Quantitative Functionalization of the Tyrosine Residues in Silk Fibroin through an Amino‐Tyrosine Intermediate

Abstract

Here, a reaction sequence that can be used to quantitatively modify the tyrosine residues in silk protein from B. mori silkworms is demonstrated. A primary amine is installed ortho to the hydroxyl group on the tyrosine ring using a diazonium coupling reaction followed by reduction of the azo bond. The resulting amine is then acylated using carboxylic acid or NHS‐ester derivatives at room temperature and neutral pH conditions. The silk derivatives are characterized using 1H NMR, UV–vis spectroscopy, ATR‐FTIR, and a unique method to follow this reaction sequence using isotopically labeled reagents and 2D NMR spectroscopy is also used. This study further demonstrates that this sequence can be used to install alkyne or azide functional groups which can undergo further bio‐orthogonal cycloaddition reactions under mild conditions. Finally, methods to carry out these modifications on solid silk microparticles and electrospun mats are also described.

Synthesis and Characterization of Highly Thiolated Silk Fibroin

“…A two‐step method previously reported by our group was first employed to increase the number of nucleophilic reaction sites in silk through the addition of an amine to the tyrosine residues ( Scheme ). [ 24 ] Silk contains an unusually high amount of tyrosine and thus this reaction sequence can provide up to 277 new nucleophilic reactive sites per silk molecule (5 mol%). Here, methods are described to further functionalize these new reactive sites to append thiol‐containing molecules onto each of the tyrosine residues (Scheme 1).…”

“…Preparation of AmTyr-Silk Solution: AmTyr-silk solution was produced as described previously. [24] Briefly, aqueous solutions of B. mori silk in borate-buffered saline were prepared. A diazonium coupling reaction was then carried out to install azo groups on the tyrosine residues in silk.…”

Silk Bioconjugates: From Chemistry and Concept to Application

“…The advantages of this amino-tyrosine group synthesis are the introduction of a nucleophilic amine, the absence of the highly colored azo group that interferes with analytical methods, and the excellent control provided for monitoring the reaction yield using 2D NMR in combination with stable isotope labeling. 62 The use of naturally reactive amino acids and their derivatives to introduce click handles can open up multistep synthetic routes due to the highly specific and efficient nature of biorthogonal click chemistry. 50 Motta and colleagues used click chemistry to synthesize dual-functional silk to create basement mimetics with both laminin IKVAV peptide and collagen functionality.…”

“…Although click chemistry was first described more than a century ago, this chemistry has only come to the fore over the past decade. Exemplary work by the Murphy lab has now established a reaction sequence to control and monitor tyrosine modification . Here, the native tyrosine was converted into azobenzene, followed by reduction of the azo bond to yield an amino-tyrosine group that can be acetylated under mild conditions with carboxylic acid, N -hydroxysuccinimide-ester derivatives, or alkyne and azide derivatives suitable for click chemistry.…”

“…Here, the native tyrosine was converted into azobenzene, followed by reduction of the azo bond to yield an amino-tyrosine group that can be acetylated under mild conditions with carboxylic acid, N -hydroxysuccinimide-ester derivatives, or alkyne and azide derivatives suitable for click chemistry. The advantages of this amino-tyrosine group synthesis are the introduction of a nucleophilic amine, the absence of the highly colored azo group that interferes with analytical methods, and the excellent control provided for monitoring the reaction yield using 2D NMR in combination with stable isotope labeling . The use of naturally reactive amino acids and their derivatives to introduce click handles can open up multistep synthetic routes due to the highly specific and efficient nature of biorthogonal click chemistry …”