Fmoc product removal

P 12] The micro channels were primed with anhydrous N,N-dimethylformamide (DMF) to remove air and moisture before carrying out the reaction [88]. A 50 pi volume of a solution of Fmoc-y9-alanine (0.1 M) in anhydrous DMF was placed in one reservoir of a micro chip, driven by electroosmotic flow. A 50 pi volume of a solution of EDCl (0.1 M) in anhydrous DMF and 50 pi of a solution of Dmab-y9-alanine (0.1 M) in anhydrous DMF were inserted in the other two reservoirs. Anhydrous DMF was placed in the fourth reservoir, for collection of the product. Room temperature was applied for reaction for a 20 min period. The voltage was set in the range 500-700 V. 

Both FMOC and its hydrolysis products have similar absorption and fluorescence spectra to FMOC-amino acids. Excess FMOC remaining after derivatization reacts with water to form 9-fluorenylmethyl alcohol (FMOC-OH), and if this is not removed prior to sample injection, it elutes as a large, broad peak in the vicinity of proline. 

During the first decade when solid-phase synthesis was executed using Fmoc/tBu chemistry, the first Fmoc-amino acid was anchored to the support by reaction of the symmetrical anhydride with the hydroxymethylphenyl group of the linker or support. Because this is an esterification reaction that does not occur readily, 4-dimethylaminopyridine was employed as catalyst. The basic catalyst caused up to 6% enantiomerization of the activated residue (see Section 4.19). Diminution of the amount of catalyst to one-tenth of an equivalent (Figure 5.21, A) reduced the isomerization substantially but did not suppress it completely. As a consequence, the products synthesized during that decade were usually contaminated with a small amount of the epimer. In addition, the basic catalyst was responsible for a second side reaction namely, the premature removal of Fmoc protector, which led to loading of some dimer of the first residue. Nothing could be done about the situation,... 

Derivatization of primary amino acids with o-phthalaldehyde (OPA) is simple and the poor reproducibility due to the instability of the reaction product can be improved by automation and the use of alternative thiols, e.g. ethanthiol in place of the 2-mercaptoethanol originally used. An alternative fluorimetric method using 9-fluoroenylmethylchloroformate (FMOC-CL) requires the removal of excess unreacted reagent prior to column chromatography. This procedure is more difficult to automate fully and results are less reproducible. However, sensitivity is comparable with the OPA method with detection at the low picomole or femtomole level, and it has the added advantage that both primary and secondary amino acids can be determined. 

The procedure described above can now he repeated. First the reaction chamber is washed with a solution containing the third unit to be added (the third amino acid). This amino acid is also protected with either a Boc or Fmoc protective group. When the third unit is added to the existing two-unit structure, a three-unit (tripeptide) product is obtained. The protecting group is then removed and a fourth group added to permit the process to continue. The process can then be repeated as often as desired. 

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