Monitoring solid phase peptide synthesis

doi:10.1038/nprot.2007.461

Method Article

Monitoring solid phase peptide synthesis

https://doi.org/10.1038/nprot.2007.461

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Synthetic chemistry

Biochemistry

Computational biology and bioinformatics

peptide synthesis

Fmoc determination

Monitoring the resin loading during peptide synthesis gives useful information about the progress of the assembly. This can easily done by quantifying the amount of Fmoc removed at each deprotection/coupling cycle via spectrophotometry.

Kaiser Test

The Kaiser test¹ is a qualitative test for the presence or absence of free primary amino groups, and it can be a useful indication about the completeness of a coupling step. The test is based on the reaction of ninhydrin with primary amines, which gives a characteristic dark blue colour. The test requires minimal amounts of analyte and is completed within a few minutes.

Microcleavage

When Fmoc removal data show anomalies, before performing a step that requires the use of particularly expensive materials, or after a critical step, and in general when assembling longer sequences, it may be useful to cleave and analyse a small amount of intermediate product.

Kaiser Test

A. 0.5 g ninhydrin in 10 mL EtOH

B. 0.4 mL of 0.001 M KCNaq in 20 ml pyridine.

UV spectrometer, UV cell (10 mm), small flasks of glass vials, measuring pipette.

Fmoc determination

Collect neatly the piperidine solution (12 mL/mg) used for deprotection (steps 4-5 of the standard protocol) in a small flask or glass vial.
Dilute 1/20 with 20% piperidine in DMF (in a small vial: 100 μL collected solution + 1.9 mL 20% piperidine). Mix.
Fill the UV cell with 2.7 mL of 20% piperidine in DMF (reference solution), place the cell into the spectrophotometer and zero at λ=301 nm.
Add 300 μL of the solution prepared at step 2 into the cell, mix and measure the absorbance.
Calculate the loading using the following equation: Loading (mmol/g) = Abs_sample x 0.4^a

abased on ε₃₀₁=6000 M^-1cm^-1 (ε depends also on the specifications of the spectrometer!); 3 mL deblocking solution, 1/200 dilution, 250mg resin.

Constance (or slight progressive decrease) of loading is an indication of good progress of the synthesis. If loading values vary irregularly or decrease drastically, a microcleavage test (described below) should be performed.

Kaiser Test

Transfer a few resin beads to a small glass tube and wash several times with ethanol.
Add 100 μL of each of the solutions above.
Mix well and place the tube in a pre-heated oven (115 °C) for 5 min. ▲ CRITICAL STEP To reduce the incidence of false negative results, it is recommendable to carry out a parallel positive control. The test is not applicable to N-terminal proline residues (secondary amine) and N-alkyl amino acids. The test may give false negative results when applied to aggregate sequences.

Microcleavage

Transfer a sample containing ~1-2 mg dry peptide-resin to a small syringe (2 mL).
Add 300 μL of the cleavage cocktail [TFA/ H₂O/ phenol/ TIPS 8.5/ 0.5/ 0.5/ 0.5] to the dried peptide resin, stir gently for 30 sec and wait for 3 h (stir gently in-between).
Collect the solution in a small HPLC vial, dilute with 400 μL ACN/water 1/1, mix.
At this point the solution can be analyzed in an analytical HPLC system (inject 20 μL) and/or further diluted (1/10) to be injected (2 μL) in LC-MS.

Kaiser, E., Colescot, R. L., Bossinge, C. D. & Cook, P. I. Color test for detection of free terminal amino groups in solid-phase synthesis of peptides. Anal. Biochem. 34, 595-598 (1970).

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Monitoring solid phase peptide synthesis

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