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Solid-phase peptide synthesis was invented by Merrifield. This was a revolution in polypeptide synthesis, and is now the accepted method for creating peptides in the lab.

The reactions that have been covered in class up to this point have all occurred in a solution, which is fine if you only want to make simple di- and tripeptides. More complex peptides are not able to be synthesized in this manner. All of the polypeptides are floating in solution, which means they only encounter each other in a stochastic (or random collision) fashion, resulting in the majority of the solution being byproducts that we don't want. A tedious purification must be done before each addition of a new peptide, and yield of the reaction drops drastically.  Solid phase peptide synthesis is the solution to these problems.

Solid Phase Beads

Solid phase peptide synthesis consists of two phases: one reactant is the C-terminus of the starting amino acid covalently bonded to an insoluble resin bead, while the other reactant is in solution.  The physical properties of the bead, and its applications, vary with the material from which the bead is constructed, the amount of crosslinking, as well as the linker and handle being used.  Solid phase sythnesis builds polypeptide chains in the C to N direction to maintain stereochemistry.


  • Polystyrene is a versatile resin and it is quite useful in multi-well, automated peptide synthesis, due to its minimal swelling in dicholoromethane.
  • Polyamide resin is also a useful and versatile resin. It seems to swell much more than polystyrene, in which case it may not be suitable for some automated synthesizers if the wells are too small.
  • PEG hybride polystyrene resin is when the base resin is polystyrene onto which there are attached long chains of polyoxyethylene (PEG). Synthesis is caried out on the distal end of the PEG spacer making it suited for long and difficult peptides. It does not expand much during synthesis, making it a preferred resin for robotic peptide synthesis.

In class, we discussed beads made of polystyrene, with a diameter about 100 μm, large enough to be filtered using filter paper.  The amino acid chain product is attached to the solid bead, which becomes separated from all of the by-products and excess reactants that flow through in solution.

These beads work well, because they have a very strong backbone that allows repeated use and contain built-in reactive sites for the amino acids. Cross-links are what give the backbone its strength and keep it from unwinding.  Cross-linked means that aromatic rings of the structure are attached to 2 strands next to each other within the structure. The reactive sites within the beads are a chlorine reactive group that is attached to 1% of the phenyl rings within the beads. There are about 100 micromils of reactive sites per bead, and each of these reactive sites can have a polypeptide chain; therefore, you can have up to 100 micromils of growing polypeptide per bead. The basic structure of the resin bead is shown below:

Download the chemdraw file above

Pros and Cons of using the beads


  • Only one final purification needed, which is an easy filtration
  • Excess reagents can be used to drive the reaction forward
  • It can be automated, saving time


  • It's hard to monitor reactions because TLC will not work (product is not in solution)
  • Poor kinetics
    • Only one reactant can move, have to wait for solution to react with the bead
    • The amino acids have to diffuse into the bead where most of the reactive sites are located
    • Soaking beads in solvent can make them expand, making it a little easier for amino acids to enter the bead
  • Works best on small scale
    • It takes a large amount of beads to get a small amount of product
  • Beads are expensive
    • However the beads can be reused several times
  • Technology for the automation is expensive

The Reaction

As mentioned above, when using the bead you can use excess amounts of reagents, helping to solve the kinetic problem of phase transition. The polypeptide chain is always kept convalently attached to the beads, and the DCC and new amino acids are then introduced in solution. DCC and amino acids are used in excess to overcome the kinetics problem of phase transition (Le Chatelier).  In solution phase synthesis, excess reagents would lead to a very tedious purification process, but here, they can simply be washed away in solution by filtration.

In this synthesis, the beginning and ending steps are unique, while the middle steps are iterative, or repetitive.  The unique beginning step is the attachment of the Boc-protected amino acid to the polystyrene bead. This initial reaction is driven by the"cesium effect" or "naked anion" effect. The cesium cation has a large diameter and is therefore more easily attacked by organic molecules, making it very soluble.  With the counter cation otherwise occupied, the amino acid becomes a much better nucleophile.  Chlorine is very good at SN2 reactions, and the amino acid easily displaces the chlorine, forming the covalent bond between the first amino acid and the polystyrene bead.  The precipitation of cesium chloride pulls the reactions forward to nearly a 100% yield (Le Chatelier's principle). Without the help of the cesium, the amino acid would remain a poor nucleophile and the reaction would then have to compete with E2 reactions.

The beginning of the solid phase synthesis reaction is shown below:

Download the chemdraw file above

These next middle steps are repeated for each amino acid added to the growing chain. TFA and DIPEA (diisopropylethylamine) are added in excess (to drive the reaction forward).  The TFA  removes the Boc protecting group from the N terminus of the amino acid. DIPEA, a tertiary amine, deprotonates the newly unprotected nitrogen to create a nucleophile and neutralize the acidic solution created by the TFA.

The next step is the DCC coupling reaction, which you've seen before. This involves taking the next Boc-protected amino acid and mixing it in another flask with DCC to create an active ester. This product is then mixed with the free amino groups on the peptide chains attached to the bead and an amide bond is formed.  The rest of the amino acids of the polypeptide chain can be added by following that exact procedure of treating them with DCC and mixing them with the bead-amino acid complex. Remember, after each addition, TFA and DIPEA need to be added again in order to remove the Boc protecting group and neutralize the solution. Each time you add an amino acid, the reaction will become easier because they won't have to invade the bead as far to attach to the growing polypeptide chain.

The coupling can be seen below:

Download the chemdraw file above

When the last amino acid of the chain has been added, it is time for the ending reaction. HF is added, finishing the reaction by removing ALL of the protecting groups. It removes the Boc-protecting groups as well as the amino acid side chain's protecting groups. Furthermore, it removes the strand from the resin bead for collection. Because of the removal from the bead, make sure to have filtered off the by-products before adding HF.  This is unique compared to other reagents, because one reagent removes ALL protecting groups. This results in a polypeptide in the solution phase, which then has to be purified using traditional methods.

The HF deprotection can be seen below:

Download the chemdraw file above

Although the reaction has a high yield, 1,000's of beads must still be used to produce the μM quantities often required for the chemists' needs. This is due to the aforementioned problems that there are only a small number of reactive sites on and within the bead (~1% of the bead's phenyl groups). Also that the active sites on the inside of the bead are harder to reach, as the reactants must snake their way through a pyrene maze to reach them, so not all sites may be in use.

Importance of Protecting Groups

Due to amino acid excesses used to ensure complete coupling during each synthesis step, polymerization of amino acids is common in reactions where each amino acid is not protected. In order to prevent this polymerization, protecting groups are used.

This adds additional deprotection phases to the synthesis reaction, creating a repeating design flow as follows:

  • Protecting group is removed from trailing amino acids in a deprotection reaction (this involves TFA and DIPEA)
  • Deprotection reagents washed away to provide clean coupling environment
  • Protected amino acids dissolved in a solvent such as DMF are combined with coupling reagents and pumped through the synthesis column
  • Coupling reagents washed away to provide clean deprotection environment

Currently, two protecting groups (t-Boc, Fmoc) are commonly used in solid-phase peptide synthesis. Their ability to act as a protecting group is caused by the carbamate group which readily releases carbon dioxide for an irreversible decoupling step.

DIPEA is an important reagent to this reaction due to its special properties. It is a nitrogen surrounded by 2 isopropyl groups and an ethyl group. This makes it a poor nucleophile and a good base, allowing it to only attack the hydrogen on the nitrogen left over on the tail end of the resin bead strand after the Boc-protecting group is removed by TFA. In this way, its protecting groups prevent strand scission or any number of other undesired reactions.

Directionality of Solid-Phase Synthesis

There is a subtle problem that makes the C -> N directionality of the polypeptide synthesis very important. If one were to try synthesizing a polypeptide in the N -> C direction, they would encounter the following:

First, DCC is used to promote the formation of a 5 membered ring, which is then expelled as DCU.

Download the chemdraw file above

Do not be tempted to have the amino acid's nitrogen directly attack the DCC adduct. This reaction does not occur. Rather, it attacks the product of the DCC elimintation step, creating a five membered ring. The correct synthesis mechanism has the nitrogen of the amino acid attacking the 5-membered ring after the expulsion of DCU. The mechnism is shown below:

Download the chemdraw file above

Furthermore, the peptide may tautomerize to form a planar, aromatic ring. The sp3 geometry of the R2 carbon is lost, which causes a loss of the stereochemistry. When the molecule flips back to its previous state and the partial positive charge of the original carboxylate carbon is attacked by the N terminus of another amino acid, the product is racemic at the R2 carbon. Differentiating between the two products would be incredibly difficult as physical properties would be the same, thus chemists opt to use the C -> N method of synthesizing polypeptides.

Download the chemdraw file above

This means that glycine and proline are the only viable amino acids for N to C solid phase synthesis.  Glycine contains a non-sterogenic carbon for the amino acid backbone, and the iminium intermediate required for racemization of proline is too strained to actually occur.  When combining larger peptides, one would have to make sure that the C terminus of the beginning peptide ended with a glycine or proline.

Problem with Synthesizing Long Chains

One last factor of solid phase synthesis that one must account for is the exponentially decreasing efficacy of the process. A solid synthesis can expect ~90% yield a step. By this logic, the following efficacies may be extrapolated for varying length polypeptide reactions.

Polypeptide Length

Percent Yield











Although it is possible to synthesize 50-mer+ polypeptides, the cost to product ratio is impractical. Therefore, chemists have devised another crafty solution: convergent synthesis. By producing multiple shorter segments and combining them in a single step at 90% efficacy, the exponential decrease in yield is bypassed. For example, two 50-mer segments produced at 0.52% efficiency will yield (0.52)(0.9) = 0.45% or a 166X improvement over synthesizing a single, long polypeptide. However, this solution doesn't work because all the protecting groups on the amino acid chain make it insoluble in aqueous or organic solvent.

Drill Problems

D7-1.  Provide a solid-phase synthesis of the following dipeptide  starting from chloromethyl polystyrene resin.  You have the following reagents available:  any appropriately protected amino acids and/or their cesium salts, trifluoroacetic acid (CF3CO2H), dicyclohexylcarbodiimide (DCC), and hydrogen fluoride (HF).  You may omit all solvents, wash steps, and neutralization steps from the synthesis.  All protecting groups must be specified, but any side chain protecting groups can be abbreviated as “P”.

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Download the chemdraw file above

D7-3.  When a polystyrene resin bead is added to organic solvent like benzene, it swells.  When the organic solvent is replaced with water, it contracts.  This process allows one to “squeeze out” any by-products not covalently connected to the resin bead during the solid phase wash steps that follow each reaction.  Which non-covalent interaction is primarily responsible for this differing behavior of resin beads in the two different solvents?  Draw a picture of the resin bead in both solvents indicating how the solvent molecules interact differently with the resin bead.

D7-5.  The reagent DCC is hygroscopic, meaning it readily absorbs water.  If DCC is improperly stored, it will not function properly in a solid-phase peptide synthesis.  Outline a mechanism that shows what goes wrong when DCC is stored in a moist environment.

D7-2.  In solid-phase peptide synthesis, it is critical that the efficiency of each coupling step be nearly quantitative (i.e.100%).  If you are trying to synthesize a 100-residue linear peptide, what would be the yield of full-length product be if:

    (a) the efficiency of each coupling were 99%?

    (b) the efficiency of each coupling were 90%?

    (c) the peptide were synthesized separately as two 50-residue pieces (99% for each step), and then joined together in a 95% yield coupling reaction?

D7-4.  Provide a mechanism for the deprotection of a benzyl-protected glutamate side chain with HF.

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