Kinetically Inert Complexes of the Siderophores in Studies of Microbial

2 Kinetically Inert Complexes of the Siderophores in Studies of Microbial Iron Transport

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KENNETH N. RAYMOND Department of Chemistry, University of California, Berkeley, CA 94720 The compounds called siderophores (earlier called sidero chromes) are low-molecular weight chelating agents which are manufactured by microbes and are involved in their cellular iron transport. Kinetically inert complexes of the siderophores have been prepared by replacing the native ferric ion, which is kinetically labile in biological systems, with the kinetically inert chromic ion. The metal-substi tuted complexes and related model compounds have then been used as chemical probes, using vis-uv and circular dichroism spectroscopy, to elucidate the coordination geome tries of siderophores, and as biological probes, using the kinetic inertness of the chromic siderophore complexes, to study the mechanisms of cellular iron transport in several microbial species. The siderophores studied include the hydroxamate-containing ferrichromes and ferrioxamines and the catechol-containing compound enterobactin. Hphe preceding and following chapters amply illustrate the reasons why microbial iron transport compounds are worthy of our attention—both from the biochemical and medical points of view. However, one might wonder what this has to do with coordination chemistry. The obvious answer is that these are, after all, coordination compounds. But more than that, when viewed from the perspective of a coordination chemistry, new experiments or new approaches suggest themselves. This is always the exciting potential of interdisciplinary research. This chapter is the result of a research project which has involved extensive collaboration between J. B. Neilands' laboratories and my own. Many of the details of the transport studies of kinetically inert, metal-substituted siderophores in 33 In Bioinorganic Chemistry—II; Raymond, K.; Advances in Chemistry; American Chemical Society: Washington, DC, 1977.

34

BIOINORGANIC CHEMISTRY

II

m i c r o b i a l systems w e r e p r e s e n t e d i n the p r e v i o u s c h a p t e r . I w i l l focus here o n the c o o r d i n a t i o n c h e m i s t r y of these c o m p o u n d s a n d h o w m e t a l s u b s t i t u t e d s i d e r o p h o r e complexes c a n b e u s e d b o t h as c h e m i c a l p r o b e s ( u s i n g spectroscopic t e c h n i q u e s ) for the structures of these m a t e r i a l s , a n d as b i o l o g i c a l probes i n m e m b r a n e t r a n s p o r t studies. T h e c o m p o u n d s c a l l e d siderophores ( e a r l i e r c a l l e d s i d e r o c h r o m e s ) are l o w - m o l e c u l a r w e i g h t m a t e r i a l s w h i c h are m a n u f a c t u r e d b y m i c r o b e s a n d are i n v o l v e d i n t h e i r c e l l u l a r i r o n t r a n s p o r t . T h e b i o c h e m i s t r y of the siderophores has b e e n d i s c u s s e d i n t h e p r e v i o u s p a p e r a n d has

been


r e v i e w e d extensively a n d r e c e n t l y ( 1 ). T h e siderophores are a l l c h e l a t i n g l i g a n d s w h i c h f o r m e x t r e m e l y stable o c t a h e d r a l complexes w i t h h i g h - s p i n f e r r i c i r o n . T w o i m p o r t a n t classes of these c o m p o u n d s — t h e f e r r i c h r o m e s and

f e r r i o x a m i n e s — a r e t r i h y d r o x a m i c acids w h i c h

( e x c e p t for

c o n t a i n i n g c h a r g e d substituents ) f o r m n e u t r a l complexes

those

u s i n g three

b i d e n t a t e h y d r o x a m a t e m o n o a n i o n s . T h e s e complexes of F e ( I I I ) are a l l k i n e t i c a l l y l a b i l e . E v e n the l a r g e hexadentate l i g a n d s s u c h as f e r r i c h r o m e , w h i c h c o m p l e t e l y enclose the f e r r i c i o n w i t h a n o c t a h e d r a l c a v i t y , h a v e exchange rates o n t h e o r d e r of several m i n u t e s at p h y s i o l o g i c a l c o n d i t i o n s of p H a n d t e m p e r a t u r e . I n contrast, complexes i n w h i c h c h r o m i c i o n is s u b s t i t u t e d for f e r r i c i o n , a l t h o u g h s t r u c t u r a l l y the same, are k i n e t i c a l l y inert.

T h i s has b e e n d e m o n s t r a t e d f o r m o d e l h y d r o x a m a t e

( 2 ) , desferriferrichromes ( 3 ) , a n d ferrioxamines ( 4 ) .

complexes

S u b s e q u e n t trans

p o r t studies h a v e b e e n c a r r i e d o u t u s i n g s e v e r a l of these k i n e t i c a l l y i n e r t complexes. A n o t h e r c o m m o n l i g a n d f u n c t i o n a l g r o u p f o u n d i n the siderophores is c a t e c h o l ( o - d i h y d r o x y b e n z e n e ).

C a t e c h o l is s i m i l a r to h y d r o x a m a t e s

i n b e i n g a b i d e n t a t e l i g a n d w h i c h coordinates t h r o u g h t w o o x y g e n atoms, b u t is a d i a n i o n . E x c e p t for the o x y g e n s e n s i t i v i t y of the c a t e c h o l c o m plexes ( b e c a u s e of the ease of o x i d a t i o n of the l i g a n d ) , they are v e r y similar

i n kinetic a n d spectroscopic

properties

to

the

hydroxamate

complexes. Structure

and Properties of Ferric

Complexes in Siderophores

General Chemistry of Iron Chelates.

T h e aqueous

c h e m i s t r y of

F e ( I I I ) is d o m i n a t e d b y its L e w i s a c i d i t y . S e v e r a l p H units b e l o w that of p h y s i o l o g i c a l solutions, h y d r o l y s i s a n d p o l y m e r i z a t i o n reactions f e r r i c i o n take p l a c e .

i n s o l u b l e as the h y d r o x i d e . T h e K the K

s p

of

A t p h y s i o l o g i c a l p H f e r r i c i o n is q u a n t i t a t i v e l y s p

for F e ( O H )

3

is 2 Χ 1 0 "

for ferrous h y d r o x i d e , F e ( O H ) , is 8 Χ 1 0 " 2

16

(6).

39

(5) while

The biological

consequences of these n u m b e r s are p r o f o u n d because, since this p l a n e t p r o d u c e d a n o x i d i z i n g a t m o s p h e r e , the u l t i m a t e source of i r o n for a l l b i o l o g i c a l systems has b e e n i n o r g a n i c F e ( I I I ) .

E v e n the complexation

of f e r r i c i o n is not a l w a y s e n o u g h to m a k e i t u s e f u l to b i o l o g i c a l systems,

In Bioinorganic Chemistry—II; Raymond, K.; Advances in Chemistry; American Chemical Society: Washington, DC, 1977.

2.

Kinetically Inert Siderophore Complexes

RAYMOND

35

since the h y d r o l y s i s of s u c h c o m p l e x e s often p r o d u c e s s u c h h i g h - m o l e c u l a r w e i g h t h y d r o x y - b r i d g e d p o l y m e r s that t r a n s p o r t across c e l l m e m branes is i m p o s s i b l e ( 7 ) . D u r i n g the last 1 0 - 1 5 years a n u m b e r of

low-molecular

weight

c o m p o u n d s of n a t u r a l o r i g i n h a v e b e e n f o u n d to b i n d F e ( I I I ) s p e c i f i c a l l y a n d t r a n s p o r t i t i n b i o l o g i c a l systems ( J ) .

M o s t i f n o t a l l of t h e c o m

p o u n d s of this t y p e i n c l u d e h y d r o x a m a t e or p h e n o l a t e groups as l i g a n d s . U p o n loss of the p r o t o n , the a n i o n is a v e r y s t r o n g c h e l a t i n g agent w i t h a n a m a z i n g s p e c i f i c i t y for F e . T h e g e n e r a l c h e m i s t r y of the h y d r o x a m i c 3 +


acids forms a p a r t of c l a s s i c a l o r g a n i c c h e m i s t r y . T h e r e a c t i o n w i t h f e r r i c i o n is a s t a n d a r d test for t h e h y d r o x a m a t e f u n c t i o n a l g r o u p .

The acid

d i s s o c i a t i o n of the h y d r o x a m i c acids t y p i c a l l y gives p K s o n the o r d e r a

Ο

OH

R _ C _ ] L _ of 9.

0

R

0"

^> R _ C _ N _ R ' + H

+

T h e s u b s e q u e n t r e a c t i o n w i t h f e r r i c i o n gives a v e r y stable

five-

m e m b e r e d r i n g ( F i g u r e 1 ) . A b o v e v e r y a c i d p H , three h y d r o x a m i c acids Fe

3 +

/-\ /

R

\

R'

Figure 1. Ferric hydroxamate complex

w i l l b i n d to f o r m a n e u t r a l , o c t a h e d r a l c o m p l e x of F e . 3 +

T h e formation

constants for e v e n the s i m p l e m o n o h y d r o x a m i c acids are v e r y l a r g e a n d q u i t e specific for F e . 3 +

the p K

a

K, and K

K

1}

F o r acetohydroxamic acid ( R =

C H , R' = 3

H)

is 9.35, a n d the l o g a r i t h m s of the stepwise f o r m a t i o n constants

2

are 11.42, 9.68, a n d 7.2, f o r a n o v e r a l l f o r m a t i o n constant,

3

/? , of the tris c o m p l e x of 2 χ 3

10

28

(8, 9).

I n contrast, t h e o v e r a l l f o r m a

t i o n constant, β , for the b i s c o m p l e x of ferrous i o n is o n l y 3 Χ 10 . T h a t 8

2

this s e n s i t i v i t y is c a u s e d m o r e b y the size of the i o n t h a n its c h a r g e c a n be seen i n the β La

3 +

(8

χ

3

10 ) 11

values for the tris c o m p l e x e s of A l (9).

3 +

T h e great d i s p a r i t y b e t w e e n

s t r e n g t h of the h y d r o x a m i c acids for F e

3 +

and F e

2 +

(3 χ 10 ) 21

the

and

complexing

is p r o b a b l y t h e i r m o s t

i m p o r t a n t p r o p e r t y f o r i r o n t r a n s p o r t , since the r e d u c t i o n of the f e r r i c i o n c o m p l e x w i t h i n the c e l l p r o v i d e s a r e a d y means of r e l e a s i n g t h e c o m p l e x e d i r o n a n d f r e e i n g the l i g a n d for a n o t h e r s h u t t l e t r i p

back

to p i c k u p F e . 3 +

T h e stabilities of the n a t u r a l l y o c c u r r i n g t r i s h y d r o x a m i c a c i d c o m plexes are a m o n g the greatest k n o w n . F o r e x a m p l e , t h e w i d e l y u s e d a n d


36

BIOINORGANIC C H E M I S T R Y

II


hi

Siderochrome

R'

R"

Ferrichrome

Η

Ferrichrysin

CH 0H

Η CH 0H

Ferricrocin

Η

II

Ferrichrome C

II

CH

Ferrichrome A

CHoOH

2

CH,

2

3

CH 0H

"CH=C(CH )-CH C0 H(/A(7/75y

2

3

2

2

Ferrirhodin

"

-CH=C(CH )-CH CH 0H(^/5)

Ferrirubin

"

-CH=C(CH )-CH CH OH {frons)

Albomycin 8,

-CH 0S0 -lO=0 ?

3

3

CH 0H

?

2

2

CH

2

2

2

3

Figure 2. Structure of the ferrichromes. The basic structural feature is a cyclic hexapeptide with the three hydroxamic acid linkages provided by a tripeptide of 8N-acyl-8N-hydroxyl-l-ornithine. The A-cis coordination isomer is shown in each case. v e r y p o w e r f u l h e x a d e n t a t e c h e l a t e E D T A has a f o r m a t i o n constant l o g Κ o f 25.1 w h i l e t h a t f o r d e s f e r r i f e r r i c h r o m e ( F i g u r e 2 ) is 29.1 a n d f o r d e s f e r r i f e r r i o x a m i n e Ε ( F i g u r e 3 ) is 32.4 ( J O ) . T h e t r i s ( h y d r o x a m a t e ) c o m p l e x e s t y p i c a l l y are w a t e r - s o l u b l e , n e u t r a l c o m p o u n d s . c o m p l e x e s t h e i r o n is h i g h - s p i n F e ( I I I )

t h e h e m e - c o n t a i n i n g p r o t e i n s , is r e a d i l y e x c h a n g e d . course, e x p e c t e d f o r h i g h - s p i n d c o m p l e x e s , 5

I n a l l o f these

a n d , i n contrast t o t h e i r o n i n This lability is, of

a l t h o u g h t h e k i n e t i c s of

e x c h a n g e f o r these h e x a d e n t a t e l i g a n d s is m u c h s l o w e r t h a n , f o r e x a m p l e , tris b i d e n t a t e c o m p l e x e s .

T h e ferric i o n can b e removed from the com

plexes o f t h e t r i h y d r o x a m i c acids b y t r e a t i n g w i t h d i l u t e base o r r e d u c tion of F e ( I I I )

toFe(II).

T h e structure of the simple hydroxamate complex droxamato) iron (III)

tris(benzohy-

(R = φ, R' = Η i n F i g u r e 1 ) has s h o w n t h e m o s t

s t a b l e c r y s t a l l i n e f o r m o f t h e s o l i d t o b e t h e r a c e m i c c i s i s o m e r (11). ( T h e c o n v e n t i o n f o r s y m b o l s o f a b s o l u t e configurations Δ a n d Λ are those of t h e I U P A C P r o p o s a l (12). T h e cis i s o m e r is d e f i n e d as t h e i s o m e r


2.

37


RAYMOND

w h i c h has C p o i n t s y m m e t r y .

See R e f . 3 f o r f u r t h e r d i s c u s s i o n ) .

3

Since

b o t h t h e cis a n d trans isomers of the c h r o m i c c o m p l e x h a v e b e e n i s o l a t e d ( 2 ) , t h e s i m i l a r geometries of t h e c h r o m i c a n d f e r r i c c o m p o u n d s

(vide

i n f r a ) w o u l d i n d i c a t e that the cis a n d trans f e r r i c c o m p l e x e s are p r o b a b l y i n s o l u t i o n i n a p p r o x i m a t e l y t h e same p r o p o r t i o n s

( 6 0 % , 4 0 % , respec-

t i v e l y ) , a n d i t is t h e p r e d o m i n a n t cis i s o m e r w h i c h c r y s t a l l i z e s out. I n t h e f e r r i c complexes, the r a p i d i s o m e r i z a t i o n o f t h e c o m p l e x e s i n s o l u t i o n therefore leads e x c l u s i v e l y to c r y s t a l l i z a t i o n o f t h e cis i s o m e r . M o s t of t h e n a t u r a l l y o c c u r r i n g h y d r o x a m i c acids h a v e three h y d r o x -


a m i c a c i d groups

p e r molecule.

T h e i r o n c o m p l e x e s o f these

trihy-

d r o x a m i c acids h a v e a c h a r a c t e r i s t i c b r o a d a b s o r p t i o n b a n d at 4 2 0 - 4 0 0 n m , a n d therefore o r i g i n a l l y w e r e g i v e n the g e n e r i c n a m e (J).

siderochromes

T h e three h y d r o x a m a t e g r o u p s are l i n k e d e i t h e r as side arms f r o m

a c y c l i c p e p t i d e (as i n t h e f e r r i c h r o m e s , F i g u r e 2 ) o r as p a r t of a l i n e a r o r c y c l i c c h a i n (as i n the f e r r i o x a m i n e s , F i g u r e 3 ) . T h o s e w i t h g r o w t h -

î

H-N

CONH

CONH

} c H ) ^ C H ) 2 ) b H )

5

2

2

η

π

OC)

ΟΟ

γ

n

η \

.0 0

/

^Fe

R

n

Ferrioxamine Β

H 0

5

R

1

CH — 3

Ferrioxamine Dj

CH d-

5

CH —

Ferrioxamine G

H

5

H0 C(CH ) -

Ferrioxamine A,

H

4

H0 C(CH ) -

5

CH ~

Ferrimycin A,

3

9

/

0

H

3

2

2

2

2

2

2

3

Figure 3. Structure of the linear ferriox amines. The basic structural feature of the ferrioxamines is repeating units of l-amino-5hydroxyaminopentane and succinic acid. Ferri oxamine Ε is cyclic with η = 5 and an amide linkage such that there are no R or R ' substituents, but just a C-N bond.


38


II

p r o m o t i n g a c t i v i t y w e r e n a m e d s i d e r a m i n e s , a n d those t h a t are a n t i b i o t i c s were named sideromycins. The

Hydroxamate-Containing

Siderophores—Ferrichromes

and

Ferrioxamines. T h e f e r r i c h r o m e s ( F i g u r e 2 ) are a l l t r i h y d r o x a m i c a c i d s p r o d u c e d b y f u n g i s u c h as Ustilago sphaerogena (1). are p r o d u c e d b y several species of Nocardia

T h e ferrioxamines

a n d Streptomyces ( J ) .

In

contrast to the f e r r i c h r o m e s , l i n e a r a n d c y c l i c f e r r i o x a m i n e s ( F i g u r e 3 ) h a v e the three h y d r o x a m a t e groups p a r t of a p o l y a m i d e c h a i n l i k e beads o n a s t r i n g . O n e other m a j o r difference is that the l i g a n d s themselves are


not o p t i c a l l y a c t i v e . O n l y if a substituent g r o u p has a n o p t i c a l center, as i n the f e r r i m y c i n s , is there o p t i c a l a c t i v i t y for the m o l e c u l e .

T h e ferri

c h r o m e s h a v e a n a t u r a l o p t i c a l a c t i v i t y associated w i t h the l i g a n d . E x c e p t i n those cases w h e r e the l i g a n d is o p t i c a l l y i n a c t i v e ( i n w h i c h case t h e c o m p l e x e s are r a c e m i c m i x t u r e s ) , t h e p r e v i o u s s i d e r o c h r o m e

com

plexes h a v e b e e n f o u n d to h a v e a Λ-cis absolute c o n f i g u r a t i o n (see

also

F i g u r e 2 i n the p r e v i o u s c h a p t e r ) . T h u s , w h i l e f e r r i o x a m i n e Ε is r a c e m i c ( 1 3 ) , x-ray s t r u c t u r e analyses of f e r r i c h r o m e A (14)

a n d f e r r i c h r y s i n (15)

h a v e s h o w n b o t h to be Λ-cis isomers. A recent s t r u c t u r e analysis of t h e m i x e d hydroxamate-/?-phenol imide siderophore manufactured by mycotic b a c t e r i a , m y c o b a c t i n , has s h o w n that f e r r i c m y c o b a c t i n also has Λ - c i s absolute c o n f i g u r a t i o n (16).

T h e other p h y s i c a l p r o p e r t i e s of the f e r r i

c h r o m e s h a v e b e e n s t u d i e d u s i n g several t e c h i q u e s . T h e N M R spectra of A l ( I I I ) a n d G a ( I I I ) d e r i v a t i v e s , as c o m p a r e d w i t h t h e free l i g a n d , h a v e s h o w n that a p r o f o u n d c o n f o r m a t i o n c h a n g e a c c o m p a n i e s c o m p l e x mation

for

(17).

H o w e v e r , despite large differences i n l i g a n d m o l e c u l a r s t r u c t u r e , a l l of t h e h y d r o x a m a t e s i d e r o p h o r e s w h o s e structures h a v e b e e n d e t e r m i n e d to date h a v e b e e n f o u n d to b e cis c o m p l e x e s w i t h a c o o r d i n a t i o n g e o m e t r y a b o u t t h e f e r r i c i o n w h i c h is s u b s t a n t i a l l y i d e n t i c a l to t h e s i m p l e t r i s (benzhydroxamato)-Fe(III)

complex.

r a c e m i c b u t w i t h a cis g e o m e t r y

(13),

Thus, while ferrioxamine Ε

is

x - r a y s t r u c t u r e analyses of f e r r i

c h r o m e A (14) a n d f e r r i c h r y s i n ( 1 5 ) h a v e s h o w n b o t h to b e Λ-cis isomers. The Catechol-Containing Siderophore—Enterobactin. T h e i s o l a t i o n and

c h a r a c t e r i z a t i o n of

the

cyclic

triester 2,3-dihydroxy-IV-benzoyl-Z-

serine, a t r i c a t e c h o l s i d e r o p h o r e ( F i g u r e 4 ), w e r e i n d e p e n d e n t l y r e p o r t e d b y b o t h P o l l a c k a n d N e i l a n d s (18)

a n d O ' B r i e n a n d G i b s o n (19).

l i g a n d w a s i s o l a t e d f r o m c u l t u r e s of SalmoneUa typhimurium

The

a n d Esche

richia coli a n d g i v e n the n a m e s e n t e r o b a c t i n a n d e n t e r o c h e l i n , r e s p e c tively.

E n t e r o b a c t i n is a n efficient c e l l u l a r t r a n s p o r t agent b u t , u n l i k e

f e r r i c h r o m e , i n t r a c e l l u l a r release of the i r o n i n v o l v e s e n z y m a t i c h y d r o l y s i s of

the

enterobactin

to

the

monomer,

2,3-dihydroxy-N-benzoyl-Z-ser-

ine(J).


2.

RAYMOND


39

It c a n b e seen f r o m m o l e c u l a r m o d e l s that t w o diastereoisomers a r e p o s s i b l e f o r t h e f e r r i c e n t e r o b a c t i n c o m p l e x , Λ-cis a n d Δ-cis.

These are

n o t m i r r o r images b e c a u s e o f t h e o p t i c a l a c t i v i t y of t h e l i g a n d . T h e s i m i l a r i t y of t h e roles p l a y e d b y t h e f e r r i c h r o m e s a n d e n t e r o b a c t i n l e n t a d d i t i o n a l s p e c u l a t i v e interest to t h e p r e f e r r e d a b s o l u t e c o n f i g u r a t i o n of t h e i r o n c o m p l e x ( 2 0 ) . T h e s t r u c t u r a l studies of t h e tris c a t e c h o l c o m


plexes

(vide infra)

a n d the spectroscopic

/ 0

v

of t h e c h r o m i c

\

0=C HO

properties

HCH

Λ" μ

"C>H

Μ

OH Figure 4.

Structural diagram of enterobactin

e n t e r o b a c t i n c o m p l e x h a v e l e d to a n assignment of g e o m e t r y f o r t h e most stable isomer of t h e f e r r i c e n t e r o b a c t i n c o m p l e x ( F i g u r e 5 ) . Replacement

of Ferric

Hydroxamate

Ion by Chromic

Siderophores.

Ion in Siderophores

GEOMETRIC

ISOMERS.

Many

of

the

questions r e g a r d i n g t h e s t r u c t u r e - f u n c t i o n r e l a t i o n s h i p o f t h e s i d e r o phores c o u l d n o t b e a n s w e r e d i n d e t a i l b e c a u s e of t h e k i n e t i c l a b i l i t y o f these h i g h - s p i n F e ( I I I ) complexes.

T h i s l a b i l i t y a l w a y s left a m b i g u o u s ,

for e x a m p l e , w h e t h e r or n o t m e t a l t r a n s p o r t occurs v i a u p t a k e of t h e intact molecular complex.

S u r p r i s i n g l y , t h e c o o r d i n a t i o n c h e m i s t r y of

t h e s i d e r o p h o r e l i g a n d s w i t h m e t a l ions other t h a n f e r r i c w a s l a r g e l y unknown.

( A b r i e f r e p o r t of t h e C D s p e c t r u m of t h e C r ( I I I )

of d e s f e r r i f e r r i c h r y s i n has a p p e a r e d (21).

complex

However, the complex appar

ently was not isolated, a n d the C D spectrum was not interpreted. ) W e therefore b e g a n to i n v e s t i g a t e t h e c o o r d i n a t i o n geometries of s i d e r o p h o r e l i g a n d s o r t h e i r l i g a n d moieties w i t h k i n e t i c a l l y i n e r t t r i v a l e n t m e t a l ions s u c h as C o ( I I I ) a n d C r ( I I I ) .

S i n c e h y d r o x a m i c acids a r e u n s y m m e t r i c a l

b i d e n t a t e l i g a n d s , there a r e b o t h g e o m e t r i c a n d o p t i c a l isomers i n t r i s -


40

BIOINORGANIC

CHEMISTRY

II


Ο

Journal of the American Chemical Society

Figure 5. A schematic of the A-cis isomer of chromic and ferric enterobactin. The metal lies at the center of a dis torted octahedron formed by the oxygen atoms of tne three catechol dianions. (hydroxamate)

complexes.

A s n o t e d e a r l i e r for a n o c t a h e d r a l

complex

f o r m e d w i t h three e q u i v a l e n t o p t i c a l l y a c t i v e h y d r o x a m a t e anions, t h e r e are t w o g e o m e t r i c isomers p o s s i b l e — t r a n s a n d cis. E a c h g e o m e t r i c

iso

m e r consists of Δ a n d Λ o p t i c a l isomers (12). O f t e n these are diastereoisomers because o f the l i g a n d o p t i c a l a c t i v i t y , i n w h i c h case there are four possible isomers—Λ-cis, Λ-trans, Δ-cis, and Δ-trans. P r e l i m i n a r y exploratory research was directed t o w a r d p r e p a r i n g a n d c h a r a c t e r i z i n g C r ( I I I ) or C o ( I I I ) complexes. d

6

T h e s e are d a n d l o w - s p i n 3

m e t a l ions, r e s p e c t i v e l y , w h i c h h a v e the greatest p o s s i b l e l i g a n d

field

s t a b i l i z a t i o n energy a n d h e n c e are k i n e t i c a l l y i n e r t t o w a r d l i g a n d s u b s t i t u t i o n a n d i s o m e r i z a t i o n reactions. T h i s is i n contrast to t h e h i g h - s p i n d

s

f e r r i c i o n w h i c h has zero l i g a n d field s t a b i l i z a t i o n e n e r g y (22). T h u s , i n contrast t o the f e r r i c s i d e r o p h o r e complexes, c h r o m i c o r c o b a l t i c - s u b s t i t u t e d c o m p l e x e s s h o u l d be k i n e t i c a l l y i n e r t . MODEL

droxamate)

HYDROXAMATE

COMPLEXES.

complexes of C o ( I I I )

Attempts

to prepare

w i t h benzohydroxamic

tris (hy

acid or its


2.


RAYMOND

41

N - m e t h y l d e r i v a t i v e r e s u l t e d i n o x i d a t i o n of t h e l i g a n d w i t h c o n c o m i t a n t r e d u c t i o n of C o ( I I I ) to C o ( I I ) . T h e p r e p a r a t i o n of tris ( b e n z o h y d r o x a mato) chromium ( I I I ) ,

Cr(benz) , 3

w a s successful

a n d resulted i n the

s e p a r a t i o n a n d c h a r a c t e r i z a t i o n of its t w o g e o m e t r i c isomers

(2).

The

h a l f - l i v e s for i s o m e r i z a t i o n of these complexes near p h y s i o l o g i c a l c o n d i tions is o n the o r d e r of h o u r s .

T o f a c i l i t a t e t h e s e p a r a t i o n of a l l f o u r

o p t i c a l isomers of a s i m p l e m o d e l tris ( h y d r o x a m a t e ) c h r o m i u m ( I I I ) c o m p l e x , w e p r e p a r e d ( u s i n g Z-menthol as a s u b s t i t u e n t ) t h e o p t i c a l l y a c t i v e hydroxamic acid, N-methyl-Z-menthoxyacethydroxamic acid ( m e n ) . This


r e s u l t e d i n t h e s e p a r a t i o n of t h e t w o cis diastereoisomers of tris ( N - m e t h y l Z - m e n t h o x y a c e t h y d r o x a m a t o ) c h r o m i u m ( I I I ) f r o m t h e trans diastereoiso mers a n d t h e i r c h a r a c t e r i z a t i o n b y e l e c t r o n i c a b s o r p t i o n a n d c i r c u l a r d i c h r o i s m spectra. T h i n layer chromatography

of t h e tris ( b e n z o h y d r o x a m a t o ) c h r o m

i u m ( I I I ) c o m p l e x r e s u l t e d i n t w o green b a n d s , c o r r e s p o n d i n g to t h e cis a n d trans isomers, w h o s e e l u t i o n R

s t

v a l u e s b r a c k e t e d t h a t of t h e one

b r o a d r e d d i s h - b r o w n b a n d of the F e ( I I I ) the g e o m e t r i c isomers of t h e F e ( I I I )

complex.

A s just d e s c r i b e d ,

c o m p l e x are i n r a p i d e q u i l i b r i u m

i n s o l u t i o n , a n d as a result, t h e m i x t u r e of these isomers elutes as one b a n d w i t h an R

s t

v a l u e that is a w e i g h t e d average of t h e t w o i n d i v i d u a l

isomers. T h e tris ( N - m e t h y l - Z - m e n t h o x y a c e t h y d r o x a m a t o ) c h r o m i u m ( I I I ) a n d - i r o n ( I I ) complexes, C r ( m e n ) layer chromatography. b a n d whose elution R

3

a n d F e ( m e n ) , w e r e also p u r i f i e d b y t h i n 3

T h e i r o n c o m p l e x gives one b r o a d r e d d i s h - b r o w n v a l u e is b r a c k e t e d b y t h e b l u i s h - g r e e n b a n d s of

s t

the cis a n d trans isomers of t h e C r ( I I I )

complex

(2).

A s w i t h the

tris ( b e n z o h y d r o x a m a t e ) complexes, this b e h a v i o r is c a u s e d b y t h e r a p i d e q u i l i b r a t i o n of the k i n e t i c a l l y l a b i l e f e r r i c c o m p l e x . T h e isomers of C r ( m e n ) s i m i l a r to t h e C r ( b e n z )

3

3

isomerize w i t h half-lives (several hours)

complex.

T h e rate of i s o m e r i z a t i o n of the t r i s -

( h y d r o x a m a t e ) c o m p l e x e s is therefore n o t p a r t i c u l a r l y sensitive to t h e s u b s t i t u e n t of t h e h y d r o x a m a t e n i t r o g e n a t o m , since t h e m e n l i g a n d c o n tains a n a l k y l a t e d n i t r o g e n a t o m , a n d the b e n z l i g a n d contains a n u n s u b stituted

nitrogen

atom.

I n t h e absence

of

an induced

strain,

the

corresponding siderophore complexes must isomerize m u c h more slowly b e c a u s e of the steric constraints of t h e l i g a n d . A l t h o u g h f o u r diastereoisomers ( Λ - c i s , Λ - t r a n s , Δ - c i s , a n d Δ-trans) a r e e x p e c t e d f o r C r ( m e n ) , t h i n l a y e r c h r o m a t o g r a p h y of t h e c o m p l e x y i e l d e d 3

o n l y three b l u i s h - g r e e n b a n d s . T w o of these are t h e r e s o l v e d Λ - c i s ( 1 0 % ) a n d Δ - c i s ( 2 1 % ) isomers, a n d t h e t h i r d ( 6 9 % ) is a n u n r e s o l v e d m i x t u r e of t h e Λ-trans ( 3 1 % ) a n d Δ - t r a n s ( 3 8 % ) isomers. O n e other k e y difference

between

t h e c h r o m i c a n d f e r r i c ions is

t h e i r spectroscopic p r o p e r t i e s . S i n c e f e r r i c i o n is a h i g h - s p i n d i o n i n t h e 5



42

II

s i d e r o p h o r e c o m p l e x e s , t h e r e a r e n o s p i n - a l l o w e d d-d e l e c t r o n i c t r a n s i tions. T h u s t h e v i s - u v a b s o r p t i o n s p e c t r a of t h e siderophores a r e n o t a l l c a u s e d b y m e t a l c h r o m o p h o r e centers b u t r a t h e r are f r o m l i g a n d - m e t a l o r l i g a n d - l i g a n d transitions ( l a r g e l y c h a r g e t r a n s f e r ) w h i c h v a r y e n o r m o u s l y f r o m o n e c o m p o u n d to a n o t h e r , e v e n t h o u g h t h e c o o r d i n a t i o n g e o m e t r y a b o u t t h e F e ( I I I ) m a y b e t h e same.

I n contrast, o c t a h e d r a l ( o r n e a r l y

o c t a h e d r a l ) c o m p l e x e s of C r ( I I I ) h a v e t w o w e l l e s t a b l i s h e d d-d a b s o r p tion bands that are l o c a l i z e d o n the m e t a l chromophore a n d thus are i n s e n s i t i v e to changes i n t h e m e t a l - l i g a n d c o m p l e x w h i c h is o u t s i d e t h e


i m m e d i a t e c o o r d i n a t i o n s p h e r e of t h e m e t a l . CHROMIC

FERRICHROME

COMPLEXES.

T h e spectra

for

the

model

c h r o m i c h y d r o x a m a t e c o m p l e x e s are r e p r o d u c e d i n F i g u r e 6. S i n c e t h e v i s i b l e a n d C D s p e c t r a of t h e isomers a r e w h o l l y d o m i n a t e d b y t h e m e t a l c o m p l e x c h r o m o p h o r e , these d a t a c a n b e u s e d to c h a r a c t e r i z e a n d to i d e n t i f y c o o r d i n a t i o n isomers of c o m p l e x e s f o r m e d b y t h e s i d e r o p h o r e s . T h e p r e p a r a t i o n a n d c h a r a c t e r i z a t i o n of t h e c h r o m i c c o m p l e x e s of d e s ferriferrichrome a n d desferriferrichrysin have been reported

(3). A l

t h o u g h a n e x a m i n a t i o n of m o l e c u l a r m o d e l s f o r b o t h c o m p l e x e s shows t w o c o o r d i n a t i o n isomers are p o s s i b l e ( Λ - c i s a n d Δ - c i s ) , b o t h c h r o m i c c o m plexes consist e x c l u s i v e l y of t h e Λ - c i s i s o m e r .

T h e s e results agree w i t h

x-ray crystallographic investigations w h i c h have s h o w n that b o t h ferri c h r y s i n a n d f e r r i c h r o m e A c r y s t a l l i z e as o n l y t h e Λ - c i s i s o m e r (14, 15). B o t h c h r o m i c c o m p l e x e s h a v e i d e n t i c a l C D s p e c t r a w h i c h a r e t h e same as t h e Λ - c i s C r ( m e n ) CHROMIC

3

spectrum (Figure 6 ) .

FERRIOXAMINE

COMPLEXES.

T h e preparation a n d charac

t e r i z a t i o n of c h r o m i c c o m p l e x e s of f e r r i o x a m i n e Β (see F i g u r e 3 ) h a v e b e e n r e p o r t e d (4).

F r o m a n e x a m i n a t i o n of m o l e c u l a r m o d e l s , t h e five

g e o m e t r i c isomers ( one cis a n d f o u r trans ) s h o w n i n F i g u r e 7 are p o s s i b l e . E a c h of these isomers exists as a r a c e m i c m i x t u r e , a n d t h e s e p a r a t i o n of t h e cis g e o m e t r i c a l i s o m e r w a s a c c o m p l i s h e d . A s e c o n d f r a c t i o n w a s i s o l a t e d w h i c h consists of o n e o r m o r e trans isomers. T h e geometries of these isomers w e r e a s s i g n e d o n t h e basis of t h e i r v i s - u v s p e c t r a ( F i g u r e 8 ) w h i c h a r e s u p e r i m p o s a b l e u p o n those of t h e c i s - a n d f r a n $ - C r ( m e n )

3

c o m p l e x e s ( F i g u r e 6 ). B o t h t h e cis a n d trans g e o m e t r i c a l isomers o f c h r o m i c f e r r i o x a m i n e Β i s o m e r i z e to e q u i l i b r i u m solutions w i t h h a l f - l i v e s of s e v e r a l days at r o o m t e m p e r a t u r e . T h i s is c o n s i d e r a b l y s l o w e r t h a n t h a t f o u n d f o r t h e s i m p l e tris h y d r o x a m a t e c o m p l e x e s s u c h as C r ( m e n )

3

a n d is c a u s e d b y

t h e s t e r i c constraints o f t h e f e r r i o x a m i n e Β l i g a n d a n d its h e x a d e n t a t e chelation. Catecholate

Siderophores.

noted earlier, the c o m m o n

SIMPLE

CATECHOL

COMPLEXES.

AS

s i d e r o p h o r e f o r e n t e r i c b a c t e r i a is t h e t r i -

catechol, enterobactin ( F i g u r e 4 ) . I n order to perfect

synthetic a n d




RAYMOND

Figure 6. Absorption spectra of Cr(benz) in 17% CH OH/ CHCl solution and both absorption and CD spectra of Cr(men) in 3% CH OH/CHCl solution. 3

s

3

3

3

3

cis-Cr(benz)sy ( ); trans-Cr(benz) , (- · -); cis-Cr(men) , ( ); tram-Cr(men)s, (· · ·)· The CD spectrum of the mixture of trans isomers (31% Λ, 38% Δ) has multiplied by eight since the net optical activity of the Λ, Δ mixture is small. The CD bands near 415 nm are assigned as the high energy *A -» E transition (point group C ) which come from the *A*„ -» *Ti absorption band in octa hedral symmetry. The large bands near 570 nm are assigned as the low energy *A -> *E transition, and the bands near 670 nm are assigned as the A* -> *At transition. Both of these transitions come from the A» -> *T absorption band in octahedral symmetry. s

s

t

s

4

g

t

4

k

9

tg



44


Λ - C - trans, trans

Λ-Ν - cis, trans

II

Λ-Ν - trans, cis

Figure 7. The five enantiomeric geometrical isomers of ferrioxamine B. The oxygen donor atoms of each hydroxamate group have been omitted for clarity. The Λ optical isomer is shown in each case. See Ref. 4 for nomenclature of these geometncal isomers. s e p a r a t i o n t e c h n i q u e s to b e u s e d w i t h t h e s m a l l a m o u n t s of e n t e r o b a c t i n a v a i l a b l e , s i m p l e c a t e c h o l complexes w e r e p r e p a r e d as m o d e l c o m p o u n d s . S p e c t r o s c o p i c d a t a of t h e s i m p l e m o d e l c o m p o u n d s t h e n c o u l d b e u s e d i n a s s i g n i n g geometries f o r e n t e r o b a c t i n isomers.

T h e previous chemical

l i t e r a t u r e of tris ( c a t e c h o l ) complexes o f t r a n s i t i o n m e t a l ions is sparse.

λ

(nm)

Figure 8. Absorption spectra of the cis isomer and trans isomers of chromic desferriferrioxamine Β in aqueous solution. Cis, ( ); trans, ( ).


2.

45


RAYMOND

T h e o n l y reference to a c h r o m i c c o m p l e x r e p o r t e d t h a t i t w a s r a p i d l y h y d r o l y z e d i n d i l u t e aqueous s o l u t i o n ( 2 3 ) . T h i s , of course, w o u l d p r e c l u d e s e p a r a t i o n of o p t i c a l isomers of t h e tris chelates.

Nevertheless,

these complexes w e r e r e i n v e s t i g a t e d b e f o r e p r e p a r i n g the c h r o m i c e n t e r o bactin complex.

I t w a s f o u n d that t h e complexes a r e v e r y stable i n t h e

absence of o x y g e n

( 2 4 ) . T h e usual oxygen sensitivity of the catechol

d i a n i o n w a s f o u n d to b e s u b s t a n t i a l l y i n c r e a s e d i n t h e c h r o m i u m c o m plex.

( T h e ease o f o x i d a t i o n of c o o r d i n a t e d c a t e c h o l a n d r e l a t e d l i g a n d s

has b e e n d e m o n s t r a t e d f o r a series of m e t a l complexes

(Ni , Cu , Zn , 2 +

2 +

2 +


etc.) b y H o l m et a l . i n r e l a t i o n to the 1,2-benzenedithiolato analogs It is this o x i d a t i o n of t h e c h r o m i u m c o m p l e x

that causes

(25)).

the green-

t o - r e d c o l o r changes r e p o r t e d p r e v i o u s l y as h y d r o l y s i s . A l l p r e p a r a t i o n s a n d h a n d l i n g of t h e c h r o m i u m c a t e c h o l c o m p l e x w e r e therefore c a r r i e d out under inert atmosphere conditions. A l t h o u g h o n l y p a r t i a l r e s o l u t i o n of solutions of [ C r ( c a t ) ] " w a s 3

3

a c h i e v e d at n e u t r a l p H , c o m p l e t e r e s o l u t i o n w a s a t t a i n e d at p H 13 a n d 5°C.

T h e rate of loss of o p t i c a l a c t i v i t y f o r r e s o l v e d [ C r ( c a t ) ] ' w a s 3

3

f o u n d to d e p e n d s t r o n g l y o n h y d r o g e n i o n c o n c e n t r a t i o n s , v a r y i n g f r o m h a l f - t i m e s of s e v e r a l m i n u t e s to several h o u r s b e t w e e n p H 7 a n d p H 13 (24). COMPARISON WITH CHROMIC ENTEROBACTIN. The

visible and

l a r d i c h r o i s m spectra of [ C r ( c a t ) ] " a n d [ C r ( e n t e r o b a c t i n ) ] " 3

3

circu

complexes

3

are s h o w n i n F i g u r e s 9 a n d 10. T h e a b s o r p t i o n s p e c t r a a r e s i m i l a r e x c e p t that t h e l i g a n d - l o c a l i z e d t r a n s i t i o n occurs at l o w e r e n e r g y i n t h e entero b a c t i n c o m p l e x , thus m a s k i n g t h e A 4

2 g

—» T 4

lg

(for D

h

symmetry)

d-d

t r a n s i t i o n , w h i c h appears as a s h o u l d e r o n t h e e d g e of t h e m o r e intense π —» 7Γ* l i g a n d transitions. T h i s is a p p a r e n t l y c a u s e d b y t h e f a c t t h a t e n t e r o b a c t i n contains o r t h o - a c y l - s u b s t i t u t e d c a t e c h o l r i n g s . T h u s t h e v i s - u v spectra of [ C r ( c a t ) ] " 3

3

and [Cr(enterobactin)] " 3

are too d i s s i m i l a r to a l l o w d e t a i l e d c o m p a r i s o n s a n d confident p r e d i c t i o n of s t r u c t u r e b a s e d o n s u c h c o m p a r i s o n s . H o w e v e r , there is a d r a m a t i c a l l y different s i t u a t i o n f o u n d i n c o m p a r i n g t h e C D s p e c t r a of

[Cr(cat) ] " 3

3

a n d [ C r ( e n t e r o b a c t i n ) ] " , w h i c h are f o u n d to b e substantially i d e n t i c a l 3

( F i g u r e 1 0 ) . T h i s is b e c a u s e t h e i n t e r f e r i n g c h a r g e transfer b a n d is n o t associated w i t h t h e c h i r a l center a n d h e n c e does n o t c o n t r i b u t e to t h e optical activity. T h e c r y s t a l a n d m o l e c u l a r s t r u c t u r e of a salt of [ C r ( c a t ) ] " a n d t h e 3

3

k n o w n [ C r ( c a t ) ] " absolute configurations g i v e the f o l l o w i n g a s s i g n m e n t : 3

3

the p r e d o m i n a n t i s o m e r of t h e c h r o m i c e n t e r o b a c t i n m o n o m e r i c has a Δ-cis absolute c o n f i g u r a t i o n ( F i g u r e 5 ) .

complex

T h e s i m i l a r i t y of t h e

c h r o m i c a n d f e r r i c complexes a l l o w s this assignment to b e m a d e f o r t h e f e r r i c c o m p l e x as w e l l . T h i s is t h e opposite absolute c o n f i g u r a t i o n of t h e other o p t i c a l l y a c t i v e siderophores c h a r a c t e r i z e d to date.

T h e opposite




46

II

50

I

I

400

I

I

I

500

I

600

λ

ι

1

500

'

I

10

700

(nm) Γ

600

'

700

x(nm)

Figure 9. (a) (top) Visible absorption spectrum of K [Cr(cat) ] in water, (bottom) Circular dichroism spectra of Δ - and Λ K [Cr(cat) ] solutions. 3

3

3

3

a b s o l u t e configurations of c h r o m i c e n t e r o b a c t i n a n d c h r o m i c f e r r i c h r o m e c a n b e seen c l e a r l y i n c o m p a r i n g t h e i r C D s p e c t r a ( F i g u r e 1 0 b ) . r o l e o f the siderophores

as c e l l u l a r permeases

for ferric i o n

The

therefore

does n o t d e p e n d o n the c o m p l e x a l w a y s h a v i n g a Λ - c i s c o n f i g u r a t i o n , a l t h o u g h t h i s c o n f i g u r a t i o n or others m a y b e s p e c i f i c a l l y t r a n s p o r t e d i n i n d i v i d u a l m i c r o b i a l - l i g a n d systems. T h e m o l e c u l a r s t r u c t u r e of e n t e r o b a c t i n has not as y e t b e e n

estab

l i s h e d b y d i f f r a c t i o n t e c h n i q u e s a n d , a l t h o u g h c o o r d i n a t i o n of f e r r i c i o n


RAYMOND

47


600

500

1400


300

200

Figure 10. (a) (left) Visible absorp tion spectrum of [NHJ [C^entero bactin)]. (b) (below) Circular dichroism spectra of A-[NH\] [Cr(enterobactin)] ( ) and chromic ferri chrome (—) (the latter from Ref. 3). 3

550

3

650

x(nm)

Ο Δε

400

500

600

700

Anericart^eraical Society Library 1155 16th St. N. W.

In Bioinorganic Chemistry—II; Raymond, K.; Advances in Chemistry; American Society: Washington, DC, 1977. Washington, D.Chemical C. 20036


48


II

Figure 11. A perspective drawing of the [M(0 C H^sl ' anions. M = Cr, Fe, as viewed down the molecular threefold axis. 2

6

3

b y e n t e r o b a c t i n p r e v i o u s l y h a d b e e n a s s u m e d to b e a n o c t a h e d r a l c o m p l e x w h i c h i n v o l v e s o n l y the c a t e c h o l m o i e t i e s of the l i g a n d , n o

firm

s t r u c t u r a l e v i d e n c e f o r this w a s a v a i l a b l e . F u r t h e r m o r e , the use of C r ( I I I ) i n p l a c e of F e ( I I I ) to e n a b l e t r a n s p o r t studies of o p t i c a l l y a c t i v e Table I.

Structural Parameters pa

Charge, η

1

=

Average M - 0 distance (Â) A v e r a g e r i n g O - M - 0 angle ( ° ) A v e r a g e 0 - 0 r i n g distance (Â) Ligand bite T r i g o n a l t w i s t angle ( ° ) P l a n e - t o - p l a n e d i s t a n c e (Â) ' e

1

a 6 0 d 9

1.723(4) 91.4(2) 2.466(6) 1.431 58.9 1.940

Ref. 27. Ref. 28. Ref. 29. Ref. 26. Ratio of the 0-0 ring distance to M - 0 distance. See Ref. 80.


2.

49


RAYMOND

siderochrome

complexes

has b e e n

justified here a n d i n t h e p r e v i o u s

c h a p t e r o n the basis that s u c h complexes w o u l d b e i s o s t r u c t u r a l . H i g h s p i n F e ( I I I ) a n d C r ( I I I ) are w i t h i n 0.03 A i n i o n i c r a d i u s of o n e another, b u t the c r y s t a l field s t a b i l i z a t i o n energy ( C F S E ) f o r the c h r o m i c c o m p l e x (12 D q ) is c o n s i d e r a b l y greater t h a n that f o r h i g h - s p i n f e r r i c i o n ( 0 D q ) . A n y shift b y the c h r o m i c c o m p l e x t o w a r d s o c t a h e d r a l f r o m t r i g o n a l p r i s m a t i c c o o r d i n a t i o n , as e v i d e n c e d

b y t h e t r i g o n a l t w i s t angle, m a y b e

a t t r i b u t e d to this c r y s t a l field effect. GEOMETRY


geometries

OFMETAL

CATECHOL

of [Fe(cat) ] ~ 3

COMPLEXES.

and [Cr(cat) ] "

3

3

s i n g l e c r y s t a l studies o f t h e salts K [ M ( c a t ) ] - 1 . 5 3

T h e coordination

have been determined b y

3

3

H

2

0 (M = C r , F e )

i n o r d e r to explore the c r y s t a l field effect o f c h r o m i c i o n o n the c o o r d i n a t i o n geometry a n d , i n d i r e c t l y , t o d e t e r m i n e the c o o r d i n a t i o n g e o m e t r y o f e n t e r o b a c t i n itself (26). T h e [ M ( c a t ) ] " complexes ( F i g u r e 1 1 ) are d i s t o r t e d f r o m o c t a h e d r a l 3

3

geometry w i t h approximately D

3

molecular point symmetry.

t u r a l parameters o f t h e tris ( c a t e c h o l ) complexes

T h e struc-

r e p o r t e d t o date a r e

c o m p a r e d i n T a b l e I . T h e l i g a n d b i t e ( r a t i o of t h e O - O r i n g d i s t a n c e to the M - O d i s t a n c e ) , the t r i g o n a l t w i s t a n g l e , a n d the t r i g o n a l p l a n e - t o p l a n e distance v a r y s m o o t h l y across the t a b l e as i o n i c r a d i i increase. T h e final

g e o m e t r y represents a b a l a n c e b e t w e e n distortions o f t h e O - M - O

a n g l e a n d O - O r i n g distance a n d v a r i a t i o n s o f t h e t w i s t a n g l e

from

octahedral to trigonal prismatic. I n comparing the chromic a n d ferric c a t e c h o l structures, the difference i n M - O b o n d l e n g t h is not large e n o u g h to cause t h e n e a r l y six-degree difference i n t w i s t angle.

This must b e

a t t r i b u t e d to t h e difference i n c r y s t a l field s t a b i l i z a t i o n e n e r g y ( A C F S E ) b e t w e e n o c t a h e d r a l a n d t r i g o n a l - p r i s m a t i c geometries.

A l t h o u g h signif-

i c a n t i n terms of the p r e c i s i o n o f t h e s t r u c t u r e d e t e r m i n a t i o n s , t h e f e r r i c a n d c h r o m i c complexes are close e n o u g h i n g e o m e t r y to r e g a r d s i m i l a r for

[M(cat) ] 3

Si

n _

Complexes (26) As

b

c

2 1.784(18) 88.7(2) 2.490(6) 1.396 55.9(5) 2.093

1 1.843(5) 88.2(5) 2.565(7) 1.392 55.2(10) 2.194

Cr*

Fe*

8

3

1.986(4) 83.56(14) 2.646(6) 1.333 50.5(6) 2.247

2.015(6) 81.26(7) 2.625(2) 1.303 44.7(10) 2.303

T h i s angle is defined b y v i e w i n g t h e c o m p l e x i n p r o j e c t i o n d o w n t h e m o l e c u l a r t h r e e - f o l d axis. I t is t h e n t h e r o t a t i o n r e q u i r e d t o b r i n g t h e t o p a n d b o t t o m planes (of three o x y g e n a t o m s each) i n t o c o i n c i d e n c e . T h i s angle is 6 0 ° f o r o c t a h e d r a l a n d 0 ° for t r i g o n a l p r i s m a t i c c o o r d i n a t i o n . P l a n e - t o - p l a n e distance f o r t h e t w o t r i g o n a l o x y g e n a t o m planes d e s c r i b e d i n . r

9

1

Journal of the American Chemical Society


50


c h r o m i c - s u b s t i t u t e d s i d e r o p h o r e complexes

II

as s t r u c t u r a l l y i d e n t i c a l to

t h e n a t u r a l f e r r i c complexes f o r b i o l o g i c a l purposes. FERRIC-CATECHOLATE

CONSTANTS.

FORMATION

The

very

high

affinity for f e r r i c i o n w h i c h a l l siderophores d i s p l a y is essential to t h e i r r o l e i n o b t a i n i n g i r o n f o r the m i c r o o r g a n i s m u s i n g t h e l i g a n d . T h i s is a l w a y s a c c o m p l i s h e d i n a n e n v i r o n m e n t w h i c h also contains m a n y other s t r o n g c o m p l e x i n g agents f o r f e r r i c i o n . constant f o r the s i d e r o p h o r e

complex

Hence a very high formation

is essential for s u r v i v a l i n the

c o m p e t i t i v e w o r l d of the m i c r o o r g a n i s m . A s d e s c r i b e d e a r l i e r a n d r e


v i e w e d elsewhere

( J ) , t h e h y d r o x a m a t e siderophores

have

formation

constants for reactions of the t y p e :

Fe

3 +

+

Ο"

I

Ο—Ν

0

II

Fe

•N—C"

Ό—C

w h i c h r a n g e b e t w e e n 1 0 a n d 1 0 . T h e s e values are o n l y t w o to three orders of m a g n i t u d e greater t h a n the o v e r a l l f o r m a t i o n constant, β , for t h e s i m p l e tris ( m o n o h y d r o x a m a t e ) complexes. 30

32

3

I n contrast to the h y d r o x a m a t e siderophores, l i t t l e or n o t h i n g is k n o w n a b o u t the s t a b i l i t y constant for t h e c a t e c h o l s i d e r o p h o r e , entero b a c t i n . P r i o r to d e t e r m i n i n g t h e f o r m a t i o n constant of e n t e r o b a c t i n ( f o r w h i c h h y d r o l y s i s of the l i g a n d presents s p e c i a l p r o b l e m s ) , the r e a c t i o n of c a t e c h o l itself w i t h f e r r i c i o n has b e e n i n v e s t i g a t e d (31). C a t e c h o l is a v e r y w e a k a c i d a n d h e n c e at l o w p H is a p o o r l i g a n d . T h e k i n e t i c s a n d e q u i l i b r i a of its reactions w i t h f e r r i c i o n u n d e r a c i d i c c o n d i t i o n s h a v e b e e n i n v e s t i g a t e d (32). U n d e r such conditions, even w i t h excess c a t e c h o l , f e r r i c i o n forms o n l y a transient 1:1 c o m p l e x w h i c h e v e n t u a l l y u n d e r g o e s a r e d o x r e a c t i o n to g i v e ferrous i o n a n d o r t h o q u i n o n e as p r o d u c t s . T h i s r e a c t i o n has a r e d o x p o t e n t i a l just greater t h a n z e r o at p H 1. A t h i g h e r p H ' s the e x t r e m e l y l a r g e f o r m a t i o n constant of the tris c a t e c h o l f e r r i c c o m p l e x s t r o n g l y reverses the p o t e n t i a l , s u c h t h a t ferrous i o n w i l l r e d u c e o r t h o q u i n o n e to f o r m t h e tris c a t e c h o l f e r r i c c o m p l e x . I n the absence of a i r , b o t h t h e c h r o m i c a n d f e r r i c tris c a t e c h o l complexes are stable i n d e f i n i t e l y i n basic aqueous s o l u t i o n (24). T h e e q u i l i b r i u m constants i n v o l v e d i n the r e a c t i o n F e + 3 c a t " ^ F e ( c a t ) ~ w e r e d e t e r m i n e d as f o l l o w s . A n aqueous s o l u t i o n of F e (5.5 X 1 0 M ) a n d c a t e c h o l (1.48 X 1 0 M ) , i n i t i a l l y m a d e b a s i c w i t h the a d d i t i o n of K O H , w a s t i t r a t e d w i t h 1 . 2 4 M H C 1 u n d e r a n oxygen-free a t m o s p h e r e at 22° a n d i o n i c s t r e n g t h ( K C 1 ) 0 . 1 6 - 0 . 2 2 M ( F i g u r e 1 2 ) . T h e a c i d d i s s o c i a t i o n constants for c a t e c h o l w e r e d e t e r m i n e d i n d e p e n d e n t l y ( u n d e r s i m i l a r e x p e r i m e n t a l c o n d i t i o n s ) to b e p K i = 9.38 a n d 3 +

3

_ 3

3

2

3 +

_ 2

a


2.


RAYMOND

pK

=

a 2

13.28.

51

( A l l s t a b i l i t y a n d association constants m e n t i o n e d

are

c o r r e c t e d to i o n i c s t r e n g t h of 0 . 1 M . ) U s i n g these constants a n d l i t e r a t u r e values for the h y d r o l y s i s constants Fe (OH) 2

2

4 +

of

Fe(III)

(FeOH

2 +

, Fe(OH

) , a c l a s s i c a l B j e r r u m η vs. p L p l o t p r o d u c e d

values of the m e t a l - l i g a n d s t a b i l i t y constants.

2

+

),

approximate

L e a s t squares refinement

of the c u m u l a t i v e s t a b i l i t y constants c o n v e r g e d at the v a l u e s l o g βχ

=


13,

I 1 2 UDLUME DF

3 4 5 6 TITRANT (HL)

?

Β

9

10

11

12

I

I

13

14

I

15

1

1

16

17

Figure 12. Titration curve. An initially basic aqueous solution (5.5 X 10~ M) and catechol (1.84 X 10~ M) is titrated with 1.24M 22° under an oxygen-free atmosphere. ( ), least squares fit to served data (discrete points). Data past pH 10 were given zero 3

21.5, l o g β

2

2

=

36.6, a n d l o g β

=

3

1 IB

of Fe HCl at the ob weights. 3+

45.9. T h e i n c l u s i o n of i r o n h y d r o l y s i s

i n the refinement m o d e l p r i m a r i l y affected the c a l c u l a t e d v a l u e of β χ. T h e d i s t r i b u t i o n of the v a r i o u s species i n s o l u t i o n as a f u n c t i o n of p H is s h o w n i n F i g u r e 13. T h e w e a k a c i d i t y of c a t e c h o l makes its effective f o r m a t i o n constant m u c h less t h a n 1 0 · near p h y s i o l o g i c a l p H . H o w e v e r , a n y chelate effect 45

9

s h o u l d t e n d to m a k e the f o r m a t i o n constant f o r e n t e r o b a c t i n i n l a r g e r than β

3

for catechol.

the r e a c t i o n F e

3 +

+

Thus 10

45

c a n b e r e g a r d e d as a l o w e r b o u n d

for

e n t ' *± F e ( e n t ) " . 6

3

Summary T h i s p a p e r has f o c u s e d o n the c o o r d i n a t i o n c h e m i s t r y of the s i d e r o phores. A t this stage i n o u r studies of m e t a l - s u b s t i t u t e d siderophores have established the f o l l o w i n g :


we



52

2

3

4

5

6

7

B

9

10

II

11

PH Figure

13.

Distribution

curve,

as a function

of pH,

of the

various

species

formed in the ferric-catechol titration experiment. (A) free Fe *; (B) Fe(cat) ; (C) Fe(cat)f; (D) Fe(cat) ~; (E) H cat; (F) (H cat)~; (G) feme hydrolysis products (FeOH' , Fe,(OH) , Fe(OH) ). Cat, catecholate dianion; ALPHA, concentration of the particular species divided by the total iron concentration. 3

+

s

3

+

i+

s

2

+

t

( 1 ) T h e c h r o m i c - s u b s t i t u t e d s i d e r o p h o r e complexes c a n b e p r e p a r e d a n d , i n contrast to the n a t u r a l l y o c c u r r i n g f e r r i c complexes, are k i n e t i c a l l y i n e r t to i s o m e r i z a t i o n or l i g a n d s u b s t i t u t i o n . ( 2 ) T h e v i s i b l e a n d c i r c u l a r d i c h r o i s m s p e c t r a of the c h r o m i c s i d e r o p h o r e complexes are closely r e l a t e d to the c o r r e s p o n d i n g s p e c t r a of s i m p l e m o d e l complexes of h y d r o x a m a t e or catecholate l i g a n d s . T h i s p r o v i d e s a s p e c t r o s c o p i c p r o b e f o r s t r u c t u r e i n a s s i g n i n g t h e geometries of the s i d e r o p h o r e complexes. (3) T h e s t r u c t u r e a n d b o n d i n g of the c h r o m i c a n d f e r r i c c o m p l e x e s ( d e s p i t e t h e i r differences i n k i n e t i c p r o p e r t i e s ) are sufficiently a l i k e to r e g a r d t h e m as i d e n t i c a l for b i o l o g i c a l systems. ( 4 ) T h e c h r o m i c - s u b s t i t u t e d siderophores c a n b e u s e d to s t u d y the m e c h a n i s m s of m i c r o b i a l i r o n transport. T h e s e studies r e l y o n t h e k i n e t i c inertness of t h e c h r o m i c c o m p l e x a n d w o u l d b e i m p o s s i b l e to c a r r y out u s i n g other t e c h n i q u e s or probes.


2. RAYMOND

Kinetically Inert Siderophore Complexes 53

Acknowledgment I am pleased to acknowledge my co-workers, past and present, whose efforts have been summarized here. They are John Leong, Stephan Isied, Alex Avdeef, Frank Fronczek, Leo Brown, Jim McArdle, Hunter Nibert, and Gilbert Kuo. The collaboration of J. B. Neilands continues to be a seminal influence. This research has been supported by USPHS grant AI-11744.


Literature Cited

1. Neilands, J. B., Ed., Microbial Iron Metabolism, Academic Press, New York, N.Y. 1974. 2. Leong, J., Raymond, Κ. N.,J.Am. Chem. Soc. (1974) 96, 1757. 3. Ibid. (1974) 96, 6628. 4. Ibid. (1975) 97, 293. 5. Biedermann, G., Schindler, Acta Chem. Scad. II (1957) 731. 6. Leussing, D. L., Kolthoff, I. M.,J.Am. Chem. Soc. (1953) 75, 2476. 7. Spiro, T.G.,Saltman, P., Struct. Bonding (1969) 6, 116. 8. Schwarzenbach, G., Schwarzenbach, K., Helv. Chim. Acta (1963) 46, 1390. 9. Anderegg, G., L'Eplattenier, F., Schwarzenbach,G.,Helv. Chim. Acta (1963) 46, 1400. 10. Ibid. (1963) 46, 1409. 11. Lindner, Von H. J., Gottlicher, S., Acta Cryst. (1969) B25, 832. 12. Inorg. Chem. (1970) 9, 1. 13. Poling, M., Van der Helm, D., Book of Abstracts, American Crystallo graphic Association, Spring Meeting, Berkeley, 1974, abstract Q7, p. 111. 14. Zalkin, Α., Forrester, J. D., Templeton, D.H.,J.Am. Chem. Soc. (1966) 88, 1810. 15. Bränden, C. I., private communication, 1974. 16. Hough,E.,Rogers, D., Biochem. Biophys. Res. Commun. (1974) 57, 7 17. Llinás, M., Struct. Bonding (1973) 17, 135. 18. Pollack, J. R., Neilands, J. B., Biochem. Biophys. Res. Commun. (1970) 38, 989. 19. O'Brien, I. G., Gibson, F., Biochim. Biophys. Acta (1970) 215, 393. 20. Neilands, J. B., "Structure of Microbial Iron Transport Compounds" in "Structure and Function of Oxidation Reduction Enzymes,"Å.Akeson andÅ.Ehrenberg, Eds., pp. 541-547, Pergamon, Oxford, 1972. 21. Buerer, T., Gulyas,E.,Proc. 9th Int. Conf. Coord. Chem., St. Morit (1966) 512. 22. Basolo, F., Pearson, R. G., "Mechanisms of Inorganic Reactions," 2nd ed., Wiley, New York, 1967. 23. Weinland, R., Walter, Ε., Z. Anorg. Allg. Chem. (1923) 126, 141. 24. Isied, S. S., Kuo, G., Raymond, Κ. N.,J.Am. Chem. Soc. (1976) 98, 1763. 25. Röhrscheid, F., Balch, A. L., Holm, R. Α., Inorg. Chem. (1966) 5, 1542. 26. Raymond, Κ. N., Isied, S. S., Brown, L. D., Fronczek, F. R., Nibert, J. H., J. Am. Chem. Soc. (1976) 98, 1767. 27. Allcock, H. R., Bissell, E.C.,J.Am. Chem. Soc. (1973) 95, 3154. 28. Flynn, J.J.,Boer, F. P.,J.Am. Chem. Soc. (1969) 91, 5767. 29. Kobayashi, Α., Ito, T., Marumo, F., Sacto, Y., Acta Crystallogr., Sect. Β (1972) 28, 3446. 30. Wentworth, R. A. D., Coor. Chem. Rev. (1972) 9, 171.


54

BIOINORGANIC CHEMISTRY-II


31. Avdeef, Α., Sofen, S. R., Bregante, T. L., Raymond, Κ. N., unpublished data. 32. Mentasti, E., Pelizzetti, E., Saini, G., J. Chem. Soc., Dalton Trans. (1973) 2605, 2609. RECEIVED July 26, 1976.


Kinetically Inert Complexes of the Siderophores in Studies of Microbial

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