Some aspects of the reactivity of amino acids coordinated to metal ions

I. inor'4 nu{ I. ( ' h e m . . 1974, Vol. 36. pp. 2133 2144. Pergamon Press. Printed in Great Britain

SOME ASPECTS OF THE R E A C T I V I T Y OF A M I N O A C I D S C O O R D I N A T E D TO M E T A L IONS ALESSANDRO PAS1NI and LU1GI CASELLA lstituto di Chimica Generale, Via Venezian, 21 20133 Milan. Italy (Received 18 December 19731

Abstract- Some features of the reactivity of the amino acids coordinated to metal ions are reviewed, Special emphasis is given to the formation and the hydrolysis of esters and peptides, to some reactions of complexes of amino acids Schiff bases, to aldol condensation and to isotopic exchange and racemization at the z-carbon atom. The change of reactivity which follows the coordination to metal atoms and some possible references to inorganic biochemistry are also outlined.

Tim CHEMISTRY of metal complexes of amino acids has recently developed not only from the inorganic point of view, but also because of possible biological interest. Most of the studies concerned the synthesis and properties of these complexes and the factors influencing their stereoselective formation, but more recent studies have emphasized the reactivity of coordinated amino acids in the hope of finding simple and easily interpretable models of reactivity of some biological systems which contain metals. Indeed, even if metals are not universal components of enzymes, many classes of enzymes either contain metals or need a metal ion as a cofactor. It is known that an enzyme acts as a catalyst not because it contains particular and unusual chemical groups, but rather because the specific three-dimensional structure of the protein generates an environment which gives to a chemical group a rather particular reactivity[l]. Such an environment can be built up by cross linkages within the protein and/or by the presence of a metal constituting a coordination compound with basic groups of the protein itself. According to Gillard[2], the principal functions of a metal ion in a metal activated enzyme are as follows: (11 It can activate, through coordination, some functional groups of the substrate or of the protein. (2t It can induce, by coordination, a specific geometry of the apo-protein binding sites (a specific "lock"), so that only certain substrates can interact with the active site of a particular framework. (31 It can induce, again by coordination, a specific geometry to the substrate, so that this can interact with the active site of the enzyme possessing the right geometry (a specific "key" for a specific "lock"). All these three factors are related to the theme of this review; when an amino acid is coordinated to a metal, will its reactivity change? Or will some particular reaction be any faster than in the free amino acid'?

Moreover, can one correlate these reactions with those of the more complicated biological systems? The material collected here can be roughly divided as follows: (1) Reactions of formation and hydrolysis of esters and peptides in the coordination sphere of a metal ion. (2) Reactions of complexes of amino acid Schiffbases. (3) Aldol condensations. (4) Isotopic exchange and racemization at the ~carbon atom of the amino acid. (5) Miscellaneous reactions. I. F O R M A T I O N A N D H Y D R O L Y S I S OF E S T E R S A N D PEPTIDES

The rate of hydrolysis of amino acid esters is greatly enhanced by the presence of metal ions such as Co(IlL Cu(II), Mn(II), Mg(II), Ni(II)[3 5] and it usually increases with increasing metal-to-ester ratio[5]. Most Table 1 Abbreviations used throughout this paper trien dien en DM F

triethylenetetramine diethylenetriamine et hylenediamine dimethylformamide is an amino acid anion

The following abbreviations for the amino acid residues arc used : gly glycine ala alanine pala phenylalanine val valine isolen isoleucine ser serine threo threonine asp aspartic acid glu glutamic acid EDTA et hylenediaminetet racetic acid EDDA ethylenediaminediacetic acid

2133

2134

ALLESSANDRO PAS1NI and LUIGI CASELLA

of the mechanistic work, however, refers to Co(III) complexes because, even if this metal in this oxidation state is quite unusual in living systems, its complexes can be easily studied, being kinetically inert towards the dissociation of the ligands. Buckingham et al. published a scheme of reactions [6] that can be assumed as to be general: when the species [CotrienX2] + reacts with the ethyl ester of glycine in anhydrous conditions (DMF) the corresponding complex of the ethyl ester of glycylglycine is obtained:

oxygen and nitrogen bases act as specific nucleophiles rather than as general bases toward type (II) or (III) species, irrespective of their basicity toward a proton" [11,121. Also, in these studies a large entropy effect was evident; i.e. AS:~ for the hydrolysis of the coordinated amino acid ester is much higher than that of the free ester. According to the author this increase is responsible for the higher rate of hydrolysis[12]. The hydrolyses of esters and peptides[13] induced by metal ions, the latter being specific for N-terminal

N

NHzCH2COO E t-

X

I C%

_

.

O J Ol:t EtOOC - C H 2 - NH 2

(i) This mechanism has been established through the isolation of (11)[7] which, when treated with ethyl glycinate, gives (III). This latter complex can be isolated, but when treated with water in basic conditions, the dipeptide hydrolyzes giving the cobalt complex of ethyl glycinate. Similar results have independently been obtained by Collman and Kimura in the case of [Coen2Xz] +, who pointed out that in similar conditions, but in the absence of the metal complex, the reaction does not take place [8]. Peptide bond formation has also been reported for some platinum(II) complexes[9]. More recently Wu and Busch[10] succeeded in preparing a tetrapeptide by means of the neutral complex [CodienCl3] which can use up to three coordination sites (by removing the three chlorine atoms). In all these cases the peptide bond is formed in the coordination sphere of the Co(Ill) ion; presumably the coordination of the ester, through the carbonyl oxygen, causes bonds polarization, thus activating the carbonyl carbon and facilitating nucleophilic attack by the amino group of another ester molecule (as in (II) and (III)). Very likely[8] the species (III) is a possible intermediate in the hydrolysis of peptides, such as (II) is of esters. In other words, (II) can react with any nucleophile such as water or, in anhydrous conditions, with another molecule of ester as already mentioned; the same applies to (III). The former reaction leads to hydrolysis, the latter to peptide bond formation. We will not consider these reactions in two separate sections, since they are only two aspects of the same nucleophilic attack. In recent studies these reactions have been investigated in detail and it appears that the lysis of (II) or (III) depend on the nucleophile, that is "all

(ll)

lie\ O jj"

NH2CH2COOEt

(ill)

amino acids, have received a great deal of attention, because of their practical and biological significance. Many detailed mechanisms have been proposed. Australian workers have reported[14] the aminolysis of ethyl glycinate coordinated to a cobalt(Ill) centre where the nucleophile is a molecule of ammonia of the coordination sphere of the metal itself: 2[(NH 3)5CoNHzCH2COOEt] 3+ pH9 14 ~- [(NH3)4CoNH2CH2CONH2]2 + + 2EtOH

+ [(NH3)4CoNH2CH 2COO]2+ This "intramolecular nucleophilic attack" could be operative also in the case of hydrolysis when a mixed amino acid ester-aquo complex is considered. These authors have related their results to some enzyme reactions catalyzed by metal ions[15, 16]. Other authors proposed different mechanisms for the hydrolysis of esters and peptides in the presence of metal ions[17]; however, they all require the coordination of the amino acid moiety to the metal through the amino group, as in (IV). The carbonyl carbon atom then can be directly attacked by a variety of nucleophiles which are outside the first coordination sphere like O H - , H 2 0 or even M O H (a hydroxo complex which may be present in the solution), or the substrate can first form a chelate (as in (II) or (III)), thus being more activated towards nucleophilic reagents. In fact, the hydrolysis of the monodentate ester of (IV) is much slower than that of the chelated species[7, 18, 19]. Moreover, it must be emphasized that coordination activates not only the amino acid derivative but also water, altering its PKa in such a way that the local concentration of coordinated O H - ions is increased. Of course all these mechanisms can be possible at

2135

Reactivity of coordinated amino acids the same time and the occurrence of one with respect to the other may depend on many factors such as the pH or the kind of complex present in the reaction conditions. For instance the hydrolysis of ethyl glycinate, promoted by [Cudien] 2÷, has been reported[20] to proceed through two different mechanisms m which the ester molecule, coordinated only through the nitrogen atom, is attacked by the two different nucleophiles OH and M O H An intramolecular attack seem very unlikely because the Cu{II) ion prefers tetracoordination, which is already present through the three nitrogens of diethylenetriamine and that of the amino group of the amino acid ester as in (IVI.

N

Nit e

/

i

('H 2

j NH~- CH, /7~, ("

Cu

~/

EIOOC (7'He NH_,

NH CH, ('O()Fil Et()'

I }

EtOOC ('H, NH,

NH, ('H,

('u

]

COOEt

"

EtOOC CH, NH~

[

NH

(

()

CH, ('()()It IX1

"N

A related typical example of intramolecular attack, is the hydrolysis of 2-bromoethylamine to give ethanolamine in the presence of the [Coen2X2] + ion[21] in basic conditions.

~ N

N///

()[I

~lX~

/

(IV}

'

EIOOC ('H e NHe

{'u N

/

copper, attacks the carbonyl carbon of another ester molecule coordinated through the amino group as in (IX-X). ~ ()

I~NH

2 ~H

11

C H 2 ~ Br

~-~ N

This mechanism, established by spectroscopic evidence. leads to the formation of tripeptides when amino acid esters with small steric hindrances are used and since it does not interfere with the chiral carbon atom of lhc amino acid, optically active peptides are obtained. A very elegant example of intramolecular attack has been reported recently[24] and it concerns the transesterification between p-nitrophenyl picolinate and N-(fl-hydroxyethylt-ethylenediamine. catalyzed by the Zn 2+ ion. In this case, an intermediate complex is probably formed between the two reagents on the zinc ion which, presumably, has two catalytic roles: it changes the pK of the hydroxylic group and it assembles the two reagents into the coordination sphere, thus facilitating the intramolecular attack of the nucleophile upon the carbonyl group of picolinic acid.

i

IV) The same authors have compared this mechanism to that of the hydrolysis of esters promoted by similar hydroxo-complexes[21]. In this case. where the ester molecule is not chelated. the coordination has a threefold role: (i) it assembles the reagents: (ill it activates the nucleophile (coordinated water dissociates at a lower pH): (iii) it stabilizes, through chelation, the intermediate and the final product (VI to VIII). //

NH2 CH2

/

,N co

Na('o 0 C OR ' H (vii

~

J

NIl (H,

CH:-----CH: NO 2

(XII

This is a very important example• In fact. according to the authors[24], (XI) can be considered as analogous

{ ]

O -C OR I q H O~ (Vll~

An intramolecular mechanism has been proposed for peptide formation from amino acid esters in the presence of CuCl 2 in ethanol solution[22, 23]. In this case it seems that the amino anion, formed by the loss of a proton from the amino group coordinated to

,

!

NH2--CH e \(.)

/

NH2

/

NH3

('He L + ROH

, N4Co x

O: ..... C \

•O (Villi

to a substrate--enzyme complex where the metal ion acts as a bridge, increasing the tendency of the substrate towards nucleophilic attack by another bound small molecule or by a particular group of the enzyme itself.

2136

ALESSANDRO PASINI and LUIGI CASELLA

In other words the metal ion acts as a "template": it increases, through coordination, the local concentration of the reagents, also modifying their electronic properties, thus making a reaction easier. A similar role for the zinc ion can be found in certain enzymes like carbonic anhydrase[25]. Another interesting example of the template action of a metal complex is the hydrolysis of the methyl ester of dipeptides[26] catalyzed by Cu 2+ in the pH range 6.5-8-0. According to the authors, in fact, the ester group can be activated only if it is coordinated and this can happen only if the amide group is ionized and coordinated to the metal (XII, XIII). O

II

C

N--CH

proposed[30] between the "template" and the "promnastic" role of a metal ion. In the latter case, the metal ion serves merely to assemble the reactants, as in certain transamination reactions, which proceed through the formation of an intermediate Schiff base between the amino acid amino group and an active carbonyl group in the presence of metal ions (XIV XV): R l CH_COOI-I +

O

2

M

O

CH, \"

N--CH

C--O + CH3OH ~ ......... NH2__Cu_ O ,' (Mill

An indirect piece of evidence for this mechanism is the fact that the hydrolysis of the methyl ester of glycylsarcosine does not seem to be catalyzed by Cu 2+ in similar conditions[26]. Very recently a series of papers has appeared on the stereoselectivity of amino acid ester hydrolysis[27 29]. When (R)- or (S)-histidine methyl ester is hydrolyzed in the presence of catalytic amounts of Ni(II) and (R)or (S)-histidine (molar ratio 1:1) the rate is increased by a factor of about 40 per cent when the ester and the amino acid are of opposite configurations. This has also been found to be true for the related tryptophan system, but not for aspartic acid and methionine[28]. The interpretation of these facts is unclear but it seem obvious that such stereoselectivity arises from the coordination to the nickel ion of bulky and almost rigid molecules for which certain geometries are more stable. From models it appears[27] that only when the ester and the amino acid are of different configurations is the ester carbonyl group in a favourable position to interact with the metal ion. A correlation with the stability constants of mixed (R)~-(S)ct-Ni(II) complexes has been proposed[29]. In any case it appears that complexation plays an important role in the stereoselectivity of this reaction. 2. R E A C T I O N O F C O M P L E X E S O F S C H I F F BASES O F AMINO ACIDS

In this section we will be concerned particularly with transamination and racemization. It is worthwhile to remember that a subtle distinction has been

/

~

M

--CH--R ~

C-R 2

(XlV)

2

//

+_2

\

(-)OOC

~L

O

O--C

/

NH 2 - C u - O (XII)

)

NH(3+

//

\C_OCH3

C

C_COO I-) + M "+

R' C CO0 (-~ + R 2 CH COO'-I + Mn+

OH

/ CH2

R2

O

O--C

\

N=C--R '

( )

I

OOC CH-R (XV)

2

However, this does not seem to be the only effect; it has been reported[31] that coordination can modify the equilibrium constant between the two tautomeric forms (XIV, XV) of the Schiff base. Pyridoxal-activated enzymes[32] act through the formation of similar Schiff bases between pyridoxal and amino acids and catalyze a variety of reactions of the activated a-carbon atom of the amino acid, such as racemization, transamination, aldol condensation and decarboxylation[32]. The Schiff base has the following structure: R CH-COO ~ ~

I

~ CH

N

N (XVl)

\ M

CH 3

R C-COO ~ II N / \ CH z M

N "CH 3 (XV[I)

where M is a metal ion. The tautomeric equilibrium X V I ~ X V I I leads to transamination, as has been demonstrated by the fact that the same metal complexes can be obtained when either pyridoxal and alanine or pyridoxamine and pyruvic acid are allowed to react together with a metal ion[33], and confirmed for other systems[34]. In this scheme the metal probably serves to maintain the planarity of the Schiff base and to facilitate the electron-withdrawing power of the heterocyclic ring[35]. Similar reactions can be reproduced in many nonenzymatic systems; for instance, when an amino acid is allowed to react with pyridoxal and different metal ions like A13+, Fe 3+ and Cu 2÷, it racemizes and/or

21~7

Reactivity of coordinated amino acids transaminates[36, 37]. Moreover, one can use a variety of aldehydes, ketones or keto acids, thus studying these reactions on a series of models[38] related to vitamin B 6. These systems are easy to investigate since most of the intermediate complexes can be isolated and their structure can be correlated with their reactivity. Both racemization and transamination require the loss of a proton attached to the a-carbon atom of the amino acid[35], which must be very reactive in these structures. /

R

CH=N--CH / --

C=O

Cu:2 ~

/

jOH, Cu \\.

CH:=N /

CH =N I R C--COOEr I H i~(Xllll

@

o

() •

/

R (' It

(' ()

tXXI\h O\,

/

Cu/2

CHiN R - - C - - ( ' H 2 COOEr

/

H (XXV)

R

/ C H = N - - C ~-~ / O--M--O IXlX)

(

/

)

R

N•oCH--N ---~C \ C

--M--O

/

0

=

(XX)

The protonation of (XIX) leads to racemization, that of (XX) to transamination. Since the two carbanionic species are in equilibrium, the two processes are competitive and the occurrence of one with respect to the other depends on pH; in acid conditions transamination is preferred when a pyridoxal-type catalyst is used (XXI, XXlI)[38]: /

R

CH--N=C

j

1 --O

(xx) R

/CH--N =C \ ' ~' N ~ ' ~ / " ' , _ H/

~

- O\

\

I--O (XVlII)

--:

~ / / ~

/c=o

F -+

O--M--O (XXl) / N ~ O H/

H -- N ~ C --~

R

\C=O

_O / (XXlt)

The electronic situation is the same for both the reactions, for instance, when the metal is Cu(ll), the rate of both reactions increases in the order[38] salicylaldehyde << 4-nitrosalicylaldehyde < pyridoxal, i.e. they are facilitated by electron-withdrawing groups in positions ortho or para to the azomethine group. In the case of a particular aldehyde, the reactions also depend on the properties of the complex formed. i.e. on the amino acid, on the number of the members of the chelated rings and on the metal. Since Pfeiffer's time, it has been recognized[39] that the racemization of an amino acid ester can be a very easy reaction, all that is necessary being to form the copper complex of its Schiff base with salicylaldehyde: in other words, (XXIII) racemizes readily and the c~-hydrogen atom of the amino acid ester exchanges readily with deuterium in heavy water, even in the absence of bases (both reactions are very slow in the absence of a metal ion)[39]. The compound (XXIVt is stable in neutral conditions but it exchanges its ~-hydrogen atom and racemizes in the presence of bases[38, 40]; finally, complexes (XXV), derived from a ,&amino acid, do not undergo any of these reactions under any conditions[38], which means that the C H group is active only when adjacent to two electronegative groups. The influence of the structure of the amino acid has been the subject of a recent report on transaminations catalyzed by the system zinc pyridoxal-5-phosphate [411. It appears that the reactivity order is ala > pala > gly > ser > isoleu > threo > val, which seems to show that an electron donation to the :~-carbon atom of t.he amino acid is rate-enhancing, and that bulky sidechains act to decrease the rates. As for the structure of the complex itself, it has been reported[42, 431 that these reactions are facilitated by aldehydes and amino acids of a kind that can form two five-membered rings, chelated to the metal ion; this is, presumably, because in such a case the Schiff base is more planar.

2138

ALESSANDROPASINIand LUIGICASELLA

An interesting paper has appeared very recently[44] on the possibility of making these reactions stereospecific, provided the carbonyl group of the Schiff base belongs to a chiral molecule, such as the oxime form of (S)-2'-nitro-2-hydroxy-5-nitroso-6,6'-dimethylbiphenyl (XXVI), which, in the presence of Cu(II), activates the racemization of L-alanine to a larger extent than that of D-alanine[44].

obtaining serine and a mixture of threonine and allothreonine respectively (XXIX - , XXX).

/NH2--CH2 + RCHO pH 12)

2/c~

(XXIX) O

C=O

NH 2-CH-CHOH-R 2/Cu \

O

OH (xxw) 3. ALDOL CONDENSATIONS In the case of threonine, together with the reactions described above, a decomposition[45] to acetaldehyde and glycine in the presence of pyridoxal and AI s÷ has been reported. The reverse reaction is.also possible. For instance the copper complex of the Schiff base between pyruvic acid and glycine undergoes a condensation with aldehydes in weakly basic conditions giving fl-hydroxyamino acids[46] according to the scheme: O C/

j

JJ

N--CH 2 + RCHO /

H20

Cu

This reaction has been extended to a variety of aldehydes to obtain longer chain, fl-hydroxyamino acids [51], and the serine formation from glycine and formaldehyde has also been reported to occur in the presence of catalytic amounts of copper sulphate [52]. The synthesis of threonine was also made stereospecific, using optically active complexes of the type L-[Coen2gly]2 +, (XXXI)[53], though with a low asymmetric yield. Also, this reaction has been used to compare the absolute configuration of L-[Coen:gly]e÷ (XXXI) and L-[Coen2(+)ser] 2÷ and L-[Coen2(+ )threo] 2÷ (XXXII)[50], since the reaction does not involve any Co-amino acid bond, so that the absolute configuration about the cobalt atom of the product is the same as that of the starting complex.

JN

CH 3

%c

C=O

(XXX)

pH8),

N,

\

] /,0~ r ~0

N""H [" / i " '

O--C=O (XXWl) O

N~N ~

CH s c /

I

O

II

\ /

H20

/ Cu

N--CH-CHOH

R

\

O--C=O (XXVIII)

These condensations seem to be general and characteristic reactions of the system amino acid Schiff basemetal ion. For instance, in the pyridoxal case, serine condenses with indole to give tryptophan[35]. Also, alkyl halides have been reacted with N-salicylideneglycinato-aquo-copper(II), obtaining longer chain amino acids such as alanine from methyl iodide and phenylalanine from benzyl bromide[47]. More simple aldol condensations have been described previously by a number of authors, mainly Japanese [48-50], who reacted glycine (not its Schiff base) coordinated to copper and cobalt with formaldehyde and acetaldehyde, in aqueous solution at a pH about 12,

HCHO

Ha+ (xxxl)

,-

N".,(N,.,,"O~c~ 0 NH./cH- CH20H (xxxll) In the case of dipeptides only the C-terminal residue is activated[54]; thus in the reaction between [Codienglygly] + and acetaldehyde at pH ca. 11, [Codienglythreo] ÷ is obtained selectively. The real mechanism of this reaction is still a subject

Reactivity of coordinated amino acids for speculation. The condensation between aldehydes and amino acids, in the absence of metal ions, with the acid and the amino group protected, has been known to occur, albeit slowly, since the beginning of this century[55]. Presumably the coordination not only protects the amino group, but also activates, through bond polarization, the methylene group that, in basic conditions, most easily forms a carbanion. Such an activation could be therefore related to the charge of the complex: in fact, for the condensation between metal-coordinated glycine and acetaldehyde, the following order of reactivity has been reported [567; ECoen2gly]-'+ > Cogly.~ > [Cogly2C204] . A similar trend has been confirmed in our laboratory [57! for the reaction with formaldehyde. The metal ions seem to play an important role; for instance, we have found this order of reactivity for the condensation of formaldehyde with the following neutral complexes [571: Cugly, > Cogly 3 > Nigly 2 > Zngly2. Quite recently a French group was able to isolate a compound from the reaction of glycine, excess of acetaldehyde and copper(ll) basic carbonate, and its structure has been found through X-ray analysis to be that of an oxazolidine (XXXIII)[58], a cyclic hemiacetal formed by a molecule of threonine and one of acetaldehyde. However, acidification leads to the threonine complex, which is the most stable, and following treatment with hydrogen sulphide only threonine can be recovered. Also, from the condensation of formaldehyde with Cu-L-ser2, compound (XXXIV) has been obtained[59]. // 2 Cu

O--C=O

/ 2/Cu

NH--CH \ /

CH CH.~

CH3--CH--O (XXXIII)

O--C=O

NH--CH '-,\ CH 2 / CH 2 0 (XXXIV)

These results seem to show that the first step in the reaction is the attack of a molecule of aldehyde to the coordinated nitrogen. This could also be confirmed by the isolation of a copper complex of N-ethylidenethreonine[60]. This class of reactions has not yet been comprehensively studied, but could well be of great interest in preparative chemistry.

4. P R O T O N E X C H A N G E A N D R A C E M I Z A T I O N

AT THE =-CAIRBON ATOM

These reactions are strictly related to those described above because they involve activation of the or-carbon atom. Such an activation has been already discussed

2130

in Section 2 for the Schiff base complexes; however, we have seen in Section 3 that the formation of a Schifl" base is probably not strictly important for such an activation to occur. Here we will be concerned with reactions of isotope exchange and racemization on the or-carbon atom of coordinated amino acids. Williams and Busch[61] first reported from NMR studies that the complex [Coen2gly] 2". dissolved in DzO, not only exchanged the amino protons, as expected, but also those of the methylene group of the glycine when N a O D was added to the solution: i.e. the methylene protons exchange with deuterium in basic conditions. No exchange can be detected for the methylene protons of ethylenediamine. Similar observations have then been made with glycine complexes of other metals such as Mo(VI)[62] and Pt(II)[631. This behaviour has not been observed in the case of free amino acids in the same conditions. For instance deuterium exchange occurs very slowly at the methylene group of N-carbobenzoxy-L-phenylalanine pentachlorophenyl ester[643, but in chloroform and in the presence of triethylamine and deuterated methanol: moreover, in this case racemization, which one ~ould expect to occur together with isotope exchange. occurs at a higher rate (kex/Kr~ ~ = 0"056t and it i, better described as an isoracemization[65L When metal complexes are considered, these reactions not only occur at a reasonable rate at room temperature, but also racemization occurs at a rate comparable to that of H-D exchangeE56,63i, as expected for a mechanism which involves the 7-carbon atom of the amino acid, probably activated by the polarization of the C H bonds due to coordination. Moreover, in some instances, the H-D exchange has been reported to possess a certain degree of stereo~selectivity; for instance in the case of Cuq.-ala-2, racemization is about four times slower than isotope exchange[65] and when D-[Coen2( +_)vail" ~ is dissolved in bases and allowed to reach equilibrium, the ratio D-[Coen2-D-vall a +/D-[Coen2-L-val] 2 + is about 1.70156! (compare this figure with the k~x/K~, ~ = 0.056 reporled above for the uncomplexed amino acid derivativel. A higher degree of stereoselectivity has been obtained for the H-D exchange reaction of A-[Coen2-1.-as p ' : for which a 77 per cent retention of contigurali~,n of the amino acid has been reported[66]. In this case the formation of the complex plays a very important role. In fact it could be that the /~carboxylate anion of the amino acid forms a hydrogen bond with an amino group of a molecule of ethylenediamine as has been shown to be the case for the similar compound A-[Coen2-L-glu ] + [671. This hydrogen bond should stabilize the diastereoisomer A-Co-L-asp {or i~s enantiomer A-Co-D-asp), thus "'locking" the configuration of the amino acid. In the case of dipeptides co-ordinated to Co(llll. the H-D exchange possesses a certain degree of selectivity: for instance [Codiengly-L-ala~ + racemizes, whilst [Codien-L-alagly]+[65] is optically unaffected in the conditions in which the methylene hydrogens exchange.

2140

ALESSANDROPASINI and LUIGI CASELLA

In the case of dipeptides, therefore, the exchange is selective for C-terminal amino acids[65, 68]. It must be said, however, that all these experiments are complicated by the fact that in the conditions where they occur (i.e. usually pH > 9) the complex itself racemizes or reacts; for example in a study of racemization of cis-f12-L-[Cotrien-L-~]121693 it has been shown [70] that racemization does not occur only on the chelated amino acid, but also on the cobalt centre, together with ligand-water exchange. In addition it must be said that the fl-alanine complex (XXXV) does not undergo hydrogen exchange on either of the two methylene groups[65] (by analogy with what is reported above in the case of (XXV)[381), nor does (XXXVI)[62] while such exchange was observed for (XXXVII)[62]; i.e. the methylene group is active when adjacent to two groups coordinated to the metal ion. CH2--NH 2 X CH 2 / \ / CH 2 Cu/2 (nCp)2Mo \ / \ C

O

For this complex two types of glycine-like rings exist: the so-called "out-of-plane" (a) and the "in-plane" (b) rings, where the plane here considered is that defined by the N ~ S o - N atoms. The two out-of-plane rings (a) are strain-free, while the in-plane ones (b) possess a certain degree of distortion from the planarity of the chelated amino acid ring[72]. Both types of methylene groups undergo H-D exchange in basic conditions, but for those of the strain-free, out-of-plane, planar rings, such a reaction has been found to be faster[61]. This system possesses another peculiarity. In the similar complex trans-[CoEDDAen] + (XXXIX), it has been found[73] that H A exchanges faster than H B, presumably because H B is protected by the ethylenediamine ring. Two different mechanisms for the H-D exchange in basic medium have been proposed: one assuming the formation of a carbanion (XL), the other through an oxazolone-type intermediate (XLI). O

NHz--CH2

II

O

X = S, NH/ (xxxvI)

(xxxv) /

X--CHR N "/

(~Cp)2Mo \

H

O--C=O X : S, N H z

(XXXVII)

H

The a-hydrogen of proline exchanges with great difficulty[62] in the molybdenum complex (XXXVII), in line with the observation that proline itself does not racemize in the conditions in which most amino acids do racemize[71]. Another interesting point, which is probably related to the mechanism, was reported by Williams and Busch[61] in their original paper, and refers to the isotope exchange in the cobalt(III) complex of ethylenediaminotetraacetic acid, (CoEDTA) (XXXVIII). O

H (a)C/C~.o (b) a %HA I~CH~

CH,~c/O lJ 0

(XXXVIII)

c¢ °

o

IXXXlXl The carbanion-type intermediate has beenproposed by Australian workers[56] on the basis of the rate law r = k[OD-][Co] for the H-D exchange of the amino acid in [Coenza] 2+ complexes. In this case the metal role should be the usual one of activating the a-carbon atom, through polarization of the C-H bonds. On the other hand Gore and GreenE62] prefer a mechanism which involves an oxazolone intermediate in the case of [(/t-Cp)zMoa] +, where the system Mo(rt-Cp)2 is able to delocalize the positive charge of the oxazolonium ion and stabilize the metal-to-nitrogen double bond. An oxazolonium type intermediate has also been proposed for the racemization of free amino acids[71]. Isotope H-D exchange of a-hydrogen atoms in amino carboxylate chelates of cobalt(III) has been reported to occur in acidic medium also[73, 74], albeit at a slower rate than in basic medium and in more drastic conditions (high temperature), in line with what is found for free amino acids[75]. Enol species like (XLIIc) have been proposed as intermediates, but the pathway is probably complicated by side reactions involving the rupture of the cobaltoxygen bond, presumably decreased in strength by

2141

Reactivity of coordinated amino acids protonation. These side reactions would explain the smaller amount of stereospecificity (about one-tenth) found in the acid-catalyzed deuteriation compared with the base-catalyzed deuteriation[73]. NH~--C--R

/ +--+M

0

NH2--C--R

\

C=O

O

C

\ O~ I

(XL)

NH:C m

R

NH--CDR

/

D+

~

M

/

O--C:O

O--C:O (XLI)

OD //

O

C

OD

/2 /

M

~

0 (

)

C

NHz--CHR (XLIIb) /

,

O

M

NH2--CHR (Xklla)

It

,+/

OD

C

/

M \\

NH 2 - C

\ R

(XLllc) 5. M I S C E L L A N E O U S R E A C T I O N S

Asperger and Liu[76] obtained ~-alanine by decarboxylation of e-amino-e-methylmalonic acid in the presence of a cobaltlIII) complex. The main interest of their work is related to the fact that when an optically active cobalt complex is used, the resulting amino acid is also optically active; in other words coordination not only activates the substrate, but it may also induce a particular stereoselective course to the reaction, especially when the coordination sphere about the metal centre is a bulky one, as when the other ligand is LL-C¢~'-dimethyltriethylenetetramine, which is known to coordinate stereospecifically to cobalt(III) giving, for the so-called[69] cis ~-isomer, only the D diastereoisomer about the cobalt (XLIII). The scheme of the reaction is as follows :

c.~

/ N

/

.N~----~--X

X

COOH

*

' OH3 NH2--C-COOH

°- °

/~

The methylaminomalonate ion is not chiral, but it becomes so when coordinated as in (XLV), its chirality being dictated by that of the cobalt ion, which for its part is chiral because of the chiral tetramine ligand. The same reaction has independently been reported by Job and Bruice[77], who showed that (XLV)is formed stereospecifically with 100 per cent chiral recognition of the prochiral carbon atom. Upon decarboxylation the corresponding complex of(St-alanine is obtained. Since the sp 3 malonate carbon becomes sp 2 during decarboxytation, one is led to conclude that the dissymmetric complex induces a chiral course to the reaction[77i. Such a recognition ofa prochiral carbon atom is well known in enzymatic reactions and presumably can be operative in this model system because of the presence of a chiral reaction centre (the cobalt ioni surrounded by a bulky dissymmetric ligand. Reactions of amino acid complexes with molecular oxygen have been also investigated. The systems Co(ll)-amino acid (or peptide) react readily with oxygen and in certain cases can act as oxygen carrier E78, 791. In any case it is known that with these ligands, in aqueous solution and in the presence of air. cobalt(l I) complexes are oxidized to Co(Ill) complexes. The oxygenation and/or oxidation of metal complexes is another area of great biological interest, but we shall not discuss it here. Many good reviews on the subject have recently been published[80]. Here it is interesting to review briefly only some reactions more strongly related to our arguments, that is oxidation reactions of coordinated amino acids. Cysteine is easily oxidized to cystine by the redox couples Mo(VI)-Mo(V)[81], Fe(Ill)Fe(lI)[82! and Cu(lbCu(l)[83]. The study of these reactions is of interest for a possible interpretation of the enzymatic formation of sulphur bridges in proteins. An important reaction is the metal-catalyzed oxidation of 3A-dihydroxyphenylalanine (Dopa), because of its possible implications in the treatment of Parkinson's disease. Dopa is easily oxidized in the presence of Fe(III) at pH ~ 5[84]. At a given pH, the rate of oxidation is first-order in Dopa. Fe(Ill) and O2, thus suggesting the formation of a 1:1:1 complex as in (XLVII), where the role of the metal is that of a bridge for electron transfer from the substrate to the oxygen molecule. If the hypothesis is correct, the substratc oxidation could be made stereospecific when optically active complexes are used. This has in fact been reported in

/I

cl.*NH2,(_____:_ /N-~O )' I / I/ \ NCH3~ ! O--C CH3 N~NH2 I

/

CH3 (XLIII)

(XLIV)

CH3

(XLV)

..o

7 --C2H:OO-

,

o/° NH2-C--H i C'H~ (XLVO

CH3

2142

ALESSANDROPASINI and LUIG1 CASELLA

0

IJ

o

\Fe / \O

CH2-CH-COOH

I

NH2

(XLVII) the case of L-Dopa in the presence of Co(III)[851, and Cu(II)[861 complexes, but the results have recently been questioned[87]. In particular, not only is the rate of oxidation of L-Dopa the same in the presence of (-)-[CoenzX2] + or of (+)-[Coen2X2] + or of the racemic complex, but presumably also the intermediate complex is always the same since the optical rotatory dispersion curves of the complex are the same in all cases[871. A last, fascinating paper is worth mention here. Gillard et al.[88, 89] have reported that certain microorganisms are able to discriminate between the glycine molecules of D-[Cogly3] vs. L-[Cogly3]. When Proteus vulgaris is fed with DL-1,2,4-[Cogly3], after a certain time only D-1,2,4-[Cogly3] can be recovered[89]. The important fact is that while the amino acid is not optically active, one of the two enantiomers of the complex is utilized preferentially by the microorganism; on complex formation the amino acid is placed in an asymmetric environment and the microorganism can exercise discriminating behaviour, as it usually does with uncomplexed racemic amino acids.

6. CONCLUSIONS

We have briefly reviewed some reactions of the complexes between metal ions and amino acids which can sometimes be related to more complicated biological systems. In many of the examples reviewed, the chemistry of the amino acid, or its derivative, has changed with respect to that of the free amino acid, and sometimes the formation of a coordination compound is the condition for a particular reaction to take place. It appears in conclusion that the principal features of a reaction which involves an amino acid complex are as follows. (1) Assembling of reactants. Two reactants coordinate to a metal in the cis position, in such a way that their active sites are very close together. The effect is similar to that of an increase of total concentration. (2) Activation. The metal ion is an electron-withdrawing agent and polarizes the bonds adjacent to the coordinating groups of the substrate, making some reactions more easy and/or stabilizing the reaction intermediates. In this context it must also be remembered that coordinated water has a lower pK, and dissociates at a lower pH, thus rendering the local concentration of coordinated hydroxyl group higher than expected at a given pH.

(3) Selectivity. This comes about either because the chemical groups which are directly involved in coordination may be protected, or because different parts of a molecule are differently activated by coordination (see for instance the specificity in peptide hydrolysis). (4) Stereoselectivity. This arises because of the different stabilities of diastereoisomeric complexes. The formation of a well-defined chiral coordination compound can lead to a particular stereochemical course of the reaction, which is defined by the chirality of the complex itself. It is expected that these effects will be large when bulky ligands are used. (5) Bridge and~or source of electrons. This is reached, in redox reactions, through the redox equilibrium between two different oxidation states of a metal or when the metal acts as a conductor, transferring electrons from one substrate to another. Moreover, in reactions with molecular oxygen, a metal ion with unpaired electrons could provide a more favourable path to the spin-forbidden oxygen-organic substrate reaction [90]. The factors above reported do not usually act separately, the role of the metal often being a manifold one. Many of the reactions described here can be taken as models of biological systems. This is fairly obvious in certain instances but less clear in others, where the reaction which takes place on the metal centre cannot be found, as such, in the living systems, as in the case of the use of Co(Ill) and dimethylformamide in the synthesis of peptides. However, we think that the problem must be seen differently. Dimethylformamidc is to be considered only as a hydrophobic system, comparable to a hydrophobic part of an enzyme, while Co(lII) is a metal centre, regardless of its occurrence in living organisms. In other words a model is something which the chemist builds up with the material at hand, trying to reproduce certain situations, but simplifying the system, that is using substances whose behaviour is easily understandable. However, it has been suggested by many authors[l, 91-931 that great care must be taken in extending the results obtained from models to biological systems; the main difference being of course the presence of a large protein portion which could alter the properties of the metal moiety. In particular, spectral data show that the geometry of the metal apoenzyme complex is highly anomalous[92]. Vallee and Williams[92] have proposed that the active site of an enzyme to which a metal ion is bound is an "entatic site" which promotes the metal ion to an "entatic state", i.e. a state of low symmetry distorted geometry, which approaches that of a transition state of a reaction[l, 931. These comments are not immateral and, presumably, this point of view will gain more importance over the old "lock-key" model, posing new interesting problems to bioinorganic chemists.

Acknowledgements--We wish to thank the Italian C.N.R. for financial help.

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