Zlata Boiarska

Helena Pérez-Peña

Anne-Catherine Abel

Dr Paola Marzullo

Beatriz Álvarez-Bernad

Dr Francesca Bonato

Dr Benedetta Santini

Dr Dragos Horvath

Dr Daniel Lucena-Agell

Prof. Dr Francesca Vasile

Prof. Dr Maurizio Sironi

J Fernando Díaz

Dr Andrea E Prota

Prof. Dr Stefano Pieraccini

Prof. Dr Daniele Passarella
email: daniele.passarella@unimi.itstefano.pieraccini@unimi.it

+ H Pérez-Peña

A.-C Abel

J F Díaz

Maytansinol Functionalization: Towards Useful Probes for Studying Microtubule Dynamics

Keywords: chemical probes, maytansinoids, maytansinol functionalization, microtubules, tubulin

Maytansinoids are a successful class of natural and semisynthetic tubulin binders, known for their potent cytotoxic activity. Their wider application as cytotoxins and chemical probes to study tubulin dynamics has been held back by the complexity of natural product chemistry. Here we report the synthesis of long-chain derivatives and maytansinoid conjugates. We confirmed that bulky substituents do not impact their high activity or the scaffold's binding mode.

These encouraging results open new avenues for the design of new maytansine-based probes.

Introduction

Maytansinoids continue to excite interest almost after a decade after their discovery as potent antimitotic tubulin binders. [1][2][3] Their highly efficient mechanism of capping MT-dynamics [START_REF] Prota | Proc. Natl. Acad. Sci[END_REF] has led to their successful application in cancer treatment as antibody-drug conjugates (ADCs). [START_REF] Wedam | [END_REF][6][7] This success has further prompted their development as targeted cancer therapeutics, for example, in the form of nanoparticles [8,9] or recently reported immune checkpoint-targeting maytansinoid conjugates. [10] However, the high-affinity of maytansine towards -tubulin (K D = 6.8-14 nM) not only makes it well suited as a cytotoxin, but also derivatives with a similar affinity could be used as molecular probes. [11,12] As of today, effortless generation of maytansine-based molecular probes is hampered by major drawbacks such as the complexity of the natural product scaffold and lack of SAR studies, which could suggest suitable points for attachment of fluorophore tags or radionuclides. Here, we report on both of these aspects: the chemistry of maytansinol (Figure 1, 1) for the creation of maytansinoid conjugates and the suitability of the C3-position to tolerate bulky substituents without affecting the binding mode. Therefore, we provide a solid basis for in-depth exploration of maytansinoids as molecular probes to study microtubule dynamics.

Previously, it has been shown that the ester at the C-3 position of ansamitocins, maytansine, and maytansinoids plays an important role in biological activity and cell permeability. [3,[START_REF] Yu | Anticancer Agents from Natural Products[END_REF][START_REF] Li | [END_REF] In our recent study, we extensively investigated a series of C-3 derivatized maytansinoids obtained by acylation of maytansinol. [12] By X-ray crystallography experiments, we observed that the studied maytansinoids retained a fundamental spatial arrangement with respect to -tubulin. The binding mode of the core structure of maytansinol remained unaltered independently of the introduced substituents. Furthermore, the binding affinities of the C3-substituted maytansinoids obtained are very similar to the one of maytansine.

Based on these observations, we decided to further investigate the potential of C-3 functionalized maytansinoids as molecular probes by creating novel tubulin binders. The design of novel maytansinoids was led by the high-resolution X-ray crystallography structures of recently obtained maytansinoidtubulin complexes (PDB IDs: 5SB9, 5SBA and 5SBB). The maytansine binding site is located in proximity of the GDP nucleotide bound to the E-site and a GDP-coordinated Mg 2 + . The first interesting factor that could be exploited to exert a novel effect on MTs is the Mg 2 + ion coordinated by the nucleotide in close proximity to the maytansine binding site. The design of bivalent compounds containing maytansinol and nucleotide mimetics would a) increase the understanding of the ability of maytansinoids to accommodate in the binding site independently of the size of the substituents, and b) investigate the ability of these molecules to interact with Mg 2 + and to displace the nucleotide from the binding site.

Tubulin inhibition by nucleotide analogues is very challenging because of GTP concentrations around 300 M inside the cells. [15] However, we sought to investigate whether the presence of the maytansinol moiety in a bivalent compound could favor binding of the nucleotide portion through an entropic effect. The maytansinol moiety would act as an anchor point holding the modified nucleotide portion in close proximity of the E-site through a flexible linker, thereby favoring the nucleotide exchange.

Accordingly, we decided to prepare long-chain maytansinol derivatives and maytansinoid conjugates to target either the Mg 2 + ion or the nucleotide binding site. Introducing different substituents at the 3-O position should allow the protein-ligand interaction to be optimized and new molecules capable of producing different effects on tubulin structure and dynamics to be found.

Results and Discussion

Design and Synthesis

Long-chain maytansinoids

Initially, we envisioned to obtain the long-chain maytansinoids directly through the acylation reaction of maytansinol, which we previously used for the synthesis of short-chain maytansinoids. [START_REF] Yu | Anticancer Agents from Natural Products[END_REF] To date, neither long-chain maytansinoids of this type nor acylation reaction of maytansinol with a dicarboxylic acid have been reported in the literature. However, initial trials to functionalize maytansinol with a peptide-like linker (2) or with a sebacic acid (3), using Steglich esterification, were not successful (Scheme 1). This prompted us to change the approach to a two step-synthesis: the preparation of shortchain maytansinoids with a terminal alkene, followed by a cross metathesis (CM) reaction with an unsaturated carboxylic acid. The acylation reaction of maytansinol to obtain 4 and 5 worked well, although lower yields were obtained using longer-chain carboxylic acids. In both cases, the time prolongation had no great influence on the formation of the desired product, and only affected the ratios of different by-products obtained (Scheme 1). that the sum of A and B should be always greater or equal to 5 and smaller or equal to 13 (Figure 3).

For the linker screening, the maytansinol and the guanosine molecules were treated as fragments and were fixed in their states seen in the crystal structure 4TV8. Then, we carried out the linker screening and used the docking software S4MPLE [16] to search for the linker that best bridged the gap between the two fragments (maytansinol and guanosine). We observed that none of the generated linkers put any strain on the anchor fragments. Therefore, the linkers were chosen upon synthetic convenience (a, Figure 4). We also explored the possibility of designing a more rigid linker by adding a phenyl ring in the side chain of maytansinol. As expected, in this case we observed that the degrees of freedom of the linker were reduced, thus better orienting the linker moiety towards the nucleotide pocket (b, Figure 4). We analyzed the validity of the linkers present in conjugates 7 and 8 by template docking. The results showed that both the linkers can flexibly connect both the maytansinol and the guanosine entities in a suitable distance, which allows the two entities to bind tubulin by maintaining a proper geometry.

The interactions established by the nucleotide withtubulin were analyzed by using computational resources. It was observed that the guanine moiety established the main interactions within the site through very specific hydrogen bonds and stacking. Therefore, we also designed conjugate The preferred length of a long-c hain maytansinoid was estimated at 10 or 11 c arbon atoms, based on doc king studies. Therefore, c ompound 5 was then used to obtain the derivative with a desired length 6 through a Ru-c atalyzed CM reaction, using the sec ond-generation Hoveyda-Grubbs c atalyst. The trans isomer was the one detec ted by 1 H NMR analysis (Scheme 2).

Importantly, the doc king studies showed that the E/Z isomerism did not seem to influenc e the outc ome sinc e both stereo isomers were able to establish the same interactions with the Mg 2 + even if the poses of the linkers slightly differed from each other (Figure S41 in the Supporting Information).

Obtaining both short-c hain maytansinoids 4 and 5 and the long-c hain maytansinoid 6 allowed us to perform more studies on maytansinoids SAR based on the length and nature of the chain. Moreover, the long-chain analogue could be exploited to attach other molecules to maytansine.

Maytansinoid conjugates

We dec ided to c onjugate the maytansinoids with guanosine mimetic s and we based the rational linker design on the distanc e of ~ 12 Å (Figure 2). In the linker-design stage, the main goal was to define a proper length and flexibility to secure the ac c ommodation of maytansinol and nuc leotide-like scaffolds in their respective -tubulin binding sites.

First of all, we foc used on the linkers whic h c ould allow the c onjugation of the c ompounds of interest by means of click chemistry. We generated a library of 54 possible linkers in which the position of the triazole group and the length of the carbon tails attac hed to it varied. Moreover, since the measured spatial distanc e between the two func tional groups of interest was of ~ 12 Å, we considered linker lengths ranging from 12-14 atoms. Therefore, the linker size was rationally c hosen by determining compounds containing acyclovir (9, 10). Docking studies confirmed that the five-membered ring of the ribose can be substituted without disturbing major interactions of the guanine moiety with -tubulin. Further, superposition of the docked structure shows that a binding mode resembling the original guanosine one can be adopted (Figure 5). In addition, this choice led to simplifications in the synthesis of maytansinoid conjugates.

Conjugate compounds 7-10 can be obtained by a CuAAC reaction between the fragment of an alkyne (11, 15, 16) and an azide (12-14) providing the corresponding triazole derivatives (Scheme 3). Both fragments can be prepared as ester starting from maytansinol and guanosine (or acyclovir) by condensation with carboxylic acids.

Maytansinoids 11 and 12 were obtained by using Steglich esterification (Scheme 4) with 6-heptynoic acid or 4-(2azidoethyl)benzoic acid, which was prepared by treating the corresponding 4-(2-bromoethyl)benzoic acid with NaN 3 . The yield and selectivity of the acylation reaction can be improved by inhibiting the formations of the undesired maytansinol byproducts through the addition of excess ZnCl 2 , as reported in our previous work. [12] To obtain the nucleotide mimetic fragments 13-16, 5'-Oand 3'-O-esterification was performed on guanosine and acyclovir, respectively (Scheme 5). The poor solubility of these compounds in most organic solvents complicates the reactivity and the handling. In this regard, 2',3'-diol or free amine protection of guanosine could be considered. [17][18][19][20][21][22][23] However, the isopropylidene ketal formation on guanosine sufficiently increased the solubility, so the NH 2 protection was not undertaken. In addition, the formation of the ketal would increase the selectivity for the subsequent 5'-O esterification reaction and the formation and removal occur easily under acid condition. Therefore, guanosine was treated with perchloric acid in acetone providing 17 quantitatively.

The literature reports wide reaction conditions useful to perform acylation at 5' position of 2',3'-isopropylidene guanosine, by acyl halides, anhydrides, and acids under coupling conditions. [17,19,24] The first approach was the coupling of carboxylic acids, therefore compound 17 and acyclovir were similarly esterified with 6-azidohexanoic acid or 6-heptynoic acid in the presence of EDC yielding 13-16. The reactions on guanosine were performed in DCM giving 71 and 78 % in the isolation of the compound 13 and 15 by reverse phase purification, while a mixture of DCM/DMF was used as solvent to overcome the solubility issues of acyclovir obtaining 14 and 16 with a yield of 65 and 43 %, respectively.

Considering that deprotection of guanosine in acidic condition could affect the maytansinol part in the final conjugate compounds, the cleavage of isopropylidene was carried out before the click reaction. Therefore, compound 13 and 15 were treated with TFA 80 % aq [20,21,24] giving the desired products 18 and 19 with 81 and 98 % of yield, respectively.

The alkyne and azide building blocks were joined by performing the CuAAC. The reactions carried out on the pairs of maytansinoid/guanosine 11/18 and 12/19 with catalytic quantities of CuSO 4 in the presence of sodium ascorbate as reducing agent in H 2 O/tBuOH (1 : 4) did not lead to any product. The triazole ring was not obtained neither performing the reaction in H 2 O/DMSO (1 : 2) as solvent nor by increasing the quantities of the reagents.

In order to better support the solubility of guanosine and the reaction, a new route to carry out the CuAAC was considered. The strategy involved performing the click using the isopropylidene protected guanosine intermediates 13 and 15 and then cleaving the ketal in the final adduct. For this purpose, the experimental conditions to remove protecting group without altering the sensitive structure of maytansinol required a careful condition screening (Table 1).

All conditions except the use of PdCl 2 did not affect maytansinol, but at the same time failed to cleave the ketal. Although the use of formic acid with the conditions reported in the literature for the specific removal of acetonide from guanosine produced no results, increasing the acid concentration and heating slightly to 40 °C achieved the goal. By 1 H NMR analysis the integrity of maytansinol and deprotection of guanosine was confirmed.

The CuAAC reaction was repeated by treating maytansinoid 11 and protected 5'-O-acylguanosine 13 with an excess of CuSO 4 and Na-ascorbate in H 2 O/DMSO 1 : 2. As expected, the protecting group improved the solubility of guanosine obtaining an excellent result because the triazole ring was formed in few hours with a yield of 89 % in the isolation of compound 20.

The same approach was used on 12/15 fragment pair to get compound 21 with 85 % of yield (Scheme 6).

The NMR elucidations were completed to confirm the structures, and the shifting of the diagnostic signals demonstrated the formation of triazole on 20 and 21 (Table 2).

Then, both products were treated with formic acid 70 % aq at 40 °C providing the final conjugate compounds 7 and 8 after 4 h with yields of 62 and 77 %, respectively. The MS and NMR analyses confirmed the isopropylidene deprotection. In case of maytansinoid-acyclovir conjugates, similar conditions were applied using catalytic CuSO 4 and Na-ascorbate in H 2 O/DMF 1 : 2 as solvent. The desired final products 9 and 10 were obtained in 35 and 55 %, respectively (Scheme 7), and fully characterized by NMR and MS analysis (see the Supporting Information).

Biochemical evaluation

Inhibition of tubulin assembly

Maytansine site ligands preclude tubulin assembly by blocking the addition of new tubulin subunits onto the microtubule. Owing to this, we decided to assess the inhibitory effect of this newly synthetized compounds on tubulin polymerization (Figure 6).

All the compounds analyzed were able to abolish tubulin polymerization.

Binding affinities

In order to correlate the tubulin assembly inhibition with the binding affinities of the compounds for the maytansine site, these were determined by means of c ompetition against Fc maytansine (Table 3). [11] Binding affinity is well conserved indic ating low or neglec table influenc e of the side c hain in the interac tion of the maytansine c ore with tubulin, but also suggesting little interaction of the nucleotide moiety.

Cytotoxicity

To c orrelate the potenc y of binding with the toxic ity, and to investigate the potential of the c ompounds to overcome membrane pumps mediated multidrug resistanc e, we deter-Consistently with the binding affinity data, compounds 4, 5, 6, 11 and 12 show very good activity against the tumoral cells, being some of them even more active than the parental compound maytansine. On the other hand, compounds containing the triazole moiety arising from the click reaction undergo a severe loss of activity without losing binding affinity, which suggests that the resulting side chain precludes internalization in the cells.

X-ray crystallography structure determination

To evaluate if the attachment of long chains to the maytansine scaffold impacts the binding mode of the molecules, we sought to determine the structures of tubulin-maytansinoid complexes by X-ray crystallography. Crystals of the T 2 R-TTL complex were grown using our well-established protocols. [START_REF] Muhlethaler | Microtubules. Methods and protocols[END_REF][START_REF] Prota | [END_REF] Maytansinoids 4-12 were either soaked into the crystals at 5 mM final concentration for 6-16 h or added to the crystallization mixture. We collected high resolution data (1.9-2.6 Å) and used DIMPLE [27] to spot densities of bound ligands.

Consistently with the biochemical data we observed ligand shaped difference density at the maytansine site for all compounds, highlighting that none of the introduced moieties precluded binding in the crystal system. However, only the central ring of the maytansine scaffold was well resolved, while the introduced linkers were not defined in any structure of the obtained maytasinoid-tubulin complexes. These results are in line with the measured binding constants.

The crystal structures of the representative compounds with different side-chain substituents and high cytotoxicity, 4, 6, and mined the c ytotoxic ity of the c ompounds both in A549 and in the isogenic pair A2780/A2780AD (pGp overexpressing) cell lines (Table 4).

12, were further refined to determine their detailed molecular interactions (PDB IDs: 8B7A, 8B7B and 8B7C). In general, all three determined structures superposed well with the structure of the parent compound (PDB ID: 4TV8; RMSD of superposition over Ca: 0.380 (1993 atoms), 0.256 (1992 atoms) and 0.330 Å (1982 atoms) for T2R-TTL 4, 6 and 12, respectively), and there were no major alterations in the binding mode (Figure 7). The insertion of long chains in a acylation reaction at the C3ÀO has no impact on the previously described interactions, namely the hydrogen bonds of the C1 carbonyl to the Val181 main chain nitrogen, the C24 carbonyl to the Lys105 and Asn102 sidechain amine groups and C9ÀOH to the Gly100 main chain carbonyl. This is as well in line with our previous observation, that insertion of a smaller group at this position has no effect on the binding mode nor on the toxicity of the maytansinoids. [12] The observed ligand density in these structures extended up to 2 or 3 carbons beyond the well resolved ester-group linking the chain to the C3 carbon, indicating that the introduced linker moieties adapt multiple conformations.

Accordingly, the carbon chains in all the three structures were modeled and refined in their most probable conformations. Our data highlight the C3ÀO position as an excellent point to introduce modifications on the maytansine scaffold, allowing the accommodation of long flexible chains or even bulky moieties.

The lack of defined density and thus a stable conformation of the linker chain showed that compound 6 is not coordinating the Mg 2 + ion, since the coordination would result in a fixed position of the carbonic acid linker. Presumably, the exposed nature of the binding site and the high flexibility of the long carbon chains precluded the stable coordination of the Mg 2 + ion. Moreover, we cannot exclude an additional impact on the interaction by the presence of GDP : Mg 2 + at the E-site in our crystal system. Further, we could not observe any evidence for the exchange of the GDP by our nucleotide mimetics neither by soaking nor in co-crystallization experiments. Likely, the very high affinity of nucleotides to tubulin prevents the exchange of the previously bound nucleotide.

Conclusions

In this work, we have established robust c hemistry that allows large substituents to be plac es on a c omplex natural product. By introduc ing alkene or alkyne moieties at position C-3 in a ac ylation reac tion, we opened up the sc affold for the straightforward attac hment of more func tional probes. This allowed a whole variety of new molec ules to be attac hed to the maytansine sc affold by either c ross metathesis or click chemistry reactions.

In this work, we used these pathways to further investigate the toleranc e towards the introduc tion of bulkier groups and their impac t on tubulin binding. Guided by computational studies, we generated a whole set of maytansinoids carrying either long c arbon c hains, c arboxylic ac ids or nucleotide mimetic s. Our c ompiled data on the inhibition of tubulin assembly and binding affinities, together with the structural information confirm that even these long and heavy groups are tolerated easily. They neither impac t the binding mode on tubulin nor dramatic ally reduc e the sc affolds affinity, with even the lowest measured K D within the nanomolar range. Unexpectedly, the c ell permeability is likely affec ted by the introduction of the larger groups, as observed in their lowered cytotoxicity.

Therefore, our work lays the foundation to further exploit the C3 position of maytansinoids by introduc ing functional modific ations, suc h as spec ific fluorophores or c helators, that could potentially serve as probes to study MT dynamics in vitro. Furthermore, these modifications could comprise other types of c hemic al entities for the development of maytansinol-based bivalent c ompounds c apable to rec ruit spec ific proteins of interest.

Experimental Section

Molecular modeling: Molec ular modeling was applied to test the suitability of the different bulky substituents attac hed to maytansinol for the generation of long-chain maytansinoids and, to support linker length design and selec tion for the development of maytansinoid c onjugates. The molec ular modeling tec hnique used in this paper was molec ular doc king. To perform the docking experiments the software used was S4MPLE, an in-house developed tool that allowed us to doc k the long-c hain maytansinoids within the maytansine binding site and its surroundings, and the maytansinoid c onjugates within the GTP and maytansine tubulin binding sites and their connecting structural environment.

Synthesis: Unless otherwise stated, reagents were purc hased from general suppliers (Sigma-Aldric h and Fluoroc hem) and used without further purific ation. All solvents were of reagent grade or HPLC grade. All reac tions were c arried out in oven-dried glassware and dry solvents, under nitrogen and were monitored by glasses or aluminium TLC on silic a gel (Merc k prec oated 60F254 plates), with detec tion by UV light (254 nm), or by TLC stains as permanganate, or by HPLC Agilent 1100. Analytical HPLC was performed on Agilent 1100 Series System RP c olumn ZORBAX SBÀ C8 (3.5 m × 4.6 × 150 mm). The pressure was about 85 bar, with a c onstant flow rate solution, 0.0084 mmol). The mixture was stirred at room temperature for 6 h. Crude was purified with Biotage Isolera™ One System (gradient H 2 O/ACN), providing the product (7 mg, 35 %) as a white powder.

Synthesis of compound 10:

To a solution of 11 (11 [START_REF] Yu | Anticancer Agents from Natural Products[END_REF] C NMR data for all the synthesized compounds is presented in the Supporting Information.

High-resolution mass spectra (HRMS) were recorded on a Water QToF Premier high resolution UPLC ES MS/MS. Data is included in the Supporting Information.

Biochemical evaluation

Protein and chemicals for biochemistry: Calf brain tubulin was obtained by means of a modification of the Weisenberg procedure. [28] Briefly, calf brains were blended prior to perform two salting out steps with ammonium sulfate. Then, a DEAE chromatography was carried out to obtain a 99 % pure tubulin. Additionally, a gel filtration chromatography was done to remove all the excess of ammonium sulphate. Finally, tubulin was collected and quantified prior to its lyophilization in trehalose buffer and storage at À80. [29] Biochemistry: Polymerization of 25 M tubulin in GAB buffer (3.4 M Glycerol, 10 mM sodium phosphate (NaPi), 1 mM EGTA, 6 mM MgCl 2 , 1 mM GTP, pH 6.7) was monitored in the presence of the desired concentration of the ligand by measuring turbidity at 350 nm employing a Thermo Appliskan plate reader (Thermo Fisher). Data for the polymerization assays were obtained in duplicates of two independent experiments.

Binding constants of the compounds to the maytansine site of tubulin were measured by competition with FcMaytansine as described. [11] DMSO was used as vehicle for maytansine derivatives where less than 0.5 % was employed. Data are the mean AE SEM values of three independent experiments with duplicates in each one.

Cell biology: Human A549 non-small-cell lung carcinoma cells, human ovarian carcinomas A2780 and A2780AD (MDR overexpressing P-glycoprotein) were cultured at 37 °C in Dulbecco's modified Eagle medium (DMEM) supplemented with 10 % fetal calf serum, 2 mM l-glutamine, 1 mM sodium pyruvate, 40 g mL À1 gentamycin, 100 IU mL À1 penicillin and 100 g mL À1 streptomycin in a 5 % CO 2 air atmosphere. Antiproliferation assays were performed as described. [30] DMSO was used as vehicle for maytansine derivatives where less than 0.5 % was employed in the assay. Data are the mean AE SEM values of three independent experiments with duplicates in each one. The statistical significance of differences in IC50 values were evaluated using the t-test option implemented in the Sigma Plot software package (version 14.5, Systat Software, Inc., San Jose, CA, USA).

Purific ation with Biotage Isolera™ One System (gradient H 2 O/ACN) provided the product (156 mg, 78 %) as a white powder.

Synthesis of com pound 16:

To a solution of ac yc lovir (150 mg, 0.666 mmol) in dry CH 2 Cl 2 (1.25 mL) and dry DMF (1.25 mL) were added 6-heptynoic ac id (135 mg, 1.07 mmol), DMAP (41 mg, 0.336 mmol), EDC•HCl (269 mg, 1.405 mmol), and triethylamine dry (196 L, 1.405 mmol) at room temperature under a nitrogen atmosphere. The reac tion was stirred overnight. The c rude was purified with Biotage Isolera™ One System (gradient H 2 O/ACN), providing the product (95.5 mg, 43 %) as a white powder.

Synthesis of com pound 18:

To a solution of 13 (49 mg, 0.11 mmol) was added aqueous TFA 80 % (1.52 mL). The mixture was stirred at room temperature for 30 min. Then, the solvent was removed in vacuum, a solution of EtOH + 1 % Et 3 N (2.5 mL) were added and the solvent was evaporated to remove the TFA salt form. Purification of the c rude with Biotage Isolera™ One System (gradient H 2 O/ACN) provided the product (46 mg, > 98 %) as a white powder.

Synthesis of com pound 19:

To a solution of 15 (52 mg, 0.12 mmol) was added aqueous TFA 80 % (1.71 mL). The mixture was stirred at room temperature for 30 min. Then, the solvent was removed in vacuum, a solution of EtOH + 1 % Et 3 N (2.5 mL) were added and the solvent was evaporated to remove the TFA salt form. Purification of the c rude with Biotage Isolera™ One System (gradient H 2 O/ACN) provided the product (46 mg, > 98 %) as a white powder.

Synthesis of com pound 20:

To a solution of 11 (21 mg, 0.031 mmol) and 13 (21 mg, 0.047 mmol) in DMSO (690 L) were added water (225 L), CuSO 4 •5 H 2 O (15 mg, 0.050 mmol), then a solution of Naascorbate (39 mg, 0.19 mmol) in H 2 O (115 L) was added dropwise. The mixture was stirred at room temperature for 2 h. Then, water was added (1.4 mL) and the aqueous layer was extrac ted in DCM. The organic layer was washed with H 2 O and brine, dried over Na 2 SO 4 and c onc entrated in vac uum. Purific ation of the c rude with Biotage Isolera™ One System (gradient H 2 O/ACN) provided the product (31 mg, 89 %) as a white powder.

Synthesis of com pound 21:

To a solution of 12 (16 mg, 0.022 mmol) and 15 (14 mg, 0.032 mmol) in DMSO (482 L) were added water (159 L), CuSO 4 •5 H 2 O (10 mg, 0.035 mmol), then a solution of Naascorbate (28 mg, 0.139 mmol) in H 2 O (82 L) was added dropwise. The mixture was stirred at room temperature for 2 h. Then, water was added (1.4 mL) and the aqueous layer was extrac ted in DCM. The organic layer was washed with H 2 O and brine, dried over Na 2 SO 4 and c onc entrated in vac uum. Purific ation of the c rude with Biotage Isolera™ One System (gradient H 2 O/ACN) provided the product (22 mg, 85 %) as a white powder.

Synthesis of com pound 7:

To c ompound 2 0 (18.3 mg, 0.016 mmol) was added aq. 70 % HCOOH (650 L) and the reac tion was stirred for 4 h at 40 °C. Then, the solvent was removed under reduced pressure and the ac id was c o-evaporated by adding and evaporating three times MeOH. Purific ation of the c rude with Biotage Isolera™ One System (gradient H 2 O/ACN) provided the product (11 mg, 62 %) as a white powder.

Synthesis of com pound 8:

To c ompound 2 1 (11 mg, 0.009 mmol) was added aq. 70 % HCOOH (235 L) and the reac tion was stirred for 4 h at 40 °C. Then, the solvent was removed under reduced pressure and the ac id was c o-evaporated by adding and evaporating three times MeOH. Purific ation of the c rude with Biotage Isolera™ One System (gradient H 2 O/ACN) provided the product (8 mg, 77 %) as a white powder.

Synthesis of com pound 9:

To a solution of 12 (14 mg, 0.02 mmol) and 14 (9.1 mg, 0.025 mmol) in H 2 O/DMF as solvents (1 : 2, 400 L) were added a solution of CuSO 4 •5 H 2 O (11.2 L of 0.75 M solution, 0.084 mmol), and a solution of Na-asc orbate (83.9 L of 1 M X-ray crystallography structure determination T 2 R-TTL crystals were prepared as previously described [START_REF] Muhlethaler | Microtubules. Methods and protocols[END_REF][START_REF] Prota | [END_REF]31] and soaked with compounds 4-12* at a final concentration of 5 mM for 6-16 h. Crystals were grown at room temperature over one week in buffer containing PEG 4 K (5 %), glycerol (8 %), MgCl 2 (30 mM), CaCl 2 (30 mM), MES/imidazole pH 6.5 (100 mM) and tyrosine (5 mM). Xray diffraction data were collected at 1 Å wavelength and 100 K at the beamline X06DA of the Swiss Light Source, Paul Scherrer Institut, 5332 Villigen PSI, Switzerland. Datasets were processed using XDS [32] and structures were determined by the difference Fourier method based on phases of a T 2 R-TTL model in the absence of ligands (PBD ID: 5LXT). Refinement and model building were done in iterative cycles of restrained refinement in PHENIX [33] and model building in Coot. [34] The ligand structures were energy minimized in Moloc, [35] before restraint generation with eLBOW (part of the PHENIX suite) and AceDRG [36] in CCP4i2. [37] The quality of the obtained models was monitored with MolProbity. [38] The structures of the T 2 R-TTL-4, 6 and 12 complexes were refined to resolution of 2.25, 2.25 and 1.90 Å with crystallographic R work /R free values of 18.04/22.31 %, 18.03/22.33 % and 18.52/21.93 %, respectively. The detailed statistics for each structure are available in the Supporting Information (Table S2). Coordinates and structure factors were deposited in the PDB with the following accession codes 8B7A, 8B7B and 8B7C. The shown molecular graphics were generated using PyMol (the PyMOL Molecular Graphics System Version 2.3.4 Schrödinger, LLC). * Tubulin-maytansinoid-complex 11 was previously described [12] (PDB ID: 5SBB)

Figure 2 .

 2 Figure 2. A) Section of the crystal structure with PDB ID: 4TV8 in which maytansine (orange), GDP (green), Mg 2 + (bright green sphere), and -tubulin (gray) are present. The black dotted line represents the distance between the C3-position of maytansine and C5-position of the GDP, which is ~12 Å. B) Schematic drawing of both the maytansinoids and the conjugates designed to target the nucleotide-bound Mg 2 + or the exchangeable nucleotide site (E-site) of tubulin.

Figure 3 .

 3 Figure 3. Linker design strategy: schematic version of the applied approach followed for the generation of 54 triazole-containing linkers of different lengths.

Figure 4 .

 4 Figure 4. Design structures of the maytansinoid-conjugate compounds to target maytansine and GDP binding sites.

Figure 5 .

 5 Figure 5. Docking result of the maytansinoid-guanosine conjugate 8 superimposed on the docking result of the maytansinoid-acyclovir conjugate 10 when bound to tubulin (gray).

Scheme 4 .

 4 Scheme 4. a) 6-Heptynoic acid (or 4-(2-azidoethyl)benzoic acid) 3 equiv., DMAP 3 equiv., DCC 3.3 equiv. in DCM, ZnCl 2 3 equiv., DCM (+ 10 % DMF), 18 h (or 24 h), 11 66 % (or 12 68 %).

Scheme 6 .

 6 Scheme 6. a) 11 1 equiv., 13 1.5 equiv., CuSO 4 •5 H 2 O 1.6 equiv., Na-ascorbate 6.4 equiv., H 2 O/DMSO 1 : 2, 3 h, 89 %; b) HCOOH 70 % aq, 40 °C, 4 h, 62 %; c) 12 1 equiv., 15 1.5 equiv., CuSO 4 •5 H 2 O 1.6 equiv., Na ascorbate 6.4 equiv., H 2 O/DMSO 1 : 2, 3 h, 85 %; d) HCOOH 70 % aq, 40 °C, 4 h, 77 %.

Scheme 7 .

 7 Scheme 7. a) 11 1 equiv., 14 1.2 equiv., CuSO 4 •5 H 2 O 0.4 equiv., Na ascorbate 4 equiv., H 2 O/DMF 1 : 2, 5 h, 35 %; b) 12 1 equiv., 16 1.2 equiv., CuSO 4 •5 H 2 O 0.4 equiv., Na ascorbate 4 equiv., H 2 O/DMF 1 : 2, 6 h, 55 %.

Figure 6 .

 6 Figure 6. Inhibition of tubulin assembly by selected compounds. All experiments were performed as duplicates of two independent experiments. Time courses of assembly of 25 M tubulin in GAB buffer in the presence of vehicle (DMSO; black lines) or 27.5 M of (A) 7 (light green), 8 (teal), 9 (dark green), 10 (dark yellow); or (B) 4 (cyan), 5 (blue), 6 (dark blue), 12 (light purple).

Figure 7 .

 7 Figure 7. Binding poses of maytansinoids 4, 6 and 12 (PDB IDs: 8B7A, 8B7B, 8B7C) and their fit into the electron density. A) The molecular interactions of the three superimposed structures of maytansinoids 4 (purple), 6 (cyan) and 12 (yellow) in complex with -tubulin. The protein backbone (gray) is shown in ribbon representation, and interacting residues and ligands are shown as sticks. Oxygen, nitrogen and chlorine atoms are colored red, blue and green, respectively; water molecules are shown as red spheres. All three maytansinoids adopt the same pose as their parent compound maytansine and establish hydrogen bonds to the main chain carbonyl and amide of Gly100 and Val181, respectively, and direct or water-mediated hydrogen bonds to the side chains of Asn101, Asn102 and Lys105, as indicated by the dashed black lines. B) The long chains attached by the esterification of C3ÀO in 6 and 12 are highly mobile and adopt many different conformations, therefore, no defined electron density is visible for these long carbon chains. The highlighted definition and extension of the electron density to just 2 or 3 atoms beyond the well-defined C3 ester moiety is representative for all the soaked maytansinoids 4-10 and 12. The electron density maps 2mF o ÀDF c (blue mesh) and mF o ÀDF c (green/red mesh) are contoured at + 1.0 and 3.0 , respectively.

Table 1 .

 1 Cleavage conditions of isopropylidene on guanosine in the presence of maytansinol.

	Cleavage conditions	T [°C]	t [h]	Result
	AcOH 80 % aq	25	48	n.r.
	AcOH 80 % aq	65	18	n.r.
	PdCl 2 (CH 3 CN) 2	60	24	degradation
	CAN	25	48	n.r.
	PPTS	25	48	n.r.
	CuCl 2 •2 H 2 O	0-25	48	n.r.
	HCOOH 50 % aq [21]	25	48	n.r.
	HCOOH 70 % aq	25	48	n.r.
	HCOOH 70 % aq	40	2	ketal cleavage

Table 2 .

 2 Diagnostic 1 H NMR spectroscopic data of triazole formation [D 6 ]DMSO.

	Atom		1 H NMR [a,b]		13 C NMR [a,c]
			from	to	from	to
	20	33	2.28 [e]	2.65	18.04 [e]	28.82
		34	-	-	84.20 [e]	146.89
		35	1.95 [e]	7.85	69.0 [e]	122.13
		18G	3.29	4.25	51.59	49.38
	21	35	3.70 [d]	4.64	52.48 [d]	50.23
		17G	2.14	2.53	18.50	25.07
		18G	-	-	85.28	146.84
		19G	2.74	7.78	72.40	121.96

[a] Chemical shifts (ppm) were determined with reference to TMS. [b] Spectra determined at 400 MHz. [c] Spectra determined at 101 MHz. [d] Solvent is [D 6 ]acetone. [e] Solvent is [D]chloroform.

Table 3 .

 3 Binding affinities of maytansinoid compounds.[a]

	Compound	Mean K b [M À1 ]	K d [nM]
	maytansine	9 AE 1x10 7	11 AE 1
	4	9 AE 2x10 6	111 AE 12
	5	6 AE 1x10 6	166 AE 24
	6	5.3 AE 0.7x10 6	188 AE 22
	7	1.2 AE 0.2x10 7	83 AE 12
	8	1.3 AE 0.1x10 7	83.4 AE 7.8
	9	7.3 AE 0.8x10 6	137 AE 14
	10	8 AE 1x10 6	125 AE 14
	11	5.4 AE 0.5x10 7[b]	19 AE 2 [b]

12 1.3 AE 0.3x10 7 77 AE 15 [a] Data are the mean AE SEM values of three independent experiments with duplicates in each one. [b] Data from Marzullo al. 2022.

Table 4 .

 4 IC 50 of maytansinoid compounds in A549 and A2780/A2780AD cell lines.[a] Data are the mean AE SEM values of three independent experiments with duplicates in each one.[b] Data fromMarzullo et al. 2022.

	Cmpd	IC 50 [nM]

  of 1 mL min À1 . UV spectra were recorded at 254 nm and 210 nm with DAD detection. The mobile phase consisted of a mixture of H 2 O/ACN and the gradient was programmed using the following method: isocratic for 1 min at 50 % ACN, then gradient for 10 min to 90 % ACN. Products were purified using Biotage Isolera™ One System and Biotage® Sfär C18 6 g D¬ Duo 30 m as cartridges (BIOTAGE).

	General maytansinol acylation procedure for the synthesis of
	maytansinoids 4, 5, 11,12: To a solution of maytansinol 1 (50 mg,
	0.088 mmol, 1 equiv) in dry CH 2 Cl 2 (440 L) were added DMF dry
	(44 L), DMAP (33 mg, 0.26 mmol), the carboxylic acid (0.26 mmol),
	and ZnCl 2 (36 mg, 0.26 mmol) at room temperature under a
	nitrogen atmosphere. Then, a solution of DCC in dry CH 2 Cl 2 (60 mg,
	0.29 mmol, 1.3 M) was slowly added. The mixture was stirred at
	room temperature for 48 h before filtering off the DCU using cold
	CH 2 Cl 2 . The organic phase was washed with H 2 O (4 × 1 mL), with
	brine (1 × 1 mL), then dried over Na 2 SO 4 , and concentrated under
	reduced pressure. The residue was purified with Biotage Isolera™
	One System (gradient H 2 O/ACN) to provide the desired product as
	a white powder (Table S1).
	Synthesis of compound 6: To a solution of 5 (20 mg, 0.0279 mmol) in
	dry CH 2 Cl 2 (1.2 mL) were added acrylic acid (5.0 mg, 0.070 mmol)
	and Hoveyda-Grubbs 2nd-gen. catalyst (2.63 mg, 15 mol%), predis-
	solved in CH 2 Cl 2 (0.2 mL). The reaction was stirred at reflux (40 °C)
	for 6 h, after which additional catalyst was added (0.88 mg, 5 mol%)
	and the reaction was stirred overnight. The solvent was evaporated
	under reduced pressure. The crude was purified with Biotage
	Isolera™ One System (gradient H 2 O/ACN) providing the product
	(9,7 mg, 46 %) as a white powder.
	Synthesis of compound 13: To a solution of 17 (100 mg, 0.309 mmol)
	in dry CH 2 Cl 2 (2.5 mL) were added 6-azidohexanoic acid (72 mg,
	0.464 mmol), DMAP (22 mg, 0.185 mmol), EDC•HCl (106 mg,
	0.557 mmol), and triethylamine dry (95 L, 0.680 mmol) at room
	temperature under a nitrogen atmosphere. The reaction was stirred
	for 18 h before H 2 O was added (1 × 2 mL). The organic layer was
	dried over Na 2 SO 4 , and concentrated under reduced pressure.
	Purification with Biotage Isolera™ One System (gradient H 2 O/ACN)
	provided the product (101 mg, 71 %) as a white powder.
	Synthesis of compound 14: To a solution of acyclovir (150 mg,
	0.666 mmol) in dry CH 2 Cl 2 (1.0 mL) and dry DMF (1.5 mL) were
	added 6-azidohexanoic acid (158 mg, 1.006 mmol), DMAP (41 mg,
	0.336 mmol), EDC•HCl (269 mg, 1.405 mmol), and triethylamine dry
	(196 L, 1.405 mmol) at room temperature under a nitrogen
	atmosphere. The reaction was stirred overnight. Then, the solvent
	was removed under reduced pressure. The crude was purified with
	Biotage Isolera™ One System (gradient H 2 O/ACN), providing the
	product (154,8 mg, 65 %) as a white powder.
	Synthesis of compound 15: To a solution of 17 (150 mg, 0.464 mmol)
	in dry CH 2 Cl 2 (3.8 mL) were added 6-heptynoic acid (88 L,
	0.696 mmol), DMAP (34 mg, 0.278 mmol), EDC•HCl (160 mg,
	0.835 mmol), and triethylamine dry (142 L, 1.021 mmol) at room
	temperature under a nitrogen atmosphere. The reaction was stirred
	for 18 h before H

Synthesis of compound 17:

To a suspension of guanosine (200 mg, 0.706 mmol) in dry acetone (11.8 mL) was added an aqueous solution of 60 % HClO 4 60 % (95 L, 0.946 mmol) and the clear reaction obtained was stirred for 2 h at room temperature. The reaction was quenched by dropping an aqueous solution of 28 % NH 4 OH (134 L) at 0 °C. The precipitate was recovered by filtration on Buchner washing with cold Et 2 O. The solid was dried in vacuum to provide 229 mg (> 98 %) of product as white solid. 2 O was added (1 × 3 mL). The organic layer was dried over Na 2 SO 4 , and concentrated under reduced pressure.

  mg, 0.015 mmol) and 16 (6 mg, 0.018 mmol) in H 2 O/DMF as solvents (1 : 2, 300 L) were added a solution of CuSO 4 •5 H 2 O (8 L of 0.75 M solution, 0.06 mmol), and a solution of Na-ascorbate (60 L of 1 M solution, 0.006 mmol). The mixture was stirred at room temperature for 5 h. Crude was purified with Biotage Isolera™ One System (gradient H 2 O/ACN), providing the product (8.8 mg, 55 %) as a white powder.

	1 H NMR and 13 C NMR spectra were recorded on a Bruker Avance
	Spectrometer 400 MHz using commercially available deuterated
	solvents ([D]chloroform, [D 4 ]methanol, [D 6 ]acetone, [D 6 ]DMSO,
	[D 2 ]dichloromethane) at room temperature.
	1 H NMR and

Acknowledgements

We thank Ganadería Fernando Díaz for calf brains. We thank the staff of the Swiss Light Source responsible for beamline X06DA for their support during X-ray data collection. This work was supported by the H2020-MSCA-ITN-2019 (860070 TUBINTRAIN), Ministerio de Ciencia e Innovación PID2019-104545RBÀI00 (JFD), Proyecto de Investigación en Neurociencia Fundación Tatiana Pérez de Guzmán el Bueno 2020, and by the European Union NextGenerationEU (J.F.D.). Open Access Funding provided by $INSTITUTION within the CRUI-CARE Agreement.

Data Availability Statement

The data that support the findings of this study are available in the supplementary material of this article.

Conflict of Interest

The authors declare no conflict of interest.


John Barzman

Glimpses of Le Havre History

Introduction

  of Le Havre History 24-8-2015 Sciences Po V. Le Havre in three wars

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