Lecture 8: Chemical Synthesis of Peptides Flashcards
All proteins and peptides are composed of which elements?
Carbon, hydrogen, oxygen, nitrogen, sulfur
How many naturally occurring amino acids exist?
Twenty
What is the only achiral amino acid?
Glycine
True or false:
The human body utilises only L-amino acids
True
The formation of a peptide bond results in the synthesis of what?
H20
What was the first synthesised peptide and who synthesised it?
Benzoyl-Gly-Gly; by Emil Fischer
What was the first temporary amino protection group and who invented it?
Benzyloxycarbonyl (Z) group; by Leonidas Zervas
How is the Benzyloxycarbonyl (Z) group removed?
By hydrogenolysis
Solid phase peptide synthesis - boc chemistry
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
In solid phase peptide synthesis Boc chemistry, the resin can be cleaved using what?
Hydrogen fluoride
In solid phase peptide synthesis Boc chemistry, Boc can be cleaved using what?
TFA
In Solid phase peptide synthesis (Fmoc chemistry), the amino terminus is protected by what?
Fluorenylmethyloxycarbonyl (Fmoc)
In Solid phase peptide synthesis (Fmoc chemistry), Fmoc is cleaved by what?
Piperidine (mild base)
Steps in solid phase peptide synthesis (Fmoc chemistry)
- 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.
- Piperidine is used to cleave Fmoc to free the amino group.
- 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. - 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.
- Final deblocking occurs when the Fmoc of the final peptide is cleaved by piperidine.
- Final cleavage and deprotection by TFA removes all sidechains (including sidechain protecting groups and resin) to form the final free peptide product.
Key properties of resins in solid phase peptide synthesis
- 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
The most commonly used resin in solid phase peptide synthesis
Polystyrene-1,4-divinylbenzene (1-2%) co-polymer
Resins used in Boc chemistry
PAM resin and Merrifield (chloromethyl) resin
PAM resin and Merrifield resins can be cleaved by what?
Hydrogen fluoride (HF) or TFMSA
True or false:
Merryfield resin is more stable in TFA than PAM resin
False.
The final cleavage in Boc chemistry of Merryfield and PAM resins results in peptides with which group at the C-terminus?
Carboxyl (COOH)
Which resins are used to create peptides with a carboxamide (CONH2) group at the C-terminus in Boc chemistry?
BHA (benzhydrylamine) and MBHA (4-methyl-benzhydrylamine)
Resins used for Fmoc chemistry
Wang resin
Wang resin can be cleaved by what?
95% TFA
Amino acid coupling activation mechanism
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
Which molecule is used in amide coupling?
DIEA
Which molecule is used to monitor deblocking and coupling and how does it work?
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.
Carbocation formation in peptide synthesis is prevented by what?
Scavengers such as TIS, water, anisole, EDT
Solution for epimerisation during peptide synthesis
Use strong activator such as HATU or oxyma
Solution for aspartimide formation in Fmoc SPPS
Use bulky side chain
Solution for Diketopiperazine (DKP) formation during SPSS
Use bulky resin such as Chlorotrityl resin or use Fmoc dipeptide
Solution for aggregation during SPPS
Pseudoproline dipeptides
Solution for incomplete deprotection during SPPS
Use strong base e.g. DBU
Solution for incomplete coupling during SPPS
Use longer coupling reaction time, strong activators or double coupling
Differences between Boc and Fmoc chemistry in SPPS
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
