chemistry of conjugation - unito.it · other organic molecules ... chemistry of conjugation amines...
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CHEMISTRY OF CONJUGATION
Targets
Peptides
Proteins
Carbohydrates
Nucleic Acids
Lipids
Other organic molecules
Conjugation (of imaging probes) to specific targets
Target
Molecule
Imaging
Probe
Conjugated
Imaging Probe
CHEMISTRY OF CONJUGATION
Strategies
TargetMolecule
FunctionalizedImagingProbe
ConjugatedImaging Probe
TargetMolecule
ModifiedTarget
Molecule
ModifierFunctionalized
ImagingProbe
ConjugatedImaging Probe
TargetMolecule
FunctionalizedImagingProbe
HomobifunctionalLinker Conjugated
Imaging Probe
TargetMolecule
FunctionalizedImagingProbe
HeterobifunctionalLinker Conjugated
Imaging Probe
Direct coupling with a suitably functionalized imaging probe
Introduction of a functional group suitable for conjugation
Coupling mediated by a homobifunctional linker
Coupling mediated by a heterobifunctional linker
CHEMISTRY OF CONJUGATION
Peptides
ProteinsAminoacids
Functional groups amenable to conjugation
+H3N
HN
NH
O
OR
R'
O-
O
R"
(Protonated) AmineCarboxylic/Carboxylate
Side chain groups
Direct Coupling
CHEMISTRY OF CONJUGATION
Side chain groups
COO- CONH2 NH3+ OH SH
OH NH
SCH3
Carboxylate
(Asp, Glu)
Carboxamide
(Asn, Gln)
Amines
(Lys, Orn)
Hydroxyl
(Ser, Thr)
Thiol
(Cys)
Phenol
(Tyr)Indol
(Trp)
Phenyl
(Phe)
Methylthio
(Met)
CHEMISTRY OF CONJUGATION
Carboxylic group
C
O
OH
C
O
O-
H+
C
O
OH
Activatingagent
C
O
X
C
O
X
pKa 2.0-4.5
NH2
C
O
HN
NHNH2
C
O
NHNH
N C N NH+Cl-
EDC
NN N
O
N
CDI
NOH NOH NOH
O
O
O
O
O
O
HO3S
NHS Sulfo-NHS NHP
“Activated form”
HO
C
O
OEster
Amide
Hydrazide
COOH COO-
pKA0 7 14
pH
CHEMISTRY OF CONJUGATIONAmines
NH3+ NH2 H+
C
X
O
NH
O
NH2
pKaH = 7.5-9.5
C
H
O
NH
NaBH3CN
NCS
NH
S
NH
Acylation with “activated”carboxylic acids (anhydrides, chlorides, imidazolides, carbodiimide adducts)
Reductive amination with aldehydes
Reaction with isothiocyanates to give thioureas
NH
O
OH
Alkylation with epoxides
NH3+
NH2
pKAH0 7 14
pH
CHEMISTRY OF CONJUGATION
Thiols
SH S- H+
SHNH
S
O
Br
HN
O
pKa ~ 9-10
S
S
N
SS
N
S
N
O
O
O
O
Michael addition to maleimide
Thiol Alkylation
Mixed disulfide formation
SH S-
0 7 14
pH
pKA
CHEMISTRY OF CONJUGATION
Phenols
OH H+O-
OH
N+
N
OH
N N
pKa ~ 10
NH2
OH
NH
, CH2O
Diazo-coupling
Mannich aminoalkylation
Carbohydrates Saccharides
Functional groups amenable to conjugation
Direct Coupling
CHEMISTRY OF CONJUGATION
OHO
HOOH
OH
OH
OHO
HO
OH
OH
OH
O
HO3SO
HONH2
OH
OH O
NH
HO
Glucose Mannose
O CH3
OH
OH
HO
HO
HOOC
Galactosamine-4-O-sulfateSialic Acid
Primaryalcohol
trans-Diol cis-Diol
(Protonated)Amine
Carboxylic /Carboxylate
(Masked)Aldehyde
CHEMISTRY OF CONJUGATION
Hydroxyls
OH
C
X
O
O
O
NCO
O
NH
O
Esterification with “activated” carboxylic acids
Reaction with isocyanate to give carbamates
Diols
OH
OHNaIO4
O
OH
H
NH2
NaBH3CN
N
Oxidative demolition with periodate followed by reductive amination
CHEMISTRY OF CONJUGATION
O
OH OH
NH2
NaBH3CN
NH
OH
OH
NHNH2 N
HN
OH
“Masked” Aldehydes (hemiacetals)
Amines and Carboxylic Acids
Reductive amination
Hydrazone formation
See slides 5-6 for conjugation fo amines and carboxylic acids
CHEMISTRY OF CONJUGATION
Oligonucleotides Nucleotides
N
NN
N
NH2
O
OH
OP-O
O-
O
NH
O
ON
O
OHOH
OP-O
O-
O
Deoxyribonucleotide Ribonucleotide
Functional groups amenable to conjugation
HydroxylDiol
Substituted Heterocycles
Hydroxyl
Diol
CHEMISTRY OF CONJUGATION
See previous slides for conjugation
of alcohols and diols
Substituted Heterocycles
Specific methods of conjugation
adapted to the single bases
CHEMISTRY OF CONJUGATION
TargetMolecule
ModifiedTarget
Molecule
ModifierFunctionalized
ImagingProbe
ConjugatedImaging Probe
The modifier introduces a new FG in the target molecule, allowing its conjugation with suitably functionalized imaging probes
The modifier converts a FG present in the target molecule into another FG, more suitable for conjugation reactions
The modifier is a (homo- or hetero-) bifunctional reagent
The modifier acts as a linker and a spacer
CHEMISTRY OF CONJUGATION
Modification of amines
NH2
S O
NH
O
SH
S NH.HCl
NH
+H2N
SH
Thiobutyrolactone
Amine → Thiol
2-Iminothiolane
Amine → Thiol
NH
O
S
O
N
O
O
O S
O
CH3
O
CH3
S-Acetylthioglycolic acid NHS ester
Amine → Protected Thiol
Deprotection of thiol is accomplished with NH2OH
CHEMISTRY OF CONJUGATION
Modification of amines
NH2 NH
O
OH
OO O
O
NH
OO
O
OOH
O
NH
OO
O
O
OH
O
Succinic Anhydride
Amine → Acid
Glutaric Anhydride
Amine → Acid
Citraconic Anhydride
Amine → Acid
Cleavable at pH 3
CHEMISTRY OF CONJUGATION
Modification of carboxylic acids
COOH
H2N (CH2)n NH2
EDC
O
HN
NH2
n
EDC
O
HN
S
SNH2
NH2
S
S
NH2SH
SHHO
HO
S
SHO
HO
O
HN
SH
DTT
Condensation with diamines
(n > 1)
(Acid → Amine)
Condensation with cystamine
(Acid → Amine)
Disulfide cleavage
(Acid → Thiol)
CHEMISTRY OF CONJUGATION
Modification of thiols
SHI COOH
S
COOH
Iodoacetic acid
Thiol → Acid
May alkylate other FGs of AAs
(His imidazole, Met thioether, N-terminal and Lys amines)
Aziridine (Ethyleneimine)
Thiol → Amine
S NH2
HN
Modification of hydroxyls
OHCl COOH
O
COOH
Chloroacetic acid (basic pH)
Hydroxyl → Acid
May alkylate other FGs of AAs
CHEMISTRY OF CONJUGATION
Modification of aldehydes (indigenous or periodate-derived)
OH
OHNaIO4
O
OH
H
NaBH3CN
N
O
OH OH
NaBH3CN
NH
OH OH
H2N NH2
nNH2
n
H2N NH2
n
NH2
n Reductive amination with diamines
Aldehyde → Amine
CHEMISTRY OF CONJUGATION
Bifunctional Linkers
Target
Molecule
Functionalized
Imaging
Probe
Homobifunctional
Linker Conjugated
Imaging Probe
Target
Molecule
Functionalized
Imaging
Probe
Heterobifunctional
Linker Conjugated
Imaging Probe
CHEMISTRY OF CONJUGATION
Target
Molecule
Functionalized
Imaging
Probe
Bifunctional
Linker Conjugated
Imaging Probe
Coupling mediated by a homobifunctional linker
Stepwise procedure → avoids self-cross-linking of target molecules
Bifunctional
Linker (xs)Target
Molecule
Functionalized
Imaging
Probe
Conjugated
Imaging Probe
CHEMISTRY OF CONJUGATION
Homobifunctional Linkers
Specific for Amine-Amine
NHS-Diesters
NH2
N O
O
O
O
N
Spacer
O
O
O
ONH
O
Spacer
O
O
N
O
O
N OO
OH
H2N
NH
O
Spacer
HN
O
N OO
OH
Sulfo-NHS-Diesters
NH2
N O
O
O
O
N
Spacer
O
O
O
ONH
O
Spacer
O
O
N
O
O
N OO
OH
HO3S SO3H
SO3H
SO3H H2N
NH
O
Spacer
HN
O
N OO
OH
SO3H
Amide linkages
CHEMISTRY OF CONJUGATION
Homobifunctional Linkers for Amine-Amine
NHS and Sulfo-NHS active diesters
O
O
N N
O
O
O
O
O O
O
N
O
O
N
O
O
O
O
O
O
N
O
O
N
O
O
O
O
HO3S
SO3H
O
O
N
O
O
N
O
O
O
O
O
O
O
O
O
O
N
O
O
N
O
O
O
O
O
O
O
O SO3H
HO3S
O
O
N
O
O
N
O
O
O
O
S
S
O
O
N
O
O
N
O
O
O
O
S
SHO3S
SO3H
O
O
N
O
O
N
O
O
O
O
OH
HO
DSC DSS BSSS
DSP
Cleavable (DTT)
DTSSP
Cleavable (DTT)
EGS Sulfo-EGS
DST
Cleavable (NaIO4)
CHEMISTRY OF CONJUGATION
Homobifunctional Linkers
Specific for Amine-Amine
Diimidates
NH2
H3CO
HCl.HN
OCH3
NH.HCl
Spacer
NH
O
Spacer
OCH3
NH2+
CH3OH
H2N
NH
+H2N
Spacer
HN
NH2+
CH3OH
NH.HCl
OCH3
HCl.HN
OCH3
NH.HCl
OCH3
HCl.HN
OCH3
NH.HCl
OCH3
HCl.HN
OCH3
NH.HCl
OCH3
HCl.HN
OCH3
SS
Dimethyl adipimidate
Dimethyl pimelimidate
Dimethyl suberimidate
DTSSP
Cleavable (DTT)
CHEMISTRY OF CONJUGATION
Homobifunctional Linkers
Specific for Amine-Amine
Diisothiocyanate
NH2
N NSpacer
HN Spacer N
CCS S
C S
SHN
H2N
HN Spacer NH
SHN
SHN
NCSSCN
SO3H
SO3HNCS
SCN
1,4-Phenylene
diisothiocyanate4,4’-Diisothiocyanato
stilbene-2,2’-disulfonic acid
SO3H SO3
-
CHEMISTRY OF CONJUGATION
Homobifunctional Linkers
Specific for Thiol-Thiol
Bis-Maleimides
SH
S
Spacer N
N NSpacer
O
O
O
ON
O
O O
O
HSS
Spacer NN
O
O O
O
S
NHN
HN N
OHO
O
O
O
N,N’-Bis(3-maleimidopropyl)-1,3-propanediamine
NN
O
O
O
O
BMH
CHEMISTRY OF CONJUGATION
Homobifunctional Linkers
Specific for Aldehyde-Aldehyde
Bis-Hydrazides
CHO Spacer NHNH2
H2NHN NHNH2Spacer HNN
HC
OHC
Spacer NHNH
NCH N CH
H2N
HN
NH
NH2
O
O
Adipic acid dihydrazide
Other Homobifunctional Linkers
CHO
CHO
OO
O
On
Glutaraldehyde
Aldehyde groups react with amines to form imine or emiaminal or aminal linkages
Oligo(Poly)ethylenglycol
diglycidyl ether
Epoxides react with nucleophilic groups (amines, thiols, alcohols)
CHEMISTRY OF CONJUGATION
Heterobifunctional Linkers
Target
Molecule
Functionalized
Imaging
Probe
Heterobifunctional
Linker Conjugated
Imaging Probe
The most reactive FG of heterobifunctional linker is usually reacted first
Different functional groups in the linker prevent self-cross-linking of target molecules
Higher specificity
Higher yields
CHEMISTRY OF CONJUGATION
Heterobifunctional Linkers
Specific for Amine-Thiols
SH
N Spacer
Spacer
S
O
O
O
O N
O
O
N
O
O
O
O N
O
O
H2N
N
O
O
OH
Spacer
S
N
O
O
O
HN
SH
NH SpacerSX
O
O
O N
O
O
HN
O
O
ON
O
O
Spacer
H2N
N
O
O
OH
Spacer
S
HN
O
O
HN
CHEMISTRY OF CONJUGATION
Heterobifunctional Linkers
Specific for Amine-Thiols
N
O
O
O
ON
O
O
N ON
OO
OO
O
NO
N
OO
OO
O
N ON
OO
OO
O
NO
N
OO
O
O
O
N
ON
OO
O
O
O
SO3H
O
OBr N
O
O
O
OI N
O
O
INH
ON
O
OO
O
Iodoacetic acid NHS ester
Bromoacetic acid NHS ester
6-Iodoacetamidocaproic acid NHS ester
Sulfo-SMCCMBS
N
O
O
ON
OO
O
SMPB
2-Maleimidoacetic acid NHS ester
3-Maleimidopropionic acid NHS ester
4-Maleimidobutyric acid NHS ester
6-Maleimidocaproic acid NHS ester
CHEMISTRY OF CONJUGATION
Heterobifunctional Photoreactive Linkers
One (or both) FG react only after irradiation with UV-light
Photoreactive
LinkerTarget
Molecule
hνννν
Functionalized
Imaging
Probe
Conjugated
Imaging Probe
N3hνννν
N:
N
NH2
N
NHAryl
azideIntermediate
NitreneIntermediate
Dehydroazepine Conjugate
ON
OO
ON3
ON
OO
O
N3
NO2
ON
OO
ONO2N3
NH
4-Azidobenzoic acid NHS ester
5-Azido-2-nitro-benzoic acid NHS ester
6-(4-Azido-2-nitrophenylamino)hexanoic
acid NHS ester
CHEMISTRY OF CONJUGATION
Recent Trends: Click Chemistry
Sharpless, K.B. et al
ACIEE 2001, 40, 2004
CHEMISTRY OF CONJUGATION
Cu-Catalyzed Azide Alkyne Cycloaddition (CuAAC)
N=N+=N-Cu+
N
NN
Very efficient reaction
Compatible with several functional groups
(Bio)Orthogonal reaction
Operational simplicity
Stable linkage
Copper ion issues
FGs not present in biomolecules
Chelating agents bind Cun+
Triazole ring effect unpredictable
CHEMISTRY OF CONJUGATION
Diels-Alder Cycloaddition
NH N
O
n
O
O
N
O
O
O
NH
Efficient reaction
Compatible with several functional groups
(Bio)Orthogonal reaction
Operational simplicity
Stable linkage
FGs not present in biomolecules
Limited stability of the diene moiety
Bulky Adducts
CHEMISTRY OF CONJUGATION
Thiol-Ene Radical
SH SR.
Very efficient reaction
(Bio)Orthogonal reaction
Operational simplicity
Quite Stable Linkage
Thiol group frequently available
<
Need for radical initiator
Presence of byproducts
Possibility of side reactions
Alkene not present in biomolecules
CHEMISTRY OF CONJUGATION
Photo-Click Cycloaddition
N N
NN
O NN
O
hν
O
OH
OH
O hν
O
O O
FGs not present in biomolecules
Photochemical hardware needed
Possibility of side reactions
Bulky adducts
Very fast reactions
(Bio)Orthogonal reactions
Triggered by irradiation
Stable Linkage
No byproducts
Giovanni B. Giovenzana
DISCAFF
Università del Piemonte Orientale “A. Avogadro”
Largo Donegani 2/3
28100 Novara
Italy
CAGE Chemicals
Via Bovio 6
28100 Novara
Italy
E-mail: [email protected]
Phone: +39-0321-375846 Skype: gbgiovenzana