1
Q
What is the point of HOBt
A
1, Makes DCC better- A Secondary nucleophile
2. Good if coupling amine which isn’t very nucleophilic
2
Q
What are the important protecting groups to remember
A
- Methoxy (carboxylic acid)
- Boc, Fmoc (amine)
3
Q
What are 4 peptide bond forming reactions
A
- DCC/HOBt
- NHS esters
- BOP/ PyBOP
- HATU
4
Q
What are problems with solid phase peptide synthesis
A
- SPPS
- Purification
- Very slow repetitive addition of single amino acids
4.
5
Q
What are protecting groups needed
A
- Contain amine and acid so not selective reaction unless you protect one
6
Q
What is the solid phase initially
A
- Chloromethyl cross linked polystyrene- Merrifield Resin
- Benzene ring and alkyl groups
- Very hydrophobic as contains aromatic groups- doesn’t like water
- Functionalised with Cl
7
Q
Describe functionalisation of cross-linked polystyrene
A
- 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
8
Q
Describe carbodiimide method of coupling
A
- 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
9
Q
What are 2 problems with carbodiimides
A
- N-acylurea formation - when not nucleophilic amine
- Racemisation
10
Q
What is the Merrifield synthesis
A
- 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
11
Q
How is ester on polystyrene formed
A
- 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
12
Q
What are disadvantages of Merrifield synthesis (solid phase)
A
- Protecting group- removal with CF3CO2H is not mild enough
- HF is very bad chemical - dissolves bone
13
Q
What are advantages of Merrifield synthesis (solid phase)
A
- Can wash polymer after each step
- Removes excess of catalyst or unreacted reagents
- Produces
14
Q
How is Boc deprotected in Merrifield synthesis
A
- CF3CO2H - deprotects
- NR3- mops up remaining acid
- Leaves free amine in polystyrene ester
15
Q
What are side products of deprotection of amine by removal of Boc
A
- CO2 + (CH3)C=CH2
- CF3CO2-R3NH+
- BocX
16
Q
How is the polystyrene amino acid polymer elongated
A
- DCC
- Amino acid protected with Boc
17
Q
How is the amino acid cleaved and Boc group removed
A
- HF
18
Q
What is a disadvantage of Polystyrene in solid phase peptide synthesis
A
- 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
19
Q
What are more hydrophilic alternatives to polystyrene
A
- Polyacrylamide
- PEG-PS
20
Q
What are advances in attachment and removal of Merrifield resin
A
- 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
21
Q
What is prefered method of solid phase peptide synthesis now
A
- 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
22
Q
How can automation be used
A
- Solid phase peptide synthesis process can be fully automated
- Can build large libraries automatically
- Can purchase preloaded resins
- Can make large number very easily
23
Q
What is segment condensation
A
- Problem with previous reaction is after a number of steps - yield drops
- Make shorter chains e.g. 10 segments
- Then join together in solution
24
Q
What are advantages of segment condensation
A
- 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
25
What is an example of a drug that is synthesised using segment condensation
1. Enfuvirtide (T-20)
2. Anti-HIV drug
3. SPPS in 3 sections
4. Final stages completed in solution-phase reactions
5. 106 reactions in total- 99 being spps
6. Overall yield - 30% very good for number of steps
7. tonnes/year production
26
What are microwaves used for
1. Associated with molecular rotations and vibrations
2. Causes molecules to move faster- speeds up reactions
3. Dedicated machines used
4. Little regions of reactants get hotter than solvent without boiling
27
How are SPPS reactions sped up
1. Fully automated microwave SPPS machines are available
2. Reactions now from hours per amino acid addition to minutes
28
What is a problem with doing reactions with polymers
1. Hard to tell if it worked
29
How can you tell if a reaction with a polymer has worked
1. Indicator
2. Off bead analysis
30
What indicator could be used for synthesis of peptides
1. Ninhydrin- test for primary amines
2. Yellow- forms deep blue colour if amine is in system
3. Shows deprotected amine in polymer
4. Destructive but only small amounts needed
31
How is off-bead analysis used to determine active species
1. Cleavage of amino acid from polymer- only small amount needed
2. Then use of analytical techniques- LC, MS, NMR
3. Requires high sensitivity and high throughput format
4. E.g LC-UV/ MS
32
If you don't want to cleave amino acid from polymer how could you tell if it has been made
1. On-bead analysis
2. FT-IR microspectrometry
3. MAS-NMR
33
Describe how FT-IR microspectrometry works
1. Works well in solid phase
2. New peak in IR appearing after a certain amount of time to show generation of new functional groups
34
Describe MAS-NMR
1. Magic angle spinning NMR
2. Typically done in solution as requires spinning of molecules
3. But rotation of solid at 54.7 degrees causes it to act as if it were a solution
