Peptide synthesis

why do we need protecting groups in peptide synthesis

to overcome the random reaction between two amino acids which yields different amounts of various products

what does the protecting group do

makes the carbox acid/ amino group unreactive

directs the reaction so only one type of product is formed

a suitable protecting group must...

be easily introduced in high yield

be stable under conditions of reaction

be easily and selectively removed without affecting new formed bond

longer pep - side chain protecting groups must stay in place

2 ways we can protect carbox acid group

make it a methyl or benzyl ester

how can we deprotect carbox protecting group

methyl - NaOH

benzyl - NaOH or H2/Pd

2 ways to protect amino group

boc

fmoc

boc

reacts with primary amino group -> carbamate

one of the carbonyl on the boc group reacts with amino and rest is cleaved

advantages of boc

easily removable

stable against NaOH, hydrogenolysis i.e. can remove carbox group without removing boc

fmoc

removed by mild NaOH

stable against TFA

can monitor with fluorescence

why does carbox acid group need to be activated

carbonyl isnt very reactive

convert into acid chloride -> electroneg effect -> delta pos = can react with amine to form peptide bond

how do we activate carbonyl group

thionyl chloride

- reaction conditions must be controlled

- potential to racemisation

- can get incomplete reactions

- HCl byproduct can cleave the peptide bond - lower yield

alternative way to activate carbox acid

DCC

catalyses reaction forming reactive intermediate with carbox acid

intermediate easily attacked by amine from another aa

problemt with DCC

product that forms has poor solubility in organic solvents

have come up with variations of DCC to overcome this

disadvantages to DCC

conversion of primary amides to nitriles

some partial racemisation - can be overcome

problem with synthesis of longer peptides

stepwise method requires purification at each step

this is difficult and time consuming

strategy to solving problem

attach growing peptide to a solid insoluble support

easy purification by washing and filtration

what is this strategy known as

merrifield synthesis

steps in merrifield synthesis

attach boc protected first amino acid

carbox acid group reacts with benzyl chloride and forms ester functional group

deprotect, then add another boc protected aa

deprotect

repeat

cleave off solid support using HF, flush out peptide

purify using IEX

problem with merrifield

build up of impurities so can only synthesize up to around 50 aa