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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:

Chemdraw
2008-F-WMG-resin bead structure.cdx

Pros and Cons of using the beads

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

Chemdraw
solidphase2011.cdx

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.

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The coupling can be seen below:

Chemdraw
2010-F-LES-DCC_Coupling.cdx

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:

Chemdraw
2010-F-LES-HF.cdx

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.

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

Chemdraw
2008-F-WMG-n2csynth1(lmfedit).cdx

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:

Chemdraw
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2008-F-WMG-n2csynth2(lmfedit).cdx

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.

Chemdraw
2008-F-WMG-n2csynth3(lmfedit).cdx

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.

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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”.

Chemdraw
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D7-

1.cdx

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.

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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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