4.6. Synthesis of Peptides

Many common peptide chains are generated on industrial scale using microorganisms (e.g. insulin). However, many other peptides are synthesized using more traditional methods for use in research or pharmaceutical production. Most of the common reactions or sequences of reactions used to generate small peptides are covered in introductory organic chemistry. While these reactions can work well for very short peptides such as dimers or trimers they become very time consuming and poor-yielding when longer chains are required. An alternative method widely used in industry forms the basis for new approaches.

4.6.1. (Very) Brief Refresher of the Basics

The primary methods of synthesizing amides involve so-called addition-elimination reactions (see Chapter 13, Sections 1-3). Most addition-elimination reactions follow a simple two-step mechanism (Scheme 4.17). The nucleophile attacks the electrophile (carbonyl). This forms a new bond and adds a lone pair to the oxygen. Then that lone pair comes back and reforms the π bond with the carbon, ejecting the leaving group. Depending on the specific reaction that is occurring there may be additional steps before or after these. For example, the nucleophile, electrophile, and/or leaving group may be activated first, or there may be a protonation or deprotonation afterwards.

Scheme 4.17 – Generalized Reaction Mechanism for Addition-Elimination Reactions.

Different carbonyl-containing functional groups have different electrophilicities and can eject different leaving groups of varying leaving-group ability. As a result, not all interconversions are possible. Of those that are, some require catalysis or special reagents, and some follow unusual and lengthy mechanisms.

Because this chapter focuses on peptide chains the schemes and discussion will depict nitrogen-containing nucleophiles from amino acids. However, it is important to remember that these reactions can involve other kinds of nitrogenous nucleophiles. See (see Chapter 13) for additional details and expanded discussion on addition-elimination reactions.

4.6.1.1. Making Amides from Esters

It is technically possible to synthesize an amide by combining an amine and an ester directly (Scheme 4.18).

Scheme 4.18 – Generalized Reaction Equation for Addition-Elimination Converting Esters to Amides for Dipeptide Synthesis.

These reactions follow the typical mechanism for addition-elimination (Scheme 4.17). However, because amines are more basic than nucleophilic and esters are only moderately electrophilic, these reactions are very slow and require extreme conditions; typically the reaction takes several days or weeks at boiling or supercritical temperatures to generate a moderate amount of product. When the reactants are amino acid-derived the product ALSO possesses an ester. As a result, the product may undergo additional reactions and form undesired side-products, further lowering the yield. Consequently, these reactions are very uncommon and most chemists instead use an alternative approach (see Section 4.6.1.3).

4.6.1.2. Making Amides from Acid Halides/Anhydrides

It is possible to synthesize an amide by combining an amine and an acid halide or anhydride directly (Scheme 4.19). This approach requires that the carboxylic acid of the nucleophile be converted to another functional group, typically an ester. It also requires that the amine of the electrophile be modified in some way (see Section 4.6.2 and subsections therein).

Scheme 4.19 – Generalized Reaction Equations for Addition-Elimination Converting Acid Halides or Anhydrides to Amides for Dipeptide Synthesis.

The side product of either reaction is an acid (HX or a carboxylic acid). Because amines are also basic this has an unintended consequence: for every molecule of amine that undergoes the addition-elimination, another molecule of the amine is consumed in an acid-base reaction (Scheme 4.20). As a result, the yield may be significantly reduced.

Scheme 4.20 – Example of Synthesis of a Dipeptide from an Acid Halide with only One Equivalent of Amine Nucleophile.

There are two common approaches to improving this (Scheme 4.21). Two equivalents of the amino acid acting as the nucleophile can be added to the reaction. Alternatively, another non-nucleophilic base can be added to the reaction. The non-nucleophilic base used cannot be a hydride such as sodium hydride (NaH). Hydrides can undergo other side reactions with carbonyl-containing functional groups. Instead, a sterically hindered/bulky tertiary amine is typically used. These amines have enough steric interactions that they cannot easily act as nucleophiles but remain bases. By far the most common amine for this purpose is diisopropylethylamine (DIPEA).

Scheme 4.21 – Generalized Reaction Equations for Addition-Elimination Converting Acid Halides to Amides Using Two Equivalents of Amine or A Second, Non-Nucleophilic, Base.

Regardless of the approach, using either an acid halide or anhydride as the electrophile has limitations for peptide synthesis. In addition to the modification(s) required to the other functional groups there are issues with the ways that these compounds may be synthesized; in general making a suitable acid halide or anhydride electrophile from an amino acid requires multiple steps, requires dangerous reagents, and/or affords the reactant in low yield.

4.6.1.3. Making Amides Using Dicyclohexylcarbodiimide (DCC)

It is possible to synthesize an amide by combining an amine and a carboxylic acid using one of several special reagents. The most common reagent used for these reactions is dicyclohexylcarbodiimide (DCC; Scheme 4.22). This approach to making short peptide chains is, by a very wide margin, the most commonly used.

Scheme 4.22 – Generalized Reaction Equation for Addition-Elimination Converting Esters to Amides for Dipeptide Synthesis.

The mechanism for this reaction is significantly more complex than unusual (Scheme 4.23). The unusual nature of DCC allows several things to occur at the same time in the first step: the electrons in the H-O σ bond become a new O-C π bond, the electrons in the other C-O π bond become a new O-C σ bond, and the electrons of the C-N π bond become a new N-H σ bond. It is often useful to re-orient this intermediate to better understand the next step; the intermediate formed is structurally similar to an anhydride, which makes it significantly more electrophilic than a carboxylic acid. Then the amine (nucleophile) attacks the activated carbonyl (electrophile). This intermediate is again able to have several things occur at the same time: the lone pair comes back and reforms the π bond with the carbon, the electrons in the other C-O σ bond become a new O-C π bond, the electrons in the C-N π bond become a new N-H σ bond, and the electrons of the H-C σ bond become a lone pair on the nitrogen. This single step is equivalent to an elimination occurring at the same time as the deprotonation that normally follows. This generates the final product and a urea.

Scheme 4.23 – Reaction Mechanism for Combining Two Glycine Molecules Using Dicyclohexylcarbodiimide (DCC).

The first and final steps are advanced and do not follow the typical patterns (acid-base, nucleophile-electrophile, leaving group, etc). These are again rearrangement steps. While it is possible to use amino acids directly (as shown), the product also contains both an amine and a carboxylic acid. As a result, modifications are normally required to avoid side-products and/or to address other issues (see Section 4.6.2).

The cyclohexyl groups of DCC do not participate in the mechanism. The most common alternatives to DCC simply change those groups (Figure 4.53). The primary reason for these alternatives is to adjust solubility; some carboxylic acids and/or amines require the use of specific solvents in which DCC may not be soluble. At an introductory level learning/memorizing the specific conditions where these may be required is not necessary. It is important only to understand that there are alternatives, why there are alternatives, and that they typically follow the same mechanism.

Figure 4.53 – Examples of Carbodiimides Used for Synthesis of Amides from Carboxylic Acids and Amines.

There are other reagents that perform the same function but have different structures and follow other mechanisms. The specific details of these vary. They are mentioned only to avoid confusion if encountered in other sources.

4.6.2. Chemoselectivity in Amide Formation Using Protecting Groups

With each of the common methods of amide formation there is an issue when coupling two amino acids (or compounds made from amino acids): chemoselectivity. Consider the combination of two different amino acids using DCC (Scheme 4.24). DCC combines an amine and a carboxylic acid to form an amide. However, when the reactants are amino acids there are two amines and two carboxylic acids in the mixture. A mixture of products is obtained. Each of the products ALSO has an amine and a carboxylic acid and may also undergo the reaction again, making the problem exponentially more complex. Similar problems exist for the other approaches to the formation of amides.

Scheme 4.24 – Example of Multiple Amide Formation Reactions and Products Possible when Combining Two Different Amino Acids Using Dicyclohexylcarbodiimide (DCC).

Similar problems occur using the other methods to generate amides.

The general approach to address this type of chemoselectivity issue is the use of “protecting groups”. A protecting group is a group temporarily added to a functional group to change it into a different functional group that has different chemical reactivities. This “protects” the group from being used in an undesired reaction.

Consider the same combination of two different amino acids using DCC but with additional modifications beforehand (Scheme 4.25). DCC combines an amine and a carboxylic acid to form an amide. If the amine and carboxylic acid that are not meant to be reacted are first changed into other functional groups then they cannot be used to form the amide. Only one product is obtained. The product also DOES NOT have an amine nor a carboxylic acid and will not undergo the reaction again, making the problem drastically simpler.

Scheme 4.25 – Example of Using Protecting Groups to Achieve Chemoselectivity when Combining Two Different Amino Acids Using Dicyclohexylcarbodiimide (DCC).

Technically, in order to obtain the desired product there are now additional steps (before and after the problem step) but if these are chosen well the overall yield will go up significantly.

4.6.2.1. Properties of Good Protecting Groups

Protecting groups are a vital tool for many organic chemists. There are hundreds of different protecting groups, with many having multiple possible reaction conditions for adding and removing them. Most institutions have one or more entire courses dedicated to understanding advanced synthetic strategies using protecting groups. At an introductory level only a simplified overview of the basics is discussed.

There are many (highly specific) considerations for choosing and using protecting groups. However, three broad factors are generally important.

In general, a good protecting group is easy to add and remove. For example, a protecting group that would require an extra eight steps to add and remove would not normally be considered an appropriate choice. “Easy” is relative; there are many widely used protecting groups that require very specific (and sometimes challenging) conditions and techniques to be added and/or removed. In general, if the extra time and effort is outweighed by the overall improvement in yield then the protecting group is acceptable.

The previous consideration must be balanced with another: in general, a good protecting group is stable to the conditions of the steps it needs to be kept on for. Being “stable” in this context means that it will not react; the new functional group made by adding the protecting group will not be destroyed and/or revert back into the original one under conditions it is stable to. Although it is beneficial for the protecting group to be easily removed it would not be helpful for the protecting group to come off before or during the step it is needed for. For example, using a protecting group that can be removed by acid would not be an appropriate choice if the conditions used in the step requiring protection had an acid.

Finally, in general a good protecting group is selective for the functional group being targeted. For example, if the hypothetical protecting group used to protect the amine above (Scheme 4.25) also protected the carboxylic acid then it would not be appropriate because the protected amino acid would not be able to form the amide. There are exceptions to this but in general protecting groups should only modify the functional group they are intended for.

Although there are many possible protecting groups for amino acids there are four that are very commonly used. The variety of protecting groups and conditions for their addition or removal is required for the synthesis of long chain peptides (see Section 4.6.4.1).

4.6.2.2. The tert-Butyloxycarbonyl (BOC) Protecting Group

The tert-butyloxycarbonyl (BOC) protecting group is a common choice for protecting amines of amino acids (Scheme 4.26). This converts the functional group from an amine (moderate nucleophile, moderate base) to a carbamate (very poor nucleophile, poor base). While it is not necessary to do so, many sources use the abbreviation in place of drawing the protecting group in schemes/figures.

Scheme 4.26 – Generalized Reaction Equations for Adding and Removing the tert-Butyloxycarbonyl (BOC) Protecting Group to an Amino Acid.

BOC protecting groups can be added several different ways. By far the most common approach is to add it using di-tert-butyl dicarbonate (sometimes called “BOC anhydride”, written as BOC₂O; Scheme 4.27). A non-nucleophilic base is often added when protecting amino acids to ensure that the amine, rather than the ammonium, is in solution. The mechanism for adding this protecting group can be approximated by ignoring the carboxylic acid. The approximation is then identical to the mechanism for formation of an amide using an anhydride. This is only an approximation, in reality there are several additional acid-base steps.

Scheme 4.27 – Simplified Reaction Mechanism for Addition of the tert-Butyloxycarbonyl (BOC) Protecting Group to Glycine Using Di-tert-butyl dicarbonate (BOC₂O) and Triethylamine.

BOC protecting groups are stable to highly basic conditions but unstable in acidic conditions. They can be removed several different ways. The most common approach is to remove it using a strong acid (Scheme 4.28). It is possible to use acids such as sulfuric acid (H₂SO₄), phosphoric acid (H₃PO₄), or nitric acid (HNO₃) but it is much more common to use hydrochloric acid (HCl) or trifluoroacetic acid (CF₃CO₂H, sometimes abbreviated as TFA). Water inhibits removal of the protecting group. However, it is possible to generate hydrochloric acid and trifluoroacetic acid without any water in it. The mechanism for removing this protecting group is an acid-catalyzed elimination followed by decarboxylation, NOT hydrolysis.

Scheme 4.28 – Reaction Mechanism for Removal of the tert-Butyloxycarbonyl (BOC) Protecting Group from N-BOC-Glycine Using Anhydrous Hydrochloric Acid.

4.6.2.3. The Fluorenylmethyloxycarbonyl (Fmoc) Protecting Group

The fluorenylmethyloxycarbonyl (Fmoc) protecting group is another common choice for protecting amines of amino acids (Scheme 4.29). This also converts the functional group from an amine (moderate nucleophile, moderate base) to a carbamate (very poor nucleophile, poor base). While it is not necessary to do so, many sources use the abbreviation in place of drawing the protecting group in schemes/figures.

Scheme 4.29 – Generalized Reaction Equations for Adding and Removing the Fluorenylmethyloxycarbonyl (Fmoc) Protecting Group to an Amino Acid.

Fmoc protecting groups can be added several different ways. By far the most common approach is to add it using fluorenylmethyloxybaronyl chloride (written as FmocCl; Scheme 4.30). A non-nucleophilic base is often added when protecting amino acids to ensure that the amine, rather than the ammonium, is in solution. The base is almost always a tertiary amine, typically with more than one equivalent. This type of base avoids issues with basic conditions (see below). The mechanism for adding this protecting group can be approximated by ignoring the carboxylic acid. The approximation is then identical to the mechanism for formation of an amide using an acid halide. This is only an approximation, in reality there are several additional acid-base steps.

Scheme 4.30 – Simplified Reaction Mechanism for Addition of the Fluorenylmethyloxycarbonyl (Fmoc) Protecting Group to Glycine Using Fluorenylmethyloxycarbonyl chloride (FmocCl) and Triethylamine.

Fmoc protecting groups are stable to highly acidic conditions but unstable in basic conditions. This is the opposite stability pattern as BOC protecting groups. They can be removed several different ways. The most common approach is to remove it using a moderate base (Scheme 4.31). It is possible to use several different kinds of bases but it is very common to use piperidine or other simple secondary amines. These are basic enough to remove the group but not so basic as to cause side reactions to occur. Strong bases such as sodium hydroxide (NaOH) cause other reactions to occur and are not suitable for removal of Fmoc. The mechanism for removing this protecting group is a base-catalyzed elimination followed by decarboxylation, NOT hydrolysis.

Scheme 4.31 – Reaction Mechanism for Removal of the Fluorenylmethyloxycarbonyl (Fmoc) Protecting Group from N-Fmoc-Glycine Using Piperidine.

4.6.2.4. The Benzyloxycarbonyl (Cbz) Protecting Group

The benzyloxycarbonyl (Cbz) protecting group is another common choice for protecting amines of amino acids (Scheme 4.32). This also converts the functional group from an amine (moderate nucleophile, moderate base) to a carbamate (very poor nucleophile, poor base). While it is not necessary to do so, many sources use the abbreviation in place of drawing the protecting group in schemes/figures.

Scheme 4.32 – Generalized Reaction Equations for Adding and Removing the Benzyloxycarbonyl (Cbz) Protecting Group to an Amino Acid.

Cbz protecting groups can be added several different ways. By far the most common approach is to add it using benzyloxycarbonyl chloride (written as CbzCl). The conditions, considerations, and mechanism for adding this group are identical to those of the Fmoc protecting group (Scheme 4.33).

Scheme 4.33 – Simplified Reaction Mechanism for Addition of the Benzyloxycarbonyl (Cbz) Protecting Group to Glycine Using Benzyloxycarbonyl chloride (CbzCl) and Triethylamine.

Cbz protecting groups are stable to highly basic conditions but unstable in acidic conditions. This is the same stability pattern as BOC protecting groups. They can be removed several different ways. It is possible to remove the group using acid as with BOC (see Section 4.6.2.2). However, the most common approach is to remove it using transition metal catalyzed hydrogenation. The mechanism for this hydrogenation involves several advanced/unusual steps that lie outside the scope of this text; the mechanism is NOT the same as that for transition metal catalyzed hydrogenation of an alkene (e.g. Scheme 4.14).

4.6.2.5. Esters as “Protecting Groups”

The formation of an ester is a common choice for protecting carboxylic acids of amino acids. Many different esters may be used, some with special properties and/or methods for adding/removing them. Two simple esters are very commonly used: methyl (Me; CH₃) and benzyl (Bn; CH₂Ph) esters (Scheme 4.34). This converts the functional group from a carboxylic acid (weak nucleophile/base, moderate acid) to an ester (weak nucleophile/base, not an acid). More importantly, because the mechanism for amide formation when using DCC requires a carboxylic acid (see Scheme 4.23) changing the functional group to an ester blocks the carbonyl from becoming an amide in these reactions.

Scheme 4.34 – Generalized Reaction Equations for Adding and Removing Methyl (Me) and Benzyl (Bn) Ester Protecting Groups to an Amino Acid.

Ester protecting groups can be added several different ways. By far the most common approach is to add them using acid-catalyzed Fischer esterification (see Interconverting Carboxylic Acids and Esters; Scheme 4.35). It is possible to use acids such as sulfuric acid (H₂SO₄), phosphoric acid (H₃PO₄), or nitric acid (HNO₃) but it is much more common to use hydrochloric acid (HCl). Water inhibits formation of the ester by affecting equilibrium. However, it is possible to generate hydrochloric acid without any water in it (sometimes referred to as anhydrous hydrochloric acid). The alcohol that will form the ester is used as the solvent. Because an acid is being used the product may be formed as the ammonium salt rather than the amine directly. Careful choice of conditions can inhibit this.

Scheme 4.35 – Generalized Reaction Mechanism for Formation of the Ester “Protecting Group” with Glycine Using Hydrochloric Acid.

Ester protecting groups are stable to moderately acidic and basic conditions but unstable in strongly acidic and basic conditions. They can be removed several different ways. One common approach is acid catalyzed hydrolysis (Scheme 4.36). This may be used for almost all esters. The mechanism is identical to Fischer esterification but with the roles of water and alcohol reversed.

Scheme 4.36 – Generalized Reaction Mechanism for Removal of the Ester “Protecting Group” with Glycine Using Hydrochloric Acid (Hydrolysis).

Another common approach is base catalyzed hydrolysis (saponification; Scheme 4.37). This may be used for almost all esters. This is generally the preferred method because strong acids may react with any amides (i.e. if a peptide has already been formed) while strong bases will not.

Scheme 4.37 – Generalized Reaction Mechanism for Removal of the Ester “Protecting Group” with Glycine Using Sodium Hydroxide (Saponification).

There are other specialized ways of removing certain esters. For example, it is possible to remove the benzyl group using hydrolysis or saponification. However, the most common approach is to remove it using transition metal catalyzed hydrogenation. This uses the same approach and proceeds through the same (complicated) mechanism as the removal of the Cbz protecting group.

4.6.3. Solution-Phase Synthesis of Dipeptides

Consider a target dipeptide made from two different amino acids such as Val-Phe (Figure 4.54).

Figure 4.54 – L-Valyl-L-phenylalanine.

Both of these amino acids are “common”. They are available on industrial scale and are relatively inexpensive. It is technically possible to synthesize this dipeptide by directly combining the two amino acids in a large-scale amide formation reaction and then separating the desired dipeptide from all of the other peptide products (Scheme 4.38). In addition to being wasteful, in practice this is extremely difficult, time consuming, and low yielding. Because the dipeptide products also have amines and carboxylic acids many compounds are formed (yield issues) and they often have similar physical properties (purification issues).

Scheme 4.38 – Generalized Amide Formation Reaction Combining L-Valine and L-Phenylalanine with Examples of Peptide Products.

It is possible to chemoselectively combine two amino acids to form a dimer peptide using traditional approaches. There are several ways to do this. The most common approach is to use protecting groups and a DCC (or similar) amide formation reaction.

First, in order to synthesize only this dipeptide and none of the other options, protecting groups are added (Scheme 4.39). The carbonyl of the desired amide comes from valine. The nitrogen of the desired amide comes from phenylalanine. To avoid any amide(s) being formed from the amine of valine it is protected. To avoid any amide(s) being formed from the carbonyl of phenylalanine its carboxylic acid is protected. Because this peptide is simple there are no special considerations and any protecting groups may be used.

Scheme 4.39 – Example of Concept and Possible Addition of Protecting Groups for Chemoselective Synthesis of L-Valyl-L-phenylalanine.

Second, the amide formation occurs (Scheme 4.40). Technically there are many options for this step, but combining the two directly using a reagent like DCC is usually the simplest option. Because the amine of valine and the carboxylic acid of phenylalanine are protected they cannot become amides and the reaction only produces one dimer peptide product.

Scheme 4.40 – Example of Possible Amide Formation Step for Chemoselective Synthesis of L-Valyl-L-phenylalanine.

Finally, the protecting groups are removed (Scheme 4.41). The specific reactions needed to remove them will vary depending on which protecting groups were used. In this example two different reactions are needed.

Scheme 4.41 – Example of Possible Removal of Protecting Groups for Chemoselective Synthesis of L-Valyl-L-phenylalanine.

The peptide synthesis takes multiple steps but the overall yield is improved (Scheme 4.42).

Scheme 4.42 – Summary and Contrast of Direct Amide Formation and a Possible Protecting Group Route for Chemoselective Synthesis of L-Valyl-L-phenylalanine.

The example above is intentionally simple to highlight the general approach. If the dimer were different, with special considerations (or made from expensive synthetic amino acids) then more care may be needed. For example, if one of the amino acids were synthetic and had a functional group sensitive to acidic and/or basic conditions different protecting groups may be selected (Scheme 4.43).

Scheme 4.43 – Example of Changes to Protecting Group Route for Chemoselective Synthesis of a Dipeptide with a Sensitive Synthetic Amino Acid.

Although the overall yield of the peptide increases the physical amount of time and effort needed to make it also increases. This presents a problem: most peptide chains needed for research or pharmaceutical applications are longer than dimers. It is technically possible to make these compounds manually but the process is very labour-intensive and time consuming (Scheme 4.44). This becomes far more challenging (and lengthy) when the peptide chain involves amino acids that have sensitive side chains that may also require protection and/or side chains that are also amines/carboxylic acids (see Section 4.6.4.1).

Scheme 4.44 – Example of Conceptual Approach for Solution Phase Synthesis of a Short Chain Peptide with Highlighted Protecting Group Concerns.

In general, when peptide chains longer than di- or tri-mers are needed a different method is used.

4.6.4. Solid-Phase Synthesis of Peptides

Moderately sized peptide chains of up to a few hundred amino acids are often required for research or pharmaceutical purposes. Synthesizing these requires dozens or hundreds of chemical reactions performed in sequence. Because they are frequently needed and tedious to prepare an alternative system for peptide synthesis was developed. This method can be done manually but is very often automated instead.

The main goal of the alternative approach is to make the steps much faster.

The speed of setting up the reactions is improved by making all of the reagents standardized and commercially available. Protected versions of all of the amino acids, where the protecting groups have already been carefully chosen and added to the amino acid, are synthesized on industrial scale and purchased by chemists. The speed of setup is often also improved by automation.

In principle the speed of the chemical reactions themselves can be improved. In practice, most methods to accomplish this result in other issues such as side reactions. The actual speed of the chemical reactions is not normally improved.

The speed of working up (purifying) the product after each step is improved by physically attaching the peptide chain to an object. There are a large variety of items made for this purpose. The most commonly used are beads made from synthetic polymers (e.g. styrene) that have useful functional groups on the surface. These objects are what gives the alternate system its name: Solid-Phase Synthesis.

Technically, some methods and machines are proprietary and use different protecting groups and/or solid supports. The specific details may vary slightly but the overall approach is always the same.

First, the C-terminus of the target peptide chain is covalently attached to the solid support. There are several different ways to do this, but a common approach is to form an ester (Scheme 4.45). Although not a traditional method, because the system already has a supply of DCC this is often used to form the ester. The mechanism is identical to amide formation (see Scheme 4.23) but with an alcohol instead of an amine. For convenience a single (small) bead with a single polymer chain is shown. In reality the beads are relatively large and their surface is covered in many peptide chains.

Scheme 4.45 – Generalized First Step of Solid Phase Synthesis of a Target Peptide Chain: Attachment to Solid Support.

Attaching the amino acids this way means that the chain will be added to “backwards” (start with the last amino acid, then add the second-last, then the third-last, etc.). This has another benefit: ester protecting groups for the amino acids (ignoring the side chains) are not needed (see below). This further improves the speed of synthesis.

Second, the peptide is added to (Scheme 4.46). The amine’s protecting group is removed and a new amide is formed. The bead is washed in between each step with solvent to remove any unreacted materials and side products. These steps are repeated as each amino acid in the chain is added to the peptide.

Scheme 4.46 – Generalized Second Step of Solid Phase Synthesis of a Target Peptide Chain: Chain Growth.

Finally, the peptide chain is detached from the solid support. There are several different ways to do this. Most systems use a strong acid in water (hydrolysis; Scheme 4.47).

Scheme 4.47 – Generalized Third Step of Solid Phase Synthesis of a Target Peptide Chain: Detachment from Solid Support.

No prior step in the peptide synthesis uses this set of conditions. As a result, an added benefit is sometimes possible. In some systems the side chains of the amino acids are protected with protecting groups that are also removed by these conditions. In a single step the covalent bead-peptide attachment is broken and all of the protecting groups of the amino acids’ side chains are removed (Scheme 4.48). This is not always true; some systems have separate dedicated steps to remove the protecting groups. The examples are chosen to highlight that protecting group variability goes far beyond the scope covered in this text. The concept (planning ahead to be able to accomplish multiple things in one step) is important, not the exact structures of the protecting groups.

Scheme 4.48 – Example of Third Step of Solid Phase Synthesis of a Target Peptide Chain with Concurrent Removal of Side Chain Protecting Groups.

4.6.4.1. Why Do We Need Multiple Types of Protecting Groups?

Solid phase peptide synthesis demonstrates another important concept for the use of protecting groups. Consider the same peptide synthesis (as Scheme 4.48) but where the amine of lysine is simply protected with a tert-butyloxycarbonyl (BOC) protecting group as well (Scheme 4.49). Because both amines of the amino acid have BOC protecting groups both groups have their protection removed during the peptide synthesis (Step 2). With two available amines the rest of the synthesis produces (MANY) multiple products and chemoselectivity is reintroduced as an issue.

Scheme 4.49 – Example of Potential Problem from Accidental Removal of a Protecting Group During Solid Phase Synthesis.

In order to avoid this problem the side chain’s amine needs a protecting group that is not removed under the same conditions as the BOC of the other amine. Different protecting groups that are removed under different conditions are sometimes referred to as orthogonal protecting groups.

In solid phase peptide synthesis the side chains of the amino acids are purposefully protected by an orthogonal protecting group to the protecting group placed on the “main” amine (Figure 4.55). Many combinations are possible. For example, with lysine two of the common amine protecting groups are orthogonal to each other: tert-butyloxycarbonyl (BOC; removed by acid, stable to base) and fluorenylmethyloxycarbonyl (Fmoc; stable to acid, removed by base). Synthesizing compounds like this, where the two similar functional groups are chemoselectively protected with different groups, is challenging.

Figure 4.55 – Example of Two Orthogonal Protecting Groups for the Amines of L-Lysine.

These two factors are why there is a very large array of possible protecting groups and one of the reasons that efficiently using protecting groups is such an advanced topic.