peptide chemistry

what are the functions of proteins

• catalysts (enzymes)

• structure of cells and organisms 

• regulation of information (receptors and ion channels)

• storage and transport

• transmission of nerve impulses

what is the main problem in mixing an amine with a carboxylic acid for peptide synthesis

• an acid base reaction occurs first

• this result in a carboxylate anion and an ammonium group

• this would require temps of 160-180 to form an amide bond

• not compatible with the multifunctional peptide chain

what forms are carboxylate anions and ammonium groups

stable salt forms

why shouldnt you use temperatures of 160-180 to make an amide from stable salt forms

you will ruin the rest of the molecule

what is the solution for requiring high temperatures to form an amide bond

the attachment of a leaving group to the acyl carbon of the acid

what are the main challenges associated with peptide coupling

• low yields 

• difficult purification

• racemisation 

what is racemisation

going from 1 stereocentre to a 50:50 mix

when does racemisation most commonly occur

when the terminal acid peptide is activated

what happens mechanistically speaking in racemisation

• the carbonyl is activated and can be attacked by a nucleophile within the chain

• this leads to a proton forming a tautomer with a double bonded oxygen

• produces a conjugated anionic intermediate

• H2N-R is the next amino acid to be coupled so the end result is the desired coupling reaction but with unwanted racemisation

what way do ribosomes synthesise peptides

from the N terminus to C terminus

what way do chemists build peptides

form C terminus to N terminus

why do we build from the C terminus to the N terminus

prevents the formation of a racemic mixture

how many times can we use a protecting group in peptide formation

as many times as we want until we get the length we want

how many amino acids are there in peptides

50 or less

how many amino acids are there in a protein

over 50

what does deprotection produce

side products but they dont ‘stick’ to resin so can just be washed away 

when are acyl chlorides used in peptide coupling 

when steric hinderance is an issue

what is the two step procedure to use an acyl chloride in peptide coupling 

1. formation of acyl chloride

2. coupling with amine 

why is an acyl chloride used when steric hinderance is an issue

the carboxylic acid can be activated with 1 atom

why can the oxygen attack the thionyl chloride

because the thionyl chloride is electrophilic enough to be attacked

what happens in acyl chloride formation 

• carboxylic acid oxygen attacks the thionyl chloride 

• chlorine leaves as a leaving group

• anhydride looking system forms

• chlorine attacks carbonyl carbon 

• formation of acyl chloride, SO2 gas, and HCl 

what happens in acyl chloride coupling

• amine attacks acyl chloride 

• tetrahedral intermediate arrows 

• tri-ethyl amine attacks the intermediate 

• formation of an amide bond 

what are the limitations of acyl chlorides in peptide coupling

1. generation of HCl is an issue if there are acid sensitive functional groups in the peptide

2. oxalyl chloride generates carbon monoxide

3. coupling with acyl chlorides under basic conditions can lead to racemisation via ketene formation

what do activated esters react easily with

a large range of nucleophiles

why are active esters more reactive than alkyl esters

because their leaving groups and radical intermediates are better stabilised 

what happens to the reaction of active esters if they are more reactive than alkyl esters

they react quickly with a wide range of nucleophiles 

what does it mean of active esters react quicker than alkyl esters

they react cleanly with amines under mild conditions usually with reduced racemisation

what is a carbodiimide

a carbon with 2 double bonded nitrogens

what happens in peptide coupling with carbodiimides

• carbodiimides react with carboxylic acids to form O-acylisourea mixed with anhydrides which react directly with amines to give amides and a urea by-product 

why is an O-acylisourea mixed anhydride better than a acyl chloride anhydride looking system 

because the nucleophile can only attack at one place because the nitrogen is less delta positive than the carbonyl carbon

what are the most common reagents in peptide coupling involving carbiimides

• DCC

• DIC

• EDC 

urea formed with DCC

• urea formed is only partially soluble in many solvents

• hard to purify by column chromatography 

urea formed with DIC

urea formed is soluble in most organics 

urea formed in EDC

water soluble urea by-product is easily removed with an aqueous workup often called WSC

what is carbiimide less basic than

am amine but it can still be protonated

what happens in peptide coupling with a carbiimide

• carbiimide attacks the carboxylic acid

• carboxylate anion attacks the carbiimide carbond

• amine attacks carbonyl carbon 

• urea by-product formed

• protonation of positively charged nitrogen 

• formation of amide bond 

what are the limitations of carboiimides

1. racemisation

2. acetyl transfer forming an unreactive N-acylurea

what does HOBt do in peptide coupling with carbiimides 

makes the reaction happen faster and is added to prevent the formation of the intermediate N-acylurea 

uronium salts in peptide coupling

a one-pot solution in which the active ester is prepared in situ as an intermediate and then reacts with the amine 

in solution what are uronium salts in equilibrium with

guanidium species

what are the most common reagents for peptide coupling with uroniumsalts 

• HATU

• HBTU

• HCTU

HATU in peptide coupling

• the most reactive uronium

• its expensive and only used as a last resort 

HBTU in peptide coupling

• more cost effective alternative

• acceptable for most coupling applications

HCTU in peptide coupling

• developed as an effective alternative to HATU on industrial scales

• safer over time as people in giant peptide factories can build an anaphylactic allergy

what do all of HATU, HBTU, and HCTU give a minimal level of

racemisation