EMMI Intensive Programme "Design, Synthesis and Validation of Imaging Probes“

Torino, 19-30 September 2011

Basic principles and procedures

of solid phase peptide synthesis

Lorenzo Tei, PhD

Dipartimento di Scienze dell’Ambiente e della Vita

Università del Piemonte Orientale “Amedeo Avogadro”
Viale T. Michel 11, 15121, Alessandria

Contents:

Introduction

Historical background

Different strategies for SPPS

Possible problems

Aminoacids and protections

Amide bond formation

Solid supports and linkers

Analytical tests

Peptide isolation and purification
 WHY PEPTIDES?
Immune peptides: Hormones:
synthetic antigens; oxytocin Neuropeptides:
vaccines vasopressin cholecystokinin
diagnostic tools insulin neurotensin
immunostimulator peptides; somatostatin
muramyl dipeptide GnRH
etc. Antibiotics:
tuftsin derivatives
tachikinin
gramicidine S

Transporter peptides: Applications of

penetratin synthetic peptides Toxins:
oligoarginine conotoxins
HIV-Tat protein spider toxins
snake toxins
Carriers: ionchanel blockers
deliver diagnostic
Peptides
or therapeutic
for structural studies: Enzymes and
agents to
turn mimicking cyclic peptides enzyme inhibitors:
targeted cells
Ribonuclease A

Advantages of utilizing
peptides as therapeutic
molecules:

High activity

High specificity

Unique 3D characteristics

No accumulation in organs

Low toxicity

Less immunogenic than antibodies

They can be made synthetically, by recombinant

methods or by chemical modification of an isolated
natural product.
 Peptides developed for
therapeutic applications:

Allergy/Asthma

Arthritis

Cancer

Diabetes

Cardiovascular diseases

Inflammation

Vaccines

Ophtalmology

Central Nervous System diseases

Antiviral

Antibacterial

Peptide synthesis:

Solution synthesis: Solid phase synthesis:

Optimisation of reaction
Anchoring of the peptide
conditions and yields. chain on a resin.

Purification of every step.
Use of excess reagents

Time consuming. removed by filtration and

Synthesis of small washing procedures.
peptides.
Simple, rapid and repetitive

Useful for large-scale steps.
manufacturing.
High yields.

Possible automation

Synthesis of long peptides.
 Beginnings of the SPPS

Merrifield in 1963 introduced the use of a

polystyrenic support for the synthesis of peptides.

Development of the Boc strategy (use of quite
strong acidic conditions)

In the ’70 different polymeric supports and more
labile protecting groups were developed.

In 1978 the Fmoc-based SPPS was introduced.

In the last 15 years the Fmoc approach has
prevailed over the Merrifield SPPS.

Solid phase peptide synthesis

The idea: P P

anchoring
T AA1 X R T AA1 R

deprotection
P
P P P
T AA2
T AA2 AA1 R AA1 R
coupling (-H2O)

R = resin T = NH2 protecting P = side chain

group (boc, Fmoc) protecting group
 P P

T AA2 AA1 R deprotection repetitive

cycle
coupling

P P P

T AAn AA2 AA1 R

cleavage
+
final deprotection

AAn AA2 AA1

Fmoc strategy in SPPS

R R
R' Polimer Attachment
Fmoc OH Fmoc O
NH
NH Polimer
O R' = OH, NH2, Cl O

Deprotection

O R R
NH O Coupling O
Fmoc NH Polimer H2N Polimer
R" O R" O
Fmoc OActivated
NH
Repeat deprotection
O
and coupling

O R
NH Cleavage
O
NH Polimer Peptide and Polimer
Fmoc n
R" O
 Fmoc deprotection

O O
OH OH
O N O N
H H H H H
H O O N+
N

O
_ OH
N O N
H H
H H2+ O
N
N
+
OH
+ CO2 + H2N
O

Boc Strategy in SPPS

R
R
Boc OH Attachment
NH HO Polimer Boc O
NH Polimer
O
O
The Boc
Deprotection approach
needs to use
NH
O R
Coupling
R
highly toxic
O O
Boc NH Polimer H2N Polimer liquid HF for
R' O
R' O
Boc OActivated
cleavage
NH
Repeat deprotection
from the
O
and coupling resin.
O R
NH Cleavage
O Peptide and Polimer
Boc NH Polimer
n
R' O
 Boc deprotection

H H
N O N O
R R
O O
H
H

R NH2 + CO2 H
N O +
R
+ H
O
H

Common AA side-chain
protecting groups used in
Fmoc SPPS
Side-chain fuctionality Commonly used Abbreviation
of amino acids protecting group

NH O
Arg H Pbf
N NH2 N
H S
O O

Asp, Glu, Ser, Thr, Tyr OH

tBu
O

Lys, Trp NH(2) N

(H)
O Boc

Asn, Gln, Cys, His XH

X trt
 Coupling methods in
Fmoc SPPS
Pre-activation method:
R O
O
FmocHN N

Pre-activation of the acid via O
activated esters. O
ONSu esters

N-hydroxysuccinimide R F
(ONSu); slow formation of O F
FmocHN
the amide bond.
O
F F

Pentafluorophenyl (OPfp) Pfp esters F
ester; in the presence of O
NHFmoc
HOBt the rate of reaction is
O
very rapid. R
N
HOBt esters N
N

Coupling methods in
Fmoc SPPS
In situ - activation method:

Carbodiimides (DCC and DIC)

Benzotriazole PF6 salts (HBTU, PyBOP, HATU)
(H3C)2N + N(CH3)2

N C N N
N PF6-
+N _
O
Dicyclohexylcarbodimide (DCC) HBTU

(H3C)2N + N(CH3)2
N
N
N P O N PF6- N PF6-
N N N
+N _ N
O
PyBOP HATU
 Coupling using Diisopropylcarbodiimide

The use of HOBt minimises the formation of unreactive N-acylurea

Mechanism of amide
bond formation using
PyBOP/HOBt/DIPEA
(DIPEA)
OH N
H O-
Fmoc N H
O Fmoc N N
O
R N P O N PF6-
R N N
N

(PyBOP)
N N O-
N N
O
H N
Fmoc N N
O
( -OBt )
R

N O
N P O
NH2 R
N
NH2

Nu

NH
H
Fmoc N
O
R

+ O P N + -OBt
3
 Solid supports:

Dry polystyrene beads have an average diameter of

50 µm, but in DMF or DCM they swell 2.5 to 6.2 fold
in volume.

SPPS takes place within a well solvated gel containing
mobile and reagent accessible peptide chains.

Small particle sized resins with low cross linking
allows rapid diffusion of reagents inside the beads.

Solid support and peptide backbone may be of
comparable polarities (polyamides, PEG).

Resins for SPPS

Resin Composition Functional group

Chloro/Aminomethyl 1% divinylbenzene cross-linked CH2Cl, CH2NH2,
polystyrene polystyrene benzhydrylamino

Tentagel Polystyrene-polyethylene glycol CH2OH, CH2NH2

graft polymer

O
Cl O n OH

Cl

O
Cl Cl O n OH

Merrifield Tentagel
 Resins for SPPS

PEGA Bis 2-acrylamidoprop-1-ylPEG, 2-acrylamidoprop- CH2NH2

1-yl[2-aminoprop-1-yl]PEG dimethylacrylamide
co-polymer
NovagelTM (4’-O-methylpolyethylene glycoxycarbonylamino- CH2NH2
methyl)polystyrene

O
NMe2
O O
O HN O
HN O NH2
O n O n OCH3

HN O
O O
NH2
NH2
HN
O O
NH2

PEGA NovagelTM

Linkers:

The purpose of the linker are:

provide a reversible linkage between the synthetic peptide
chain and the solid support.

Protect the C-terminal α-carboxyl group during the process
of chain extension.

Most linkers release peptide acids or amides upon
treatment with TFA.

Some linkers permit the cleavage with nucleophiles, used
for the preparation of C-terminal modified peptides such as
esters secondary amides, aldehydes, alcohol.
 Linkers:
Hydroxymethyl- Aminomethyl-based Trityl chloride-
based linkers linkers based linkers
OH
OMe NH2

O
Cl P

P Cl
MeO O P
Wang linker
(Hydroxymethylphenoxy) Rink linker (trialcoxybenzhydrylamine)

OMe 2-Chlorotrityl chloride

OMe
P O P O

OH
NH2 P
Sasrin linker OMe
NH
PAL linker (trialcoxybenzylamine) HO

H OH O
P N NH2

HMBA linker Bayer's 4-carboxytrityl linker

O O P
OCH3 OH

Sieber linker (9-Aminoxanthenyl)

H3CO O P

Rink acid linker

Resin handling procedures

Apart from PEGA resin, all resins have to be

swollen before use.

Underivatized polystyrene resins swell only
in dichloromethane.

All the other are swollen in DMF.

Resins (except PEGA) can be dried under
vacuum without damage.

Resins are fragile and has to be handled
carefully (do not use magnetic stirrers!)
 Attachment of 1st amino acid
For hydroxymethyl based linkers quite harsh
conditions has to be employed:
1. use of the symmetrical anhydride of the Fmoc
amino acid and DMAP in DMF.
2. Use of the Fmoc amino acid and dichlorobenzoyl
chloride in DMF/pyridine
HNFmoc O

R 0.1 eq. DMAP, DMF

1) O R O
R
OH
HNFmoc O
HNFmoc O
5 eq.

OH
R DCB (5 eq.), pyridine (8.25 eq.) O
2) R
HNFmoc O DCM OH
HNFmoc O
5 eq.

Attachment of 1st amino acid

Trityl based linkers:
OH
R 3 eq. DIPEA, DCM
1) R O
HNFmoc O
Cl HNFmoc O
1.2 eq
Cl

O Toluene, AcCl O
3 eq. DIPEA, DCM R
2) HN
OH
60°C 3h OH
R HNFmoc O

HNFmoc O
1.2 eq.

Aminomethyl-based linkers:

Generally the amino function is Fmoc protected

Standard piperidine deprotection and subsequent
coupling of the 1st amino acid
 Synthesis protocol:

Swelling of the resin

1st Fmoc amino acid attachment

Deprotection Fmoc group with piperidine in DMF

Washings with DMF (between 5 to 10 times)

Fmoc amino acid coupling (Activators/DIPEA/DMF)

Washings with DMF

Deprotection Fmoc and so on…

After last coupling the resin has to be dried in
vacuo before cleavage

Analytical tests

Qualitative amine tests:

Kaiser test (ninhydrin test)
http://www.natureprotocols.com/2007/10/24/monitoring_solid_phase_peptide.php

TNBS test

Chloranil test
http://www.chempep.com/ChemPep-Fmoc-Solid-Phase-Peptide-Synthesis.htm

Fmoc determination for estimation of first residue

attachment:

The loading of the 1st AA can be calculated
determining the absorbance at 290 nm
(dibenzofulvene formed in the Fmoc deprotection)
 Possible problems

Aggregation
Peptide chains on resin
can form secondary Enantiomerization
structures or aggregates During coupling the acidity of the H
either with other peptide atom on the α-carbon is enhanced
chains or with the by the carboxy group activation.
polymer support. Mainly May occur on attachment of Cys,
caused by H-bonding and His, Pro, Met, or Trp on
hydrophobic interactions hydroxymethyl-based resins
(high proportion of Ala,
Ile, Asn or Gln)

Side reactions
Rare if the AA are protected and the
correct procedures are followed.
Aspartimide formation can occur if Asp
is coupled to Gly, Ser, Asn, Thr or Arg.

Aspartimide formation
O
O Nu O
X Nu
O NH
NH N
N N NH O
H H N N
O O H H
O Nu

Hydrolysis

O O

OH NH
NH OH
N N
H H
O O

Base catalyzed aspartimide formation involves attack by the nitrogen atom

attached to the α-carboxy group of either aspartic acid or asparagine on the
side chain ester or amide group, respectively.
Nucleophilic attack then causes subsequent ring opening, which gives rise
to a mixture of α-aspartyl and β-aspartyl peptides.
 Diketopiperazine formation

Peptides containing proline or N-alkylated amino acids

attached via benzyl ester to the resin may have the
problem of diketopiperazine formation

This not only causes a yield reduction but may also lead
to the generation of truncated sequences.

In these cases is better to use a more hindered trityl resin
R R
O O
R'
N R'
H N
N N
O HO
O
O

Cleavage from the resin

95% TFA for 1-3h cleaves the peptide from the
resin and deprotect t-butyl groups, Pbf group from
Arg, Trt group from Cys, Gln, Asn and His.

Highly reactive cationic species are generated that
can react with amino acids containing electron rich
functional groups: Tyr, Trp, Met and Cys.

Nucleophilic reagents (scavengers) are added to the
TFA to quench these ions: water, 1,2-Ethandithiol
(EDT), triisopropylsilane (TIS), thioanisole.
 Cleavage cocktails

If Cys is absent we normally use TFA, TIS 95:5 v/v.

With Cys we add EDT (TFA, TIS, EDT; 95:3:2 v/v).

With Met we add thioanisole (TFA, TIS, Thioanisole; 95:3:2 v/v)

Reagent K (TFA, thioanisole, H2O, phenol, EDT; 82.5:5:5:5:2.5
v/v).

Reagent R (TFA, thioanisole, anisole, EDT; 90:5:3:2).

With Chlorotrityl chloride resins protections on the AA side-
chains can be mantained using DCM, AcOH, trifluoroethanol
80:10:10 v/v.

With Trp a further step with a 5% water solution of AcOH is
required to obtain the fully deprotected peptide.

Peptide isolation and purification

Precipitated by addition of cold diethyl ether

Directly from TFA solution or following
evaporation of TFA and volatile scavengers.

Centrifuge and wash with Et2O 3 times

Dry the solid in vacuo

Purification by HPLC

Characterisation by MALDI-TOF mass
spectrum, NMR spectrometry.
 Multisyntech SYRO II

Syro II automated peptide synthesizer

CEM Liberty

Liberty automated peptide synthesizer shown with single-mode

microwave reaction vessel