Peptides 1

peptide bond

links one amino acid to another and is formed between carbonyl carbon and amide nitrogen

KNOW HOW TO DRAW***

how are peptides named

a.b - 3 letter abbreviation

a = aa with free amino group on the left

b = aa with free carbox group on the right

advantages and disadvantages of peptide drugs

peptides are natural biological messengers for most endocrine signalling pathways

they are membrane impermeable

they are biologically unstable

2 types of peptide drugs

insulin type

non insulin type

optimisation of peptide drugs

- identify minimum active sequence

- determine critical residues

- protection from degradation at termini

- identify proteolysis sites

- proteolytic stabilisation

- optimise side chain modification

- cyclisation

- improve physical properties

what is the aim of chemical modifications in peptide drug developments

increase metabolic stability and bioavailability

give an example of a chemically modified peptide drug

ozempic

- first 6 aa taken off

- a-amino isobutyric acid added to position 8

- fatty acid with PEG at pos 26

- arginine in pos 24

what did the chemical modifications to ozempic do

pos 8 - increase stability and protect against degradation

pos 26 - increase half life

pos 24 - prevents c18 FA binding on the wrong side

3 steps in peptide analysis and sequencing

1. purify pp by IEX

2. determine which aa are present

3. determine aa sequence

how do we determine which aa are present

complete hydrolysis using strong HCl and boil for 24 hours

the pre/post column derivatisation - can tell aa based on retention time

3 methods in determining peptide sequence

end group analysis

fragmentation

complete sequence

end group analysis

use sanger reagent

react amino acid with reagent - a-amino group get labelled

cleave and hydrolyse aa acid with acid

Then we get all of the different amino acids but one of them has a dinitro benzene attached - UV spec

complete sequence - edman sequencing

PITC reacts with amino end and forms thiol urea

hydrolysis to cleave the terminal aa -> phenylthiohydantoin

phenylthiohydantoin identified using HPLC

cycle repeats to identify each aa

problem with complete sequencing

build up of impurities due to incomplete reaction/ hydrolysis

enzymatic fragmentation - peptide mapping

sequencing of longer pp chains

-> digest with multiple enzymes -> cleave into shorter fragments

-. leads to overlapping fragments

-> each fragment isolated and sequenced using edman sequencing

-> arrange fragments with info - like a puzzle