Solid-Phase Synthesis of an Insect Pyrokinin Analog Incorporating an Imidazoline Ring as Isosteric Replacement of a trans Peptide Bond

A facile solid-phase synthetic method for incorporating the imidazoline ring motif, a surrogate for a trans peptide bond, into bioactive peptides is reported. The example described is the synthesis of an imidazoline peptidomimetic analog of an insect pyrokinin neuropeptide via a cyclization reaction of an iminium salt generated from the preceding amino acid and 2,4-diaminopropanoic acid (Dap).

Solid Phase Synthesis of an Insect Pyrokinin Analog Incorporating an Imidazoline Ring as Isosteric Replacement of a trans Peptide Bond

A facile solid-phase synthetic method for incorporating the imidazoline ring motif, a surrogate for a trans peptide bond, into bioactive peptides is reported. The example described is the synthesis of an imidazoline peptidomimetic analog of an insect pyrokinin neuropeptide via a cyclization reaction of an iminium salt generated from the preceding amino acid and 2,4-diaminopropanoic acid (Dap).

In-peptide amino acid racemization via inter-residue oxazoline intermediates during acidic hydrolysis

Isopedopeptins are antibiotic cyclic lipodepsipeptides containing the subsequence L-Thr—L-2,3-diaminopropanoic acid—D-Phe—L-Val/L-3-hydroxyvaline. Acidic hydrolysis of isopedopeptins in D2O showed the D-Phe residues to racemize extensively in peptides with L-3-hydroxyvaline but not in peptides with L-Val. Similarly, one Leu residue in pedopeptins, which are related peptides containing the subsequence Leu—2,3-diaminopropanoic acid—Leu—L-Val/L-3-hydroxyvaline, was found to racemize in peptides with L-3-hydroxyvaline. Model tetrapeptides, L-Ala—L-Phe—L-Val/3-hydroxyvaline—L-Ala, gave the corresponding results, i.e. racemization of L-Phe only when linked to a L-3-hydroxyvaline. We propose the racemization to proceed via an oxazoline intermediate involving Phe/Leu and the L-3-hydroxyvaline residues. The 3-hydroxyvaline residue may form a stable tertiary carbocation by loss of the sidechain hydroxyl group as water after protonation. Elimination of the Phe/Leu H-2 and ring-closure from the carbonyl oxygen onto the carbocation results in the suggested oxazoline intermediate. The reversed reaction leads to either retained or inversed configuration of Phe/Leu. Such racemization during acidic hydrolysis may occur whenever a 3-hydroxyvaline residue or any amino acid that can form a stable carbocation on the C-3, is present in a peptide. The proposed mechanism for racemization was supported by incorporation of 18O in the 3-hydroxyvaline sidechain when the acidic hydrolysis was performed in H2O/H218O (1:1). The 2,3-diaminopropanoic residues of isopedopeptins and pedopeptins were also found to racemize during acidic hydrolysis, as previously described. Based on the results, the configuration of the Leu and 2,3-diaminopropanoic acid residues of the pedopeptins were reassigned to be L-Leu and D-Leu, and 2 × L-2,3-diaminopropanoic acid.

Evidence of protein binding by a nucleopeptide based on a thymine-decorated L-diaminopropanoic acid through CD and in silico studies

Background: Nucleopeptides are chimeric compounds of biomedical importance carrying DNA nucleobases anchored to peptide backbones with ascertained capacity to bind nucleic acids. However, their ability to interact with proteins involved in pathologies of social relevance is a feature that still requires investigation. The worrying situation currently observed worldwide for the COVID-19 pandemic urgently requires the research of novel anti-SARS-CoV-2 molecular weapons, whose discovery can be aided by in silico predictive studies. Objective: The aim of this work is to explore by spectroscopic methods novel features of a thymine-bearing nucleopeptide based on L-diaminopropanoic acid, including conformational aspects as well as its ability to bind proteins, starting from bovine serum albumin (BSA) as a model protein. Moreover, in consideration of the importance of targeting viral proteins in the current fight against COVID-19, we evaluated in silico the interaction of the nucleopeptide with some of the most relevant coronavirus protein targets. Methods: First, we investigated via circular dichroism (CD) the conformational behaviour of this thymine-bearing nucleopeptide with temperature: we observed CD spectral changes particularly passing from 15 to 35 °C. Scanning Electron Microscopy (SEM) analysis of the nucleopeptide was also conducted on nucleopeptide solid samples. Additionally, CD binding and in silico investigations were performed with BSA as a model protein. In particular, molecular dockings were run using as targets some of the main SARS-CoV-2 proteins. Results: The temperature-dependent CD behaviour reflected the three-dimensional rearrangement of the nucleopeptide at different temperatures, with a higher exposure to the solvent of its chromophores at higher temperatures compared to a more stacked structure at low temperature. SEM analysis of nucleopeptide samples in the solid state showed a granular morphology, with a low roughness and some thread structures. Moreover, we found through spectroscopic studies that the modified peptide bound the albumin target by inducing significant changes to the protein secondary structure. Conclusion: CD and in silico studies suggested that the nucleopeptide bound the BSA protein with high affinity according to different binding modes, as testified by binding energy scores lower than -11 kcal/mol. Interestingly, a predictive study performed on 3CLpro and other SARS-CoV-2 protein targets suggested the potential ability of the nucleopeptide to bind with good affinity the main protease of the virus and other relevant targets including the RNA-dependent RNA polymerase, especially when complexed with RNA, the papain-like protease, and the coronavirus helicase at the nucleic acid binding site.

2,3-Diaminopropanols Obtained from d-Serine as Intermediates in the Synthesis of Protected 2,3-l-Diaminopropanoic Acid (l-Dap) Methyl Esters

A synthetic strategy for the preparation of two orthogonally protected methyl esters of the non-proteinogenic amino acid 2,3-l-diaminopropanoic acid (l-Dap) was developed. In these structures, the base-labile protecting group 9-fluorenylmethyloxycarbonyl (Fmoc) was paired to the p-toluensulfonyl (tosyl, Ts) or acid-labile tert-butyloxycarbonyl (Boc) moieties. The synthetic approach to protected l-Dap methyl esters uses appropriately masked 2,3-diaminopropanols, which are obtained via reductive amination of an aldehyde prepared from the commercial amino acid Nα-Fmoc-O-tert-butyl-d-serine, used as the starting material. Reductive amination is carried out with primary amines and sulfonamides, and the process is assisted by the Lewis acid Ti(OiPr)4. The required carboxyl group is installed by oxidizing the alcoholic function of 2,3-diaminopropanols bearing the tosyl or benzyl protecting group on the 3-NH2 site. The procedure can easily be applied using the crude product obtained after each step, minimizing the need for chromatographic purifications. Chirality of the carbon atom of the starting d-serine template is preserved throughout all synthetic steps.

Environmental distribution of the neurotoxin l-BMAA in Paenibacillus species

The environmental distribution of the neurotoxic amino acid, 3-N-methyl-2,3-diaminopropanoic acid (BMAA), first isolated in 1967, was initially believed to be limited to tropical and subtropical plants of the genus Cycas. The seeds of one such species, which had been used historically on the Pacific island of Guam as a foodstuff, had a reputation for neurotoxicity. Some 40 years later the amino acid was detected in terrestrial and aquatic cyanobacteria and in other aquatic organisms. Overlooked was the discovery of BMAA in peptides of bizarre structure that had been isolated in 1975 from Paenibacillus pulvifaciens during a search for antibiotics. More recently (2014), peptides of similar structure were isolated from Paenibacillus larvae; this organism is causative of American Foulbrood, a lethal disease of honeybee colonies. These are interesting chemical and environmental observations, but knowledge of the bacterial distribution of BMAA is limited to just these two species of Paenibacillus, while more than 200 Paenibacillus spp. are known. Paenibacillus spp. are ever present naturally in the environment and are used agriculturally; recent research reports that some species infect human foods – including cow's milk – and have been isolated from human body fluids. We wish to stimulate interest in the environmental distribution of the neurotoxic BMAA in Paenibacillus spp. by drawing together previously isolated streams of research and by proposing experimental approaches by which this matter might be resolved.