Bioconjugates: an introduction · Bioconjugates: an introduction Ferenc Hudecz1,2 1Department of...
Transcript of Bioconjugates: an introduction · Bioconjugates: an introduction Ferenc Hudecz1,2 1Department of...
Bioconjugates: an introduction
Ferenc Hudecz1,2
1Department of Organic Chemistry, 2Research Group of Peptide Chemistry
Hungarian Academy of Sciences, Eötvös Loránd University Budapest
Conditions
1. 5-7 lectures plus substantial homework
2. Participation at lectures > 70 %
3. Examination: 25-30 min presentation based on literature Website: http://szerves.chem.elte.hu/oktatas/ea/Hudecz/index.htm#biokonjugatumok Email: [email protected]
Outline: Protein bioconjugates
1. Historical background
2. Functional groups of proteins/glycoproteins N-nucleophiles: -NH2, imidazole, indole, guanidino S-nucleophiles: -SH, CH2-S-CH3
O-nucleophile: -OH O/C-nucleophiles: -CHO, -COOH, -CONH2
3. Creation of reactive groups - Limited reactivity (eg. – OH vs. - CHO) - Improved selectivity (e.g. - NH2 vs. - SH)
- Space considerations - Convenient chemistry (e.g. - COOH vs. - NH2)
4. Detection of reactive groups sensitive quantitative quick small sample
5. Conjugation - Chemical synthesis - Enzymatic synthesis (e.g. - NH2 vs. - SH)
- Gene technology
6. Analysis of conjugates Purification Structure determination
Introduction
Transformation
Destructive
Non-destructive
Design of bioconjugates
Why?
What?
With What?
How?
Synthetic antigens or drug targeting
Peptide epitope, drug, reporter molecules
Protein, DNA, liposome
Covalent bond
Introduction
Application of bioconjugates: immunoassays
a) competitive
b) direct
Introduction
Application of bioconjugates: histo- and cytochemistry
Introduction
Application of bioconjugates
Western blotting FACS analysis
Introduction
Application of bioconjugates: DNA detection
Introduction
Ionisation status of proteins as function of pH
Ionisation of aspartic acid
Reactivity and transformation of amino group
Reactivity and transformation of hydroxyl group
Reactivity and transformation of thiol group
1) Establishment of protein structure – function relationship (1925 –
Solubility Sumner, J.B., Graham V.A.: The nature of insoluble crease Proc Soc Exp Biol Med 22 504 (1925)
Identification of amino acid side chain(s) necessary for the protein function
Olcott, H.S., Fraenkel-Conrat, H.: Specific group reagents for proteins Chem Rev 41 151 (1947) Herriot, R.M.: Reactions of native proteins with chemical reagents Adv Prot Chem 3 161 (1947)
Historical background
2) Structural studies Proteins
Determination of N-terminal amino acid
Sequencing (1956)
Edman, P., Begg, G.: A protein sequenator Eur J Biochem 1 80 (1967)
Determination of amino acid composition
(1960) Moore, S., Stein, W.H.: Chromatographic determination of amino acids … Methods in Enzymol 6 819 (1963)
F
O2N NO2
N
SO2Cl
CH3CH3
= 254 nm s = 360 nm
e = 480 nm
NC
Sop.: -21 oC
O
O
OH
OH
CHO
CHO
s = 340 nm
e = 455 nm
Carbohydrates E. Fischer (1884) Reducing monosaccharides
O
OH
H
OH
OH
OH
OH
NHNH2
fenil-hidrazin
N
OH
H
OH
OH
OH
OH
NH
D-Glükóz Glükóz-1-fenilhidrazon
NHNH2
fenil-hidrazinN
OH
H
OH
OH
OH
N
NH
NH
Glükóz fenil-oszazon
phenyl-hydrazine
D-glucose-1-phenylhydrazone
phenyl-hydrazine
D-glucose–bis–phenylhydrazone (osazone)
Carbohydrates Periodate oxidation to form oxo-function
oxo-group from (secondary) hydroxyl-function
O
OH
O
OH OH
O
O
OH
O O
O O
10 mM Nátrium-perjodát
-D-Mannóz részlet C - C kötés hasadása
poliszacharid láncban aldehid részlet oxidálásávalNaIO4
10 mM Sodium periodate
-D-mannosyl unit of a polysaccaride chain
C – C bond cleavage and oxidation of the OH groups
Nucleic acids Maxam, A.M., Gilbert, W … et al. A new method for
sequencing DNA PNAS 74 560-564 (1977)
Sanger, F. et al. PNAS 74 5463 (1977)
Smith, L.M. et al. Nature 321 674 (1986)
N
N
N
N
NH2
R
1
2
3
4
56 7
8
9
1
2
3
4
56 7
8
9N
NH
N
N
NH2
O
R
N+
N
N
N
NH2
RCH3
N
NH
N
N+
NH2
O
R
CH3
(CH3)2SO4
a) 0.1 M HCl
b) pH 7, 100 oC
N
N
O
H
O H
NH (CH2)5
O
NH
O
O
H
H
OH
H
H
H
O O
O-
O
P O
O-
O
P OH
O-
O
P
O OOH
O
OH
3 Na+
Structure of ChromaTide fluorescein-12-dUTP
(C-7604)
2) Detection of biopolymers in cell (tissue) Labelling proteins with radioactive isotope
Li, C.H.: Iodination of tyrosine groups in serum albumin and pepsin JACS 67 1065 (1945)
I131 I3- I
2 + I-
I2 + H
2O H
2OI+ + I-
Tyr His
I
O-
COO-
NH3
+
I
O-
I
NH+
NH
I
COO-
NH3
+
Hnatowich, D.J. et al.:
The preparation and labeling of DTPA-coupled albumin Int J Appl Radiat Isot 33 327-332 (1982)
N
O
NH+ N
O
O
O
O
O
O-
O
NNH
+ N
O-
O
NH OR
O
O-
O-
O
O-
O
R NH2
amin tartalmú molekula
DTPA
Labelling of proteins with biotin Bayer, E.A., Wilehek, M.: The use of avidin-biotin complex
Methods Biochem Anal 26 1 (1980)
Chaiet, L., Wolf, F.J.: The properties of streptavidin, a biotin-binding protein produced by streptomyces Arch Biochem Biophys 106 1 (1964)
Green, N.M.: Avidin Adv Protein Chem 29 85 (1975)
NH NH
S
O
COOH
2
4*
D-Biotin
[Hexahidro-2-oxo-1H-tieno[3,4-d]imidazol-4-pentánsav]
Ka = 1015 M-1
op.: 232-233 oC
Hexahydro-2-oxo-1H-thieno[3,4-d]imidazole-4-pentanoic acid; (+)-Biotin
m.p.
Labelling of biopolymers with fluorophore
McKinney, R. et al.
Factors affecting the rate of reaction of fluorescein isothiocyanat
with serum proteins J Immunol 93 232 (1964)
OO-
O
NC
S
O-
O
R NH2
amin tartalmú molekula+
OO-
O
NHC
S
O-
O
NHR
Fluoreszcein izotiocianát Tiourea kötés létrehozása
fluorescein isothiocyanate thiourea linkage
amino functional group
2) Affinity chromatography Bethell, G.S. et al. A novel method of activation of cross-
linked agarose with 1,1’-carbonyldiimidazol which gives a matrix for affinity chromatography
J Biol Chem 254 2572 (1979)
O
OH
OH
OO
CH2OH
O
O
O
O
CH2
CHOH
CH2
O
CH2
O
OH
OH
OOO
O
O
OH
CNBr
C NH
O
O
C NO
L NH2C NHO
NH
L
Drug research
Analysis (chemical, medical)
Proteins: structure - function
Separation science
Structural studies Definitions
1. Two or more active components 2. Covalent linkage
Approaches 1. Size of the partners
Small – small
Small – big
Big – big
2. Type of linkage Direct
NH2 HOOC
Indirect
NH2 HOOC
spacer
3. Type of linkage
Acid amide
Acid ester
Acid anhydride
C NH
O
S NH
O
O
O P NH
O-
O
O P O
O-
CH3O
C O
O
carboxylic acid sulphonic acid phosphoric acid amid
carboxylic acid phosphoric acid
CO
O
C
O
PO
O
P
OOH
OH
CO
O
P
O
OH
carboxylic acid phosphoric acid mixed
Hydrazone
Schiff base
Ether
C NH
O NAldehyde + hydrazine Aldehyde + amine Alcohol
NC
H
CH2
OCH2
Carbamate
Secunder amine (N-glycoside)
Isourea
Isothiourea
CH2
OC
NH
O
CH2 Alcohol Amine + aldehyde + aryl-halogenide Amine Amine
CH2
NH CH2
NHCH2
CH2
NHC
NH
O
CH2
CH2
NHC
NH
S
CH2
Thioether
Thioester
Disulphide
Diazo
C – X
Thiol Thiol Thiol Azide
CH2
S CH2
SCH2
C
S CH2
O
CH2S
SCH2
SS
CH2
NN
C I
adenylic acid +
„carboxylic acid” mixed anhydride
1. Example: small–small, direct, anhydride bond „Firefly” luminescence
©nationalgeographic.com,
Radim Schreiber
http://www.photobiology.info/Branchini2.html
Step a: formation of the enzyme-bound luciferyl adenylate; Step b: a proton is abstracted from the C-4 carbon of the adenylate by a basic side chain amino acid residue of luciferase; Step c: molecular oxygen adds to the newly formed anion; Step d: a highly reactive dioxetanone intermediate is formed; Step e: an electronically excited state oxyluciferin molecule, CO2 and red light emission (λmax = 615 nm) are produced (at pH 6); Step f: the enolate dianion formed by tautomerization from the exited form of oxyluciferin and yellow-green light emission (λmax = 560 nm) occurs at pH 8.
Yellow-Green light Red light Green to red light
D-Luciferin (LH2) Luciferyl-AMP)
oxyluciferin (keto) oxyluciferin (enol)
CO2
+ AMP
+ PPi + H+
dioxetanone
2. Example: small – big, indirect, amide bond
Liposome – hapten conjugate
NH2
NH2
O
O
O
O
CH3
O
CH3
O
O-
O
P
NH3
+
PE liposzóma Foszfatidil etanolaminphosphatidylethanolamine
PE liposome
2. Example
O
O
O
O
CH3
O
CH3
O
O-
O
P
NH3
+
+ HOOC GAFA
NH
CH3
O
NC
N
CH3
NH+
CH3
CH3
EDC
1-etil-3-(3-dimetil-aminopropil)karbodiimid
O
O
O
O
CH3
O
CH3
O
O-
O
P
NH NH CH3
O
GAFACO
+ EDC HOH
NH CO GAFA NHAc
NH CO GAFA NHAc
NH2
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide
HOOC-GAFA +
tetrapeptide
3. Example: big–big, indirect, disulphide bond
Immunotoxin conjugate
1. step: introduction of protected -SH group(s) of
antibody (Ab + SPDP → amide bond
2. step: creation of free -SH groups of the toxin subunits (cleavage of the -S-S- bond, DTT)
3. step: free –SH partner reacts with the component having „protected” SH function (AB-SSP + Toxin-SH → disulphid linkage)
3. Example Carlsson, J. et al. Biochem J 173 723 (1978)
Cleland W. Biochemistry 3 480 (1964)
NH2
+ N O
O S
S
N
O
O
N OH
O
O
NH
O S
S
Namino csoportot tartalmazó SPDP
antitest NHS
SPDP aktivált antitest
NH
O S
S
B chain
A chain DTT
B chain A chain
Toxin with A and B subunits
Reduction, free SH groups
Immunotoxin formation with disulphide bridge
N
S
Pyridine-2-thione = 343 nm
Antibody with free NH2 group(s)
SPDP modified Ab
+
R
O
O
R1 (C, P)
R
S
O
R1
R
NH
O
R1
R
O
O
R1
O
R
NH
O
NH2
R
N3
O
R
NH
O
N
R2
R
OH
O
R2
H
O
R2
H
N
N N
CH2
CH2R
R3 OH
R2 O
R4NH
R4
OR3
O
esther
anhydride hydrazide azide
hydrazone
diazo
Schiff base
ether carbamate
reduction
oxidation
Map tioesther amide
R5 NH2
R5 NH
R (Ar)
R
NHR5
O
CH
NR5NH
R
NHR5
O
NH
R
NHR5
S
Map
Schiff-base isourea isothiourea
secunder amine amide (SO, POH)
R6 SH
R6 S
R6
S R
SR6
tioether disulphid bridge
1) Introduction of functional groups ? (-COOH, -CHO, -OH, -SH, -NH2)
2) Elimination of functional groups?