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. it is often more efficient to obtain a protein sequence from a nucleic acid sequence than by sequencing the protein itself. The antibodies is useful for several application including purifying the protein itself from the cell. 2. Synthetic peptides can be used to isolate receptors for many hormones and other signal molecules. 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 (also called antidiuretic hormone -ADH), is a peptide hormone that stimulates the reabsorption of water in the distal tubules of the kidney. Patients deficient in vasopressin (types of diabetes), 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 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 a helix, b strand ….

Vasopressin and Synthetic Vasopressin. Structural formulas of (A) vasopressin, a peptide hormone that stimulates water resorption. (B) 1-desamino-8-d-arginine vasopressin, a more stable synthetic analog of this antidiuretic hormone.

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. 1. The a-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). 2. 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. 3. 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.

The solid-phase method for peptide synthesis 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 (a) 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. (b) All reactions are carried out in a single vessel, eliminating losses caused by repeated transfers of products. STEPS The carboxyl-terminal amino acid of the desired peptide sequence is first anchored to the polystyrene beads. The t-Boc protecting group of this amino acid is then removed. The next amino acid (in the protected t-Boc form) and DCC, 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.

Amino Acid Activation. DCC is used to activate carboxyl groups for the formation of peptide bonds.

Solid-Phase Peptide Synthesis. The sequence of steps in solid-phase synthesis is: anchoring of the Cterminal amino acid. (2) deprotection of the amino terminus. (3) coupling of the next residue. Steps 2 and 3 are repeated for each added amino acid. (4) the completed peptide is released from the resin.

NOTE, this method is not restricted to peptides, DNA Probes and Genes also Can Be Synthesized by Automated Solid-Phase Methods.

1. Chemical synthesis of peptides: a simple example

2. Ligation of peptide fragments

2a. Native chemical ligation

non-canonical amino acids fluorescent tags spin resonance labels Applications Introduction of: non-canonical amino acids fluorescent tags spin resonance labels cross-linking reagents Fragmental isotope labelling for NMR studies Generation of cytotoxic proteins The chemically synthesized section can be as small as possible whereas the expressed part is not limited in size

Non-natural amino acids incorporation into protein

Examples of non-natural amino acids

In vivo translation : microinjection Study on ionic channels How is the translation terminated?? From D.L. Beene, D.A. Dougherty & H.A. Lester, Curr. Opin. Neurol 3:264-270 (2003)

Codons that can be assigned to non-natural amino acids. (a) Amber codon. An amber codon UAG is decoded by aa-tRNA containing a CUA anticodon. (b) Four-base codon. CGGG is shown as an example of four-base codons. The CGGG codon is decoded by aa-tRNA containing the corresponding anticodon CCCG. (c) Non-natural codon. A recently developed non-natural codon-anticodon pair yAG-CUs and structures of y and s are shown. The yAG codon makes a pair with the CUs anticodon exclusively through a cellfree translation. The gray oval represents an amino acid.