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Synthesis and Screening of New Enantiomeracally Enriched Non-Protein (S) and (R)-α -Amino Acids, Peptides and Polymers

#A-2141


Synthesis and Screening of New Potentially Pharmacologically Active Enantiomerically Enriched Non-Protein (S) and (R)-α -Amino Acids, Peptides and Polymers Containing Acetylenic Groups in the Side-Chain Radical

Tech Area / Field

  • BIO-CHM/Biochemistry/Biotechnology
  • CHE-SYN/Basic and Synthetic Chemistry/Chemistry
  • MED-DRG/Drug Discovery/Medicine

Status
3 Approved without Funding

Registration date
29.08.2014

Leading Institute
Scientific and Production Center "Armbiotechnology" NAS RA, Armenia, Yerevan

Supporting institutes

  • Agricultural University of Georgia, Georgia, Tbilisi

Collaborators

  • Universite de Nantes / Laboratoire de Biotechnologie, France, Nantes\nUniversite de Paris-Sud / Institut de Chimie Moleculaire et des Materiaux D’Orsay, France, Orsay\nRostok University, Germany, Rostock\nUniversitat Politècnica de Catalunya / Departament d'Enginyeria Quimica, Spain, Barcelona

Project summary

The goal of the project is development of methods for the synthesis of new enantiomerically enriched (S) and (R)-α-amino acids, containing acetylenic groups in the side chain, and peptides based thereon; study of antibacterial, antitumor properties to reveal potentially pharmacologically active preparations.
Amino acids playing a central role in all process of a live cell are widely used in biosynthesis of molecules that have specific biological activity. As a rule, in synthesis of peptides and other amino acid-based biopreparations only natural amino acids were previously used [1]. However, in the last 20 years in production of peptides and other medications enantiomerically enriched non-protein amino acids containing different functional groups (aliphatic, aromatic and heterocyclic) in the side chain have been used. Among similar compounds amino acids containing acetylenic bond in the side chain radical occupy a special place [2]. Such amino acids were used as selective inhibitors of Endothelin-converting enzymes, inhibitors of thrombin and cathepsin B [3,4], inactivators of pyridoxalphosphate-dependent γ-cystathionase, growth inhibitors of B. Subtillis B-50, etc. In particular, propargylglycylchloroalanyl dipeptides have a strong antibacterial effect, 2-aminohex-5-ynoic acid inhibits growth of B. Subtillis B-50 [5-9], arylacetylene amino phosphonate dipeptide inhibits Endothelin-Converting Enzyme-1 (ECE 1) and Neutral Endopeptidase (NEP) [10], etc.
Within the framework of the project it is also envisaged to synthesize non-protein amino acids containing acetylenic bonds in the side chain radical as well as to study their antibacterial and antitumor properties. The proposed project is a continuation of the research fulfilled in the framework of two ISTC projects #A-1247 and #A-1677. In the course of these projects realization about 100 novel aliphatic and heterocycle-substituted non-protein alpha amino acids and peptides containing non-protein amino acids have been synthesized. Synthesized compounds were screened on microbiological, biochemical and cytological models to reveal their biological activity. As a result new inhibitors of serine proteases as well as compounds with antibacterial activity were revealed.
Non-protein α-amino acids containing acetylenic bond belong to a class of substrates that irreversibly inhibit the enzyme (suicide substrate). There are three ways of “suicidal” effect of the acetylenic substrate on the active site of the enzyme: 1) by carbanion formation at an adjacent carbon followed by propargylic rearrangement to an allene, 2) by oxidation of the side group to ketone and 3) by monoxygeneration to oxypropene [11]. This fact is the crux in selection of biological models to reveal pharmacological activities of new non-protein α-amino acids and peptides that are scheduled to be synthesized within the frame of this project.
Antibacterial compounds are inhibitors of enzymes involved in the biosynthesis of peptidoglycan – component of the cell wall. Such enzymes are e.g. alanine racemase, DAla:Dala ligase and aminotransferase of D-amino acids. All these enzymes are inhibited by alanine analogs. It is especially important to take into account the microbiological specificity of the enzyme, i.e. search for inhibitors should be conducted considering the enzyme origin. Due to the discovery of the phenomenon of inactivation (irreversible inhibition) of the enzyme based on the operating mechanism of the enzyme itself (substrate interacting with the active site of the enzyme causes enzyme suicide), the search for new inhibitors among such compounds is especially attractive. Non-protein amino acids and peptides containing acetylenic bond in the side-chain radical are expected to have high level of specificity since they are suicide substrates [12].
Another targeted study of biological activity of new non-protein α-amino acids containing acetylenic bond in the side-chain radical and peptides are inhibitors of metalloproteases. Matrix metalloproteases (ММРs) belong to zinc-dependent metalloproteases. They play an important role in degradation of extracellular matrix in both norm and at various pathologies [13]. The interaction of cells with extracellular matrix is a key moment for the human body normal development and functioning. However, matrix metalloproteases are also responsible for many proteolytic processes that lead to tumor development. They are involved in matrix hydrolysis thus promoting spread of metastases in oncological patients. Metalloprotease inhibitors are able to prevent this process. In this connection, metalloproteases are targets for creation of antitumor drugs. Unfortunately, clinical trials of gelatinize inhibitors on oncological patients have not revealed therapeutic effect so far; moreover undesirable side effects were registered. The search for new highly specific compounds able to inhibit metalloproteases is one of the directions in creation of antitumor drugs. At the preliminary stage our enantiomerically enriched (S)-2-amino-2-(benzyl)pent-4-ynoic and (S)-2-amino-2(3,4-dichlorobenzyl)pent-4-ynoic new α-amino acids were studied on biological targets and proved to have inhibiting activity in relation to metalloproteinases. It should be mentioned that non-protein α-amino acids containing acetylenic bond in the side-chain radical are key units in the synthesis of compounds that are inhibitors of thrombin and cathepsin B [3,4].
In addition, the incorporation of non-protein amino acids (NPAAs) into the polymeric backbones can result in multiple positive effects such as: enhanced stability compared to NPAAs based peptides, better bioavailability (e.g. via the preparation of nanoparticles), increased membrane penetration (e.g. via imparting cationic nature to macromolecules), prolonged action and decreased immunogenicity. It is important that these polymers will be degraded after the fulfilment of their functions, and the degradation fragments can be either assimilated or cleared from the body. One of the most popular ways to render amino acid based polymers biodegradable is the incorporation of easily hydrolysable ester bonds into their backbones [14]. It is also important for the realization of biological activities that the polymers be water soluble. This can be achieved by incorporating ether bonds in the polymeric backbones as it has been demonstrated for arginine-based biodegradable ester polymers [15], or by attaching to the polymeric chains solubilizing (e.g. PEG) groups via lateral functional groups. In other words, we plan to construct NPAAs based ester and ether-ester polymers, and these polymers will be four classes – poly(ester amide)s & poly(ether ester amide)s, and poly(ester urethane)s & poly(ether ester urethane)s. Some cationic polymers to enhance membrane penetration and water insoluble polymers for making nanoparticle are included in the research plan as well.
There are several methods to obtain non-protein amino acids containing acetylenic bond in the side chain: isolation from some fungi species [14], alkylation of O’Donnell substrate by unsaturated alkyl halogenide, Strecker and Sonogashira reactions followed by enzymic cleavage [4]. Thus, for example, to synthesize unsaturated amino acids, Crisp and Robinson for the first time used Pd-catalytic system in the reactions of С-alkylation of unsaturated groups, in particular, of propargylglycine [15]. Using this approach, Li and coworkers succeeded in producing a number of compounds with acetylenic bond that are thrombin inhibitors [6].
Later on the Procter and Gamble Pharmaceutical Company using the Sonogashira reaction has generated a series of phenyl substituted propargylglycine derivatives that became the basis for metalloproteases inhibition [4]. Similar strategy was employed by the researchers of the Novartis Company. Using the Sonogashira reaction they succeeded in producing enantiomerically pure compounds with a propargyl fragment that are inhibitors of Endothelium-converting enzymes. Using phenyl substituted derivatives of propargylglycine they also managed to produce dipeptide nitrile, which was successfully tested as an inhibitor of lysosomal cysteine protease cathepsin B [16-18].
However, in the majority of cases, racemic precursors were used in synthesis of unsaturated α-amino acids and end products were produced in the form of optically inactive racemates. Only in some cases it became possible to provide the enantiomeric yield by enzymic hydrolysis.
Obviously, the search and development of efficient methods for the asymmetric synthesis of enantiomerically enriched non-protein amino acids containing acetylenic bond in the side chain and synthesis of peptides, polymers and other preparations based on these amino acids, is an urgent task for the pharmaceutical industry.
This project aims to develop effective methods for the asymmetric synthesis of (R)- and (S)- non-protein α-amino acids containing acetylenic bond in the side chain with the use of our previously developed methodologies for the asymmetric synthesis of amino acids based on the unique abilities of NiII square-planar complexes of Schiff’s bases of amino acids with chiral auxiliary (S)- and (R)- 2-N-(N`-benzylprollyl)aminobenzophenone (BPB) and its derivative analogs.
New amino acids, peptides and polymers synthesized in the frames of these projects were subjected to screening on microbiological, biochemical and cytological models and this resulted in detection of potentially active antibacterial, antiviral, antitumor and other biologically active compounds.
In the framework of this project as a starting amino acid synthon to produce complex non-protein α-amino acid containing acetylenic bond in the side chain radical, NiII complexes of Schiff’s bases of propargylglycine with chiral (S)- or (R)- (BPB) auxiliary as well as with its analogs (2-CBPB, 2-FBPB, etc.) will be used. Considering data on antitumor, antiviral, antibacterial and other effects of non-protein amino acids and peptides obtained earlier in the frame of Project # A-1677, to achieve the posed goal within the framework of this project it is envisaged to fulfill the following scope of researches:
· To develop efficient methods for the asymmetric synthesis of amino acids containing non-protein groups in the side chain.
· To develop methods for the synthesis of peptides (di-, tri-, etc.) containing amino acids with acetylenic groups in the side-chain radicals.
· To develop methods for the synthesis of non-protein amino acid based polymers;
· To synthesize enantiomerically enriched non-protein α-amino acids containing acetylenic bond in the side chain.
· To study antibacterial activity of the obtained new non-protein α-amino acids, peptides and polymers containing acetylenic bond in the side chain using a collection of microorganisms’ test cultures including gram positive and gram negative bacteria.
To study the effect of enantiomerically enriched non-protein α-amino acids, peptides and polymers containing acetylenic bond in the side chain on the activity of metalloproteinases.


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