Solid phase peptide synthesis (SPPS), strategies, resins and comparison with

Fmoc-strategy

General scheme of SPPS

attach to linker

deprotect amino function

couple

n times deprotection and coupling

cleave

N-α-protecting groups

• two mainly used N-α-protecting groups

Fmoc Boc

Boc-protecting group

Boc =tert. Butyloxycarbonylor tert. Butoxycarbonyl

�stable to bases and nucleophiles

�unaffected by catalytic hydrogenation

�deprotection with TFA is rapid

Introduction of the Boc-group

N

N

OO

O

• both commercially available• storage in refrigerator for extended periods• (t-boc)2O is more expensive• preparation: „Kates S. A., Albericio F. (ed): Solid-Phase Synthesis, A

practical guide, Marcel Dekker Inc. 2000, p. 105-107“

O O O

O O

Di-tert-butyl-dicarbonate = Boc-anhydride = (t-boc)2O

2-(tert. Butoxycarbonyl-oxyimino)-2-phenylacetonitril = Boc-NO

N-α-Boc protected amino acids

• already N-α-Boc protected amino acids can simply be bought from firms like „Novabiochem“

O

O

NH

COOHe.g. Boc-Ala-OH

Cleavage of the Boc-group

TFA

E1-elimination

CO2 cleavage

TFA is volatile and can be easily removed in vacuum!

Resins for Boc SPPS

� Merrifield (Chloromethylstyrene-divinylbenzene)- was the standard support for the synthesis of peptide acids by Boc SPPS- now only used in the synthesis of small to medium sized peptides, because the

benzylic ester resin linkage is not completely stable towards repetitive treatment with TFA

� Resins for preparing peptide acids

Cl O

O

R

NH

boc, DMF

+ BocAS-Cs+, KI

- attachment of the C-terminal residue is achieved by heating the resin in DMF withthe appropiate amino acid cesium salt in the presence of KI

- cleavage is affected by treatment of resin with HF or TFMSA, or by hydrogenolysis

- alcohols can be released using reducing agents like DIBALH or LiBH4

- methyl esters can be produced by transesterification with NaOMe

Resins for Boc SPPS

� PAM (4-Hydroxymethylphenylacetamidomethyl)- also a standard support for Boc SPPS- stabilizing effect of the phenylacetamidomethyl function on the ester linkage

� reduction of losses during repetitive TFA acidolysis

coupling:a) first addition of the PAM-linker on to the aminomethyl resin and then

coupling of the Boc-protected amino acidb) first coupling of the Boc-protected amino acid to the PAM-linker and then

reaction with the aminomethyl resin followed by end-capping of unreacted aminomethyl groups

cleavage:- treatment with HF or TFMSA releases the peptide acid

NH

O

O

O

NH

bocR

� Resins for preparing peptide acids

Resins for Boc SPPS

� Brominated Wang (Brominated α-Methylphenylacyl resin)

� Resins for preparing peptide acids

, DMF

+ BocAS-Cs+, KI

BrO

OO

ONH

R

boc

hν (350 nm)

ONH

Rboc OH

Resins for Boc SPPS

� BHA / MBHA (Benzhydrylamine / 4-Methylbenzhydrylamine)- used for the synthesis of peptide amides by Boc SPPS- attachment of the first amino acid with standard methods of amide bond

formation- cleavage of the carboxamides with HF or TFMSA- MBHA is more acid sensitive and the peptide amide can be released with HF

or TFMSA under less drastic conditions

� Resins for preparing peptide amides

NH2 NH2

Resins for Boc SPPS

� Brominated PPOA (Brominated [4-Propionylphenoxy]-acetic acid)- versatile resin for the Boc SPPS of peptide acids, esters and hydrazides by

photolytic or nucleophilic cleavage

� Resins for preparing C-terminally modified peptide fragments

, DMF

+ BocAS-Cs+, KI

hν or NaOH in dioxane

ONH

R

boc O

NEt3 in methanol/dioxane

ONH

R

boc OMe

NH2NH2/ DMF

ONH

R

boc NH

NH2

OBr

ONH

O

O

ONH

O

O

ONH

R

boc

Resins for Boc SPPS

� Oxime resin- attachment of the first amino acid with DCC- afterwards acetylation of unreacted oxime groups- cleavage from the support by various nucleophiles like NaOH, NH3, R1NH2,

NaBH4, MeOH, NH2NH2

� Resins for preparing C-terminally modified peptide fragments

amino acid, DCC

NOH

NO2

N

NO2

O

O

NH

R

• cleavage of the N-α-Boc-protection group with TFA (usually 25-50% (v/v) in DCM)

• side chain protecting groups must be orthogonal(!), that means:• stable against TFA during N-α-Boc deprotection• removable at the end of peptide synthesis

• release of the peptide from the resin by treatment with HF

General aspects of Boc strategy

Side chain protecting groups for Boc strategyArg: Toluolsulfonyl- (Tos) or

Mesitylen-2-sulfonyl-group (Mts)cleavage: HF/anisole (Tos)

thioanisole (Mts)Ser, Thr, Tyr: Benzyl (Bzl)

cleavage: HFAsp, Glu: Bzl N

HCOOHboc

R

O

Lys: Fmoc or 2-Chlorobenzyloxycarbonyl (2ClZ)cleavage: piperidine (Fmoc), TFA (2ClZ) NH

ONH

OH

boc

O

ONH

ONH

boc

O

O

ClOH

Cys: Acetamidomethyl (Acm) or4-Methoxybenzyl (MeOBzl)cleavage: Hg2+- or Ag+-salts (Acm), HF (MeOBzl)

NH

OS

OHMeO

boc

His: Dinitrophenyl-group (DNP)cleavage: thioles

DNP

Advantages and disadvantages of Boc- and Fmoc-strategy

� easy to introduce� Boc-amino acids are stable at room

temp. for extended periods (but storage at 4°C is recommended)

� deprotection with TFA is rapid� successful strategy for many peptide

synthesis applications� good coupling results

� temporary and permanent (side chain) protecting groups are both acid labile �side chain deprotection during repeated TFA treatment can occur

� repeated TFA-mediated N-α-deprotection over the course of a long synthesis may lead to modification and/or degradation of sensitive peptide sequences

� difficulties for fragile peptides that don‘t survive the relatively harsh final HF cleavage

� Boc-strategy requires the use of “dangerous“ HF and expensive laboratory apparates

� side reactions are possible:t-Bu+ reacts with nucleophilic side chains like trp, tyr, met, his� side chain protecting groups / adding of scavengers (1,2-Ethanedithiole) to the deprotection reagent

BocAdvantages Disadvantages

Advantages and disadvantages of Boc- and Fmoc-strategy

� orthogonal protection sheme� Fmoc-amino acids are easy to

prepare in crystalline form in high yield and stable when stored at 4°C

� milder reaction conditions:milde base (piperidine) for N-αdeprotection, TFA only for the final resin cleavage and deprotection

� progress of each deprotection reaction can be followed by real time spectrophotometric monitoring the release of the cleaved Fmoc-group at 300-320 nm

� Piperidine: harmful vapor, toxic� side reactions:

• aspartimide formation at Asp-X residues like Asp-Gly, -Ser, -Thr, -Asn, -Gln

• linker-bound C-terminal Cys undergoes significant racemisation (ca. 0,5%) with each cycle of Piperidine-treatment

Advantages DisadvantagesFmoc

Literature-Novabiochem 2002/3 Catalog

-www.cup.unimuenchen.de/oc/carell/lehre/peptide2.pdf

-Kates S. A., Albericio F. (ed): Solid-Phase Synthesis, A practicalguide, Marcel Dekker Inc. 2000