Introduction:
The thiol-maleimide reaction 1 also known as the thiol-Michael addition 2, is included among the chemical reactions classified as click-chemistry 2,3and is one of the most widely used methods for conjugation4,5 and the synthesis of antibody drug conjugates (ADC) and conjugate vaccines 4.
As a result of this chemical reaction, the cytotoxic drug and the antigen are linked to the carrier protein through a thioether bond, generally between a cysteine residue, a five-membered succinimide ring and an N -acyl group that is linked to primary amino groups (structure I, Figure 1). The resultant five membered thiosuccinimide ring is considered to be unstable in vivo due to the fact that a retro-Michael addition can occur 6,7 (Figure S1). In serum, the presence of molecules with free thiols (e.g. free Cys, reduced glutathione, albumin, etc.), through a thiol-exchange reaction, promotes the release of the antigen or the cytotoxic drug linked to the thiosuccinimide ring in the conjugate vaccine and ADC, respectively. This reaction, which is undesirable for the development of a biotechnology product, increases the cytotoxicity of the ADC8, and it could also lead to the partial or complete loss of the biological activity of the conjugate vaccine. To overcome this potential in vivo instability of the resultant five-membered thiosuccinimide ring, it can be stabilized through hydrolysis9-13 or via Michael-transcyclization reactions14,15.
Hydrolysis of the five-membered thiosuccinimide could proceed through two pathways (see pathways (a) and (b) in structure (I), Figure 1) that would yield two thiosuccinamic acid isomers (structures (II) and (III), Figure 1) 10. These isomers are stable in vivoand are not subject to the retro Michael addition. This hydrolysis of the thiosuccinimide linker proceeds more rapidly at basic pH, and also takes place during the sample preparation and proteolysis of conjugates regardless of the position of the cysteine residue that is involved in the thioether bond 16-18. The thiosuccinamic acids, hydrolysis products derived from a thiosuccinimide, can be readily identified by the MS-mode, based on the fact that the molecular masses of these species differ by 18 Da.
In the case of the peptide with a free cysteine at the Nterminus, the transcyclization predominantly takes place by a nucleophilic attack of the amino terminal group of the cysteine residue to the carbonyl group in the five-membered ring of the thiosuccinimide, which, through an internal rearrangement, results in the formation of a more stable six-membered thiazine ring and a new pseudopeptide bond as well (structure (IV), in Figure 1) 14.
The conjugate peptides linked through thiazine or thiosuccinimide are isobaric, and thus, cannot be differentiated based on their molecular masses. However, the thiazine form can be identified by CID or HCD fragmentation15. This is because the thiazine form can undergo cleavage at new pseudo-peptide bond that was formed through the transcyclization process14 (see structure (IV), in Figure 1).
The findings reported in this study show that C-S linkage between the cysteine sulfur atom and the succinimide at the conjugation site is amenable to cleavage by MALDI-MS, MALDI-MS/MS or UVPD/HCD-MS/MS, which yields two component peptides, namely, a free-thiol-containing peptide and its counterpart linked to the original N -propionyl maleimide and N -propionyl succinimide groups. The thiazine form, however, is stable and no fragment ions derived from it are produced in MALDI-MS, but fragment ions for the newly formed peptide bond are observed in MALDI-MS/MS.