Understanding base‐assisted desulfurization using a variety of disulfide‐bridged peptides

Abstract

A recently rediscovered reaction of base-assisted lanthionine formation has been applied to several systems of disulfide-bridged peptides. In addition to previously described nonapeptides consisting of i, i+3 cystine linkages, the reaction has now been extended to systems consisting of shorter (i, i+2) and longer (i, i+4) disulfide bridges. The desulfurization reaction is also compatible with disulfide bridges formed through homocysteines and penicillamines, yielding unusual amino acids such as cystathionine and beta,beta-dimethyl lanthionine (referred to as "penthionine") in a peptide chain, respectively. Systematic study of this transformation has provided several new insights into its mechanism. We have observed formation of dehydroalanine and dehydrovaline residues resulting from i, i+2-bridged cysteines and i, i+3-bridged cysteine/penicillamine peptides, respectively, thereby supporting a beta-elimination/Michael-addition mechanism for this transformation. Amino acid analysis and NMR data from total correlation spectroscopy (TOCSY) and (1)H-(13)C heteronuclear single quantum correlation (HSQC) experiments show three diastereomeric lanthionine-bridged peptides in the product mixture. But in the case of desulfurization of a cysteine/homocysteine containing disulfide-bridged peptide, Michael addition appears to be stereoselective, yielding a single stereoisomer of cystathionine within the peptide. According to molecular modeling and CD spectroscopy, constrained peptides such as those containing penicillamine are less likely to undergo facile desulfurization. Flexibility of the torsional angles (C(alpha)H-C(alpha)-C(beta)-S) corresponding to the cysteine residues and temperature appear to be contributing factors determining the extent of desulfurization.

Analysis of reduced monoclonal antibodies using size exclusion chromatography coupled with mass spectrometry

“…The mass spectra of peaks a to c are shown in Figure 4b. The theoretical molecular weights of antibody-A heavy chain without C-terminal Lys and with a core-fucosylated biantennary complex oligosaccharide structure without terminal galactose ( linkage has been well documented in the literature [31][32][33] and reported for monoclonal antibodies [17,28]. No interpretable mass spectrum was obtained for the shoulder that eluted in front of peak a.…”

Characterization of alkali induced formation of lanthionine, trisulfides, and tetrasulfides from peptide disulfides using negative ion mass spectrometry

“…The use of negative ion mass spectrometry [13][14][15][16][17][18][19][20][21][22][23][24][25][26][27][28][29] for the study of disulfide containing peptide natural products has been established in an extensive series of investigations by Bowie and coworkers [30 -35] and other groups [36 -38]. During the course of recent attempts to fragment the negative ions of disulfide bridged peptides under mass spectrometric conditions, we noted that the cleavage reactions closely resemble those observed during breakage of disulfide bonds under alkaline conditions [39,40]. Under basic conditions, both ␣-and ␣ ␤-hydrogens of cysteine residues can be abstracted ␤ leading to two distinct modes of cleavage of the disulfide bonds, as noted by Parker and Kharasch in their comprehensive review of the mechanism of scission of sulfur-sulfur bonds [41].…”

“…The chemistry of the base catalyzed degradation of protein disulfides has been the subject of considerable study [44 -53]. More recently, Spatola and coworkers demonstrated the formation of lanthionine peptides by alkali treatment of several synthetic disulfide peptides [39,40]. Interestingly, reports of trisulfide formation when proteins are exposed Address reprint requests to Professor P. Balaram, Molecular…”

Topology of the disulfide bonds in the antiviral lectin scytovirin

“…The topology of disulfides in SVN was originally assigned by matching the masses of its tryptic fragments obtained by LC-MS, 1 as well as of fragments of expressed SD1. 2,5 Starting with assignment of species ionizing as m/z ¼ 1318.6 (residues 30-43; C32-C38; SD1), m/z ¼ 1553.6 (residues 79-95; C80-C86; SD2), and m/z ¼ 3157.5 (residues [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19] to [51-59]; C7-55), the remaining two species, termed here ''cystine clusters'' of m/z ¼ 2511.0 and m/z ¼ 2719.1, each containing two disulfides, C20-C26/C32-C38 (SD1) and C68-C74/C80-C86 (SD2), were assigned by process of elimination.…”

“…As can be seen from fragment assignments [ Fig. 2(A), Table II], a heterodimeric peptide, residues [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19]-[51-60], containing C7-C55, has been identified (fragment 10). Its Edman degradation yielded two N-terminal sequences, including di-PTH-Cystine in Cycle 7, confirming the presence of a disulfide bridge.…”