vinyl sulfones: click applications in bioconjugation. the resurgence of a chemical function

glycohcembio.ugr.es

R SO

O

Javier Lopez-‐Jaramillo /[email protected]

Francisco Santoyo-‐Gonzalez [email protected]

Permanet Members

Fernando Hernandez-‐Mateo [email protected]

Applied Science

Basic Science

Scien6fic Ac6vity

SO2

R SO

O

Research Lines

NN

N

Synthesis of Neoglycoconjugates

Cyclodextrins & Glycocyclodextrins

Carbon Nanopar6cles

1,3-‐Dipolar Cycloaddi6ons

Cyclic Sulfates

Gene Transfec6on

Polymeric matrix from polysaccharides

“Chemistry tailored to generate substances quickly and reliably by joining units together” H. C. Kolb, M. G. Finn, K. B. Sharpless, Angew. Chem. Inter. Ed. 2001, 40, 2004-‐2021

The click-‐chemistry philosophy

Modular and wide in scope

High chemical yield

Inoffensive byproducts

Readily available star>ng materials and reagents

Stereospecific

Simple reac>on condi>ons

No solvent or benign solvents H2O

Easy product isola>on. Not chromatography

Physiologically stable

High atom economy

Click-‐Chemistry

Click-‐chemistry Reac>ons

Cycloaddi>ons of unsaturated species

1,3-‐Dipolar cycloaddi>on reac>ons

Nucleophilic subs>tu>on chemistry Ring-‐opening reac>ons of strained heterocyclic

electrophiles

Epoxides

Aziridines

Aziridinium ions

Episulfonium ions

Non-‐aldol type carbonyl chemistry

Ureas

Thioureas

Aroma>c heterocycles

Oxime

Ethers

Hydrazones

Amides

Addi>ons to C-‐C mul>ple bonds

Epoxida>on

Dihydroxyla>on

Aziridina>on

Sulfenyl halide addi>on

Michael addi>ons of Nu-‐H reactants

Click-‐Chemistry

Azide-‐Alkyne Cycloaddi6on (ACC)

NN

NR

R'

R

R' N3

1-‐3 Dipolar cycloaddi6ons NN

N

Cu (I) catalyzed click-‐chemistry

(CuAAC)

Metal-‐free click-‐chemistry

“Click-‐Chemistry”

(Ph3P)3·∙CuBr (EtO)3P·∙CuI

NN

NR

R'Cu(I)

R R' N3

Cu(I) complex as organic soluble catalyst for CuAAC

MW MW

Org. Le4, 2000, 2, 2499 Org. Le4, 2003, 5, 1951 cited by 240

NN

N 1-‐3 Dipolar cycloaddi6ons

Featured contribu6ons in CuAAC CarboUGR

Glyco clusters (Homo and Hetero)

Glyco dendrons

Glyco dendrimers

Glyco polymers

Glyco Cyclodextrins

Glyco-‐silicas Bio>n-‐silica

Func>onal Glycoconjugates

NN

N 1-‐3 Dipolar cycloaddi6ons Featured applica6ons of CuAAC CarboUGR

NN

NR

R'

Catalysis Methodology

Applications Chem. Soc. Rev., 2009, 38, 3449

Tetrahedron., 2005, 61, 9338 Org. Biomol. Chem., 2007, 2291

Adv. Synth. Catal., 2006, 348, 2410 Adv. Synth. Catal., 2010, 352, 3306 Adv. Synth. Catal., 2012, en prensa

Org. Le4, 2000, 2, 2499 Org. Le4, 2003, 5, 1951

J. Org. Chem., 2008, 73, 7768 J. Org. Chem., 2008, 73, 7772

Eur. J. Org. Chem., 2009, 2441 Eur. J. Org. Chem., 2009, 2454

NN

N 1-‐3 Dipolar cycloaddi6ons

Featured Publica6ons

CarboUGR

Non-‐AAC Click-‐reac6ons

“Thiol-‐Ene ”

R SH

R'

RS

R'

R SH

R'

“Thiol-‐Yne ”

Catalyst Catalyst

“Michael-‐Type Addi6on ” (1,4-‐ addi6on or conjugate addi6on)

RS

R'

SR

R''

H

R'R

acceptor

donor

R

R''

R'

H

R, R’ and R’’ electron-‐withdrawing groups Base B:

Click-‐Chemistry

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O

Vinyl Sulfones

Conjugated addi6ons

ü Efficiency ü High yields ü No sub-‐products

Reac6vity

R SO

OVinyl Sulfones

R SO

O

RS

O O

NuRS

O ONu-+

Nu- = C-, N-, y S-Nucleófilos

Cycloaddi6on reac6ons ü  [2+2] Cycloaddi6ons ü  [3+2] Cycloaddi6ons ü  [4+2] Cycloaddi6ons (Diels-‐Alder

Mixed reac6ons ü  Epoxida6on ü  Reduc6ons ü  Methatesis with olefines ü  Asimetrid dyhydroxila6ons

Bioconjuga6on Labelling

Cross-‐linkers Catalysis

Immobiliza6on

R SO

O

R SO

OVinyl Sulfones

Func6onal Labelling

Non-‐Magnetic and Magnetic heterogeneous catalyst

Cu(I) Scavengers

NN

NR1

R2

R1

R2 N3

+

Lm Cu+

Lm1) Cu(I), 2)

CuAAC

Lm

Lm Cu+

Cu+ NN

NR1

R2

R1

R2 N3

+

Lm Cu+

Lm1) Cu(I), 2)

CuAAC

Lm

Lm Cu+

Cu+

Cu(I) Sorbents based in soported chelating agents

ü  Robust ü  Efficient ü  Low doses ü Negligible Cu Leaching ü  Simple operational protocols: Filtration or magnetic decantation ü  Recyclability

ü  High efficiency ü  Isolation of metal-‐free clicked compounds

LmLm Cu+

Heterogeneous Catalyst Scavengers

Catalysis R SO

OVinyl Sulfones

N N

Nn n n = 0, 1N N

NH

NH

NSO2

LmLm

R

SiO2O2S

H

SO2

LmNH2 LmR= NH2R= SO2CH=CH2

Lm

Removal of heavy metals

α,β-‐CD

Homogeneous Matrix

Heterogeneous matrix

Starch Dextrin

Cationic matrix Anionic matrix

Cr3+, Pb2+ y Cd2+

Removal of organic dyes

Removal of organic contaminants: Fenols

S OO

N+HO

H2NOH

OH2N

OH

O

HO

ONH

OH

O

OH

OHS

CO3-‐2 , 0,5 M, pH 12

Polymeric adsorbents based on polysaccharides and cyclodextrins for water purification ES 2334756 A1

R SO

OVinyl Sulfones

Cross-‐linkers

R SO

OVinyl Sulfones Bioconjuga6on

R SO

O

From Chemical reac6vity to Bioconjuga6on

RS

O O

NuRS

O O Nu-

Nu- = C-, N-, y S-Nucleófilosü  Efficiency ü  High yields ü No sub-‐products

ü  Physiological conditions -‐ Aqueous media, -‐ Mild alkaline pH, -‐ Room temperature

ü No catalyst and/or subproducts

Nu-‐ = Lys, Cys, His

Bioconjuga6on

Labelling Immobiliza6on

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OVinyl Sulfones

Func6onal Labelling

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O

Glycosyda6on Drug delivery

Lipida6on

Sensors

Glycoarrays

Hybrid materials

Fluorophores

Redox

Bio6n

Bioconjuga6on

Labelling

R SO

OVinyl Sulfones

R SO

O

Fluorophores

Redox

Bio6n

Biotine Fluorophore

Fluorescent Labelling

SX

O

O

X

R

SO2

OSO2

2

ON N

N

O

+

Rhodamina B VS

N

SO O

RO2S

O

O2S

2N

Dansilo VS

S

NHHNHH

O

O3

R

SO2

OSO2

2N

Biotina VS

SX

O

O

SO

OSO

O

R

SO2

OSO2

2

ON N

N

O

+

Rhodamina B VS

N

SO O

RO2S

O

O2S

2N

Dansilo VS

S

NHHNHH

O

O3

R

SO2

OSO2

2N

Biotina VS

Biotinylation Redox labelling SO

O

SO

O

FeSX

O

O

Fe

S

O

O

X = NH2, SH ConA, BSA, lisozime, Avidine

Fe

O SO

O

Fe

S

O

O

R SO

OVinyl Sulfones Labelling

Labelling Reagents

SO

O

Bio6n Fluorophore

HRP AVST

BTSAP BTSAP

Ruta a

Ruta b

Ruta b

Bifunctional labelling

Triazolyl containing vinyl sulfones as double-‐labeling agents and their preparation and use in the marking of biomolecules WO 2009/106665 A1 (PCT Int. Appl.)

R SO

OVinyl Sulfones

Route A

Route B

Bioconjuga6on

R SO

OVinyl Sulfones

Func6onal Labelling

R SO

O

Glycosyla6on Drug delivery

Lipida6on

SO

OSS

OOS

Neoglicoproteins

Glycosyla6on

BSA, ConA, Lisozyme Lumazine, Apoferritin VS azúcares

O S SO

O

O SH

HO nAcO n

α-D-Man, β-D-Glc, β-D-GlcNAc, α-L-Fucβ-Lac, β-Mal, β-Mel

R SO

OVinyl Sulfones

Func6onal labelling: Glycosyla6on

Carbohydrate-‐func6onalized Magne6c Nanopar6cles

Maghemite (γ-‐Fe2O3) nanopar6cles

Apoferritin

pH-‐Driven templated assembly (Encapsula6on) Glycosyla6on

ü High efficiency ü Low toxicity ü Maintenance of properties in the presence of serum ü Different cell lines

Improved transfection efficiency

(NH2)16

(NH2)16-n

R1SO

O

NHn

(NH2)16-n

SO

O

NH

n

R1

O

NHY

(NH2)16-n

SO

O

NH

n

O

R1 SO

O

NH

(NH2)15.5-n

SO

O

NH0.5n

R1 SO

O

PAMAN G2

Rho

PAMAM-‐G2-‐based amphiphilic non-‐viral gene vectors

R1 SO

O

R1 = (CH2)2Me, (CH2)10Me, (CH2)16Me,

O SO2

R1

O

NH SO

O

Y

Y = O, S

z(CH2)7CH=CH(CH2)7Me

O

N

ON+ N

SO2

O SO2

R

+

2

Rhodamine B VS

Rho

R SO

OVinyl Sulfones Func6onal labelling: Lipida6on

Preparation of lipids with vinylsulfonyl funcionality for therapeutic use in the transport of therapeuticals ES 2337226 A1

Targeted delivery of ISCOM

Inmunolabelling essay for ISCOM targe6ng delivery to Trypanosoma cruzi

R SO

OVinyl Sulfones Func6onal labelling: Drug delivery

CO3-‐2 buffer 0,125 M

pH 8, 12 h, 4º C

ISCOM-‐Ficocianine ISCOM-‐ drug

Mono VS-‐Cyclodextrin

R SO


O

OHHO

N

O

OOH

HOOHO

OOH

OH

OH

O

O

OHOH

OH

OO

OH

OH

HOO

O OH

OHHO

O

OTs

OOH

HO

HO

O

SO2

HOSO

O

SO

O

O2S

Conjuga6on

Nanobody

ONHN

HN OH

OO2N

Nitrofurazone

Encapsula6on

SO

O

O2S

Therapeu6c func6on

Carrier func6on

Targe6ng func6on

Tripanosoma Cruzi

ü Payload flexibility ü Target flexibility by adequate selection of the recognizable biomolecule ü Multifunctional decoupling: therapeutic, carrier and targeting

Modular design

Poly-‐His

Mono VS-‐Cyclodextrin R S

O


SO

OConjuga6on

Protein A

Encapsula6on

Therapeu6c func6on

Carrier func6on

Targe6ng func6on

Tripanosoma Cruzi

SO

O

O2S

ON

O

OHON

Camptotecine (Cpt)

SO

O

O2S

5-‐chloro-‐1H-‐benzo [d]imidazole-‐2-‐thiol

N

HN

SHCl

SO

O

O2S

Modular design

ü Payload flexibility ü Target flexibility by adequate selection of the IgG ü Multifunctional decoupling: therapeutic, carrier and targeting

IgG

Bioconjuga6on

Immobiliza6on

R SO

OVinyl Sulfones

R SO

O

Sensors

Glycoarrays

Hybrid materials

PROTEOMIC “Ad-‐hoc” chromatographic supports

INDUSTRIAL PROCESS Immobilization of Enzymes

FUNCTIONAL HYBRID MATERIALS

VS func6onalized silica

X

SiO2

OOO

O

O

X

Y = NMe, S

Y3Si S

Y= NMe, Y = S

SiO2

OO

OSi Y3

SO

OX = Lys, Cys, His

ü Symplicity ü Efficiency ü Green chemistry

Silica-‐vinylsulfone compound, synthesis and uses as immobilization surface WO 2009/040460 A1 (PCT Int. Appl.)

R SO

OVinyl Sulfones Immobiliza6on: Hybrid materials

A”Ready to Use” pre-‐ac6vated material

SO

OSiO2


R SO


SiO2OOO

Si SO

OO

NH

SH

O

HN

O

OH

O

HONH2

Glutathione (GSH)

h-‐Type Thioredoxins

u  Silica-‐glutathione for the isolation of proteins fused with GST

u  Pull-‐down experiments for proteins interacting whit thioredoxine

“Ad-‐hoc” chromatographic

supports

SO

OSiO2


R SO


SO

O SO

OS

OO

SO

O Apoferri6n Lactoferrin Immobiliza6on

An6microbial ac6vity against E. Coli

High Fe-‐Uptake capacity

Immobilization of Enzymes

SO

OSiO2

Functional Hybrid Materials

X

X = Lys, Cys, His SO

OSiO2 S

O

OSiO2 X

Invertase, Lactase, Lysozyme

Super-‐paramagne6c Hybrid Nanopar6cles

Polymer Compounds Having Immobilizing Properties WO 2012/085303 A1 (PCT Int. Appl.)

O

O

NHSS

O

O

O N

Me

O OH

H2N

Invertase, Avidin HRP

Vinyl sulfone SP-‐NPs

R SO


SO

O S

S

OOS

HN

LECTIN COMMERCIALLY LABELLED LECTIN LECTIN COMERCIALLY

LABELLED LECTIN

S

OOS

HN

S

OOS

Glyco-‐array

Immobiliza6on

DETECTION

ISOLATION

VS Sugars

NH2 NH2

NH2 NH2

NH2 NH2NH2NH2placas de

micropozillos

placas de vidrio

placas de vidrioinjertadas con polisina

SO

O S

S

OOS

HN

R SO


Glass slides

Microwell plates

Polylysine coated glass slides

O

NH

S

HNHN

H H

3O

R

R SO

OVinyl Sulfones Immobiliza6on: Sensors

R

Bio6nylated pyrrols

NH2 O

NHNH

O

Luminol

R

NSO2

NSO2

HN

Bio6nyla6on Electropolymeriza6on

Electrochemiluminiscence bio6nylated polymers

Electropolymeriza6on

R

SO2

OSO2

2

ON N

N

O

+

Rhodamina B VS

N

SO O

RO2S

O

O2S

2N

Dansilo VS

S

NHHNHH

O

O3

R

SO2

OSO2

2N

Biotina VS

SO

O

Bio6nyla6on

Bio6nylated Cholesterol Oxidase

HO Cholesterol

Electrochemiluminiscent disposable cholesterol biosensor

Avidin

Gold

Acta Cryst., 2005, F61, 435

J. Org. Chem., 2010, 75, 4039

J. Mat. Chem., 2010, 20, 7189

Org. Biomol. Chem, 2013, 11, 2586 Org. Biomol. Chem., 2011, 9, 851 Org. Biomol. Chem., 2010, 8, 7189 Org. Biomol. Chem., 2010, 8, 667

Bioconjugate Chem., 2012, 846

Scien6fic Produc6on R S

O

OVinyl Sulfones

ChemMedChem., 2014, 9, 383 2013, 13, 61

Int. J. Nanomed., 2012, 7, 5491

Anal, Chim. Acta., 2012, 754, 91 J. Am. Chem. Soc., 2011, 133, 4889

Adv. Synth. Catal., 2010, 3306

J. Plant Physiol., 2010, 423

R SO

OKnowledge Transfer

Triazolyl containing vinyl sulfones as double-‐labeling agents and their preparation and use in the marking of biomolecules WO 2009/106665 A1 (PCT Int. Appl.)

Single-‐labeling agents based on vinyl sulfones and their preparation and use in marking biomolecules such as protein WO 2009/106664 A1 (PCT Int. Appl.)

Compound for labeling biomolecules based on vinyl sulfone, their preparation and use in marking biomolecules such as proteins WO/2009144344 A2 (PCT Int. Appl.)

Silica-‐vinylsulfone compound, synthesis and uses as immobilization surface WO 2009/040460 A1 (PCT Int. Appl.)

Polymeric adsorbents based on polysaccharides and cyclodextrins for water purification ES 2334756 A1

Drug delivery and transfection agents based on alkylsulfonate functionalized PAMAM dendrimers ES 2351909 A1

R SO

OVinyl Sulfones

R SO

O

Simple and efficient chemistry

R SO

OVinyl Sulfones

for smart applica6ons

Collabora6ons

CTS-‐183 Bioquímica y Parasitología Molecular A. Osuna Carillo Parasitología

J. F. Corpas Bioquímica, Biología Celular y

Molecular de Plantas

FQM-‐118 Solid Phase Spectrometry Group Luis F. Capitan Valley

Química Analí6ca

FQM-‐368 BionNanoMetal José M. Domínguez Vera Química Inorgánica

CVI-‐212 Regulación Bioquímica y Gené6ca Rafael Salto González

Bioquímica y Biología Molecular II

BIO-‐286 Bioquimica y Señalizacion Celular J. B. Barroso-‐Albarran Bioquimica y Biologia Celular

Former Ph fellows

• Alicia Megia-‐Fernández • Julia Morales-‐SanFrutos • Francisco Pérez Balderas

CarboUGR

Non-‐Permanent Members • Mariano Ortega-‐Muñoz

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O

Research Lines