Stereospecific Alkylation with Asymmetric Reagents

Abstract

1.When undergoing nucleophilic reactions with proteins, a-iodopropionic acid and its amide show greater specificity for SH groups than do iodoacetic acid and its amide.2. The kinetics and stereochemistry of the reactions of the D( +) and L( -) antipodes of a-iodopropionic acid and its amide with both cysteine and papain were investigated. The antipodes of the SH reagents reacted with these asymmetric SH compounds a t different rates. I n the reaction with papain, the L( -) antipode of the acid reacted faster than the D(+) antipode; the stereochemical preference was inverted when the amide was used, the D(+)antipode reacting faster than the L( -)antipode. 3.Arguing from the differing reactivities of the SH reagents and their antipodes and the pH dependence of the reaction rates, it has been suggested, that L( -)a-iodopropionic acid is specifically oriented onto the reaction center through the concerted attractive and repulsive actions of a cationic and an anionic group in the protein. These have been tentatively identified as a protonated imidazole residue and a carboxylate group respectively.4. Parallels between these stereospecific alkylations and the catalytic action of the enzyme are discussed.Gundlach, Stein, and Moore [1] demonstrated in their studies of the alkylation of ribonuclease with iodoacetic acid and iodoacetamide, that these "classic" sulfhydryl reagents do not react only with SH groups. Other nucleophiles present in proteins, such as amino, imidazolyl or thioether groups may also be alkylated. The extent of these side reactions depends on the reaction conditions. Our purpose in undertaking this work was to find SH reagents with higher selectivity for SH groups.Reasoning from a theory of Bunnett [a] it seemed likely, that this could be achieved by the introduction of polarizable substituents alpha to the halogen of halogen-alkyles. The possibility for the action of dispersion forces, which is introduced with the polarizable substituent, should specially favor the transition state for the reaction with the highly polarizable SH group. Steric, inductive or mesomeric effects of the a substituent would be expected to show the same influence on the reactions with all different nucleophilic groups.We tested this hypothesis using a series of derivatives of iodoacetamide with different substituents alpha to the iodine. With the methyl group as CI substituent i e . in the reaction of amino acids with a-iodopropionamide we observed an enhanced selectivity for SH groups as predicted by Bunnett's theory A further possibility for selective reactivity is contributed by a-substitution in iodoacetic acid derivatives ; the leaving group of the reagent is attached to an asymmetric carbon atom. One may expect specific interactions if the substrate itself is asymmetric since diastereomeric transition states are involved. This type of stereoselective reaction was shown for the first time by Heinrikson et al. [4,5]. They found, that the essential histidine (residue 12) of ribonuclease reacts preferentially with the D-...

Aziridine‐2‐carboxylic acid A reactive amino acid unit for a new class of cysteine proteinase inhibitors

“…Similarly lowered rate constants have previously been reported for the inactivation of papain by g-iodopropicnic acid amide [12] and ¢hloroacetamide [18], a fact which ha~, been interpreted in terms of loss of electrostatic interaction between the carboxyl group of the thiol reagents and the active-site imidazole function. In view of these findings even the carboxyl group of azi.…”

“…Studies on irreversible inhibition of papain by ~-iodopropionic acid [12,13] and chloroacetic acid [14] clearly revealed a bell-shaped, pH-dependent reaction rate of these thiol reagents with a maximum around pH 6.0. This behaviour was assigned to intraprotein electrostatic interactions between sulfhydryl, imidazolium and carboxylate groups causing a remarkably increased nucleophilicity of the active-site sulfur anion.…”

“…The experimental data are therefore consistant with the model of aziridine-2-carboxylic acid representing primarily a non-specific irreversible inhibitor with an inactivation potency which is significantly superior to that of other thiol-trapping reagents, e.g. N-ethylmaleimide (I M-~.s -~, at 40°C and pH 6.0 with cathepsin B) [3], L(-)-0~-iodopropionic acid (4.9 M-~.s -~ at 25°C and pH 7.0 with papain) [12] and chloroacetie acid (4.23 M-1.s -~ at 30.5°C with papain) [14]. The enzyme-specificity of this thiol alkylating agent in presence of the large excess of cysteine, used as an additive in the assay medium, relies mainly on the different pK, values of the active site and cysteine thiol group and thus on their markedly different nucleophilicity.…”

On the Mode of Activation of the Catalytically Essential Sulfhydryl Group of Papain

“…The pHdependence of the reaction of papain with chloroacetamide [I] and 2-iodopropionamide [2] is similar to that of an ordinary SH-compound. The second-order rate constants with the enzyme are about 10 times higher [I, 21.…”

Benzofuroxan as a thiol-specific reactivity probe. Kinetics of its reactions with papain, ficin, bromelain and low-molecular-weight thiols

“…Particularly pronounced effects can be provided by using two-protonic-state electrophiles such as 2,2'-dipyridyl disulphide (see Brocklehurst, 1974;Malthouse & Brocklehurst, 1976;Norris & Brocklehurst, 1976), but other types of probe can also yield valuable information, e.g. anionic alkylating agents (Wallenfels & Eisele, 1968;Chaiken & Smith, 1969) and 4-chloro-7-nitrobenzofurazan [see Shipton et al (1976), Baines et al (1977) and references therein].…”