90% TFA solution for effective removal.
-SH (Cys)
trityl (Trt)
For selective deprotection
CH2 NH C CH3 O
acetamidomethyl (Acm)
Racemisation during the attachment of Cys derivatives to the resins
C H3 Trt group can be used if on-resin derivatization is required (glycosylation,
phosphorylation). Trt can be cleaved with diluted TFA, while tBu needs
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.
Applied side chain protecting groups in Fmoc-chemistry
Side chain functional group protecting group name (abbreviation)
-OH (Ser, Thr, Tyr)
CH3 H3C C
tert-butyl (tBu)
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:
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.
Outline
Resins; Protecting groups; Synthetic protocol; Monitoring; Cleavage technics; Side reactions;
Fmoc/tBu:
Fmoc
tert-butyl
H3C
H3C H3C C CH3
OCH2-P
Rink Amide-AM and Rink Amide-MBHA resins:
OCH3
H3CO Fmoc-HN CH
OCH2-CO-Nle-R
H2N CH2
P
Aminomethyl-PS-DVB
CH3
Peptide cleavage with 90-95% TFA solution.
enantiomerisation!
Determination of loading
(Calculation of the resin capacity) 1.
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);
peptides (available for fragment condensation).
ClTrt resin prevents the diketopiperazine formation ! Attachment of Cys and His derivatives to the resin is free from
Similar to Wang resin
4-carboxytrityl linker
O
NovaSyn R TGT alcohol resin
HO
C OH Before use the resin must be converted
to the chloride form by heating with
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
Solid phrt II
Application of Fmoc/tBu strategy
Gábor Mező
Research Group of Peptide Chemistry Hungarian Academy of Sciences Eötvös L. University Budapest, Hungary
AcCl or SOCl2 in toluene. Similar to ClTrt resin.
OCH3 H3CO
H2N CH
2,4-dimethoxy-benzhydryl linker NovaSyn R TGR resin
Similar to Rink Amide MBHA resin O
OCH2 C OH
ca. 2 mg Fmoc-Gly-OH +400 mL 50% piperidine/DMF 30 min at RT dilute with MeOH to 25 mL
Capacity of the resin (mmol/g) =
1000.mgly.A3r0es1 in Mgly.mresin.A3g0ly1
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
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
OCH3
the linker
Cl Cl
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
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 CH2
O CH2
P
Acm-S CH2 O
SASRIN (Super Acid Sensitive ResIN) (2-methoxy-4-alkoxybenzyl-alcohol resin)
HO CH2
O CH2
P
CH3O
Peptide is cleavable with 0.5-1.0% TFA in DCM resulted in protected peptide fragments.
The basic polymer support is aminomethyl PEG-PS-DVB (NovaSyn R TG) NH2 PEG
HO C H2
O O CH2 C OH
4-hydroxymethylphenoxyacetic acid linker
NovaSyn R TGA resin
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.
HO CH2 CH3O
O (CH2)3 C OOH Attach to aminomethyl PS-DVB resin
Removal of the peptide with diluted TFA
2-Chlorotrityl chloride (ClTrt) resin: