- Limits of miniaturization: Assessing ITP performance in sub-micron and nanochannels
- Janssen, Kjeld G. H.; Li, Jiajie; Hoang, Hanh T.; Vulto, Paul; van den Berg, Richard J. B. H. N.; Overkleeft, Herman S.; Eijkel, Jan C. T.; Tas, Niels R.; van der Linden, Heiko J.; Hankemeier, Thomas
The feasibility of isotachophoresis in channels of sub micrometer and nanometer dimension is investigated. A sample injection volume of 0.4 pL is focused and separated in a 330 nm deep channel. The sample consists of a biomatrix containing the fluorescently-labeled amino acids glutamate and phenylalanine, 20 attomoles of each. Isotachophoretic focusing is successfully demonstrated in a 50 nm deep channel. Separation of the two amino acids in the 50 nm deep channel however, could not be performed as the maximum applicable voltage was insufficient. This limit is imposed by bubble formation that we contribute to cavitation as a result of the mismatch in electro-osmotic flow, so called electrocavitation. This represents an unexpected limit on the miniaturization of ITP. Nonetheless, we report the smallest isotachophoretic separation and focusing experiment to date, both in terms of controlled sample injection volume and channel height.
- Synthesis of Eight 1-Deoxynojirimycin Isomers from a Single Chiral Cyanohydrin
- van den Nieuwendijk, Adrianus M. C. H.; van den Berg, Richard J. B. H. N.; Ruben, Mark; Witte, Martin D.; Brussee, Johannes; Boot, Rolf G.; van der Marel, Gijsbert A.; Aerts, Johannes M. F. G.; Overkleeft, Herman S.
Eight configurational 1-deoxynojirimycin isomers have been synthesized starting from a chiral cyanohydrin as the common precursor. The cyanohydrin chiral pool building block is easily accessible in large quantities by using almond hydroxynitrile lyase as the chiral catalyst in condensing hydrogen cyanide and crotonaldehyde. Our work complements the large body of literature on the synthesis of 1-deoxynojirimycin derivatives with the distinguishing feature that eight stereoisomers of this important class of glycosidase inhibitors can be derived from a common precursor in an efficient manner.
- Quantification of Globotriaosylsphingosine in Plasma and Urine of Fabry Patients by Stable Isotope Ultraperformance Liquid Chromatography-Tandem Mass Spectrometry
- Gold, H.; Mirzaian, M.; Dekker, N.; Joao Ferraz, M.; Lugtenburg, J.; Codee, J. D.; van der Marel, G. A.; Overkleeft, H. S.; Linthorst, G. E.; Groener, J. E.; Aerts, J. M.; Poorthuis, B. J.
BACKGROUND: Biochemical markers that accurately reflect the severity and progression of disease in patients with Fabry disease and their response to treatment are urgently needed. Globotriaosylsphingosine, also called lysoglobotriaosylceramide (lysoGb3), is a promising candidate biomarker.METHODS: We synthesized lysoGb3 and isotope-labeled [5,6,7,8,9] (13)C(5)-lysoGb3 (internal standard). After addition of the internal standard to 25 muL plasma or 400 muL urine from patients with Fabry disease and healthy controls, samples were extracted with organic solvents and the lysoGb3 concentration was quantified by UPLC-ESI-MS/MS (ultraperformance liquid chromatography-electrospray ionization-tandem mass spectrometry). Calibration curves were constructed with control plasma and urine supplemented with lysoGb3. In addition to lysoGb3, lyso-ene-Gb3 was quantified. Quantification was achieved by multiple reaction monitoring of the transitions m/z 786.4 > 282.3 [M+H](+) for lysoGb3, m/z 791.4 > 287.3 [M+H](+) for [5,6,7,8,9] (13)C(5)-lysoGb3, and 784.4 > 280.3 [M+H](+) for lyso-ene-Gb3.RESULTS: The mean (SD) plasma lysoGb3 concentration from 10 classically affected Fabry hemizygotes was 94.4 (25.8) pmol/mL (range 52.7-136.8 pmol/mL), from 10 classically affected Fabry heterozygotes 9.6 (5.8) pmol/mL (range 4.1-23.5 pmol/mL), and from 20 healthy controls 0.4 (0.1) pmol/mL (range 0.3-0.5 pmol/mL). Lyso-ene-Gb3 concentrations were 10%-25% of total lysoGb3. The urine concentration of lysoGb3 was 40-480 times lower than in corresponding plasma samples. Lyso-ene-Gb3 concentrations in urine were comparable or even higher than the corresponding lysoGb3 concentrations.CONCLUSIONS: This assay for the quantification of lysoGb3 and lyso-ene-Gb3 in human plasma and urine samples will be an important tool in the diagnosis of Fabry disease and for monitoring the effect of enzyme replacement therapy in patients with Fabry disease.
- Identification and isolation of lantibiotics from culture: a bioorthogonal chemistry approach
- Li, J.; Girard, G.; Florea, B. I.; Geurink, P. P.; Li, N.; van der Marel, G. A.; Overhand, M.; Overkleeft, H. S.; van Wezel, G. P.
A distinguishing feature of the lantibiotic family of cyclic peptides is the presence of thioethers. Treatment of a lantibiotic with an alkaline solution at high pH gives rise to a beta-elimination reaction yielding the corresponding ring opened precursor, containing a dehydro-amino acid residue. We here reveal in a proof-of-concept study that a ring opened lantibiotic (mersacidin) can be captured for pull-down from a culture broth, subsequently released and identified by mass spectrometry.
- A pH-Sensitive, Colorful, Lanthanide-Chelating Paramagnetic NMR Probe
- Liu, W. M.; Keizers, P. H. J.; Hass, M. A. S.; Blok, A.; Tirnmer, M.; Sarris, A. J. C.; Overhand, M.; Ubbink, M.
Paramagnetic lanthanides ions are broadly used in NMR spectroscopy. The effects of unpaired electrons on NMR spectral parameters provide a powerful tool for the characterization of macromolecular structures and dynamics. Here, a new lanthanide-chelating NMR probe, Caged Lanthanide NMR Probe-7 (CLaNP-7), is presented. It can be attached to protein surfaces via two disulfide bridges, yielding a probe that is rigid relative to the protein backbone. CLaNP-7 extends the application range of available probes. It has a yellow color, which is helpful for sample preparation. Its effects are comparable to those of CLaNP-5, but its charge is two units lower (+1) than that of CLaNP-5 (+3), reducing the change in surface potential after probe attachment. It also has a different magnetic susceptibility tensor, so by using both tags, two sets of structural restraints can be obtained per engineered cysteine pair. Moreover, it was found that the orientation of the magnetic susceptibility tensor is pH dependent (pK(a) approximate to 7) when a histidine residue is located in the neighborhood of the probe attachment site. The results show that the His imidazole group interacts with the CLaNP-7 tag. It is proposed that the histidine residue forms a hydrogen bond to a water/hydroxyl molecule that occupies the ninth coordination position on the lanthanide, thus breaking the two-fold symmetry of the CLaNP tag in a pH-dependent way.
- Novel protecting groups in carbohydrate chemistry
- Codee, J. D. C.; Ali, A.; Overkleeft, H. S.; van der Marel, G. A.
Protecting groups play a pivotal role in carbohydrate chemistry. This review describes a selection of protecting groups and protecting group strategies that have been introduced since the beginning of this century. (C) 2010 Academie des sciences. Published by Elsevier Masson SAS. All rights reserved.
- A practical synthesis of capped 4-methylumbelliferyl hyaluronan disaccharides and tetrasaccharides as potential hyaluronidase substrates
- Gold, H.; Munneke, S.; Dinkelaar, J.; Overkleeft, H. S.; Aerts, J. M.; Codee, J. D.; van der Marel, G. A.
The synthesis of hyaluronan dimers and tetramers equipped with a 4-methylumbelliferyl group at the reducing end to potentially allow monitoring of hyaluronidase activities is described. The 4-OH at the non-reducing glucuronate in the presented series is either removed or methylated to prohibit transglycosylase reactions, leading to a total of four probes.
- A Concise Synthesis of Globotriaosylsphingosine
- Gold, H.; Boot, R. G.; Aerts, J. M. F. G.; Overkleeft, H. S.; Codee, J. D. C.; van der Marel, G. A.
Globotriaosylsphingosine (lysoCTH) is produced in the cell by deacylation of the globo-sphingolipid globotriaosylceramide. The latter compound is the major storage material encountered in Fabry patients, an inherited lysosomal storage disorder characterized by partially impaired a-galactosidase A (GLA) activity. Recent findings suggest that lysoCTH, next to its acylated precursor, is an important causative of Fabry disease symptoms. The glycolipid is thus a relevant synthetic target, and we here report on its efficient synthesis. Key to our strategy is the use of 4,6-O-di-tert-butylsilyleneprotected D-galactose donors to yield D-Gal-a-D-Gal linkages with high stereoselectivity. In our optimized route we make use of acyl protecting groups to mask most of the hydroxy functions in the carbohydrate building blocks to facilitate straightforward global deprotection.
- Synthesis of pH-Activatable Red Fluorescent BODIPY Dyes with Distinct Functionalities
- Hoogendoorn, S.; Blom, A. E. M.; Willems, L. I.; van der Marel, G. A.; Overkleeft, H. S.
A series of tunable pH-dependent BODIPY dyes were synthesized and further functionalized in a Knoevenagel condensation reaction with various aldehydes. In this fashion, monofunctional dyes containing an alkyne, azide, or carboxylic acid (masked as its methyl ester) as ligation sites as well as asymmetrical bifunctional dyes were obtained, without compromising their pH-dependency. In addition, fluorescence excitation and emission maxima for these dyes were shown to be significantly red-shifted in comparison to their tetramethyl precursors.
- Bioorthogonal Chemistry: Applications in Activity-Based Protein Profiling
- Willems, L. I.; Van der Linden, W. A.; Li, N.; Li, K. Y.; Liu, N.; Hoogendoorn, S.; Van der Marel, G. A.; Florea, B. I.; Overkleeft, H. S.
The close Interaction between organic chemistry and biology goes back to the late 18th century, when the modern natural sciences began to take shape. After synthetic organic chemistry arose as a discipline, organic chemists almost immediately began to pursue the synthesis of naturally occurring compounds, thereby contributing to the understanding of their functions in biological processes. Research in those days was often remarkably interdisciplinary; in fact, it constituted chemical biology research before the phrase even existed. For example, histological dyes, both of an organic and inorganic nature, were developed and applied by independent researchers (Gram and Golgi) with the aim of visualizing cellular substructures (the bacterial cell wall and the Golgi apparatus).Over the years, as knowledge within the various fields of the natural sciences deepened, research disciplines drifted apart, becoming rather monodisciplinary. In these years, broadly ranging from the end of World War II to about the 1980s, organic chemistry continued to Impact life sciences research, but contributions were of a more indirect nature. As an example, the development of the polymerase chain reaction, from which molecular biology and genetics research have greatly profited, was partly predicated on the availability of synthetic oligonucleotides. These molecules first became available in the late 1960s, the result of organic chemists pursuing the synthesis of DNA oligomers primarily because of the synthetic challenges involved.Today, academic natural sciences research Is again becoming more interdisciplinary, and sometimes even multidisciplinary. What was termed "chemical biology" by Stuart Schreiber at the end of the last century can be roughly described as the use of Intellectually chemical approaches to shed light on processes that are fundamentally rooted in biology. Chemical tools and techniques that are developed for biological studies In the exciting and rapidly evolving field of chemical biology research Include contributions from many areas of the multifaceted discipline of chemistry, and particularly from organic chemistry. Researchers apply knowledge inherent to organic chemistry, such as reactivity and selectivity, to the manipulation of specific biomolecules in biological samples (cell extracts, living cells, and sometimes even animal models) to gain insight into the biological phenomena in which these molecules participate.In this Account, we highlight some of the recent developments in chemical biology research driven by organic chemistry, with a focus on bioorthogonal chemistry In relation to activity-based protein profiling. The rigorous demands of bioorthogonality have not yet been realized in a truly bioorthogonal reagent pair, but remarkable progress has afforded a range of tangible contributions to chemical biology research. Activity-based protein profiling, which aims to obtain information on the workings of a protein (or protein family) within the larger context of the full biological system, has in particular benefited from these advances. Both activity-based protein profiling and bioorthogonal chemistry have been around for approximately 15 years, and about 8 years ago the two fields very profitably intersected. We expect that each discipline, both separately and in concert, will continue to make important contributions to chemical biology research.