advances-in-protein-chemistry

ability to selectively target cells or penetrate the blood-brain barrier. Albiglutide (GlaxoSmithKline), dulaglutide (Eli Lilly & Company),
NGR-hTNF (Mol Med S.P.A), and davunetide (Allon Therapeutics) are prime examples of such peptides that have progressed to Phase 3
studies. Recent report suggests that there are almost 80 peptides in the market, about 200 are in clinical phases and 400 are in advanced
preclinical development stages [12,13], with over 75 per cent of these arriving in the last three decades. Sales for marketed peptide
(glatiramer acetate (Copaxone®), leuprolide (Lupron®), octreotide acetate (Sandostatin®), goserelin acetate (Zoladex®), teriparatide
(Forteo®), exenatide (Byetta®) therapeutics have also grown substantially in last decades. Towards the end of the previous century, the
use of peptide drugs was very limited for the treatment of metabolic diseases, but recent reviews showing metabolism as the largest
therapeutic field (25%) for peptides, followed by cancer (16%) and other clinical fields [14] revealing a significant and promising change
in peptide growth trends.
Advantages and Disadvantages
The most important advantage of peptide therapeutics over all small molecule drug candidates is the structural relationships
between the constructed peptide and the physiologically active parent molecules from which they are derived, an attribute that help in
depicting the risk of unforeseen side‐reactions. As compared to the small molecules that show the advantages of small size, low price,
oral availability, easy synthesis, membrane-penetrating ability and stability, peptides are at a drawback [15-19]. However, in comparison
to large molecules such as proteins and antibodies, peptides are still small. It is owing to its smaller size that the peptides can be easily
synthesized, optimized, and evaluated to prevent any possible side effects. Moreover, the new drugs targeting protein–protein interactions
often require larger interaction sites than small molecules can offer. The main benefit of peptides as therapeutics lies in their high activity,
high specificity and affinity which are often in the nanomolar range, minimal drug-drug interactions, biological and chemical diversity
etc. One of the important aspects of using peptides as drug candidate is their ability not to get accumulated in specific organs such as
the kidney and liver, and thus help in minimizing their toxic side effects. In contrast, small molecules are not particular and can build
up in various organs, ultimately leading to severe toxic side effects. Generally, small molecule peptides are not directly linked to severe
side effects since they are composed of naturally occurring amino acids and which are metabolically endurable too. It is their dosage or
delivery forms which are to be considered while evaluating the side effects.
Peptide drugs have also disadvantages mainly related to their in vivo instability such as, short half-life and low bioavailability,
susceptibility to proteases, formulation and manufacturing challenges etc, resulting in restricted use until last few years. Major
disadvantage of using peptides is the high production cost and the market price as compared to that of the small molecules. However,
the cost of manufacturing peptides has been minimized with the rise in its production scale and efficiency due to developments in
synthesizer, synthesis and purification strategies.
Chemical Synthesis of Peptides:
Concept of synthetic peptides was started in the first halves of twentieth century. After 1950, there had become a significant
development in the field of peptide chemistry in order to define wide role of peptides in all life processes. It had been more interesting to
analyse different peptides by the development of sensitive analytical techniques. It was the group of Friedrich Wessely (1897-1967) which
systematically synthesized peptides using N-carboxyanhydride (NCA) method. The invention of solid phase peptide synthesis (SPPS)
by Bruce Merrifield in 1963 was the revolutionary development in peptide synthesis. This led to the acceleration of peptide synthesis
resulting in production of thousands of peptides and peptide analogues in a short time. At the time it was clear that peptides were a
very important biologically active class of naturally occurring compounds. It involved the stepwise addition of protected amino acids to
a growing peptide chain which was attached to a solid resin particle. This approach provided an easy procedure whereby reagents and
by-products can be easily removed simply by filtration. The advantages of the new method were speed and simplicity of operation [20].
Prior to the development of SPSS, peptides were synthesized via solution phase method, which was tedious and required high level of
safety and skill. SPPS also allows the synthesis of natural peptides which are difficult to express in bacteria, the incorporation of unnatural
amino acids, peptide/protein backbone modification, and the synthesis of D-proteins, which consist of D-amino acids. In 1971, R. B.
Merrifield synthesized and characterized Ribonuclease a, linear polypeptide of 124 amino acid residues, by the solid phase method using
t-Boc (N-tert-butoxycarbonyl) protecting amino acids on styrene-divinyl-benzene resin. Basic concept of SPSS is to covalently attach the
first amino acid to an insoluble support (Resin) and to extend the peptide chain from this support bound residue and desired length of
peptide is completed through a series of coupling and deprotection steps. After completion of the desired length of peptide, the peptide
is removed from solid support by using appropriate cleaving reaction. One of the most important advantages of SPSS is that the growing
peptide chain can be washed to remove uncoupled amino acid by using suitable solvent, which makes sure that unwanted amino acids
are not attaching in the growing peptide chain [21].
SPSS normally proceeds in the C→N direction. Solid support (resin) holds entire growing peptide chain. A linker, in between the
resin and the first amino acid, can be attached to avoid stearic hindrance and facilitate smooth synthesis of peptide. All amino acids
used are orthogonally protected with temporary protecting group at N-terminal and permanent protecting groups at side chain with
C-terminal free to form peptide bond with N- terminal of previously attached amino acid, which make sure that peptide is growing at C-
to N-terminal orientation. Once the first amino acid is attached to the resin, its N-terminal is deprotected using appropriate solvent and
next amino acid is attached. Solid supports for SPSS generally increase significantly in size, i.e. “swell” when solvated and reactions take
place throughout the support (interior as well as surface) [22,23]. Polystyrene, cross-linked with divinylbenzene has been widely used
[24]. Polyethylene glycol (PEG) grafted onto polystyrene or ethylene oxide polymerized onto the polystyrene (Tentagel and ArgoGel)
[25,26]. Several other polymeric supports are now available which can be derivatized with functional groups to produce a highly stable
linkage to the peptide being synthesized [27] and peptides with different functionalities in the terminal carboxyl group (i.e. amide,
acid, thioester). Some more resins can be used such as p-methoxybenzhydrylamine (MBHA), 4-hydroxymethylphenylacetamidomethyl
(PAM), 4-(2’, 4’-dimethoxyphenyl-amino methyl) -phenoxymethylpolystyrene (Rink), 2-chlorotrityl chloride, 4-alkoxybenzyl alcohol
(WANG) and diphenyldiazomethane based resins.
In the last few decades, several protecting groups have been proposed to make peptides synthesis easy and to avoid use of harmful
chemicals in the synthesis [28]. Currently, two main schemes of protection, which are known as t-Boc/Bzl and Fmoc/tBu have been
used [29]. The Boc-benzyl strategy depends on graduated acid lability. The N-terminal Boc group is removed by Triflouroacetic acid
(TFA) in dichloromethane (DCM). TFA treatment produces isobutylene, carbon dioxide, and a protonated amine, which must be
neutralized before the next coupling. In case of t-Boc strategy, side chain protecting groups need to be TFA resistant in order to prevent
premature cleavage of side chain. Removal of side chain protecting groups occurs with strong acids such as hydrofluoric acid (HF) or
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