Azapeptide Synthesis Methods for Expanding Side-Chain Diversity for

Article pubs.acs.org/accounts

Azapeptide Synthesis Methods for Expanding Side-Chain Diversity for Biomedical Applications Published as part of the Accounts of Chemical Research special issue “Chemical Biology of Peptides”. Ramesh Chingle, Caroline Proulx,† and William D. Lubell* Department of Chemistry, Université de Montréal, C. P. 6128, Succursale Centre-Ville, Montreal, Quebec, Canada H3C 3J7 CONSPECTUS: Mimicry of bioactive conformations is critical for peptide-based medicinal chemistry because such peptidomimetics may augment stability, enhance affinity, and increase specificity. Azapeptides are peptidomimetics in which the αcarbon(s) of one or more amino acid residues are substituted by nitrogen. The resulting semicarbazide analogues have been shown to reinforce β-turn conformation through the combination of lone pair−lone pair repulsion of the adjacent hydrazine nitrogen and urea planarity. Substitution of a semicarbazide for an amino amide residue in a peptide may retain biological activity and add benefits such as improved metabolic stability. The applications of azapeptides include receptor ligands, enzyme inhibitors, prodrugs, probes, and imaging agents. Moreover, azapeptides have proven therapeutic utility. For example, the aza-glycinamide analogue of the luteinizing hormone-releasing hormone analogue Zoladex is a potent long-acting agonist currently used in the clinic for the treatment of prostate and breast cancer. However, the use of azapeptides was hampered by tedious solution-phase synthetic routes for selective hydrazine functionalization. A remarkable stride to overcome this bottleneck was made in 2009 through the introduction of the submonomer procedure for azapeptide synthesis, which enabled addition of diverse side chains onto a common semicarbazone intermediate, providing a means to construct azapeptide libraries by solutionand solid-phase chemistry. In brief, aza residues are introduced into the peptide chain using the submonomer strategy by semicarbazone incorporation, deprotonation, N-alkylation, and orthogonal deprotection. Amino acylation of the resulting semicarbazide and elongation gives the desired azapeptide. Since the initial report, a number of chemical transformations have taken advantage of the orthogonal chemistry of semicarbazone residues (e.g., Michael additions and N-arylations). In addition, libraries have been synthesized from libraries by diversification of aza-propargylglycine (e.g., A3 coupling reactions, [1,3]-dipolar cycloadditions, and 5-exo-dig cyclizations) and aza-chloroalkylglycine residues. In addition, oxidation of aza-glycine residues has afforded azopeptides that react in pericyclic reactions (e.g., Diels−Alder and Alder−ene chemistry). The bulk of these transformations of aza-glycine residues have been developed by the Lubell laboratory, which has applied such chemistry in the synthesis of ligands with promising biological activity for treating diseases such as cancer and age-related macular degeneration. Azapeptide analogues of growth hormone-releasing peptide-6 (His-D-Trp-Ala-Trp-D-Phe-Lys-NH2, GHRP-6) have for example been pursued as ligands of the cluster of differentiation 36 receptor (CD36) and show promising activity for the development of treatments for angiogenesis-related diseases, such as age-related macular degeneration, as well as for atherosclerosis. Azapeptides have also been employed to make a series of conformationally constrained second mitochondria-derived activator of caspase (Smac) mimetics that exhibit promising apoptosis-inducing activity in cancer cells. The synthesis of cyclic azapeptide derivatives was used to make an aza scan to study the conformation−activity relationships of the anticancer agent cilengitide, cyclo(RGDfN(Me)V), and its parent counterpart cyclo(RGDfV), which exhibit potency against human tumor metastasis and tumor-induced angiogenesis. Innovations in the synthesis and application of azapeptides will be presented in this Account, focusing on the creation and use of side-chain diversity in medicinal chemistry.

1. INTRODUCTION Azapeptides employ semicarbazides as amino amide surrogates in which the backbone CHα is replaced by nitrogen.1,2 Side chains can be preserved on aza residues in potentially optimal orientations because of adaptive chirality about the αnitrogen.1,2 Moreover, semicarbazides favor backbone β-turn geometry because of a combination of urea planarity and © 2017 American Chemical Society

hydrazine nitrogen lone pair−lone pair repulsion, as ascertained by computation,3 X-ray crystallography,4 and spectroscopy.2 Relative to amides, semicarbazides have reduced electrophilicity, enhanced chemical stability,5,6 and protease resistReceived: March 8, 2017 Published: June 9, 2017 1541

DOI: 10.1021/acs.accounts.7b00114 Acc. Chem. Res. 2017, 50, 1541−1556

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Accounts of Chemical Research

Scheme 1. (a) Submonomer Azapeptide Synthesis of [azaPhe4]GHRP-6; (b) HPLC Trace of a Crude Cleaved Sample Monitored at 254 nm

ance.7 Although pure semicarbazide oligomers have been synthesized,8 crystallized,9 and later termed “azatides”,10,11 they have exhibited reduced receptor affinities in receptor binding assays. On the other hand, the consequences of single Cα to Nα substitution have enabled serine and cysteine protease inhibitor design by insertion of aza residues at the P1 position of peptide substrates12−14 and selective receptor ligand conception, culminating in clinically approved drugs with prolonged duration of action, such as [D-Ser(t-Bu)6-azaGly10]LHRH.2,15,16 Capacity to restrict the ϕ and ψ dihedral angle geometry and enhance the backbone stability without compromising the sidechain position bestows strong utility on aza residues for peptide mimicry. However, the synthesis, activation, and coupling of Nprotected N′-alkyl carbazate building blocks has encumbered

azapeptide synthesis. To enable studies of structure−activity relationships (SARs) with aza residues, a strategy was devised for introducing and adding side chains onto aza-glycines in peptides by a so-called “submonomer” approach.17,18 Submonomer strategies have streamlined peptidomimetic (e.g., peptoid) production by facilitating oligomer assembly.19 Enabling azapeptide library construction, submonomer synthesis has been particularly useful for studying growth hormone-releasing peptide-6 (His-D-Trp-Ala-Trp-D-Phe-LysNH2, GHRP-6) to develop modulators of the cluster of differentiation-36 scavenger receptor (CD36).18,20 Furthermore, aza-glycine N-arylation and functionalization with reactive ω-chloroalkyl and propargyl side chains by submonomer synthesis have expanded the diversity of aza residues through robust chemistry, including nucleophilic displace1542


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Accounts of Chemical Research Scheme 2. Solution-Phase Submonomer Synthesis and C- and N-Terminal Elongation of Benzhydrylidene Azaphenylalaninylproline tert-Butyl Ester

ments,21 copper-catalyzed Mannich additions, with A3 reactions,22 and [1,3]-dipolar cycloadditions.23 Oxidation of azaglycine residues to their azo counterparts has permitted pericyclic chemistry,24 that was applied to study the second mitochondria-derived activator of caspase (Smac) protein AlaVal-Pro-Ile (AVPI) sequence with aza analogues that induced apoptosis in breast cancer cells.25,26 Cyclic azapeptide analogues of GHRP-6 and cilengitide have also provided potent CD36 modulators27 and antagonists of integrin receptors,28 respectively. This Account summarizes recent advances in azapeptide chemisty since debut of submonomer synthesis 8 years ago, focusing on amplified aza residue sidechain diversity, and highlights the use of aza analogues to study biologically relevant peptides (e.g., GHRP-6, Smac AVPI, and cilengitide).

of the resulting semicarbazone 4, and (c) orthogonal liberation and amino acylation of semicarbazide 5 (Scheme 1).17 The GHRP-6 sequence was selected to develop the submonomer method, in part because of interest in CD36 modulators to control blood vessel growth (vide infra) as well as to optimize the procedure in the presence of nucleophilic side chains (e.g., His, Trp, Lys). Benzaldehyde hydrazone was activated using p-nitrophenyl chloroformate, and carbazate 2 was effectively coupled to the resin-bound peptide as ascertained by LCMS analysis of the cleaved product. Potassium tert-butoxide served to deprotonate semicarbazone 4, which was alkylated with benzyl bromide in THF without modification of the other amide, carbamate, and urea nitrogens, giving aza-phenylalanine 5 with 89% conversion. Orthogonal semicarbazone deprotection in the presence of Boc protection was ultimately achieved by trans-imination using 1.5 M hydroxylamine hydrochloride in pyridine at 60 °C for 12 h with sonication. Pyridine proved to be critical for resin swelling, prevention of premature azapeptide cleavage, and avoiding sidechain Boc group removal. Amino acid activation for coupling onto the resulting semicarbazide has been accomplished by a variety of methods, including the use of symmetric anhydrides generated with diisopropylcarbodiimide (DIC),17 amino acid chlorides prepared using triphosgene (BTC),30−32 and the latter under microwave irradiation.33 Standard Fmoc-based solid-phase peptide synthesis protocols34 were subsequently employed to complete the [azaPhe4]GHRP-6 sequence (Scheme 1). Nine other aza-GHRP-6 analogues were made by selective introduction of aromatic and aliphatic side chains at the D-Trp2, Ala3, and Trp4 positions.17 The submonomer method gave access to a wide array of aza residues, including

2. DEVELOPMENT OF SUBMONOMER AZAPEPTIDE SYNTHESIS 2.1. Solid-Phase Submonomer Azapeptide Synthesis

The conception of a submonomer strategy for constructing azapeptides was inspired by earlier applications of glycine Schiff bases to synthesize di- and tripeptides by chemoselective alkylation with side-chain components.29 In the azapeptide strategy, side-chain installation on the more acidic semicarbazone was successful in longer peptide chains without issues of chiral induction and racemization, in part because of the absence of amide and carbamate deprotonation. To insert the aza residue, three steps were added into the standard solidphase peptide synthesis protocol: (a) activation and coupling of hydrazone 1, (b) chemoselective deprotonation and alkylation 1543


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Scheme 3. (a) Alkylation of Carbazates 15a−c Using KOtBu versus Et4NOH; (b) Alkylation of Azadipeptides 17a and 17b Using Et4NOH without Detectable Racemization

Scheme 4. Cu-Mediated N-Arylation in the Submonomer Synthesis of [Aza(3-indolyl)Gly4]GHRP-6

diketopiperazine byproducts during N-alkylation and semicarbazone deprotection, respectively. Benzophenone-derived semicarbazones were relatively more stable than their benzaldehyde counterparts.

aza-propargyl- and aza-allylglycines, which were relatively inaccessible by previous methods. 2.2. Solution-Phase Submonomer Azapeptide Synthesis

In parallel to the development of solid-phase chemistry, solution-phase azapeptide synthesis was achieved on a gram scale by employing benzophenone semicarbazones as azaglycine surrogates.35 Activated methylidine carbazates (e.g., 10) circumvented the formation of oxadiazalone and hydantoin byproducts that arise from undesired intramolecular cyclization reactions during strategies using Fmoc-aza-glycine acid chlorides and the activation of the peptide terminus as an isocyanate or activated carbamate, respectively.36−38 Benzhydrylidene azadipeptides were diversified using submonomer protocols similar to those described for solid-phase synthesis (Scheme 2).39 Application of azadipeptide tert-butyl esters avoided competitive formation of hydantoin and aza-

2.3. Improvements to Submonomer Azapeptide Synthesis

In solution, ester racemization during alkylation of aza-glycinyl dipeptides (e.g., 11) was investigated through detection of minor isomers by HPLC analysis of diastereomeric azatripeptides [e.g., (S,S)- and (R,S)-14; Scheme 2]39 and supercritical fluid chromatography (SFC) of azadipeptide substrates on a chiral column.40,41 Ester epimerization varied with the choice of semicarbazone, base, and reaction temperature. Fluorenylidene tert-butyl carbazates were alkylated using milder bases than benzylidene and benzhydrylidine counterparts as a result of resonance stabilization by the aromatic fluorenyl anion (Scheme 3a).40 Aqueous tetraethylammonium hydroxide 1544


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Accounts of Chemical Research Scheme 5. Conjugate Reactions with (a) Michael Acceptors and (b) Activated Allylic Acetates

Scheme 6. “Libraries from Libraries” Using Aza-propargylglycine and (A) CuAAC, (B) A3 Reaction, (C) Sonogashira, and (D) 5-Exo-Dig Cyclization Chemistry

benzophenone hydrazone, N,N′-disuccinimidyl carbonate

proved to be superior to KOtBu for avoiding racemization, which was minimized at 0 °C. Microwave irradiation improved the alkylation yield from 43 to 73% with benzophenone semicarbazone 17b (Scheme 3b).42 In the activation of

(DSC) gave purer product by eliminating removal of the pnitrophenol byproduct using p-nitrophenyl chloroformate.42 1545


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Scheme 7. Cyclic Azapeptide Synthesis by A3 Macrocyclization after Aza-propargylglycine Introduction (e.g., 44−48) and after Sequence Completion (e.g., 52−55)

3. EXPANDING SIDE-CHAIN DIVERSITY

counterparts of aspartate, glutamate, asparagine, and glutamine all have demonstrated utility in enzyme inhibitors and ligands of the MHC II protein.45−49 Mimics of phosphate and sulfate esters of serine and cysteine garner interest for inhibition of kinases, phosphatases, and sulfatases. To install such side chains on aza residues, conjugate alkylation of aza-glycine using Michael acceptors proved to be effective and was best achieved on resin using the non-nucleophilic phosphazene base tertbutyliminotris(pyrrolidino)phosphorene (BTPP) instead of KOtBu (Scheme 5a).18 For example, [aza-Glu4]- and [azaGlu3]GHRP-6 (i.e., 22a and 22b) were synthesized using tertbutyl acrylate and BTPP. The aza-Gln side chain was installed (e.g., 23b) using acrylamide as the Michael acceptor; however, the corresponding aza-pyroglutamate (aza-pGlu) was obtained from intramolecular cyclization during semicarbazone removal and incorporated into [aza-pGlu1]GHRP-6. Acrylonitrile, vinyl phosphonate, and vinyl sulfonate electrophiles afforded Michael adducts 24−27 in 29−80% conversion (Scheme 5a). Phosphonate diester solvolysis during semicarbazone deprotection gave phosphonate monoester analogues. Peptide

3.1. Semicarbazone N-Arylation

Beyond N-alkylation, submononer azapeptide chemistry was extended to N-arylation on-resin to prepare aza-arylglycines. NArylation of semicarbazone 4 was accomplished by Cu(I)mediated reactions with aryl and heteroaryl iodides (Scheme 4),43 including N-Boc-3-iodoindole and N-trityl-4-iodoimidazole, which furnished aza-indolyl- and aza-imazolylglycine residues, respectively, as acid-stable aza-Trp and aza-His mimics. Attempts to synthesize [aza-Trp4]GHRP-6 failed previously because of loss of the indolylmethyl side chain during resin cleavage with trifluoroacetic acid (TFA), giving the corresponding aza-glycine analogue.30 Aza-arylglycines circumvent issues of racemization associated with arylglycines.44 Copper-mediated N-arylations at the D-Trp2 and Trp4 positions provided 13 aza-arylglycine GHRP-6 analogues. 3.2. N-Alkylation with Michael Acceptors and Allylic Acetates

Polar and ionic side chains attract interest because of their potential to engage in hydrogen bonds and salt bridges. Aza 1546


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Scheme 8. Cyclic Azapeptide Synthesis by Modification of Nε-(o-NBS)-lysine Using Mitsunobu Chemistry in the Center of the Peptide Sequence

4.1.2. A3 Reactions. Aza-lysines with diverse Nε substituents and restricted side-chain flexibility due to the presence of a triple bond were synthesized by copper-catalyzed Mannich reactions on aza-propargylglycine peptides anchored on Rink amide resin using aqueous formaldehyde, secondary amine, and CuI in DMSO at room temperature.22 Eighteen [aza-Lys]GHRP-6 analogues possessing aza residues at the 3-, 4-, and 5positions were made with crude purities of 17−72% in overall yields of 1−11%. In solution, A3 reactions were successful with paraformaldehyde at 80 °C in dioxane; however, after exposure to such conditions on the solid phase, semicarbazone removal with hydroxylamine hydrochloride in pyridine gave product mixtures that were avoided using aqueous formaldehyde. 4.1.3. Azacyclopeptides and A3 Macrocyclization. A dearth of methods exists to make cyclic azapeptides. For example, application of aza-amino acid chlorides and final headto-tail cyclization gave azacyclopeptide analogues of the anticancer cyclic peptide cilengitide (vide infra).28 No method had previously explored the potential for using aza residues in side chain-to-side chain cyclization. In view of the merits of A3 chemistry for aza-lysine synthesis,22 A3 cyclization was perceived to be favorable because the aza residue promotes turn geometry and the copper catalysis has potential to bring together the acetylene and amine prior to ring closure.52 The multicomponent strategy offers the opportunity to introduce diversity for SAR studies, in part because of its potential for making related macrocycles by acetylene reduction. The resulting tertiary amine cross-link could also favor salt bridges, bioavailability, and molecular recognition. By means of Cu-catalyzed A3 macrocyclizations, tertiary propargylamine cross-links were installed using formaldehyde as the linchpin between aza-propargylglycine and lysine residue side chains to provide macrocycles with ring sizes of 16−25 atoms, e.g., 44−48, 52−55, 58a−c, and 62−64 (Schemes 7 and 8). Diverse Nε-alkyllysines were introduced by Mitsunobu alkylation of Nε-o-nitrobenzenesulfonyl residues with various alcohols in the presence of aza-glycine residues prior to propargylation of the latter with propargyl bromide and cesium carbonate. On longer sequences, A3 macrocyclization as the penultimate step before simultaneous deprotection and resin

elongation, cleavage, and HPLC purification resulted in four [aza-Glu4]GHRP-6 and five [aza-Glu3]GHRP-6 analogues. Conjugate addition/elimination reactions onto aza-glycine 4 using reactive allylic acetates in the presence of BTPP were also successful, affording 4-alkylidine aza-glutamate residues 28−31 as mixtures of (E)- and (Z)-olefin isomers (Scheme 5b). Semicarbazide liberation and elongation converted azapeptides 28−30 into [4-alkylidene-aza-Glu4]GHRP-6 analogues; however, semicarbazone 31 failed to react under the trans-imination conditions.

4. “LIBRARIES FROM LIBRARIES” 4.1. Aza-propargylglycine Modification

The concept of building a library of analogues from a member of a combinatorial library has been employed to expand diversity from groups of common intermediates.50,51 Azapropargylglycine and aza-ω-chloroalkylglycine proved to be particularly promising for building “libraries from libraries”. Specifically, aza-propargylglycine was diversified through copper-catalyzed azide−alkyne cycloaddition (CuAAC)23 and Mannich reagent additions (A3 reaction)22 to generate aza1,2,3-triazole-3-alanine and aza-lysine residues, respectively (Scheme 6A,B). In solution, Sonogashira cross-coupling to aza-propargylglycine followed by base-promoted 5-exo-dig cyclization gave novel turn-inducing 4-substituted N-aminoimidazolin-2-ones (Scheme 6C,D).41 4.1.1. 1,3-Dipolar Cycloadditions. Aza-propargylglycine resin swollen in DMSO reacted in a one-pot aryl azide formation/CuAAC sequence to give aza-1,2,3-triazole-3alanines using CuI, aryl iodide, sodium azide, triethylamine, Lproline, and powdered molecular sieves at 80 °C for 24 h (Scheme 6A).23 The powdered sieves proved to be critical for high conversion (61−82%). Moreover, removal of trace amounts of copper by resin agitation with 0.1 N HCl in DMF facilitated subsequent semicarbazide liberation and azapeptide elongation. Electron-poor and -rich aryl iodides were used to produce seven [aza-1,2,3-triazole-3-alanine4]GHRP-6 analogues with crude purities ranging from 52 to 69%.23 1547


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Figure 1. Nai peptide X-ray structures.

Scheme 9. [Aza-Lys4]-, [Aza-Orn4]-, and [Aza-Arg4]GHRP-6 from Aza-ω-chloroalkylglycines

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Accounts of Chemical Research Scheme 10. [Aid4]GHRP-6 Synthesis by Bisalkylation with 1,3-Dibromoethane

butoxycarbonyl)-2-methyl-2-thiopseudourea gave access to azaArg analogues (e.g., 73b). Alternatively, chloride displacement with primary, secondary, and tertiary amine nucleophiles in DMF provided various ω-N-alkyl aza-lysine and aza-ornithine residues. By this method, 13 GHRP-6 analogues possessing azaLys and aza-Orn residues at the Ala3, Trp4, and Lys6 positions were synthesized with crude purities of 23−63% in isolated yields of 2−9%.21 Similarly, chloride displacement from aza-ωchlorobutenylglycines gave aza-Lys mimics with restricted (E)and (Z)-alkene side chains that were used to study GHRP-654 as well as peptide inhibitors of chromobox homologue 7.55

cleavage proved to be more effective than peptide elongation after cyclization. In total, 15 diverse cyclic aza-GHRP-6 analogues were synthesized, and some exhibited unprecedented affinity for CD36 and the capacity to modulate Toll-like receptor agonist-induced signaling in macrophages. 4.1.4. 5-Exo-Dig Cyclization: Nai Residues. Targeting peptidomimetic scaffolds that combine electronic (e.g., azapeptide) and cyclic (e.g., Freidinger lactam) constraints, N-aminoimidazolin-2-one (Nai) analogues were synthesized by 5-exo-dig intramolecular cyclization of aza-propargylglycinyl dipeptides (Scheme 6D).41 Although transition metal catalysis failed to induce cyclization, Nai analogues were made effectively using NaH in acetonitrile, albeit with racemization of Cterminal carboxylates. Mixtures of exo and endo double-bond isomers were converted to the latter by olefin migration during tert-butyl ester removal with TFA, semicarbazone cleavage with hydroxylamine hydrochloride, and sitting in chloroform-d6 during examination by NMR spectroscopy. Sonogashira reactions on aza-propargylglycinyl dipeptide using various aryl iodides prior to cyclization provided 4-substituted Nai residues.41 X-ray crystallographic analysis showed that 4methyl- and 4-benzyl-Nai peptides 65 and 66 adopted type II β- and γ-turns, respectively, in the solid state (Figure 1).53

4.3. Semicarbazone Bisalkylation: Aid Residues

N-Aminoimidazolidin-2-one (Aid) peptide mimics are the saturated counterparts of Nai residues (vide supra) and were synthesized by bisalkylation of aza-glycine with 1,2-dibromoethane and Et4NOH (Scheme 10 and Figure 2).56 In an

4.2. Aza-ω-chloroalkylglycine Modifications

Aza-lysines possessing acetylene, olefin, and saturated side chains could in principle be obtained by hydrogenation of A3 reaction products from aza-propargylglycine.22 However, alkylation of aza-glycine with α,ω-dihaloalkanes proved to be more straightforward for generating libraries of aza-lysine residues possessing olefin and saturated side chains. Aza-glycine alkylation with 1-bromo-3-chloropropane, 1-bromo-4-chlorobutane, and (E)- and (Z)-1,4-dichloro-2-butene effectively provides the corresponding aza-ω-chloroalkylglycines.21,54,55 Displacement of the ω-chloride with azide and amines gave ultimate access to aza residues possessing basic side chains, i.e., aza-Lys (saturated and E- and Z-unsaturated), aza-Orn, and azaArg mimics (Scheme 9).21,54,55 Both aza-Lys and aza-Orn GHRP-6 analogues (e.g., 73a) were prepared by displacement of the corresponding aza-ωchloroalkylglycine (e.g., 67) with sodium azide, elongation to the full-length azapeptide, chemoselective azide reduction using tris(2-carboxyethyl)phosphine (TCEP), and resin cleavage. Guanidinylation of aza-ornithine (e.g., 70) using 1,3-bis(tert-

Figure 2. X-ray analysis of Aid peptide 76.

investigation of bisalkylation of nine different N-terminal azaglycine dipeptides, Aid residues were installed in quantitative conversions on Rink amide resin, except in the case of the cysteine residue, which yielded a product mixture; side-chain benzyl ester hydrolysis and Wang resin cleavage using Et4NOH were avoided using BTPP as the base. The optimized conditions were utilized to Aid scan positions 1−5 of the sequence, giving five [Aid]GHRP-6 analogues in 6−16% yield. 1549


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Accounts of Chemical Research Scheme 11. Aza-glycine Peptide Synthesis by Carbazate Coupling Using DSC

1765 cm−1, and crystallized in the case of 83e. X-ray analysis of 83e showed an (E)-diazine with NN bond length of ∼1.24 Å and dihedral angle of −173° (Figure 3).24

X-ray crystallography of Aid peptide 76 indicated two pairs of 10-membered hydrogen-bonded conformers in the unit cell, each pair having respectively dihedral angles approaching ideal type II′ and type II β-turns (Figure 2).57 In solution, NMR titration studies on 76 supported a 10-membered hydrogen bond between the benzamide carbonyl and the solvent-shielded isopropylamide NH. Like its Nai counterpart, the saturated Aid residue was situated at the i + 1 position in the β-turn but adopted both type II′ and type II geometries because of greater ring pucker flexibility. Solid-phase installation of Aid residues into peptides thus offers an effective means for β-turn mimicry, albeit without ring substituents to mimic side-chain function.

5.2. Diels−Alder Cycloadditions

Cycloadditions of azodicarboxylates with dienes were notably described by Diels58 and later with Alder59 prior to the Nobel Prize discovery of the all-carbon variation that bears their names.60 So-called Diels−Alder cyclizations on azopeptides using 1,3-butadiene, 2,3-dimethylbutadiene, and 2-methyl-1,3butadiene yielded aza-pipecolates 85−88 in 92−99% yield, 88c as a 1:1 regioisomeric mixture (Scheme 13). Cycloadditions with cyclopentadiene similarly gave aza-methanopipecolates 89b−e and 90c in 96−98% yield as 1:1−4:1 mixtures of diastereomers as ascertained by SFC.24

5. AZOPEPTIDE SYNTHESIS AND PERICYCLIC CHEMISTRY

5.3. Alder−Ene Reaction

5.1. Aza-glycine Oxidation

Alder−ene chemistry on azopeptides was explored employing 1,3-cyclohexadiene, isobutylene, and cycloheptatriene under two different sets of conditions. Aza-allylglycines 91−94 were typically obtained upon treatment of azoglycines 83c−e with cyclohexadiene and isobutylene in CH2Cl2, but cycloheptatriene did not react under these conditions. Cycloheptatriene and isobutylene reacted with azopeptide in the presence of NBS and pyridine at −78 °C in CH2Cl2 to give β-substituted analogues 93c, and 94c, respectively. Although α-substituted product was usually obtained using both sets of conditions (e.g., 91c and 92d), in cases in which no reaction was observed without NBS and pyridine, β-substituted product resulted from adding these reagents (Scheme 14).24 The location of the side chain after Alder−ene reaction was determined by a combination of methods: NMR spectroscopy, MS fragmentation, and X-ray analysis. The latter established αand β-substitution for cyclohexadiene and cycloheptatriene analogues 91c and 93c, which adopted conformers similar to ideal type I and type VI β-turns, respectively (Figure 3). Azopeptides are thus useful for synthesizing aza-pipecolate and aza-allylglycine peptides that adopt biologically relevant turn structures.24

Azopeptides possess an imino urea as an amino amide surrogate. In view of the rich chemistry of azodicarboxylates, azopeptides were pursued to study particularly their pericyclic chemistry. Alkyl carbazates 77 and 78 were activated with DSC and coupled with amino esters to give good yields of carbamate-protected aza-glycine dipeptides 81a and 82a and tripeptides 81b and 82b with minimal oxadiazole formation (Scheme 11).42 Alternatively, semicarbazide protection gave NCbz- and N-Boc-aza-glycines 81c−e and 82c−e, respectively (Scheme 12).24 Scheme 12. Azapeptide Oxidation to Azopeptide

Azopeptides were made by oxidation of aza-glycines 81 and 82 using N-bromosuccinimide and pyridine in dichloromethane at −78 °C to room temperature, isolated in 97−99% yield after an aqueous bicarbonate workup, and used without further purification.24 Albeit relatively unstable, the azopeptides appeared as single yellow spots on TLC, exhibited IR spectra with characteristic NN stretching bands between 1700 and

6. BIOMEDICAL APPLICATIONS 6.1. CD36 Receptor Ligands

Over 80 aza analogues of GHRP-6 were synthesized while developing submonomer methods. Certain aza-GHRP-6 analogues exhibited selectivity for CD36 without significant affinity for the growth hormone secretagogue receptor 1a 1550


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Figure 3. X-ray structures of (a) azopeptide 83e and (b) azapeptides 91c and 93c. The dotted line indicates the inferred hydrogen bond.

Scheme 13. Aza-pipecolyl Peptide Synthesis by Diels−Alder Cycloaddions of Azopeptides

Scheme 14. Aza-allylglycine Peptide Synthesis by Alder−Ene Reactions of Azopeptides

(GHS-R1a).61 The selective CD36 modulators affected angiogenesis (Figure 4c) and serve as prototypes toward treatments of age-related macular degeneration, tumors, and diabetic retinopathy. Aza-amino acid chlorides were initially used to synthesize a library of ∼50 aza-GHRP-6 analogues.20 An aza-amino acid scan was first performed using N′-substituted fluorenylmethyl carbazates, which were prepared in solution, activated with phosgene in toluene, and coupled onto peptides linked to Rink amide resin.20,30 A set of azapeptide candidates were subjected

to an alanine scan of their non-aza residues. Receptor-selective ligands were identified that maintained affinity for CD36 but exhibited a >104-fold decrease in GHS-R1a receptor affinity (Figure 4a). Reinforcement of a β-turn geometry about the DTrp2-Ala3-Trp4 region of GHRP-6 appeared to be critical for receptor selectivity.18 In a microvascular sprouting assay on choroidal explants, [azaTyr4]GHRP-6 and [Ala1, azaPhe4]GHRP-6 inhibited and promoted blood vessel growth, respectively, despite the fact that the two azapeptides exhibited 1551


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Figure 4. Selected data on GHRP-6 analogues exhibiting CD36 receptor selectivity: (a) IC50 values for binding to GHS-R1a and CD36 receptors; (b) CD spectra in water; (c) microvascular sprouting from choroidal explants treated with azapeptides (10−5 M, scale bar 50 μm) for 4 days.

Figure 5. AVPI 95, indolizidinone 96, and aza-Smac mimics 97−106 (Val side chain highlighted).

similar circular dichroism (CD) spectra indicative of β-turn conformations (Figure 4b,c). Submonomer synthesis was employed to study lead azaGHRP-6 analogues to gain understanding of their divergent biology and ideally to improve their CD36 affinity. Analogues having aza residues with carboxylate side chains at the Trp4 position exhibited generally better affinity than the corresponding azapeptides possessing aliphatic (i.e., aza-Leu) and polar side chains (i.e., phophonate and cyano groups),18 which was rationalized in terms of potential to form salt bridges at the lysine-rich domain of CD36.61 A breakthrough in CD36 affinity was achieved using A3 macrocyclization. Cyclic aza-GHRP-6 analogues 54 and 55 exhibited improved CD36 affinity, with IC50 values of 0.08 and 0.49 μM, respectively, compared with 2.03 μM for GHRP-6, as well as the ability to inhibit Toll-like receptor-2 agonist-induced nitric oxide formation and release of pro-inflammatory cytokines and chemokines in macrophages.27

6.2. Smac Mimicry

Programmed cell death (apoptosis) maintains tissue integrity by eliminating old and abnormal cells. Smac is an inhibitor of apoptosis protein (IAP)-binding protein. In response to apoptotic stimuli, Smac is released to bind IAPs and promote apoptosis by liberating caspases.62 Accordingly, Smac mimics have garnered interest as anticancer agents. Constraint of the N-terminal AVPI tetrapeptide sequence of Smac (95) using azabicycloalkanone amino acids has produced inhibitors such as indolizidinone 96 that promote apoptosis by blocking interactions between certain IAPs and caspases,63 likely through mimicry of a preferred β-turn binding geometry.64 However, azabicycloalkanone Smac mimic synthesis is relatively lengthy, requiring multiple steps. To facilitate SAR studies, azapeptide analogues of the AVPI sequence (e.g., 97−101) were designed, synthesized effectively by submonomer methods, and tested for their ability to induce apoptosis in cancer cells.25,65 In MDAMB-231 breast cancer cells, aza-Gly and aza-Phe analogues 97 1552


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Accounts of Chemical Research Scheme 15. [azaF4]Cilengitide Synthesis

Figure 6. Cilengitide, cyclo[RGDfV], and azacyclopeptides 111−117.

without removal of side-chain protection, cyclized in solution, and deprotected to give seven cyclic azapeptides 111−117 possessing an aza-glycine (azaG), aza-aspartic acid (azaD), azaphenylalanine (azaF), or aza-valine (azaV) residue (Scheme 15 and Figure 6). The aza-glycine analogues retained nanomolar activity, and cyclo[R-azaG-DfV] (112) exhibited better receptor selectivity than cilengitide, but the azacyclopeptides exhibited generally decreased affinities for the αvβ3 and α5β1 integrin receptors, likely because of the influence of the aza residue on the backbone geometry.

and 99 efficiently induced cell death by a caspase-9-mediated pathway. The relationship between active azabicycloalkanone and azapeptide AVPI mimics may be due to a similar preferred turn geometry.65 From azopeptides, AVPI analogues possessing constrained aza-valine residues (e.g., 102−106; Figure 5) were made using pericyclic chemistry and methods for effective coupling on relatively bulky electron-deficient semicarbazides.26 In MCF-7 breast cancer cells, aza-cyclohexanylglycine 106 induced cell death more efficiently than the parent tetrapeptide, likely by a caspase-9-mediated apoptotic pathway.25 6.3. Azacyclopeptide Integrin Ligands

7. CONCLUSION Azapeptide libraries are valuable for SAR studies in ligand− receptor chemical biology. Submonomer methods have facilitated azapeptide library synthesis, providing effective side-chain incorporation on the aza residue using chemoselective N-alkylations, N-arylations, and conjugate additions. Submonomer construction of aza-propargylglycine and aza-ωchloroalkylglycine residues has enabled synthesis of libraries from libraries using A3 coupling, CuAAC, Sonogashira, and nucleophilic substitution chemistry. Moreover, submonomer

Integrin receptors play key roles in cell adhesion and development. Implicated in tumor metastasis and angiogenesis, they are targets for treating cancer.66 The cyclic peptide cilengitide [cyclo(RGDf-N(Me)V)] is an αvβ3 and αvβ5 integrin antagonist that has shown activity with few side effects in clinical trials of patients with glioblastoma.67 A general approach for azacyclopeptide synthesis was developed and applied to study cilengitide and cyclo(RGDfV). 28 On chlorotrityl resin, N-Fmoc-aza-amino acid chlorides were used to make linear azapeptides,37 which were cleaved from the resin 1553


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Accounts of Chemical Research methods have provided novel heterocyclic turn mimics (e.g., Nai and Aid residues) that combine electronic and covalent constraints to control the peptide conformation. Oxidation of aza-glycine residues has facilitated exploration of the pericyclic chemistry of azopeptides, providing access to aza-pipecolate and aza-allylglycine derivatives through Diels−Alder and Alder−ene reactions. Applying such azapeptide chemistry to study GHRP-6, Smac mimetics, and cilengitide has provided critical information about conformational and side-chain requirements to improve activity and receptor selectivity. Importantly, A3 macrocyclization afforded azacyclopeptides exhibiting up to 25-fold-increased affinity toward CD36 relative to GHRP-6. Overall, these practical synthetic methods for preparing azapeptides have expanded side-chain diversity and facilitated the application of libraries to study the biochemical, physical, and medicinal properties of peptide architypes.

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in drug discovery, including submonomer azapeptide synthesis, aminolactam scanning, and azabicycloalkane amino acid libraries for the evolution of peptide leads into peptidomimetic drug candidates.

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ACKNOWLEDGMENTS We thank the NSERC of Canada, the CIHR, the Ministère du développement économique de l’innovation et de l’exportation du Quebec (878-2012), Amorchem, and Mperia Therapeutics Inc. for support.

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ABBREVIATIONS GHRP-6, growth hormone-releasing peptide 6; CD36, cluster of differentiation 36; GHS-R1a, growth hormone secretagogue receptor 1a; BTTP, (tert-butyliminotris(pyrrolidino)phosphorene; DSC, N,N′-disuccinimidyl carbonate; Smac, second mitochondria-derived activator of caspase; IAP, inhibitor of apoptosis protein

AUTHOR INFORMATION

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Corresponding Author

*E-mail: [email protected].

REFERENCES

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ORCID

Ramesh Chingle: 0000-0003-1175-2454 Caroline Proulx: 0000-0003-3851-793X Present Address †

C.P.: Department of Chemistry, North Carolina State University, Raleigh, North Carolina 27695, USA. Author Contributions

The manuscript was written through contributions of all authors, who have given approval of the final version. Notes

The authors declare no competing financial interest. Biographies Ramesh Chingle received his M.Sc. with honors in Organic Chemistry (2006) from Goa University, where he studied the synthesis of antidiabetic drugs and coumarin natural products with Dr. Santosh Tilve. After a six-year tenure as research scientist at Bristol Myers Squibb Biocon Research Center with Syngene International Ltd., India, where he was distinguished for his productivity and problemsolving skills, in 2013 he began graduate studies at the Université de Montréal under the supervision of Professor William D. Lubell. His scientific research interests focus on the design, synthesis, pericyclic chemistry, and biomedical applications of azopeptides. Dr. Caroline Proulx obtained her Ph.D. in Chemistry in 2012 from the Université de Montréal under the guidance of Professor William D. Lubell, where she developed methods for combinatorial azapeptide synthesis. As an NSERC postdoctoral fellow (2012−2016) at the Molecular Foundry, Lawrence Berkeley National Laboratory, she studied peptoid synthesis and self-assembly with Dr. Ronald N. Zuckermann. In July 2016, she joined the faculty at North Carolina State University as an assistant professor, focusing on the design, synthesis, folding, and function of peptide mimics. Dr. William D. Lubell received his B.A. from Columbia College in 1984 and his Ph.D. from the University of California, Berkeley, in 1989 under the supervision of Professor Henry Rapoport, after which he studied as a postdoctoral fellow with Professor Ryoji Noyori at Nagoya University in Japan. In 1991, he joined the Chemistry Department of the Université de Montréal, where he is a full professor. Studying novel approaches for educating and performing research on medicinal chemistry, he has made seminal advances toward employing peptides 1554


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Azapeptide Synthesis Methods for Expanding Side-Chain Diversity for

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