Metal Ion Macrocyclic Complexes as Artificial ... - ACS Publications

16 Metal Ion Macrocyclic Complexes as Artificial Ribonucleases Janet R. Morrow, Kimberly A. Kolasa, Shahid Amin, and K. O . Aileen Chin

Downloaded by CORNELL UNIV on July 27, 2016 | http://pubs.acs.org Publication Date: May 5, 1996 | doi: 10.1021/ba-1995-0246.ch016

State University of New York at Buffalo, Department of Chemistry, Buffalo, NY 14214

Macrocyclic ligands that form thermodynamically stable or kinetically inert complexes with metal ions are used in the design of metal complexes that behave as artificial ribonucleases. Artificial ribonucleases are compounds that cleave RNA by transesterification of the phosphate diester linkages. Zn(II) complexes of tetraaza and triaza macrocycles promote RNA cleavage, but cleavage rates are modest. Hexadentate Schiff base macrocyclic complexes of the trivalent lanthanides promote rapid cleavage of RNA oligomers. New ligands that encapsulate the trivalent lanthanides have been constructed from the addition offour pendent ligating groups to the 1,4,7,10-tetraazacyclododecane macrocycle. Several of the new lanthanide complexes are resistant to metal ion release and show promise as artificial ribonucleases. The mechanism of RNA cleavage by metal complexes and the effect of RNA structure on cleavage are discussed.

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D E S I G N O F I N O R G A N I C C O M P O U N D S t h a t m a y b e u s e f u l as t h e r a p e u t i c o r d i a g n o s t i c agents i s a t o p i c o f g r e a t i n t e r e s t i n b i o i n o r g a n i c chemistry. Because free m e t a l ions m a y b e h i g h l y toxic, m a n y p h a r maceutical applications r e q u i r e the use o f strong chelates. M a c r o c y c l i c ligands are useful w h e r e strong chelates are r e q u i r e d . P h a r m a c e u t i c a l applications where macrocycles have b e e n used include the design o f m a g n e t i c r e s o n a n c e i m a g i n g a g e n t s (1-3) a n d t h e c o n s t r u c t i o n o f m e t a l c o m p l e x - a n t i b o d y c o n j u g a t e s f o r u s e as n e w r a d i o p h a r m a c e u t i c a l s ( 4 6). T h e u t i l i t y o f m a c r o c y c l i c l i g a n d s i n c o n t r o l l i n g t h e r e a c t i v i t y o f m e t a l ions a n d i n f o r m i n g h i g h l y stable m e t a l c o m p l e x e s m a k e t h e m ideal for use i n the design o f n e w metallodrugs.

0065-2393/95/0246-0431$08.00/0 © 1995 American Chemical Society

Thorp and Pecoraro; Mechanistic Bioinorganic Chemistry Advances in Chemistry; American Chemical Society: Washington, DC, 1996.


432

MECHANISTIC BIOINORGANIC CHEMISTRY

T h e u s e o f m a c r o c y c l e s as l i g a n d s m a y b e a n i m p o r t a n t s t r a t e g y i n the design o f m e t a l complexes for R N A cleavage (artificial ribonucleases). Inertness to m e t a l i o n release u n d e r p h y s i o l o g i c a l c o n d i t i o n s a n d a h i g h d e g r e e o f t h e r m o d y n a m i c stability are i m p o r t a n t p r o p e r t i e s that must b e c o n s i d e r e d i f m e t a l ions are to be u s e d for cleavage. A m o t i v a t i o n for the study of m e t a l complexes that catalyze R N A cleavage lies i n the d e v e l o p m e n t of sequence-specific c l e a v i n g agents for R N A . T h e a t t a c h m e n t o f a c o m p l e x that catalyzes R N A cleavage to an o l i g o n u c l e o t i d e t h a t is c o m p l e m e n t a r y i n s e q u e n c e to a m e s s a g e R N A (an a n t i s e n s e o l i g o n u c l e o t i d e ) m a y b e o n e m e t h o d to p r o m o t e s e q u e n c e - s p e c i f i c c l e a v a g e of R N A . Antisense oligonucleotides bearing cleaving groups may be m o r e effective i n p r o m o t i n g translation arrest than are other types of a n t i s e n s e o l i g o n u c l e o t i d e s (7). T h e R N A cleavage reaction of interest involves transesterification o f t h e p h o s p h a t e esters o f R N A a n d is a n a l o g o u s t o t h e first s t e p o f t h e r e a c t i o n c a t a l y z e d b y e n z y m e s s u c h as R N a s e A . S m a l l m o l e c u l e s t h a t catalyze this t y p e o f cleavage r e a c t i o n are m o r e l i k e l y to b e useful for t h e r a p e u t i c a p p l i c a t i o n s b e c a u s e t h e r e a c t i o n is s p e c i f i c f o r R N A . H o w ever, one of the difficulties i n the use o f m o l e c u l e s that c a t a l y z e R N A c l e a v a g e b y t r a n s e s t e r i f i c a t i o n is t h e s l o w r a t e o f t h e r e a c t i o n . F o r e x a m p l e , s l o w rates o f cleavage are o b s e r v e d for c l e a v i n g agents that c o n t a i n n o m e t a l i o n s i n c l u d i n g p h o s p h a t e e s t e r r e c e p t o r s ( 3 5 % . A n a l y t i c a l data ( C , H , N analysis) f o r the c o m p l e x e s w e r e satisfactory. L a ( T C E C ) ( C F S 0 ) ( C H C N ) : F A B M S m/e: 8 9 3 ( c o m p l e x - C F S 0 ) . L a ( T C M C ) ( C F S 0 ) ( C H C H O H ) : F A B M S m/e: 8 3 7 . 0 ( c o m p l e x - C F S 0 ) . E u ( T C M C ) ( C F S 0 ) : a n a l y t i c a l data satisfactory. E u ( T H E D ) ( C F S 0 ) : A n a l y s i s c a l c u l a t e d f o r C H N 0 F E u : C , 2 4 . 1 0 ; H , 3.80; N , 5 . 9 1 . F o u n d C , 2 3 . 8 2 ; H , 3 . 8 2 ; N , 5.79. F A B M S m/e: 7 9 8 . L a ( T H E D ) ( C F S 0 ) : F A B M S m / e 7 8 5 ( c o m p l e x - C F S 0 ) . 3

3

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1 9

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3 6

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1 3

9

3

K i n e t i c s . T h e rate o f d i s s o c i a t i o n o f L a or E u f r o m the m a c r o c y c l i c c o m p l e x was m o n i t o r e d at 3 7 ° C i n the p r e s e n c e o f C u b y f o l l o w i n g the increase i n absorbance c h a r a c t e r i s t i c or t h e Cu(II) m a c r o c y c l i c c o m p l e x . B e e r ' s l a w plots w i t h v a r y i n g c o n c e n t r a t i o n s o f the Cu(II) c o m p l e x ( 0 . 1 0 0 1.00 m M ) gave an e x t i n c t i o n coefficient o f 6 8 3 0 M c m " (312 n m , T H E D ) , 6,500 M c m " (304 nm, T C E C ) , and 5500 M " c m " (312 n m , T C M C ) . Solutions for e x p e r i m e n t s to m o n i t o r the rate o f d i s s o c i a t i o n o f L n from the m a c r o c y c l i c c o m p l e x c o n t a i n e d t h e c o m p l e x (0.1 m M ) , 10 m M M e s buffer, p H 6.0, w i t h different c o n c e n t r a t i o n s o f C u C l . T h e C u concent r a t i o n was v a r i e d f r o m 0.1 to 1.0 m M f o r c e r t a i n e x p e r i m e n t s . I n most experiments the C u c o n c e n t r a t i o n was m a i n t a i n e d i n 1 0 - f o l d excess to complex. 3 +

3 +

2 +

1

1

1

1

1

1

3 +

2

2 +

2 +

T r a n s f e r - R N A Experiments. Transfer R N A was p u r c h a s e d f r o m S i g m a a n d u s e d as r e c e i v e d . T h e R N A was 3'-end l a b e l e d w i t h c y t i d i n e b i s p h o s p h a t e b y use o f T R N A ligase as d e s c r i b e d p r e v i o u s l y (18). T h e l a b e l e d t R N A was p u r i f i e d b y p o l y a c r y l a m i d e g e l electrophoresis. T h e bands w e r e cut out a n d the R N A was e l u t e d at r o o m t e m p e r a t u r e o v e r n i g h t a n d p h e

4


434


r e c o v e r e d b y e t h a n o l p r e c i p i t a t i o n . A l l s t a n d a r d p r e c a u t i o n s w e r e t a k e n to a v o i d r i b o n u c l e a s e c o n t a m i n a t i o n . A l l solutions w e r e a u t o c l a v e d a n d gloves w e r e w o r n for a l l m a n i p u l a t i o n s . R e c r y s t a l l i z a t i o n o f the e u r o p i u m c o m p l e x [ E u ( L ) ] ( O A c ) C l f r o m c h l o r o f o r m was p e r f o r m e d m u l t i p l e t i m e s . T h i s h a d no effect o n the e u r o p i u m p r o m o t e d R N A cleavage reactions. 1

2

Complexes of the Transition Metals and Zn(II) Several complexes of the transition metals a n d Zn(II) p r o m o t e transest e r i f i c a t i o n o f R N A (12, 13, 19, 20). A f e w o f t h e s e c o m p l e x e s h a v e macrocyclic ligands. Z n ( C R ) , Z n - N - m e t h y l - ( C R ) , Z n ( c y c l a m ) , Zn([12]aneN ) , and Zn([9]aneN ) promote R N A transesterification a l t h o u g h rates a r e m o d e s t ( C R = 2 , 1 2 - d i m e t h y l - 3 , 7 , l l , 1 7 - t e t r a a z a b i cyclo[11.3.1]heptadeca-l(17)2,ll,13,15-pentaene; N - m e t h y l - C R = 7( N - m e t h y l ) - 2 , 1 2 - d i m e t h y l - 3 , 7 , 1 1 , 1 7 - t e t r a a z a b i c y c l o [11.3.1] h e p t a d e c a 1(17)2,11,13,15-pentaene; [12]aneN = 1,5,9-triazacyclododecane; [9]aneN = 1,4,7-triazacyclononane). Z n ( C R ) (0.160 m M ) promotes 7 0 % cleavage of the R N A oligomer A - A o v e r a 2 0 - h p e r i o d (12). D i n u c l e o t i d e c l e a v a g e is a c c e l e r a t e d b y Z n ( c y c l a m ) , Z n ( [ 9 ] a n e N ) , and Zn([12]aneN ) at 6 4 ° C . C l e a v a g e w a s t o o s l o w t o o b s e r v e at 3 7 ° C ( i 9 ) . C l e a v a g e o f d i n u c l e o t i d e s w i t h m e t a l c o m p l e x e s is g e n e r a l l y m u c h m o r e d i f f i c u l t t h a n is c l e a v a g e o f l o n g e r o l i g o m e r s o f R N A . 2 +


3

2 +

2 +

3

2 +

2 +

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3

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S t r o n g l y c h e l a t i n g ligands m a y serve to m a i n t a i n the m e t a l i o n i n solution i n an active f o r m . T h e initial rate of cleavage of dinucleotides by Zn([9]aneN ) is s l o w e r t h a n c l e a v a g e b y Z n ( N 0 ) . H o w e v e r , o v e r t i m e a g r e a t e r e x t e n t o f c l e a v a g e is o b s e r v e d w i t h t h e m e t a l c o m p l e x t h a n w i t h t h e Z n ( I I ) salt. T h e m e t a l c o m p l e x s h o w e d c a t a l y t i c t u r n o v e r , w h e r e a s t h e Z n ( I I ) salt p r e c i p i t a t e s f r o m s o l u t i o n (19). F o r a Z n ( I I ) c o m plex, the addition of nitrogen donor ligands may reduce the n u m b e r of c o o r d i n a t i o n sites f o r c a t a l y s i s b u t m a y f a v o r a b l y m o d i f y t h e L e w i s a c i d ity of the Zn(II) center. U s i n g the p K of the m e t a l - b o u n d water of the Z n ( I I ) c o m p l e x e s as a m e a s u r e o f t h e i r L e w i s a c i d i t y , o n e w o u l d p r e d i c t that the t r i a z a m a c r o c y c l i c c o m p l e x e s w o u l d p r o m o t e R N A cleavage m o r e r a p i d l y t h a n w o u l d Z n ( c y c l a m ) (19). T h e f o l l o w i n g o r d e r o f c a t a l y t i c e f f i c i e n c y is o b s e r v e d f o r d i n u c l e o t i d e c l e a v a g e : ( Z n ( [ 9 ] a n e N ) « (Zn[12]aneN ) > Zn(cyclam) . F o r transition metals and Zn(II), h o w might metal complexes be d e s i g n e d t o p r o m o t e r a p i d c l e a v a g e o f R N A ? O n e a p p r o a c h is t h e f u n c t i o n a l i z a t i o n o f l i g a n d s to p a r t i c i p a t e i n c a t a l y s i s . T h e N - m e t h y l - C R l i g a n d was m o d i f i e d to c o n t a i n a basic g r o u p for b i f u n c t i o n a l catalysis (21). A Z n ( I I ) c o m p l e x o f o n e o f t h e m o d i f i e d m a c r o c y c l e s w a s s h o w n t o a c c e l e r a t e c y c l i z a t i o n o f t h e R N A m o d e l s u b s t r a t e 1 (1 = 4n i t r o p h e n y l p h o s p h a t e ester of p r o p y l e n e glycol) 2 0 - f o l d m o r e r a p i d l y than the Zn(II) c o m p l e x of N - m e t h y l - C R . 2 +

3

3

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a

2 +

3

3

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2 +


2 +

16.

M O R R O W E T AL.

Metal Ion Macrocyclic Complexes

435

OH +

"O—P=0

4-N0 Ph0" 2

+

H

+

4-N0 Ph0 2

1 Trivalent Lanthanides

in RNA Cleavage


Several k i n e t i c studies of phosphate diester transesterification b y m e t a l i o n s i n d i c a t e that l a n t h a n i d e i o n s are a m o n g t h e m o s t efficient p r o m o t e r s (17, 22, 23). T h e t r i v a l e n t l a n t h a n i d e s a r e g o o d L e w i s a c i d s a n d h a v e flexible

coordination geometries and a high cationic charge. Inasmuch

as l a n t h a n i d e h y d r o x i d e s f o r m r e a d i l y at n e a r n e u t r a l p H , t h e o v e r a l l charge of the complex w i l l decrease. A metal h y d r o x i d e ligand, h o w e v e r , m a y s e r v e as a g e n e r a l b a s e c a t a l y s t . T h e l a n t h a n i d e s a r e c o n s i d e r e d t o b e o x o p h i l i c m e t a l ions a n d b i n d w e l l to p h o s p h a t e d i e s t e r s . O x o p h i l i c i t y is a n i m p o r t a n t p r o p e r t y f o r a n a r t i f i c i a l n u c l e a s e , as it is d e s i r a b l e t h a t the m e t a l i o n b i n d to the p h o s p h a t e ester i n p r e f e r e n c e to b i n d i n g a nitrogenous base of R N A . T h u s , strong coordination of a metal c o m p l e x t o o n e o r m o r e o f t h e n i t r o g e n o u s bases o f R N A m a y i n h i b i t m e t a l c o m plex p r o m o t e d transesterification. L i g a n d s for the lanthanides must strongly chelate l a n t h a n i d e ions b u t n o t i n a c t i v a t e t h e m as c a t a l y s t s . C o o r d i n a t i o n sites m u s t b e a v a i l a b l e for catalysis a n d the m e t a l i o n s h o u l d r e t a i n a h i g h d e g r e e o f L e w i s acidity. A n overall positive charge on the complex may a i d i n catalysis, as d i s c u s s e d i n s u b s e q u e n t p a r a g r a p h s . M a c r o c y c l i c c o m p l e x e s o f t h e l a n t h a n i d e s a b o u n d (24). F e w l a n t h a n i d e m a c r o c y c l i c c o m p l e x e s ,

how-

ever, are i n e r t to m e t a l i o n release i n w a t e r . F o r e x a m p l e , p e r h a p s the m o s t w e l l - k n o w n class o f l a n t h a n i d e m a c r o c y c l i c c o m p o u n d s

are the

c r o w n ethers. C r o w n ether complexes are s y n t h e s i z e d u n d e r a n h y d r o u s conditions a n d are k n o w n to h y d r o l y z e i n water. W h e n w e b e g a n to s t u d y l a n t h a n i d e m a c r o c y c l i c c o m p l e x e s f o r R N A cleavage, w e b e g a n our search w i t h complexes that w e r e effective

for

another biomedical application: magnetic resonance imaging (MRI). M R I agents m u s t b e s t a b l e u n d e r p h y s i o l o g i c a l c o n d i t i o n s a n d m u s t also h a v e at least o n e c o o r d i n a t i o n site a v a i l a b l e f o r c o o r d i n a t i o n t o w a t e r ( J ) . Properties of artificial ribonucleases may be similar. L a n t h a n i d e c o m plexes that w i l l efficiently p r o m o t e R N A cleavage w i l l r e q u i r e available c o o r d i n a t i o n sites f o r c a t a l y s i s a n d m u s t b e i n e r t t o m e t a l i o n r e l e a s e o r have large formation constants.


436

M E C H A N I S T I C BIOINORGANIC CHEMISTRY

Hexadentate SchiffBase Macrocyclic

Complexes

T h e hexadentate Schiffbase complexes [ L i ^ L ) * , L n ( L ) ] have b e e n dev e l o p e d b y s e v e r a l g r o u p s (2, 25, 26). A p p l i c a t i o n s f o r these c o m p l e x e s i n c l u d e t h e i r u s e as fluorescent agents a n d as M R I agents. T h e n e u t r a l S c h i f f b a s e l i g a n d i m p a r t s a + 3 c h a r g e t o t h e c o m p l e x at n e u t r a l p H (27). S i x - c o o r d i n a t i o n sites are o c c u p i e d b y t h e n i t r o g e n d o n o r s , l e a v i n g t h r e e to f o u r c o o r d i n a t i o n sites for w a t e r o r c o u n t e r ions. I n t h e s o l i d state, t h e m a c r o c y c l e is not p l a n a r , b u t is best d e s c r i b e d as b o w l - o r b u t t e r f l y - s h a p e d . C a n d * H N M R studies i n d i c a t e a h i g h e r d e g r e e o f s y m m e t r y i n s o l u t i o n . Luminescence decay measurements i n H 0 and i n D 0 indicate approxim a t e l y t h r e e b o u n d w a t e r m o l e c u l e s for E u ( L ) at n e u t r a l p H (28). 1

3

2

3 +

1 3

2

2


x

LniL )

3 +

LnfL )

1 43

2 43

S e v e r a l l a n t h a n i d e c o m p l e x e s o f L p r o m o t e r a p i d cleavage o f o l i g o m e r s o r d i n u c l e o t i d e s o f R N A (14). T h e G d ( f f l ) , E u ( I I I ) , T b ( I I I ) , a n d La(III) c o m p l e x e s a l l p r o m o t e g r e a t e r t h a n 7 0 % cleavage o f A i - A after 4 h at 3 7 ° C , p H 7.00. P s e u d o - f i r s t - o r d e r rate constants for t h e cleavage o f A p U p by 0.490 m M E u ( L ) or of A - A b y 0 . 1 6 0 m M E u i L ) * are 0 . 1 4 a n d 1.5 h " , r e s p e c t i v e l y . C a t a l y t i c t u r n o v e r is o b s e r v e d for t h e cleavage o f a dinucleotide i n the presence o f the e u r o p i u m complex. 1

2

x

3 +

1 2

1 8

1

1 8

3

1

RNA

Structure

M o s t substrates e x a m i n e d t o date a r e flexible fragments o f R N A . H o w m i g h t R N A s t r u c t u r e m o d u l a t e t h e rate o f R N A cleavage b y artificial r i bonucleases? C e r t a i n l y i n t h e cleavage o f transfer R N A ( t R N A ) b y m e t a l ions, R N A s t r u c t u r e has a d r a m a t i c effect o n t h e site o f cleavage (29). R N A s t r u c t u r e dictates t h e h i g h l y specific cleavage o b s e r v e d i n s e l f - c l e a v i n g R N A s that r e q u i r e m e t a l ions (30). F o r m e t a l c o m p l e x e s t h e r e is l i t t l e i n f o r m a t i o n o n w h e t h e r t r a n s e s t e r i f i c a t i o n catalysts that r e a d i l y c l e a v e s i n g l e - s t r a n d e d R N A are able t o c l e a v e R N A w i t h a l a r g e d e g r e e o f s e c o n d a r y a n d t e r t i a r y s t r u c t u r e . I n a d d i t i o n , b e f o r e a m e t a l c o m p l e x is a t t a c h e d t o


16.

Metal Ion Macrocyclic

M O R R O W E T AL.

437

Complexes

an o l i g o d e o x y n u c l e o t i d e f o r s e q u e n c e - s p e c i f i c R N A c l e a v a g e , i t w o u l d b e u s e f u l t o k n o w w h e t h e r t h e m e t a l c o m p l e x is a b l e t o c l e a v e a D N A - R N A hybrid. Attachment o f the metal complex cleaver to the middle o f the o l i g o d e o x y n u c l e o t i d e w o u l d necessitate c l e a v a g e o f a d o u b l e - s t r a n d e d D N A - R N A h y b r i d . I n f o r m a t i o n o n t h e effect o f R N A s t r u c t u r e o n cleavage rates m a y have a significant effect o n t h e d e s i g n o f o l i g o n u c l e o t i d e - a r t i f i c i a l r i b o n u c l e a s e conjugates. T o p r o b e t h e effect o f R N A s t r u c t u r e o n c l e a v a g e b y E u ( L ) , t R N A (yeast) 3'-end l a b e l e d w i t h a P l a b e l w a s i n c u b a t e d w i t h t h e e u r o p i u m complex. Fragments from cleavage reactions w e r e resolved b y high-res o l u t i o n g e l e l e c t r o p h o r e s i s as s h o w n i n F i g u r e 1. C l e a v a g e o f t R N A by E u ( L * ) o c c u r r e d at sites t h a t a r e q u i t e d i s t i n c t f r o m t h o s e o b s e r v e d f o r E u ( C H C 0 ) (31, 32) as s h o w n i n a r e c e n t p u b l i c a t i o n (33). L a ( L ) gave a n i d e n t i c a l p a t t e r n (data n o t s h o w n ) . B a n d s c o - m i g r a t e d w i t h t h o s e p r o d u c e d b y alkaline hydrolysis or b y digestion w i t h R N a s e Ύ . O v e r l o n g t i m e p e r i o d s , t h e E u ( L ) c o m p l e x i n d u c e d c l e a v a g e at n e a r l y e v e r y n u c l e o t i d e t o p r o d u c e a l a d d e r o f c l e a v a g e sites. A 20-base oligodeoxynucleotide c o m p l e m e n t a r y to A to G of tRNA was annealed to t - R N A b y heating the oligodeoxynucleotide w i t h t h e t R N A to 6 5 ° C f o l l o w e d b y c o o l i n g to 0 ° C . Sites i n t h e R N A sequence complementary to t h e D N A strand w e r e protected from c l e a v a g e b y E u ( L * ) w i t h t h e e x c e p t i o n o f sites at t h e e n d s o f t h e h y b r i d w h e r e fraying probably occurs. L o n g e r incubation times l e d to cleavage at n e a r l y e v e r y n u c l e o t i d e o f t h e t R N A w i t h t h e e x c e p t i o n o f t h o s e p r o t e c t e d b y t h e o l i g o d e o x y n u c l e o t i d e . T h e s e q u e n c e is as f o l l o w s : 1

3 +

p h e

3 2

p h e


3 +

3

2

1

3

λ

1

3 +

3

p h e

8

p h e

3 +

C

GACACC mA u

A D

G

A

9

I 11 I I

8

CUCm G

m CUGUG

2

5

G T

ψ OJ

complementary oligonucleotide (5'-CGAACACAGGACCTCCAGAT) Cm

A

U

Gm

A

A

Y


5

7

3 +

438



1 2

3

4

Figure 1. Autoradiogram showing the effect of annealing a complementary oligodeoxynucleotide (complementary to RNA bases A to G ) to tRNA followed by treatment with Eu(L*) . L is 2J 13 18-tetramethyl-3 6 14 17 23M-hexaazatricyclo[17.3J:l]tetracosa-l(23) 2,6,8,10J2(24)J3 17 19 21-decane). Autoradio grams are of 8 M urea denaturing polyacrylamide sequencing gels of tRNA labeled with P at the 3' end. Approximately 1 X 10 cpm of labeled tRNA was loaded onto the gel for each sample. Cold tRNA was added to give a total concentration of tRNA of 20 μΜ (1.3 mM nucleotide). Metal complex concentrations were 1 mM and HEPES buffer was 0.4 M. Reactions were run at pH 7.86, 37 °C for the times indicated. Lane 1: control, 5 h; lane 2: control and oligonucleotide (20 μΜ), 5 h; lane 3: EuiL ) *, 5 h; and lane 4: Eu(L ) and oligonucleotide (20 μΜ), 5 h. (Reproduced from reference 33. Copyright 1993 American Chemical Society.) 38

3+

1

y

y

phe

s

phe

57

f

>

y

f

r

32

phe

phe

1 3

2 3+


y

y

r

16.

439


M O R R O W ET AL.

T h e s e r e s u l t s i n d i c a t e t h a t R N A i n a D N A - R N A h y b r i d is p r o t e c t e d f r o m c l e a v a g e L y E u ( L ) u n d e r c o n d i t i o n s w h e r e n e a r l y a l l o t h e r sites in t R N A are cleaved. W e cannot r u l e out p o o r e r b i n d i n g of the metal c o m p l e x to t h e h y b r i d t h a n t o R N A a l o n e . T h e r e is a m p l e p r e c e d e n c e f o r b i n d i n g o f E u (III) i o n s t o d o u b l e - s t r a n d e d n u c l e i c a c i d s (34). E u ( I I I ) is also k n o w n t o b i n d w e l l t o p o c k e t s i n h i g h l y s t r u c t u r e d R N A s s u c h as t R N A (31). It is n o t k n o w n h o w t h e e u r o p i u m c o m p l e x w i l l b i n d t o t h e s e different structures. 1

3 +


p h e

E a r l i e r studies suggest that the c o n f o r m a t i o n o f R N A w i l l p r o b a b l y play a major role i n phosphate ester transesterification reactions. T h e flexibility o f R N A i n t h e D N A - R N A h y b r i d w i l l b e l i m i t e d i n c o m p a r i s o n to single-stranded R N A . Studies have s h o w n that R N A cleavage w i t h e t h y l e n e d i a m i n e as a c a t a l y s t is i n h i b i t e d f o r R N A i n a t r i p l e h e l i x r e l a t i v e t o s i n g l e - s t r a n d e d R N A (35). M o r e r e c e n t w o r k (36) d e m o n s t r a t e d that p o l y v i n y l p y r r o l y d o n e p r o m o t e s h y d r o l y s i s o f s i n g l e - s t r a n d e d oligoribonucleotides but not those i n double-stranded f o r m . O n e explan a t i o n f o r t h i s b e h a v i o r is b a s e d o n t h e o r i e n t a t i o n o f t h e 3 ' - h y d r o x y l group and the phosphate diester i n an R N A double helix; n u c l e o p h i l i c attack of the 3'-hydroxyl a n d d i s p l a c e m e n t of the 5 ' - h y d r o x y l cannot o c c u r b y a n i n - l i n e d i s p l a c e m e n t m e c h a n i s m (37). M o s t D N A - R N A d o u b l e h e l i c e s a r e s t r u c t u r a l l y s i m i l a r t o R N A - R N A d o u b l e h e l i c e s (38) a n d g e o m e t r i c c o n s t r a i n t s m a y also b e s i m i l a r . F u r t h e r s t u d y is u n d e r w a y i n t h e a u t h o r ' s l a b o r a t o r y t o p r o b e t h e effect o f s t r u c t u r e o n c l e a v a g e .

Decomposition of Lanthanide(lll)

SchiffBase

Macrocycles

T h e m a c r o c y c l i c l i g a n d i n the Schiff base m a c r o c y c l i c c o m p l e x e s L n ( L ) slowly hydrolyzes i n water. D e c o m p o s i t i o n products of the lanthan i d e ( I I I ) c o m p l e x e s o f L a n d L m a c r o c y c l e s as d e t e r m i n e d b y u s e o f * H N M R are p y r i d i n e d i c a r b o x a l d e h y d e or d i a c e t y l p y r i d i n e , r e s p e c t i v e l y , a n d e t h y l e n e d i a m i n e . F o r t h e L n ( L ) c o m p l e x e s , it is n o t k n o w n w h e t h e r t h e c o o r d i n a t e d m a c r o c y c l e is i n i t i a l l y h y d r o l y z e d , f o l l o w e d by metal ion dissociation, or whether dissociation of the lanthanide ion is f o l l o w e d b y h y d r o l y s i s o f t h e f r e e m a c r o c y c l e . T h e first p a t h w a y , hydrolysis of the Schiff base complex, may o c c u r t h r o u g h the formation of a carbinolamine, followed b y expulsion of the amine and cleavage of the C - N b o n d ; c a r b i n o l a m i n e c o m p l e x e s are intermediates i n the h y d r o l y s i s o f i m i n e b o n d s for c e r t a i n S c h i f f b a s e l i g a n d s (39). C a r b i n o l a m i n e c o m p l e x e s (40) o f L h a v e b e e n r e p o r t e d f o r a l l l a n t h a n i d e s f r o m N d ( I I I ) t o L u (III) [ w i t h t h e e x c e p t i o n o f E u (III)]. T h e s e c o n d p a t h w a y , d i s s o c i a t i o n o f t h e l a n t h a n i d e f o l l o w e d b y h y d r o l y s i s o f t h e f r e e l i g a n d , has n o t b e e n r u l e d o u t . T e s t i n g t h i s p o s s i b i l i t y is m a d e m o r e d i f f i c u l t b y t h e fact t h a t t h e f r e e L l i g a n d has n o t b e e n i s o l a t e d . L i g a n d s s u c h as L a r e g e n e r a l l y s u s c e p t i b l e to h y d r o l y s i s a n d d i f f i c u l t t o i s o l a t e . H o w e v e r , i f 1

1

2

1

3 +

2

1


1

3 +

440


it w e r e to o c c u r , d i s s o c i a t i o n o f L n f r o m t h e m a c r o c y c l e is p r o b a b l y n o t r e v e r s i b l e . I n c u b a t i o n o f L a ( L ) w i t h excess C e or Y for several hours does not result i n i n c o r p o r a t i o n of the C e or Y label into the m a c r o c y c l e (26, 28). 3 +

1

3 +

3 +

3 +

3 +

3 +

T h e L complexes of the middle lanthanides Gd(III), Eu(III), and T b ( I I I ) d e c o m p o s e less r a p i d l y at p H 7.4, 3 7 ° C t h a n d o t h e L c o m p l e x e s o f L a ( I I I ) o r L u ( I I I ) (14). T h e fit o f t h e l a n t h a n i d e i o n i n t o t h e m a c r o c y c l e m a y b e i m p o r t a n t h e r e . C e r t a i n l y , t h e m a c r o c y c l e fit w i l l v a r y f o r L a (116 p M ) c o m p a r e d to L u ( 9 7 . 7 p M ) (41). A r e c e n t s t u d y u s i n g l u m i n e s c e n c e m e a s u r e m e n t s suggests a g r e a t e r l a b i l i t y o f t h e E u ( L ) c o m p l e x t h a n p r e v i o u s l y r e p o r t e d (28). D e t e c t i o n o f t h e E u ( D P T A ) complex produced upon addition of diethylenetriaminepentaacetic acid ( D T P A ) to E u ( L ) indicates that the c o m p l e x d e c o m p o s e s a p p r o x i m a t e l y 1 2 % i n 4 8 h at 3 7 ° C , p H 7.4. It is n o t e w o r t h y t h a t s o l u t i o n s o f E u i L ) * c o n t a i n t w o d i f f e r e n t s p e c i e s (28). O n e o f t h e m , p o s s i b l y a h y d r o x y - b r i d g e d d i m e r , is p r e s e n t i n g r e a t e r a m o u n t s at h i g h c o n c e n trations of E u i L ) * . 1

1

3 +

3 +

1

3 +


-

1

1

3 +

3

1

Encapsulated

3

Lanthanide Ions

T h e most t h e r m o d y n a m i c a l l y stable a n d k i n e t i c a l l y i n e r t c o m p l e x e s of the trivalent lanthanides are those of the l i g a n d D O T A ( 1 , 4 , 7 , 1 0 tetraazacyclododecane-l,4,7,10-tetraacetate) (42, 43). O u r s e a r c h f o r l a n t h a n i d e m a c r o c y c l i c c o m p l e x e s that w o u l d r e m a i n i n t a c t f o r l o n g e r t i m e p e r i o d s l e d us to e x a m i n e d e r i v a t i v e s o f D O T A . T h e r e a r e t w o p o t e n t i a l difficulties w i t h the use o f D O T A c o m p l e x e s o f the t r i v a l e n t lanthanides for R N A cleavage. F i r s t , the o v e r a l l negative charge o n the c o m p l e x is n o t c o n d u c i v e t o a n i o n b i n d i n g ; f o r e x a m p l e , G d ( D O T A ) " d o e s n o t b i n d h y d r o x i d e w e l l (44). S e c o n d , D O T A c o m p l e x e s o f t h e m i d d l e lanthanides Eu(III) and Gd(III) have only one available c o o r d i n a t i o n site f o r c a t a l y s i s . T h e p r e v i o u s l a n t h a n i d e c o m p l e x e s t h a t w e u s e d , e.g., E u ( L ) , w e r e g o o d c a t a l y s t s a n d h a d at least t w o a v a i l a b l e c o o r d i n a t i o n sites. 1

3 +

A n e u t r a l l i g a n d was p r e p a r e d b y r e p l a c e m e n t of the negatively c h a r g e d acetate groups w i t h n e u t r a l a m i d e or h y d r o x y a l k y l groups. T h r e e o f t h e s e l i g a n d s a r e s h o w n i n F i g u r e 2. A l l l a n t h a n i d e c o m p l e x e s w e r e c h a r a c t e r i z e d f u l l y b y e l e m e n t a l a n a l y s i s a n d mass s p e c t r o m e t r y (see E x p e r i m e n t a l D e t a i l s ) a n d b y H a n d C N M R . I n a d d i t i o n L a ( T C M C ) ( C F S 0 ) - ( E t O H ) (45) a n d L a ( T C E C ) ( C F S 0 ) (46) h a v e b e e n c h a r a c t e r i z e d b y single-crystal X - r a y diffraction studies. A diagram o f t h e [ L a ( T C E C ) ] c a t i o n is s h o w n i n F i g u r e 3. Ή a n d C N M R d a t a in d -acetonitrile or d - m e t h a n o l support lanthanide chelation of the ligands through all four nitrogen donors and all four oxygen donors i n a m a n n e r s i m i l a r t o that o b s e r v e d i n t h e s o l i d - s t a t e s t r u c t u r e s o f t h e l

3

3

3

3

3 +

3

1 3

3

3

1 3

4



16.

M O R R O W ET AL.


Figure 2.

441

Encapsulated lanthanide complexes.

c o m p l e x e s (45,46). V a r i a b l e t e m p e r a t u r e s t u d i e s f o r a l l l a n t h a n u m c o m p l e x e s i n d i c a t e d a l a r g e d e g r e e o f l i g a n d r i g i d i t y at t e m p e r a t u r e s b e l o w 0 ° C , a p r o p e r t y also a c h a r a c t e r i s t i c o f L n ( D O T A ) " c o m p l e x e s (42, 47). T h e O R T E P of the cation of L a ( T C E C ) is s h o w n i n F i g u r e 3 (46). 3 +

T h e [ L a ( T C E C ) ] c a t i o n ( F i g u r e 3) c o n s i s t s o f a n e n c a p s u l a t e d e i g h t c o o r d i n a t e l a n t h a n u m ( I I I ) i o n (46). T h e p r i m a r y c o o r d i n a t i o n p o l y h e d r o n o f t h e l a n t h a n u m c a t i o n c a n b e d e s c r i b e d as a d i s t o r t e d s q u a r e antiprism. T h e a m i d e substituents are a r r a n g e d i n a c l o c k w i s e fashion a r o u n d t h e l a n t h a n u m i o n . L a n t h a n u m - n i t r o g e n b o n d d i s t a n c e s a r e as follows: L a ( l ) - N ( l ) = 2.727(6), L a ( l ) - N ( 2 ) = 2.711(6), L a ( l ) - N ( 3 ) = 2.710(7), and L a ( l ) - N ( 4 ) = 2.724(6). L a n t h a n u m - o x y g e n b o n d dis3 +


442



N41

Figure 3.

The [La(TCEC)]

3+

cation (46).

t a n c e s a r e as f o l l o w s : L a ( l ) - 0 ( 1 1 ) = 2 . 3 9 0 ( 5 ) , L a ( l ) - 0 ( 2 1 ) = 2 . 4 3 4 ( 5 ) , L a ( l ) - 0 ( 3 1 ) = 2 . 4 1 1 ( 5 ) , a n d L a ( l ) - 0 ( 4 1 ) = 2 . 4 5 5 ( 5 ) [average L a - N = 2 . 7 1 8 ( ± 0 . 0 0 9 ) , L a - O = 2 . 4 2 3 ( ± 0 . 0 3 2 ) ] . C o m p a r i s o n o f t h e t w o sets of b o n d lengths indicates that the L a - N b o n d lengths are l o n g e r t h a n w o u l d b e p r e d i c t e d f r o m t h e i r i o n i c r a d i i (48), w h i l e t h e L a - O b o n d lengths are shorter t h a n w o u l d b e p r e d i c t e d f r o m t h e i r i o n i c r a d i i . T h e t r e n d o f s h o r t L a - O b o n d d i s t a n c e s a n d l o n g L a - N b o n d d i s t a n c e s is also f o u n d i n s t r u c t u r e s o f m a c r o c y c l i c p o l y a m i n o c a r b o x y l a t e c o m p l e x e s o f e u r o p i u m (49) a n d g a d o l i n i u m (50). T h e L a cation, w h i c h cannot fit i n t o t h e c a v i t y o f t h e s m a l l 1 2 - m e m b e r e d r i n g [trans Ν t o Ν d i s t a n c e is 4 . 3 5 6 ( ± 0 . 0 0 3 ) ] , is f o u n d a b o v e t h e r i n g w i t h t h e a m i d e g r o u p s f o l d i n g o v e r to e n c a p s u l a t e t h e i o n . 3 +

N M R studies i n d i c a t e d that dissociation of the encapsulated l a n thanum complexes varied dramatically w i t h the ligand. T h e L a ( T C E C ) c o m p l e x contains p e n d e n t groups that f o r m a s i x - m e m b e r e d r i n g . T h i s 3 +


16.

443


M O R R O W E T AL.

c o m p l e x u n d e r g o e s d e c o m p o s i t i o n w i t h i n m i n u t e s i n D 0 at 3 7 ° C , initial p H 6.5. Resonances a t t r i b u t e d to free l i g a n d r a p i d l y appear. T h e p e n d e n t a m i d e g r o u p of the T C M C l i g a n d differs f r o m that o f the T C E C ligand b y a single m e t h y l e n e group, yet the lanthanum(III) complex of t h e T C M C l i g a n d is i n e r t t o m e t a l i o n r e l e a s e ; t h e m a j o r i t y o f t h e c o m p l e x w a s i n t a c t a f t e r 4 d a y s at 3 7 ° C , n e u t r a l p H . I n c o n t r a s t , d i s s o c i a t i o n o f La(THED) is e x t e n s i v e o v e r a p e r i o d o f a d a y . 2

3 +

Several of the complexes i n F i g u r e 2 w e r e e x a m i n e d further for t h e i r resistance to dissociation. T h e e u r o p i u m c o m p l e x e s E u ( T H E D ) a n d E u ( T C M C ) w e r e m o r e difficult to study q u a n t i t a t i v e l y b y H N M R because of t h e i r b r o a d H resonances. D e c o m p o s i t i o n was m o n i t o r e d b y use o f a U V - v i s assay. E x c e s s C u was a d d e d to solutions c o n t a i n i n g the lanthanide macrocycles. T h e C u ion served the dual purpose of t r a p p i n g t h e f r e e m a c r o c y c l e a n d as a n i n d i c a t o r t o m o n i t o r t h e a m o u n t of m a c r o c y c l e that h a d dissociated. A l l Cu(II) m a c r o c y c l i c c o m p l e x e s gave an absorbance peak i n the U V - v i s s p e c t r u m that was characteristic of the Cu(II) macrocycle complex. F o r all macrocycles, C u was an effective trap; formation of the Cu(II) m a c r o c y c l i c c o m p l e x w e n t to completion i n the presence of 0.10 m M L a or 0.10 m M E u , 0.10 m M l i g a n d a n d excess C u (1.0 m M ) . T h e increase i n the c o n c e n t r a t i o n o f C u ( I I ) m a c r o c y c l e c o m p l e x o v e r t i m e is a m e a s u r e o f t h e i n e r t n e s s of the lanthanide c o m p l e x to dissociation. F o r the L a ( T H E D ) complex, t h e r e a c t i o n r a t e (5J) w a s i n d e p e n d e n t o f t h e c o n c e n t r a t i o n o f C u , consistent w i t h the f o l l o w i n g mechanism: 3 +

l

3 +

1


2 +

2 +

2 +

3 +

3 +

2 +

3 +

2 +

La(THED) THED + Cu

THED + La

3 +

2 +

— Cu(THED)

3 +

2 +

(k ) x

(rapid)

(1)

F o r L a ( T H E D ) a n d E u ( T H E D ) , r a t e c o n s t a n t s (fc ) o f 9.2 ( ± 0.5) Χ 1 0 " s " a n d 7.1 ( ± 0.4) Χ 1 0 " s " c o r r e s p o n d i n g t o h a l f - l i v e s o f 2 1 h a n d 11 d a y s , r e s p e c t i v e l y , w e r e d e t e r m i n e d at p H 6 . 0 , 3 7 ° C . T h e La(TCMC) c o m p l e x h a d a h a l f - l i f e o f 6.7 d a y s (k = 1.2 X 1 0 ~ s" ) at p H 6.0, 37 ° C . E u ( T C M C ) s h o w e d < 1 % d e c o m p o s i t i o n at p H 6 . 0 , 3 7 ° C after 6 w e e k s . T h a t t h e E u ( T C M C ) c o m p l e x a p p e a r s to b e h i g h l y i n e r t t o m e t a l i o n r e l e a s e is n o t s u r p r i s i n g g i v e n its s i m i l a r i t y t o G d ( D O T A ) " . T h e half-life for the dissociation of G d f r o m D O T A at p H 5.0 is a p p r o x i m a t e l y 2 0 0 d a y s (43). 3 +

6

3 +

1

7

x

1

3 +

6

x

1

3 +

3 +

3 +

Several of the encapsulated lanthanide complexes p r o m o t e transes t e r i f i c a t i o n o f i ( T a b l e I). T h e E u ( T C M C ) " c o m p l e x a l o n e d i d n o t p r o m o t e c y c l i z a t i o n o f 1 o v e r a 1-h p e r i o d w i t h 1.00 m M e u r o p i u m c o m p l e x . P r e l i m i n a r y studies w i t h t R N A indicate substantial R N A cleavage b y Eu(THED) a f t e r 1 h at 3 7 ° C , p H 7.4. 3 4

p h e

3 +

Mechanism of RNA

Cleavage

F u r t h e r studies are u n d e r w a y to c h a r a c t e r i z e the solution p r o p e r t i e s of the encapsulated macrocyclic complexes of the lanthanides. O n e of the


444


Table I.

Apparent Second-Order Rate Constants for the Transesterification of 1 by Lanthanide(III) Complexes at 37 °C, p H 7.4

Complex LaOL ) ** EuOL ) * La(TCMC) Eu(THED) 1

k

(M-'s- ) 1

0.046 0.12 0.016 0.058

3

1

2

3

3+ 3+

p H 6.85. All reactions contained 0.10 M NaCl, 0.010 M HEPES buffer. [1 ] = 5 Χ Ι Ο " M to 1 Χ Ι Ο M , [complex] = 2 Χ 10" to 1 X 1 0 " M .


a

5

4

- 4

3

p r o p e r t i e s t h a t is o f i n t e r e s t is t h e n u m b e r o f c o o r d i n a t i o n sites t h a t a r e available for b i n d i n g small m o l e c u l e s . M o s t o f the b e t t e r catalysts such as E u ( L ) o r C o ( I I I ) a m i n e c o m p l e x e s (9) h a v e at least t w o a v a i l a b l e a d j a c e n t c o o r d i n a t i o n sites. O n e o f t h e m m a y b e o c c u p i e d b y a h y d r o x i d e l i g a n d , a n d t h e other may b e available for b i n d i n g t h e phosphate ester. A m e t a l h y d r o x i d e m a y p a r t i c i p a t e i n t h e r e a c t i o n b y a c t i n g as a g e n e r a l b a s e t o d e p r o t o n a t e t h e 2 ' - h y d r o x y l g r o u p (12, 19, 2 0 ) . E v i d e n c e f o r s u c h a m e c h a n i s m arises f r o m t h e p H p r o f i l e o b s e r v e d f o r R N A t r a n s e s t e r i f i c a t i o n b y m e t a l c o m p l e x e s (19). T h r e e p o s s i b l e p a t h w a y s a r e s h o w n i n S c h e m e 1. I t is c l e a r t h a t t h e m e t a l d o e s n o t p a r t i c i p a t e p u r e l y as a g e n e r a l b a s e (a). O r g a n i c bases w i t h s i m i l a r p K s s h o w m u c h l o w e r activity than observed for metal complexes with similar p K s . Pathways b a n d c w i t h t h e m e t a l h y d r o x i d e p a r t i c i p a t i n g as a b a s e o r h y d r o x i d e a c t i n g as a b a s e , r e s p e c t i v e l y , a r e k i n e t i c a l l y i n d i s t i n g u i s h a b l e . H o w m a n y o p e n c o o r d i n a t i o n sites a r e o p t i m a l t o p r o d u c e c a t a l y t i c a l l y a c t i v e l a n t h a n i d e c o m p l e x e s ? I n t h e s o l i d state (45), t h e 1

3 +

a

a

Scheme 1


16.

M O R R O W E T AL.

445


La(TCMC) c o m p l e x has t w o c o o r d i n a t i o n sites o c c u p i e d b y l i g a n d s other than t h e macrocycle. E u ( T C M C ) , similar to E u ( D O T A ) " , has o n l y o n e a v a i l a b l e c o o r d i n a t i o n site (52). T h u s f o r t h e T C M C l i g a n d , t h e l a r g e r l a n t h a n i d e s t h a t h a v e m o r e o p e n c o o r d i n a t i o n sites m a y b e catalytically active, whereas the smaller, heavier lanthanides may lack a c t i v i t y . O n t h e basis o f its s i m i l a r i t y t o E u ( D O T A ) " , t h e T H E D c o m p l e x o f E u ( I I I ) p r o b a b l y has o n l y o n e a v a i l a b l e c o o r d i n a t i o n s i t e . W h y t h e n is t h i s c o m p l e x a c t i v e ? O n e p o s s i b l e e x p l a n a t i o n is t h a t t h e a c t i v e catalyst is p r o d u c e d u p o n d i s s o c i a t i o n o f o n e o f t h e h y d r o x y e t h y l g r o u p s . A n o t h e r p o s s i b i l i t y is t h e p a r t i c i p a t i o n o f a n h y d r o x y e t h y l g r o u p as a g e n e r a l base catalyst. F u r t h e r studies are n e e d to delineate these possibilities. Studies are i n progress t o determine h o w many water molecules are b o u n d to the e u r o p i u m complexes i n solution and to determine the p K of the b o u n d water molecules. 3 +


3 +

a

T h e o v e r a l l charge o n t h e c o m p l e x may b e i m p o r t a n t i n catalyst d e s i g n . I t is w e l l - k n o w n t h a t i n r e a c t i o n s w h e r e a m e t a l i o n acts as a L e w i s a c i d , the a d d i t i o n o f anionic ligands to the metal i o n catalyst may d e c r e a s e t h e r a t e o f t h e r e a c t i o n (53). F o r p h o s p h a t e e s t e r s u b s t i t u t i o n reactions i n v o l v i n g a n i o n i c phosphate esters, charge n e u t r a l i z a t i o n o f t h e p h o s p h a t e d i e s t e r b y t h e c a t a l y s t m a y b e e s p e c i a l l y i m p o r t a n t (54). I n s u p p o r t o f t h i s h y p o t h e s i s , p o l y a m i n o c a r b o x y l a t e l i g a n d s s u c h as E D T A d o n o t f o r m complexes w i t h La(III) that are active i n cleavage (14). T h e l a n t h a n i d e c o m p l e x e s d i s c u s s e d h e r e t h a t a r e a c t i v e t r a n s e s terification catalysts have n e u t r a l ligands. C o n d u c t i v i t y m e a s u r e m e n t s indicate a n overall + 3 charge o n L a ( L ) (27), E u ( T H E D ) , a n d La(TCMC) (45, 51). 1

3 +

3 +

3 +

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