INTRODUCTION:

Peptides are short polymers of amino acid monomers linked by peptide bonds. They are distinguished from proteins on the basis of size, typically containing fewer than 50 monomer units. The shortest peptides are dipeptides, consisting of two amino acids joined by a single peptide bond. There are also tripe tides, tetra peptides, etc. Amino acids which have been incorporated into a peptide are termed "residues"; every peptide has an N-terminus and C-terminus residue on the ends of the peptide (except for cyclic peptides).

A polypeptide is a long, continuous, and unbranched peptide. Proteins consist of one or more polypeptides arranged in a biologically functional way and are often bound to cofactors, or other proteins. The size boundaries which distinguish peptides, polypeptides, and proteins are arbitrary. Long peptides such as amyloid beta can be considered proteins, whereas small proteins such as insulin can be considered peptides.

PEPTIDE CLASSIFICATION:

Peptide Classification based on:

1. Function

2. Synthesis

PEPTIDE CLASSIFICATION BY FUNCTION:

Peptides are involved into many processes in the living organisms and it is possible to classify them on the basis of their function.

· Hormones: Hormones are involved into carrying signals between cells.

Classical examples of hormones are: bradykinins, gastrins, oxytocin etc.

· Neuropeptides: Neuropeptides found in neural tissues. Usually these peptides are produced in the brain and involved into regulatory and signaling processes.

Classical examples of neuropeptides are: endorphins, vasopressin, atrial-natriuretic peptide etc.

· Alkaloids: Alkaloids are peptides, usually from plants, fungi and some animals like shellfish. Alkaloids involved into defend of one organism from consuming by other organisms.

Classical examples of peptide alkaloid are: ergotamine, panda mine, dynorphin A-(1-8)-octapeptide, N beta-(D-Leu-D-Arg-D-Arg-D-Leu-D-Phe)-naltrexamie,etc.

· Antibiotics: Antibiotics are inhibits the grows of microorganisms, usually bacterial cells and occasionally fungi and protozoa.

Classical examples of peptide antibiotics are: tyrothricinm bacitracin, gramicidin, valinomicin etc.

· Toxins: Toxin is the poison substance. Peptide toxins are the most poison substances.

Classical Examples of peptide toxins are: palutoxins, agatoxins, curtatoxins etc.

· Regulation peptides: The group of regulation peptides is not well defined because almost anypeptides can regulate some processes in organisms, but this group is used to classify peptides which are not clearly belongs to other groups.

Classical examples of regulatory peptides are: anserine, carnosine, etc.

PEPTIDE CLASSIFICATION BY SYNTHESIS:

· Ribosomal peptides:

Ribosomal peptides are synthesized by translation of mRNA. They are often subjected to proteolysis to generate the mature form. These function, typically in higher organisms, as hormones and signaling molecules. Some organisms produce peptides as antibiotics, such as microcins.^[1] Since they are translated, the amino acid residues involved are restricted to those utilized by the ribosome. However, these peptides frequently have post-translational modifications, such as phosphorylation, hydroxylation, sulfonation, palmitylation, glycosylation and disulfide formation.

· Non-Ribosomal peptides:

These peptides are assembled by enzymes that are specific to each peptide, rather than by the ribosome.The most common non-ribosomal peptide is glutathione, which is a component of the antioxidant defenses of most aerobic organisms. Other nonribosomal peptides are most common in unicellular organisms, plants, and fungi and are synthesized by modular enzyme complexes called nonribosomal peptide synthetases^[2].

· Peptones :

Peptones are derived from animal milk or meat digested by proteolytic digestion. In addition to containing small peptides, the resulting spray-dried material includes fats, metals, salts, vitamins and many other biological compounds. Peptone is used in nutrient media for growing bacteria and fungi^[3].

· Peptide fragments :

Peptide fragments refer to fragments of proteins that are used to identify or quantify the source protein^[4] often these are the products of enzymatic degradation performed in the laboratory on a controlled sample, but can also be forensic or paleontological samples that have been degraded by natural effects.

PEPTIDE SYNTHESIS:

Peptide synthesis is a chemical process of coupling of the carboxyl group of one amino acid to the amino group of another amino acid. Usually chemical techniques are used to synthesize peptides of up to 30-40 amino acids length. The first peptide synthesis was carries out by T. Curtius 1882 via reaction between benzyl chloride and silver salt of glycine. During this process Curtius produce crystals of N-benzyl-glycile-glycine. In 1950-1960 the first biological peptides, like oxytocin, vasopressin, insulin and others were synthesized.

TYPES OF PEPTIDE SYNTHESIS

Peptide synthesis process can be classified on the basis of used techniques and type of the final product.

· Liquid-phase peptide synthesis:

Liquid-phase, or classical peptide synthesis can be divided into two classes - step-by-step peptide synthesis with subsequent adding of one amino acid at ones from C-terminal to N-terminal and block-synthesis with coupling of polypeptide fragments.

· Solid phase peptide synthesis:

Peptide synthesis is much more complicated than simply forming amide bonds by mixing the desired amino acids together in a test tube with twenty natural amino acids and a number of unnatural ones as well the possible combinations formed with this technique are numerous^[5]. If solutions containing two amino acids are mixed together, four different dipeptides (as well as other longer peptides) will be formed.

E.g. for a mixture of glycine and alanine the four dipeptides would be gly-gly, gly-ala, ala-gly, ala-ala.

In this representation of peptides the free amino group or N-terminus is on the left-hand amino acid and the free carboxylic acid group, the C-terminus is at the righthand end. To ensure that only the desired dipeptide is formed the basic group of one amino acid and the acidic group of the other must both be made unable to react. This 'deactivation' is known as the protection of reactive groups, and a group that is unable to react is spoken of as a protected group. In classical organic synthesis the acids are protected, allowed to react and deprotected, then one end of the dipeptide is protected and reacted with a new protected acid and so on. In SPPS the amino acid that will be at one end of the peptide is attached to a water-insoluble polymer and remains protected throughout the formation of the peptide, meaning both that fewer protection / deprotection steps are necessary and that the reagents can easily be rinsed away without losing any of the peptide.

Step 1 - Attaching an amino acid to the polymer:

Peptide chains have two ends, known respectively as the N-terminus and the C-terminus3, and which end is attached to the polymer depends on the polymer used. Polyamide beads are used and hence that the the C-terminus of the peptide is attached to the polymer. The attachment is done by reacting the amino acid with a linkage agent and then reacting the other end of the linkage agent with the polymer. This means that a peptide-polyamide link can be formed that will not be hydrolyzed during the subsequent peptide-forming reactions. Common linkage agents are di- and tri-substituted benzenes such as those shown below

These then join the C-terminus amino acid and resin together as follows:

Step 2 Protection:

The next amino acid also needs to have its amino group protected to prevent the acids reacting with each other. This is done by protecting it with FMOC (9-fluorenylmethoxy- carbonyl). In addition, any amino acid side chains that are aromatic, acid, basic or highly polar are likely to be reactive. These must also be protected to prevent unwanted

branched chains from forming. There are four main groups used in this way: tBu(a tertiary butyl group), Trt (a triphenylmethyl group), tBOC (a tertiary butyloxycarbonyl group) and PMC (a 2,2,5,7,8-pentamethylchroman-6-sufonyl group).Examples of a carboxyl group protected with FMOC and examples of the different types of side chain protection are given in figure 1.

Figure 1

Step 3 - Coupling:

The FMOC protected amino acid is then reacted with the last amino acid attached to the polyamide. The reaction is catalyzed by DCC (1,3dicyclohexylcarbodiimide), which is itself reduced to DCU (1,3-dicyclohexylurea). The reaction is shown in Figure 1 (RCOOH represents the FMOC protected acid and H2NR represents the reactive end of the growing peptide chain).

Step 4 - Deprotection:

Excess DCC is washed off the insoluble polymer with water, and then the FMOC group removed with piperidine (a cyclic secondary amine). This is a transamidification reaction.

PIPERIDINE

Steps 2 to 4 are repeated as each new amino acid is added onto the chain until the desired

Peptide has been formed.

Step 5 - Polymer removal:

Once the peptide is complete it must be removed from the polyamide(figure 2). This is done by cleaving the polyamide - peptide bond with a 95% solution of tri-fluoroacetic acid (TFA). The side-chain protecting groups are also removed at this stage.

Figure 2

SOLID SUPPORT:^[6]

There are three primary types of solid supports:

1. Gel-type supports: These are highly solvated polymers with an equal distribution of functional groups. This type of support is the most common which includes:

· Polystyrene: Styrene cross-linked with 1-2% divinylbenzene

· Polyacrylamide: A hydrophilic alternative to polystyrene

· Polyethylene glycol (PEG): PEG-Polystyrene (PEG-PS) is more stable than polystyrene and spaces the site of synthesis from the polymer backbone

· PEG-based supports: Composed of a PEG-polypropylene glycol network or PEG with polyamide or polystyrene

2. Surface-type supports: Many materials have been developed for surface functionalization, including controlled pore glass, cellulose fibers, and highly cross-linked polystyrene.

3. Composites: Gel-type polymers supported by rigid matrices.

PROTECTION GROUPS:

Amino acids have reactive moieties at the N- and C-termini, which facilitates amino acid coupling during synthesis. To facilitate proper amino acid synthesis with minimal side chain reactivity, chemical groups have been developed to bind to specific amino acid functional groups and block, or protect the functional group from nonspecific reactions. These protecting groups can be separated into three groups, as follows:

1. N-terminal protecting groups

2. C-terminal protecting groups (mostly used in liquid-phase synthesis)

3. Side chain protecting groups

§ N-terminal protecting groups:

Amino acids are added to excess to ensure complete coupling during each synthesis step, and without N-terminal protection, polymerization of unprotected amino acids could occur, resulting in low peptide yield or synthesis failure. N-terminal protection requires an additional step of removing the protecting group, termed deprotection, prior to the coupling step, creating a repeating design flow as follows^[6]:

a. Protecting group is removed from the trailing amino acids in a deprotection reaction.

b. Deprotection reagents are washed away to provide a clean coupling environment.

c. Protected amino acids dissolved in a solvent such as dimethylformamide (DMF) combined with coupling reagents are pumped through the synthesis column.

d. Coupling reagents are washed away to provide clean deprotection environment.

Currently, two protecting groups:

1. t-Boc,

2. Fmocare

1. t-Boc(tert-butyloxycarbonyl)protecting group:

The Boc group covalently bound to the amino group to suppress its nucleophilicity. The C-terminal amino acid is covalently linked to the resin through a linker. Next, the Boc group is removed with acid, such as trifluoroacetic acid (TFA). This forms a positively-charged amino group (in the presence of excess TFA) which is neutralized (via in-situ or non in-situ methods) and coupled to the incoming activated amino acid.^[13] Reactions are driven to completion by the use of excess (two- to four-fold) activated amino acid. After each deprotection and coupling step, a wash with N,N-dimethylformamide (DMF) is performed to remove excess reagents, allowing for high yields (~99%) during each cycle.

2. Fmoc protecting group:

The Fmoc method allows for a milder deprotection scheme and utilizes a base, usually piperidine (20-50%) in DMF in order to remove the Fmoc group to expose the α-amino group for reaction with an incoming activated amino acid^[6].Therefore, no neutralization of the peptide-resin is required, but the lack of electrostatic repulsions between the peptides can lead to increased aggregation. Because the liberated fluorenyl group is a chromophore, deprotection by Fmoc can be monitored by UV absorbance of the run-off, a strategy which is employed in automated synthesizers.

§ Side chain protecting groups:

Amino acid side chains represent a broad range of functional groups and are sites of nonspecific reactivity during peptide synthesis. Because of this, many different protecting groups are required that are usually based on the benzyl (Bzl) or tert-butyl (tBu) group. Side chain protecting groups are known as permanent or semi-permanent protecting groups.

ACTIVATING GROUPS:

For coupling the peptides the carboxyl group is usually activated. This is important for speeding up the reaction.

There are two main types of activating groups:

1.Carbodiimides and

2.Triazolols.

However the use of pentafluorophenyl esters (FDPP,^[7] PFPOH^[8]) and BOP-Cl^[9] are useful for cyclisingpeptides.

1.Carbodiimides:

These activating agents were first developed. Most common are

o dicyclohexylcarbodiimide (DCC) and

o diisopropylcarbodiimide (DIC).

Reaction with a carboxylic acid yields a highly reactive o-acyl-urea. To enhance the electrophilicity of carboxylate group, the negatively charged oxygen must first be "activated" into a better leaving group, DCC is used for this purpose. DCC is temporarily attached to the former carboxylate group (which is now an ester group), making nucleophilic attack by an amino group (on the attaching amino acid) to the former C-terminus (carbonyl group) more efficient. The problem with carbodiimides too reactive and can cause racemization of the amino acid.

2.Triazoles:

To solve the problem of racemization, triazoles were introduced. The most important are

1-hydroxy-benzotriazole (HOBt) and 1-hydroxy-7-aza-benzotriazole (HOAt).

These substances can react with the o-acylurea to form an active ester which is less reactive and less in danger of racemization. HOAt is especially favorable because of a neighboring group effect. HOBt may be explosive, when allowed to fully dehydrate and shipment by air or sea is heavily restricted. Alternatives to HOBt and HOAt have been introduced. One of the most promising and inexpensive is ethyl 2-cyano-2-(hydroxyimino)acetate (trade name Oxyma Pure), which is not explosive and has a reactivity of that in between HOBt and HOAt

OTHER PEPTIDE SYNTHESIS:

ü Homo-polymerization: Homo-polymerization is used to synthesize homo-polymeric chains of amino acids.

ü Enzymatic peptide synthesis:Enzymatic peptide synthesis is based on enzymes which are able to formate peptide bond. Unfortunately this technique is very complicated and there is no remarkable results were achieved yet.

ü Partial peptide synthesis: In partial peptide synthesis technique the natural peptides and proteins are used as a source of peptide fragments.

ü Cyclopeptidesynthesis:Cyclopeptide synthesis is the cyclization of linear peptides.

ü Non-standard peptide synthesis: Non-standard peptide synthesis is used to produce peptides with non-standard peptide bonds, for example ester-bonds etc.

PEPTIDE ARRAY SYNTHESIS:

Currently, there are two common ways to prepare peptide arrays^[10][11]:

(i) synthesize functionalized peptides in advance and then covalently attaching them to the support;

(ii) synthesize the peptides sequentially directly on the solid support, usually by using SPOT synthesis or photolithography.

MICROARRAY TECHNIQUE:

The gene fragments (probes) are fixed on a glass slide instead of a membrane support; the size and density of the arrays are smaller and higher. The analysis of genetic expression of the hybridized RNA (target) is not done with isotopes but with florescent dye markets. The number of probes and number of genes can be increased, and it is possible to analyze the gene expression of the comprised genes. Conversely, the probes used in the microarrays and microarrays give no information on the expression of unfixed genes. Therefore, from the point of view of analyzing the expression of the genes in the genome, the number of probes required should be maximum.

Currently there are two methods that are used for the microarrays.:

1. The Gene chip

2.cDNA microarray

PEPTIDE MICROARRAY – RETEROSPECT:

The first development of peptide microarray was conceived in as early as 1984, when the concept of combinatorial approach in chemical synthesis was developed^[12]. Several years later, Frank et al. modified this approach and further developed the SPOT synthesis, which synthesizes and analyzes multiple peptides as spots on a cellulose paper. In 1991, Fodor et al. brought forward the light directed, spatially addressable parallel chemical synthesis method and generated 1024 peptides on a 1.6 cm2 glass surface.

Further development of this innovative technology was hampered by the low quality of peptides synthesized on the glass surface, primarily due to the relatively low efficiency in peptide deptrotection/coupling steps. Alternatively, Lam et al. developed a ‘one-bead one compound’ (OBOC) combinatorial peptide library method^[13]. This spatially separable, but non addressable, array was screened by an enzyme-linked calorimetric method. Following Fodor’s ingenious concept, the next few years saw an explosion in the use of photolithographic techniques for the development of Gene Chip.

By taking advantage of Brown’s spotting method, Schreiber etal.first described the successful generation of high-density microarrays made of non-nucleic acid biomolecules, including peptides^[14]. By taking advantage of the chemistry involved in the native chemical ligation and biotin-avidin interaction, Yao et al. offered two new approaches for the site-specific immobilization of peptides in a microarray^[15a]. They exploited the resulting peptide array and subsequently developed fluorescence-based assay for the rapid profiling of kinase activity in a microarray format^[15b]. In their case, the peptides used to generate the corresponding array were made by recombinant approaches, rather than chemical synthesis. Recently, supramolecular hydrogels were reported as an array platform to provide a semi-wet environment for the immobilized peptides/proteins, making them more compatible with enzyme assays. A few more recent reports which are relevant to the field are the construction of spatially defined arrays of peptoids using photolithographic synthesis by Li et al.^[16] and the synthesis of metal chelating hexapeptide on a chip by Cheng et al..

PEPTIDE MICROARRAY CLASSIFICATION:

ü Immobilization of pre-synthesized peptides

ü In-situ synthesis of peptides directly on the array surface^[17].

This method can provide miniaturized spatially addressed peptide arrays more rapidly and economically.

1. Immobilization of pre-synthesized peptides:

The immobilization of pre-synthesized peptides is sometimes a more effective method than the in situ synthesis. Therefore, chemoselective immobilization methods have been widely used for the peptide microarray preparation (Fig. 3). Especially, they play an important role in the preparation of long-chain peptide arrays. Even though the pre-synthesized peptides are contaminated with their byproduct, only the peptide derivatives containing the chemoselective functional group can be attached to the appropriately modified chip surface. Moreover, the chemoselective immobilization can provide a useful method for controlling the orientation and the density of the immobilized peptides. However, the chemoselective immobilization methods have an intrinsic limitation in that it involves a laborious purification process of each peptide probes for the preparation of a high-density peptide array.

SPOT synthesis:

In the SPOT synthesis, the peptides arrays are usually synthesized in a stepwise manner on a flat solid substrate such as functionalized cellulose membrane, polypropylene, and glass, following the standard Fmoc-based peptide chemistry (Frank, 1992; Min et al., 2004). Firstly, small volumes of solutions containing activated amino acid are spotted on the solid substrate to make addressable array. Each spot can be considered as an independent micro reactor composed of delivered droplet so that the functional surfaces react with the spotted amino acid to carry out solid-phase synthesis(figure 4). In the SPOT synthesis, the employed solvent should be nonvolatile so as to maintain the wet state on the spot.

Figure 4

PEPTIDE-DNA INTERACTIONS:

ü Many proteins can bind and interact with DNA or RNA, including polymerases, gyrases, helicases, ribosomes, transcriptional factors and restriction endonucleases.

ü There is a great potential to study peptide-DNA or peptide-RNA interactions usingthe SPOT technology to increase our understanding of these proteins’ functions. Combinatorial peptide libraries on cellulose membranes were successfully used to study peptide-DNA interactions.

Similarly the region of theendonucleaseEcoRII involved in DNA recognition was identified using the peptidescan method, with the peptides synthesized on cellulose^.[24]

PEPTIDE-RECEPTOR INTERACTIONS:

ü Communication and regulation within multi-cellular organisms is also often mediated by ligand-receptor interactions. Therefore, studying such interactions is a major focus for understanding many different biological processes.This information can also lead to the development of new drugs against many different diseases.

ü Vascular endothelial growth factor (VEGF) directly stimulates endothelial cell proliferation and migration through tyrosine kinase receptors. The potential binding site of VEGF with its receptor was identified using SPOT technology (peptide scan)^[25].

ü A substitution analysis of VEGF-derived peptides was used to study this interaction.

KINASE DETECTION:

ü Schreiber first described the peptide array-based kinase detection method in 2000 . they detected the enzymatic activity of three different kinases using a microarray containing their corresponding peptide substrates. Phosphorylation of the kinase peptide substrate on the microarray was successfully detected by using the radioactive [γ33P]-ATP in the kinase reaction.

ü Later, Zhuadopted the same strategy and successfully characterized the substrate specificity of over ahundred yeast kinases using an array of peptides immobilized in microwells. The major limitations of this radioisotope-based method are the usage of hazardous radioactive reagents and the long exposure time needed for sensitive detection of peptide phosphorylation.

ü To overcome these limitations, we and others independently developed fluorescence-based approaches to detect the phosphorylation of kinases in a peptide microarray^[26].

ü In these methods, fluorescently labeled antibodies which specially recognized phosphorylated peptides were used to detect peptide phosphorylation in a microarray.

For a real-time detection of kinase activity in a microarray format, explored the utility of the aforementioned fluorescence-based method inboth concentration- and time-dependent detection of peptide phosphorylation on a chip: the fluorescence intensity was directly proportional to the concentration of the substrate, showing the feasibility for the determination of concentration-dependent kinase activity.

DETECTION OF HYDROLASES AND OTHER ENZYMES

ü Another emerging application of peptide-based microarray is the substrate/inhibition detection of hydrolases which include proteases and other types of hydrolytic enzymes, such as lipases and esterase’s, etc.

ü The global profiling methods such as phage-displayed peptide libraries , positional scanning libraries and mixturebased oriented peptide libraries have obtained a greatsuccess in the protease substrate specificity study^.[27]

ü Other related strategies include fluorescence-quenched peptide substrate libraries and end-labeled peptide substrate libraries

ü We recently explored a protein microarraybased strategy which utilizes mechanism-based suicide probes to detect different enzymatic activities in a microarray format.

ü A 361-member spatiallyaddressable peptide library was synthesized and immobilized on an aldehyde-derivatized glass slide.

OTHER APPLICATIONS OF PEPTIDE ARRAYS:

ü Mihara et al. developed knowledge about peptide/protein and protein/protein interactions is essential for a better understanding of important biological processes, as well as providing essential information which lead to drug discovery and development.

ü For example, techniques which allow the accurate identification of specific peptide binding sequences of a protein could provide insights into how enzyme/substrate, enzyme/inhibitor, antibody/antigen and protein/protein interact. Recently, Duburcqet al. used a peptide/protein microarray to study pathogen infection in human lymphocytes^[28].

ü The microarray-based technique displayed high sensitivity and specificity for the detection of antibodies directed against different pathogens.

CONCULSION:

Peptide microarray technologies allow site-specific and stable immobilization of peptides on a variety of solid surfaces. With a peptide array, allow for the rapid identification, design and selection of effective enzyme substrates/inhibitors, as well as potential drug candidates. In addition, such arrays may also be employed for the determination of ligand-receptor interactions, the assessment of antigen-antibody affinities and the establishment of other similar interactions. Compared with other microarray-based technologies, peptide arrays are highly versatile, in that they provide an easy access to a large number of molecular entities. Despite the great progress made in only a few years, the field of peptide microarray is still expanding with a remarkable pace

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Received on 26.06.2012 Modified on 203.07.2012

Research J. Pharm. and Tech. 5(8): August 2012; Page 1015-1024