EMMI Intensive Programme "Design, Synthesis and Validation of Imaging Probes Torino, September 2011

Basic principles and procedures

of solid phase peptide synthesis

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

Torino, 19-30 September 2011

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

Applications of synthetic peptides Immune peptides: synthetic antigens; vaccines diagnostic tools immunostimulator peptides; muramyl dipeptide tuftsin derivatives Hormones: oxytocin vasopressin insulin somatostatin GnRH etc. Neuropeptides: cholecystokinin neurotensin Antibiotics: tachikinin gramicidine S Toxins: conotoxins spider toxins snake toxins ionchanel blockers Enzymes and enzyme inhibitors: Ribonuclease A Carriers: deliver diagnostic or therapeutic agents to targeted cells Transporter peptides: penetratin oligoarginine HIV-Tat protein Peptides for structural studies: turn mimicking cyclic peptides

WHY PEPTIDES? WHY PEPTIDES?

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:

Optimisation of reaction

conditions and yields.

Purification of every step. Time consuming.

Synthesis of small

peptides.

Useful for large-scale

manufacturing.

Solid phase synthesis:

Anchoring of the peptide

chain on a resin.

Use of excess reagents

removed by filtration and washing procedures.

Simple, rapid and repetitive

steps.

High yields.

Possible automation

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.

The idea:

T P AA1 X

R

anchoring _T P AA1

R

deprotection P AA1

R

T P AA2 coupling (-H₂O) T P AA2 P AA1

R

Solid phase peptide synthesis

R

= resin _T = NH₂ protecting _P

group (boc, Fmoc)

= side chain

T P AA2 P AA1

R

deprotection coupling repetitive cycle T P AAn P AA2 P AA1

R

AAn _AA2 _AA1 cleavage + final deprotection

Fmoc strategy in SPPS

NH OH Fmoc R O R' Polimer R' = OH, NH2, Cl NH O Fmoc R O Polimer Attachment H2N O R O Polimer Deprotection NH OActivated Fmoc R" O Coupling NH Fmoc R" O NH O R O Polimer NH Fmoc R" O NH O R O Polimer n Repeat deprotection and coupling

Peptide and Polimer Cleavage

Fmoc deprotection

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

Boc Strategy in SPPS

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

Peptide and Polimer Cleavage

Boc deprotection

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

Common AA side-chain

protecting groups used in

Fmoc SPPS

Side-chain fuctionality of amino acids Commonly used protecting group Abbreviation N H NH2 NH Arg S H N O O O Pbf OH

Asp, Glu, Ser, Thr, Tyr

O

Asn, Gln, Cys, His XH

X Lys, Trp NH(2) N O O (H) tBu Boc trt

Coupling methods in

Fmoc SPPS

Pre-activation method:

Pre-activation of the acid via

activated esters.

N-hydroxysuccinimide

(ONSu); slow formation of the amide bond.

Pentafluorophenyl (OPfp)

ester; in the presence of HOBt the rate of reaction is very rapid. FmocHN R O O F F F F F Pfp esters FmocHN R O O ONSu esters N O O N N N O O NHFmoc R HOBt esters

Coupling methods in

Fmoc SPPS

In situ

- activation method:

Carbodiimides (DCC and DIC)

Benzotriazole PF₆salts (HBTU, PyBOP, HATU)

N C N Dicyclohexylcarbodimide (DCC) N P N O N N N N PF6 -PyBOP N N N _N PF6 -O + _ N(CH3)2 (H3C)2N + HATU N N N PF6 -O + _ N(CH3)2 (H3C)2N + HBTU

The use of HOBt minimises the formation of unreactiveN-acylurea

Coupling using Diisopropylcarbodiimide

Mechanism of amide

bond formation using

PyBOP/HOBt/DIPEA

N P N O N N N N N P N N O R O N Fmoc HN R OH O Fmoc HN R O -O N N N O -NH2 Fmoc HN R O O N N N Fmoc HN R NH O NH2 -_OBt PF6 -P O N -OBt (DIPEA) (PyBOP) ( ) 3 Nu + +

Solid supports:

Dry polystyrene beads have an average diameter of

50

µ

m, 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).

Resin Composition Functional group

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

Tentagel Polystyrene-polyethylene glycol

graft polymer CH2OH, CH2NH2 O O O O OH OH n n Tentagel

Resins for SPPS

Merrifield Cl Cl Cl Cl

Resins for SPPS

O O HN NMe2 O O NH2 O HN O O NH2 O HN O O NH2 n PEGA HN NovagelTM NH2 O O On OCH3 PEGA Bis 1-ylPEG,

2-acrylamidoprop-1-yl[2-aminoprop-1-yl]PEG dimethylacrylamide co-polymer

CH2NH2

NovagelTM _{(4’-O-methylpolyethylene}

glycoxycarbonylamino-methyl)polystyrene

CH2NH2

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:

P OMe OH P OH (Hydroxymethylphenoxy) NH2 OMe MeO O P

Rink linker (trialcoxybenzhydrylamine)

O O P HN O OH OH OCH3 H3CO O P

Rink acid linker Sasrin linker HMBA linker Wang linker P OMe NH2 O OMe

PAL linker (trialcoxybenzylamine)

NH2

O P

Sieber linker (9-Aminoxanthenyl)

O P Cl Cl 2-Chlorotrityl chloride P NH O

Bayer's 4-carboxytrityl linker

HO Hydroxymethyl-based linkers Aminomethyl-based linkers Trityl chloride-based linkers

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

Attachment of 1

st

_{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 OH HNFmoc R O O O HNFmoc R 5 eq. 0.1 eq. DMAP, DMF HNFmoc R O O OH HNFmoc R O OH 5 eq.

DCB (5 eq.), pyridine (8.25 eq.) DCM HNFmoc R O O 1) 2)

Trityl based linkers:

Aminomethyl-based linkers:

Generally the amino function is Fmoc protected Standard piperidine deprotection and subsequent

coupling of the 1st amino acid

HNFmoc R O OH 1.2 eq 3 eq. DIPEA, DCM HNFmoc R O O HNFmoc R O OH 1.2 eq. HNFmoc R O O 1) 2) ClCl Toluene, AcCl 60°C 3h 3 eq. DIPEA, DCM HN O OH

Synthesis protocol:

Swelling of the resin

1

st

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 1

st

AA can be calculated

determining the absorbance at 290 nm

Possible problems

Aggregation

Peptide chains on resin can form secondary structures or aggregates either with other peptide chains or with the polymer support. Mainly caused by H-bonding and hydrophobic interactions (high proportion of Ala, Ile, Asn or Gln)

Enantiomerization

During coupling the acidity of the H atom on the αααα-carbon is enhanced

by the carboxy group activation. May occur on attachment of Cys, His, Pro, Met, or Trp on hydroxymethyl-based resins

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 X N H O NH N H O N O O OH N H O NH O NH N H O OH O N H O NH O NH N H Nu O Nu Nu Hydrolysis

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

N O O O N H R' R N HO O N R' R 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 Et

₂

O 3 times

Dry the solid in vacuo

Purification by HPLC

Characterisation by MALDI-TOF mass

Multisyntech SYRO II

Syro II automated peptide synthesizer

Liberty automated peptide synthesizer shown with single-mode microwave reaction vessel