Crazy about biomedicineJúlia García Pindado

Designing peptides: Designing peptides: from nature to benchfrom nature to bench

Organic chemistry: 5 elements to build us up

A brief reminder about proteins and peptides

What’s an aminoacid?What’s an aminoacid?

ChiralityChirality

A brief reminder about proteins and peptides

A brief reminder about proteins and peptides

A brief reminder about proteins and peptides

How amino acids form proteins?How amino acids form proteins?

A brief reminder about proteins and peptides

ProteinsProteins

A brief reminder about proteins and peptides

Protein structureProtein structure

PeptidesPeptides

A brief reminder about proteins and peptides

Peptides as drugs

40 marketed peptides (only 1%)

270 in clinical trials 15% year growth

400 in advanced preclinical trials

Peptides as drugs

Chemically well defined

Low cost of production

No immunogenicity

Specificity

Oral administration

Permeability through biological barriers

Small moleculesSmall molecules PeptidesPeptides BiologicsBiologics

Chemically well definedLow production costChemical synthesisOral administrationSpecificity

SpecificityHigh production costNon chemical synthesisImmunogenicityNo oral administration

Ideal drug candidate

Permeability through biological barriers reach the target

Stability

Solubility

Active (nM range)

Non toxic

Which is the appearance of peptides?

Peptide synthesis

Solid phase peptide synthesis (SPPS)

Developed by Merrifield in 1963

Manual SPPSMicrowave assisted

SPPS

o Fast development

o Complex peptides can be reached

o 60-70% efficiency

Solid phase peptide synthesis (SPPS)

Solid phase peptide synthesis (SPPS)

Temporary protecting group

Permanent protecting groupBoc/BzlBoc/Bzl Fmoc/tBuFmoc/tBu

ResinsResins

Size of the resin beads (80-200 µm)

Mechanical and thermal stability

Price

Swelling

Solid phase peptide synthesis (SPPS)

Types: Cross-linked polysterene Polyamide

Tentagel

Soluble

Solid phase peptide synthesis (SPPS)

ResinsResinsWang

2-Chlorotrityl chloride

Rink amide Chemmatrix

SWELLINGSWELLING

Solid phase peptide synthesis (SPPS)

Coupling reagentsCoupling reagents Name Structure

DIPCDI

N

C

N

HOAt

N

N

NN

OH

PyBOP

N

NN

O P

N

N

N

PF6

TBTU

N

N

N

O

NN

BF4

DIEA

N

Solid phase peptide synthesis (SPPS)

Protecting groupsProtecting groupsName Structure Removal conditions

(SPPS)

Stable to the removal of

Fmoc OO

20% piperidine in DMF Boc, Trt, Alloc, pNZ

Boc

O O

20-50% TFA in DCM Fmoc, Trt, Alloc, pNZ

pNZ

O2N

O

O

1-6M SnCl2, 1.6mM HCl

(dioxane) in DMF

Boc, Fmoc, Trt, Alloc

Trt

1% TFA in DCM Fmoc, Alloc

Alloc O

O

Pd(PPh)3 cat., scavengers

PhSiH3 in organic solvents

Boc, Fmoc, Trt, pNZ

Solid phase peptide synthesis (SPPS)

Protecting groupsProtecting groupsAlloc

Pd(PPh)3 cat., scavengers

PhSiH3 in organic solvents

Boc, Fmoc, Trt, pNZ

iVDde

O O

2% N2H4·H2O - DMF Boc, Fmoc, Trt, Alloc

Azide N3 PMe3 in dioxane -

pNB

O2N

SnCl2 in DMF Boc, Fmoc, Trt, Alloc

Acm

NH

O

I2 (S-S)

Hg(II) (SH)

Trt

Bzl

NaOH in organic solvents Boc, Fmoc, pNZ, Trt,

Alloc

Al

0.1 equiv Pd(PPh)3, 10

equiv PhSiH3

Boc, Fmoc, pNZ, Trt

Name Structure Removal conditions

(SPPS)

Stable to the removal of

Solid phase peptide synthesis (SPPS)

The concept of orthogonalityThe concept of orthogonality

Remove only the Remove only the desired PG without desired PG without affecting the othersaffecting the others

Solid phase peptides synthesis (SPPS)

Tests to check coupling reactionsTests to check coupling reactions

Chloranil

Kaiser

Free amines

O

OH

OH

O

+

NH2

R

OH

O

HO

O

N

O

O

Solid phase peptides synthesis (SPPS)

Side reactionsSide reactions

Diketopiperazine (DKP)

Epimerization: losing the stereochemistry

Solid phase peptides synthesis (SPPS)

Other inconveniencesOther inconveniences

Aggregation

Deletion of residues

Problems during the cleavage process

Solid phase peptides synthesis (SPPS)

PurificationPurification

Traditional chromatographic purification

Solid phase peptides synthesis (SPPS)

PurificationPurification

SPPS enables us to avoid performing several purification steps

ReagentsSolventImpuritiesBeads with the peptide wash

Reagents and

byproducts

Beads of resin

Solid phase peptides synthesis (SPPS)

PurificationPurification

HPLC can be used to separate the drug from all the other impurities

How can we improve synthetic peptides?

To…

- Increase stability

- Enhance cell uptake and permeability through biological barriers

- Improve resistance against proteases

WHY?WHY?

N-methylated peptides Non natural aminoacids Cyclic peptides Stapled peptides

How can we improve synthetic peptides?

ProteasesProteases

N-methylated peptides

Cyclic peptides

Stapled peptides

Designing peptides

Synthesis of natural peptides

Synthesis of novel peptides

Mimicking nature

Aromatic AA(Phe)

Variable AA

1) Envisaging the desired product

2) Proper selection of the strategy

1) Resin

2) PG

3) Final removal of all the PG?

4) Further reactions and/or deprotections after cleavage

3) Carrying out the synthesis

Creating new sequences

Selecting the resin…

2-Chlorotrityl: 1% TFA in DCMProtected peptide acid

Wang: 95% TFAPeptide acid

Rink amide Chemmatrix: 95% TFAPeptide amide

Choosing the suitable PG...

Removal in acidic conditions:Removal in acidic conditions:

Boc:25-50% TFA tBu: 90% TFApNZ:1-6M SnCl2, 1.6mM HCl (dioxane) in DMF

Trt: 1% TFA in DCM

O2N

O

O

Removal in basic conditions:Removal in basic conditions:

Fmoc:20% piperidine/DMF Bzl: NaOH in organic solvents

Removal in other conditions:Removal in other conditions:

Alloc: Pd(PPh)3 cat., scavengers PhSiH3 in organic solvents

Al: Pd(PPh)3 cat., scavengers PhSiH3 in organic solvents

NH

O

Acm: I2 (S-S) Hg(II) (SH)

Azide: PMe3 in dioxane

N3

Choosing the suitable PG...

Choosing the suitable PG...

Analysis of the obtained product

High Performance Liquid Chromatography (HPLC)High Performance Liquid Chromatography (HPLC)

Analysis of the obtained product

Mass spectrometry (MS)Mass spectrometry (MS)

Our lab (Giralt’s group)

Design, synthesis and structure of peptides and proteinsDesign, synthesis and structure of peptides and proteins