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Amino acidic scaffolds bearing unnatural side chains: An old idea generates new and versatile tools for the life sciences
Bioorganic & Medicinal Chemistry Letters 24 (2014) 5349–5356
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Amino acidic scaffolds bearing unnatural side chains: An old idea generates new and versatile tools for the life sciences Andrea Stevenazzi ⇑, Mattia Marchini, Giovanni Sandrone, Barbara Vergani, Maria Lattanzio Italfarmaco Research Centre, Italfarmaco SpA, Via dei Lavoratori 54, I-20092 Cinisello Balsamo, Milan, Italy
a r t i c l e
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Article history: Received 29 May 2014 Revised 26 September 2014 Accepted 2 October 2014 Available online 13 October 2014 Keywords: UAA Unnatural amino acid Synthesis Drugs Biomaterials Bioconjugates
a b s t r a c t The unnatural amino acids (UAAs) are members of a class of molecules with relevant impacts in the life sciences. Due to the role of these molecules in the modulation of the chemical and physical properties of biological and inorganic materials, UAAs have attracted increasing interest in recent years. The aim of this review is to highlight (i) the most recent and innovative synthetic routes for the preparation of UAAs, (ii) the recently marketed UAA-based drugs, and (iii) the most promising technological applications involving novel UAA-containing molecular entities. Ó 2014 Published by Elsevier Ltd. This is an open access article under the CC BY-NC-SA license (http:// creativecommons.org/licenses/by-nc-sa/3.0/).
Proteinogenic amino acids are considered pivotal chiral pool material. In addition to the 20 well-known amino acids, a plethora of analogues with modified side chains and backbones or atypical stereochemistry can also be considered, widening the scope of investigation and increasing the chances for success. Although some of these non-standard amino acids, such as ornithine, which is present in the urea cycle, are naturally occurring, these amino acids are generally termed ‘non-natural’ or ‘unnatural’ amino acids (UAAs). Unlike the proteinogenic amino acids, for which the preferred collection method is extraction from natural sources, most non-standard amino acid analogues must be synthesized. For this reason, as well as the growing awareness of the relevance of these molecules in several technological contexts, UAAs have been the subject of a relatively large number of scientific investigations in recent decades (Fig. 1). In addition to the use of amino acid scaf-
Abbreviations: AA, amino acid; UAA, unnatural amino acid; ADC, antibody-drug conjugate; DOPA, L-3,4-dihydroxyphenylalanine; flp, 4-(R)-fluoroproline; GnRH, gonadotropin-releasing hormone; hyp, hydroxyproline; LAR, long acting release; MOF, metal–organic framework; 4-DMAP, 4-N,N-dimethylaminophthalimide; 6DMN, 6-N,N-dimethylamino-2,3-naphthalimide; TOAC, 4-amino-1-oxyl-2,2,6,6-tetramethyl-piperidine-4-carboxylic acid; SASA, solvent-accessible surface area; SLP, silk-like properties; 3D, tridimensional. ⇑ Corresponding author. Tel.: +39 02 64433097. E-mail addresses: [email protected] (A. Stevenazzi), m.marchini@ italfarmaco.com (M. Marchini), [email protected] (G. Sandrone), [email protected] (B. Vergani), [email protected] (M. Lattanzio).
Figure 1. Growing interest towards unnatural amino acids within the last decades (number of annual publications between 1980 and 2012, SciFinder Scholar, research topic search: unnatural amino acids).
folds as building blocks during drug discovery1 and the unusual role of amino acids as reactants for important drug-like compounds2 (e.g., benzodiazepines), the conserved backbones and their variable side chains are potentially relevant for a wide number of applications, which span from probing protein function to adhesive agents and biomedical products. Among the increasing number of publications on this subject, very few general reviews
http://dx.doi.org/10.1016/j.bmcl.2014.10.016 0960-894X/Ó 2014 Published by Elsevier Ltd. This is an open access article under the CC BY-NC-SA license (http://creativecommons.org/licenses/by-nc-sa/3.0/).
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addressing UAAs, including their synthesis and their use in diverse fields, are currently available in the literature. The current review will focus on a-UAAs, providing an overview of the most recent and the most innovative synthetic routes for the preparation of these molecules and an overview of recently marketed UAA-based drugs. This review will also include a discussion of the most promising technological applications that involve novel UAA-containing molecular entities in the life science disciplines and beyond. Synthesis of UAAs: Access to UAAs has attracted attention in recent years.3 The synthetic routes that are currently available can be reduced to a small number of diversity-oriented transformation categories, which differ from one another with respect to the starting materials employed and the number of bonds formed (see Fig. 2). The most intuitive procedures are based on alkylations that involve glycine equivalents or the widest range of modifications to amino acid side chains. Multicomponent or tandem reactions that proceed via imines or other activated intermediates are likely the most synthetically attractive routes, even though these reactions often lack efficiency or stereoselectivity. Due to the large number of methodologies and examples available, we chose to focus our discussion on only the most innovative approaches, including (but not limited to) the stereoselective multicomponent synthesis of non-proteinogenic a-amino acids. Several multicomponent access routes to UAAs involving an imine as the key intermediate (see Fig. 2a) were discussed in the literature. The asymmetric Petasis borono–Mannich reaction represents one of the most powerful and straightforward approaches for the stereoselective preparation of a-amino acids. Strategies involving chiral amines, chiral boronate esters, and chiral organocatalysts have been employed, but to date, none of these strategies has yielded satisfactory results in the synthesis of a-amino acids. Li and Xu4 developed an extremely mild and practical approach for the asymmetric synthesis of b,c-unsaturated a-amino acids that
employs a highly diastereoselective Lewis acid-promoted Petasis three-component-reaction of N-tert-butanesulfinamide in the presence of glyoxylic acid and a vinylboronic acid. This reaction was conducted at room temperature and optimized with respect to Lewis acid (InBr3), solvent (CH2Cl2), concentration (0.3 M) and time (12 h), with the aim of enhancing diastereoselectivity and yield (Fig. 3a). This method has proven efficient and general with respect to substrate scope and stereocontrol. Not only styryl boronic acids but also alkenyl and benzo-condensed aryl boronic acids performed well under the optimized conditions. After establishing carboxylation reactions of mono-protected N-Boc-a-amido stannanes using CO2 as a C1 source, Mita and Sato5 developed a one-pot synthesis of a-amino acids based on imine formation, stannylation or silylation, and final carboxylation (Fig. 3b). This reaction was made possible by the combination of CsF and Me3Si–SnBu3 or PhMe2Si–Bpin reagents and gave products with yields up to 91%. a,a-Dialkylglycines are sterically constrained amino acids that are of great interest due to their properties when incorporated into peptides. In fact, these amino acids increase proteolytic stability and confer specific conformations. The synthesis of several a,a-dialkylglycines was achieved using a solid phase Ugi reaction performed over isocyanide functionalized resins, with phenyl acetic acid as the acid component, 4-methoxybenzylamine as the amine component and diverse ketones to afford the desired N-acylated a,a-dialkylglycines in good overall yields (60–80%) after acidolytic cleavage from the resin (Fig. 3c).6 A recent diversity-oriented approach envisaged a solvent/catalyst-free Biginelli condensation to prepare a series of propargylated dihydropyrimidinone scaffolds, which may be considered cyclic b-amino acids.7 These precursors were further decorated through cycloaddition reactions with small peptide-like azides synthesized from Ugi or Mannich reactions (Fig. 3d). Various hydroxyaldehydes were functionalized via base-catalyzed condensation with propargyl bromide before performing the Biginelli
Figure 2. Retrosynthetic approaches to a-amino acids: (a) multicomponent reactions via imine; (b) other tandem reactions; (c) glycine equivalents; (d) side chain modifications.
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Figure 3. Multicomponent access to a-amino acids through an imine intermediate: (a) an asymmetric Petasis borono-Mannich access to UAA; (b) Mita and Sato one pot approach to a-amino acids, based on imine formation, stannylation or silylation, and final carboxylation; (c) synthesis of N-acylated a,a-dialkylglycines via solid phase Ugi reaction.
Figure 4. Alternative multicomponent/tandem/domino approaches to a-amino acids: (a) multicomponent one-pot approach to a-aryl serine derivatives; (b) synthesis of N-protected a-amino acid esters; (c) synthesis of a and b homophenylalanine precursors via sequential allylic C–H amination/vinylic C–H arylation; (d) access to bicyclic b-prolines and a,b,c-triamino acids via a combination of anionic aza-Michael additions, a diazo transfer and 1,3-dipolar cycloadditions.
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reaction in the presence of methyl acetoacetate or ethyl cyanoacetate as the CH acidic oxo components and urea as the diamide component. Other multicomponent routes envisage the formation of alternative activated species as an intermediate. Wang et al. proposed a multicomponent one-pot approach to a-aryl serine derivatives.8 The reaction involves the in situ Rh-catalyzed generation of ammonium ylides from aryl-substituted diazoacetates and anilines and the trapping of these molecules by formaldehyde (Fig. 4a). However, yields were only moderate (30–69%), the substrate scope was limited to 2-substituted anilines and aryldiazoacetates, and an asymmetric version of the reaction remains to be investigated. The synthesis of a series of N-protected non-proteinogenic a-amino acid esters from glyoxylates and carbamates was reported by Ali and Fathalla.9 The key hemiacetal intermediates were activated to a-substitution as trichloroacetimidates or acetates, giving the desired products in good yields with short reaction times (Fig. 4b). Also fascinating cascade approaches led to the core formation of novel UAAs (see Fig. 2b). White et al. exploited Pd(II)/sulfoxide catalysis to promote a sequential allylic C–H amination/vinylic C–H arylation, which started from inexpensive commercially available a-olefins and boronic acids, to provide a wide range of a- and b-homophenylalanine precursors (Fig. 4c).10 Apart from the outstanding regio- and E:Z selectivities of this first tandem reaction step, the route to the desired enantiomerically pure amino acids required several steps, with low overall yields. An unprecedented combination of anionic aza-Michael additions, a diazo transfer and 1,3-dipolar cycloadditions was reported by Jahn et al. to approach bicyclic b-prolines and a,b,c-triamino acids.11 Several b-substituted Michael acceptors, such as esters or amides, could be employed, and nonaflyl azide was identified as the reagent of choice for the diazo transfer (Fig. 4d). Lithium amides were generated in situ via deprotonation of the corresponding amine with n-BuLi. The use of chiral amines provided highly diastereoselective products. a,b,c-Triamino acids were obtained via hydrogenolytic opening of the pyrazoline ring. Access to UAAs using glycine derivatives as starting reagents (see Fig. 2c) or by modifying other amino acids side chains (see Fig. 2d) has been extensively reviewed in recent years.12–15 However, noteworthy examples continue to be reported in the literature. An efficient, enantioselective preparation of fluorinated a-amino acids via Ni-catalyzed Mitsunobu–Tsunoda alkylation of a chiral nucleophilic glycine equivalent (Fig. 5) with alcohol
Figure 5. Ni-catalyzed asymmetric synthesis of fluorinated a-amino acids from glycine.
substrates was recently discussed by Brimble et al.16 Several diversity-oriented strategies that started from glycine equivalents were described by Kotha et al.17 Highly atom-economic processes, such as the Diels–Alder reaction, [2+2+2] cycloaddition, Suzuki– Miyaura cross-coupling, and olefin metathesis, were employed to access UAA derivatives and peptides. Side chain modifications may be considered the most obvious transformations for accessing UAAs; nonetheless, some examples are worth mentioning. The Catellani reaction is one of these transformations. Göbel and Lautens reported the synthesis of simple to more complex substituted aromatic systems in a Pd-catalyzed cascade fashion; these products were used as the precursors for novel amino acids with a basic side chain (Fig. 6a).18 Sinha et al. reported the stereoselective synthesis of 2- and 3-indolylglycine derivatives and their oxygen analogues starting from 2-iodoheteroarenes.19 A tandem Sonogashira/cyclization reaction with ethynyloxazolidinone gave access to 2-indoylglycine analogues, while 3-indolylglycine derivatives were synthesized from silylated internal alkyne using Larock’s heteroannulation as the key reaction (Fig. 6b). UAA-based Drugs Available on the Market: In recent years, the design of peptidomimetics and peptide analogues has attracted increasing interest in the field of drug research. The discovery of a multitude of naturally occurring bioactive peptides represented a source of enlightment for medicinal chemists in the development of new therapeutic drugs. Although natural amino acid-based peptides find wide applications as drugs,20 major drawbacks, such as rapid proteolytic metabolism and poorly selective interactions, limit the application of these peptides. Hence, peptidomimetics could serve as privileged surrogates, maintaining the ability to modulate and control biological functions while offering several advantages, such as improved in vivo stability, enhanced potency, better oral absorption, improved tissue distribution and increased selectivity during biological responses.21,22 Thus, UAAs have been employed as building blocks with the aim of introducing conformational constraints and optimizing fit (i.e., tertiary structure) and interactions (e.g., hydrogen bonding and electrostatic or hydrophobic interactions) with the target active site. Of the several classes of UAAs that are available, examples of cyclic and bicyclic proline-like amino acids and non-proteinogenic AAs bearing aromatic or cyclic aliphatic side chains are herein discussed in more depth. Natural amino acids bearing heavy atoms (i.e., S, O, and N) suitable for further derivatization provide a large number of possible UAAs. Serine, threonine, cysteine and tyrosine, along with acidic and basic residues, as already discussed in the previous section, could potentially undergo easy transformations and derivatizations. Although this strategy represents the most intuitive and facile approach for introducing structural and/or chemical diversity, only a small number of UAAs of this class were successfully incorporated into approved drugs. Abarelix23 (Fig. 7, compound 1, GlaxoSmithKline) is a gonadotropin-releasing hormone (GnRH) antagonist that contains an N-isopropyl lysine, Lacosamide24 (Fig. 7, compound 2, NewBridge Pharmaceuticals) displays an O-methyl-serine residue, Pasireotide (Fig. 7, compound 3) contains an O-benzyl-tyrosine, and the orphan drug Histrelin25 (Fig. 7, compound 4, Ortho-McNeil-Janssen) has a benzyl-bearing histidine. Amino acidic scaffolds that exhibit tunable annular side chains are well known features in peptidomimetic design. In fact, the relevance of proline-like UAAs to drug discovery is primarily attributed to their typical characteristics, which can influence the secondary structure of a molecule. Because they contain no amide hydrogen, these molecules cannot donate a hydrogen bond and they possess a certain structural rigidity. Moreover, (i) the modulation of the ‘backbone’ rigidity by increasing the number of atoms in the ring (see the Sanofi products Bupivacaine26 and Quinupristin,27 Fig. 7, compounds 5 and 6, respectively) and (ii) the ability to
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Figure 6. Synthesis of UAAs by side chain modification of natural AAs: (a) synthesis of precursors for novel amino acids with a basic side chain via Catellani reaction; (b) stereoselective synthesis of 2- and 3-indolylglycine derivatives and their oxygen analogues starting from 2-iodoheteroarenes.
expand the molecular diversity of the scaffold when starting from a derivatized substrate (i.e., hydroxyproline, Hyp) represent key strengths of these molecular tools. Several antiviral drugs exhibit an unnatural derivatized proline; the Hepatitis C virus-Nonstructural protein 3 (HCV-NS3) protease inhibitors Asunaprevir28 (Fig. 7, compound 7, Bristol-Myers Squibb) and Faldaprevir29 (Fig. 7, compound 8, Boehringer Ingelheim, pre-registered) share a large portion of their structures and differ primarily with respect to the substituent at the hydroxyl group of the Hyp. Another substitution of the Hyp’s OH group is observed in the previously cited drug Pasireotide30 (Fig. 7, compound 3, Novartis), which was launched in 2012 for the treatment of Cushing’s disease. Other very common features are fused bicyclic UAAs, which display proline-like five- or six-membered side chains fused with either aliphatic or aromatic rings. A fused proline side chain is clearly detectable in Boceprevir31 (Fig. 7, compound 9, Merck), which is a new HCV NS3 protease inhibitor, in the bradykinin B2 antagonist Icatibant (Fig. 7, compound 10, Sanofi),32 and in Telaprevir (Fig. 7, compound 11).33 These UAAs provide increased steric hindrance and enhanced favorable hydrophobic interactions compared to the corresponding monocyclic analogues. In addition to proline-like UAAs, several approved drugs have been synthesized using specific (ad hoc) non-proteinogenic residues, primarily with the aim of enhancing binding activity or improving drug profiles. Among these drugs, amino acidic scaffolds bearing aromatic side chains are quite common. Phenylglycine (in either the L- or D-form) is one of the most widely employed aromatic UAAs. As the inferior homologue of phenylalanine, this amino acid takes advantage of the main features of phenylalanine with less degrees of freedom. Phenylglycine is embedded in the antibiotics Apalcillin (Fig. 7, compound 12, Boehringer Ingelheim), Mezlocillin (Fig. 7, compound 13, Bayer) and Pivampicillin (Fig. 7, compound 14, LEO Pharma) and in the cyclic hexapeptide Pasireotide (Fig. 7, compound 3). Besides phenylglycine, the depsipeptide Quinupristin (Fig. 7, compound 6) displays an N,N-dimethyl-4-aminophenylalanine. Higher phenylalanine homologues, such as homophenylalanine, are also detectable in approved drugs. As an example, see the proteasome inhibitor Kyprolis34 (Fig. 7, compound 15, Amgen). Naphthylalanine is often present in self-assembled compounds (see below in the text) and is frequently enclosed in peptidic scaffolds to modulate weak, non-local, protein–ligand interactions, such as p–p or cation–p. The condensed rings represent efficient fragments that can fit into deep and flat hydrophobic binding pockets that display narrow breaches. Lanreotide (Fig. 7, compound 16), which is a somatostatin receptor agonist, and the radioligand
[68Ga]-DOTA-NOC35 (Fig. 7, compound 17) both exhibit a cyclic fragment with a sulfur bridge and a D-naphthylalanine in the peptide sequence. In contrast, the L-enantiomer residue is one of the building blocks of Abarelix23 (Fig. 7, compound 1, GlaxoSmithKline), which is a GnRH antagonist. In addition to one naphthylalanine residue, Abarelix contains the L-3-pyridin-3-ylalanine residue in its peptide sequence. This residue as well as 3-(2-thienyl)alanine, which is incorporated into Icatibant (Fig. 7, compound 10, Sanofi),32 are interesting aromatic building blocks characterized by a heterocyclic side chain. UAAs with side chains that bear non-aromatic cycles, with and without heteroatoms, exhibit common six-membered or more complex architectures. Boceprevir (Fig. 7, compound 9, Merck)31 includes a cyclobutylalanine in its peptide sequence, Telaprevir (Fig. 7, compound 11)33 exhibits an a-cyclohexylglycine, and Cobicistat (Fig. 7, compound 18),36 which is an HIV protease inhibitor, has 2-amino-4-(4-morpholinyl) butanoic acid as a building block. The dipeptidyl peptidase IV inhibitor Saxagliptin37 (Fig. 7, compound 19, Bristol-Myers Squibb) displays the bulky and hydrophobic (S)-2-amino-2-(3-hydroxy-1-adamantyl)-acetic acid substructure, in an amount that is equivalent to nearly 50% of the total molecular weight. Applications: Engineered amino acid entities are typically associated with conventional drug discoveries, but in the last decade, an increasing number of applications involving UAAs have been reported in the field of life sciences. Indeed the manipulation of the amino acidic scaffold can be regarded as a potentially powerful tool in both biochemical investigations and the development of biomedical devices, whereas the unique properties of peptides motivated their use in apparently exotic scientific fields, not directly related to the pharmaceutical or healthcare industries (see Fig. 8). The aim of the current section is to compile a concise but hopefully complete overview of the non-trivial applications of UAAs and to highlight potential future developments. The central idea is to illustrate all reported applications involving UAAs, neglecting the most obvious applications related to traditional drug discovery (see Fig. 8). We focus on two main subjects: (i) the replacement of natural residues to expand the lexicon related to proteins, (ii) ‘soft’ and/or ‘wet’ materials that relate to the ability of peptides to build spontaneously complex supramolecular structures. The discovery of the amber codon suppression technique in recent decades38 revealed a new and intriguing role of UAAs: the opportunity to introduce in proteins residues bearing different side chains that were designed a priori. This technique provides a
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Figure 7. Examples of currently available registered drugs displaying UAA substructures.
remarkable chance (i) to expand the number of reactions that a protein can undergo at the post-translational level and (ii) to locally manipulate the physical and chemical properties of the
proteins. Although several conditions should be satisfied (i.e., a large perturbation induced by the engineered side chain should be avoided), more than 70 new chemical entities have been
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Figure 8. Use of UAAs in life sciences: soft matter (self-assembly), bioconjugates (fluorescent probe, spin labeled amino acids, antibody-drug conjugates, ADCs) and biomedical materials (bone clays).
successfully inserted into a range of different proteins recently and the number of adoptable UAAs in proteic lexicon expansion is actually unknown.39 New synthetic routes in combination with a more rigorous stoichiometry and localization of the involved reactive sites suggest an imminent and relevant improvement of bioconjugate devices, in which a protein is covalently linked to a drug, a probe, or a generic chemical entity through a specific linker that can release or activate the payload under particular environmental stimuli. The well-known antibody-drug conjugates40,41 (ADCs) typically exhibit a drug (i.e., payload) that is connected to defined residues of the antibody through a linker. The drawbacks related to the post-translational antibody modifications restricted to proteinogenic residues are (i) the limited number of reactions available and (ii) the poor control of the stoichiometry that is caused by the presence of multiple reacting amino acids in the proteic chains, which each have the potential to exhibit different behavior. Axup and coworkers42 recently reported a new bioconjugate of Trastuzumab in which exposed lysines and cysteines on the light chain (or alanines in the antibody’s sequence) are replaced with a paraacetylphenylalanine, where the new reactive group permitted the covalent ligation of Auristatin using an alkoxy-amine precursor of the cytotoxic compound. It is worth noting that several ADC platforms based on unnatural residues are currently under active development, although the potential immunogenicity of the resulting engineered proteins must still be assessed.43 The increasing number of available synthetic routes is apparently improving even the activity of ADCs, as uncleavable bonds between anticancer agents and related moieties may result in an enhanced stability of the antibody towards lysosome degradation. The ability to introduce exogenous residues into engineered proteins accelerated the discovery of new fluorescent probes,44 which
are precious tools in both imaging and the elucidation of the molecular mechanisms of drugs. A relatively large number of papers describing amino acidic entities bearing fluorophores (such as amino acid derivatives of 4-N,N-dimethylaminophthalimide (4DMAP) and 6-N,N-dimethylamino-2,3-naphthalimide (6-DMN) on their side chains is currently available.45 Peptides or proteins bearing paramagnetic UAAs (although due to a posteriori derivatization of natural residues) can be monitored using electron spin resonance spectroscopy: TOAC (4-amino-1-oxyl-2,2,6,6-tetramethylpiperidine-4-carboxylic acid) and/or pyrroline nitroxide-containing amino acids have been successfully inserted into oligopeptides via a chemoselective ligation of the paramagnetic core structure at the unique lysine that is available in the test peptide. One of the most interesting properties of several polymeric chains is their ability to originate supramolecular architectures.46 Due to the particular intermolecular interactions that occur between monomers and in the presence of specific conditions (ionic strength, temperature and solvent effects), the mesoscale structures can exhibit several complex morphologies: flat assemblies can bend and originate single or multi-layer wall cylinders or evolve toward microspheres. The resulting intermolecular interaction networks show the pivotal role of side chains in the control of the assembly at the molecular level: modulation of chemical and physical properties of residues can allow change of shape and size of the final nano/micro-objects. Cyclopeptides with D-amino acids in their sequence are well known monomers for self-assembled nanostructures due to their unique ability to stack along a privileged direction in long fibers.47 The inclusion of UAAs bearing unnatural side chains in the sequence of the monomer can enhance or induce assembly: naphthylalanine is likely to be the key feature in the self-assembly of the somatostatin agonist Lanreotide in long
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cylinders:48 the resulting macroscopic gel exhibits a very high degree of viscosity and was adopted as a long acting release (LAR) formulation for the treatment of acromegaly. An intriguing application of peptide-based soft matter is the use as tridimensional template for cell cultures. A slab of the self-assembled material is spread onto a plastic support with a peculiar 3D morphology, allowing cell growth on the exposed surface, whereas a continuous layer of cellular entities, which eventually correspond to a tissue, can grow on the exposed surface. Several hydrogels (i.e., Fmoc diphenylalanine in water) were investigated as a potential framework for 3D cell culture: although the most relevant examples are made of natural amino acid polymers, several attempts were made using D-enantiomers of proteinogenic residues.49 Other interesting studies related to the adoption of unnatural amino acids have been conducted in the field of tissue engineering concern the development of new collagen-mimetic peptides.50 Besides the obvious peptides made from naturally occurring amino acids, oligopeptides that bear one or more unnatural residues, such as 4-(R)-fluoroproline (Flp), were designed in the last decade as a tool for elucidating the crucial role of the naturally occurring molecule 4-(R)-hydroxyproline (Hyp), which is intrinsically a UAA entity, in the triple helix stability of collagen. Tailored side chains also allow new strategies in the search of a balance between mechanical properties and biocompatibility of several biomaterials: Katti et al. reported an increased flexibility of montmorillonite-based bone cements after the introduction of amino acids with aliphatic side chains into the free volume between two adjacent layers.51 Intercalation is not the only example of inorganic frameworks that can profitably host natural and unnatural amino acids, allowing possible future applications in life science: both UAAs and naturally occurring residues may be included52,53 in the synthesis of metal–organic frameworks (MOFs) as organic ligands of the target metal ion/atom, an emerging class of materials, with a low density, a large surface area and an adjustable pore size.54 In summary, the brief overview provided herein clearly describes the large amount interest that simple molecules, such as amino acids and their UAA analogues, continue to attract. These molecules continue to represent important and versatile tools that can be used by chemists for the preparation of chiral and variously substituted scaffolds that can be applied in areas that span from drug discovery to catalysis and from solid phase synthesis to biopolymers. Sophisticated techniques, such as amber codon suppression and the intercalation or encapsulation of UAAs, allow the integration of new chemistry into biological or inorganic materials, offering advanced approaches for the chemical manipulation of the structure and function of those materials. The use of novel UAAs widens the exploration of the chemical space and opens exciting new opportunities in basic and applied science research.
11. 12. 13. 14. 15. 16.
References and notes
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Bioorg. Med. Chem. 1999, 7, 2381. Schenken, R. S. Am. J. Obstet. Gynecol. 1990, 162, 579. Adger, B.; Dyer, U.; Hutton, G.; Woods, M. Tetrahedron Lett. 1996, 37, 6399. Lamb, H. M.; Figgitt, D. P.; Faulds, D. Drugs 1999, 58, 1061. Scola, P. M.; Sun, L. Q.; Wang, A. X.; Chen, J.; Sin, N.; Venables, B. L.; Sit, S. Y.; Chen, Y.; Cocuzza, A.; Bilder, D. M.; D’Andrea, S. V.; Zheng, B.; Hewawasam, P.; Tu, Y.; Friborg, J.; Falk, P.; Hernandez, D.; Levine, S.; Chen, C.; Yu, F.; Sheaffer, A. K.; Zhai, G.; Barry, D.; Knipe, J. O.; Han, Y. H.; Schartman, R.; Donoso, M.; Mosure, K.; Sinz, M. W.; Zvyaga, T.; Good, A. C.; Rajamani, R.; Kish, K.; Tredup, J.; Klei, H. E.; Gao, Q.; Mueller, L.; Colonno, R. J.; Grasela, D. M.; Adams, S. P.; Loy, J.; Levesque, P. C.; Sun, H.; Shi, H.; Sun, L.; Warner, W.; Li, D.; Zhu, J.; Meanwell, N. A.; McPhee, F. J. Med. Chem. 2014, 57, 1730. Llinas-Brunet, M.; Bailey, M. D.; Goudreau, N.; Bhardwaj, P. K.; Bordeleau, J.; Bos, M.; Bousquet, Y.; Cordingley, M. G.; Duan, J.; Forgione, P.; Garneau, M.; Ghiro, E.; Gorys, V.; Goulet, S.; Halmos, T.; Kawai, S. H.; Naud, J.; Poupart, M. A.; White, P. W. J. Med. Chem. 2010, 53, 6466. Lewis, I.; Bauer, W.; Albert, R.; Chandramouli, N.; Pless, J.; Weckbecker, G.; Bruns, C. J. Med. Chem. 2003, 46, 2334. Rotella, D. P. Expert Opin. Drug Discov. 2013, 8, 1439. Abe, Y.; Kayakiri, H.; Satoh, S.; Inoue, T.; Sawada, Y.; Inamura, N.; Asano, M.; Aramori, I.; Hatori, C.; Sawai, H.; Oku, T.; Tanaka, H. J. Med. Chem. 1998, 41, 4062. Kwong, A. D.; Kauffman, R. S.; Hurter, P.; Mueller, P. Nat. Biotechnol. 2011, 29, 993. Kim, K. B.; Crews, C. M. Nat. Prod. Rep. 2013, 30, 600. Decristoforo, C.; Knopp, R.; von Guggenberg, E.; Rupprich, M.; Dreger, T.; Hess, A.; Virgolini, I.; Haubner, R. Nucl. Med. Commun. 2007, 28, 870. Xu, L.; Liu, H.; Hong, A.; Vivian, R.; Murray, B. P.; Callebaut, C.; Choi, Y. C.; Lee, M. S.; Chau, J.; Tsai, L. K.; Stray, K. M.; Strickley, R. G.; Wang, J.; Tong, L.; Swaminathan, S.; Rhodes, G. R.; Desai, M. C. Bioorg. Med. Chem. Lett. 2014, 24, 995. Augeri, D. J.; Robl, J. A.; Betebenner, D. A.; Magnin, D. R.; Khanna, A.; Robertson, J. G.; Wang, A.; Simpkins, L. M.; Taunk, P.; Huang, Q.; Han, S. P.; Abboa-Offei, B.; Cap, M.; Xin, L.; Tao, L.; Tozzo, E.; Welzel, G. E.; Egan, D. M.; Marcinkeviciene, J.; Chang, S. Y.; Biller, S. A.; Kirby, M. S.; Parker, R. A.; Hamann, L. G. J. Med. Chem. 2005, 48, 5025. Noren, C. J.; Anthony-Cahill, S. J.; Griffith, M. C.; Schultz, P. G. Science 1989, 244, 182. Liu, C. C.; Schultz, P. G. Annu. Rev. Biochem. 2010, 79, 413. Sammet, B.; Steinkuhler, C.; Sewald, N. Pharm Pat Anal 2012, 1, 65. Teicher, B. A. Curr Opin Oncol 2014. Axup, J. Y.; Bajjuri, K. M.; Ritland, M.; Hutchins, B. M.; Kim, C. H.; Kazane, S. A.; Halder, R.; Forsyth, J. S.; Santidrian, A. F.; Stafin, K. Proc. Natl. Acad. Sci. U.S.A. 2012, 109, 16101. Zimmerman, E. S.; Heibeck, T. H.; Gill, A.; Li, X.; Murray, C. J.; Madlansacay, M. R.; Tran, C.; Uter, N. T.; Yin, G.; Rivers, P. J.; Yam, A. Y.; Wang, W. D.; Steiner, A. R.; Bajad, S. U.; Penta, K.; Yang, W.; Hallam, T. J.; Thanos, C. D.; Sato, A. K. Bioconjugate Chem. 2014, 25, 351. Katritzky, A. R.; Narindoshvili, T. Org. Biomol. Chem. 2009, 7, 627. Krueger, A. T.; Imperiali, B. Chembiochem 2013, 14, 788. Gazit, E. Chem. Soc. Rev. 2007, 36, 1263. Lihi, A.-A.; Ehud, G. Self-Assembled Peptide Nanostructures. In Handbook of Nanophysics; CRC Press, 2010; p 1. Valery, C.; Pouget, E.; Pandit, A.; Verbavatz, J. M.; Bordes, L.; Boisde, I.; CherifCheikh, R.; Artzner, F.; Paternostre, M. Biophys. J. 2008, 94, 1782. Luo, Z.; Wang, S.; Zhang, S. Biomaterials 2011, 32, 2013. Chow, D.; Nunalee, M. L.; Lim, D. W.; Simnick, A. J.; Chilkoti, A. Mater. Sci. Eng., R 2008, 62, 125. Katti, K. S.; Ambre, A. H.; Peterka, N.; Katti, D. R. Philos. Trans. R. Soc. London, Ser. A 2010, 368, 1963. Naik, A. D.; Marchand-Brynaert, J.; Garcia, Y. Synthesis 2008, 2008, 149. Dong, L.; Chu, W.; Zhu, Q.; Huang, R. Cryst. 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Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com/locate/bmcl
BMCL Digest
Amino acidic scaffolds bearing unnatural side chains: An old idea generates new and versatile tools for the life sciences Andrea Stevenazzi ⇑, Mattia Marchini, Giovanni Sandrone, Barbara Vergani, Maria Lattanzio Italfarmaco Research Centre, Italfarmaco SpA, Via dei Lavoratori 54, I-20092 Cinisello Balsamo, Milan, Italy
a r t i c l e
i n f o
Article history: Received 29 May 2014 Revised 26 September 2014 Accepted 2 October 2014 Available online 13 October 2014 Keywords: UAA Unnatural amino acid Synthesis Drugs Biomaterials Bioconjugates
a b s t r a c t The unnatural amino acids (UAAs) are members of a class of molecules with relevant impacts in the life sciences. Due to the role of these molecules in the modulation of the chemical and physical properties of biological and inorganic materials, UAAs have attracted increasing interest in recent years. The aim of this review is to highlight (i) the most recent and innovative synthetic routes for the preparation of UAAs, (ii) the recently marketed UAA-based drugs, and (iii) the most promising technological applications involving novel UAA-containing molecular entities. Ó 2014 Published by Elsevier Ltd. This is an open access article under the CC BY-NC-SA license (http:// creativecommons.org/licenses/by-nc-sa/3.0/).
Proteinogenic amino acids are considered pivotal chiral pool material. In addition to the 20 well-known amino acids, a plethora of analogues with modified side chains and backbones or atypical stereochemistry can also be considered, widening the scope of investigation and increasing the chances for success. Although some of these non-standard amino acids, such as ornithine, which is present in the urea cycle, are naturally occurring, these amino acids are generally termed ‘non-natural’ or ‘unnatural’ amino acids (UAAs). Unlike the proteinogenic amino acids, for which the preferred collection method is extraction from natural sources, most non-standard amino acid analogues must be synthesized. For this reason, as well as the growing awareness of the relevance of these molecules in several technological contexts, UAAs have been the subject of a relatively large number of scientific investigations in recent decades (Fig. 1). In addition to the use of amino acid scaf-
Abbreviations: AA, amino acid; UAA, unnatural amino acid; ADC, antibody-drug conjugate; DOPA, L-3,4-dihydroxyphenylalanine; flp, 4-(R)-fluoroproline; GnRH, gonadotropin-releasing hormone; hyp, hydroxyproline; LAR, long acting release; MOF, metal–organic framework; 4-DMAP, 4-N,N-dimethylaminophthalimide; 6DMN, 6-N,N-dimethylamino-2,3-naphthalimide; TOAC, 4-amino-1-oxyl-2,2,6,6-tetramethyl-piperidine-4-carboxylic acid; SASA, solvent-accessible surface area; SLP, silk-like properties; 3D, tridimensional. ⇑ Corresponding author. Tel.: +39 02 64433097. E-mail addresses: [email protected] (A. Stevenazzi), m.marchini@ italfarmaco.com (M. Marchini), [email protected] (G. Sandrone), [email protected] (B. Vergani), [email protected] (M. Lattanzio).
Figure 1. Growing interest towards unnatural amino acids within the last decades (number of annual publications between 1980 and 2012, SciFinder Scholar, research topic search: unnatural amino acids).
folds as building blocks during drug discovery1 and the unusual role of amino acids as reactants for important drug-like compounds2 (e.g., benzodiazepines), the conserved backbones and their variable side chains are potentially relevant for a wide number of applications, which span from probing protein function to adhesive agents and biomedical products. Among the increasing number of publications on this subject, very few general reviews
http://dx.doi.org/10.1016/j.bmcl.2014.10.016 0960-894X/Ó 2014 Published by Elsevier Ltd. This is an open access article under the CC BY-NC-SA license (http://creativecommons.org/licenses/by-nc-sa/3.0/).
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addressing UAAs, including their synthesis and their use in diverse fields, are currently available in the literature. The current review will focus on a-UAAs, providing an overview of the most recent and the most innovative synthetic routes for the preparation of these molecules and an overview of recently marketed UAA-based drugs. This review will also include a discussion of the most promising technological applications that involve novel UAA-containing molecular entities in the life science disciplines and beyond. Synthesis of UAAs: Access to UAAs has attracted attention in recent years.3 The synthetic routes that are currently available can be reduced to a small number of diversity-oriented transformation categories, which differ from one another with respect to the starting materials employed and the number of bonds formed (see Fig. 2). The most intuitive procedures are based on alkylations that involve glycine equivalents or the widest range of modifications to amino acid side chains. Multicomponent or tandem reactions that proceed via imines or other activated intermediates are likely the most synthetically attractive routes, even though these reactions often lack efficiency or stereoselectivity. Due to the large number of methodologies and examples available, we chose to focus our discussion on only the most innovative approaches, including (but not limited to) the stereoselective multicomponent synthesis of non-proteinogenic a-amino acids. Several multicomponent access routes to UAAs involving an imine as the key intermediate (see Fig. 2a) were discussed in the literature. The asymmetric Petasis borono–Mannich reaction represents one of the most powerful and straightforward approaches for the stereoselective preparation of a-amino acids. Strategies involving chiral amines, chiral boronate esters, and chiral organocatalysts have been employed, but to date, none of these strategies has yielded satisfactory results in the synthesis of a-amino acids. Li and Xu4 developed an extremely mild and practical approach for the asymmetric synthesis of b,c-unsaturated a-amino acids that
employs a highly diastereoselective Lewis acid-promoted Petasis three-component-reaction of N-tert-butanesulfinamide in the presence of glyoxylic acid and a vinylboronic acid. This reaction was conducted at room temperature and optimized with respect to Lewis acid (InBr3), solvent (CH2Cl2), concentration (0.3 M) and time (12 h), with the aim of enhancing diastereoselectivity and yield (Fig. 3a). This method has proven efficient and general with respect to substrate scope and stereocontrol. Not only styryl boronic acids but also alkenyl and benzo-condensed aryl boronic acids performed well under the optimized conditions. After establishing carboxylation reactions of mono-protected N-Boc-a-amido stannanes using CO2 as a C1 source, Mita and Sato5 developed a one-pot synthesis of a-amino acids based on imine formation, stannylation or silylation, and final carboxylation (Fig. 3b). This reaction was made possible by the combination of CsF and Me3Si–SnBu3 or PhMe2Si–Bpin reagents and gave products with yields up to 91%. a,a-Dialkylglycines are sterically constrained amino acids that are of great interest due to their properties when incorporated into peptides. In fact, these amino acids increase proteolytic stability and confer specific conformations. The synthesis of several a,a-dialkylglycines was achieved using a solid phase Ugi reaction performed over isocyanide functionalized resins, with phenyl acetic acid as the acid component, 4-methoxybenzylamine as the amine component and diverse ketones to afford the desired N-acylated a,a-dialkylglycines in good overall yields (60–80%) after acidolytic cleavage from the resin (Fig. 3c).6 A recent diversity-oriented approach envisaged a solvent/catalyst-free Biginelli condensation to prepare a series of propargylated dihydropyrimidinone scaffolds, which may be considered cyclic b-amino acids.7 These precursors were further decorated through cycloaddition reactions with small peptide-like azides synthesized from Ugi or Mannich reactions (Fig. 3d). Various hydroxyaldehydes were functionalized via base-catalyzed condensation with propargyl bromide before performing the Biginelli
Figure 2. Retrosynthetic approaches to a-amino acids: (a) multicomponent reactions via imine; (b) other tandem reactions; (c) glycine equivalents; (d) side chain modifications.
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Figure 3. Multicomponent access to a-amino acids through an imine intermediate: (a) an asymmetric Petasis borono-Mannich access to UAA; (b) Mita and Sato one pot approach to a-amino acids, based on imine formation, stannylation or silylation, and final carboxylation; (c) synthesis of N-acylated a,a-dialkylglycines via solid phase Ugi reaction.
Figure 4. Alternative multicomponent/tandem/domino approaches to a-amino acids: (a) multicomponent one-pot approach to a-aryl serine derivatives; (b) synthesis of N-protected a-amino acid esters; (c) synthesis of a and b homophenylalanine precursors via sequential allylic C–H amination/vinylic C–H arylation; (d) access to bicyclic b-prolines and a,b,c-triamino acids via a combination of anionic aza-Michael additions, a diazo transfer and 1,3-dipolar cycloadditions.
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reaction in the presence of methyl acetoacetate or ethyl cyanoacetate as the CH acidic oxo components and urea as the diamide component. Other multicomponent routes envisage the formation of alternative activated species as an intermediate. Wang et al. proposed a multicomponent one-pot approach to a-aryl serine derivatives.8 The reaction involves the in situ Rh-catalyzed generation of ammonium ylides from aryl-substituted diazoacetates and anilines and the trapping of these molecules by formaldehyde (Fig. 4a). However, yields were only moderate (30–69%), the substrate scope was limited to 2-substituted anilines and aryldiazoacetates, and an asymmetric version of the reaction remains to be investigated. The synthesis of a series of N-protected non-proteinogenic a-amino acid esters from glyoxylates and carbamates was reported by Ali and Fathalla.9 The key hemiacetal intermediates were activated to a-substitution as trichloroacetimidates or acetates, giving the desired products in good yields with short reaction times (Fig. 4b). Also fascinating cascade approaches led to the core formation of novel UAAs (see Fig. 2b). White et al. exploited Pd(II)/sulfoxide catalysis to promote a sequential allylic C–H amination/vinylic C–H arylation, which started from inexpensive commercially available a-olefins and boronic acids, to provide a wide range of a- and b-homophenylalanine precursors (Fig. 4c).10 Apart from the outstanding regio- and E:Z selectivities of this first tandem reaction step, the route to the desired enantiomerically pure amino acids required several steps, with low overall yields. An unprecedented combination of anionic aza-Michael additions, a diazo transfer and 1,3-dipolar cycloadditions was reported by Jahn et al. to approach bicyclic b-prolines and a,b,c-triamino acids.11 Several b-substituted Michael acceptors, such as esters or amides, could be employed, and nonaflyl azide was identified as the reagent of choice for the diazo transfer (Fig. 4d). Lithium amides were generated in situ via deprotonation of the corresponding amine with n-BuLi. The use of chiral amines provided highly diastereoselective products. a,b,c-Triamino acids were obtained via hydrogenolytic opening of the pyrazoline ring. Access to UAAs using glycine derivatives as starting reagents (see Fig. 2c) or by modifying other amino acids side chains (see Fig. 2d) has been extensively reviewed in recent years.12–15 However, noteworthy examples continue to be reported in the literature. An efficient, enantioselective preparation of fluorinated a-amino acids via Ni-catalyzed Mitsunobu–Tsunoda alkylation of a chiral nucleophilic glycine equivalent (Fig. 5) with alcohol
Figure 5. Ni-catalyzed asymmetric synthesis of fluorinated a-amino acids from glycine.
substrates was recently discussed by Brimble et al.16 Several diversity-oriented strategies that started from glycine equivalents were described by Kotha et al.17 Highly atom-economic processes, such as the Diels–Alder reaction, [2+2+2] cycloaddition, Suzuki– Miyaura cross-coupling, and olefin metathesis, were employed to access UAA derivatives and peptides. Side chain modifications may be considered the most obvious transformations for accessing UAAs; nonetheless, some examples are worth mentioning. The Catellani reaction is one of these transformations. Göbel and Lautens reported the synthesis of simple to more complex substituted aromatic systems in a Pd-catalyzed cascade fashion; these products were used as the precursors for novel amino acids with a basic side chain (Fig. 6a).18 Sinha et al. reported the stereoselective synthesis of 2- and 3-indolylglycine derivatives and their oxygen analogues starting from 2-iodoheteroarenes.19 A tandem Sonogashira/cyclization reaction with ethynyloxazolidinone gave access to 2-indoylglycine analogues, while 3-indolylglycine derivatives were synthesized from silylated internal alkyne using Larock’s heteroannulation as the key reaction (Fig. 6b). UAA-based Drugs Available on the Market: In recent years, the design of peptidomimetics and peptide analogues has attracted increasing interest in the field of drug research. The discovery of a multitude of naturally occurring bioactive peptides represented a source of enlightment for medicinal chemists in the development of new therapeutic drugs. Although natural amino acid-based peptides find wide applications as drugs,20 major drawbacks, such as rapid proteolytic metabolism and poorly selective interactions, limit the application of these peptides. Hence, peptidomimetics could serve as privileged surrogates, maintaining the ability to modulate and control biological functions while offering several advantages, such as improved in vivo stability, enhanced potency, better oral absorption, improved tissue distribution and increased selectivity during biological responses.21,22 Thus, UAAs have been employed as building blocks with the aim of introducing conformational constraints and optimizing fit (i.e., tertiary structure) and interactions (e.g., hydrogen bonding and electrostatic or hydrophobic interactions) with the target active site. Of the several classes of UAAs that are available, examples of cyclic and bicyclic proline-like amino acids and non-proteinogenic AAs bearing aromatic or cyclic aliphatic side chains are herein discussed in more depth. Natural amino acids bearing heavy atoms (i.e., S, O, and N) suitable for further derivatization provide a large number of possible UAAs. Serine, threonine, cysteine and tyrosine, along with acidic and basic residues, as already discussed in the previous section, could potentially undergo easy transformations and derivatizations. Although this strategy represents the most intuitive and facile approach for introducing structural and/or chemical diversity, only a small number of UAAs of this class were successfully incorporated into approved drugs. Abarelix23 (Fig. 7, compound 1, GlaxoSmithKline) is a gonadotropin-releasing hormone (GnRH) antagonist that contains an N-isopropyl lysine, Lacosamide24 (Fig. 7, compound 2, NewBridge Pharmaceuticals) displays an O-methyl-serine residue, Pasireotide (Fig. 7, compound 3) contains an O-benzyl-tyrosine, and the orphan drug Histrelin25 (Fig. 7, compound 4, Ortho-McNeil-Janssen) has a benzyl-bearing histidine. Amino acidic scaffolds that exhibit tunable annular side chains are well known features in peptidomimetic design. In fact, the relevance of proline-like UAAs to drug discovery is primarily attributed to their typical characteristics, which can influence the secondary structure of a molecule. Because they contain no amide hydrogen, these molecules cannot donate a hydrogen bond and they possess a certain structural rigidity. Moreover, (i) the modulation of the ‘backbone’ rigidity by increasing the number of atoms in the ring (see the Sanofi products Bupivacaine26 and Quinupristin,27 Fig. 7, compounds 5 and 6, respectively) and (ii) the ability to
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Figure 6. Synthesis of UAAs by side chain modification of natural AAs: (a) synthesis of precursors for novel amino acids with a basic side chain via Catellani reaction; (b) stereoselective synthesis of 2- and 3-indolylglycine derivatives and their oxygen analogues starting from 2-iodoheteroarenes.
expand the molecular diversity of the scaffold when starting from a derivatized substrate (i.e., hydroxyproline, Hyp) represent key strengths of these molecular tools. Several antiviral drugs exhibit an unnatural derivatized proline; the Hepatitis C virus-Nonstructural protein 3 (HCV-NS3) protease inhibitors Asunaprevir28 (Fig. 7, compound 7, Bristol-Myers Squibb) and Faldaprevir29 (Fig. 7, compound 8, Boehringer Ingelheim, pre-registered) share a large portion of their structures and differ primarily with respect to the substituent at the hydroxyl group of the Hyp. Another substitution of the Hyp’s OH group is observed in the previously cited drug Pasireotide30 (Fig. 7, compound 3, Novartis), which was launched in 2012 for the treatment of Cushing’s disease. Other very common features are fused bicyclic UAAs, which display proline-like five- or six-membered side chains fused with either aliphatic or aromatic rings. A fused proline side chain is clearly detectable in Boceprevir31 (Fig. 7, compound 9, Merck), which is a new HCV NS3 protease inhibitor, in the bradykinin B2 antagonist Icatibant (Fig. 7, compound 10, Sanofi),32 and in Telaprevir (Fig. 7, compound 11).33 These UAAs provide increased steric hindrance and enhanced favorable hydrophobic interactions compared to the corresponding monocyclic analogues. In addition to proline-like UAAs, several approved drugs have been synthesized using specific (ad hoc) non-proteinogenic residues, primarily with the aim of enhancing binding activity or improving drug profiles. Among these drugs, amino acidic scaffolds bearing aromatic side chains are quite common. Phenylglycine (in either the L- or D-form) is one of the most widely employed aromatic UAAs. As the inferior homologue of phenylalanine, this amino acid takes advantage of the main features of phenylalanine with less degrees of freedom. Phenylglycine is embedded in the antibiotics Apalcillin (Fig. 7, compound 12, Boehringer Ingelheim), Mezlocillin (Fig. 7, compound 13, Bayer) and Pivampicillin (Fig. 7, compound 14, LEO Pharma) and in the cyclic hexapeptide Pasireotide (Fig. 7, compound 3). Besides phenylglycine, the depsipeptide Quinupristin (Fig. 7, compound 6) displays an N,N-dimethyl-4-aminophenylalanine. Higher phenylalanine homologues, such as homophenylalanine, are also detectable in approved drugs. As an example, see the proteasome inhibitor Kyprolis34 (Fig. 7, compound 15, Amgen). Naphthylalanine is often present in self-assembled compounds (see below in the text) and is frequently enclosed in peptidic scaffolds to modulate weak, non-local, protein–ligand interactions, such as p–p or cation–p. The condensed rings represent efficient fragments that can fit into deep and flat hydrophobic binding pockets that display narrow breaches. Lanreotide (Fig. 7, compound 16), which is a somatostatin receptor agonist, and the radioligand
[68Ga]-DOTA-NOC35 (Fig. 7, compound 17) both exhibit a cyclic fragment with a sulfur bridge and a D-naphthylalanine in the peptide sequence. In contrast, the L-enantiomer residue is one of the building blocks of Abarelix23 (Fig. 7, compound 1, GlaxoSmithKline), which is a GnRH antagonist. In addition to one naphthylalanine residue, Abarelix contains the L-3-pyridin-3-ylalanine residue in its peptide sequence. This residue as well as 3-(2-thienyl)alanine, which is incorporated into Icatibant (Fig. 7, compound 10, Sanofi),32 are interesting aromatic building blocks characterized by a heterocyclic side chain. UAAs with side chains that bear non-aromatic cycles, with and without heteroatoms, exhibit common six-membered or more complex architectures. Boceprevir (Fig. 7, compound 9, Merck)31 includes a cyclobutylalanine in its peptide sequence, Telaprevir (Fig. 7, compound 11)33 exhibits an a-cyclohexylglycine, and Cobicistat (Fig. 7, compound 18),36 which is an HIV protease inhibitor, has 2-amino-4-(4-morpholinyl) butanoic acid as a building block. The dipeptidyl peptidase IV inhibitor Saxagliptin37 (Fig. 7, compound 19, Bristol-Myers Squibb) displays the bulky and hydrophobic (S)-2-amino-2-(3-hydroxy-1-adamantyl)-acetic acid substructure, in an amount that is equivalent to nearly 50% of the total molecular weight. Applications: Engineered amino acid entities are typically associated with conventional drug discoveries, but in the last decade, an increasing number of applications involving UAAs have been reported in the field of life sciences. Indeed the manipulation of the amino acidic scaffold can be regarded as a potentially powerful tool in both biochemical investigations and the development of biomedical devices, whereas the unique properties of peptides motivated their use in apparently exotic scientific fields, not directly related to the pharmaceutical or healthcare industries (see Fig. 8). The aim of the current section is to compile a concise but hopefully complete overview of the non-trivial applications of UAAs and to highlight potential future developments. The central idea is to illustrate all reported applications involving UAAs, neglecting the most obvious applications related to traditional drug discovery (see Fig. 8). We focus on two main subjects: (i) the replacement of natural residues to expand the lexicon related to proteins, (ii) ‘soft’ and/or ‘wet’ materials that relate to the ability of peptides to build spontaneously complex supramolecular structures. The discovery of the amber codon suppression technique in recent decades38 revealed a new and intriguing role of UAAs: the opportunity to introduce in proteins residues bearing different side chains that were designed a priori. This technique provides a
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Figure 7. Examples of currently available registered drugs displaying UAA substructures.
remarkable chance (i) to expand the number of reactions that a protein can undergo at the post-translational level and (ii) to locally manipulate the physical and chemical properties of the
proteins. Although several conditions should be satisfied (i.e., a large perturbation induced by the engineered side chain should be avoided), more than 70 new chemical entities have been
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Figure 8. Use of UAAs in life sciences: soft matter (self-assembly), bioconjugates (fluorescent probe, spin labeled amino acids, antibody-drug conjugates, ADCs) and biomedical materials (bone clays).
successfully inserted into a range of different proteins recently and the number of adoptable UAAs in proteic lexicon expansion is actually unknown.39 New synthetic routes in combination with a more rigorous stoichiometry and localization of the involved reactive sites suggest an imminent and relevant improvement of bioconjugate devices, in which a protein is covalently linked to a drug, a probe, or a generic chemical entity through a specific linker that can release or activate the payload under particular environmental stimuli. The well-known antibody-drug conjugates40,41 (ADCs) typically exhibit a drug (i.e., payload) that is connected to defined residues of the antibody through a linker. The drawbacks related to the post-translational antibody modifications restricted to proteinogenic residues are (i) the limited number of reactions available and (ii) the poor control of the stoichiometry that is caused by the presence of multiple reacting amino acids in the proteic chains, which each have the potential to exhibit different behavior. Axup and coworkers42 recently reported a new bioconjugate of Trastuzumab in which exposed lysines and cysteines on the light chain (or alanines in the antibody’s sequence) are replaced with a paraacetylphenylalanine, where the new reactive group permitted the covalent ligation of Auristatin using an alkoxy-amine precursor of the cytotoxic compound. It is worth noting that several ADC platforms based on unnatural residues are currently under active development, although the potential immunogenicity of the resulting engineered proteins must still be assessed.43 The increasing number of available synthetic routes is apparently improving even the activity of ADCs, as uncleavable bonds between anticancer agents and related moieties may result in an enhanced stability of the antibody towards lysosome degradation. The ability to introduce exogenous residues into engineered proteins accelerated the discovery of new fluorescent probes,44 which
are precious tools in both imaging and the elucidation of the molecular mechanisms of drugs. A relatively large number of papers describing amino acidic entities bearing fluorophores (such as amino acid derivatives of 4-N,N-dimethylaminophthalimide (4DMAP) and 6-N,N-dimethylamino-2,3-naphthalimide (6-DMN) on their side chains is currently available.45 Peptides or proteins bearing paramagnetic UAAs (although due to a posteriori derivatization of natural residues) can be monitored using electron spin resonance spectroscopy: TOAC (4-amino-1-oxyl-2,2,6,6-tetramethylpiperidine-4-carboxylic acid) and/or pyrroline nitroxide-containing amino acids have been successfully inserted into oligopeptides via a chemoselective ligation of the paramagnetic core structure at the unique lysine that is available in the test peptide. One of the most interesting properties of several polymeric chains is their ability to originate supramolecular architectures.46 Due to the particular intermolecular interactions that occur between monomers and in the presence of specific conditions (ionic strength, temperature and solvent effects), the mesoscale structures can exhibit several complex morphologies: flat assemblies can bend and originate single or multi-layer wall cylinders or evolve toward microspheres. The resulting intermolecular interaction networks show the pivotal role of side chains in the control of the assembly at the molecular level: modulation of chemical and physical properties of residues can allow change of shape and size of the final nano/micro-objects. Cyclopeptides with D-amino acids in their sequence are well known monomers for self-assembled nanostructures due to their unique ability to stack along a privileged direction in long fibers.47 The inclusion of UAAs bearing unnatural side chains in the sequence of the monomer can enhance or induce assembly: naphthylalanine is likely to be the key feature in the self-assembly of the somatostatin agonist Lanreotide in long
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cylinders:48 the resulting macroscopic gel exhibits a very high degree of viscosity and was adopted as a long acting release (LAR) formulation for the treatment of acromegaly. An intriguing application of peptide-based soft matter is the use as tridimensional template for cell cultures. A slab of the self-assembled material is spread onto a plastic support with a peculiar 3D morphology, allowing cell growth on the exposed surface, whereas a continuous layer of cellular entities, which eventually correspond to a tissue, can grow on the exposed surface. Several hydrogels (i.e., Fmoc diphenylalanine in water) were investigated as a potential framework for 3D cell culture: although the most relevant examples are made of natural amino acid polymers, several attempts were made using D-enantiomers of proteinogenic residues.49 Other interesting studies related to the adoption of unnatural amino acids have been conducted in the field of tissue engineering concern the development of new collagen-mimetic peptides.50 Besides the obvious peptides made from naturally occurring amino acids, oligopeptides that bear one or more unnatural residues, such as 4-(R)-fluoroproline (Flp), were designed in the last decade as a tool for elucidating the crucial role of the naturally occurring molecule 4-(R)-hydroxyproline (Hyp), which is intrinsically a UAA entity, in the triple helix stability of collagen. Tailored side chains also allow new strategies in the search of a balance between mechanical properties and biocompatibility of several biomaterials: Katti et al. reported an increased flexibility of montmorillonite-based bone cements after the introduction of amino acids with aliphatic side chains into the free volume between two adjacent layers.51 Intercalation is not the only example of inorganic frameworks that can profitably host natural and unnatural amino acids, allowing possible future applications in life science: both UAAs and naturally occurring residues may be included52,53 in the synthesis of metal–organic frameworks (MOFs) as organic ligands of the target metal ion/atom, an emerging class of materials, with a low density, a large surface area and an adjustable pore size.54 In summary, the brief overview provided herein clearly describes the large amount interest that simple molecules, such as amino acids and their UAA analogues, continue to attract. These molecules continue to represent important and versatile tools that can be used by chemists for the preparation of chiral and variously substituted scaffolds that can be applied in areas that span from drug discovery to catalysis and from solid phase synthesis to biopolymers. Sophisticated techniques, such as amber codon suppression and the intercalation or encapsulation of UAAs, allow the integration of new chemistry into biological or inorganic materials, offering advanced approaches for the chemical manipulation of the structure and function of those materials. The use of novel UAAs widens the exploration of the chemical space and opens exciting new opportunities in basic and applied science research.
11. 12. 13. 14. 15. 16.
References and notes
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