Peptides: Synthesis and Biological Interest
Therapeutic Agents
Therapeutic peptides approved by the FDA (2009-2011) 3
Proteins Biopolymers of α-amino acids. Amino acids are joined by peptide bond. They serve a variety of functions: Structure Enzymes Transport Protection Hormones 4
Structure of Amino Acids 5
Amino Acids NH 2 on the carbon next to CH. Glycine, NH 2 CH 2 CH, is simplest. With side chain, molecule is chiral. Most natural amino acids are L-amino acids, related to L-(-)-glyceraldehyde. Direction of optical rotation, (+) or (-), must be determined experimentally. 6
Standard Amino Acids Twenty standard α-amino acids. Differ in side-chain characteristics: H or alkyl Contains an H Contains sulfur Contains a nonbasic nitrogen Has CH Has a basic nitrogen 7
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9
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Properties of Amino Acids High melting points, over 200 C. More soluble in water than in ether. Larger dipole moments than simple acids or simple amines. Less acidic than most carboxylic acids; less basic than most amines. + _ H 3 N CH C pk a = 10 pk b = 12 11
esonance Stabilization The peptide bond is an amide bond. Amides are very stable and neutral. 12
Human Hormone Bradykinin An oligopeptide is made out of four to ten amino acids. Peptide structures are drawn with the N-terminal end at the left. Peptides are named from left to right: arginylprolylprolyl arginine. 13
Peptide Bond Formation The amino group of one molecule condenses with the acid group of another. Polypeptides usually have molecular weight less than 5,000. Protein molecular weight is 6,000 40,000,000. 14
Solution Phase Peptide Synthesis First, protect the amino group at the N terminus with benzyl chloroformate. Activate the carboxyl group with ethyl chloroformate to form anhydride of carbonic acid. Couple the next amino acid. epeat activation and coupling until all amino acids needed have been added. emove the protecting group. 15
Peptide Synthesis: Solid Phase X Y NH X NH 1) Deprot. 2) Coupling X H N NH X : Cl, Br, SH, H, NH 2 X H N NH 2 Cleavage X H N NH 2 n n 16
Peptide Synthesis: Solid Phase X NH H N H X NH - X NH 2 H + + + C 2 X NH - 17
Peptide Synthesis: Solid Phase HN H NH N C N HN N C N H HN + - N C N + N C N H H H 2 N 1 HN HN HN 1 NH H 2 N 1 + H N C H N 18
Peptide Synthesis: Solid Phase 19
Peptide Synthesis: eaction Monitoring General Considerations Constant monitoring of the reaction to facilitate optimization of the yield and minimize side reactions is necessary n-bead Methods: - Fast, reliable and sensitive - Use of common analytical techniques (Colorimetric / Fluorescent detection) Destructive Methods ff-bead Methods: Cleavage of resin bound materials and analysis by traditional rg Chem methods Advantage: Highly Accurate Drawbacks: - esin beads are lost (lost of material and difficult to quantify) - Analysis requires time (hours) Taddei, M. et al. J. Comb. Chem. 2004, 6, 805-810 20
Peptide Synthesis: eaction Monitoring NH 2 Ninhydrin C 2 H H H Blue beads (+) Free amines HN Complete coupling? Kaiser Test Yellow beads (-) Complete Coupling Colorimetric tests for : H, C 2 H, CH and more. 21
eaction with Ninhydrin Used to visualize spots or bands of amino acids separated by chromatography or electrophoresis. Deep purple color formed with traces of any amino acid. 22
Solid Phase Synthesis: MAS-NM General Considerations Magic Angle Spinning (MAS-NM), derives from the observation that spinning a heterogenous NM sample at the magic angle (54.736o) reduces the linebroadening of solid/swollen polymer samples. Using a high-resolution NM probe of reduced volume (Nanoprobe) that holds all the sample in the active region of the receiver coil, allows acquisition of SP MAS-1H NM spectra with line widths as small as 1Hz using a 500 MHz spectrometer. - The quality of the MAS- 1 H NM spectrum in primarily influenced by the nature of the polymer matrix - Special techniques can be employed with PEG resins (high mobility and flexibility) that will provide spectra of comparable quality to their solution-phase counterpart. - - Hydrophobic PS resins with short tether (e.g., wang linker) give poor qulity spectra - 2D MAS techniques such as 2D-CSY AND TCSY has also been developed MS AND I are also very useful tools Kiefer, P.A. Drugs Future 1998, 23, 301 Luo Y. et al. J. rg. Chem 1998, 63, 8719 23
Advantages of Solid Phase Synthesis Growing chain, built from C to N terminus, is attached to polystyrene beads. Intermediates do not have to be purified. Excess reagents are washed away with a solvent rinse. Process can be automated. Larger peptides can be constructed. Chapter 24 24
We know how to synthesize peptides on solid phase, but how could we synthesize several peptides at a time? 25
Peptide Synthesis: Parallel Synthesis Parallel Synthesis of 27 tripeptides requires 3 + 9 + 27 = 39 Coupling eactions What can we do if we want to synthesize 1 million of peptides? 26
Peptide Synthesis: Mixture Libraries Parallel Synthesis of 27 tripeptides requires 3 eactions!!!!! Problems : - Different monomers will have different reactivities - Different reactivities of certain dimeric sequences - Complex mixtures on resin beads precludes the on-resin assays - Complex deconvolution process for in-solution assays 27
Peptide Synthesis: Split and Mix A B C A B C A B C A B C A B C A B C D E F AD BD CD AE BE CE AF BF CF 28
Split and Mix Libraries: Screening ADG AEG AFG ADH AEH AFH ADI AEI AFI BDG BEG BFG BDH BEH BFH BDI BEI BFI CDG CEG CFG CDH CEH CFH CDI CEI CFI Fluorescently Labeled Protein Structure of the biologically active peptide? Peptide sequencing by Edman Degradation - 29
Sequencing from the N Terminus Edman degradation: The reaction with phenyl isothiocyanate followed by hydrolysis removes the N terminus amino acid. The phenylthiohydantoin derivative is identified by chromatography. Use for peptides with < 30 amino acids. 30
Edman Degradation 31
Edman Degradation (Continued) In the final step (step 3) the thiazoline isomerizes to the more stable phenylthiohydantoin. 32
Edman Degradation (Continued) 33