Amino acid reactions  
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Amino acid reactions
 
Introduction
Reactive groups in amino acids include -NH2 and -COOH groups and groups present on side chains. In peptides and proteins only the side chain is available for reactions (besides amino and carboxylic groups at the terminal ends).
Please note that in peptides and proteins:
  • Compounds reacting with amino groups can affect both the amino group at N-terminal end and the epsilon-amino group of Lysine.
  • Compounds reacting with carboxyl groups can affect the C-terminal carboxyl group and carboxylic groups of aspartic and glutamic acid side chains.

    •  
     
    NH2 group


    Ninhydrin. Ninhydrin (triketohydrindene hydrate) is an oxidating agent which leads to the oxidative deamination of alpha-amino groups. It is very important for the detection and the quantitative analysis of amino acids. Ninhydrin also reacts with primary amines however the formation of carbon dioxide is quite diagnostic for amino acids. Alpha amino acids yield a purple substance that absorbs maximally at 570 nm. Imino acids (proline) yield a yellow product (absorption maximum 440 nm).

     
     


    Sanger Reagent (FDNB). The arylation (dinitrophenylation, DNP) reaction of amino groups with FDNB (1-Fluoro-2,4-dinitrobenzene) was used by Sanger et al. (1955) for the determination of the primary structure of insulin. The DNP-derivatives are stable to acid hydrolysis.

    The reaction of FDNB with peptides/proteins leads to the formation of the DNP-derivative of the terminal alpha-amino group and the DNP-derivative of other reactive groups (e.g., the epsilon amino group of lysine). After acid hydrolysis only one amino acid will result modified at the alpha-amino group, i.e., the N-terminal residue. The DNP derivatives after acid hydrolysis can be extracted with organic solvents and identified by cromathography.


    Dansyl Chloride. 1-dimethylaminonaphtalene-5-sulphonyl chloride (Dansyl chloride) is useful for the determination of the N-terminal residue in peptides and proteins. The dansyl derivative is highly fluorescent (much more sensitive than FDNB) and stable to acid hydrolysis.

     

    Cyanate


    It is worth noting that cyanate ion is formed when urea is dissolved in water:

    NH2CONH2NH4+ + NCO-
    thus proteins in urea solutions can be carbamylated.
    Edman Degradation. Phenylisotiocyanate (Edman Reagent) is the must in protein and peptide sequencing ..........


    After treatment with anidrous trifluoroacetic acid (TFA, F3CCOOH) the peptide bond involving the carboxyl of the N-terminal residue is cleaved (cleavage reaction) with the formation of the thiazolinone derivative. The treatment with TFA does not affect the other peptide bonds leaving a peptide chain with n-1 amino acid residues.

    The treatment of the thiazolinone derivative with aqueous acid (conversion reaction) leads to the formation of Phenylthiohydantoin (PTH) derivative of N-terminal amino acid which can be identified by chromatography.
     
    The residue peptide chain can be now submitted to a new coupling reaction and the next amino acid can be identified.

    Phosgene and Carbon Disulfide. The reaction of these compounds with alpha-amino acids yields N-carboxy anhydrydes. These compounds react with nucleophilic reagents and have been used as intermediates in the synthesis of peptides.

    Maleic Anydride. Maleic anydride is used to modify lysine residues in a protein. Trypsin does not recognize these modified residues thus cleaving only at arginyl bonds.


    Experimental conditions. Add slowly a 4-5 fold excess of reactive at pH 8.5-9.5. This pH maximize the NH2 form of amino groups and minimize the hydrolysis of maleic anydride by H2O and OH-. Keep pH to the initial value by adding dilute NaOH. The reaction can be considered terminated when no further variations in pH occur. Do not use buffer containing amino groups. Urea and guanidine hydrocloride are allowed. Amino groups can be restored lowering the pH at 3.5.
    Formaldehyde. Formaldehyde, the most simple aldehyde, is a colorless gas (boiling point -21 oC) with a pungent odor. It is also known as formalin, embalming fluid, or formol. The term formalin is referred to aqueous solutions. Formaldehyde can also occur in solid state as a polymer (paraformaldehyde and trioxymethylene, see figure below). The heating of these polymers allows to obtain anydrous formaldehyde. At high levels, formaldehyde is a probable human carcinogen.

    It reacts with amino groups and it is used to fix protein by means of a methylenic bridge (see reaction below).

    It is also used in the so called formol titration, where the reaction of formaldehyde with the unprotonated form of the amino acid leads to the formation of methylol derivatives:
     

    2HCHO + --NH2 = (HOCH2)2 - N ---
    As a consequence of this reaction the equilibrium:
    -NH3+ = -NH2 + H+
    is moved to the right with liberation of protons which can be titrated with NaOH. During the digestion of protein by proteases there is the liberation of amino groups thus the titration with formol can be used to follow the extent of the reaction.


     
     

     
     
    SH group


    Elman Reagent. DTNB (5,5' - dithio - bis -(2-nitrobenzoate) reacts with -SH groups to yield a mixed disulfide and thionitrobenzoate (molar extinction coefficient=13600 at 412 nm). It is very specific for SH groups.


     

    2,2'- Dipyridyl disulfide (DPDS). DPDS reacts with -SH groups to yield a mixed disulfide and 2-thiopyridone (molar extinction coefficient=7060 at 343 nm). It is very specific for SH groups.
     


    Cyanylation
     
     
     

    S-amino ethilation. Ethylamine Bromide reacts with SH groups to yield S-aminoethylated cysteines. The S-aminoethil derivative of cysteine resembles lysine. Trypsin is then able to cleave peptide bonds involving carboxyl group of this cysteine derivative.


     

    Iodoacetic acid and Iodoacetamide. These two reagents react with SH groups to yield carboxymethyl and carbamidomethyl cysteine. They are used to protect SH group of cysteine. They are stable to acid hydrolysis

     
     

    -CH2- SH + I-CH2-COOH    		  --->
    -CH2-S-CH2-COOH
    cysteine   Iodoacetic acid S-carboxymethylcysteine

    -CH2- SH + I-CH2-CONH2
    --->
    -CH2-S-CH2-CONH2
    cysteine   Iodoacetamide S-carbamidomethyl cysteine


    Heavy-metal ions. Heavy-metal ions reacts with SH groups to give mercaptides and are responsible for the irreversible inactivation of many enzymes. In particular Ag+ have a high affinity for sulphydryl groups and can be used to titrate SH groups quantitatively.
     
    E-SH + Ag+ ----> E-S-Ag + H+



    Tetranitromethane. Tetranitromethane is a reagent for the nitration of phenolic compounds (such as tyrosine and tyrosyl residues of proteins).The reaction leads to the formation of 3-nitro tyrosine (molar extinction coefficient=14400 at 428 nm). It is very specific for phenolic compounds.
     


    References:

    Riordan, J.F., Wacker, W.E.C. & Vallee, B.L. Tetranitromethane. A reagent for the nitration of tyrosine and tyrosyl residues of proteins. J Am. Chem. Soc. 88, 4104-4105, 1966

    M. Froschle, W. Ulmer, and K.D. Jany. Tyrosine modification of glucose dehydrogenase from bacillus megaterium . Effect of tetranitromethane on the enzyme in the tetrameric and monomeric state. Eur. J. Biochem. 142, 533-540, 1984.


    Cleavage of S-S bond. Because the S-S bond, during protein sequence studies, can easily undergo to a number of rearrangement reactions it is opportune to destroy it before starting the hydrolysis of the protein.

    The S-S bond can be reduced to SH by many reducing agents such as mercaptoethanol, dithiothreitol, sodium borohydride. However SH groups, because of their reactivity, need to be blocked (see reactions of cysteine side chain).

    Oxidation of S-S bonds have been also used: Performic acid leads to the formation of cysteic acid which is very stable. Performic acid also oxidizes methionine to the corresponding sulfone (a vantage, see reactions of methionine side chain) and leads to the destruction of tryptophan (a disadvantage). Oxidation of S-S bridges with sulfite does not destroy any other amino acids but yields a SH group and a S-sulfonate group which are unstable.
     


     
    Cyanogen Bromide (CNBr). CNBr reacts with methionine to yield homoserine, homoserine lactone and metylisotiocyanate. If the carboxyl group of methionine is involved in a peptide bond, the bond is hydrolized leading to peptides having homoserine or homoserine lactone at the C-terminus.

     N-terminal methionine and Met-Met sequences will yield free homoserine.

    The oxidation of methionine prevents CNBr cleavage.

     

    Selectivity

    At alkaline pH reacts also with basic groups.

    At acid pH reacts only with Met and Cys.

    Cysteine is slowly oxidized to cysteic acid without cleavage of the peptide bond. It can be protected by carboxymethylation, carboxymethylcysteine does not react with CNBr.

     Experimental conditions
    possible development of HCN (hydrogen cyanide). Be very careful when using a cyanide salt at acid pH.

     The most used solvents to perform this reaction are: formic acid (70%), HCl (0.1 N) or trifluoracetic acid (TFA) anydrous.

    Equilibrium homoserine-homoserine lactone

     The purification of peptides resulting from CnBr cleavage can be complicated by the presence of homoserine and homoserine lactone. This problem can be solved by converting homoserine in its lactone. Ambler, R.P. Biochem. J. 96,32,1965

     
     


    Methionine Oxidation. The oxidation of methionine to sulphone and sulphoxide prevents CNBr cleavage.

     
    Qualitative Detection of Amino Acids


    The following reactions can be useful to detect specific amino acids after paper cromathography and electrophoresis.

    Millon reaction. HgNO3 in nitric acid with trace of nitrous acid reacts with tyrosine yielding a red product.

    Xanthoproteic reaction. Boiling concentraded nitric acid reacts with tyr, trp and phe to yield yellows products.

    Hopkins-Cole reaction. Glyoxylic acid (OHC-COOH) in concentrated H2SO4 reacts with trp to yield a purple product.

    Folin-Ciocalteu reaction. phosphomolybdotungstic acid react with tyrosine yielding a blue product.

    Nitroprusside reaction. Sodium nitroprusside in diluted ammonia react with cysteine (red product).

    Sakaguchi reaction: a-naphthol and sodium hypoclorite react with arginine (red product).

    Sullivan reaction: sodium 1,2 naphthoquinine-4-sulfonate and sodium hydrosulfite react with cysteine (red product).

    Pauly reaction: reacts with the hymidazolic ring of histidine and the phenol group of tyrosine.

  • A : Sulfanilic acid 4.5g + 5 ml of concentrated HCl for 500 ml of solution (in water).
  • B : Sodium nitrite 4.5 g/100 ml of water
  • C : Sodium carbonate 10%
  • Store solution A and B at 4 ¡C.
  • Spraying solution: 10 ml A + 10 ml B + 10 C
  • His: yellow spot (with a red halo), Tyr: purple.
    • Ehrlich reaction: react with the indole ring of trp. Spray with a fresh solution of p-dimethylaminobenzaldehyde (500 mg in 45 of acetone and 5 ml of concentrated HCl. Trp and trp-containing peptides yield blue products. Citrulline and kynurenine also reacts yielding yellows products. Spray with a solution of 1% methionine to stabilize color. Successive coloration with ninhidrin is possible after exposition to acetone-HCl 90:10 (V:V) to bleach spots. 1% p-dimethylaminobenzaldehyde in 1 N HCl (ethanolic solution) can be also used.

    a-nitrous-b-naphtol: reacts with phenol compounds. Phenols substituted in 3 or in 5 also reacts. Phenols substituted in orto or substituted in 3 and 5 do not reacts.

  • A: a-nitrous-b-naphtol 0.1 % in 95% ethanol
  • B: Nitric acid 10% in water
    • Spray with soluton A and dry in the air, spray with solution B and dry for three minutes at 100o C. Tyrosine gives a red coloured compound. If the reactif is used after the ninhydrin reaction (paper electrophoresis or paper chromathography) tyrosine change from violet to red while other coulered spots disappears.