How Is The Bond Created?

When two amino acids form a covalent bond, it creates a peptide bond.^[1] The Carboxyl group of one amino acid will react with the second amino group of the other amino acid. This essentially forms a peptide bond. Due to this process, a molecule (amide) filled with water is released, and this reaction is known as a condensation reaction. This creates the peptide bond (CO-NH bond).

Now how exactly are the bonds formed? Firstly, the amino acids needed must be oriented. This is so that the carboxylic acid group from the amino acid can react with the other amino acids amine. These create a dipeptide which we know as the smallest peptide composed of two amino acids.

It is important to note that new peptides can also be formed by amino acids joining together in chains. To be considered a peptide, there must be 50 or less amino acids connected.^[2] Scientists also report that “As peptide chains form between joining of the primary structure of amino acids, they may enlarge to become an oligopeptide when there are between 10 to 20 amino acids in the chain.” To be considered a polypeptide, there must be about 50 to 100 amino acids connected and if there are over 100 amino acids connected, that would be called a protein.

A peptide bond can be broken down from hydrolysis. Hydrolysis is a result of a chemical breakdown reaction to water.^[3] This reaction is unhurried, and the peptide bonds, which can be formed in three ways (peptides, polypeptides, or protein), are vulnerable to breakage if they were to come in contact with metastable bonds (water). 

Looking At The Structure and Polarity Of Peptide Bonds

To discover the physical characteristics of compound bonds, researchers perform x-ray diffraction studies.^[4] With this study, researchers have proven that peptide bonds are both rigid and planar.^[5] The researchers describe techniques for determining the X-ray crystallographic structures of peptides: incorporation of amino acids containing heavy atoms for crystallographic phase determination, commercially available kits to crystallize peptides, modern techniques for X-ray crystallographic data collection, and free user-friendly software for data processing and producing a crystallographic structure.”
These results are due to the interaction of the amide, which is able to delocalize its sole pair of electrons into carbonyl oxygen. This primarily will affect a peptide bond structure. The N–C bond is shorter than the N–Cα bond, while the C=0 bond is longer than the carbonyl bond. There is no cis configuration in the peptide, but there is a trans configuration between the carbonyl oxygen and amide hydrogen in the peptide. A trans configuration is preferred over a cis configuration as a Cis configuration can cause a steric interaction.

In the structure of a peptide bond, free rotation can occur. Free rotations are the process of a single bond coming between a carbonyl carbon and amide nitrogen. The nitrogen, however, has a free set of electrons, and thus resonance structure can be drawn where a double bond can connect the carbon and nitrogen. The oxygen, in this case, will have a negative charge while nitrogen has a positive charge, and thus, the rotation of the peptide bond will be inhibited from the resonance structure.

Disclaimer: The products mentioned are not intended for human or animal consumption. Research chemicals are intended solely for laboratory experimentation and/or in-vitro testing. Bodily introduction of any sort is strictly prohibited by law. All purchases are limited to licensed researchers and/or qualified professionals. All information shared in this article is for educational purposes only.

References

1. Alberts B, Johnson A, Lewis J, et al. Molecular Biology of the Cell. 4th edition. New York: Garland Science; 2002. The Shape and Structure of Proteins. Available from: https://www.ncbi.nlm.nih.gov/books/NBK26830/
2. Forbes J, Krishnamurthy K. Biochemistry, Peptide. [Updated 2022 Aug 29]. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2022 Jan-. Available from: https://www.ncbi.nlm.nih.gov/books/NBK562260/
3. Bhimrao Muley, A., Bhalchandra Pandit, A., Satishchandra Singhal, R., & Govind Dalvi, S. (2021). Production of biologically active peptides by hydrolysis of whey protein isolates using hydrodynamic cavitation. Ultrasonics sonochemistry, 71, 105385. doi:10.1016/j.ultsonch.2020.105385.
4. Mikusinska-Planner, A., & Surma, M. (2000). X-ray diffraction study of human serum. Spectrochimica acta. Part A, Molecular and biomolecular spectroscopy, 56A(9), 1835–1841. doi:10.1016/s1386-1425(00)00262-6.
5. Spencer, R. K., & Nowick, J. S. (2015). A Newcomer’s Guide to Peptide Crystallography. Israel journal of chemistry, 55(6-7), 698–710. doi:10.1002/ijch.201400179.

How To Find The Best Peptide Solubility Option

Figuring out the most effective solvent to dissolve peptides with is possibly one of the most difficult components when working with peptides and conducting research. Aqueous solutions–also known as sterile waters–are one way to dissolve peptides. Problems do, however, still arise with this method. Some issues you may encounter are related to low solubility or even solubility. This matter is more common when working with peptides containing long hydrophobic amino acid sequences. Though there are difficulties, in this day and age, researchers can predict a peptide’s solubility just by studying its known characteristics and its amino acid.

An Amino Acid’s physical properties are what predominantly determines a peptide’s solubility. Amino Acids classification can be any one of the following four:
1. Basic
2. Acidic
3. Polar uncharged
4. Non-polar (hydrophobic-do not dissolve in aqueous solutions)

Researchers report that “The polar amino acids are: R, S (codons AGC and AGU), K, N, Q, H, W, C, Y, G, E, D; apolar ones are: T, M, I, P, L, S (codons UCN)”^[1]. A large number of non-polar or polar uncharged amino acids dissolve more effectively with organic solvents such as:
1. DMSO
2. Propanol
3. Isopropanol
4. Methanol
5. DMF

Basic solvents (ammonium hydroxide) are of better use for peptides with high content amino acids. It is important to note that ammonium hydroxide should not be used with peptides having Cys. Acidic solvents, such as acetic acid solution, are also better paired with peptides that contain a high number of basic amino acids.^[2] Each peptide dissolves more easily and effectively when paired with the correct solvent. The first step to take into account is that researchers should first attempt to dissolve peptides in sterile water. This is important, especially if a peptide has less than 5 amino acids since they dissolve more easily in water.

Steps To Remember For Proper Peptide Solubility

1. In order to achieve the ideal solubility, researchers must test a small amount of peptide in peptide solubility. This helps the researcher decipher whether the solution is a good match with the peptide.

2. Allow time for the peptide to reach room temperature before attempting to dissolve it in solution.
*Utilize solutions that are able to be removed by lyophilization if sterile water solution does not work when trying to dissolve the peptide. If none of the options are successful, then it can be removed by the lyophilization which allows the researcher to start the process again without losing or affecting the peptide.^[3]

3. A slightly warmer solution, for example less than 40 Celsius or 104 Fahrenheit, would aid in solubility. Sonication techniques can also be of use. It is vital to note that this only helps in dissolving a peptide. It will not change the peptides natural characteristics.

Researchers should evaluate an amino acids composition of the peptide to predict the solubility characteristics of a peptide. It is essential to check the number and type of ionic charges of a peptide influence solubility. It is imperative as it helps tell whether the peptide is acidic, basic, or neutral. To figure this out, one would assign a value of -1 to amino acids (also known as residues). For example, you may see Asp (D), Glu (E), and C-terminal (COOH). Then you would assign +1 value to each basic amino acid which includes Lys (K), Arg (R), and N-terminal NH2. You would also assign value to +1 to each His (H) amino acid at pH6. And finally calculate the net charge overall of the peptide. This is done by adding up the peptide’s total number.

What To Do When It Is Time To Conduct Peptide Solubility

Only when the net charge of the peptide is calculated can solubility predictions be made.^[4] The scientists report that “the pH of minimum solubility varies with the pI of the protein, but that the pH of maximum activity and the pH of maximum stability do not.” This allows researchers to begin dissolving the peptides in solution. As mentioned previously it is also essential to try to dissolve peptides in sterile water before attempting to use other solutions. If and only when the water is ineffective, researchers may follow these directions.

First, you attempt to dissolve a peptide in acetic acid solution (10-30%) only if the overall net charge is positive. If this is not the case, try TFA (<50μl). Alternatively, if there is a negatively charged peptide, you may experiment with using ammonium hydroxide (NH4OH; < 50 μl) to dissolve the peptide. Note that if Cys is in the peptide, add a small amount of DMF and do not work with ammonium hydroxide. Lastly, If the overall net charge is 0, meaning the peptide is neutral, the most effective solvent would be organic solvents. Organic solvents consist of acetonitrile, methanol, or isopropanol. Use a small amount of DMSO if a peptide is highly hydrophobic.

CAUTION: DMSO can oxidize peptides containing cysteine, methionine, or tryptophan.^[5] Some peptides also tend to aggregate (gel); in regard to these peptides, add 6 M guanidine•HCl or 8 M urea.

Dilute the peptide solution to the desired concentrate by slowly adding the peptide solution into a buffered solution only when the peptide has successfully dissolved. It is critical to use gentle but, most importantly, constant agitation while combining it to monitor and avoid localized concentration of the peptide in an aqueous solution. The experimental assay also suggests preparing peptide stock solution at higher concentrations than what is typically required. This can help dilute the stock more with use of the assay buffer.

When the process is finished and the solution is prepared correctly, it should be moved and stored at -20C (-4F). Peptides that contain cysteine, methionine, or tryptophan should be stored
In an oxygen-free environment to prevent oxidative damage.

Disclaimer: The products mentioned are not intended for human or animal consumption. Research chemicals are intended solely for laboratory experimentation and/or in-vitro testing. Bodily introduction of any sort is strictly prohibited by law. All purchases are limited to licensed researchers and/or qualified professionals. All information shared in this article is for educational purposes only.

References

1. Chipens, G. I., Balodis, I.uI.u, & Gnilomedova, L. E. (1991). Poliarnost’ i gidropatichnye svoĭstva prirodnykh aminokislot [Polarity and hydropathic properties of natural amino acids]. Ukrainskii biokhimicheskii zhurnal (1978), 63(4), 20–29.
2. Sikora, K., Jaśkiewicz, M., Neubauer, D., Migoń, D., & Kamysz, W. (2020). The Role of Counter-Ions in Peptides-An Overview. Pharmaceuticals (Basel, Switzerland), 13(12), 442. doi:10.3390/ph13120442
3. Jameel, F., Alexeenko, A., Bhambhani, A., Sacha, G., Zhu, T., Tchessalov, S., Kumar, L., Sharma, P., Moussa, E., Iyer, L., Fang, R., Srinivasan, J., Tharp, T., Azzarella, J., Kazarin, P., & Jalal, M. (2021). Recommended Best Practices for Lyophilization Validation-2021 Part I: Process Design and Modeling. AAPS PharmSciTech, 22(7), 221. doi:10.1208/s12249-021-02086-8
4. Shaw, K. L., Grimsley, G. R., Yakovlev, G. I., Makarov, A. A., & Pace, C. N. (2001). The effect of net charge on the solubility, activity, and stability of ribonuclease Sa. Protein science : a publication of the Protein Society, 10(6), 1206–1215. doi:10.1110/ps.440101
5. Savige, W. E., & Fontana, A. (1980). Oxidation of tryptophan to oxindolylalanine by dimethyl sulfoxide-hydrochloric acid. Selective modification of tryptophan containing peptides. International journal of peptide and protein research, 15(3), 285–297. doi:10.1111/j.1399-3011.1980.tb02579.x

Advancements in today’s world have driven the scientific field of peptide synthesis to what it is today. Peptide synthesis has allowed for an immense production of custom peptides. Due to having more peptides for research, it is imperative and critical to have and implement effective peptide purification procedures. Here you will find information on the different aspects of peptide purification that take place during the synthesis, different strategies and methods, and possible mistakes, such as impurities that can actually be removed just by purification during the synthesis.

They are unique and complex. Due to this complexity, they can affect purification methods that work for other organic compounds. To maximize efficiency and yield to supply the purest peptides possible for an affordable cost, special attention must be made when they are in synthesis. Chromatography, specifically high reverse phase chromatography, is what many purification processes like to utilize. However, purification based on crystallization is also effective with other compounds.

The BluePrint and Process for Peptide Purification

The purification process should be simple and have as few steps as possible to achieve purity.
You can still get your targeted results even with two or more purification processes done right after another. One example is when ion exchange chromatography is used in alliance with reverse-phase chromatography. This can result in a highly pure product when completed.

The first step to be taken is the capturing step. This step will remove more than half of the impurities found in a synthetic mixture. The impurities that are eliminated are typically produced in the final deprotection stage of peptide synthesis. Those impurities are typically uncharged and are considered small due to their molecular weight.

If a higher purity level is needed, a second purification can be done as well to achieve the target purity. This second step is referred to as the polishing step. It is effective when working with a complementary chromatographic principle.

Some multiple subsystems and units make up the system for peptide purification. These include:
1. Buffer Preparation Systems
2. Solvent Delivery Systems
3. Fraction Systems
4. Data Collection Systems.
*some columns and detectors could be pivotal.

The heart of the purification system is the column. The column’s features are vital to the effectiveness of the process. Columns have features made from glass or steel with static or dynamic compression modes. Any of those features can affect the outcome of the final purification. It is crucial that every purification practice is carried out following Good Manufacturing Practices (GMP), and priority is given to sanitation.

Purification Process Types:

1. Ion Exchange Chromatography (IEX)
The Ion Exchange Chromatography excels with different charges among peptides in a mixture. When peptides with opposite charges meet in a chromatographic medium, one charge begins to isolate. Columns and binds are what peptides are loaded into. So that bound substances are eluted differently, conditions are continuously changed. What is being manipulated during this is levels of salt concentration (which is used to elute the mixture) or the pH level. During the binding process, the peptide is concentrated and then collected in its purified form. This process has high resolution and high capacity as well.

2. Hydrophobic Interaction Chromatography (HIC)
Hydrophobicity is the principle; this purification process follows; the selected peptides will then isolate due to the interaction between a peptide and a hydrophobic surface of a chromatic medium. Due to this also being a reversible action, a peptide can be concentrated and purified. HIC could be an effective purification after a previous salt-in-elution (IEX) purification method. To do this, a high ionic strength buffer is needed to magnify the process. During this process, they bind together in a high ionic strength solution. As they bind, they are loaded onto columns. Following this, the salt concentration is decreased, which causes elution. This results in bound substances eluting differentially. Using ammonium sulfate is one way to implement this process typically. It is used to dilute samples on a reduced gradient. Ultimately, the peptide is collected in the final concentrated and purified form. HIC provides good levels of resolution and sample capacity.

3. Affinity Chromatography (AC)
The first note about Affinity Chromatography is that it offers high resolution and sample capacity. It isolates them by capitalizing the interaction between peptides and a particular ligand attached to a chromatographic matrix. The peptide then bonds to a ligand, cleaning the unbound material. One crucial piece of information to know about this binding is that it is reversible. This means the condition is changed to be more complementary to desorption. Desorption could also be performed specifically or not specifically. Specific desorption uses a competitive ligand, while nonspecific uses an altered pH, polarity, or ionic strength. Once that is completed, the peptide of choice is collected in its purified form.

4. Reversed Phase Chromatography (RPC)
This method separates peptides from contaminants by reversible interactions between the selected molecules and chromatographic mediums on a hydrophobic surface. This method also offers high resolution. The sample could then be loaded on columns or even bound together. After they have done one of these steps, the conditions are changed to the bound substances elute differently. The organic solvents and additional additives are needed for elution. Due to the reversed-phase metrics, initial bonding is considered to be strong. Typically, organic solvent concentration, known as acetonitrile, needs to be increased to achieve elution. The molecules are gathered through the binding process while in their pure form. Note this method is primarily used as a clean-up step with peptides and oligonucleotide samples. RPC is successful when working with analytical separations such as peptide mapping. It is still not the perfect method, as organic solvents can denature peptides. Thus, it is not always the preferred method as requirements may call for recovery of activity and the need to return the peptides to a correct tertiary structure.

5. Gel Filtration (GF)
This purification method can isolate peptides by taking advantage of the different molecular sizes between the chosen peptides and impurities. This method, however, is only used when dealing with small-volume samples. It does still offer good resolution as well.

The Importance Of Following Good Manufacturing Practices

To ensure the final peptides are pure and high quality, researchers must follow the guidelines given by Good Manufacturing Practices (GMP). It is a requirement that chemical and analytical procedures that have been executed are properly documented. All testing, research methods, and specifications must be known and accepted before any work begins. This will promise that the process is reproducible and regulated correctly.

The requirements for the purification of peptide synthesis are careful and diligent. This is partly due to the step’s huge impact on the final results of the quality of the peptides. Crucial steps and parameters must be pinpointed, and limits must be set for those parameters. This is required to duplicate the process, and others could replicate these steps and produce the same results. Examples of the important parameters of the purification process include the following:
1. Column Loading
2. Flow Rate
3. Column Performance
4. Column Cleaning Procedures
5. Composition of Elution Buffer
6. Storage Time in Process
7. Pooling of Fractions

How To Rid Peptides Of Specific Impurities

The goal for synthesis is to have the purest peptide possible for researchers to use. A minimum of acceptable purity levels are welcomed and used for different research purposes. Take an in vitro study, for example. They have a need for a high standard of purity (>95%). ELISA, on the other hand, has a minimal acceptability of >70% for measuring titers of antibodies. Therefore the minimum level is essential to attain.

It is also important to understand and know the types of impurities that can come forth along with their characteristics. This way, researchers know the most effective purification method as each situation differs.

Specific impurities during peptide synthesis can happen; These consist of:
1. Hydrolysis products of labile amide bonds
2. Deletion sequences generated in solid-phase peptide synthesis (SPPS)
3. Diastereomers
4. Insertion peptides and by-products formed during removal of protection groups

Other than impurities, polymeric forms of peptides may also occur. This transpires due to a byproduct resulting from the formation of cyclic peptides which have disulfide bonds. Therefore, the chosen purification process method must make certain to isolate the selected peptide while in a multifaceted mixture of compounds and impurities.

At Biotech Peptides, we adhere to the industry’s most stringent synthesis and purification practices. 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.

Disclaimer: The products mentioned are not intended for human or animal consumption. Research chemicals are intended solely for laboratory experimentation and/or in-vitro testing. Bodily introduction of any sort is strictly prohibited by law. All purchases are limited to licensed researchers and/or qualified professionals. All information shared in this article is for educational purposes only.

The Process of Peptide Synthesis

Solution Phase Synthesis (SPS) was the original approach to peptide synthesis. Though this process still has merit in this modern age, for large-scale peptide production, Solid-Phase Peptide Synthesis (SPPS) has proven to be the method of choice. SPPS’s advantages are the reason for this. SPPS creates peptides with higher purity, high yield, and faster production time.

There are five steps performed in a cyclical manner SPPS involves. The first step involves the attachment of an amino acid to a polymer. The second step is the protection of this attachment to prevent unwanted reactions. The next step is coupling the protected amino acids. Once that is done, they deprotect them to allow attachment acids to react to the following amino acid that will be added. And finally, they engage in polymer removal, allowing there to be a free peptide.

Microwave-assisted SPPS can also enhance SPPS synthesis. This could be helpful when synthesizing long peptide sequences due to improved yield and speed. Microwave-assisted SPPS can, however, be the more expensive option compared to the traditional SPPS synthesis process.

Though SPSS can offer excellent purity and yield standards, it can still become a victim of impurities and imperfections during the process. The chances increase with a lengthier peptide sequence as more steps are needed to complete the synthesis process. Thus in order to secure optimal quality, specific purification techniques would need to be utilized. Examples would be:

1. Reverse-Phase Chromatography (RPC)
2. High-Performance Liquid Chromatography (HPLC)

Reverse-Phase Chromatography (RPC) is today’s most widely used peptide purification method. These purification methods can separate the impure peptides from the desired peptide. Thus benefiting peptides’ physicochemical properties.

Linking two amino acids together is how Peptides are synthesized. Most of the time, this is 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.

While there are 20 amino acids commonly occurring in the natural world, such as arginine, lysine, and glutamine,  many other amino acids are also being synthesized. This creates abundant possibilities for the creation of new peptides. However, amino acids have multiple reactive groups that can negatively interact during synthesis. This could lead to unwanted truncating or branching of the peptide chain or cause suboptimal purity or yield. Due to these negative effects, peptide synthesis must be expertly carried out as it is a complex process.

Protecting Groups

Scientists have engineered special and specific chemical groups to secure the preferred outcome from the synthesis process and avoid extraneous, unwelcome reactions. To accomplish this goal, certain amino acid reactive groups must be deactivated or protected from reacting. These special groups are known as protecting groups and can be separated into three categories: N-terminal, C-terminal, and Sidechain. The N-terminal protecting group protects the N-termini of amino acids. Known as the temporary protecting groups, they are removed easily to aid the formation of peptide bonds. Two frequently used N-terminal protecting groups are:

1. 1. Tert-butoxy carbonyl (Boc)
   2. 9-fluorenyl methoxycarbonyl (Fmoc).

The C-terminal protecting group protects the C-terminus of amino acids. C-terminal protecting groups are needed in liquid-phase peptide synthesis but not solid-phase synthesis. Lastly, side chains various and unique protecting groups are needed to protect against unwanted reactions because amino acid side chains are conducive to reactivity during peptide synthesis. It can stay intact during the numerous cycles of chemical treatment during synthesis. These side-chain protecting groups are known as permanent protecting groups. Once peptide synthesis is done, side chains are only removed with strong acids.

Disclaimer: The products mentioned are not intended for human or animal consumption. Research chemicals are intended solely for laboratory experimentation and/or in-vitro testing. Bodily introduction of any sort is strictly prohibited by law. All purchases are limited to licensed researchers and/or qualified professionals. All information shared in this article is for educational purposes only.