Racemization proton abstraction

Mandelate racemase, another pertinent example, catalyzes the kinetically and thermodynamically unfavorable a-carbon proton abstraction. Bearne and Wolfenden measured deuterium incorporation rates into the a-posi-tion of mandelate and the rate of (i )-mandelate racemi-zation upon incubation at elevated temperatures. From an Arrhenius plot, they obtained a for racemization and deuterium exchange rate was estimated to be around 35 kcal/mol at 25°C under neutral conditions. The magnitude of the latter indicated mandelate racemase achieves the remarkable rate enhancement of 1.7 X 10, and a level of transition state affinity (K x = 2 X 10 M). These investigators also estimated the effective concentrations of the catalytic side chains in the native protein for Lys-166, the effective concentration was 622 M for His-297, they obtained a value 3 X 10 M and for Glu-317, the value was 3 X 10 M. The authors state that their observations are consistent with the idea that general acid-general base catalysis is efficient mode of catalysis when enzyme s structure is optimally complementary with their substrates in the transition-state. See Reference Reaction Catalytic Enhancement... 

There are numerous studies of control of racemization for specific subsets of amino acids. For example, Benoiton has carried out extensive studies on racemization of Al-methyl amino acids. In particular, McDermott and Benoiton1261 demonstrated that the presence of tertiary amine salts in coupling reactions had a profound effect on activated TV-methyl amino acids in contrast to the nonmethylated form. In one example, when Z-Ala-MeLeu-OH was coupled to the tosylate salt of Gly-OBzl with ethyl 2-ethoxy-l,2-dihydroquinoline-l-carboxylate (EEDQ) in the presence of TEA, 15% of the l-d dipeptide was formed with a yield of 68%, compared to 0.5% for Z-Ala-Leu-OH with a yield of 78%. When the free base of Gly-OBzl was utilized in a DCC/HOSu coupling in the absence of tertiary amine, no l-d products were detected. The authors attributed this increased susceptibility to epimerization to an ox-azolonium intermediate, which can epimerize by proton abstraction or merely by tauto-merization (Scheme 11). 

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... 

Many base catalysed isotope exchange or racemization processes involve either rate-determining proton abstraction or rapid preequilibrium formation of the anion (19) followed by ratedetermining reaction with solvent (20) (L is an isotope of hydrogen). 

Reaction of an enantiomeric hydrosilane with KH yields a racemic silyl anion (77). Thus, treatment of (-l-)-(l-Np)PhMeSiH with KH at 50°C in DME for 24 hours and subsequent addition of n-BuBr give racemic ( )-(l-Np)PhMeSi-n-Bu, via the formation of the corresponding silyl anion with loss of optical activity. Hydrolysis or deuterolysis also gives a racemic product. These observations clearly rule out a mechanism in which the silyl anion is formed by proton abstraction from the hydrosilane, because retention of configuration would be expected. A possible mechanism involves a pentacoordinate dihydrosilyl anion formed via coordination of H in an initial fast, reversible process, and its decomposition to the racemic silyl anion with loss of molecular hydrogen [Eq. (7)]. A gas-... 

Independent of the thiol protecting groups used in the synthesis of cysteine peptides, the facile racemization of the Cys residue has been recognized as a serious problem since the early days of peptide chemistry (see Section 1.2.1.2 and Vol. E22b, Section 7.4).It is well established that N,S-protected cysteine active esters are unusually prone to racemization via a C -proton-abstraction mechanism in the presence of excess amines (Scheme More... 

Though less attention has been paid to the intramolecular proton abstraction from the intermediate O-acylisoureas, it represents an obvious alternative racemization pathway (Scheme 4). The resulting isouronium-enolate betaine might be further stabilized by formation of the corresponding enol.P l... 

The proton-abstraction mechanism is the pathway only in very special cases such as the rapid racemization of derivatives of phenylglycine (Scheme 3). Racemization through the proton-abstraction mechanism can be prevented by employing suitable reaction conditions, in particular by controlling the use of tertiary amine.f " ... 

Histidine and cysteine are both very susceptible to racemization during routine SPPS. If not optimized, microwave SPPS has been shown capable of causing substantial racemization during the coupling of both histidine and cysteine [36]. Cysteine racemization has been attributed to a-carbon proton abstraction by... 

Intramolecular proton migration results in isomerization with mutarotation, whereas formation and protonation of a symmetrically solvated carbanion results in simultaneous racemization and isomerization. In all cases studied, the kinetic data support the assumption that proton abstraction by the basic catalyst is rate-limiting. 

The reaction of dimethyl sulfoxide, oxalyl chloride and an alcohol is normally carried out at -78 or —60 °C, since the formation of the alkoxysulfonium salt 29 is rapid at this low temperature. After addition of the base triethylamine, the mixture may be warmed to —30 °C or higher to promote proton abstraction and fragmentation. The use of diisopropylethylamine instead of triethylamine as the base or addition of pH 7 phosphate buffer can, in the rare cases when it does occur, reduce the extent of enoUzation and therefore minimize any racemization or rearrangement of p,7-double bonds. 

C. Pepper, Racemization of drug enantiomers by benzylic proton abstraction at physiological pH, Chirality, 5(1994)372. 

Pyridoxal 5 -phosphate is also a coenzyme for the enzyme-catalyzed racemization of amino acids. The key reaction is proton abstraction from the a carbon of the amino acid imine of PLP. This step converts the a carbon, which is a chirality center, from sp to sp. ... 

In the most commonly detected pathway of racemization azlactones (5(4if)-oxazolones, 5-oxazolinones) are implicated. Proton abstraction from these cyclic intermediates results in a resonance stabilized carbanion... 

The presence of a second nucleophile in the reaction mixture reduces the concentration of the O-acylisourea and thereby the extent of racemization. Also, HOBt, a weak acid, prevents proton abstraction from the chiral carbon atom and thus contributes to the conservation of chiral purity in a second manner as well. Last, but not least the availability of the auxiliary nucleophile (HOBt) efficiently shortens the lifetime of the overactivated O-acyl-isourea intermediate and thus diminishes the extent of O N acyl-migration leading to... 

The general approach for carboxyl protection is esterification. The simplest solution, the use of methyl or ethyl esters, is suitable for semipermanent blocking, although the commonly applied process of unmasking, alkaline hydrolysis, is far from unequivocal. It is accompanied by racemization, partial hydrolysis of carboxamide groups in the side chain of asparagine and glutamine residues and by several other side reactions which are initiated by proton abstraction (Cf. Chapter VII). Nevertheless, perhaps because of the attractively simple esterification of amino acids... 

This kind of simple proton abstraction is, however, not the sole and not even the most common mechanism of racemization. The most frequently invoked pathway involves cyclic intermediates, 4,5-dihydro-oxazole-5-ones or azlac-tones ... 

The mechanisms described in the preceding paragraphs are the ones generally proposed for the explanation of racemization, but it is far from certain that other pathways are not involved. For instance it seems to be possible that the repeatedly observed loss of chiral integrity of the activated residue in coupling of peptides with the aid of dicyclohexylcarbodiimide is due to intramolecular proton abstraction by the basic center in the reactive O-acylisourea intermediate ... 

Various side chains affect the extent of racemization in different ways. Thus, the benzyl side chain in phenylalanine contributes to the stabilization of a carbanion and can thereby facilitate proton abstraction from the a-carbon atom. This effect is much more pronounced in phenylglycine (which is not a protein constituent but occurs in microbial peptides) because its chiral carbon atom is benzylic ... 

The aliphatic side chains in alanine and leucine have no major influence but branching at the ) -carbon atom in valine and isoleucine can enhance racemization because the combination of electron release and steric hindrance results in reduced coupling rates. The ensuing increase in the life-time of the reactive intermediate provides an extended opportunity for proton abstraction by base. It is obvious from these examples that the effect of individual side chains, the influence of various methods of coupling and the conditions of the peptide bond forming reaction (solvents, concentration, temperature, additives) must be studied in well designed experiments. Several model systems have been proposed for this purpose. 

It is not surprising that a process that involves proton abstraction is influenced by the polarity of the solvent. Base catalyzed racemization of active esters is fast in polar solvents such as dimethylformamide and slow in non-polar media, for instance in toluene. It is rather unfortunate that such non-polar solvents are more often than not impractical in peptide synthesis. The poor solubility of most blocked intermediates in the commonly used organic solvents severely limits their use and in the preparation of larger peptides indeed dimethylformamide is most frequently applied. The problem of solubility is less serious in solid phase peptide synthesis (cf. Chapter X), where no real solvent is needed but merely a medium in which the polymeric support properly swells. This function is fulfilled by dichloromethane its effect on racemization lies between the extremes mentioned. 

Comparing the activation mode of iminium and enamine catalysis, iminium catalysis is based on a LUMO-activation mode of the electrophile whereas enamine catalysis is based on a HOMO-activation of the nucleophile. Keeping in mind the fact that enamine and iminium species are rapidly interconverted via a two-electron redox process (proton abstraction of an iminium species results in an enamine), MacMillan and co-workers reasoned that it should be possible to interrupt this equilibrium chemically by carrying out just a one-electron oxidation of an enamine. This would then generate a three-7i-electron radical cation with a singly occupied molecular orbital (SOMO) that should be activated towards catalytic transfomiatirHis (racemic or asymmetric) not possible using classical enamine or iminium activation (Scheme 80) 316). 

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