2
Tetrahedron
1. Introduction
S2.1, and Table S1). Please also note that in all these studies, we
have taken proper care of the reduction in thiol concentration due
to disulfide formation (see Supporting Information, section S2.2
and Table S2).
Bioconjugation is a major research theme since the 70s, best
known for linking antibodies to drugs1 or prodrugs, or therapeutic
proteins to poly(ethylene glycol) (PEG)2 or other polymers.3
By measuring the thiol concentration as a function of time
(Figure 3A, left), it is possible to calculate the effective rate
constant keff for the various Michael-type addition reactions
(slopes of the graphs in Figure 3A, right); as it is apparent in the
3-MPA / HEA example, the reaction is more rapid at high pH
(higher concentration of thiolates) and at high acceptor
concentration. keff can then be used to calculate a kinetic constant
independent of the initial concentrations of the reactants, kobs
(Figure 3B); this highlights that at any given pH, as it should be
expected, the reaction is faster for acceptors bearing esters,
Michael-type addition is a most popular bioconjugation reaction,4
most commonly with the use of thiols as nucleophiles and
electron-poor double bonds as electrophiles. For clarity, Michael-
type addition differs from Michael addition, where the
nucleophile is a (stabilized) carbanion, and from thiol-ene
reactions, where thiols add through a free radical mechanism
onto non-electron- poor olefins. Thiol-based Michael-type
addition’s popularity for bioconjugation and beyond (e.g. in thiol
recognition,5 surface functionalization,6 synthesis of polymers4 or
biomaterials7 etc.) is due to a) the mild reaction conditions, b) the
absence of byproducts and c) its bio-orthogonal character, i.e. the
reaction has hardly any competition by other biologically
occurring nucleophiles. This selectivity has a kinetic origin:
thiols have an appreciable acidity, hence anionic and thus
strongly nucleophilic thiolates are present already at neutral pH;
the more acid the thiol, the more rapid the reaction,8 a feature
also shared with disulfide formation.9 There are, however, still
areas of poor mechanistic understanding for this reaction, which
to date often hinder the accurate prediction of e.g. thiol
reactivity10 or of the stability of the products . For example, we
still do not know if thiol pKa is one or the main controlling factor
of the reaction kinetics, and how this may depend on the structure
of the acceptor. Another point to clarify is which one of the two
main degradative paths and to which extent can undermine the
stability of the conjugation (Figure 1); it is known that sulfur in γ
Figure 1. Thiol-based Michael-type addition (left) and the two
degradation reactions that its products may undergo (right).
slower with amides or methacrylates, and slowest for
methacrylamides, which combine steric hindrance with the poor
electronegativity of amides (see also Supporting Information,
Section S2.3 and Table S3). Please note that the maleimide-based
AcAEMi was excluded from this analysis, because of its almost
instantaneous reactivity with both thiols, and also because of its
interference with the Ellman’s reagent (see Supporting
Information, section S2.5). For similar reasons, we have not
considered vinyl sulfones: if on one hand they react with thiols so
rapidly to be kinetically selective over acrylates, on the other
hand this reactivity is marred by the parasite addition of OH- at
even mildly basic pH.
12,13
position,11 even more when oxidized as sulfoxide or sulfone,
accelerates ester hydrolysis, but there appears to be no
quantitative relation nor extension to other hydrolysable groups;
it is also known that retro-Michael-type addition can occur,
allowing for an exchange with more reactive/more concentrated
thiols, but this has been shown only on maleimides.14,15
Here we carried out a comprehensive investigation on the effects
of the Michael-type donors’ and acceptors’ structures, as well as
the reaction environment on the rate constant and the stability of
the final Michael-type adducts.
NAC is a stronger acid than 3-MPA; this means that, at a
given pH, NAC has a higher proportion of thiolates and therefore
2. Results and discussion
We have employed a small library of α,β-unsaturated acceptors
(Figure 2), varying strength of the electron-withdrawing group
(ester, amide, maleimide), hindrance on the double bond (CH3 vs.
H) and polarity of the side chain potentially linking a payload
(alcohol vs. amide). Since amino- or NHS-ester-terminated
heterobifunctional linkers are routinely used in conjugation
reactions which both result in amide bond formation, five of the
seven Michael-type acceptors (AcAEA, AcAEMA, AcAEAm,
AcAEMAm and AcAEMi) featured a terminal N-acetyl group,
thereby better mimicking the structures (and thus, the kinetic and
hydrolysis properties) of these Michael-type acceptors. The other
two commercially available compounds (HEA and HEMA)
simply contained a terminal hydroxyl group to serve as controls.
Using these compounds, we have determined A) the rate constant
for the addition of two model thiols, i.e. 3-mercaptopropionic
acid (3-MPA) and N-acetylcysteine (NAC), separately analysing
thiol and thiolate reactivity; B) the stability of their products
towards hydrolysis and exchange with the most common thiol in
biological fluids, i.e. glutathione (G-SH). It is noteworthy that
the three thiols used in this study differ in size, polarity and
above all acidity, pka values being >11 (3-MPA), 10-11 (NAC)
and 8-9 (G-SH) (for their determination through the thiolate UV
absorption, see Supporting Information, sections S1.3.2. and
Figure 2. Summary of the reactants used in this study: two
nucleophiles (NAC and 3-MPA) and a small library of
acceptors. A third thiol (G-SH) was used in retro-Michael
studies.