The carbanion mechanism

Instead of the reaction being initiated by C -X bond rupture, a mechanism in which C -H bond breaking occurred first would seem reasonable in the presence of a highly basic reagent. Such a mechanism, called the ElcB (or carbanion) mechanism (18) was proposed before the unimolecular process and has recently been reviewed .  

From a steady-state approximation for the carbanion concentration, expression (19) is obtained. 

Two limiting conditions can be applied, the first in which a rapid equilibrium producing the carbanion is followed by a slow unimolecular decomposition. This is the circumstance for which the mechanism El cB was originally defined, and the kinetics are first-order in each reactant, provided that if the base B is not the lyate ion of the solvent, its conjugate acid,, is present in excess , viz. 

A second condition is achieved when the carbanion decomposes to the olefin more rapidly than it protonates, viz- 

Under this circumstance the ElcB mechanism degenerates into the E2 mechanism and this part of the topic is considered more fully under discussion of the concerted processes (Section 2.2). 

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Bunnett (1991) expresses doubts that the aryldiazene can be formed at all under these strongly basic conditions, after Huang and Kosower (1968) showed that phenyldiazene is destroyed in water at pH 13.8 (25 °C) with a half-life of <10s, doubtless via C6H5-N2. In addition, Broxton s proposal (Scheme 8-53) does not provide a satisfying explanation of why the ortho halogen has such a strong effect in favor of the carbanion mechanism. 

In the El mechanism, X leaves first, and then H. In the E2 mechanism, the two groups leave at the same time. There is a third possibility the H leaves first, and then the X. This is a two-step process, called the ElcB mechanism, or the carbanion mechanism, since the intermediate is a carbanion ... 

Biochemical reactions often involve addition to C = C bonds that are not conjugated with a true carbonyl group but with die poorer electron acceptor - COO. While held on an enzyme a carboxylate group may be protonated, making it a better electron acceptor. Nevertheless, there has been some doubt as to whether the carbanion mechanism of Eq. 13-6 holds for these enzymes. Some experimental data suggested a quite different mechanism, one that has been established for the nonenzymatic hydration of alkenes. An example is the hydration of ethylene by hot water with dilute sulfuric acid as a catalyst (Eq. 13-11), an industrial method of preparation of ethanol. The electrons of the double bond form the point of attack by a proton, and the resulting carbocation readily abstracts a hydroxyl... 

Experimental support for the mechanism of Eq. 15-26 has been obtained using D-chloroalanine as a substrate for D-amino acid oxidase.252-254 Chloro-pyruvate is the expected product, but under anaerobic conditions pyruvate was formed. Kinetic data obtained with a-2H and a-3H substrates suggested a common intermediate for formation of both pyruvate and chloro-pyruvate. This intermediate could be an anion formed by loss of H+ either from alanine or from a C-4a adduct. The anion could eliminate chloride ion as indicated by the dashed arrows in the following structure. This would lead to formation of pyruvate without reduction of the flavin. Alternatively, the electrons from the carbanion could flow into the flavin (green arrows), reducing it as in Eq. 15-26. A similar mechanism has been suggested for other flavoenzymes 249/255 Objections to the carbanion mechanism are the expected... 

In the other three variations of the carbanion mechanism, an equilibrium concentration of carbanion is formed, which then either returns to starting material or decomposes to products. 

In the carbanionic mechanisms for elimination, if the substrate has two proton-bearing ft carbons, the more acidic protons will be removed. Thus in alkylated substrates the double bond will be oriented toward the less substituted carbon and Hofmann elimination is obtained. 

A similar difference in reactivity is evident in the N-oxides pyrazine N-oxide is even more reactive than pyrimidine or pyridine N-oxide. Substituents in the 3-position appear to act mainly through their inductive effects (Table 10.3). Log k2 varies linearly with p/Ca, and the carbanion mechanism probably operates (there is, however, doubt about the mode of substitution at C-6 in pyrazine N-oxides) (70JOC3467). All four hydrogens of pyrazine N,AT-dioxide are exchanged in MeOD at 65°C (68MI2). 

In NaOD-D20 (or with added DMSO) 1,2,4-triazine and its 5- or 6-methyl and 5-phenyl derivatives exchange H-3 for deuterium with an opposite reactivity order to that observed under acid catalysis (vide supra) (73T2495). The observation that the 5-phenyl derivative reacts 15 times faster than the unsubstituted parent (Table 10.4 comparison was made in D,0-DMS0 because of solubility problems) clearly indicates that the carbanion mechanism operates in basic media rather than a process involving covalent hydration. 

FIGURE 5. Proposed reaction mechanisms for flavin-catalysed oxidation of amines. A, the carbanion mechanism initially proposed for TMADH (Rohlfs and Hille, 1994). B, the amminium cation radical mechanism, as originally proposed for monoamine oxidase (Silver-man, 1995) although only the pathway passing through a transient covalent intermediate is shown, several alternative pathways for breakdown of the initial flavin semiquinone/... 

The first step in the catalytic cycle of flavocytochrome i>2 is the oxidation of L-lactate to pyruvate and the reduction of the flavin. Our understanding of how this occurs has been dominated by what can only be described as the dogma of the carbanion mechanism. Although this mechanism for flavoprotein catalysed substrate oxidations is accepted by many, doubts remain, and the alternative hydride transfer process cannot be ruled out. The carbanion mechanism has been extensively surveyed in the past, reviews by Lederer (1997 and 1991) and Ghisla and Massey (1989) are recommended, and for this reason there is little point in covering the same ground in the present article in any great detail. 

The fundamentals on which the carbanion mechanism is founded are the early studies on the related enzj mes D-amino acid oxidase (Walsh et al., 1972 and 1971), lactate oxidase (Walsh et al., 1973) and flavocytochrome bj (Urban and Lederer, 1985 Pompon and Lederer, 1985). It is interesting that all of the key work, which established the carbanion mechanism, was done before any 3-dimensional structures were available on the respective enzymes. 

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-... 

The conversion of L-lactate to pyruvate is a two-electron redox process. One could consider this occurring as two one-electron steps (a radical mechanism) or as one two-electron step. There are two options for a single two-electron step, and these are hydride transfer (H ) or proton (H+) abstraction followed by a two-electron transfer from a carbanion intermediate. These two alternatives for lactate are shown formally in Eqs. (1) and (2) for hydride transfer and the carbanion mechanism, respectively. 

In the case of flavocytochrome 62 and related flavoenzymes, there is a sufficient body of evidence to indicate that the carbanion mechanism operates. The formation of a carbanion is not, of course, an oxidation and two electrons need to be transferred from the carbanion intermediate to the flavin cofactor. This could occur possibly via a covalent intermediate or by sequential one-electron transfers. These possibilities will be discussed in detail later in this section. 

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. 

There is, however, another reasonable mechanism that we must consider the carbanion mechanism, which has as its first step the abstraction of a hydrogen... 

It has been pointed out by Joseph Bunnett (of the University of California, Santa Cruz) that evidence against the carbanion mechanism is available in the element effect. 

Manchester) in 1904, showed for the first time how kinetics could be used to reveal the mechanism of an organic reaction. The carbanion mechanism has since been confirmed not only by the iodination work, but also by studies of stereochemistry and isotopic exchange. 

Problem 21.5 Show in detail exactly how each of the following facts provides evidence for the carbanion mechanism of base-promoted halogenation of ketones. 

Problem 21.7 Suppose, as an alternative to the carbanion mechanism, that hydrogen exchange and racemization were both to arise by some kind of direct displacement of one hydrogen (H) by another (D) with inversion of configuration. What relationship would you then expect between the rates of racemization and exchange (Hint Take one molecule at a time, and see what happens when H is replaced by D with inversion.)... 

This reaction type is called the ElcB mechanism, which stands for unimolecular elimination conjugate base reaction, because the conjugate base of the starting material is being formed as the reactive intermediate. It is sometimes called the carbanion mechanism. As this mechanism results from the removal of a proton, it is not surprising that it is favoured by those substrates that possess an acidic hydrogen atom. Thus, would you expect the ElcB mechanism to be more prevalent in reactions that result in a carbon/carbon double bond or in reactions that result in a carbon/carbon triple bond ... 

R.E Pratt and TC. Bruice, The Carbanion Mechanism (El b) of Ester Hydrolysis. III. Some Structure-Reactivity Studies and the Ketene Intermediate, J. Am. Chem. Soc., 1970, 92, 5956. 

Actually, secondary isotope effects are more likely to give useful information on transition state structures than primary ones, except where coupled hydrogen motions distort the secondary isotope effects. Secondary isotope effects were quite useful in determining the carbanion mechanism for ftimarase (709). The interested reader is referred to a recent review which gives a number of other examples (96). 

The decarboxylation affords dibromoacetic acid, but in the presence of bromine the end-product is tribromoacetic acid since bromine has no effect on the rate of reaction, and since dibromoacetic acid is not brominated under the reaction conditions, the carbanion mechanism is conclusively proved for this case. 

Although the carbanion mechanism offers an attractive alternative to the bimolecular process, it has proved extremely elusive and has been demonstrated... 

When the carbanion is formed in a rapid pre-equilibrium, isotopic exchange between the substrate and the solvent should be a faster process than formation of the olefin. The elimination reaction of 2-(p-nitrophenyl)ethyltrimethyl-ammonium ion in aqueous solution is accelerated by hydroxide ion but retarded by acid. This observation led to the postulation of the carbanion mechanism acid depressing the rate by reversing the equilibrium step (22). 

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