Abstract

Glycoproteins are important for cellular processes including cell recognition and protein cellular quality control [1]. They are estimated to make up > 50% of proteins [2]. Additionally, they may be the key to unlocking new therapeutic vaccines and cancer immunotherapy treatments [3]. However, glycoproteins remain understudied due to the difficulty of obtaining homogenous samples. A recently published glycoligation method may ease this difficulty if improved [1]. The method required peptides to be created containing trityl protected aspartic thioester residues at intended ligation sites [4]. Using these thioacid precursors can induce an unwanted cyclization reaction and aspartimide formation [4]. A method to eliminate the cyclization reaction and aspartimide formation must be discovered. Aspartyl nitrobenzothiazine (Asp(NBT)) does not undergo any cyclization reaction, should be resistant to aspartimide formation, and be compatible with Fmoc solid phase peptide synthesis. During this study four peptides containing Asp(NBT) were synthesized and analyzed finding no evidence of aspartmide formation. These peptides have been given to the Garner Lab at Washington State to test the removal of NBT, producing a thioacid primed for glycoligation. If successful, the amber suppression technique will be used to genetically encode proteins containing Asp(NBT) for use in the first protein glycosylation attempt using the new glycoligation method [5]. The project described was supported by a SURF award provided by the University of Idaho Office of Undergraduate Research.

[1] Joseph R, Dyer FB, Garner P (2013) Rapid formation of N-Glycopeptides via Cu(II)promoted glycosylative ligation. Org Lett 15:732–735. doi: 10.1021/ol302961s

[2] Apweiler R, Hermjakob H, Sharon N (1999) On the frequency of protein glycosylation, as deduced from analysis of the SWISS-PROT database, Biochimica et Biophysica Acta (BBA), 1473 (1):4-8.

[3] Kent SBH (2015) The critical role of peptide chemistry in the life sciences. J Pept Sci 21:136–138. doi: 10.1002/psc.2754

[4] Joseph R, Morales Padilla M, Garner P (2015) Solid phase synthesis of ω-aspartic thioacid containing peptides. Tetrahedron Lett 29: 4302-4304. doi: 10.1016/j.tetlet.2015.05.064

[5] Liu CC, Schultz PG (2010) Adding new chemistries to the genetic code. Annu Rev Biochem 79:413–444. doi: 10.1146/annurev.biochem.052308.105824

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Using Aspartyl Nitrobenzothiazine to Improve Glycoligation Strategy

Glycoproteins are important for cellular processes including cell recognition and protein cellular quality control [1]. They are estimated to make up > 50% of proteins [2]. Additionally, they may be the key to unlocking new therapeutic vaccines and cancer immunotherapy treatments [3]. However, glycoproteins remain understudied due to the difficulty of obtaining homogenous samples. A recently published glycoligation method may ease this difficulty if improved [1]. The method required peptides to be created containing trityl protected aspartic thioester residues at intended ligation sites [4]. Using these thioacid precursors can induce an unwanted cyclization reaction and aspartimide formation [4]. A method to eliminate the cyclization reaction and aspartimide formation must be discovered. Aspartyl nitrobenzothiazine (Asp(NBT)) does not undergo any cyclization reaction, should be resistant to aspartimide formation, and be compatible with Fmoc solid phase peptide synthesis. During this study four peptides containing Asp(NBT) were synthesized and analyzed finding no evidence of aspartmide formation. These peptides have been given to the Garner Lab at Washington State to test the removal of NBT, producing a thioacid primed for glycoligation. If successful, the amber suppression technique will be used to genetically encode proteins containing Asp(NBT) for use in the first protein glycosylation attempt using the new glycoligation method [5]. The project described was supported by a SURF award provided by the University of Idaho Office of Undergraduate Research.

[1] Joseph R, Dyer FB, Garner P (2013) Rapid formation of N-Glycopeptides via Cu(II)promoted glycosylative ligation. Org Lett 15:732–735. doi: 10.1021/ol302961s

[2] Apweiler R, Hermjakob H, Sharon N (1999) On the frequency of protein glycosylation, as deduced from analysis of the SWISS-PROT database, Biochimica et Biophysica Acta (BBA), 1473 (1):4-8.

[3] Kent SBH (2015) The critical role of peptide chemistry in the life sciences. J Pept Sci 21:136–138. doi: 10.1002/psc.2754

[4] Joseph R, Morales Padilla M, Garner P (2015) Solid phase synthesis of ω-aspartic thioacid containing peptides. Tetrahedron Lett 29: 4302-4304. doi: 10.1016/j.tetlet.2015.05.064

[5] Liu CC, Schultz PG (2010) Adding new chemistries to the genetic code. Annu Rev Biochem 79:413–444. doi: 10.1146/annurev.biochem.052308.105824