Non-natural Amino Acids Research Articles

Trends in the Development of Antibody-Drug Conjugates for Cancer Therapy.

In cancer treatment, the first-generation, cytotoxic drugs, though effective against cancer cells, also harmed healthy ones. The second-generation targeted cancer cells precisely to inhibit their growth. Enter the third-generation, consisting of immuno-oncology drugs, designed to combat drug resistance and bolster the immune system's defenses. These advanced therapies operate by obstructing the uncontrolled growth and spread of cancer cells through the body, ultimately eliminating them effectively. Within the arsenal of cancer treatment, monoclonal antibodies offer several advantages, including inducing cancer cell apoptosis, precise targeting, prolonged presence in the body, and minimal side effects. A recent development in cancer therapy is Antibody-Drug Conjugates (ADCs), initially developed in the mid-20th century. The second generation of ADCs addressed this issue through innovative antibody modification techniques, such as DAR regulation, amino acid substitutions, incorporation of non-natural amino acids, and enzymatic drug attachment. Currently, a third generation of ADCs is in development. This study presents an overview of 12 available ADCs, reviews 71 recent research papers, and analyzes 128 clinical trial reports. The overarching objective is to gain insights into the prevailing trends in ADC research and development, with a particular focus on emerging frontiers like potential targets, linkers, and drug payloads within the realm of cancer treatment.

Desymmetrization of Geminal Difluoromethylenes using a Palladium/Copper/Lithium Ternary System for the Stereodivergent Synthesis of Fluorinated Amino Acids.

Fluorinated amino acids and related peptides/proteins have been found widespread applications in pharmaceutical and agricultural compounds. However, strategies for introducing a C-F bond into amino acids in an enantioselective manner are still limited and no such asymmetric catalysis strategy has been reported. Herein, we have successfully developed a Pd/Cu/Li ternary system for stereodivergent synthesis of chiral fluorinated amino acids. This method involves a sequential desymmetrization of geminal difluoromethylenes and allylic substitution with amino acid Schiff bases via Pd/Li and Pd/Cu dual activation, respectively. A series of non-natural amino acids bearing a chiral allylic/benzylic fluorine motif are easily synthesized in high yields with excellent regio-, diastereo-, and enantioselectivities (up to >20 : 1 dr and >99 % ee). A density functional theory (DFT) study revealed the F-Cu interaction of the allylic substrate and the Cu catalyst significantly influence the stereoselectivity.

PyPept: a python library to generate atomistic 2D and 3D representations of peptides

We present pyPept, a set of executables and underlying python-language classes to easily create, manipulate, and analyze peptide molecules using the FASTA, HELM, or recently-developed BILN notations. The framework enables the analysis of both pure proteinogenic peptides as well as those with non-natural amino acids, including support to assemble a customizable monomer library, without requiring programming. From line notations, a peptide is transformed into a molecular graph for 2D depiction tasks, the calculation of physicochemical properties, and other systematic analyses or processing pipelines. The package includes a module to rapidly generate approximate peptide conformers by incorporating secondary structure restraints either given by the user or predicted via pyPept, and a wrapper tool is also provided to automate the generation and output of 2D and 3D representations of a peptide directly from the line notation. HELM and BILN notations that include circular, branched, or stapled peptides are fully supported, eliminating errors in structure creation that are prone during manual drawing and connecting. The framework and common workflows followed in pyPept are described together with illustrative examples. pyPept has been released at: https://github.com/Boehringer-Ingelheim/pyPept.Graphical

Advances in enzymatic production of L-homophenylalanine

L-homophenylalanine (L-HPA) is an important non-natural amino acid that has been used as a key intermediate for the synthesis of Puli drugs for the treatment of hypertension. At present, L-HPA is synthesized using chemical methods, which has the disadvantages of expensive raw materials, tedious steps and serious pollution. Therefore, researchers have conducted in-depth research on the enzymatic production of L-HPA. This review summarizes the research progress on the enzymatic synthesis of L-HPA, including the dehydrogenase process, the transaminase process, the hydantoinase process, and the decarboxylase process, with the hope to facilitate the industrial production of L-HPA.

Recent developments in amino acid-derived imidazole-, imidazolium- and N-heterocyclic carbene-carboxylate complexes

Compounds based on imidazole or imidazolium moieties are excellent building blocks for the preparation of a plethora of ligand precursors. The introduction of chirality in these compounds is relatively easy through the Debus-Radziszewski reaction, in which the chiral pools are natural or non-natural amino acids. Using this synthetic route, enantiopure imidazole-carboxylate, imidazolium-carboxylate, and imidazolium-dicarboxylate ligand precursors can be prepared, from which N-heterocyclic carbenes (NHC) with one or two carboxylate groups can also be obtained. This review is a personal account that summarizes our recent results regarding the use of all these compounds as chiral ligands coordinated to several transition metals (Cu, Zn, Ag and Mo). The structural features of the new synthesized coordination polymers with novel bonding modes will be described, and the bonding capabilities of these ligands rationalized on the basis of theoretical calculations. The efficiency of homochiral imidazolium-dicarboxylate compounds as chiral inductors in asymmetric catalysis will also be discussed. Finally, the antimicrobial and antitumoral activities of imidazolium- and NHC-carboxylate silver complexes and the found chirality-activity and structure–activity relationships will be highlighted.

On the Role of a Conserved Tryptophan in the Chromophore Pocket of Cyanobacteriochrome

The cyanobacteriochrome Slr1393 can be photoconverted between a red (Pr) and green absorbing form (Pg). The recently determined crystal structures of both states suggest a major movement of Trp496 from a stacking interaction with ring D of the phycocyanobilin (PCB) chromophore in Pr to a position outside the chromophore pocket in Pg. Here, we investigated the role of this amino acid during photoconversion in solution using engineered protein variants in which Trp496 was substituted by natural and non-natural amino acids. These variants and the native protein were studied by various spectroscopic techniques (UV–vis absorption, fluorescence, IR, NIR and UV resonance Raman) complemented by theoretical approaches. Trp496 is shown to affect the electronic transition of PCB and to be essential for the thermal equilibrium between Pr and an intermediate state O600. However, Trp496 is not required to stabilize the tilted orientation of ring D in Pr, and does not play a role in the secondary structure changes of Slr1393 during the Pr/Pg transition. The present results confirm the re-orientation of Trp496 upon Pr → Pg conversion, but do not provide evidence of a major change in the microenvironment of this residue. Structural models indicate the penetration of water molecules into the chromophore pocket in both Pr and Pg states and thus water-Trp contacts, which can readily account for the subtle spectral changes between Pr and Pg. Thus, we conclude that reorientation of Trp496 during the Pr-to-Pg photoconversion in solution is not associated with a major change in the dielectric environment in the two states.

Artificial Small Molecules as Cofactors and Biomacromolecular Building Blocks in Synthetic Biology: Design, Synthesis, Applications, and Challenges.

Enzymes are essential catalysts for various chemical reactions in biological systems and often rely on metal ions or cofactors to stabilize their structure or perform functions. Improving enzyme performance has always been an important direction of protein engineering. In recent years, various artificial small molecules have been successfully used in enzyme engineering. The types of enzymatic reactions and metabolic pathways in cells can be expanded by the incorporation of these artificial small molecules either as cofactors or as building blocks of proteins and nucleic acids, which greatly promotes the development and application of biotechnology. In this review, we summarized research on artificial small molecules including biological metal cluster mimics, coenzyme analogs (mNADs), designer cofactors, non-natural nucleotides (XNAs), and non-natural amino acids (nnAAs), focusing on their design, synthesis, and applications as well as the current challenges in synthetic biology.

Enzyme and photocatalyst team up to make nonnatural amino acids

Enzyme and photocatalyst team up to make nonnatural amino acids

Enzyme and photocatalyst team up to make nonnatural amino acids

Enzyme and photocatalyst team up to make nonnatural amino acids

Non-natural amino acids into LfcinB-derived peptides: effect in their (i) proteolytic degradation and (ii) cytotoxic activity against cancer cells.

The dimeric peptide 26[F]: (RRWQWRFKKLG)2-K-Ahx has exhibited a potent cytotoxic effect against breast cancer cell lines, with position 26 (F) being the most relevant for anti-cancer activity. In this investigation, six analogues of the 26[F] peptide were synthesized in which the 26th position was replaced by non-natural hydrophobic amino acids, finding that some modifications increased the resistance to proteolytic degradation exerted by trypsin or pepsin. Additionally, these modifications increased the cytotoxic effect against breast cancer cells and generated cell death mediated by apoptosis pathways, activating caspases 8 and 9, and did not compromise the integrity of the cytoplasmic membrane. Finally, it was found that the modified peptides have a broad spectrum of action, since they also have a cytotoxic effect against the HeLa human cervical cancer cell line. Peptide 26[F] was inoculated in mice by ip administration and the lethal dose 50 (LD50) was between 70 and 140 mg kg-1. While for the 26[1-Nal]: (RRWQWR-1-Nal-KKLG)2-K-Ahx peptide, a dose-response test was performed, and the survival rate was 100%. These results suggested that these peptides are safe in this animal model and could be considered as promissory to develop a treatment against breast cancer.

Pepfun 2.0: Improved Protocols for the Analysis of Natural and Modified Peptides

Aim: The role of peptides is nowadays relevant in fields, such as drug discovery and biotechnology. Computational analyses are required to study their properties and gain insights into rational design strategies. Materials & methods: PepFun 2.0 is a new version of the python package for the study of peptides using a set of modules to analyze the sequence and structure of the molecules. Both natural and modified peptides containing non-natural amino acids can be studied based on the provided functionalities. Results: PepFun 2.0 comprises five main modules for tasks such as sequence alignments, prediction of properties, generation of conformers, detection of interactions and extra functions to include peptides containing non-natural amino acids. Conclusion: The code, tutorial and specific examples are open source and available at: https://github.com/rochoa85/PepFun2 .

Microwave Assisted Aza-michael Additions Towards β-amino Acids

Background: β-amino acids are non-natural amino acids, where the amino group is attached to the β-carbon instead of the α-carbon. Peptides containing β-amino acids present with particular secondary structures and remarkable chemical and biological properties. β-peptides are used as peptidomimetics, based on the resistance to hydrolysis in vivo, with various important applications in the field of Medicine. Methods: Several synthetic methods have appeared in recent years, with the Aza- Michael conjugate addition reactions, being a very effective approach towards β-amino acids. Microwave irradiation mediated reactions have also attracted much interest since they significantly improve the reaction yields while reducing the reaction time and avoiding by-products formation. The aim of this project has been the development of a reliable and general synthetic methodology towards β2 and β3 amino acids with the use of conjugate additions of N-nucleophiles to substituted acrylate derivatives. The application of effective catalysts has been also examined here. Results: The results show that the acrylate precursors of β2 and β3 amino acids with side chains corresponding to Phe, Asp, Ile, Leu, Val, and Tyr have been synthesized successfully by using a one-pot Horner-Wadsworth-Emmons reaction, and they have been used for the addition of Nnucleophiles, with a study on conditions and yields optimization. Conclusion: With the main challenge for β-amino acid synthesis being that there is still no general method to synthesize different types of β-amino acids corresponding to all-natural α-amino acids, the proposed synthetic methodology may offer this possibility.

Impact of Organ Impairment on the Pharmacokinetics of Therapeutic Peptides and Proteins.

The kidneys and liver are major organs involved in eliminating small-molecule drugs from the body. Characterization of the effects of renal impairment (RI) and hepatic impairment (HI) on pharmacokinetics (PK) have informed dosing in patients with these organ impairments. However, the knowledge about the impact of organ impairment on therapeutic peptides and proteins is still evolving. In this study, we reviewed how often therapeutic peptides and proteins were assessed for the effect of RI and HI on PK, the findings, and the resulting labeling recommendations. RI effects were reported in labeling for 30 (57%) peptides and 98 (39%) proteins and HI effects for 20 (38%) peptides and 55 (22%) proteins. Dose adjustments were recommended for RI in 11 of the 30 (37%) peptides and 10 of the 98 (10%) proteins and for HI in 7 of the 20 (35%) peptides and 3 of the 55 (5%) proteins. Additional actionable labeling includes risk mitigation strategies; for example,someproduct labels have recommendedavoid use or monitor toxicitiesin patients with HI. Over time, there is an increasing structural diversity of therapeutic peptides and proteins, including the use of non-natural amino acids and conjugation technologies, which suggests a potential need for reassessing the need to evaluate the effect of RI and HI. Herein, we discuss scientific considerations for weighing the risk of PK alteration due to RI or HI for peptide and protein products. We briefly discuss other organs that may affect the PK of peptides and proteins administered via other delivery routes.

An ultrafast phototrigger of the Trp5CN-Trp motif in a β-hairpin peptide.

Phototriggers are useful molecular tools to initiate reactions in enzymes by light for the purpose of photoenzymatic design and mechanistic investigations. Here, we incorporated the non-natural amino acid 5-cyanotryptophan (W5CN) in a polypeptide scaffold and resolved the photochemical reaction of the W5CN-W motif using femtosecond transient UV/Vis and mid-IR spectroscopy. We identified a marker band of ∼2037cm-1 from the CN stretch of the electron transfer intermediate W5CN·- in the transient IR measurement and found UV/Vis spectroscopic evidence for the W·+ radical at 580nm. Through kinetic analysis, we characterized that the charge separation between the excited W5CN and W occurs in 253ps, with a charge-recombination lifetime of 862ps. Our study highlights the potential use of the W5CN-W pair as an ultrafast phototrigger to initiate reactions in enzymes that are not light-sensitive, making downstream reactions accessible to femtosecond spectroscopic detection.

Hydrogen Bonding Compensation on the Convex Solvent-Exposed Helical Face of IA3, an Intrinsically Disordered Protein.

Saccharomyces cerevisiae IA3 is a 68 amino acid peptide inhibitor of yeast proteinase A (YPRA) characterized as a random coil when in solution, folding into an N-terminal amphipathic alpha helix for residues 2-32 when bound to YPRA, with residues 33-68 unresolved in the crystal complex. Circular dichroism (CD) spectroscopy results show that amino acid substitutions that remove hydrogen-bonding interactions observed within the hydrophilic face of the N-terminal domain (NTD) of IA3-YPRA crystal complex reduce the 2,2,2-trifluoroethanol (TFE)-induced helical transition in solution. Although nearly all substitutions decreased TFE-induced helicity compared to wild-type (WT), each construct did retain helical character in the presence of 30% (v/v) TFE and retained disorder in the absence of TFE. The NTDs of 8 different Saccharomyces species have nearly identical amino acid sequences, indicating that the NTD of IA3 may be highly evolved to adopt a helical fold when bound to YPRA and in the presence of TFE but remain unstructured in solution. Only one natural amino acid substitution explored within the solvent-exposed face of the NTD of IA3 induced TFE-helicity greater than the WT sequence. However, chemical modification of a cysteine by a nitroxide spin label that contains an acetamide side chain did enhance TFE-induced helicity. This finding suggests that non-natural amino acids that can increase hydrogen bonding or alter hydration through side-chain interactions may be important to consider when rationally designing intrinsically disordered proteins (IDPs) with varied biotechnological applications.

Photophysics of Two Indole-Based Cyan Fluorophores

For the purpose of searching for new biological fluorophore, we assess the photophysical properties of two indole derivatives, 4-cyano-7-azaindole (4CN7AI) and 1-methyl-4-cyano-7-azaindole (1M4CN7AI), in a series of solvents. We find that (1) the absorption spectra of both derivatives are insensitive to solvents and are red-shifted from that of indole, having a maximum absorption wavelength of ca. 318 nm and a broad profile that extends beyond 370 nm; (2) both derivatives emit in the blue to green spectral range with a large Stokes shift, for example, in H2O, the maximum emission wavelength of 4CN7AI (1M4CN7AI) is at ca. 455 nm (470 nm); (3) 4CN7AI has a higher fluorescence quantum yield (QY) and a longer fluorescence lifetime (τF) in aprotic solvents than in protic solvents, for example, QY (τF) = 0.72 ± 0.04 (7.6 ± 0.8 ns) in tetrahydrofuran and QY (τF) = 0.29 ± 0.03 (6.2 ± 0.6 ns) in H2O; (4) in all of the solvents used except H2O, the fluorescence QY (τF) of 1M4CN7AI is equal to or higher (longer) than 0.69 ± 0.03 (11.2 ± 0.7 ns). Taken together, these results suggest that the corresponding non-natural amino acids, 4-cyano-7-azatryptophan and 1-methyl-4-cyano-7-azatryptophan, could be useful as biological fluorophores.

A Novel Shigella O-Polysaccharide-IpaB Conjugate Vaccine Elicits Robust Antibody Responses and Confers Protection against Multiple Shigella Serotypes.

Shigella is responsible for high burdens of diarrhea and dysentery globally. Children living in areas of endemicity are the most affected, and currently, there are no licensed vaccines to prevent shigellosis. Vaccine approaches have traditionally targeted the bacterial lipopolysaccharide as a protective antigen. Shigella O-polysaccharide (OPS) conjugated to recombinant Pseudomonas aeruginosa exotoxin A (rEPA) or tetanus toxoid (TT) is advanced in clinical evaluation. Adequate efficacy of these vaccines, particularly in the infant target group, remains to be demonstrated. A major limitation of the OPS-glycoconjugate concept is its limited coverage, since immunity to the O antigen is serotype specific, and there are multiple disease-causing serotypes. Another concern is the use of protein carriers already included in multiple other childhood vaccines. This study reports a novel Shigella OPS conjugate vaccine that uses the Shigella invasion plasmid antigen B (IpaB) as the carrier protein. IpaB is a virulence factor component of the Shigella type III secretion system and highly conserved among Shigella serotypes. It is robustly immunogenic and a protective antigen. IpaB and IpaB containing nonnative amino acids (nnAA) were produced at large scale using cell-free protein synthesis. Incorporation of nnAA enabled site-specific conjugation of IpaB to Shigella flexneri 2a OPS using click chemistry, yielding OPS-IpaB glycoconjugate. Parenteral immunization of mice with the OPS-IpaB vaccine resulted in high levels of OPS- and IpaB-specific serum IgG and robust protection against lethal S. flexneri 2a or Shigella sonnei challenge. The OPS-IpaB vaccine is a promising new vaccine candidate with the capacity to confer broad protection against clinically relevant Shigella serotypes. IMPORTANCE Diarrhea caused by Shigella species results in long-term disability and mortality globally, disproportionally affecting younger children living in poor countries. Although it is treatable by antibiotics, the rapid and widespread emergence of resistant strains and the highly contagious nature of the disease compel the development of preventive tools. Currently, several Shigella OPS conjugate vaccines are being evaluated in clinical studies, but these rely exclusively on immunity against the bacterial O antigen, which limits their coverage to only the immunizing serotype; multivalent vaccines are needed to protect against the most prevalent serotypes. This is the first report of a novel Shigella OPS-conjugate vaccine that uses Shigella IpaB as a carrier and protective antigen. This vaccine, administered parenterally, elicited robust immunity and protected mice against lethal infection by S. flexneri 2a or S. sonnei. The OPS-IpaB vaccine is a promising candidate for evaluation in vulnerable populations.

Natural Plasmodium falciparum Infection Stimulates Human Antibodies to MSP1 Epitopes Identified in Mice Infection Models upon Non-Natural Modified Peptidomimetic Vaccination.

(1) Background: Malaria, a vector-borne infectious disease, is caused by parasites of the Plasmodium genus, responsible for increased extreme morbidity and mortality rates. Despite advances in approved vaccines, full protection has not yet been achieved upon vaccination, thus the development of more potent and safe immuno-stimulating agents for malaria prevention is a goal to be urgently accomplished. We have focused our research on a strategy to identify Plasmodium spp. epitopes by naturally acquired human antibodies and rodent malaria infection models immunized with site-directed non-natural antigens. (2) Methods: Some predictive algorithms and bioinformatics tools resembling different biological environments, such as phagosome-lysosome proteolytic degradation, affinity, and the high frequency of malaria-resistant and -sensitive HLA-II alleles were regarded for the proper selection of epitopes and potential testing. Each epitope's binding profile to both host cells and HLA-II molecules was considered for such initial screening. (3) Results: Once selected, we define each epitope-peptide to be synthesized in terms of size and hydrophobicity, and introduced peptide-bond surrogates and non-natural amino acids in a site-directed fashion, and then they were produced by solid-phase peptide synthesis. Molecules were then tested by their antigenic and immunogenic properties compared to human sera from Colombian malaria-endemic areas. The antigenicity and protective capacity of each epitope-peptide in a rodent infection model were examined. The ability of vaccinated mice after being challenged with P. berghei ANKA and P. yoelii 17XL to control malaria led to the determination of an immune stimulation involving Th1 and Th1/Th2 mechanisms. In silico molecular dynamics and modeling provided some interactions insights, leading to possible explanations for protection due to immunization. (4) Conclusions: We have found evidence for proposing MSP1-modified epitopes to be considered as neutralizing antibody stimulators that are useful as probes for the detection of Plasmodium parasites, as well as for sub-unit components of a site-directed designed malaria vaccine candidate.

Expanding the genetic code: a non-natural amino acid story

From enzymes to hormones, proteins are the most versatile macromolecules that serve a vital function throughout all biological systems. In nature, organisms are restricted to a 20 amino acid repertoire, which in turn limits the chemistry. Nature evolves and adapts but currently does so under a “chemistry-limited” circumstance. This in turn has limited the aspirations of protein designers and engineers that aim to expand the functional and structural properties of proteins into new and exciting realms. One way nature has circumvented this problem is to recruit non-proteinaceous cofactors. Another way is to change one of the most fundamental concepts underlying biology: the genetic code. Pyrrolysine and selenocysteine are the two main genetically encodable proteinogenic “21st” amino acids; stop codons are recruited to encode the incorporation during ribosomal polypeptide synthesis. This ability of nature to go beyond the standard 20 amino acid repertoire inspired researchers to engineer the fundamental elements of ribosomal polypeptide synthesis and allow full genetic encoding of non-natural amino acids. This in turn helped advance synthetic biology and protein engineering in ways that were not possible by using the standard 20 amino acids. To this day, a vast repertoire of non-natural amino acids is available and is continuously expanding with increasing scientific needs.

An Integrated In Vivo/In Vitro Protein Production Platform for Site-Specific Antibody Drug Conjugates

The XpressCF+® cell-free protein synthesis system is a robust platform for the production of non-natural amino acids containing antibodies, which enable the site-specific conjugation of homogeneous antibody drug conjugates (ADCs) via click chemistry. Here, we present a robust and scalable means of achieving a 50-100% increase in IgG titers by combining the high productivity of cell-based protein synthesis with the unique ability of XpressCF+® reactions to produce correctly folded and assembled IgGs containing multiple non-natural amino acids at defined positions. This hybrid technology involves the pre-expression of an IgG light-chain (LC) protein in a conventional recombinant E. coli expression system, engineered to have an oxidizing cytoplasm. The prefabricated LC subunit is then added as a reagent to the cell-free protein synthesis reaction. Prefabricated LC increases IgG titers primarily by reducing the protein synthesis burden per IgG since the cell free translation machinery is only responsible for synthesizing the HC protein. Titer increases were demonstrated in four IgG products in scales ranging from 100-µL microplate reactions to 0.25-L stirred tank bioreactors. Similar titer increases with prefabricated LC were also demonstrated for a bispecific antibody in the scFvFc-FabFc format, demonstrating the generality of this approach. Prefabricated LC also increases robustness in cell-free reactions since it eliminates the need to fine-tune the HC-to-LC plasmid ratio, a critical parameter influencing IgG assembly and quality when the two IgG subunits are co-expressed in a single reaction. ADCs produced using prefabricated LC were shown to be identical to IgGs produced in cell-free alone by comparing product quality, in vitro cell killing, and FcRn receptor binding assays. This approach represents a significant step towards improving IgG titers and the robustness of cell-free protein synthesis reactions by integrating in vivo and in vitro protein production platforms.