Selective Modification of Ribosomally Synthesized and Post‐Translationally Modified Peptides (RiPPs) through Diels–Alder Cycloadditions on Dehydroalanine Residues

Abstract We report the late‐stage chemical modification of ribosomally synthesized and post‐translationally modified peptides (RIPPs) by Diels–Alder cycloadditions to naturally occurring dehydroalanines. The tail region of the thiopeptide thiostrepton could be modified selectively and efficiently under microwave heating and transition‐metal‐free conditions. The Diels–Alder adducts were isolated and the different site‐ and endo/exo isomers were identified by 1D/2D 1H NMR. Via efficient modification of the thiopeptide nosiheptide and the lanthipeptide nisin Z the generality of the method was established. Minimum inhibitory concentration (MIC) assays of the purified thiostrepton Diels–Alder products against thiostrepton‐susceptible strains displayed high activities comparable to that of native thiostrepton. These Diels–Alder products were also subjected successfully to inverse‐electron‐demand Diels–Alder reactions with a variety of functionalized tetrazines, demonstrating the utility of this method for labeling of RiPPs.

Abstract: We report the late-stage chemical modification of ribosomally synthesizeda nd post-translationally modified peptides (RIPPs)b yD iels-Alder cycloadditions to naturally occurring dehydroalanines. The tail region of the thiopeptide thiostrepton could be modified selectively and efficiently under microwaveh eatinga nd transitionmetal-free conditions. The Diels-Alder adducts were isolated and the different site-and endo/exo isomerswere identified by 1D/2D 1 HNMR. Via efficient modification of the thiopeptide nosiheptide and the lanthipeptide nisin Zt he generality of the methodw as established. Minimum inhibitory concentration (MIC)a ssays of the purified thiostrepton Diels-Alder products against thiostrepton-susceptible strains displayed high activities comparable to that of native thiostrepton.T hese Diels-Alder products were also subjected successfully to inverse-electron-demandD iels-Alder reactions with av ariety of functionalized tetrazines, demonstrating the utility of this method for labeling of RiPPs.
Here, we now report the Diels-Alder reaction with cyclopentadiene as am ild and selective modification reaction for of dehydroalanine residues in antimicrobialR iPPs (Scheme1). Furthermore, the unactivated, strained alkene in the formed norbornenep roduct could be employed in Inverse Electron Demand Diels-Alder (IEDDA, "click") reactions with tetrazines (Scheme 1), ap opular labelingt ool in chemical biology. [31] As as tartingp oint, the Diels-Alderr eactionb etween cyclopentadiene and ap rotected dehydroalanine substrate (1)w as studied (SupportingI nformation, SI-7). In previouss tudies only anhydrous conditions and also high temperatures had been re-ported for this reaction. [32] The Diels-Alder reactioni sk nown to be significantly accelerated in water. [33] Indeed, appreciable conversion was observed in water at room temperature after 48 h, whereas no product waso bservedw hen using dichloromethanea ss olvent(SI-7).
Next, different co-solvents that are tolerated by peptides were tested in ordert oh elp solubilize the cyclopentadiene and thereby increase the conversion.I tw as found that 2,2,2trifluoroethanol (TFE) gave the best results, likely due to its mild Brønsted acidity,w hich can give rise to activation of the dienophile. [34] Using 20 mol %S c(OTf) 3 to activatet he dienophile improved the conversion further, up to 88 %a fter 48 h with 10 equiv.c yclopentadiene.
The endo/exo ratio was % 40:60 in all cases, which is in agreement with previous reports about the secondary orbital interactions between this particular Dha substrate (1)a nd cyclopentadiene. [32] 1,3-cyclohexadiene, 1,3-dimethylbutadiene, and furan were also evaluated as dienes, but did not give any conversion at room temperature (SI-7).
The conditions established with the protectedD ha substrate appeared suitable for modificationo ft he thiopeptide thiostrepton ( Figure 1A), given its high solubility in TFE. During initial screening and subsequent LC-MS analysis, it was found that addition of Sc(OTf) 3 did not give rise to increased conversions compared to reactions performed without the scandium salt.
On the contrary,t he transition metal free conditions gave rise to the cleanest transformations, giving mainly single-and double modified thiostrepton( Figure 1B). After seven days of reactiont ime (whilea dding freshly distilled cyclopentadiene daily) 64 %c onversion to single-and double-modified thiostrepton was obtained as based on peak integrationo ft he startingm aterial and the products in analytical HPLC.
Performing the reactiona t5 0 8Ci namicrowaver eactor greatly improved the conversion to 72 %a fter only 16 ho fr eaction time, compared to 28 %c onversion after 16 ha tr oom temperature and 50 %c onversion when heating the reaction at 50 8Ci na no il bath.Amixture of single-and double-modified products was obtained and the starting material and the products provedt ob es table under the microwave conditions. Even hydrolytic cleavage of the Dha-tail, which is ac ommon side reaction in thiostrepton modification, [23] was not observed.
The reactionw as performed on a2 5mgs cale, after which the three major singlem odified products (2a-c)w ere isolated using preparative HPLC ( Figure 1C). Products 2a-c,o btained as mixtures of diastereomers that could not be separated, were analyzed by NMR. When comparing the 1 HNMR spectra of unmodified thiostreptona nd the products, with particular focus on the region between 5.00 ppm and 7.00 ppm (Figure 1D,o nly showingp roduct 2b for this example, see SI-10-12, 33-37 for all spectra)i tc an be seen that the methylene signals of Dha3 (purple) and Dhb8 (yellow) are conserved in product 2b.F rom the two sets of signals originating from the methylenes in the tail, that is, Dha16 (blue) and Dha17 (green), one set of signals has disappeareda nd the other has shifted upfield, indicating that the reactionh as taken place in the tail region of thiostrepton. Moreover,t he appearance of two doublets of doublets (red) is characteristicf or the formation of the alkene of norbornene. The NMR spectra of 2a and 2c showed similar changes in signals (SI-11, 33-37).
Using 1 H-1 HT OCSY NMR,p roducts 2a and 2b were both identified as Dha16-modified thiostrepton (see SI-11f or ad etailed explanation). By comparing the methylene signals of Dha17 in products 2a and 2b,t hereby taking into account the shielding effect of the newly formed carbon-carbond ouble bond in the norbornene,i tw as established that product 2a is Dha16-endo and product 2b is Dha16-exo (see SI-11). In as imilar manner,u sing 1 HNMR and 1 H-1 HT OCSY NMR techniques, product 2c could be identified as Dha17-modifiedt hiostrepton (SI-12).
To furtherd emonstrate the selectivity for the tail region,a truncated variant of thiostrepton( 3)w as synthesized via selective base-mediated cleavage of Dha17 from the tail of thiostrepton using Et 2 NH, leaving only Dha16 as ar eactive site (Scheme 2, SI-6). [23] When 3 was subjected to the optimizedr eaction conditions, only two major single modified products (4a and 4b,S cheme 2) were obtained. Using analytical HPLC a 41 %t otal conversion was observed (SI-13). Both products were isolated as mixtures of diastereomers and identified (SI-16-17) as endo-( 4a)a nd exo (4b)i somerso fD ha16-modified 3 (SI-13)u sing NMR analysis analogoust ot he identification of products 2a-c.
Collectively,t hese results show that the reaction is highly selective for the tail region of thiostrepton. Also, the LC-MS UV signal areas of products 2a and 2b compared to product 2c ( Figure 1B)i ndicate as ignificant preference for modificationa t Dha16, which can be explained by the fact that this residue is the most electron-poor site due to the neighboring thiazole15 and Dha17, both electron-withdrawingm oieties.
The scope of the reactionw as evaluated by performing the reactiono nd ifferentR iPPs. The Diels-Alder reactiono fc yclopentadiene and the thiopeptide nosiheptide was performed under the optimized conditions and after microwave-assisted heatinga t5 08Cf or 32 haconversion of 75 %t os ingle modified nosiheptide waso bserved (Scheme 3A,S I-18). The commercial nosiheptide starting material contained as mall amounto fn osiheptide that lacks the terminal Dha, having a terminal amide instead. The product of the reactiono ft his im-purity with cyclopentadienew as not observed in the LC-MS analysis,c onfirming that the reaction is selectivef or the terminal Dha over the internal Dhb residue, which is consistentw ith the results obtained using thiostrepton and 3.
The reaction betweenc yclopentadiene and the lanthipeptide nisin Zw as investigated next (Scheme 3B). In this case, the same conditions as for the thiopeptides wereu sed, except for the substitution of ddH 2 Of or 0.1 %A cOH (aq.) due to solubility-and stabilityi ssues of nisin at pH > 5. In addition to the inevitable, but well-documented addition of water to Dha in nisin, [35] a5 2% conversion to single Diels-Alder modified product was observed after 16 ho fm icrowavei rradiation at 50 8C (SI- [19][20]. For nisin Z, which bears one Dhb and two Dha residues, the site selectivity could not be determined due to poor separation of isomerso nL C-MSa nd HPLC. However,g ood stabilitiesu nder microwave irradiation were observed for both nosiheptide and nisin Z, demonstrating the general applicability of our approachf or the modification of Dha-containing RiPPs. Previous studies have shown that modification of the tail region of thiostrepton does not severelyi mpact its activity. [23,26] To confirmt hat this is also true for the norbornene modifications, thiostrepton and purified derivatives 2a-c, 3,a nd 4a,b were tested against S. aureus (ATCC29213) and E. faecalis (ATCC29212) strains in aM IC-assay (SI-21). The results (Table 1) Scheme2.Synthesis and Diels-Alder reaction of truncated thiostrepton (3).
Scheme3.Diels-Alder reactionsofc yclopentadiene with A) the thiopeptide nosiheptide and B) the lanthipeptide nisin Z. For nisin Zonly one of the possible products is shown. show that all derivatives have excellent antimicrobial activity, with aM IC value that is within one order of magnitude compared to native thiostrepton for both strains.M oreover,v ariations in activity towards both strainsa nd between the different site-and endo/exo isomersr emained limited to af actor of 4. The activity of 3 also very closely resembles that of thiostrepton, showing that even removingp art of the tail regionh as little effecto nits activity.
The selectivei ncorporation of the norbornene functionality in the tail of thiostreptonw hile leaving the inherenta ctivity intact enables furtherd erivatization through IEDDA click reactions with tetrazines. [31] Purified 2a was treated with di-2-pyridyl tetrazine (5)i nH 2 O/ACN 1:1a tr oom temperature (Figure 2A)a nd after overnight reaction full conversion to singly labeled dihydropyridazine (m/z = 1938) and pyridazine (m/z = 1936) products was observed by MALDI-TOF MS of the crude reactionm ixture ( Figure 2B). As ac ontrol,u nmodified thiostrepton was subjected to the same conditions, after which only starting material (m/z = 1664, Figure 2B inset) was observed, illustrating the high chemoselectivity for the norbornene moiety over the other unsaturated motifs in thiostrepton.
AB ODIPY-labeled tetrazine (12)w ith fluorescencet urn-on properties was synthesized using ap rocedure by Carlson et al. with minor modifications (SI-3). [37] The fluorescence of 12 is quenched almost completely by the tetrazine motif. However, this effect is lifted upon reaction of the tetrazine in the IEDDA click reaction( Figure 3A). [37] Upon addition of 2a to as olution of 12,f luorescencem easurements indeed showedarapid increase in fluorescencec ompared to an identical solution of 12 where only DMSO was added as ac ontrol (SI-23). Thisf luorescence turn-on effect could even be visualized by shining UV light (365 nm) on the undiluted samples ( Figure 3B), which shows the potential for using this two-step labeling method in the detection of new Dha-containing peptides.
We have established the Diels-Alder reaction as ap owerful tool for efficient and selectivel ate-stagec hemical editing of peptidea ntibiotics. This approach, which only requires cyclo-pentadiene as ar eagent and microwave-assisted heating, allows for straightforward and transition-metal-free installation of the norbornene functionality on these complex natural products by reactingw ith the naturally occurring Dha residues under mild conditions. Especially attractive is the possibility of employing the norbornene product in InverseE lectron Demand Diels-Alder reactions with tetrazines, which gives   access to av ariety of new semisynthetic derivatives. Additionally,t he norbornene moiety couldp otentially be used in other labeling reactions. [38,39] These results demonstratet he potential of this methodology for the tailoringo fR iPPs.