Amino acid N-carboxyanhydride

Amino acid N-carboxyanhydrides, also called Leuchs' anhydrides, are reactive derivatives of amino acids. They are used in the field of biomaterials for their applications in drug delivery, gene therapy, antibiotics, and tissue engineering.^[1][2] Typically, these compounds are derived from amino acids by treatment with triphosgene, phosgene, PCl₅, and other halogenating reagents. NCAs can be used to prepare polypeptides through ring-opening polymerization, a practical approach for large-scale production of polypeptides where sequence specificity is less important than bulk physical properties.^[1][2] These compounds find application in biochemistry, biomedical engineering, and nanotechnology due to their propensity for polymerization upon treatment with nucleophiles, hexamethyldisilazane, and transition metals to create polypeptides.^[3][4][5][6] Evidence suggests that these compounds might be involved in abiogenesis.^[3]

Amino acid N-carboxyanhydride

N-Carboxyanhydrides

NCA molecule undergoes ring opening reaction to create amino acids and polypeptide chains.

Preparation and History[]

NCAs were first synthesized in 1906 by Hermann Leuchs by heating an N-ethoxycarbonyl or N-methoxycarbonyl amino acid chloride in a vacuum at 50-70 °C.^[7][8]

This synthesis of NCAs is sometimes called the Leuchs method. The relatively high temperatures necessary for this cyclization results in the decomposition of several NCAs. Of several improvements, one notable procedure involves treating an unprotected amino acid with phosgene or its trimer.^[9][10][11]

Initially, research into NCA’s was impeded by a myriad of issues, including a lack of technology needed  to characterize compounds.^[3][12] As time went on, NCA’s were mostly used as monomers in polymerization techniques beginning around the 1950’s.^[8] These methods were inefficient however due to multiple side reactions that would lead to early termination or undesired products.^[3][12][8] Due to the poor dispersity of these products, focus turned to tuning the NCA polymerization process.

NCAs are precursors to amino acid homopolymers. Ephraim Katzir first used this method to synthesize poly-L-lysine from N-carbobenzyloxy-α-N-carboxy-L-lysine anhydride, followed by deprotection with phosphonium iodide.^[13]

Use in peptide synthesis[]

Peptide synthesis reactions with NCAs do not require protection of the amino acid functional groups. NCA's are highly reactive and their use can cogenerate many side products. N-Substituted NCAs, such as sulfenamide derivatives, solve some of these problems.^[14]

Organometallic Initiators[]

NCA’s suffered issues with consistency and dispersity due to common side reactions.^{[3][8][12][15]} Thus, methods were needed that produced consistent products of a controllable length. Before 1985, metal salts and other covalent compounds were investigated as potential initiators.^[3] However, these showed limited upside and so research then turned to other methods. Finally, in 1998, T.J Deming discovered organometallic initiators as a method to produce low dispersity products.^[8][12][15] By using a complex of nickel and cyclooctadiene, Deming was able to produce a product that showed a dispersity of about 1.15, which is very good for most reactions.^[15] The organometal complex works by incorporating nickel into the NCA ring and creating a temporary 6 membered ring instead of 5. This produced a living polymerization which allows high control of molecular weight and length of the product.^[3][15] Further research involved using iron and cobalt as initiators; however, only cobalt showed similar, if not better, efficacy than nickel.^[8][12][15]

Polymerization-induced self-assembly[]

Amphiphilic block copolypeptides have been used to form self-assembled structures including hydrogels, vesicles, and micelles. These have been applied as drug delivery carriers and as scaffolds for tissue repair.

A challenge in nanomedicine is eliminating residues of toxic solvents such as dimethylformamide in products, while finding products that are simple and efficient to manufacture. NCA-derived polypeptides may self-assemble in aqueous solutions, allowing for versatile nanomaterials applications and improving application in vivo by avoiding the need to use toxic solvents and nanoprecipitation compared to their use in methods employing separate polymerization and precipitation steps.^[16][17] The one-step process demonstrates an increased rate of reaction with a decreased sensitivity to external conditions such as humidity. In 2019, researchers found that biodegradable nanoparticles may be created with the use of NCA-induced self assembly without the restriction of oxygen-free conditions.^[17]

Biomedical applications of NCA-derived polypeptides[]

NCAs in siRNA delivery platform synthesis[]

One major drawback of siRNA-based therapies is that they lack sufficient in-vivo delivery of siRNA into the target cells because of the large size and negative charge density of siRNA. Efficient transfer of siRNA to the liver has been achieved by lipid nanoparticles, polymer conjugates, and peptide conjugates. One of the major challenges of current methods of transfer is the nonbiodegradability of the vessel once it is inside the body. This challenge is solved by using biodegradable poly(amide) polymers derived from polymerization of NCAs.^[18]

Synthetic Mucins[]

Kramer and Bertozzi recently described synthesis of mucin mimic glycopolypeptides based on native N-acetylgalactosylated poly(serines) (GalNAc-PS).^[19] They described synthesis of controlled molecular weight glycopolypeptides in the massive size range of native mucins and prepared a small library of structures with varied chain lengths and glycosylation densities using NCA polymerization. These glycopolypeptides were found to have physical properties similar to native mucins in that their circular dichroism (CD) spectra indicated a very rigid extended conformation. Atomic force microscopy (AFM) studies of persistence length revealed that native glycosylation indeed results in a rigidification of the peptide backbone and these polymers have a nanorod-like structure

Imine-NCA condensation[]

NCAs have been used for the synthesis of imidazolidinones, which are of interest in the pharmaceutical industry.^[20]

Origins of Life[]

A specific area of research that is garnering much attention is the role of NCA’s in the origins of life. It has been hypothesized that NCA’s may be involved in how the very first polypeptides came to be.^[3] Evidence has strongly suggested that NCA’s were used as an intermediary step as amino acids formed polypeptide structures. This phenomenon has been observed by multiple different research groups utilizing multiple different methods.^[3][21] One of the most recent and strongest pieces of evidence supporting this is the observation that carbamoyl amino acids (CAAs) will easily react with NO/O2 gas mixtures and form an NCA intermediary.^[3][21] CAAs have been shown to result from the reaction of regular amino acids with cyanate ions which is a prebiotic situation that is known to have occurred.^[3][21] Thus, reacting CAAs with NO/O2 gas, which is known to have been present, to then form NCA’s is a valid scenario.^[3] However, these methods only describe how short polypeptides can be developed. To begin and sustain life, it has been hypothesized that a polypeptide chain must be at least 60 peptides long.^[22] Studies have shown the formation of NCA’s using previously described methods only produce peptides of roughly 10 or so monomers.^[22] Since this is not sufficient to sustain life, research began into the possibility of natural solid supports. To mimic this approach, it was hypothesized that mineral surfaces that would have been present at the time acted as solid supports for amino acids to polymerize through the NCA intermediate step method. Through this method, oligomers up to 55 monomers long were produced, close to the hypothesized length required to support life. This process would have taken years, as hydrolysis of the amino acids would have been a common side reaction prohibiting the continued chain growth.^[22]

Current Drugs on the Market[]

Copaxone, which was released in 2014, is a treatment for multiple sclerosis marketed by TEVA Pharmaceuticals, and is the only drug prepared by NCA-polymerization that is FDA approved. Ring opening polymerization is a more practical approach for large-scale production of polypeptides where sequence specificity is less important than bulk physical properties.^[1][2]

See also[]

• Dakin–West reaction

References[]