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eaction towards the thermodynamically most stable assembly of host and guest and allows its isolation. In a series of seminal papers Sanders has demonstrated the viability of the concept. [5-9] In this contribution, we aim to develop a novel antibiotic compound using DCC using real-time mass spectrometry to monitor the DCL. [11-14] As a biological target mimic, we have chosen a peptide sequence unique for bacteria in the synthesis of their cell walls. A bacterial cell wall obtains mechanic stability by cross linking carbohydrate co-polymer chains build from N-acetyl glucosamine and muraminic acid with peptide bridges to form murein. β-Lactam antibiotics inhibit this process by inhibition of the transpeptidase enzyme carrying out the cross linking, [15] while Vancomycin binds specifically to the interpeptide sequence. [16] The compounds to be developed here should share the same biological activity and mechanism as Vancomycin being selected to bind to the specific amino acid sequence of the interpeptide linkage. In terms of DCL building blocks we have chosen aromatic dialdehydes in combination with dihydrazides based on tartaric acid. We have shown that dialdehydes in combination with diamines are able to form trianglimine macrocycles allowing large variability in the dialdehyde building block. [17-25] Recently, we have shown that using real-time mass spectrometry, we could detect all intermediates in a DCL formation along with probing the dynamic reversibility of the process. [25] In a further contribution we have shown that diamines can be replaced by dihydrazides allowing increased versatility in building block selection with dihydrazides bearing useful functionalities, facilitating molecular recognition and enhancing solubility in polar solvents. [26,27] When analysing a DCL by mass spectrometry, the most important question arising is whether the members amplified and selected in DCL are observable as individual compounds or as complex ions formed from host and guest. The majority of DCL studies using MS (Mass spectrometry) as a prime analytical tool have reported the detection of the DCL host compounds rather than supramolecular complex ions, with a notable exception published by Poulsen using a peptide library. [28] The ability to detect intact complex ions is of particular importance for two reasons. Firstly, the positive observation of a complex ion is evidence that the chosen guest molecule amplifies the host rather than any other building block present in the library. Secondly, modern tandem MS techniques using energy resolved mass spectrometry would allow in principle to screen the binding energy between each individual host-guest system during the MS experiment. 3 | P a g e
EXPERIMENTAL All the solvents and reagents used for the reactions were purchased from Sigma-Aldrich (Munich, Germany) or Applichem (Darmstadt, Germany) and were used as obtained. Solvents and reagents were used without further purification. Whenever possible the reactions were monitored by thin layer chromatography on aluminum-backed plates pre-coated with silica gel 60 (UV 254 ) (Macherey-Nagel, Düren, Germany). Preloaded wang resin, N,N-diisopropylethylamine (DIEA), O-(Benzotriazol-1-yl)-hydroxybenzotriazole (HOBt), N,N,N',N'-tetramethyluranium hexafluorophosphate (HBTU), 1-Fmoc (9-fluorenylmethoxycarbonyl)amino acid derivatives, dimethylformamide (DMF), N-methylpyrrolidone (NMP), trifluoroacetic acid (TFA), 1,2-ethanedithiol (EDT) and other chemicals required for peptide synthesis were bought from Iris Biotech GmbH (Marktredwitz, Germany). Peptides were synthesised with standard solid phase peptide synthesis technique and it was carried out on an automated peptide synthesiser (ABI-433A, Applied Biosystems, Foster city, USA). Fmoc protected preloaded resin was used to grow peptide chain on it. In detail, deprotection of the N-terminal of resin bound amino acid was performed by 20% piperidine in NMP and the C-terminal of other protected amino acid was activated using HBTU/HOBt/DIEA (1:1:2) in DMF for coupling with the free N- terminal of the resin bound amino acid. A mixture of 82.5% TFA, 5% phenol, 5% water, 5% thioanisol and 2.5% EDT was used to separate the peptide from resin as well as to remove the side chain protecting groups. Subsequently, the peptide was isolated through precipitation in cold (˗20 o C) diethyl ether and lyophilised by Christ freeze dryers (Martin Christ Gefriertrocknungsanlagen GmbH, Osterode am Harz, Germany). Peptides were purified by high performance liquid chromatography (HPLC) and analysed by high resolution mass spectrometry (HRMS). Compounds 1 and 4-7 (Scheme 1) were synthesised following our synthetic procedure. [26,27] Mass spectra of the samples dissolved in DMF and acetonitrile (ACN) were acquired by a high resolution (exceeding 15000 FWHM) micrOTOF Focus mass spectrometer equipped with an electrospray ionisation (ESI) source in the positive ion mode (Bruker Daltonics, Bremen, Germany). Calibration was carried out using a 0.1 M solution of sodium formate in the enhanced quadratic mode prior to each experimental run. Samples were directly infused into the ESI-TOF mass spectrometer. The results of the measurements were processed using Compass 1.3 data analysis software for a Bruker Daltonics 4 | P a g e
Page 1 and 2:
The Development of Novel Antibiotic
Page 3 and 4:
Declaration of Authorship I, Hany N
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Abstract Over the past few decades,
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Chapter 2 Trianglimine Chemistry
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Figure 2.4 Trianglimines 10-12 with
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Abbreviations ACN AcOH Ala Acetonit
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RMS ROESY SjGST TFA THF TLC TMS TOC
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Introduction complete remodel of th
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Introduction following protonation.
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Introduction Lehn and co-workers re
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Introduction Figure 1.8 Generation
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Introduction Figure 1.11 Synthetic
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Introduction Figure 1.13 Generation
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Introduction 1.3.11 Boronic ester e
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Introduction References 1. E. Mouli
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Introduction 51. B. Shi, M. F. Grea
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Introduction Figure 2.2 shows compu
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Introduction incorporate β-cyclode
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Introduction Trianglamine-Zn(R) 2 c
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Introduction 24. J. Gajewy, J. Gawr
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Scope of Work Scope of Work In this
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Scope of Work can be obtained from
Page 45 and 46: Scope of Work Figure 3.4 Structures
Page 47 and 48: Scope of Work References 1. G. Wrig
Page 49 and 50: Organic & Biomolecular Chemistry Ci
Page 51 and 52: Downloaded by Jacobs University Bre
Page 53 and 54: View Online Downloaded by Jacobs Un
Page 63 and 64: Paper 2 Rapid Commun. Mass Spectrom
Page 65 and 66: Trianglimine formation mechanism by
Page 75 and 76: Paper 3 Org. Biomol. Chem., 2012, 1
Page 81 and 82: View Online Table 2 Distances (Å)
Page 85 and 86: Paper 4 Chem. Eur. J., 2012, submit
Page 87 and 88: (4R,5R)- and (4S,5S)-dimethyl-2,2-d
Page 89 and 90: that two distinct conformational is
Page 91 and 92: the thimble was recharged with fres
Page 93 and 94: Paper 5 Rapid Commu. Mass Spectrom.
Page 95: INTRODUCTION No single class of dru
Page 99 and 100: (b) Refluxing macrocycles 5, 6 and
Page 101 and 102: Scheme 1. Graphical representation
Page 103 and 104: Figure 1. A plot of relative intens
Page 105 and 106: In contrast, formation of library m
Page 107 and 108: Figure 5. Generated dynamic library
Page 109 and 110: macrocycle 32 from the dynamic libr
Page 111 and 112: The polar carbamate group participa
Page 113 and 114: [2] WHO World health report 2003. h
Page 115 and 116: [32] B. Lienard, N. Selevsek, N. Ol
Page 117 and 118: FULL PAPER Synthesis and ESI-MS Com
Page 119 and 120: ability of the technique to provide
Page 121 and 122: aimed at assessing molecular recogn
Page 123 and 124: Paper 7 manuscript Synthesis, self-
Page 125 and 126: 2 Similar to the case of trianglimi
Page 127 and 128: 4 Receptor 7 showed enhanced recogn
Page 129 and 130: 6 The newly synthesized receptors s
Page 131 and 132: 8 27. Nour, H. F.; Hourani, N.; Kuh
Page 133 and 134: 120| P a g e Appendix
Page 135 and 136: Supplementary Information: Suppleme
Page 137 and 138: Supplementary Material (ESI) for Or
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Supplementary Material (ESI) for Or
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Page 151 and 152:
Page 153 and 154:
Monitoring the [3+3]-cyclocondensat
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Monitoring the cyclocondensation re
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Product ions appeared as [M+H] + Fi
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Scheme 1. Assigned higher oligomers
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Supplementary Information: Synthesi
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Scheme 4. Proposed fragmentation me
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DMSO Figure 1. 1 H-NMR spectrum for
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Figure 6. 13 C-NMR spectrum for (4S
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Intens. 7 x10 1.5 200.7 1.0 0.5 0.0
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Intens. 7 x10 200.7 1.5 1.0 0.5 0.0
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Intens. 4 x10 577.2 2.0 1.5 1.0 451
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HN N O O R R NH O O N N HN R O O O
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Intens. 5 x10 727.2 2.0 1.5 1.0 O O
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Intens. 5 x10 697.1 1.5 1.0 728.2 0
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O O O O HN N O O NH N HN N O O NH N
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Figure 38. 1 H-NMR spectrum for mac
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Intens. 5000 276.8 4000 3000 2000 1
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Intens. 7 x10 599.1 1.5 1.0 0.5 0.0
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(a) HN N O O NH O O N (b) HN N O NH
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Scheme 11. Proposed fragmentation m
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Scheme 14. Proposed fragmentation m
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Intens. 4 x10 550.9 3 2 1 0 500 100
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Figure 68. 2D-ROESY spectrum for ma
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Supplementary Information: Novel sy
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Intens. 6 x10 0.8 0.6 0.4 240.9 459
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Figure 9. 1 H-NMR spectrum for macr
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Intens. 5 x10 1.5 968.1 1.0 0.5 813
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Intens. 5 x10 897.4 4 2 335.3 707.3
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Scheme 3. Suggested fragmentation m
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Page 217 and 218:
Molecular modelling data for the co
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Figure 1. 1 H NMR spectrum for dica
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Intens. 2000 1493.6091 Host-Guest 1
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Intens. [%] 100 775.3 80 388.1 60 4
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Intens. 600 N HN O O 467.1916 400 O
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Intens. 6000 5000 HN N O O O O NH N
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Table 1. High resolution ESI-TOF da
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i Figure 1. 1 H NMR spectrum for ma
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i Figure 5. 1 H NMR spectrum for ma
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i j Figure 9. 1 H NMR spectrum for
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i Figure 13. DEPT-135 spectrum for
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Scheme 1. General mechanism of frag
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Intens. [%] 100 80 60 40 20 0 O H N
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Intens. [%] 100 1131.3 80 60 40 20
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Intens. 1500 1528.6088 1000 500 150
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Intens. [%] 100 80 60 40 Free guest
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Intens. 4 x10 1085.3575 1.25 1.00 0
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Intens. [%] 100 80 Free guest 586.1
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Synthesis, self-assembly and ESI-MS
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O O HN NH HN O O NH HN O 11 O NH O
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O O HN NH HN O O NH HN O 9 O NH O O
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H 2 O DMSO DMSO Figure 6. 1 H-NMR a
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Intens. 5 x10 5 -MS 487.1588 4 3 2
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Intens. -MS 6 x10 0.8 0.6 0.4 0.2 1
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Intens. -MS 6 x10 O O 350.9 1.5 1.0
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Intens. [%] 100 -MS 486.9 Exact str
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Intens. [%] -MS 100 486.9 80 60 975
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Intens. [%] +MS 100 80 517.2 Exact
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Intens. [%] 100 80 60 +MS 388.1 775
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Intens. [%] 100 +MS 517.2 Exact str
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Intens. [%] 100 80 +MS 1031.4939 7
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Intens. [%] 100 +MS 517.2 Exact str
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Intens. [%] 100 -MS 2 586.1 80 60 4
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Intens. [%] 100 -MS 2 875.2 Exact s
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Table 1. High resolution ESI-TOF/MS
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274 | P a g e
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276 | P a g e
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4. H. Nour, A. López-Periago, N. K
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