A method of highly selective N-terminal modification of proteins as well as peptides by an isolated ketene was developed. Modification of a library of unprotected peptides XSKFR (X varies over 20 natural amino acids) by an alkyne-functionalized ketene (1) at room temperature at pH 6.3 resulted in excellent N-terminal selectivity (modified α-amino group/modified ε-amino group = >99:1) for 13 out of the 20 peptides and moderate-to-high N-terminal selectivity (4:1 to 48:1) for 6 of the 7 remaining peptides. Using an alkyne-functionalized N-hydroxysuccinimide (NHS) ester (2) instead of 1, the modification of peptides XSKFR gave internal lysine-modified peptides for 5 out of the 20 peptides and moderate-to-low N-terminal selectivity (5:1 to 1:4) for 13 out of the 20 peptides. Proteins including insulin, lysozyme, RNaseA, and a therapeutic protein BCArg were selectively N-terminally modified at room temperature using ketene 1, in contrast to the formation of significant or major amounts of di-, tri-, or tetra-modified proteins in the modification by NHS ester 2. The 1-modified proteins were further functionalized by a dansyl azide compound through click chemistry without the need for prior treatment.

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Background: Protein used in medicine, e.g. interferon, are immunogenic and quickly broken down by the body. Pegylation is a recognized way of preserving their integrity and reducing immune reactions, and works well with enzymes used to degrade amino acids, a recent focus of attention in controlling cancer growth. Of the two arginine-degrading enzymes being explored clinically, arginine deiminase is a decidedly foreign mycoplasm-derived enzyme, whereas human arginase 1 is a native liver enzyme. Both have been pegylated, the former with adjuncts of 20 kD, the latter with 5 kD PEG. Pegylation is done by several different methods, not all of which are satisfactory or desirable.

It is demonstrated that engineered arginase with a single protruded site of cysteine deliberately placed away from its active centre by site-directed mutagenesis can facilitate its attachment on a gold-nanoparticle surface with atomic precision, resulting in no apparent loss in enzymatic activity.

Gastric cancer is one of the most common malignant solid tumors in the world, especially in Asia with high mortality due to a lack of effective treatment. The potential usage of the newly constructed arginine-depleting enzyme—mono-PEGylated Bacillus caldovelox arginase mutant (BCA-M-PEG20), an effective drug against multiple cancer cell lines such as cervical and lung cancers, for the treatment of gastric cancer was demonstrated. Our results indicated that BCA-M-PEG20 significantly inhibited argininosuccinate synthetase (ASS)-positive gastric cancer cells, MKN-45 and BGC-823, while another arginine-depleting enzyme, arginine deiminase (ADI, currently under Phase III clinical trial), failed to suppress the growth of gastric cancer cells. In vitro studies demonstrated that BCA-M-PEG20 inhibited MKN-45 cells by inducing autophagy and cell cycle arrest at the S phase under 0.58 U/mL (IC50 values). Significant caspase-dependent apoptosis was induced in MKN-45 after the treatment with 2.32 U/mL of BCA-M-PEG20. In vivo studies showed that administrations of BCA-M-PEG20 at 250 U/mouse twice per week significantly suppressed about 50% of tumor growth in the MKN-45 gastric cancer xenograft model. Taken together, BCA-M-PEG20 demonstrated a superior potential to be an anti-gastric cancer drug.