Synthesis of Hybrid Cyclopeptides through Enzymatic Macrocyclization

Abstract Natural products comprise a diverse array of molecules, many of which are biologically active. Most natural products are derived from combinations of polyketides, peptides, sugars, and fatty‐acid building blocks. Peptidic macrocycles have attracted attention as potential therapeutics possessing cell permeability, stability, and easy‐to‐control variability. Here, we show that enzymes from the patellamide biosynthetic pathway can be harnessed to make macrocycles that are hybrids of amino acids and a variety of manmade chemical building blocks, including aryl rings, polyethers, and alkyl chains. We have made macrocycles with only three amino acids, one of which can be converted to a thiazoline or a thiazole ring. We report the synthesis of 18 peptide hybrid macrocycles, nine of which have been isolated and fully characterized.

Natural products, in particulars econdary metabolites, derived from variousn aturalr esources such as bacteria, fungi, marine sources, and plants, have been used as mediciness ince ancient times. Today they have utility directly as drugs, for example paclitaxel, or as actives caffolds,f or example the b-lactam ring. [1] The advent of high-throughputc ombinatorial chemistry has coincided with ad ecline in attention towards natural products by pharmaceutical companies. [2] Recent progress in phenotypic screening and metabolomic technologies has rekindled interest in natural products. [1a, 2-3] In addition to vast structural and chemicald iversity,n atural productsa re often stereochemically complex; this feature has allowed them to evolve both specificity and potency. [4] Macrocycles, in particular,w hether peptidic or polyketides, are especially appealing, owing to their inherent chemical stability and structural rigidity, [5] the later proper-ty reducing the entropic penalty for binding, thus raising affinity.
Several naturalm acrocyclesp ossess useful biological and medicinal activities. [6] Hybrid peptidem acrocycless how increasedc hemical and structural diversity,f or example, largazole (PKS/NRPS hybrid), zizyphines (cyclopeptide alkaloids), and maytansin (macrolide) [6c, 7] (Figure 1). Ad rawback of peptidic macrocycles is their polarity,b oth in the side chain and the backbone, where it is an inescapable consequence of the peptide bond.H igh polarityh inders transport across lipid bilayers; [8] strategies such as methylation, prenylation, andh eterocyclization aim to overcome these problems. [9] The synthesis of macrocyclic compounds where the backbonei sah ybrid of both peptidic and non-peptidic scaffolds is desirable, as it permits diversification and exquisite control of the physicochemical properties. [10] PatGmac, the macrocyclizationdomain of PatG from the biosynthetic pathway of patellamides, [12] recognizes octapeptides containing at hiazoline ring at the C-terminal adopting a cis conformation followed by the recognition sequence AYD(GE). [12b] PatGmac is as av ersatile tool capable of tolerating ar ange of different precursor peptides; [11,13] ap rolinec an replace the thiazoline ring, [11, 13a] the amino acids of the core peptide are variable almost at will, [11,13] PatGmachas been reported to make macrocycles of up to 22 amino acids, [13b] and can tolerate non-naturala mino acids andt riazole amide bond mimics. [11a, 13a] In an elegant paper by Schmidta nd co-workers, [11b] the chemistry of the N-terminus of the core peptidewas exten- Figure 1. Structures of hybrid molecules. A) Naturalhybrid macrocyclic compounds. [6c, 7] B) Hybrid cyclopeptides previously synthesized using PatGmac. [ KGaA. This is an openaccessarticleunder the termsoft he Creative Commons AttributionL icense, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. sively evaluated to study the cyclization patternso fl ysine and its analogues.D epending on the length of the sequence and the amino acid side chain, both backbone and side-chain cyclization were observeda long with al inear product. The synthetic utility of PatGmacd erives precisely from its promiscuity towards the core sequence. The cyclic dehydratases,P atD and LynD, which make thiazoline and oxazoline rings from cysteine and serine (threonine), respectively,h ave also been shown to tolerate the presence of non-natural amino acids, [13a] but it is not knownh ow they toleratenon-amino acidg roups. Here, we have used enzymes from cyanobactin biosynthesis to generate eighteen hybrid peptidic/non-peptidicm acrocyclest hat explore aw ide range of chemical variation. Nine of these macrocycles (1-6,7a-c,F igure 2) were made at sufficient scale to allow full characterization.
Nineteena nalogues (8-26)w ere synthesized based on ac ommonp recursorp eptide VGAGIGFP,i nw hich one or more amino acids were substituted with non-natural, non-aminoacid scaffolds. All but one of the substrates finished with aP ro in the core peptide( one had aC ys) and all possessed aC -terminal AYD, the minimal recognition sequence for PatGmac, or AYD-Doc (8-amino-3,6-dioxaoctanoic acid;T able 1). Peptides 8-10 contain ao ne-residue substitution involving small non-natural amino acids b-Ala, GABA, and Doc (8-amino-3,6-dioxaoctanoic acid), respectively.P eptide 11 has an N-Me amino acid. Peptides 12-18 contain either an isomer of amino benzoic acid (Abz) at different positions or ar ibose-derived sugar amino acid (Rib). Finally,p eptides 19-26 represent the hybrid peptides, where ap olyketide chain 7-aminoheptanoic acid (7Ahp), 8-aminooctanoic acid (8Aoc), or ap olyether amino acid (PEG) 4 is introduced at different positionso ft he peptide. Peptide 26 has aC ys, which requiresaheterocyclization reaction prior to macrocylization. The peptides werea ll synthesized throughs olid-phase peptide synthesis by using the Fmoc strategy and commercially available reagents (see the Supporting Information). The fully protected Fmoc-sugar amino acid 27 was synthesized in as imilar approach to the one reported in the literature [14] for the synthesis of the Boc-protected analogue startingf rom d-ribose. Minor changes,s uch as the use of benzyle ster protection in the synthesis, were required to produce the desired compound 27 in good yields (see Scheme1 and the Supporting Information). The azide of compound 28 wass electively reduced under the Staudinger reaction conditions to afford the corresponding amine, which was immediately Fmoc protected to yield 29.B enzyl deprotection [15] gave 27 in 62 %y ield. Compound 27 was used for peptide synthesis, and deprotection of the alcohols was accomplished during the final acidic cleavage of the peptide.
Substrates 12-26 were purified before reactionw ith the enzyme(s), and substrates 8-11 were used without any further purification after cleavage from the resin.
[ c] The corresponding isolated macrocycle number. The macrocylization reactions were set up on small scale (< 1mLr eactions) with PatGmaci nb icine buffer at pH 8.1 and 37 8C. The reactions were followedb yM ALDI-MS to monitor progress. MS-MS fragmentation data established the structure of the different productswith the presence of the PV dipeptide fragment being indicative of macrocycle formation.T able 1 summarizes the results of the reactions;i nb rief, only peptide 21 gave no reaction, whereas 20 and 23 yieldedalinear cleaved product. The reactionr ate for these transformations was still slow,w ith reactiont imes ranging between 2-4 weeks. Nine representative cyclic hybrid peptides (1-6, 7a-c; Figure 2), products of peptides 11, 14,16,18,24,25,a nd 26, were purified from al arge-scale reaction and fully characterized by using NMR spectroscopy.O ne of the products of peptide 26 (7a)w as subsequently oxidized.
Substrates with b-Ala and GABA (8, 9)p rocessed essentially as native peptides, perhaps unsurprisingly because these are very similar to amino acids. More interestingly,alonger chain in that position, namely Doc (10), alsog ave only the macrocycle. The inclusion of a N-methylated amino acid in peptide 11 proceeded to afford the corresponding macrocycle 1.T he incorporation of ar igid aromatic scaffold, 2-,3 -, or 4-aminobenzoic acid at either position 4o r5of the core peptide (12-17), proceeded normally.R emarkably,t he presence of as ugar amino acid( Rib, 18), known to cause conformationalr estraints in peptidic analogues, [16] afforded exclusivelyt he macrocyclic product 4.
Schmidta nd co-workers have previously shown the tolerance of PatGmacf or polyketide-like scaffolds such as alkyl chains as part of the core peptide. [11b] Peptide 19,h aving a Nterminal 8Aoc unit, gave only linear peptide,w hereas 20, which hasaN-terminal Valp recedingt he alkyl chain of 7Ahp, yielded exclusively the cyclic product. Comparing substrates 19 and 20 indicatedt hat an amino acid at the N-terminus was important with our sequence for efficient macrocyclization (Table 1). Schmidt andc o-workers, [11b] showedb oth cyclic and linear products were obtained with an N-terminal amino-alkyl chain of ad ifferent peptidic sequence (Figure 1). Peptide 21 with the 7Ahp chain immediatelyb efore the required Pro in the core peptidef ailed to react, but when aP he was introduced in between 8Aoc and Pro (22), the macrocycle was obtained. It seems that an additional amino acid is required N-adjacent to the terminal Pro or thiazoline of the core peptide for it to be processed by the enzyme (see Table 1a nd the Supporting Information).
We observed al ength requirement for the macrocyclization reaction. Compound 23,w hichh as an 8Aoc unit between a Nterminal Vala nd the Phe-Pro at the C-terminus,g ave only al inear product, whereas 24 with two molecules of 8Aoc, yielded only macrocycle 5.S ubstrate 25 was designedw ith ap egylated chain (PEG) 4 and yielded the corresponding macrocycle 6.F ull conversion of substrates 25 and 26 was difficult to achieve, even after long incubation times (4 weeks, adding more enzyme), reaching only to about7 0% conversion to macrocycle (the remainder was unreacted starting material). In 26,t he Pro was replaced with aC ys, which was converted to thiazoline with the engineered LynD enzyme [17] (establishing this enzymet olerated PEG) and subsequently macrocyclized to 7 (Scheme 2).
Compounds 1 and 2 were present as as ingle conformer, whereas compounds 3-6 were all present as am ixture of two conformers in approximately 2:1( 3, 4)o r3:2( 5, 6)r atios, as confirmed by exchange spectroscopy (EXSY) experiments (see the Supporting Information). Compound 7,containing the thiazoline ring, shows two separable peaks in the HPLC spectrum, in an 8:2( 7a/7b)r atio. Both compounds have identicalH RMS and MS-MS fragmentation data. The NMR spectra of these two compounds are, nonetheless, different,b ut confirm the same peptidic sequence [cyclic V(PEG) 4 FThz];h owever,a nE XSYe xperiment showed no exchange. We postulate that 7a and 7b are epimers. As thiazoline with the natural( R) l-configuration is as ubstrate for the oxidasee nzyme, [12d] 7a and 7b werei ncubated in the presence of this enzymeo vernight. 7a afforded compound 7c,w hich was isolated, purified,a nd characterized by NMRa st he thiazole,c onfirming the oxidasee nzyme also tolerates non-peptide groups( Scheme 2). 7 b was, however, not enzymatically oxidized. We suggest the thiazoline stereocentero f7a is (R) l,w hereas it has inverted to a( S) d-configuration in 7b.T his has precedent;t he thiazoline moietyo fp iperidine thiopeptides is known to be spontaneously epimerized from the (R) l-to the (S) d-configuration. [18] Natural peptideh ybrid macrocycles have interesting and usefulb iological properties, [6c] but are, of course,l imited to natural buildingb locks. The routine synthesis of macrocycles composed of peptide and non-peptideb uildingb locks has wide potentiali nm edicinal chemistry,a llowing accesst on ovel scaffolds and the fine-tuning of properties. The patellamide biosynthetic pathway has enzymes that catalyze an umber of valuablec hemical reactions including heterocylization, oxidation, and macrocyclization. The enzymesi nthe pathway largely separater ecognition from catalysis, and are thusa ssumed to have ah ighd egree of promiscuity. [12c] We have used these enzymes to synthesize diverse macrocyclesc ontainingm ethylat-Scheme2.Generalreaction scheme of the enzymatic transformations.