Protein Chemistry. Structure, Properties, Research Methods - Shendryk A.N. 2022

Amino Acids
Amino Acids
Physical and Chemical Properties of Amino Acids — Reactions at the α-Amino Group

N-Alkylation. This reaction occurs when Amino Acids interact with alkyl halides or dimethyl sulfate ((CH₃)₂SO₄). In the latter case, an N-monomethyl or N,N-dimethyl derivative of the corresponding amino acid is formed:

Class="center">HOOC-CH(R)-NH₂ + (CH₃)₂SO₄→ HOOC-CH(R)-N(CH₃)₂ + H₂SO₄

The aforementioned Reagents can also substitute hydrogen in the carboxyl group or the R radical, but The rate of these reactions is significantly lower. This reaction is sometimes used for research purposes to determine The Effect of hydrogen Substitution at the terminal amino groups of a protein on its biological activity.

Arylation. Examples of this type of reaction include: a) the well-known Sanger reaction with 2,4-dinitrofluorobenzene (DNFB) and b) the Edman reaction with phenylisothiocyanate (PITC).

The Sanger reaction proceeds in an alcohol-alkaline medium at T = 35-37 °C, yielding dinitrophenyl Amino Acid Derivatives (2,4-dinitrophenylamino acid - DNP-derivative of The amino acid):

F. Sanger was the first to use this reaction for the quantitative Introduction of a label into the amino groups of Amino Acids and Peptides.

All amino acids attach one DNFB radical via their a-NH₂ group. Lysine, Histidine, Cysteine, and Tyrosine also attach a second radical via their ε-NH₂ group, imidazole ring, SH group, and hydroxyl group, respectively. All DNP derivatives, except for O-mono-DNP-tyrosine and S-mono-DNP-cysteine, are yellow in color (λ_max = 360 nm). All of them, with the exception of di-DNP-lysine, are readily soluble in ether. The latter is soluble in butyl alcohol.

The Significance of the dinitrophenylation reaction lies in the fact that it is quantitative and leads to The formation of yellow crystalline compounds. They are easily isolated chromatographically and quantified using Spectrophotometric Methods. Furthermore, the DNP-amino acid bond is not hydrolyzed by acid in the products of the dinitrophenylation reaction, making it suitable for determining N-terminal residues in Proteins.

Later, alongside DNFB, researchers began using a similar reaction between the amino group and 1-dimethylaminonaphthalene-5-sulfonyl chloride (dansyl chloride - DNS). The DNS derivative of the amino acid (dansyl amino acid) formed in this reaction exhibits intense fluorescence, which is attributed to the dansyl groups. This allows for the analysis of trace amounts of amino acids using fluorimetry.

The Edman reaction. Phenylisothiocyanate readily reacts with amino acids in a pyridine-Water medium at pH = 8-9 and T = 40 ⁰C to form phenylthiocarbamoyl amino acid derivatives. In non-aqueous media, these derivatives cyclize to yield phenylthiohydantoins of amino acids. We will examine the equations of these reactions in detail in the section dedicated to peptide chain sequencing methods. Phenylthiohydantoins of various Amino acids are easily separated by Chromatography. Upon alkaline Hydrolysis, they yield the original Amino Acid and PITC.

Acylation. Acylation of Amino acids refers to a broad range of reactions aimed at introducing carboxylic acid residues, arylsulfonic acid residues, or various acid esters into amino acids.

Examples of amino acid acylation include their interaction with formic acid, acetic anhydride, or gaseous ketene upon heating.

The reaction takes place in glacial acetic acid.

The introduction of an organic acid residue is frequently used to evaluate the effect of acetylating the terminal a-NH₂ and ε-NH₂ groups of lysine on the Functional Properties of a protein.

Benzyl chloroformate esters. These have proven useful for protecting free amino groups (Z-protection or CBZ-protection, via carbobenzoxy chlorides) during Peptide Synthesis.

The Van Slyke reaction. The amino group of an amino acid reacts with nitrous acid to form a hydroxy acid and release nitrogen gas:

This reaction is classical for monitoring the progress of Protein Hydrolysis based on The amount of evolved nitrogen, which is readily determined by manometric methods.

Advantages of the Van Slyke method: the reaction proceeds rapidly (5-10 min.); only the a-NH₂ groups of amino acids and the N-Terminal Groups of peptides react. Neither the ε-NH₂ group of lysine nor the ammonia cleaved from the amides of aspartic and glutamic acids has time to react with nitrous acid in such a short period.

Reaction with formaldehyde (formol titration). This reaction was first proposed by Sörensen. It is widely used to assess the degree of protein hydrolysis by measuring the amount of liberated carboxyl groups. The Amino groups are blocked with formaldehyde, and the carboxyl groups are titrated with alkali:

CH₂O + H₂N-CHR-COOH → CH₂=N-CHR-COOH + H₂O

CH₂=N-CHR-COOH + NaOH → CH₂=N-CHR-COONa + H₂O

Formaldehyde is also capable of forming methylene bridges between amino groups and other reactive groups containing mobile hydrogen atoms (such as imidazole, guanidine, indole, SH, and OH groups). This reaction is widely used to obtain modified proteins.

Transamination. When aqueous solutions of a-amino acids are boiled with a-keto acids, the a-amino group is transferred from the amino acid to the a-keto acid:

Transamination is frequently accompanied by decarboxylation. In the presence of oxygen, Oxidative Deamination and decarboxylation take place:

R-CHNH₂-COOH + O₂ + H₂O → RCHO + NH₃ + H₂O + CO₂

In Cells, transamination reactions are catalyzed by Enzymes known as aminotransferases.

Formation of Schiff bases. Amino acids interact with aldehydes similarly to primary amines, yielding Schiff bases:

The reaction is carried out in glacial acetic acid, and the resulting Schiff bases are analyzed using electronic spectroscopy or photocolorimetry.

Reaction with phosgene. Phosgene reacts with a-amino acids to form N-carboxy anhydrides (Leuchs' anhydrides):

Leuchs' anhydrides interact with nucleophilic reagents and play a crucial role as intermediates in peptide synthesis.