5/28: FMOC Protecting Groups: 1) We need to protect these side groups in these amino acids because they are reactive.

Recall: Put on FMOC.

(FMOC) Mechanism is acid chloride + amine amide.

Deprotection Mechanism:

Mechanism for Releasing these groups?

Linker Groups:

1) This is created via an SN2 reaction with the chloride. 2) After we create the linker, we can add our first amino acid that is protected with FMOC

(amine end) and activated on the carboxy end (will discuss later what we use). 3) Add PiP to remove FMOC to free the amine for further attack.

4) In the last step, add TFA to remove the Wang Linker. a. Key Principle: The Wang linker is good because we can selectively remove the

wang linker and any side chain protecting groups with TFA.

(removal of wang resin)

(Removal of Side Chains Protectors)

Removal of Carbocation with Scavengers:

1) Notice that we get carbocations floating around as byproducts. We currently have electron rich side chains that can react with the carbocations, so we have scavengers in the TFA to get rid of the carbocations.

a. Examples: silicon (TIS) donates a hydride (forms a more stable carbocation that does not react), sulfur attacks as a nucleophile, anisole does an EAS that outcompetes the reaction that would occur within the side chains.

Activating Carboxyl Group: Add Things to C-Terminus 1) Why do we not use SOCl2?

a. First, we need to activate because C.A. and amine will not react by itself. b. We get racemization if we use SOCl2 to activate the C.A.

2) Solution: We use a mixture of DIC, HOBt, and new amine to prevent racemization.

HOBt: DIC:

Purpose of DIC: Makes the oxygen on the C.A. a better leaving group (substitute for acyl chloride)

Purpose of HOBt: Supresses intermolecular cyclization. It is a good nucleophile (alpha effect) that prevents formation of unwanted product.

Overall Reaction: We create our desired attachment to the C.A. end.

3) Normally, we have the activated amine end attacking this carbonyl that is now activated.

Mechanism for DIC Attachment: Example uses DCC, but same idea.

Problem of Racemization:

1) Five membered ring favorable. 2) After we form this five membered ring, we can get racemization because deprotonation

will form the aromatic 6 e- system.

3) The nucleophile amine can still add in to this carbonyl, though, forming a racemized product.

4) Using HOBT suppresses this reaction because it is a better nucleophile (alpha effect). It leads to our desired product after addition.

5/30: Example Synthesis with all steps 1) Protect with FMOC on one, leave CA “open” – we will activate it with HOBT and DIC. 2) Make the ester to protect the carboxylate. 3) Note free A.A. is a zwitterion: amine protonated (more basic) and C.A. deprotonated (more

acidic).

Another step by step example: 1) Once we have the wang linker in place, add in FMOC protected amine with DIC and HOBT.

a. This activates C.A. and adds the first A.A. in! 2) Add PiP to remove the FMOC protected amine (excess) to drive the reaction forward. 3) Then, add in excess FMOC protected AA with DIC and HOBT to activate C.A. and add in our

new A.A. 4) Repeat for as many A.A. we need to add (PiP, FMOC-A.A.) 5) Finally, add TFA + scavengers (TIS, anisole) to take off protecting groups and wang linker.

a. Scavengers make sure that we quench carbocations because side chains can react with carbocations.

Making C-Terminal Amide: 1) Linker: Use Rink amide linker. 2) FMOC-protected amine, DIC, HOBT: activate C.A. and allow growing

chain amine to attack. 3) Why we make amide instead of C.A.? 4) After each step, use PiP to remove FMOC. 5) TFA cleavage for last step to get our peptide. Note that when we cleave,

we end with an amide instead of a carboxy.

Ribosomes and Peptide Synthesis:

1) Amino Acid tRNA synthetase enzyme: facilitates addition of ATP, which activates the A.A. (serves as our DIC/HOBT combination).

2) A.A. tRNA Synthetase Enzyme: then this enzyme adds on the activated ester onto the tRNA.

a. Reaction is downhill because we go from activated ester to ester, and the EWGs on the ring stabilize

3) tRNA and mRNA: each tRNA attaches to an mRNA; the mRNA tells what tRNA to come by and bring what A.A.

4) Mechanism of attachment:

4) Mechanism of detachment: Stop codon releases the final amino acid to make the C-terminus carboxylate.

Load unnatural amino acid?

1) Amber suppression stop codon: artificial TRNA that uses this stop codon, so the peptide keeps on growing past the stop codo. Enzyme needed to put this AA onto this artifcla TRNA