The rate of reduction of oxidized glutathione (GSSG) to glutathione (GSH) by thiolate anions (RS-) follows a Brjánsted relation in pXas of the conjugate thiols (RSH): /5nuc a 0.5. This value is similar to that for reduction of Ellman's reagent: jSnue ~0.4-0.5. Analysis of a number of rate and equilibrium data, taken both from this work and from the literature, indicates that rate constants, k, for a range of thiolate-disulfide interchange reactions are correlated well by equations of the form log k = C + |6nucPXanu<: + /3cpAfac + 0igpKjg (nuc = nucleophile, c = central, and Ig = leaving group sulfur): eq [36][37][38] give representative values of the Br^nsted coefficients. The values of these Br^nsted coefficients are not sharply defined by the available experimental data, although eq 36-38 provide useful kinetic models for rates of thiolate-disulfide interchange reactions. The uncertainty in these parameters is such that their detailed mechanistic interpretation is not worthwhile, but their qualitative interpretation-that all three sulfur atoms experience a significant effective negative charge in the transition state, but that the charge is concentrated on the terminal sulfurs-is justified. Equilibrium constants for reduction of GSSG using , -dithiols have been measured. The reducing potential of the dithiol is strongly influenced by the size of the cyclic disulfide formed on its oxidation: the most strongly reducing dithiols are those which can form six-membered cyclic disulfides. Separate equilibrium constants for thiolate anion-disulfide interchange (As~) and for thiol-disulfide interchange (A'SH) have been estimated from literature data: Ks~is roughly proportional to 2 Xa is the difference between the pKas of the two thiols involved in the interchange. The contributions of thiol pA"a values to the observed equilibrium constants for reduction of GSSG with , -dithiols appear to be much smaller than those ascribable to the influence of structure on intramolecular ring formation. These equilibrium and rate constants are helpful in choosing dithiols for use as antioxidants in solutions containing proteins: dithiothreitol (DTT), 1,3-dimercapto-2-propanol (DMP), and 2-mercaptoethanol have especially useful properties.

The rate constants for thiol-disulfide interchange between 21 mono-and dithiols and Ellman's reagent correlate with the pKu's of the thiol groups with a Bronsted coefficient of p = 0.36. The maximum rates ofreduction are observed for thiols having pK, values close to the pH of the solution in which the reactions were carried out. In the dilute solutions examined (10-4-10-6 M in each reagent), the rate of the second, intramolecular interchange step in reactions of dithiols was faster than that of the first, intermolecular interchange, regardless of the size of the cyclic disulfide formed. A convenient synthesis of a mixture of diastereomers of 1,4-dimercapto-2,3-butanediol (i.e., of a mixture of dithiothreitol, DTT, and dithioerythritol, DTE) has been developed from 1,2:,4-diepoxybutane and thiolacetic acid.Oxidation of cysteine sulfhydryl groups during isolation, storage, and use of proteins is often an important contributor to their deactivation.2 Although the rate of oxidation can be decreased by limiting access of oxygen to the enzyme, it is usually impractical to exclude oxygen completely, particularly in practical synthetic and analytical applications. The most effective and widely used reagents for protecting the cysteine moieties of enzymes against oxidation by adventitious oxygen, and for activating partially oxidized and deactivated enzymes by reduction, are thiols, particularly dithiothreitol (DTT, Cleland's reagent);r and B-mercaptoethanol. Each has its advantages and disadvantages: DTT reduces protein disulfide groups rapidly and completely and is convenient to handle, but is exorbitantly expensive; p-mercaptoethanol is readily available and inexpensive, but reacts less rapidly and completely.As part of a project designed to develop techniques to permit the use of enzymes as catalysts in large-scale organic synthesis, we required an agent that would reduce disulfide moieties more rapidly and completely than B-mercaptoethanol but which would be less expensive than DTT. The design of an appropriate reagent is not straightforward for several reasons. First, the mechanism of reduction (illustrated in Scheme I for DTT) involves multiple acid-base and sulfhydryl-disulfide interchange equilibria, and the dependence of the overall rate and equilibrium position on the structure of the reducing agent (and possibly of the protein) is difficult to predict. An important part of the difference in reactivity between DTT and /3-mercaptoethanol can, however, plausibly be attributed to the rate of release of the second equivalent of CysS-(or CysSH) from initially formed mixed disulfides: since (l-mercaptoethanol is commonly used in enzymology at concentrations of ca.

Brønsted coefficients have been determined for the rates of thiol-disulfide interchange between low molecular weight thiols and the disulfide groups of four native or modified proteins: DNase (beta nuc congruent to 0.36), lysozyme (beta nuc congruent to 0.55), adenylate kinase(SSCH3)2 (beta nuc congruent to 0.65), and papain(SSCH3) (beta nuc congruent to 0.45). These values are similar to those observed for reductions of oxidized glutathione and Ellman's reagent by a similar set of thiols (beta nuc congruent to 0.5). Glutathione is anomalously slow in reduction of certain protein disulfide groups: although this effect may in part reflect steric hinderance to attack by the relatively large glutathione molecule at disulfides shielded by protein tertiary structure, other (presently undefined) factors appear also to be important, at least in the case of DNase. The rates of reduction of several disulfide derivatives of papain(SSR) by DTT were determined. These data provide estimates of the Brønsted coefficient for the "central" thiol in thiol-disulfide interchange: these estimates fall in the range beta c congruent to -0.25 to -0.43. Rates of reduction of protein disulfide moieties were analyzed by using a Brønsted equation developed previously [Szajewski, R. P., & Whitesides, G. M. (1980) J. Am. Chem. Soc. 102, 2011] to yield pKa values for the participating thiol moieties: in particular, for papain, pKa(Cys-25) = 8.4 at pH 9 and pKa (Cys-25) = 4.1 at pH 6. The thiols of the structurally essential cysteine group of lysozyme seem to have pKa congruent to 11. The advantages and disadvantages of this method for estimating thiol pKa values are discussed.

The potential and limitations of the photochemical ring contraction of 3-diazo-2,4-pyrrolidinediones as a route to carboxy p-lactams (2-azetidinone-3-carboxylic acids) are explored. Although the method seems to be a fairly general route from a-amino acids to p-lactams, the difficulty of achieving steric control makes the process not especially promising as a route to natural penicillins and cephalosporins.

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