protocols in SPPS- Side Chain Reactions.

1. Presented by:
SantoshSantosh KumarKumar SahooSahoo
MC-Ph. D/2016/08

2. Protecting groups
Strategies
Comparison
Procedure
Approaches in synthesis
Protocol
Side reactions
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Flow of Presentation

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Solid Phase Peptide Synthesis
Principle

4. Transient protecting groups
for amino groups that
form the peptide bond
Permanent protecting groups
for functional groups within
the amino acid side chains
tBoc Fmoc
1963: Merrifield
acid labile, Nα-protecting group
1970: Carpino & Han
a base labile Nα-protecting group
Synthesis, 1979, 955.Biochemistry, 1964, 3, 1385.
Protecting groups
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Transient protecting groups
tBoc Protection

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tBoc Protection
tBoc deprotection

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Fmoc
protection
Fmoc deprotection
Based upon the graduated acid lability of
the side-chain protecting groups.
In this approach,
• Boc group is removed by neat TFA or TFA in DCM
• Side-chain protecting groups
• peptide–resin linkages
tBoc/ Bzl Fmoc/tBu
Removed by strong
acid like HF
•Use of highly toxic HF
•Need for PTFE-lined apparatus
•Specialists job
•Use of strongly acidic conditions
can damage peptides
In this approach,
•Base labile Fmoc group is used for protection
•Acid labile side-chain protecting groups
•Acid labile linkers that constitute the C-terminal
amino acid protecting group
Advantage:
 Temporary / permanent orthogonal protections
are removed by different mechanisms allowing the
use of milder acidic conditions for final deprotection
and cleavage of the peptide from the resin.
For all these reasons, Fmoc-based SPPS
Method is now the method of choice for
the routine synthesis of peptides
Based upon an orthogonal
protecting group strategy
But not generally used, Why?
Strategies
Mol. Biotech., 2006, 33, 239-54.

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Boc / Bn → Based upon the graduated acid
lability of the side-chain protecting groups.
Fmoc / tBu → Based upon an orthogonal
protecting group strategy
niper_H
Strategies

8. 8
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Comparison at synthesis level

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Resin + BOC AA (Cs/ TEA salt)
Carboxamide resin by DCC/DMAP
Benzoylation / acetylation of
unreacted –OH group of PAM
BOC deprotection by TFA / DCM
+ Dithioerythritol (DTE)
scavenger for Cys, Met, Trp
Washing DCM, IPA
AA as Trifluoro acetate
+ TEA / DCM neutralization
Washing DCM, IPA
Coupling of activated BOC AA
Deprotection, Neutralization, coupling
Dinytrophenylethyl (DNPE)
PG of His, Formyl PG of Trp
removed by Piperidine / DMF
Cleavage from resin by HF
Anisole, Thiocresol, Dimethyl
sulfide as Scavenger for side
chain PG alkylating agent
BOC-SPPS

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PAM resin
Phenyl acetamido
methyl linker
BHA resin MBHA resinMerrifield resin
Partial cleavage during
deprotection of BOC
Balances Stability to TFA, lability to HF
N
H
R
O
H
N
O
H+
HO X
N
H
R
HO
HN
O
O X
H+
N
H
R NH3
O
XO O
X = H or CH3
Acid catalyzed acyl
NO migration
Side Reactions for BOC-PSSP
Resins BOC-SPPS
Diketopiperazine
Formation
Protonated N → Less prone
Reversed by Base
Aspartamide Formation
Peptide having
Asp-Gly
Asp-Ala
Asp-Ser

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HN
NH
Asp-Pro cleavage with HF
Homoserine Lactone Formation
C-terminal Met
cyclize to
homoserine lactone
γ-COOH lose water in acid
↓
acylium ion
↓
cyclization
Side chain involving Glu

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Resins for peptide amide synthesis.
Resins for peptide acid synthesis.
(Wang) resin:
Fmoc-Aaa(X)-OH:DIC:DMAP 2:2:0.2
equiv to the resin OH content) in DMF
diketopiperazine formation side reaction
SASRIN (Super Acid Sensitive ResIN)
Peptide is cleavable with
0.5-1.0% TFA in DCM
1 g ClTrt-resin + 2 mmol Fmoc-Aaa(X)-OH
+ 8 mmol DIEA in 3-5 mL DCM, for 1.5 h
0.8 mL MeOH to block unreacted groups
washing with DCM, iPrOH, MeOH, ether
prevents the diketopiperazine formation
Attachment of Cys and His derivative
to the resin is free from enantiomerisation
Rink Amide-AM (Aminomethyl) and
Rink Amide-MBHA (4-methylbenz
hydrylamine) resins.
Fmoc-SPPS

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Linkers are bifunctional molecules anchoring the
growing peptide to the insoluble carrier. Linkers may
be coupled to any carrier suitable for SPPS, an
important option if alternatives to polystyrene-based
resins (PS-DVB) have to be considered.
Ramage linker
Rink linker
HMP linker
4-Formyl-3-methoxy
Phenoxyacetic acid
LinkersFmoc-SPPS

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Mechanism of base-catalyzed
racemization during activation.
More potent coupling reagents such as HATU or very active Fmoc
amino acid derivatives such as the acid fluorides may drive the
coupling to completion.
Path A
Base
O
X
H
N
O
R'
BH+
O
X
H
N
H RO
R'
R
O
N
H
H
N
H RO
R'
H2N
Coupling Reagents

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O
OR1
N
N
R' R'
H
Carbodiimide
N
H
N
R'
R'
O
O
R1
O-acylisourea
N
N
DCC
O
OXtR1
HOXt
HOXt
R2
-NH2
N
N
N
OHHOBt
Problems with Carbodiimides Coupling Reagents

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Carbodiimide reagents:
Additives:
N N N N N N
N
HCl
N N N N
N NN N
N
N
O
O
O
O
N NN N
DCC DIC EDC CIC BMC
BEMCPC BDDC PECPIC
N
N
N
OH
HOBt
N
N
N
OH
O2N
6-NO2-HOBt
N
N
N
OH
F3C
6-CF3-HOBt
N N
N
N
OH
HOAt
N
N
N
OH
Cl
6-Cl-HOBt
N
N
N
O
OH
HODhbt
N
N
N
N
O
OH
HODhat
N
N
OH
Ph
HOBI
N
O
O
OH
HOSu
N
NC
OH
O
O
Oxyma

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HOXt-based coupling reagents

18. Resin beads + DCM filtration
under vacuum Wash reaction vessel, resin with
DMF → MeOH → DCM→ DMF
Desired Fmoc-amino-acid in dry DCM
+ DMAP in DMF
↓
Cap the remaining hydroxyl groups
by adding benzoic or acetic anhydride
And pyridine in DMF.
Mol. Biotech., 2006, 33, 239-54.
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Protocol 1: Resin swelling
Protocol 3: Attachment to hydroxy
methyl based resin
Protocol 2: Standard
washing procedures
Fmoc amino acid + DIPEA in dry DCM
↓
Wash the resin with DMF
↓
+ DCM/MeOH/ DIPEA (80:15:5) to cap
remaining reactive chloride group.
↓
Wash with DMF and DCM
After drying in vacuum, the
Substitution can be measured from
Fmoc release.
Protocol 4: Attachment to
trityl based resin
Fmoc-SPPS

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Loading 2-chlorotrityl
chloride resin.
capping of 2-chlorotrityl
chloride resin.
Loading Rink
Amide resin.
Capping unreacted sites
on Rink Amide resin.

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X X = Cl, OH, NH2
N
H
O
OH
R1
PG1
Fmoc
N
H
O
O(NH)
R1
PG1
Fmoc
piperidine
H
N
H2N
O
O(NH)
R1
PG1
Fmoc
H
N
O
OH
R2
PG2
coupling
reagent
Fmoc
H
N
O
O
R2
PG2
Y
Y: activating group
N
H
O
O(NH)
R1
PG1
Fmoc
H
N
O
R2
PG2
Fmoc deprotection
Fmoc-Amino acid coupling
repetitive Fmoc deprotection and AA coupling
N
H
O
O(NH)
R1
PG1
H
N
O
R2
PG2
Peptide segment
PG3
PG4
O
PGn-1
H2N
Rn
PGn
TFA/Scavenger
N
H
O
OH(NH2)
R1
H
N
O
R2
Peptide segment
O
H2N
Rn
Loading
Cleavage and Global deprotection
Peptide Assembly
Fmoc-SPPS

21. wash the resin with DMF + N-α Fmoc protected amino acid
↓
+ HBTU / HCTU coupling → filtration → Wash the resin.
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Protocol 5: Standard coupling procedure
Activating amino acid
Coupling amino acid
Swell the resin in DCM → filtration+ 50/50
DCM/acetic anhydride solution Remove
the capping solution by filtration → Wash
with DCM Check the disappearance of free
amino groups by colorimetry
Resin is washed once with DMF
↓
80/20 DMF/piperidine solution
↓
Filtration → Wash the resin.
Protocol 6: Capping Protocol 7: Removal of
Nα Fmoc protection

22. (Scavengers) Trifluoroacetic acid/water/triisopropyl silane 95/2.5/2.5
per 100 mg of resin Filter + MTBE to precipitate the peptide. Solubilize the
peptide in acetonitrile/water/ TFA 50/50/0.1 and lyophilize.
Dissolve the lyophilized peptide in a 5% acetic acid solution
+ 10% volume of DMSO pre-adjusted pH to 6.0 - 7.0 with a 0.5 M NH4OAc
Disulfide bridge formation should be monitored by reverse phase HPLC.
Dissolve linear peptide in buffer (0.1–0.2 M Tris-HCl, pH 7.7–8.7)
+ 1 mM EDTA and reduced (1–10 mM) and oxidized (0.1–1 mM) glutathione
Lyophilize the solution after acidification with a TFA solution (pH 2.0)
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Protocol 8: Standard TFA cleavage
Protocol 9: Standard formation of disulfide bridges by air oxidation
Protocol 10: Formation of disulfide bridges using oxidative folding in redox buffer

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Cleaving a side-chain protected peptide form
2- chlorotrityl chloride resin with HFIP
Formation of disulde bond with DMSO.

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Side-chain Protecting Groups for Fmoc Amino Acids
Acm: acetamidomethyl
Al: allyl
Aloc: allyloxycarbonyl
Boc: t-butyloxycarbonyl
Fmoc: 9-fluorenyl methoxycarbonyl
Mbh: 4,4-dimethyloxybenzhydryl
Mtr: methoxytrimethyl benzene sulfonyl
OAl: allyl ester
OtBu: t-butyl ester
Pbf: 2,2,4,6,7-pentamethyl-
dihydrobenzofuran-5-sulfonyl
Pmc: 2,2,5,7,8-pentamethyl-
chroman-6-sulfonyl chloride
tBu: t-butyl ether
Tmob: 2,4,6-trimethoxybenzyl
Trt: trityl or triphenyl

25. Diketopiperazine formation after
deprotection of the penultimate amino acid
N-Terminal guanidinylation
by the coupling reagent
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Reaction occurs during couplings
mediated by uronium/ aminium
reagents or carbodiimides. Avoided
by preactivation of the amino acid
(i.e. the coupling reagent is
consumed). The side reaction can’t
occur when activating with
phosphonium salts (Bop, PyBop).
By using 2-chlorotrityl chloride
resin or other bulky resins such
as DHPP-Resin.
By coupling the appropriate
Fmoc-dipeptide in lieu of the
penultimate amino acid.
By coupling the appropiate
Trt-amino acid → Deblocking
with dilute TFA yields the
protonated dipeptide
Fmoc-SPPS – Side chain reactions

26. Base-catalyzed aspartimide
Formation and subsequent reactions.
Chem. Commun., 1994, 20, 2343–2344.10/26/2016 26

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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)
NH CH C
O
N CH2 C
O
CH2
C OtBu
O
(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
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.

28. N-O shift involving
serine or threonine
Base-induced β-elimination
of C- terminal cysteine
J. Pept. Sci., 2005, 11, 441.10/26/2016 28niper_H
Reversal is induced by
bases, e.g. aq. NH3.
This side reaction is minimized (but not
avoided!) when trityl is used for the
protection of the C-terminal Cys.
Deguanidination side reaction on Arg
If the guanidino moiety from Arg side chain is acylated by amino
acid derivatives, it could be decomposed into Orn side product.

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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
N and side chain protecting groups
fully protected sequences can be
prepared.
Comparision

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