Several reports on amino acids and peptides functionalization have been reported in the month of May-June 2021. Two high-standard methods among those represented direct synthesis of novel unnatural amino acids, representing Cysteine Borylation and Enantioselective Difluoromethylation of natural amino acids.
Both of these discoveries represent first-ever reports for the corresponding analogs and are expected to have potent applicability in the medicinal field.
Let’s explore the reason and importance behind the discoveries.
An Organometallic Strategy for Cysteine Borylation
Synthetic bioconjugation as well as modifications of natural biological systems has emerged as a powerful tool in enhancing the therapeutic efficiency of medicinal natural products. So far such semi-synthetic approaches have been applied to many natural products by creating C−S, C−O, C−N, and C−C, among which conversion of natural to unnatural amino acids is one of the most fascinating areas.
Owing to the soft nucleophilic site, Cysteine (Cys) residues in proteins and peptides have gained potent attraction for synthetic bioconjugation. There are 100s of reports of C-S bond formation and many of them become emerging tools in the development of peptide therapeutics.
Previous Research and Drawbacks
In natural peptides and proteins, S-S bond formation via attachment of 2 cysteine residues is pretty common. As mentioned above, extensive research followed by successful C-S bond formation on cysteine via synthetic approach is also performed. But till date, there was no existing method for effective borylation toward peptide and protein.
Although borylation could be done on free thiols, the known methods often lack selectivity for thiols over other competing nucleophilic centers, which eventually restricts successful bioconjugation reactions.
New discovery: Accessing borylated novel unnatural amino acids
Alexander M. Spokoyny and co-researchers from MIT, USA hypothesized and executed the first-ever methodology that could effectively perform borylation on peptides and proteins. As a part of their successful hypothesis, they targeted the three-dimensional delocalized aromaticity of Icosahedral boranes that can represent a promising platform to overcome past failures.
They selected a specific boron-bound carboranyl cluster supported Pt(II) complex which successfully performed chemoselective Cysteine borylation on several unprotected peptide substrates, generating first-ever B−S bond linkages post synthetically. Notably, the borylated peptides exhibited high stability toward the excess base, acid, and external thiol! In addition, the borylated peptides were shown to be non-toxic up to 50 μM in cell culture.
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Importance of the discovery
The area of cysteine borylation was an unexplored area of medicinal chemistry research, however, this approach is selectively borylated the cysteine sulfur in free peptides, which is a remarkable success in synthetic.
This new approach opens up opportunities in areas of multivalent binding and the tuning of ligand−receptor interactions in biomolecules, revealing an important discovery in medicinal chemistry.
Enantioselective Difluoromethylation of Amino Acids
First, let’s talk about the importance of fluorination or fluoroalkylation. Fluorine is a very fascinating group in medicinal chemistry mainly due to 2 reasons:
- Introduction of fluorine in a certain site of specific molecules can dramatically change the pharmaceutical value of the functionalized molecule.
- Despite such remarkable efficiency of fluorinated/fluoroalkyl moieties, they are rarely available in nature.
Unnatural amino acids gained immense attention in drug development research due to their promising and diversified bioactivities. It is proven that the introduction of fluorine-rich groups in amino acids enhances the bioactivities of novel unnatural amino acids remarkably, and hence the development of fluorinated amino acids has become a top research interest to the scientist from this specific field.
Difluoromethyl amino acids (DFAA) are generally designed to inactivate their target enzymes by losing the fluoride atom via the E2 elimination mechanism. Thus, DFAA can act as suicide inhibitors for enzymes from unwanted/external bodies as well as potential drug candidates. For instance, fluorinated ornithine analog α-difluoromethylornithine (DFMO) can inhibit pyridoxal phosphate (PLP)-dependent ODC, acting as a potential anticancer and chemo-preventive agent.
Past problems/ deficiencies
The sincere efforts of the scientist fetched many methodologies for deriving structurally diverse gem-difluorinated compounds, though accessing chiral difluoromethylated compounds remained a long-standing problem. In most cases, difluorocarbene is used for deriving difluoroalkylated compounds, while the intrinsic instability and high reactivity of the difluorocarbene species are the prime facts that restrict the enantioselective synthesis of such moieties.
Now, focusing on the present research, it’s almost decades passed when Bey and co-researchers pioneered the method for difluoromethylation of aldimine esters using HCFC-22, generating racemic DFAA. Other existing protocols require stoichiometric quantities of strong bases, involvement of multistep sequences, and harsh conditions to generate racemic DFAA.
Eventually, the lack of enantioselective methodologies for synthesizing DFAA lowers the pharmaceutical value of derived DFAAs; therefore, sustainable asymmetric synthesis of DFAA is highly demanding.
New discovery: Enantioselective Difluoromethylation to access novel unnatural amino acids
Prof. Chan Guo and co-researchers from the University of Science and Technology of China hypothesized, under a milder condition, the generation of N-metallated azomethine ylide may act as a binding cavity to enhance the enantioselectivity by overcoming the previous challenges.
Based on the hypothesis they proceeded further and developed a copper-catalyzed difluoromethylation on aldimine esters in an asymmetric fashion, deliver chiral DFAA. HCFC-22 is used as difluoromethyl source for the direct and one-pot access of structurally diverse DFAA with high enantiomeric excess.
Besides the successful enantioselective approach for synthesizing free difluorocarbene species, this method has several other benefits including readily available feedstock, easy substrate preparation, and single-step synthesis.
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Importance of the discovery
Uncovering the method for the direct access of chiral DFAA is a remarkable success in synthetic organic chemistry, especially due to the high-end medicinal value of the final product.
In general, the efficiency and activities of any two enantiomers differ a lot; in drug discovery, scientists are keeping to get only the more active enantiomers. The purification of pure enantiomers from racemic mixtures tends to atom-economic losses, and the same thing happens in the case of multi-step synthesis. However, this one-step catalytic approach offers flexibility in catalyst choice by inversion of the catalyst configuration on demand, which affords the single enantiomer rapidly. Eventually, it becomes more atom-economic and reduces the cost of medicine where DFAA is incorporated (if developed).
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Conventional methods for deriving unnatural amino acids require multi-step procedures, which are not atom economic. Besides, those methods mostly experience failures during late-stage functionalizations, producing plenty of unwanted impurities as well as degrading the precursor.
The massive demand for unnatural amino acids in drug discovery encouraged synthetic chemists to search for the simplest methodologies. Since the last decade, the continuous effort of modern scientists successfully promoted the field, developing several one-step methods to access variety of unnatural amino acids. Still, many important functionalizations remain unexplored due to certain difficulties.
The new discoveries exploring one-step borylation and enantioselective difluoromethylation on natural amino acids undoubtfully open a new direction in the drug development field.