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Typical approaches in classical peptide synthesis: segment condensation

Q - NH. CO-X. H. N. CO – Q ,. Q - NH. CO-X. H. N. CO - Q ,. 2. 2. CO - Q ,. CO - Q ,. Q - NH. Q - NH. Partial deprotection /. Partial deprotection. activation. CO-X. CO – Q ,. Prot - NH. H. N. 2. Q - NH. CO – Q ,. Deprotection. H. N. COOH. 2.

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Typical approaches in classical peptide synthesis: segment condensation

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  1. Q - NH CO-X H N CO – Q, Q - NH CO-X H N CO - Q, 2 2 CO - Q, CO - Q, Q - NH Q - NH Partial deprotection / Partial deprotection activation CO-X CO – Q, Prot - NH H N 2 Q - NH CO – Q, Deprotection H N COOH 2 Typical approaches in classical peptide synthesis: segment condensation

  2. H N 2 Partial deprotection / activation CO-X H N CO – Q, Q - NH 2 Q - NH CO – Q, etc. H N 2 H N 2 Typical approaches in classical peptide synthesis: stepwise elongation A) C-terminal stepwise elongation CO-X Q - NH CO – Q, CO – Q, Q - NH B) N-terminal stepwise elongation CO-Q, Q -NH CO-X CO-Q, Q -NH N-terminal deprotection CO-Q, Q -NH CO-X etc.

  3. Bruce R. Merrifield

  4. The Origins There is a need for rapid, quantitative, automatic method for the synthesis of long peptides. A possible approach may be the use of chromatographic columns Where the peptide is attached to the polymer packing and added to by an activated amino acid followed by removal of protecting group, with repetition of the process Until the desire peptide is built up. Finally the peptide must be removed from the supporting medium. R.B.Merrifield Laboratory note book (1959) JACS 85, 2149 (1963)

  5. H N 2 The solid phase principle Functionalize Anchor Resin Resin X . Resin Couple . Resin S COOH S Cleave, purify Elongate . . . Resin Target peptide . . .

  6. 1. Contains reactive sites that allow functionalization 2. Peptide-polymer bond must be cleaved efficiently 3. Stable to physical and chemical synthesis conditions 4. Good accessibility of the growing peptide chain to solvents and reagents Essetial aspects of solid phase peptide synthesis A. The solid support B. The protection scheme A combination of protecting groups (N-, C-, side chains) that ensures: 1. Peptide-polymer bond stable during synthesis 2. Temporary protection of the a-amino group 3. Permanent protection of the side chains 4. Efficient cleavage with simultaneous side chain removal

  7. H N-CHR -CO Deprotect / 2 1 Q-NH-CHR -CO 1 Neutralize H N-CHR -CO 2 1 Q-NH-CHR -CO 1 H N-CHR -CO Q-NH-CHR -CO 2 1 1 Q-NH-CHR -COOH 2 Q -NH-CHR - CONH -CHR -CO 2 1 Q-NH-CHR - CONH -CHR -CO 2 1 Q-NH-CHR - CONH -CHR -CO 2 1 ...... Q-NH-CHR -CO NH-CHR -CO n 1 ...... Q NH-CHR -CO NH-CHR -CO n 1 ...... Q-NH-CHR -CO NH-CHR -CO n 1 Solid phase synthesis on polystyrene-divinylbenzene X Functionalize Anchor X X Carboxyl activation Repeat, n times 1,4-divinylbenzene Cleave, purify, etc... Q = N-terminal amino-protecting group

  8. a a. N - Protecting group ("temporary") b. Side-chain protecting groups ("permanent") c. Resin-peptide anchorage Protection in solid phase synthesis b c a R

  9. CH 3 CH OCH 3 2 CH 3 CH 2 labile to medium labile to strong acid acid (TFA / CH Cl ) (HF, TFMSA) 2 2 Boc/benzyl chemistry - Merrifield method P OCO-NH CH CO O

  10. Robert C. Sheppard

  11. OCH OCH 2 2 CH 2 CH CH 3 3 The para alkoxy substituent CH 3 decreases the acid resistance Removed by base of the peptide-resin linkage (piperidine / DMF) OCH 3 CH O 3 4-(2',4'-dimethoxyphenylaminomethyl)- phenoxy resin Barlos et al., TL , 30 , 3947 (1989) Rink, TL 28, 3787 (1987) Fmoc/t-butyl chemistry - Sheppard method H OCO-NH CH CO P O Labile to TFA P Cl P CH O Cl Fmoc NH 2-chlorotrityl resin HOAc/free acid HOAc, dilute TFA/amide

  12. Linear solid phase synthesis

  13. Convergent solid phase synthesis

  14. Solid-Phase Synthesis of Peptides: A Summary Solid phase Fully protected Functionalized peptide synthesis peptide-resin polymer deprotection * peptide-resin cleavage AAA HPLC purification Characterization HPCE Crude free peptide Purified peptide enzyme digestion MS (control by HPLC, AAA, etc.) What can I expect to find in a synthetic peptide crude ? The desired peptide (!) Ac-DEFGHIK Deprotection scavengers Terminated Ac-HIK Non-peptide Wrong peptides Protecting group derivatives Peptide ABCDEFHIK (minus G) Anything else... ABCDFGHIK (minus E) Deleted ABCEFGHIK (minus D) ACDEFGHIK (minus B) Incompletely deprotected peptide products

  15. Formats Multiple Mixture Parallel T-bag synthesis Pin – technology Spot synthesis (1988) Photolitography/chips Combinatorial libraries

  16. Combinatorial synthesis: portioning-mixing principle Furka et al. Int. J.Pept. Prot. Res. (1991) T- R 6 1 2 3 4 5 7 8 9 10 11 12 13 15 16 17 18 19 14 A D E F G H I K L M N P Q R S T V W Y 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 X 1 T- R 6 1 2 3 4 5 7 8 9 10 11 12 13 14 15 16 17 18 19 A D E F G H I K L M N P Q R S T V W Y 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 R X2X1T - X2X1T

  17. T- R 6 1 2 3 4 5 7 8 9 10 11 12 13 15 16 17 18 19 14 A D E F G H I K L M N P Q R S T V W Y 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 X 2 T- R T TX 2 T- R 6 1 2 3 4 5 7 8 9 10 11 12 13 14 15 16 17 18 19 A D E F G H I K L N P Q R S T V W Y M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 X X X X X X X X X X X X X X X X X X X T T T T T T T T T T T T T T T T T T T A D E F G H I K L M N P Q R S T V W Y T T T T T T T T T T T T T T T T T T T Combinatorial synthesis of TX1TX2T peptide library

  18. T T T T T T T T T T T T T T T T T T T Q Q Q Q Q Q Q Q Q Q Q Q Q Q Q Q Q Q Q Paralell synthesis of TQTX2T peptide sub-library T- R 6 1 2 3 4 5 7 8 9 10 11 12 13 15 16 17 18 19 14 A D E F G H I K L M N P Q R S T V W Y 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 6 1 2 3 4 5 7 8 9 10 11 12 13 14 15 16 17 18 19 Q Q Q Q Q Q Q Q Q Q Q Q Q Q Q Q Q Q Q 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T A D E F G H I K L M N P Q R S T V W Y T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T

  19. SPOT peptide synthesis

  20. Applications

  21. More applications

  22. Cys-Tyr-Ile-Gln-Asn-Cys-Pro-Leu-Gly-NH2 Peptidhormon: tejelválasztás, uterus kontrakció Szerkezet: 1953 Duvigneaud (Nobel díj 1955) Szintézis: 1954 Duvigneau Cys-Tyr-Phe-Gln-Asn-Cys-Pro-Lys-Gly-NH2 Vérnyomás szabályozása, Duvigneau Glu Izolálás: 1921 Hopkins Cys-Gly Szerkezet: 1930 Szintézis: 1935 Cisztin Cisztein g-L-glutamil-L-ciszteinil-glicin Néhány „fontos” peptid Karnozin (N-b-alanilhisztidin) Glutation Oxitocin Vazopresszin

  23. 6-7 11 20-21 • 1 7 19 30 Inzulin Hasnyálmirigy hormonja A lánc B-lánc Izolálás: 1922 Banting Primer Szerkezet: 1953 Sanger Szintézis: 1969 Zahn, Wang, Katsoyannis Térszerkezet: 1965 Hodgkin ATCH 39 aminosav Sertés: szintézis Schwitzer, 1963 Humán: szintézis Bajusz, Kisfaludy, Medzihradszky 1971

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