Derivatisable Cyanobactin Analogues: A Semisynthetic Approach

Abstract Many natural cyclic peptides have potent and potentially useful biological activities. Their use as therapeutic starting points is often limited by the quantities available, the lack of known biological targets and the practical limits on diversification to fine‐tune their properties. We report the use of enzymes from the cyanobactin family to heterocyclise and macrocyclise chemically synthesised substrates so as to allow larger‐scale syntheses and better control over derivatisation. We have made cyclic peptides containing orthogonal reactive groups, azide or dehydroalanine, that allow chemical diversification, including the use of fluorescent labels that can help in target identification. We show that the enzymes are compatible and efficient with such unnatural substrates. The combination of chemical synthesis and enzymatic transformation could help renew interest in investigating natural cyclic peptides with biological activity, as well as their unnatural analogues, as therapeutics.

Ribosomally synthesised and post-translationally modified peptides (RiPPs) make up aw ideg roup of naturalc ompounds with variousb iological activities. [1] The biosynthetic pathway of RiPPs involves the action of many tailoringe nzymes on as pecific precursor peptide to yield the highly modified final product. Cyanobactins are al arge family of RiPPs that includes patellamides, ulithiacyclamides, and trunkamides (Scheme 1) and that are produced by ad iverses electiono fc yanobacteria. [2] Amongst the best-studied cyanobactinsa re the patellamides: cyclic octapeptides produced by Prochloron didemni,t he cyanobacterial symbiont of Lissoclinum patella.T he biosynthetic pathway of patellamidesc onsists of as even-gene cluster (patA-G; Figure 1) that encodes the precursor peptide(PatE) as well as the altering enzymes. Common modifications of patellamides include heterocyclisation, oxidation, epimerisation and macrocyclisation. [3] Cyanobactins, including patellamides, have diverse and valuable biological activities. [2b] Patellamides B, Ca nd Dh ave shown ar eversal of the multidrug resistances een for vinblastine, colchicine and adriamycin treatment in the CEM/VLB 100 humanl eukemic cell line, [4] and patellamide Di scytotoxic towards fibroblast (MRC5V1) and bladder calcinoma( T24) tumour cell lines. [5] However,t he development of these compounds requiresl arge-scale synthesis in order to identify their exact biological targets and ascertain structure-activity relationships to fine-tune their properties. The chemical synthesis of such compounds is challenging, even more so (> 16 syn-Many natural cyclic peptides have potent and potentially useful biological activities. Their use as therapeutics tarting points is often limited by the quantities available, the lack of known biological targets and the practical limits on diversification to fine-tune their properties. We report the use of enzymes from the cyanobactin family to heterocyclise and macrocyclise chemically synthesised substrates so as to allow larger-scale syntheses and better control over derivatisation. We have made cyclic peptides containing orthogonal reactive groups,a zide or dehydroalanine, that allow chemical diversification, includingt he use of fluorescent labels that can help in target identification. We show that the enzymes are compatible and efficient with such unnatural substrates. The combination of chemical synthesis and enzymatic transformation could help renew interest in investigating natural cyclic peptides with biological activity,a sw ell as their unnatural analogues,a s therapeutics. thetic steps) for those containing thiazoline and/or oxazoline moieties. [6] Biochemical studies have shown that PatG mac (macrocyclisation domain of PatG) tolerates significant diversity in the amino acids in the core peptide, [8] as long as the core peptide ends with af ive-membered heterocyclicr ing (eithert he naturally occurring thiazoline/oxazoline motif or cis-proline) and is flankedb yaC-terminal macrocyclisation signature AYD. [9] More recently, an engineered heterocyclase that can completely process peptidesubstrates lacking the leader peptidehas been reported. [10] Ta king advantage of this efficient biosynthetic machinery,w es how that pairing chemical and enzymatic syntheses is efficient for the generationo fu nnatural patellamide-like cyclic peptides. We reporth erein the macrocyclisation of synthetic peptidest hat contain unnatural amino acids by using PatG mac as well as the selective derivatisation of the subsequent cyclic peptides.W ea lso report the synthesis of ap atellamide-like cyclic peptidec ontaining both ah eterocycle and an unnatural amino acid in ao ne-pot procedure.
The introduction of bio-orthogonal or specific reacting groups on the side chains of linear/cyclic peptider esidues would be highly desirable, as they would allow regiospecific and facile derivatisation.T his approachh as been previously used to study binding and/or activity, [11] to link af luorescent probe in order to investigate biological processes or pharmacokinetic behaviour, [12] and to connecto ther building blocks for activitye nhancement or drug delivery, [13] among others. Althougho rthogonal reacting groups have been previouslyi ntroduced in vivo on precursor peptides of RiPPs through stopcodon suppression (SCS) and supplementation-basedi ncorporation (SPI), [14] these strategies lack the control for better selectivity,s pecificity and flexibility that chemical synthesis permits. Three patellamide-like cyclic peptides were made with either an azidoalanine A(N 3 )o radehydroalanine( Dha) reactive group. The azide moiety is aw ell known bio-orthogonal group that reacts with alkynes both ex and in vivo. [15] Although not fully bio-orthogonal, Dha is extensivelyu sed for bioconjugation purposes. [16] The two groups, A(N 3 )a nd Dha, were introduced at different positions (4 and 2, respectively)i nt heir respectivec ore peptides to explore whether their incorporation presenteda ny challenges forP atG mac processing. For simplicity, we made the first two compounds from peptides that lacked any cysteine residues, and therefore heterocycles. Havinge stablished as uitable approach, we advanced to adding the azide to ac ysteine-containing peptide. We were able to enzymatically heterocyclise the cysteinet oathiazoline within the sequence and then macrocyclise the resulting product in a one-pot process, to make agenuine patellamide analogue.
The measured maximum absorbance (l = 649 nm) ande mission (l = 671 nm) properties of conjugate 8 werei ng ood agreement with those of the parent Cy5 molecule ( Figure S33). As these types of compounds could potentially be used for target identification by fluorescence microscopy,c onjugate 8 (dark blue colour) was tested to ensuret here was no unexpected behaviour (such as quenching or precipitation) in cells. When incubated with permeabilised HeLa cells, ad iffuse staining pattern of 8 (red colour) throughout the cytoplasm and nucleusw as visualised by fluorescentm icroscopy (Figure 2); this showedt hat the molecule behaves as expected in biological buffers. Figure 1. A) The pat gene cluster codes for PatA, which cleaves N-terminal to core peptide, PatB and PatC (unknown function), PatD, which heterocyclases cysteine (serine, threonine)r esidues in the core peptide, PatE (precursor peptide), PatF (inactive prenylase [7] )a nd PatG,w hich cleaves and macrocyclises to the core peptide and oxidises thiazolines.B)PatE precursorp eptide withi ts key regions highlighted.
Scheme2.Macrocyclisation of the unnatural amino acid-containing precursor peptides 1 and 3 with PatGmac affordsc yclic peptides 2 (45 %y ield) and 4 (63 %y ield). a) PatG mac ,3 78C, pH 8.1 ChemBioChem 2015ChemBioChem , 16,2646ChemBioChem -2650 www.chembiochem.org Cyclic peptide 4 underwent at hio-Michael addition with the cysteine-containingg lutathione peptide 9 (Scheme 4) with an excess of triethylamine in water and methanol. [18] The corresponding compound 10 was obtained in 43 %y ield. Following the successful addition of glutathione, we investigated whether the reactionc ould be carried out directly after the macro-cyclisation reaction as ao ne-pot process. Once peptide 3 has been fully macrocyclised, 100 equivalents of mercaptoethanol were directly added into the reaction mixture,a nd this was left at 37 8Co vernight.T he reactionw as judged to be complete by MS, and the final compound 11 was obtained in 60 %y ield. The final product purifies as two separablepeaks, which we attributet od ifferent diastereoisomers ( Figure S34).
As PatG mac processes substrates with unnatural amino acids at similar rates to other sequences, [9] we next tested the feasibility of introducing heterocycles into such unnatural substrates. The proline residue in peptide 1 was replaced with a cysteine( peptide 12)t hat could be enzymatically heterocyclised.L ike PatG mac ,t he heterocyclase enzymes of the cyanobactin pathways (known as the De nzymes) [3a, 19] have been shown to be tolerant of aw ide range of sequences within the core peptide.
We incubated peptide 12 overnight with the engineered heterocyclase LynDfusion (from the aestuaramide pathway (Lyngbya sp.)) in the presence of ATPa nd MgCl 2 .T he fully heterocyclysed product 13 was detected by MS but not isolated (Scheme 5; Figures S35-S36). Subsequent addition of PatG mac Scheme3.Copper-free azide-alkyne cycloaddition of 2 with strained cyclooctynes affords conjugates 6 (95 %y ield) and 8 (30 %y ield). a) CH 3 CN, H 2 O. to the reaction mixture afforded the patellamide-like analogue 14 cyclo(-ITAA(N 3 )ITA het C-) in 58 %yield. [20] Milligram quantities of cyanobactin derivatives with fluorescent componentsw ill greatlyf acilitatet he target identification of many of these naturalb iologically active products. [2b] Ta rget identification will not only provideabasis forr edesign of the natural product but could also disclosen ew opportunities for therapy.T he expense and complexity of these labels means in practical terms that they are better introducedl ate in the synthesis. In the case of macrocyclic peptides, this meansi deally after the macrocycle is made. Introducing chemical diversity to probe or fine tune the pharmacokinetic and biological proper-ties of natural products is likewise most desirable when performed asaf inal step on ac ommon scaffold.
We have demonstrated that both the heterocyclases and macrocyclases from the patellamide (and ar elated) pathway can be used in vitro with entirely synthetic substrates that contain such chemically reactive unnatural amino acids. Moreover, we have shown that the resulting macrocyclesc an be derivatised with high efficiency.T he ability to combine the diversity of chemical synthesis with the exquisite catalysis of enzymes is well knownand recognised to be powerful in developing natural products into therapeutics. [21] This approachc an be extended to peptidic macrocycles and might likewise enable their further development. (094476), the MALDI TOF-TOF Analyser( 079272AIA), 700 NMR and supported G.M. (097831)). J.H.N. is aR oyal Society Wolfson Merit Award Holder and 1000 talent scholar at Sichuan University.