Protein reductive alkylation

See Section B for protein reduction, alkylation, digestion, and HPLC. 

Heilman, EJ., Wiksell, E., and Karlsson, B.-M. (1990). A new approach to micropreparative desalting exemplified by desalting a reduction/alkylation mixture, presenred ar Eighr International Conference on Methods in Protein Sequence Analysis, Kiruna, Sweden, July 1-6. 

Cabacungan, J.C., Ahmed, A.I., and Feeney, R.E. (1982) Amine boranes as alternative reducing agents for reductive alkylation of proteins. Anal. Biochem. 124, 272-278. 

Dottavio-Martin, D., and Ravel, J.M. (1978) Radiolabeling of proteins by reductive alkylation with [14C]-formaldehyde and sodium cyanoborohydride. Anal. Biochem. 87, 562. 

Figure 6.5 LC/MS/MS strategy for mapping glycoproteins and proteins via reduction, alkylation, and enzymatic digestion, followed by three LC/MS and LC/MS/MS experiments. (Reprinted with permission from Carr et al., 1993. Copyright 1993 Cold Spring Harbor Laboratory Press.)...

Bertrand-Harb, C., Charrier, B., Dalgalarrondo, M., Chobert, J.-M., and Haertle, T. 1990. Condensation of glycosidic and aromatic structures on amino groups of (3-lactoglobulin B via reductive alkylation. Solubility and emulsifying properties of the protein derivatives. Lait 71, 205-215. 

The amino groups of ovomucoid, lysozyme, and ovotransferrin were alkylated extensively (40-100%) with various carbonyl reagents in the presence of sodium borohydride. Monosubstitution was observed with acetone, cyclopentanone, cyclohexanone, and benzaldehyde, while 20-50% disubstitution was observed with 1-butanal and nearly 100% disubstitution was observed with formaldehyde. The methylated and isopropylated derivatives of all three proteins were soluble and retained almost full biochemical activities. Recently amine boranes have been shown to be possible alternative reducing agents for reductive alkylation... 

Reversible Modifications. Reversibility of a modification is frequently a desired characteristic in order to regain the protein in its original native form. A variety of methods (I) is available for this purpose. Sometimes a further intermediate modification is necessary as used recently in the reductive alkylation of amino groups with an a-hydroxy aldehyde or ketone and subsequent removal of the hydroxyalkyl group by periodate cleavage (85) (see Figure 18). 

Substances also may be attached covalently to proteins for transporting the substances to targets in the body. Examples of this approach have been transporting radioactive iodine or toxic substances attached to antibodies in attempts to kill cancer cells and attaching radioactive metals to proteins and bleomycin for similar purposes (122). A recent novel approach has been that of Wu and Means (123) who have attached insulin to artificially formed liposomes by a reductive alkylation modification. The insulin-liposome aggregate reacts with insulin receptors. 

Gallenbeck et al. (25) has summarized the work on reductive alkylation using formaldehyde and sodium borohydride to yield dimethylated proteins. Oxidation and reduction reactions involving thiol and disulfide groups have been discussed by Ryan (13) and Feeney (11). 

As an alternative strategy, lysine residues can be modified through reductive alkylation. Fig. 2e. This method is most frequently carried out by exposing the protein to aldehydes in the presence of hydride-containing agents that reduce the transiently formed imines. NaB(CN)H3 and NaB(OAc)3H are commonly used for this purpose. As an alternative, transfer hydrogenation can be carried out in the presence of an Ir(III)[Cp ]2(bipyridyl) catalyst, which allows imine reduction to occur under mild conditions using buffered formate as the hydride source (14). 

Relative to acylation strategies, modification via reductive alkylation preserves the overall charge state (and thus the solubility) of the protein. In general, this technique also suffers from poor site selectivity. 

McFarland JM, Francis MB. Reductive alkylation of proteins using iridium catalyzed transfer hydrogenation. J. Am. Chem. Soc. 2005 127 13490-13491. 

It may be noted that reductive alkylation of proteins with acetaldehyde, or acetone, results in the formation of only the corresponding monoalkyl derivatives of lysine. 

Auto-oxidation of cysteine residues during cleavage of the disulfide bridge-containing proteins is a potential concern. This concern can be addressed by first reducing those proteins at alkaline pH ( 8.0) with either 2-mercaptoethanol or dithiothreitol (Equation (1)) and then alkylating with iodoacetic acid to S-carboxymethyl derivatives (Equation (2)) The reduction-alkylation process also disrupts the 3D structure of proteins to allow more sites accessible for cleavage. 

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