Dipeptide Fragment Research Articles

Design of sustainable ionic liquids based on l-phenylalanine and l-alanine dipeptides: Synthesis, toxicity and biodegradation studies

A series of dipeptide ionic liquids (ILs) with l-phenylalanine and l-alanine fragments in structure were synthesized and their possible degradation pathways were analyzed. Based on this analysis, potential transformation products (PTPs) were proposed and synthesized. All of these compounds (25 in total) went through microbial toxicity screening and aerobic biodegradation testing. Obtained results demonstrated that by investigating ILs and PTPs with a dipeptide fragment (in tandem with single amino acid analogues), the design of ILs with high biodegradation values in closed bottle test can be accomplished. One finding was that within the scope of the compounds studied, l-phenylalanine containing compounds were more biodegradable than l-alanine derivatives. In addition to the choice of amino acid residue, its position in the dipeptide IL structure also had a significant effect on biodegradability. PyCH2CO-Phe-Ala-OEt IL, where l-phenylalanine was in close proximity to the positively charged pyridinium sub-unit, gave higher biodegradation percentages compared to PyCH2CO-Ala-Phe-OEt IL, where alanine was closer to pyridinium than the phenylalanine residue. Analysis of PTPs data showed that the presence of an alanine residue resulted in undesirable (less green) PTPs more often compared to PTPs containing phenylalanine, especially when alanine was in close proximity to the pyridinium headgroup. Based on both toxicity and biodegradation testing results preferable and less preferable subunits can be chosen for the design of new sustainable chemicals based on amino acids. Results from this study demonstrate a potential of designing new sustainable chemicals using amino acid moieties as part of their structure.

Влияние новых производных 6,7-диметоксихиназолин-4(3H)-она с остатками аминокислот и дипептидов на активность главной (Mpro) и папаиноподобной (PLpro) протеаз SARS-CoV-2

Introduction. A novel coronavirus infection caused by the SARS-CoV-2 virus is a highly contagious disease with a significant risk of complications up to mortality. The development of anti-SARS-CoV-2 drugs began almost immediately after the identification of the virus, while two promising pharmacological targets were identified: the main and papain-like proteases. Aim. Evaluation of the effect of new derivatives of 6,7-dimethoxyquinazoline-4(3H)-one with amino acid residues and dipeptide fragments on the activity of SARS-CoV-2 proteases in vitro. Materials and methods. The studied substances were obtained from commercially available 2-amino-4,5-dimethoxybenzoic acid which were cyclized into 2-alkyl-6,7-dimethoxy-3,1-benzoxazine-4-ones, followed by reaction with amino acids and dipeptides. The changes in the activity of viral proteases were evaluated with spectrophotometry using the appropriate substrates. During the study, the half maximal inhibitory concentration (IC50) was calculated. Results. As a result, it was found that the obtained compounds exhibit pronounced inhibitory activity with respect to both the main and papain-like protease of SARS-CoV-2 with an IC50 range of 0.012–0.100 and 0.013-0.100 µM/ml, respectively. The most pronounced effect was shown for the 3h compound with a fragment of the glycyl-tryptophan dipeptide with the isopropyl radical, which reduced the activity of the main protease (IC50 0.012 ± 0.005 µM/ml) and the papain-like protease (IC50 0.013 ± 0.009 µM/ml). Conclusion. Compound 3h containing the isopropyl radical and the glycyl-tryptophan dipeptide may become a promising object for further study and development of a new antiviral agent based on it.

Design and synthesis of efficient fluororethylene-peptidomimetic inhibitors of dipeptidyl peptidase III (DPP3)

Dipeptidyl peptidase III (DPP3) is a ubiquitously expressed zinc-dependent peptide cutting enzyme and selectively hydrolyses amide bonds to cleave N-terminal dipeptide fragments off of physiologically important oligopeptides. DPP3 has been found in a multitude of different types of cells and appears to be involved in various physiological processes (e.g. nociception, blood pressure control, protein turnover). Using the slowly converted peptide substrate tynorphin (VVYPW) as starting point, we have replaced the scissile bond with a fluoroethylene bioisostere to design ground state inhibitors, which led to the so far most effective peptide-based inhibitor of DPP3.

Design and Synthesis of Dipeptide Nerve Growth Factor Loop 1 Mimetics and In Vitro Studies of their Neuroprotective and Differentiation-Inducing Activities

Previous studies at the V. V. Zakusov Science Research Institute of Pharmacology created a dimeric dipeptide mimetic of the most exposed fourth loop of nerve growth factor (NGF), bis-(N-monosuccinyl-L-glutamyl-Llysine) hexamethylenediamide (GK-2), which activates specific TrkA receptors and has neuroprotective activity in vitro (at 105 to 109 M) and in vivo (0.1 – 10 mg/kg, i.p., or p.o.). The present report describes the construction and synthesis of a mimetic of the first loop of NGF based on the β-turn (-Lys32-Gly33-Lys34-Glu35-), bis-(N-aminocaproyl-glycyl-L-lysine) hexamethylenediamide (GK-6). The structure of GK-6 preserves the central dipeptide fragment of the β-turn -Gly33-Lys34-, the Lys32 residue being substituted by its bioisostere – a 6-aminocaproic acid residue - and the dimeric structure of NGF being reproduced by dimerization at the C-terminal using a bivalent hexamethylenediamine spacer. Structure-activity relationships of GK-6 were studied by sequential substitution of the side groups of the peptide by hydrogen to produce bis-(N-acetyl-glycyl-Llysine) hexamethylenediamide (GTS-611) and bis-(N-aminocaproyl-glycyl-glycine) hexamethylenediamine (GTS-613). Mimetic GL-6 and its analogs GTS-613, which contains the N-aminocaproyl radical, had neuroprotective effects at concentrations of 106 and 105M in conditions of oxidative stress in HT-22 neuron cultures. Dipeptide GTS-611, in which the aminocaproyl fragment was replaced by an acetyl residue, had no neuroprotective activity in these conditions, pointing to the importance of Lys32 in NGF for this property. In contrast to the loop 4 mimetic GK-2, the loop 1 mimetics GK-6 and GTS-611 showed differentiation-inducing activity on PC12 cells.

Synthesis and In Vitro Neuroprotective Activity of Glycine Analogs of Gk-2 Dimeric Dipeptide Mimetic of Nerve Growth Factor 4th Loop

A dimeric dipeptide mimetic of nerve growth factor (NGF), bis-(N-monosuccinyl-L-glutamyl-L-lysine) hexamethylenediamide (GK-2), was previously developed at V. V. Zakusov State Institute of Pharmacology, activated specific TrkA receptors, and exhibited neuroprotective activity in vitro (10–5 – 10–9 M) and in vivo (0.1 – 10 mg/kg i.p. and p.o.). GK-2 was designed based on the beta-turn (-Asp94-Glu95-Lys96-Gln97-) of the NGF 4th loop and preserved the central dipeptide fragment (-Glu95-Lys96-). The Asp94 residue was replaced by its monosuccinyl bioisostere. The dimeric structure of NGF was reproduced using a bivalent hexamethylenediamine spacer. The structure—activity (neuroprotective) relationship for GK-2 was studied in the present work using a glycine scan, i.e., successive replacement of the peptide side groups by H. The bis-(N-acetyl-L-glutamyl-L-lysine) (GK-2Ac), bis-(N-monosuccinylglycyl-L-lysine) (GK-2-Gly1), and bis-(N-monosuccinyl-L-glutamylglycine) hexamethylenediamides (GK-2-Gly2) were less active with neuroprotective activity in vitro under oxidative stress for HT22 cells at concentrations 10 – 100 times greater than GK-2. The conclusion was drawn that each side radical of GK-2 was important for manifestation of the full neuroprotective activity of dimeric dipeptide GK-2, a mimetic of the NGF 4th loop. However, removal of any of the side radicals would probably not change the active structure of the beta-turn so that the two remaining side radicals should retain the ability to bind to their TrkA subsites. This could explain the retention of neuroprotective activity in the GK-2 glycine analogs.

Targeted Delivery of Adamantylated Peptidoglycan Immunomodulators in Lipid Nanocarriers: NMR Shows That Cargo Fragments Are Available on the Surface.

We present an in-depth investigation of the membrane interactions of peptidoglycan (PGN)-based immune adjuvants designed for lipid-based delivery systems using NMR spectroscopy. The derivatives contain a cargo peptidoglycan (PGN) dipeptide fragment and an adamantyl group, which serves as an anchor to the lipid bilayer. Furthermore, derivatives with a mannose group that can actively target cell surface receptors on immune cells are also studied. We showed that the targeting mannose group and the cargo PGN fragment are both available on the lipid bilayer surface, thereby enabling interactions with cognate receptors. We found that the nonmannosylated compounds are incorporated stronger into the lipid assemblies than the mannosylated ones, but the latter compounds penetrate deeper in the bilayer. This might be explained by stronger electrostatic interactions available for zwitterionic nonmannosylated derivatives as opposed to the compounds in which the charged N-terminus is capped by mannose groups. The higher incorporation efficiency of the nonmannosylated compounds correlated with a larger relative enhancement in immune stimulation activities upon lipid incorporation compared to that of the derivatives with the mannose group. The chirality of the adamantyl group also influenced the incorporation efficiency, which in turn correlated with membrane-associated conformations that affect possible intermolecular interactions with lipid molecules. These findings will help in improving the development of PGN-based immune adjuvants suitable for delivery in lipid nanoparticles.

Synthesis, bioactivities and phloem uptake of dipeptide-chlorantraniliprole derivatives

Phloem systemicity is a desirable property for insecticides to control sucking insects. However, the development of phloem systemic insecticides is challenging. One possible strategy is to link existed insecticides with endogenous substances so that the resulting conjugates can be transported by specific transporters into the phloem. In this study, novel dipeptide promoieties were introduced into chlorantraniliprole, which is an efficient and broad-spectrum anthranilic diamide insecticide without phloem mobility. Twenty-two new dipeptide-chlorantraniliprole conjugates have been synthesized. Systemic tests showed that all conjugates exhibited phloem mobility in Ricinus communis. In particular, compound 4g with alanyl-alanine dipeptide fragment was able to accumulate in phloem sap (114.49 ± 11.10 μM) in the form of its hydrolysis product 5g. Results of bioassay showed that conjugates 4g and 5g were able to exhibit comparable insecticidal activity against Plutella xylostella L. and Spodoptera exigua compared to its parent compound chlorantraniliprole. This work demonstrated that the dipeptide structures were able to contribute to the improvement of the uptake and phloem mobility of chlorantraniliprole, and two phloem mobile conjugates with satisfactory in vivo insecticidal effect was obtained as new candidates for high-efficient insecticides.

Characterization of a Citrulline 4-Hydroxylase from Nonribosomal Peptide GE81112 Biosynthesis and Engineering of Its Substrate Specificity for the Chemoenzymatic Synthesis of Enduracididine.

The GE81112 tetrapeptides are a small family of unusual nonribosomal peptide congeners with potent inhibitory activity against prokaryotic translation initiation. With the exception of the 3-hydroxy-l-pipecolic acid unit, little is known about the biosynthetic origins of the non-proteinogenic amino acid monomers of the natural product family. Here, we elucidate the biogenesis of the 4-hydroxy-l-citrulline unit and establish the role of an iron- and α-ketoglutarate-dependent enzyme (Fe/αKG) in the pathway. Homology modelling and sequence alignment analysis further facilitate the rational engineering of this enzyme to become a specific 4-arginine hydroxylase. We subsequently demonstrate the utility of this engineered enzyme in the synthesis of a dipeptide fragment of the antibiotic enduracidin. This work highlights the value of applying a bioinformatics-guided approach in the discovery of novel enzymes and engineering of new catalytic activity into existing ones.

Characterization of a Citrulline 4‐Hydroxylase from Nonribosomal Peptide GE81112 Biosynthesis and Engineering of Its Substrate Specificity for the Chemoenzymatic Synthesis of Enduracididine

AbstractThe GE81112 tetrapeptides are a small family of unusual nonribosomal peptide congeners with potent inhibitory activity against prokaryotic translation initiation. With the exception of the 3‐hydroxy‐l‐pipecolic acid unit, little is known about the biosynthetic origins of the non‐proteinogenic amino acid monomers of the natural product family. Here, we elucidate the biogenesis of the 4‐hydroxy‐l‐citrulline unit and establish the role of an iron‐ and α‐ketoglutarate‐dependent enzyme (Fe/αKG) in the pathway. Homology modelling and sequence alignment analysis further facilitate the rational engineering of this enzyme to become a specific 4‐arginine hydroxylase. We subsequently demonstrate the utility of this engineered enzyme in the synthesis of a dipeptide fragment of the antibiotic enduracidin. This work highlights the value of applying a bioinformatics‐guided approach in the discovery of novel enzymes and engineering of new catalytic activity into existing ones.

Plant Coumarins: XVII. Synthesis and Transformations of 7-Hydroxy-2-oxo-2H-chromene-6-carboxamides

Umbelliferone-6-carbonyl chloride reacted with amines and α-, β-, and ω-amino acid methyl esters to afford the corresponding N-substituted 7-hydroxy-2-oxo-2H-chromene-6-carboxamides. The reaction of umbelliferone-6-carbonyl chloride with glycine gave 2-(7-hydroxy-2-oxo-2H-chromene-6-carboxamido)acetic acid which was converted into acid chloride, and the latter reacted with benzylamines and α-amino acid methyl esters to produce compounds containing a dipeptide fragment.

Stabilization of Cyclic β-Hairpins by Ugi-Reaction-Derived N-Alkylated Peptides: The Quest for Functionalized β-Turns

A solid-phase approach including on-resin Ugi reactions was developed for the construction of β-hairpins. Various N-alkylated dipeptide fragments proved capable of aligning antiparallel β-sheets in a macrocyclic scaffold, thus serving as β-hairpin templates. Gramicidin S was used as the model β-hairpin to compare the Ugi-derived β-turns with the type-II' β-turn. The results show that the multicomponent incorporation of such N-alkylated residues allows for the simultaneous stabilization and exo-cyclic functionalization of cyclic β-hairpins.

Synthesis and In Vitro Neuroprotective Activity of Analogs with Various Spacer Lengths of a Dimeric Dipeptide Mimetic (GK-2) of Nerve Growth Factor

Dimeric dipeptide mimetic GK-2 or hexamethylenediamide bis-(N-monosuccinyl-L-glutamyl-L-lysine), which activated TrkA receptors and exhibited neuroprotective activity in vitro (10–5 – 10–9 M) and in vivo (0.05 – 5 mg/kg i.p.), was designed by us earlier based on the beta-turn of the nerve growth factor (NGF) fourth loop. The effect of dimerizing spacer length on the manifestation of neuroprotective activity was studied by synthesizing GK-2 (11 σ-bonds) analogs tetramethylenediamide bis-(N-monosuccinyl-L-glutamyl-Llysine) (GK-2c); pentamethylenediamide bis-(N-monosuccinyl-L-glutamyl-L-lysine) (GK-2b); and trimethylenediamide bis-(N-monosuccinyl-L-glutamyl-L-lysyl-6-aminohexanoic acid) (GK-2a) containing 9, 10, and 22 σ-bonds, respectively, between the lysine Cα-atoms of the dipeptide fragments. Neuroprotective activity was investigated in vitro using HT-22 mouse hippocampal neuronal cell culture under H2O2-induced oxidative stress and showed that the neuroprotective activity as measured by the minimum effective concentration was 104 times less if the distance between the dipeptide fragments increased from 11 to 22 σ-bonds. The neuroprotective activity decreased insignificantly if this distance was decreased from 11 to 10 σ bonds (GK-2b) and disappeared with 9 σ -bonds (GK-2c). Therefore, the hexamethylene spacer was optimal although the distance between the corresponding amino-acid moieties of natural NGF from an x-ray structure analysis was considerably greater at 33 σ-bonds. This contradiction could have arisen because two ionically bound mimetic molecules interacted with TrkA receptors. Another possible explanation was that TrkA molecules could approach closer in the presence of bis-dipeptide GK-2 than NGF.

Синтез и нейропротекторная активность <i>in vitro</i> аналогов димерного дипептидного миметика фактора роста нервов ГК-2 с разной длиной спейсера

Ранее нами на основе бета-изгиба 4-й петли фактора роста нервов получен димерный дипептидный миметик ГК-2, гексаметилендиамид бис-(N-моносукцинил-L-глутамил-L-лизина), который активировал TrkA-рецептор и проявлял нейропротекторную активность in vitro (10–5 – 10–9 М) и in vivo (0,05 – 5 мг/кг внутрибрюшинно). Для изучения влияния длины димеризующего спейсера на проявление нейропротекторной активности были синтезированы и исследованы аналоги ГК-2 (11 сигма-связей): тетраметилендиамид бис-(N-моносукцинил-L-глутамил-L-лизина) (ГК-2в); пентаметилендиамид бис-(N-моносукцинил-L-глутамил-L-лизина) (ГК-2б); триметилендиамид бис-(N-моносукцинил-L-глутамил-L-лизил-6-аминогексановой кислоты) (ГК-2а), содержащие соответственно 9, 10 и 22 сигма-связи между Ca-атомами лизина дипептидных фрагментов. Исследование нейропротекторной активности in vitro на нейрональной культуре HT-22 в условиях окислительного стресса, вызванного перекисью водорода, позволило установить, что при увеличении расстояния между дипептидными фрагментами от 11 до 22 сигма-связей происходит снижение нейропротекторного действия в 10000 раз по минимальной действующей концентрации. При уменьшении этого расстояния от 11 до 10 сигма-связей (ГК-2б) выраженность нейропротекторного действия незначительно снижается, а при 9 сигма связях (ГК-2г) активность исчезает. Таким образом, оптимальным является гексаметиленовый спейсер, несмотря на то, что расстояние по данным рентгеноструктурного анализа между соответствующими аминокислотными остатками в природном NGF существенно больше и соответствует 33 сигма-связям. Это противоречие может быть связано с тем, что во взаимодействии с TrkA-рецептором участвуют 2 молекулы миметика, связанные солеобразованием. Другое возможное объяснение состоит в том, что молекулы TrkA способны сближаться в присутствии бис-дипептида ГК-2 теснее, чем при участии NGF.

Diastereoselective Radical Couplings Enable the Asymmetric Synthesis of anti‐β‐Amino‐α‐hydroxy Carboxylic Acid Derivatives

anti‐β‐Amino‐α‐(aminoxy) esters or amides are synthesized by merging polar asymmetric aza‐Michael additions of lithium 1‐phenylethylamides to α,β‐unsaturated carboxylic acid derivatives and diastereoselective radical couplings with the persistent free radical TEMPO mediated or catalyzed by ferrocenium hexafluorophosphate. Aliphatic α,β‐unsaturated carboxylic derivatives gave good to excellent anti‐diastereoselectivity for the radical coupling step, whereas the selectivity remained lower for cinnamic acid derivatives. The method allows the convenient introduction of a protected oxygen functionality, which is stable to acidic, basic, hydride and hydrogenolytic reductive conditions, but can be deprotected with zinc and acetic acid in the presence of TBDMS and Boc groups without noticeable epimerization. The tandem aza‐Michael/oxygenation strategy was applied in total syntheses of the TH2 cytokine secretion modulator cytoxazone, and dipeptide fragments of the anti‐β‐amino‐α‐hydroxy acid containing macrocyclic peptides perthamide C and largamide H.

Novel Technologies for Dipeptide Drugs Design and their Implantation.

The article is an overview of author’s data obtained in the framework of the project “The Creation of dipeptide preparations” at the V.V. Zakusov Institute of Pharmacology, Moscow, Russia. Advantages of dipeptides over longer peptides consist in that they are orally active owing to higher sta-bility and ability to penetrate biological barriers due to the presence of specific ATP–dependent trans-porters in enterocytes and blood-brain barrier. Two original approaches for dipeptide drugs design have been developed. Both of them are based on the idea of a leading role of central dipeptide fragment of the peptide chain beta-turn in the peptide-receptor interaction. The first approach, named “peptide drug-based design” represents the transformation of known nonpeptide drug into its dipeptide topological analog. The latter usually corresponds to a beta-turn of some regulatory peptide. The second approach represents the design of tripeptoide mimetic of the beta-turn of regulatory peptide or protein. The results of the studies, which led to the discovery of endogenous prototypes of the known non-peptide drugs piracetam and sulpiride, are presented herein. The paper discusses the process, based on the above-mentioned principles, that was used in designing of nontoxic, orally available, highly effective dipeptide drugs: nootropic noopept, dipeptide analog of piracetam; antipsychotic dilept, neurotensin tripeptoid analog; selective anxiolytic GB-115, tripeptoid analog of CCK-4, and potential neuroprotector GK-2, homodimeric dipeptide analog of NGF.

Synthesis and structure of the first representative of pentacoordinate C,O-chelates with a dipeptide fragment, the fluorosilane Ts—Gly—(S)-Pro—N(Me)CH2SiMe2F

A reaction of bicyclic 2-sila-5-piperazinone, 2,2,4-trimethyl-1,4-diaza-2-silabicyclo-[4.3.0]nonan-5-one containing a proline moiety, and N-tosylglycine acyl chloride with subsequent hydrolysis of the primary unstable product to the intermediate disiloxane and its treatment with BF3•OEt2 furnished a first representative of pentacoordinate C,O-chelate halosilanes with a dipeptide fragment, namely, Ts—Gly—(S)-Pro—N(Me)CH2SiMe2F.

Materials from a peptide soup

Small dipeptide fragments enzymatically combine and split to form sequences that self-assemble into nanomaterials.

Theoretical grounds and technologies for dipeptide drug development

Two original technologies for dipeptide drug development are described. Both are based on the theoretical idea about major role of the central dipeptide fragment of polypeptide chain β-turn in the peptide–receptor interaction. The first technology named Drug-Based Peptide Design includes movement from the structure of a known non-peptide drug toward its topological peptide analog, usually coinciding with the central region of the β-turn. The other technology represents movement from the β-turn of the regulatory peptide or protein to its dipeptide mimetic. This theoretical view is illustrated by examples of discovery of endogenous peptide prototypes of the well-known non-peptide drugs Piracetam and Sulpiride. The development of highly effective, non-toxic, orally administrable dipeptide drugs such as Noopept and Dilept with nootropic and neuroleptic activities, respectively, as well as dipeptide anxyolytic GB-115 and dipeptide anti-stroke drug candidate GK-2 on the basis of this approach is described.

Communication: Quantitative multi-site frequency maps for amide I vibrational spectroscopy.

An accurate method for predicting the amide I vibrational spectrum of a given protein structure has been sought for many years. Significant progress has been made recently by sampling structures from molecular dynamics simulations and mapping local electrostatic variables onto the frequencies of individual amide bonds. Agreement with experiment, however, has remained largely qualitative. Previously, we used dipeptide fragments and isotope-labeled constructs of the protein G mimic NuG2b as experimental standards for developing and testing amide I frequency maps. Here, we combine these datasets to test different frequency-map models and develop a novel method to produce an optimized four-site potential (4P) map based on the CHARMM27 force field. Together with a charge correction for glycine residues, the optimized map accurately describes both experimental datasets, with average frequency errors of 2-3 cm(-1). This 4P map is shown to be convertible to a three-site field map which provides equivalent performance, highlighting the viability of both field- and potential-based maps for amide I spectral modeling. The use of multiple sampling points for local electrostatics is found to be essential for accurate map performance.

Synthesis of a Cyclic Analogue of Tuv N-Methyl Tubulysin

Tubulysins are the most potent antimitotic agents known so far. We are interested in the conformational effect of tubulysin and herein we report the design and synthesis of a conformationally rigid cyclic analogue of Tuv N-methyl tubulysin. A conformationally rigid tetrahydropyran moiety was incorporated into the Tuv fragment via enantioselective hetero Diels–Alder reaction to prevent the rotation of the Tuv chain. The following diastereoselective reductive amination yielded the (4-methylamino)tetrahydropyranyl Tuv fragment, which was coupled to d-Mep-l-Ile dipeptide fragment and Tup fragment sequentially.