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A peptide is a chemical compound containing two or more amino acids (amino acid polymers) that are coupled by a Peptide Bond peptide bond. This bond is a special linkage in which the nitrogen atom of one amino acid binds to the carboxyl carbon atom of another.
Peptides are often classified according to the number of amino acid residues. Oligopeptides have 10 or fewer amino acids. Molecules consisting from 10 to 50 amino acids are called peptides. The term protein describes molecules with more than 50 amino acids.
Molecules with molecular weights ranging from several thousand to several million daltons (D) are called polypeptides. The terms polypeptide and protein are frequently used interchangeably.
In 1901, the first synthetic peptide glycyl-glycine (see picture) was discovered by Emil Fischer, in collaboration with Ernest Fourneau. The first polypeptide (oxytocin - nine amino acid sequence) was synthesized by Vincent du Vigneaud in 1953.
Peptides have a wide range of applications in medicine and biotechnology. They regulate most physiological processes, acting at some sites as endocrine or paracrine signals and at others as neurotransmitters or growth factors.
Insulin was the first therapeutic protein to be introduced to treat insulin-dependent diabetes in the 1920s. There are sixty FDA approved peptide drugs in the market. About 140 peptide drugs are in clinical trials and over 500 are in pre-clinical development.
A number of peptides are used for diagnostic purposes, for example C-peptide is used to monitor insulin production and to help determine the cause of low blood sugar (hypoglycemia).
Peptides are synthesized by coupling the carboxyl group or C-terminus of one amino acid to the amino group or N-terminus of another. There are two strategies for peptide synthesis: liquid-phase peptide synthesis and solid-phase peptide synthesis (SPPS).
The goal of this project is to collect all information about peptides and peptide synthesis in one place.
A peptide bond (sometimes mistakenly called amino bond) is a covalent bond that is formed between two molecules when the carboxyl group of one molecule reacts with the amino group of the another molecule, releasing a molecule of water. This is a a condensation reaction and usually occurs between amino acids. The resulting CO-NH bond is called a peptide bond, and the resulting molecule is an amide.
The molecules must be orientated so that the carboxylic acid group of one can react with the amine group of the other. For example, two amino acids combining through the formation of a peptide bond to form a dipeptide.
Any number of amino acids can be joined together in chains of 50 amino acids called peptides, 50-100 amino acids called polypeptides, and over 100 amino acids called proteins. A number of hormones, antibiotics, antitumor agents and neurotransmitters are peptides (proteins).
A peptide bond can be broken down by hydrolysis (the adding of water). The peptide bonds that are formed within proteins have a tendency to break spontaneously when subjected to the presence of water (metastable bonds) releasing about 10 kJ/mol of free energy. This process, however, is very slow. Living organisms use enzymes to broken down or to form peptide bonds. The wavelength of absorbance for a peptide bond is 190-230 nm.
X-ray diffraction studies of crystals of small peptides by Linus Pauling and R. B. Corey indicated that the peptide bond is rigid, and planer. Pauling pointed out that this is largely a consequence of the resonance interaction of the amide, or the ability of the amide nitrogen to delocalize its lone pair of electrons onto the carbonyl oxygen.
Because of this resonance, the C=O bond is actually longer than normal carbonyl bonds, and the N–C bond of the peptide bond is shorter than the N–Cα bond. Notice that the carbonyl oxygen and amide hydrogen are in a trans configuration, as opposed to a cis configuration. This configuration is energetically more favorable because of possible steric interactions in the other.
The peptide bond is usually portrayed as a single bond between the carbonyl carbon and the amide nitrogen. Normally, this should allow free rotation about than bond. However, notice that the nitrogen has a lone pair of electrons, which are adjacent to a carbon-oxygen bond. Therefore, a reasonable resonance structure can be draw with a double bond linking the carbon and nitrogen, and which result in a negative charge on the oxygen and a positive charge on the nitrogen.
The resonance structure prevents rotation around the peptide bond. The real structure of course is a weighted hybrid of these two structures. Therefore, the question is how significant is the resonance structure in depicting the true electron distribution. It is know that the peptide bond has approximately 40% double-bond character and therefore it is rigid.
Charges give the peptide bond a permanent dipole. Because of the resonance, the oxygen has a -0.28 charge, while the nitrogen bears a +0.28 charge.
The word "protein" comes from Greek "prota", meaning "of primary importance". Back in 1838, Jons Jakob Berzelius introduced the word "protein" to name large organic compounds featuring almost equivalent empirical formulas. This word was used as the studied organic compounds were considered primitive while extremely important for animal nutrition.
Another significant step of the protein study was performed in 1926 by James B. Sumner, who showed that enzymes can be isolated and crystallized. Later, in 1955, Frederick Sanger determined the entire amino acid sequence of the first protein, known today as insulin. This was the first proof of the fact that all proteins have specific structure. Three years later, the 3-dimensional structures of haemoglobin and myoglobin were determined by Max Perutz and Sir John Cowdery Kendrew, respectively, through X-ray diffraction analysis.
The terms "protein", "polypeptide", and "peptide" are a bit ambiguous and are able to overlap in meaning. Protein is usually used to specify the entire biological molecule in a stable conformation, while peptide refers to short amino acid oligomers, in most cases lacking a stable 3-dimensional structure. Nevertheless, the difference between them is not well defined and generally lies near 20-30 residues. Polypeptide can be envisaged as any single linear chain of amino acids, generally regardless of length. However, it usually implies an absence of a defined conformation.
Actually, proteins are the basic building blocks of all life. For instance, the human body contains around 100,000 various proteins, and every cell in our body contains protein, which is a major part of our skin, organs, muscles, and glands. It can also be found in almost all body fluids. Humans need protein in their diet in order to help the body repair cells and produce new ones. In addition, protein is also important for growth and development of humans during childhood, adolescence, and pregnancy.
Proteins can be of different length and structure and consist of polymers of twenty different amino acids that fold upon themselves in order to create a shape which is characteristic for each protein. The function and properties of various proteins are defined by the sequence of twenty different amino acids. The sequence in question depends on the genetic data, which is contained in the DNA. A lot of proteins are encoded on each piece of DNA, and that is why it is vital for biologists to find out where the protein code begins and ends. Nevertheless, this is quite complicated as the human genome features more DNA than is needed to encode proteins.
As it has already been said, the structure of a protein is created by the folding of a peptide chain back on itself, and sometimes the association of multiple peptide chains. Such folding happens because of the rotation of bonds within the amino acids and bonds joining various amino acids. In their attempts to understand the structural complexities of proteins, scientists decided to divide them by the structure, which can be primary (only a sequence of amino acids in the protein); secondary (like helical or sheet-like structures and beta sheets); tertiary (comprised of super secondary structures); and quarternary (multiply-folded polypeptide chains). Finally, the development of a tertiary structure turns a linear amino acid sequence into a 3-dimensional structure.
Most part of proteins will lose their structure if they are put in unsuitable chemical conditions, like high or low pH, high salt concentrations, or hydrophobic environment, as well as physical conditions like high temperature or pressure. Such process can be called denaturation. Those proteins that have been denatured have no defined structure and, particularly if concentrated, in most cases aggregate into insoluble masses. Of course, protein denaturation is an outstanding event: for example, a boiled egg will become solid only because of denaturation and subsequent aggregation of its proteins. In some cases denatured proteins are able to refold if put again in the correct environment, but in some cases the process is irreversible (particularly after aggregation, which has a good example of the boiled egg again). The proteins are also responsible for susceptibility or resistance to a pathogen or parasite.
Dipeptide (DP) is actually one molecule, which consists of 2 amino acids that are joined by a single peptide bond. Since it consists of 2 different amino acids, it can have different sequences, like Ala-Gly or Gly-Ala. Such dipeptides would have different chemical and biological properties. The matter is that if written left to right in Gly-Ala, the glycine has the "free" amine terminal end, while alanine has the "free" carboxyl acid terminal end.
Dipeptide can be produced in different ways, which are generally divided into 3 methods: chemical synthesis, chemoenzymatic synthesis, and enzymatic synthesis.
Meanwhile, the function of di-peptides can be viewed from two points: as a derivative of amino acid and as the dipeptide itself. The first one is quite easy to understand, since although DPs and the constitutive amino acids feature different physicochemical properties, they should still share the same physiological effects, because dipeptides are degraded into the individual amino acids in organisms.
For instance, L-Glutamine is heat labile, but L-alanyl-L-glutamine is more tolerant to heat. Another good example is solubility: while Tyr is practically insoluble, you can dissolve Ala-Tyr up to 14 g/L. The most interesting fact is that a number of dipeptides are more soluble than each of the constitutive amino acids. For example, the solubilities of Ala and Gln are 89 and 36 g/L accordingly, but that of Ala-Gln is 586 g/L! Considering these properties and the fact that Ala-Gln and Gly-Tyr are quickly degraded into the individual amino acids when taken into the body, these dipeptides are used as components of patient infusions.
A number of dipeptides feature really unique functions that you would not find in the constitutive amino acids. For example, Carnosine and the related DPs anserine have appeared to exist in many tissues of mammalian, bird, and fish origin. A lot of functions have been anticipated to such dipeptides, like antioxidation and maintenance of cellular pH. Since di peptides and their derivatives reflect such possible functions, they are used in many ways - in sport nutrition, for example, which is based on the fact that the muscle of a fastswimming fish contains the dipeptides in question in higher concentrations than anything else. Other compounds, like Zinc carnosine, can be used as an antiulcer drug, while N-acetyl carnosine is used as an agent for cataracts.
The taste of dipeptides has also been studied for a long time. The research into the taste of synthetic di-peptides revealed that most of them appeared to be bitter, and the relationship between their physicochemical properties and bitterness has attracted the attention of the researchers. If we consider commercial application, aspartame is the only DPs of outstanding importance. Today almost 20,000 tons of aspartame, which is hundreds of times sweeter than sugar, is consumed annually throughout the globe as a low-calorie sweetener.
Another thing that captured the researchers attention was the antihypertensive effect of DPs. Extracts or hydrolysates of fish meat, seaweed, or mushrooms were found out to exert a blood pressure lowering effect, with the active agents being identified as a few kinds of dipeptides like Ile-Tyr, Ile-Trp, and Lys-Trp. Such effects of the dipeptides in question were considered to be derived from their inhibitory effect on angiotensin-I-converting enzyme. As a result, in Japan the extracts or hydrolysates with such dipeptides were approved as foods for specified health uses.
Aside from the DPs that are mostly applied in industry, there are a number of dipeptides that are not used practically, but their functions are still well-known. For example, Kyotorphin was isolated from bovine brain to feature analgesic effects; a synthetic DP Lys-Glu was found to show antitumor activity, while Leu-Ile appeared to have a neuroprotective effect, and Tyr-Gly is known for enhancing proliferation of peripheral blood lymphocytes.
Finally, transport mechanisms for dipeptides and amino acids differ, which means that they may exert different nutritional impacts on your body if taken orally.
The combinatorial peptide library is a powerful tool in which a huge number of diverse peptides are produced. It is widely used in drug discovery and development, epitope mapping, target validation, structure-activity studies.
This method has been successfully used to identify biologically active peptides, including antibacterial peptides, ligands for cell surface receptors, opioid receptor antagonists, protein kinase inhibitors and substrates.
The first use of combinatorial peptide library (96 peptides) was reported by Geysen and colleagues in 1984. The peptide library was generated with the multipin technology. Since then several different methods of synthesizing and screening peptide libraries have been developed.
Currently, there are five main approaches in peptide library methods:
1) the biologic peptide library method such as phage-display peptide library
2) the spatially addressable parallel library method
3) the combinatorial library method that requires deconvolution
4) the one-bead one-compound (OBOC) combinatorial library method
5) the synthetic library method that requires chromatography selection
Each peptide library method has its own advantages and disadvantages. The phage-display peptide library method is simple and economical. It can provide a large number of peptide entities to be screened (106-108) and fewer restrictions exist in the length of peptides that can be achieved. However, this biologic approach generally is limited to peptides that contain only 20 natural L-amino acids. The synthetic methods (2-5) can use D-amino acids, unnatural amino acids, nucleotides, monosaccharides, lipids and small organic moieties, which makes these approaches highly versatile.
The peptide library methods offer great potential for facilitating the drug discovery and development process and also provide a powerful tool for basic research.
The Protein Builder constructs text representations for simple peptides and proteins from a library of 20 standard amino acids. This program support on-the-fly calculation of Molecular Weight and Molecular Formula.
It is often important to know the molecular weight of a peptide or protein sequence. This program will take a protein sequence and calculate the molecular weight. The program will ignore numbers, spaces or characters like U or X which do not correspond to one of the amino acids from the table below.
The first stage of the technique consists of peptide chain assembly with protected amino acid derivatives on a polymeric support. The second stage of the technique is the cleavage of the peptide from the resin support with the concurrent cleavage of all side chain protecting groups to give the crude free peptide.
The general principle of SPPS is one of repeated cycles of coupling-deprotection. The free N-terminal amine of a solid-phase attached peptide is coupled to a single N-protected amino acid unit. This unit is then deprotected, revealing a new N-terminal amine to which a further amino acid may be attached.
There are two major used forms of solid phase peptide synthesis – Fmoc (base labile alpha-amino protecting group) and t-Boc (acid labile protecting group). Each method involves different resins and amino acid side-chain protection and consequent cleavage/deprotection steps. Fmoc chemistry is known for generating peptides of higher quality and in greater yield than t-Boc chemistry. Impurities in t-Boc-synthesized peptides are mostly attributed to cleavage problems, dehydration and t-butylation.
After cleavage from the resin, peptides are usually purified by reverse phase HPLC using columns such as C-18, C-8, and C-4.
The primary advantage of SPPS is its high yield. As peptides consists of many amino acids, if the yield for each amino acid addition is much less than 100%, overall peptide yields are negligible. For example, if each amino acid addition has a 90% yield then the overall yield of a 50 amino acid peptide is only 0.5%. Modern SPPS instrumentation pushes coupling and deprotection yields to greater than 99.99%, giving an overall yield of greater than 99% for a 50 amino acid peptide.
The following is an outline of the synthetic steps for peptide synthesis on Wang resin as the solid support, using the base labile 9-fluorenylmethyloxycarbonyl (Fmoc) protecting group.
Load 0.08 mmol of Fmoc-Val-Wang resin into a fritted column equipped with a plastic cap. Wash the resin with 2 x 3-ml portions of DMF (dimethylformamide) for 1 minute each. Next, add about 3 ml of 20% piperidine in DMF and allow the deprotection to continue for 15 minutes. During this time, gently swirl or agitate the column to assure a complete mixing. After the reaction is complete (about 15 min.), drain the reaction column and wash the resin again with DMF (4 x 3ml).
Amide bond coupling
In a small vial, pre-activate the 3 equivalents Fmoc amino acid by combining it with 3 equivalents of HBTU, 6 equivalents of DIPEA (N,N -Diisopropylethylamine), and 3 ml of DMF. Make sure this solution is fully dissolved and then allow it to react for an additional 3-5 minutes. Then, add this coupling solution to the resin, place the cap on the reaction column, and agitate the resin slurry every 2-3 minutes over a period of 20 minutes.
In order obtain the peptide in the free acid form, the ester linkage is cleaved using strongly acidic conditions such as TFA (trifluoroacetic acid). Treat the resin with 2-3 ml of a solution of trifluoroacetic acid and water 95:5. Gently agitate the resin over a period of 25. Next, drain the column and carefully collect the filtrate into a glass collection vessel.
Because of amino acid is an acid with a basic group at one end and an acid group at the other, polymerization of amino acids is common in reactions where each amino acid is not protected. In order to prevent this polymerization, protective groups are used.
Currently, two protecting groups are commonly used in Solid Phase Peptide Synthesis – Fmoc (9-fluorenylmethyl carbamate) and t-Boc (Di-tert-butyl dicarbonate).
The use of Fmoc chemistry for protection of the alpha amino group has become the preferred method for most contemporary solid and solution phase peptide synthetic processes. Fmoc has also been shown to be more reliable and produce higher quality peptides than Boc chemistry.
The advantage of Fmoc is that it is cleaved under very mild basic conditions (e.g. piperidine), but stable under acidic conditions. After base treatment, the nascent peptide is typically washed and then a mixture including an activated amino acid and coupling co-reagents is placed in contact with the nascent peptide to couple the next amino acid. After coupling, non-coupled reagents can be washed away and then the protecting group on the N-terminus of the nascent peptide can be removed, allowing additional amino acids or peptide material to be added to the nascent peptide in a similar fashion.
Before the Fmoc group became popular, the t-Boc group was commonly used for protecting the terminal amine of the peptide, requiring the use of more acid stable groups for side chain protection in orthogonal strategies. Boc groups can be added to amino acids with Di-tert-butyl dicarbonate (Boc anhydride) and a suitable base.
The t-Boc protecting group is removed by exposing the Boc-protected residue on the chain to a strong acid. Typical reagents of choice for deprotection in existing methods are trifluoroacetic acid (TFA) in dichloromethane, hydrochloric acid or methanesulfonic acid in dioxane. The acid used to remove the Boc protecting group is typically neutralized with a tertiary amine such as N-methylmorpholine, N-diisopropylethylamine (DIEA) or triethylamine (TEA).
Synthetic peptides are widely used for the following purposes:
- To verify the structure of naturally occurring peptides as determined by degradation techniques
- To study the relationship between structure and activity of biologically active protein and peptides and establish their molecular mechanisms
- To develop new peptide-based immunogens, hormones, vaccines, etc
Peptide drugs are either naturally-occurring peptides or altered natural peptides. There are many naturally-occurring peptides that are biologically active. If a patient does not naturally produce a peptide that they need, this peptide can be synthesized and given to them. In addition, the amino acids in an active peptide can be altered to make analogues of the original peptide. If the analogue is more biochemical active than the original peptide it is known as an agonist and if it has the reverse effect is known as an antagonist.
Peptides can be designed that change color under certain conditions, and these can be used for diagnostic purposes. For example, a chromogenic peptide substrate can readily detect the presence, absence and varying blood levels of enzymes that control blood pressure and blood clotting ability.
Here you have an access to the related page: Peptides in Drug Discovery
Peptide research on drug design and drug discovery is one of the most promising fields in the development of the new drugs. Peptide sequences are constituents of larger proteins, where they are responsible for molecular recognition and biological activities. Inhibition of protein-protein interactions by peptides and the evolution of peptide ligands to small molecule mimetics is a major goal of the field, with several notable successes. Peptides would therefore seem to be ideal drug leads. However, peptides are limited in that they are metabolically unstable due to the protease cleavage of the peptide backbone and have poor bioavailability, in part due to low membrane transport characteristics of the peptide is amide backbone structure.
The starting point for a peptide mimetics research is the identification of a peptide or peptide sequence within a protein context that is activ in the relevant assay. The process involves deconstructing the original peptide and reassembling the essential features on a new, mimetic scaffold that retains the ability to interact with the biological target, but circumvents the problems associated with a natural peptide. The deconstruction process begins by developing structure-activity relationships, then designing analogs to define a minimal active sequence and to identify the key residues and portions of the backbone in the peptide that are responsible for the biological effect. The structural constraints are added to check the effectivity of these features.
The interaction of a peptide with a biological target may occur via a direct binding of a linear sequence in any number of conformations accessible to a peptide. The modern peptide mimetics approach incorporates a production of small molecules which mimic peptides in order to overcome their ineffectiveness as drugs when administered orally. The small molecules mimetics retain the desired biological properties of the peptide lead, but are metabolically stable, have unlimited diversity, and can be designed to provide the new drugs.
By this process, the peptide has been reduced to its information content, the basis for a pharmacophore model that defines the critical features and their arrangement in space. This model supports the re-assembly of the critical elements and non-peptide variants on a modified scaffold that presents the optimized pharmacophore to the receptor. The optimized peptide-hybrid may be valuable as a first drug candidate, in addition to its role as a tool for further evolution to a mimetic. Mimetic scaffolds are designed to be resistant to the proteases that would destroy a natural peptide, and would have pharmaceutical properties consistent with a drug candidate.
It is possible to represent the biologically active sites of the peptides in the form of orally administered small-molecule mimietics that take all the advantages of evolutionally designed peptides on the one hand and have good drug properties, are stable, bioavailable, inexpensive in manufacture and convenient in use, on the other hand. There is no way to get involved in modern drug discovery and drug design without peptide and their small molecule mimetics research.
This glossary lists terms you may come across when reading about peptides, peptide synthesis and amino acids.
Two or more amino acids chained together by a bond called a "peptide bond".
A biological or chemical process in which amino acids are added stepwise to a chain by the formation of a peptide bond between a carboxyl group on one amino acid and a free amino group on another amino acid. The formation of each peptide bond is energetically favorable because the growing carboxyl terminus is activated by the covalent attachment of a tRNA molecule.
An organic high polymer that is an amphoteric biopolymer consisting of amino acids joined by peptide linkages. Genetic code determines the order of the twenty possible amino acids used in protein synthesis, and thus the protein is structure and function. Proteins are the principal constituents of cellular material and serve as enzymes, hormones, structural elements, and antibodies. Protein is involved in electron and oxygen transport, muscle contraction, and other bodily activities.
Peptides in which the amino-acid sequence forms a ring structure rather than a straight chain, such as the antibiotics tyrocidin and gramicidin.
An amino acid is any molecule that contains both amine and carboxyl functional groups. Alpha-amino acids are the building blocks from which proteins are constructed.
Synthesis of a protein, directed by the genetic code, which occurs by translation of mRNA into protein via tRNA. The ribosome attaches to the mRNA, using it as a template.
The order in which amino acid residues connected by peptide bonds.
The amide linkage between the alpha-amino group of one amino acid and the alpha-carboxyl group of another, with the elimination of a molecule of water.
A general term for methods used to identify unique proteins or nucleic acids by breaking them up with enzymes and looking at the resulting pattern of their amino acid or nucleotide base sequences.
The design of structurally similar organic compounds.
A chromatographic pattern produced by partial hydrolysis of a protein and 2-D mapping of the resulting peptide fragments.
A systematic combination of different peptides in a large number. It is a powerful tool for drug discovery, structural studies and other applications. Solid phase peptide synthesis, along with other methods, has been successfully used to prepare peptide libraries.
Oleg Larin, Postgraduate Student (D. Mendeleev University of Chemical Technology of Russia).>
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Peptide Guide provides a basic introduction to the field of peptide chemistry. It includes an overview of amino acids, a protein builder that constructs text representations for simple peptides and proteins, a peptide mass calculator, and a glossary as well as information about peptide bonds and synthesis.
A basic introduction to the field of peptide chemistry along with some applications of peptides. It includes an overview of amino acids, a peptide mass calculator, and a glossary as well as information about peptide bonds and synthesis.
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