Amino acid nucleophilic

Pharmacologically active allenic steroids have already been examined intensively for about 30 years [5], Thus, the only naturally occurring allenic steroid 107 had been synthesized 3 years before its isolation from Callyspongia diffusa and it had been identified as an inhibitor of the sterol biosynthesis of the silkworm Bombyx mori (Scheme 18.34) [86d], At this early stage, allenic 3-oxo-5,10-secosteroids of type 108 were also used for the irreversible inhibition of ketosteroid isomerases in bacteria, assuming that their activity is probably caused by Michael addition of a nucleophilic amino acid side chain of the enzyme at the 5-position of the steroid [103, 104]. Since this activity is also observed in the corresponding /3,y-acetylenic ketones, it can be rationalized that the latter are converted in vivo into the allenic steroids 108 by enzymatic isomerization [104, 105],... 

A similar bait and switch approach has been exploited for acyl-transfer reactions (Janda et al., 1990b, 1991c). The design of hapten [10] incorporates both a transition state mimic and the cationic pyridinium moiety, designed to induce the presence of a potential general acid/base or nucleophilic amino acid residue in the combining site, able to assist in catalysis of the hydrolysis of substrate [11] (Appendix entry 2.6). 

Lysine is the most common site for N-methylation, but methylation can also occur on arginine, histidine, glutamine, and asparagine. The enzymes responsible for N-methylation are known as N-methyltransferases aided with SAM as a cosubstrate. All forms of methylation share the same mechanism the nucleophilic amino acid side chain attacks the electrophilic methyl group of SAM and releases S-adenosylhomocysteine (SAH) (Scheme 7). 

The enzymes used to generate reactive quinone methides often undergo inactivation by addition of this electrophile to essential nucleophilic amino acid side chains of the protein catalyst. This is a type of suicide enzyme inhibition.80 This was observed for the acid phosphatase and ribonuclease catalysts used to generate 43.76 79 Alkaline phosphatase has been used to remove the phosphate protecting group from a derivative of an o-difluoromethyl phenyl phosphate that was covalently attached to a solid support. Breakdown of the immobilized 4-hydroxybenzyl difluoride gives an immobilized quinone methide that, in principle, will react irreversibly with proteins and lead to their attachment to the solid support.81... 

Covalent reaction with nucleophilic amino acid residues in the active site (45). 

T-cell lymphoma. Additionally, 8-methoxypsoralen has been shown to inhibit CYP2A6 (13). The mechanism of inhibition by this compound appears to be an initial oxidation to generate an epoxide that reacts with a nucleophilic amino acid at the active site (14). 

The a-chloroacetamide group has features that are beneficial for undirected ABPP. Its small size does not bias binding elements towards a specific class of enzyme, and it possesses reactivity towards a broad variety of nucleophilic amino acid residues. A library of a-chloroacetamide-based probes were synthesized by Cravatt s group. The binding element in these probes was a dipeptide that was varied with small, large, hydrophobic, and charged side chains, and a biotin or rhodamine tag was appended as a reporter tag. Upon screening of eukaryotic proteomes with this library, many enzymes previously unaddressed by directed ABPP probes were uncovered. These included fatty acid synthase, hydro-xypyruvate reductase, malic enzyme, and the nitrilase superfamily [163, 164]. In contrast to the sulfonate esters, a-chloroacetamides react preferentially with cysteine residues in the proteome. 

Figure 1. A simplified representation of the possible process for the papain-catalyzed hydrolysis and aminolysis E, enzyme (papain) S, substrate (protein) ES, Michaelis complex ES, peptidyl enzyme N, nucleophile (amino acid ester) P and P2, products formed from S by hydrolysis ...

Figure 3.5. Reversible and irreversible inhibition of a-adren-ergic receptors in the spleen, a Contractile tension developed by spleen slices in response to norepinephrine, in the presence of tolazoline and phenoxybenzamine. b Stmctures of norepinephrine, tolazoline, and phenoxybenzamine. c Reaction of phenoxybenzamine with the a-adrenergic receptor. The initial formation of the aziridine ring occurs in solution. The aziridine then reacts with a nucleophilic amino acid side chain (most probably a cysteine) in the binding site of the receptor.

The acidic cleavage of the Boc group is most probably proceeded by the protonation of the carbamate carbonyl with subsequent elimination of isobutene, either by an open or, more likely, by a cyclic transition state. Protonation leads to the liberated anoine or the annmonium salt, respectively (Scheme 35). The byproduct isobutene is either protonated to give the stable carbenium ion, or generates the tcrt-butyl trifluoroacetate, both of which act as terf-butylating agents, especially, when nucleophilic amino acid side chains are present in the molecule (vide infra). 

The perturbation of the acid dissociation constant of an amino acid residue as a consequence of its environment within a protein represents another mechanism for enhancing its reactivity relative to a free amino acid in solution. Most amino acid residues react with their respective modification reagents in their unprotonated form instead of in their conjugate acid form. Eq. (4.10) describes the pH dependence of the simple bimolecular reaction (eq. 4.9) of the free base form of nucleophilic amino acid side chain with a non-ionizable modification reagent where is the acid dissociation constant and Aj is the total concentration of amino acid. 

The striking successes achieved by Shaw (1970a) and his coworkers with haloketone derivatives of N-tosyl-phenylalanine and a-N-tosyl-lysine as affinity labels for chymotrypsin and trypsin, respectively, have stimulated their use in a large number of affinity labels. Haloketones are potentially reactive with all the nucleophilic amino acid residues in proteins. Examples of residues modified by haloketones include methionine (Sigman et al. 1970), glutamate (Visser et al. 1971), cysteine (Porter et al. 1971), histidine (Schoellman and Shaw 1963) and serine (Schroeder and Shaw 1971). 

Amides and esters of haloacids have been frequently used in the synthesis of affinity labels. Like haloketones, these derivatives react with all the nucleophilic amino acids. Their advantages as modification reagents are two-fold. First, they are relatively more easy to synthetize than haloketones. Any substrate or reversible inhibitor with a free amino or hydroxyl group can be potentially converted into an affinity labelling reagent. Second upon acid hydrolysis, the modified protein yields carboxymethylated derivatives which are known and therefore readily identifiable and quantifiable. 

As already pointed out, the chief advantage of using derivatives of haloacetates as modification reagents is that the carboxymethyl derivatives of most of the nucleophilic amino acids have been described. The chromatographic behavior of the carboxymethylated derivatives of lysine, cysteine and histidine have been described in ch. 2. O-Carboxymethyl tyrosine appears as a single peak in the long column... 

One of the chief drawbacks of epoxides is that it is difficult to predict the structure of the derivative which might be formed with the various nucleophilic amino acid residues. All epoxides incorporated into affinity labels will have the general structure (IV). Nucleophiles can therefore react at either of the carbon atoms and since it is generally unlikely that the R and R will be identical, the formation of at least two types of derivatives might be expected. 

Reactive aldehydes derived from lipid peroxidation, which are able to bind to several amino acid residues, are also capable of generating novel amino acid oxidation products. By means of specific polyclonal or monoclonal antibodies, the occurrence of malonaldehyde (MDA) and 4-hydroxynonenal (4-HNE) bound to cellular protein has been shown. Lysine modification by lipid peroxidation products (linoleic hydroperoxide) can yield neo-antigenic determinants such as N-c-hexanoyl lysine. Both histidine and lysine are nucleophilic amino acids and therefore vulnerable to modification by lipid peroxidation-derived electrophiles, such as 2-alkenals, 4-hydroxy-2-alkenals, and ketoaldehydes, derived from lipid peroxidation. Histidine shows specific reactivity toward 2-alkenals and 4-hydroxy-2-alkenals, whereas lysine is an ubiquitous target of aldehydes, generating various types of adducts. Covalent binding of reactive aldehydes to histidine and lysine is associated with the appearance of carbonyl reactivity and antigenicity of proteins [125]. 

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