Amino acid synthesis

Of course, amino acids can be extracted from living organisms. It is convenient to be able to synthesize them, as well. There are several methods of more or less general applicability that use chemistry we are familiar with. The first is just an S_N2 reaction of ammonia with an a -bromo acid.
 
 
 
Under these conditions, the second alkylation is relatively slow, and so we can stop at the a -amino acid. Remember that we can prepare the brominated acid by treatment of the carboxylic acid itself with HBr/Br₂ (see ch. 13). Of course, there is no stereospecificity in this reaction, so the amino acid is a racemate.

Amino acids can also be prepared by what is essentially a malonic ester synthesis. Instead of the simple malonate diester, we start with the brominated ester. This reacts with phthalimide, which is unable to overalkylate, in a kind of Gabriel synthesis. The remaining a proton can be removed in base, and the a -carbon alkylated in the usual way. Vigorous acid hydrolysis of the esters and amides releases the amino acid.
 
 
 
 
Of course, the side chains, or groups, that can be added in this reaction are somewhat limited. Primary unbranched alkyl groups (as in methionine) or a ,b -unsaturated carbonyls will work as electrophiles.

The Strecker synthesis puts together two reactions that we learned in carbonyl chemistry. The first is the reaction of ammonia with an aldehyde. The second is the addition of HCN to a carbonyl, only in this case, we use an imine (the N analog of carbonyl).

Reactions of amino acids

Amino acid reactions are essentially those we have seen before for amines and carboxylic acids. Esters and amides can be formed in the usual ways. Side chain reactions are, of course, dependent on the functional groups of the particular side chains.

Structural analysis of polypeptides

Polypeptides are composed of hundreds or thousands of atoms. Determining the structure of such a compound is a formidable task, except in the case of polymers. Although the monomeric units are not identical, they are very similar and there are a limited number of them. The problem reduces to determining the order of monomers in order to determine the Lewis structure or connectivity of the molecule.

The usual first step is to determine the amino acid composition of the polypeptide. This is analogous to determining the atomic composition of a simpler compound. This requires complete hydrolysis in order to get a reliable quantitative estimate of the proportions of amino acids found in the polypeptide. The polypeptide is heated at 100° in 6 N HCl for 24 hours. The amino acids are separated by ion exchange chromatography and detected by their reaction with ninhydrin, a phthalic acid derivative. This reaction produces a purple color that is proportional to the amount of amino acid and allows quantitation as the amino acids come off the column.

The most direct method for determining the order of amino acids is to take advantage of the fact that there is only one free a -amino group. This group will react with the so-called Edman's reagent, phenyl isothiocyanate:

Each amino acid produces a different phenylthiohydantoin, which can be identified by HPLC (high pressure liquid chromatography). The Edman degradation can be applied sequentially to identify amino acids from the amino end of the polypeptide. It is unusual for this to successfully determine more than about 40 amino acids. Typical protein molecules have several hundred amino acids. The Edman degradation cannot determine an entire protein amino acid sequence. However, it can be used to obtain a long enough sequence to do a library screen, looking for the gene that codes for a particular protein. This is the most common way to determine the complete sequence of a protein.

It should be remembered that these analytical techniques require a pure protein. Even small amounts of impurity can decrease the efficiency of the Edman method and make it difficult to determine very many amino acids in the sequence.

Another tool that is used to help determine protein primary structure is proteolysis. There are many enzymes that catalyze the hydrolysis of peptide bonds. Some of these enzymes are more specific than others and will only catalyze the reaction if a particular amino acid is found on one side or the other of the peptide bond. For instance, trypsin requires that lysine or arginine (basic amino acids) contribute the acyl group in the peptide linkage. Chymotrypsin requires an aromatic amino acid (phenylalanine, tyrosine or tryptophan). Cleavage then indicates the presence of these amino acids and the sizes of the products can tell where in the sequence these amino acids appear. In order to determine the cleavage sites, two proteolyses with differing specificities are required. In this case, each cleavage site of one will be contained completely within one fragment generated in the other. Some of these fragments will be small enough for the Edman sequencing. Others will need to be fragmented yet again.

Deciphering the three dimensional structure of proteins requires more sophisticated physical and chemical techniques. By far the most productive has been x-ray crystallography. This requires protein crystals, which can be difficult to obtain. X-ray crystallography can produce atomic resolution. Multidimensional NMR can be used to obtain similar information, but is more difficult to interpret. On the other hand, no crystals are required. Electron microscopy has been used in a limited number of cases; it would be more accurate to call it electron diffraction. This works best with proteins that form two-dimensional crystals, usually membrane proteins. Neutron scattering and distance measurements within the protein structure have also produced some useful results in isolated cases.