Typical approaches in classical peptide synthesis: segment condensation

CO - Q,Q - NH CO - Q,Q - NH

CO-X CO – Q,H2NQ - NH CO-X CO - Q,H2NQ - NH

Partial deprotectionPartial deprotection / activation

CO-XProt - NH CO – Q,H2N

Q - NH CO – Q,

H2N COOH

Deprotection

A) C-terminal stepwise elongation

Partial deprotection / activation

B) N-terminal stepwise elongation

N-terminal deprotection

etc.

CO-XQ - NH H2N CO – Q,

Q - NH CO – Q,

etc.

Q - NH CO-X H2N CO – Q,

Q - NH CO – Q,

CO-Q,H2NQ -NH CO-X

Q -NH

H2N

CO-Q,

CO-Q,Q -NH CO-X

Typical approaches in classical peptide synthesis: stepwise elongation

Bruce R. Merrifield

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)

Cleave, purify Elongate

Couple

AnchorFunctionalize

..

..

..

Resin

Resin

Resin

.

.

ResinResinX

The solid phase principle

Target peptide

S S

H2N

COOH

1. Peptide-polymer bond stable during synthesis2. 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 functionalization2. Peptide-polymer bond must be cleaved efficiently3. 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

Solid phase synthesis on polystyrene-divinylbenzene

Functionalize AnchorDeprotect /Neutralize

1,4-divinylbenzene

Q-NH-CHR2-COOHCarboxyl activation

Repeat, n times

Cleave, purify, etc...

X

X

X

Q-NH-CHR1-CO

Q-NH-CHR1-CO

Q-NH-CHR1-CO

H2N-CHR1-CO

H2N-CHR1-CO

H2N-CHR1-CO

Q-NH-CHR2-CONH -CHR1-CO

Q -NH-CHR2-CONH -CHR1-CO

Q-NH-CHR2-CONH -CHR1-CO

Q-NH-CHRn-CO ...... NH-CHR 1-CO

Q-NH-CHRn-CO ...... NH-CHR 1-CO

Q NH-CHR n-CO ...... NH-CHR 1-CO

Q = N-terminal amino-protecting group

Protection in solid phase synthesis

R

ab

c

a. N- Protecting group ("temporary")b. Side-chain protecting groups ("permanent")c. Resin-peptide anchorage

Boc/benzyl chemistry - Merrifield method

CH3

CH3

CH3

OCO-NH CH

CH2

O

OCH2CO P

labile to mediumacid (TFA / CH2Cl2)

labile to strong acid (HF, TFMSA)

Robert C. Sheppard

Fmoc/t-butyl chemistry - Sheppard method

OCO-NH CH

CH2

O

OCH2 OCH2H

CH3 CH3

CH3

CO

Removed by base(piperidine / DMF)

P

Labile to TFA The para alkoxy substituentdecreases the acid resistance of the peptide-resin linkage

4-(2',4'-dimethoxyphenylaminomethyl)- phenoxy resin HOAc, dilute TFA/amide

CH3O CH O

NHFmoc

OCH3

HOAc/free acid

2-chlorotrityl resin

Barlos et al., TL, 30, 3947 (1989)

Cl

Cl P P

Rink, TL 28, 3787 (1987)

X

X

X

X

O

XH

H2N

O

X

O

O

OH

O

OH

O

OH

O

O

H2NO

O

X

H2N

R

R

R

R

R

R

R

+

+

Amino acid coupling

Free peptide

Acidolysis

Resin-bound fullyprotected peptide

Repetitive cycles of N

deprotection and

amino acid coupling

N Deprotection

Amino acid coupling

N Deprotection

Anchoring of firstamino acid

+

X=O for peptide acidsX=NH for peptide amides

Linearsolid phase synthesis

H2NCOOH

COOHH2N

COOH H2N R

R

R

R

R

Detachment of protected peptidesegment from resin, and purification

Segment coupling on a solid support

Fully protectedpeptide on solidsupport

Free peptide

Convergent solid phase synthesis

*

Solid phasepeptide synthesis

peptide-resincleavage

Solid-Phase Synthesis of Peptides: A Summary

Functionalized polymer

Fully protected peptide-resin

Crude free peptide

deprotection

Characterization

AAAHPLC

MS

HPCEenzyme digestion

purification

(control by HPLC, AAA, etc.)

Purified peptide

What can I expect to find in a synthetic peptide crude ?

The desired peptide (!)

Wrong peptidesTerminated

Ac-DEFGHIKAc-HIK

Deleted

ABCDEFHIK (minus G) ABCDFGHIK (minus E)ABCEFGHIK (minus D)ACDEFGHIK (minus B)

Incompletely deprotected peptide products

Pepti

de

Deprotection scavengers

Protecting group derivatives

Anything else...Non-p

epti

de

Formats

Parallel

Combinatorial libraries

Multiple

Mixture

T-bag synthesis Pin – technologySpot synthesis (1988)Photolitography/chips

T- R

7 98 10 12111 32 4 65 1913 1514 16 17 18

YA D E F G H I K L M N P Q R S T V W

7 98 10 12111 32 4 65 1913 1514 16 17 18

X1T- R

7 98 10 12111 32 4 65 1913 1514 16 17 18

Combinatorial synthesis: portioning-mixing principleFurka et al. Int. J.Pept. Prot. Res. (1991)

X2X1T - R

X2X1T

YA D E F G H I K L M N P Q R S T V W

7 98 10 12111 32 4 65 1913 1514 16 17 18

T T T T T T T T T T T T T T T T T T T

T- R

T

7 98 10 12111 32 4 65 1913 1514 16 17 18

YA D E F G H I K L M N P Q R S T V W

7 98 10 12111 32 4 65 1913 1514 16 17 18

X2T- R

TXTA

TXTD

TXTE

TXTF

TXTG

TXTH

TXTI

TXTK

TXTL

TXTM

TXTN

TXTP

TXTQ

TXTR

TXTS

TXTT

TXTV

TXTW

TX2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2

TY

7 98 10 12111 32 4 65 1913 1514 16 17 18

YA D E F G H I K L M N P Q R S T V W

7 98 10 12111 32 4 65 1913 1514 16 17 18

TT T T T T T T T T T T T T T T T T T

TX2T- R

Combinatorial synthesis of TX1TX2T peptide library

T- R

7 98 10 12111 32 4 65 1913 1514 16 17 18

YA D E F G H I K L M N P Q R S T V W

7 98 10 12111 32 4 65 1913 1514 16 17 18

T

TA

T

T

TD

T

T

TE

T

T

TF

T

T

TG

T

T

TH

T

T

TI

T

T

TK

T

T

TL

T

T

TM

T

T

TN

T

T

TP

T

T

TQ

T

T

TR

T

T

TS

T

T

TT

T

T

TV

T

T

TW

T

T

T

T

7 98 10 12111 32 4 65 1913 1514 16 17 18

QQ Q Q Q Q Q Q Q Q Q Q Q Q Q Q Q Q Q

7 98 10 12111 32 4 65 1913 1514 16 17 18

TT T T T T T T T T T T T T T T T T T

TT 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

Y

Paralell synthesis of TQTX2T peptide sub-library

SPOT peptide synthesis

Applications

More applications

Oxitocin Cys-Tyr-Ile-Gln-Asn-Cys-Pro-Leu-Gly-NH2

Peptidhormon: tejelválasztás, uterus kontrakcióSzerkezet: 1953 Duvigneaud (Nobel díj 1955)Szintézis: 1954 Duvigneau

Vazopresszin Cys-Tyr-Phe-Gln-Asn-Cys-Pro-Lys-Gly-NH2

Vérnyomás szabályozása, Duvigneau

Glutation Glu Izolálás: 1921 HopkinsCys-Gly Szerkezet: 1930

Szintézis: 1935 Cisztin

Cisztein-L-glutamil-L-ciszteinil-glicin

NH2 CH2 CH2 CO NH CH COOH

CH2

C

CH

NH

N

CH

Karnozin(N-b-alanilhisztidin)

Néhány „fontos” peptid

Inzulin

1 6-7 11 20-21

1 7 19 30

Hasnyálmirigy hormonja

A lánc

B-lánc

Izolálás: 1922 Banting Primer Szerkezet: 1953 SangerSzintézis: 1969 Zahn, Wang, KatsoyannisTérszerkezet: 1965 Hodgkin

ATCH 39 aminosavSertés: szintézis Schwitzer, 1963Humán: szintézis Bajusz, Kisfaludy, Medzihradszky 1971