1. Solid phase peptide synthesis Application of Fmoc/tBu strategy
Part II
Application of Fmoc/tBu strategy
Gábor Mező
Research Group of Peptide Chemistry
Hungarian Academy of Sciences
Eötvös L. University
Budapest, Hungary

2. Outline Resins; Protecting groups; Synthetic protocol; Monitoring;
Cleavage technics;
Side reactions;

3. Fmoc/tBu: .. tert-butyl TFA Fmoc Wang-resin piperidineH C 2 O 3 R
Fmoc
tert-butyl ..
piperidine
TFA
Wang-resin
Fmoc-Asp(OtBu)-Tyr(tBu)-Wang resin

4. 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: C H 2 O P HO
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 !

5. SASRIN (Super Acid Sensitive ResIN)
(2-methoxy-4-alkoxybenzyl-alcohol resin) C H 2 O P HO 3
Peptide is cleavable with
% TFA in DCM
resulted in protected
peptide fragments.
OOH
4-Hydroxymethylphenoxyacetic acid (HMPA) linker:
Attach to aminomethyl PS-DVB resin
(CH2)3
4-(4-Hydroxymethyl-3-methoxyphenoxy)butyric acid (HMPB) linker:
Removal of the peptide with TFA
Removal of the peptide with diluted TFA

6. 2-Chlorotrityl chloride (ClTrt) resin:P
Attachment of the first amino acid:
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!
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

7. NH2(mmol/g) = [A301.V(ml)/e301.m(mg)].106
(Calculation of the resin capacity)
Determination of loading
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 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 1. 2.
ca. 4-6 mg Fmoc-Aaa-resin ca. 2 mg Fmoc-Gly-OH
+400 mL 50% piperidine/DMF mL 50% piperidine/DMF
30 min at RT, then filtration min at RT
dilute with MeOH to 25 mL dilute with MeOH to 25 mL
Capacity of the resin (mmol/g) =
1000.mgly.Aresin
301
Mgly.mresin.Agly
Mgly =297

8. Rink Amide Resin: synthesis of peptides with CONH2 C-terminus3
Fmoc-H
OCH2-P OC CO
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.
Peptide-resin bond can be detached
with 5% TFA. Removal of protecting
groups needs a separate step.
OCH2-CO-Nle-R
Rink Amide-AM and Rink Amide-MBHA resins:
Aminomethyl-PS-DVB
4-methylbenzhydrylamine-PS-DVB
Peptide cleavage with 90-95% TFA solution.
Nle is a reference for quantitation.

9. 4-hydroxymethylphenoxyacetic
Pegylated resins: composition of polyethylene glycol (Mw: )
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 ( 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)
PEG
NH2 C H 2 O HO OH
4-hydroxymethylphenoxyacetic
acid linker
NovaSyn R TGA resin
Similar to Wang resin

10. 4-carboxytrityl linker NovaSyn R TGT alcohol resin
Before use the resin must be converted
to the chloride form by heating with
AcCl or SOCl2 in toluene.
Similar to ClTrt resin. H N H2
OCH2 OC 3 CO
2,4-dimethoxy-benzhydryl linker
NovaSyn R TGR resin
Similar to Rink Amide MBHA resin

11. Applied side chain protecting groups in Fmoc-chemistry
-OH (Ser, Thr, Tyr)
Side chain functional group protecting group name (abbreviation) C H 3
tert-butyl (tBu)
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)
trityl (Trt) 2 NH O
acetamidomethyl
(Acm)
For selective deprotection

12. 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: C H 2 O P N
Acm-S
piperidine
H2O HO
Cys Mcalc
DAla Mcalc – 34
DL-Ala(Pip) Mcalc+ 41
DL-Ser Mcalc – 16

13. Side chain functional group protecting group name (abbreviation)
tert-butyloxycarbonyl
(Boc)
eNH2 (Lys) O C H 3
Selectively removable protecting groups for preparation of modified
peptides (labeled, functionalised, branched or cyclic peptides):
4-methytrityl
(Mtt)
Mtt can be removed selectively with 1%TFA/DCM solution in
the presence of 3-5% TES (triethylsilane) at RT in min.
Trt groups may be not stable enough under this condition.

14. ylidene)-3-methylbutyl
Side chain functional group protecting group name (abbreviation)
eNH2 (Lys) O C H 3 R
R = metil1, isopropyl2
1-(4,4-dimethyl-
2,6-dioxocyclohex-1-
ylidene)ethyl (Dde)1
ylidene)-3-methylbutyl
(ivDde)2
ivDde is more stable in basic cleavage mixture
applied for Fmoc removal than Dde.
Both protecting groups can be removed with 2% NH2-NH2 in DMF 2
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.

15. wCOOH (Asp, Glu)
Side chain functional group protecting group name (abbreviation) O C H 3
tert-butyl ester (OtBu)
Selectively removable protecting groups for preparation of cyclic peptides:
(pairs of amino and carboxyl protecting groups: Dde-ODmab, Aloc-OAll) N 2
4-{N-[1-(4,4-dimethyl-
2,6-dioxocyclohexylidene)
-3-methylbutyl]-amino}
Benzyl ester
(ODmab)
Similarly to Dde and ivDde, the Dmab protecting group can be removed
with 2% hydrazine in DMF.
allyl ester (OAll)
It can be removed in P(Ph)3 by Pd(0) catalysis.

16. 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:
-Asp-Gly- N H C O 2
tBu
-Asu-Gly-
- tBuOH
piperidine
M = Mcalc+ 57

17. Fmoc-(Fmoc-Hmb)Gly-OH
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) N H C O 2
tBu 3 HO
(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.
Fmoc-(Fmoc-Hmb)Gly-OH
1g = 370 EUR (NovaBiochem)
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.

18. Side chain functional group protecting group name (abbreviation)
wCONH2 (Asn, Gln)
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. N H
(His) p t
imidazol group
tert-butyloxymethyl
(Bum) (p) C 2 O 3
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 !

19. Side chain functional group protecting group name (abbreviation)
(His) p t
imidazol group
trityl (Trt)
(t)
Trt group protects the tN. However, its application prevents both
epimerisaton (not in case of attachment to resins) and alkylation.
-NH-C-NH2 NH
guanidino group C 3 S O
4-methoxy-2,3,6-
trimethylbenzene-
sulfonyl (Mtr)
(Arg)
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.

20. Side chain functional group protecting group name (abbreviation)
-NH-C-NH2 NH
guanidino group
(Arg)
2,2,5,7,8-pentamethyl-
chroman-5-sulfonyl
(Pmc)
2,2,4,6,7-pentamethyl-
dihydrobenzofurane-
6-sulfonyl (Pbf) S O C H 3
Pmc can be cleaved with TFA in 2-3 hrs, but Pbf protecting group can
be removed 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 !

21. tert-butyloxycarbonyl
Side chain functional group protecting group name (abbreviation)
indole N H
(Trp)
tert-butyloxycarbonyl
(Boc) O C 3
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.

22. Synthetic protocol of Fmoc-strategy
Wash the resin 3x with DMF; min each
Cleavage of Fmoc protection with
2% piperidine + 2%DBU/DMF; min*
Wash the resin 8x with DMF; min each**
Coupling: Fmoc-amino acid derivative-DIC-HOBt in DMF***
(3 equiv each calculated to the resin capacity); 60 min
Wash the resin 2x with DMF; min each
Wash the resin 2x with DCM; min each
Ninhydrin monitoring
Synthetic protocol of Fmoc-strategy
(-) yellow
(+) blue
* 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.

23. Coupling agents + CH3 HC CH N C H3C NH O X-NH R OBt
N,N’-diisopropylcarbodiimide (DIC, DIPCDI))
Coupling agents N C
N,N’-dicyclohexylcarbodiimide (DCC) CH HC
CH3
H3C NH O
X-NH R
X: Boc, Fmoc
O-acyl-isourea derivatives
N-acyl-urea derivatives
O-N acyl shift
urea derivatives: DCU, DIU
OBt
HOBt
in situ active ester +

24. > N OH O PF6- P+ (CH3)2N N(CH3)2 1-hydroxybenzotriazole (HOBt)
1-hydroxy-7-aza-benzotriazole
(HOAt) O P+
(CH3)2N
N(CH3)2
PF6-
benzotriazol-1-yl-oxy-tris(dimethylamino)-
phosphonium hexafluoro phosphate (BOP)
benzotriazol-1-yl-oxy-
tris(pyrrolidino)phosphonium
hexafluoro phosphate (PyBOP)
They don’t need
DCC or DIC for
preparation of
in situ active ester
Hexamethylphosphoramide (carcinogen)!
>
AOP
PyAOP

25. According to NMR and röntgen diffraction studies a new structure
2-(1H-benzotriazol-1-yl)-1,1,3,3,-
tetramethyluronium hexafluorophosphate
HBTU N O C+
(CH3)2N
N(CH3)2
PF6-
BF4-
tetramethyluronium tetrafluoroborate
TBTU + -
N-[(1H-benzotriazol-1-yl)(dimethylamino)-
methylene]-N-methanaminium
hexafluorophosphate N-oxide
According to NMR and röntgen
diffraction studies a new structure
was suggested:
HATU, TATU, HBPyU, HAPyU, etc.

26. Guanylation with uronium type coupling reagents
(CH3)2N
N(CH3)2
PF6- C CH
HNH R
NH-PEPTIDE + NH
N+(CH3)2
+ 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.

27. contain N-terminal Fmoc group?
Fmoc cleavage flow chart
Does the peptide
contain N-terminal Fmoc group?
yes no
Remove Fmoc
contain Arg, Met, Trp or Trt?
contain Arg, Met?
Use cleavage mixture A
Use cleavage mixture B
contain Trp or Trt?
Use cleavage mixture C
A: 0.5 mL d.i. water
9.5 mL TFA
C: 0.25 mL EDT
0.25 mL d.i. Water
9.50 mL TFA
B: 0.75 g cryst. phenol
0.25 mL EDT
0.50 mL thioanisole
0.50 mL d.i. water
10 mL TFA

28. 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 EUR
Fmoc-Ala-OH (Mw: 311) 5g 11 EUR, 1mmol EUR
Boc-Arg(Tos)-OH (Mw: 429) 5g 32 EUR, 1mmol EUR
Fmoc-Arg(Pbf)-OH (Mw: 649) 5g 90 EUR, 1mmol EUR
MBHA resin ( mmol/g) g 49 EUR
Rink Amide MBHA resin ( 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.

29. Boc Fmoc 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.
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.