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Solid phase peptide synthesis
Part II
Application of Fmoc/tBu strategy
Gábor Mező
Research Group of Peptide Chemistry
Hungarian Academy of Sciences
Eötvös L. University
Budapest, Hungary
Outline
 Resins;
 Protecting groups;
 Synthetic protocol;
 Monitoring;
 Cleavage technics;
 Side reactions;
Fmoc/tBu:
H3C
tert-butyl
Fmoc
C
H3C
H3C
C
H3C
CH3
TFA
O
CH3
R
C
O
C O
O
C
H
..
NH
CH2
O
C
H2C
NH
CH
H2C
C
O
NH
CH
C
O
CH2
O
CH2
O
Wang-resin
piperidine
Fmoc-Asp(OtBu)-Tyr(tBu)-Wang resin
Type of resins for Fmoc-chemistry
There are many different resins and most of them are used for special
cases and in individual laboratories. Here only the most widely applied
resins will be presented. Resins are based on PS-DVB (1%) copolymer.
4-Alkoxybenzyl alcohol (Wang) resin:
HO
CH2
O CH2
P
Attachment of the first amino acid:
Fmoc-Aaa(X)-OH:DIC:DMAP (2:2:0.2 equiv to the resin OH content)
in DMF, 1h at RT.
The final cleavage results in peptides with COOH group at the C-terminus
The resin is not available for the synthesis of peptides with a sequence
on the C-terminal that is sensitive for diketopiperazine formation !
SASRIN (Super Acid Sensitive ResIN)
(2-methoxy-4-alkoxybenzyl-alcohol resin)
HO
CH2
O CH2
P
CH3 O
Peptide is cleavable with
0.5-1.0% TFA in DCM
resulted in protected
peptide fragments.
4-Hydroxymethylphenoxyacetic acid (HMPA) linker:
HO
CH2
O CH2 COOH
Attach to aminomethyl PS-DVB resin
Removal of the peptide with TFA
4-(4-Hydroxymethyl-3-methoxyphenoxy)butyric acid (HMPB) linker:
HO
CH2
CH3 O
O
(CH2)3 COOH Attach to aminomethyl PS-DVB resin
Removal of the peptide with diluted TFA
2-Chlorotrityl chloride (ClTrt) resin:
P
Attachment of the first amino acid:
Cl
Cl
1 g ClTrt-resin + 2 mmol Fmoc-Aaa(X)-OH
+ 8 mmol DIEA in 3-5 mL DCM, for 1.5 h
then 0.8 mL MeOH to block the unreacted groups
washing with DCM, iPrOH, MeOH, ether
The final cleavage results in peptides with COOH group at the C-terminus
Cleavage with 90-95% TFA + scavangers results in free peptides
Cleavage with AcOH:MeOH(TFE):DCM (1:1:8 or 2:2:6) results in protected
peptides (available for fragment condensation).
ClTrt resin prevents the diketopiperazine formation !
Attachment of Cys and His derivatives to the resin is free from
enantiomerisation!
Determination of loading
1.
(Calculation of the resin capacity)
 10-20 mg of dried resin are weighted exactly into a 100 mL
measuring flask (for a load of ca. 0.5 meq/g 20 mg is sufficient);
 Piperidine/DMF (1:4, V/V) is added to the mark;
 The mixture is shaken thoroughly and left for 25-30 min;
 The resin is filtered off and the absorbance of the filtrate is
measured at 301 nm (e = 7800).
NH2(mmol/g) = [A301.V(ml)/e301.m(mg)].106
2.
ca. 4-6 mg Fmoc-Aaa-resin
+400 mL 50% piperidine/DMF
30 min at RT, then filtration
dilute with MeOH to 25 mL
ca. 2 mg Fmoc-Gly-OH
+400 mL 50% piperidine/DMF
30 min at RT
dilute with MeOH to 25 mL
301
Capacity of the resin (mmol/g) =
1000.mgly.Aresin
Mgly
.m
.A301
resin
gly
Mgly =297
Rink Amide Resin: synthesis of peptides with CONH2 C-terminus
Cleavage with high concentration of
TFA can lead to the break down of
the linker
byproducts.
Use low TFA concentration and/or
trialkylsilanes in the cleavage mixture.
H3CO
Peptide-resin bond can be detached
Fmoc-HN
with 5% TFA. Removal of protecting
groups needs a separate step.
OCH3
CH
OCH2-P
Rink Amide-AM and Rink Amide-MBHA resins:
OCH3
H2N CH2
P
Aminomethyl-PS-DVB
CH3
H3CO
Fmoc-HN
CH
OCH2-CO-Nle-R
Peptide cleavage with 90-95% TFA solution.
Nle is a reference for quantitation.
H2N CH
P
4-methylbenzhydrylamine-PS-DVB
Pegylated resins: composition of polyethylene glycol (Mw:3000-4000)
and low-cross linker polystyrene gel-type resins.
Advantages:
excellent pressure stability (continuous flow synthesis)
excellent swelling properties (also in water)
high diffusion rates
available with many types of functional groups
low capacity (0.2-0.6 mmol/g), suitable for the synthesis
of aggregating peptides, for on resin cyclisation and
fragment condansation.
The basic polymer support is aminomethyl PEG-PS-DVB (NovaSyn R TG)
NH2
PEG
O
HO C H2
O CH 2 C
OH
4-hydroxymethylphenoxyacetic
acid linker
NovaSyn R TGA resin
Similar to Wang resin
4-carboxytrityl linker
NovaSyn R TGT alcohol resin
O
HO
C
OH
Before use the resin must be converted
to the chloride form by heating with
AcCl or SOCl2 in toluene.
Similar to ClTrt resin.
OCH
3
H CO
3
H2 N
2,4-dimethoxy-benzhydryl linker
NovaSyn R TGR resin
O
CH
OCH2
C
Similar to Rink Amide MBHA resin
OH
Applied side chain protecting groups in Fmoc-chemistry
Side chain functional group
-OH (Ser, Thr, Tyr)
protecting group
CH3
H 3C C
name (abbreviation)
tert-butyl (tBu)
CH3
Trt group can be used if on-resin derivatization is required (glycosylation,
phosphorylation). Trt can be cleaved with diluted TFA, while tBu needs
90% TFA solution for effective removal.
-SH (Cys)
For selective deprotection
trityl (Trt)
CH2 NH C CH3
O
acetamidomethyl
(Acm)
Racemisation during the attachment of Cys derivatives to the resins
in the presence of DMAP: Fmoc-Cys(Trt)-OH > Fmoc-Cys(Acm)-OH
However, Fmoc-Cys(Acm) at the C-terminal resultes in side reaction:
NH
CH
C
O
Acm-S CH2 O
NH
C
C
H2C O
piperidine
CH2
O CH2
P
O CH2
P
piperidine
O
CH2
H2O
Cys
C
NH
CH
C
NH
CH
N
CH2 O
HO
CH2 O
DAla
Mcalc
Mcalc – 34
DL-Ala(Pip) Mcalc+ 41
DL-Ser
Mcalc – 16
Side chain functional group protecting group
eNH (Lys)
2
O
CH3
C O C
CH3
name (abbreviation)
tert-butyloxycarbonyl
(Boc)
CH3
Selectively removable protecting groups for preparation of modified
peptides (labeled, functionalised, branched or cyclic peptides):
eNH (Lys)
2
CH3
4-methytrityl
(Mtt)
Mtt can be removed selectively with 1%TFA/DCM solution in
the presence of 3-5% TES (triethylsilane) at RT in 15-30 min.
Trt groups may be not stable enough under this condition.
Side chain functional group protecting group
O
R
CH3
eNH (Lys)
C
2
CH3
O
R = metil1, isopropyl2
ivDde is more stable in basic cleavage mixture
applied for Fmoc removal than Dde.
name (abbreviation)
1-(4,4-dimethyl2,6-dioxocyclohex-1ylidene)ethyl (Dde)1
1-(4,4-dimethyl2,6-dioxocyclohex-1ylidene)-3-methylbutyl
(ivDde)2
Both protecting groups can be removed with 2% NH2-NH2 in DMF
eNH (Lys)
2
O
C O CH2
CH
CH2
allyloxycarbonyl
(Aloc)
Aloc protecting group is compatible with Boc as well as Fmoc-chemistry.
It is stable in acids and bases. It can be removed in P(Ph)3 by Pd(0)
catalysis. To prevent addition on double bond under other cleavage
conditions application of allyl alcohol in cleavage mixtures is recommended.
Side chain functional group protecting group
CH3
wCOOH (Asp, Glu)
O C
CH3
name (abbreviation)
tert-butyl ester (OtBu)
CH3
Selectively removable protecting groups for preparation of cyclic peptides:
(pairs of amino and carboxyl protecting groups: Dde-ODmab, Aloc-OAll)
O CH2
CH
C
NH
O
CH3
CH3
O
4-{N-[1-(4,4-dimethyl2,6-dioxocyclohexylidene)
-3-methylbutyl]-amino}
Benzyl ester
(ODmab)
Similarly to Dde and ivDde, the Dmab protecting group can be removed
with 2% hydrazine in DMF.
O CH2
CH
CH2
It can be removed in P(Ph)3 by Pd(0) catalysis.
allyl ester (OAll)
Succinimide ring formation (Asp):
Acid catalised reaction results in a or b-Asp-peptides, however, piperidine
catalised side reaction under Fmoc cleavage procedure results in piperidide:
O
C
H 2C
NH
O
CH
OtBu
C
NH CH2
O
C
C
H2C
- tBuOH
NH
CH2 C
N
CH
O
C
O
O
-Asp-Gly-
-Asu-Gly-
piperidine
piperidine
O
H 2C
NH
CH
C
C
O
M = Mcalc+ 57
O
N
NH CH2
C
O
C
H2C
NH
N
CH2 C
O
CH
C
O
N
Application of other cleavage reagents (DBU, TBAF, DEA, morpholine)
eliminate the piperidide formation, but not the succinimide formation.
Addition of HOBt to the cleavage mixture can reduce the succinimide
ring closure. But the best results may get with the use of Fmoc-(Hmb)amino acid derivatives:
Hmb: 2-hydroxy-4-methoxybenzyl (removable with TFA)
O
C
OtBu
H 2C
NH
CH
C
O
N CH2
C
O
Fmoc-(Fmoc-Hmb)Gly-OH
1g = 370 EUR (NovaBiochem)
(Hmb)amino acid derivatives are
secundary amines:
Removal of Fmoc group and the
attachement of the next Asp
derivative is difficult, needs
longer time.
Ninhydrin test can’t detect the
efficacy of the coupling.
The increasing of the solubility of protected peptide fragments
as well as preventing of aggregation of ”difficult” sequences can
be reach by the application of Hmb groups.
Side chain functional group
protecting group
wCONH (Asn, Gln)
2
name (abbreviation)
trityl (Trt)
The solubility of Fmoc-Asn-OH and Fmoc-Gln-OH is extremely bad.
The Trt protecting group increases the solubility and prevents the
dehydratation as well as ring closure side reactions during the synthesis.
N-terminal Gln or Asn-Gly (Arg, Ser, Ala, Asn) sequence may cause
problems after the cleavage of the protecting group.
(His)
p
N
N
H
t
imidazol group
C H3
CH2
O
C
C H3
tert-butyloxymethyl
(Bum) (p)
C H3
The same problem as in case of Bom in Boc startegy. Don’t use it for the
synthesis of peptides containing Cys at the N-terminal !
Side chain functional group
protecting group
(His)
p
N
N
name (abbreviation)
trityl (Trt) (t)
H
t
imidazol group
Trt group protects the tN. However, its application prevents both
epimerisaton (not in case of attachment to resins) and alkylation.
-NH-C-NH2
(Arg)
NH
guanidino group
O
CH3
OCH3
S
O
CH3
CH3
4-methoxy-2,3,6trimethylbenzenesulfonyl (Mtr)
Mtr is too stable in TFA. Elevated temperature (30oC) and/or increased
time (4-6 hrs) is necessary for effective cleavage.
1M TMSOBr-thioanisol/TFA mixture is an alternative cleavage mixture
that can remove Mtr more effectively.
Side chain functional group
-NH-C-NH2
(Arg)
NH
guanidino group
protecting group
O
CH3
CH3
O
S
O
CH3
O
CH3
CH3
2,2,5,7,8-pentamethylchroman-5-sulfonyl
(Pmc)
CH3
CH3
O
S
O
CH3
name (abbreviation)
CH3
CH3
2,2,4,6,7-pentamethyldihydrobenzofurane6-sulfonyl (Pbf)
Pmc can be cleaved with TFA in 2-3 hrs, but Pbf protecting group can
be removed 1.5-2 times faster than Pmc. Pbf also gives rise to less
sulfonated Trp byproduct than Pmc or Mtr.
Use Fmoc-Arg(Pbf)-OH for the synthesis of oligo-Arg as a
cell penetrating peptide !
Side chain functional group
(Trp)
N
H
indole
protecting group
O
CH3
C O C
CH3
name (abbreviation)
tert-butyloxycarbonyl
(Boc)
CH3
The protection of indole side chain of Trp is not necessary, but the
application of Boc group is recommended. Under TFA cleavage the
appearance of inN-carboxy indole protects Trp vs alkylation and sulfonation.
inN-carboxy group is removed under aqueous condition in working up
procedure.
Protection of the side chain of Met is not needed in Fmoc-strategy.
Fmoc/Bzl (benzyl type protecting groups for blocking of side chains)
strategy is applied for the synthesis of protected peptide fragments,
because of the better solubility of benzyl protected fragments over
tert-butyl and trityl protected fragments.
Synthetic protocol of Fmoc-strategy
(-) yellow
1) Wash the resin 3x with DMF; 0.5-1.0 min each
2) Cleavage of Fmoc protection with
2% piperidine + 2%DBU/DMF; 2+2+5+10 min*
3) Wash the resin 8x with DMF; 0.5-1.0 min each**
(+) blue
4) Coupling: Fmoc-amino acid derivative-DIC-HOBt in DMF***
(3 equiv each calculated to the resin capacity); 60 min
5) Wash the resin 2x with DMF; 0.5-1.0 min each
6) Wash the resin 2x with DCM; 0.5-1.0 min each
7) Ninhydrin monitoring
*
DBU is the cleavage reagent, piperidine is for the capture of dibenzofulvene
20% or 50% piperidine in DMF, 50% morpholine or DEA in DMF and 20mM
TBAF in DMF are also used as cleavage mixture.
** After 4 DMF washing, IPA washing may be applied for shrinking the resin.
An unefficient removal of base from the resin may cause Fmoc cleavage in the
next coupling step.
*** DIC is used instead of DCC in this method, because of the limited solubility
of DCU in the applied solvents.
Coupling agents
H3C
N
C
CH3
HC
N
N
N
C
CH
H3C
CH3
N,N’-diisopropylcarbodiimide (DIC, DIPCDI))
N,N’-dicyclohexylcarbodiimide (DCC)
O
N
X-NH
CH
R
C
C
NH
O
O
X-NH
X: Boc, Fmoc
X-NH
CH
C
R
C
NH
O
N-acyl-urea derivatives
O-acyl-isourea derivatives
HOBt
N
O-N acyl shift
CH
R
C
OBt
O
in situ active ester
+
NH
C
O
NH
urea derivatives: DCU, DIU
N
N
N
N
N
N
OH
1-hydroxy-7-aza-benzotriazole
(HOAt)
PyAOP
1-hydroxybenzotriazole
(HOBt)
N
N
O
(CH3)2N
OH
AOP
N
N
N
They don’t need
DCC or DIC for
preparation of
in situ active ester
PF6-
P+
N(CH3)2
N(CH3)2
benzotriazol-1-yl-oxy-tris(dimethylamino)phosphonium hexafluoro phosphate (BOP)
Hexamethylphosphoramide (carcinogen)!
>
N
N
O
N
P+
N
PF6N
benzotriazol-1-yl-oxytris(pyrrolidino)phosphonium
hexafluoro phosphate (PyBOP)
N
N
N
N
N
N
O
C+
(CH3)2N
O
PF6N(CH3)2
(CH3)2N
2-(1H-benzotriazol-1-yl)-1,1,3,3,tetramethyluronium hexafluorophosphate
HBTU
PF6-
C+
N
+
N
O-
N
N(CH3)2
N(CH3)2
C+
BF4N(CH3)2
2-(1H-benzotriazol-1-yl)-1,1,3,3,tetramethyluronium tetrafluoroborate
TBTU
According to NMR and röntgen
diffraction studies a new structure
was suggested:
N-[(1H-benzotriazol-1-yl)(dimethylamino)methylene]-N-methanaminium
hexafluorophosphate N-oxide
HATU, TATU, HBPyU, HAPyU, etc.
Guanylation with uronium type coupling reagents
N
N
N
O
(CH3)2N
PF6-
C
C
O
N(CH3)2
NH
N+(CH
CH
R
PF6-
C+
(CH3)2N
HNH
+
NH-PEPTIDE
CH
3)2
R
C
NH-PEPTIDE
O
+ HOBt
Don’t use excess of coupling agent (cyclisation, fragment condensation);
Make preactivation of the incoming amino acid;
Apply: X-Aaa-OH: HBTU: DIEA = 3:2.9:3 (equiv) to the resin capacity.
Fmoc cleavage flow chart
Does the peptide
contain N-terminal Fmoc group?
yes
no
Remove Fmoc
Does the peptide
contain Arg, Met, Trp or Trt?
yes
Does the peptide
contain Arg, Met?
no
Use cleavage mixture A
no
yes
Use cleavage mixture B
yes
Use cleavage mixture C
A: 0.5 mL d.i. water
9.5 mL TFA
Does the peptide
contain Trp or Trt?
no
B: 0.75 g cryst. phenol
0.25 mL EDT
C: 0.25 mL EDT
0.50 mL thioanisole
0.25 mL d.i. Water
0.50 mL d.i. water
9.50 mL TFA
10 mL TFA
Boc/Bzl or Fmoc/tBu strategy
Amino acid derivatives and resins for Boc-strategy is still cheaper:
Boc-Ala-OH
(Mw: 189)
5g 11 EUR, 1mmol
Fmoc-Ala-OH
(Mw: 311)
5g 11 EUR, 1mmol 0.684 EUR
Boc-Arg(Tos)-OH
(Mw: 429)
5g 32 EUR, 1mmol 2.746 EUR
Fmoc-Arg(Pbf)-OH (Mw: 649)
5g 90 EUR, 1mmol 11.682 EUR
MBHA resin (0.4-1.2 mmol/g)
0.416 EUR
5g 49 EUR
Rink Amide MBHA resin (0.4-0.8 mmol/g) 5g 168 EUR
Cleavage of protecting groups (decapeptide): 15 EUR (Boc), 5 EUR (Fmoc)
DCM (for peptide synthesis) 49 EUR/L
DMF (for peptide synthesis) 111 EUR/L
However, application of Boc-strategy needs a special HF cleavage apparatus!
Many synthesizers are designed for Fmoc chemistry. They are TFA sensitive.
Ordering of piperidine might need allowance, because it is the starting material
in the synthesis of morphine.
Boc
 It is better for avoiding DKP formation;
 There is no problem with the Boc
cleavage, so it is better in case of
peptides that aggregate easily.
Aggregates are destroyed in every
TFA cleavage step;
 Because of the extra neutralisation
step, the synthetic cycle takes longer
time;
 Resins for Boc-strategy are available
for Fmoc-chemistry, too. Two steps
cleavage procedure may results in
better crude product. First step TFA
cleavage (side chain protecting groups)
then HF (peptide-resin bond). More
suitable for preparation of branched
peptides.
Fmoc
 ClTrt resin must be used to
prevent DKP formation;
 Incomplete Fmoc deprotection
in case of aggregating peptides;
 It is better for acid sensitive
peptides (Trp, Met), oxidation,
alkylation can be avoided. Asp-Pro
bond is highly acid sensitive.
 especially recommended for
O-glycosylated or sulfated
peptides;
 Because of the orthogonality of
Na and side chain protecting groups
fully protected sequences can be
prepared.