Disulfides by reduction

Add 1-10 mg of a protein or antibody containing an available thiol group to the particle suspension. Alternatively, add the protein to be coupled to the particle suspension in an amount equal to 1-10 X molar excess over the calculated monolayer for the protein type to be coupled. The optimal amount of protein to be added should be determined experimentally. Creating thiol groups on proteins or peptides may be done from disulfides by reduction. Alternatively, a thiolation reagent may be used to add thiols to the protein surface for coupling (see the protocols in Chapter 1, Section 4.1). 

Disulfides. As shown in Figure 4, the and h-chains of insulin are connected by two disulfide bridges and there is an intrachain cycHc disulfide link on the -chain (see Insulin and other antidiabetic drugs). Vasopressin [9034-50-8] and oxytocin [50-56-6] also contain disulfide links (48). Oxidation of thiols to disulfides and reduction of the latter back to thiols are quite common and important in biological systems, eg, cysteine to cystine or reduced Hpoic acid to oxidized Hpoic acid. Many enzymes depend on free SH groups for activation—deactivation reactions. The oxidation—reduction of glutathione (Glu-Cys-Gly) depends on the sulfhydryl group from cysteine. 

Pyrrolethiols, readily obtained from the corresponding thiocyanates by reduction or treatment with alkali, rapidly oxidize to the corresponding disulfides. They are converted into thioethers by reaction with alkyl halides in the presence of base. Both furan- and thiophene-thiols exist predominantly as such rather than in tautomeric thione forms. 

A thiol can be protected by oxidation (with O2 H2O2 I2. - ) to the corresponding symmetrical disulfide, which subsequently can be cleaved by reduction [Sn/HCl Na/xylene, Et20, or NH3 LiAlH4 NaBH4 or thiols such as HO(CH2)2SH]. Un-symmetrical disulfides have also been prepared and are discussed. 

Three substituted 5-phenyl unsymmetrical disulfides have been prepared, i, ii, and iii —compounds i and ii by reaction of a thiol with a sulfenyl halide, compound iii from a thiol and an aiyl thiosulfonate (ArS02SAr). The disulfides are cleaved by reduction (NaBH4) or by treatment with excess thiol (HSCH2CH2OH). 

Easily oxidised in air to give diquinolyl-8,8 -disulfide (which is stable). It is more convenient to make 8-mercaptoquinoline by reduction of the material. [Nakamura and Sekido Talanta 17 515 1970.]... 

FIGURE 5.18 Methods for cleavage of disulfide bonds in proteins, (a) Oxidative cleavage by reaction with performic acid, (b) Reductive cleavage with snlfliydryl compounds. Disulfide bridges can be broken by reduction of the S—S link with snlfliydryl agents such as 2-mercaptoethanol or dithiothreitol. Because reaction between the newly reduced —SH groups to re-establish disulfide bonds is a likelihood, S—S reduction must be followed by —SH modification (1) alkylation with iodoac-etate (ICH,COOH) or (2) modification with 3-bromopropylamine (Br— (CH,)3—NH,). 

V. Reduction of the disulfide by a reductase exposes the thiol, which then closes to give an episulfide, releasing the phosphate. 

Sulfinyl chlorides may be prepared from the corresponding thiols or disulfides by oxidative reactions rather than by reductions of sulfonyl compounds. A recent example, which improves the earlier procedure of Douglass, is given by equation (3)50. The chemistry of sulfinic acids and their derivatives has been reviewed51 52,53. 

Nitriles from aromatic aldehydes, diammonium hydrogen phosphate, and 1-nitropropane, 43, 59 w-Nitrobenzenesulfonyl chloride, reduction to m-nitrophenyl disulfide by hydriodic acid, 40, 80 2 Nitro-2,3-dimethylbutane, 43, 89... 

Schneemeyer LF, Wrighton MS (1979) Flat-band potential of n-type semiconducting molybdenum disulfide by cyclic voltammetry of two-electron reductants Interface energetics and the sustained photooxidation of chloride. J Am Chem Soc 101 6496-6500... 

Fig. 6.22. Folate-FRET sensor structure and its application to measure disulfide bond reduction in endosomes. The molecule contains the folate moiety which is recognized by the folate receptor situated at the plasma membrane. This recognition leads to endocytosis and after some time to cleavage of the probe. 

Fig. 3 Chemistry of site-specific PEGylation developed by Brocchini et al. [5, 6]. After cleavage of the native disulfide bond between two cysteine thiols by reduction, the free cysteines are reacted with an a, (3-unsaturated PEG derivative to produce a PEG conjugate via a three-carbon bridge...

One of the most convenient ways of generating sulfhydryl groups is by reduction of indigenous disulfides. Many proteins contain cystine disulfides that are not critical to structure or activity. 

The reduction of disulfides by 2-mercaptoethanol proceeds through a mixed disulfide intermediate. 

Figure 4.4 The reaction of DSP with amine-containing molecules yields amide bond crosslinks. The conjugates may be cleaved by reduction of the disulfide bond in the cross-bridge with DTT.

Figure 5.23 SAND can be used to modify amine-containing molecules, and then photo-initiate crosslinking to another amine-containing molecule via a ring-expansion process. The conjugates may be disrupted by reduction of the cross-bridge disulfide with DTT.

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