General introduction ............................................ 8
Chapter I Theoretical part ................................... 11
I.1 Hydrogen bond ............................................ 11
I.1.1 Geometry of hydrogen bond ........................ 11
I.1.2 Classification of homo-nuclear formally
symmetric H-bonds ................................ 14
I.2 NMR for H-bond investigation ............................. 15
I.2.1 NMR in the liquid state .......................... 15
I.2.2 NMR in the solid state ........................... 16
I.3 Correlation of some NMR parameters with hydrogen bond
geometry ................................................. 18
I.3.1 1H chemical shift ................................ 18
I.3.2 15N chemical shift ............................... 19
I.3.3 1JNH coupling constant ........................... 20
I.3.4 Primary H/D isotope effect on chemical shift ..... 21
I.4 Carbonic anhydrase ....................................... 23
I.4.1 Structure ........................................ 24
I.4.2 Functionality .................................... 25
I.4.3 Models for carbonic anhydrase .................... 28
Chapter II Model complexes containing single NHN H-bond ....... 30
II.1 Anions ................................................... 32
II.2 NMR line shape analysis .................................. 33
II.3 Homoconjugated cations ................................... 35
II.3.1 Comparison of Py, 4MePy and 4DMAPy ............... 36
II.3.2 Homoconjugated cation of pyridine ................ 39
II.4 Protonated bases ......................................... 40
II.5 Counteranion-dependent intra- vs. intermolecular
hydrogen bonding and proton transfer ..................... 42
II.5.1 Results .......................................... 45
II.5.2 Discussion ....................................... 48
II.6 Poly-4-vinylpyridine ..................................... 51
II.6.1 Results and Discussion ........................... 54
II.7 Conclusions .............................................. 57
Chapter III Models of the carbonic anhydrase active site ...... 60
III.1 Hydroxide complex [TpPh,me-Zn-OH-] ........................ 60
III.1.1 Assignment of OH- signal ......................... 60
III.1.2 Interactions with bases .......................... 62
III.2 Bicarbonate complex ...................................... 63
III.2.1 Identification of the bicarbonate complex ........ 64
III.2.2 Acid properties of [TpPh,meZn-OC(0)OH-] ........... 66
III.2.3 Formation of covalently bound dimers ............. 70
III.3 Aqua complex [TpPh,meZn-CH2] .............................. 72
III.3.1 Results .......................................... 74
III.3.2 Discussion ....................................... 76
III.4 Conclusions .............................................. 79
Chapter IV Wild and mutant types of native carbonic
anhydrase .......................................... 80
IV.1 4-Methylimidazole ........................................ 80
IV.2 Native carbonic anhydrase ................................ 81
IV.3 Conclusions .............................................. 85
Chapter V Experimental part .................................. 86
V.1 Spectral methods ......................................... 86
V.1.1 Liquid state NMR ................................. 86
V.1.2 Solid state NMR .................................. 86
V.1.3 Molecular Weight Analysis ........................ 87
V.1.4 Multiwavelength UV irradiation ................... 87
V.2 Chemicals and materials .................................. 87
V.3 Samples preparation ...................................... 88
V.3.1 Small models containing H-bond ................... 88
V.3.2 Models of carbonic anhydrase active site ......... 89
V.4 Synthesis of the selectively labeled target molecules .... 91
V.4.1 15N-Pyridine ..................................... 91
V.4.2 4-Methyl-15N-pyridine ............................ 92
V.4.3 4-N,N-dimethylamino-15N-pyridine ................. 93
V.4.4 2,2'-bi-15N-pyridine ............................. 95
V.4.5 Poly-4-vinyl-15N-pyridine ........................ 97
V.4.6 15N,13C-4-methylimidazole ........................ 102
V.5 Conclusions ............................................. 103
Summary of the work ........................................... 104
Literature .................................................... 107
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