The solid phase peptide synthesis (SPPS) was first introduced by Merrifield in 1963 (Merrifield, 1963). The first protected amino acid is attached to an insoluble polystyrene solid support via an acid labile linker. The amino acids are protected by a temporary acid labile protecting group, t-butoxycarbonyl (t-Boc), on the a-amino position, and by a more acid stable benzyl type protecting group on the functionality of the side chain. The t-Boc group is deprotected by trifluoroacetic acid (TFA) followed by the neutralization and washing steps, and then the next protected amino acid couples to the amino peptide resin in the presence of activator. The deprotection and coupling steps are repeated until the desired sequence of the peptide is assembled. The final peptide is cleaved and deprotected from the resin simultaneously by liquid hydrogen fluoride which requires a special apparatus for its safe handling.

The SPPS strategy with a temporary base labile a-amino protecting group, 9-fluorenylmethoxycarbonyl (Fmoc), was introduced in by Carpino in 1972. Generally speaking in Fmoc SPPS, a-amino group is protected by Fmoc and the side chain functionality is protected by the acid labile t-butyl type protecting groups. Fmoc-based SPPS provided an alternative to the t-Boc SPPS and offered the advantage of a milder acid cleavage process. The main focus of this article is to describe the advances in the Fmoc SPPS with which many long peptides have been synthesized successfully. Examples include human parathyroid hormone (84 residues), HIV-1 aspartyl protease (99 residues) and interleukin-3 (140 residues).
The developments in Fmoc SPPS (Fields and Noble, 1990) can be summarized by the following categories: solid supports, linkers, the first residue attachment, protecting groups, Fmoc deprotections, coupling reagents, monitoring, cleavage and removal of protecting groups, peptide evaluation, peptide modifications and peptide ligation.

Solid support:
The SPPS requires a well-solvated gel to allow the reactions to take place between reagents in the mobile phase and functional groups on chains throughout the interior of a resin. The original resin was developed as a polystyrene polymer cross-linked with 1% of 1,3-divinylbenzene with a swelling capacity 3 fold in volume in DMF. A polyamide resin was introduced by Atherton and Sheppard (Atherton and Sheppard, 1989) under the concept that the solid support and peptide backbone should be of comparable polarities. Recently, resins based on grafting of polyethylene glycol (PEG) to low cross-linked polystyrene was developed such as Tentagel (Bayer and Rapp, 1986) and PEG-PS resins (Barany et al, 1992) with a swelling capacity 5 fold in volume in DMF. More recently, resins based on cross-linked PEG have also been available such as PEGA (Meldal M, 1992) and CLERA resins (Kempe and Barany, 1996) with a swelling capacity 11 and 6.5 fold in volume, respectively. Due to their excellent swelling property, Tetagel and PEGA resins have shown superior performance in our laboratory, especially on peptides with long and difficult sequences.

Linkers:
The function of the linker is to provide a reversible linkage between the peptide chain and the solid support, and to protect the C-terminal a-carboxyl group. The commonly used resins to provide peptides acid are Wang, Hydroxymethyl-phenoxy acetyl (HMPA), Rink acid, 2-Chlorotrityl chloride, SASRIN. The most commonly used resin for peptide amide is Rink amide resin.

The first residue attachment:
The esterification of the first amino acid to the hydroxyl group on the resin is one of the key steps to produce a high quality peptide. The incomplete loading and racemization will cause truncated and epimeric peptides respectively, as a result of slow esterification reaction. The commonly used loading methods are the HOBt active ester, symmetrical anhydride and dichlorobenzoyl chloride procedures. The first amino acid residue can be loaded to trityl-based resins with no racemization.

Protecting groups:
For routine synthesis, the global protecting strategy is employed to all reactive functionalities of the side chains. For instance, hydroxyl and carboxyl functionalities are protected by t-butyl group, lysine and trptophan are protected by t-Boc group, and asparagines, glutamine, cysteine and histidine are protected by trityl group, and arginine is protected by the pbf group. A wide range of protecting groups are also available for different applications such as Hmb group used as an amide protecting group to alleviate aggregation during SPPS.

Fmoc deprotection:
The removal of the Fmoc group is usually accomplished by treatment with 20-50% piperidine in DMF for 20 minutes. In the case of incomplete Fmoc deprotection, a stronger base such as 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) with 2% piperidine can be used.

Coupling:
Amide bond formation involves activation of the carboxyl group of the amino acid. There are four major coupling techniques: (a) in situ coupling reagents such as carbodiimide-mediated coupling, BOP, HBTU as well as HATU, (b) preformed active esters such as Opfp, Osu, Onp, (c) preformed symmetrical anhydrides, (d) acid Halides such as acyl fluoride as well as acyl chloride.

Monitoring:
The completion of the deprotection and coupling needs to be monitored to ensure the success of the SPPS. The most widely used monitoring reaction is the Ninhydrin test to examine the presence of free amino group as a result of incomplete coupling. Other methods such as the TNBS and the Chloranil test can be used as complementary methods to the Ninhydin test.

Cleavage and removal of the protecting groups:
Fmoc SPPS is designed for simultaneous cleavage of the anchoring linkage and global deprotection of side-chain-protecting groups with TFA. The most commonly used cleavage cocktail is Reagent K (TFA/thioanisol/water/phenol/EDT: 82.5:5:5:5:2.5 v/v).

Peptide evaluation:
Nowadays, the peptide quality is examined routinely by the analytical HPLC to determine the purity in conjunction with mass spectral analysis to determine the identity. Most of the crude peptides can be purified alone by the reversed phase HPLC to achieve the desired purity. The combinations of anion or cation HPLC purification followed by the reversed phase HPLC purification provide a powerful technique to purify a crude peptide with inferior quality. The peptide purity needs to be determined by analytical HPLC with two different buffer systems or even further by capilliary Electrophoresis (CE). Data from sequence analysis and amino acid analysis can provide further detailed information on peptide homogeneity.

Peptide modifications:
By using of orthogonal protecting group strategy, resins with novel linkers and customized cleavage protocols, modified peptides can be synthesized routinely. These modified peptides can be catagorized as biotinylated, branched, chromogenic, C-terminal modified, fatty acid containing, fluorescent, glycosylated, isoprenated, cyclic lactam , multiple disulfide, peptide mimetics, phosphorated and sulfation peptides

Peptide ligation:
The introduction of the ligation strategy (chemoselective coupling of two unprotected peptide fragments) by Kent (Schnolzer and Kent, 1992) provides the tremendous potential to achieve protein synthesis which is beyond the scope of SPPS. Many proteins with the size of 100-300 residues have been synthesized successfully by this method. Synthetic peptides have continued to play an ever increasing crucial role in the research fields of biochemistry, pharmacology, neurobiology, enzymology and molecular biology because of the enormous advances in the SPPS. The ligation approach further enhances the capacity for synthetic peptides. With future developments in the SPPS and ligation methodology, synthetic peptides will continue to be an indispensable tool for the research communities.