The generation of protein sequences through chemical synthesis is proving an especially attractive complement to recombinant DNA-based approaches. In this post-genomic era, the pivotal role of proteins in biological processes and diseases has fuelled the development of technologies for their production. In particular, the ability to incorporate unnatural amino acids and chemical modifications into proteins in a site-specific manner is highly prized.
The chemical synthesis of proteins is therefore highly attractive, as the control afforded by such an approach enables all manner of backbone and side-chain modifications to be inserted specifically into the primary sequence. For example, biochemical and biophysical probes can be incorporated to investigate protein structure and function and to facilitate readouts and detection in biological assays and diagnostics. Non-proteinogenic amino acids, including D-stereoisomers and b-amino acids, can be introduced for sophisticated structure function studies and to generate therapeutics with enhanced pharmacokinetic properties.
In addition, sequences containing sophisticated patterns of post-translational modifications can be synthesised in a defined fashion, which is a prerequisite for generating all constituent members of the human proteome. The ability to engineer protein sequences in such a site-specific manner opens up many powerful new opportunities across basic research, drug discovery, diagnostics and therapeutics.
In addition, chemical protein synthesis enables protein sequences to be generated rapidly from the genome sequence. Advantageously, these peptide and protein products are free from endotoxins. This latter characteristic of synthetic polypeptides is particularly beneficial when the protein product is to be administered for in vivo applications, for example high dose regimes of protein therapeutics or diagnostic imaging, where the preparation must be essentially free from endotoxins. The necessary removal of endotoxins from proteins expressed in cultivated bacteria is traditionally a laborious and expensive process. Direct production of endotoxin-free protein preparations through chemical synthesis is clearly beneficial for such applications.
Peptide synthesis
The field of peptide and protein synthesis is more than 100 years old. During this period there have been major advances in the synthetic methodologies, purification techniques and analytical approaches. A combination of all these factors has contributed to the increased efficiency and robustness of chemical synthesis and, importantly, to the size of target that can be accessed.
In this regard, one of the defining moments in the field was the development of solid phase peptide synthesis (SPPS) for the step-wise assembly of peptide sequences on a solid support. This ingenious approach, initially reported by Dr Robert Bruce Merrifield in 19633, has been described in detail elsewhere4 and is also briefly outlined in Figure 1.
In SPPS the peptide is constructed on a solid resin support, starting from the C-terminus, through the step-wise addition of chemically activated amino acids. Protecting groups are required for reactive amino acid side-chain functionalities, which do not participate in the chain assembly. Similarly, the Na amino group of the reacting amino acid is protected during the coupling reaction. The properties of this protecting group are orthogonal to the side chain protecting groups. This enables it to be selectively removed from the growing peptide chain to facilitate the chemoselective addition of the next amino acid.
In general, one of two synthetic protocols is used for SPPS, based on either an acid labile Na protection strategy (Boc group) or a base labile Na protecting group (Fmoc or less commonly Nsc). Once the target sequence has been assembled, the peptide is chemically cleaved from the resin, usually with concomitant side-chain deprotection, to liberate the desired material. As the assembly is performed on the solid phase, the reactions can be driven to completion by the addition of large excesses of reagents. These can simply be washed away and there is no need to handle and isolate the intermediates.
Assembling the peptide sequence is a series of repetitive cycles. Hence the whole process lends itself to automation and there are now a variety of automated peptide synthesisers on the market.
Consequently automated synthesis is, by and large, the default approach for synthesising polypeptides and is the strategy that CSS-Albachem uses for the chemical synthesis of peptides and proteins.
Synthesis methods
To gain access to larger synthetic protein molecules researchers have focused on increasing the efficiency of each individual step in the overall SPPS process; namely the assembly of the protected primary sequence, minimisation of unwanted side reactions during assembly and cleavage with improved methods for purification of the polypeptide product. In addition, methods for selectively joining together two or more synthetic peptide fragments, through their N- and C-termini, have also been investigated.
After chain assembly the polypeptide is then cleaved from the resin. A number of optimised cleavage protocols have been developed depending on the nature of the resin support and the polypeptide sequence4,6. Despite this, a variety of unwanted side reactions may occur during the cleavage procedure so that new side-chain protecting groups are continually being developed to facilitate their ease of removal and minimise such side reactions.
Purification
Almost without exception the default procedure for purifying synthetic peptides is reverse-phase HPLC (RPHPLC). As the behaviour of the full-length peptide in RPHPLC is, in general, sufficiently different from those of the associated truncated and deletion sequences, the desired material can be isolated pure.
However, with increasing peptide length there is a greater likelihood that a portion of these chromatographically similar by products co-elute with the desired material. As a consequence, the isolation of homogeneous long synthetic peptides and synthetic proteins often represents a significant technical challenge. In order to combat this phenomenon, a number of different purification strategies have been developed to facilitate the production of long synthetic proteins.
One particularly successful approach uses cleavable chemical tags that are appended to the N-termini of the full-length sequence as a final step in the chain assembly. Due to the nature of the SPPS procedure, incomplete sequences can be capped through an acetylation cycle after each coupling step. So, in principle, only the desired full-length sequence is modified with the tag.
This provides a unique handle that is exploited for purification purposes. The tag is designed in such a manner that, after purification, it can be removed cleanly from the purified peptide, so is in fact traceless. One can envisage that there are a variety of different moieties whose physical or chemical properties could be exploited for such a purification approach. For example, hydrophobic tags have proved to be very effective for the purification of synthetic proteins.
Through selective tagging of the full-length sequence the hydrophobicity is sufficiently altered to enable its separation from untagged deletion and truncated sequences using RPHPLC. One such hydrophobic tag is the Tbfmoc group, which can be used for the production of long synthetic peptides.
By using such techniques the desired full-length sequence can often be purified to a high degree of homogeneity in a simple 'one-step' purification. In an extension of this approach, CSS-Albachem is developing traceless tags for peptide and protein purification procedures that are orthogonal to RPHPLC techniques.
Fragment coupling
Fragment coupling approa-ches enable large peptides and proteins to be synthesised by chemically linking smaller purified synthetic peptides. To effect such a strategy requires the coupling reaction to be specific for the C-terminal of one fragment and the N-terminal of another.
In the classical fragment condensation approach, this non-ambiguity is achieved by using maximally protected fragments and these fragments are joined together via an amide bond-forming reaction in organic solvents. Through differential protection strategies, multiple protected segments can be linked together to construct the target sequence.
The difficulty in handling maximally protected fragments, particularly with respect to their poor solubility, has led to the development of approaches that utilise minimally protected peptide fragments. With this aim we developed a transfer-active ester condensation (TAEC) technique for peptide fragment couplings
In this approach, a peptide hydrazide is initially converted into the corresponding peptide azide. The azide can then be transformed in situ into its active ester with 1-hydroxy-7-azabenzotriazole (HOAt) or ethyl 1-hydroxy-1H-1,2,3-triazole-4-carboxylate (HOCt), and coupled directly with another peptide fragment to obtain the target product.
- (The author is R&D Group Leader CSS-Albachem Ltd, Elvingston Science Centre)