What is a Peptide?

A peptide is a biologically occurring chemical compound containing two or more amino acids connected to one another by peptide bonds. A peptide bond is a covalent bond that is formed between two amino acids when a carboxyl group or C-terminus of one amino acid reacts with the amino group or N-terminus of another amino acid in a condensation reaction (a molecule of water is released during the reaction). The resulting bond is a CO-NH bond and forms a peptide, or amide molecule. Likewise, peptide bonds are amide bonds.

(More information about peptide bonds.)

The word “peptide” itself comes from πέσσειν, the Greek word meaning “to digest.” Peptides are an essential part of nature and biochemistry, and thousands of peptides occur naturally in the human body and in animals. In addition, new peptides are being discovered and synthesized regularly in the laboratory as well. Indeed, this discovery and innovation in the study of peptides holds great promise for the future in the fields of health and pharmaceutical development.

How Are Peptides Formed?

Peptides are formed both naturally within the body and synthetically in the laboratory. The body manufactures some peptides organically, such as ribosomal and non-ribosomal peptides. In the laboratory, modern peptide synthesis processes can create a virtually boundless number of peptides using peptide synthesis techniques like liquid phase peptide synthesis or solid phase peptide synthesis. While liquid phase peptide synthesis has some advantages, solid phase peptide synthesis is the standard peptide synthesis process used today. Read more about peptide synthesis.

Peptide Formation

The first synthetic peptide was discovered in 1901 by Emil Fischer in collaboration with Ernest Fourneau. Oxytocin, the first polypeptide, was synthesized in 1953 by Vincent du Vigneaud.

Peptide Terminology

Peptides are generally classified according to the amount of amino acids contained within them. The shortest peptide, one composed of just two amino acids, is termed a “dipeptide.” Likewise, a peptide with 3 amino acids is referred to as a “tripeptide.” Oligopeptides refer to shorter peptides made up of relatively small numbers of amino acids, generally less than ten. Polypeptides, conversely, are typically composed of more than at least ten amino acids. Much larger peptides (those composed of more than 40-50 amino acids) are generally referred to as proteins.

While the number of amino acids contained is a main determinate when it comes to differentiating between peptides and proteins, exceptions are sometimes made. For example, certain longer peptides have been considered proteins (like amyloid beta), and certain smaller proteins are referred to as peptides in some cases (such as insulin). For more information about the similarities and differences among peptides and proteins, read our Peptides Vs. Proteins page.

Classification of Peptides

Peptides are generally divided into several classes. These classes vary with how the peptides themselves are produced. For example, ribosomal peptides are produced from the translation of mRNA. Ribosomal peptides often function as hormones and signaling molecules in organisms. These can include tachykinin peptides, vasoactive intestinal peptides, opioid peptides, pancreatic peptides, and calcitonin peptides. Antibiotics like microcins are ribosomal peptides produced by certain organisms. Ribosomal peptides often go through the process of proteolysis (the breakdown of proteins into smaller peptides or amino acids) to reach the mature form.

Conversely, nonribosomal peptides are produced by peptide-specific enzymes, not by the ribosome (as in ribosomal peptides). Nonribosomal peptides are frequently cyclic rather than linear, although linear nonribosomal peptides can often occur. Nonribosomal peptides can develop extremely intricate cyclic structures. Nonribosomal peptides frequently appear in plants, fungi, and one-celled organisms. Glutathione, a key part of antioxidant defenses in aerobic organisms, is the most common nonribosomal peptide.

Milk peptides in organisms are formed from milk proteins. They can be produced by enzymatic breakdown by digestive enzymes or by the proteinases formed by lactobacilli during the fermentation of milk. Additionally, peptones are peptides derived from animal milk or meat that have been digested by proteolytic digestion. Peptones are often used in the laboratory as nutrients for growing fungi and bacteria.

Peptide fragments, moreover, are most commonly found as the products of enzymatic degradation performed in the laboratory on a controlled sample. However, peptide fragments can also occur naturally as a result of degradation by natural effects.

Important Peptide Terms

There are some basic peptide-related terms that are key to a general understanding of peptides, peptide synthesis, and the use of peptides for research and experimentation:

• Amino Acids – Peptides are composed of amino acids. An amino acid is any molecule that contains both amine and carboxyl functional groups. Alpha-amino acids are the building blocks from which peptides are constructed.
• Cyclic Peptides – A cyclic peptide is a peptide in which the amino acid sequence forms a ring structure instead of a straight chain. Examples of cyclic peptides include melanotan-2 and PT-141 (Bremelanotide).
• Peptide Sequence – The peptide sequence is simply the order in which amino acid residues are connected by peptide bonds in the peptide.
• Peptide Bond – A peptide bond is a covalent bond that is formed between two amino acids when a carboxyl group of one amino acid reacts with the amino group of another amino acid. This reaction is a condensation reaction (a molecule of water is released during the reaction).
• Peptide Mapping – Peptide mapping is a process that can be used to validate or discover the amino acid sequence of specific peptides or proteins. Peptide mapping methods can accomplish this by breaking up the peptide or protein with enzymes and examining the resulting pattern of their amino acid or nucleotide base sequences.
• Peptide Mimetics – A peptide mimetic is a molecule that biologically mimics active ligands of hormones, cytokines, enzyme substrates, viruses or other bio-molecules. Peptide mimetics can be natural peptides, a synthetically modified peptide, or any other molecule that performs the aforementioned function.
• Peptide Fingerprint – A peptide fingerprint is a chromatographic pattern of the peptide. A peptide fingerprint is produced by partially hydrolyzing the peptide, which breaks up the peptide into fragments, and then 2-D mapping those resulting fragments.
• Peptide Library – A peptide library is composed of a large number of peptides that contain a systematic combination of amino acids. Peptide libraries are often utilized in the study of proteins for biochemical and pharmaceutical purposes. Solid phase peptide synthesis is the most frequent peptide synthesis technique used to prepare peptide libraries.

What are Research Peptides?

Simply, research peptides are any peptides that are used in scientific research. In recent years, peptides have gained recognition as being highly selective and effective in therapeutic applications, all while being relatively safe and well tolerated in subjects and patients. As a result, there has been a huge uptick in interest in peptides for pharmaceutical research and development. With the promising potential that peptides present for medical applications, more and more research, study, and experimentation with peptides is necessitated in order to unlock the pharmaceuticals and therapeutics of today and tomorrow. Consequently, there has been a surge of demand for research peptides to fuel progress in these new avenues of research.

Research Peptides vs Medicines?

Importantly, research peptides are only made available for in-vitro study and experimentation. From the Latin for “in glass,” in-vitro refers to studies performed outside of the body. Hundreds of peptide therapeutics have been evaluated in clinical trials, and scientists and researchers around the globe are using research peptides in the lab to explore beyond the realm of traditional peptide design, pushing the boundaries to discover peptide variants that can be used as pharmaceuticals in the future. Already, there are over 60 peptide-based medicines on the market that have received approval from the US Food and Drug Administration (FDA). Among these are LupronTM, a treatment for prostate cancer, and VictozaTM, a treatment for type 2 diabetes. Both pharmaceuticals have achieved sales in the billions. However, it is essential to recognize that such FDA-approved drugs are NOT research peptides, they are just that: FDA-approved medications able to be prescribed by a healthcare professional for the treatment of a specific condition. Research peptides, on the other hand, are only intended for in-vitro study and research: they are not FDA approved for the treatment, prevention, or curing of any medical condition, disease, or ailment. Research peptides are those synthesized for study in the lab that can lead to new breakthroughs and future pharmaceuticals, but they become medicines only after undergoing rigorous study, clinical trial, and, crucially, the FDA approval process.

Research Peptides as Future Therapeutics

Over 7,000 naturally occurring peptides have been discovered. These peptides can often play vital roles in the human body as hormones, growth factors, neurotransmitters, ion channel ligands, and anti-infectives. Generally, peptides are effective and selective signaling molecules that bind to specific cell surface receptors, triggering intracellular effects. Additionally, in clinical trials, peptides have shown exceptional safety and tolerability in study subjects, while maintaining high selectivity and potency as well as a predictable metabolism. Consequently, peptides clearly present an enormous area of opportunity for therapeutic development.

Currently, the primary areas of disease fueling the research and use of peptide-based pharmaceuticals are metabolic diseases (such as type 2 diabetes) and oncology. The huge increases in obesity and type 2 diabetes in North America and other parts of the world have driven the development of peptide therapeutics for treatment of these conditions. Increases in cancer mortality and calls for alternatives to chemotherapy have spurred peptide research focused on oncological remedies. In addition, peptide research has expanded into the areas of infectious diseases, inflammation, and rare diseases. Crucially, all of the research and study focused on unlocking the therapeutic potential of peptides for future medicines is reliant upon research peptides to serve as the basis for experimentation and development in the laboratory.

What are the Differences?

Peptides and proteins, while similar in many regards, have several key differences that are important to understand. Oftentimes the terms “peptide” and “protein” are used synonymously, but differing characteristics and biological activities between the two compounds prevent the terms from being totally interchangeable. To fully appreciate the differences between proteins and peptides, it is important to understand amino acids, the building blocks of both, and how all three (amino acids, peptides, and proteins) relate to one another.

Peptides vs Proteins

Amino Acids

Amino acids are small but biologically vital compounds containing an amino group (NH2) and a carboxylic acid group (COOH) as well as a side-chain structure that varies between different amino acids. While hundreds of amino acids are known, only twenty are genetically combined into peptides (such as arginine, lysine, and glutamine), while others can be combined synthetically.

Importantly, amino acids make up the building blocks of peptides. When amine and carboxylic acid functional groups in amino acids join to form amide bonds, a peptide is formed. Combining two or more amino acids, whether naturally or synthetically, results in the formation of a peptide. The shortest peptide, containing two amino acids, can be referred to as a “dipeptide.” A peptide three amino acids in length is a “tripeptide,” and so on.

Peptides

Peptides are short chains of amino acids that have been linked by amide, or peptide, bonds. While the term “peptide” generally refers to a compound made up of two or more amino acids, peptides can be further classified as oligopeptides and polypeptides. Meaning “few,” “oligo” denotes that oligopeptides are made up of relatively small numbers of amino acids, generally less than ten. Polypeptides, on the other hand, are composed of more than ten amino acids.

Polypeptides and Proteins

Scientists commonly differentiate between proteins and polypeptides based on their size and structure. Regarding size, a polypeptide composed of more than 50 amino acids is generally classified as a protein, though the minimum categorization threshold can range from around 40-100 amino acids. However, 50 is a general guideline.

Secondly, proteins and polypeptides tend to differ in regards to their structure. Typically, polypeptides shorter than about 40-50 amino acids in length do not fold into a fixed structure. Proteins, however, are able to fold into a three-dimensional stable fixed structure. Proteins tend to have a fixed structure for a certain function (i.e. hemoglobin, a protein responsible for transporting oxygen in the blood). Polypeptides shorter than 40-50 amino acids, on the other hand, generally do not have enough cooperative interactions to form a stable native structure.

Peptide Vs. Protein: Which Term to Use?

Importantly, all proteins are technically polypeptides. However, as a researcher, it can sometimes be useful to differentiate between the two and reserve the term “proteins” to refer to relatively long and structurally fixed amino acid chains. Accordingly, peptides will generally refer to shorter (sub-50) amino acid chains.

In the modern era, huge leaps forward in the scientific field of peptide synthesis have enabled the production of custom peptides on an immense scale. With the increased production of synthetic peptides for research, the implementation of effective peptide purification methods has only become more critical. For more information on how Peptide Sciences ensures that every peptide on our website exceeds 99% purity, see our Peptide Purity page. This page will detail various aspects of peptide purification that take place during peptide synthesis, different methods of peptide purification and strategy, and possible impurities that can be removed by purification during synthesis.

Removing Specific Impurities From Peptides

As mentioned before, it is vital that the final synthesized peptide is as pure as possible for research use. Minimum acceptable purity levels can vary among different research purposes; for example, in vitro studies generally require a much higher standard of purity (greater than 95%) than, say, performing an ELISA standard for measuring titers of antibodies (minimum acceptable purity greater than 70%). Nonetheless, the minimum purity level must be achieved. In order to ensure that purity standards are met, it is vital to recognize the types of impurities than can arise as well as their nature. Then the appropriate purification method (or methods) can be implemented.

During peptide synthesis, specific impurities that can occur include hydrolysis products of labile amide bonds, deletion sequences generated mainly in solid-phase peptide synthesis (SPPS), diastereomers, and insertion peptides and by-products formed during removal of protection groups. This latter impurity can occur in the last step of peptide synthesis. Additionally, polymeric forms of the peptide intended to be synthesized can also occur, often arising as a byproduct resulting from the formation of cyclic peptides that have disulphide bonds.

Certainly, the purification process employed must be able to effectively isolate the targeted peptide in a multifaceted mixture of compounds and potential impurities.

Peptide Purification Strategy

Ideally, the purification method should be as simple as possible, achieving targeted purity in as few steps as possible. Often, two or more purification processes conducted sequentially can give excellent results, particularly when each process operates through differing principles of chromatography. For example, ion exchange chromatography utilized in conjunction with reversed phase chromatography can result in a very highly pure final product.

Generally, the first step in peptide purification is a capturing step that removes the majority of impurities from the synthetic peptide mixture. Many of the impurities removed in this phase are produced in the final deprotection step of peptide synthesis and are mostly uncharged and have a small molecular weight. While a significant amount of impurities can be removed during this initial step, a second purification step can be added if a higher purity level is required. This second step can be referred to as a polishing step and is highly effective, especially when operating on a complementary chromatographic principle as previously mentioned.

Peptide Purification Processes

Peptide purification systems are composed of several integral subsystems and units, which can include buffer preparation systems, solvent delivery systems, fractionation systems, and data collection systems, along with the crucial columns and detectors. Indeed, the column is the heart of the purification system and its selected features can be critical to the process’s efficaciousness. A column may have features constructed of glass or steel along with static or dynamic modes of compression, any of which can affect the final purification outcome.

Additionally, it is vital that all purification methods are carried out in accordance with current Good Manufacturing Practices (cGMP) and sanitization is given the highest priority.

Affinity Chromatography (AC)

This process isolates peptides by capitalizing on the interaction between a peptide and a particular ligand attached to a chromatographic matrix. The desired peptide binds to the ligand, and unbound material is washed away. Importantly, this binding is reversible. Conditions are changed to become favorable to desorption, which can be performed specifically or nonspecifically. Specific desorption is performed using a competitive ligand, and nonspecific desorption is accomplished by altering pH, polarity, or ionic strength. The targeted peptide is then collected in a purified form. AC offers both high resolution and sample capacity.

Ion Exchange Chromatography (IEX)

This purification process capitalizes on differences in charge among peptides in a mixture. Peptides of one charge are isolated when faced with a chromatographic medium with the opposite charge. Peptides are loaded into a column and bind; conditions are subsequently changed so that bound substances are eluted differentially. Conditions manipulated are level of salt concentration or pH level alteration. Typically, salt (NaCl) is used to elute the mixture. The desired peptide is concentrated during the binding process and then collected in purified form. IEX is a high resolution and high capacity process.

Hydrophobic Interaction Chromatography (HIC)

This process operates on the principle of hydrophobicity. The targeted peptides are able to be isolated as a result of the interaction between a peptide and the hydrophobic surface of a chromatic medium. This interaction is reversible, allowing the peptide to be concentrated and purified. A high ionic strength buffer enhances the process, making HIC a highly effective purification method to implement after an initial purification method that utilizes salt in elution (like the IEX technique).

During HIC, samples in the high ionic strength solution bind together as they are loaded onto a column. Next, elution implemented via decreases in salt concentration results in the bound substances being eluted differentially. A typical method of implementation involves using ammonium sulfate to dilute the sample on a decreasing gradient. The desired peptide is then collected in a concentrated and purified form. HIC offers good levels of resolution and sample capacity.

Gel Filtration (GF)

Gel filtration isolates peptides by capitalizing on differences in molecular size between the targeted peptides and impurities. GF is only utilized on small volume samples. However, this process does offer very good resolution.

Reversed Phase Chromatography (RPC)

This purification process offers very high resolution and separates peptides from contaminants by utilizing the reversible interaction between target molecules and a chromatographic medium’s hydrophobic surface. Samples are loaded onto a column and bind together. Next, conditions are altered so that these bound substances are eluted differentially. Organic solvents and other additives are often required for elution, as the initial binding is very strong as a result of the nature of the reversed phase matrices. Generally, elution is accomplished by increasing the concentration of organic solvents, typically acetonitrile. Concentrated molecules resulting from the binding process are then collected in a purified form. RPC is often utilized as a polishing step with peptides and oligonucleotide samples. It is very effective for analytical separations like peptide mapping. However, because organic solvents can denature many peptides, RPC is not an ideal purification process if requirements call for recovery of activity and a return to a correct tertiary structure.

Compliance with GMP

Throughout the processes of peptide synthesis and purification, special attention must be given to following GMP. This is to ensure that the final peptide is pure and of high quality. GMP requires that chemical and analytical procedures performed are well documented. Test methods and specifications are required to be established beforehand, ensuring that the manufacturing process is under control and reproducible.

GMP requirements for the purification phase of peptide synthesis are particularly rigorous. This is because this process is a late step in the overall synthesis process and has a large impact on the quality of the final peptide. Critical steps and parameters must be identified, along with limits for those parameters, so that the process is reproducible within those predetermined limits. Vital parameters of the peptide purification process can include column loading, flow rate, column performance, column cleaning procedures, composition of elution buffer, storage time in process, and pooling of fractions.

At Peptide Sciences, we adhere to the most stringent synthesis and purification practices in the industry. Through our dedication to these standards, our company is able to provide peptides that exceed 99% purity and are fit for any research study or application.

What is Peptide Synthesis?

Characterized by the formation of a peptide bond between two amino acids, peptide synthesis is, essentially, the production of peptides. Though peptide synthesis was somewhat hampered by relatively inefficient production practices at its inception, advancements in chemistry and technology have led to vastly improved synthesis methods. With the strong growth of the field of peptide science, it is clear that synthetic peptides will continue to play vital roles in areas of scientific and medical progress in the modern age.

Steps of Peptide Synthesis

How Peptides are Synthesized?

Peptides are synthesized by linking two amino acids together. This is most often accomplished by attaching the C-terminus, or carboxyl group, of one amino acid to the N-terminus, or amino group, of another. Unlike protein biosynthesis, which involves N-terminus to C-terminus linkage, peptide synthesis occurs in this C-to-N fashion.

Peptide Synthesizer

While there are twenty amino acids that occur commonly in the natural world (such as arginine, lysine, and glutamine), many other amino acids have also been synthesized. This allows for abundant possibilities in the creation of new peptides. However, amino acids have numerous reactive groups that can negatively interact during the synthesis process, leading to unwanted truncating or branching of the peptide chain or causing suboptimal purity or yield. As a result, peptide synthesis is a complex process that must be expertly carried out.

In order to ensure the desired outcome from the synthesis process and avoid extraneous, unwelcome reactions, certain amino acid reactive groups must be deactivated, or protected, from reacting. Thus, scientists have engineered special chemical groups designed to do just that. Called “protecting groups,” they can be separated into three categories:

• N-terminal protecting groups – These groups protect the N-termini of amino acids. Referred to as temporary protecting groups, they are removed relatively easily to facilitate the formation of peptide bonds. Tert-butoxycarbonyl (Boc) and 9-fluorenylmethoxycarbonyl (Fmoc) are two frequently used N-terminal protecting groups.
• C-terminal protecting groups – These groups protect the C-terminus of amino acids. The use of C-terminal protecting groups is warranted in liquid-phase peptide synthesis but not solid-phase peptide synthesis.
• Side chain protecting groups – As amino acid side chains are quite conducive to reactivity during peptide synthesis, various unique side chain protecting groups are needed to protect against unwanted reactions. Able to remain intact during the many cycles of chemical treatment during synthesis, side chain protecting groups are known as permanent protecting groups. They are only removed with strong acids after the conclusion of peptide synthesis.

Peptide Synthesis Processes

The original approach to peptide synthesis was through a process known as solution phase synthesis (SPS). While SPS does have some merit today, notably in large-scale peptide production, it has some advantages, it has largely been supplanted by solid-phase peptide synthesis, or SPPS. This is because SPPS offers several advantages, including high yield, purity, and speed of production.

SPPS involves five steps performed in a cyclical manner:

1. Attaching an amino acid to the polymer
2. Protection (to prevent unwanted reactions)
3. Coupling
4. Deprotection (to allow the attached acid to react to the next amino acid to be added on)
5. Polymer removal (resulting in a free peptide)

Additionally, SPPS synthesis can be further enhanced by the use of microwave-assisted SPPS. This is particularly useful when synthesizing long peptide sequences, as yield and speed can both be improved. However, microwave-assisted SPPS can be more expensive than traditional SPPS.

While peptide synthesis processes like SPPS offer excellent purity and yield standards, impurities and imperfections can still occur along the way. This likelihood increases with the length of the peptide sequence, as more steps are needed to complete synthesis. Therefore, certain purification techniques are utilized in order to ensure optimal quality. Among these are reverse-phase chromatography (RPC) and high-performance liquid chromatography (HPLC). Capitalizing on peptides’ physiochemical properties, these purification methods are able to separate impurities from the desired peptide. RPC is the most widely used peptide purification method today.

The Value of Synthetic Peptides

Peptides have proven to be critical elements of biomedical research, and peptide synthesis continues to fuel scientific progress worldwide. The therapeutic potential of peptides has drawn the attention of various pharmaceutical companies, and several drugs made from peptides have received FDA approval and reached the market. The efficacy, specificity, and low toxicity of peptides assures us that peptides will continue to be pursued and developed for pharmaceutical and diagnostic purposes and will remain a growing area of biochemical research.

What is a Peptide Bond?

A peptide bond is a covalent bond that is formed between two amino acids. To form a peptide bond, a carboxyl group of one amino acid reacts with the amino group of another amino acid. As a result, a molecule of water is also released. This is referred to as a condensation reaction. The resulting bond is a CO-NH bond and is henceforth referred to as a peptide bond. Additionally, the resulting molecule is termed an amide.

Peptide Bond Formation

In order to form a peptide bond, the molecules of the amino acids in question must be orientated so that the carboxylic acid group of one amino acid is able to react with the amine group of another amino acid. At its most basic, this can be illustrated by two lone amino acids combining through the formation of a peptide bond to form a dipeptide, the smallest peptide (i.e. only composed of 2 amino acids).

Peptide Bond Formation

Further, any number of amino acids can be joined together in chains to form new peptides: as a general guideline, 50 or less amino acids are referred to as peptides, 50 – 100 are termed polypeptides, and peptides with over 100 amino acids are generally referred to as proteins. For a more detailed description of peptides, polypeptides, and proteins, refer to the Peptides Vs. Proteins page of our peptide glossary.

Hydrolysis (a chemical breakdown of a compound resulting from a reaction with water) can break down a peptide bond. Though the reaction itself is quite slow, the peptide bonds formed within peptides, polypeptides, and proteins are susceptible to breakage when they come into contact with water (metastable bonds). The reaction between a peptide bond and water releases about 10kJ/mol of free energy. The wavelength of absorbance for a peptide bond is 190-230 nm.

In the biological realm, enzymes inside living organisms can both form and break down peptide bonds. A number of hormones, antibiotics, antitumor agents and neurotransmitters are peptides, most of which are referred to as proteins (due to the number of amino acids contained).

Structure of the Peptide Bond

Amino Acid and Peptide Bonds

Scientists have conducted x-ray diffraction studies of several small peptides in order to ascertain the physical characteristics of peptide bonds. Such studies have indicated that peptide bonds are rigid and planer. These physical characteristics are principally derived as a result of the resonance interaction of the amide: the amide nitrogen is able to delocalize its sole pair of electrons into the carbonyl oxygen.

This resonance directly affects the structure of the peptide bond. Indeed, the N–C bond of the peptide bond is actually shorter than the N–Cα bond, and the C=O bond is longer than normal carbonyl bonds. In the peptide, the carbonyl oxygen and amide hydrogen are in a trans configuration, not a cis configuration; such a configuration is more energetically favorable due to the possibility of steric interactions in a cis configuration.

The Polarity of the Peptide Bond

Usually, free rotation should be able to take place about a single bond between a carbonyl carbon and amide nitrogen, the structure of a peptide bond. However, the nitrogen in this case has a lone pair of electrons. These electrons are near a carbon-oxygen bond. As a result, a reasonable resonance structure can be drawn, in which a double bond links the carbon and nitrogen. Consequently, the oxygen has a negative charge and the nitrogen has a positive charge. Rotation around the peptide bond is therefore inhibited by the resonance structure. Additionally, the real structure is a weighted hybrid of these two structures. The resonance structure is a significant factor in depicting the true electron distribution: the peptide bond has approximately 40% double-bond character. As a result, it is rigid.

Charges result in the peptide bond having a permanent dipole. The oxygen has a -0.28 charge, and the nitrogen has a +0.28 charge as a result of the resonance.