scholarly journals Use of an asparaginyl endopeptidase for chemo-enzymatic peptide and protein labeling

2020 ◽  
Vol 11 (23) ◽  
pp. 5881-5888 ◽  
Author(s):  
T. M. Simon Tang ◽  
Davide Cardella ◽  
Alexander J. Lander ◽  
Xuefei Li ◽  
Jorge S. Escudero ◽  
...  
Keyword(s):  
Chemical Reaction ◽  
Protein Labeling ◽  
Asparaginyl Endopeptidase ◽  
Simple Chemical

Asparaginyl endopeptidases (AEP) are ideal for peptide and protein labeling. Its pairing with a simple chemical reaction significantly lowers the amount of label needed for effective bioconjugation.

scholarly journals Use of an Asparaginyl Endopeptidase for Chemo-enzymatic Peptide and Protein Labeling

2020 ◽  
Author(s):  
Simon Tang ◽  
Davide Cardella ◽  
Alexander J. Lander ◽  
Xuefei Li ◽  
Yu-Hsuan Tsai ◽  
...  
Keyword(s):  
Phenylboronic Acid ◽  
Product Formation ◽  
Protein Labeling ◽  
Recognition Sequence ◽  
Reaction Conditions ◽  
Site Specific ◽  
Asparaginyl Endopeptidase ◽  
Coupling System ◽  
Labeling System ◽  
Specific Peptide

Transpeptidases are ideal biocatalysts for site-specific peptide and protein labeling, whereas reactions that target N-terminus cysteine with commercially available reagents have become common practice. However, a versatile approach that allows bioconjugation at the terminus of choice (N or C), while avoiding the use of backbone-modified substrates (<i>e.g.</i> depsipeptide) or large excess of reagent, is highly desirable. Aiming to meet these benchmarks, we have combined the advantages of asparaginyl endopeptidase (AEP) catalysis with a N-terminal cysteine trapping reaction and created a chemo-enzymatic labeling system. In this approach, polypeptide with a Asn-Cys-Leu recognition sequence are ligated with a counterpart possessing an N-terminal Gly-Leu by AEP; the byproduct Cys-Leu is subsequently trapped by a stable and inexpensive scavenger, 2-formyl phenylboronic acid (FPBA), to yield an inert thiazolidine derivative, thereby driving the reaction forward to product formation. By carefully screening the reaction conditions for optimal compatibility and minimal hydrolysis, conversion to the ligated product in the model reaction resulted in excellent yields. The versatility of this AEP ligation/FPBA coupling system was further demonstrated by site-specific labeling the N- or C-termini of various proteins.

scholarly journals THE RELATIONSHIP BETWEEN SPREADING FACTOR AND HYALURONIDASE

1941 ◽  
Vol 73 (1) ◽  
pp. 109-123 ◽  
Author(s):  
Gladys L. Hobby ◽  
Martin H. Dawson ◽  
Karl Meyer ◽  
Eleanor Chaffee
Keyword(s):  
Hyaluronic Acid ◽  
Comparative Study ◽  
Chemical Reaction ◽  
Spreading Factor ◽  
Hyaluronidase Activity ◽  
Comparable Effect ◽  
Simple Chemical ◽  
The Relationship ◽  
Homologous Enzyme

A comparative study of spreading factor and hyaluronidase in preparations from various sources revealed the following points of similarity and dissimilarity in the two reactions. 1. Similarities: (a) All preparations containing hyaluronidase also produced spreading. (b) Heating at 65° and 100°C. for 30 minutes produced a comparable effect on both reactions. (c) The demonstration of the presence of hyaluronic acid in skin offers a plausible explanation for the mechanism of spreading on the basis of hyaluronidase activity. 2. Dissimilarities: (a) No parallelism was observed in the degree of activity of spreading factor and hyaluronidase in the same preparations. (b) All preparations which produced spreading did not contain hyaluronidase. (c) Antisera to hyaluronidase preparations specifically and completely inhibited the activity of the homologous enzyme but did not inhibit the spreading factor in the same preparations. The significance of the similarities and dissimilarities between the two reactions is discussed. It is concluded that while hyaluronidase may play a rôle in the spreading reaction the phenomenon is a complex one and cannot be explained on the basis of a simple chemical reaction.

The Acetic Anhydride-Sulphuric Reaction for General Paresis

1927 ◽  
Vol 73 (302) ◽  
pp. 419-421 ◽  
Author(s):  
A. G. Duncan
Keyword(s):  
Acetic Anhydride ◽  
Mental Disorder ◽  
Sulphuric Acid ◽  
Positive Reaction ◽  
Chemical Reaction ◽  
Spinal Fluid ◽  
General Paresis ◽  
Simple Chemical

While elaborating a test for cholesterol in the cerebro-spinal fluid, 0. H. Boltz (I) observed and investigated a reaction which occurred predominantly in cases of neuro-syphilis, and which he named the acetic anhydride-sulphuric test. To I c.c. of spinal fluid was added 03 c.c. of acetic anhydride, drop by drop, and after mixing the fluids by shaking, [email protected]. of concentrated sulphuric acid was added in drops, and the mixture shaken again. The development of a blue pink or lilac colour characterized a positive reaction. The test has been studied in this country, and it has been claimed that the reaction is positive in almost every case of general paresis, and negative in almost every other type of mental disorder, except certain cases of non-paretic neuro-syphilis. As this constitutes a surprising degree of specificity of an apparently simple chemical reaction for a single disease, I have carried out the reaction in association with the routine tests in a series of cerebro-spinal fluids with a view to ascertaining its value. One hundred and sixty fluids were examined—a comparatively small number on which to base conclusions, but the results will show that the claims previously advanced for the value of the test require considerable modification.

scholarly journals An Isotope Effect in a Simple Chemical Reaction

1948 ◽  
Author(s):  
Peter E. Yankwich ◽  
Melvin Calvin
Keyword(s):  
Isotope Effect ◽  
Chemical Reaction ◽  
Simple Chemical

Site-Specific C-Terminal Labeling of Peptides and Proteins using Asparaginyl Endopeptidase in a Chemo-Enzymatic Sequence

2019 ◽  
Author(s):  
Simon Tang ◽  
Davide Cardella ◽  
Alexander J. Lander ◽  
Xuefei Li ◽  
Yu-Hsuan Tsai ◽  
...  
Keyword(s):  
Phenylboronic Acid ◽  
Catalytic Efficiency ◽  
Model Reaction ◽  
Protein Labeling ◽  
Recognition Sequence ◽  
Site Specific ◽  
Asparaginyl Endopeptidase ◽  
Leaving Group ◽  
Reaction Proceeds ◽  
Potential Use

Asparaginyl endopeptides (AEP) are recognized for their catalytic efficiency, presenting as ideal tools for protein bioconjugation. However, the peptide ligation catalyzed by AEP is reversible. In an attempt to obtain high reaction yields, thiodepsipeptides have been used as substrates but found to be highly unstable, and labeling is only limited to the N-terminus. To maximize the potential use of AEP, here we developed a novel chemo-enzymatic sequence for protein bioconjugation at both the N- and C-termini. In this system, an alternative recognition sequence, Asn-Cys-Leu, was used. Upon ligation, the reaction yields Cys-Leu as leaving group, and its reactive 1,2-aminothiol functionality was quenched by an effective and affordable electrophile, 2-formyl phenylboronic acid (FPBA), to yield a non-reactive cyclic byproduct. In the presence of FPBA our model reaction proceeds with ~95% yield using only 1.2 equivalent of substrate, whereas the yield remains at ~50% in the absence of this additive. This “quenching” approach enables protein labeling at both the N- and C-termini ranging from 75 to 85% (five examples). The simplicity and versatility of this quenching approach will enhance the future use of AEPs in protein bioconjugation.

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