Substrate carbanions, intermediate

Prototropic interconversions have been the subject of much detailed study, as they lend themselves particularly well to investigation by deuterium labelling, both in solvent and substrate, and by charting the stereochemical fate of optically active substrates having a chiral centre at the site of proton departure. Possible limiting mechanisms (cf. SNl/SN2) are those (a) in which proton removal and proton acceptance (from the solvent) are separate operations, and a carbanion intermediate is involved, i.e. an intermolecular pathway and (b) in which one and the same proton is transferred intramolecularly ... 

FIGURE 14-26 Carbanion intermediates stabilized by covalent interactions with transketolase and transaldolase, (a) The ring of TPP stabilizes the two-carbon carbanion carried by transketolase see Fig. 14-13 for the chemistry of TPP action, (b) In the transaldolase reaction, the protonated Schiff base formed between the e-amino group of a Lys side chain and the substrate stabilizes a three-carbon carbanion. 

An example of an a-ketol formation that does not involve decarboxylation is provided by the reaction catalyzed by transketolase, an enzyme that plays an essential role in the pentose phosphate pathway and in photosynthesis (equation 21) (B-77MI11001). The mechanism of the reaction of equation (21) is similar to that of acetolactate synthesis (equation 20). The addition of (39) to the carbonyl group of (44) is followed by aldol cleavage to give a TPP-stabilized carbanion (analogous to (41)). The condensation of this carbanionic intermediate with the second substrate, followed by the elimination of (39), accounts for the observed products (B-7IMIHOO1). 

Figure 14-5 Some reactions of Schiff bases of pyridoxal phosphate, (a) Formation of the quinonoid intermediate, (b) elimination of a (3 substituent, and (c) transamination. The quinonoid-carbanionic intermediate can react in four ways (1—4) if enzyme specificity and substrate structure allow.

When the substrate is substituted at the /3 carbon with a potential leaving group, such as —OH, —SH, —0P033-(see fig. 10.3d), the corresponding a-carbanion intermediate (see fig. 10.4d) can eliminate the group. This is an essential step in a,/3 eliminations. Upon hydrolysis, the elimination intermediate produces pyridoxal-5 -phosphate and the substrate-derived enamine, which spontaneously hydrolyzes to ammonia and an a-keto acid. 

Cyclopropanations are known for several other carbanionic intermediates of the general type (7), in which the substituent G is ultimately lost as an anionic leaving group in the last step of the ring-forming pathway (see Scheme 3 above). The substituent G is most often a functional group based upon sulfur, selenium or nitrogen. Halide-substituted derivatives probably react via the a-elimination pathway in most cases (see Section 4.6.3.1), but in some reactions with electron deficient alkenes as substrates, the normal order of steps may be altered (e.g. Table 10, ref. 162). 

The alanine racemization catalyzed by alanine racemase is considered to be initiated by the transaldimination (Fig. 8.5).26) In this step, PLP bound to the active-site lysine residue forms the external Schiff base with a substrate alanine (Fig. 8.5, 1). The following a-proton abstraction produces the resonance-stabilized carbanion intermediates (Fig. 8.5, 2). If the reprotonation occurs on the opposite face of the substrate-PLP complex on which the proton-abstraction proceeds, the antipodal aldimine is formed (Fig. 8.5,3). The subsequent hydrolysis of the aldimine complex gives the isomerized alanine and PLP-form racemase. The random return of hydrogen to the carbanion intermediate is the distinguishing feature that differentiates racemization from reactions catalyzed by other pyridoxal enzymes such as transaminases. Transaminases catalyze the transfer of amino group between amino acid and keto acid, and the reaction is initiated by the transaldimination, followed by the a-proton abstraction from the substrate-PLP aldimine to form a resonance-stabilized carbanion. This step is common to racemases and transaminases. However, in the transamination the abstracted proton is then tranferred to C4 carbon of PLP in a highly stereospecific manner The re-protonation occurs on the same face of the PLP-substrate aldimine on which the a-proton is abstracted. With only a few exceptions,27,28) each step of pyridoxal enzymes-catalyzed reaction proceeds on only one side of the planar PLP-substrate complex. However, in the amino acid racemase... 

The implication of carbanion intermediates in the mechanisms of flavoenzyme oxidations of substrates of the second class has been dependent upon the determination of the competition between /3-halide release and oxidation (e.g., Equation 11) (4, 5, 6, 7). In model studies,... 

FIGURE 13. Species identified in the reaction cycle of copper amine oxidases. Oxidised, resting state enzyme (1) reacts with substrate to form a substrate Schiff base (2). Proton abstraction by the active site base (Asp383 in ECAO) leads, via a carbanion intermediate (3) to the product Schiff base (4). Hydrolysis releases the product aldehyde, leaving reduced cofactor in equilibrium between aminoquinol/Cu (S) and semiquinone/Cu (6). The reduced cofactor is reoxidised by molecular oxygen, releasing ammonium ions and hydrogen peroxide. (Modified from Wilmot et al., 1999 with permission). 

The core requirement for the carbanion mechanism to operate is that an active-site base must abstract the a-carbon hydrogen of the substrate, as a proton, forming a carbanion intermediate (Lederer, 1991). This would then require the equivalent of two electrons to be transferred to the flavin either with or without the formation of a covalent intermediate between the a-carbon and the flavin N-5 (Ghisla and Massey, 1989). With this in mind, it is intriguing to find that the crystal structure of D-amino acid oxidase reveals that there is no residue correctly located to act as the active-site base required for the carbanion mechanism (Mattevi et al., 1996 Mizu-tani et al., 1996). In fact, the crystallographic information available is far more consistent with this enzyme operating a hydride transfer mechanism (Mattevi et al., 1996). If this is correct then the earlier experiments on d-amino acid oxidase, which were claimed to be diagnostic of a carbanion mechanism, are ealled into question. It is important to note that similar experiments were used to provide support for a carbanion mechanism in the ease of flavocytochrome b2-... 

Studies of nitroalkane oxidation by n-amino acid oxidase (55) and glucose oxidase 49, 56) have provided strong evidence both for intermediate substrate carbanions and for subsequent covalent adduct formation between these and the N position of the flavin nucleus. The rationale for using nitroalkanes can be seen in the following reaction stoichiometries for D-amino acid oxidase (55) ... 

Two new lines of evidence suggest that proton abstraction comes first. A careful study of both and H isotope effects supports the carbanion intermediate, as does the strong inhibition by anions of 3-nitropropionate and 3-nitro-2-hydroxypropionate. To provide a good electron sink the carboxylate group adjacent to the proton that is removed by fumarate hydratase must either be actually protonated in the enzyme-substrate (ES) complex or paired with and hydrogen bonded to a positively charged group. 

To understand the carbanion mechanism in flavocytochrome 62 it is useful to first consider work carried out on related flavoenzymes. An investigation into o-amino acid oxidase by Walsh et al. 107), revealed that pyruvate was produced as a by-product of the oxidation of )8-chloroalanine to chloropyruvate. This observation was interpreted as evidence for a mechanism in which the initial step was C -H abstraction to form a carbanion intermediate. This intermediate would then be oxidized to form chloropyruvate or would undergo halogen elimination to form an enamine with subsequent ketonization to yield pyruvate. The analogous reaction of lactate oxidase with jS-chlorolactate gave similar results 108) and it was proposed that these flavoenzymes worked by a common mechanism. Further evidence consistent with these proposals was obtained by inactivation studies of flavin oxidases with acetylenic substrates, wherein the carbanion intermediate can lead to an allenic carbanion, which can then form a stable covalent adduct with the flavin group 109). Finally, it was noted that preformed nitroalkane carbanions, such as ethane nitronate, acted as substrates of D-amino acid oxidase 110). Thus three lines of experimental evidence were consistent with a carbanion mechanism in flavoenzymes such as D-amino acid oxidase. 

Fig. 10. Proposed mechanism for halosubstrate oxidation (route 1) or elimination (route 2) via a carbanion intermediate. The formation of the enzyme-substrate complex is followed by abstraction of the hydrogen at C-2 by the active site base. The carbanion intermediate can then undergo oxidation to form halopyruvate via route 1, or can eliminate halide to form pyruvate via route 2. E, Enzyme S, substrate FI, flavin B, active site base.

The mechanism of the Krapcho dealkoxycarbonylation is dependent on the structure of the substrate ester and the type of anion used. In the case of a,a-disubstituted diesters (especially the methyl esters), the anion from the salt (cyanide ion in the scheme) attacks the alkyl group of the ester in an Sn2 fashion and the decarboxylation results in the formation of a carbanionic intermediate that is quenched by the water. In the case of a-monosubstituted diesters the cyanide attacks the carbonyl group to form a tetrahedral intermediate, which breaks down to give the same carbanionic intermediate and a cyanoformate, which is hydrolyzed to give carbon dioxide and an alcohol. 

Figure 17-9. Mechanism of glutamate racemase reaction. Cys 70 and Cysl78 serve as the bases to abstract an a-proton from the substrate, and a carbanion intermediate is formed. Alternatively, the racemization may proceed through a concerted mechanism. Reprinted from Hwang et al. 951.

Whereas sodium indenide displays a stereoselectivity similar to phenylzinc chloride (see Table 26), sodium cyclopentadienide and 3-trimethylsilylindene act like soft carbanions. Both substrates 10 and 12 undergo allylic substitution with net retention of configuration to 1127 and 1328, respectively, via attack on the ally ligands of the intermediate 7t-allylpalladium complexes anti to the metal. Again, the ratio of the two possible diastereomers of structure 13 is unknown. 

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