Computational Study of the Addition of Methanethiol to 40+ Michael Acceptors as a Model for the Bioconjugation of Cysteines

A long series of Michael acceptors are studied computationally as potential alternatives to the maleimides that are used in most antibody–drug conjugates to link Cys of mAbs with cytotoxic drugs. The products of the reaction of methanethiol (CH3SH/MeSH, as a simple model of Cys) with N-methylated ethynesulfonamide, 2-ethynylpyridinium ion, propynamide, and methyl ethynephosphonamidate (that is, with HC≡C–EWG) are predicted by the M06-2X/6-311+G(d,p) method to be thermodynamically more stable, in relation to their precursors, than that of MeSH with N-methylmaleimide and, in general, with H2C=CH–EWG; calculations with AcCysOMe and tBuSH are also included. However, for the addition of the anion (MeS–), which is the reactive species, the order changes and N-methylated 2-vinylpyridinium ion, 2,3-butadienamide, and maleimide may give more easily the anionic adducts than several activated triple bonds; moreover, the calculated ΔG⧧ values increase following the order HC≡C–SO2NHMe, N-methylmaleimide, HC≡C–PO(OMe)NHMe, and HC≡C–CONHMe. In other words, MeS– is predicted to react more rapidly with maleimides than with ethynephosphonamidates and with propynamides, in agreement with the experimental results. New mechanistic details are disclosed regarding the advantageous use of some amides, especially of ethynesulfonamides, which, however, are more prone to double additions and exchange reactions.


SI-4
Extended Scheme 3. The total energies that appear in the first row are directly obtained by the M06-2X/ 6-311+G(d,p) method, while those in the second row have been optimized in water as the implicit solvent (CPCM) with the same method. They belong to the lowest-energy conformer of each molecule. This was determined after a standard conformational search by empirical methods, selection of the low energy species from M06-2X/ 6-31G(d) or MP2/6-31G(d)//B3LYP/ 6-31(G), and final ordering by means of M06-2X/ 6-311+G(d,p) or M06-2X/6-311+G(d,p)·w (CPCM), as just mentioned. Not always the lowest-energy conformer of the isolated species (gas phase, in vacuo) was coincident with that obtained in water; such cases are commented in due course.
The reaction energies are given in kcal/mol. All the reactions are exoergic (around -24 kcal/mol for the maleimide, around -40 for the activated triple bonds, in accordance with what was observed for the addition of MeSH). We assume that the corresponding enthalpy energies (Gibbs free energies) would generally be ca. 16 kcal/mol less favorable.
The phosphonamidate case is also included in the expanded Scheme 3. The energy difference between the RR and RS stereoisomers is predicted to be small, in water. As indicated in Figure S1, there are several conformers very close in energy (a much higher number than in any other case examined by us), due to the tetrahedral arrangement of the substituents bound to the P atom. Another feature is that the energy difference between the adduct of MeSH and that of AcCysOMe was in this case reversed, which we attribute to the network of intramolecular hydrogen bonds (involving the P=O, NHAc, CO groups, NHMe, and OMe groups) that occur in the latter and overstabilize it, especially in the gas phase. Actually, the phosphonamidate case (alkynephosphonamidates, alkenephosphonamidates, allenephosphonamidates...) would require an independent study.
Extended Figure 2. The M06-2X-predicted values for the reaction of addition of MeS -(methanethiolate ion) with optimization in water were collected in Figure 2 of the main text, the details of which are included below. This Figure can be expanded up to 40-50 pairs as we did for the equilibria involving neutral species (Figure 1), but the selection of samples is illustrative of the changes that occur when the anion (MeS -) is involved instead of MeSH: the activated double bonds follow a pattern different from that of activated triple bonds. As pointed out in the main text, the energy for the reaction with MeSH is indicative of the thermodynamic stability of the adduct with regard to the retro-Michael reaction (the corresponding E2 mechanism can be favored under basic catalysis and on heating), whereas the energy for the reaction with the anion to give an anionic intermediate is indicative of the kinetics of the process (the lower the energy of such an intermediate, the lower the barrier to reach it).
The total energies in au or Hartrees as directly obtained by M06-2X, that is, by optimization of geometries at the M06-2X/6-311+G(d,p) level, are given in the following extended Figure 2 for each Michael acceptor in the gas phase and optimized in w (CPCM/Spartan'18, in blue), which are the two upper values, and for each adduct, which are the two lower values.

SI-5
Quite often the lowest-energy anionic adducts adopted a conformation with the MeS folded (due to the electrostatic interaction between the Me group and the partially delocalized negative charge), whereas in water a zigzag conformation (ap) of the SMe group turned out often to be that of lowest energy.
Since, as indicated in the main text, the reliability of some implicit-solvent models is a hot topic under debate, especially when charged molecules are involved, we include below the scale in the gas phase. The relative values are essentially maintained, with a few exchanges of positions (in the next Figure, as shown by the crossing lines, with regard to the preceding Figure, which is Figure 2 of the main text). This may be understood assuming that disparate delocalized anions will not be similarly stabilized by a polar solvent.
Finally, the following Figure shows the reaction energies in water/CPCM, obtained with Gaussian 16. Despite the fact that the total energies were different, there are almost no changes of position with regard to the first Figure, which indicates that the CPCM parameters implemented in the two packages are qualitatively consistent. ∆E in kcal/mol, from M06-2X/ 6-311+G(d,p), in water (in blue) and in the gas phase, for the addition reaction of CH 3 Sto the depicted acceptors --438

SI-6
Extended Scheme 9. Kinetically, the second addition of MeSto N-methylpropynamide is unfavorable (see ∆E(w) = 6.6 kcal/mol in Scheme 9, hence ∆G˚(w) [22][23] kcal/mol, and see ∆G ‡ (w) = 25 kcal/mol; it is even more unfavorable for the case of t BuS -(see Scheme 6). Simple steric and electronic effects explain these differences. Extended Scheme 10. The M06-2X total energies for the lowest-energy species involved in the first addition of MeSH to representative triple bonds and in the second addition are given in au. The reaction energies obtained from these values are in kcal/mol. In all cases, the second addition, as expected, is predicted to be many kcal/mol less feasible.

Relevant M06-2X/6-311+G(d,p) equilibrium geometries (Cartesian coordinates)
The Cartesian coordinates (in Å) of the lowest energy conformers for a selection of the more relevant molecular entities and anionic species are given below. N

Treatment of 1 with NaH
Sodium hydride (60% dispersion in mineral oil, ca. 4.00 mg, 0.10 mmol, 0.8 equiv) was added to a solution of 1 (47 mg, 0.13 mmol) in CD3CN (1.0 mL) (Scheme 5 of the main text). The solution turned dark red. Within 10 min its 1 H NMR spectrum was registered, which was compared with that of the starting material (1). The signal at 3.95 ppm (two overlapped dd corresponding to the two diastereomers of 1) quickly and significantly diminished, but also some percentage of deprotonation of the signals at 2.50 ppm (succinimide methylene) was noted. Although not indicated in the drawings, the NH protons of the acetamido groups of the two diastereomers also disappeared partially.

Exchange of thiolates in the maleimide adducts (mechanisms)
Formally, an exchange of MeSH between the methylsulfanylsuccinimide shown in Scheme S1 and any propynamide is feasible. The hypothetical equilibrium must be shifted very far to the right; it will suffice to find a suitable catalyst and/or to reach the appropriate temperature.
In vitro, when succinimide 1 and N-benzylpropynamide (2) were mixed in CH3CN at rt plus a catalytic amount of Na + DMSOor in THF at -78 ºC plus t BuLi (either in catalytic or substoichiometric amounts), the formation of 3 (Z/E mixture) was rapidly and clearly observed by TLC and NMR. The exchange takes place, in agreement with the predictions, but in the absence of a source of protons it cannot be avoided that the generated allenolate-type ion, during the workup, gives rise to the Z/E mixture instead of the Z isomer. The coproduct from the exchange, N-benzylmaleimide, could not be isolated as it undergoes a rapid polymerization (formation of a dark red polymer) in non-aqueous basic media. S7 Scheme S1. Plausible exchange reaction on the basis of M06-2X calculations. ∆E and ∆G˚ values in kcal/mol, in bold. Reaction of 1 and 2 in the presence of strong bases If the elimination of RS -(E1cB, from the minor succinimide anion with the negative charge at C4, see Scheme 4) or of RSH (E2, from the neutral adduct, under basic catalysis) occurs, the generated species, RS -, will immediately react with the ynamide. In aqueous solutions, this reversal or retro-Michael reactions is assumed for the exchange of thiols in Cysmaleimide-containing ADCs. As shown in Scheme S2, there is a series of equilibrium reactions with very small barriers (as predicted in Scheme 4).

Scheme S2. Exchange reactions via elimination-addition steps
Other reactions of alkylsulfanyl derivatives are possible. When the acceptors are strong, as in the case of 4-phenyl-1,2,4triazoline-3,5-dione (PTAD), and the new bond (S-N) is weaker than a standard S-C bond, it is known that a second unity of thiol or thiolate causes the formation of dialkyl disulfide, S8 as shown in Scheme 12.
We have calculated the energies of these species for the case of the methanethiolate ion (MeS -). The overall reaction is highly exothermic (Scheme S3), as are all the individual steps. In contrast, for the parallel reaction of MeSwith the adduct from N-methylmaleimide, the reaction cannot work: the value of ∆E(g) was 3.7 kcal/mol and that of ∆E(w) 18.7 kcal/mol (both positive). Scheme S3. Known reaction of dimerization of thiols, mediated by very strong acceptors, which probably pass through the Michael adduct, followed by the formation of the S-S bond. Values of ∆E in kcal/mol Another possibility is that a SN2 mechanism is involved, such as the identity reaction S9 depicted in Scheme S4, where the substitution would be relatively favored as it occurs at position a to a carbonyl group. We investigated the TS for such a simple SN2 reaction (Scheme S4, with R = R* = Me). With M06-2X, in the gas phase, the energy of the located TS was around 3 kcal/mol above the sum of the energies of the individual components (quite a low barrier), whereas in water it was 26 kcal/mol above them (quite a high barrier). The nature of the TS, which is much less polar than MeS -, explains this large effect of the solvent polarity. With larger thiolate ions the effect of water is expected to be less significant.

SI-14
Scheme S4. Alternative mechanism (a standard SN2) for the exchange of thiols Attempted experiments to gain more insight into the exchange mechanisms in vitro, with partially deuterated adducts and lithium 1-dodecanethiolate, were not conclusive. The known tendency of succinimide intermediates to undergo ring opening in basic aqueous media and of maleimides to undergo anionic polymerization in non-protic media were prevalent.