lecture 1: key concepts in stereoselective synthesis · web viewthe concerted huisgen cycloaddition...

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This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License . OC VI (HS 2015) Bode Research Group http://www.bode.ethz.ch/ Topic: Ligation and Bioconjugation 1. 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. 2. 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 1

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Lecture 1: Key Concepts in Stereoselective Synthesis

OC VI (HS 2015) Bode Research Group http://www.bode.ethz.ch/

This work is licensed under aCreative Commons Attribution-NonCommercial-ShareAlike 4.0 International License.

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