Matriz de categorización inductiva

As mentioned in section 7.2, the strategy for synthesising inhibitors with the warhead appended to the C-terminus commences with the synthesis of the warhead building block using solution phase chemistry before attachment to the solid support for further peptide elongation. The details for each warhead type will be treated separately in the following sections.

7.4.1 Synthesis of Michael acceptor inhibitors

Revisiting the cleavage site of SlpA, it was noted that P1 of the substrate is either an alanine or a serine in most of the strains. The synthesis of inhibitors with warhead appended to the C-terminus using SPPS proved difficult to adapt for a number of reasons, including restriction on the number of amino acids with the properties required for transformation into the corresponding warhead functionality and/or loading onto resin. For example: a functionalised side chain is required to enable the attachment on the resin, and acid and/or base stability is typically essential to survive the harsh condition during solution phase warhead synthesis.

Ac-AA1-AA2-AA3-AA4-MA

No. AA1 AA2 AA3 AA4 Abbr. Name

TD 90 Leu Glu Thr Lys Ac-LETK-MA

TD 91 - - Lys(Biotin) Tyr Ac-K(Biotin)-Y-MA TD 92 - - Lys(Biotin) Leu Ac-K(Biotin)-L-MA TD 93 - - Lys(Biotin) Glu Ac-K(Biotin)-E-MA TD 94 - - Lys(Biotin) Met Ac-K(Biotin)-M-MA

Table 7. 3: Inhibitors with Michael acceptor warhead appended to the C-terminus of the specificity element.

COOEt N

H HO AA1-AA2-AA3-AA4 O

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Figure 7. 10: Synthesis of inhibitors with an MA warhead appended on the C-terminus. Reagents and conditions: IX) DCC (1.05eq.), DMAP (0.10eq.), BnSH (2eq.), RT, o/n; X) Et3SiH (5eq.), Pd/C (10%), in acetone RT,1h; XI) Ph3PCHCOOEt (1.5eq.) in THF, RT, 24h; XII) 20%TFA in DCM, RT, 2h; loading on resin in pyridine and THF, 60°C, 6h; peptide elongation using standard SPPS; cleavage with 95% TFA, 2.5%H2O, 2.5% TIS, 2h, RT.

The synthesis of MA inhibitors commenced with Fmoc-Ser(tBu)-OH, which consists of three functional groups utilised for conversion into the MA warhead building block: the carboxyl group for modifying to the corresponding MA, the alcohol side chain to enable attachment onto the solid support and the Fmoc protected amine for subsequent peptide elongation. The reaction conditions were adapted from the literature procedure [62] (Figure 7. 10). Firstly, serine was converted into S-benzyl thioester (15) by coupling to benzyl mercaptan. Reduction of this thioester (15) using palladium over carbon and a source of hydrogen (Et3SiH in this case) provided the corresponding aldehyde intermediate (16). Crude 16 was then condensed with the carbethoxymethylene triphenylphosphorane Wittig reagent to afford the α,β-unsaturated ester (17). Removal of the tBu protective group using 20% TFA supplied the corresponding side chain deprotected ester (18) allowing the attachment of the modified serine onto the solid support, which is a trityl resin in this case. Subsequently, the peptidyl specificity element was built up on the loaded resin by exploiting standard Fmoc/tBu SPPS. The biotin label was introduced to the inhibitor by incorporating a side

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chain biotinylated lysine in the peptidic specificity element, and the free amine at the N-terminus was acetylated to prevent additional charges in the molecule. Under standard TFA cleavage conditions as used previously, MA inhibitors was obtained and presented in Table 7. 3. The warhead of MA inhibitors is defined as the combination of the remaining serine side chain and the α,β-unsaturated ester (indicated in red).

7.4.2 Synthesis of AOMK- and CMK- inhibitors

A general method for synthesising AOMK and CMK probes was developed by Bogyo and co- workers [53], and this involved the attachment of Fmoc-protected CMK derivatives on hydrazine preloaded resin. Based on this work, an adapted synthetic route was proposed for the production of CMK inhibitors (Figure 7. 11).

Like the synthesis of the MA inhibitors, the initial amino acid building block was modified to contain the CMK functionality using solution chemistry. CMK derivative syntheses have been widely reported in the literature [53, 442-443]. The conventional route involves multiple transformations of the corresponding amino acid via diazomethyl ketones as intermediates [444]. These methods utilised diazomethane as the key reagent to generate the diazomethyl ketone moiety. However, diazomethane suffers from numerous safety issues due to its high toxicity, shock sensitivity and explosiveness [444-446]. Alternative routes avoiding the use of diazomethane and employing other reagents of a significantly reduced toxicity were explored, such as the use of chloromethyl lithium enolate, silyl enol ether, or a bromination step followed by decarboxylation, and a sulfur ylide. However, only the sulfur ylide route, which was based on the work of Wang et al. [444], proved to be simple and reliable with only three steps in total to obtain the CMK derivative. This procedure was adapted to synthesise the CMK building block used in the present study. Firstly, Z-protected alanine, where the ‘Z’ group is stable under acidic conditions, was converted into the corresponding methyl ester (20) Figure 7. 12. Treatment of this ester (20) with trimethylsulfoxonium iodide and potassium tert-butoxide afforded the trimethylsulfoxonium ylide (21), which, without further purification, was subsequently converted into Z-Ala-CMK (22) utilising HCl generated in situ via heating a mixture of lithium chloride and methanesulfonic acid in THF. One part of 22 was transformed into the AOMK derivative (23) by treatment with methoxybenzoic acid

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in the presence of potassium fluoride in DMF; a protocol adapted from the work of Wood et al. [443].

Figure 7. 11: Proposed synthetic route for CMK and AOMK inhibitors [53]. The Fmoc-protected building blocks are loaded onto hydrazine-functionalised resin in THF at 50°C. Standard SPPS was used for peptide elongation and final cleavage was performed with 95% TFA, 2.5% TIS, 2.5% H2O, 2h at RT.

Prior to loading these building blocks onto the solid support, via a hydrazone linkage as depicted in Figure 7. 11, removal of the Z protecting group was attempted in order to enable subsequent substitution by an Fmoc group. Unfortunately, classical Z-deprotection methods such as catalytic (Pd/C) hydrogenlysis led to decomposition for both the CMK and AOMK building blocks, and therefore replacement with an Fmoc group was not possible. Due to time constraints, persistent decomposition upon deprotection meant that these building blocks could not be coupled onto the solid support and hence the desired final inhibitors could not be synthesised. The Z-Ala-CMK and Z-Ala-AOMK compounds were

FmocHN N O NH N H O R O FmocHN O Cl H2N NH N H O + FmocHN O O R O FmocHN N Cl NH N H O N H N O NH N H O R O N H N Cl NH N H O AA1_-AA2_-AA3 AA1-AA2-AA3 O O SPPS N H O Cl AA1_-AA2_-AA3 O N H O O R O AA1_-AA2_-AA3 O THF, 50o_C

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nevertheless taken through to in vivo testing as representatives for these classes of inhibitors.

Figure 7. 12: Synthesis of CMK and AOMK building blocks. Reagents and conditions: XIV) K2CO3 (1eq.), MeI (2eq.) in DMF, 0°C, 30min; XV) KOtBu (3eq.), Me3SOI (3eq.) in THF, 2h reflux, then cooled down to 0°C before adding XIV (1eq.), 4h; XVI) MeSO3H (1.1eq.), LiCl (1.14 (eq.) in THF, 0°C-> 70°C, 2h, and quenched with H2O; XVII) 4-MeOBnCOOH (2eq.), KF (4eq.) in DMF, RT, o/n.

7.4.3 Activity and labelling effect of inhibitors with the warhead appended to C- terminus

Testing all inhibitors with the warhead appended at the C-terminus at 100 µM against C. difficile 630 revealed that all inhibitors possessed moderate inhibition activity towards SlpA processing with the exception of Z-Ala-CMK (Figure 7. 13). This suggests that the strength of the electrophilic trap may play an important role since the corresponding amides were not active at the concentration tested. In addition, the resultant significant change in structure may also contribute to activity variation between inhibitors with the warhead appended to different ends of the peptidic chain. Intriguingly, although Z-Ala-CMK did not possess activity against SlpA processing, it did prove effective at killing C. difficile. Further investigation of this compound is discussed in a later section.

The labelling effect was observed on the NeutrAvidin blot; however, it was observed that there was a similar tendency to that found for other inhibitor types. Generally, the labelled protein pattern of Michael acceptor inhibitors (Ac-XXS-MA) was almost identical to the one of the control sample EtEP-R-PEG3-Biotin though less intense. Since no distinct bands were

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observed in comparison with the epoxysuccinyl control sample, no post proteomics study was carried out on this type of inhibitor.

Figure 7. 13: Activity and labelling effect of inhibitors with the warhead appended to the C-terminus. C. difficile 630 cultures were treated with indicated inhibitors/probes at 100 μM, as described standard inhibition assays (see section 2.1.1).