Solid-Phase Synthesis
Introduction
Section titled “Introduction”Solid-phase peptide synthesis (SPPS) is the standard method for laboratory-scale peptide synthesis. Developed by Bruce Merrifield in the 1960s (Nobel Prize, 1984), SPPS allows efficient assembly of peptide chains on an insoluble polymer support.
Fmoc Strategy
Section titled “Fmoc Strategy”The Fmoc (9-fluorenylmethyloxycarbonyl) strategy is the most widely used approach in modern SPPS. It employs base-labile Fmoc protection for the alpha-amino group and acid-labile side chain protecting groups.
Fmoc deprotection uses 20% piperidine in DMF via beta-elimination (5-20 minutes). Monitoring is done by UV absorbance at 301 nm. Advantages include mild deprotection conditions, compatibility with acid-labile side chain protection, and compatibility with automated synthesizers.
Coupling Reagents
Section titled “Coupling Reagents”Efficient amide bond formation requires activating reagents. Modern coupling reagents overcome the low reactivity of carboxylic acids toward amines.
| Reagent | Type | Key Features |
|---|---|---|
| HBTU | Uronium | Most commonly used, fast coupling, low racemization |
| HATU | Uronium | Most powerful, extremely fast, used for difficult couplings |
| DIC | Carbodiimide | Water-soluble, easier byproduct removal |
| PyBOP | Phosphonium | Good for sterically hindered amino acids |
Additives like HOBt and HOAt prevent racemization and accelerate coupling. Oxyma Pure is a newer non-explosive alternative.
Protecting Groups
Section titled “Protecting Groups”The Fmoc strategy provides orthogonal protection: Fmoc is removed by base (piperidine), while side chain groups are removed by acid (TFA). Common protecting groups include OtBu for Asp/Glu, Boc for Lys/Trp, Pbf for Arg, Trt for Cys/His/Asn/Gln, and tBu for Ser/Thr.
Resin Types
Section titled “Resin Types”| Resin | C-Terminal | Application |
|---|---|---|
| Wang Resin | Free acid | Standard SPPS |
| Rink Amide Resin | Amide | Peptide amides |
| 2-Chlorotrityl | Free acid or ester | Fragments, sensitive sequences |
Synthesis Cycle
Section titled “Synthesis Cycle”- Deprotection: 20% piperidine in DMF, 2 x 5 minutes
- Washing: DMF (5 x 30 seconds)
- Activation: Amino acid (5 equiv), HBTU/HATU (4.5 equiv), DIPEA (10 equiv)
- Coupling: 15-60 minutes, monitor by Kaiser test
- Washing: DMF (5 x 30 seconds)
- Repeat from step 1 for next amino acid
Cleavage and Deprotection
Section titled “Cleavage and Deprotection”Standard cleavage uses TFA (95%) with TIS (2.5%) and water (2.5%) as scavengers. The mechanism involves protonation of the linker, cleavage from resin, removal of acid-labile protecting groups, and scavenging of carbocations. Mild cleavage (1% TFA in DCM) is available for 2-chlorotrityl resin.