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Peptides Can Be Synthesized by Automated SolidPhase Methods

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Peptides Can Be Synthesized by Automated SolidPhase Methods
Figure 4.38. Nuclear Localization of a Steroid Receptor. (A) The receptor, made visible by attachment of the green
fluorescent protein, is located predominantly in the cytoplasm of the cultured cell. (B) Subsequent to the addition of
corticosterone (a glucocorticoid steroid), the receptor moves into the nucleus. [Courtesy of Professor William B. Pratt/
Department of Pharmacology, University of Michigan.]
I. The Molecular Design of Life
4. Exploring Proteins
4.3. Immunology Provides Important Techniques with Which to Investigate Proteins
Figure 4.39. Immunoelectron Microscopy. The opaque particles (150-Å, or 15-nm, diameter) in this electron
micrograph are clusters of gold atoms bound to antibody molecules. These membrane vesicles from the synapses of
neurons contain a channel protein that is recognized by the specific antibody. [Courtesy of Dr. Peter Sargent.]
I. The Molecular Design of Life
4. Exploring Proteins
4.4. Peptides Can Be Synthesized by Automated Solid-Phase Methods
The ability to synthesize peptides of defined sequence is a powerful technique for extending biochemical analysis for
several reasons.
1. Synthetic peptides can serve as antigens to stimulate the formation of specific antibodies. For instance, as discussed
earlier, it is often more efficient to obtain a protein sequence from a nucleic acid sequence than by sequencing the protein
itself (see also Chapter 6). Peptides can be synthesized on the basis of the nucleic acid sequence, and antibodies can be
raised that target these peptides. These antibodies can then be used to isolate the intact protein from the cell.
2. Synthetic peptides can be used to isolate receptors for many hormones and other signal molecules. For example, white
blood cells are attracted to bacteria by formylmethionyl (fMet) peptides released in the breakdown of bacterial proteins.
Synthetic formylmethionyl peptides have been useful in identifying the cell-surface receptor for this class of peptide.
Moreover, synthetic peptides can be attached to agarose beads to prepare affinity chromatography columns for the
purification of receptor proteins that specifically recognize the peptides.
3. Synthetic peptides can serve as drugs. Vasopressin is a peptide hormone that stimulates the reabsorption of water in
the distal tubules of the kidney, leading to the formation of more concentrated urine. Patients with diabetes
insipidus are deficient in vasopressin (also called antidiuretic hormone), and so they excrete large volumes of
urine (more than 5 liters per day) and are continually thirsty. This defect can be treated by administering 1-desamino-8-darginine vasopressin, a synthetic analog of the missing hormone (Figure 4.40). This synthetic peptide is degraded in vivo
much more slowly than vasopressin and, additionally, does not increase the blood pressure.
4. Finally, studying synthetic peptides can help define the rules governing the three-dimensional structure of proteins.
We can ask whether a particular sequence by itself folds into an α helix, β strand, or hairpin turn or behaves as a random
coil.
How are these peptides constructed? The amino group of one amino acid is linked to the carboxyl group of another.
However, a unique product is formed only if a single amino group and a single carboxyl group are available for reaction.
Therefore, it is necessary to block some groups and to activate others to prevent unwanted reactions. The α -amino group
of the first amino acid of the desired peptide is blocked with a tert-butyloxycarbonyl (t-Boc) group, yielding a t-Boc
amino acid. The carboxyl group of this same amino acid is activated by reacting it with a reagent such as
dicyclohexylcarbodiimide (DCC), as illustrated in Figure 4.41. The free amino group of the next amino acid to be linked
attacks the activated carboxyl, leading to the formation of a peptide bond and the release of dicyclohexylurea. The
carboxyl group of the resulting dipeptide is activated with DCC and reacted with the free amino group of the amino acid
that will be the third residue in the peptide. This process is repeated until the desired peptide is synthesized. Exposing the
peptide to dilute acid removes the t-Boc protecting group from the first amino acid while leaving peptide bonds intact.
Peptides containing more than 100 amino acids can be synthesized by sequential repetition of the preceding reactions.
Linking the growing peptide chain to an insoluble matrix, such as polystyrene beads, further enhances efficiency. A
major advantage of this solid-phase method is that the desired product at each stage is bound to beads that can be rapidly
filtered and washed, and so there is no need to purify intermediates. All reactions are carried out in a single vessel,
eliminating losses caused by repeated transfers of products. The carboxyl-terminal amino acid of the desired peptide
sequence is first anchored to the polystyrene beads (Figure 4.42). The t-Boc protecting group of this amino acid is then
removed. The next amino acid (in the protected t-Boc form) and dicyclohexylcarbodiimide, the coupling agent, are
added together. After the peptide bond forms, excess reagents and dicyclohexylurea are washed away, leaving the
desired dipeptide product attached to the beads. Additional amino acids are linked by the same sequence of reactions. At
the end of the synthesis, the peptide is released from the beads by adding hydrofluoric acid (HF), which cleaves the
carboxyl ester anchor without disrupting peptide bonds. Protecting groups on potentially reactive side chains, such as
that of lysine, also are removed at this time. This cycle of reactions can be readily automated, which makes it feasible to
routinely synthesize peptides containing about 50 residues in good yield and purity. In fact, the solid-phase method has
been used to synthesize interferons (155 residues) that have antiviral activity and ribonuclease (124 residues) that is
catalytically active.
I. The Molecular Design of Life
4. Exploring Proteins
4.4. Peptides Can Be Synthesized by Automated Solid-Phase Methods
Figure 4.40. Vasopressin and Synthetic Vasopressin. Structural formulas of (A) vasopressin, a peptide hormone that
stimulates water resorption, and (B) 1-desamino-8-d-arginine vasopressin, a more stable synthetic analog of this
antidiuretic hormone.
I. The Molecular Design of Life
4. Exploring Proteins
4.4. Peptides Can Be Synthesized by Automated Solid-Phase Methods
Figure 4.41. Amino Acid Activation. Dicyclohexylcarbodiimide is used to activate carboxyl groups for the formation
of peptide bonds.
I. The Molecular Design of Life
4. Exploring Proteins
4.4. Peptides Can Be Synthesized by Automated Solid-Phase Methods
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