By Kayleigh Schouren
Summary Chemical Biology
TABLE OF CONTENTS
Introduction ................................................................................................................................. 4
Carbohydrate Synthesis ................................................................................................................ 5
Nomenclature ..................................................................................................................................... 6
Fischer projection rules....................................................................................................................... 7
The anomeric effect ............................................................................................................................ 8
Ring conformations ............................................................................................................................. 9
Protecting groups................................................................................................................................ 9
Ethers ................................................................................................................................................ 10
Acetal / Ketal ..................................................................................................................................... 13
Carbonates ........................................................................................................................................ 13
Amine protecting groups .................................................................................................................. 14
Protecting the anomeric centre ........................................................................................................ 16
Relative reactivity of sugar alcohols ................................................................................................. 17
Regioselective deprotection ............................................................................................................. 21
Functionalization of monosaccharides ............................................................................................. 22
Halogenation ..................................................................................................................................... 22
Inverse the stereochemistry of alcohols ........................................................................................... 23
What to do with the modified/halogenated sugars? ....................................................................... 24
Glycosyl epoxides: ............................................................................................................................. 25
Oligosaccharide Synthesis .......................................................................................................... 27
Anomeric leaving groups .................................................................................................................. 27
Stereochemistry of the glycosidic bond............................................................................................ 30
Glycosidic linkages ............................................................................................................................ 34
Solvent participation ......................................................................................................................... 35
Peptide Synthesis....................................................................................................................... 41
Nomenclature ................................................................................................................................... 41
Peptide Protecting Groups................................................................................................................ 42
Nα amino protection ......................................................................................................................... 43
Cα carboxy protection ....................................................................................................................... 44
Orthogonality .................................................................................................................................... 47
1
, By Kayleigh Schouren
Solution Phase Peptide Chemistry .................................................................................................... 48
carbodiimides.................................................................................................................................... 48
Phosphonium reagents ..................................................................................................................... 49
Uranium reagents ............................................................................................................................. 49
Racemization..................................................................................................................................... 49
Solid Phase Peptide Synthesis........................................................................................................... 50
Peptide Ligations ............................................................................................................................... 54
Larger proteins .................................................................................................................................. 54
Native Chemical Ligation .................................................................................................................. 54
Nature’s ligation................................................................................................................................ 56
Non-native chemical ligation ............................................................................................................ 57
Peptide Modifications ....................................................................................................................... 58
Cyclization ......................................................................................................................................... 58
Disulphide bonds .............................................................................................................................. 59
Labels and tags .................................................................................................................................. 59
Phosphorylation ................................................................................................................................ 59
Close analogues: ............................................................................................................................... 60
PNA ................................................................................................................................................... 61
Synthesis of Oligonucleotides ..................................................................................................... 61
Molecular Structure of Oligonucleotides.......................................................................................... 62
Phosphoramidite coupling ................................................................................................................ 62
Side reactions .................................................................................................................................... 65
Synthesis of RNA ............................................................................................................................... 65
Common modifications ..................................................................................................................... 66
DNA-peptide conjugates ................................................................................................................... 67
Bioorthogonal Chemistry ........................................................................................................... 69
Fluorescent proteins ......................................................................................................................... 69
Metabolic labelling............................................................................................................................ 69
Staudinger Ligation ........................................................................................................................... 70
CuAAC (Huisgen 3+2 dipolar cycloaddition) ..................................................................................... 71
SPAAC (strain-promoted alkyne-azide click ...................................................................................... 71
Tetrazine ligation .............................................................................................................................. 73
Orthogonal bioorthogonal reactions ................................................................................................ 73
Click to release .................................................................................................................................. 73
Protein Engineering : Incorporation of Unnatural Amino Acids .................................................... 74
Method 1: residue specific replacement .......................................................................................... 75
2
, By Kayleigh Schouren
Method 2: site specific incorporation of a 21st amino acid .............................................................. 76
Orthogonal tRNA............................................................................................................................... 77
Expanding the genetic code to 4-letter codons ................................................................................ 80
Protein Engineering : (Semi)Synthetic Protein Modification ........................................................ 82
Adding modified cofactors ................................................................................................................ 84
Use the functionality of protein splicing: .......................................................................................... 85
Enzyme tagging (fusion proteins) ..................................................................................................... 87
Post Translational Modifications (PTMs) ..................................................................................... 88
Phosphorylation ................................................................................................................................ 89
Acetylation ........................................................................................................................................ 91
Methylation....................................................................................................................................... 93
Chemical Glycobiology ............................................................................................................... 95
Glycan labelling ................................................................................................................................. 95
Functional modifications................................................................................................................... 99
Enzymatic labelling ......................................................................................................................... 101
Inhibitors ......................................................................................................................................... 102
Make probes for recycling enzymes ............................................................................................... 104
Glycoprotein synthesis .................................................................................................................... 104
Activity Based Protein Profiling ................................................................................................ 106
Covalent catalysis ............................................................................................................................ 107
Non-covalent catalysis .................................................................................................................... 108
Reactive Cysteine/Lysine profiling .................................................................................................. 109
Cysteine cathepsins in cancer ......................................................................................................... 110
Regulation of Protein Levels ..................................................................................................... 112
DNA modifying techniques ............................................................................................................. 115
Hijack the ubiquitin proteasome system ........................................................................................ 116
Chemical Biology of Oligonucleotides ....................................................................................... 117
Nucleic acid Templated Chemistry ................................................................................................. 118
Expanded genetic alphabet ............................................................................................................ 119
Fluorescent RNA aptamers ............................................................................................................. 120
RNA Structure Profiling ................................................................................................................... 120
Metabolic labelling of DNA ............................................................................................................. 120
3
, By Kayleigh Schouren
INTRODUCTION
What is chemical biology?
‘ use of chemistry to advance a molecular understanding of biology and the harnessing of biology to
advance chemistry’
It is a relatively new field in which the synthesis of the biomolecules is crucial to study their function.
The central dogma on its own cannot explain life, for that we need to understand epigenetics and
post translational modifications (PTM) as well (ubiquitination, cleavage, phosphorylation etc.).
Reversible PTMs are installed by enzymes, whereas irreversible PTMs stem from environmental
factors. PTMs are often specific for amino acids →
Functions of PTMs include regulation of:
- protein maturation
- enzymatic activity
- cellular localization
- stability / turnover
- interactions with other molecules
The most complex PTM is glycosylation, for which
no direct blueprint is available. Glycans can
be N-linked (Arg) and O-linked (Ser or Thr).
50% of all proteins are glycosylated, 90%
of extracellular proteins are glycosylated.
N-glycan biosynthesis starts in the ER
lumen with GlcNAc, Mannose and
eventually Glucose. When proteins are
misfolded and remain in the ER lumen,
their glycan chain will degrade. This is
recognized which leads to elimination by
the ER-associated degradation pathway
(ERAD). The glycan processing continues in
the Golgi. From where proteins will go to
lysosomes or the extracellular
environment.
4
Summary Chemical Biology
TABLE OF CONTENTS
Introduction ................................................................................................................................. 4
Carbohydrate Synthesis ................................................................................................................ 5
Nomenclature ..................................................................................................................................... 6
Fischer projection rules....................................................................................................................... 7
The anomeric effect ............................................................................................................................ 8
Ring conformations ............................................................................................................................. 9
Protecting groups................................................................................................................................ 9
Ethers ................................................................................................................................................ 10
Acetal / Ketal ..................................................................................................................................... 13
Carbonates ........................................................................................................................................ 13
Amine protecting groups .................................................................................................................. 14
Protecting the anomeric centre ........................................................................................................ 16
Relative reactivity of sugar alcohols ................................................................................................. 17
Regioselective deprotection ............................................................................................................. 21
Functionalization of monosaccharides ............................................................................................. 22
Halogenation ..................................................................................................................................... 22
Inverse the stereochemistry of alcohols ........................................................................................... 23
What to do with the modified/halogenated sugars? ....................................................................... 24
Glycosyl epoxides: ............................................................................................................................. 25
Oligosaccharide Synthesis .......................................................................................................... 27
Anomeric leaving groups .................................................................................................................. 27
Stereochemistry of the glycosidic bond............................................................................................ 30
Glycosidic linkages ............................................................................................................................ 34
Solvent participation ......................................................................................................................... 35
Peptide Synthesis....................................................................................................................... 41
Nomenclature ................................................................................................................................... 41
Peptide Protecting Groups................................................................................................................ 42
Nα amino protection ......................................................................................................................... 43
Cα carboxy protection ....................................................................................................................... 44
Orthogonality .................................................................................................................................... 47
1
, By Kayleigh Schouren
Solution Phase Peptide Chemistry .................................................................................................... 48
carbodiimides.................................................................................................................................... 48
Phosphonium reagents ..................................................................................................................... 49
Uranium reagents ............................................................................................................................. 49
Racemization..................................................................................................................................... 49
Solid Phase Peptide Synthesis........................................................................................................... 50
Peptide Ligations ............................................................................................................................... 54
Larger proteins .................................................................................................................................. 54
Native Chemical Ligation .................................................................................................................. 54
Nature’s ligation................................................................................................................................ 56
Non-native chemical ligation ............................................................................................................ 57
Peptide Modifications ....................................................................................................................... 58
Cyclization ......................................................................................................................................... 58
Disulphide bonds .............................................................................................................................. 59
Labels and tags .................................................................................................................................. 59
Phosphorylation ................................................................................................................................ 59
Close analogues: ............................................................................................................................... 60
PNA ................................................................................................................................................... 61
Synthesis of Oligonucleotides ..................................................................................................... 61
Molecular Structure of Oligonucleotides.......................................................................................... 62
Phosphoramidite coupling ................................................................................................................ 62
Side reactions .................................................................................................................................... 65
Synthesis of RNA ............................................................................................................................... 65
Common modifications ..................................................................................................................... 66
DNA-peptide conjugates ................................................................................................................... 67
Bioorthogonal Chemistry ........................................................................................................... 69
Fluorescent proteins ......................................................................................................................... 69
Metabolic labelling............................................................................................................................ 69
Staudinger Ligation ........................................................................................................................... 70
CuAAC (Huisgen 3+2 dipolar cycloaddition) ..................................................................................... 71
SPAAC (strain-promoted alkyne-azide click ...................................................................................... 71
Tetrazine ligation .............................................................................................................................. 73
Orthogonal bioorthogonal reactions ................................................................................................ 73
Click to release .................................................................................................................................. 73
Protein Engineering : Incorporation of Unnatural Amino Acids .................................................... 74
Method 1: residue specific replacement .......................................................................................... 75
2
, By Kayleigh Schouren
Method 2: site specific incorporation of a 21st amino acid .............................................................. 76
Orthogonal tRNA............................................................................................................................... 77
Expanding the genetic code to 4-letter codons ................................................................................ 80
Protein Engineering : (Semi)Synthetic Protein Modification ........................................................ 82
Adding modified cofactors ................................................................................................................ 84
Use the functionality of protein splicing: .......................................................................................... 85
Enzyme tagging (fusion proteins) ..................................................................................................... 87
Post Translational Modifications (PTMs) ..................................................................................... 88
Phosphorylation ................................................................................................................................ 89
Acetylation ........................................................................................................................................ 91
Methylation....................................................................................................................................... 93
Chemical Glycobiology ............................................................................................................... 95
Glycan labelling ................................................................................................................................. 95
Functional modifications................................................................................................................... 99
Enzymatic labelling ......................................................................................................................... 101
Inhibitors ......................................................................................................................................... 102
Make probes for recycling enzymes ............................................................................................... 104
Glycoprotein synthesis .................................................................................................................... 104
Activity Based Protein Profiling ................................................................................................ 106
Covalent catalysis ............................................................................................................................ 107
Non-covalent catalysis .................................................................................................................... 108
Reactive Cysteine/Lysine profiling .................................................................................................. 109
Cysteine cathepsins in cancer ......................................................................................................... 110
Regulation of Protein Levels ..................................................................................................... 112
DNA modifying techniques ............................................................................................................. 115
Hijack the ubiquitin proteasome system ........................................................................................ 116
Chemical Biology of Oligonucleotides ....................................................................................... 117
Nucleic acid Templated Chemistry ................................................................................................. 118
Expanded genetic alphabet ............................................................................................................ 119
Fluorescent RNA aptamers ............................................................................................................. 120
RNA Structure Profiling ................................................................................................................... 120
Metabolic labelling of DNA ............................................................................................................. 120
3
, By Kayleigh Schouren
INTRODUCTION
What is chemical biology?
‘ use of chemistry to advance a molecular understanding of biology and the harnessing of biology to
advance chemistry’
It is a relatively new field in which the synthesis of the biomolecules is crucial to study their function.
The central dogma on its own cannot explain life, for that we need to understand epigenetics and
post translational modifications (PTM) as well (ubiquitination, cleavage, phosphorylation etc.).
Reversible PTMs are installed by enzymes, whereas irreversible PTMs stem from environmental
factors. PTMs are often specific for amino acids →
Functions of PTMs include regulation of:
- protein maturation
- enzymatic activity
- cellular localization
- stability / turnover
- interactions with other molecules
The most complex PTM is glycosylation, for which
no direct blueprint is available. Glycans can
be N-linked (Arg) and O-linked (Ser or Thr).
50% of all proteins are glycosylated, 90%
of extracellular proteins are glycosylated.
N-glycan biosynthesis starts in the ER
lumen with GlcNAc, Mannose and
eventually Glucose. When proteins are
misfolded and remain in the ER lumen,
their glycan chain will degrade. This is
recognized which leads to elimination by
the ER-associated degradation pathway
(ERAD). The glycan processing continues in
the Golgi. From where proteins will go to
lysosomes or the extracellular
environment.
4
