What is peptide: A Simple Introduction of Peptide Synthesis.Peptides and proteins are linear polymers of amino acids linked by amide "peptide bonds" (Fig. 1). Thepeptide bond is formed by linking an amino group to a caroboxyl group on another amino acid. Aminoacids are primary amines that contain an alpha carbon that is connected to an amino (NH3) group, acarboxyl group (COOH), and a variable side group (R). Carboxylic acid and carboxylate groups arenormally not very reactive. It requires activating the carboxylic acid for the formation of the amidepeptide bond (Fig. 2).

Fig. 1: Peptide Structure

The classical approaches to peptides production are called liquid-phase peptide synthesis and solid-phase peptide synthesis (SPPS). These two methods can be combined in a process called nativechemical ligation. LifeTein’s standard peptide synthesis process involves the solid phase. The liquid-phase approach is used for the synthesis of short peptides, such as di- and tripeptides, and C-terminally modified peptides, such as enzyme substrates. The controlled peptide synthesis requiresselective protection and deprotection of the various functional groups: the amino group, the -carboxylgroup or the side chain functional groups. The side group gives each amino acid its distinctiveproperties and helps to dictate the folding of the protein.

Fig. 2: Peptide bonds created with peptide coupling agents

Principles of Solid-phase Peptide Synthesis

SPPS involves repeated cycles of coupling, washing, deprotection, and washing. A single cycle consistsof the following:

Cleavage of the alpha-amino protecting groupWashing to remove the cleavage reagentCoupling of the protected amino acidWashing to remove excess material

Unlike ribosome protein synthesis, artificial synthesis builds peptides in the C to N direction. Duringsolid-phase peptide synthesis, each peptide is anchored to an insoluble polymer at the C-terminus. Thefree N-terminal amine is coupled to a single N-protected amino acid unit. This unit is thendeprotected, revealing a new N-terminal amine to which another amino acid may be attached. Thenthe peptide is assembled by the successive addition of protected amino acids. Once synthesis iscomplete, the desired peptide is cleaved from the resin. Usually, this cleavage step is performed with

acids of varying strength. The by-products can be removed by repetitive washings with appropriatesolvents.

Solid SupportPolystyrene is styrene cross-linked with 1–2% divinylbenzene. It is a popular carrier resin in SPPS.Other common gel-type supports include polyacrylamide and polyethylene glycol (PEG). Polystyrene ischemically inert under SPSS conditions. It is physically stable and permits the rapid filtration ofliquids, such as excess reagents. The solid support allows the introduction of a large variety ofanchoring groups. This causes the resin to swell in solvents suitable for SPPS. Highly cross-linked(50%) polystyrene offers increased mechanical stability, better filtration of reagents and solvents, andrapid reaction kinetics.

Protecting GroupsAmino acids have reactive moieties at the N-termini, C-termini, and side chains. One category ofprotecting group allows temporary protection of the α-amino group. Another type of protecting groupcan permanently protect groups by blocking the amino acids’ side-chain functionalities. Permanentprotecting groups have to withstand the repeated cleavages of the temporary protecting group. Theyare removed only during the cleavage from the carrier resin. Untimely removal of protecting groupscan cause the formation of undesirable by-products. Once purified, individual amino acids are reactedwith these protecting groups and then selectively removed during specific steps of peptide synthesis.Two protecting groups, t-Boc and Fmoc, are common in solid-phase peptide synthesis.

Fmoc/tBu and Boc/Bzl StrategiesThe Fmoc method offers a mild deprotection scheme. This method involves a base, usuallypiperidine(20–50%) in DMF in order to remove the Fmoc group and expose the α-amino group so thatit may react with an incoming activated amino acid. The advantage of Fmoc is that it is cleaved undervery mildly basic conditions, but it remains stable under acidic conditions. This allows the use of mildacid-labile protecting groups, such as Boc and benzyl groups, to be used on the side-chains of aminoacid residues of the target peptide. Fmoc is often preferred over Boc because of its ease of cleavage. TheBoc/Bzl-strategy requires anchoring groups, which tolerate repetitive TFA treatment. Usually, theinorganic acid HF is used for the final cleavage, which limits batch size and choice of reactor. TheFmoc/tBu-strategy is the most popular of theses. It can be automated far more conveniently than theBoc/Bzl-strategy and it can be scaled as needed.

Fmoc BocRoutine synthesis Requires special equipmentRelatively safe Potentially dangerousAcid-sensitive peptides andderivatives

Base-labile peptides

Frequent aggregation Moderate aggregationTFA final deprotection HF final deprotection

The following graphs show the Fmoc (base labile, Fig. 3) and the Boc (acid labile, Fig. 4) for the amineterminus of the amino acid.

Fig. 3: Fmoc Strategy (Wang resin)

Fig. 4: Boc Strategy (Merifield resin)

Solid phase synthesis consists of assembling amino acids from the C-terminal to the N-terminal. SPPSallows efficient removal of excess reagents and soluble byproducts after each reaction cycle because thepeptide remains anchored to an insoluble solid resin support. Resins commonly used are composed ofpolystyrene.

The controlled synthesis of peptides and formation of amide bonds requires the use of reversible ion ofthe amino group. Three common protection chemistries are: tert-Butoxycarbonyl (tBoc), 9-Fluorenylmethyloxycarbonyl (Fmoc) and N-Allyloxycarbonyl (Alloc).

These represent different protection and deprotection chemistries. It is also necessary to reversiblymask reactive side chain functional groups. After the anchoring of the first amino acid onto the resin,the Fmoc group is removed. To confirm that the Fmoc protecting groups are removed, a kaiser test isperformed. Then another Fmoc amino acid is attached by activation of its carboxyl group (Coupling).See examples of the coupling agents at Fig. 5.

Fig. 5: Examples of coupling agents.

A kaiser test is needed to confirm the complete coupling. The process is repeated through a cycle ofdeprotection, coupling and washing until the peptide is completely synthesized. Then the synthesizedpeptide is cleaved from the resin and side chain protection groups are removed. The synthetic peptidepurification is usually including the peptide precipitation from the cleavage reaction mixture by theaddition of diethyl ether. The peptide and resin mixture can be suspended in water or aqueous acidand filtered to remove the resin. Further purification can be by gel-filtration, ion exchangechromatography and reversed-phase HPLC.

Fig. 6: Polypeptide formation

Long Peptides (up to 120 Amino Acids)

Many types of research require peptides of 80–120 amino acids. These large molecules can besynthesized successfully using LifeTein’s PeptideSynTM strategy, which incorporates chemical ligationtechnology. The unprotected peptide chains react chemo-selectively in aqueous solution. The most

common form of chemical ligation involves a peptide thioester that reacts with a terminal cysteineresidue.

Most synthetic strategies may yield crude products that cannot be purified by standardchromatographic protocols. The PeptideSynTM technology allows selective coupling of unprotectedpeptide fragments. Subsequent purification involves only the removal of unreacted fragments. Thistechnology is routine at LifeTein in the synthesis of long peptides for research purposes. LifeTein hasalready succeeded in producing very long and complex peptides that could not be produced elsewhere.

Peptide PurificationSolid-phase peptide synthesis involves only a limited number of undesirable components—by-products. However, the identification and removal of these undesirable components can beproblematic. Acidolytic cleavage yields a crude product containing both the desired peptide andimpurities, such as deletion peptides, truncated peptides, incompletely deprotected peptides, modifiedpeptides, scavengers, and by-products derived from the cleaved protecting groups. All thesecontaminants have to be removed.

The impurities are very structurally similar to the desired peptide product. This necessitates high-performance methods such as UV peak detection and reversed phase high-performance liquidchromatography (RP-HPLC), which uses C18-modified silica as the stationary phase. The purificationof relatively small numbers of peptides requires a universal HPLC material. The process must be verychemically stable so that the same column can be for the acidic, basic, and neutral eluents. The poresmust be large enough to allow for good mass transfer characteristics for the largest of the syntheticpeptides but not so large so that the loadings are low for the smaller peptides. The particles should bespherical so that they can be packed using dynamic axial compression and should have a narrowparticle size distribution to promote permeability and packed bed stability.

The properties of an individual peptide depend on the composition and sequence of amino acids. Thetarget peptide and impurities are retained by the stationary phase depending on their hydrophobicity.Very polar contaminants are eluted at the beginning of the process, with aqueous 0.1% TFA. Then thepolarity of the eluent is gradually reduced by continuously increases of the proportion of acetonitrile.The elution of material is monitored at 220 nm. Fractions containing sufficiently pure target peptide,as determined by analytical HPLC, are pooled and lyophilized.

Quality Control of Peptides

Peptide PurityThe purity of the lyophilized target peptide is determined by analytical RP-HPLC followed by UVdetection at 220 nm. Amide bonds and other chromophores absorb light at 220nm, but water andresidual salts are not detectable under UV-spectrophotometry.

Normally, the peptide is delivered as in TFA salt form, which is the natural results of the RP-HPLCpurification. The side-chain functionalities of Arg, Lys, and His and the free N-terminus formtrifluoroacetates, and small amounts of TFA may adhere to the peptide. These contaminants cannot bedetected by analytical HPLC.

The resolution of analytical HPLC expressed as relative area because it corresponds to the area of themain peak over the total area of all peaks. The resolution of analytical HPLC can be improved byselecting an optimal buffer system, stationary phase, gradient, and column temperature. The expertiseof the analyst is of the utmost importance in the analysis of this type of data.

Net Peptide Content (NPC)Net peptide content is defined as the number of peptides relative to nonpeptide material, which usuallyconsists mostly of counterions and moisture. Hydrophilic peptides can absorb considerable amounts ofmoisture and, depending on the conditions of final purification and lyophilization, the NPC can varyfrom batch to batch. Low net peptide content could be expected for peptides containing large numbersof basic amino acids. NPC can be determined by amino acid analysis.

ImpuritiesPeptide impurities may include deletion sequences (peptides lacking at least one of the required aminoacids), incompletely deprotected sequences, truncated peptides, and by-products formed duringpeptide synthesis or cleavage.

Batch-to-batch VariabilityThe purity of a peptide can vary from batch to batch. When a peptide is ordered at 90% purity, thequality of the product can range between 90% and 99%. Lower-purity orders may have a wider range.Low-purity batches contain a considerable number of peptide by-products. These peptide by-productsmay show biological activity, but it will not necessarily match the activity of the target peptide. In somecases the impurities may interfere with the assay. The net peptide content in the low-purity peptidesvaries. It can be affected by the polarity of the amino acids, the lyophilization process, storageconditions, and other conditions.

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What is peptide: A Simple Introduction of Peptide Synthesis.

Peptides and proteins are linear polymers of amino acids linked by amide "peptide bonds" (Fig. 1). Thepeptide bond is formed by linking an amino group to a caroboxyl group on another amino acid. Aminoacids are primary amines that contain an alpha carbon that is connected to an amino (NH3) group, acarboxyl group (COOH), and a variable side group (R). Carboxylic acid and carboxylate groups arenormally not very reactive. It requires activating the carboxylic acid for the formation of the amidepeptide bond (Fig. 2).

Fig. 1: Peptide Structure

The classical approaches to peptides production are called liquid-phase peptide synthesis and solid-phase peptide synthesis (SPPS). These two methods can be combined in a process called nativechemical ligation. LifeTein’s standard peptide synthesis process involves the solid phase. The liquid-phase approach is used for the synthesis of short peptides, such as di- and tripeptides, and C-terminally modified peptides, such as enzyme substrates. The controlled peptide synthesis requiresselective protection and deprotection of the various functional groups: the amino group, the -carboxylgroup or the side chain functional groups. The side group gives each amino acid its distinctiveproperties and helps to dictate the folding of the protein.

Fig. 2: Peptide bonds created with peptide coupling agents

Principles of Solid-phase Peptide Synthesis

SPPS involves repeated cycles of coupling, washing, deprotection, and washing. A single cycle consistsof the following:

Cleavage of the alpha-amino protecting groupWashing to remove the cleavage reagentCoupling of the protected amino acidWashing to remove excess material

Unlike ribosome protein synthesis, artificial synthesis builds peptides in the C to N direction. Duringsolid-phase peptide synthesis, each peptide is anchored to an insoluble polymer at the C-terminus. Thefree N-terminal amine is coupled to a single N-protected amino acid unit. This unit is thendeprotected, revealing a new N-terminal amine to which another amino acid may be attached. Thenthe peptide is assembled by the successive addition of protected amino acids. Once synthesis iscomplete, the desired peptide is cleaved from the resin. Usually, this cleavage step is performed withacids of varying strength. The by-products can be removed by repetitive washings with appropriatesolvents.

Solid SupportPolystyrene is styrene cross-linked with 1–2% divinylbenzene. It is a popular carrier resin in SPPS.Other common gel-type supports include polyacrylamide and polyethylene glycol (PEG). Polystyrene ischemically inert under SPSS conditions. It is physically stable and permits the rapid filtration ofliquids, such as excess reagents. The solid support allows the introduction of a large variety ofanchoring groups. This causes the resin to swell in solvents suitable for SPPS. Highly cross-linked(50%) polystyrene offers increased mechanical stability, better filtration of reagents and solvents, and

rapid reaction kinetics.

Protecting GroupsAmino acids have reactive moieties at the N-termini, C-termini, and side chains. One category ofprotecting group allows temporary protection of the α-amino group. Another type of protecting groupcan permanently protect groups by blocking the amino acids’ side-chain functionalities. Permanentprotecting groups have to withstand the repeated cleavages of the temporary protecting group. Theyare removed only during the cleavage from the carrier resin. Untimely removal of protecting groupscan cause the formation of undesirable by-products. Once purified, individual amino acids are reactedwith these protecting groups and then selectively removed during specific steps of peptide synthesis.Two protecting groups, t-Boc and Fmoc, are common in solid-phase peptide synthesis.

Fmoc/tBu and Boc/Bzl StrategiesThe Fmoc method offers a mild deprotection scheme. This method involves a base, usuallypiperidine(20–50%) in DMF in order to remove the Fmoc group and expose the α-amino group so thatit may react with an incoming activated amino acid. The advantage of Fmoc is that it is cleaved undervery mildly basic conditions, but it remains stable under acidic conditions. This allows the use of mildacid-labile protecting groups, such as Boc and benzyl groups, to be used on the side-chains of aminoacid residues of the target peptide. Fmoc is often preferred over Boc because of its ease of cleavage. TheBoc/Bzl-strategy requires anchoring groups, which tolerate repetitive TFA treatment. Usually, theinorganic acid HF is used for the final cleavage, which limits batch size and choice of reactor. TheFmoc/tBu-strategy is the most popular of theses. It can be automated far more conveniently than theBoc/Bzl-strategy and it can be scaled as needed.

Fmoc BocRoutine synthesis Requires special equipmentRelatively safe Potentially dangerousAcid-sensitive peptides andderivatives

Base-labile peptides

Frequent aggregation Moderate aggregationTFA final deprotection HF final deprotection

The following graphs show the Fmoc (base labile, Fig. 3) and the Boc (acid labile, Fig. 4) for the amineterminus of the amino acid.

Fig. 3: Fmoc Strategy (Wang resin)

Fig. 4: Boc Strategy (Merifield resin)

Solid phase synthesis consists of assembling amino acids from the C-terminal to the N-terminal. SPPSallows efficient removal of excess reagents and soluble byproducts after each reaction cycle because the

peptide remains anchored to an insoluble solid resin support. Resins commonly used are composed ofpolystyrene.

The controlled synthesis of peptides and formation of amide bonds requires the use of reversible ion ofthe amino group. Three common protection chemistries are: tert-Butoxycarbonyl (tBoc), 9-Fluorenylmethyloxycarbonyl (Fmoc) and N-Allyloxycarbonyl (Alloc).

These represent different protection and deprotection chemistries. It is also necessary to reversiblymask reactive side chain functional groups. After the anchoring of the first amino acid onto the resin,the Fmoc group is removed. To confirm that the Fmoc protecting groups are removed, a kaiser test isperformed. Then another Fmoc amino acid is attached by activation of its carboxyl group (Coupling).See examples of the coupling agents at Fig. 5.

Fig. 5: Examples of coupling agents.

A kaiser test is needed to confirm the complete coupling. The process is repeated through a cycle ofdeprotection, coupling and washing until the peptide is completely synthesized. Then the synthesizedpeptide is cleaved from the resin and side chain protection groups are removed. The synthetic peptidepurification is usually including the peptide precipitation from the cleavage reaction mixture by theaddition of diethyl ether. The peptide and resin mixture can be suspended in water or aqueous acidand filtered to remove the resin. Further purification can be by gel-filtration, ion exchangechromatography and reversed-phase HPLC.

Fig. 6: Polypeptide formation

Long Peptides (up to 120 Amino Acids)

Many types of research require peptides of 80–120 amino acids. These large molecules can besynthesized successfully using LifeTein’s PeptideSynTM strategy, which incorporates chemical ligationtechnology. The unprotected peptide chains react chemo-selectively in aqueous solution. The mostcommon form of chemical ligation involves a peptide thioester that reacts with a terminal cysteineresidue.

Most synthetic strategies may yield crude products that cannot be purified by standardchromatographic protocols. The PeptideSynTM technology allows selective coupling of unprotectedpeptide fragments. Subsequent purification involves only the removal of unreacted fragments. Thistechnology is routine at LifeTein in the synthesis of long peptides for research purposes. LifeTein hasalready succeeded in producing very long and complex peptides that could not be produced elsewhere.

Peptide PurificationSolid-phase peptide synthesis involves only a limited number of undesirable components—by-

products. However, the identification and removal of these undesirable components can beproblematic. Acidolytic cleavage yields a crude product containing both the desired peptide andimpurities, such as deletion peptides, truncated peptides, incompletely deprotected peptides, modifiedpeptides, scavengers, and by-products derived from the cleaved protecting groups. All thesecontaminants have to be removed.

The impurities are very structurally similar to the desired peptide product. This necessitates high-performance methods such as UV peak detection and reversed phase high-performance liquidchromatography (RP-HPLC), which uses C18-modified silica as the stationary phase. The purificationof relatively small numbers of peptides requires a universal HPLC material. The process must be verychemically stable so that the same column can be for the acidic, basic, and neutral eluents. The poresmust be large enough to allow for good mass transfer characteristics for the largest of the syntheticpeptides but not so large so that the loadings are low for the smaller peptides. The particles should bespherical so that they can be packed using dynamic axial compression and should have a narrowparticle size distribution to promote permeability and packed bed stability.

The properties of an individual peptide depend on the composition and sequence of amino acids. Thetarget peptide and impurities are retained by the stationary phase depending on their hydrophobicity.Very polar contaminants are eluted at the beginning of the process, with aqueous 0.1% TFA. Then thepolarity of the eluent is gradually reduced by continuously increases of the proportion of acetonitrile.The elution of material is monitored at 220 nm. Fractions containing sufficiently pure target peptide,as determined by analytical HPLC, are pooled and lyophilized.

Quality Control of Peptides

Peptide PurityThe purity of the lyophilized target peptide is determined by analytical RP-HPLC followed by UVdetection at 220 nm. Amide bonds and other chromophores absorb light at 220nm, but water andresidual salts are not detectable under UV-spectrophotometry.

Normally, the peptide is delivered as in TFA salt form, which is the natural results of the RP-HPLCpurification. The side-chain functionalities of Arg, Lys, and His and the free N-terminus formtrifluoroacetates, and small amounts of TFA may adhere to the peptide. These contaminants cannot bedetected by analytical HPLC.

The resolution of analytical HPLC expressed as relative area because it corresponds to the area of themain peak over the total area of all peaks. The resolution of analytical HPLC can be improved byselecting an optimal buffer system, stationary phase, gradient, and column temperature. The expertiseof the analyst is of the utmost importance in the analysis of this type of data.

Net Peptide Content (NPC)

Net peptide content is defined as the number of peptides relative to nonpeptide material, which usuallyconsists mostly of counterions and moisture. Hydrophilic peptides can absorb considerable amounts ofmoisture and, depending on the conditions of final purification and lyophilization, the NPC can varyfrom batch to batch. Low net peptide content could be expected for peptides containing large numbersof basic amino acids. NPC can be determined by amino acid analysis.

ImpuritiesPeptide impurities may include deletion sequences (peptides lacking at least one of the required aminoacids), incompletely deprotected sequences, truncated peptides, and by-products formed duringpeptide synthesis or cleavage.

Batch-to-batch VariabilityThe purity of a peptide can vary from batch to batch. When a peptide is ordered at 90% purity, thequality of the product can range between 90% and 99%. Lower-purity orders may have a wider range.Low-purity batches contain a considerable number of peptide by-products. These peptide by-productsmay show biological activity, but it will not necessarily match the activity of the target peptide. In somecases the impurities may interfere with the assay. The net peptide content in the low-purity peptidesvaries. It can be affected by the polarity of the amino acids, the lyophilization process, storageconditions, and other conditions.