What is the role of N Hydroxysuccinimide?
NHS (N-hydroxysuccinimide), the uncharged analog of Sulfo-NHS (N-hydroxysulfosuccinimide), enables control and modification of carbodiimide crosslinking reactions involving activation of carboxylates (—COOH) for conjugation with primary amines (—NH2).
What is a primary amine?
Primary amine (1o amine): An amine in which the amino group is directly bonded to one carbon of any hybridization which cannot be a carbonyl group carbon. General primary amine molecular structure. X = any atom but carbon; usually hydrogen. C = any carbon group except carbonyl.
What is EDC NHS?
EDC couples NHS to carboxyls, forming an NHS ester that is considerably more stable than the O-acylisourea intermediate while allowing for efficient conjugation to primary amines at physiologic pH. Sulfo-NHS plus EDC (carbodiimide) crosslinking reaction scheme.
How do you quench NHS esters?
NHS ester crosslinking reactions are most commonly performed in phosphate, carbonate/bicarbonate, HEPES and borate buffers. Other buffers may also be used, provided they do not contain primary amines such as Tris or glycine. Using a large excess of Tris or glycine at neutral-to-basic pH can quench the reaction.
Is N Hydroxysuccinimide water soluble?
Solubility : Soluble in water (50 mg/ml), DMSO (100 mM), methanol, acetone (50 mg/ml), and DMF.
How do you make N Hydroxysuccinimide?
It is a white solid that is used as a reagent for preparing active esters in peptide synthesis. It can be synthesized by heating succinic anhydride with hydroxylamine or hydroxylamine hydrochloride.
What is primary and secondary amine?
Amines are classified according to the number of carbon atoms bonded directly to the nitrogen atom. A primary (1°) amine has one alkyl (or aryl) group on the nitrogen atom, a secondary (2°) amine has two, and a tertiary (3°) amine has three (Figure 15.10. 1).
What is secondary amine?
Secondary (2°) amines—Secondary amines have two organic substituents (alkyl, aryl or both) bound to the nitrogen together with one hydrogen. Important representatives include dimethylamine, while an example of an aromatic amine would be diphenylamine.
What is EDC NHS coupling reaction?
EDC/NHS coupling chemistry is commonly used to alter the surface chemistry of NPs with biological molecules to reduce their toxicity [31], [32]. EDC is a water-soluble cross-linker agent, which forms amide bonds between carboxyl and amine groups.
How do you stop an EDC reaction?
Add 1.2µl 2-mercaptoethanol to quench the EDC. Add an equimolar amount of Protein #2 compared to Protein#1 and allow to react at room temperature for 2 hours. Quench the reaction with the addition of hydroxylamine to a 10mM final concentration.
Can NHS ester react with thiol?
Similarly to isothiocyanates, the reversible reactions of thiol and alcohol groups with NHS-esters largely increase the rate of hydrolysis thereof. Bioconjugation methodologies have proven to play a central enabling role in the recent development of biotherapeutics and chemical biology approaches.
Does NHS dissolve in water?
Both NHS and Sulfo-NHS are soluble in aqueous and organic solvents. Activation with NHS, however, decreases water- solubility of the modified carboxylate molecule, while activation with Sulfo-NHS preserves or increases water-solubility of the modified molecule, by virtue of the charged sulfonate group.
How do imidoester crosslinkers react with primary amines?
Imidoester crosslinkers react with primary amines to form amidine bonds. Imidoester crosslinkers react rapidly with amines at alkaline pH but have short half-lives. As the pH becomes more alkaline, the half life and reactivity with amines increases; therefore, crosslinking is more efficient when performed at pH 10 than at pH 8.
How does pH affect imidoester reaction?
Imidoester reaction chemistry. As the pH becomes more alkaline, the half life and reactivity with amines increases; therefore, crosslinking is more efficient when performed at pH 10 than at pH 8. Reaction conditions below pH 10 may result in side reactions, although amidine formation is favored between pH 8-10.
How do NHS esters react with primary amines?
NHS ester-activated crosslinkers and labeling compounds react with primary amines in physiologic to slightly alkaline conditions (pH 7.2 to 9) to yield stable amide bonds. The reaction releases N-hydroxysuccinimide (NHS). NHS ester reaction scheme for chemical conjugation to a primary amine.
What is the half life of imidoester crosslinker?
Imidoester crosslinkers react rapidly with amines at alkaline pH but have short half-lives. As the pH becomes more alkaline, the half life and reactivity with amines increases; therefore, crosslinking is more efficient when performed at pH 10 than at pH 8.