Lecture 1: Key Concepts in Stereoselective Synthesis
OC VI (HS 2015) Bode Research Group http://www.bode.ethz.ch/
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Topic: Ligation and Bioconjugation
Introduction
Macromolecules, especially peptides up to 30-40 amino acids can reliably synthetized by solid phase peptide synthesis. Longer sequences can be accessed with fragment coupling methods. The first possibility is to couple partially protected peptide fragments followed by the removal of protecting groups. This strategy is limited by the low solubility of the fragments and by the possible epimerization of the activated C terminus.
To overcome these limitations new approaches were reported. These are called chemoselective ligations of unprotected peptides or macromolecules.
The term chemoselective ligation refers to the coupling of two mutually and uniquely reactive functional groups in aqueous environment or in the presence of biological material. Thus, even among a multitude of potentially reactive functional groups, two chemoselective partners will react only with each other.
Bertozzi Trends Biotech. 1998, 16, 506
The ligation reactions can be used for the chemical synthesis macromolecules or for the modification/labeling of biomolecules. On the scheme above A and B functional groups are chemoselective reaction partners.
The criteria to perform chemoselective ligation/bioconjugation in biological systems
Ligation and bioconjugation are essentially used to bind high molecular mass molecules together in aqueous media or biological systems. To fulfill these challenges, ligation/bioconjugation reactions have to respect certain criteria:
The functionalities of the ligation reaction must react selectively with each other under mild conditions
The reaction must yield covalent bonds and no or harmless side products like water or carbon dioxide
The reactants must be stable before the ligation reaction and not toxic
The reaction must proceed with a reasonable rate
Bode ACS Chem. Biol. 2015, 10, 1026
The graph on the right illustrates the limitations of the current ligation reactions. The low solubility of macromolecules requires working in diluted conditions (M concentrations). To achieve good yields under reasonable time at these conditions fast reactions are required.
Types of ligation reactions
Ligation at cysteine junctions
The first type of protein ligation reactions are based on the unique reactivity of the thiol function on the side chain of cysteine. This allows the chemoselective ligation of unprotected protein fragments. Unfortunately cysteine is the second rarest amino acid in natural proteins.
Lau Justus Liebigs Ann. Chem. 1953, 583, 129
Native Chemical Ligation (NCL)
In 1994 Kent and co-workers reported the reaction of unprotected peptides with thioesters on the N peptide and cysteine on the C peptide.
Kent Science 1994, 266, 776
The method can be applied to unprotected peptide fragments, and importantly, additional cysteine residues do not interfere with the overall reaction, since the irreversibile S -> N shift occurs uniquely on the N terminal of the cysteine residue.
NCL is a highly chemoselective reaction and forms native amide bonds. The reactions proceed at low (mM) concentration and in neutral buffered aqueous solutions. NCL even tolerates denaturing reagents during the hydrophobic peptide synthesis. Compared with solution phase peptide synthesis ( 10,000 M1 s1).
Lin JACS 2014, 136, 4153
Inverse electron-demand Diels-Alder reaction
The normal Diels-Alder reaction usually requires EWG-activated dienophiles (e.g. maleimides). Those dienophiles are not suitable because of their Michael acceptor properties to various nucleophiles commonly found in biological systems.
trans-Cyclooctenes and tetrazines
Bioorthogonal reactions between tetrazines and trans-cyclooctenes proceed very fast in water (k = 102-104 M1s1) without any catalyst and produce N2 as a sole byproduct. The fast kinetics result from the ring strain of trans-cyclooctenes (16 kcal/mol). However, there is also a concern of the isomerization of the double bond from trans to cis over time. Also cyclooctenes and tetrazines are large in size and may complicate the metabolic or enzymatic incorporation into biomolecules.
Fox JACS 2008, 130, 13518
Norbornenes and tetrazines
This cycloaddition is based on the inverse electron-demand Diels-Alder reaction of tetrazines to norbornenes, followed by retro-Diels-Alder elimination of molecular nitrogen to afford the expected dihydropyridazines and their regioisomers. Although the reaction kinetics is slower (k = 101-10 M1 s1) compared to the reaction with trans-cyclooctenes, norbornenes are bench stable.
Hilderbrand Bioconjugate Chem. 2008, 19, 2297
Cyclopropenes and tetrazines
Functionalized cyclopropenes were recently found to be good substrates for IED-DA reactions with tetrazines. Cyclopropenes are stable yet reactive and compatible with various metabolic pathways sue to their small size. The reaction rate is, however, slower than that of the reaction with trans-cyclooctenes.
Prescher JACS 2012, 134, 18638
Metal-mediated bioconjugation reactions
Ru-mediated cross-metathesis
Cross-metathesis appears as an attractive method to install modifications onto biomolecules through the formation of a stable C=C bond. Ruthenium-catalyzed metathesis is a remarkable reaction for its high selectivity and tolerance toward different functional groups. However, the reactions are usually run with organic co-solvents. In the case below, the method was applied to the serine protease subtilisin Bacillus lentus (SBL).
Davis JACS 2008, 130, 9642
Pd-mediated Suzuki-Miyaura cross-coupling
The Suzuki-Miyaura cross-coupling reaction is an emerging tool for bioconjugations. The reaction can be utilized such as for modification of the protein surface and DNA. This bioconjugation method does not require organic solvents.
Davis JACS 2009, 131, 16346
Mutually orthogonal bioconjugation
A new challenge in bioconjugation areas is identification of the mutually orthogonal bioconjugation reactions. Those reactions allow the simultaneous monitoring of the multiple biomolecules in a single biological system.
- Some cyclooctynes are reactive toward tetrazines, and they cannot be used for the multilabeling purpose. However, the judicious choice of the substrate structure allowed double [3 + 2] cycloaddition simultaneously in the cell imaging by taking advantage of the difference in reaction rates. The substituents on the tetrazine was critical. Hilderbrand demonstrated the simultaneous labeling of two different cancer cell types.
Hilderbrand ACIE 2012, 51, 920
- Houk and Prescher have recently reported the unique reactivity of the substituted cyclopropenes. 1,3-disubstituted cyclopropenes showed high reactivity toward tetrazines to undergo the IED-DA reaction while 3,3-disubstituted cyclopropenes did not react with tetratines. Instead, 3,3-disubstituted cyclopropenes showed the facile reaction with nitrile imines (generated by photolysis) to undergo the 1,3-dipolar cycloaddition. They tested the mutual orthogonality by labeling the model protein, BSA (Scheme below).
Houk & Prescher JACS 2013, 135, 13680
1
MgCl2, 30% tBuOH, pH 8.0, 2-5 h, 37 C
MesN NMes
RuClCl
iPrOS SBL
HOO
O3
S SBLHO
OO
3
SBL S
I
SBL SB(OH)2
37 C, pH 8.0 phosphate
N
N
NaO
NaO
NH2 Pd(OAc)2
N
OR
NNR
N
HO
NN N
N
Me
(2.1 0.2) M-1s-1
(210 10) M-1s-1
no reaction observed
(0.0064 0.002) M-1s-1
N
OR
NN
N R
NHN
MeHO
N
OR
NHNMe
HO
NN
N
R* Buffer pH 7.4, 37 C
NH25
NH2
NH25
5
R = PEG4COOH
N
O
R
N
N
R
N
HO
N
NN
N
Me
(2.1 0.2) M
-1
s
-1
(210 10) M
-1
s
-1
no reaction observed
(0.0064 0.002) M
-1
s
-1
N
O
R
N
N
N
R
NH
N
Me
HO
N
O
R
NHN
Me
HO
N
N
N
R
* Buffer pH 7.4, 37 C
NH
2
5
NH
2
NH
2
5
5
R = PEG
4
COOH
BSANH
O
Me
BSANH
OMe
O
NN
NNN
rhodamine
N
NN
NOMe
hv
1)
2)
BSANH
O
NN
Me
N
Orhodamine
BSANH
O
NN
Me
OMe
H2NO
SR
H2N
HS
O
OH - HSR
+ HSR
H2NO
H2N
S
O
OHRearrangement H2N
O
NH
O
OH
SH
Peptide-1
COOH
NH2
Peptide-2
OH
SH
SR
O OH3N
S
capture
Peptide-1
CH2OOH
NH2
Peptide-2
OH
SHO
OH2N
S
S -> N shiftrearrangement
thiol-thioester exchange
Peptide-1
COOH
NH2
Peptide-2
OH
SH
NH
O
O
SH
H2N COOH
H2N
H2N
COOH
COOH
SAryl
O OH2N
HS
1. kinetic controlled NCL (buffer pH6.3, 6 M GnHCl, 0.2 M NaHPO4, 19 mM TCEP)2. arylthiol additive; Cys41 capped with 2-bromoacetamide3. purification
Y-Gln41
SAlkyl
O
SAryl
O
HIV (B1-B40) HIV (B42-B99)SAryl
O OH2N
HS
HIV (B1-B40) HIV (B42-B99)
1. NCL2. Cys41 Cys41 capped with 2-bromoacetamide; Thz deprotection3. purification
Y-Gln41
(Gly)4-Cys(Thz)
1. NCL2. Cys modification
Y-Gln41 HIV (B1-B40) HIV (B42-B99)Y-Gln41Y-Gln201
Cys-(Gly)4
Cys-(Gly)4
HIV (A1-A40) HIV (A42-A99)
HIV (A1-A40) HIV (A42-A99)
HIV (A1-A40) HIV (A42-A99)
H2N
H2N
H2N
COOH
COOH
COOH
Peptide-1 Peptide-2NH
O
O
SHPeptide-1 Peptide-2N
H
O
O
CH3S
ACM
H2N
H2N
COOH
COOHPd/Al2O3
Phosphate buffer
H2
Peptide-1 Peptide-2NH
O
O
CH3S
ACM
H2N COOH
TCEPAIBN
SACM
Peptide-1
SR
O OHN
R
N
O
O
R
AuxHS
Aux
SH
NH
O
O
R
Peptide-2
Peptide-1
Peptide-1 Peptide-2
Peptide-2H2N
H2N
H2N
COOH
COOH
COOH
NCL
Peptide-1 Peptide-2SR
OO
H2N
SeH
Peptide-1 Peptide-2NH
O
O
SeH
Peptide-1 Se
OPeptide-2
OH2N
Ligation
Se - N shift
Peptide-1 Peptide-2SR
O OH2N
SH
Homocysteine
Peptide-1 Peptide-2NH
O
O
SMeMethionine
H2N
H2N
COOH
COOH
1. Ligation2. S - N shift3. S methylation
OH
OH
NH2
NH2
COOH
COOH
R1O
OH
O
NH
HOR2
R1NH
R2
O
- H2O- CO2
H2N
O
OHO
NH
R1
HOHN
NH
O
R2
O
OH
H2NO
NH
R1HN
NH
O
R2
O
OH
Type I KAHA-ligation
Unprotected peptide 1 Unprotected peptide 2
Unprotected peptide 1 Unprotected peptide 2
OHHNR2
NR2OH O
OHR1
O
OHO
R1
OH R1
NO
R2
O
OHR1
N
O
OH
OR2- H2O H+
O
O
R1
NHO
R2
NH
R2
O R1
NH
R2O
R1O
O
hemiaminal Z- and E-nitrones
amide
-lactone oxaziridine
hydroxylamine and ketoacid
NH
OHN
HONH2
O
ONH
COOH
OH
O
H2N
oxalic acid3:1 DMA/DMSO60 C, 20 h 51 % yield
ONH
COOH
H2N NH
OHN
NH2
O
GLP-1 (7-20) GLP-1 (23-36)
GLP-1 (23-36)GLP-1 (7-20) GLP-1 (23-36)
GLP-1 (23-36)
H2N
O
OHO
NH
R1
OHN
NH
OO
OH
H2NO
NH
R1HN
NH
OO
OH
Type II KAHA-ligation
Unprotected peptide 1 Unprotected peptide 2
Unprotected peptide 1 Unprotected peptide 2
OH
H2NO
NH
R1
Unprotected peptide 1
H2N NH
OO
OHUnprotected peptide 2
O
Depsipeptide
O - N shift
O
NH
HN
OR1
OOH
O
R2
O
NH
NO
R2H
CO2HHO
R1
NH
NO
R2R1 H2O
+ H2O
O
HO
OO
NH
NHO
R2CR1
CO2
O
NH
R2HNR1
O
+ H2O / - H+
Path A
OH
amide
O
NH
R2HN
O
R1
Path B
O
NH
R2H2N
O
R1HO
O
NH
R2H3N
O + H2O
H2OOR1
ester
iminium nitrilium
iminium ether
ketoacid and hydroxylamine
O
NH
HN
OO
H2N
O
O
NH
HN
NH
O
OH
Me
Me
NH OH
O
1) KAHA ligation DMSO:H2O 0.1 M oxalic acid 60 C, 20 h 2) O to N acyl shift pH 9.5, buffer
H2NOH
OMe
Me
OH
Pup (2-31) Pup (34-63)
Pup (34-63)Pup (2-31)
Pup (2-63) (T33T)
Pup (2-31) Pup (34-63)
R1 BF3K
OHN
R2 R1 NH
OR2
tBuOH/H2ORT
OBz
H2NHN
NH
O
ONH
HNO
N
O
EtEt
MeO
OH
H2NHN
NH
O
ONH
HN
MeO
OH
O
OO
MeOn
OO
MeOn
PEG20,000
BF3K
0.1 M oxalic acidtBuOH/H2O
NHN
COOH
OH
OH
COOH CONH2
COOHOH
COOH
NH
HN
H2N
NH
HN
NH2
NH
COOH HN
H2N
NH
NH
HN
NH2
NH
NHN
COOH
OH
OH
COOH CONH2
COOHOH
HAEGTFTSDVSSYLEGQA
HAEGTFTSDVSSYLEGQA EFIAWLVRGRG
EFIAWLVRGRG
O
unprotected peptide unprotected peptideO
O
CHOH2N
RHO
OHH unprotected peptideO
H unprotected peptide OHN
O R
OH
unprotected peptideO
H NH
unprotected peptide OH
OHR
SAL ester Ser or Thr
N,O-benzylidine acetal cleavageTFA/H2O 10 min
unprotected peptide unprotected peptide
O
O
CHO
H
2
N
R
HO
OH
H
unprotected peptide
O
H
unprotected peptide
OH
N
O
R
OH
unprotected peptide
O
H
N
H
unprotected peptide
OH
OH
R
SAL esterSer or Thr
N,O-benzylidine acetal cleavage
TFA/H
2
O 10 min
Peptide I
Peptide II
NH2HS
O
S R1. ICL2. S - N shift
Peptide I
Peptide II
NHHS
O
Peptide I
Peptide II
NH
O
Desulphurisation
Biomolecule N3MeO
O
Ph2PBiomolecule
MeO
O
PPhPh
N-N2
NH
O
Ph2P
Biomolecule
O-MeOH
+H2O
Fluorophore
Cystein proteas inhibitor
Fluorescence microscopy
Living cell NH
O
Ph2P
Streptavidin + Fluorophore
O
SH
OLiving cell
Cystein proteas inhibitor
HO
SN3
N3
MeO
O
Ph2P Biotin
Biotin
Living cell
Cystein proteas inhibitor
HO
S
NH
O
Ph2PO
Biotin
Living cell
Cystein proteas inhibitor
HO
S
Streptavidin + Fluorophore
X
PPh2
O
R2
N N N R1+
N2
X
PPh2
O
R2
N R1PPh2
N
X OR2
R1 PPh2
N
R2X
R1
O
XH
PPh2
O + HN
R2
R1
O
+ H2O
X
PPh2
O
R2
= S
PPh2
OR2
O
PPh2
OR2
S
PPh2
OR2
iminophosphorane(aza-ylide) tetrahedral intermediate
amidophosphonium saltstabilizedphosphine
R1 = proteins, peptides, lipidsR2 = peptides, proteins, fatty acids, fluorophores, biotin, FLAG
N N NR1
+(R2O)3P
N2R1
N PO
R2
OO
R2
R2 R1HN P
OO
OR2
R2
+ H2O
-R2OHtrialkyl phosphite
R1N P
OR2
OO
R2
R2
phosphorimidate phosphoramidate
R1 = proteins, peptides, lipids, carbohydratesR2 = H, PEG, photocaged PEG, peptides
ProteinH2N CO2H
N3
OP
O2N
O
OO
CH3
CH3
16
3Photocaged PEG750
28 C, 12 h
ProteinH2N CO2H
HN
PO
O
O
PPEG
PPEG
h (355nm)
pH 6.8, 0 C, 1 min
ProteinH2N CO2H
HN
PHO
OH
O
R1 H
O+
NH2H
H2O R1 H
NPh
R1 H
NPhHH
protonatedSchiff-Base
H2N O R2
H2N NH
R3
O
R1 H
NO R2
oxime
+
NH2
+
NH2
R1 H
NN R3
hydrazone
O
NH
OEt
OEt
Ub
0.5 M aq. HCl
NH
O
Ub H
peptide
NH
OO
H2N
0.5 M aq. HCl
peptide
NH
OO
NNH
Ub
peptide
NHHN
NH
UbO
O
native isopeptide
NNR1
N
R2
60-120 NN
N NN
N
R2
R1 R1
R2
1
4
1
5
regioisomers
hours-days
H2NHN
OHO
OR2
R1
H2N N
R2
N N O
OHR1
NH
O
NH
O
N3n
NH
N
O
O
HN
O n
, Cu(I), buffer pH 8
N N
NaO3S SO3Nay
NN
NR
CPMVCPMV
transferrin
NNH
O NH
O
SN
O
O DyeN N
NNN N
N3
O
O
paclitaxel
CuSO4sodium ascorbate
in living cells
N
NH
O
NH
O
NN
N
NN N
O
O
paclitaxel
NNN
S
NDyeO
O
RN3N
NN R
160
O
COOHO
FF
COOH
NOMe
k = 2.4 x 103 M1 s1 k = 7.6 x 102 M1 s1 k = 0.96 M1 s1
ONH2
OH
O NO
Me
RO
OO
NHO
MeO
50
1. NaIO4, pH 6.9 buffer, RT2. MeNHOHHCl, RT
3. RT
IL-8
IL-8
R N O
NO
ON
R
RDirectly on the DNA synthesizer or directly on DNA solid support
+ regioisomers
HN
HNNH
NH
NH
O
O
OO
O
HN NH2
NH
OHO
Ph
N3
R
MeOD/D2O,37 C, 4-5 days
OF3C
O
HN
OHN
ON
N
O
OHN
HO O
O
HOOOH
HN
HNNH
NH
NH
O
O
O
O
O
HN NH2
NH
OHOPh
N
NN
F3C
O
HNO
HN
ON
N
O
OHN
HOO
O
HOOOH
handle for radiolabling
+ another regioisomer
N NN
N
R1 X
N2
hN N
R1X + R2 NN
X R
R2
+ regioisomer
pyrazoline
Segment I.
NH2
OH
SH
A B
COOH
OH
NHN
Segment II. Segment I.
NH2
OH
SH
COOH
OH
NHN
Segment II.
O
N NN
NR
O
MeO
302 nm
ONN
R
R2O
OMe
E. Coli
protein
protein
E. Coli
sfGFP
HN
O
O
sfGFP
HN
O
O
NN
ONHBocNaO3S
H H
NNN
NO
NHBocNaO3S
302 nm, phosphate buffer
k = 10420 810 M1 s1
O
NN N
N
N
N
NN
N
N
RO
H
H
N2
NNH
N
N
RO isomers
NNN
N
RO
N
N
H
H
O
HN
N
O
Othioredxin
+
HN
O NN N
N
N
HN
N2
HNO
NN
NH
HNO
NHN
NH
NNN
N
HNO
NH
O
O
antibody
fluorophore
antibody
fluorophore
Ofluorophore
antibody
Ofluorophore
antibody
isomers+
CellMe
NN
NN
N
O
biotin
1)
2)Cell
NN
Me N
Obiotin
APC
APC
Avidin-APC