Total Synthesis and Biological Evaluation of Modified Ilamycin Derivatives

Ilamycins/rufomycins are marine cycloheptapeptides containing unusual amino acids. Produced by Streptomyces sp., these compounds show potent activity against a range of mycobacteria, including multidrug-resistant strains of Mycobacterium tuberculosis. The cyclic peptides target the AAA+ protein ClpC1 that, together with the peptidases ClpP1/ClpP2, forms an essential ATP-driven protease. Derivatives of the ilamycins with a simplified tryptophane unit are synthesized in a straightforward manner. The ilamycin derivative 26 with a cyclic hemiaminal structure is active in the nM-range against several mycobacterial strains and shows no significant cytotoxicity. In contrast, derivative 27, with a glutamic acid at this position, is significantly less active, with MICs in the mid µM-range. Detailed investigations of the mode of action of 26 indicate that 26 deregulates ClpC1 activity and strongly enhances ClpC1-WT ATPase activity. The consequences of 26 on ClpC1 proteolytic activities were substrate-specific, suggesting dual effects of 26 on ClpC1-WT function. The positive effect relates to ClpC1-WT ATPase activation, and the negative to competition with substrates for binding to the ClpC1 NTD.


Introduction
Marine organisms are a rich source of natural products, producing a plethora of fascinating new chemical structures [1,2]. Many of these natural products show interesting biological activities, e.g., against cancer cell lines or bacteria, making them good candidates for drug development against infectious diseases such as tuberculosis, among others [3][4][5]. Tuberculosis is a serious health issue: in 2019, approximately 10 million people fell ill with the disease, and 1.5 million died [6]. A major problem is the continuous development of resistant strains of Mycobacterium tuberculosis, the bacteria responsible for this disease. In 2018, 500,000 people demonstrated resistance to the most effective first-line drug rifampicin, and 80% of these suffered from multidrug-resistant tuberculosis (MDR-TB). This clearly illustrates the high demand for new drugs that would work via new modes of action against the largely drug-resistant tuberculosis (XDR-TB) strains, which are resistant to almost all known drugs [7]. In this context, natural products are excellent candidates for developing new anti-TB drugs, since more than 60% of drugs under current development are natural products or derived from natural products [8][9][10].
In 1962, Japanese researchers isolated interesting cyclic peptides from marine Streptomyces sp. that showed interesting activities against Mycobacteria. Takita et al. isolated two antibiotics, named ilamycin A and B, from the culture filtrate of Streptomyces insulates (A-165-Z1), a new strain later renamed as Streptomyces islandicus, that inhibited the growth of Mycobacterium 607 and Mycobacterium phlei [11,12]. In the same year, Shibata et al. isolated two new antibiotics, rufomycin A and B, from the new strain Streptomyces atratus (46408) [13,14], found to be especially active against Mycobacterium tuberculosis Unfortunately, CymA as a natural product lacks satisfactory pharmacokinetic properties, making it challenging for optimization into an (orally) bioavailable drug. Therefore, we decided to develop an independent synthetic route towards the cyclomarins [35,36] and derivatives for SAR studies [37], with the goal of simplifying the rather complex structure without losing too much biological activity [38,39]. Because we were interested to modify the cyclomarins mainly at the tryptophan unit and the unsaturated amino acid, we decided to incorporate these two building blocks at the end of the linear peptide synthesis and to undertake the ring closure at the same position as Yao et al. [33]. Interestingly, neither the epoxide nor the prenyl substituent are required for good activity [39]. Interestingly, the β-OH-functionality can also be removed [38], which allowed a significant simplification of the synthesis, because the βhydroxytryptophan was found to be extremely sensitive to acid as well as base, which caused severe problems during natural product synthesis. Without the β-OHfunctionality, this building block is the same as in the ilamycins.

Results and Discussion
Based on these results, we started to develop a synthesis of simplified ilamycin derivatives and to investigate their biological properties. We decided to use the same ring closing position as in the previous cyclomarin synthesis [35,36]. The N-prenylated tryptophan was replaced by an N-methylated analogue, since this structural change had no significant influence on the activity in the case of the cyclomarins and since N-methyl tryptophan is commercially available. We decided to use N-Alloc-protected amino acids as building blocks [40,41] in the linear peptide synthesis, because this protecting group can be removed via Pd-catalysis [42] and is therefore compatible with the other protecting groups used during the synthesis. In 1999, the groups of Fenical and Clardy reported the isolation of three cyclomarins (Cym) A−C from extracts of a Streptomyces sp. (CNB-982) [26]. These compounds are structurally related to the ilamycins/rufomycins. Very similar amino acid building blocks are incorporated, but, interestingly, in a different sequence. As in the ilamycins, an Nprenylated tryptophan (CymC) is found as a unique building block that can also be epoxidized (CymA), as in Ruf I, but in the case of the cyclomarins these tryptophan units are β-hydroxylated (Figure 1). At the N-terminus of the tryptophan, a γ,δ-unsaturated amino acid is also incorporated and one of the leucines is also oxidized at the δ position, but at another position, as in the ilamycins. Most obvious is the replacement of the unique nitrotyrosine by another aromatic amino acid, syn-β-methoxyphenylalanine. Some more, slightly modified, derivatives have been isolated in recent years [27,28]. The cyclomarins were also found to be active against Mycobacterium tuberculosis (M. tub.) in the higher nM range [29][30][31].
Their excellent biological activities drove the development of syntheses of these interesting cyclic peptides. So far, only one synthesis has been reported for the ilamycins E1 and F, by Guo and Ye et al. in 2018, closing the macrolactame ring between the tryptophan unit and the unsaturated amino acid [32]. The first synthesis of CymC was reported in 2004 by Yao and colleagues [33]. They failed in their attempts to close the peptide ring at the same position [34] but were successful between the unsaturated amino acid and the N-methylated leucine [33].
Unfortunately, CymA as a natural product lacks satisfactory pharmacokinetic properties, making it challenging for optimization into an (orally) bioavailable drug. Therefore, we decided to develop an independent synthetic route towards the cyclomarins [35,36] and derivatives for SAR studies [37], with the goal of simplifying the rather complex structure without losing too much biological activity [38,39]. Because we were interested to modify the cyclomarins mainly at the tryptophan unit and the unsaturated amino acid, we decided to incorporate these two building blocks at the end of the linear peptide synthesis and to undertake the ring closure at the same position as Yao et al. [33]. Interestingly, neither the epoxide nor the prenyl substituent are required for good activity [39]. Interestingly, the β-OH-functionality can also be removed [38], which allowed a significant simplification of the synthesis, because the β-hydroxytryptophan was found to be extremely sensitive to acid as well as base, which caused severe problems during natural product synthesis. Without the β-OH-functionality, this building block is the same as in the ilamycins.

Results and Discussion
Based on these results, we started to develop a synthesis of simplified ilamycin derivatives and to investigate their biological properties. We decided to use the same ring closing position as in the previous cyclomarin synthesis [35,36]. The N-prenylated tryptophan was replaced by an N-methylated analogue, since this structural change had no significant influence on the activity in the case of the cyclomarins and since N-methyl tryptophan is commercially available. We decided to use N-Alloc-protected amino acids as building blocks [40,41] in the linear peptide synthesis, because this protecting group can be removed via Pd-catalysis [42] and is therefore compatible with the other protecting groups used during the synthesis.

Preparation of the Amino Acid Building Blocks
The OH-functionality of the commercially available nitrotyrosine (1) had to be protected to avoid side reactions during subsequent peptide coupling steps. In a previous synthesis we used an allyl protecting group [43,44], which can easily be removed with ruthenium catalysts [45], but because we were not sure if this protecting group would create problems during Alloc deprotection we decided to use a methoxymethyl (MEM) protecting group in this case (Scheme 1). After N-Alloc protection of 1 [46] the carboxylic acid 2 was converted into methyl ester 3 to allow regioselective MEM-protection of the phenolic OH-functionality [47]. Subsequent saponification of 4 provided the desired amino acid building block 5 in an overall very high yield. phenolic OH-functionality [47]. Subsequent saponification of 4 provided the desired amino acid building block 5 in an overall very high yield. The desired N-methylated δ-hydroxyleucine was prepared from commercially available (R)-Roche ester 6 (Scheme 2). After TBS-protection the ester functionality of 7 was reduced with DIBALH and the aldehyde formed was directly reacted with Schmidt's phosphonoglycinate 8, giving rise to the desired dehydroamino acid ester 9 as a 3:1 Z/Emixture [48]. A separation of the two isomers was not required in this case, because in the next step (the asymmetric catalytic hydrogenation with (R)-Monophos ® [49,50]) the Zisomer reacted much faster, almost exclusively generating the desired amino acid ester 10 as a single stereoisomer. Saponification of the methyl ester and subsequent N-methylation of 11 under standard conditions [51] delivered the required building block 12. The desired N-methylated δ-hydroxyleucine was prepared from commercially available (R)-Roche ester 6 (Scheme 2). After TBS-protection the ester functionality of 7 was reduced with DIBALH and the aldehyde formed was directly reacted with Schmidt's phosphonoglycinate 8, giving rise to the desired dehydroamino acid ester 9 as a 3:1 Z/Emixture [48]. A separation of the two isomers was not required in this case, because in the next step (the asymmetric catalytic hydrogenation with (R)-Monophos ® [49,50]) the Zisomer reacted much faster, almost exclusively generating the desired amino acid ester 10 as a single stereoisomer. Saponification of the methyl ester and subsequent N-methylation of 11 under standard conditions [51] delivered the required building block 12.
was reduced with DIBALH and the aldehyde formed was directly reacted with Schmidt's phosphonoglycinate 8, giving rise to the desired dehydroamino acid ester 9 as a 3:1 Z/Emixture [48]. A separation of the two isomers was not required in this case, because in the next step (the asymmetric catalytic hydrogenation with (R)-Monophos ® [49,50]) the Zisomer reacted much faster, almost exclusively generating the desired amino acid ester 10 as a single stereoisomer. Saponification of the methyl ester and subsequent N-methylation of 11 under standard conditions [51] delivered the required building block 12. Scheme 2. Synthesis of protected hydroxyleucine building block 12.
The unique unsaturated 2-amino-4 hexenoic acid was also synthesized in Allocprotected form from glycine derivative 13 and commercially available (S)-but-3-yn-2-ol using a modified Steglich protocol (Scheme 3) [52]. Lindlar hydrogenation of 14 gave rise to allyl ester 15, which was subjected to a chelated ester enolate Claisen rearrangement [53,54]. Based on a chair like transition state a perfect chirality transfer from the allyl alcohol to the α-stereogenic center of the amino acid 16 was observed [55,56]. This approach allows the introduction of all kinds of γ,δ-unsaturated side chains. The unique unsaturated 2-amino-4 hexenoic acid was also synthesized in Allocprotected form from glycine derivative 13 and commercially available (S)-but-3-yn-2-ol using a modified Steglich protocol (Scheme 3) [52]. Lindlar hydrogenation of 14 gave rise to allyl ester 15, which was subjected to a chelated ester enolate Claisen rearrangement [53,54]. Based on a chair like transition state a perfect chirality transfer from the allyl alcohol to the α-stereogenic center of the amino acid 16 was observed [55,56]. This approach allows the introduction of all kinds of γ,δ-unsaturated side chains. phosphonoglycinate 8, giving rise to the desired dehydroamino acid ester 9 as a 3:1 Z/Emixture [48]. A separation of the two isomers was not required in this case, because in the next step (the asymmetric catalytic hydrogenation with (R)-Monophos ® [49,50]) the Zisomer reacted much faster, almost exclusively generating the desired amino acid ester 10 as a single stereoisomer. Saponification of the methyl ester and subsequent N-methylation of 11 under standard conditions [51] delivered the required building block 12. Scheme 2. Synthesis of protected hydroxyleucine building block 12.
The unique unsaturated 2-amino-4 hexenoic acid was also synthesized in Allocprotected form from glycine derivative 13 and commercially available (S)-but-3-yn-2-ol using a modified Steglich protocol (Scheme 3) [52]. Lindlar hydrogenation of 14 gave rise to allyl ester 15, which was subjected to a chelated ester enolate Claisen rearrangement [53,54]. Based on a chair like transition state a perfect chirality transfer from the allyl alcohol to the α-stereogenic center of the amino acid 16 was observed [55,56]. This approach allows the introduction of all kinds of γ,δ-unsaturated side chains.

Synthesis of the Linear Heptapeptide
With the suitable protected building blocks in hand, we started the synthesis of the linear peptide chain with the coupling of hydroxyleucine derivative 12 with Leu-OMe to 17 (Scheme 4). Subsequent cleavage of the Cbz-protecting group and coupling with Alloc-Ala-OH provided tripeptide 18 in acceptable yield. While EDC/HOBt was used as a coupling reagent in the first step, BEP was found to be the reagent of choice in the second step. Palladium-catalyzed removal of the Alloc protecting group using the water-soluble phosphine ligand TPPTS [57,58] provided the free tripeptide which was reacted with the protected nitrotyrosine 5 to tetrapeptide 19. The nitrotyrosine was found to be the most critical building block of the whole series. Both couplings, at the Cas well as on the N-terminus, required an optimization of the reaction conditions to obtain good yields. Activation of 5 with TBTU/DIPEA was the method of choice to obtain 19, and the best results in the subsequent coupling with N-MeLeu to 20 were obtained with PyBOP/DIPEA [59]. The continuing prolongation of the peptide chain provided no further problems and the linear heptapeptide 22 was subjected to ring closing reaction. Interestingly, cleavage of the Alloc-protecting group from 21 under the usual conditions gave only a moderate yield, but it could be improved significantly by using dimethylbarbiturate (DMBA) as the cleaving reagent [60][61][62].
Mar. Drugs 2022, 20, 632 5 of 28 Activation of 5 with TBTU/DIPEA was the method of choice to obtain 19, and the best results in the subsequent coupling with N-MeLeu to 20 were obtained with PyBOP/DIPEA [59]. The continuing prolongation of the peptide chain provided no further problems and the linear heptapeptide 22 was subjected to ring closing reaction. Interestingly, cleavage of the Alloc-protecting group from 21 under the usual conditions gave only a moderate yield, but it could be improved significantly by using dimethylbarbiturate (DMBA) as the cleaving reagent [60][61][62].

Synthesis of Cyclic Ilamycin Derivatives
After saponification of the C-terminal ester moiety of 22 and removal of the N-terminal Alloc protecting group, the cyclization was performed using HATU/DIPEA, providing the desired cyclopeptide 23 in an acceptable yield of 47% over the last three steps (Scheme 5). The final TBS and MEM-deprotection was performed with ethanedithiol and BF 3 etherate [63], before the hydroxyleucine subunit of 24 was oxidized to the desired ilamycin derivatives. Dess-Martin periodinane oxidation [64] in the presence of an excess pyridine provided aldehyde 25 as an intermediate, which could be converted either into hemiaminal 26 or further oxidized under Pinnick conditions [65] to carboxylic acid 27.
terminal Alloc protecting group, the cyclization was performed using HATU/DIPEA, providing the desired cyclopeptide 23 in an acceptable yield of 47% over the last three steps (Scheme 5). The final TBS and MEM-deprotection was performed with ethanedithiol and BF3 etherate [63], before the hydroxyleucine subunit of 24 was oxidized to the desired ilamycin derivatives. Dess-Martin periodinane oxidation [64] in the presence of an excess pyridine provided aldehyde 25 as an intermediate, which could be converted either into hemiaminal 26 or further oxidized under Pinnick conditions [65] to carboxylic acid 27. Scheme 5. Synthesis of cyclic ilamycin derivatives.

Biological Evaluation
The new ilamycin derivatives were tested against several mycobacterial strains for anti-mycobacterial activity. The growth inhibition was determined by a resazurin reduction microtiter assay (REMA) [66], and the minimal inhibitory concentrations (MICs) are summarized in Table 1. Compound 26 with the hemiaminal subunit was found to be significantly more active than derivative 27 with the carboxylic acid side chain. While the latter derivative showed MICs in the mid-micromolar range for all tested mycobacterial strains, the highest activity for 26 was observed against M. tuberculosis H37Ra strain (MIC: 50 nM). Although 26 was less active compared to the most active ilamycin E, it was twice as active as cyclomarin A. Only moderate cytotoxicity against cancer cell lines (e.g., HepG2) was observed.

Biological Evaluation
The new ilamycin derivatives were tested against several mycobacterial strains for anti-mycobacterial activity. The growth inhibition was determined by a resazurin reduction microtiter assay (REMA) [66], and the minimal inhibitory concentrations (MICs) are summarized in Table 1. Compound 26 with the hemiaminal subunit was found to be significantly more active than derivative 27 with the carboxylic acid side chain. While the latter derivative showed MICs in the mid-micromolar range for all tested mycobacterial strains, the highest activity for 26 was observed against M. tuberculosis H37Ra strain (MIC: 50 nM). Although 26 was less active compared to the most active ilamycin E, it was twice as active as cyclomarin A. Only moderate cytotoxicity against cancer cell lines (e.g., HepG2) was observed.

Mode of Action
To elucidate the molecular basis of the antibacterial activity of the potent ilamycin derivative 26, we determined its impact on ATPase and proteolytic activities of its cellular target ClpC1. ClpC1 is an AAA+ protein that forms, together with the peptidase complex ClpP1/ClpP2, an essential ATP-dependent protease in M. tuberculosis (Mtb). ClpC1 is We next determined the consequences of 26 on the degradation of the alternative substrate GFP-SsrA, which harbors the SsrA degron for Hsp100 recognition [69]. GFP-SsrA does not require the NTD for recognition as it directly binds to the pore site of Hsp100 proteins [70]. 26 did not inhibit the fast proteolysis of GFP-SsrA by ClpC1-F444S/SaClpP, demonstrating that its impact on ClpC1-F444S proteolytic activity is substrate-specific. Similarly, 26 strongly (5.3-fold) enhanced degradation of GFP-SsrA by ClpC1-WT/SaClpP and was even more efficient than CymA, which stimulated degradation 2.4-fold ( Figures 2C and S1D). Together these findings demonstrate that 26 deregulates ClpC1 proteolytic activities in a substrate-specific manner.
Finally, we determined how 26 modifies the ClpC1 assembly state by dynamic light scattering (DLS) (Figure 3). Here, we used ATPase-deficient E288A/E626A mutants (termed DWB), which harbor mutations in the Walker B motifs of both ATPase domains, to stabilize assembly states in presence of 2 mM ATP. The hydrodynamic radii of ClpC1-DWB and ClpC1-DWB-F444S were 11.1 ± 0.5 nm and 9.5 ± 0.3 nm, respectively, indicating the presence of a decameric resting state (WT) and hexamers (F444S). Addition of CymA to ClpC1-DWB induced the formation of larger assemblies (18.7 ± 1.9 nm radius), which likely represent the tetrahedral assembly of CymA-bound ClpC1-DWB hexamers as described before [67,71]. Presence of 26 caused a smaller, yet significant increase in the ClpC1-DWB radius to 12.2 ± 0.8 nm ( Figure 3A,C). Since 26 increases ClpC1-WT ATPase but also proteolytic activity (GFP-SsrA), we suggest that this assembly state consists of interacting hexamers. No significant changes in radii were observed for ClpC1-DWB-F444S in the presence of the cyclic peptides ( Figure 3B,C). This suggests that the change of ClpC1-DWB particle size triggered by 26 involves interactions of the coiled-coil MDs. Next, we monitored the consequences of 26 on the degradation of the disordered model substrate FITC-casein. We used S. aureus ClpP (SaClpP) as a cooperating peptidase, which functions with ClpC1 similarly to Mtb ClpP1/P2, but without requiring a dipeptide activator [67]. 26 did not enhance FITC-casein degradation by ClpC1/SaClpP in contrast to CymA, but caused a minor, 1.24-fold reduction at 10 µM concentration ( Figure 2B, Supporting informations, Figure S1A). Notably, 26 strongly inhibited the fast FITC-casein degradation by ClpC1-F444S/SaClpP, whereas CymA hardly changed degradation kinetics. These findings indicate fundamental differences in the consequences of 26 and CymA on ClpC1 proteolytic activity, although both compounds enhance ClpC1 ATPase activity to comparable degrees. ATPase stimulation of ClpC1-WT or ClpC1-F444S by 26 was not affected in the presence of ClpP, excluding an altered impact of 26 on ClpC1/SaClpP complexes ( Figure S1B). Cyclic peptides bind to a hydrophobic groove of the N-terminal domain (NTD) of ClpC1, a site that has also been identified as a casein binding site in ClpB, a close homolog of ClpC [68]. This raised the possibility that 26 inhibits FITCcasein degradation by ClpC1-F444S by competing with the substrate for binding to the NTD groove. We monitored the binding of FITC-casein to ClpC1-F444S by anisotropy measurements in the absence and presence of 26. Binding of FITC-casein was 2-fold reduced upon addition of 26, while CymA had no effect ( Figure S1C). This suggests that reduced binding of FITC-casein to ClpC1-F444S contributes to the inhibitory effect of 26 on substrate degradation. Additionally, 26 might modulate NTD dynamics and hamper the delivery of NTD-bound FITC-casein to the processing ClpC1 pore site.
We next determined the consequences of 26 on the degradation of the alternative substrate GFP-SsrA, which harbors the SsrA degron for Hsp100 recognition [69]. GFP-SsrA does not require the NTD for recognition as it directly binds to the pore site of Hsp100 proteins [70]. 26 did not inhibit the fast proteolysis of GFP-SsrA by ClpC1-F444S/SaClpP, demonstrating that its impact on ClpC1-F444S proteolytic activity is substrate-specific. Similarly, 26 strongly (5.3-fold) enhanced degradation of GFP-SsrA by ClpC1-WT/SaClpP and was even more efficient than CymA, which stimulated degradation 2.4-fold ( Figure 2C and Figure S1D). Together these findings demonstrate that 26 deregulates ClpC1 proteolytic activities in a substrate-specific manner.
Finally, we determined how 26 modifies the ClpC1 assembly state by dynamic light scattering (DLS) ( Figure 3). Here, we used ATPase-deficient E288A/E626A mutants (termed DWB), which harbor mutations in the Walker B motifs of both ATPase domains, to stabilize assembly states in presence of 2 mM ATP. The hydrodynamic radii of ClpC1-DWB and ClpC1-DWB-F444S were 11.1 ± 0.5 nm and 9.5 ± 0.3 nm, respectively, indicating the presence of a decameric resting state (WT) and hexamers (F444S). Addition of CymA to ClpC1-DWB induced the formation of larger assemblies (18.7 ± 1.9 nm radius), which likely represent the tetrahedral assembly of CymA-bound ClpC1-DWB hexamers as described before [67,71]. Presence of 26 caused a smaller, yet significant increase in the ClpC1-DWB radius to 12.2 ± 0.8 nm ( Figure 3A,C). Since 26 increases ClpC1-WT ATPase but also proteolytic activity (GFP-SsrA), we suggest that this assembly state consists of interacting hexamers. No significant changes in radii were observed for ClpC1-DWB-F444S in the presence of the cyclic peptides ( Figure 3B,C). This suggests that the change of ClpC1-DWB particle size triggered by 26 involves interactions of the coiled-coil MDs. . The relative frequencies of particle sizes were determined for 0-50 nm. Larger particle sizes were not considered as those were also observed for peptide only controls. (C) Radii of peak fractions of indicated ClpC1 assemblies were determined. An unpaired t-test (two-tailed) was used to assess the statistical significance of size differences (n ≥ 3, *, p < 0.05; ****, p < 0.0001, ns: not significant). . The relative frequencies of particle sizes were determined for 0-50 nm. Larger particle sizes were not considered as those were also observed for peptide only controls. (C) Radii of peak fractions of indicated ClpC1 assemblies were determined. An unpaired t-test (two-tailed) was used to assess the statistical significance of size differences (n ≥ 3, *, p < 0.05; ****, p < 0.0001, ns: not significant).

Discussion
How does ilamycin exert its antibacterial activity? Here, we show that ilamycin derivative 26 deregulates ClpC1 activity and strongly enhances ClpC1-WT ATPase activity. The consequences on proteolytic activities are diverse and depend on substrate identity. Degradation of FITC-casein, which binds to the NTD like ClpC1 targeting cyclic peptides, remained largely unaffected by 26. Notably, a strong inhibition of FITC-casein proteolysis by 26 is observed for ClpC1-F444S, which is constitutively hexameric and thus has a high basal ATPase activity. We therefore speculate that 26 has dual effects, negative and positive, on ClpC1-WT function. The positive effect relates to ClpC1-WT ATPase activation, and the negative to substrate binding to the NTD. In case of ClpC1-WT, both effects cancel each other out, while ClpC1-F444S, which is already activated, experiences only the negative impact. This model is consistent with our finding that 26 strongly increases degradation of GFP-SsrA, which does not require NTD for recognition, by ClpC1-WT. Accordingly, GFP-SsrA degradation by activated ClpC1-F444S is not affected by 26.
Our findings support a model that ClpC1-targeting cyclic peptides in general function by overriding ClpC1 activity control. This explains ClpC1 ATPase activation by lassomycin and ecumicin [72,73]. The consequences of cyclic peptides on substrate degradation in vivo will be diverse and depend on the mechanism of substrate recognition. While we anticipate enhanced degradation of substrates that do not require the ClpC1 NTD for processing, the consequences on NTD-dependent substrates (e.g., FITC-casein) seem diverse, as stimulatory (CymA) but also inhibitory (lassomycin, ecumicin) effects have been described [67,72,73]. Overall, all these peptides bind with similar affinities and in a similar manner to the ClpC1 NTD, though there are differences in their interaction details [74][75][76]. Why peptides can exhibit opposing consequences on substrate degradation is currently unclear. We speculate that peptides might differ in their interaction dynamics or the number of peptide-bound NTDs in a ClpC1 hexamer. We also noticed that 26 induces a specific change in the ClpC1 assembly state that is smaller compared to the tetrahedral hexamers described for CymAbound ClpC1 [67,71]. We speculate that this state represents a dimer of ClpC1 hexamers, similar to assemblies described for other AAA+ proteins [77,78]. The induction of distinct ClpC1 assembly states triggered by the diverse peptides might also help to explain their specific effects on total ClpC1 activities.
In summary, we show that 26 can exert opposing effects on ClpC1 proteolytic activities. We suggest that both effects massively change the ClpC1 substrate spectrum and amplify 26 antibacterial potency.

Materials and Methods
General Synthetic Methods: Air or moisture sensitive reactions were performed in oven-dried reaction flasks (80 • C) under nitrogen or argon atmosphere. Reactions at room temperature were performed at 25 • C. Dry DMF and DCM were purchased from Acros Organics and stored under nitrogen. THF was dried over sodium/benzophenone and diisopropylamine over CaH 2 . Ethyl acetate, pentane and petroleum ether (40-60 • C; PE) were distilled before use. Analytical TLC was carried out with pre-coated silica gel plates from Marcherey Nagel (Polygram ® SIL G/UV 254 ; Dueren, NRW, Germany). Detection was done using UV light at 254 nm, a KMnO 4 solution or a ninhydrin solution. LC-MS measurements were carried out either with the following LC unit from Shimadzu (controller: SCL-10Avp, pump: LC-10ATvp, diode array detector SPD-M10Avp; Kyōto, Japan) or with the LC unit LC-2030C 3D Plus, which is also from Shimadzu. The Shimadzu mass spectrometer (LCMS-2020) (Kyōto, Japan) was used for detection in each case. In addition to a Luna ® C18 column (50 × 4.6 mm, particle size 3 mm) from Phenomenex (Torrance, CA, USA), an Onxy ® Monolithic C18 130 Å column (50 × 4.6 mm) from the same company was used as stationary phase. The crude products were purified either by flash chromatography on silica gel (Macherey-Nagel 60, 0.063-0.2 mm or 0.04-0.063 mm; Dueren, NRW, Germany)) or by using a Büchi Reveleris ® Prep Chromatography System (Flawil, Switzerland) with Büchi FlashPure Select C18 (30 mm spherical) columns. Preparative HPLC was also performed on a Büchi Reveleris ® Prep Chromatography System using a Phenomenex Luna ® C18 (2) 6 . CHCl 3 , MeOD-d 3 or DMSO-d 5 were used as an internal standard. The chemical shifts are given in ppm (δ) compared to TMS. Peaks were assigned using 1 H, 1 H COSY, 1 H, 13 C HSQC, 1 H, 13 C HMBC and TOCSY spectra. Mass spectra were recorded using a Finnigan MAT 95 sector field spectrometer (HRMS, CI) or a Daltonics maXis 4G hr-ToF spectrometer (HRMS, ESI) from Bruker (Billerica, MA, USA). The Alloc-protected amino acids (Alloc-AlaOH [79], Alloc-N-Me-LeuOH [80,81] and Alloc-N'-Me-TrpOH [39]) were synthesized according to protocols known from the literature.
Strains, plasmids and proteins: Mtb ClpC1-WT and ClpC1-F444S were overproduced from pET24a expression vector in E. coli BL21 cells at 30 • C. The proteins were purified using Ni-IDA (Macherey-Nagel) using 50 mM Na-phosphate pH 8.0, 300 mM NaCl, 5 mM β-mercaptoethanol as lysis and washing buffer and the same buffer supplemented with 250 mM imidazole as elution buffer. Elution fractions were pooled and further purified by size exclusion chromatography (Superdex S200, GE Healthcare) in buffer A (50 mM HEPES pH 7.5, 150 mM KCl, 20 mM MgCl 2 , 2 mM DTT, 5% (v/v) glycerol). Sa ClpP was purified from E. coli MC4100 ∆clpB cells after overproduction from pDS56 expression vector. Sa ClpP was purified using the same protocol as Mtb ClpC1. Protein concentrations refer to monomers and were determined with the Bio-Rad Bradford assay using BSA as standard.
ATPase Assay: The ATPase activity of ClpC1 was determined using coupled reactions of pyruvate kinase (PK) and lactate dehydrogenase (LDH) in the presence of phosphoenolpyruvate (PEP) and NADH in buffer A. The reactions included 1 M ClpC1, 1.5 µM Sa ClpP and 1-10 µM 26 or cyclomarin A as indicated. Reactions were started with the injection of 2 mM ATP and changes in NADH absorbance was followed at 340 nm in a BMG Biotech CLARIOstar plate reader. ATPase rates were calculated from the linear decrease in A 340 in at least three independent experiments and standard deviations were calculated. All reactions included 1% (v/v) DMSO to equal buffer conditions in presence of 26 or cyclomarin A. The presence of DMSO did not affect ClpC1 activities.
Degradation Assays: All degradation assays were performed in buffer A in presence of an ATP regenerating system (20 ng/µL pyruvate kinase, 3 mM PEP, 2 mM ATP). All reactions included 1% (v/v) DMSO to equal buffer conditions in presence of 26 or cyclomarin A. 0.1 µM FITC-casein was incubated with 1 µM ClpC1, 1.5 µM Sa ClpP and 1-10 µM 26 or cyclomarin A as indicated. The increase in FITC-casein fluorescence upon its degradation was monitored in BMG Biotech CLARIOstar plate reader by using 483 and 520/530 nm as excitation and emission wavelengths, respectively. For data processing the initial fluorescence intensities were set to 100. FITC-casein degradation rates were determined from the initial slopes of the fluorescence signal increase in at least three independent experiments and standard deviations were calculated. Degradation of 0.5 µM GFP-SsrA was performed in presence of 3 µM ClpC1, 4.5 µM Sa ClpP and 20 µM 26 or cyclomarin A as indicated in a Perkin Elmer LS55 Spectrofluorometer. Degradation was initiated by addition of an ATP regenerating system. GFP fluorescence was monitored by using 400 and 510 nm as excitation and emission wavelengths, respectively. Initial GFP fluorescence was set to 100. Degradation rates were determined from the initial slopes of fluorescence signal decrease in at least three independent experiments and standard deviations were calculated.
Anisotropy measurements: Binding of ClpC1 to FITC-casein (100 nM) was monitored by fluorescence anisotropy measurements using a BMG Biotech CLARIOstar plate reader.  (2) Alloc-Cl (ρ = 1.134 g/mL, 0.26 mL, 2.4 mmol) was slowly added to a solution of 3-nitrotyrosine (500 mg, 2.21 mmol) (1) in 1.2 mL (4.8 mmol) 4 M aq. NaOH solution at 0 • C. After the reaction mixture had been stirred for 10 min at this temperature, the ice bath was removed and 1.2 mL (4.8 mmol) 4 M aq. NaOH solution and 0.2 mL water were added. Alloc-Cl (94 µL, 0.88 mmol) was added again to the reaction mixture after 1 h at room temperature and 2.4 mL MeOH after 2 h. After three days the solution was diluted with 10 mL sat. aq. NaHCO 3 solution. The aqueous phase was washed with diethyl ether, cooled to 0 • C and acidified to pH 3 with conc. HCl solution. After the aqueous phase had been extracted three times with EtOAc, the combined organic phases were dried over Na 2 SO 4 and concentrated under reduced pressure. For further purification, the crude product was lyophilized to yield 639 mg (2.06 mmol, 93%) of Alloc-protected tyrosine (2) (5) To a solution of methyl ester (4) (4.82 g, 11.7 mmol) in 146 mL THF was added dropwise a 1 M aq. LiOH solution (14.0 mL, 14.0 mmol) at 0 • C. During the reaction, the reaction mixture was slowly warmed to room temperature (2 h). The solvent was removed in vacuo. The residue was dissolved in water and the aqueous phase was acidified with 1 M aq. KHSO 4 solution to pH 3-4. Subsequently, the aqueous phase was extracted three times with DCM. After the combined organic phases had been dried over Na 2 SO 4 , the solvent was removed under reduced pressure to obtain 5 (4. To a solution of (R)-Roche ester (6) (5.00 g, 42.3 mmol) in anhydrous DMF was added imidazole (3.31 g, 48.7 mmol) at room temperature. Subsequent addition of TBS-Cl (7.02 g, 46.6 mmol) was carried out at 0 • C. The reaction mixture was slowly warmed to room temperature overnight. After 19 h (TLC), the solution was dissolved in diethyl ether. The organic phase was washed successively with water and brine and dried over Na 2 SO 4 . After the solvent was removed under reduced pressure, the crude product was purified by flash chromatography (silica gel, PE/Et 2 O 95:5). The TBS-protected Roche ester (7)

Synthesis of Methyl (S,Z)-2-{[(benzyloxy)carbonyl]amino}-5-[(tertbutyldimethylsilyl)oxy]-4-methylpent-2-enoate (9)
To a solution of TBS-protected Roche ester (7) (9.80 g, 42.2 mmol) in 422 mL anhydrous DCM, a DIBALH solution (ρ = 0.701 g/mL, 46.4 mL, 46.4 mmol, 1 M in hexane) was slowly added at -78 • C within 60 min. Stirring was continued at this temperature for 40 min. After complete conversion (1 h, TLC), 21 mL of MeOH and 120 mL of a 10% Na/K tartrate solution were added to the reaction mixture at -78 • C. The cooling bath was removed and the reaction mixture was allowed to warm to room temperature. After the DCM layer became clear, the phases were separated and the aqueous phase was extracted three times with DCM. The combined organic phases were washed with 1 M aq. KHSO 4 solution as well as with sat. aq. NaHCO 3 solution and dried over Na 2 SO 4 . The solvent was removed in vacuo and the crude aldehyde was dissolved in 320 mL dry THF. The Cbz-protected phosphonate (8) (16.7 g, 46.4 mmol) was dissolved in 100 mL anhydrous THF and cooled to -78 • C. Tetramethylguanidine (ρ = 0.918 g/mL, 5.6 mL, 44 mmol) was added. After the solution was stirred for 20 min at this temperature, the aldehyde solution was slowly added. Subsequently the reaction mixture was warmed to room temperature overnight. The solution was diluted with diethyl ether and water. The aqueous phase was extracted three times with diethyl ether and the combined organic phases were dried over Na 2 SO 4 . To determine the enantiomeric excess of amino acid 10, the Cbz protecting group was replaced by an acetate group to allow gas chromatographic measurement. For this purpose, hydroxyleucine (10) (30 mg, 73 µmol) was dissolved in 0.7 mL dry THF and 10 wt-% Pd-C (3 mg, 10% on activated charcoal) was added to the reaction mixture. Stirring was carried out at 1 bar H 2 atmosphere for 26 h at room temperature. The mixture was filtered through Celite and the solvent was removed in vacuo. Subsequently, the residue was dissolved in 1.  (11) To a solution of methyl ester 10 (2.35 g, 5.74 mmol) in 57 mL THF was added a 1 M aq. LiOH solution (6.9 mL, 6.9 mmol) at 0 • C. With stirring, the reaction mixture was slowly warmed to room temperature (19 h). The solvent was removed under reduced pressure. The residue dissolved in water and acidified with 1 M aq. KHSO 4 solution to pH 3. The aqueous phase was extracted three times with DCM. After the combined organic phases were dried over Na 2 SO 4 , the solvent was removed in vacuo again. Without further purification, 2.  (12) Cbz-protected amino acid 11 (5.58 g, 14.1 mmol) was dissolved in 141 mL anhydrous THF. After cooling down to -10 • C, NaH (2.26 g, 56.5 mmol, 60% in mineral oil) was added to the reaction mixture in portions, followed by methyl iodide (ρ = 2.28 g/mL, 7.1 mL, 113 mmol). After 43 h (LCMS) at this temperature, the reaction mixture was diluted with EtOAc and poured onto water. The organic phase was extracted three times with water. The combined aqueous phases were acidified to pH 3 using 1 M aq. KHSO 4 solution and extracted three times with DCM. Subsequently, the combined organic phases were washed with 5% Na 2 SO 3 solution and brine and dried over Na 2 SO 4 . The solvent was removed under reduced pressure to obtain the desired N-methylated amino acid 12 (4. (14) To a solution of propargyl alcohol (1.61 g, 23.0 mmol) in 23 mL anhydrous DCM was added Alloc glycine (13) (4.03 g, 25.3 mmol) and DMAP (141 mg, 1.15 mmol) at room temperature. The reaction mixture was cooled to 0 • C, followed by the addition of DCC (5.23 g, 25.3 mmol) and 8 mL dry DMF. Overnight it was warmed to room temperature (16 h). The reaction mixture was filtered through Celite and the solvent was subsequently removed under reduced pressure. The residue was diluted with EtOAc and the organic phase was washed three times with water and once with brine. After drying over Na 2 SO 4 , the crude product was concentrated under reduced pressure. Flash chromatographic purification (silica gel, PE/EtOAc 8:2) afforded the desired propargyl ester 14 (3. To a solution of hexapeptide 21 (226 mg, 190 µmol) in 1.9 mL dry DCM was added 1,3-dimethylbarbituric acid (90 mg, 0.57 mmol) and Pd(Ph 3 P) 4 (6.6 mg, 5.7 µmol) at room temperature. After 45 min the reaction mixture was diluted with EtOAc and the organic phase was washed three times with sat. aq. NaHCO 3 solution. Subsequently, a back extraction from the aqueous phase was carried out with EtOAc. The combined organic phases were dried over Na 2 SO 4 and concentrated under reduced pressure. The residue was dissolved again in 1.9 mL dry DCM and Alloc-crotylglycine 16 (44.9 mg, 210 µmol) was added. The reaction mixture was cooled to 0 To a solution of protected cyclic heptapeptide 23 (77 mg, 65 µmol) in 6.5 mL dry DCM was added 1,2-ethanedithiol (ρ = 1.12 g/mL, 165 mL, 1.96 mmol) as well as BF 3 ·OEt 2 (ρ = 1.12 g/mL, 166 µL, 1.31 mmol) at 0 • C. After complete conversion (2 h, LCMS) at this temperature, the reaction mixture was quenched with approx. 10 mL sat. aq. NaHCO 3 solution. The aqueous phase was extracted three times with DCM and the combined organic phases were dried over Na 2 SO 4 and concentrated under reduced pressure. The crude product was first purified by flash chromatography (silica gel, DCM/MeOH 95:5 → 9:1). Further purification was carried out by preparative HPLC (  at 0 • C. The ice bath was removed and the reaction mixture was stirred for 1 h at room temperature. After complete conversion (LCMS), the reaction solution was quenched with approx. 1.6 mL sat. aq. Na 2 SO 3 solution. The aqueous phase was extracted three times with EtOAc and the combined organic phases were washed with sat. aq. CuSO 4 solution and brine, dried over Na 2 SO 4 and concentrated. After dissolving the residue in 2 mL MeOH, the reaction mixture was cooled again to 0 • C and K 2 CO 3 (3.6 mg, 26 µmol) was added. Overnight the reaction solution was slowly warmed to room temperature (18 h). The reaction mixture was quenched with 1.5 mL sat. aq. NH 4 Cl solution and the aqueous phase was extracted three times with EtOAc. The combined organic phases were washed with 1 M aq. NH 4 Cl solution and brine and dried over Na 2 SO 4 . The solvent was removed in vacuo and the crude product was purified by reverse phase flash chromatography (C18 silica gel, H 2 O/MeCN 9:1 → MeCN). Further purification was carried out by preparative HPLC (Luna,H 2  at 0 • C. The ice bath was removed and the reaction mixture was stirred for 1 h at room temperature. After complete conversion (LCMS), the reaction solution was quenched with approx. 1.6 mL sat. aq. Na 2 SO 3 solution. The aqueous phase was extracted three times with EtOAc and the combined organic phases were washed with sat. aq. CuSO 4 solution and brine, dried over Na 2 SO 4 and concentrated. After the residue was dissolved in 0.3 mL of t-BuOH and water, NaH 2 PO 4 dihydrate (16 mg, 0.1 mmol) and 2-methyl-2-butene (ρ = 0.66 g/mL, 27 µL, 0.26 mmol) were added to the reaction mixture at room temperature followed by the addition of sodium chlorite (5.8 mg, 51 µmol) at 0 • C. The ice bath was removed and stirring was continued for two hours at room temperature. The reaction mixture was diluted with approx. 2.6 mL EtOAc and 0.6 mL water and the phases were separated. The aqueous phase was extracted three times with EtOAc and the combined organic phases were washed with water and brine, dried over Na 2 SO 4 and concentrated under reduced pressure. After purification by reverse phase flash chromatography (C18 silica gel, H 2 O/MeCN 9:1 → MeCN), the crude product was further purified via prepara-

Conclusions
The ilamycins/rufomycins are highly interesting marine cycloheptapeptides characterized by their incorporation of unusual amino acids. The natural products are produced by Streptomyces sp. and show potent activity against a range of mycobacteria, including multidrug-resistant strains of Mycobacterium tuberculosis. Simplified derivatives 26 and 27 of the ilamycins are synthesized. Key steps in the synthesis of the unusual amino acid building brocks are an asymmetric hydrogenation for the synthesis of the required δ-hydroxyleucine derivative and a chelate enolate Claisen rearrangement to generate the γ,δ-unsaturated amino acid. The ilamycin derivative 26 with a cyclic hemiaminal structure is highly active against M. tuberculosis (MIC: 50 nM) as well as M. smegmatis and M. marinum (MIC: 0.26 µM) and shown no significant cytotoxicity. In contrast, derivative 27, with a glutamic acid at this position was significantly less active with MIC's in the mid µM-range. Detailed investigations of the mode of action of 26 indicate that 26 deregulates ClpC1 activity and strongly enhances ClpC1-WT ATPase activity. Degradation of FITC-casein, which binds to the NTD like ClpC1 targeting cyclic peptides, remained largely unaffected by 26. Notably, a strong inhibition of FITC-casein proteolysis by 26 is observed for ClpC1-F444S, a ClpC1 MD mutant, which is constitutively hexameric and thus has a high basal ATPase activity. Probably, 26 has dual effects, negative and positive ones, on ClpC1-WT function. The positive effect relates to ClpC1-WT ATPase activation and the negative one to substrate binding to the NTD.

Supplementary Materials:
The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/md20100632/s1, Figure S1: Biochemical analysis of 26 impact on ClpC1 activity; Copies of NMR spectra of all new compounds.
Author Contributions: J.G. was performing the synthesis of ilamycin derivatives and was involved in writing the manuscript. A.M. performed the biological studies and was involved in writing the manuscript. U.K. coordinated the project and synthesis and was involved in writing the manuscript. All authors have read and agreed to the published version of the manuscript.