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.