Cell Separation Science and Technology - American Chemical Society

Chapter 3

Rare-Earth Chelates as Fluorescent Markers in Cell Separation and Analysis 1

2

Downloaded by NORTH CAROLINA STATE UNIV on August 2, 2012 | http://pubs.acs.org Publication Date: June 10, 1991 | doi: 10.1021/bk-1991-0464.ch003

R. C. Leif and L. M. Vallarino 1

Coulter Electronics, Inc., 690 West 20th Street, Hialeah, FL 33010 Department of Chemistry, Virginia Commonwealth University, Richmond, VA 23284 2

The synthesis of a novel series of functionalized macrocyclic complexes of the lanthanide (III) ions is reported. The Eu(III) complexes possess a set of properties (water solubility, inertness to metal release, ligand-sensitized luminescence, reactive peripheral functionalities) that make them suitable as luminescent markers for bio-substrates. Currently employed organic fluorophores give efficient signal production, but this is accompanied by interference from background fluorescence and, if multiple fluorophores are used, from spectral overlap. The long lifetimes and narrow-band emissions of the luminescent lanthanide complexes will minimize background interference; however, long lifetimes will also result in a significantly reduced signal for flow cytometry or cell sorting. C e l l s e p a r a t i o n and c e l l a n a l y s i s are mutually dependent branches of c y t o p h y s i c s . The q u a l i t y o f a c e l l s e p a r a t i o n can be a s c e r t a i n e d by analyzing t h e i n d i v i d u a l f r a c t i o n s t o determine t h e i r c e l l u l a r composition. In t u r n , t h e accuracy o f a c e l l a n a l y s i s procedure can be documented most e a s i l y f o r a c e l l population t h a t has been e n r i c h e d i n a s p e c i f i c f r a c t i o n by an e f f e c t i v e s e p a r a t i o n procedure. In t h e past, i t was s u f f i c i e n t t o r e p o r t t h e r e s u l t s o f a c e l l s e p a r a t i o n i n terms of morphology (2-3). Recently, however, the development of monoclonal a n t i b o d i e s and of recombinant DNA techniques has g r e a t l y i n c r e a s e d t h e c a p a b i l i t y t o analyze c e l l s f o r s p e c i f i c antigens and even t o observe s i n g l e genes (4) thus p e r m i t t i n g t h e use of these advanced d e t e c t i o n procedures.

0097H5156/91/0464-0041$06.00/0 © 1991 American Chemical Society

In Cell Separation Science and Technology; Kompala, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

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E m i t t i n g Fluorophores

Current f l u o r e s c e n t h i s t o c h e m i c a l techniques, whether based on flow a n a l y s i s or on image cytometry, are l i m i t e d by t h e chemical nature of the f l u o r e s c e n t markers. Traditional f l u o r o p h o r e s are large, r i g i d organic molecules i n which both the ground and the e x c i t e d e l e c t r o n i c s t a t e s a r e comprised o f a multitude of c l o s e l y spaced v i b r a t i o n a l l e v e l s . In these molecules the e l e c t r o n s i n v o l v e d i n l i g h t a b s o r p t i o n are outer s h e l l (valence) e l e c t r o n s ; t h e i r energies a r e a f f e c t e d by atomic v i b r a t i o n s . The photons absorbed t h e r e f o r e have an energy d i s t r i b u t i o n which corresponds t o the many p o s s i b l e values o f the energy d i f f e r e n c e between the lowest v i b r a t i o n a l l e v e l ( s ) o f t h e ground e l e c t r o n i c s t a t e and v a r i o u s v i b r a t i o n a l l e v e l s o f the e x c i t e d e l e c t r o n i c s t a t e . Almost immediately a f t e r l i g h t e x c i t a t i o n , these molecules r e l a x t o the lowest v i b r a t i o n a l l e v e l of the f i r s t e x c i t e d s t a t e . When f l u o r e s c e n c e emission occurs, the energy d i s t r i b u t i o n o f the emitted photons corresponds t o the many p o s s i b l e v a l u e s of t h e energy d i f f e r e n c e between t h i s lowest v i b r a t i o n a l l e v e l o f the f i r s t e x c i t e d s t a t e and v a r i o u s v i b r a t i o n a l levels o f the ground state. At room temperature, t h e r e f o r e , both the e x c i t a t i o n and the emission s p e c t r a a r e broad (5) . With t r a d i t i o n a l organic f l u o r o p h o r e s , o v e r l a p between the f l u o r e s c e n c e of the marker and the broad autofluorescence o f the c e l l u l a r s u b s t r a t e r e p r e s e n t s a major l i m i t a t i o n i n the s e n s i t i v i t y of the o p t i c a l measurements (6-7). The broadness of t h e e m i s s i o n e x c i t a t i o n s p e c t r a a l s o severely reduces t h e number of f l u o r o p h o r e s t h a t may be measured simultaneously (8) . The emissions o f f l u o r e s c e i n and rhodamine, f o r example, o v e r l a p t o such an extent that i n flow microfluorometers and other analytical instruments special electronic c i r c u i t r y must be employed t o separate t h e i n d i v i d u a l s i g n a l s (9). Although t h i s type of dual measurement has been c o n s i d e r a b l y f a c i l i t a t e d by the s u b s t i t u t i o n o f rhodamine with p h y c o e r y t h r i n (10), which absorbs a t lower wavelengths, the l i m i t a t i o n remains. The preceding c o n s i d e r a t i o n s l e d L e i f e t a l . t o propose the use o f r a r e - e a r t h c h e l a t e s as luminescent reporter molecules (11). In these compounds, t h e luminescence results from t r a n s i t i o n s w i t h i n t h e 4f e l e c t r o n manifold o f the metal i o n . Since t h e i n n e r s h e l l 4f e l e c t r o n s are non-bonding and are e f f e c t i v e l y s h i e l d e d from t h e environment by the outer e l e c t r o n s h e l l s , these emissions are f r e e from v i b r a t i o n a l broadening (12). F i g u r e 1 shows the most intense emissions o f Eu(III) and T b ( I I I ) superimposed f o r comparison on the emissions o f fluorescein and rhodamine; c l e a r l y , these l a n t h a n i d e emissions do not overlap. The Question o f Long L i f e t i m e s . Another distinctive f e a t u r e o f the luminescent lanthanide c h e l a t e s i s t h e i r long e x c i t e d - s t a t e l i f e t i m e , u s u a l l y more than 300 με (13) . Since t h e l i f e t i m e s o f n a t u r a l as w e l l as c o n v e n t i o n a l s y n t h e t i c fluorophores are u s u a l l y o f the order o f 10 ns In Cell Separation Science and Technology; Kompala, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.


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WAVELENGTH F i g u r e 1. Emission p r o f i l e s of f l u o r e s c e i n , rhodamine, and of Eu (III) and Tb(III) n i t r a t e s i n aqueous solution. (Emission intensities on a r b i t r a r y scales.)

(5) , the lanthanide luminescence may be completely f r e e d from background i n t e r f e r e n c e by s h o r t - p u l s e e x c i t a t i o n of the sample, followed by s l i g h t l y delayed s i g n a l d e t e c t i o n (delay, ca. 1 μβ) . These time-gated measurements based on the l o n g - l i v e d luminescence of the lanthanide marker are advantageous i n automated immunoassays (14) and would a l s o be of value i n the microscopic examination of c e l l s as w e l l as i n flow cytometry or flow c e l l s o r t i n g . In the l a t t e r case, however, previous a n a l y s i s (25) has shown t h a t the s i g n a l i n t e n s i t y w i l l a l s o be diminished. Even i n the f a v o r a b l e s i t u a t i o n of a 10 με e x c i t a t i o n p u l s e and a lanthanide c h e l a t e with a r e l a t i v e l y short l i f e t i m e of 38 με, the percentage of l i g h t emitted w i t h i n the o r i f i c e w i l l be only 34%. T h i s i n c l u d e s the l i g h t s i g n a l c o l l e c t e d while the c e l l i s s t i l l being i l l u m i n a t e d . True dark f i e l d o b s e r v a t i o n with a conventional continuous wave l a s e r would decrease t h i s t o l e s s than 25%. With l o n g - l i v e d lanthanide fluorophores as markers, slowing the flow r a t e of the sample would s i g n i f i c a n t l y increase the e f f i c i e n c y of signal collection. In Cell Separation Science and Technology; Kompala, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.


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A pulsed l i g h t source would a l s o provide a s i g n i f i c a n t i n c r e a s e i n c o l l e c t e d s i g n a l (16). Pulsed e x c i t a t i o n would reduce the time, during the observation of the c e l l i n the orifice, when the d e t e c t i o n system must be either e l e c t r o n i c a l l y gated o f f or o p t i c a l l y obscured i n order t o achieve dark f i e l d i l l u m i n a t i o n . Two s u i t a b l e p u l s e d l i g h t sources are s h o r t a r c xenon strobe lamps and noble gas lasers. Both argon and neon l a s e with markedly g r e a t e r e f f i c i e n c y when pulsed and produce u l t r a v i o l e t emissions s u i t a b l e f o r the e x c i t a t i o n of lanthanide complexes. The luminescence l i f e t i m e of europium c h e l a t e s i s longer than the t r a n s i t time through a transducer (10-30 Ms) . Therefore, the question may be r a i s e d of whether such c h e l a t e s , when used as markers i n a flow system, would produce a s i g n i f i c a n t l y lower s i g n a l than c o n v e n t i o n a l s h o r t - l i v e d fluorophores. Mathies and S t r y e r (17) have stated that, under saturating laser excitation, a fluorophore i s i n p r i n c i p l e capable of being e x c i t e d once f o r each l i f e t i m e ; f o r f l u o r e s c e i n , t h i s would mean approximately 200 times per με. The e x c i t a t i o n c o n d i t i o n s assumed by Mathies and S t r y e r i n t h e i r c a l c u l a t i o n s were d i f f e r e n t from those employed i n a modern flow cytometer such as the EPICS E l i t e (EPICS i s a trademark of the C o u l t e r Corp.). These authors assumed a beam w i t h a c i r c u l a r 4 μιη c r o s s - s e c t i o n at the 1/e p o i n t s and 65 mW of 488 nm r a d i a t i o n energy. With these parameters, Mathies and S t r y e r c a l c u l a t e d t h a t one f l u o r e s c e i n molecule c o u l d be e x c i t e d approximately 97 times per με. The 8 i t u a t i o n ίε q u i t e d i f f e r e n t i n a flow cytometer. At the flow rate8 r e q u i r e d t o o b t a i n s t a t i s t i c a l l y 8 i g n i f i c a n t data, the thickne88 of the sample stream i s s u f f i c i e n t t o permit some l a t e r a l p o s i t i o n d i s p e r s i o n of the c e l l s i n the flow chamber. To provide c o n s i s t e n t l a s e r e x c i t a t i o n of c e l l s with imperfect t r a j e c t o r y , the gaus8ian l a 8 e r beam i s broadened i n the d i r e c t i o n p e r p e n d i c u l a r t o the flow. The s i z e of the l a s e r beam at the 1/e p o i n t s i n an EPICS E l i t e i s approximately 35X75 μπι , an area 164 times g r e a t e r than t h a t as8umed i n the c a l c u l a t i o n by Mathie8 and S t r y e r . The a i r - c o o l e d argon i o n l a s e r i n the EPICS E l i t e i s U8ually 8et t o produce 15 mW of 488 nm radiation. C o r r e c t i o n f o r both the i n c r e a s e d area of the beam c r o s 8 - 8 e c t i o n and the decrea8ed r a d i a t i o n i n t e n s i t y r e 8 u l t 8 i n a 711 f o l d decrease i n e x c i t a t i o n energy t o the sample. Under the best c o n d i t i o n s , t h i s i s e q u i v a l e n t t o 0.129 e x c i t i n g photons per molecule per με. With the same geometric parameters, a 50 mW water-cooled UV Argon l a 8 e r would e x c i t e 0.32 molecules per με and a 9 mW UV He-Cd l a s e r would e x c i t e 0.05 molecules per με. I f a combined DNA and Eu-chelate a n a l y s i 8 were t o be performed, the beam would be broadened. Multimode He-Cd Ia8er8 produce l a r g e r beams than gaus8ian Ia8er8. In a t e n με e x c i t a t i o n i n t e r v a l with the 50 mw UV Argon la8er, approximately (10x0.32)=3.2 photons would be a v a i l a b l e t o e x c i t e each chelate. T h i s number could be increa8ed by employing a 2

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high powered continuous or pulsed l a s e r ; however, such sources are not as yet a v a i l a b l e i n c l i n i c a l flow cytometers. The above c a l c u l a t i o n s , based on the c i t e d work of Mathies and S t r y e r , i n d i c a t e t h a t the i n a b i l i t y of the Eumacrocycle t o undergo m u l t i p l e c y c l e s of e x c i t a t i o n and emission during the time of flow d e t e c t i o n may r e s u l t i n an approximate 3 - f o l d decrease i n u s e f u l s i g n a l . However, preliminary studies indicate that luminescent Eumacrocycles undergo l e s s concentration quenching than t r a d i t i o n a l organic fluorophores. Thus, t h i s l i m i t a t i o n may i n p r i n c i p l e be overcome by greater l o a d i n g of the luminescent marker on the s u b s t r a t e . Work by Other I n v e s t i g a t o r s The u s e f u l n e s s of lanthanide complexes as biological markers has been recognized by other workers. S o i n i and Hemmila (18) and l a t e r S o i n i and Lovgren (14) have d e s c r i b e d an a n a l y t i c a l procedure i n v o l v i n g the Eu(III) complex of the polyamino-carboxylate l i g a n d DTPA (19) ( F i g . 2 ( a ) ) , which i s non-luminescent i n aqueous s o l u t i o n . In t h i s procedure, the Eu-DTPA c h e l a t e was decomposed with a c i d and the s o l u b i l i z e d Eu (III) was complexed with a Bdiketonate i n a m i c e l l a r phase (20). These d i s s o c i a t i o n combination steps complicated the a n a l y t i c a l procedure and i n c r e a s e d the volume of s o l u t i o n t o be monitored f o r Eu(III) emission, with consequent decrease i n o p t i c a l sensitivity. Since t h i s p r o t o c o l i n v o l v e d s e p a r a t i o n of the fluorophore from the s p e c i f i c b i o - s u b s t r a t e , the technique was u n s u i t a b l e f o r immuno-luminescence and other measurements on s i n g l e c e l l s or p a r t i c l e s by e i t h e r flow cytometry or microscopy. Another example of luminescence a n a l y s i s based on a Eu(III) complex has been reported by E v a n g e l i s t a e t a l . (21) . In t h i s case the l i g a n d was the d i n e g a t i v e i o n of 4,7-bis(chlorosulfenyl)-l,10-phenanthroline-2,9d i c a r b o x y l i c a c i d , BCPDA ( F i g . 2 ( b ) ) , which formed a luminescent c h e l a t e with E u ( I I I ) . The luminescence, however, was not detectable i n aqueous s o l u t i o n a t the c o n c e n t r a t i o n s of the analytes of i n t e r e s t and the sample had t o be d r i e d p r i o r t o measurement. Furthermore, the i n t r i n s i c luminescence of the Eu-BCPDA complex was r a t h e r low; i n order to measure analytes present i n minor q u a n t i t i e s , i t was necessary t o increase s e n s i t i v i t y by b i n d i n g the c h e l a t e t o t h y r o g l o b u l i n (22) i n a m u l t i l a y e r system. S o i n i e t a l . (23) have observed luminescence from a c h e l a t e attached by antibody t o a c e l l , but do not r e p o r t the chemical s t r u c t u r e of the c h e l a t e . Beverloo e t a l . (24) have reported the use of m i c r o c r y s t a l s of the phosphor y t t r i u m ( I I I ) o x y s u l f i d e doped with europium(III). Cells s p e c i f i c a l l y l a b e l e d with t h i s phosphor c o u l d be detected i n a time- gated mode even i n the presence of the very b r i g h t , red e m i t t i n g DNA s t a i n , ethidium bromide. A disadvantage of these 50-500 nm phosphor p a r t i c l e s i s t h a t they must be coated t o prevent a g g l u t i n a t i o n with a


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Figure 2. Schematic formulas o f : (a) Diethylenetriamine-pentacetate, DTPA; (b) 4,7bis(chlorosulfenyl)-1,lO-phenanthroline-2,9d i c a r b o x y l a t e , BCPA; (c) A g e n e r i c B-diketonate (R and R* may be equal or d i f f e r e n t ) ; (d) 5isothiocyanato-1,10-phenanthroline; (e) Eu-L complex, where L represent the m a c r o c y c l i c ligand C 2H N . 1

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p o l y c a r b o x y l i c a c i d , which may then be d i s p l a c e d by n e g a t i v e l y charged ions such as phosphate, c i t r a t e , or i o n i z e d ethylenediamine t e t r a c a r b o x y l i c a c i d (EDTA). The negative charge of these coated phosphors a l s o causes n o n s p e c i f i c binding t o s l i d e c o a t i n g agents such as p o l y - 1 l y s i n e or bovine serum albumin.


R e s u l t s and

Discussion

T h i s s e c t i o n describes our systematic e f f o r t s t o design and synthesize water-soluble, chemically s t a b l e , luminescent complexes of the Eu(III) and Tb(III) ions, equipped with p e r i p h e r a l f u n c t i o n a l groups f o r c o u p l i n g t o a b i o s u b s t r a t e . D e t a i l s of the experimental methods and r e s u l t s are t o be found i n the references c i t e d . Our search f o r lanthanide d e r i v a t i v e s s u i t a b l e as luminescent markers i n i t i a l l y focused (11,15) on the t r i s c h e l a t e complexes of B-diketonates ( F i g . 2 ( c ) ) . These compounds possessed three important f a v o r a b l e f e a t u r e s . F i r s t , the t r i s - B - d i k e t o n a t e s of both Eu(III) and Tb(III) were known t o e x h i b i t l i g a n d - s e n s i t i z e d luminescence, with emission i n t e n s i t y depending l a r g e l y on the e f f i c i e n c y of the energy t r a n s f e r from an e x c i t e d t r i p l e t s t a t e of the B-diketonate l i g a n d to the emission l e v e l ( s ) of the metal ion (25). Second, the lanthanide t r i s - B - d i k e t o n a t e s were thermodynamically stable, with cumulative stability constants, B , ranging from 18 t o 21 l o g u n i t s (26-27). T h i r d , the t r i s - B - d i k e t o n a t e s contained s e v e r a l coordinated water molecules that could be replaced by uncharged N-donor heterocyclic ligands (28) to give complexes having decreased v i b r a t i o n a l quenching and hence increased luminescence. I t was reasonable to conclude t h a t , i f the N-donor h e t e r o c y c l e were a c h e l a t i n g l i g a n d p r e v i o u s l y coupled t o a b i o - s u b s t r a t e , t h i s s u b s t i t u t i o n r e a c t i o n c o u l d provide a s t r a i g h t f o r w a r d way of a t t a c h i n g the luminescent complex to the intended t a r g e t . To explore this possibility, we developed the s y n t h e s i s of the p r e v i o u s l y unknown b i f u n c t i o n a l c h e l a t e , 5-isothiocyanato-l,10-phenanthroline ( F i g . 2(d)) (29) . T h i s was coupled t o a model p r o t e i n (BSA and γ-globulin) v i a a t h i o u r e a linkage, f o l l o w i n g the procedure e s t a b l i s h e d for f l u o r e s c e i n isothiocyanate. We then proceeded t o a t t a c h a preformed Eu(III) or Tb(III) t r i s - B - d i k e t o n a t e t o the protein-coupled phenanthroline. However, t h i s l a s t step met with f a i l u r e , i n that the h i g h l y luminescent p r o t e i n - l a n t h a n i d e conjugate i n i t i a l l y formed d i s s o c i a t e d upon washing or d i a l y s i s (29). The phenanthroline moiety remained c o v a l e n t l y attached t o the p r o t e i n , w h i l e the lanthanide complex broke down and luminescence was l o s t . Obviously, the thermodynamic s t a b i l i t y of these complexes, however high, was not sufficient to prevent their d i s s o c i a t i o n i n a very d i l u t e , buffered aqueous s o l u t i o n . These f i r s t unsuccessful attempts emphasized an important g e n e r a l i z a t i o n , namely, t h a t the time frame of l i g a n d exchange and metal exchange k i n e t i c s becomes a major c o n s i d e r a t i o n f o r complexes t o be used as probes i n b i o l o g i c a l systems. .In the very d i l u t e aqueous or aqueous3

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o r g a n i c media r e q u i r e d f o r such systems, o f t e n i n v o l v i n g contact with p o t e n t i a l l y competing l i g a n d s , even o r d i n a r i l y " s t a b l e " metal complexes, i f l a b i l e , may d i s s o c i a t e . When such d i s s o c i a t i o n occurs, the value of t h e probe i s diminished or l o s t . The task, t h e r e f o r e , was t o design and s y n t h e s i z e a new c l a s s o f Eu(III) and Tb(III) complexes t h a t would r e t a i n t h e i r i d e n t i t y i n d i l u t e aqueous s o l u t i o n through k i n e t i c i n e r t n e s s r a t h e r than through thermodynamic s t a b i l i t y alone. To achieve t h i s o b j e c t i v e , we decided t o take advantage o f the s o - c a l l e d macrocyclic e f f e c t . Examples o f meta1-macrocyclie complexes t h a t remain u n d i s s o c i a t e d i n s o l u t i o n , even though c o n t a i n i n g i n h e r e n t l y l a b i l e metal ions, abound both i n nature and i n t h e s y n t h e t i c chemical l i t e r a t u r e (30). The vast majority of these m a c r o c y c l i c complexes c o n t a i n a "small" metal i o n bound i n s i d e t h e four-donor-atom c a v i t y of an organic l i g a n d , a s t r i k i n g example being the Mg(II) o f c h l o r o p h y l l . A l i m i t e d number of i n e r t complexes of l a r g e r metal ions with f i v e - a n d s i x donor-atom macrocyclic ligands have a l s o been r e p o r t e d . Among these, the most r e l e v a n t were the complexes o f L a ( I I I ) and Ce(III) with the s i x - n i t r o g e n l i g a n d L ( F i g . 2 ( e ) ) , obtained by Backer-Dirks e t a l . (31) from the metaltemplated cyclic Schiff-base condensation o f 1,2diaminoethane and 2 , 6 - d i a c e t y l p y r i d i n e . Although these authors had been unable t o s i m i l a r l y s y n t h e s i z e the corresponding complexes of other lanthanides, an a p p r o p r i a t e change i n experimental procedure, together with use o f the lanthanide acetates i n s t e a d of the n i t r a t e s as templates, allowed us t o obtain the Eu(III) and Tb(III) complexes i n high y i e l d s and e x c e l l e n t p u r i t y (32). These complexes were white crystalline solids, t h e r m a l l y very s t a b l e and s o l u b l e i n water as w e l l as common organic s o l v e n t s . They were unique among the d e r i v a t i v e s of the lanthanide(III) ions i n t h a t the metalmacrocycle e n t i t i e s remained u n d i s s o c i a t e d i n dilute aqueous s o l u t i o n , even under c o n d i t i o n s t h a t would r e s u l t i n r a p i d decomposition of most other lanthanide complexes. In c o n t r a s t , the e x o c y c l i c l i g a n d s , whether anions or s o l v e n t molecules, were l a b i l e and r e a d i l y exchangeable. For example, prolonged contact of the Eu-L a c e t a t e complex with a t e n - f o l d excess of h y d r o c h l o r i c a c i d i n methanol r e s u l t e d i n replacement o f the acetates by c h l o r i d e s without degradation of the Eu-L e n t i t y . The behavior of the M-L complexes i n s o l u t i o n i s c o n s i s t e n t with t h e i r s t r u c t u r e as e s t a b l i s h e d by s i n g l e - c r y s t a l X-ray a n a l y s i s (Benetollo, F.; Bombieri, G.; Fonda, K.K.; Polo, A.; V a l l a r i n o , L.M. Polyhedron, i n p r e s s ) . In EuL (CH C00) Cl-4H 0 ( F i g . 3), the Eu i o n i s bound t o the s i x Ν atoms of the L macrocycle i n a n e a r l y p l a n a r arrangement, with Eu-N i n t e r n u c l e a r d i s t a n c e s corresponding t o t h e sum of the i n d i v i d u a l r a d i i . The two b i d e n t a t e chelating acetate ligands occupy a x i a l p o s i t i o n s on opposite s i d e s of the Eu-macrocyle u n i t , t h e o r g a n i c p o r t i o n o f which i s s l i g h t l y f o l d e d i n a " b u t t e r f l y c o n f i g u r a t i o n " t h a t minimizes s t e r i c s t r a i n . 1

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Figure 3. Structure of the [EuL (CH COO) ]Cl* 4H 0 complex i n the c r y s t a l l i n e s t a t e , showing the atom l a b e l i n g scheme. The oxygens of the c l a t h r a t e d water molecules are omitted f o r c l a r i t y . 1

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The ease of exchange of the e x o c y c l i c anions proved t o be a major asset i n view of the p o t e n t i a l use of these compounds as luminescent markers. The Eu-L and Tb-L a c e t a t e - c h l o r i d e s a l t s , i n i t i a l l y obtained from the metaltemplated s y n t h e s i s , e x h i b i t e d only a weak luminescence (33-34); however, s u b s t i t u t i o n of the acetates by a v a r i e t y of c h e l a t i n g carboxylates or β-diketonates r e s u l t e d i n highly luminescent species. The most effective luminescence enhancers were mononegative l i g a n d s with rigid, π-bonded s t r u c t u r e s and hard donor atoms (0, aromatic N) (35). Figure 4 i l l u s t r a t e s the i n c r e a s e i n emission i n t e n s i t y observed upon a d d i t i o n of 2 - f u r o i c a c i d t o the Eu-L diacetate-chloride. The stoichiometric c h a r a c t e r of t h i s e f f e c t i s evident from the luminescence t i t r a t i o n graph. Substituted macrocycle-enhancer complexes were a l s o i s o l a t e d and c h a r a c t e r i z e d as pure c r y s t a l l i n e s o l i d s (34-35). 1

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Having succeeded i n synthesizing water-soluble complexes of Eu(III) and Tb(III) t h a t could r e t a i n t h e i r i d e n t i t y i n d i l u t e s o l u t i o n and e x h i b i t l i g a n d - s e n s i t i z e d luminescence, our next goal was t o introduce i n t o t h e backbone of the macrocycle a f u n c t i o n a l group s u i t a b l e f o r c o u p l i n g t o a b i o - s u b s t r a t e . We chose the s y n t h e t i c scheme shown i n F i g . 5, which u t i l i z e d a c a r b o n - s u b s t i t u t e d 1,2diaminoethane precursor. T h i s s y n t h e s i s had the advantage of i n t r o d u c i n g the coupling groups i n t o t h e f l e x i b l e -CH CH - s i d e - c h a i n s , a t a p o s i t i o n and a t a d i s t a n c e where i t would be l e s s l i k e l y t o d i s t u r b e i t h e r the conformation o f the m a c r o c y c l i c l i g a n d or the e l e c t r o n i c c h a r a c t e r o f the ligand-metal bonding. There was no precedent i n the l i t e r a t u r e f o r t h i s k i n d of s y n t h e t i c procedure. However, we a n t i c i p a t e d t h a t under appropriate c o n d i t i o n s the template a c t i o n of the metal i o n , f a v o r i n g the formation of the m a c r o c y c l i c complex, would p r e v a i l over the competitive s i d e - r e a c t i o n s a r i s i n g from the f u n c t i o n a l group o f the diamine precursor. T h i s expectation proved t o be t r u e and lanthanide complexes were obtained i n good y i e l d s f o r the t h r e e m a c r o c y c l i c ligands L (with f u n c t i o n a l group X = CH -0H) , L (with X = -CH -C H -0H) , and L (with X = -CH C H -NH ). ?


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These functionalized metal-macrocycles closely resembled t h e i r n o n - f u n c t i o n a l i z e d analogs. They were m i c r o c r y s t a l l i n e s o l i d s with e x c e l l e n t thermal s t a b i l i t y . They were s o l u b l e i n water as w e l l as i n common o r g a n i c s o l v e n t s , and they e x h i b i t e d the same i n e r t n e s s t o metal r e l e a s e i n s o l u t i o n as w e l l as the same l a b i l i t y o f the e x o c y c l i c anions. Also, s i m i l a r t o t h e i r L analogs, the lanthanide complexes of the L , L , and L macrocyclic l i g a n d s were only modestly luminescent as the a c e t a t e s a l t s but became intense emitters i n the presence o f a s u i t a b l e enhancer (36-37.). Figure 6 i l l u s t r a t e s the marked i n c r e a s e i n emission i n t e n s i t y t h a t occurs when the Eu-L t r i a c e t a t e i s t r e a t e d with g r a d u a l l y i n c r e a s i n g amounts o f 4 , 4 , 4 - t r i f l u o r o - l ( 2 - t h i e n y l ) b u t a n e - l , 3 - d i o n e . The s h i f t i n the e x c i t a t i o n maximum of the macrocycle-enhancer system, r e l a t i v e t o t h a t of the o r i g i n a l macrocycle acetate alone, c l e a r l y shows t h a t the c h e l a t i n g enhancer not only provides b e t t e r p r o t e c t i o n from v i b r a t i o n a l quenching by the s o l v e n t but a l s o a c t s as a r a d i a t i o n absorber and promotes e f f e c t i v e energy t r a n s f e r t o the metal i o n . I t should be noted t h a t a d i s u b s t i t u t e d macrocycle such as L , L , or L can e x i s t as two regioisomers, as i l l u s t r a t e d i n F i g . 7. One isomer i s the c i s form, i n which the two f u n c t i o n a l groups X occupy p o s i t i o n s adjacent t o t h e same p y r i d i n e r i n g and thus are "on t h e same s i d e " r e l a t i v e t o the metal center. The other isomer i s the t r a n s form, i n which the two X groups occupy p o s i t i o n s adjacent t o d i f f e r e n t p y r i d i n e r i n g s and thus are "on opposite s i d e s " r e l a t i v e t o the metal center. Furthermore, the carbon atoms of the diimine s i d e - c h a i n s which c a r r y t h e X groups are c h i r a l ; thus, a d d i t i o n a l isomers become p o s s i b l e i f the diamine precursor i s racemic. The presence of isomers most l i k e l y i s r e s p o n s i b l e f o r the f a i l u r e we 1

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Figure 4. Luminescence "titration" of [EuL (CH C00) ]Cl-4H 0 with 2-furoic acid i n methanol. Inset shows the e x c i t a t i o n s p e c t r a o f : (a) o r i g i n a l complex and (b) complex with added enhancer (1:1 mole r a t i o ) . 1

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F i g u r e 5. Synthetic scheme f o r f unet i ona1i ζed lanthanide macrocyclic complexes The f u n c t i o n a l -CH -C H -OH group X i s -CH -OH f o r l i g a n d L , f o r l i g a n d L , and CH -C H -NH f o r l i g a n d L 2

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Figure 6. Luminescence "titration" of EuL (CH COO) - nH 0 with 4 , 4 , 4 - t r i f luoro-1 (2thienyl)butane-1,3-dione in methanol: (a) emission spectra of solutions containing i n c r e a s i n g Eu t o enhancer mole r a t i o s , (b) excitation spectrum of 1:1 complex-enhancer solution. 3

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F i g u r e 7. Computer-generated schematic formulas of the two [S,S]-regioisomers of the Eu-L macrocycle, i n which X = -CH -OH. S t r u c t u r e s (a) and (b) are f r o n t and s i d e views, r e s p e c t i v e l y , of the c i s isomer; (c) and (d) are the corresponding views of the t r a n s isomer. For these molecules, the f u n c t i o n a l groups of the c i s isomer occupy opposite "hemispheres" r e l a t i v e t o the plane of the macrocycle, whereas those of the t r a n s isomer occupy the same "hemisphere". The carbon-attached hydrogen atoms of the macrocycle and the e x o c y c l i c ligands are omitted f o r clarity. 2

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have so f a r encountered i n obtaining s i n g l e c r y s t a l s of these f u n c t i o n a l i z e d macrocycles, suitable f o r X-ray analysis. The p e r i p h e r a l -OH and -NH f u n c t i o n a l groups of the macrocycles were found t o e x h i b i t t h e i r normal r e a c t i v i t y . For example, r e a c t i o n of the metal macrocycle a c e t a t e s with acetic anhydride, in the presence of 4dimethylaminopyridine as c a t a l y s t , gave the corresponding e s t e r o r amide i n good y i e l d s (38) . No degradation o f t h e metal-macrocycle e n t i t y occurred even under the d r a s t i c c o n d i t i o n s r e q u i r e d f o r t h i s r e a c t i o n and the r e s u l t i n g d e r i v a t i v e s were s o l u b l e and i n e r t t o metal release. F i g u r e 8 shows the i n f r a r e d spectrum of the a c e t a t e e s t e r of t h e M-L t r i a c e t a t e complex and i l l u s t r a t e s the c h a r a c t e r i s t i c features of t h i s type of compound.


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F i g u r e 8. I n f r a r e d spectrum of the d i a c e t y l e s t e r of EuL (CH COO) -nH 0. The peak a t 1750 cm" represents the s t r e t c h i n g absorption of the carbonyl e s t e r group. The two peaks a t 1634 and 1589 cm" represent the C=N stretching absorptions of the p y r i d i n e and imine groups, r e s p e c t i v e l y , and are a d i a g n o s t i c f e a t u r e of t h i s k i n d of macrocycle. 3

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Conclusions The s e r i e s of f u n c t i o n a l i z e d m a c r o c y c l i c complexes of Eu(III) and Tb(III) reported i n t h i s paper f u l f i l l t h e fundamental requirements of a luminescent marker f o r c y t o l o g y and immunology. These complexes a r e s o l u b l e i n water and water-compatible s o l v e n t s ; they do not r e l e a s e the metal i o n even i n the presence of a c i d s , bases, o r competing l i g a n d s ; they can be made i n s t a n t a n e o u s l y luminescent i n aqueous s o l u t i o n by t h e simple a d d i t i o n o f an enhancer; f i n a l l y , they c o n t a i n primary hydroxy o r amino f u n c t i o n a l i t i e s t h a t can be used f o r c o u p l i n g t o a d e s i r e d bio-substrate. These f u n c t i o n a l i z e d macrocycles e x h i b i t the narrow-emission, long-lived luminescence that i s t y p i c a l of t r a d i t i o n a l Eu(III) and Tb(III) c h e l a t e s , and thus o f f e r the same advantages i n regard t o s e n s i t i v i t y and l a c k o f mutual o r background i n t e r f e r e n c e . Future work will focus on the attachment of the f u n c t i o n a l i z e d macrocycles t o b i o - s u b s t r a t e s and on the e v a l u a t i o n o f t h e s t a b i l i t y and luminescence of the r e s u l t i n g conjugates. Acknowledgments T h i s work has been supported by C o u l t e r E l e c t r o n i c s , H i a l e a h , FL, by V i r g i n i a Commonwealth U n i v e r s i t y , and by Ν.Α.Τ.Ο. B i l a t e r a l P r o j e c t No. 185-85. We wish t o thank Dr. M. L. Cayer f o r e d i t o r i a l a s s i s t a n c e and Mrs. M. Warren f o r her a s s i s t a n c e i n manuscript p r e p a r a t i o n .

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Leif, R.C.; Smith, S.; Warters, R . L . ; Dunlap, L . A . ; Leif, S.B. J. Histochem. Cytochem. 1975, 23, p. 378. (2) Hirsch, M.A.; Lipner, H.; Leif, R.C. Cell Biophys. 1979, 1, p. 93. (3) Pretlow II, T.G.; Pretlow, T.P. In Cell Separation Methods and Selected Applications; Pretlow II, T. G.; Pretlow, T . P . , Eds.; Academic Press; San Diego, 1982, Vol.1; Chapt.2. (4) Stewart, C.C. International Conference on Analytical Cytology XIV, 1990, Abstr. 16. (5) Lakowicz, J.R. Principles of Fluorescence Spectroscopy; Plenum, N.Y., 1983; p. 5. (6) Benson, R. C.; Meyer, R. Α.; Zaruba; M.E, McKhann G.M. J. Histochem. Cytochem. 1979, 27, p. 44. (7) Aubin, J . E. J. Histochem. Cytochem. 1979. 27, p.36. (8) Shapiro, H.M. Practical Flow Cytometry, 2nd Ed., Alan R. Liss, Wiley; New York, N.Y, 1988. (9) Loken, M.R.; Parks, D.R.; Hertzenberg, L.A. J. Histochem. Cytochem. 1977, 25, p. 899. (10) Glazer, A.N.; Stryer, L. Biophys J. 1983, 43, p. 383. (11) Leif, R.C.; Clay, S.P.; Gratzner, H.G.; Haines, H.G.; Rao K.V.; Vallarino, L.M. In The Automation of Uterine Cancer Cytology, Wied, G . L . ; Bhar, G.F.; Bartels, P.H. Eds.; Tutorials of Cytology; Chicago, IL, 1976; p. 313. (12) Crosby, G.A. Molecular Crystals, 1966, 1, p. 37. In Cell Separation Science and Technology; Kompala, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.


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(13) Bhaumik, M.L. J. Chem. Phys. 1964 40, p. 3711. (14) Soini, E . ; Lovgren T. In CRC Critical Reviews in Analytical Chemistry, 1987, 18, Issue 2, p. 105. (15) Vallarino, L.M.; Watson, B.D.; Hindman, D.H.K.; Jagodic V . ; Leif R.C. In The Automation of Cancer Cytology and Cell Image Analysis, Pressman, H . J . ; Wied, G.L. Eds.; Tokyo, 1979; p. 53. (16) Leif, R.C. In Automated Cell Identification and Sorting, G. L. Wied and G. F. Bahr, Eds, Academic Press, New York, 1970, p. 131. (17) Mathies, R. A; Stryer, L. In Applications in Biomedical Sciences, Alan R. Liss, Inc. 1986, p. 129. (18) Soini, E . ; Hemmila, I. U.S. Patent 4,374,120, 1983. (19) Mikola, H . ; Mukkala, V.-M.; Hemmila, I . , WO Patent 03698, 1984. (20) Hemmila, I.; Dakubu, S.; Mukkala, V.M.; S i i t a r i , H . ; Lovgren, T. Anal. Biochem, 1984, 137, p. 335. (21) Evangelista, R.A.; Pollak, Α.; Allore, B. Clin. Biochem, 1988, 21, p. 173. (22) Khosravi, M.J.; Diamandis, E.P. Clin. Chem. 1989, 35, p. 181. (23) Soni, E . J ; Pelliniemi, L. J.; Hemmila, I. A.; Mukkalva, V-M, Kankare, J. J.; Frojdman, K. J. Histochem Cytochem, 1988, 36, p. 1449. (24) Beverloo, Η. B.; van Schadewijk, Α.; van GelderenBoele, S.; Tanke, H. J. Cytometry, 1990, 11, p. 784. (25) Filipescu, N.; Sager, W.F.; Serafin, F.A. J. Phys. Chem., 1964, 68, p. 3324. (26) Dutt, N.K.; Bandyopadhyay, P. J. Inorg. Nucl. Chem. 1964, 26, p.729. (27) Gent, N.J. Determination of Stability Constants of Lanthanide Complexes by Fluorescence Spectroscopy, M.S. Thesis, Virginia Commonwealth University, 1983. (28) Melby, L.R.; Rose, N . J . ; Abramson, E . ; Caris, J.C. J. Am. Chem. Soc., 1964, p. 5117. (29) McGuire, A.A.P.; Vallarino, L.M. 30th South Eastern Regional Meeting, American Chemical Society; Savannah, GA, Nov. 1978; Abstr. 152 (30) Melson, G.A. in Coordination Chemistry of Macrocyclic Compounds, Melson, G.A. Ed.; Plenum; New York, 1979; p. 17. (31) Backer-Dirks, J.D.J.; Gray, C.H.; Hart, F.A.; Hursthouse, M.B.; Schoop, B.C. J. Chem. Soc. Chem. Commun. 1979, p.774. (32) Smailes, D.L.; Vallarino, L.M. Inorg. Chem. 1986, 25, p. 1729. (33) Sabbatini, N.; De Cola, L . ; Vallarino, L.M.; Blasse, G.J. J. Phys. Chem, 1987, 91, p.4681. (34) Smailes, D.L. Hexa-aza Macrocyclic Complexes of the Lanthanides: Synthesis and Properties, M.S. Thesis. Virginia Commonwealth University, 1986. (35) De Cola, L.; Smailes, D.L.; Vallarino, L.M. X Convegno Nazionale di Fotochimica, Ravenna, Italy, 1985; p. 4. (36) Gootee, W.A; Pham, K.T.; Vallarino, L.M. 41st South Eastern Regional Meeting, American Chemical Society, Winston-Salem, NC., Nov. 1989; Abstr. 313. In Cell Separation Science and Technology; Kompala, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

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(37) Gootee, W.A. Study of Metal Ions in Polymers: Model Compounds for the Inclusion of Lanthanides in Polyimides and Bifunctional Lanthanide(III) Macrocyclic Complexes, Ph.D. Dissertation, Virginia Commonwealth University, 1989. (38) Gribi, C . ; Smailes, D.L.; Twiford, Α.; Vallarino, L.M. 41st South Eastern Regional Meeting, American Chemical Society, Winston-Salem, NC., Nov. 1989; Abstr. 312. March 15, 1991


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