Combinational Therapy (Peptide therapeutics)

Question 1

How do you detect amino acids? What is the exception to this and why?

Answer 1

React with ninhydrin and it should produce a purple colour. However, with proline, it produces a more yellow colour due to subsitution of the alpha amino group

Question 2

How do you write a peptide sequence?

Answer 2

N terminus on the left C terminus on the right

Question 3

Where does chymotrypsin cleave on a peptide sequence during partial hydrolysis?

Answer 3

Phe/Lys Tyr/Gly Trp/Leu Recognises carboxy aromatic side chain

Question 4

Where does trypsin cleave on a peptide sequence during partial hydrolysis?

Answer 4

Lys/Pro Arg/Ser Recognises carboxy on basic chains and chops it down (lysine and arginine)

Question 5

What is the problem with combining amino acids together in a mixture to produce peptides?

Answer 5

• Two amino acids can react, other they can react with each other separately - Amino acids are zwitterionic giving them more flexibility with what to react with - This produces lots of different reactions as polymers can react with each other - Cannot be sure what products you are going to get and there is no control

Question 6

How would you control peptide synthesis to produce a more selective process?

Answer 6

• Introduce protecting groups (easily introduced and removed, and are stable to the chemical reactions) 1. Two amino acids that you want to react with each other, each containing an amino and a carboxy group 2. Protect an amino group of one and a carboxy group of the other to ensure that one amino with react with one carbosy 3. After the amino acids have joint via amide bond, then you can deprotect the remaining groups

Question 7

What are possible protecting groups for carboxylic acids? (any disadvantages?)

Answer 7

• Methyl ester- not used as you need NaOH or HCl which are too harsh - Benzyl ester is good for small molecules, however peptides are macromolecules - TFA= Trifluoroacetic acid, which is a mild acid and undergoes Sn1 reaction. There are no byproducts apart from isobutylene which is a gas.

Question 8

What are possible protecting groups for alcohols, thiols and phenols?

Answer 8

• Benzyl ether - t-Butyl ether - Tityl group

Question 9

How do you protect an amine group?

Answer 9

• Cannot convert it to an amide as peptides are composed of amide bonds and cannot differentiate between the protecting group and actual peptide. Reaction conditions are too harsh - Instead, convert to a carbamate. Through ester hydrolysis, it can easily be converted back to an amine

Question 10

What is benzylchloroformate used for?

Answer 10

• Protecting amino acid - Too harsh conditions so not useful in peptide synthesis - Removed with HF

Question 11

What groups need protection in peptides?

Answer 11

• Amine - Amide - Thiol (susceptible to oxidation) - Imidazole - Thioether (susceptible to oxidation) - OH - Phenol - COOH - Guanidine - Indole = reacts with electrophiles

Question 12

What can you use to activate carboxylic acids?

Answer 12

• Carbodiimde - Activated ester - Symmetrical anhydride Involves conversion of hydroxyl group into a good leaving group

Question 13

Outline amide bond formation with DCC

Answer 13

1. DCC functions as a base and abstracts a proton from the acid 2. Carboxylate attacks the carbon 3. Using R2-NH2 it can produce N-acylurea in the 1st step which cannot react and is not ideal

Question 14

How do you produce a tripeptide?

Answer 14

DCC1 and TFA reaction however this is not a practical way to make a peptide

Question 15

What are the advantages of solid phase peptide synthesis?

Answer 15

• Optimises reaction kinetics - Allows the use of a large XS of reagents (however you usually use a 1:1 ratio otherwise you will have to purify to get rid of the XS) - Improves efficiency of transformations

Question 16

In solid phase synthesis, how do you purify the compound bound to the solid support from those in solution?

Answer 16

Simple filtration

Question 17

What is the process of solid phase peptide synthesis?

Answer 17

1. Anchor 1st amino acid including side chain protecting group (SPG) and amino protecting group (NPG). NPG is t-BOC 2. Deprotect the amino group by removing t-BOC to allow it to react(but SPG stays) 3. Formation of peptide bond with another amino acid 4. Repeat deprotection and peptide bond formation 5. Repeat 6. Remove all SPG and cleavage from support using TFA Small porous beads are treated with functional units (‘linkers’) on which peptide chains can be built. The peptide will remain covalently attached to the bead until cleaved from it by TFA

Question 18

What are the three types of protecting groups?

Answer 18

1. Carboxy protecting group 2. Amino protecting group 3. Side chain protecting group

Question 19

Why is it important to anchor the growing polypeptide chain is solid phase peptide synthesis?

Answer 19

To prevent it form being washed away when it is being treated with protecting and deprotecting agents

Question 20

What is DCC?

Answer 20

Activating agent that activates the carbon atom on carboxyl group on the amino acid

Question 21

What is t-BOC?

Answer 21

Deactivating agent that blocks amino group on the amino acid

Question 22

What is the solid support in peptide synthesis and what characteristics should it have?

Answer 22

• Cross-linked polymer support - Resin cannot be rigid/static - Must be able to swell and shrink - Resins must be resistant to chemical reactions - Peptide is attached to the support via a linker

Question 23

How is the Sheppard approach different to solid phase peptide synthesis?

Answer 23

Milder than solid phase peptide synthesis

Question 24

What compound is used as a linker in peptide synthesis?

Answer 24

-HPMA - Makes an ester bond, prone to racemicism - Covalently connects the peptide to the solid support and provides a means for their chemical attachment and cleavage

Question 25

Name an example of a compound that can be used as a polymer support in peptide synthesis

Answer 25

Polystyrene High loading resin that swells

Question 26

How do you cleave the peptide chain from its solid support?

Answer 26

1. Place resin in flask and add cleavage mixture and scavengers (TFA/water) and allow to react at room temperature for 3 hours 2. Remove resin by filtration and wash with TFA 3. Extract peptide with water-ether 4. Resuspend in water and freeze dry 5. Analyse HPLC and MS 6. Preparative purification

Question 27

What is the difference between orthodox synthesis and combinatorial synthesis?

Answer 27

Orthodox synthesis- Stepwise directed synthesis to produce one specific product using solution based chemistry Combinatorial synthesis- Produces large numbers of molecules in parallel or simultaneously in mixtures. It is faster, more efficient and cheaper. e.g. Tea Bag methodology