Lecture 8: Chemical Synthesis of Peptides

Question 1

All proteins and peptides are composed of which elements?

Answer 1

Carbon, hydrogen, oxygen, nitrogen, sulfur

Question 2

How many naturally occurring amino acids exist?

Answer 2

Twenty

Question 3

What is the only achiral amino acid?

Answer 3

Glycine

Question 4

True or false:

The human body utilises only L-amino acids

Answer 4

True

Question 5

The formation of a peptide bond results in the synthesis of what?

Answer 5

H20

Question 6

What was the first synthesised peptide and who synthesised it?

Answer 6

Benzoyl-Gly-Gly; by Emil Fischer

Question 7

What was the first temporary amino protection group and who invented it?

Answer 7

Benzyloxycarbonyl (Z) group; by Leonidas Zervas

Question 8

How is the Benzyloxycarbonyl (Z) group removed?

Answer 8

By hydrogenolysis

Question 9

Solid phase peptide synthesis - boc chemistry

Answer 9

BMF or NMP is added to swell the resin, allowing the functional side chain of resin to be open. The carboxyl group is the only available group as the amino group is blocked by Boc

Question 10

In solid phase peptide synthesis Boc chemistry, the resin can be cleaved using what?

Answer 10

Hydrogen fluoride

Question 11

In solid phase peptide synthesis Boc chemistry, Boc can be cleaved using what?

Answer 11

TFA

Question 12

In Solid phase peptide synthesis (Fmoc chemistry), the amino terminus is protected by what?

Answer 12

Fluorenylmethyloxycarbonyl (Fmoc)

Question 13

In Solid phase peptide synthesis (Fmoc chemistry), Fmoc is cleaved by what?

Answer 13

Piperidine (mild base)

Question 14

Steps in solid phase peptide synthesis (Fmoc chemistry)

Answer 14

1. The first amino acid is attached to resin (solid phase), its amino terminus protected by a Fmoc group. If the side chain is reactive (e.g. glutamic acid, serine), it must be protected.
2. Piperidine is used to cleave Fmoc to free the amino group.
3. The second amino acid has a Fmoc-protected amino
   group but a free carboxy-terminal. A coupling agent (activator) is attached to the carboxyl group to activate the C-terminus.
4. When coupled together in the presence of a base, the free carboxy terminal of the second amino acid will bind to the freed amino group of the first amino acid, forming an amide bond. The coupling releases the activator bound to the C-terminus of the second amino acid. The previous steps can be repeated for each amino acid addition.
5. Final deblocking occurs when the Fmoc of the final peptide is cleaved by piperidine.
6. Final cleavage and deprotection by TFA removes all sidechains (including sidechain protecting groups and resin) to form the final free peptide product.

Question 15

Key properties of resins in solid phase peptide synthesis

Answer 15

• Must contain a functional group
• Chemical stable (must be inert to all applied chemicals)
• Mechanically stable (shouldn’t break under stirring)
• Must swell extensively in the solvents used for the synthesis
• Peptide-resin bond should be stable during synthesis
• Peptide-resin bond can be cleaved effectively at the end of synthesis

Question 16

The most commonly used resin in solid phase peptide synthesis

Answer 16

Polystyrene-1,4-divinylbenzene (1-2%) co-polymer

Question 17

Resins used in Boc chemistry

Answer 17

PAM resin and Merrifield (chloromethyl) resin

Question 18

PAM resin and Merrifield resins can be cleaved by what?

Answer 18

Hydrogen fluoride (HF) or TFMSA

Question 19

True or false:

Merryfield resin is more stable in TFA than PAM resin

Answer 19

False.

Question 20

The final cleavage in Boc chemistry of Merryfield and PAM resins results in peptides with which group at the C-terminus?

Answer 20

Carboxyl (COOH)

Question 21

Which resins are used to create peptides with a carboxamide (CONH2) group at the C-terminus in Boc chemistry?

Answer 21

BHA (benzhydrylamine) and MBHA (4-methyl-benzhydrylamine)

Question 22

Resins used for Fmoc chemistry

Answer 22

Wang resin

Question 23

Wang resin can be cleaved by what?

Answer 23

95% TFA

Question 24

Amino acid coupling activation mechanism

Answer 24

A coupling agent (activator) such as Carbodiimide (DCC) serves as both a proton acceptor and donor. The nitrogen of the DCC accepts a proton from the carboxyl group of the C-terminus of the amino acid. This leaves a negatively charged oxygen. The lone pair of

Question 25

Which molecule is used in amide coupling?

Answer 25

DIEA

Question 26

Which molecule is used to monitor deblocking and coupling and how does it work?

Answer 26

Ninhydrin is used in the Kaiser Test to monitor deblocking and coupling. A blue colour indicates a free amino group and that coupling/amide bond formation hasn’t occured.

Question 27

Carbocation formation in peptide synthesis is prevented by what?

Answer 27

Scavengers such as TIS, water, anisole, EDT

Question 28

Solution for epimerisation during peptide synthesis

Answer 28

Use strong activator such as HATU or oxyma

Question 29

Solution for aspartimide formation in Fmoc SPPS

Answer 29

Use bulky side chain

Question 30

Solution for Diketopiperazine (DKP) formation during SPSS

Answer 30

Use bulky resin such as Chlorotrityl resin or use Fmoc dipeptide

Question 31

Solution for aggregation during SPPS

Answer 31

Pseudoproline dipeptides

Question 32

Solution for incomplete deprotection during SPPS

Answer 32

Use strong base e.g. DBU

Question 33

Solution for incomplete coupling during SPPS

Answer 33

Use longer coupling reaction time, strong activators or double coupling

Question 34

Differences between Boc and Fmoc chemistry in SPPS

Answer 34

Boc:

• Requires special equipment (to handle HF)
• Cost of reagents: lower
• Solubility of peptides: higher
• Purity of hydrophobic peptides: high
• Problems with aggregation: less frequently
• Synthesis time: ~20min/amino acid
• Final deprotection: HF
• Safety: potentially dangerous

Fmoc:

• Does not require special equipment
• Cost of reagents: higher
• Solubility of peptides: lower
• Purity of hydrophobic peptides: may be lower
• Problems with aggregation: more frequently
• Synthesis time: ~20-60min/amino acid
• Final deprotection: TFA
• Safety: relatively safe