We introduced the dipeptide LeuGly as an example because it appears at one end of the peptide hormone oxytocin. The first step in the synthesis of oxytocin is indeed the coupling of glycine (through its amino group) with leucine. This is how it was done by du Vigneaud and Bodanszky. First, the carboxylic acid of the glycine was protected as an ethyl ester. Making an ester is the obvious way to stop CO₂H groups interfering as acids or as nucleophiles. However, simple methyl and ethyl esters may pose problems—they can still react with such nucleophiles as amines. Ethyl esters of amino acids are therefore stable only if the NH₂ group is protected. The glycine ethyl ester had to be stored as its hydrochloride salt: in effect, the –NH₂ group is ‘protected’ as –NH₃⁺.

A more commonly used carboxylic-acid-protecting group that is rather more stable towards attack by nucleophiles is the t-butyl ester. t-Butyl esters can be made by reacting the carboxylic acid with the cation generated from isobutene in sulfuric acid.

Steric bulk means that t-butyl esters are resistant to nucleophilic attack at the carbonyl group, and that includes hydrolysis under basic conditions (nucleophilic attack by HO⁻). But they do hydrolyse relatively easily in acid because the mechanism of hydrolysis of t-butyl esters in acid is quite different. Instead of undergoing nucleophilic attack at the carbonyl group, the t-butyl ester loses a stable carbocation, which is either captured by solvent in an SN1 reaction or loses a proton in an E1 reaction.

In the event, the chemists needed a group that they could later react with ammonia to make the primary amide that is present in oxytocin. They also wanted a group that was stable to mild acid—so they chose the ethyl ester. As for the leucine residue, it had to have its NH₂ group protected using a base-stable protect ing group because base would be needed to release the NH₂ group of the glycine hydrochloride salt. The group that was used is one of the most important nitrogen-protecting groups and is known as the Cbz group (Cbz stands for carboxybenzyl). Cbz groups are put on by treating with benzyl chloroformate (BnOCOCl) and weak base.

Cbz-protected amines are actually carbamates: just like amides they are no longer nucleophilic because the nitrogen’s lone pair is tied up in conjugation with the carbonyl group. They are resistant to both aqueous acid and aqueous base, but they have, to use the analogy we developed earlier, an Achilles’ heel—the benzyl group. Removal of the benzyl group under the same two sets of conditions that remove benzyl ethers releases the safety catch and removes Cbz:

The carboxylic acid of the Cbz-protected leucine next has to be activated to allow it to react with the glycine. The acyl chloride won’t do as it is unstable, and an alternative in peptide chemistry is to make a p-nitrophenyl or 2,4,6-trichlorophenyl ester. Phenoxide, especially when substituted with electron-withdrawing substituents, is a good leaving group, and Cbz leucine p-nitrophenyl ester reacts with the glycine hydrochloride ethyl ester in the presence of a weak base (triethylamine, to release the glycine’s NH₂ group).

The dipeptide is now coupled—but is still protected. Deprotection (HBr/AcOH) gave the HCl salt of LeuGly ethyl ester for further reaction. The rest of the peptide was built up in much the same way—each amino acid being introduced as the Cbz-protected p-nitrophenyl ester before being deprotected ready for the next coupling, until all nine of oxytocin’s amino acids had been introduced.

مواضيع ذات صلة

The Boc protecting group—gastrin and aspartame

Peptide synthesis

A survey of protecting groups

How to react the less reactive group (II): protecting groups

Chemoselectivity in the reactions of dianions

How to react the less reactive group (I): react both then ‘unreact’ one

Competing reactivity: choosing which group reacts

How to oxidize primary alcohols to aldehydes

How to oxidize secondary alcohols to ketones

Birch reduction of arenes

Dissolving metal reductions

Getting rid of functional groups