Peptide hydrolysis cobalt complexes

Owing to the snccess of cobalt(III)-mediated hydrolysis of amino acid esters, the next step was to examine how these complexes reacted with peptides. If similar hydrolytic results could be obtained with peptides, then one of the potential uses of cobalt(III) complexes would be in the N-terminal determination and seqnential analysis of polypeptides. This area has been investigated by several gronps. Peptides... 

The rate of hydrolysis of peptides by cobalt(III) complexes is 10" times faster than hydrolysis with no metal present. " Unlike the hydrolysis of amino acid esters, where the rate of hydrolysis is dependent on how the ester is bonded to the cobalt(III) complex, peptides are hydrolyzed equally if they are bound to the cobalt(III) complex in a monodentate fashion (throngh the carbonyl oxygen) or in a bidentate fashion (throngh the amino nitrogen and carbonyl oxygen). 

The active site of methionine aminopeptidase contains a binuclear cobalt complex that is required for activity, although a number of divalent metal ions support turnover to varying degrees. X-ray crystallographic studies on the enzyme in complexes with transition state analogs suggests that the binuclear metal cluster serves to stabilize the tetrahedral intermediate in peptide hydrolysis. ... 

Even though this dipeptide is turned over quite slowly, the complex examined is probably a non-productive one. Furthermore an analogous ester substrate has not been found, and it is known that carboxypeptidase behaves quite differently toward ester and peptide substrates. In particular, the kinetic parameters for peptide hydrolysis for a series of metal substituted carboxypeptidases indicate that fccat values can range from 6000 min for the cobalt enzyme down to 43 min for the cadmium enzyme 66). The values on the other hand are almost totally independent of the particular metal present. The exact opposite is true for ester hydrolysis. Km varies from 3300 M for the cobalt enzyme to 120 M for the cadmium enzyme while k<.at is essentially unchanged. 

Co(in) complexes promote similar reactions. When four of the six octahedral positions are occupied by amine ligands and two cis positions are available for further reactions, it is possible to study not only the hydrolysis itself, but the steric preferences of the complexes. In general, these compounds catalyze the hydrolysis of N-terminal amino acids from peptides, and the amino acid that is removed remains as part of the complex. The reactions apparently proceed by coordination of the free amine to cobalt, followed either by coordination of the carbonyl to cobalt and subsequent reaction with OH or H2O from the solution (path A in Figure 12-15) or reaction of the carbonyl carbon with coordinated hydroxide (path B). As a result, the N-terminal amino acid is removed from the peptide and left as part of the cobalt complex in which the a-amino nitrogen and the carbonyl oxygen are bonded to the cobalt. Esters and amides are also hydrolyzed by the same mechanism, with the relative importance of the two pathways dependent on the specific compoimds used. 

The cobalt(III)-promoted hydrolysis of amino acid esters and peptides and the application of cobalt(III) complexes to the synthesis of small peptides has been reviewed. The ability of a metal ion to cooperate with various inter- and intramolecular acids and bases and promote amide hydrolysis has been investigated. The cobalt complexes (5-10) were prepared as potential substrates for amide hydrolysis. Phenolic and carboxylic functional groups were placed within the vicinity of cobalt(III) chelated amides, to provide models for zinc-containing peptidases such as carboxypeplidase A. The incorporation of a phenol group as in (5) and (6) enhanced the rate of base hydrolysis of the amide function by a factor of 10 -fold above that due to the metal alone. Intramolecular catalysis by the carboxyl group in the complexes (5) and (8) was not observed. The results are interpreted in terms of a bifunctional mechanism for tetrahedral intermediate breakdown by phenol. 

Coordinated a-amino amides can be formed by the nucleophilic addition of amines to coordinated a-amino esters (see Chapter 7.4). This reaction forms the basis of attempts to use suitable metal coordination to promote peptide synthesis. Again, studies have been carried out using coordination of several metals and an interesting early example is amide formation on an amino acid imine complex of magnesium (equation 75).355 However, cobalt(III) complexes, because of their high kinetic stability, have received most serious investigation. These studies have been closely associated with those previously described for the hydrolysis of esters, amides and peptides. Whereas hydrolysis is observed when reactions are carried out in water, reactions in dimethyl-formamide or dimethyl sulfoxide result in peptide bond formation. These comparative results are illustrated in Scheme 91.356-358 The key intermediate (126) has also been reacted with dipeptide... 

The copper(II)-promoted hydrolysis of glycylglycine has been studied in some detail.120 Copper(II) ions catalyze the hydrolysis of glycylglycine in the pH range 3.5 to 6 at 85 °C.120 The pH rate profile has a maximum at pH 4.2, consistent with the view that the catalytically active species in the reaction is the carbonyl-bonded complex. The decrease in rate at higher pH is associated with the formation of a catalytically inactive complex produced by ionization of the peptide hydrogen atom. This view has subsequently been confirmed by other workers,121 in conjunction with an IR investigation of the structures of the copper(II) and zinc(II) complexes in D20 solution.122 Catalysis by cobalt(II),123 and zinc(II), nickel(II) and manganese(II) has also been studied.124-126... 

The above studies indicate that metal ions catalyze the hydrolysis of amides and peptides at pH values where the carbonyl-bonded species (25) is present. At higher pH values where deprotonated complexes (26) can be formed the hydrolysis is inhibited. These conclusions have been amply confirmed in subsequent studies involving inert cobalt(III) complexes (Section 61.4.2.2.2). Zinc(II)-promoted amide ionization is uncommon, and the first example of such a reaction was only reported in 1981.103 Zinc(II) does not inhibit the hydrolysis of glycylglycine at high pH, and amide deprotonation does not appear to occur at quite high pH values. Presumably this is one important reason for the widespread occurrence of zinc(Il) in metallopeptidases. Other metal ions such as copper(II) would induce amide deprotonation at relatively low pH values leading to catalytically inactive complexes. 

The use of kinetically inert cobalt(III) complexes has led to important developments in our understanding of the metal ion-promoted hydrolysis of esters, amides and peptides. These complexes have been particularly useful in helping to define the mechanistic pathways available in reactions of this type. Work in this area has been the subject of a number of reviews.21-24 Although most of the initial work was connected with cobalt(III), investigations are now being extended to other kinetically inert metal centres such as Rhin, lrni and Ru111. 

A variety of N-O-chelated glycine amide and peptide complexes of the type [CoN4(GlyNR R2)]3+ have been prepared and their rates of base hydrolysis studied.169 The kinetics are consistent with Scheme 8. Attack of solvent hydroxide occurs at the carbonyl carbon of the chelated amide or peptide. Amide deprotonation gives an unreactive complex. Rate constants kOH are summarized in Table 16. Direct activation of the carbonyl group by cobalt(III) leads to rate accelerations of ca. 104-106-fold. More recent investigations160-161 have dealt with... 

Table 16 Rate Constants for the Base Hydrolysis of Ester, Amide and Peptide Bonds in Various Cobalt(III) Complexes (25 °C, / = 1.0 M)a...

Two mechanisms of cobalt(III)-mediated peptide-bond cleavage have been investigated. The first one involves hydrolysis of a directly activated amino acid ester, or peptide (equation 4). The other mechanism involves the intramolecular attack of an amino acid ester or peptide by a cis coordinated hydroxide or water molecule (equation 5). In both cases, the cobalt(III) complex must have two open coordination sites cis to each other. For the directly activated mechanism, these sites are needed to bind the amino acid ester or peptide. The intramolecular reaction requires one site for coordination of the ester or peptide, and one site for the coordination of the hydroxy or water molecnle. One of the initial cobalt(III) complexes to be investigated was... 

The peptide bond that is cleaved is the bond between Leu-189 and Asp-190. There are two peptide bonds in close proximity to the iron chelate on Cys-212. The other peptide bond is between He-144 and Gly-145. The Cys-212 sulfur is 5.1 A from the carbonyl carbon of Gly-145, and 5.3 A from the carbonyl carbon of Leu-189. However, the main difference is that the peptide bond of Leu-189-Asp-190 is oriented parallel to Cys-212, while the peptide bond of lie-144-Gly-145 is oriented away for Cys-212. As was seen with cobalt(lll) hydrolysis of peptide bonds, the proximity and orientation of the carbonyl carbon is important for hydrolysis. This approach has been extended to the cleavage of multisubunit proteins. Palladium(n) and platinum(ll) complexes as synthetic peptidases have been reviewed elsewhere. ... 

The use of kinetically inert cobalt(lll) complexes has led to very significant developments in our understanding of metal-ion-promoted hydrolysis of esters amides and peptides. Extensive reviews on the topic are available [1,2,24-26]. 

Table 7.6 Rate constants for the base hydrolysis of ester, amide and peptide bonds in various cobalt(III) complexes (25°C, 1 = 1.0 M)...

With the complex ion catalyst cis-)5-hydroxoaquatriethylenetetramine-cobalt(III), abbreviated [Co(trien)(H20)OH], peptide bond hydrolysis of the dipeptide L-aspartylglycine takes place, but only in the productive binding mode. 

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