Selective Aza‐Michael Addition to Dehydrated Amino Acids in Natural Antimicrobial Peptides

We report the efficient and site selective modification of non‐canonical dehydroamino acids in ribosomally synthesized and post‐transationally modified peptides (RiPPs) by β‐amination. The singly modified thiopeptide Thiostrepton showed an up to 35‐fold increase in water solubility, and minimum inhibitory concentration (MIC) assays showed that antimicrobial activity remained good, albeit lower than the unmodified peptide. Also the lanthipeptide nisin could be modified using this method.


Introduction
The increasing number of bacteria resistant against antibiotics is leading to the urgent need to discover new antibiotics to fight drug-resistant pathogens. [1,2]Ribosomally synthesized and post-translationally modified antimicrobial peptides (RiPPs) are a promising new class of antibiotics, as they often show high activity and, up to date, no resistance has been reported. [3,4]nfortunately, clinical application of RiPPs is often hampered by poor pharmacokinetics, low water solubility, as well as poor oral bioavailability.For this reason, new semi-synthetic approaches are desirable in order to fine-tune RiPPs properties. [5]As a result of biosynthetic post-translational modifications, RiPPs display unique structural features.These include dehydroalanine (Dha) and dehydrobutyrine (Dhb), which are versatile, non-canonical, α,β-unsaturated amino acids resulting from dehydration of serine and threonine residues. [6,7,8]They are carbon electrophiles because of their unsaturated side chain, which permit siteselective chemical modification.In recent years several chemoand site selective modifications of dehydroamino acids in RiPPs have been established, including: conjugate phospa-Michael additions, [9] β-silylation, [10] radical carbonÀ carbon bond formation, [11,12] cross-coupling reactions, [13,14] amidations, [15,16] cyclopropanations [17] and cycloadditions. [18,19]Recently, we reported the Cu(II) catalysed β-borylation of RiPPs (Scheme 1A), [20] with a particular focus on the thiopeptide Thiostrepton.The double borylated Thiostrepton, modified on the bis-dehydroala-nine tail, was isolated and its water solubility and bioactivity were tested.Despite a significant increase in water solubility (up to 84-fold, in basic environment), the doubly borylated Thiostrepton proved to be less bio-active compared to its unmodified counterpart.Since a previous report suggested that the bis-dehydroalanine tail seems not to play a crucial role in the bio-activity of Thiostrepton, [21] we hypothesized that the negative charge arising from the reaction of the Lewis acid boronic acids with water to form the corresponding boronates could adversely interact with the negatively charged bacterial membrane. [22]A way to overcome this problem and simultaneously obtain an orthogonal water-solubility in acidic environments could be adding a basic moiety to the peptides.A good reaction to achieve this would be the aza-Michael addition, which has been reported for Dha residues introduced chemically into proteins, [23][24] but never for native Dha and Dhb residues in natural products such as RiPPs.Here were report the exploitation of the electrophilicity of Dha and Dhb to perform a selective aza-Michael addition of morpholine (Scheme 1B), resulting in the introduction of a tertiary amine in the peptide side chains.

Results and Discussion
First, the β-amination of Dha and Dhb acceptors in the thiopeptide Thiostrepton was investigated (Figure 2A).Generally, peptide modifications are performed in aqueous environment, but due to the poor water solubility of Thiostrepton we chose to use an organic solvent, employing 1,4-dioxane for the reaction.A variety of primary and secondary amines were tested (Figure S2, ESI † for a detailed explanation), but most either gave no reaction because the amine was not nucleophilic enough, or were too basic and, hence, caused cleavage of the terminal amino acids of the tail, which is base-labile. [19,25]owever, morpholine shows nucleophilic character typical of secondary amines, although the presence of the ether oxygen makes it less basic than structurally similar amines (pKa morpholine is 8.3), but nucleophilic enough to perform the Michael type addition.Indeed, using morpholine gave rise to the desired modification and produced only a small amount of truncated Thiostrepton (derived from the cleavage of the terminal amino acids of the tail, Figure S3C, ESI † for a detailed explanation).The first experiments showed that employing 50 equivalents of morpholine, the highest conversion was obtained after 24 h (Figure S3, ESI †).Testing different equivalents of morpholine (summarized in Figure 1) showed that the best selectivity toward the singly modified peptide was obtained using 20 equivalents of morpholine.
To demonstrate both the chemo-and site-selectivity of this approach for the different dehydrated residues within Thiostrepton, the singly modified product 2 was isolated using preparative reversed-phase HPLC (Figure S5 and S6, ESI †) and subsequently characterized via HRMS and 1D and 2D NMR spectroscopy.From inspection of the region between 5.00 ppm and 7.00 ppm of the 1 H NMR spectra of unmodified Thiostrepton and product 2, it is clear that the methylene signals of Dha3 and Dhb8 are preserved in the modified peptide.From the two sets of signals deriving from the methylenes in the tail, one has disappeared and the other has shifted upfield, suggesting that the reaction takes place in the tail, which is the solvent-exposed region of Thiostrepton (Figure 2B, S7).Analyzing 1 H-1 H TOCSY NMR, product 2 was identified as Dha16-modified Thiostrepton (Figures S8, ESI † for a detailed explanation), hence the functionalization of the subterminal Dha16 is favored.This selectivity is in agreement with that observed previously in Thiostrepton modifications using Diels-Alder, photocatalytic radical and rhodium catalyzed conjugate addition of boronic acid reactions. [11,13,19]This is attributed to the alkene in Dha 16 being the most electron deficient due to the adjacent electronwithdrawing thiazole15 and Dha17 moieties.Shorter retention times of 2 in the reversed phase HPLC chromatogram compared to unmodified Thiostrepton qualitatively indicated that aqueous solubility was improved after incorporation of morpholine moiety.To obtain quantitative information, both unmodified Thiostrepton and the singly modified product 2 were submitted to a water-solubility assay (Table 1, ESI † for a detailed explanation).The results obtained demonstrate that introduction of a single morpholine group improves the water solubility at neutral pH of Thiostrepton 25-fold, which was comparable to the improvement reported before for the double borylated Thiostrepton. [20]Moreover, the basic moiety introduced in this work permits to increase the solubility in acidic aqueous environment.pH is a feature that can significantly affect both drug absorption and bioavailability as it may have serious influence on drug dissolution and solubility, drug release as well as drug stability. [26]Therefore, the solubility of 2 was also determined at pH 4.0 and 2.0, which is prototypical of the vaginal environment and the stomach, respectively. [27]Indeed, a significantly increased aqueous solubility, up to 35-fold, was found at these pH values (Table 1).
The antimicrobial activity of Thiostrepton and singly modified Thiostrepton 2 against two Gram-positive strains (S.Aureus and E. Faecalis) was evaluated in a MIC assay and compared to the results already obtained for the double borylated Thiostrepton (Table 2).
The results depict that although 2 has a decreased activity compared to the unmodified Thiostrepton, relatively high bioactivity is preserved, which is better than those previously found for the double borylated peptide.Work from our and other groups suggests that, although the tail of the peptide appears to have no direct impact on its bioactivity, [21] adding a group that will generate a charge will decrease the activity of Thiostrepton, whereas uncharged residues seem to have less impact. [13,16,19]On the other hand, this has proven to be the most effective method to date to significantly increase watersolubility. [20]To assess the generality of the method, also another RiPP was investigated as a substrate for the aza-Michael addition: Nisin Z, a member of the lanthipeptide family. [28]n contrast to Thiostrepton, Nisin Z has a high aqueous solubility, so the amination was conducted in pure water (Figure 3A).The reaction mixture was analysed by LC-MS (Figure S4, ESI †).Using 10, 50 or 100 equivalents of morpholine, both after 24 and 48 hours, a considerable amount of unreacted Nisin Z was detected, as well as singly and doubly modified Nisin Z products (Figure 3B).Employing instead a thousand equivalents of the nucleophile, after 24 hours complete conversion was obtained, with a strong selectivity toward the doubly modified product.In contrast to Thiostrepton, where a good regio-selectivity of the modification was found, the dehydroamino acids in Nisin Z are all reactive and accessible, resulting in a mixture of regioisomers impossible to be resolved employing UPLC-MS.Consequently, the isolation of the different modified peptides was not achievable.

Conclusions
In conclusion, we developed a new selective modification of dehydroamino acids in RiPPs based on the aza-Michael addition of morpholine.The method is practical and experimentally straightforward: the reagent is commercially available and the reaction occurs under mild conditions and ambient conditions.Singly modified Thiostrepton 2 was isolated and NMR analysis showed that modification occurred selectively on Dha16.The aza-Michael product 2 was significantly better water-soluble (up to 35-fold) than the unmodified peptide and Minimum Inhibitory Concentration (MIC) assays showed that antimicrobial activity was maintained.These results demonstrate the utility of aza Michael addition to dehydroamino acids as a means to alter, and potentially improve the properties of RiPPs.

Table 2 .
MIC assay results in μg/mL of Thiostrepton and the modified peptides.