Lecture 3 Flashcards
What is the point of HOBt
1, Makes DCC better- A Secondary nucleophile
2. Good if coupling amine which isn’t very nucleophilic
What are the important protecting groups to remember
- Methoxy (carboxylic acid)
- Boc, Fmoc (amine)
What are 4 peptide bond forming reactions
- DCC/HOBt
- NHS esters
- BOP/ PyBOP
- HATU
What are problems with solid phase peptide synthesis
- SPPS
- Purification
- Very slow repetitive addition of single amino acids
4.
What are protecting groups needed
- Contain amine and acid so not selective reaction unless you protect one
What is the solid phase initially
- Chloromethyl cross linked polystyrene- Merrifield Resin
- Benzene ring and alkyl groups
- Very hydrophobic as contains aromatic groups- doesn’t like water
- Functionalised with Cl
Describe functionalisation of cross-linked polystyrene
- Add ClCH2OMe, CH2Cl2, ZnCl2
- Can add just one or multiple MeCl to benzenes - percentage loading
- Don’t want on every benzene
- Want on around 10% of benzenes to have chloromethyl attached
Describe carbodiimide method of coupling
- Used in solution
- acid attacks carbodiimide + protonation to generate o-acylurea
- If amine is not very nucleophilic get migration of carboxylate to nitrogen
- Why HOBt is needed
- Then attacks activated carboxylic acid to generate peptide bond
- Driving force is very stable urea formation
What are 2 problems with carbodiimides
- N-acylurea formation - when not nucleophilic amine
- Racemisation
What is the Merrifield synthesis
- Have chloromethylpolystyrene - Solid phase peptide synthesis
- Make ester attached to polystyrene
- Then Boc group is deprotected
- Extend polymer- using another Boc protected amino acid + DCC- not using HOBt
- Then remove Boc group
- Continue adding amino acids
- Or can cleave off peptide from polymer + Boc group to give amino acid
How is ester on polystyrene formed
- Cs+, DMF
- Forms ester with amino acid OH- Cl removed
- Boc group protecting the amine
- Cs+ is a big cation that doesn’t coordinate strongly- it enhances nucleophilicity of C=O
What are disadvantages of Merrifield synthesis (solid phase)
- Protecting group- removal with CF3CO2H is not mild enough
- HF is very bad chemical - dissolves bone
What are advantages of Merrifield synthesis (solid phase)
- Can wash polymer after each step
- Removes excess of catalyst or unreacted reagents
- Produces
How is Boc deprotected in Merrifield synthesis
- CF3CO2H - deprotects
- NR3- mops up remaining acid
- Leaves free amine in polystyrene ester
What are side products of deprotection of amine by removal of Boc
- CO2 + (CH3)C=CH2
- CF3CO2-R3NH+
- BocX
How is the polystyrene amino acid polymer elongated
- DCC
- Amino acid protected with Boc
How is the amino acid cleaved and Boc group removed
- HF
What is a disadvantage of Polystyrene in solid phase peptide synthesis
- Peptide can coil up
- As polystyrene is hydrophobic
- Result in reactive end group being hidden- reaction speed drops
- May want to use more hydrophilic groups but not as cheap
What are more hydrophilic alternatives to polystyrene
- Polyacrylamide
- PEG-PS
What are advances in attachment and removal of Merrifield resin
- Requires very harsh conditions- HF
- Resin with ether bond between two benzene rings- facilitates cleavage
- Or Rink amide- methoxy group attached with facilitates cleavage- amide formed to amino acid instead of ester
What is prefered method of solid phase peptide synthesis now
- Fmoc PG + Wang resin (more hydrophilic)
- Used anhydride amino acid to start- produces ester + fmoc-amino acid which can be reconverted to anhydride and recycled
- Weak base cleaves off fmoc
- Fmoc-amino acid + Bop used to add another amino acid
- Cleaved off from resin using milder acid- CF3COOH
How can automation be used
- Solid phase peptide synthesis process can be fully automated
- Can build large libraries automatically
- Can purchase preloaded resins
- Can make large number very easily
What is segment condensation
- Problem with previous reaction is after a number of steps - yield drops
- Make shorter chains e.g. 10 segments
- Then join together in solution
What are advantages of segment condensation
- Have advantage over linear synthesis for very long sequences
- Can be faster + higher yield
- could use pre-synthesised short chain peptides which are commercially available- saves time and money
What is an example of a drug that is synthesised using segment condensation
- Enfuvirtide (T-20)
- Anti-HIV drug
- SPPS in 3 sections
- Final stages completed in solution-phase reactions
- 106 reactions in total- 99 being spps
- Overall yield - 30% very good for number of steps
- tonnes/year production
What are microwaves used for
- Associated with molecular rotations and vibrations
- Causes molecules to move faster- speeds up reactions
- Dedicated machines used
- Little regions of reactants get hotter than solvent without boiling
How are SPPS reactions sped up
- Fully automated microwave SPPS machines are available
- Reactions now from hours per amino acid addition to minutes
What is a problem with doing reactions with polymers
- Hard to tell if it worked
How can you tell if a reaction with a polymer has worked
- Indicator
- Off bead analysis
What indicator could be used for synthesis of peptides
- Ninhydrin- test for primary amines
- Yellow- forms deep blue colour if amine is in system
- Shows deprotected amine in polymer
- Destructive but only small amounts needed
How is off-bead analysis used to determine active species
- Cleavage of amino acid from polymer- only small amount needed
- Then use of analytical techniques- LC, MS, NMR
- Requires high sensitivity and high throughput format
- E.g LC-UV/ MS
If you don’t want to cleave amino acid from polymer how could you tell if it has been made
- On-bead analysis
- FT-IR microspectrometry
- MAS-NMR
Describe how FT-IR microspectrometry works
- Works well in solid phase
- New peak in IR appearing after a certain amount of time to show generation of new functional groups
Describe MAS-NMR
- Magic angle spinning NMR
- Typically done in solution as requires spinning of molecules
- But rotation of solid at 54.7 degrees causes it to act as if it were a solution
