# Development of Low-Nanomolar Covalent Epoxide Inhibitors of Tubulin Detyrosinating Enzymes VASH1&2

Anthony Feral, Guillaume Marcellin, Yoann Lannay, Camille Hennebert, Yazid Souf, Aicha Talha, Aline Haetty, Matthieu Simon, Pascal Verdié, Maxime Louet, et al.

<figure><img src="image_1.png" /></figure>

## To cite this version:

Anthony Feral, Guillaume Marcellin, Yoann Lannay, Camille Hennebert, Yazid Souf, et al., *Development of Low-Nanomolar Covalent Epoxide Inhibitors of Tubulin Detyrosinating Enzymes VASH1&2*. Journal of Medicinal Chemistry, 2025, 68 (17), pp.18335-18355. 10.1021/acs.jmedchem.5c00980. *hal-05328434*

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# Development of Low-Nanomolar Covalent Epoxide Inhibitors of Tubulin Detyrosinating Enzymes VASH1&2

Anthony Feral<sup>2,1</sup>, Guillaume Marcellin<sup>2</sup>, Yoann Lannay<sup>2</sup>, Camille Hennebert<sup>1</sup>, Yazid Souf<sup>1</sup>, Aicha Talha<sup>1</sup>, Aline Haetty<sup>3</sup>, Matthieu Simon<sup>1</sup>, Pascal Verdié<sup>1</sup>, Maxime Louet<sup>1</sup>, Khaled Hached<sup>2</sup>, Muriel Amblard<sup>1</sup>, Krzysztof Rogowski<sup>3*</sup>, Siem van der Laan<sup>2*</sup>, Lubomir Vezenkov<sup>1*</sup>

## Abstract:

The development of small molecule inhibitors targeting the set of microtubules' post-translational modifications, also known as the "tubulin code", remains an underexplored area in medicinal chemistry. The recent discovery of the VASH1 and VASH2 enzymes, which are responsible for tubulin detyrosination—a modification associated with neurodegeneration and cancer—prompted us to develop new molecules that inhibit their activity. In this study, we conducted the first QSAR analysis of VASH proteases. Through rational substrate-based design, we identified our lead compound, LV87, as a potent epoxide-based covalent inhibitor of tubulin detyrosination in *cellulo*. Specificity assessments against other cysteine proteases and incubations with nonspecific nucleophiles demonstrated that LV87 selectively targets VASH1/2. Additionally, safety data, serum and microsome stability tests support the notion that LV87 is a selective and potent inhibitor of tubulin detyrosination, paving the way for further optimization and applications.

## INTRODUCTION

Microtubules (MTs) are important cytoskeletal elements of all eukaryotic cells and are particularly abundant in neurons, where they play a critical role in regulating cellular structure and function over time.<sup>1</sup> MTs contribute to development and wiring of the central nervous system by actively controlling the morphology of neurons, which typically have a single long axon involved in transmitting electrochemical impulses and numerous dendrites that receive them.<sup>2</sup> The rigidity and stability of axonal MTs contribute to the active intracellular cargo transport over long distances orchestrated by motor proteins. A growing body of evidence suggests that a wide variety of post-translational modifications (PTMs) of MTs, mostly consisting of the addition or removal of amino acids (AA) on the C-terminal tails protruding at the surface of MTs, actively regulate the direction and pace of intracellular cargo transport.<sup>3-5</sup> Arguably tubulin detyrosination, that consists of proteolytic removal of the hydrophobic C-terminal tyrosine (Tyr) residue from α-tubulin (Figure 1A), represents one of the most functionally relevant MTs modification. This is most likely due to the major changes in the physicochemical properties at the surface of MTs due to removal of this hydrophobic AA, which unmasks negatively charged glutamates. Tubulin detyrosination is reversible and ligation of a Tyr residue on the detyrosinated C-terminus of α-tubulin is achieved by tubulin tyrosine ligase (TTL).<sup>6</sup>

<footnote n="1"><sup>1</sup> IBMM, Université de Montpellier, CNRS, ENSCM 34293 Montpellier, France</footnote>

<footnote n="2"><sup>2</sup> MT-act, CRBM, Montpellier 34293, France</footnote>

<footnote n="3"><sup>3</sup> IGH, CNRS, Université de Montpellier, 34090 Montpellier, France</footnote>
<figure><img src="image_2.png" /><figcaption>**Figure 1.** (A) Schematic representation of α-tubulin in VASH enzymes active sites. The substrate position preferences are illustrated as reported in the publications of Nieuwenhuis *et al.* 2017 and Adamopoulos *et al.* 2019. (B) Schematic representation of EpoY in VASH enzymes active sites. (C) Schematic representation of 11 in VASH enzymes active sites.</figcaption></figure>

While tubulin detyrosination was originally described over 40 years ago, the first class of enzymes responsible for this modification were only discovered in 2017. Using different approaches, two independent studies found that members of the vasohibin family, VASH1 and VASH2, act as tubulin detyrosinases in humans.⁷,⁸ More recently, the discovery of a second type of detyrosinating enzyme named MATCAP/TMCP1 has been reported by two independent labs.⁹⁻¹⁰ However, unlike VASHs which are cysteine proteases, the newly-identified detyrosinase is a metalloprotease. Furthermore, knockout studies in mice revealed that MATCAP/TMCP1 has only a minor contribution to the overall detyrosination level, which is mostly generated by VASHs.¹⁰

The VASH1/2 enzymes (VE) are unusual cysteine (Cys) proteases with a non-canonical active triad in the catalytic cleft consisting of three residues: leucine (Leu), histidine (His) and Cys. Their catalytic pockets are highly similar as highlighted by superposition of their X-ray structures (see Figure SI.1C). In the context of recombinant proteins, the enzymatic activity of VE is increased by interaction with a chaperone protein termed small vasohibin-binding protein (SVBP). While SVBP binding is essential for VASH1 activity, VASH2 exhibits low tubulin detyrosinase activity even in the absence of SVBP.¹¹ Of note, knockout animals lacking the reverse enzyme TTL are not viable due to abnormally high levels of

<figure><img src="image_3.png" /><figcaption>**Chart 1.** Identification of compound 11 via substrate-based VASH enzyme design.</figcaption></figure>
tubulin detyrosination that are responsible for severe defects in neuronal wiring of the brain.¹² The importance of detyrosination in neurons was further supported by a recent report showing an increase in the levels of this modification in the brains of Alzheimer's patients that correlates with disease progression.¹³

In this context, we aimed to develop efficient VE inhibitors using rational substrate-based design. VE recognize and cleave the C-terminal Tyr residue of α-tubulin, which is followed by two anionic glutamic acid (Glu) residues then a glycine (Gly) and another Glu residue (Figure 1A).⁸,¹⁴ Articles by Nieuwenhuis et al.⁸ (2017) and Adamopoulos et al.¹⁴ (2019), that were published at the beginning of our QSAR program, showed that another aromatic amino acid, the Phenylalanine (Phe), was tolerated at position S₁'. Interestingly the Glu residue at position S₁ could be substituted by non-charged Alanine or hindered Proline. Finally, an anionic Glu residue at position S₂ was crucial for the substrate recognition. As our hit molecule, we selected the previously designed irreversible VE inhibitor **EpoY** (Figure 1B and compound 1 in Chart 1) which was instrumental in identifying VE.⁷ This inhibitor comprises the crucial C-terminal Tyr residue connected to the electrophilic ethyl (S,S)trans-2,3-epoxysuccinate (TES) moiety.⁷,¹¹ Crystal structures of VE showed that the TES group forms a covalent bond with the thiol function of the Cys residue present in VE active sites (Figure SI.2). This binding mechanism results in an irreversible VE inhibition with a moderate IC₅₀ of about 20 µM on human CHL-1 cells (Table 1), which were shown to be >95% dependent on VE for tubulin detyrosination.⁸ Irreversible inhibitors offer several advantages including long target deactivation times and thus lower therapeutic doses,¹⁵⁻¹⁸ as highlighted by the recent success of several covalent tyrosine kinase inhibitors.¹⁹,²⁰ Nevertheless, a small epoxide molecule such as **EpoY** might hypothetically interact with free thiols present on other proteins including in their active site²¹ or with other nucleophilic residues such as primary amines present in the lysine (Lys) residues.²² Therefore, improving the specificity, potency, and bioavailability of **EpoY** is needed for conducting studies on the cellular role of VE and for further considerations in biomedical applications. Our research aims to address these challenges and develop more effective VE inhibitors based on the **EpoY** scaffold.

# RESULTS AND DISCUSSION

## Substrate-based design of VE inhibitors

Our strategy to develop potent VE inhibitors was centered on improving the previously described **EpoY** by substrate-based design (Chart 1). At the beginning of this study, no 3D structures of the enzymes were available, preventing design by *in silico* structural analysis. Consequently, the design of the inhibitors was based on the structure of **EpoY** and the C-terminal tail of α-tubulin. The C-terminal tail of α-tubulin contains a GlyGluGluTyr motif with two anionic Glu residues at positions P₁ and P₂ (Figure 1A), whereas **EpoY** features an epoxide function at position P₁, and an ethyl ester function instead of the Glu residue at position P₂, with the P₁' Tyr residue being conserved (Figure 1B, Chart 1). We hypothesized that the addition of a second anionic residue in place of the ethyl ester group could engage in ionic or hydrogen bond interactions at positions S₁ or S₂ of the VE. Thus, we substituted the ethyl ester group of **EpoY** with Glu residue to obtain compound 2 (Chart 1) as a first modification in our QSAR study. During the first phase of the optimization process, we used racemic (+,-)-TES instead of the enantiomerically pure (S,S)-TES or (R,R)-TES to allow faster screening of the VE inhibitors. Compounds were initially screened using our well-characterized recombinant detyrosination assay with VASH2. Compound 2 showed a significantly lower VASH2 inhibition compared to **EpoY**, with an IC₅₀ of 41.63 µM. This result was not completely surprising, as the rigid TES moiety added an additional carbon and a specific steric constraint, compared to the Glu residue at position P₁ of the α-tubulin
sequence. Thus, the introduced second Glu residue at position P₂ in compound **2** might not be at the required distance to form favorable molecular interactions inside the VE active sites. Moreover, it can even create unfavorable interactions that can explain the loss of inhibitory activity of compound **2**.

<table><caption>**Table 1.** Structures, inhibition data and physicochemical properties of the substrate-based designed compounds.</caption><thead><tr><th rowspan="2">Compound</th><th rowspan="2">Structure</th><th rowspan="2">MW (g/mol)</th><th colspan="2">IC<sub>50</sub> (µM)</th><th rowspan="2">Rt<sup>c</sup> (min)</th><th rowspan="2">LogD<sub>7.4</sub> LogP<sup>d</sup></th></tr><tr><th>In vitro<sup>a</sup></th><th>In cell.</th></tr></thead><tbody><tr><td>EpoY (1)</td><td><img src="epoY1.png" alt="Chemical structure of EpoY (1)"></td><td>323.3</td><td>VASH1 0.21<br>VASH2 1.08</td><td>20.7</td><td>1.73</td><td>LogD<sub>7.4</sub> = N/A<sup>e</sup><br>LogP = -0.29±0.00</td></tr><tr><td>2</td><td><img src="epoY2.png" alt="Chemical structure of EpoY (2)"></td><td>423.4</td><td>41.63</td><td>X<sup>b</sup></td><td>0.84</td><td>-</td></tr><tr><td>3</td><td><img src="epoY3.png" alt="Chemical structure of EpoY (3)"></td><td>366.3</td><td>7.24</td><td>X<sup>b</sup></td><td>1.30</td><td>-</td></tr><tr><td>4</td><td><img src="epoY4.png" alt="Chemical structure of EpoY (4)"></td><td>380.4</td><td>10.08</td><td>X<sup>b</sup></td><td>1.43</td><td>-</td></tr><tr><td>5</td><td><img src="epoY5.png" alt="Chemical structure of EpoY (5)"></td><td>394.4</td><td>3.15</td><td>19.9</td><td>1.51</td><td>-</td></tr><tr><td>6</td><td><img src="epoY6.png" alt="Chemical structure of EpoY (6)"></td><td>408.4</td><td>3.48</td><td>80.4</td><td>1.68</td><td>-</td></tr><tr><td>7</td><td><img src="epoY7.png" alt="Chemical structure of EpoY (7)"></td><td>365.3</td><td>13.1</td><td>74.7</td><td>1.21</td><td>-</td></tr><tr><td>8</td><td><img src="epoY8.png" alt="Chemical structure of EpoY (8)"></td><td>379.4</td><td>23.31</td><td>67.8</td><td>1.26</td><td>-</td></tr><tr><td>9</td><td><img src="epoY9.png" alt="Chemical structure of EpoY (9)"></td><td>393.4</td><td>6.97</td><td>66.3</td><td>1.33</td><td>-</td></tr></tbody></table>
<table><tr><td>10</td><td><img src="image_10.png" alt="Chemical structure of 10"></td><td>407.4</td><td>10.05</td><td>X<sup>b</sup></td><td>1.43</td><td>-</td></tr><tr><td>11</td><td><img src="image_11.png" alt="Chemical structure of 11"></td><td>437.4</td><td>2.13</td><td>26.1</td><td>1.31</td><td>-</td></tr></table>

<sup>a</sup>Assays were made for VASH2 except if mentioned. See Figure SI.7 for graphics of inhibitory activity. <sup>b</sup>X: no inhibition observed. <sup>c</sup>Retention time obtained using a Waters UPLC-MS. H<sub>2</sub>O/Formic Acid (1000/1) (v/v) and AcN/Formic Acid (1000/1) (v/v) solutions as mobile phase and C-18 reversed phase chromatography column. <sup>d</sup>See Figure SI.8 and SI.9 for chromatograms and raw data. <sup>e</sup>Not applicable.

To determine the optimal length of the hydrocarbon spacer between the TES-Tyr moiety and the introduced carboxylic acid function, a series of compounds (3 to 6) were synthesized, with the spacer length ranging from 2 to 5 methylene groups respectively (Table 1 and Chart 1). Although the analogues incorporating the shortest spacers, compounds 3 and 4, exhibited improved in vitro detyrosination inhibition of VASH2 as compared to compound 2, they remained less potent than the hit compound EpoY. The two most efficient compounds of this series were 5 and 6 containing spacers of 4 and 5 methylene groups respectively. Interestingly, when the COOH function at the R<sub>2</sub> position is substituted with an amide moiety (compounds 7 to 10), the in vitro activity was reduced by a factor ranging from 1.8 to 2.9. These results support our hypothesis that an anionic residue was required at position R<sub>2</sub> for better interaction with VE, leading to improved inhibitory activity. Compound 5 was further modified by substituting the non-chiral aminopentanoic acid moiety with the chiral α-amino adipic acid (Aad) residue, generating compound 11 with an IC<sub>50</sub> of 2.13 µM in vitro (Table 1 and Chart 1). Interestingly, compounds EpoY, 5, and 11 exhibited quite similar inhibitory activities in vitro (IC<sub>50</sub> of 1.08, 3.15, and 2.13 µM, respectively). While the activities of the majority of EpoY analogues were very low in cell assays, in the case of compounds 5 and 11 they were comparable with the IC<sub>50</sub> value of EpoY (Table 1). More importantly, although compound 11 does not show superior inhibitory activity in vitro and in cellulo compared to EpoY, it allows for the introduction of a series of new modifications. Therefore, this compound represents an easily modifiable starting point for further derivatization, notably at positions R<sub>3</sub> (Chart 2). Indeed, different substitutions could simply be linked via an amide bond at this position to search for further interactions within the S<sub>3</sub> and S<sub>4</sub> pockets of the VE's active sites (Figure 1C). Interactions at these positions are reported to provide both activity and selectivity to protease inhibitors for their respective targets.<sup>23,24</sup> The publication of several crystal structures of VE bound to EpoY in 2019 (Figure SI.2)<sup>25,26</sup>, significantly contributed to the enhancement of compound 11 through a rational design approach using molecular docking studies (AutoDock4.6 software) and structural analysis of VE catalytic pocket (UCSF Chimera1.15 software).

## Molecular Docking Studies for the improvement of compound 11
In this study, we first performed a docking simulation of **EpoY** prior to the ring opening reaction within the active site. Interestingly, we observed that the intact epoxide ring only slightly altered the position of the ester moiety of the docked inhibitor as compared to co-crystallized **EpoY** (Figure 2A and SI.2). Docking simulation of compound 11 positioned in the catalytic cleft of VASH2 highlighted several

<figure><img src="image_4.png" /><figcaption>**Figure 2.** In silico drug development studies. (A) Selected low-energy poses for **EpoY** (in gold) in VASH2 and 2D diagram of their interactions with VASH2 amino-acids. (B) Selected low-energy poses for compound 11 (in gold) in VASH2 and 2D diagram of their interactions with VASH2 amino-acids. (C) Selected low-energy poses for **LV80** (in gold) in VASH2 and 2D diagram of their interactions with VASH2 amino-acids. Residues in 3D views are colored accordingly to 2D diagrams, cyan-colored residues do not directly interact with the inhibitors. VE catalytic cysteine is colored in magenta. Inhibitors were docked to VASH2 [PDB: 6J4P] using AutoDock4.6 software, and modeling was rendered using UCSF Chimera1.15 software. 2D diagrams of inhibitor-enzyme's interactions were processed and rendered using BIOVIA Discovery Studio 2021. The rendering of these diagrams was modified to be sharper.</figcaption></figure>
interactions including hydrogen bonds, π-π stacking with Phe191 and π-alkyl interactions with Arg211/Arg212 (Figure 2B). Furthermore, we observed that significant molecular space was available in the vicinity of both R₃ and R₄ positions of our inhibitors (Figure 2B), allowing for the accommodation of hydrophobic substituents. These modifications were undertaken with the dual aim of improving VE affinity through more hydrophobic interactions or π-π stacking, and simultaneously enhancing the lipophilicity of compound **11** to increase its cellular penetration. This molecule possesses two carboxylic acid groups that are predominantly deprotonated at physiological pH, resulting in polar carboxylates. As such, it may limit the cell penetration and consequently *in cellulo* activity of this molecule.

## Improving cellular penetration for biological application

We introduced four different hydrophobic side chains at the R₃ position (Chart 2) using Val (12), Leu (13), Phe (14) and Trp (15) residues. Compounds **12** and **13** exhibited similar *in vitro* inhibition to that of compound **11**, while compounds **14** and **15** were approximatively 3 times more potent than **11** with IC₅₀ of about 0.6 µM *in vitro*. However, *in cellulo* their inhibitory activity remained as modest as **11** and **EpoY** (IC₅₀ > 20 µM). The substituents at position R₃ for compounds **12** to **15** are quite bulky, suggesting the presence of a notable hydrophobic pocket in the S₃ position of VE. This is rather surprising given that, in the case of α-tubulin, S₃ is occupied by the non-bulky and very flexible glycine. However, this hypothesis is supported by the molecular docking studies of compound **11** (Figure 2B), suggesting that the incorporation of hydrophobic substituents at this specific position is important for both affinity and the eventual selectivity of our class of inhibitors.

<figure><img src="image_5.png" /><figcaption>**Chart 2.** Road to LV80 and LV87.</figcaption></figure>

Given its improved potency, inhibitor **14** was selected as the new lead molecule for further modifications. The logP of compound **14** was measured experimentally to be -0.79, indicating that even after the introduction of a second aromatic group, it remained rather hydrophilic. LogD<sub>7.4</sub> could not be measured, as the compound was completely partitioned in PBS. Subsequently, to enhance the lipophilicity of **14** by suppressing hydrogen bond donors (HBD) and hydrogen bond acceptors (HBA), the polar terminal amide group at position R₃ was removed, leading to compound **16**. As expected, the removal of the amide group resulted in increased lipophilicity, with logP of 0.17 for **16** compared to -0.79 for **14** (Table 2). With IC₅₀ of 9.1 µM compound **16** was 3 times more efficient *in cellulo* than **14** suggesting that the increase of lipophilicity improved the cell penetration of this analogue.

Then, spacers of different lengths between the newly introduced phenyl group and the Aad moiety were explored, resulting in the synthesis of two new compounds **17** and **18**. All compounds (**16-18**) are slightly more potent than **14** *in vitro* and 12 to 18 times more potent than **EpoY**. Importantly,
compounds **16** to **18** exhibit stronger inhibitory activity *in cellulo* than **EpoY** (>2 times) which, as stated previously, can be explained by their higher lipophilicity.

We then focused on compound **16** for further optimization. However, this compound was still quite polar and completely partitioned in PBS when we attempted to measure a logD at pH 7.4. Starting from this compound, the objective was to increase lipophilicity and remove one HBD of the Tyr residue to further improve its *in cellulo* activity. In addition, substitutions on the phenolic OH of Tyr (R₄ position), the last natural AA residue within the structure of compound **16**, could further reduce its “peptide character” and improve its stability in biological media. Various lipophilic substituents were introduced at the position R₄, yielding compounds **19** to **21**. Introduction of methyl and ethyl groups (compounds **19** and **20**) only slightly improved the *in cellulo* efficacy compared to **16**. Remarkably, the introduction of a rather bulky benzyl group at the R₄ position resulted in a separable crude diastereomeric mixture

<figure><img src="image_6.png" /><figcaption>**Figure 3.** Biological evaluation of **EpoY**, **LV80** and **LV87**. (A) *In vitro* evaluation of **EpoY**, **LV80** and **LV87**. IC₅₀ of the compounds were determined for VASH1. (B) *In vitro* evaluation of **EpoY**, **LV80** and **LV87**. IC₅₀ of the compounds were determined for VASH2. (C) *In cellulo* VASHs detyrosinase activity inhibition evaluation using **EpoY**, **LV80** and **LV87**. (D) Kᵢ and kᵢinact determination of **LV80** for VASH2.</figcaption></figure>

of the (*S*,*S*)-TES and (*R*,*R*)-TES containing peptides. The separation of these diastereomers by preparative HPLC enabled the identification of the most active compound, **LV80** (compound **21**) containing the (*S*,*S*)-TES moiety. While the (*R*,*R*)-TES inhibitor proved to be inactive, **LV80** showed potent inhibition both *in vitro* and *in cellulo*. **LV80** exhibited an *in vitro* IC₅₀ approximately 30 and 12-fold lower on VASH1 and VASH2 respectively, compared to **EpoY** (Figure **3A** and **3B**). Equally, its *in cellulo* IC₅₀ of 830 nM was around 25-fold lower compared to **EpoY** (Figure **3C**). The inactivity of the (*R*,*R*)-TES containing diastereomer is possibly due to the fact that the C-O TES nonbonding orbital σ* is not accessible for a S<sub>N</sub>2 reaction with the thiol function of the VE active sites. The high *in vitro* activity of **LV80** could mainly be explained by the occupation of the hydrophobic pocket at S<sub>3</sub> and S<sub>4</sub>, as emphasized by compound **16**, in addition to the ionic interaction identified with compound **11** (Figure **2B** and **2C**).

Another crucial positive interaction is provided by the benzyl group at R₄ position. Indeed, according to our molecular docking study, the S₁' position of VASH2 could form π interactions between the Bn group at R₄ position and Arg212 or Glu50 (Figure **2C**). This compound probably benefits from its considerably enhanced lipophilicity compared with **14** and thus improved cell penetration: the HPLC
retention time (Rt) of LV80 was significantly higher compared to 14, as was its experimental logP of 0.37 (Table 2). As the next step, we decided to study the kinetic parameters of the VASH inhibition by LV80. Indeed, the IC50 values for covalent irreversible inhibitors are highly influenced by the preincubation and incubation times of the enzyme with the inhibitor, as well as the concentration of the enzyme in the assay.²⁷ Thus, although we always performed the assays in the same condition, it is challenging to compare IC50 values from different studies. Hence, the most appropriate kinetic study for our class of inhibitors is probably the measurement of the inactivation constant (KI) and the maximum rate of inactivation (kinact). KI accounts for the initial stage of reversible slow binding, while kinact assesses the maximal rate of the covalent interaction between the catalytic Cys and the epoxide (Figure 3D). Therefore, the higher the kinact/KI ratio, the more efficient the inhibitor. In this context, we applied the method 3A (2-step IRREV) reported by Mons *et al*.²⁷ to measure the KI and kinact of LV80. We obtained a KI of 0.173 µM and a kinact of 0.001 s⁻¹, consistent with covalent irreversible mode of enzyme inactivation, (Figure 3D and SI.3) and compared them with the data acquired for the inhibition of Calpain by the E-64 natural inhibitor. We chose Calpain for comparison as it is similarly to the VE enzyme a cytosolic cysteine protease functioning at neutral pH. E-64 on the other hand is widely used as potent but unselective inhibitor of cysteine proteases. A KI of 3.96 µM was obtained for E-64 inhibition of Calpain,²⁸ which suggests that LV80 possesses a much higher affinity for VASH compared to E-64 for Calpain. The kinact/KI ratio, which considers both affinity and rate of the covalent inactivation of the enzyme, was slightly lower for LV80 (0.6*10⁻⁴ s⁻¹M⁻¹) compared to the 3.02*10⁻⁴ s⁻¹M⁻¹ reported for the inactivation of Calpain by E-64. This result is explained by the significantly higher kinact of E-64 which means that once inside the active site, LV80 reacts less quickly with the catalytic cysteine of VASH2 compared to E-64 within the active site of Calpain. This could be explained by the larger size of LV80, which renders it less mobile and thus less reactive as electrophile compared to the smaller E-64. LV80 emerged as a promising candidate for exploring the role of tubulin detyrosination in general and of the VE in particular. Nevertheless, at physiological pH, the compound still contains two polar carboxylate moieties which could limit its cellular penetration and consequently its activity. This hypothesis was supported by the rather low logD7.4 value of LV80 of -1.97. Transforming one of the two carboxylic acids into a less polar bioisostere could lower LV80 polarity, thus increasing cellular penetration and hence activity. Since our previous experience with this series of inhibitors highlighted that the COOH of Tyr is a highly important pharmacophore for the inhibition of *Trypanosoma brucei* VE, we postulated that the same effect could be observed in the case to human VE, as sequence homology in the catalytic domain is high.¹¹ Consequently, only the carboxyl function at the R5 position (Chart 2) was substituted with a hydroxamic acid moiety to obtain compound LV87 (compound 22). Hydroxamic acids are recognized as isosteres of the carboxylic function, characterized by a higher pKa value (8.5 – 9.5), that could confer a reduced polarity in physiological media.²⁹ Consequently, they generally exhibit an enhanced capability for cellular penetration.³⁰ Accordingly, LV87 remained a very good inhibitor *in vitro*, while exhibiting very strong inhibition *in cellulo* with an IC50 of 60 nM, around 350 folds lower than EpoY (Figure 3A-C and Table 2). LogD7.4 value of LV87 was 4 times higher than LV80, supporting our hypothesis that the introduction of hydroxamic isostere will lower the hydrophilicity of the latter in physiological medium (Table 2). Finally, we attempted to determine the covalent engagement of LV87 within the VASH1 active site. The VASH1/SVBP complex was incubated with LV87, then treated with iodoacetamide and subjected to trypsin digestion. The resulting samples were analyzed by LC-MS/MS. Notably, the ionization intensity of the peptide fragment containing the active site cysteine decreased significantly following pre-incubation with LV87 (Figure SI.4). However, we were unable to identify the LV87-Cys-peptide adduct. A possible explanation for this may be the poor ionization or fragmentation efficiency of the adduct in mass spectrometry. Another explanation is a difficult to predict inhibitor-specific fragmentation behavior of the adduct.³¹ Further investigations to clarify the exact nature of this covalent engagement will be reported in due course.
<table><caption>Table 2. Structures, inhibition data and physicochemical properties of rationally designed compounds.</caption><thead><tr><th rowspan="2">Compound</th><th rowspan="2">Structure</th><th rowspan="2">MW (g/mol)</th><th colspan="2">IC<sub>50</sub> (µM)</th><th rowspan="2">Rt<sup>c</sup> (min)</th><th rowspan="2">LogD<sub>7.5</sub> LogP<sup>d</sup></th></tr><tr><th>In vitro<sup>a</sup></th><th>In cell.</th></tr></thead><tbody><tr><td>12</td><td><img src="12_structure.png" alt="Chemical structure of 12"></td><td>536.5</td><td>1.91</td><td>X<sup>b</sup></td><td>1.55</td><td>-</td></tr><tr><td>13</td><td><img src="13_structure.png" alt="Chemical structure of 13"></td><td>550.6</td><td>1.54</td><td>X<sup>b</sup></td><td>1.78</td><td>-</td></tr><tr><td>14</td><td><img src="14_structure.png" alt="Chemical structure of 14"></td><td>584.6</td><td>0.66</td><td>28.8</td><td>1.86</td><td>LogD<sub>7.4</sub> = N/A<sup>e</sup><br>LogP = -0.79±0.03</td></tr><tr><td>15</td><td><img src="15_structure.png" alt="Chemical structure of 15"></td><td>623.6</td><td>0.59</td><td>22.8</td><td>2.00</td><td>-</td></tr><tr><td>16</td><td><img src="16_structure.png" alt="Chemical structure of 16"></td><td>541.6</td><td>0.37</td><td>9.1</td><td>2.14</td><td>LogD<sub>7.4</sub> = N/A<sup>e</sup><br>LogP = 0.17±0.02</td></tr><tr><td>17</td><td><img src="17_structure.png" alt="Chemical structure of 17"></td><td>555.6</td><td>0.21</td><td>11.0</td><td>2.34</td><td>-</td></tr><tr><td>18</td><td><img src="18_structure.png" alt="Chemical structure of 18"></td><td>569.6</td><td>0.18</td><td>10.4</td><td>2.52</td><td>-</td></tr><tr><td>19</td><td><img src="19_structure.png" alt="Chemical structure of 19"></td><td>555.6</td><td>0.54</td><td>7.1</td><td>2.47</td><td>-</td></tr></tbody></table>
In this complex context, we approached EpoY, expecting difficult derivatization and purification due to the high reactivity of the epoxide moiety. To our surprise, our class of epoxide inhibitors showed very low chemical reactivity and high chemical selectivity. Purifications were straightforward and ring-opening reactions of the epoxide were very seldomly observed. This prompted us to perform several experiments to assess the reactivity of our lead inhibitors towards different types of nucleophiles and their selectivity towards Calpain, Caspase-3, and Papain cysteine proteases. Calpain and Caspase-3 were chosen as they are cytosolic cysteine protease indispensable for correct cell homeostasis and cell cycle maintenance. Papain, on the other hand, while not clinically relevant, is considered a very good model for measuring inhibitor specificity as it possesses a very broad substrate specificity.²⁸ When assessing the reactivity of our lead inhibitors towards nonspecific nucleophiles, we compared their

<figure><img src="image_7.png" /><figcaption>**Figure 4.** (A) Stability of LV80 and LV87 in the presence of 2 mM GSH (left panel) and 0.2 mM Ac-Phe-Lys-Leu-NH₂ (right panel), compared to Ibrutinib, Afatinib, and Carfilzomib. Afatinib and Boc-Phe-Osu were used as positive controls to assess reactivity toward GSH and Ac-Phe-Lys-Leu-NH₂, respectively. Data are presented as mean ± SD (n = 2). (B) Enzymatic activity of Calpain (left panel), Caspase-3 (middle panel), and Papain (right panel) in the presence of increasing concentrations of EpoY and LV87. ALLN, Z-VAD-FMK, and E-64 were used as known reference inhibitors for Calpain, Caspase-3, and Papain, respectively. Data are presented as mean ± SD (n = 3). (C) Serum stability of LV87 was evaluated in human (solid bars) and rat (striped bars) serum (left panel). Data are presented as mean ± SD (n = 3). Microsomal stability of LV80 and LV87 in human (solid bars) and rat (striped bars) liver microsomes. Verapamil was used as a positive control (right panel). Data are presented as mean ± SD (n = 2).</figcaption></figure>
reactivity with FDA-approved covalent drugs: Ibrutinib, Afatinib and Carfilzomib. Ibrutinib and Afatinib are successful acrylamide anti-cancer drugs, while Carfilzomib is a proteasome epoxide-based inhibitor. Comparing our lead compounds to FDA-approved drugs containing different warheads should allow us to correctly evaluate and position their nonspecific reactivity.

First, we measured the stability of our lead compounds **LV80** and **LV87**, while incubated in a 2 mM glutathione (GSH) PBS pH 7.4 solution, as a standard test of the reactivity of covalent modifiers.⁴⁰,⁴¹ Indeed, one of the many functions of GSH is to react with electrophiles to protect cells from toxicity. **LV80** and **LV87** reacted rather slowly with GSH and both compounds were more than 80% intact after 4 hours of incubation (Figure **4A** and **SI.5**). During the same period, Afatinib was almost 100% conjugated to GSH and its half-life was less than 1 hour in accordance with previous results from Shibata's group.⁴¹ The other acrylamide compound standard, Ibrutinib, reacted in a slower pace, also in accordance with the results from Shibata's group. However, it still reacted faster than the epoxide drug Carfilzomib and our two lead compounds **LV80** and **LV87**. After 48 hours, **LV80** was more than 50% intact, while Carfilzomib and **LV87** were around 40% intact. Ibrutinib's reactivity remained higher and it was around 25% intact (Figure **SI.5**). This stability of the TES moiety is in line with results presented by Mons *et al.*,⁴² where Ibrutinib was less reactive with GSH compared to Afatinib but more reactive than the **E-64** trans-epoxysuccinate inhibitor. Additionally, this result is consistent with previous tests of GSH stability performed by the Thatcher group with Calpain epoxide inhibitors.²⁸

The low reactivity of GSH with our inhibitors can be explained by a poor match between the nucleophile and the electrophile, as GSH is a soft nucleophile, whereas the TES moiety could be considered as a hard electrophile. To test this hypothesis, we incubated **LV80** and **LV87** with an Ac-Phe-Lys-Leu-NH₂ model lysine containing peptide. We also measured the activity of this peptide towards our panel of covalent drugs. Once again, **LV80** and **LV87**, as well as the FDA-approved drugs, proved to be very stable: after 48 hours no-adduct could be detected by HPLC and LC-MS analysis (Figure **4A**). On the other hand, our model electrophile Boc-Phe-OSu reacted quickly and no-intact compound could be detected after 1 hour of incubation.

We were also interested in the enzymatic selectivity of our inhibitors towards different cysteine proteases. We tested **EpoY** and **LV87** for their inhibition of the cysteine proteases Calpain-1, Caspase-3 and Papain. While Calpain-1 and Caspase-3 were chosen as highly important cysteine proteases for the cell cycle, Papain was chosen as model for inhibitor specificity, as this enzyme is known for very broad substrate specificity. **EpoY** slightly inhibited Calpain at high concentration of 100 µM, while **LV87** showed no inhibition at the highest concentration tested of 100 µM (Figure **4B**). Concerning the Caspase-3 assay, both **EpoY** and **LV87** did not show significative inhibition even at high concentrations (Figure **4B**). Both **LV87** and **EpoY** showed significant inhibition of Papain, but at very high dose of 100 µM. **E-64** inhibited Papain completely at 1 µM and its IC₅₀ is reported to be in the low nM range according to the literature.⁴³,⁴⁴ In this context, inhibiting Papain with an IC₅₀ greater than 10 µM further establishes **LV87** as a specific inhibitor of the VASH Enzymes. These results once again support our hypothesis that targeted covalent inhibitors can be highly specific if carefully designed to fit their target. However, testing with larger panels of other proteases would be necessary in the future to fully validate our hypothesis.

Next, we evaluated the toxicity of **LV80** and **LV87** on CHL-1 cells incubated with increasing concentrations of inhibitors. CHL-1 cells were chosen as they were used for the *in cellulo* evaluation of our inhibitors. No significant cytotoxicity was observed up to 10 µM for **LV80** and **LV87**, further supporting the positive safety profile of our class of molecules while engaging the target (see Figure **SI.6**).
With future in vivo applications in mind, we tested the stability of **LV87** in rat and human serum. Our lead inhibitor showed very good stability in these assays, with only around 20% and 30% degradation in human and rat serum, respectively, after 6 hours of incubation (Figure 4C).

<figure><img src="image_8.png" /><figcaption>**Scheme 1**<sup>a</sup></figcaption></figure>

<sup>a</sup>Reagents: (i) HATU, DIEA, DMF anhydrous; (ii) DMF/piperidine (8/2); (iii) DIC/OxymaPure/DMAP, DIEA, DMF anhydrous; (iv) TFA/TIS/H<sub>2</sub>O, DCM.

Finally, we tested the stability of **LV80** and **LV87** in a human and rat liver microsomal assay. We compared the stability of our lead compounds, **LV80** and **LV87**, to Verapamil, an FDA-approved drug, often used as positive control in such assays.<sup>45</sup> While Verapamil's half-life was less than 10 min in human microsomes and around 10 min in rat microsomes, both **LV80** and **LV87** showed a very good stability with half-lives greater than 2 hours. These results are especially encouraging, considering that microsomal epoxide hydrolase is an enzyme heavily involved in metabolism of epoxide drugs.<sup>46</sup>

Overall, our results in this section suggest that **LV87** is a selective inhibitor with very good stability in different media, including serum and microsomes, and a good safety profile.

## CHEMISTRY

A variety of different synthetic procedures using TES or *cis*-epoxysuccinate moieties to produce affinity-based probes or cysteine protease inhibitors can be found in the literature. One of the most famous epoxide-containing compound is arguably the **E-64**, where the coupling of the epoxysuccinate monomethyl ester was historically performed using diethylphosphoryl cyanide in DMF in the presence of triethylamine.<sup>47</sup> Nowadays, the coupling of epoxysuccinic acid or epoxysuccinate monoesters with amine compounds (including AAs) is performed with EDC/HOBT,<sup>38,48–50</sup> HATU,<sup>51–53</sup> *p*-nitrophenol activated ester,<sup>54–56</sup> PyBOP,<sup>55,57</sup> DCC/HOBT,<sup>54</sup> EDC,<sup>58</sup> EDC/HOBT/DMAP,<sup>59</sup> DCC/DMAP,<sup>60</sup> PCl<sub>5</sub>,<sup>60</sup> or DIC/HOBT.<sup>61</sup> Interestingly, ester-amide epoxysuccinate is much more common in the literature than diamide epoxysuccinate. Moreover, our bibliographic search reveled that “double-headed” epoxysuccinate compounds represent only a few examples where alkyl ester-amide and alkyl amide-ester compounds predominate. Several methods are available to obtain “double-headed” epoxysuccinyl-peptides from epoxysuccinate monoamides. Activations by HATU,<sup>51</sup> *p*-nitrophenol
activated ester,⁵⁸,⁶² EDC/HOBT⁴⁸⁻⁵⁰,⁶³ or PyBOP⁵⁵,⁵⁶ are the most commonly used. Of note, other methods using HBTU/HOBT,⁵⁷,⁶⁴ BOP⁶⁵ or pentafluoroester⁶⁶ are also described in the literature. According to the latter, several of these methods can be used either in solution and solid-support.⁵⁵,⁵⁶,⁶¹,⁶⁴

<figure><img src="image_9.png" /><figcaption>Scheme 2ª</figcaption></figure>

ªReagents: (i) Allyl bromide, Cs₂CO₃, DMF anhydrous; (ii) TFA/TIS/H₂O, DCM; (iii) HATU, DIEA, DMF anhydrous; (iv) Et₂NH, DMF; (v) EDCI/OxymaPure/DMAP, DIEA, DMF anhydrous; (vi) NaOH (1M), AcN/H₂O (8/2).

In our case, we have chosen to synthesize most of the compounds presented in this study on solid phase to speed up their synthesis. As an example, we choose to present the synthesis of **14** in Scheme 1. One of the key steps was the preparation of large quantities of the building block **23**, as it was used to carry out the solid phase synthesis of 14 compounds reported in this publication. Racemic (+,-)-TES was used in the presence of HCl.H-Tyr(tBu)-OtBu, HATU and DIEA. This *quasi*-instantaneous reaction gave not only the desired compound **23**, but also its dimeric form **24** in a non-negligible amount. Optimization of this step, using two equivalents of (+,-)-TES instead of one, allowed sufficient reduction of **24** formation to use **23** as a building block without further purification (ratio **23/24** : 95/5). Once compound **23** in hand, peptide **14** was synthesized according to conventional Fmoc/tBu SPPS strategy using AmphiSpheres 40 RAM resin. HATU and DIEA were used for anchoring Fmoc-Phe-OH to the resin and to couple Fmoc-Aad(tBu)-OH. After Fmoc deprotection with DMF/piperidine, compound **23** was coupled to compound **28** using a DIC/OxymaPure/DMAP (1.5/1/0.1) (equiv/equiv/equiv) cocktail. For other compounds, this step was also performed using PyAOP and DIEA. The final step consisted of conventional cleavage of the molecule from the resin and simultaneous removal of the three tBu protecting groups to afford compound **14** after purification on preparative HPLC.

The most complex compounds (**16** to **LV87**) were synthesized in the solution phase due, on the one hand, to the impossibility to anchor the phenylalkyl moiety on a solid support, and on the other hand to some difficulties encountered in activating the carboxylic acid of the epoxide when the TES-Tyr synthone was anchored on a resin. As an example of the solution phase synthesis, we choose to present the preparation of **LV87** (Scheme 2). Compounds **16** to **20** synthesis is presented in Figure **SI.11**. An important point for the synthesis of **L87** is the preparation of compound **32** as an enantiomerically pure (S,S)-TES derivative. First, the (S,S)-epoxide was obtained according to Tamai *et al.* resolution
methodology.⁶⁷ Then, using the same optimized coupling protocol that yielded **23** (and its dimeric form **24**), **32** was obtained with a conversion of 88% while its dimeric form **33** was obtained with only 12% of conversion. Of note, these dimeric side products and some derivatives, such as **LV104**⁶⁸, were tested and gave promising results *in cellulo* and *in vivo*.⁶⁹ Compounds **32** and **35**, obtained from Fmoc-Aad(tBu)-OH as depicted in Scheme **2**, were synthesized in parallel and subsequently condensed to afford compound **36**. The latter is an interesting intermediate as it has two orthogonally protected carboxylic acids available for further derivatization. In addition, the simple deprotection of allyl and tert-butyl esters allows rapid access to **LV80** avoiding the diastereomers separation pathway. Compound **36** was then deprotected on its Aad moiety, using a TFA/TIS/H₂O (95/2.5/2.5) (v/v/v) solution, to release its carboxylic acid function. Without further purification, compound **37** was then coupled to O-(tetrahydropyran-2-yl) hydroxylamine (H₂NO-THP) using the previously described cocktail modified by the use of EDCI instead of DIC. Without further purification, the allyl ester of **38** was removed using a NaOH (1 M) solution followed by a classical treatment under acidic TFA conditions to give, after a purification step, **LV87**.

## CONCLUSION

The set of post-translational modifications of microtubules, also known as “tubulin code”, has been established as one of the central topic in fundamental biology.⁴ Several of these tubulin posttranslational modifications are emerging as valuable therapeutic targets.⁷⁰ However, there are currently few efficient and selective inhibitors for the various proteases and ligases implicated in this complex modification system. Thus, the “tubulin code” remains an underexplored research area within medicinal chemistry and chemical biology. Notably, while the detyrosination of microtubules was discovered 40 years ago, the enzymes involved were only identified recently.⁷ To our knowledge, we report here the first structure activity study of the VASH1&2 enzymes. We used **EpoY**, a small epoxide inhibitor initially employed for the identification of these two proteases, as our lead molecule. **EpoY** explored only the S₁' position within the VE's active sites. Through rational substrate-based design, that explored both primed and unprimed positions within the VE's active sites, we significantly improved **EpoY**. In particular, our QSAR study aimed at increasing affinity, specificity, and cell penetration of the inhibitors. This study led to the identification of two lead compounds, namely **LV80** and **LV87**, that are nanomolar inhibitors effective both *in vitro* and *in cellulo*. **LV87** is especially potent *in cellulo*, with IC₅₀ of 60 nM. The K₁ of **LV80** for the VASH2 enzyme was measured at 0.173 µM, which compares favorably with the value of 3.96 µM for **E-64** inhibition of Calpain-1. Increasing evidence shows that with careful design, epoxide inhibitors could be both potent and selective.²⁸,³⁸ In this context, we studied the inhibition of Calpain-1, Caspase-3 and Papain by **LV87**. Neither of the first two proteases was significantly inhibited even at very high **LV87** concentration of 100 µM. **LV87** inhibited Papain at high doses superior to 10 µM. Considering the very broad substrate specificity of Papain and the fact that **LV87** inhibits VASH1 and VASH2 with IC₅₀ of 10 nM and 60 nM respectively, this result further establishes **LV87** as selective VE inhibitor. In general, these results support our hypothesis that epoxide inhibitors could be selective, although tests on larger protease panels are required to confirm it. During the realization of this QSAR study, we observed the surprising chemical stability of the TES epoxide moiety. We performed further experiments to evaluate whether the perceived reactivity of this group is overstated. We incubated **LV87** as well as a panel of FDA-approved covalent inhibitors for 48 hours with a high 2 mM concentration of the nucleophilic GSH. Remarkably, only around 15% of **LV87** were conjugated to GSH after 4 hours of incubation, highlighting its low reactivity toward unspecific nucleophiles and high hydrolytic stability in general. Most of the compounds tested from the panel of FDA-approved covalent drugs, with the exception of Afatinib, showed equally high
stability, suggesting that low reactivity towards nonspecific inhibitors is a feature of successful covalent
inhibitors. We performed a similar experiment with a peptide-lysine model and **LV87** proved yet again
to be very stable. Furthermore, **LV87** was equally not a subject of rapid degradation in human or rat
sera and liver microsomal solutions. Lastly, **LV80** and **LV87**, did not show significant cellular toxicity for
doses lower than 10 µM. Based on all the evidence presented, we believe that **LV80** and **LV87**
represent valuable tools in the study of the role of the VE in particular and the "tubulin code" in
general. They can also be used to evaluate the benefits of targeting VE in the context of
neurodegenerative diseases such as Alzheimer's.

## EXPERIMENTAL SECTION

**VASH inhibition in vitro assay.** In vitro analysis of detyrosination activities was performed using the recombinant hVASH1/SVBP or hVASH2/SVBP complex. Both direct ELISA assays were used following manufacturer's recommendations (MT-act). Both the ELISA assays have the following technical specifications Z'>0.8 ; S/B>12. Briefly, 96-well plates were protein-coated with substrate in coating buffer. Next, the plates were washed 3 times with 200 µL of wash buffer and ready to use for the detyrosination assay. The detyrosination assay was performed by addition of the enzymatic mix (recombinant hVASH1/SVBP or hVASH2/SVBP and compounds) to each well, and the plate incubated 5 min at 37 °C. Subsequently the reactions were blocked with 200 µL of blocking buffer at room temperature and washed off. The primary anti-detyrosinated tubulin antibody was diluted in blocking buffer and added to each well. Following 60 min incubation at room temperature, the antibody mix was washed off and the secondary HRP-conjugated antibody added to each well. Following 30 min incubation the antibody was washed off and 100 µL of TMB (3,3',5,5'-Tetramethylbenzidine) added to each well for coloration. The plate was incubated 30 min at room temperature and color development stopped by addition of 100 µL of 0,5 M sulfuric acid. The optical density (OD) measured at 450 nm.

**VASH inhibition in cellulo assay.** A previously described cellular system that is >95% dependent on VASH-mediated tubulin detyrosination was adapted to high content screening technology and analyzed using the Opera Phenix® Plus High-Content Screening System from PerkinElmer. Briefly, following treatments, CHL-1 cells were fixed and permeabilized using ice cold methanol, then washed twice in PBS 0.1% Triton X100 (v/v) and blocking was performed with PBS containing 3% FBS (v/v), 0.3% Triton X100 (v/v). Next, cells were incubated with tubulin detyrosinated affinity purified primary antibodies. Cells were washed in PBS 0.1% Triton X100 (v/v) and incubated with secondary antibody Alexa Fluor 488-conjugated goat anti-rabbit (Invitrogen) containing DAPI for nuclear staining. The wells plate was analyzed on Opera Phenix® Plus High-Content Screening System from PerkinElmer with optimized parameters.

**Compound stability in serum.** Serum reaction sample contained 125 µL of Human or rat serum and 375 µL of PBS (pH 7.4) (Sigma Aldrich). The reactions were initiated by addition of 25 µL peptide (10 mM stock in DMSO). The assays were performed in a Thermo-Shaker for Eppendorf tubes at 37 °C with an agitation of 600 rpm. 50 µL samples were collected at known time intervals and added to 100 µL of AcN (acetonitrile) 0.1% TFA to precipitate serum proteins. Cloudy sample was cooled to 4 °C for at least 15 min and then submit to centrifugation for 10 min at 13,000 rpm to pellet the precipitated serum proteins. 100 µL of the clear supernatants were collected and peptides were finally analyzed by RP-HPLC and LC-MS.⁷¹

**Inhibitors' human and rat liver microsomal stability.** Microsomal reaction sample contained 197 µL of a 0.63 mg/ml human or rat liver microsomal solution in PBS and 3 µL of a 0.1 mM compound solution
in DMSO. The reactions were initiated by addition of 50 µL of NADPH (5 mM PBS). The assays were performed in a Thermo-Shaker for Eppendorf tubes at 37 °C with an agitation of 1200 rpm. 50 µL samples were collected at known time intervals and added to 150 µL of AcN to precipitate microsomes. Sample was cooled to 4 °C for at least 15 min and then submitted to centrifugation for 15 min at 3,000 rpm to pellet the precipitate. 100 µL of the clear supernatants were collected and analyzed by LC-MS/MS.

**Calpain-1 activity assay.** Calpain-1 activity in presence of **EpoY** and **LV87** was measured using the Human Calpain-1 Inhibitor Screening Kit (MAK210) following manufacturer's instructions (Sigma Aldrich). Detection of the cleavage of Calpain substrate Ac-LLY-AFC by Calpain-1 lead to liberation of free AFC (7-amino-4-trifluoromethylcoumarin) that was quantified fluorometrically (Ex/Em = 400/505 nm) using a SpectraMax i3x (Molecular Devices) microplate Reader. In the presence of potent Calpain-1 inhibitor, the hydrolyzation of substrate will be impeded. ALLN (MAK210D) was used as an inhibitor positive control. Fluorescence (FLU) was plotted versus time. The slope for each well, in the linear range, was calculated to determine the slope of the plot (FLU/min). After corresponding slope blank subtraction, Calpain activity was calculated using the following equation:

$$ \% Relative Inhibition = \frac{(SlopeEC - SlopeSC)}{SlopeEC} \times 100 \quad Calpain activity \% = 100 - \% Relative Inhibition $$

Where Slope SC is the slope of the Sample with the compound and Slope EC is the slope of the Enzyme Control.

**Caspase-3 activity assay.** Caspase-3 activity in presence of **EpoY** and **LV87** was measured using Abcam's Caspase-3 Inhibitor Drug Detection Kit (ab102491) and also following manufacturer's instructions. The protocol used to detect and calculate enzyme activity is also based on the cleavage of a fluorogenic substrate (DEVD-AFC) and is similar to the one used to determine Calpain activity.

**Papain activity assay.** Papain activity in the presence of **EpoY** and **LV87** was assessed using Papain (Sigma P3125, 29 mg/mL) diluted to 0.37 mg/mL and pre-activated by incubation for 15 min at room temperature in an activation buffer consisting of 100 mM sodium phosphate (pH 6.6), 2 mM EDTA, and 5 mM L-cysteine. For inhibitor testing, 89 µL of the activation buffer was mixed with 1 µL of **EpoY** or **LV87** from DMSO stock solutions (10 mM, 1 mM, or 100 µM), followed by the addition of 10 µL of the diluted Papain solution. The mixture was incubated at 37 °C for 30 min. The enzymatic reaction was then initiated by adding 1 µL of Z-Arg-AMC.HCl (MedChemExpress, HY-134435, 10 mM stock in DMSO) to each well. Fluorescence was measured every minute over a 30 min period using a SpectraMax i3x microplate reader (Molecular Devices) with excitation/emission set to 355/455 nm. All tested samples contained 1% final DMSO. **E-64** was used as a positive inhibitor control at a final concentration of 1 µM. Papain activity was calculated using the same method as for Calpain-1 and Caspase-3 assays.

**Cell viability assay.** The CellTiter-Glo Luminescent Cell Viability Assay was employed following manufacturer's guidelines (Promega). The homogeneous assay procedure involves adding a single reagent (CellTiter-Glo Reagent) directly to cells cultured in serum-supplemented medium. The CellTiter-Glo assay is a homogeneous method of determining the number of viable cells in culture based on quantitation of the ATP present, an indicator of metabolically active cells. The amount of ATP is directly proportional to the number of cells present in culture.

**GSH-Conjugation assay.** An aliquot of stock solution of GSH (100 mM, adjusted to pH 7.0 with 0.1 M NaOH) was added to potassium phosphate buffer (pH 7.4) to prepare an incubation solution containing 2 mM GSH. An aliquot (6 µL) of stock compound solution (2 mM) in 100% AcN (Afatinib, Carfilzomib, Boc-Phe-OSu)) or 100% methanol (MeOH) (Ibrutinib, **LV80**, **LV87**) was added to the buffer (1200 µL) to achieve a final concentration a final concentration of 10 µM and the resultant mixture was incubated
at 37°C with shaking at 120 rpm. Samples (300 µL) were taken from the plate at 0 min, 10 min, 30 min, 60 min, 120 min, 240 min, 24 hours, and 48 hours after the onset of incubation. The sample was then immediately analyzed by HPLC analysis on the Agilent 1220 LC system with a Chromolith HighResolution RP-C18 column (50 x 4.6 mm, 5 µm) as stationary phase, the mobile phase consisted of solvent A, water with 0.1% TFA, and solvent B, AcN with 0.1% TFA. A linear gradient of 4 min with a flow rate of 4 mL/min was used and a 214 nm UV lamp was used to detect the different compounds. The intact compound was quantified as the remaining proportion (%) of the parent compound relative to the GSH adduct using the ratio of the areas found by HPLC analysis. T 0 min and T 48 hours were further analyzed by Acquity I class UPLC system, which was coupled to a high-resolution mass spectrometer equipped with Acquity UPLC BEH C18 2.1 × 50 mm column at 40 °C with a flow rate of 0.8 mL/min and a gradient of 0-100% B over 3.5 min. The mobile phase consisted of solvent A, water with 0.1% HCOOH, and solvent B, AcN with 0.1% HCOOH. The LC-MS analysis could be found in Figure SI.5.

**AcPheLysLeuNH₂-Conjugation assay.** An aliquot of stock solution of AcPheLysLeuNH₂ (10 mM, adjusted to pH 7.0 with 0.1 M NaOH) was added to potassium phosphate buffer (pH 7.4) to prepare an incubation solution containing 0.2 mM GSH. An aliquot (6 µL) of stock compound solution (2 mM) in 100% AcN (Afatinib, Carfilzomib, Boc-Phe-OSu)) or 100% MeOH (Ibrutinib, **LV80, LV87**) was added to the buffer (1200 µL) to achieve a final concentration = of 10 µM and the resultant mixture was incubated at 37°C with shaking at 120 rpm. Samples (300 µL) were taken from the plate at 0 min, 60 min, and 48 hours after the onset of incubation. The sample was then immediately analyzed by HPLC analysis on the Agilent 1220 LC system used for GSH-Conjugation assay. The intact compound was quantified as the remaining proportion (%) of the parent compound relative to the GSH adduct using the ratio of the areas found by HPLC analysis. T 0 min and T 48 hours were further analyzed by Acquity I class UPLC system, which was coupled to a high-resolution mass spectrometer equipped with Acquity UPLC BEH C18 2.1 × 50 mm column at 40 °C with a flow rate of 0.8 mL/min and a gradient of 0-100% B over 3.5 min. The mobile phase consisted of solvent A, water with 0.1% HCOOH, and solvent B, AcN with 0.1% HCOOH. The LC-MS analysis could be found in Figure SI.5.

**Molecular Docking.** Molecular docking using Autodock4.6⁷² was used to predict the binding poses of all the biologically tested compounds presented in this study. Although these compounds bind covalently to VASH1 and VASH2, we chose to dock them in their non-covalent forms (closed epoxide ring) to determine their affinity just before the irreversible formation of the S-C bond and the opening of the epoxide. Furthermore, we decided to dock their (S,S)-TES form to facilitate the analysis. The protonation states of the ligands were calculated using MarvinSketch® at pH 7. The major microspecies described charged carboxyl groups, while the rests of the molecules were neutral. The 3D conformations of ligands were generated by MarvinSketch®. The structures of the targets were retrieved from the Protein Data Bank (PDB), and chose due to the presence of **EpoY** in order to shape the binding pocket to this series of compound (VASH1 [PDB: 6J7B]²⁵ ; VASH2 [PDB: 6J4P]²⁶). The protonation state of the targets was predicted with PROPKA.⁷³ All protein residues and termini were found in their canonical state at pH 7 and set so for molecular docking. The ligands and targets were prepared using Autodock Tools.⁷² During the docking procedure, atoms of the proteins were rigid while all the rotatable dihedral angles of the compounds were set free to move as in typical Autodock4 fashion. Regarding the flexibility of the N-terminal region assessed by our Molecular Dynamic simulations, confirmed by the lack of structural information in all other experimental structure of VASH1 in the PDB, and its proximity with the binding site, residues Gly57 to Pro59 for VASH1 were removed and not considered in the docking procedure. No residue was removed from the VASH2 structure. To take into account the flexibility of Arg222 and Arg211 observed in different experimental structures of respectively VASH1 and VASH2, and due to their close proximity to the tyrosine carboxylic
group of our compounds, Arg222 (and Arg211) was set flexible in our docking procedure. The box size for grid mapping was set to 20.25 Å, 33.0 Å and 23.25 Å in x, y and z direction respectively for docking in VASH1, and 26.5 Å, 34.0 Å and 23.0 Å for docking in VASH2 (a bigger box has been chosen for VASH2 in order to accommodate slightly larger compounds), and centered near Phe202 and Arg222 (Phe191 and Arg211 for VASH2) to encompass largely the binding cleft. The grid spacing was kept to its default value (0.375 Å). In order to restraint the conformational space, we used Autodock4⁷² with a covalent docking procedure. For each compound, the carbon covalently bound to Cys169 of VASH1 (Cys158 of VASH2) was chosen accordingly to the Xray structure of VASH enzymes and EpoY. A well of 3 Å wide and an energy barrier of 10 kcal/mol nearby the sulfur of Cys169 (Cys158) were used to build the covalent grid map. Regarding docking parameters, we generated 200 poses for each ligand-target couples using the genetic algorithm with 27000 generations, a population size of 300 and 50000000 energy evaluations which could be considered as a deep search regarding the complexity of the system. Finally, the poses were clustered according to their cross RMSD and the best poses in term of binding free energy (score) were further analyzed.

**Determination of logD<sub>7.4</sub>/logP.** Aqueous medium (AM) was chosen depending on logD<sub>7.4</sub> (PBS, pH 7.4) or logP (Ultra-Pure Water, pH 5-6) determination. In both cases, this procedure was followed. A pre-saturation step of n-octan-1-ol and AM was performed using a biphasic solution of the two liquid (1/1). The latter was spun during 24 hours at 80 rpm using a tube rotator. An overnight decanting step was then performed before the separation of the two phases. 50 mM solutions of the test compounds were prepared in DMSO. 4 µL of these solutions were placed in 1,996 mL of pre-saturated AM/n-octan-1-ol (1/1) and the resultant was spun during 2 hours using the same tube rotator. After 2 hours decanting step, 10 µL of each phase were diluted in 490 µL of MeOH and analyzed using the Agilent 1220 HPLC system used for GSH-Conjugation assay. The different log were then calculated using the ratio of the areas found by HPLC analysis:

$$logD_{7.4}(\frac{AUC_{Octanol}}{AUC_{PBS}}); logP(\frac{AUC_{Octanol}}{AUC_{Water}})$$

For more detailed data and calculations, please see Figure SI.8 and SI.9.

**Synthesis and analysis. General methods.** All reactions were performed at room temperature (rt, 25 °C). Solvents and chemicals were purchased from Sigma-Aldrich, Across, TCI Chemicals or Iris-Biotech GmbH and used as received. Purifications of the final compounds were performed on a Waters Delta Prep 4000 reversed phase preparative HPLC using solvent A, water with 0.1% TFA, and solvent B, AcN with 0.1% TFA as mobile phases. The resulting pure fractions were lyophilized using an L-300 Buchi freeze dryer. Visualization for purification and analysis was accomplished using a 214 nm UV lamp. ¹H and ¹³C NMR spectra were recorded on either a Bruker 600 MHz or a Bruker 500 MHz spectrometer. Chemical shifts are reported in ppm with the solvent resonance as the internal standard (DMSO-d6 at 2.5 and 39.52 ppm for ¹H and ¹³C, respectively). Data are reported as follows: chemical shift, number of protons, multiplicity (s = singlet, d = doublet, dd = doublet of doublet, ddd = doublet of doublet of doublet, t = triplet, q = quartet, quint = quintuplet, sxt = sextuplet, br = broad, m = multiplet) and coupling constants. High resolution mass spectrometry (HRMS) data were obtained using a Bruker ESI-micrOTOF-Q. All reported novel compounds tested in biological models were fully characterized by 1H NMR and 13C NMR. The structures of the majority of novel final compounds were supported by HRMS. All compounds submitted for biological testing were confirmed to be >95% pure by analytical Waters UPLC-MS (1) or HPLC-MS (2) using solvent A, water with 0.1% HCOOH, and solvent B, AcN with 0.1% HCOOH as mobile phase and C-18 reversed phase chromatography column. (1) Acquity H-Class UPLC system coupled to a Waters SQ Detector-2 mass spectrometer equipped with a Waters Acquity UPLC BEH C18 column (50 × 2.1 mm, 1.8 µm), a 5 min gradient and a flow rate of 0.8 mL/min; (2) Quattro micro ESI triple quadrupole mass spectrometer equipped with a Chromolith Speed Rod RP-C18 185 Pm
column (50 × 4.6 mm, 5 µm) and an Alliance HPLC system with a 2.5 min gradient and a flow rate of 3 mL/min. the synthetic procedures, tabulated spectral data, HRMS, and purity analysis of the final compounds are described below.

**General procedure for Solid Phase synthesis.** The solid support (resin) is placed in a classic fritted syringe and swelled 5 min in DCM (dichloromethane) under mechanical stirring using a vortex mixer coupled to a pump for filtration.

**Coupling.** All the necessary compounds to perform the desired coupling step were solubilized in NMP (N-methyl-2-pyrrolidone) or DMF (104 equiv) and then added to the resin. The reaction was stopped after 1-18 hours of mechanical stirring depending on the substrate and the reagent used. HPLC analysis was used to determine the end of the coupling step reaction.

**Washings.** Performed after each step. The resin was washed 3 times with DCM, 3 times with DMF and finally 3 times with DCM.

**Capping.** Performed after the washes of the anchoring step. Depending on the resin used: if 2-Chlorotrityl PS resin, a MeOH/DIEA/DCM (1/2/17) (v/v/v) solution was used with stirring for 2x20 min; if AmphiSpheres 40 RAM resin, Ac₂O (acetic anhydride, 2 equiv) and DIEA (3 equiv) in DCM was used with stirring for 15 min.

**Fmoc removal.** Performed between each coupling step. A 20% piperidine solution in DMF was added to the resin and stirred for 2x10 min.

**Cleavage.** Performed at the end of the compound synthesis. A TFA/TIS/H₂O (95/2.5/2.5) (v/v/v) solution was added to the resin and magnetic stirring was performed for 1 hour. This procedure was repeated twice when AmphiSpheres 40 RAM resin was used. The resulting solution was collected in a round bottom flask and concentrated under vacuum.

**General Solution Phase tert-Butyl(tBu)/tert-Butoxycarbonyl(Boc)/Tetrahydropyran(THP) removal.** The reactant was dissolved or suspended in DCM. A solution of TFA/TIS/H₂O (95/2.5/2.5) (v/v/v) (30 equiv TFA) was then added to the reaction mixture and was stirred for 30 min to give the desired deprotected crude product after concentration under vacuum.

**General Solution Phase Fluorenylmethoxycarbonyl(Fmoc) removal.** The reactant was dissolved in DMF (30 equiv) and diethylamine (1.5 equiv) was added. The reaction mixture was stirred for 1-2 hours and then diluted in water. The solution was acidified to pH 2 with KHSO₄ (1 M) and was then washed 3 times with Et₂O. The aqueous layer was then basified to pH 12 using a NaOH (1 M) solution and extracted 3 times with AcOEt (ethyl acetate). The combined organic layers were then washed with brine, dried on MgSO₄, filtered on cotton and concentrated under vacuum to give the desired crude product.

**(trans)-3-(((S)-1-(tert-butoxy)-3-(4-(tert-butoxy)phenyl)-1-oxopropan-2-yl)carbamoyl)oxirane-2-carboxylic acid (23).** Commercial HCl.H-Tyr-(tBu)-OtBu (5.39 mmol) was dissolved in 12 mL of anhydrous DMF and (+,-)-trans-epoxysuccinic acid (10.8 mmol) was added. DIEA (43.12 mmol) and HATU (8.09 mmol) were added in this order and the mixture was stirred for 30 min before 150 mL H₂O and 50 mL AcOEt were added. The aqueous phase was acidified to pH 2 with KHSO₄ (1 M) solution. The extraction was continued with 3x50 mL AcOEt, the organic layer was washed with 3x30 mL KHSO₄ (1 M) and 2x50 mL brine then dried on MgSO₄, filtered on cotton and concentrated under vacuum to give the desired product (23) and its dimer form (24) with a ratio of 95:5. This component was used without further purification (crude: 2.1 g, 95%).

**(trans)-(3-(ethoxycarbonyl)oxirane-2-carbonyl)-L-tyrosine (EpoY; 1).** EpoY was obtained as previously reported procedure.⁷ ¹H NMR (DMSO-d6, 600 MHz): δH 12.87 (1H, br s), 9.23 (1H, d, J = 3.2 Hz), 8.63 (1H, t, J = 9.3 Hz), 6.99 (2H, t, J = 8.1 Hz), 6.65 (2H, dd, J = 4.4 Hz, J = 8.5 Hz), 4.41-4.35 (1H, m), 4.20-4.15 (2H, m), 3.67 (0.5H, d, J = 1.9 Hz), 3.66 (0.5H, d, J = 1.9 Hz), 3.48 (0.5H, d, J = 1.8 Hz), 3.45 (0.5H, d, J = 1.9 Hz), 2.97 (1H, ddd, J = 14.0 Hz, J = 5.1 Hz, J = 1.9 Hz), 2.79 (1H, dd, J = 13.9 Hz, J = 9.1
Hz), 1.22 (3H, dt, *J* = 7.1 *Hz*, *J* = 2.3 *Hz*). ESI(+)-HRMS (m/z) [M+H⁺] calcd for C₁₅H₁₈NO₇: 324.1078.
Observed: 324.1079. HPLC method 1: tR = 1.73 min, purity = 98.3%.

**(S)-5-amino-4-((trans)-3-(((S)-1-carboxy-2-(4-hydroxyphenyl)ethyl)carbamoyl)oxirane-2-carboxamido)-5-oxopentanoic acid (2).** The general procedure for solid phase synthesis was performed using AmphiSpheres 40 RAM (0.4 mmol/g; 300 mg), Fmoc-Glu(tBu)-OH (0.44 mmol), HATU (0.44 mmol) and DIEA (0.67 mmol) were added for the anchoring step followed by **23** (0.22 mmol) in the same conditions as the anchoring step as coupling step. The crude product was then purified to afford **2** as a white solid (13.7 mg, 27%). ¹H NMR (DMSO-d6, 600 MHz): δH 12.37 (1H, br s), 9.22 (1H, br s), 8.61 (0.5H, br s), 8.52 (0.5H, d, *J* = 7.9 *Hz*), 8.46 (0.5H, d, *J* = 8.3 *Hz*), 7.49 (0.5H, s), 7.43 (0.5H, s), 7.09 (1H, s), 6.99 (2H, dd, *J* = 8.4 *Hz*, *J* = 5.6 *Hz*), 6.65 (2H, dd, *J* = 8.5 *Hz*, *J* = 5.7 *Hz*), 4.36-4.29 (1H, m), 4.21 (1H, tdd, *J* = 25.2 *Hz*, *J* = 8.5 *Hz*, *J* = 5.0 *Hz*), 3.59 (1H, dd, *J* = 2.7 *Hz*, *J* = 2.1 *Hz*), 3.57 (0.5H, d, *J* = 1.9 *Hz*), 3.52 (0.5H, d, *J* = 1.9 *Hz*), 2.97-2.94 (1H, m), 2.83-2.77 (1H, m), 2.22 (2H, dt, *J* = 8.3 *Hz*, *J* = 2.2 *Hz*), 1.96-1.89 (1H, m), 1.79-1.73 (1H, m). ¹³C NMR (DMSO-d6, 150 MHz): δC 173.8, 172.6, 172.5, 172.4, 172.4, 165.7, 165.5, 155.9, 130.1, 130.0, 127.4, 115.0, 54.1, 54.0, 52.7, 52.6, 52.5, 52.0, 51.8, 35.9, 35.7, 30.2, 30.2, 27.2, 27.1. ESI(+)-HRMS (m/z) [M+H⁺] calcd for C₁₈H₂₂N₃O₈: 424.1351. Observed: 424.1349. HPLC method 2: tR = 0.86 min, purity >99%.

**((trans)-3-((2-carboxyethyl)carbamoyl)oxirane-2-carbonyl)-L-tyrosine (3).** The general procedure for solid phase synthesis was performed using 2-Chlorotrityl PS resin (1.6 mmol/g; 250 mg), Fmoc-β-Ala-OH (0.4 mmol) and DIEA (0.8 mmol) for the anchoring step followed by **23** (0.8 mmol), OxymaPure (0.4 mmol), DIC (0.52 mmol), DMAP (0.04 mmol) and NMM (3.2 mmol) as coupling step. The crude product was then purified to afford **3** as a white solid (13.2 mg, 9%). ¹H NMR (DMSO-d6, 600 MHz): δH 12.28 (1H, br s), 9.24 (1H, br s), 8.53 (0.5H, br s), 8.47 (0.5H, br s), 8.42 (1H, q, *J* = 5.8 *Hz*), 6.98 (2H, t, *J* = 7.3), 6.65 (2H, dd, *J* = 8.5 *Hz*, *J* = 3.2 *Hz*), 4.34-4.29 (1H, m), 3.55 (1H, s), 3.44 (0.5H, s), 3.38 (0.5H, s), 3.28 (2H, q, *J* = 6.7 *Hz*), 2.96 (1H, dd, *J* = 14.4 *Hz*, *J* = 4.3 *Hz*), 2.79 (1H, dt, *J* = 13.3 *Hz*, *J* = 9.1 *Hz*), 2.40 (2H, t, *J* = 6.7 *Hz*). ¹³C NMR (DMSO-d6, 150 MHz): δC 172.8, 165.7, 165.7, 165.6, 165.4, 155.9, 130.1, 130.0, 127.5, 115.0, 115.0, 54.2, 54.1, 52.8, 52.7, 52.5, 35.9, 35.7, 34.9, 33.5. ESI(+)-HRMS (m/z) [M+H⁺] calcd for C₁₆H₁₉N₂O₈: 367.1136. Observed: 367.1138. HPLC method 1: tR = 1.30 min, purity = 98.9%.

**4-((trans)-3-(((S)-1-carboxy-2-(4-hydroxyphenyl)ethyl)carbamoyl)oxirane-2-carboxamido)butanoic acid (4).** The general procedure for solid phase synthesis was performed using 2-Chlorotrityl PS resin (1.6 mmol/g; 250 mg), Fmoc-GABA-OH (0.4 mmol) and DIEA (0.8 mmol) for the anchoring step followed by **23** (0.8 mmol), OxymaPure (0.4 mmol), DIC (0.52 mmol), DMAP (0.04 mmol) and NMM (3.2 mmol) as coupling step. The crude product was then purified to afford **4** as a white solid (12.5 mg, 8.2%). ¹H NMR (DMSO-d6, 600 MHz): δH 12.42 (1H, br s), 9.24 (1H, br s), 8.50 (0.5H, br s), 8.44 (0.5H, br s), 8.36 (1H, dt, *J* = 13.4 *Hz*, *J* = 5.7 *Hz*), 6.98 (2H, dd, *J* = 8.6 *Hz*, *J* = 5.4 *Hz*), 6.65 (2H, dd, *J* = 8.5 *Hz*, *J* = 4.7 *Hz*), 4.34-4.28 (1H, m), 3.56 (1H, d, *J* = 1.9 *Hz*), 3.41 (0.5H, d, *J* = 1.9 *Hz*), 3.36 (0.5H, d, *J* = 1.9 *Hz*), 3.14-3.05 (2H, m), 2.97 (1H, dd, *J* = 13.9 *Hz*, *J* = 4.7 *Hz*), 2.82-2.75 (1H, m), 2.21 (2H, t, *J* = 7.4 *Hz*), 1.63 (2H, quint, *J* = 7.3 *Hz*). ¹³C NMR (DMSO-d6, 150 MHz): δC 174.2, 172.8, 165.7, 165.6, 165.5, 165.3, 155.9, 130.1, 130.0, 127.6, 115.0, 54.3, 52.9, 52.8, 52.6, 52.5, 38.0, 38.0, 36.0, 35.8, 31.0, 24.3, 24.3. ESI(+)-HRMS (m/z) [M+H⁺] calcd for C₁₇H₂₁N₂O₈: 381.1292. Observed: 381.1294. HPLC method 1: tR = 1.43 min, purity = 97%.

**5-((trans)-3-(((S)-1-carboxy-2-(4-hydroxyphenyl)ethyl)carbamoyl)oxirane-2-carboxamido)pentanoic acid (5).** The general procedure for solid phase synthesis was performed using 2-Chlorotrityl PS resin (1.6 mmol/g; 250 mg), Fmoc-5-Ava-OH (0.4 mmol) and DIEA (0.8 mmol) for the anchoring step followed by **23** (0.8 mmol), OxymaPure (0.4 mmol), DIC (0.52 mmol), DMAP (0.04 mmol) and NMM (3.2 mmol) as coupling step. The crude product was then purified to afford **5** as a white solid (10.9 mg,
6.9%). ¹H NMR (DMSO-d6, 600 MHz): δ<sub>H</sub> 12.36 (1H, br s), 9.22 (1H, br s), 8.61 (0.5H, d, *J* = 8.1 *Hz*), 8.54 (0.5H, d, *J* = 7.3 *Hz*), 8.35 (1H, dt, *J* = 10.9 *Hz*, *J* = 5.6 *Hz*), 6.99 (2H, dd, *J* = 8.3 *Hz*, *J* = 6.6 *Hz*), 6.65 (2H, dd, *J* = 8.4 *Hz*, *J* = 4.8 *Hz*), 4.38-4.29 (1H, m), 3.55 (1H, t, *J* = 1.9 *Hz*), 3.42 (0.5H, d, *J* = 1.9 *Hz*), 3.38 (0.5H, d, *J* = 1.9 *Hz*), 3.12-3.03 (2H, m), 2.96 (1H, dd, *J* = 13.9 *Hz*, *J* = 4.7 *Hz*), 2.83-2.75 (1H, m), 2.21 (2H, t, *J* = 7.3 *Hz*), 1.47 (2H, quint, *J* = 7.1 *Hz*), 1.41 (2H, quint, *J* = 6.9 *Hz*). ¹³C NMR (DMSO-d6, 150 MHz): δ<sub>C</sub> 174.4, 172.6, 172.5, 165.7, 165.6, 165.5, 165.5, 155.9, 130.0, 130.0, 127.4, 115.0, 54.0, 54.0, 52.9, 52.8, 52.5, 38.3, 38.2, 35.9, 35.7, 33.2, 28.4, 28.3, 21.8. ESI(+)-HRMS (m/z) [M+H<sup>+</sup>] calcd for C<sub>18</sub>H<sub>23</sub>N<sub>2</sub>O<sub>8</sub>: 395.1449. Observed: 395.1451. HPLC method 1: t<sub>R</sub> = 1.51 min, purity = 100%.

**6-((trans)-3-(((S)-1-carboxy-2-(4-hydroxyphenyl)ethyl)carbamoyl)oxirane-2-carboxamido)hexanoic acid (6).** The general procedure for solid phase synthesis was performed using 2-Chlorotrityl PS resin (1.6 mmol/g ; 250 mg), Fmoc-6-Ahx-OH (0.4 mmol) and DIEA (0.8 mmol) for the anchoring step followed by **23** (0.8 mmol), OxymaPure (0.4 mmol), DIC (0.52 mmol), DMAP (0.04 mmol) and NMM (3.2 mmol) as coupling step. The crude product was then purified to afford **6** as a white solid (11.1 mg, 6.8%). ¹H NMR (DMSO-d6, 600 MHz): δ<sub>H</sub> 12.47 (1H, br s), 9.21 (1H, br s), 8.58 (0.5H, d, *J* = 7.2 *Hz*), 8.50 (0.5H, d, *J* = 5.7 *Hz*), 8.32 (1H, dt, *J* = 15.5 *Hz*, *J* = 5.6 *Hz*), 6.99 (2H, dd, *J* = 8.3 *Hz*, *J* = 5.4 *Hz*), 6.65 (2H, dd, *J* = 8.4 *Hz*, *J* = 4.8 *Hz*), 4.35-4.27 (1H, m), 3.54 (1H, s), 3.41 (0.5H, d, *J* = 1.9 *Hz*), 3.37 (0.5H, d, *J* = 1.9 *Hz*), 3.06 (2H, q, *J* = 6.5 *Hz*), 2.96 (1H, dd, *J* = 13.9 *Hz*, *J* = 4.7 *Hz*), 2.79 (1H, dt, *J* = 12.9 *Hz*, *J* = 9.2 *Hz*), 2.19 (2H, t, *J* = 7.3 *Hz*), 1.48 (2H, quint, *J* = 7.6 *Hz*), 1.40 (2H, quint, *J* = 7.6 *Hz*), 1.24 (2H, quint, *J* = 7.7 *Hz*). ¹³C NMR (DMSO-d6, 150 MHz): δ<sub>C</sub> 174.5, 174.5, 172.6, 172.5, 165.7, 165.6, 165.4, 155.9, 130.1, 130.0, 127.6, 127.5, 115.0, 54.1, 52.9, 52.8, 52.5, 38.4, 38.4, 35.9, 35.7, 33.6, 28.6, 28.5, 25.8, 24.1. ESI(+)-HRMS (m/z) [M+H<sup>+</sup>] calcd for C<sub>19</sub>H<sub>25</sub>N<sub>2</sub>O<sub>8</sub>: 409.1605. Observed: 409.1608. HPLC method 1: t<sub>R</sub> = 1.68 min, purity = 99.6%.

((trans)-3-((3-amino-3-oxopropyl)carbamoyl)oxirane-2-carbonyl)-L-tyrosine (7). The general procedure for Solid Phase synthesis was performed using AmphiSpheres 40 RAM resin (0.4 mmol/g ; 200 mg), Fmoc-β-Ala-OH (0.22 mmol), HATU (0.22 mmol) and DIEA (0.29 mmol) for the anchoring step followed by **23** (0.22 mmol), PyAOP (0.22 mmol) and DIEA (0.63 mmol) as coupling step. The crude product was then purified to afford **7** as a white solid (10.0 mg, 38%). ¹H NMR (DMSO-d6, 600 MHz): δ<sub>H</sub> 12.87 (1H, br s), 9.22 (1H, s), 8.57 (0.5H, d, *J* = 6.3 *Hz*), 8.53 (0.5H, d, *J* = 6.1 *Hz*), 8.39 (1H, q, *J* = 5.8 *Hz*), 7.34 (1H, br s), 6.99 (2H, dd, *J* = 8.8 *Hz*, *J* = 5.1 *Hz*), 6.84 (1H, br s), 6.65 (2H, dd, *J* = 8.3 *Hz*, *J* = 3.5 *Hz*), 4.36-4.30 (1H, m), 3.54 (1H, t, *J* = 1.7 *Hz*), 3.44 (0.5H, d, *J* = 1.8 *Hz*), 3.39 (0.5H, d, *J* = 1.9 *Hz*), 3.28-3.24 (2H, m), 2.96 (1H, ddd, *J* = 14.0 *Hz*, *J* = 4.9 *Hz*, *J* = 2.3 *Hz*), 2.79 (1H, td, *J* = 13.9 *Hz*, *J* = 9.8 *Hz*), 2.24 (2H, dt, *J* = 7.1 *Hz*, *J* = 1.8 *Hz*). ¹³C NMR (DMSO-d6, 150 MHz): δ<sub>C</sub> 172.5, 172.3, 165.7, 165.6, 165.5, 165.5, 155.9, 130.0, 130.0, 127.4, 115.0, 115.0, 54.0, 53.9, 52.8, 52.7, 52.5, 35.8, 35.6, 35.3, 34.6. ESI(+)-HRMS (m/z) [M+H<sup>+</sup>] calcd for C<sub>16</sub>H<sub>20</sub>N<sub>3</sub>O<sub>7</sub>: 366.1296. Observed: 366.1296. HPLC method 2: t<sub>R</sub> = 0.79 min, purity >99%.

((trans)-3-((4-amino-4-oxobutyl)carbamoyl)oxirane-2-carbonyl)-L-tyrosine (8). The general procedure for solid phase synthesis was performed using AmphiSpheres 40 RAM resin (0.4 mmol/g ; 200 mg), Fmoc-GABA-OH (0.22 mmol), HATU (0.22 mmol) and DIEA (0.29 mmol) for the anchoring step followed by **23** (0.22 mmol), PyAOP (0.22 mmol) and DIEA (0.63 mmol) as coupling step. The crude product was then purified to afford **8** as a white solid (10.4 mg, 45%). ¹H NMR (DMSO-d6, 600 MHz): δ<sub>H</sub> 12.85 (1H, br s), 9.22 (1H, s), 8.62 (0.5H, d, *J* = 6.5 *Hz*), 8.56 (0.5H, d, *J* = 7.1 *Hz*), 8.35 (1H, q, *J* = 6.2 *Hz*), 7.26 (1H, br s), 6.99 (2H, dd, *J* = 8.6 *Hz*, *J* = 5.4 *Hz*), 6.74 (1H, br s), 6.65 (2H, dd, *J* = 7.8 *Hz*, *J* = 4.4 *Hz*), 4.37-4.32 (1H, m), 3.55 (1H, t, *J* = 2.1 *Hz*), 3.41 (0.5H, d, *J* = 1.9 *Hz*), 3.37 (0.5H, d, *J* = 1.9 *Hz*), 3.10-3.03 (2H, m), 2.96 (1H, dd, *J* = 13.8 *Hz*, *J* = 5.0 *Hz*), 2.79 (1H, td, *J* = 14.1 *Hz*, *J* = 9.8 *Hz*), 2.04 (2H, t, *J* = 7.5 *Hz*), 1.62 (2H, quint, *J* = 7.3 *Hz*). ¹³C NMR (DMSO-d6, 150 MHz): δ<sub>C</sub> 173.8, 173.8, 172.5, 165.7, 165.6,
165.5, 155.9, 130.0, 130.0, 127.4, 115.0, 54.0, 53.9, 52.9, 52.8, 52.5, 38.4, 35.8, 35.6, 32.4, 24.9, 24.9.
ESI(+)-HRMS (m/z) [M+H⁺] calcd for C₁₇H₂₂N₃O₇: 380.1452. Observed: 380.1452. HPLC method 2: tR =
0.82 min, purity = 99.5%.

**((trans)-3-((5-amino-5-oxopentyl)carbamoyl)oxirane-2-carbonyl)-L-tyrosine (9).** The general procedure for solid phase synthesis was performed using AmphiSpheres 40 RAM resin (0.4 mmol/g; 200 mg), Fmoc-5-Ava-OH (0.22 mmol), HATU (0.22 mmol) and DIEA (0.29 mmol) for the anchoring step followed by **23** (0.22 mmol), PyAOP (0.22 mmol) and DIEA (0.63 mmol) as coupling step. The crude product was then purified to afford **9** as a white solid (7.1 mg, 25%). ¹H NMR (DMSO-d6, 500 MHz): δH 12.90 (1H, br s), 9.24 (1H, s), 8.68-8.50 (1H, m), 8.38 (1H, td, *J* = 8.8 *Hz*, *J* = 5.7 *Hz*), 7.25 (1H, s), 6.99 (2H, dd, *J* = 7.5 *Hz*, *J* = 5.3 *Hz*), 6.73 (1H, s), 6.65 (2H, dd, *J* = 8.4 *Hz*, *J* = 3.8 *Hz*), 4.31 (1H, m), 3.55-3.54 (1H, m), 3.42 (0.5H, d, *J* = 1.9 *Hz*), 3.36 (under water peak), 3.06 (2H, q, *J* = 6.4 *Hz*), 2.96 (1H, dd, *J* = 13.9 *Hz*, *J* = 5.1 *Hz*), 2.78 (1H, td, *J* = 13.9 *Hz*, *J* = 9.7 *Hz*), 2.03 (2H, t, *J* = 7.2 *Hz*), 1.46 (2H, quint, *J* = 7.6 *Hz*), 1.38 (2H, quint, *J* = 7.6 *Hz*). ¹³C NMR (DMSO-d6, 150 MHz): δC 174.1, 172.5, 165.6, 165.5, 155.9, 130.1, 130.0, 127.5, 115.0, 54.1, 52.9, 52.8, 52.5, 38.4, 35.9, 35.7, 34.7, 28.6, 28.5, 22.5. ESI(+)-HRMS (m/z) [M+H⁺] calcd for C₁₈H₂₄N₃O₇: 394.1609. Observed: 394.1604. HPLC method 2: tR = 0.84 min, purity = 99.6%.

**((trans)-3-((6-amino-6-oxohexyl)carbamoyl)oxirane-2-carbonyl)-L-tyrosine (10).** The general procedure for solid phase synthesis was performed using AmphiSpheres 40 RAM resin (0.4 mmol/g; 200 mg), Fmoc-6-Ahx-OH (0.22 mmol), HATU (0.22 mmol) and DIEA (0.29 mmol) for the anchoring step followed by **23** (0.22 mmol), PyAOP (0.22 mmol) and DIEA (0.63 mmol) as coupling step. The crude product was then purified to afford **10** as a white solid (11.1 mg, 38%). ¹H NMR (DMSO-d6, 600 MHz): δH 12.83 (1H, br s), 9.24 (1H, s), 8.68 (0.5H, d, *J* = 8.1 *Hz*), 8.61 (0.5H, d, *J* = 8.1 *Hz*), 8.34 (1H, td, *J* = 10.9 *Hz*, *J* = 5.7 *Hz*), 7.22 (1H, br s), 6.99 (2H, dd, *J* = 8.6 *Hz*, *J* = 7.1 *Hz*), 6.68 (1H, br s), 6.66 (2H, dd, *J* = 8.6 *Hz*, *J* = 4.6 *Hz*), 4.39-4.33 (1H, m), 3.55 (1H, dd, *J* = 2.6 *Hz*, *J* = 1.9 *Hz*), 3.42 (0.5H, d, *J* = 1.8 *Hz*), 3.36 (0.5H, d, *J* = 1.8 *Hz*), 3.06 (2H, q, *J* = 6.5 *Hz*), 2.96 (1H, dd, *J* = 13.9 *Hz*, *J* = 4.8 *Hz*), 2.79 (1H, td, *J* = 13.9 *Hz*, *J* = 9.8 *Hz*), 2.02 (2H, t, *J* = 7.4 *Hz*), 1.47 (2H, quint, *J* = 7.6 *Hz*), 1.40 (2H, quint, *J* = 7.1 *Hz*), 1.22 (2H, quint, *J* = 8.1 *Hz*). ¹³C NMR (DMSO-d6, 150 MHz): δC 174.2, 172.5, 172.5, 165.8, 165.6, 165.5, 165.4 156.0, 130.0, 130.0, 127.3, 115.1, 53.9, 53.8, 52.8, 52.7, 52.4, 40.1, 38.5, 35.8, 35.6, 35.0, 28.7, 28.6, 26.0, 24.8. ESI(+)-HRMS (m/z) [M+H⁺] calcd for C₁₉H₂₆N₃O₇: 408.1765. Observed: 408.1765. HPLC method 2: tR = 0.89 min, purity = 97.8%.

**(S)-6-amino-5-((trans)-3-(((S)-1-carboxy-2-(4-hydroxyphenyl)ethyl)carbamoyl)oxirane-2-carboxamido)-6-oxohexanoic acid (11).** The general procedure for solid phase synthesis was performed using AmphiSpheres 40 RAM resin (0.4 mmol/g; 200 mg), Fmoc-Aad(tBu)-OH (0.22 mmol), HATU (0.22 mmol) and DIEA (0.29 mmol) for the anchoring step followed by **23** (0.22 mmol), PyAOP (0.22 mmol) and DIEA (0.63 mmol) as coupling step. The crude product was then purified to afford **11** as a white solid (10.7 mg, 34%). ¹H NMR (DMSO-d6, 600 MHz): δH 12.44 (1H, m), 9.23 (1H, s), 8.66 (0.5H, d, *J* = 7.2 *Hz*), 8.56 (0.5H, d, *J* = 6.8 *Hz*), 8.49 (1H, dd, *J* = 8.2 *Hz*, *J* = 2.0 *Hz*), 7.51 (0.5H, d, *J* = 1.4 *Hz*), 7.45 (0.5H, d, *J* = 1.4 *Hz*), 7.06 (1H, dd, *J* = 5.7 *Hz*, *J* = 1.5 *Hz*), 7.00 (2H, t, *J* = 8.0 *Hz*), 6.65 (2H, dd, *J* = 8.8 *Hz*, *J* = 6.7 *Hz*), 4.38-4.31 (1H, m), 4.20 (1H, tdd, *J* = 28.3 *Hz*, *J* = 8.3 *Hz*, *J* = 5.1 *Hz*), 3.59 (1H, s), 3.58 (0.5H, d, *J* = 1.9 *Hz*), 3.56 (0.5H, d, *J* = 1.9 *Hz*), 2.96 (1H, dt, *J* = 12.6 *Hz*, *J* = 3.3 *Hz*), 2.79 (1H, dt, *J* = 13.9 *Hz*, *J* = 9.1 *Hz*), 2.2 (2H, t, *J* = 7.4 *Hz*), 1.70-1.64 (1H, m), 1.57-1.43 (3H, m). ¹³C NMR (DMSO-d6, 150 MHz): δC 174.2, 172.8, 172.5, 172.4, 165.7, 165.6, 165.5, 156.0, 130.0, 130.0, 127.3, 115.0, 54.0, 53.9, 52.6, 52.5, 52.5, 52.4, 52.4, 52.1, 35.9, 35.6, 33.3, 31.5, 31.3, 20.9, 20.9. ESI(+)-HRMS (m/z) [M+H⁺] calcd for C₁₉H₂₄N₃O₉: 438.1507. Observed: 438.1502. HPLC method 1: tR = 1.31 min, purity = 98%.
**(S)-6-(((S)-1-amino-3-methyl-1-oxobutan-2-yl)amino)-5-((trans)-3-(((S)-1-carboxy-2-(4-hydroxyphenyl)ethyl)carbamoyl)oxirane-2-carboxamido)-6-oxohexanoic acid (12).** The general procedure for solid phase synthesis was performed using AmphiSpheres 40 RAM resin (0.4 mmol/g; 500 mg), Fmoc-Val-OH (0.72 mmol), HATU (0.72 mmol) and DIEA (1.44 mmol) for the anchoring step followed by Fmoc-Aad(tBu)-OH (0.72 mmol), HATU (0.72 mmol) and DIEA (1.44 mmol) as first coupling step. Then, **23** (0.2 mmol), OxymaPure (0.2 mmol), DIC (0.3 mmol), DMAP (0.02 mmol) and DIEA (1.44 mmol) were used for the final coupling step. The crude product was then purified to afford **12** as a white solid (8.3 mg, 7.7%). <sup>1</sup>H NMR (DMSO-d6, 500 MHz): $\delta_H$ 12.46 (1H, br s), 9.23 (1H, br s), 8.63 (0.5H, d, *J* = 8.0 Hz), 8.54 (0.5H, d, *J* = 8.0 Hz), 7.92 (0.5H, d, *J* = 9.2 Hz), 7.82 (0.5H, d, *J* = 8.9 Hz), 7.38 (1H, d, *J* = 8.3 Hz), 7.04 (1H, d, *J* = 6.2 Hz), 6.99 (2H, dd, *J* = 7.8 Hz, *J* = 6.3 Hz), 6.65 (2H, dd, *J* = 8.6 Hz, *J* = 6.1 Hz), 4.42-4.25 (2H, m), 4.11-4.05 (1H, m), 3.59 (0.5H, d, *J* = 1.8 Hz), 3.57 (1H, d, *J* = 1.6 Hz), 3.54 (0.5H, d, *J* = 1.5 Hz) 2.96 (1H, dd, *J* = 13.9 Hz, *J* = 4.8 Hz), 2.79 (1H, dt, *J* = 14.1 Hz, *J* = 9.0 Hz), 2.20 (2H, t, *J* = 7.0 Hz), 1.98-1.87 (1H, m), 1.70-1.61 (1H, m), 1.58-1.40 (3H, m), 0.86-0.80 (6H, m). <sup>13</sup>C NMR (DMSO-d6, 125 MHz): $\delta_C$ 174.4, 172.7, 170.9, 170.8, 165.6, 165.4, 157.8, 157.6, 155.9, 130.1, 127.6, 115.0, 57.5, 57.4, 54.3, 52.6, 52.5, 52.4, 36.0, 35.7, 33.4, 31.6, 31.3, 30.6, 30.5, 21.0, 20.9, 19.3, 18.1, 18.0. ESI(+)-HRMS (m/z) [M+H<sup>+</sup>] calcd for C<sub>24</sub>H<sub>33</sub>N<sub>4</sub>O<sub>10</sub>: 537.2191. Observed: 537.8185. HPLC method 2: *t*<sub>R</sub> = 0.96 min, purity >99%.

**(S)-6-(((S)-1-amino-4-methyl-1-oxopentan-2-yl)amino)-5-((trans)-3-(((S)-1-carboxy-2-(4-hydroxyphenyl)ethyl)carbamoyl)oxirane-2-carboxamido)-6-oxohexanoic acid (13).** The general procedure for solid phase synthesis was performed using AmphiSpheres 40 RAM resin (0.4 mmol/g; 500 mg), Fmoc-Leu-OH (0.72 mmol), HATU (0.72 mmol) and DIEA (1.44 mmol) for the anchoring step followed by Fmoc-Aad(tBu)-OH (0.72 mmol), HATU (0.72 mmol) and DIEA (1.44 mmol) as first coupling step. Then, **23** (0.2 mmol), OxymaPure (0.2 mmol), DIC (0.3 mmol), DMAP (0.02 mmol) and DIEA (1.44 mmol) were used for the final coupling step. The crude product was then purified to afford **13** as a white solid (4.9 mg, 4.5%). <sup>1</sup>H NMR (DMSO-d6, 500 MHz): $\delta_H$ 8.68 (0.5H, d, *J* = 7.7 Hz), 8.52 (0.5H, d, *J* = 7.2 Hz), 8.30 (1H, s), 8.19 (0.5H, d, *J* = 6.8 Hz), 8.10-8.01 (1.5H, m), 7.26 (1H, d, *J* = 20.0 Hz), 6.99-6.97 (1H, m), 6.95 (2H, t, *J* = 8.5 Hz), 6.62 (2H, t, *J* = 8.1 Hz), 4.31-4.14 (3H, m), 3.62-3.51 (2H, m), 2.96 (1H, dt, *J* = 14.1 Hz, *J* = 5.6 Hz), 2.79 (1H, dt, *J* = 12.5 Hz, *J* = 7.9 Hz), 2.17 (2H, t, *J* = 7.3 Hz), 1.71-1.64 (1H, m), 1.61-1.41 (6H, m), 0.87 (3H, dd, *J* = 6.6 Hz, *J* = 1.3 Hz), 0.82 (3H, dd, *J* = 6.6 Hz, *J* = 1.6 Hz). <sup>13</sup>C NMR (DMSO-d6, 125 MHz): $\delta_C$ 174.0, 174.0, 170.8, 166.0, 165.9, 165.1, 164.9, 164.9, 155.8, 155.7, 130.2, 128.4, 128.2, 114.9, 55.1, 53.0, 52.8, 52.8, 52.7, 52.5, 50.9, 50.9, 40.8, 36.5, 36.0, 33.9, 33.7, 31.5, 24.3, 23.1, 21.6, 21.6, 21.0, 20.9. ESI(+)-HRMS (m/z) [M+H<sup>+</sup>] calcd for C<sub>25</sub>H<sub>35</sub>N<sub>2</sub>O<sub>10</sub>: 551.2348. Observed: 551.2349. HPLC method 1: *t*<sub>R</sub> = 1.78 min, purity = 96.5%.

**(S)-6-(((S)-1-amino-1-oxo-3-phenylpropan-2-yl)amino)-5-((trans)-3-(((S)-1-carboxy-2-(4-hydroxyphenyl)ethyl)carbamoyl)oxirane-2-carboxamido)-6-oxohexanoic acid (14).** The general procedure for solid phase synthesis was performed using AmphiSpheres 40 RAM resin (0.4 mmol/g; 500 mg), Fmoc-Phe-OH (0.72 mmol), HATU (0.72 mmol) and DIEA (1.44 mmol) for the anchoring step followed by Fmoc-Aad(tBu)-OH (0.72 mmol), HATU (0.72 mmol) and DIEA (1.44 mmol) as first coupling step. Then, **23** (0.2 mmol), OxymaPure (0.2 mmol), DIC (0.3 mmol), DMAP (0.02 mmol) and DIEA (1.44 mmol) were used for the final coupling step. The crude product was then purified to afford **14** as a white solid (5.1 mg, 4.4%). <sup>1</sup>H NMR (DMSO-d6, 500 MHz): $\delta_H$ 8.71 (0.5H, br s), 8.45 (0.5H, br s), 8.17 (0.5H, d, *J* = 7.9 Hz), 8.10 (0.5H, d, *J* = 6.9 Hz), 7.35 (1H, d, *J* = 26.8 Hz), 7.26-7.15 (5H, m), 7.10 (1H, d, *J* = 10.8 Hz), 6.93 (2H, t, *J* = 8.9 Hz), 6.61 (2H, t, *J* = 8.1 Hz), 4.43-4.38 (1H, m), 4.26-4.05 (2H, m), 3.62 (1H, d, *J* = 1.1 Hz), 3.54 (0.5H, d, *J* = 1.1 Hz), 3.46 (0.5H, d, *J* = 1.1 Hz), 3.03-2.93 (2H, m), 2.84-2.76 (2H, m), 2.12-2.05 (2H, m), 1.60-1.31 (5H, m). <sup>13</sup>C NMR (DMSO-d6, 125 MHz): $\delta_C$ 172.8, 170.7, 166.0, 166.0, 165.0, 164.6, 155.8, 155.6, 138.0, 137.9, 130.2, 129.2, 128.1, 126.3, 126.3, 114.8, 55.2, 53.8, 53.0, 52.8,
52.7, 37.5, 37.4, 36.5, 36.0, 34.2, 31.4, 31.2, 29.2, 29.1, 21.0, 20.9. ESI(+)-HRMS (m/z) [M+H⁺] calcd for C₂₈H₃₃N₄O₁₀: 585.2191. Observed: 585.2191. HPLC method 1: tR = 1.86 min, purity = 96.5%.

(S)-6-(((S)-1-amino-3-(1H-indol-3-yl)-1-oxopropan-2-yl)amino)-5-((trans)-3-(((S)-1-carboxy-2-(4-hydroxyphenyl)ethyl)carbamoyl)oxirane-2-carboxamido)-6-oxohexanoic acid (15). The general procedure for solid phase synthesis was performed using AmphiSpheres 40 RAM resin (0.4 mmol/g; 500 mg), Fmoc-Trp(Boc)-OH (0.72 mmol), HATU (0.72 mmol) and DIEA (1.44 mmol) for the anchoring step followed by Fmoc-Aad(tBu)-OH (0.72 mmol), HATU (0.72 mmol) and DIEA (1.44 mmol) as first coupling step. Then, 23 (0.2 mmol), OxymaPure (0.2 mmol), DIC (0.3 mmol), DMAP (0.02 mmol) and DIEA (1.44 mmol) were used for the final coupling step. The crude product was then purified to afford 15 as a white solid (5.2 mg, 4.2%).¹H NMR (DMSO-d6, 500 MHz): δH 10.86 (1H, d, *J* = 37.4*Hz*), 9.21 (0.5H, br s), 8.63 (0.5H, d, *J* = 6.7 *Hz*), 8.55-8.46 (0.5H, m), 8.30 (0.5H, br s), 8.13 (0.5H, d, *J* = 8.1 *Hz*), 8.09 (0.5H, d, *J* = 8.0 *Hz*), 7.58 (1H, t, *J* = 7.3 *Hz*), 7.36 (1H, d, *J* = 24.0 *Hz*), 7.31 (1H, dd, *J* = 8.1 *Hz*, *J* = 6.2 *Hz*), 7.11 (1H, dd, *J* = 5.9 *Hz*, *J* = 1.9 *Hz*), 7.08-7.02 (2H, m), 6.98-6.93 (3H, m), 6.63 (2H, dd, *J* = 8.1 *Hz*, *J* = 6.9 *Hz*), 4.44 (1H, q, *J* = 5.7 *Hz*), 4.33-4.15 (2H, m), 3.61 (1H, s), 3.56 (0.5H, d, *J* = 1.3 *Hz*), 3.50 (0.5H, d, *J* = 1.1 *Hz*), 3.13-3.08 (1H, m), 2.98-2.89 (2H, m), 2.83-2.74 (1H, m), 2.14 (2H, q, *J* = 4.6 *Hz*), 1.66-1.58 (1H, m), 1.53-1.37 (3H, m). ¹³C NMR (DMSO-d6, 125 MHz): δC 173.3, 173.2, 170.7, 165.9, 165.8, 165.2, 165.0, 155.8, 155.7, 136.1, 136.0, 130.2, 127.4, 123.6, 123.5, 120.8, 118.5, 118.2, 114.9, 111.4, 111.3, 110.1, 110.0, 53.3, 52.8, 52.6, 52.3, 36.4, 35.9, 33.6, 31.5, 31.4, 27.7, 27.6, 20.9, 20.9. ESI(+)-UPLCMS (m/z) [M+H⁺] observed: 624.4. HPLC method 1: tR = 2.00 min, purity = 95.8%.

tert-butyl (S)-5-amino-6-oxo-6-(phenethylamino)hexanoate (35). 2-phenethylamine (0.74 mmol) and Fmoc-Aad(tBu)-OH (0.57 mmol) were dissolved in 10 mL anhydrous DMF. DIEA (1.71 mmol) was added followed by HATU (0.57 mmol). The mixture was stirred for 15 min. 40 mL of water was added to the mixture before extraction with 3x15 mL AcOEt. The organic layer was then washed with 3x15 mL of a 1 M solution of KHSO₄, 2x20 mL of brine then dried on MgSO₄, filtrated on cotton and concentrated under vacuum to give **34**. The general solution phase Fmoc removal procedure was then used to give **35** as a pale yellowish oil (crude: 102.3 mg, 56%) which was used without further purification.

(S)-5-((trans)-3-(((S)-1-carboxy-2-(4-hydroxyphenyl)ethyl)carbamoyl)oxirane-2-carboxamido)-6-oxo-6-(phenethylamino)hexanoic acid (16). Compounds 35 (0.5 mmol) and 23 (1.0 mmol) were dissolved in 4 mL anhydrous DMF. DMAP (0.05 mmol), OxymaPure (0.5 mmol) and DIEA (2.0 mmol) and finally DIC (0.65 mmol) were added. The mixture was left overnight under magnetic stirring. 50 mL water was added to the mixture before extraction with 3x30 mL AcOEt. The organic layer was then washed with 2x30 mL NaHCO₃ (sat), 2x30 mL KHSO₄ (1 M) and 2x50 mL brine then dried on MgSO₄, filtrated on cotton and concentrated under vacuum. The general solution phase tBu removal procedure was then used to give, after a purification step, **16** as a white powder (121.9 mg, 45%). ¹H NMR (DMSO-d6, 500 MHz): δH 12.80 (1H, br s), 12.09 (1H, br s), 9.25 (1H, br s), 8.76 (0.5H, d, *J* = 8.3 *Hz*), 8.64 (0.5H, d, *J* = 8.1 *Hz*), 8.59 (1H, d, *J* = 8.1 *Hz*), 8.16 (1H, dt, *J* = 23.8 *Hz*, *J* = 5.7 *Hz*), 7.28 (2H, t, *J* = 7.6 *Hz*), 7.22-7.16 (3H, m), 7.01 (2H, dd, *J* = 8.7 *Hz*, *J* = 6.7 *Hz*), 6.66 (2H, dd, *J* = 8.5 *Hz*, *J* = 5.8 *Hz*), 4.42-4.31 (1H, m), 4.26-4.15 (1H, m), 3.60 (0.5H, d, *J* = 1.7 *Hz*), 3.59 (1H, d, *J* = 1.6 *Hz*), 3.57 (0.5H, d, *J* = 1.7 *Hz*), 3.36-3.18 (2H, m), 2.96 (1H, dt, *J* = 13.9 *Hz*, *J* = 4.7 *Hz*), 2.80 (1H, dt, *J* = 13.2 *Hz*, *J* = 9.5 *Hz*), 2.69 (2H, t, *J* = 7.3*Hz*), 2.18 (2H, t, *J* = 7.0*Hz*), 1.65-1.34 (4H, m). ¹³C NMR (DMSO-d6, 125 MHz): δC 174.2, 172.6, 172.5, 170.7, 170.7, 165.9, 165.5, 156.0, 139.4, 139.3, 130.1, 130.0, 128.7, 128.3, 127.3, 126.1, 115.1, 54.0, 53.9, 52.6, 52.5, 52.4, 52.4, 35.8, 35.6, 35.1, 33.2, 31.6, 31.5, 20.9. ESI(+)-HRMS (m/z) [M+H⁺] calcd for C₂₇H₃₂N₃O₉: 542.2139. Observed: 542.2139. HPLC method 1: tR = 2.14 min, purity = 99%.
**tert-butyl (S)-5-amino-6-oxo-6-((3-phenylpropyl)amino)hexanoate (Intermediate A).** The same procedure as described for the synthesis of **35** was performed using 3-phenylpropylamine (0.74 mmol) and Fmoc-Aad(tBu)-OH (0.57 mmol) to give intermediate **A** as a pale yellowish oil (crude: 76.3 mg, 40%) which was used without further purification.

**(S)-5-((trans)-3-(((S)-1-carboxy-2-(4-hydroxyphenyl)ethyl)carbamoyl)oxirane-2-carboxamido)-6-oxo-6-((3-phenylpropyl)amino)hexanoic acid (17).** The same procedure as described for the synthesis of **16** was performed using intermediate **A** (0.4 mmol) and **23** (0.8 mmol) to give, after a purification step, **17** as a white powder (82.2 mg, 37%). <sup>1</sup>H NMR (DMSO-d6, 500 MHz): $\delta_H$ 12.83 (1H, br s), 12.09 (1H, br s), 9.24 (1H, br s), 8.74 (0.5H, d, $J = 8.1$ Hz), 8.65-8.58 (1.5H, m), 8.13 (1H, dt, $J = 27.2$ Hz, $J = 5.2$ Hz), 7.27 (2H, t, $J = 7.5$ Hz), 7.20-7.15 (3H, m), 7.00 (2H, dd, $J = 8.5$ Hz, $J = 6.3$ Hz), 6.66 (2H, dd, $J = 8.5$ Hz, $J = 5.2$ Hz), 4.40-4.32 (1H, m), 4.28-4.19 (1H, m), 3.60 (1H, dd, $J = 11.0$ Hz, $J = 1.7$ Hz), 3.58-3.57 (1H, m), 3.13-2.98 (2H, m), 2.95 (1H, dt, $J = 13.9$ Hz, $J = 4.6$ Hz), 2.79 (1H, dt, $J = 13.9$ Hz, $J = 9.2$ Hz), 2.56 (2H, t, $J = 7.2$ Hz), 2.21 (2H, t, $J = 7.2$ Hz), 1.71-1.62 (3H, m), 1.59-1.42 (3H, m). <sup>13</sup>C NMR (DMSO-d6, 125 MHz): $\delta_c$ 174.3, 172.5, 170.7, 165.5, 156.0, 141.7, 130.1, 130.0, 128.4, 128.3, 127.3, 125.8, 115.1, 54.0, 52.4, 52.4, 38.1, 35.8, 35.6, 33.2, 32.5, 31.6, 31.5, 30.9, 20.9. ESI(+)-HRMS (m/z) [M+H<sup>+</sup>] calcd for C<sub>28</sub>H<sub>34</sub>N<sub>3</sub>O<sub>9</sub>: 556.2295. Observed: 556.2289. HPLC method 1: t<sub>R</sub> = 2.34 min, purity = 98.2%.

**tert-butyl (S)-5-amino-6-oxo-6-((4-phenylbutyl)amino)hexanoate (Intermediate B).** The same procedure as described for the synthesis of **35** was performed using 4-phenylbutylamine (0.74 mmol) and Fmoc-Aad(tBu)-OH (0.57 mmol) to give intermediate **B** as a pale yellowish oil (crude: 91.4 mg, 46%) which was used without further purification.

**(S)-5-((trans)-3-(((S)-1-carboxy-2-(4-hydroxyphenyl)ethyl)carbamoyl)oxirane-2-carboxamido)-6-oxo-6-((4-phenylbutyl)amino)hexanoic acid (18).** Intermediate **B** (0.1 mmol) and **23** (0.08 mmol) were dissolved in 1mL of NMP. PyAOP (0.08 mmol) and DIEA (0.32 mmol) were added and the mixture was left overnight under magnetic stirring. 20 mL of water was added to the mixture before extraction with 3x10 mL of AcOEt. The organic layer was then washed with 2x10 mL NaHCO<sub>3</sub> (sat), 2x10 mL KHSO<sub>4</sub> (1 M) and 2x15 mL brine then dried on MgSO<sub>4</sub>, filtrated on cotton and concentrated under vacuum. The general solution phase tBu removal procedure was then used to give, after a purification step, **18** as a white powder (17.1 mg, 30%). <sup>1</sup>H NMR (DMSO-d6, 500 MHz): $\delta_H$ 12.51 (1H, m), 9.25 (1H, br s), 8.68-8.55 (2H, m), 8.06 (1H, dt, $J = 26.8$ Hz, $J = 5.2$ Hz), 7.26 (2H, t, $J = 7.6$ Hz), 7.19-7.14 (3H, m), 7.00 (2H, dd, $J = 7.7$ Hz, $J = 6.6$ Hz), 6.66 (2H, dd, $J = 8.4$ Hz, $J = 5.7$ Hz), 4.37-4.29 (1H, m), 4.26-4.17 (1H, m), 3.60 (0.5H, d, $J = 1.7$ Hz), 3.57 (1H, dd, $J = 2.9$ Hz, $J = 1.9$ Hz), 3.56 (0.5H, d, $J = 1.7$ Hz), 3.13-3.00 (2H, m), 2.97-2.93 (1H, m), 2.79 (1H, dt, $J = 13.9$ Hz, $J = 9.1$ Hz), 2.56 (2H, t, $J = 7.4$ Hz), 2.20 (2H, t, $J = 7.4$ Hz), 1.67-1.59 (1H, m), 1.57-1.36 (7H, m). <sup>13</sup>C NMR (DMSO-d6, 125 MHz): $\delta_c$ 174.3, 172.6, 172.5, 170.6, 170.6, 165.8, 165.6, 165.5, 156.0, 142.1, 130.1, 128.3, 128.3, 127.4, 125.7, 115.1, 54.1, 52.6, 52.5, 52.4, 38.3, 35.9, 35.7, 34.8, 33.3, 31.7, 31.6, 28.7, 28.3, 20.9, 20.9. ESI(+)-HRMS (m/z) [M+H<sup>+</sup>] calcd for C<sub>29</sub>H<sub>36</sub>N<sub>3</sub>O<sub>9</sub>: 570.2446. Observed: 570.2443. HPLC method 1: t<sub>R</sub> = 2.52 min, purity = 95.4%.

**((trans)-3-(((S)-6-(tert-butoxy)-1,6-dioxo-1-(phenethylamino)hexan-2-yl)carbamoyl)oxirane-2-carboxylic acid (Intermediate C).** Compound **35** (0.5 mmol), (+,-)-trans-epoxysuccinic acid (0.5 mmol) and DIEA (4.5 mmol) were dissolved in 3 mL NMP followed by PyAOP (4.5 mmol). The reaction mixture was stirred for 1 hour and then poured in 50 mL of ice-cold water. The obtained white precipitate was removed by filtration. The pH of the filtrate was lowered to 2 by adding 1 M KHSO<sub>4</sub> solution. Then the water phase was extracted with 2x30 mL AcOEt. The combined organic phases were washed with brine, dried over Na<sub>2</sub>SO<sub>4</sub> and concentrated dry to provide intermediate **C** as white powder (300 mg, 91%). The crude product was used without any further purification.
tert-butyl-(*S*)-5-((trans)-3-(((*S*)-1-(tert-butoxy)-3-(4-hydroxyphenyl)-1-oxopropan-2-yl)carbamoyl)oxirane-2-carboxamido)-6-oxo-6-(phenethylamino)hexanoate (Intermediate D). Intermediate C (0.35 mmol), HCl.H-Tyr-OtBu (0.52 mmol) and DIEA (1.38 mmol) were dissolved in 3 mL NMP followed by PyAOP (0.41 mmol). After 5 min of stirring the reaction mixture was poured in 20 mL of AcOEt. The organic phase was washed 1 time with a 1/1 v/v water brine mixture, 3 times with saturated NaHCO₃ solution, 3 times with 1 M KHSO₄ solution, one time with brine, dried over MgSO₄ and evaporated dry to provide 66mg of intermediate D as a crude oil that was used without further purification.

(*S*)-5-((trans)-3-(((*S*)-1-carboxy-2-(4-methoxyphenyl)ethyl)carbamoyl)oxirane-2-carboxamido)-6-oxo-6-(phenethylamino)hexanoic acid (Compound 19). 0.03 mmol of intermediate D were dissolved in 300 µL of NMP. Then, 0.3 mmol of Cs₂CO₃ were suspended and the reaction mixture was stirred for 10 min. 0.12 mmol of methyl iodide (Mel) were added and the reaction mixture was stirred overnight. The Mel was evaporated under nitrogen flux and reaction mixture was poured in 20 mL of AcOEt. The organic phase was washed 1 time with water/brine, 1 time with 1 M KHSO₄, 1 time with brine, dried over MgSO₄ and concentrated dry. The general solution phase tBu removal procedure was then used. The remaining oil was purified by preparative HPLC to obtain, after lyophilization, compound 19 as white solid (7.6mg, 46%). ¹H NMR (DMSO-d6, 500 MHz): δ<sub>H</sub> 12.51 (1H, m), 8.70 (0.5H, br s), 8.61-8.57 (1.5H, m), 8.16 (1H, dt, *J* = 24.6 *Hz*, *J* = 5.6 *Hz*), 7.28 (2H, t, *J* = 8.1 *Hz*), 7.20-7.17 (3H, m), 7.14-7.10 (2H, m), 6.85-6.82 (2H, m), 4.40-4.31 (1H, m), 4.25-4.17 (1H, m), 3.71 (3H, d, *J* = 3.3 *Hz*), 3.30-3.19 (2H, m), 3.03-2.99 (1H, m), 2.84 (1H, dt, *J* = 13.8 *Hz*, *J* = 8.9 *Hz*), 2.69 (2H, t, *J* = 7.4*Hz*), 2.17 (2H, t, *J* = 7.3Hz), 1.63-1.56 (1H, m), 1.52-1.36 (3H, m). ¹³C NMR (DMSO-d6, 125 MHz): δ<sub>C</sub> 174.3, 172.6, 172.5, 170.7, 165.8, 165.6, 165.5, 157.9, 139.4, 130.2, 130.2, 130.1, 129.3, 128.7, 128.3, 126.1, 113.6, 55.0, 54.0, 52.6, 52.5, 52.4, 45.9, 45.9, 35.8, 35.6, 35.1, 33.3, 31.6, 31.5, 26.0, 25.9, 20.9, 20.9. ESI(+)-HRMS (m/z) [M+H<sup>+</sup>] calcd for C<sub>28</sub>H<sub>34</sub>N<sub>3</sub>O<sub>9</sub>: 556.2290. Observed: 556.2295. HPLC method 1: t<sub>R</sub> = 2.47 min, purity = >98%.

(*S*)-5-((trans)-3-(((*S*)-1-carboxy-2-(4-ethoxyphenyl)ethyl)carbamoyl)oxirane-2-carboxamido)-6-oxo-6-(phenethylamino)hexanoic acid (Compound 20). 0.03 mmol of intermediate D were dissolved in 300 µL of NMP. Then, 0.3 mmol of Cs₂CO₃ were suspended and the reaction mixture was stirred for 10 min. 0.12 mmol of ethyl iodide (EtI) were added and the reaction mixture was stirred overnight. The EtI was evaporated under nitrogen flux and reaction mixture was poured in 20 mL of AcOEt. The organic phase was washed 1 time with water/brine, 1 time with 1 M KHSO₄, 1 time with brine, dried over MgSO₄ and concentrated dry. The general solution phase tBu removal procedure was then used. The remaining oil was purified by preparative HPLC to obtain, after lyophilization, compound 20 as white solid (6.1mg, 37%). ¹H NMR (DMSO-d6, 500 MHz): δ<sub>H</sub> 12.72 (1.5H, br s), 8.75 (0.5H, d, *J* = 8.3 *Hz*), 8.64 (0.5H, d, *J* = 8.1 *Hz*), 8.55 (1H, dd, *J* = 8.3 *Hz*, *J* = 5.0 *Hz*), 7.28 (2H, t, *J* = 7.9 *Hz*), 7.20-7.17 (3H, m), 7.12 (2H, t, *J* = 8.2 *Hz*), 6.82 (2H, t, *J* = 8.7 *Hz*), 4.43-4.36 (1H, m), 4.25-4.17 (1H, m), 4.00-3.95 (2H, m), 3.59-3.57 (2H, m), 3.35-3.20 (2H, m), 3.01 (1H, ddd, *J* = 14.1 *Hz*, *J* = 5.1 *Hz*, *J* = 2.1 *Hz*), 2.84 (1H, ddd, *J* = 14.1 *Hz*, *J* = 8.9 *Hz*, *J* = 6.9 *Hz*), 2.70 (2H, t, *J* = 7.4Hz), 2.17 (2H, t, *J* = 7.3Hz), 1.64-1.57 (1H, m), 1.53-1.41 (3H, m), 1.30 (3H, t, *J* = 7.0 *Hz*). ¹³C NMR (DMSO-d6, 125 MHz): δ<sub>C</sub> 174.3, 172.6, 170.7, 165.5, 157.2, 139.4, 130.2, 130.2, 128.7, 128.3, 126.1, 114.1, 62.8, 52.6, 52.5, 52.4, 45.9, 45.9, 35.1, 33.3, 31.6, 31.5, 26.0, 25.9, 20.9, 14.6. ESI(+)-HRMS (m/z) [M+H<sup>+</sup>] calcd for C<sub>29</sub>H<sub>36</sub>N<sub>3</sub>O<sub>9</sub>: 570.2446. Observed: 570.2451. HPLC method 1: t<sub>R</sub> = 2.64 min, purity = 97.2%.

TFA salt of allyl (*S*)-2-amino-3-(4-(benzyloxy)phenyl)propanoate (31). Boc-Tyr(Bn)-OH (5.39 mmol) was dissolved in 5 mL anhydrous DMF with Cs₂CO₃ (5.39 mmol) and stirred for 10 min before adding allyl bromide (8.08 mmol). The mixture was stirred for 2 hours. 60 mL of a 1:1 H₂O/brine solution was added to the mixture which was then extracted with 3x20 mL AcOEt. The organic layer was then
washed with 2x20 mL NaHCO₃(sat), 2x20 mL KHSO₄ (1 M), 2x30 mL brine then dried on MgSO₄, filtrated on cotton and concentrated under vacuum. The general solution phase Boc removal procedure was then used to give **31** as a colorless oil (crude: 2.3 g, 99%) which was used without further purification.

**(2S,3S)-3-(((S)-1-(allyloxy)-3-(4-(benzyloxy)phenyl)-1-oxopropan-2-yl)carbamoyl)oxirane-2-carboxylic acid (32).** Compound **31** (5.39 mmol) was dissolved in 12 mL of anhydrous DMF and (S,S)-TES (10.8 mmol) was added. DIEA (43.12 mmol) and HATU (8.09 mmol) were added in this order. The mixture was stirred for 30 min before 150 mL of H₂O was added. The extraction was continued with 3x50 mL AcOEt, the organic layer was washed with 3x30 mL KHSO₄ (1 M) and 2x50 mL brine then dried on MgSO₄, filtrated on cotton and concentrated under vacuum to give the desired product **32** and its dimer form **33** (crude: 1.8 g, 80%; ratio (32/33): 88/12) were used without further purification.

**(S)-5-((2S,3S)-3-(((S)-2-(4-(benzyloxy)phenyl)-1-carboxyethyl)carbamoyl)oxirane-2-carboxamido)-6-oxo-6-(phenethylamino)hexanoic acid (LV80; 21).** Compounds **35** (2.18 mmol) and **32** (4.36 mmol) were dissolved in 10 mL of anhydrous DMF. DMAP (0.22 mmol), OxymaPure (4.36 mmol) and DIEA (8.72 mmol) and finally EDCi (5.67 mmol) were added. The mixture was then left overnight under magnetic stirring. 100 mL of H₂O was added to the mixture before extraction with 3x30 mL AcOEt. The organic layer was then washed with 2x30 mL KHSO₄ (1 M), 2x50 mL brine then dried on MgSO₄, filtrated on cotton and concentrated under vacuum. A purification step gave the desired product **36** as a white powder (793.4 mg, 50%). The general solution phase Boc removal procedure was then used to give **37** as a yellowish oil. Without further purification, **37** was dissolved in AcN/H₂O (8/2) (v/v) and a solution of NaOH (1M) (2.18 mmol) was added to the reaction mixture which was stirred for 30 min. A solution of KHSO₄ (1 M) was then added until pH 5-6 to quench the reaction. The solution was then immediately purified by preparative HPLC with a gradient going from 30/70/0.1 AcN/H₂O/TFA to 60/40/0.1 AcN/H₂O/TFA in 30 min. The fraction containing the pure product were identified by HPLC, regrouped, and lyophilized to give the desired **LV80** as a white powder (413.1 mg, 60%). ¹H NMR (DMSO-d6, 500 MHz): δ<sub>H</sub> 12.22 (2H, br s), 8.73 (1H, br s), 8.55 (1H, d, *J* = 8.4 Hz), 8.16 (1H, t, *J* = 5.5 Hz), 7.44 (2H, d, *J* = 7.1 Hz), 7.38 (2H, t, *J* = 7.2 Hz), 7.32 (1H, t, *J* = 7.2 Hz), 7.27 (2H, t, *J* = 7.5 Hz), 7.20-7.14 (5H, m), 6.93 (2H, d, *J* = 8.6 Hz), 5.06 (2H, s), 4.40 (1H, q, *J* = 4.7 Hz), 4.24 (1H, q, *J* = 5.8 Hz), 3.58 (2H, dd, *J* = 7.1 Hz, *J* = 1.6 Hz), 3.30 (1H, s), 3.26-3.19 (1H, m), 3.02 (1H, dd, *J* = 13.9 Hz, *J* = 4.7 Hz), 2.84 (1H, dd, *J* = 14.1 Hz, *J* = 9.2 Hz), 2.69 (2H, t, *J* = 7.3 Hz), 2.17 (2H, t, *J* = 7.3 Hz), 1.64-1.57 (1H, m), 1.53-1.37 (3H, m). ¹³C NMR (DMSO-d6, 125 MHz): δ<sub>C</sub> 174.2, 172.5, 170.7, 165.6, 165.4, 165.3, 157.1, 139.3, 137.2, 130.2, 129.5, 128.7, 128.6, 128.5, 128.4, 128.4, 128.3, 128.3, 127.8, 127.8, 127.7, 126.1, 114.5, 69.1, 53.8, 52.5, 52.4, 45.7, 35.7, 35.0, 33.2, 31.6, 20.8. ESI(+)-HRMS (m/z) [M+H<sup>+</sup>] calcd for C₃₄H₃₇N₃O₉: 632.2603. Observed: 632.2606. HPLC method 1: t<sub>R</sub> = 3.17 min, purity = 99.8%.

**(S)-3-(4-(benzyloxy)phenyl)-2-((2S,3S)-3-(((S)-6-(hydroxyamino)-1,6-dioxo-1(phenethylamino)hexan-2-yl)carbamoyl)oxirane-2-carboxamido)propanoic acid (LV87; 22).** Compound **37** (0.09 mmol) was dissolved in 10 mL DMF then DMAP (0.01 mmol), OxymaPure (0.09 mmol) and DIEA (0.36 mmol) were added. Finally, EDCi (22.4 mg, 0.12 mmol) and O-(tetrahydropyran-2-yl)hydroxylamine (H₂NO-THP) (0.1 mmol) were added. After stirring for 27 hours, as the reaction wasn't complete, H₂NO-THP (0.1 mmol), DIEA (0.18 mmol) and EDCi (0.06 mmol) were introduced. After 3 hours, the reaction was stopped, 50 mL H₂O was added and extraction was performed with 3x30 mL AcOEt. The organic layer was then washed with 2x30 mL KHSO₄ (1 M), 2x50 mL brine then dried on MgSO₄, filtrated on cotton, and concentrated under vacuum to give **38** as a white fluffy solid (crude: 61.1 mg, 88%). Without further purification, **38** was dissolved in AcN/H₂O (8/2) (v/v) and a solution of NaOH (1 M) (0.16 mmol) was added to the reaction mixture which was stirred for 5 hours. A solution of KHSO₄ (1 M) was then added until pH 5-6 to quench the reaction. The solution was then immediately purified by preparative HPLC with a gradient going from 35/65/0.1 AcN/H₂O/TFA to
65/35/0.1 AcN/H₂O/TFA in 30 min. The fraction containing the pure product were identified by HPLC, regrouped, and lyophilized to provide **39** as a white powder (16.4 mg, 28%). The general solution phase THP removal procedure was performed followed by a preparative HPLC with a gradient going from 30/70/0.1 AcN/H₂O/TFA to 60/40/0.1 AcN/H₂O/TFA in 30 min. The fraction containing the pure product were identified by HPLC, regrouped, and lyophilized to give the desired **LV87** as a white powder. (3.3 mg, 23%). ¹H NMR (DMSO-d6, 500 MHz): δ<sub>H</sub> 13.05 (1H, br s), 10.37 (1H, s), 8.72 (1H, s), 8.59 (1H, d, *J* = 8.2 *Hz*), 8.17 (1H, t, *J* = 5.6 *Hz*), 7.44 (2H, d, *J* = 7.7 *Hz*), 7.38 (2H, t, *J* = 7.7 *Hz*), 7.34-7.30 (1H, m), 7.29-7.25 (2H, m), 7.20-7.16 (3H, m), 7.15-7.11 (2H, m), 6.92 (2H, d, *J* = 8.0 *Hz*), 5.05 (2H, s), 4.32 (1H, br s), 4.22 (1H, q, *J* = 6.9 *Hz*), 3.61-3.53 (2H, m), 3.30-3.19 (2H, m), 3.01 (1H, dd, *J* = 13.9 *Hz*, *J* = 4.7 *Hz*), 2.84 (1H, dd, *J* = 13.9 *Hz*, *J* = 8.9 *Hz*), 2.69 (2H, t, *J* = 7.4 *Hz*), 1.91 (2H, t, *J* = 7.2 *Hz*), 1.63-1.52 (1H, m), 1.51-1.33 (3H, m). ¹³C NMR (DMSO-d6, 125 MHz): δ<sub>C</sub> 172.4, 170.8, 170.7, 168.7, 165.5, 157.0, 139.4, 139.3, 137.2, 130.2, 128.7, 128.4, 128.3, 127.8, 127.8, 126.1, 114.4, 69.1, 52.6, 52.6, 35.9, 35.1, 32.0, 31.8, 21.6. ESI(+)-HRMS (m/z) [M+H<sup>+</sup>] calcd for C<sub>34</sub>H<sub>39</sub>N<sub>4</sub>O<sub>9</sub>: 647.2712. Observed: 647.2714. HPLC method 1: *t*<sub>R</sub> = 2.89 min, purity = 95%.

## ASSOCIATED CONTENT

### Supporting Information

The Supporting Information is available free of charge at ...

Additional representative data (MTT, stability, *K*<sub>i</sub>, ...) and additional descriptive figures; ¹H NMR, ¹³C NMR, and HRMS of tested compounds **1 – 22**; UV-purity HPLC traces of representative compounds (PDF)

## AUTHOR INFORMATION

### Corresponding Authors

**Lubomir Vezenkov** - IBMM, Université de Montpellier, CNRS, ENSCM, 34293 Montpellier, France

https://orcid.org/0000-0001-6348-7985

**Siem Van Der Laan** - MT-act, CRBM, Montpellier 34293, France

https://orcid.org/0000-0001-8169-1963

**Krzysztof Rogowski** - IGH, CNRS, Université de Montpellier, 34090 Montpellier, France

https://orcid.org/0000-0002-0206-3289

### Authors:

**Anthony Feral** – 1) MT-act, CRBM, Montpellier 34293, France 2) IBMM, Université de Montpellier, CNRS, ENSCM, 34293 Montpellier, France

https://orcid.org/0000-0001-8896-9710

**Guillaume Marcellin** - MT-act, CRBM, Montpellier 34293, France

https://orcid.org/0009-0006-2831-7845

**Yoann Lannay** - MT-act, CRBM, Montpellier 34293, France
https://orcid.org/0009-0006-6472-5088

**Camille Hennebert** - IBMM, Université de Montpellier, CNRS, ENSCM, 34293 Montpellier, France

**Yazid Souf** - IBMM, Université de Montpellier, CNRS, ENSCM, 34293 Montpellier, France

https://orcid.org/0000-0002-1717-9137

**Aicha Talha** - IBMM, Université de Montpellier, CNRS, ENSCM, 34293 Montpellier, France

https://orcid.org/0000-0001-8250-5333

**Aline Haetty** - IGH, CNRS, Université de Montpellier, 34090 Montpellier, France

**Matthieu Simon** - IBMM, Université de Montpellier, CNRS, ENSCM, 34293 Montpellier, France

https://orcid.org/0000-0002-9202-1583

**Pascal Verdié** - IBMM, Université de Montpellier, CNRS, ENSCM, 34293 Montpellier, France

https://orcid.org/0000-0002-5807-0293

**Maxime Louet** - IBMM, Université de Montpellier, CNRS, ENSCM, 34293 Montpellier, France

https://orcid.org/0000-0002-5840-2147

**Khaled Hached** - MT-act, CRBM, Montpellier 34293, France

https://orcid.org/0000-0003-1143-6929

**Muriel Amblard** - IBMM, ENSCM, Université de Montpellier, CNRS, 34293 Montpellier, France

https://orcid.org/0000-0001-8922-1755

## Author Contributions

The study was designed by L.V., S.v.d.L, K.R. M.A. and A.F.; Synthesis of the inhibitors were performed by L.V., A.F., Y.S. and C.H.; Molecular docking studies were performed by A.F and M.L.; Stability, selectivity and lipophilicity of the compounds were studied by P.V., A.T., M.S. and A.F.; The biological evaluations of the compounds were performed by G.M., Y.L., K.H. and A.H.

## Notes

S.vdL., K.H., Y.L., G.M., and A.F. are part of MT-act society which has patented financial interest on the molecules presented in this study.

## ACKNOWLEDGMENTS

The authors acknowledge the financial support of the Agence Nationale de Recherche et Technologie (ANRt), Université de Montpellier, CNRS and ENSCM. We also thank France Alzheimer, Région Occitanie Pre-maturation DeTyrKit and ANR-21-CE16-0026-01. We acknowledge the imaging facility MRI, member of the France-Biolmaging national infrastructure supported by the French National
Research Agency (ANR-10-INBS-04, «Investments for the future»). We thank the PAC Chimie Balard (UAR CNRS 2041), SYNBIO3 (supported by IBiSa & Chimie Balard Cirimat Carnot Institute).

## REFERENCES

(1) Park, J. H.; Roll-Mecak, A. The Tubulin Code in Neuronal Polarity. *Curr. Opin. Neurobiol.* **2018**, *51*, 95-102. https://doi.org/10.1016/j.conb.2018.03.001.

(2) Witte, H.; Neukirchen, D.; Bradke, F. Microtubule Stabilization Specifies Initial Neuronal Polarization. *J. Cell Biol.* **2008**, *180* (3), 619-632. https://doi.org/10.1083/jcb.200707042.

(3) Song, Y.; Brady, S. T. Posttranslational Modifications of Tubulin: Pathways to Functional Diversity of Microtubules. *Trends Cell Biol.* **2015**, *25* (3), 125-136. https://doi.org/10.1016/j.tcb.2014.10.004.

(4) Janke, C.; Magiera, M. M. The Tubulin Code and Its Role in Controlling Microtubule Properties and Functions. *Nat. Rev. Mol. Cell Biol.* **2020**, *21* (6), 307-326. https://doi.org/10.1038/s41580-020-0214-3.

(5) Roll-Mecak, A. The Tubulin Code in Microtubule Dynamics and Information Encoding. *Dev. Cell* **2020**, *54* (1), 7-20. https://doi.org/10.1016/j.devcel.2020.06.008.

(6) Schröder, H. C.; Wehland, J.; Weber, K. Purification of Brain Tubulin-Tyrosine Ligase by Biochemical and Immunological Methods. *J. Cell Biol.* **1985**, *100* (1), 276. https://doi.org/10.1083/jcb.100.1.276.

(7) Aillaud, C.; Bosc, C.; Peris, L.; Bosson, A.; Heemeryck, P.; Van Dijk, J.; Le Friec, J.; Boulan, B.; Vossier, F.; Sanman, L. E.; Syed, S.; Amara, N.; Couté, Y.; Lafanechère, L.; Denarier, E.; Delphin, C.; Pelletier, L.; Humbert, S.; Bogyo, M.; Andrieux, A.; Rogowski, K.; Moutin, M.-J. Vasohibins/SVBP Are Tubulin Carboxypeptidases (TCPs) That Regulate Neuron Differentiation. *Science* **2017**, *358* (6369), 1448-1453. https://doi.org/10.1126/science.aao4165.

(8) Nieuwenhuis, J.; Adamopoulos, A.; Bleijerveld, O. B.; Mazouzi, A.; Stickel, E.; Celie, P.; Altelaar, M.; Knipscheer, P.; Perrakis, A.; Blomen, V. A.; Brummelkamp, T. R. Vasohibins Encode Tubulin Detyrosinating Activity. *Science* **2017**, *358* (6369), 1453-1456. https://doi.org/10.1126/science.aao5676.

(9) Nicot, S.; Gillard, G.; Impheng, H.; Joachimiak, E.; Urbach, S.; Mochizuki, K.; Wloga, D.; Juge, F.; Rogowski, K. A Family of Carboxypeptidases Catalyzing α- and β-Tubulin Tail Processing and Deglutamylation. *Sci. Adv.* **2023**, *9* (37), eadi7838. https://doi.org/10.1126/sciadv.adi7838.

(10) Landskron, L.; Bak, J.; Adamopoulos, A.; Kaplani, K.; Moraiti, M.; Van Den Hengel, L. G.; Song, J.-Y.; Bleijerveld, O. B.; Nieuwenhuis, J.; Heidebrecht, T.; Henneman, L.; Moutin, M.-J.; Barisic, M.; Taraviras, S.; Perrakis, A.; Brummelkamp, T. R. Posttranslational Modification of Microtubules by the MATCAP Detyrosinase. *Science* **2022**, *376* (6595), eabn6020. https://doi.org/10.1126/science.abn6020.

(11) van der Laan, S.; Lévêque, M. F.; Marcellin, G.; Vezenkov, L.; Lannay, Y.; Dubra, G.; Bompard, G.; Ovejero, S.; Urbach, S.; Burgess, A.; Amblard, M.; Sterkers, Y.; Bastien, P.; Rogowski, K. Evolutionary Divergence of Enzymatic Mechanisms for Tubulin Detyrosination. *Cell Rep.* **2019**, *29* (12), 4159-4171.e6. https://doi.org/10.1016/j.celrep.2019.11.074.

(12) Erck, C.; Peris, L.; Andrieux, A.; Meissirel, C.; Gruber, A. D.; Vernet, M.; Schweitzer, A.; Saoudi, Y.; Pointu, H.; Bosc, C.; Salin, P. A.; Job, D.; Wehland, J. A Vital Role of Tubulin-Tyrosine-Ligase for Neuronal Organization. *Proc. Natl. Acad. Sci.* **2005**, *102* (22), 7853-7858. https://doi.org/10.1073/pnas.0409626102.

(13) Peris, L.; Parato, J.; Qu, X.; Soleilhac, J. M.; Lanté, F.; Kumar, A.; Pero, M. E.; Martínez-Hernández, J.; Corrao, C.; Falivelli, G.; Payet, F.; Gory-Fauré, S.; Bosc, C.; Blanca Ramirez, M.; Sproul, A.; Brocard, J.; Di Cara, B.; Delagrange, P.; Buisson, A.; Goldberg, Y.; Moutin, M. J.; Bartolini, F.;
Andrieux, A. Tubulin Tyrosination Regulates Synaptic Function and Is Disrupted in Alzheimer's Disease. *Brain* **2022**, *145* (7), 2486-2506. https://doi.org/10.1093/brain/awab436.

(14) Adamopoulos, A.; Landskron, L.; Heidebrecht, T.; Tsakou, F.; Bleijerveld, O. B.; Altelaar, M.; Nieuwenhuis, J.; Celie, P. H. N.; Brummelkamp, T. R.; Perrakis, A. Crystal Structure of the Tubulin Tyrosine Carboxypeptidase Complex VASH1-SVBP. *Nat. Struct. Mol. Biol.* **2019**, *26* (7), 567-570. https://doi.org/10.1038/s41594-019-0254-6.

(15) Aljoundi, A.; Bjij, I.; El Rashedy, A.; Soliman, M. E. S. Covalent Versus Non-Covalent Enzyme Inhibition: Which Route Should We Take? A Justification of the Good and Bad from Molecular Modelling Perspective. *Protein J.* **2020**, *39* (2), 97-105. https://doi.org/10.1007/s10930-020-09884-2.

(16) Singh, J.; Petter, R. C.; Baillie, T. A.; Whitty, A. The Resurgence of Covalent Drugs. *Nat. Rev. Drug Discov.* **2011**, *10* (4), 307-317. https://doi.org/10.1038/nrd3410.

(17) Mutanto, F.; Konstantinidou, M.; Dömling, A. Covalent Inhibitors: A Rational Approach to Drug Discovery. *RSC Med. Chem.* **2020**, *11* (8), 876-884. https://doi.org/10.1039/D0MD00154F.

(18) Hillebrand, L.; Liang, X. J.; Serafim, R. A. M.; Gehringer, M. Emerging and Re-Emerging Warheads for Targeted Covalent Inhibitors: An Update. *J. Med. Chem.* **2024**, *67* (10), 7668-7758. https://doi.org/10.1021/acs.jmedchem.3c01825.

(19) Wang, J.; Lam, D.; Yang, J.; Hu, L. Discovery of Mobocertinib, a New Irreversible Tyrosine Kinase Inhibitor Indicated for the Treatment of Non-Small-Cell Lung Cancer Harboring EGFR Exon 20 Insertion Mutations. *Med. Chem. Res.* **2022**, *31* (10), 1647-1662. https://doi.org/10.1007/s00044-022-02952-5.

(20) Sun, S.-L.; Wu, S.-H.; Kang, J.-B.; Ma, Y.-Y.; Chen, L.; Cao, P.; Chang, L.; Ding, N.; Xue, X.; Li, N.-G.; Shi, Z.-H. Medicinal Chemistry Strategies for the Development of Bruton's Tyrosine Kinase Inhibitors against Resistance. *J. Med. Chem.* **2022**, *65* (11), 7415-7437. https://doi.org/10.1021/acs.jmedchem.2c00030.

(21) Powers, J. C.; Asgian, J. L.; Ekici, Ö. D.; James, K. E. Irreversible Inhibitors of Serine, Cysteine, and Threonine Proteases. *Chem. Rev.* **2002**, *102* (12), 4639-4750. https://doi.org/10.1021/cr010182v.

(22) Kaur, B.; Singh, P. Epoxides: Developability as Active Pharmaceutical Ingredients and Biochemical Probes. *Bioorganic Chem.* **2022**, *125*, 105862. https://doi.org/10.1016/j.bioorg.2022.105862.

(23) Poreba, M.; Salvesen, G. S.; Drag, M. Synthesis of a HyCoSuL Peptide Substrate Library to Dissect Protease Substrate Specificity. *Nat. Protoc.* **2017**, *12* (10), 2189-2214. https://doi.org/10.1038/nprot.2017.091.

(24) Rut, W.; Lv, Z.; Zmudzinski, M.; Patchett, S.; Nayak, D.; Snipas, S. J.; El Oualid, F.; Huang, T. T.; Bekes, M.; Drag, M.; Olsen, S. K. Activity Profiling and Crystal Structures of Inhibitor-Bound SARS-CoV-2 Papain-like Protease: A Framework for Anti-COVID-19 Drug Design. *Sci. Adv.* **2020**, *6* (42), eabd4596. https://doi.org/10.1126/sciadv.abd4596.

(25) Liao, S.; Rajendraprasad, G.; Wang, N.; Eibes, S.; Gao, J.; Yu, H.; Wu, G.; Tu, X.; Huang, H.; Barisic, M.; Xu, C. Molecular Basis of Vasohibins-Mediated Detyrosination and Its Impact on Spindle Function and Mitosis. *Cell Res.* **2019**, *29* (7), 533-547. https://doi.org/10.1038/s41422-019-0187-y.

(26) Wang, N.; Bosc, C.; Ryul Choi, S.; Boulan, B.; Peris, L.; Olieric, N.; Bao, H.; Krichen, F.; Chen, L.; Andrieux, A.; Olieric, V.; Moutin, M.-J.; Steinmetz, M. O.; Huang, H. Structural Basis of Tubulin Detyrosination by the Vasohibin-SVBP Enzyme Complex. *Nat. Struct. Mol. Biol.* **2019**, *26* (7), 571-582. https://doi.org/10.1038/s41594-019-0241-y.

(27) Mons, E.; Roet, S.; Kim, R. Q.; Mulder, M. P. C. A Comprehensive Guide for Assessing Covalent Inhibition in Enzymatic Assays Illustrated with Kinetic Simulations. *Curr. Protoc.* **2022**, *2* (6). https://doi.org/10.1002/cpz1.419.

(28) Schiefer, I. T.; Tapadar, S.; Litosh, V.; Siklos, M.; Scism, R.; Wijewickrama, G. T.; Chandrasena, E. P.; Sinha, V.; Tavassoli, E.; Brunsteiner, M.; Fa', M.; Arancio, O.; Petukhov, P.; Thatcher, G. R. J. Design, Synthesis, and Optimization of Novel Epoxide Incorporating Peptidomimetics as Selective Calpain Inhibitors. *J. Med. Chem.* **2013**, *56* (15), 6054-6068. https://doi.org/10.1021/jm4006719.
(29) Citarella, A.; Moi, D.; Pinzi, L.; Bonanni, D.; Rastelli, G. Hydroxamic Acid Derivatives: From Synthetic Strategies to Medicinal Chemistry Applications. *ACS Omega* **2021**, *6* (34), 21843–21849. https://doi.org/10.1021/acsomega.1c03628.

(30) Zhang, R.; Qin, X.; Kong, F.; Chen, P.; Pan, G. Improving Cellular Uptake of Therapeutic Entities through Interaction with Components of Cell Membrane. *Drug Deliv.* **2019**, *26* (1), 328–342. https://doi.org/10.1080/10717544.2019.1582730.

(31) Chan, W. C.; Sharifzadeh, S.; Buhrlage, S. J.; Marto, J. A. Chemoproteomic Methods for Covalent Drug Discovery. *Chem. Soc. Rev.* **2021**, *50* (15), 8361–8381. https://doi.org/10.1039/D1CS00231G.

(32) Gomes, A. R.; Varela, C. L.; Tavares-da-Silva, E. J.; Roleira, F. M. F. Epoxide Containing Molecules: A Good or a Bad Drug Design Approach. *Eur. J. Med. Chem.* **2020**, *201*, 112327. https://doi.org/10.1016/j.ejmech.2020.112327.

(33) Baillie, T. A. Approaches to Mitigate the Risk of Serious Adverse Reactions in Covalent Drug Design. *Expert Opin. Drug Discov.* **2021**, *16* (3), 275–287. https://doi.org/10.1080/17460441.2021.1832079.

(34) Péczka, N.; Orgován, Z.; Ábrányi-Balogh, P.; Keserű, G. M. Electrophilic Warheads in Covalent Drug Discovery: An Overview. *Expert Opin. Drug Discov.* **2022**, *17* (4), 413–422. https://doi.org/10.1080/17460441.2022.2034783.

(35) Gehringer, M. Covalent Inhibitors: Back on Track? *Future Med. Chem.* **2020**, *12* (15), 1363–1368. https://doi.org/10.4155/fmc-2020-0118.

(36) Cross, D. A. E.; Ashton, S. E.; Ghiorghiu, S.; Eberlein, C.; Nebhan, C. A.; Spitzler, P. J.; Orme, J. P.; Finlay, M. R. V.; Ward, R. A.; Mellor, M. J.; Hughes, G.; Rahi, A.; Jacobs, V. N.; Brewer, M. R.; Ichihara, E.; Sun, J.; Jin, H.; Ballard, P.; Al-Kadhimi, K.; Rowlinson, R.; Klinowska, T.; Richmond, G. H. P.; Cantarini, M.; Kim, D.-W.; Ranson, M. R.; Pao, W. AZD9291, an Irreversible EGFR TKI, Overcomes T790M-Mediated Resistance to EGFR Inhibitors in Lung Cancer. *Cancer Discov.* **2014**, *4* (9), 1046–1061. https://doi.org/10.1158/2159-8290.CD-14-0337.

(37) Honigberg, L. A.; Smith, A. M.; Sirisawad, M.; Verner, E.; Loury, D.; Chang, B.; Li, S.; Pan, Z.; Thamm, D. H.; Miller, R. A.; Buggy, J. J. The Bruton Tyrosine Kinase Inhibitor PCI-32765 Blocks B-Cell Activation and Is Efficacious in Models of Autoimmune Disease and B-Cell Malignancy. *Proc. Natl. Acad. Sci.* **2010**, *107* (29), 13075–13080. https://doi.org/10.1073/pnas.1004594107.

(38) James, K. E.; Asgian, J. L.; Li, Z. Z.; Ekici, Ö. D.; Rubin, J. R.; Mikolajczyk, J.; Salvesen, G. S.; Powers, J. C. Design, Synthesis, and Evaluation of Aza-Peptide Epoxides as Selective and Potent Inhibitors of Caspases-1, -3, -6, and -8. *J. Med. Chem.* **2004**, *47* (6), 1553–1574. https://doi.org/10.1021/jm0305016.

(39) Klett, T.; Schwer, M.; Ernst, L.; Engelhardt, M.; Jaag, S.; Masberg, B.; Knappe, C.; Lämmerhofer, M.; Gehringer, M.; Boeckler, F. Evaluation of a Covalent Library of Diverse Warheads (CovLib) Binding to JNK3, USP7, or P53. *Drug Des. Devel. Ther.* **2024**, *Volume 18*, 2653–2679. https://doi.org/10.2147/DDDT.S466829.

(40) Flanagan, M. E.; Abramite, J. A.; Anderson, D. P.; Aulabaugh, A.; Dahal, U. P.; Gilbert, A. M.; Li, C.; Montgomery, J.; Oppenheimer, S. R.; Ryder, T.; Schuff, B. P.; Uccello, D. P.; Walker, G. S.; Wu, Y.; Brown, M. F.; Chen, J. M.; Hayward, M. M.; Noe, M. C.; Obach, R. S.; Philippe, L.; Shanmugasundaram, V.; Shapiro, M. J.; Starr, J.; Stroh, J.; Che, Y. Chemical and Computational Methods for the Characterization of Covalent Reactive Groups for the Prospective Design of Irreversible Inhibitors. *J. Med. Chem.* **2014**, *57* (23), 10072–10079. https://doi.org/10.1021/jm501412a.

(41) Shibata, Y.; Chiba, M. The Role of Extrahepatic Metabolism in the Pharmacokinetics of the Targeted Covalent Inhibitors Afatinib, Ibrutinib, and Neratinib. *Drug Metab. Dispos.* **2015**, *43* (3), 375–384. https://doi.org/10.1124/dmd.114.061424.

(42) Mons, E.; Jansen, I. D. C.; Loboda, J.; van Doodewaerd, B. R.; Hermans, J.; Verdoes, M.; van Boeckel, C. A. A.; van Veelen, P. A.; Turk, B.; Turk, D.; Ovaa, H. The Alkyne Moiety as a Latent Electrophile in Irreversible Covalent Small Molecule Inhibitors of Cathepsin K. *J. Am. Chem. Soc.* **2019**, *141* (8), 3507–3514. https://doi.org/10.1021/jacs.8b11027.
(43) Abd El-latif, A. O. Isolation and Purification of a Papain Inhibitor from Egyptian Genotypes of Barley Seeds and Its *in Vitro* and *in Vivo* Effects on the Cowpea Bruchid, *Callosobruchus Maculatus* (F.). *Pestic. Biochem. Physiol.* **2015**, *118*, 26–32.
https://doi.org/10.1016/j.pestbp.2014.10.016.

(44) Vuorinen, E.; Valtonen, S.; Hassan, N.; Mahran, R.; Habib, H.; Malakoutikhah, M.; Kopra, K.; Härmä, H. Protease Substrate-Independent Universal Assay for Monitoring Digestion of Native Unmodified Proteins. *Int. J. Mol. Sci.* **2021**, *22* (12), 6362. https://doi.org/10.3390/ijms22126362

(45) Chen, P.; Ren, S.; Song, H.; Chen, C.; Chen, F.; Xu, Q.; Kong, Y.; Sun, H. Synthesis and Biological Evaluation of BMS-986120 and Its Deuterated Derivatives as PAR4 Antagonists. *Bioorg. Med. Chem.* **2019**, *27* (1), 116–124. https://doi.org/10.1016/j.bmc.2018.11.024.

(46) Gautheron, J.; Jéru, I. The Multifaceted Role of Epoxide Hydrolases in Human Health and Disease. *Int. J. Mol. Sci.* **2020**, *22* (1), 13. https://doi.org/10.3390/ijms22010013.

(47) Hanada, K.; Tamai, M.; Ohmura, S.; Sawada, J.; Seki, T.; Tanaka, I. Structure and Synthesis of E-64, a New Thiol Protease Inhibitor. *Agric. Biol. Chem.* **1978**, *42* (3), 529–536.
https://doi.org/10.1080/00021369.1978.10863015.

(48) Therrien, C.; Lachance, P.; Sulea, T.; Purisima, E. O.; Qi, H.; Ziomek, E.; Alvarez-Hernandez, A.; Roush, W. R.; Ménard, R. Cathepsins X and B Can Be Differentiated through Their Respective Mono- and Dipeptidyl Carboxypeptidase Activities. *Biochemistry* **2001**, *40* (9), 2702–2711.
https://doi.org/10.1021/bi002460a.

(49) Schaschke, N.; Assfalg-Machleidt, I.; Machleidt, W.; Turk, D.; Moroder, L. E-64 Analogues as Inhibitors of Cathepsin B. On the Role of the Absolute Configuration of the Epoxysuccinyl Group. *Bioorg. Med. Chem.* **1997**, *5* (9), 1789–1797. https://doi.org/10.1016/S0968-0896(97)00105-3.

(50) Huang, Z.; McGowan, E. B.; Detwiler, T. C. Ester and Amide Derivatives of E64c as Inhibitors of Platelet Calpains. *J. Med. Chem.* **1992**, *35* (11), 2048–2054.
https://doi.org/10.1021/jm00089a015.

(51) Li, S.; Xiao, J.; Zhang, X.; Zhong, W.; Zheng, Z.; Wang, L.; Xie, Y.; Li, X.; Zhou, X.; Wang, X. 2,3-Epoxy Succinamide Compound, Preparation Method and Purpose Thereof. CN104211666A, December 17, 2014.

(52) Rochat, V.; Marti, R.; Descloux, M.; Demotz, S.; Mutel, V. COMPOSÉS ET LEUR UTILISATION POUR LE TRAITEMENT DE MALADIES ASSOCIÉES À UNE ACTIVITÉ DE β-GALACTOSIDASE ALTÉRÉE. WO2023135480A1, July 20, 2023.

(53) Ternansky, R. J.; Allan, A.; Hook, G. Compositions Et Procédés Pour Traiter Des Maladies Associées Au Bêta-Amyloïde. WO2011017600A2, February 10, 2011.

(54) Zhang, X.; Yang, X.; Wang, H.; Li, S.; Guo, K.; Jiang, D.; Xiao, J.; Liang, D. Design, Synthesis, and Structure-Activity Relationship Study of Epoxysuccinyl-Peptide Derivatives as Cathepsin B Inhibitors. *Biol. Pharm. Bull.* **2017**, *40* (8), 1240–1246. https://doi.org/10.1248/bpb.b17-00075.

(55) Sadaghiani, A. M.; Verhelst, S. H. L.; Bogyo, M. Solid-Phase Methods for the Preparation of Epoxysuccinate-Based Inhibitors of Cysteine Proteases. *J. Comb. Chem.* **2006**, *8* (6), 802–804.
https://doi.org/10.1021/cc0601027.

(56) Verhelst, S. H. L.; Bogyo, M. Solid-Phase Synthesis of Double-Headed Epoxysuccinyl Activity-Based Probes for Selective Targeting of Papain Family Cysteine Proteases. *ChemBioChem* **2005**, *6* (5), 824–827. https://doi.org/10.1002/cbic.200400377.

(57) Pfizer, J. M.; Assfalg-Machleidt, I.; Machleidt, W.; Moroder, L.; Schaschke, N. Primed-Site Probing of Papain-like Cysteine Proteases. *Int. J. Pept. Res. Ther.* **2007**, *13* (1), 93–104.
https://doi.org/10.1007/s10989-006-9050-3.

(58) Arora, K.; Herroon, M.; Al-Afyouni, M. H.; Toupin, N. P.; Rohrabaugh, T. N. Jr.; Loftus, L. M.; Podgorski, I.; Turro, C.; Kodanko, J. J. Catch and Release Photosensitizers: Combining Dual-Action Ruthenium Complexes with Protease Inactivation for Targeting Invasive Cancers. *J. Am. Chem. Soc.* **2018**, *140* (43), 14367–14380. https://doi.org/10.1021/jacs.8b08853.

(59) Ternansky, R. Nouveaux Medicaments Contre La Demence. WO2007038772A1, April 5, 2007.

(60) Moseley, J. D.; Staunton, J. Preparation of Dicarboxylate Analogues of Cerulenin. *J. Heterocycl. Chem.* **2005**, *42* (5), 819–830. https://doi.org/10.1002/jhet.5570420511.
(61) Dókus, L. E.; Menyhárd, D. K.; Tantos, Á.; Hudecz, F.; Bánóczi, Z. Probing of Primed and Unprimed Sites of Calpains: Design, Synthesis and Evaluation of Epoxysuccinyl-Peptide Derivatives as Selective Inhibitors. *Eur. J. Med. Chem.* **2014**, *82*, 274-280.
https://doi.org/10.1016/j.ejmech.2014.05.058.

(62) Huisman, M.; Kodanko, J. P.; Arora, K.; Herroon, M.; Alnaed, M.; Endicott, J.; Podgorski, I.; Kodanko, J. J. Affinity-Enhanced Luminescent Re(I)- and Ru(II)-Based Inhibitors of the Cysteine Protease Cathepsin L. *Inorg. Chem.* **2018**, *57* (13), 7881-7891.
https://doi.org/10.1021/acs.inorgchem.8b00978.

(63) Schaschke, N.; Assfalg-Machleidt, I.; Machleidt, W.; Moroder, L. Substrate/Propeptide-Derived Endo-Epoxysuccinyl Peptides as Highly Potent and Selective Cathepsin B Inhibitors. *FEBS Lett.* **1998**, *421* (1), 80-82.
https://doi.org/10.1016/S0014-5793(97)01538-X.

(64) Schaschke, N.; Deluca, D.; Assfalg-Machleidt, I.; Höhneke, C.; Sommerhoff, C. P.; Machleidt, W. Epoxysuccinyl Peptide-Derived Cathepsin B Inhibitors: Modulating Membrane Permeability by Conjugation with the C-Terminal Heptapeptide Segment of Penetratin. **2002**, *383* (5), 849-852.
https://doi.org/10.1515/BC.2002.090.

(65) Sarabia, F.; Sánchez-Ruiz, A.; Chammaa, S. Stereoselective Synthesis of E-64 and Related Cysteine Proteases Inhibitors from 2,3-Epoxyamides. *Bioorg. Med. Chem.* **2005**, *13* (5), 1691-1705.
https://doi.org/10.1016/j.bmc.2004.12.018.

(66) Jantke, D.; Marziale, A. N.; Reiner, T.; Kraus, F.; Herdtweck, E.; Raba, A.; Eppinger, J. Synthetic Strategies for Efficient Conjugation of Organometallic Complexes with Pendant Protein Reactive Markers. *J. Organomet. Chem.* **2013**, *744*, 82-91.
https://doi.org/10.1016/j.jorganchem.2013.05.027.

(67) Tamai, M.; Yokoo, C.; Murata, M.; Oguma, K.; Sota, K.; Sato, E.; Kanaoka, Y. Efficient Synthetic Method for Ethyl (+) - (2S, 3S)-3-[(S)-3-Methyl-1-(3-Methylbutylcarbamoyl) Butylcarbamoyl]-2-Oxiranecarboxylate (EST), a New Inhibitor of Cysteine Proteinases. *Chem. Pharm. Bull.* (Tokyo) **1987**, *35* (3), 1098-1104.
https://doi.org/10.1248/cpb.35.1098.

(68) Rogowski, K.; Amblard-Caussil, M.; Van, D. L. S.; Vezenkov, L.; Marcellin, G.; Lannay, Y.; Hached, K. New Vashs Inhibitors, Conjugates Thereof and Their Uses as Drugs or as Research Tools. *CA3230297* (A1), March 2, 2023.
https://worldwide.espacenet.com/publicationDetails/biblio?FT=D&date=20230302&DB=&locale=fr EP&CC=CA&NR=3230297A1&KC=A1&ND=4 (accessed 2024-07-30).

(69) Eaton, D. M.; Lee, B. W.; Caporizzo, M. A.; Iyengar, A.; Chen, C. Y.; Uchida, K.; Marcellin, G.; Lannay, Y.; Vite, A.; Bedi, K. C.; Brady, C. F.; Smolyak, J. N.; Meldrum, D.; Dominic, J.; Weingarten, N.; Patel, M.; Belec, A.; Hached, K.; Atluri, P.; Van Der Laan, S.; Prosser, B. L.; Margulies, K. B. Vasohibin Inhibition Improves Myocardial Relaxation in a Rat Model of Heart Failure with Preserved Ejection Fraction. *Sci. Transl. Med.* **2024**, *16* (756), eadmm842.
https://doi.org/10.1126/scitranslmed.adm8842.

(70) Rogowski, K.; Hached, K.; Crozet, C.; van der Laan, S. Tubulin Modifying Enzymes as Target for the Treatment of Tau-Related Diseases. *Pharmacol. Ther.* **2021**, *218*, 107681.
https://doi.org/10.1016/j.pharmthera.2020.107681.

(71) Jenssen, H.; Aspmo, S. I. Serum Stability of Peptides. *Methods Mol. Biol. Clifton NJ 2008*, *494*, 177-186.
https://doi.org/10.1007/978-1-59745-419-3_10.

(72) Morris, G. M.; Huey, R.; Lindstrom, W.; Sanner, M. F.; Belew, R. K.; Goodsell, D. S.; Olson, A. J. AutoDock4 and AutoDockTools4: Automated Docking with Selective Receptor Flexibility. *J. Comput. Chem.* **2009**, *30* (16), 2785-2791.
https://doi.org/10.1002/jcc.21256.

(73) Olsson, M. H. M.; Søndergaard, C. R.; Rostkowski, M.; Jensen, J. H. PROPKA3: Consistent Treatment of Internal and Surface Residues in Empirical pKa Predictions. *J. Chem. Theory Comput.* **2011**, *7* (2), 525-537.
https://doi.org/10.1021/ct100578z.