Amino Acids and Peptides

Last time we described amino acids, their structures, names, properties, and procedures we use to separate mixtures of them. Today we will discuss some of the carachteristic chemical reactions these molecules undergo, and we'll hopefully finish describing how peptides are formed.

Making amino acids derivatives

Since amino acids have both amino and carboxyl groups, they will undergo reactions that these two groups normally undergo, such as acylation and esterification. The reactions we will analyze are those that make life easy if you are trying to analyze a mixture of amino acids. If they are in small concentrations, detecting and identifying isolated amino acids can be very difficult. We use a number of reactions to make derivatives which are easier to identify, and for which properties are well studied.

The oldest trick in the book is the ninhydrin reaction. When a solution of an amino acid is heated in the presence of  ninhydrin, this compound reacts with the free a-amino group forming a compound that is purple. For Proline, which has a secondary amine, the reacion is not complete and the final product is yellow:

In theory, if conditions are carefully controlled the amount of amino acid present in a sample can be determined spectrophotometrically from the color (optical density) of the solution after the ninhydrin reaction. Unfortunately, this one literally pulls the nitrogen away from the amino acid, so all amino acids give the same reaction, and although it supposedly reacts only with a-amino acids, it is promiscuous as hell, and anything with an amine will react.

A series of more specific reagents are used to detect and quantify amino acids. They are 1-fluoro-2,4-dinitrobenzene, fluorescamine, dansyl chloride, and dabsyl chloride. They all react with the amino group of the amino acid, forming a complex that retains the amino acid side chain. Therefore, they will all be different, and we will be able to distinguish them because they will have different physical proeperties. Since they have several conjugated aromatic rings, they are either highly coloured or fluorecent, which allows to determine their concentrations even if we have minute ammounts. Furthermore, the complexes are very stable to the normal conditions of peptide digestion (6M HCl, heat).

What are the mechanisms of the reaction between the amino group and these reagents?

Linking amino acids - Peptides

Perhaps the most remarkable 'reaction' an amino acid undergoes is a condensation reaction with another amino acid, to form an amino acid polymer called a peptide. Correspondingly, the bond formed between both amino acids is called the peptide bond. Depending on the size, we will call them dipeptide, tripeptide, tetrapeptide, etc. (two, three, four, etc., amino acids), oligopeptide (for a few amino acids), or polypeptide (anything bigger).

As a pseudo-rule, if they are relativelly big and have some sort of biological function we will refer to them as proteins. We can have poly-alanine with hundred of alanine residues, but since it has no specific biological activity we would call this a polypeptide, or peptide in general.

As we saw before, the amino end of one amino acid combines with the carboxyl end of another one forming a peptide bond and loosing one molecule of water in the process:

Now, not to be a pest, but how can we make this reaction go? What happens when we mix and acid with an amine is aqueous solution? Do they react to form a peptide bond? Fortunatelly, they don't. When two amino acids come together in solution they form a salt (one is negative, the other one positive). If they did, they would eventually form a protein which has no encoding whatsoever. The formation of the bond itself is energetically unfavourable (DG > 0).

In living systems, the energy barrier is overcome because we have enzymes that will catalyze the reactions. As we saw at the very begining, the enzymes responsible for the sysnthesis of peptides and proteins (the formation of peptide bonds) are located in the ribosomes, were the transcription of genetic material to protein sequence takes place. The enzymes activate the carboxyl group, making its carbonyl carbon a better electrophile (the carbon of the -COO^- group alone is not a good electrophile), which reacts with the free amine of the other amino acid.

Chemically, we have to do something similar to activate the carbonly carbon and make it more electrophilic. We first react the amino acid with dicyclohexyl-carbodiimide (DCC). The carboxylate attacks the carbon atom between both nitrogens (it is very electropositive):

Since the activating group is very electron withdrawing, the carbonyl carbon becomes highly electropositive, and has no negative charge. Now we add our amino acid to the pot, and it will react with the activated carboxylic acid:

Note that at all times we had a funny looking group in the amino (first step) and the carboxylic acid (coupling). These are called protective groups (therefore the PG), and they are chosen so that we can avoid self-coupling of the amino acid when we activate the carboxylic acid.

If we want to make a dipeptide this route is OK, but if we are trying to make a 100 residue protein, we'll run into trouble. Even for a modestly sized peptide, doing one at a time is tedious, and very time consuming if we think that each single step will involve a purification. What we do is the synthesis on a solid support. One of the amino acids is covalently attached through its amino or carboxy end to a polymer bead, so that we can wash the stuff easily.

Peptide bonds are very important, as they link covalently different amino acids in a protein. As we will see in the near future, it imposes certain conformational constraints to the flexibility  of the peptide backbone that will be very important when we study peptide and protein structure. Why?

We also said that the formation of a peptide bond has a DG > 0). The reverse process (hydrolysis) is, therefore, spontaneous thermodynamically. However, it has a huge activation energy, which means that kinetically it does not happen right away. Again, we will need an enzyme to make this reaction work in a reasonable time (milliseconds...). These enzymes are called peptidases (or proteases), and the catalyze the hydrolysis of the peptide bond to a free amino and carboxylic acid, choping the peptide in two (or more) pieces.

If we are in the lab, we do this in strongly acidic conditions (6M HCl), because, as you probably know from organic chemistry, the hydrolysis of amides is catalyzed by strong acids or strong bases. We use this reactions in the characterization of peptide composition. First, we react the peptide with F-2,4-DNB. The reagent will react with all the free amino groups on the molecule. We then digest it (hydrolyze it) with 6M HCl, and we then look for the 'marked' amino acid. It will correspond to the free amine we had in the original peptide.

Now that we master peptide synthesis, lets get some more nomenclature about peptides:

1) Once two or more amino acids react and form a peptide we do not refer to them as amino acids anymore, but as resiudes of the polypepeptide chain. We change the -ine termination to -yl. Therefore, an alanine amino acids forming part of a peptide will be the alanyl residue.

2) You will sometimes see that the the atoms forming the peptide bond, two of which belong to one amino acid (C and O from the carboxyl end) and two from the other one (N and H from the mine end), are called a peptide group.

3) After we form a peptide we will still have one end with a free amine and the other with a free carboxylic acid. We call them the amino-terminus and the carboxy-terminus.

4) By convention, the polypeptides are written starting from the amino-terminus to the carboxy-terminus, from left to right.

We can clearly see why this starts getting hairyier and hairyier as we start adding more and more amino acids to our polypeptide. As we know, we can use either of the shorthand notations, the three-letter or the one-letter codes, to represents them:

In some cases, people will include the free amino-end and the carboxyl-end in the sequence:

We'll try to use the first of the two shorthand notations whenever possible. Again, the more amino acids we have, this notation will be hard to, and we have to use the one-letter code.

Peptides with biological activity

Even with a few amino acids we can get interesting biological activities. There are many molecules that are used as signals by cells. We already saw one of them: Enkephalin, which is the natural opioid of the brain, and can affect the perception of pain. We have a whole set of hormones, which are cellular messengers, that are small peptides. Additionally:

1) Many natural peptides found in fungi (and therefore unnatural to vertebrates) have antibiotic activity (gramicidin).

2) Most of the toxins found in snake (i.e., cobratoxin), spider, scorpion, and conesnail (i.e., conotoxins) venoms are small (13 to 50 residues) peptides that interfere with the acetylcholine receptors in the nerves. That is how they induce paralysis (the creeppy bastards like to eat you while you are still alive...).

3) Some man-made peptides also have interesting properties. Aspartame (L-aspartyl-L-phenylalanyl methyl ester) is the sweetener used in NutraSweet.

Prepared by Guillermo Moyna, 1999.