Stereoselective Synthesis of ß-Branched Aromatic α-Amino Acids by Biocatalytic Dynamic Kinetic Resolution*.

ß-Branched noncanonical amino acids are valuable molecules in modern drug development efforts. However, they are still challenging to prepare due to the need to set multiple stereocenters in a stereoselective fashion, and contemporary methods for the synthesis of such compounds often rely on the use of rare-transition-metal catalysts with designer ligands. Herein, we report a highly diastereo- and enantioselective biocatalytic transamination method to prepare a broad range of aromatic ß-branched α-amino acids. Mechanistic studies show that the transformation proceeds through dynamic kinetic resolution that is unique to the optimal enzyme. To highlight its utility and practicality, the biocatalytic reaction was applied to the synthesis of several sp3 -rich cyclic fragments and the first total synthesis of jomthonic acid A.

Reductant-Induced Free Radical Fluoroalkylation of Nitrogen Heterocycles and Innate Aromatic Amino Acid Residues in Peptides and Proteins.

A series of fluoroalkylated cyclic λ3 -iodanes and their hydrochloride salts was prepared and used in a combination with sodium ascorbate in buffer or aqueous methanol mixtures for radical fluoroalkylation of a range of substituted indoles, pyrroles, tryptophan or its derivatives, and Trp residues in peptides. As demonstrated on several peptides, the aromatic amino acid residues of Trp, Tyr, Phe, and His are targeted with high selectivity to Trp. The functionalization method is biocompatible, mild, rapid, and transition-metal-free. The proteins myoglobin, ubiquitin, and human carbonic anhydrase I were also successfully functionalized.

Visible light-promoted CO2 fixation with imines to synthesize diaryl α-amino acids.

Light-mediated transformations with CO2 have recently attracted great attention, with the focus on CO2 incorporation into C-C double and triple bonds, organohalides and amines. Herein is demonstrated visible light -mediated umpolung imine reactivity capable of engaging CO2 to afford α-amino acid derivatives. By employing benzophenone ketimine derivatives, CO2 fixation by hydrocarboxylation of C=N double bonds is achieved. Good to excellent yields of a broad range of α,α-disubstituted α-amino acid derivatives are obtained under mild conditions (rt, atmospheric pressure of CO2, visible light). A procedure that avoids tedious chromatographic purification and uses sustainable sunlight is developed to highlight the simplicity of this method.

Discovery of a 29-Amino-Acid Reactive Abiotic Peptide for Selective Cysteine Arylation.

The regio- and chemoselective modification of proteins or peptides with chemical reagents is often challenging. One approach to overcome this problem involves identifying abiotic polypeptide sequences that react with specific small molecules. Toward this goal, we profiled ∼5 × 1013 randomized 30-mer peptides using mRNA display and high-throughput sequencing in search of polypeptides that can undergo cysteine arylation with a water-soluble perfluoroarene. Within this vast chemical space, we discovered a cysteine-containing sequence with a second-order rate constant of 0.29 M-1 s-1 for arylation. An N- and C-terminal truncation reduced the reaction rate, as did the addition of denaturants. When the reactive peptide was covalently fused to the enzyme Sortase A, we observed regiospecific arylation at a single cysteine site, leaving the enzyme's active site cysteine unchanged. Taken together, these results demonstrate that long polypeptides of defined sequence, when matched with the appropriate reactive group, can be used for selective arylation of cysteine in water.

Design, Synthesis, and Biological Evaluation of New Peptide Analogues as Selective COX-2 Inhibitors.

A new class of peptide derivatives possessing SO2 Me and N3 pharmacophores at the para position of a phenyl ring bound to different aromatic amino acids were synthesized based on solid-phase synthesis methodology, and evaluated as selective cyclooxygenase-2 (COX-2) inhibitors. One of the analogues, i.e., compound 2a as the representative of this series, was recognized as the highest selective COX-2 inhibitor with a COX-2 selectivity index of >500. The structure-activity relationships (SARs) acquired indicated that compound 2a containing a 4-(methylsulfonyl)benzoyl group as a pharmacophore and tyrosine as a ring bearing amino acid in the second position and glutamic acid as the C-terminal amino acid can give the essential geometry to provide selective COX-2 inhibitory activity. Antiproliferative activity of the synthesized peptides (1a-7b) was also determined against four different human cancer cell lines, including MCF-7, HepG2, A549, and HeLa. According to our results, A549, HepG2, and MCF7 seemed to be more sensitive cell lines than HeLa cells encountering these compounds, which gave inhibitory action with IC50 values from 4.8 to 64.4 µM. In this regard, compounds 3a and 2b displayed the best inhibitory activity against the cell lines. Moreover, a good correlation was observed between the antiproliferative potency and the COX-2 inhibitory activity of compounds 1a, 2a, 2b, and 5b. Such findings suggest that one of the mechanism of anticancer activity of these peptides may be through the COX-2 inhibitory action.

Asymmetric synthesis of aromatic ß-amino acids using ω-transaminase: Optimizing the lipase concentration to obtain thermodynamically unstable ß-keto acids.

Synthesized aromatic ß-amino acids have recently attracted considerable attention for their application as precursors in many pharmacologically relevant compounds. Previous studies on asymmetric synthesis of aromatic ß-amino acids using ω-transaminases could not be done efficiently due to the instability of ß-keto acids. In this study, a strategy to circumvent the instability problem of ß-keto acids was utilized to generate ß-amino acids efficiently via asymmetric synthesis. In this work, thermodynamically stable ß-ketoesters were initially converted to ß-keto acids using lipase, and the ß-keto acids were subsequently aminated using ω-transaminase. By optimizing the lipase concentration, we successfully overcame the instability problem of ß-keto acids and enhanced the production of ß-amino acids. This strategy can be used as a general approach to efficiently generate ß-amino acids from ß-ketoesters.

A concise synthesis of tubuphenylalanine and epi-tubuphenylalanine via a diastereoselective Mukaiyama aldol reaction of silyl ketene acetal.

We have developed a straightforward and auxiliary-free synthetic route towards tBu-tubuphenylalanine (tBu-Tup) and tBu-epi-tubuphenylalanine (tBu-epi-Tup), which are the key components of tubulysins and their analogs. A Lewis acid-mediated diastereoselective Mukaiyama aldol reaction using silyl ketene acetal and N-Boc-L-phenylalaninal provided γ-amino-ß-hydroxyl-α-methyl esters, which were deoxygenated to γ-amino-α-methyl esters under Barton-McCombie deoxygenation conditions. Notably, the desired tBu-Tup and tBu-epi-Tup were obtained in good overall yields in four steps.

Design and synthesis of novel 19F-amino acid: a promising 19F NMR label for peptide studies.

Novel aliphatic (19)F-substituted amino acid was designed as a (19)F NMR label for peptide studies. The synthesis was performed in 11 steps and 9% overall yield from a commercially available starting material. The key transformation was a decarboxylative fluorination of an aliphatic carboxylic acid with XeF2 in C6F6.

Synthesis and evaluation of 2-amino-5-(4-[(18)F]fluorophenyl)pent-4-ynoic acid ([(18)F]FPhPA): a novel (18)F-labeled amino acid for oncologic PET imaging.

INTRODUCTION: (18)F-labeled amino acids are important PET radiotracers for molecular imaging of cancer. This study describes synthesis and radiopharmacological evaluation of 2-amino-5-(4-[(18)F]fluorophenyl)pent-4-ynoic acid ([(18)F]FPhPA) as a novel amino acid radiotracer for oncologic imaging. METHODS: (18)F]FPhPA was prepared using Pd-mediated Sonogashira cross-coupling reaction between 4-[(18)F]fluoroiodobenzene ([(18)F]FIB) and propargylglycine. The radiopharmacological profile of [(18)F]FPhPA was evaluated in comparison with O-(2-[(18)F]fluoroethyl)-L-tyrosine ([(18)F]FET) using the murine breast cancer cell line EMT6 involving cellular uptake studies, radiotracer uptake competitive inhibition experiments and small animal PET imaging. RESULTS: (18)F]FPhPA was prepared in 42±10% decay-corrected radiochemical yield with high radiochemical purity >95% after semi-preparative HPLC purification. Cellular uptake of L-[(18)F]FPhPA reached a maximum of 58±14 % radioactivity/mg protein at 90 min. Lower uptake was observed for racemic and D-[(18)F]FPhPA. Radiotracer uptake inhibition studies by synthetic and naturally occurring amino acids suggested that Na(+)-dependent system ASC, especially ASCT2, and Na(+)-independent system L are important amino acid transporters for [(18)F]FPhPA uptake into EMT6 cells. Small animal PET studies demonstrated similar high tumor uptake of [(18)F]FPhPA in EMT6 tumor-bearing mice compared to [(18)F]FET reaching a maximum standardized uptake value (SUV) of 1.35 after 60 min p.i.. Muscle uptake of [(18)F]FPhPA was higher (SUV30min=0.65) compared to [(18)F]FET (SUV30min=0.40), whereas [(18)F]FPhPA showed a more rapid uptake and clearance from the brain compared to [(18)F]FET. CONCLUSION: L-[(18)F]FPhPA is the first (18)F-labeled amino acid prepared through Pd-mediated cross-coupling reaction. ADVANCES IN KNOWLEDGE AND IMPLICATIONS FOR PATIENT CARE: L-[(18)F]FPhPA displayed promising properties as a novel amino acid radiotracer for molecular imaging of system ASC and system L amino acid transporters in cancer.

Thiophenyl-substituted triazolyl-thione L-alanine: asymmetric synthesis, aggregation and biological properties.

In this work, we report the asymmetric synthesis and characterization of an artificial amino acid based on triazolyl-thione L-alanine, which was modified with a thiophenyl-substituted moiety, as well as in vitro studies of its nucleic acid-binding ability. We found, by dynamic light scattering studies, that the synthetic amino acid was able to form supramolecular aggregates having a hydrodynamic diameter higher than 200 nm. Furthermore, we demonstrated, by UV and CD experiments, that the heteroaromatic amino acid, whose enzymatic stability was demonstrated by HPLC analysis also after 24 h of incubation in human serum, was able to bind a RNA complex, which is a feature of biomedical interest in view of innovative antiviral strategies based on modulation of RNA-RNA molecular recognition.

Aromatic interactions with naphthylalanine in a ß-hairpin peptide.

Stable peptides have been explored as epitope mimics for protein-protein and protein-nucleic acid interactions; however, presentation of a regular structure is critical. Aromatic interactions are ubiquitous and are competent at stabilizing a ß-hairpin fold. The greatest stabilization has been reported from pairs of tryptophan side chains. Naphthylalanine residues are often used as tryptophan replacements, but it is not clear if 1-naphthylalanine or 2-naphthylalanine is adequate at replicating the geometry and stability observed with tryptophan aromatic interactions. Herein, a 12-residue peptide has been constructed with laterally disposed aromatic amino acids. A direct comparison is made between tryptophan and other bicyclic, unnatural amino acids. Significant stabilization is gained from all bicyclic amino acids; however, geometric analysis shows that only 1-naphthylalanine adopts a similar edge to face geometry as tryptophan, whereas the 2-naphthylalanine appears most similar to a substituted phenylalanine.

Design, synthesis and anti-itch activity evaluation of aromatic amino acid derivatives as gastrin-releasing peptide receptor antagonists.

Eight aromatic amino acid derivatives (9a-9h) as gastrin-releasing peptide receptor (GRPR) antagonists were designed and synthesized. For the design, the tertiary structure of GRPR was predicted by blast searching in the premise of 1u19 protein as the template. Eight target compounds were docked into the binding pocket to investigate their possible binding interactions. Their anti-itch activities were tested by intrathecal injection using Kunming mice as experimental animals and chloroquine phosphate as a modeling medium. Compounds 9e and 9f significantly inhibited scratching behaviors. The anti-itch activities of these compounds decreased with the following sequence: 9e > 9f = 9d > 9b > 9g > 9a > 9h > 9c predicted by computer-aided drug design (CADD) and 9e > 9f > 9b > 9h > 9g > 9d > 9a > 9c evaluated by preliminary test. They were broadly in line with activity order pretested by CADD. It showed that the predicted tertiary structure of GRPR could be used for antipruritic drug design.

Long-range effects on the capture and release of a chiral guest by a helical molecular capsule.

Helically folded molecular capsules based on oligoamide sequences of aromatic amino acids which are capable of binding tartaric acid in organic solvents with high affinity and diastereoselectivity have been synthesized, and their structures and binding properties investigated by (1)H NMR, X-ray crystallography, circular dichroism, and molecular modeling. We found that elongating the helices at their extremities by adding monomers remote from the tartaric binding site results in a strong increase of the overall helix stability, but it does not influence the host-guest complex stability. The effect of this elongation on the binding and release rates of the guest molecules follows an unexpected non-monotonous trend. Three independent observations (direct monitoring of exchange over time, 2D-EXSY NMR, and molecular modeling) concur and show that guest exchange rates tend to first increase upon increasing helix length and then decrease when helix length is increased further. This investigation thus reveals the complex effects of adding monomers in a helically folded sequence on a binding event that occurs at a remote site and sheds light on possible binding and release mechanisms.

Synthesis of tetrafluorinated aromatic amino acids with distinct signatures in 19F NMR.

Fluorinated amino acids serve as powerful tools in protein chemistry. We synthesized a series of para-substituted tetrafluorophenylalanines via the regioselective S(NAr) chemistry of the commercially available pentafluorophenylalanine Boc-Z. These novel unnatural amino acids display distinct (19)F NMR signatures, making them powerful tools for analyzing protein-membrane interactions with NMR spectroscopy.

Synthesis and characterization of a novel ester-based nucleoamino acid for the assembly of aromatic nucleopeptides for biomedical applications.

In this work, we report a technological approach to a novel Fmoc-protected nucleoamino acid, based on l-tyrosine, carrying the DNA nucleobase on the hydroxyl group by means of an ester bond, suitable for the solid-phase synthesis of novel aromatic nucleopeptides of potential interest in biomedicine. After ESI-MS and NMR characterization this building block was used for the assembly of a thymine-functionalized tetrapeptide, composed of nucleobase-containing and underivatized l-tyrosine moieties alternated in the backbone.

Design of novel neurokinin 1 receptor antagonists based on conformationally constrained aromatic amino acids and discovery of a potent chimeric opioid agonist-neurokinin 1 receptor antagonist.

A screening of conformationally constrained aromatic amino acids as base cores for the preparation of new NK1 receptor antagonists resulted in the discovery of three new NK1 receptor antagonists, 19 [Ac-Aba-Gly-NH-3',5'-(CF(3))(2)-Bn], 20 [Ac-Aba-Gly-NMe-3',5'-(CF(3))(2)-Bn], and 23 [Ac-Tic-NMe-3',5'-(CF(3))(2)-Bn], which were able to counteract the agonist effect of substance P, the endogenous ligand of NK1R. The most active NK1 antagonist of the series, 20 [Ac-Aba-Gly-NMe-3',5'-(CF(3))(2)-Bn], was then used in the design of a novel, potent chimeric opioid agonist-NK1 receptor antagonist, 35 [Dmt-D-Arg-Aba-Gly-NMe-3',5'-(CF(3))(2)-Bn], which combines the N terminus of the established Dmt(1)-DALDA agonist opioid pharmacophore (H-Dmt-D-Arg-Phe-Lys-NH(2)) and 20, the NK1R ligand. The opioid component of the chimeric compound 35, that is, Dmt-D-Arg-Aba-Gly-NH(2) (36), also proved to be an extremely potent and balanced µ and δ opioid receptor agonist with subnanomolar binding and in vitro functional activity.

Synthesis and properties of MIDA boronate containing aromatic amino acids: new peptide building blocks.

Herein, we report the synthesis of novel phenylalanine and tyrosine derivatives containing a N-methyliminodiacetic acid boronate group. These compounds can be prepared enantiomerically pure, they are stable to column chromatography and they can be stored in air for two months without degradation occurring. This new class of boronate containing aromatic amino acids has potential applications in both peptide chemistry and natural product synthesis.

Functionalization of aromatic amino acids via direct C-H activation: generation of versatile building blocks for accessing novel peptide space.

Functionalized alpha-amino acid building blocks have been prepared in good yield with high regiocontrol and preservation of stereochemistry via iridium-catalyzed borylation of suitably protected aromatic alpha-amino acid derivatives. The utility of these systems in peptide couplings and Suzuki reactions has been demonstrated.

Simple synthesis of ruthenium pi complexes of aromatic amino acids and small peptides.

The interaction of [Ru(eta(6)-C(10)H(8))(Cp)](+) (Cp=C(5)H(5)) with aromatic amino acids (L-phenylalanine, L-tyrosine, L-tryptophane, D-phenylglycine, and L-threo-3-phenylserine) under visible-light irradiation gives the corresponding [Ru(eta(6)-amino acid)(Cp)](+) complexes in near-quantitative yield. The reaction proceeds in air at room temperature in water and tolerates the presence of non-aromatic amino acids (except those which are sulfur containing), monosaccharides, and nucleotides. The complex [Ru(eta(6)-C(10)H(8))(Cp)](+) was also used for selective labeling of Tyr and Phe residues of small peptides, namely, angiotensin I and II derivatives.

Phenylalanine aminomutase-catalyzed addition of ammonia to substituted cinnamic acids: a route to enantiopure alpha- and beta-amino acids.

An approach is described for the synthesis of aromatic alpha- and beta-amino acids that uses phenylalanine aminomutase to catalyze a highly enantioselective addition of ammonia to substituted cinnamic acids. The reaction has a broad scope and yields substituted alpha- and beta-phenylalanines with excellent enantiomeric excess. The regioselectivity of the conversion is determined by substituents present at the aromatic ring. A box model for the enzyme active site is proposed, derived from the influence of the hydrophobicity of substituents on the enzyme affinity toward various substrates.