Solid phase peptide synthesis (SPPS) was introduced by R. B. Merrifield of Rockefeller University (N — Organic Chemistry Chemistry Question

Solid phase peptide synthesis (SPPS) was introduced by R. B. Merrifield of Rockefeller University (Nobel Prize 1984). This method is based on sequential addition of α–amino and side-chain protected amino acid residues to an insoluble polymeric support. 2–Chlorotrityl chloride resin, whose use has been pioneered by K. Barlos, is an acid labile resin. The steric bulk and the mild acidic conditions required for cleavage make this resin useful in many applications. The base-labile Fmoc–group is used for N^-α-protection of amino acids. After removal of this protecting group, the next protected amino–acid is added using either a coupling reagent or pre–activated protected amino–acid derivative. The resulting peptide is attached to the resin through its C-terminus and may be cleaved to yield a peptide acid or amide (depending on the linker used). Side–chain protecting groups are often chosen so as to be cleaved simultaneously with detachment of the peptide from the resin.

[VISUAL]
Figure 35.1: 2–Chlorotrityl chloride group attached to a polymeric support of 1% divinyl–benzene cross–linked polystyrene.

Synthesis of the dipeptide H₂N^-SER^-ALA^-OH
The synthesis of the above dipeptide is accomplished by the Fmoc–strategy using 2–chlorotrityl chloride resin as the solid support. The reactions that take place are illustrated in Figure 35.2.

[VISUAL]
Figure 35.2a: Synthesis of H₂N–Ser–Ala–OH using the Fmoc strategy on 2–chlorotrityl chloride resin (Step 1).

[VISUAL]
Figure 35.2b: Synthesis of H₂N^-Ser^-Ala^-OH using the Fmoc strategy on 2-chlorotrityl chloride resin (Steps 2–5).
Abbreviations: TFA = CF₃COOH, DCM = CH₂Cl₂, DMF = HCON(CH₃)₂

Estimation of level of first residue attachment
This procedure takes place after the completion of Step 1.
a) Take a UV cell
b) Weigh 2 mg dry Fmoc-amino acid-resin into the UV cell. Dispense freshly prepared 20% solution piperidine/DMF (3 cm 3) into the cell.
c) Agitate the resin mixture using a Pasteur pipette for 2 – 3 minutes.
d) Place the cell in a spectrophotometer. Read the absorbance (Asample) at 290 nm.
e) Using another UV cell, read the absorbance of 20% solution piperidine/DMF (3 cm 3) at the same wavelength (Ablank)
f) Estimate the level of first residue attachment using the equation:
Fmoc loading = (Asample - Ablank) / (1.75 * m), where n is the amount of Fmoc–amino acid-resin (in mmol) and m is the mass of resin (in g).

Experimental
Step 1:
Dissolve Fmoc^-Ala^-OH (62 mg, 0.2 mmol) in dry CH₂Cl₂ (DCM, 2 cm 3) by adding DIPEA (N,N-diisopropylethylamine) (139×10–6 dm 3, 0.8 mmol). Add this solution to a fine sintered glass manual SPPS reaction vessel containing 2-chlorotrityl chloride resin (200 mg, 0.2 mmol; loading 1.0 eq Cl/g resin) in 1 cm 3 DCM, shake for 30 minutes and then filter. To end-cap any remaining reactive trityl groups, add HPLC (high performance liquid chromatography) grade methanol (0.2 cm 3) and mix for 15 minutes. Filter the resin and wash it three times with 2 cm 3 of a mixture of DCM / MeOH / DIPEA = 17 / 2 / 1, then three times with 2 cm 3 of DCM and dry the resin in a desiccator over KOH.

Step 2:
Estimate the level of first residue attachment as described before. Remove the Fmoc-protecting group using freshly prepared 20% solution piperidine/dimethyl formamide (DMF, 3 cm 3). Filter after 5 minutes and repeat with another 3 cm 3 of 20% solution piperidine/DMF. Wash the resin with DMF (3×2 cm 3) and DCM (3×2 cm 3).

Step 3:
Dissolve Fmoc^-Ser(OBut^-OH (383 mg, 1 mmol) and HOBt (135 mg, 1 mmol) in a sample vial in the minimum volume of DMF. Then add dropwise DIC (156 μL, 1 mmol) and stir the mixture for 20 minutes. This results in the formation of the corresponding active ester, which has the structure shown below:
[VISUAL]
Add this solution to the resin, which has been swollen in 1 cm 3 DMF. Agitate gently for 1 hour and then filter, wash with DMF (3x₂ cm 3) and DCM (3x₂ cm 3). Then, perform a Kaiser test to ascertain the completeness of the reaction, as described below.

Kaiser test:
Prepare the following solutions:
1. Dissolve 5 g of ninhydrin in 100 cm 3 EtOH.
2. Dissolve 80 g of liquefied phenol in 20 cm 3 of EtOH.
3. Add 2 cm 3 of a 0.001 M aqueous solution of potassium cyanide to 98 cm 3 pyridine.
4. Transfer a few resin beads to a small glass tube and add 2 drops of each of the solutions above.
5. Mix well and heat in boiling water for 5 minutes. A positive test indicated by blue resin beads means that the coupling step should be repeated until a negative Kaiser test is achieved.

Step 4:
Removal of Fmoc-protecting group is accomplished using the same procedure described in Step 2.

Step 5:
Wash resin with isopropanol and ether (3×2 cm 3) and air-dry it by application of vacuum for 10 minutes. Add to the dry resin the cleavage reagent (3 cm 3 0.5% TFA/DCM) and leave to stand at room temperature with occasional agitation for 1 1/2 hours. Collect the filtrate and wash the resin with the cleavage reagent (3×2 cm 3). Evaporate the combined filtrates until dryness. Add cold ether (5 cm 3) and agitate the solid precipitated with a spatula. Leave for 10 minutes, decant the supernatant liquid and repeat once more with 5 cm 3 of ether. The product H₂N–Ser–Ala–OH is obtained by filtration through a sintered glass funnel.