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

� Peptide isolation and purification

Applications of synthetic peptides

Immune peptides:synthetic antigens;

vaccinesdiagnostic tools

immunostimulator peptides;muramyl dipeptidetuftsin derivatives

Hormones:oxytocin

vasopressininsulin

somatostatinGnRHetc.

Neuropeptides:cholecystokininneurotensin

Antibiotics:tachikinin

gramicidine S

Toxins:conotoxinsspider toxinssnake toxins

ionchanel blockers

Enzymes and enzyme inhibitors:Ribonuclease A

Carriers:deliver diagnostic or therapeutic

agents to targeted cells

Transporter peptides:penetratinoligoarginine

HIV-Tat protein

Peptides for structural studies:

turn mimicking cyclic peptides

WHY PEPTIDES?WHY PEPTIDES?

Advantages of utilizingpeptides 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 recombinantmethods or by chemical modification of an isolatednatural product.

Peptides developed fortherapeutic 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� 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.

The idea:

T

P

AA1 RXanchoring

T

P

AA1 R

deprotection

P

AA1 R

T

P

AA2

coupling (-H2O)T

P

AA2

P

AA1 R

Solid phase peptide synthesis

R T P= resin = NH2 protectinggroup (boc, Fmoc)

= side chainprotecting group

T

P

AA2

P

AA1 Rdeprotection

coupling

repetitive cycle

T

P

AAn

P

AA2

P

AA1 R

AAn AA2 AA1

cleavage+

final deprotection

Fmoc strategy in SPPS

NHOHFmoc

R

O

R' Polimer

R' = OH, NH2, Cl

NHOFmoc

R

O

Polimer

Attachment

H2NO

R

O

Polimer

Deprotection

NHOActivatedFmoc

R"

O

CouplingNHFmoc

R"

O

NHO

R

O

Polimer

NH

Fmoc

R"

O

NHO

R

O

Polimern

Repeat deprotectionand coupling

Peptide and Polimer

Cleavage

Fmoc deprotection

HO

O

NH

O

OHO

O

NH

O

OH

NH

O

+ CO2 +

OHN

HN

NH

O

OH

NH2

O

OHH2N

N

H

+

_

+

+

H

Boc Strategy in SPPS

The Boc approach needs to use highly toxic liquid HF for cleavage from the resin.

NHOHBoc

R

O

HO PolimerNH

OBoc

R

O

Polimer

Attachment

H2NO

R

O

Polimer

Deprotection

NHOActivatedBoc

R'

O

CouplingNHBoc

R'

O

NHO

R

O

Polimer

NHBoc

R'

O

NHO

R

O

Polimern

Repeat deprotectionand coupling

Peptide and PolimerCleavage

Boc deprotection

R

HN

O

OR

HN

O

O

HH

R

HN

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

NH

NH2

NH

Arg

S

HN

O O

O

Pbf

OHAsp, Glu, Ser, Thr, TyrO

Asn, Gln, Cys, His XHX

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

ONHFmoc

R

HOBt esters

Coupling methods in Fmoc SPPS

In situ - activation method:

� Carbodiimides (DCC and DIC)

� Benzotriazole PF6 salts (HBTU, PyBOP, HATU)

N C N

Dicyclohexylcarbodimide (DCC)

N P

N

O

N

N

N N PF6-

PyBOP

N

N

N NPF6

-

O

+ _

N(CH3)2(H3C)2N +

HATU

N

N

NPF6

-

O

+ _

N(CH3)2(H3C)2N +

HBTU

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

Coupling using Diisopropylcarbodiimide

Mechanism of amidebond formation using PyBOP/HOBt/DIPEA

N P

N

O

N

NN N

N P

N

N

OR

O

N

FmocHN

R

OH

O FmocHN

R

O-

O

NN

N

O-

NH2

FmocHN

R

O

O

N

NN

FmocHN

R

NH

O

NH2

-OBt

PF6-

PO N -OBt

(DIPEA)

(PyBOP)

( )

3

Nu

+ +

Solid supports:

� Dry polystyrene beads have an average diameter of50 µm, but in DMF or DCM they swell 2.5 to 6.2 fold

in volume.

� SPPS takes place within a well solvated gel containingmobile and reagent accessible peptide chains.

� Small particle sized resins with low cross linkingallows rapid diffusion of reagents inside the beads.

� Solid support and peptide backbone may be ofcomparable 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

OOH

OHn

n

Tentagel

Resins for SPPS

Merrifield

Cl

ClCl

Cl

Resins for SPPS

O

O

HN

NMe2

OO

NH2O

HNO O

NH2O

HNO O

NH2

n

PEGA

HN

NovagelTM

NH2

O

OO

OCH3n

PEGA Bis 2-acrylamidoprop-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)

NH2OMe

MeO O P

Rink linker (trialcoxybenzhydrylamine)

O

O

PHN

O

OH

OHOCH3

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

PCl

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 undervacuum 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

OH

HNFmoc

R

O

O

OHNFmoc

R

5 eq.

0.1 eq. DMAP, DMF

HNFmoc

R

O

O

OHHNFmoc

R

O

OH

5 eq.

DCB (5 eq.), pyridine (8.25 eq.)

DCMHNFmoc

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

Attachment of 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 testhttp://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

AggregationPeptide 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)

EnantiomerizationDuring 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 reactionsRare 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

NH

O

NHNH

O

N

O

O

OH

NH

O

NH

O

NH

NH

O

OH

O

NH

O

NH

O

NH

NH

Nu

O

NuNu

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

NH

R'

R

N

HO

O

NR'

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 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

Liberty automated peptide synthesizer shown with single-mode

microwave reaction vessel

CEM Liberty