Download Solid phase peptide synthesis (SPPS), strategies, resins and

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. Butyloxycarbonyl
or
tert. Butoxycarbonyl
stable to bases and nucleophiles
unaffected by catalytic hydrogenation
deprotection with TFA is rapid
Introduction of the Boc-group
Di-tert-butyl-dicarbonate
= Boc-anhydride = (t-boc)2O
2-(tert. Butoxycarbonyloxyimino)-2-phenylacetonitril
= Boc-NO
O
O
O
O
O
O
O
O
N
N
•
•
•
•
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“
N-α-Boc protected amino acids
• already N-α-Boc protected amino acids can simply be
bought from firms like „Novabiochem“
O
e.g. Boc-Ala-OH
O
N
H
COOH
Cleavage of the Boc-group
TFA
E1-elimination
CO2 cleavage
TFA is volatile and can be easily removed in vacuum!
Resins for Boc SPPS
Resins for preparing peptide acids
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
, DMF
Cl
+
BocAS-Cs+,
boc
KI
O
H
N
O
R
- attachment of the C-terminal residue is achieved by heating the resin in DMF with
the 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
Resins for preparing peptide acids
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
boc
O
H
N
O
R
O
N
coupling:
H
a)
first addition of the PAM-linker on to the aminomethyl resin and then
coupling of the Boc-protected amino acid
b)
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
Resins for Boc SPPS
Resins for preparing peptide acids
Brominated Wang (Brominated α-Methylphenylacyl resin)
O
Br
R
, DMF
+ BocAS-Cs+, KI
boc
O
O
N
H
O
hν (350 nm)
boc
O
H
N
OH
R
Resins for Boc SPPS
Resins for preparing peptide amides
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
NH2
NH2
Resins for Boc SPPS
Resins for preparing C-terminally modified peptide fragments
Brominated PPOA (Brominated [4-Propionylphenoxy]-acetic acid)
-
versatile resin for the Boc SPPS of peptide acids, esters and hydrazides by
photolytic or nucleophilic cleavage
O
O
Br
O
N
H
, DMF
+ BocAS-Cs+, KI
boc
O
O
O
H
N
O
R
O
NEt3 in
methanol/
dioxane
hν or NaOH in
dioxane
boc
O
H
N
O
R
N
H
boc
NH2NH2/
DMF
O
H
N
OMe
R
boc
O
H
N
R
N
H
NH2
Resins for Boc SPPS
Resins for preparing C-terminally modified peptide fragments
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
NO2
NO2
amino acid, DCC
HO
N
R
H
N
O
O
N
General aspects of Boc strategy
• 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
Side chain protecting groups for Boc strategy
Arg: Toluolsulfonyl- (Tos) or
Mesitylen-2-sulfonyl-group (Mts)
cleavage: HF/anisole (Tos)
thioanisole (Mts)
Ser, Thr, Tyr: Benzyl (Bzl)
O
cleavage: HF
R
boc N
Asp, Glu: Bzl
COOH
H
O
Lys: Fmoc or
2-Chlorobenzyloxycarbonyl (2ClZ)
cleavage: piperidine (Fmoc), TFA (2ClZ)
O
O
Cl
H
N
Cys: Acetamidomethyl (Acm) or
MeO
4-Methoxybenzyl (MeOBzl)
cleavage: Hg2+- or Ag+-salts (Acm), HF (MeOBzl)
His: Dinitrophenyl-group (DNP)
cleavage: thioles
DNP
O
O
H
N
OH
O
HN
OH
HN
boc
O
S
OH
boc
NH
boc
Advantages and disadvantages of Boc- and Fmoc-strategy
Advantages
Boc
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
Disadvantages
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
Advantages and disadvantages of Boc- and Fmoc-strategy
Advantages
Fmoc
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-α
deprotection, 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
Disadvantages
Piperidine: harmful vapor, toxic
side reactions:
• aspartimide formation at AspX residues like Asp-Gly, -Ser,
-Thr, -Asn, -Gln
• linker-bound C-terminal Cys
undergoes significant
racemisation (ca. 0,5%) with
each cycle of Piperidinetreatment
Literature
-Novabiochem 2002/3 Catalog
-www.cup.unimuenchen.de/oc/carell/lehre/peptide2.pdf
-Kates S. A., Albericio F. (ed): Solid-Phase Synthesis, A practical
guide, Marcel Dekker Inc. 2000