CHAPTER 6: CHAPTER 6: APPLICATION OF AROMATIC TRIAZOLYL AMINO ACID SCAFFOLD AND ITS MONO- AND BIS-
1.1. Introduction
Amino acids are indispensable ingredients of life system and protein function.
Proteins play a vital role in all living organisms to maintain the cell structures, properties and functions. However, in all organisms, the building blocks of all the translated proteins are the same 20 natural amino acids. Moreover, natural selection/evolution has generated a large no of proteins with more or less common structures and functions in a population.1 Therefore, these proteins are highly specialized for performing specific jobs and thus, are not suitable for a different function other than they use to do the specific job. To perform a complex additional function by a protein, it needs other functionalities within its framework.1b Post- translational modifications, cofactor-dependent catalysis, and pyrrolysine/selenocystein incorporation in bacteria proves that the natural evolutionary movement needs extra chemical functionalities other than those present within the 20 natural amino acids.2
Figure 1.1. Presentation of naturally occurring 21st and 22nd amino acids.
Thus, the approach “directed evolution”, have come up with proteins of altered structural and functional properties that do not occur in nature.3 As for example, researchers are trying to generate new proteins to function as potent therapeutic with high oral viability or as a sensitive probe for visualizing intracellular events and understanding molecular interactions inside a cell. Now a day, scientists are involved in designing the proteins via rational approach of directed evolution to produce new proteins with desirable properties. Therefore, inspired by Natures’ post-translational modification to include different functional units or molecules into the proteins, chemoselective conjugation methods have been developed to attach probe to proteins;
thereby facilitating the study of structure and functions at molecular level.4 However, conventional bioconjugation reaction has several drawbacks and mostly exploited the only nine canonical amino acids with limited functional groups and abundance for modification/ligation within a protein. Therefore, site-specific conjugation of an additional functionality/probe molecule to a desired canonical amino acid within a protein’s framework is a challenging task.5 To circumvent this problem, several chemical and biochemical methodology have been developed to site-specifically
incorporate “designer amino acids” (the unnatural amino acids) with desired functionalities to probe protein structure, conformation and function and to generate proteins/enzyme for several novel biochemical applications, such as for chemical synthesis, biomedical research or even as therapeutics.6 Therefore, the research in the field of design and synthesis of non-natural amino acids with novel properties, to encode it genetically and to incorporate it site-specifically into a protein via bio- orthogonal strategy, is growing at a fast space for the growing demand of proteins of potential therapeutic and many other diversified novel biotechnological applications.6,
7 Therefore, an expanded genetic code when site-specifically incorporated into protein would allow us to study proteins’ physicochemical/biophysical properties which would otherwise be extremely difficult-(a) probing protein structure, conformation, function and interbiomolecular interaction, (b) regulating protein activity, (c) monitoring the mode of action, (d) improving immunogenicity, and (e) very recent development of a protein with a “chemical warhead” which target specific cellular components.8, 9
The historical development of synthesis or semisynthetic methods for introduction of unnatural amino acids into peptide and proteins by Offord and Kaiser has paved the way to develop methods for site specific incorporation of more unnatural amino acids into proteins by Schultz et al. towards expanding the genetic code and reaching the goal of semisynthetic organism.10 Towards this end, an increasing amount of interest from various research groups has resulted in the acceleration of progress of design of unnatural amino acids for application in protein engineering. Several non-natural amino acids were reported looking after the steric and electronic properties and incorporated into proteins site specifically.11 Moreover, for the growing demand of proteins of diverse and improved functionality, several unnatural amino acids (UNAA) with novel functionalities have been incorporated within a protein’s framework via the developed strategies of bioconjugations like translational incorporation, semisynthetic incorporation, bio-orthogonal conjugation and most recently developed recombinant introduction of amino acids into protein.12 Among the various examples of UNAAs, the work of Schultz et al. about the encoding an amino acid with a chemical warhead which target specific cellular components, is highly appreciable. Therefore, the synthesis of unnatural amino acids with novel functionality is highly demanding and is currently an emerging area of research because of the currently developed technology, “recombinant introduction” of amino acid into proteins.13 However, many of the reported unnatural amino acids are not suitable for giving novel biological properties of the proteins or not containing functionality/signaling elements for labeling the proteins that can offer information about protein’s structure, function and dynamics with a fast and easily detectable signal generation.
Sensing of proteins’ microenvironment needs the help of highly sensitive fluorescence probe. Extremely sensitive fluorescence based detection techniques find widespread applications for probing of structure, function, dynamics of biomolecules,
for visualizing intracellular events, understanding molecular interactions inside a cell and for enabling experiments to be measured in solution.14 Many such aspects have been studied either by exploiting intrinsic fluorescence of tryptophan amino acid in a protein or extrinsic fluorescent label. However, intrinsic fluorescence limits the interpretation of the results in presence of multiple tryptophan in a protein.Moreover, the limitation of labeling with large size of extrinsic fluorescent probe has led a strong demand for generating the smaller protein tag or site-specific incorporation of unnatural fluorescent amino acids into a protein.15 Many of the problems for generating extrinsic fluorescently labeled proteins can be solved if a microenvironment sensitive intrinsic fluorescent amino acid can be synthesized and incorporated into a protein site-specifically.15
Fluorescent amino acids can be used for in vitro and cellular imaging of protein localization, biomolecular interactions, and conformational changes with the ability to place these small probes at virtually any site in the proteome. The fluorescent proteins are valuable tools in cell biology for monitoring molecular localization and activities of proteins and gene expression in live cells. As for an example, green fluorescent protein (GFP) despite of having few shortcomings is a powerful probe of protein expression, localization, and studying biomolecular interactions.16 The most important and novel method for the generation of fluorescent proteins is to genetically encoded incorporation of fluorescent amino acids site-specifically.16c Thus, Schultz et al., have exploited a polarity sensitive fluorescent amino acid, L-(7-hydroxycoumarin-4- yl) ethylglycine, as a probe of the urea dependent denaturation of holomyoglobin. An environmentally sensitive dansyl amino acid (DansA) was also genetically encoded and site-specifically incorporated into proteins by the same group and used as an environmentally sensitive reporter of protein unfolding.17 A fluorescent unnatural amino acid, 6-propionyl-2-(N,N-dimethyl)aminonaphthalene (Anap), has been incorporated into proteins to study ligand-induced local conformational changes in proteins and biomolecular interactions in vitro and to localize proteins in living mammalian cells.13 Christiane Garbay et al., have reported the incorporation of fluorescent coumaryl amino acid into short peptide to generate fluorescent peptide and studied the cell internalization behavior.17c There are also reports wherein “click reaction” has been utilized for fluorescent labeling of proteins.18 Multiple chromophore labeled peptides/proteins have also been developed for investigating folding mechanism, detection of a target protein, enzyme activity detection and studying protein-protein/protein-drug interactions utilising various fluorescence phenomena such as FRET, excimer and exciplex emission.19, 20 Till the date only a very limited number of such amino acids as discussed have been synthesized or encoded genetically. Therefore, there is a high demand to develop fluorescent unnatural amino acids (FUAA) or fluorescently labelled UAA with high microenvironment sensitivity for genetic encoding or to generate labelled proteins/peptide for studying conformational or diverse functional realm.
Despite the availability of a few of such fluorescent amino acids, no special attentions were taken to develop amino acids with tunable photophysical property and specific conformational adoptability. Therefore, the synthesis of unnatural amino acids with novel photophysical property is currently an emerging area of research which would address the following aspects: (a) labeling a protein via site specific incorporation or a short peptide with such fluorescent “designer amino acid” would enable to study the specific conformational adaptability, functions and dynamics of a protein; (b) these fluorescent unnatural peptide/proteins can be utilized as sensitive probes for visualizing intracellular events and understanding molecular interactions inside a cell such as receptor-ligand binding,monitoring enzyme activity,elucidating protein’s structures, functions and dynamics, high-throughput screening,diagnostics and proteomics.15, 16
Figure 1.2. Graphical presentation of some reported unnatural amino acids and their applications.
During the past decade, peptides have gained a wide range of applications in medicine and biotechnology. Thus, therapeutic peptide research is currently experiencing a renaissance for commercial reasons as peptides are selective and efficient signaling molecules and trigger intracellular effects through binding to specific cell surface receptors, such as G protein-coupled receptors (GPCRs) or ion channels.21 Given their attractive pharmacological profile and intrinsic properties, peptides represent an excellent starting point for the design of novel therapeutics.
Moreover, high specificity would make peptides as excellently safe, tolerable and effective in humans. Thus, in various aspects of drug market, peptides are in the sweet spot between small molecules and biopharmaceuticals. However, natural peptides seldom can be used therapeutically as drugs, because of the problems associated with
low absorption, rapid metabolism and low oral bioavailability, many efforts aimed to modify the natural sequence of the amino acids of bioactive peptides achieved a desired and a focused effect.22a Therefore, harnessing for peptide based therapeutics a great number of researchers all over the world are engaged in the design and synthesis of peptidomimetics or peptide like molecules of clinical importance.
Conformationally constrained small, molecular scaffold/ non-peptide isostere are attracting much research interest in this regard because it could induce a particular conformation of a peptide which is the key for showing biological activity by the peptide with high oral viability.22 Considerable efforts have thus been invested in studying the impact of appended molecular scaffolds in one hand and nucleating β- sheet/β-turn mimics on the other hand, on the conformational preferences of proteins and peptides in solution. Though, an exponential growth on the development of constrained non-peptidic molecular scaffolds as peptidomimetics is observed, very few peptide-based drugs have been developed. Therefore, there is a need to renovate the existing design principles with newer concepts, chemistry and protocols to introduce conformationally constrained nonpeptide isosteres or small molecule scaffold into peptide backbones in order to achieve desirable secondary structures along with pharmacologically viable peptide-based drug candidates of enhanced metabolic stability.23
The following sections of this chapter would represent a critical survey of applications of unnatural amino acids and conformationally constrained small, molecular scaffold/ non-peptide isostere in the context of peptidomimetics and generation of functional peptides with novel photophysical properties.