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# Maleimide–thiol adducts stabilized through stretching

## Abstract

Maleimide–thiol reactions are widely used to produce protein–polymer conjugates for therapeutics. However, maleimide–thiol adducts are unstable in vivo or in the presence of thiol-containing compounds because of the elimination of the thiosuccinimide linkage through a retro-Michael reaction or thiol exchange. Here, using single-molecule force spectroscopy, we show that applying an appropriate stretching force to the thiosuccinimide linkage can considerably stabilize the maleimide–thiol adducts, in effect using conventional mechanochemistry of force-accelerated bond dissociation to unconventionally stabilize an adjacent bond. Single-molecule kinetic analysis and bulk structural characterizations suggest that hydrolysis of the succinimide ring is dominant over the retro-Michael reaction through a force-dependent kinetic control mechanism, and this leads to a product that is resistant to elimination. This unconventional mechanochemical approach enabled us to produce stable polymer–protein conjugates by simply applying a mechanical force to the maleimide–thiol adducts through mild ultrasonication. Our results demonstrate the great potential of mechanical force for stimulating important productive chemical transformations.

## Main

Maleimides are broadly applicable for coupling with cysteines or reactive thiol moieties in proteins, peptides and drugs via Michael-type addition reactions1,2. Owing to its high selectivity, fast reaction kinetics and mild reaction conditions, this specific covalent conjugation has been commonly employed in the field of bio-labelling3,4, surface science5,6, materials science7,8,9 and drug delivery10,11,12,13,14. In particular, the use of maleimide–thiol adducts in antibody–drug conjugates (ADCs), which combine the high selectivity of therapeutic antibodies with the high potency of drugs, has proven to be an efficient strategy in cancer therapy15,16,17. Notably, several FDA-approved ADCs incorporate a maleimide–thiol-formed thiosuccinimide linkage as the means of conjugation, such as ado-trastuzumab emtansine (Kadcyla) for Her2-positive breast cancer18 and brentuximab vedotin (Adcentris) for relapsed Hodgkin lymphoma and anaplastic large-cell lymphoma19,20,21, along with the approved antibody PEGylated conjugate (Cimzia)22.

Even so, the thiosuccinimide linkage is well recognized as being unstable under physiological conditions or in the presence of free thiols, as it is eliminated through a retro-Michael reaction or thiol exchange23,24,25. Nearly all maleimide–thiol adducts in ADCs suffer from measurable drug loss, limiting their in vivo stability and efficacy17,21. Yet the stability of maleimide–thiol adducts can also be dramatically increased through the hydrolysis of the thiosuccinimide five-membered ring, resulting in a stable hydrolysate that is resistant to elimination of the maleimide–thiol bond23,25,26,27. These two competing reaction pathways lead to products with disparate chemical stabilities (Fig. 1a).

Considerable efforts have been devoted to accelerating the spontaneous ring-opening hydrolysis of maleimide–thiol adducts after conjugation to stabilize the bonds. The addition of special catalysts28 or treatment with base23,25,29 can efficiently trigger ring-opening hydrolysis, but the harsh reaction conditions are not biocompatible. Introducing an electron-withdrawing group to the thiosuccinimide ring26,30 or selectively engineering cysteines in the positively charged environment of proteins27 can also promote the self-hydrolysis of the thiosuccinimide ring, successfully preventing elimination of the maleimide–thiol bond; these methods are biocompatible but require complex molecular designs and syntheses. Thus, there is currently no general and practical way to stabilize maleimide–thiol conjugates. A versatile and broadly applicable method for tuning the chemistry of maleimide–thiol adducts remains in high demand.

We considered whether we could stabilize the maleimide–thiol adducts by simply applying force to the bonds. Tensile force is usually considered a destructive factor, as it can cause bond rupture31, but recently this force has been demonstrated to be productive if the compounds are properly designed32,33,34,35,36,37. For example, some force-responsive compounds (or mechanophores) with specific structural elements, such as strained rings38,39,40,41,42,43, weak bonds44,45,46,47 or isomerizable bonds48,49, are force-activable to change colour46, emit light44, release small molecules47 or trigger new reactions39 if an appropriate force is applied. Moreover, force can bias reaction pathways and generate products that are otherwise inaccessible through thermal- or light-activated processes39,50. A recent study further showed that the effect of force on the stability of a bond depends on the direction of the force relative to the reaction coordinate51. Inspired by these findings, we reasoned that the ring-open hydrolysis and retro-Michael reaction may have different force dependencies because the force directions with respect to the two reaction coordinates are altered by the five-membered succinimide ring. Although the force-accelerated rupture of maleimide–thiol adducts through the retro-Michael reaction is well recognized, we reasoned that if the ring-opening reaction can be kinetically accelerated over the retro-Michael reaction by force, we should unconventionally be able to obtain stable hydrolysates that are resistant to elimination.

Using atomic force microscopy (AFM)-based single-molecule force spectroscopy, we show here that force can largely stabilize maleimide–thiol adducts through ring-opening hydrolysis. This observation was verified at the bulk level by applying solvodynamic forces to the polymers containing maleimide–thiol conjugates, through ultrasonication. We further demonstrated the applicability of this method to stabilize maleimide–thiol linked antibody–polyethylene glycol (PEG) conjugates using ultrasonication. This method demanded neither additional chemical synthesis steps nor harsh reaction conditions. Therefore, we anticipate this method will be widely applicable in drug delivery, bio-labelling, surface modification and soft-materials manufacture. The discovery of these counter-intuitive force-stabilized maleimide–thiol adducts may also broaden the scope of using force as a productive means of accelerating chemical transformations.

## Results

### Distinct mechanical stability of unhydrolysed and hydrolysed maleimide–thiol adducts

AFM-based single-molecule force spectroscopy has been widely used to measure the mechanical strength of both weak biomolecular interactions (for example, receptor–ligand interactions and intercellular adhesion molecules) and strong chemical-bond cleavage52,53,54,55. We used this technique to directly measure the bond strength of maleimide–thiol adducts under different conditions to reveal the effect of a stretching force on the mechanical stability of the bonds. We show here that, in aqueous solution, the application of force to the unhydrolysed maleimide–thiol adducts can substantially accelerate ring-opening hydrolysis to the point where it is favoured over the retro-Michael pathway.

Three experiments were designed to apply a stretching force to the maleimide–thiol conjugate under various conditions (Fig. 1b): (1) anhydrous, where the maleimide–thiol conjugates are stretched in anhydrous acetonitrile, in which maleimide–thiol adducts cannot be hydrolysed and rupture can only occur via the retro-Michael pathway; (2) aqueous, where the maleimide–thiol adducts are stretched in neutral aqueous PBS, in which some newly formed adducts can be hydrolysed before the final detachment; (3) alkaline-pretreated, where the maleimide–thiol adducts are first completely hydrolysed by treatment with basic PBS and then stretched in neutral aqueous PBS. The detailed experimental conditions and surface modification schemes are provided in the Methods and Supplementary Fig. 1. Typical force traces from the three experiments are shown in Fig. 1c and Supplementary Fig. 2a–c. We have employed practical controls to ensure that these traces indeed correspond to single molecule events (Supplementary Note 1). Worm-like chain (WLC) fitting of the force curves (red curves) provided the contour length Lc (~35 nm) and the persistence length p (~0.38 nm, Supplementary Fig. 2d,e) of the PEG linker used to attach the maleimide and thiol groups to the cantilever tip and the substrate, which were consistent with the values reported in the literature for a single PEG chain56. The sharp peak on each retraction trace was assigned as the rupture peak for the maleimide–thiol adduct, which was further confirmed by a few control experiments using a quenching molecules-treated cantilever or different surface modifications (Supplementary Note 2 and Supplementary Figs. 3 and 4). The rupture-force histograms are presented in Fig. 1d. The contact time, pressure or attaching geometry of the maleimide–thiol conjugate between the cantilever tip and the substrate did not affect the rupture forces (Supplementary Figs. 5 and 6). We have also carried out single-molecule experiments using four repeats of protein GB1 as the fingerprint to unambiguously identify single molecule events57. The rupture forces measured in this way agreed well with those obtained using PEG linkers (Supplementary Note 3 and Supplementary Fig. 7). The rupture forces of newly formed thiosuccinimides in aqueous solution (900 ± 516 pN; hereafter written as mean ± s.d. unless otherwise indicated) were higher than those observed in anhydrous solution (389 ± 207 pN) at the same pulling speed (4 μm s−1), suggesting that some maleimide–thiol adducts were hydrolysed before being ruptured. The rupture forces (1,072 ± 359 pN) for the completely hydrolysed samples were the highest under these conditions and were independent of the solvent condition (Supplementary Fig. 8). Moreover, the force distribution obtained in an aqueous solution can be deconvoluted into two peaks corresponding to the retro-Michael pathway (black) and the rupture of the ring-opened hydrolysates of the maleimide–thiol adducts (green) (Fig. 1d). Note that, in aqueous solution, the maleimide–thiol adducts were stretched immediately after they were formed, yet a significant portion of the ring-opened hydrolysates were detected, indicating that a stretching force can reduce the reaction’s half-life from >100 h (ref. 23) to a number of seconds.

To further confirm that the hydrolysates gave rise to higher rupture forces, we performed the single-molecule experiments in aqueous solutions at different pH levels (pH 6.4, 7.4 and 8.1). As expected, more high rupture-force events were observed at higher pH levels due to the elevated rate of hydrolysis (Supplementary Fig. 9).

Because of the distinct reversibility of the two reaction pathways, the single-molecule pick-up ratios were dramatically affected by buffer conditions. In a continuous, long-term single-molecule force spectroscopy measurement conducted in anhydrous acetonitrile, cleavage of the thiosuccinimide underwent a reversible retro-Michael reaction, leading to low rupture forces with steady single-molecule pick-up ratios throughout the single-molecule experiment (Supplementary Fig. 10). However, the single-molecule pick-up ratio dropped gradually when the buffer was changed to aqueous PBS, indicating that the maleimide–thiol conjugates were irreversibly hydrolysed and the broken bonds were not able to reform (Supplementary Fig. 10 and Supplementary Note 4). It is worth mentioning that the gradual decrease in the single-molecule pick-up ratio in a timescale of a few hours was due to the force-activated hydrolysis of maleimide–thiol adduct instead of the spontaneous degradation of the maleimide in water, as the spontaneous degradation has a half-life of ~48 h (ref. 30). The single-molecule pick-up ratios of all experiments are provided in Supplementary Table 1.

### Force-dependent rupture kinetics of the unhydrolysed and hydrolysed maleimide–thiol adducts

Dynamic force spectroscopy was used to map the free-energy landscape of the force-induced rupture of the maleimide–thiol conjugates54. The rupture-force distributions of the newly formed (unhydrolysed) maleimide–thiol conjugates in acetonitrile and the hydrolysates in PBS at different loading rates are shown in Supplementary Fig. 11. The former data set corresponds to the retro-Michael pathway and the latter corresponds to the rupture of the ring-opened hydrolysates. The average rupture forces (f*) under both conditions increased logarithmically with increasing loading rates (Supplementary Fig. 12), and these data could be fitted by an equation derived from the Bell–Evans model58,59

$$f^ \ast = f_\beta {\mathrm{ln}}\frac{{r_{\mathrm{F}}}}{{kf_\beta }} = f_\beta {\mathrm{ln}}r_{\mathrm{F}} - f_\beta {\mathrm{ln}}kf_\beta$$

where fβ = kBT/∆x (kB is the Boltzmann constant, T is the temperature and ∆x is the extension of the bond along the force axis in the transition state), k is the rate constant at zero force, and rF is the loading rate. The fitting parameters are summarized in Supplementary Table 2. The value of ∆x for the retro-Michael cleavage is 0.559 Å, which is close to that of the rupture of the ring-opened hydrolysate (0.505 Å). The values of ∆x for both pathways are shorter than those of other mechanically activated reactions50,60,61, and they are close to the transition-state distance for the rupture of covalent bonds62. The rate constant of the hydrolysate obtained by the Bell–Evans model is 3.5 × 10−4 s−1, which is far lower than the retro-Michael reaction rate constant (2.1 s−1), indicating that the mechanical stability is enhanced by hydrolysis of the thiosuccinimide.

The parameters from dynamic force spectroscopy were further confirmed by the model-free analysis method introduced by Craig and Oesterhelt63,64 (Supplementary Fig. 13 and Supplementary Table 2). Moreover, the obtained kinetic parameters can be used to adequately reproduce the rupture force distributions at different force loading rates by Monte Carlo simulation (Supplementary Fig. 14).

The rupture-force distributions for the newly formed maleimide–thiol adducts at different loading rates can also be roughly fitted by two Gaussian distributions, corresponding to the retro-Michael reaction and the rupture of the hydrolysates (Supplementary Fig. 15). This further indicated that a substantial amount of thiosuccinimide was hydrolysed in PBS by force on an experimental timescale of <1 s.

### Force-dependent ring-opening hydrolysis of maleimide–thiol conjugates by pre-stretching

To elucidate how force accelerates the ring-opening hydrolysis of thiosuccinimide, we designed a pre-stretching experiment in aqueous solution (Fig. 2a). We found that applying a pre-stretching force did accelerate the ring-opening hydrolysis, leading to more stable hydrolysed maleimide–thiol conjugates. The final rupture-force distributions of the maleimide–thiol conjugates after different pre-stretching forces (from ~60 pN to ~600 pN for 100 ms) are summarized in Fig. 2b. The histograms can be deconvoluted by two Gaussian distributions corresponding to events from the retro-Michael reaction and the cleavage of hydrolysate, respectively. With the increase of pre-stretching force, the relative ratio between the two peaks decreased gradually, leading to an increase in the overall rupture forces. This indicates that the application of a pre-stretching force could accelerate the ring-opening hydrolysis. When a pre-stretching force of more than 300 pN was applied for 100 ms, the average rupture forces reached a plateau at ~1,200 pN, which was close to the rupture force of the hydrolysate (Fig. 2c). This indicated complete conversion of the thiosuccinimide to the hydrolysate by force. However, if the solvent was changed to anhydrous acetonitrile, the final average rupture force was close to the retro-Michael pathway and became independent of the pre-stretching force (Supplementary Fig. 16). Moreover, the final rupture force also increased with increasing pre-stretching time at different pre-stretching forces in aqueous solution (Fig. 2d).

Assuming that the hydrolysis of thiosuccinimide is a first-order reaction, the lifetimes of the substrate at different pre-stretching forces can be obtained by exponentially fitting the time-dependent pre-stretching data or bimodal fitting the pre-stretching distributions, as summarized in Fig. 2e. The lifetimes of the retro-Michael dissociation of the thiosuccinimide and the rupture of the hydrolysates at different forces obtained from dynamic force spectroscopy experiments are also plotted in the same figure for comparison. All lines were generated based on the Bell–Evans model. At forces lower than 270 pN, the retro-Michael reaction is the major pathway (Fig. 2e, inset), and force destabilizes the maleimide–thiol adducts. However, if the stretching forces are higher than 270 pN, the hydrolysis pathway becomes dominant. Once the thiosuccinimide is hydrolysed, the lifetime of the adduct is greatly increased (Fig. 2e), leading to force-stabilized maleimide–thiol conjugates.

### Force-induced ring-opening hydrolysis of maleimide–thiol adducts by ultrasound

To further confirm that force can induce hydrolysis of maleimide–thiol adducts and lead to stable conjugates at the bulk level, we directly characterized the force-induced hydrolysates at the bulk level using ultrasound-based mechanochemistry. The solvodynamic shear force along the polymer chain in solution was generated by solvent cavitation, or the rapid nucleation, growth and collapse of microbubbles under ultrasound, which has been used to explore a number of mechanically responsive polymers65.

We coupled a maleimide-terminated PEG (Mw of 5 kDa) and a thiol-terminated PEG (Mw of 5 kDa) to yield P1, which contained a maleimide–thiol adduct in the middle (see Supplementary Methods for details) (Fig. 3a). The small-molecule maleimide–thiol adduct, S1, was also synthesized as a control by coupling N-ethylmaleimide and 2-mercaptoethanol. P1 and S1 were treated with basic PBS buffer (pH 8.1) at 37 °C for 5 days until the ring-opening process was complete to obtain completed hydrolysed products AP1 and AS1, respectively, for comparison. Ultrasound-treated P1, UP1, was obtained by applying pulsed ultrasound for just 30 min (Fig. 3a).

These small-molecule compounds and polymers were then characterized by Fourier-transform infrared (FT-IR) and 1H NMR spectroscopy. FT-IR spectroscopy showed the appearance of new transmittance peaks at 1,695, 1,650 and 1,550 cm−1 (two asymmetric carbonyls after ring-opening and the NH of the CONH moiety, respectively) after alkaline treatment (AS1), whereas the spectrum of the as-synthesized model compounds (S1) showed only one peak at 1,685 cm−1 in this region (two symmetric carbonyls on the maleimide five-membered ring) (Supplementary Fig. 17). The spectra of the polymers containing a central thiosuccinimide after alkaline treatment (AP1) or ultrasonication (UP1) also showed a peak at 1,650 cm−1, whereas the peak at 1,550 cm−1 was covered by the peak from the PEG (Fig. 3b). 1H NMR spectroscopy could not be used to identify the hydrolysis product, as the signal from the PEG chain dominated the 3.0–4.0 ppm region, masking the signals from the hydrolysed thiosuccinimide (Supplementary Fig. 18).

To separate the signals from the thiosuccinimide and PEG in the 1H NMR spectra, another model compound was synthesized, P2, containing two symmetric thiosuccinimides with adjacent benzene rings, along with its small-molecule analogue (S2) (see Supplementary Methods for details). Formation of the Michael-type adduct (S2 and P2) and its ring-opened hydrolysate (AS2 and AP2) can easily be identified in the 1H NMR spectra, providing a facile means of following the addition and hydrolysis reactions (Fig. 3c). Following the addition of 2-mercaptoethanol and thiol-PEG, the aromatic protons shifted downfield and upfield, respectively (two peaks centred at 7.370 and 7.190 ppm), while the 7.165 ppm peak vanished, indicating production of the thiosuccinimide. After alkaline treatment, the ring-opened product (AS2 and AP2) was evidenced by the shifts in aromatic protons from 7.370 and 7.190 ppm to 7.475 and 7.146 ppm, respectively (Fig. 3d and Supplementary Fig. 19). Trace amounts of ring-opened hydrolysates were observed before any treatment due to the hydrolysis of P2 by moisture in the air. The two thiosuccinimide rings can be hydrolysed either symmetrically or asymmetrically, leading to the splitting of each resonance peak to four separate peaks. Following ultrasonication, similar chemical shifts were observed for ultrasound-treated P2, UP2 (Fig. 3d). Approximately 75% of the thiosuccinimide in the polymers was mechanically hydrolysed by ultrasonication (based on the integrals of the b and d peak areas, Supplementary Fig. 20), while a portion of the thiosuccinimide underwent retro-Michael cleavage as indicated by the appearance of small peaks at ~7.310 ppm. This finding was further confirmed by UV–vis (Supplementary Fig. 21) and FT-IR spectra, which showed the appearance of new transmittance peaks between 1,500 and 1,700 cm−1 (Supplementary Fig. 22). Furthermore, the hydrolysis reaction was confirmed by the pH change of the solution after ultrasonication (Supplementary Fig. 23). The hydrolysis generated an additional hydroxyl group, which is expected to decrease the pH of the solution.

NMR spectra also show that the force-induced ring-opening might give two regio-isomers (four peaks splitting of the aromatic protons), while alkaline treatment favours breaking the distal, instead of proximal, N–C bonds (two peaks splitting of the aromatic protons) (Fig. 3c and Supplementary Fig. 19). In the alkaline treatment, the distal N–C bond has less steric hindrance than the proximal one for attack by OH. Therefore, the major product is the distal N–C bond hydrolysed product. However, force may greatly change the reactivity of the two N–C bonds, leading to a hydrolysis rate of the proximal bond comparable to the distal one in ultrasound experiments.

### Force-induced hydrolysis improves the stability of maleimide–thiol-based antibody–PEG conjugates

Under physiological conditions, the relatively high concentration of exogenous thiol nucleophiles (R-SH) (for example, cysteine, glutathione and serum albumin) slowly reduces the loading efficiency, which becomes the key therapeutic limitation of maleimide–thiol-based antibody conjugates13,23,25,26,27,29,30. Encouraged by the finding that solvodynamic shear forces counter-intuitively strengthen and stabilize maleimide–thiol conjugates in polymers, we endeavoured to engineer stable maleimide–thiol-based antibody–PEG (Ab-PEG) conjugates via ultrasonication (Fig. 4a).

Compared with the precise stretching forces provided by AFM at the single-molecule level, the solvodynamic shear forces generated by ultrasound are not homogeneous65 and may lead to bond scission through the retro-Michael pathway, as seen for P2 (Fig. 3d). Fortunately, because the Michael addition and retro-Michael reaction are reversible, the dissociated maleimide–thiol adducts can re-form in the absence of force (Fig. 1a). Thus, we used excess maleimide–PEG and multiple ultrasonication cycles to generate Ab–PEG conjugates with a majority of the maleimide–thiol linkers hydrolysed.

We used trastuzumab, a major antibody targeting Her2-positive breast cancer, as the model antibody. Trastuzumab was reduced to expose free cysteine residues. Then, the reduced antibody was treated with maleimide–PEG to yield the Ab–PEG conjugates. A dilute solution of the purified Ab–PEG conjugates (~2 mg ml−1) was subjected to pulsed ultrasound (switched on and off every second) in the presence of a 10-fold excess of maleimide–PEG (Mw, 5 kDa) for 30 min in an ice-water bath to generate the ultrasound-treated sample. The sample was then purified and condensed to the same concentration as that of the untreated sample. The stabilities of the ultrasound-treated and untreated Ab–PEG samples were evaluated in vitro in a PBS solution (pH 7.4) containing 1 mM of reduced glutathione at 37 °C. The binding curve of ultrasound-treated Ab–PEG was similar to untreated conjugates as well as the unmodified trastuzumab. The half maximal effective concentrations (EC50) of ultrasound-treated and untreated Ab–PEG were in a range of 0.03–0.04 nM, on the same scale as that of the unmodified trastuzumab (0.02 nM) (Fig. 4b). Enzyme-linked immunosorbent assay (ELISA) results indicate that ultrasonication did not cause the unfolding and loss of function of the antibody conjugates. The loss of PEG chains from the antibody can be evaluated by the decrease in molecular weight based on western blot analysis (Supplementary Fig. 24). The Ab–PEG samples not subjected to ultrasonication lost ~50% of their payload in one week, whereas the Ab–PEG samples treated by ultrasound retained over 90% of their payload over the same period for both light and heavy chains (Fig. 4c). The stability of ultrasound-treated antibody conjugates was comparable to that of the alkaline-treated samples29 (Supplementary Fig. 25). These results clearly show that force-induced ring-opening hydrolysis by ultrasonication can be used as an efficient means of improving the stability of maleimide–thiol-based antibody conjugates.

## Discussion

In this work, we report the force-induced hydrolysis of maleimide–thiol adducts, leading to products with improved mechanical and chemical stabilities under physiological conditions. Due to the presence of a competing retro-Michael reaction, this reaction pathway is not accessible in the absence of force and only becomes dominant when the applied force reaches a certain threshold.

Although there are many previous theoretical and experimental examples in which force is used to alter reaction pathways, the mechanism of the force-induced hydrolysis of maleimide–thiol adducts presented here is fundamentally different. In this reaction, force does not change the reaction mechanism of either the retro-Michael reaction or the hydrolysis of the thiosuccinimide, but it differentially alters their reaction kinetics. The selectivity of the final product is kinetically controlled instead of thermodynamically controlled due to the force-dependent kinetics of the two competing reaction pathways. In other words, our results show that the application of force can selectively favour the pathway that shows a stronger force-dependency. If the effects observed in this work can be shown to be general, then such a mechanism of force-dependent kinetic control expands our current understanding of using force to control mechanochemistry and may inspire the implementation of different mechanophores in the same polymer to achieve a complex mechanical response. It is expected that the activation of multiple mechanophores can be finely controlled by the amplitude and direction of force.

The discovery of the stabilization of maleimide–thiol adducts by applying a stretching force provides a potentially convenient solution to the instability of maleimide–thiol conjugates by simple ultrasonication. Notably, the apparent solvodynamic shear forces introduced by ultrasonication depend largely on the size of the polymers and proteins, as well as the position of the maleimide–thiol linkage39,65. On ultrasonication, although the forces exerted on the thiosuccinimide ring can be as high as a few hundred piconewtons35, the forces on the antibody protein are much lower because they are not at the centre of the adducts, and they are in a compact folded conformation. We did not observe any measurable loss in the functionality of the protein–polymer conjugates following the ultrasonication procedure used in our experiments. Moreover, the undesired retro-Michael reaction products can be avoided by using excess maleimide-containing polymers. Despite these advantages, practically controlling the force applied to the maleimide–thiol adducts for different systems remains challenging. Rigorous optimization of this method is currently underway for practical translation.

## Conclusion

We discovered that the application of force to maleimide–thiol adducts can counter-intuitively increase their mechanical and chemical stability. This was achieved through the force-accelerated ring-opening hydrolysis of the thiosuccinimide ring, which generates a product that is resistant to retro-Michael reactions and thiol exchange. Using single-molecule force spectroscopy, we have quantitatively illustrated how and to what extent force can favour one of the two competing pathways and afford products that are not observed in thermally activated pathways. Our proof-of-principle experiments also demonstrated that this force-controlled reaction may be used to produce stable antibody–polymer conjugates using ultrasound-induced solvodynamic force. We envision that other types of force are also applicable to stabilize maleimide–thiol conjugates, including hydrodynamic force from flow, compressive or stretching forces from materials deformation, and the friction forces at diverse interfaces. The concept of using force-dependent kinetic control to obtain products inaccessible by conventional chemical reaction pathways may also greatly extend the current scope of mechanochemistry.

## Methods

### Single-molecule AFM measurements

Single-molecule force spectroscopy experiments were carried out on a commercial AFM instrument (JPK Force Robot 300) at room temperature (~22 °C). Standard silicon nitride (Si3N4) cantilevers were purchased from Bruker (type: MLCT). D cantilevers (spring constant of ~0.05 N m−1) were used in all experiments, and the spring constant was calibrated using an equipartition theorem for each experiment. Three experiments were undertaken in different solvents to measure the rupture force of the thiosuccinimide and its hydrolysate. In the first two experiments, a maleimide-functionalized cantilever was brought into contact with a thiol-functionalized glass substrate at a set-point force of 1.0 nN and left there for 1.0 s to trigger the formation of a maleimide–thiol conjugate between the cantilever tip and the substrate. In the third experiment, the maleimide–thiol conjugate-functionalized substrate was incubated in basic PBS buffer (pH 8.1, 5 days, 37 °C) until the five-membered ring was completely hydrolysed. An NHS (N-hydroxysuccinimide)–PEG functionalized cantilever was used to pick up the amino group at the very end of the hydrolysate-containing linker using a constant contact force of 1.0 nN for 1.0 s. The cantilever was then retracted from the substrate at a constant speed of 4.0 μm s−1 to obtain the force–extension curves. All force curves were collected by commercial software (JPK) and analysed using a custom-written protocol in Igor 6.35 (Wavemetrics). Details on the AFM measurements and analysis procedures are provided in the Supplementary Information.

### Pre-stretching single-molecule force spectroscopy experiments

The pre-stretching single-molecule force spectroscopy experiments were carried out using a maleimide-functionalized cantilever and a thiol-coated substrate in anhydrous acetonitrile and PBS (pH 7.4) solution. The maleimide-functionalized cantilever was first brought into contact with the thiol-coated glass surface at a constant force of 1.0 nN for 1.0 s. The cantilever was then retracted to a preset force (pre-stretching force) and held there for a certain duration (pre-stretching time). Next, the cantilever was relaxed to zero force (close but not touching the substrate). Finally, the cantilever was fully retracted to rupture the maleimide–thiol conjugate. The pre-stretching force was set between 60 and 600 pN, and the pre-stretching time was varied from 50 ms to 2.0 s. The relaxation and stretching speeds were 4.0 μm s−1. Events that did not survive the pre-stretching procedure were excluded from data analysis. Because the molecule was always relaxed to zero force before the final stretch, the pre-stretching protocol does not artificially shift the force population to a higher force if no chemical reaction occurs during the pre-stretching. (Supplementary Fig. 26)

### Synthesis and characterization of maleimide–thiol-containing model compounds and polymers

Model compound small-molecule adducts S1 and S2, polymers P1 and P2 and their alkaline-treated hydrolysates (AS1, AS2, AP1, AP2) were synthesized following previous reports1,23,29 (for details see Supplementary Information). Compounds and polymers were dissolved at high concentrations (~20 mg ml−1 for small-molecule compounds and ~100 mg ml−1 for polymers) before chemical characterization. 1H NMR spectra were recorded on a Bruker 500 MHz NMR spectrometer in dimethyl sulfoxide-d6 (DMSO-d6). 1H NMR spectra of the polymers were merged from more than 128 scans to improve the signal-to-noise ratio. FT-IR spectra were recorded on a Nicolet iS50 FT-IR (Thermo Scientific) with ATR (attenuated total reflection). Samples for FT-IR were dissolved in CDCl3, and ~20 µl of solution was added to the detector after baseline correction. The spectra were recorded from 400 to 4,000 cm−1 with a bandwidth of 0.4 cm−1.

### Pulsed ultrasonication

Ultrasonication was performed using an ultrasonic apparatus XQ-1000D operating at 20 kHz with a 6 mm tip probe. The homemade Suslick-like cells were oven-dried before use. The ultrasonication experiments were carried out in ~10 ml PBS (pH 7.4) containing 2.0 mg ml−1 of P1 or P2 in an ice/water bath to maintain a temperature of ~6–9 °C during ultrasonication. Nitrogen was introduced into the cell using a syringe needle, and the solution was degassed with bubbling N2 for 30 min prior to and during each experiment. The ultrasound was pulsed (1.0 s on and 1.0 s off, 11.81 W cm−2) in each experiment to prevent the temperature from increasing. After 30 min of ultrasonication (total ultrasound time of 15 min), the resulting solution was withdrawn from the Suslick-like cell and filtered through a 0.22 µm syringe filter. The crude product was purified using a desalination column. Purified fractions were collected and freeze-dried before characterization.

### Stability of the Ab–PEG conjugate

The monoclonal antibody anti-Her2 trastuzumab was diluted to 200 μM in PBS (pH 7.4, containing 2 mM EDTA). The resulting antibody was treated with ~4–5 equiv. of tris(2-carboxyethyl)phosphine hydrochloride (TCEP, 10 mM in distilled water) and incubated at 37 °C for 2 h. The reduced antibodies with a mixture of heavy chains (containing three free cysteine groups) and light chains (containing one free cysteine group) were treated with 20 equiv. of maleimide–PEG (Mw, 5 kDa). The reaction was stirred at 4 °C for 12 h to generate the Ab–PEG conjugates. Due to the steric hindrance, we obtained the Ab–PEG with different conjugation ratios. For the heavy chain, the conjugation ratios were in the range of 0–3. For the light chain, the conjugation ratios were in the range of 0–1.

The conjugates were diluted to ~2.0 mg ml−1 and combined with a 10-fold excess of maleimide–PEG (Mw, 5 kDa). The solution used for pulsed ultrasonication was kept in an ice/water bath and degassed with bubbling N2 for 30 min before as well as during the experiment. The ultrasound treatment lasted for 30 min (11.81 W cm−2; switched on and off every second). Both the ultrasound-treated and untreated conjugates were purified using Millipore ultrafiltration centrifugal tubes and then concentrated to ~25 μM. The constructions of the Ab–PEG conjugates were reproducible in phosphate-free buffer (Tris buffer saline, pH 7.4) (Supplementary Fig. 27).

The stability of the ultrasound-treated and untreated Ab–PEG samples was evaluated in vitro at a concentration of 20 μM in a PBS solution (pH 7.4) containing 1 mM reduced glutathione. GB1 protein (Mw, ~8 kDa) as an internal reference was added into the solutions to monitor the protein degradation. The solutions were filtered using a 0.22 μm filter and incubated at 37 °C. At each time point, a 100 μl aliquot was removed and frozen at −80 °C. After the final time point, the binding activity of samples from day 0 and day 6 were evaluated by ELISA assay. All samples were fully reduced before the western blot analysis. The loss of PEG chains from the antibody could be evaluated by the decrease in molecular weights based on western blot analysis using Gelpro. The average drug:antibody ratios (DAR) of the light chains and heavy chains were obtained and the Ab–PEG remaining (%) each day was calculated and normalized to the average DAR at day 0. Details on the ELISA and western blot analysis are provided in the Supplementary Information.

### Statistics and reproducibility

The numbers of biological replicates are indicated in the figure legends and Methods. Data are presented as mean ± s.d., as indicated in the legends of the figures and Supplementary figures. Student’s t-test was performed with GraphPad Prism 5.0. The reproducibility of repeated independent experiments is indicated in the legends of the figures and Supplementary figures.

### Reporting summary

Further information on research design is available in the Nature Research Reporting Summary linked to this article.

## Code availability

Igor procedures for single-molecule experiment analyses of this study are available from the corresponding author upon request.

## Data availability

All data generated and analysed during this study are included in this article and its Supplementary Information, and are also available from the authors upon reasonable request.

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## Acknowledgements

This research is supported mainly by the National Natural Science Foundation of China (grants nos. 21522402, 11674153, 11374148, 21774057 and 11334004) and the Fundamental Research Funds for the Central Universities (grant no. 020414380080). The authors thank Y. Li for discussions.

## Author information

Authors

### Contributions

Yi.C., W.H. and X.G. conceived the project and designed the experiments. W.H., X.G. and H.L. performed the single-molecule experiments and analysed the data. W.H., Y.Y., Z.Z., Y.L.C. and Y.S. performed the ultrasound experiments and analysed the data. W.H., X.W. and Y.Y. performed the antibody stability experiments. Yi.C., W.W. and M.Q. supervised the project. W.H. and Yi.C. wrote the paper with contributions from all authors.

### Corresponding authors

Correspondence to Wei Wang or Yi Cao.

## Ethics declarations

### Competing interests

The authors declare no competing interests.

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## Supplementary information

### Supplementary Information

Supplementary Methods, Supplementary Notes, Supplementary Figures 1–27, Supplementary Tables 1 and 2.

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Huang, W., Wu, X., Gao, X. et al. Maleimide–thiol adducts stabilized through stretching. Nat. Chem. 11, 310–319 (2019). https://doi.org/10.1038/s41557-018-0209-2

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