Área de Infraestructura de Comunicaciones

M errifield was awarded a Nobel Prize in 1984 for his seminal work on solid phase peptide synthesis (SPPS).^^ The principal o f solid phase synthesis is that an insoluble polym eric matrix in the form o f resin beads, appropriately functionalised with a linker or handle, can be used to host reactions forming amide or ester bonds with the linker (Figure 2.1). The functional groups on the amino acids need to be suitably protected. The a - N protection needs to be temporary, being easily removed without affecting any other protecting groups, to allow for the formation o f repeated amide bonds to form the peptide. The side chain protecting groups, on the other hand, are

sem i-perm anent as they need to be able to w ithstand the conditions applied throughout the peptide synthesis and are only removed at the end o f the synthesis, often concurrently with peptidyl-resin cleavage to yield the fully deprotected peptide.

F u n c tio n a lity c o n t a in i n g s e m i- p e r m a n e n t p r o te c ti n g g ro u p * X = O o r N H X - lin k e r -I Y> O T e m p o r a r y p ro te c tin g g ro u p

Figure 2.1. Different degrees o f amino acid protection, allowing for repeated amide bond formation

Due to the insolubility o f the resins, which could be placed on sinters, excess reagents, solvents and by-products for the removal of the temporary protection and amide bond formation could simply be filtered through once the reactions have taken place, avoiding tedious aqueous work-up procedures required after each amino acid coupling carried out in solution.

The Boc methodology employed by Merrifield involves the use o f a copolymer o f styrene and 2 % divinylbenzene as the insoluble support, with chlorométhylation to provide the active site for first residue coupling. The temporary Boc 7V-protection used is cleaved in relatively mild acidic conditions (HCl/AcOH) whereas the peptidyl-resin cleavage is carried out using hydrogen fluoride, which also cleaves semi-permanent side chain protections such as benzyl or Z. The serious hazards of the use o f hydrogen fluoride in the cleavage o f peptides from Merrifield-type resins led Sheppard in the 1970s to develop a different approach to SPPS.^^^’ Instead of the acid-based Boc methodology employed by Merrifield, a new methodology was developed: Fmoc-based C ^ N methodology used in conjunction with acid labile semi-permanent side chain protections and peptide cleavage from the linker-resin system. Figure 2.2 shows the general scheme for an Fmoc SPPS using a resin with an amino-linker yielding a C-terminal peptide amide upon cleavage.

F m o c - N H - [ l i n k e r |-l i) F m o c d e p r o te c tio n , 0 H ( a c tiv a tio n ) F m o c H N ‘ ii) A m in o a c id c o u p lin g .N - lin k e r - I iii) C a p p in g ,N - lin k e r -I F m o c H N N - lin k e r -I F m o c H N - ( a a ) „ - C O N M ' iv ) P e p tid e c l e a v a g e N il F m o c l lN - ( a a ) „ - C O N H ' O Figure 2.2. General scheme for Fmoc SPPS

In step (i) the temporary Fmoc protecting group is removed with a mild base, typically with 20 % piperidine solution in DMF. In step (ii) the first amino acid, pre­ activated to form an active ester, is coupled onto the resin. This is followed by the N-

acetylation step (iii), carried out to “cap” any unreacted amino group, minimising the amount o f by-products - in the form o f peptides with varying lengths - formed during the synthesis. This sequence is then repeated as necessary to form the required peptide sequence. The manual repeated cycle o f SPPS has been adapted for automation in the development of the solid phase peptide synthesiser.

Following each Fmoc deprotection and amino acid coupling steps in a manual synthesis, a small amount o f resin is normally removed, washed and treated with solutions o f Kaiser test.^^ The Kaiser test is a mixture o f solutions which essentially

is a ninhydrin test for free amines, which indicates a dark blue to purple colouration. A positive Kaiser test should be observed after an Fmoc deprotection step and a negative one (colour o f resulting solution remains unchanged) after an amino acid coupling step. In automated SPPS - either batch (automated delivery o f reagents to sintered column containing resin and agitation during reaction) or continuous flow (automated delivery to column followed by continuous flow o f reagents in a loop) - each Fmoc deprotection and amino acid coupling is m onitored by U.V. instead. Finally, in step (iv) the peptide is cleaved from the linker-resin.