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Typical approaches in classical peptide synthesis: segment condensation Partial deprotection Partial deprotection / activation.

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Presentation on theme: "Typical approaches in classical peptide synthesis: segment condensation Partial deprotection Partial deprotection / activation."— Presentation transcript:

1 Typical approaches in classical peptide synthesis: segment condensation Partial deprotection Partial deprotection / activation

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

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.MerrifieldLaboratory note book (1959) JACS 85, 2149 (1963)

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

6 1. Peptide-polymer bond stable during synthesis 2. Temporary protection of the  -amino group 3. Permanent protection of the side chains 4. Efficient cleavage with simultaneous side chain removal A combination of protecting groups (N-, C-, side chains) that ensures: B. The protection scheme 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 A. The solid support Essetial aspects of solid phase peptide synthesis

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

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

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

10 Robert C. Sheppard

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

12 Linear solid phase synthesis

13 Convergent solid phase synthesis

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

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

16 T- R YADEFGHIKLMNPQRSTVW X 1 T- R Combinatorial synthesis: portioning-mixing principle Furka et al. Int. J.Pept. Prot. Res. (1991) X 2 X 1 T - R X 2 X 1 T YADEFGHIKLMNPQRSTVW

17 TTTTTTTTTTTTTTTTTTT T- R T YADEFGHIKLMNPQRSTVW X 2 T- R T X T A T X T D T X T E T X T F T X T G T X T H T X T I T X T K T X T L T X T M T X T N T X T P T X T Q T X T R T X T S T X T T T X T V T X T W T X T Y YADEFGHIKL M NPQRSTVW TTTTTTTTTTTTTTTTTTT TX 2 T- R Combinatorial synthesis of TX 1 TX 2 T peptide library

18 T- R YADEFGHIKLMNPQRSTVW T T A T T T D T T T E T T T F T T T G T T T H T T T I T T T K T T T L T T T M T T T N T T T P T T T Q T T T R T T T S T T T T T T T V T T T W T T T T QQQQQQQQQQ Q QQQQQQQQ TTTTTTTTTTTTTTTTTTT TTTTTTTTTTTTTTTTTTT QQQQQQQQQQQQQQQQQQQ Y Paralell synthesis of TQTX 2 T peptide sub-library

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21 SPOT peptide synthesis

22 Applications

23 More applications

24 Oxitocin Cys-Tyr-Ile-Gln-Asn-Cys-Pro-Leu-Gly-NH 2 Peptidhormon: tejelválasztás, uterus kontrakció Szerkezet: 1953 Duvigneaud (Nobel díj 1955) Szintézis: 1954 Duvigneau Vazopresszin Cys-Tyr-Phe-Gln-Asn-Cys-Pro-Lys-Gly-NH 2 Vérnyomás szabályozása, Duvigneau Glutation Glu Izolálás: 1921 Hopkins Cys-Gly Szerkezet: 1930 Szintézis: 1935 Cisztin Cisztein  -L-glutamil-L-ciszteinil-glicin Karnozin (N-b-alanilhisztidin) Néhány „fontos” peptid

25 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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