e n more importantly, coumarins absorb l i g h t between 3 0 0 - 4 0 0 nm w h i c h i s a s u i t a b l e range f o r use i n o p t i c a l f i b e r s . In addition, c o u m a r i n s a r e more s o l u b l e i n w a t e r t h a n f u s e d a r o m a t i c s s u c h a s n a p h t h a l e n e and a n t h r a c e n e (20). F i g u r e 1 shows t h e s t r u c t u r e o f c o u m a r i n a n d the numbering system. I t a l s o shows t h e g r o u n d s t a t e a n d t h e dipolar excited state of the 7-hydroxy and 6,7dihydroxycoumarins (18). T h e r e p o r t e d f l u o r e s c e n c e quantum yields of t h e s e two compounds a r e h i g h (18.19). As d i s c u s s e d e a r l i e r , t h e way t h e f l u o r e s c i n g s i g n a l g r o u p i s attached t o the ionophore i s of great importance. The efficiency of transduction of the free energy of supramolecular interaction (between t h e metal ion and l i g a t i n g heteroatoms) depends o f t h e ease o f t h e e l e c t r o n transport from the heteroatoms to the fluorescing chromophore. In our d e s i g n , t h e oxygen a t p o s i t i o n 7 o f the coumarin w i l l p l a y a c r i t i c a l r o l e i n such e l e c t r o n transports. T h i s o x y g e n atom w i l l c o n v e r t t h e " p a s s i v e ionophore" i n t o i o n - r e c o g n i z i n g chemosensor. We u s e t h e term chemosensor t o emphasize t h e t o t a l l y s y n t h e t i c n a t u r e o f our s e n s o r as opposed t o the n a t u r a l o r semi-natural s y s t e m s w h i c h d e p e n d a t l e a s t i n p a r t o n n a t u r e t o do t h e d e s i g n and s y n t h e s i s .

Materials and Methods 1

N- [ 2-Hydroxyethy 1 ] piperaz i n e - N - [ 2 - e t h a n e s u l f o n i c acid ] (HEPES) was o b t a i n e d f r o m S i g m a . N H C 1 , C a C l , M g C l , L i C l , N a C l , K C 1 , e t h a n o l and m e t h a n o l w e r e p u r c h a s e d f r o m F i s h e r . DMSO a n d q u i n i n e s u l f a t e were p r o c u r e d f r o m A l d r i c h . The water used was purified by MILLI-Q water system (Millipore). Stock solutions (2 mM) of the 4m e t h y l c o u m a r o - c r y p t a n d s w e r e p r e p a r e d i n DMSO a n d d i l u t e d with water or methanol. Stock s o l u t i o n s (0.1 M) were p r e p a r e d i n 50:50 e t h a n o l - w a t e r f o r 4-methylcoumaro-crown e t h e r s a n d d i l u t e d w i t h t h e same s o l v e n t m i x t u r e . 4

2


2

168


Table m. Crown Ethers Cavity Diameter


^

Cavity Diameter

log(Kaj (2.66A)

K+

log(Ka)N (1.90A)

>+

log(Ka)

LI+

( 1 . 2 0 A )

2.8A

9.74 (W)

7.20 (W)

1.25 (W)

22 .A

3.95 (W)

5.40 (W)

2.50 (W)

2.0 (W)

3.20 (W)

5.5 (W)

1.6Â (W)Water

4

^Y^v^^pOH OH Figure

1

(M)Methanol

5

O^Ovx^

+ OH

^ ^ O H Coumarins




169

S p e c t r a l measurements were p e r f o r m e d o n a P e r k i n - E l m e r L S - 5 f l u o r o m e t e r u s i n g 5-nm s l i t s . R e l a t i v e quantum y i e l d measurements were d e t e r m i n e d on a P e r k i n - E l m e r LS-100 fluorometer. A l l s t u d i e s were p e r f o r m e d a t 2 2 . The s p e c t r a were n o t c o r r e c t e d f o r the spectral v a r i a t i o n i n t h e e x c i t a t i o n source and p h o t o m u l t i p l i e r tube sensitivity. Background scattered light was z e r o e d e l e c t r o n i c a l l y before the acquisition of the fluorescence of 4-methylcoumaro-[222]cryptand. A b s o r p t i o n s p e c t r a were obtained using Perkin Elmer Lambda 7 UV/VIS Spectrophotometer. T h e f a s t atom bombardment mass s p e c t r o m e t e r (FABMS) was a VG A n a l y t i c a l 2AB-HF r e v e r s e g e o m e t r y d o u b l e f o c u s i n g i n s t r u m e n t w i t h a n i o n s o u r c e u s i n g 7 KEV X e n o n . Spectra w e r e r e c o r d e d o n a VG 1 1 - 2 5 0 i n s t r u m e n t c o n t r o l a n d a n i o n e n e r g y o f 8 K E V . P o s i t i v e i o n s p e c t r a were more i n t e n s e and g e n e r a l l y of great analytical value. Competitive a l k a l i i o n b i n d i n g w i t h t h e 4 - m e t h y l c o u m a r o - c r y p t a n d s were c o n d u c t e d i n g l y c e r o l m a t r i x c o n t a i n i n g KC1 a n d N a C l .


e

Results and Discussion Evaluating the F e a s i b i l i t y of Using

4-Methylcoumarin i n Chemosensors f o r K*. We s y n t h e s i z e d two c l a s s e s of chemosensors: 4-methylcoumaro-crown ethers and 4methylcoumaro-cryptands. T h e s e a r e shown i n F i g u r e s 2 a n d 3. T h e f e a t u r e t h a t i s common t o a l l t h e s e c h e m o s e n s o r s i s the b r i d g i n g o f t h e ionophores t o t h e coumarin through o x y g e n s a t p o s i t i o n 6 a n d 7 . We h a v e a l r e a d y d i s c u s s e d t h e i m p o r t a n t r o l e a s s i g n e d t o t h e oxygen a t p o s i t i o n 7. It may a l s o b e a p p r o p r i a t e t o p o i n t o u t t h a t t h e n i t r o g e n s o f the cryptand series play a role in "switching on" fluorescence. T h e crown e t h e r s e r i e s w h i c h d o n o t c o n t a i n n i t r o g e n s tend t o "switch o f f " f l u o r e s c e n c e (Vide i n f r a ) . The m e t h y l group i n t h e s e chemosensors w i l l b e u s e d l a t e r f o r a t t a c h i n g them t o o p t i c a l f i b e r . In developing a new t e c h n o l o g y for fluorescent chemosensors, it i s important to establish that the observed changes i n t h e f l u o r e s c e n c e e m i s s i o n t r u l y r e f l e c t t h e a n t i c i p a t e d b e h a v i o r o f t h e i r i o n o p h o r e component. The i o n o p h o r e components i n t h e s i x c h e m o s e n s o r s ( F i g u r e s 2 a n d 3) h a v e b e e n d i s c u s s e d e a r l i e r . T h e i r l o g Ka v a l u e s f o r b i n d i n g K*, Na* a n d L i * h a v e b e e n l i s t e d i n T a b l e s I I I a n d IV. T h e f e a s i b i l i t y s t u d i e s were f i r s t c a r r i e d o u t w i t h the 4-methylcoumaro-crown e t h e r s . A 10' M s o l u t i o n o f 4m e t h y l c o u m a r o - 1 8 - c r o w n - 6 i n 5 0 : 5 0 e t h a n o l - w a t e r was e x c i t e d a t 330 nm. T h e f l u o r e s c e n c e e m i s s i o n was o b s e r v e d a t 410 nm. A p r o n o u n c e d q u e n c h i n g o f t h e f l u o r e s c e n c e was observed with a d d i t i o n o f KC1. T h e q u e n c h i n g was l e s s pronounced f o r N a C l . L i C l a n d C a C l showed n o q u e n c h i n g a t all. A plot of Io/I (the r a t i o o f i n i t i a l fluorescence i n t e n s i t y t o t h e i n t e n s i t y o f t h e quenched f l u o r e s c e n c e ) 5

2



170


4 - (Methyl) Coumaro -18 - Crown - 6

4 - (Methyl) Coumaro - 21 - Crown - 7

4 - (Methyl) Coumaro-15-Crown-5 Figure 2

4-Methylcoumaro-crown

4 - (Methyl) Coumaro (222) Cryptand

ethers

4 - (Methyl) Coumaro (221) Cryptand

4 - (Methyl) Coumaro (211) Cryptand Figure 3

4-Methylcoumaro-cryptands





171

a g a i n s t t h e c o n c e n t r a t i o n o f t h e a d d e d s a l t s (KC1 a n d N a C l ) i s shown i n F i g u r e 4 . The a n t i c i p a t e d s e l e c t i v i t y o f t h i s c h e m o s e n s o r f o r K* i s c l e a r l y d e m o n s t r a t e d . We were p a r t i c u l a r l y i n t e r e s t e d i n t h e b e h a v i o r o f 4 methylcoumaro-21-crown-7. We h a v e d i s c u s s e d e a r l i e r t h a t t h e p a r e n t 2 1 - c r o w n - 7 i s more s e l e c t i v e f o r K* o v e r Na* t h a n 18-crown-6 (12). T h e l a t t e r however was more s e n s i t i v e i n i t s r e s p o n s e t o K* (12) . T h i s i s c l e a r l y demonstrated i n F i g u r e 4 which a l s o i n c l u d e s a p l o t of Io/I against c o n c e n t r a t i o n o f t h e added s a l t s f o r t h e 4-methylcoumaro21-crown-7. The r a t i o o f I o / I f o r K* a n d Na* f o r t h i s c h e m o s e n s o r i s much l a r g e r t h a n t h e c o r r e s p o n d i n g r a t i o f o r the 4-methylcoumaro-18-crown-6, indicating higher s e l e c t i v i t y o f t h e former system f o r K*. However, t h e 4 m e t h y l c o u m a r o - 1 8 - c r o w n - 6 i s d e f i n i t e l y more s e n s i t i v e t o K* binding. These r e s u l t s demonstrate beyond doubt t h a t t h e 4-methylcoumarin is indeed an excellent fluorescent transducer that accurately r e f l e c t s the i o n s e l e c t i v i t i e s and sensitivities o f the ionophores t o which it is conjugated. Even though t h e f e a s i b i l i t y o f u s i n g coumarin i n chemosensors h a s been e s t a b l i s h e d w i t h t h e crown e t h e r s , t h e s e systems a r e n o t s u f f i c i e n t l y s e n s i t i v e t o be used i n commercial d e v i c e s . Since cryptands are several orders of m a g n i t u d e more s e l e c t i v e a n d s e n s i t i v e t h a n c r o w n e t h e r s ( 9 . 1 3 . 1 4 ) . we s y n t h e s i z e d t h r e e 4 - ^ m e t h y l c o u m a r o - c r y p t a n d s shown i n F i g u r e 3 a n d e v a l u a t e d t h e i r p e r f o r m a n c e . 4-Methylcoumaro-[222] C r y p t a n d a s F l u o r e s c e n t C h e m o s e n s o r f o r K*. A l l t h r e e c r y p t a n d - b a s e d c h e m o s e n s o r s shown i n F i g u r e 3 performed f a r beyond o u r e x p e c t a t i o n . Unlike the c r o w n e t h e r s y s t e m s w h i c h showed s e l e c t i v e f l u o r e s c e n c e quenching, t h e c r y p t a n d s showed s e l e c t i v e fluorescence enhancement. A 4 μΜ s o l u t i o n o f t h e 4-methylcoumaro-[222] c r y p t a n d (MCC222) i n w a t e r (pH 7 . 4 ) showed a t h r e e - f o l d enhancement o f i t s f l u o r e s c e n c e e m i s s i o n when t r e a t e d w i t h 20 mM K C 1 . T h e a d d i t i o n o f N a * , M g * , C a * a n d N H * d i d n o t change its fluorescence. Similarly, 4-methylcoumaro[221]cryptand and 4-methylcoumaro[211]cryptand showed selective fluorescence enhancement for Na* a n d L i * , respectively. T h e l a t t e r two compounds however w i l l n o t b e discussed i n t h i s chapter. F i g u r e s 5a a n d 5b show t h e f l u o r e s c e n t e x c i t a t i o n a n d e m i s s i o n s p e c t r a o f MCC222 a t v a r y i n g c o n c e n t r a t i o n s ( 0 - 2 0 mM) o f KC1 i n a n a q u e o u s medium c o n t a i n i n g HEPES (pH 7 . 4 ) . S i m i l a r r e s u l t s were o b t a i n e d i n 5 0 : 5 0 e t h a n o l - w a t e r as w e l l as i n methanol. The e x c i t a t i o n and e m i s s i o n peaks were a t 340 nm a n d 420 nm, r e s p e c t i v e l y . T h e r e i s no spectral shift either i n the excitation or emission spectra. The d o s e - r e s p o n s e c u r v e for concentrations r a n g i n g f r o m 0 - 1 0 mM o f KC1 i s shown i n F i g u r e 6 . The r a n g e o f c o n c e n t r a t i o n o f K* i n human b l o o d i s b e t w e e n 3 . 5 5 . 3 mM. T h i s f a l l s b e a u t i f u l l y w i t h i n t h e d y n a m i c r a n g e o f MCC222. 2

2



172


[ K ] or [Na ] +

Figure 4

+

Selectivities and Sensitivities of 4M e t h y l c o u m a r o - c r o w n e t h e r s t o w a r d s K*" a n d N a

+

240 280 320 360 400 Wavelength (nanometers) F i g u r e 5 a E x c i t a t i o n s p e c t r a o f 4 μΜ 4 - M e t h y l c o u m a r o [222] C r y p t a n d i n a q u e o u s s o l u t i o n c o n t a i n i n g 5 mM HEPES (pH 7 . 4 ) a n d 0 - 2 0 mM K C 1 . E m i s s i o n 420 nm (5 nm s l i t s ) .





173

350 390 430 470 510 550 Wavelength (nanometers) F i g u r e 5b E m i s s i o n s p e c t r a described above. slits).

2

4

o f t h e aqueous solutions Excitation 340 nm (5 nm

6

8

K° Concentration (mM) Figure

6

Calibration of 4 μΜ 4 - M e t h y l c o u m a r o [ 2 2 2 ] C r y p t a n d i n A q u e o u s S o l u t i o n (pH 7 . 4 )


174


T h e p r o p e r t i e s o f MCC222 a r e s u m m a r i z e d i n T a b l e V . T h e l o g Ka v a l u e s were m e a s u r e d f r o m t h e c h a n g e s i n t h e fluorescence. T h e l o g Ka i n m e t h a n o l was m e a s u r e d i n t h e p r e s e n c e o f 1.4 mM N a C l . T h i s v a l u e i s r o u g h l y two o r d e r s o f magnitude l a r g e r than t h a t observed i n water. K* i s b o u n d much s t r o n g e r t o MCC222 i n m e t h a n o l . T h e r e s p o n s e o f MCC222 f o r o t h e r i o n s was i n v e s t i g a t e d i n a s y s t e m a t i c manner b y two s e t s o f e x p e r i m e n t s . First, a 4 μΜ s o l u t i o n o f MCC222 was t r e a t e d i n c r e m e n t a l l y w i t h Na* ( r a n g i n g f r o m 0-140 mM), C a * ( r a n g i n g f r o m 0-10 mM) a n d NH * ( r a n g i n g f r o m 0-10 mM) . No c h a n g e i n t h e fluorescence e m i s s i o n was o b s e r v e d f o r t h e s e s a l t s . In the second s e r i e s o f e x p e r i m e n t s , a 4 μΜ s o l u t i o n o f MCC222 was f i r s t t r e a t e d w i t h 4 mM K C 1 , w h i c h c a u s e d a n instantaneous enhancement o f t h e f l u o r e s c e n c e a n d t h e n r e m a i n e d c o n s t a n t at that l e v e l . The Na*, Ca * and N H / s a l t s were added i n i n c r e m e n t a l amounts a s b e f o r e . C a * a n d NH * showed no f u r t h e r change i n t h e f l u o r e s c e n c e . However, t h e a d d i t i o n o f Na* d e c r e a s e d t h e f l u o r e s c e n c e i n c r e m e n t a l l y without altering the spectral properties of the probe. This s u g g e s t s t h a t Na* c a n d i s p l a c e K* f r o m i t s b i n d i n g s i t e , y e t t h e b o u n d Na* d o e s n o t show any c h a n g e i n t h e f l u o r e s c e n c e o f the chemosensor. The s e l e c t i v i t y o f t h e p a r e n t [222] c r y p t a n d f o r K* o v e r Na* h a s b e e n r e p o r t e d t o b e 350 (14) corresponding to a difference i n l o g Ka o f 2 . 5 4 . Our c h e m o s e n s o r i s more r i g i d b e c a u s e o f t h e f u s i o n o f the aromatic r i n g o f coumarin t o the [222] cryptand. The s e l e c t i v i t y i s e x p e c t e d t o be lower f o r K \ T h e l o g Ka f o r Na* c a n n o t b e d e t e r m i n e d f l u o r o m e t r i c a l l y s i n c e Na* d o e s n o t a f f e c t t h e f l u o r e s c e n c e o f t h i s chemosensor i n t h e absence o f K*. H o w e v e r , we c a n d e t e r m i n e t h e c h a n g e i n t h e l o g Ka f o r K* c a u s e d b y t h e a d d i t i o n o f N a * . T h i s i s s u m m a r i z e d i n Table VI. We now h a v e a s i t u a t i o n where t h e competitive b i n d i n g b y Na* d o e s n o t a f f e c t t h e s i g n a l b u t d e c r e a s e s t h e l o g K a v a l u e s f o r K* b i n d i n g . D e c r e a s e s i n t h e l o g Ka implies that the i o n o p h o r e b i n d s K* w e a k l y but more reversibly. T h u s , MCC222 p e r f o r m s b e t t e r a s a s e n s o r i n t h e p r e s e n c e o f Na* t h a n i t d o e s i n i t s a b s e n c e . However, i t w i l l be l e s s s e n s i t i v e . I n m e d i c i n a l c h e m i s t r y , Na* w i l l b e d e s c r i b e d a s an a n t a g o n i s t s i n c e i t p r o d u c e s no e f f e c t o f i t s own o n t h e f l u o r e s c e n c e a n d y e t c o m p e t e s w i t h K* w h i c h i s a n a g o n i s t . K* d o e s a f f e c t t h e f l u o r e s c e n c e when b o u n d t o t h e s e n s o r (see Appendix I I ) . T h e a p p a r e n t Ka v a l u e s f o r K* b i n d i n g i n T a b l e V I w e r e u s e d t o c a l c u l a t e t h e s e l e c t i v i t y o f J? o v e r Na* f o r b i n d i n g MCC222. Using a linear regression a n a l y s i s (see Appendix I I ) , t h i s v a l u e was d e t e r m i n e d t o b e 15. T h e c a l c u l a t e d Ka f o r Na* i s 35 M" w h i c h c o r r e s p o n d s t o l o g Ka o f 1 . 5 4 . E a r l i e r i t was i n d i c a t e d t h a t the s e l e c t i v i t y o f K* o v e r Na* o f t h e o r d e r o f 500 was n e c e s s a r y for the fluorescent chemosensors t o be effective in continuous monitoring devices. However, t h i s constraint was b a s e d on t h e a s s u m p t i o n t h a t Na* w i l l b e h a v e l i k e a n 2


4

2

2

4

1




agonist. S i n c e i t i s an a n t a g o n i s t i t does n o t i n t e r f e r e w i t h t h e t r a n s d u c t i o n a n d t h e r e f o r e , a s e l e c t i v i t y o f 15 i s sufficient for practical applications. Further, f l u c t u a t i o n s i n the c o n c e n t r a t i o n o f Na d u r i n g o p e n - h e a r t s u r g e r y w i l l n o t a f f e c t t h e a p p a r e n t Ka v a l u e f o r K* a s s e e n i n Table VI. I f n e c e s s a r y , c o r r e c t i o n c a n b e made f o r s u c h fluctuations. T h e c o m p e t i t i v e b i n d i n g o f Na* t o MCC222 was c o n f i r m e d b y FABMS m e t h o d . The a d d i t i o n o f N a C l a n d KC1 t o MCC222 i n the g l y c e r o l m a t r i x showed a p r e d o m i n a n t p e a k a t 545 c o r r e s p o n d i n g t o (M+K*) a n d m i n o r p e a k a t 529 (M+Na ) . We s e l e c t e d MCC222 f o r d e v e l o p i n g t h e f i b e r o p t i c s b a s e d s e n s o r f o r m o n i t o r i n g K* i n t h e e x t r a c o r p o r e a l b l o o d during open-heart surgery. The t e c h n o l o g y has been l i c e n s e d t o a d i a g n o s t i c company a n d c o m m e r c i a l i z a t i o n i s expected w i t h i n the next couple of y e a r s . +


+

M i c r o f l u o r o m e t r i c Assay f o r K*. We h a v e a l r e a d y shown t h a t t h e l o g Ka v a l u e f o r K* i n m e t h a n o l f o r MCC222 i s two o r d e r s o f magnitude l a r g e r than the v a l u e i n water. This implies t h a t t h e i o n o p h o r e i s more s t r o n g l y b o u n d t o K* i n m e t h a n o l than i n water. T h e r e f o r e , i t may b e h a v e a s a r e a g e n t i n methanol. A s a f l u o r e s c e n t r e a g e n t , MCC222 c a n b e u s e d i n a continuous flow microfluorometric assay (21). This technique i s so s e n s i t i v e t h a t o n l y n a n o l i t e r q u a n t i t i e s o f samples a r e needed f o r a n a l y s i s . The f e a s i b i l i t y o f u s i n g MCC222 a s a r e a g e n t was d e m o n s t r a t e d a s f o l l o w s : the flow a s s a y s o l u t i o n was p r e p a r e d b y d i s s o l v i n g MCC222 ( 7 . 6 μΜ) a n d N a C l ( 1 . 4 mM) i n 100% m e t h a n o l . Two s e t s o f a n a l y t i c a l standards were prepared in water. These standards r e p r e s e n t t h e b l o o d serum s a m p l e s . I n b o t h s e t s , t h e l e v e l of KC1 v a r i e d f r o m 1 . 0 - 6 . 0 mM. The f i r s t set also c o n t a i n e d N a C l a t a c o n s t a n t l e v e l o f 50 mM. I n t h e s e c o n d s e t , t h e l e v e l o f N a C l was m a i n t a i n e d a t 150 mM. T h e l e v e l c o n c e n t r a t i o n o f Na* i n human b l o o d i s a p p r o x i m a t e l y 135 mM. T h e 50 mM a n d 150 mM l e v e l s o f N a C l t h e r e f o r e represent extreme l i m i t s i n NaCl c o n t e n t . Our p u r p o s e h e r e i s t o d e m o n s t r a t e t h a t c h a n g e s i n t h e l e v e l o f Na* i n b l o o d samples w i l l n o t a f f e c t o u r a s s a y , and t h a t t h e reagent (MCC222) w i l l r e s p o n d o n l y t o c o n c e n t r a t i o n c h a n g e s i n K*. B e c a u s e o f t h e s t r o n g b i n d i n g o f t h e r e a g e n t t o K* i n m e t h a n o l , t h e s t a n d a r d samples had t o be d i l u t e d 2000:1 t o b r i n g t h e c o n c e n t r a t i o n o f K* w i t h i n t h e " d y n a m i c r a n g e " . The r e s u l t s a r e summarized i n T a b l e V I I . Whether t h e l e v e l c o n c e n t r a t i o n o f Na* i s 50 mM o r 150 mM, t h e f l u o r e s c e n c e i n t e n s i t y d e p e n d e d o n l y o n t h e c o n c e n t r a t i o n o f K*. The c o e f f i c i e n t o f v a r i a t i o n was o f t h e o r d e r o f 0 . 9 6 . T a b l e VII a l s o shows t h e ratios of the reagent ionophore concentrations to K* c o n c e n t r a t i o n s for the s a m p l e s w e r e >1. T h i s i s a p r o o f t h a t t h e r e a g e n t i s now consumed i n s t o i c h i o m e t r i c q u a n t i t i e s . Further, i n Table VII, t h e r a t i o c o n c e n t r a t i o n s o f Na* t o K* f o r t h e s a m p l e s v a r i e s f r o m 3000 t o 5 0 0 . This confirms the fact that i n m e t h a n o l , MCC222 i s more s e l e c t i v e a n d s e n s i t i v e t o w a r d s K* than i n water.


175


176

Table V. Properties of 4-Methykoumaro [222] Cryptand


LOG

EXCITATION PEAK, NM

EMISSION PEAK, NM

LOG ( K A ) (H 0)

340

420

2.72

(KA)

= LOG ASSOCIATION

K+

LOG (KA) *

K+

QUANTUM YIELD* (RELATIVE)

K+

:

4.92

0.045

CONSTANT

* = THIS MEASUREMENT WAS MADE IN THE PRESENCE OF 1.4 MM NA* IN 100% MEOH 4

= RELATIVE TO QUININE

SULFATE

( 0 . 1 ABSORBANCE IN 1 Ν SULFURIC ACID)

Table VI. Effect of N a on Log ( K A ) +

K+

of 4-Methykoumaro [222] Cryptand

NA* C O N C E N T R A T I O N . 0 LOG

(KA)

K +

2.72

10 2.64

50 2.34

2.03

Table Υ Π . Microfluorometric Assay for K Ratio

MM 100

140 1.97

+

Fluorescence Intensity (arbitrary units) Assay Solution (Γ) SOmMNa*

Assay Solution (II) 150 mM Na*

Assay Solution (mM)

After Dilution' (mM)

1.0

0.0005

15.2

18.1 ± 0 . 2 5

2.0

0.0010

7.6

35.6 ± 0 . 3 2

35.610.24

3.0

0.0015

5.1

52.510.44

52.410.48

4.0

0.0020

3.8

68.9±0.75

68.410.53

5.0

0.0025

3.0

84.710.42

83.710.36

6.0

0.0030

2.5

Ionophore /K*

2

100.310.42

3

4

17.610.46

99.6i0.47

5

1. Diluted 1:2000 in aflowingassay solution of 4-methylcoumaro[222] cryptand (7.6 μΜ) and N a d (1.4 mM) in methanol 2. Total Na 1.425 M 3. Total Na 1.475 M 4. Na upper limit » 3000-fold excess 5. Na lower limit- 500-fold excess +

+

+

+



Potassium in Blood ami across Membranes

177

+

K Transport Across Biological Membranes.

MCC222 i s a l s o an e x c e l l e n t research tool for studying the rate of t r a n s p o r t o f K* a c r o s s b i o l o g i c a l membranes. T h i s work was c a r r i e d out i n c o l l a b o r a t i o n w i t h P r o f . Ira Kurtz of the UCLA C o l l e g e o f M e d i c i n e a n d p u b l i s h e d e a r l i e r ( 2 2 ) . This work w i l l n o t b e d i s c u s s e d h e r e . M e c h a n i s m of Fluorescence Enhancement i n K -Bound 4Methylcourmaro[222]-cryptand. I o n - b i n d i n g quenches the f l u o r e s c e n c e i n coumaro-crown e t h e r s w h i l e i t enhances t h e fluorescence in coumaro-cryptands. Between the two effects, f l u o r e s c e n t enhancement i s more d r a m a t i c than quenching. Even though h i g h e r selectivities of the c r y p t a n d s may a c c o u n t i n p a r t f o r t h e h i g h e r s e n s i t i v i t i e s , t h e r e a s o n why t h e s e e f f e c t s a r e o p p o s i t e f o r t h e two systems i s i n t r i g u i n g . We h a v e a l r e a d y i n d i c a t e d t h a t t h e two n i t r o g e n s i n t h e c r y p t a n d s e r i e s may a f f e c t t h e f l u o r e s c e n c e i n ways d i f f e r e n t from t h e crown e t h e r s e r i e s . The c r o w n e t h e r s a r e not s e n s i t i v e t o changes i n pH. However, c h a n g i n g t h e pH f r o m 7 . 0 t o 7 . 4 d e c r e a s e s t h e f l u o r e s c e n c e e m i s s i o n o f MCC222 b y 16% i n t h e a b s e n c e o f K*. In i t s p r e s e n c e , t h e f l u o r e s c e n c e was i n c r e a s e d b y 12%. This lends further support t h a t the nitrogens indeed play a c r i t i c a l r o l e i n f l u o r e s c e n c e enhancement. Deprotonation of the nitrogens (due t o pH i n c r e a s e ) w o u l d e x p o s e t h e l o n e e l e c t r o n p a i r s of these nitrogens. Such d e p r o t o n a t i o n s u p p r e s s e s t h e fluorescence. P r e s u m a b l y , d e p r o t o n a t i o n a l s o p r o m o t e s K* b i n d i n g ; the lone p a i r s of electrons of the nitrogens are no l o n g e r e x p o s e d a n d a n enhancement in fluorescence results· The e x t i n c t i o n c o e f f i c i e n t ( 6 ) o f a l l t h e s i x c o u m a r o i o n o p h o r e s a r e o f t h e same o r d e r (10~ ) a n d a r e therefore equally efficient in absorbing l i g h t . However, the c o u m a r o - c r y p t a n d s a r e an o r d e r o f magnitude l e s s e f f i c i e n t in their fluorescence emission than the coumaro-crown ethers. T h e quantum y i e l d ( r e l a t i v e t o q u i n i n e sulfate) f o r MCC222 i n w a t e r i s 0 . 0 4 5 . (See T a b l e V I . ) The optimum c o n c e n t r a t i o n o f t h e coumaro-crown e t h e r s i n 50:50 e t h a n o l w a t e r i s 10' M w h i l e i t i s Ι Ο " M f o r c o u m a r o - c r y p t a n d s . Our e x p l a n a t i o n a s t o why t h e f l u o r e s c e n t enhancement o f MCC222 i s d r a m a t i c a l l y i n c r e a s e d on a d d i t i o n o f K* i s summarized i n F i g u r e 7. When t h i s c r y p t a n d i s n o t b o u n d t o K \ t h e s i n g l e t d i p o l a r e x c i t e d s t a t e (17.18) i s s u s c e p t i b l e t o t h e t r a n s f e r o f a s i n g l e e l e c t r o n from t h e nitrogen p r o x i m a l t o t h e p o s i t i v e l y charged oxygen a t p o s i t i o n 7. This photo-induced electron transfer (PET) c o n v e r t s t h e dipolar singlet excited state to a radical ion pair, in w h i c h t h e r a d i c a l c a t i o n r e s i d e s on t h e n i t r o g e n w h i l e t h e anion r a d i c a l i s d e l o c a l i z e d at the α,β-unsaturated ester functionality. These two radicals are no longer c o n j u g a t e d , t h e r e f o r e undergo a r a d i a t i o n l e s s d e c a y . T h u s , i n t r a m o l e c u l a r PET c a u s e s t h e f l u o r e s c e n c e t o b e s u p p r e s s e d i n the f r e e ionophore.


+

4

5

4



II

η

Figure

7

A Mechanism f o r F l u o r e s c e n c e - E n h a n c e m e n t bound 4-Methylcoumaro[222] C r y p t a n d

II

i n Κ*





179

When t h e c r y p t a n d i s bound t o K*, t h e n i t r o g e n l o n e p a i r s a r e s t a b i l i z e d and t h e i r i o n i z a t i o n p o t e n t i a l s are increased. PET i s no l o n g e r f a v o r e d a n d f l u o r e s c e n c e emission i s "turned-on" since the pathway suppressing fluorescence i s "turned-off". Similar chelation-enhanced f l u o r e s c e n c e h a s b e e n r e p o r t e d b y C z a r n i k (23) a n d de S i l v a (24) . However, t h i s t r i g g e r i n g o f f l u o r e s c e n c e does n o t h a p p e n f o r Na* s i n c e MCC222 i s n o t s e l e c t i v e f o r t h i s i o n . T h e Na* b i n d i n g d o e s n o t s t a b i l i z e t h e n i t r o g e n l o n e p a i r s s u f f i c i e n t l y t o p r e v e n t P E T . F l u o r e s c e n c e enhancement i s , t h e r e f o r e , not observed f o r t h i s i o n . I n t h e c a s e o f c o u m a r o - c r o w n e t h e r s , t h e r e a r e no nitrogens available for PET and, therefore, the f l u o r e s c e n c e i s not suppressed i n the free ionophore. However, metal ion binding quenches t h e fluorescence t h r o u g h d i s t o r t i o n s c a u s e d by t h e r e p u l s i v e interaction between t h e p o s i t i v e l y c h a r g e d oxygen a t p o s i t i o n 7 and t h e metal i o n . Of c o u r s e , t h i s e f f e c t i s present in the cryptands a l s o , but i s overwhelmed b y t h e PET i n the unbound s t a t e .

Conclusion We h a v e i n v e n t e d a f l u o r e s c e n t c h e m o s e n s o r f o r Au vivo m o n i t o r i n g o f K* i n b l o o d . By c h a n g i n g t h e s o l v e n t , the same c h e m o s e n s o r c a n b e u s e d a s a f l u o r e s c e n t r e a g e n t in a u t o m a t e d b a t c h a n a l y s i s o f K*. The c h e m o s e n s o r a l s o l e n d s i t s e l f a s a r e s e a r c h t o o l i n s t u d y i n g K* t r a n s p o r t a c r o s s b i o l o g i c a l a n d s y n t h e t i c membranes. B u t most i m p o r t a n t l y , we h a v e d e m o n s t r a t e d t h a t t h i s i n f o r m a t i o n p e r t a i n i n g to ion-binding in receptors can be communicated instantaneously in a cost-efficient way. This u n d e r s t a n d i n g opens t h e d o o r f o r f u r t h e r a d v e n t u r e into a r e a s o f m o l e c u l a r e l e c t r o n i c s , m o l e c u l a r r e c o g n i t i o n and i n f o r m a t i o n s t o r a g e and r e t r i e v a l .

Acknowledgements D r . I r a K u r t z o f t h e UCLA C o l l e g e o f M e d i c i n e was a n a c t i v e collaborator in this project a n d p r o v i d e d many o f the spectral data. His contribution is gratefully acknowledged. We t h a n k Ray B r a m b i l l a who p e r f o r m e d t h e NMR work. We a p p r e c i a t e t h e work o f D a v i d H i n d e n l a n g a n d Don S e d g w i c k who p r o v i d e d FAB mass s p e c t r a l d a t a . Ken L e g g s u p p o r t e d o u r work a n d r e c o g n i z e d t h e i m p o r t a n c e o f the f l u o r e s c e n c e a p p r o a c h t o K* s e n s i n g . G e o r g e S . Hammond s h e p h e r d e d t h e p r o j e c t a n d was a s o u r c e o f inspiration. The contributions of Ken and George are greatly appreciated. M a r y l o u Grumka i s a c k n o w l e d g e d f o r t y p i n g t h e manuscript·



180


Appendix I. Synthesis of Coumarin-Fused Ionophores

n s 1 or 2


11.

MASILAMANI & LUCAS



Appendix II The referee who reviewed this paper brought to attention terms "agonist" and antagonist" used s c i e n t i s t s i n the f i e l d of medicine. The referee also helped us to calculate s e l e c t i v i t y o f K* o v e r Na* b y NCC222 u s i n g t h e a p p a r e n t Ka v a l u e s f o r K* d e t e r m i n e d i n t h e p r e s e n c e o f Na* reported i n Table VI. Because V A p p a r e n t Ka f o r Κ* = Γ Κ* Ί Γ F r e e Licrand+Na*. L i a a n d 1 [Κ*. L i g a n d ] a n d s i n c e [ N a * . L i g a n d ] = [ F r e e L i g a n d ] [Na*] (Ka)

our by the log and

N a + #

V A p p a r e n t Ka f o r K* - 1 + TNa*l ( K a i , where ( K a ) i s the (Ka) t r u e Ka f o r K*. T h u s , a p l o t o f / A p p a r e n t Ka f o r K* v s [Na*] should g i v e a s t r a i g h t l i n e and t h e s l o p e o f t h i s l i n e w i l l g i v e s e l e c t i v i t y Na*/K*. This linear regression analysis using d a t a from T a b l e VI gave a s l o p e o f 0.067. The r e c i p r o c a l o f t h i s v a l u e , which i s 15, corresponds t o the s e l e c t i v i t y o f K* o v e r Na* f o r MCC222. Using t h i s value, (Ka) for MCC222 was c a l c u l a t e d t o b e 35 M" ( o r , t h e l o g ( K a ) = 1.54). We t h a n k t h e r e f e r e e f o r t h i s c o n t r i b u t i o n . M a

K +

K +

1

N a +

1

N a +

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RECEIVED

July 9, 1993


Fluorescent Chemosensors for Monitoring Potassium in Blood and

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