Peptide macrocycles with embedded heterocycles

Abstract

Substituted 2-aminocarbonyl co-substrates are used to provide quinoline-peptide hybrids, or other heterocycle-peptide hybrids, possessing stable and / or conformationally mobile biaryl atropisomeric axes, in methods for the synthesis of peptide macrocycles with embedded heterocycles.

Description

Peptide Macrocycles with Embedded Heterocycles

[0001] Government Support Clause

[0002] This invention was made with government support under grant number 2002182 awarded by the National Science Foundation. The government has certain rights in the invention.

[0003] Introduction

[0004] From the first clinical application of insulin in 19211,2to the recent triumph of GLP-1 agonists34, peptides have proven a valuable therapeutic modality. More recently, advances in genetic code reprogramming, biological display, and computational design have galvanized development of the next generation of peptide-derived therapeutics5. The first wave of these compounds represents some of the most exciting therapeutic candidates of the past decade because of their antibody-like affinity for traditionally “undruggable” targets while simultaneously showing promise for oral or intracellular delivery. Several therapeutic candidates derived from de novo peptide leads have progressed into clinical trials or onto the market6-13. In each of these cases, the initial lead identified by mRNA / cDNA or phage display required extensive modification to improve its physicochemical properties10. Modifications introduced during lead optimization often include the installation of one or more macrocyclic rings and other backbone alterations that enhance protease resistance, target affinity, bioavailability, and / or cell permeability14 15. Chemical strategies to install these modifications earlier in the drug discovery process-ideally, prior to biological display-would offer distinct advantages.

[0005] Many strategies exist to install macrocyclic rings within peptides before or after biological display. Common methods include disulfide formation between Cys side chains, thioether formation between a Cys side chain and a terminal chloroacetamide or benzyl halide, copper-catalyzed azide-alkyne cycloaddition, amide bond-forming reactions16-18, and a variety of metal-catalyzed macrocyclizations19-21. Although widely employed, each of these methods suffers from at least one liability with respect to downstream applications and none provide added value. Disulfide bonds are prone to reduction, while thioethers are flexible and, like click reaction products, prone to oxidation. Newer strategies employ nitrile-aminothiol condensations to generate thiazolines” or enzymatic macrocyclizations with tyrosinase” , transglutaminases , or RiPP enzymes . Alternative macrocyclization strategies that generate stable and drug-like linkers are widely recognized as an unmet need9,31-34. Chemistry that exploits the macrocyclization event to establish a known pharmacophore within the peptide backbone prior to biological display is a promising strategy towards this end35.

[0006] Quinolines are an established class of aromatic pharmacophores replete in natural products, FDA-approved drugs, and high-affinity ligands for both protein and RNA. Quinoline- containing small molecules that engage proteins include FDA-approved therapeutics such as chloroquine, moxifloxacin, topotecan, lenvatinib, and saquinavir, which is itself a peptidomimetic with an TV-terminal quinoline36’37. Quinolines are especially privileged withrespect to RNA38^11: Quinoline -peptide conjugates function as helix-threading intercalators42'43, and substituted quinolines are found within small molecules that target pre-mRNA to modulate splicing44. Perhaps the most unique aspect of the quinoline pharmacophore is its ability to support axial chirality and exist, in certain cases, as stable atropisomers. This stereochemical complexity would add a new level of topological diversity to genetically encoded or synthetic peptides and could impart long-range conformational control over the peptide structure.

[0007] Recently, we reported that linear peptides with a P-dicarbonyl functionality at the N- terminus could be synthesized ribosomally using in vitro translation to generate novel [>- dicarbonyl-peptide hybrid molecules45. Other P-dicarbonyl functionality could be introduced into polypeptides using orthogonal aminoacyl-tRNA synthetases46. In both cases, the P- dicarbonyl species reported were derivatives of malonic acid, which are among the least reactive of all 1,3-dicarbonyl species, especially under mild conditions. We hypothesized that if we could ribosomally incorporate a more reactive P-dicarbonyl species at the A-terminus, then the resulting peptide or protein could be modified post-translationally into a heterocyclic pharmacophore using any one of a number of classic or modern chemical transformations. If the post-translational modification made use of a side chain as a co-substrate, then the resulting macrocycle would contain an embedded heterocycle. Such a macrocyclization strategy embodies the post-translational modification logic of RiPP natural products while appropriating chemical intermediates produced by polyketide synthase modules.

[0008] Summary of the Invention

[0009] We disclose that linear peptides with a reactive - or y-keto amide at their A-termini can be synthesized ribosomally using in vitro translation methods. Peptides carrying an A-terminal P-keto amide can be converted into diverse quinoline-peptide hybrids via Friedlander reactions with a variety of 2-aminoarylcarbonyl substrates. Reactions with appropriately substituted 2- aminobenzophenones generated quinoline-peptide hybrids with stable biaryl atropisomeric axes. In vitro-translated peptides carrying both an A-terminal P-keto amide and an internal 2- aminoacetophenone motif undergo intramolecular Friedlander macrocyclization reactions that embed a quinoline pharmacophore directly within the macrocyclic backbone. Friedlander macrocyclization reactions proceed on unprotected linear peptides, tolerate proteinogenic functional groups, are mild enough to be employed on in vitro-translated peptides, and do not degrade DNA. When the A-terminal P-keto amide is A-methylated, the Friedlander macrocyclization generates a pair of separable conformational isomers; the structure of one was determined using MicroED. The introduction of A-terminal ketone building blocks into genetically encoded materials and their post-translational derivatization expands the chemical diversity and structural complexity of genetically encoded materials and provides a paradigm for the generation of a wide array of genetically encoded and chemically modified peptides, and the programmed synthesis of peptide-derived materials that more closely resemble complex natural products.

[0010] In enumerated aspects and embodiments the invention provides:[Oil] 1. A method of peptide synthesis comprising:

[0012] providing a translation system with a tRNA acylated with a reactive 1,3-dicarbonyl motif under conditions wherein the tRNA introduces the 1,3-dicarbonyl motif as a P- or y-keto amide at the N-terminus of a nascent peptide, providing a substrate for Friedlander reactions for generating quinoline -peptide hybrid products.

[0013] 2. The method of claim 1 , further comprising converting the A-tenni nal keto amide into a quinoline-peptide hybrid via a Friedlander reaction with a 2-aminoarylcarbonyl substrate.

[0014] 3. The method of claim 1 , further comprising reacting the -lerminal keto amide with a substituted 2-aminobenzophenone to provide a Friedlander product possessing a stable biaryl atropisomeric axis.

[0015] 4. The method of claim 1 , wherein the peptide comprises the A-terminal keto amide and an internal 2- aminoacetophenone motif, and undergoes an intramolecular Friedlander macrocyclization reaction that embeds a quinoline pharmacophore directly within the macrocyclic backbone.

[0016] 6. A method of chemo-ribosomal synthesis of atropisomeric and macrocyclic peptides with embedded heterocycles or pharmacophores, including quinolines, substantially as described herein.

[0017] 7. A method of ribosomally synthesizing genetically encoded linear peptides with reactive N-terminal - and y-ketoamide functionality using in vitro translation.

[0018] 8. A method herein wherein resultant peptides carrying an N-terminal P-ketoamide provide substrates for Friedlander reactions that convert the P-ketoamide into diverse heterocyclic quinoline-peptide hybrids or heterocycle-peptide hybrids.

[0019] 9. A method herein wherein use of substituted 2- aminocarbonyl co-substrates provide quinoline-peptide hybrids, or other heterocycle-peptide hybrids, possessing stable and / or conformationally mobile biaryl atropisomeric axes, further expanding stereochemical diversity in genetically encoded peptides, including substituted 2-aminobenzophenone co-substrates that provide quinoline-peptide hybrids.

[0020] 10. A method herein wherein intramolecular Friedlander reaction of peptides carrying an N-terminal P-ketoamide with an internal 2-aminocarbonyl co-substrate (such as an internal 2- aminobenzophenone co-substrate) or kynurenine residue provide substrates for intramolecular Friedlander cyclization reactions (an effective peptide macrocyclization strategy) that effectively embeds quinoline pharmacophores into the peptide or macrocyclic backbone.

[0021] The invention encompasses all combinations of the particular embodiments recited herein, as if each combination had been laboriously recited.

[0022] Brief Description of the Drawings

[0023] Fig. 1A-B. Introduction of a reactive 1,3-dicarbonyl motif at the .V-terminus via in vitro translation, a, Scheme illustrating dFx-promoted acylation of lRNAIMlwith 3,5- dinitrobenzyl (DNB) esters and use of these tRNAs to initiate the in vitro translation of peptide Pl, where X represents A-l'ormyl methionine (fM) or monomers 1-4. DYKDDDDK is a FLAG-tag that enables peptide isolation and enrichment prior to LC-HRMS analysis, b, Yields for flexizyme-promoted acylation of MH and tRNAtMetby the DNB esters of fM and monomers 1-4. Yields were determined using intact tRNA LC-MS46’48.

[0024] Fig. 2A-C. Friedlander reactions of P-keto amides proceed readily in aqueous acetic acid, a, Scheme illustrating a classic Friedlander reaction between 2- aminobenzaldehyde and a substituted ketone, b, Model system used to evaluate more mild conditions for the Friedlander reaction of P-keto amide dipeptide 5 and 2-aminoacetophenone 6 (0.33 M each), c, Representative substituted quinoline products generated from Friedlander reactions performed in AcOH at 60 °C for 48 h. Isolated yields are shown in parentheses.

[0025] Figure 3A-F. Post- translational Friedlander reactions on IVT products proceed under mild conditions, a, Post- translational Friedlander reactions of IVT peptide 2-P1 generate diverse quinoline-peptide hybrid molecules denoted Qi-Pl, where i identifies the 2- aminocarbonyl substrate, b-e, Reaction scope. Shown is the extracted ion chromatogram (EIC) for the expected major ion produced when 2-P1 is treated under the conditions shown in the presence and absence of substrates 6, 8, 9, or 12 to generate Q6-P1, Q8-P1, Q9-P1, and Q12- Pl, respectively. The mass spectrum of each major ion is inset, f, eFx -promoted acylation of tRNAMetwith quinoline cyanomethyl ester 25-CME led to no detectable acylated tRNA product under any condition tested.

[0026] Figure 4A-C. Friedlander reactions of -keto amides generate stable atropisomers. a, Friedlander reaction of substrate 26 and dipeptide 28 in the presence of either Brpnsied or Lewis acids generates quinoline-dipeptide 29, which exists as a pair of stable atropisomers with a diastereomeric ratio (d.r.) of -41:59 regardless of the conditions used for its preparation, b, X- ray crystal structure of (P, 5)-29 (left, CCDC: 2341553) and (M, S)-29 (right, CCDC: 2341552) as their respective hydrochloride salts, c, Friedlander reaction of 2-P1, prepared using IVT, with aminocarbonyl 26 in the presence of 100 mM Yb(OTf)3 in EtOH generates quinoline-peptide Q26-P1. Extracted ion chromatograms for product formation in the presence of Yb(OTf)3 in EtOH with 26 (teal), acetic acid with 26 (red), or Yb(OTf)3 in EtOH without 26 (black) for 24 h at 40 °C. Significantly less product is observed when the reaction is performed using AcOH in place of Yb(OTf)3in EtOH.

[0027] Fig. 5A-F. Intramolecular post-translational Friedlander macrocyclization reactions, a, Preparative scale intramolecular Friedlander reactions to generate peptide macrocycles with a backbone-embedded quinoline. Each linear peptide contains monomer 2 at the V-terminus and a single internal kynurenine residue, b-e, Structures and total ion chromatograms of crude reaction mixtures containing the Friedlander macrocyclization products CPI-4. The peak containing the cyclic peptide is highlighted in teal. Atom count of the macrocycle backbone, HPLC254nmyield, and isolated yield (in parentheses) are shown, f, Incorporation of both 2 and kynurenine into a single IVT peptide ivt.LP5 and subsequent Friedlander cyclization into ivt.CP5.

[0028] Fig. 6A-B. Aqueous Friedlander macrocyclization of V-methyl p-keto amide peptide LPa generates two distinct isomers, a, V-substitution at the z+7 position in LPa producesstable conformational isomers, CPal and (P)-CPa, upon Friedlander cyclization in aqueous sodium phosphate at pH 3.5, favoring the kinetic product CPal with a diastereomeric ratio of 76:24. The ostensible atropisomeric axis between the quinoline 3’-carbon and N-methyl amide is highlighted in red. b, MicroED crystal structure (CCDC: 2395208) of (P)-CPa depicting a unique double beta-turn motif. Intramolecular H-bonds shown as dashed orange lines. Cyclic backbone carbon atoms are highlighted in green.

[0029] Fig. 7A-B. Synthesis of activated 3,5-dinitrobenzyl (DNB) esters used for microhelix (MH) and tRNA acylation as well as model P-keto amide dipeptide 5 used for Friedlander reaction optimization, a, Schemes illustrating the synthesis of 3,5-dinitrobenzyl (DNB) esters of fMet (fM) and monomers 1 - 4 used for Flexizyme-promoted acylation of microhelix (MH) and tRNA substrates, b, Scheme showing the synthesis of model P-keto amide dipeptide 5..

[0030] Fig. 8. Friedlander reaction mechanism. The Friedlander reaction is a double condensation reaction and can proceed along the paths shown. Precisely which pathway is ■7 chosen is dependent on the reaction conditions.

[0031] Fig. 9. Substrates and products of Friedlander reactions.

[0032] Fig. 10A-B Additional synthetic routes. Synthetic routes used to prepare the cyanomethyl esters of a, quinoline 25, and b, kynurenine 14.

[0033] Fig. 11. Friedlander reaction of model P-keto amide peptide 5 and 2-amino(o- tolyl)phenone (26) to generate 27. Reaction scheme for the generation of atropisomer 27.

[0034] Fig. 12. Structures of linear peptides LP1-LP5 containing an N- terminal P-keto amide and an internal kynurenine residue. Linear peptides were prepared using solid phase synthesis, purified, and characterized.

[0035] Fig. 13. Cyclic products generated via Friedlander macrocyclization of LP1-5.

[0036] Description of Particular Embodiments of the Invention

[0037] Unless contraindicated or noted otherwise, in these descriptions and throughout this specification, the terms “a” and “an” mean one or more, the term “or” means and / or. It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein, including citations therein, are hereby incorporated by reference in their entirety for all purposes.

[0038] Chemical and Ribosomal synthesis of atropisomeric and macrocyclic peptides with embedded quinolines

[0039] In these examples we show that linear peptides with a reactive / / -terminal |3-keto amide functionality can be synthesized ribosomally using in vitro translation (IVT) and that peptides carrying an N-terminal P-keto amide are substrates for Friedlander reactions capable of generating a diverse set of quinoline-peptide hybrid products. Reaction of the / / -terminal P-keto amide with an appropriately substituted 2-aminobenzophenone provides a Friedlander product possessing a stable biaryl atropisomeric axis whose structure could be characterized by NMRand X-ray crystallography. Synthetic or IVT peptides carrying both an N- terminal P-keto amide and an internal kynurenine residue are substrates for intramolecular Friedlander macrocyclization reactions that embed a quinoline pharmacophore into the peptide backbone. When the / / -terminal -keto amide is / -methylated, the Friedlander macrocyclization generates a pair of separable conformational isomers; the structure of one isomer was determined using MicroED. The introduction of simple ketide building blocks into ribosomal synthesis and their post-translational derivatization with carbonyl chemistry expands the structural and stereochemical diversity available to genetically encoded peptides. Overall, this work provides a paradigm for the programmed synthesis of peptide-derived materials that more closely resemble complex natural products.

[0040] Introduction of a reactive 1,3-dicarbonyl motif at the / -terminus via in vitro translation

[0041] We began by exploring the extent to which tRNAs could be acylated with monomers carrying a reactive dicarbonyl motif and the extent to which these acylated tRNAs could initiate the in vitro translation of short peptides (Fig. la), / / -formyl methionine (fM) and monomers 1-4 (Fig. lb) were converted into activated 3,5-dinitrobenzyl (DNB) esters (Fig. 7a) and their ability to acylate a model tRNA microhelix (MH) in the presence of flexizyme dFx47was evaluated as a function of time and pH. The extent of MH acylation was monitored using both acidic polyacrylamide gel electrophoresis (aPAGE) and intact tRNA LC-MS46’48. Although gel electrophoresis failed to resolve any acylated MH product from the non-acylated MH substrate, presumably due to similarity in mass and charge, intact tRNA LC-MS revealed that both 2-DNB and 3-DNB were substrates for dFx in MH-acylation reactions, especially at higher pH and longer incubation times. It is likely that the activity of 3-DNB as a substrate for dFx was previously missed because of the low resolving power of gel electrophoresis . Although we were unable to identify conditions that led to detectable MH acylation by the 3,5-dinitrobenzyl ester of monomer 1 (Fig. lb), in the case of monomer 4, intact tRNA LC-MS detected formation of acylated MH in 0.6% yield. In vitro-transcribed E. coli initiator tRNA (tRNAMet) was subsequently acylated on a preparative scale with the 3,5-dinitrobenzyl esters of monomers 2 and 3; in each case, product identity was confirmed using intact tRNA LC-MS.

[0042] With acylated tRNAMetin hand, we next evaluated the extent to which monomers 2 and 3 could be introduced at the / / -termini of short peptides prepared using in vitro translation (IVT). We made use of a commercial IVT kit (PURExpress® A (aa, tRNA) Kit), supplemented with the requisite amino acids and tRNAs, tRN Alk tpre-acylated with fM or monomers 2 or 3, and a duplex DNA template encoding the FLAG-containing polypeptide MGVDYKDDDDK (MGV-flag). Peptides initiated with monomers 2 or 3 were translated at levels comparable to those initiated with tRNAIK Ithat had been pre-acylated with / / -formyl methionine (fM).

[0043] Friedlander reactions of P-keto amides proceed readily under mild aqueous conditions

[0044] - and y-keto carbonyl groups participate in an array of chemical transformations that generate carbocyclic and heterocyclic frameworks, often with elements of stereocontrol50"52.One classic transformation is the Friedlander reaction53, a double condensation of a 2- aminoarylcarbonyl and an aldehyde or ketone to form a quinoline, a fused [6,6] heterocyclic aromatic ring, via the formation of one C-N bond and one C-C bond (Fig. 8). Friedlander reactions are typically performed under harsh conditions, requiring a strongly acidic or basic additive and / or reaction temperatures greater than 150 °C (Fig. 2a)54’55; these conditions would likely degrade sensitive peptides. Although we were encouraged by several reports of high- yielding Friedlander reactions of [3-keto esters performed in acetic acid56and acetic acid / water mixtures , we could identify only limited literature examples of Friedlander reactions of [3-keto amides58-61, and none that proceeded under mild conditions that would be appropriate for peptides and with a wide substrate scope.

[0045] We thus sought to identify mild conditions that would support the Friedlander reaction of model [3-keto amide 5 (Fig. 2b), and apply these conditions to peptides generated using IVT. We examined the reaction of 5 with 2-aminoacetophenone 6 to generate quinoline 15 in acetic acid and acetic acid / water mixtures at temperatures between RT and 60 °C. Reactions were performed using 330 mM of 5 and 6 and the formation of quinoline 15 was monitored as a function of time using LC-MS. We found that all conditions tested supported the Friedlander reaction of [3-keto amide 5 to generate the expected quinoline product 15. Conversion was most rapid at 60 °C in AcOH or H2O / AcOH mixtures or at 40 °C in AcOH. To evaluate reaction scope under these conditions, we treated |3-keto amide 5 with a collection of nine structurally diverse 2-aminoarylcarbonyl substrates 6-14 (Fig. 2c and Fig. 9). Substrates 6 and 9-13 reacted cleanly with [3-keto amide 5 with full conversion to the expected quinoline products 15 and 18- 22 after 48 h at 60 °C in AcOH. Reactions with 2-aminobenzaldehydes 7 and 8, which polymerize under acidic conditions62, and kynurenine 14, led to lower levels of product formation. Quinolines 15-20 were synthesized on a preparative scale and the products were characterized using LC-HRMS and NMR.

[0046] Post-translational Friedlander reactions of IVT products proceed under mild conditions

[0047] We next asked whether Friedlander reactions would also proceed efficiently when performed on IVT peptides initiated with tRNAMetacylated with monomer 2. Performing reactions on in vitro translation products is challenging because the reaction must proceed cleanly at low peptide concentration (< 1 pM) and in complex reaction mixtures. [3-keto amide peptide 2-P1 was generated using IVT, isolated using anti-FLAG magnetic beads, lyophilized, and treated with 2-aminoarylcarbonyl substrates 6, 8, 9, or 12 in acetic acid at 40 °C for 24 h. The formation of Friedlander products Q6-P1, Q8-P1, Q9-P1, and Q12-P1 was monitored using LC-HRMS. In each case, the post-translational Friedlander reaction of 2-P1 proceeded to generate peptides with quinoline derivatives at their A-lermini (Fig. 3b-e). In theory, identical quinoline-peptide hybrids could be prepared ribosomally using tRNAMetacylated with a preassembled quinoline. Yet treatment of tRNAMctwith eFx and dimethyl quinoline cyanomethyl ester 25-CME (Fig. 10A-B) led to no detectable acyl-tRNA product under any condition tested(Fig. 3f). We conclude that post-translational Friedlander reactions represent a simple path to ribosomally synthesized peptides with diverse quinoline functionality at the Wterminus.

[0048] Friedlander reactions of P-keto amides generate stable atropisomers

[0049] Appropriately substituted quinolines can exist as atropisomers. Canonical atropisomers are stereoisomers that arise from hindered rotation around a o-bond63. Like stereoisomers with point chirality, atropisomers can bind macromolecular targets with distinct kinetics and affinities64. Atropisomeric quinolines and quinoline-like heterocycles are well known. They are found in pharmaceuticals6"67such as Sotorasib, a first-in-class mutant KRAS G12C inhibitor ’ , as well as novel HIV-I integrase inhibitors . Atropisomeric quinolines also serve as the molecular framework for catalysts with widespread use in organic synthesis and process research, such as QUINAP65. Although many macrocyclic peptide natural products contain unusual structural isomers, sometimes called atropisomers , non-canonical atropisomers , or ansamers ' , that result from threaded- loop or knot- like structures ' ' , true canonical atropisomers are also important constituents of this class of compounds, such as the famous case of the cyclic glycopeptide vancomycin76. Peptides or cyclic peptides containing atropoisomeric quinolines would add a sophisticated layer of stereochemical complexity to macrocyclic peptide libraries.

[0050] To assess whether atropisomeric quinolines could be introduced within ribosomal peptides, we first confirmed that Friedlander reaction of peptide 28 with (2-aminophenyl)(o- tolyl)methanone (26) in AcOH at 60 °C for 48 h produced the expected product 29 as a roughly 41:59 mixture of atropisomeric products, as determined by1H NMR (Fig. 4a). However, when we attempted to reproduce a post-translational Friedlander reaction between (2- aminophenyl )(o- tolyl)methanone (26) and IVT-generated peptide 2-P1 in acetic acid, the expected product Q26- P1 was detected, but in low abundance.

[0051] Lewis acid catalysis of post-translational Friedlander reactions generates atropisomeric products

[0052] We hypothesized that Friedlander reactions of P-keto amide-containing peptides would proceed under mild Lewis acid catalysis, conditions that would be unlikely to harm ribosomal peptides generated by IVT. Indeed, Lewis acids such as the chloride salts of Ce(III)77and Fe(III)78as well as the triflate salts of Yb(III)79, Y(III)80, and Zn(II)81promote Friedlander reactions of -keto esters55. To evaluate if they could improve atropisomer-forming reactions of P-keto amide 2-P1, we screened their effect (at 20 mol%) on the Friedlander reaction of dipeptide 5 and (2-aminophenyl)(<9-tolyl)methanone (26) to generate the quinoline 27 (Fig. 11). Of all Lewis acids tested, the highest yields were obtained with Yb(OTf)3 in ethanol, but Yb(OTf)3also performed well in DCM, MeCN, THF, and AcOH. Although strong Brpnsted acids such as triflic acid, sulfuric acid, phosphoric acid, and trifluoroacetic acid also outperformed acetic acid with regard to the preparation of 27, these conditions are far less mild.

[0053] When the Friedlander reaction of 28 with 26 was repeated, this time in the presence of 20 mol% Yb(OTf)3 in EtOH at 60 °C for 48 h, we isolated quinoline 29 in 65% yield with a similar 41 :59 diastereomeric ratio (Fig. 4a). The diastereomers were separated and characterized by 'HNMR and X-ray structure determination (Fig. 4b), revealing the (P, .S') diastereomer as the major product. The stability of the atropisomeric axis was assessed by heating a pure sample of the minor diastereomer (i.e., (M, S)-29) in DMSO-dg at 100 °C for 18 h82. Subsequent H NMR analysis revealed minimal atropisomerization (< 1%). With optimized conditions in hand, we again attempted modification of a ribosomal peptide generated by IVT. Indeed, when treated with 26 in EtOH containing Yb(OTf)3, the IVT synthesized P-keto amide peptide 2-P1 was transformed cleanly into the quinoline-peptide hybrid Q26-P1 after 24 h at 40 °C (Fig. 4c). Q26-P1 is presumed to exist as a mixture of atropisomers in analogy to 29, but chromatographic separation of distinct isomers by reverse phase UPLC-MS was not observed on this larger IVT peptide.

[0054] Friedlander macrocyclizations

[0055] We envisioned that Lewis acids might also aid post-translational Friedlander macrocyclization reactions to generate products with a quinoline pharmacophore embedded within the peptide backbone. To test this idea, we prepared four linear peptides containing both an N-terminal P-keto amide and an internal 2-aminoarylcarbonyl side chain provided by the non- canonical amino acid kynurenine (Fig. 5a and Fig. 12). The linear peptide LP1 is a heptapeptide related to the FDA-approved drug octreotide5’83, while LP2 is a hexapeptide containing a turninducing L-proline, LP3 is a decapeptide bearing diverse functional groups, and LP4 is a longer diverse tridecapeptide related to the hormone somatostatin84. All four linear peptides LP1-4 (1 mM) underwent intramolecular Friedlander reactions to generate macrocycles CPI-4 (Fig. 13) in the presence of 1-2 mM Yb(OTf)3 in EtOH or MeCN at 60 °C, as supported by LC-HRMS (Fig. 5b-e) and NMR experiments). The smaller peptides LP1 and LP2 cyclized cleanly and the macrocyclic products CPI (72% HPLC and 60% isolated yield) and CP2 (91% HPLC and 62% isolated yield) could be isolated with efficiencies that tracked well with LC-MS and HPLC chromatograms of reaction progress. Cyclization of the larger peptides LP3 and LP4 proceeded under analogous conditions to deliver their corresponding macrocycles with reduced, but still notable efficiency: CP3 (58% HPLC and 37% isolated yield) and CP4 (58% HPLC and 27% isolated yield). LC-HRMS analysis of the crude macrocyclization product revealed residual unreacted linear peptide as well as (in the case of LP3) single-dehydration byproducts, indicating incomplete conversion under the conditions tested. Cyclization of long linear peptides is known to be challenging in the absence of turn-inducing elements85. The presence of singledehydration byproducts in the reaction of LP3 could result from side reactions, such as aspartimide formation, or indicate the accumulation of Friedlander reaction intermediates. For comparison, macrocyclization reactions of LP1-4 were also performed under aqueous conditions (80 mM glycine pH 2.8 at 60 °C); in all cases, cyclization proceeded faster using the Lewis acid Yb(OTf)3. The isolated macrocycles CP1-CP4 were also characterized by MALDI-TOF MS to confirm the monomeric mass and the absence of significant quantities of higher order oligomers.

[0056] Next, we examined whether Lewis acid-catalyzed Friedlander macrocyclization conditions were compatible with the nucleic acids used to establish genetically encoded libraries. We screened a series of Friedlander reaction conditions to identify those that wouldsupport both the cyclization of LP2 and the stability of a 28-mer DNA:RNA duplex. We find that the Yb(OTf)3-promoted Friedlander macrocyclization of LP2 proceeds well in MeCN containing 20% H2O, a minimum requirement for oligonucleotide solubility in genetically encoded libraries86. Although Friedlander macrocyclization efficiency is lower in MeCN containing 50% H2O, it can be rescued using super- stoichiometric Yb(OTf)3. These conditions result in substantial degradation of the RNA strand, but the DNA remains intact. This observation indicates that Friedlander reactions could be used in the context of either DNA-encoded or mRNA / cDNA-display libraries ’ .

[0057] Finally, we asked whether a Lewis acid-catalyzed Friedlander macrocyclization reaction was compatible with the challenging conditions required for post-translational modifications of in vitro translation (IVT) products. First, the cyanomethyl ester of kynurenine (Kyn-CME) and the flexizyme eFx were used to acylate tRNAAsnE2#2GCU, a previously reported engineered tRNA89. The resulting Kyn-tRNAAsnE2#2GCU was added to the IVT reaction while excluding serine and sequestering native tRNA GCU with an antisense oligonucleotide (M5-1) to suppress near cognate read-through22,90. We confirmed that P-keto acid 2 and kynurenine (Kyn) could independently and effectively recode methionine at position 1 and serine at position 6, respectively, of a peptide prepared using IVT. Using an identical strategy, we then used IVT to generate tetradecapeptide ivt.LP5, which contains both an V-terminal P-keto amide as well as an internal kynurenine residue (Fig. 5f). Subsequent reaction of ivt.LP5 with 100 mM Yb(OTf3) in MeCN at 60 °C for 24 h yielded quinoline-cyclized peptide ivt.CP5 (Fig. 5f) whose retention time and mass spectrum were identical to that of an authentic standard produced by SPPS. This result confirms that mild, post-translational Lewis acid-catalyzed Friedlander reactions can effectively generate macrocyclic quinoline-peptide hybrids even at very low peptide concentrations and in the presence of the high concentrations of salts and biomolecules that are intrinsic to in vitro translation reactions.

[0058] Atroposelective Friedlander macrocyclizations

[0059] Aryl-tertiary amide atropisomers are well-studied conformational isomers that have garnered significant attention owing to their room- temperature stability and complex stereodynamics91. Yet, these systems have never been employed in peptides. Noting the steric congestion around the quinoline-amide bond in macrocycles CPI-5, we hypothesized that N- methylation of the N- termi nal P-keto amide would increase the barrier to rotation about the quinoline-amide bond and lead to isolable diastereomeric quinoline products. If so, the quinoline-amide bond would function as a “dominant rotor” to stabilize a unique peptide conformation92,93.

[0060] To test this hypothesis, we synthesized peptide LPa, which contains V-methyl-alanine at position 2, and evaluated its macrocyclization under various conditions (Fig. 6a). When heated in aqueous sodium phosphate buffer at pH 3.5, LPa was converted into a pair of isobaric products that were readily separable by reverse phase HPLC. Both bore the characteristic]H NMR and absorbance signatures of a quinoline heterocycle. The two products were isolated in 42% yield (86% HPLC conversion) and a ratio of 76:24 (HPLC). The minor isomer formedsmall needle-like crystals during methanol evaporation and was characterized using MicroED94. The resulting structure (Fig. 6b, CCDC: 2395208), solved at a resolution of 0.9 A, shows a (P)- configured atropisomer about the quinoline-amide C-C axis, along with tran -amides at each amide linkage. In this structure, the A-methyl group points away from the rest of the peptide macrocycle and the tertiary amide oxygen points inward to accept an H-bond from the i+3 Phe N-H. (P)-CPa adopts a unique double beta-turn motif (type II’ followed by type I), with two H- bonds that cross in the interior of the structure. This type of double turn is rare in proteins, accounting for only 2.2% of double turns seen in the PDB95. This observation emphasizes the ability for formation of unique macrocyclization motifs in these Friedlander cyclizations; moreover, the installation of an axis of chirality clearly introduces an element of stereogenicity to the conformational landscape of these peptidic structures.

[0061] Discussion

[0062] Peptides and their derivatives are a rapidly expanding class of therapeutic molecules, in part because they condense the expansive recognition properties of a protein into a more compact molecular framework. The extended binding surface of a peptide provides a better complement to the extended and featureless surfaces that comprise many traditionally “undruggable” targets96. Perhaps the greatest attribute of peptides, whether linear or cyclic, is that they are genetically encodable, and hence their properties can be optimized using directed evolution methods performed in vitro or in vivo. Many of these methods depend on ribosomal translation and reliably return peptide leads for challenging and clinically relevant targets. For these reasons, de novo peptides have attracted significant attention as the next frontier in pharmaceutical development97.

[0063] Yet the difference between the de novo peptide lead and the final compound advanced to clinical trials is often profound, and the modifications introduced demand exceptional commitments of labor and state-of-the-art medicinal chemistry98. Introducing these modifications earlier in the discovery process would be advantageous. Some important progress has been made, including the introduction of A-Me groups99, D-amino acids100, p2- and p3-amino acids, as well as linkers that introduce rings7or even appended pharmacophores24'35. Even with these developments, the chemistry available to ribosomally translated peptides remains limited when compared to the vast array of transformations, functional groups, and architectures that are emblematic of small molecule pharmaceuticals and bioactive natural products. Yudin and others have shown that the introduction of heterocyclic grafts into peptide macrocycles can have profound impacts on passive permeability, bioavailability, and even topological complexity92 101. Quinolines, however, are underrepresented among these grafts; thus, the properties imbued by these motifs represent an area ripe for further exploration. Well-known properties of quinolines, such as turn-on fluorescence under acidic conditions102’103, oligonucleotide binding38, and atropisomer scaffolding82, are valuable additions to peptide chemistry.

[0064] This work was initiated in an attempt to generate ribosomally synthesized peptides with modifications that resemble those found in peptide-derived clinical candidates. We accomplished this goal by installing a reactive P-keto amide functional group at the A-termi nusand developing mild Friedlander reaction conditions to convert the P-keto amide into quinolinepeptide hybrids, including those that exist in distinct atropisomeric forms. The same mild Friedlander conditions were deployed as a macrocyclization strategy, embedding a quinoline pharmacophore directly into the backbone of a sequence-encoded macrocycle. Peptides containing iV-methyl P-keto amides undergo intramolecular Friedlander reactions to generate separable conformational isomers, providing a new strategy to achieve conformational control and increase the stereochemical diversity of a macrocyclic peptide.Finally, we note the ^ -terminal P-keto amide and y-keto amide peptides described here are potential substrates for many other macrocyclization or late-stage diversification reactions51. Potential reactions of Mterminal P- and y-keto amides include those that generate pyridines (Hantzsch reaction104), pyrazoles (Knorr105), pyrroles (Hantzsch, Knorr, Paal-Knorr106), dihydropyrimidinones (Biginelli ), and carbocycles (Conia-Ene ), in addition to Michael additions, alkylations, acylations, and arylations. Some of these reactions also have the potential to install atropisomeric axes into the macrocyclic product. These modifications could be used alone or combined with more traditional backbone diversification strategies to generate biased libraries containing pharmacophores and drug-like motifs. We envision the ribosomal incorporation of P- and y-keto amides as an important development in the late-stage functionalization and macrocyclization of peptides with validated drug-like features. The simplicity of the substrates, tolerance of diverse functional groups, and mildness of the chemistry indicate that Friedlander reactions find utility beyond peptides, in protein functionalization or macromolecule bioconjugation.

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Claims

CLAIMS1. A method of peptide synthesis comprising: providing a translation system with a tRNA acylated with a reactive 1,3-dicarbonyl motif under conditions wherein the tRNA introduces the 1,3-dicarbonyl motif as a P- or y-keto amide at the N-terminus of a nascent peptide, providing a substrate for Friedlander reactions for generating quinoline-peptide hybrid products.

2. The method of claim 1 , further comprising converting the A-termi nal keto amide into a quinoline-peptide hybrid via a Friedlander reaction with a 2-aminoarylcarbonyl substrate.

3. The method of claim 1, further comprising reacting the N-terminal keto amide with a substituted 2-aminobenzophenone to provide a Friedlander product possessing a stable biaryl atropisomeric axis.

4. The method of claim 1, wherein the peptide comprises the N-terminal keto amide and an internal 2-aminoacetophenone motif, and undergoes an intramolecular Friedlander macrocyclization reaction that embeds a quinoline pharmacophore directly within the macrocyclic backbone.

6. A method comprising chemo-ribosomal synthesizing atropisomeric and macrocyclic peptides with embedded heterocycles or pharmacophores, including quinolines.

7. A method of ribosomally synthesizing a genetically encoded linear peptide with a reactive N- terminal P- and y-ketoamide functionality using in vitro translation.

8. A method of claim 1, 2, 3, 4, 5, 6 or 7, wherein the resultant peptide carrying an N-terminal P- ketoamide is deployed as a substrate for Friedlander reaction that converts the -ketoamide into a heterocyclic quinoline-peptide hybrid or heterocycle-peptide hybrid.

9. A method of claim 1, 2, 3, 4, 5, 6 or 7, further comprising use of a substituted 2- aminocarbonyl co- substrate to provide a quinoline-peptide hybrid, or other heterocycle-peptide hybrid, possessing a stable and conformationally mobile biaryl atropisomeric axis, further expanding stereochemical diversity in genetically encoded peptides, including substituted 2- aminobenzophenone co-substrates that provide quinoline-peptide hybrids.

10. A method of claim 1, 2, 3, 4, 5, 6 or 7, further comprising an intramolecular Friedlander reaction of the peptide carrying an N-terminal P-ketoamide with an internal 2-aminocarbonyl cosubstrate or kynurenine residue, providing a substrate for an intramolecular Friedlander cyclization reaction that effectively embeds a quinoline pharmacophore into the peptide or macrocyclic backbone.

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