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chapter 3

Protein Isolation and Determination of Amino Acid Sequence

Coupling of the

C-terminal residue

to the solid support

Removal of the protective

group by acidification

Covalent joining of two

amino acids by using the

condensing agent

Removal of the protective

group

Dipeptidal group anchored

to the resin and ready for

another cycle of amino

acid addition

FIGURE 3-14

Steps in the chemical synthesis of a polypeptide by the solid-phase technique.

Fmoc Solid-Phase Peptide Synthesis

Solid-phase polypeptide synthesis that uses the t-BOC

group to protect the N"-amino group is a major proce-

dure used by protein chemists. However, another solid-

phase procedure is frequently used for the chemical

synthesis of peptides and proteins of interest; this is the

9-fluorenylmethoxycarbonyl (Fmoc) procedure (Figure

3-15). The Fmoc method for solid-phase peptide synthesis

uses the Fmoc group for protecting the N“-amino group.

In contrast to the t-BOC group method which requires acid

for removal of the N“-amino protective group, the Fmoc

group can be removed by a mild base. A piperidine solution

in N,N-dimethylformamide or N-methylpyrrolidone is

generally used for the removal of the Fmoc group; an-

hydrous trifluoroacetic acid is used to remove the t-BOC

group. This Fmoc procedure is technically simpler and

chemically less complex than the t-BOC procedure. Be-

cause the Fmoc protecting group can be removed by base,

the linkage of the peptide to the resin support does not

have to be stable under acidic conditions as in the t-BOC

procedure.

The Fmoc procedure offers more flexible reaction con-

ditions and more reagent options. Because of the milder

conditions of peptide synthesis, the Fmoc procedure

is widely used for the synthesis of modified polypep-

tides that are phosphorylated, sulfated, or glycosylated.