Short Peptide Synthesis

Short Peptide Synthesis

Keith ó Proinsias

Introduction

• Amide bond and basic amide synthesis

• Solution phase peptide synthesis

• Protecting groups required for peptide synthesis

• Coupling reagents used in peptide synthesis

The Amide Bond

H₂N R' + R COOH _R O H N R -H₂0

• Basic amide synthesis is the reaction of a carboxylic

acid and an amine with the loss of water.

• Some of the main properties of the amide bond is it’s

low basicity, which is useful in purification, and it’s

stability, due to resonance.

O N H O N H

Basic Synthesis

• At the beginning acid chlorides where used.

•

Not good for peptide synthesis mainly due to

racemisation occurring.

R OH O SO₂Cl R Cl O + H₂N R' R N H O R' + HCl

Advanced Organic Chemistry- Reaction Mechanisms by Brukner, R., 2002, 216

Solution Phase Peptide Synthesis

H₂N COOH H2N COOPG R R PGHN COOH R' PGHN R' O H N COOPG R H2N R' O H N COOH R H2N R' O H N COOPG R PGHN COOH R' N H R' O H N COOPG R O PGHN R N H R' O H N COOH R O H2N R Coupling Deprotection Dimer Triemer Acid Protection Coupling

Planning For Solution Phase Peptide Synthesis

Before starting….

•

Choose the C-terminal protecting group

•

Choose the N-terminal protecting group

•

Choose protecting groups for any other R-group on the

amino acids

Protecting Groups

Acid Protection

H

₂

N

O

OH

R

Protecting Groups

•

The most common acid protecting group used is the methyl ester.

• It is stable in most coupling reaction and deprotection reaction conditions.

• Difficult to selectively remove.

• Depending on what type of coupling reaction and with what amino acids will be used other acid protecting groups can be used, such as the allyl ester.

Alanine methyl ester

H₂N O O H₂N O O

Protecting Groups

Amine Protection

H

₂

N

O

OH

R

Protecting Groups

N-Boc cleavage H₂N COOH BocHN COOH Glycine 50% TFA in DCM N-Fmoc cleavage H₂N COOH FmocHN COOH Glycine 20% Piperidine in DMF

•

There are two standard types of N-protecting groups used, the Boc and Fmoc group.

O O _{H C} 3 _CH 3 CH₃ O O

Protecting Groups

•

Other protecting groups that can be used are Cbz and more recently the Nosyl group.

Leggio, A.; Gioia, M.L.D.; Perri, F.; Liguori, A. Tetrahedron, 2007, 63, 8164-8173

O

O CBZ

Protecting Groups

Protection of the R-group

H

₂

N

O

OH

R

Protecting Groups

• Some of the different R-groups that must be protected before coupling are hydroxyl groups (Ser), thiol groups (Cys), amines (Lys) and

carboxylic acids (Asp).

HO NH2 COOH HS NH2 COOH NH2 COOH HOOC NH2 COOH H2N

Serine (Ser) Cysteine (Cys)

Lysine (Lys) Aspartic Acid (Asp)

HO NH2 COOH HS NH2 COOH NH2 COOH HOOC NH2 COOH H₂N

Serine (Ser) Cysteine (Cys)

Protecting Groups

Base Sensitive Protecting Groups

• Used in N-Boc protected peptide synthesis.

Cleavage : 2M NaOH (aq) EtOH (1:1)

Cleavage : 20% Piperidine in DMF O NH₂ COOH Si Ph Ph TBDPS S NH2 COOH Fm NH2 COOH H N O O Fmoc NH2 COOH O O Fm

Cleavage : 20% Piperidine in DMF _{Cleavage : 20% Piperidine in DMF}

Protecting Groups

Acid Sensitive Protecting Groups

• Used in N-Fmoc protected peptide synthesis.

• Normally the t-Butyl, Boc or Trityl group is used.

• Cleaved using 5-50% TFA in DCM.

O NH₂ COOH Ph Ph Ph Trityl NH₂ COOH H N Boc O O S NH₂ COOH t-Butyl NH2 COOH O O t-Butyl

Protecting Groups

Other Protecting Groups

O O Alloc S O O NO2 Nosyl Allyl O PMB Cleavage: Pd(PPh₃)₄

Cleavage: Thiophenol Cleavage: Ceric Ammonium Nitrate (CAN)

Protecting Groups

• There are some exception were an unprotected amino

acid, such as serine, can be used without being

protected.

Coupling Reagents

N-(3-Dimethylaminopropyl)-N’-ethylcarbonate (EDC) N C N N C N N N C N N N,N’-Dicyclohexylcarbodimide (DCC)

N

N

DMAP

Coupling Reagents

Coupling Reagents

PG O N X O R H O N O PG R _O N O PG R base O N O PG R _O N O PG R _O N O PG R

Racemisation of an activated amino acid

X= Activator

Coupling Reagents

N N N O Me2N NMe2 PF₆ (BF₄ ) N N N N O Me₂N NMe₂

HBTU (TBTU) HATU

PF₆ N N N OH N N N N OH HOBt _HOAt N N N O P N N N PF₆ N N N O P N N N PF₆ BOP PyBOP P N N N PF₆ PyBrOP Br

Coupling Reagents

R O O N N N O N Me2N + R O O N N N O N NMe2 R O O N N N O NMe₂ NMe₂ R O O N N N Me2N NMe2 O + R'-NH R _HO O N N N N H R' +

Coupling reagents

Brink, H.T.; Rijkers, D.T.S.; J. Org. Chem., 2006, 71, 1824

Coupling reagents

Anderson, R.J.; Coleman, J.E.; Tetrahedron Lett., 1997, 38, 317-320

Coupling reagents

R. Wischnat, Tetrahedron Lett., 2003, 44, 4393-4394

Coupling reagents

El-Faham, A.; Albericio, F., Org. Lett., 2007, 9, 4475-4477

Solid Phase Peptide Synthesis

Solid Phase Vs Solution Phase

Advantages

Disadvantages

• Fast production of long peptides by increasing the amount of reactant. • Quick purification by filtration.

• Automated or manual option.

• Easily scaled up from mg to kg. • No need for excess reactants or

expensive machinery.

• Expensive resin and can require specialised equipment.

• Limited scale-up.

• More difficult to purify. • Longer reaction time

Solid Phase Peptide Synthesis

Chan, W.; White, P. Fmoc Solid Phase Peptide Synthesis, Oxford, New York, 2000.

By deprotecting the final -amino group and cleaving the peptide from the resin after peptide chain elongation, using the appropriate cleavage conditions, the peptide is isolated.

It can then be coupled to another N-protected amino acid.

The first step is to deprotect the amino group to produce a free amine.

Solid Phase Peptide Synthesis

•

The resin is not completely spherical, with the reaction only occur on the surface of the resin.

• There are cavities were the coupling takes place and is the reason why swelling is very important before coupling can occur.

Types of Resin.

• You buy commercially available pre-loaded resins.

• Different resins can be cleaved under basic (N-Boc) or acidic (N-Fmoc) conditions.

• Depending on the resin the final peptide can have an amide or acid C-terminal.

Solid Phase Peptide Synthesis

Nitrogen _Vacuum

Resin - Amino acids - Coupling reagents - Base - Solevnt Sinter

Merrifield Bubbler

1. Add resin to column.

2. Swell resin using DMF and bubbling with N₂. 3. Remove solvent using vacuum.

4. To cleave the first N-protecting group the appropriate deprotecting reagent is added.

5. Bubble N₂ and then remove solvent under vacuum and wash with DCM. 6. Add DMF plus coupling reagent, base and N-protected amino acid. 7. Bubble N₂ until reaction is complete.

8. Remove solvent and wash with DCM.

Thank You For

Your

Attention