Lecture 19 November 29, 2011 Today we will continue our discussion of peptide chemistry, and in particular, focus on two nontraditional ways to make amide bonds. 1. Jeff Bode chemistry: Until this point, we ve been talking about making peptides from the constituent amino acids, which contain a free amino group and a free carboxylic acid group. Professor Bode realized that you can use different starting materials, and if you choose them carefully, the reaction will proceed rapidly and under mild conditions. In particular, let s look at the following reaction: This is a reaction between a ketoacid and a hydroxylamine (learn new nomenclature wherever possible!) to generate the new dipeptide. The major advantage to this reaction is that it proceeds just by heating it in DMF to 40oC no acids, no base, no high temperatures and gives very high yields. The mild conditions also will prevent the alpha carbon from racemizing (you may remember from our chirality discussion that the proton on the alpha carbon is somewhat prone to racemization, depending on reaction conditions). The major disadvantage is that it is not always so easy to make these starting materials. The mechanism for this reaction is shown below: Here are a few examples that utilize Jeff Bode s chemistry: 1. Draw the product of the reaction shown below: Basically all you have to realize is that you should pull off CO2 from the ketoacid, and pull off MeO (formally MeOH) from the other molecule, and form a new amide bond that connects the two
compounds: 2. Draw the product of the reaction shown below: The novelty here is that two relatively complicated, unprotected peptides can be coupled in high yield using this methodology: 2. Danishefsky isonitrile coupling: Researchers found that they could directly couple an isonitrile and a carboxylic acid to generate a new amide bond. The overall reaction is shown below: Nomenclature note: Isonitrile = compounds where the substituents are attached to the nitrogen of the N=C bond. Nitrile/Cyanide = compounds where the substituents are attached to the carbon of the C=N bond. One possible mechanism for this reaction is: The first step is a proton transfer from the carboxylic acid to the isocyanide. Then the carboxylate acts as a nucleophile, then the nitrogen attacks the carbonyl to generate the four membered ring. In the final step, the ring breaks apart while breaking the bond between the carbonyl and the oxygen. Formally it s a 1,3 acyl transfer because the acyl group (carbonyl) ends up attached to the nitrogen when it used to be attached to the oxygen. The resulting product has a formyl group attached to the nitrogen that s what the C(=O)H group is called. We can remove that formyl group in a separate step to generate a secondary amide
Another benefit is that the formyl group can be partially reduced to a methyl: This makes this procedure very useful because it provides access to N-methyl peptides, which are not so easy to access by other methods. It is very easy to couple a primary amine with a carboxylic acid to generate a secondary amide: But less easy to couple a secondary amine (like proline) (or an N-methyl amine) with a carboxylic acid to generate a tertiary amide: It requires expensive reagents like PyBop or PyBrop. However, here you can just use partial reduction of the formyl groups which is very convenient. Retrosynthetically, you should be able to look at a dipeptide like this one and disconnect it back in two ways: Path A disconnects the amide bond in a traditional way to give the amine and carboxylic acid. Path B disconnects using this new methodology to give an isocyanide and carboxylic acid as starting materials. Your third option, which is a variation on path A, is to add a methyl group to the nitrogen after the amide bond is already formed: This is useful in this case, but if you had a complicated molecule where some nitrogens were methylated and some were not, it would make things very difficult to selectively add methyl groups after forming the amide bonds. Let s look at another example: Predict the product of the following reaction:
So here, the free carboxylic acid group gets converted into an amide bond, and the isonitrile gets converted into a N-formyl group, to give you the following product: You can also use a thioester (instead of a free carboxylic acid) as the coupling partner with the isonitrile. Researchers used this sort of chemistry to synthesize N-methyl peptides en route to the synthesis of cyclosporine: The first step here is the regular isonitrile coupling (with the thioacid) and then they did a partial reduction of the THIO-FORMYL group, so that instead of completely eliminating it, they reduced it to a methyl group. Let s move on now to a new sub-topic in chemistry: Native Chemical Ligation (NCL) The point of NCL is to figure out an efficient way to couple two large peptides together. The problem is that large peptides (more than 16 amino acids) are very insoluble, especially if they are unprotected, and people were having a really hard time making large synthetic peptides in the lab. In 1994, Professor Kent came up with this idea of Native Chemical Ligation to solve this problem. The reaction and mechanism are shown below: This reaction is between a peptide that has a thioester on its C terminus, and a second peptide that has a cysteine amino acid on its N terminus. The structure of cysteine is shown below: There are two steps to this reaction. The first is a transthioesterification between the thioester and the cysteine residue to form a new thioester.
The second step is called an S->N acyl shift, where the carbonyl group migrates from being attached to the sulfur to being attached to the nitrogen: The big advantages here are: mild conditions, high functional group tolerance, ability to couple large unprotected peptides. Big disadvantage is that it requires the presence of a cysteine residue. So if you are trying to make a peptide that is found in natural sources, you need to identify where there is a cysteine residue and make that the amino acid that is involved in the ligation. You have one option: The cysteine can be desulfurized sulfur removed to generate the free methyl group (which is the substituent of alanine). This now allows you to look in the target peptide for alanine residues, and make them from cysteine, then do native chemical ligation, then take the cysteine off. The reagent that you use for converting cysteine to alanine (or in general for removing sulfur atoms) is called Raney Nickel. Next time we are going to do more peptide chemistry, and focus particularly on ways to adapt native chemical ligation so that you do not actually need the cysteine residue.