Exploring the Binding Site of Ubiquinone in Complex I by EPR/DEER
Exploring the Binding Site of Ubiquinone in Complex I by EPR/DEER
Exploring the Binding Site of Ubiquinone in Complex I by EPR/DEER
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<strong>Explor<strong>in</strong>g</strong> <strong>the</strong> <strong>B<strong>in</strong>d<strong>in</strong>g</strong> <strong>Site</strong> <strong>of</strong> <strong>Ubiqu<strong>in</strong>one</strong> <strong>in</strong> <strong>Complex</strong> I <strong>by</strong> <strong>EPR</strong>/<strong>DEER</strong><br />
Müge Aksoyoglu*, Udo Glessner**, Thomas Spatzal**, Erik Schleicher*, Stefan Weber*<br />
* Department <strong>of</strong> Physical Chemistry, Albert-Ludwigs-Universität Freiburg, Germany<br />
** Department <strong>of</strong> Biochemistry, Albert-Ludwigs-Universität Freiburg, Germany<br />
The proton pump<strong>in</strong>g NADH:ubiqu<strong>in</strong>one oxidoreductase, <strong>the</strong> respiratory complex I, couples<br />
electron transfer from NADH to ubiqu<strong>in</strong>one with <strong>the</strong> translocation <strong>of</strong> protons across <strong>the</strong><br />
membrane [1]. <strong>Ubiqu<strong>in</strong>one</strong>, which b<strong>in</strong>ds to complex I, is a key component <strong>of</strong> <strong>the</strong> electrontransport<br />
cha<strong>in</strong> because it delivers electrons from complex I to complex III. Recently, <strong>the</strong><br />
three-dimensional structure <strong>of</strong> <strong>the</strong> entire complex I from Thermus <strong>the</strong>rmophilius was<br />
determ<strong>in</strong>ed [2]. However, <strong>the</strong> structure does not show <strong>the</strong> presence <strong>of</strong> bound qu<strong>in</strong>one but a<br />
large cavity at <strong>the</strong> end <strong>of</strong> <strong>the</strong> peripheral arm was proposed as b<strong>in</strong>d<strong>in</strong>g site. In this study, sitedirected<br />
sp<strong>in</strong> labell<strong>in</strong>g (SDSL) <strong>in</strong> comb<strong>in</strong>ation with <strong>EPR</strong>/<strong>DEER</strong> spectroscopy is used to<br />
localize <strong>the</strong> ubiqu<strong>in</strong>one b<strong>in</strong>d<strong>in</strong>g site <strong>in</strong> complex I on a molecular level. Various positions <strong>in</strong><br />
<strong>the</strong> prote<strong>in</strong> as well as decyl-ubiqu<strong>in</strong>one (Q10) were sp<strong>in</strong> labelled with MTSL to measure<br />
distances between <strong>the</strong> two labels via cw-<strong>EPR</strong> and <strong>DEER</strong> experiments. We present some <strong>of</strong><br />
our results consider<strong>in</strong>g <strong>the</strong> b<strong>in</strong>d<strong>in</strong>g site <strong>of</strong> ubiqu<strong>in</strong>one.<br />
Moreover, we present prelim<strong>in</strong>ary data on <strong>the</strong> FeMo c<strong>of</strong>actor from nitrogenase, which reduces<br />
<strong>the</strong> d<strong>in</strong>itrogen triple bond under atmospheric pressure and temperature, <strong>the</strong>re<strong>by</strong> form<strong>in</strong>g<br />
ammonia <strong>in</strong> a reaction coupled to <strong>the</strong> hydrolysis <strong>of</strong> ATP [3]. Various <strong>EPR</strong> and HYSCORE<br />
methods were used to <strong>in</strong>vestigate <strong>the</strong> FeMo c<strong>of</strong>actor, which is not yet fully understood <strong>in</strong><br />
terms <strong>of</strong> its composition.<br />
[1] Pohl, T., Spatzal, T., Aksoyoglu, M., Schleicher, E., Rostas, A.M., Lay, H., Glessner, U.,<br />
Boudon, C., Hellwig, P., Weber, S., Friedrich, T., Sp<strong>in</strong> labell<strong>in</strong>g <strong>of</strong> <strong>the</strong> Escherichia coli<br />
NADH ubiqu<strong>in</strong>one oxidoreductase (complex I). Biochim. Biophys. Acta 2010, 1797,<br />
1894–1900.<br />
[2] Efremov, R.G., Baradaran, R., Sazanov, L.A., The architecture <strong>of</strong> respiratory complex I.<br />
Nature 2010, 465, 441–447.<br />
[3] Lee, H., Benton, P., Laryukh<strong>in</strong>, M., Igarashi, R., Dean, D.R., Seefeldt, L.C., H<strong>of</strong>fmann,<br />
B.M., The <strong>in</strong>terstitial atom <strong>of</strong> <strong>the</strong> nitrogenase FeMo-c<strong>of</strong>actor: ENDOR and ESEEM show<br />
it is not exchangeable nitrogen. J. Am. Chem. Soc. 2003, 125, 5604–5605.<br />
1
PELDOR Study <strong>of</strong> Nitroxide-Labeled Prote<strong>in</strong>s<br />
Florian Altvater, Stephan Re<strong>in</strong>, Stefan Weber and Sylwia Kacprzak<br />
Investigat<strong>in</strong>g biomacromolecules <strong>in</strong> solution with pulsed electron-electron double<br />
resonance (PELDOR) spectroscopy yields distance distributions, which provide<br />
<strong>in</strong>formation about structure and dynamics. The distances are measured between two<br />
paramagnetic centers, e. g. nitroxides, that are <strong>in</strong>troduced <strong>in</strong>to <strong>the</strong> desired positions<br />
via site-directed sp<strong>in</strong> label<strong>in</strong>g. PELDOR is a method <strong>of</strong> choice especially when no<br />
crystal structure is available. Based on <strong>the</strong> results <strong>of</strong> distance measurements <strong>in</strong>itial<br />
model structures can be ref<strong>in</strong>ed or <strong>the</strong> conformational changes dur<strong>in</strong>g enzymatic<br />
actions can be followed.<br />
We present <strong>the</strong> application <strong>of</strong> PELDOR method on <strong>the</strong> examples <strong>of</strong> two dist<strong>in</strong>ct<br />
prote<strong>in</strong>s, Gyrase <strong>of</strong> Bacillus subtilis and Agp1 <strong>of</strong> Agrobacterium tumefaciens. Gyrase<br />
is a heterotetramer consist<strong>in</strong>g <strong>of</strong> two A and two B subunits. Prelim<strong>in</strong>ary results <strong>of</strong> <strong>the</strong><br />
distance distribution <strong>in</strong> <strong>the</strong> Gyrase A dimmer <strong>of</strong> <strong>the</strong> mutant N399C are shown. The<br />
second example present molecular dynamics (MD) simulation approach to ref<strong>in</strong>e <strong>the</strong><br />
model structure <strong>of</strong> phytochrome Agp1 <strong>of</strong> Agrobacterium tumefaciens.<br />
2
A Flexible Approach to Benzotropolones<br />
Deniz Arican and Re<strong>in</strong>hard Brückner<br />
Institut für Organische Chemie und Biochemie, Albert-Ludwigs-Universität, Albertstraße 21,<br />
79104 Freiburg, Germany, e-mail: deniz.arican@uranus.uni-freiburg.de<br />
Benzotropolones (1) are bicyclic aromatic compounds, which are found <strong>in</strong> plants and fungi.<br />
Besides <strong>the</strong>y are <strong>in</strong>terest<strong>in</strong>g from an <strong>in</strong>dustrial po<strong>in</strong>t <strong>of</strong> view. With<strong>in</strong> <strong>the</strong> last six years patents<br />
have been granted regard<strong>in</strong>g <strong>the</strong>ir use as anti-<strong>in</strong>flammatory agents, anti-obesity medication,<br />
UV-absorbers <strong>in</strong> sunscreens,<br />
antimicrobial<br />
agents, and for <strong>the</strong><br />
stabilization <strong>of</strong> household,<br />
cosmetic, and<br />
nutritional products.<br />
Perceiv<strong>in</strong>g benzotropolone (1) not as a benz-annulated aromatic but ra<strong>the</strong>r as <strong>the</strong> enol<br />
tautomer <strong>of</strong> 1,2-diketone 5 gave rise to <strong>the</strong> idea <strong>of</strong> trac<strong>in</strong>g back <strong>the</strong> latter to <strong>the</strong> monoketal 6.<br />
Be<strong>in</strong>g a substituted cycloheptenone <strong>of</strong> sorts 6 should be accessible <strong>by</strong> a r<strong>in</strong>g-clos<strong>in</strong>g<br />
meta<strong>the</strong>sis <strong>of</strong> diolef<strong>in</strong><br />
7. The<br />
latter could<br />
orig<strong>in</strong>ate from<br />
<strong>the</strong> aldehyde 8<br />
and <strong>the</strong> aromatic<br />
compound 9.<br />
Follow<strong>in</strong>g this strategy benzotropolones with 1-3 substituents at various locations were<br />
syn<strong>the</strong>sized <strong>in</strong> overall yields from 18% over 8 steps to 78% over 6 steps.<br />
3
Enantiomerically Pure para-Benzoqu<strong>in</strong>one Spiroketals –<br />
Novel Build<strong>in</strong>g-Blocks for Stereochemically Versatile<br />
Syn<strong>the</strong>ses <strong>of</strong> Cyclohexitols and Carbasugars<br />
Johannes Aucktor, a Chiara Anselmi, a,b Re<strong>in</strong>hard Brückner,* a and Manfred Keller a<br />
a) Institut für Organische Chemie und Biochemie, Albert-Ludwigs-Universität, Albertstraße 21, 79104 Freiburg, Germany<br />
b) New address: Dipartimento di Chimica, Università degli Studi di Perugia, Via Elce di Sotto 8, 06123 Perugia, Italy<br />
Conduritols (1), quercitols (2), and <strong>in</strong>ositols (3) are structurally diverse polyhydroxy-<br />
cyclohexanes (“cyclohexitols”) from nature. Carbapyranoses (4) are analogously<br />
structured unnatural compounds. The great stereochemical variety <strong>of</strong> <strong>the</strong>se species<br />
makes <strong>the</strong>m challeng<strong>in</strong>g syn<strong>the</strong>tic targets.<br />
HO<br />
1<br />
*<br />
OH<br />
*<br />
* OH<br />
* OH<br />
HO<br />
* * OH<br />
HO * * * OH<br />
2 OH<br />
O<br />
5<br />
HO<br />
*<br />
O<br />
O O *<br />
*<br />
H/R<br />
*<br />
OH<br />
HO<br />
*<br />
OH<br />
*<br />
* OH<br />
HO * * * OH<br />
3 OH<br />
OH<br />
* * OH<br />
HO * * * OH<br />
4 OH<br />
We recognized that (almost) all such compounds may stem from one common pre-<br />
cursor 5. The latter was accessible from hydroqu<strong>in</strong>one with �99% ee. 5 conta<strong>in</strong>s a<br />
rigid tricyclic scaffold. It allows to functionalize <strong>the</strong> cyclohexenone-moiety with a high<br />
degree <strong>of</strong> diastereocontrol. Well-work<strong>in</strong>g functionalizations <strong>in</strong>clude cis-vic-dihydroxy-<br />
lation, nucleophilic epoxidation, carbonyl group reduction, and enolate hydroxylation.<br />
Precursors <strong>of</strong> <strong>the</strong> above-mentioned polyhydroxycyclohexanes are accessible under a<br />
variety <strong>of</strong> conditions from such functionalized scaffolds.<br />
4
Biaxial Liquid Crystall<strong>in</strong>e Polymers<br />
F. Brömmel 1 , D. Kramer 1 , P. Benzie 2 , G. Osterw<strong>in</strong>ter 1 , H. F<strong>in</strong>kelmann 1 , S. Elston 2 , A. H<strong>of</strong>fmann 1<br />
1.) Institut für Makromolekulare Chemie, Albert-Ludwigs-Universität Freiburg, 79104 Freiburg<br />
2.) Department <strong>of</strong> Eng<strong>in</strong>eer<strong>in</strong>g Science, University <strong>of</strong> Oxford, Oxford, Parks Road, Oxford, OX1<br />
3PJ, UK<br />
The recent experimental discovery <strong>of</strong> biaxial liquid crystal<br />
(LC) phases provides <strong>the</strong> opportunity to develop <strong>the</strong> technology<br />
for a new generation <strong>of</strong> faster biaxial LC displays.<br />
These expectations are based on <strong>the</strong> existence <strong>of</strong> a secondary<br />
director that can be controlled <strong>by</strong> external fields.<br />
It is estimated that <strong>the</strong> reorientation <strong>of</strong> <strong>the</strong> secondary director<br />
can be more than one order <strong>of</strong> magnitude faster<br />
than <strong>the</strong> reorientation <strong>of</strong> <strong>the</strong> uniaxial nematic director.<br />
However, fundamental questions concern<strong>in</strong>g <strong>the</strong> biaxial<br />
nematic phase behaviour, its alignment with respect to<br />
external fields and its relation to <strong>the</strong> molecular architecture<br />
are still open.<br />
So far, a large number <strong>of</strong> nematic polymers and elastomers have been found to form biaxial nematic<br />
phases. We deuterated <strong>the</strong>se systems <strong>in</strong> various positions. This allowed us to compare <strong>the</strong> obta<strong>in</strong>ed<br />
biaxiality parameters from 2 H NMR experiments on directly deutrated monomers and deuterated probe<br />
molecules, that are commonly used to determ<strong>in</strong>e biaxiality <strong>in</strong> order to overcome <strong>the</strong> syn<strong>the</strong>tical effort<br />
<strong>of</strong> deuteration. Also, it is possible to vary <strong>the</strong> molecular geometry <strong>of</strong> <strong>the</strong> mesogens while preserv<strong>in</strong>g<br />
<strong>the</strong> biaxiality. This way <strong>the</strong> <strong>in</strong>fluence <strong>of</strong> <strong>the</strong> molecular geometry on biaxiality was studied.<br />
The temperature dependence <strong>of</strong> <strong>the</strong> biaxiality for all polymers <strong>in</strong>vestigated so far suggests that <strong>the</strong><br />
formation <strong>of</strong> <strong>the</strong> biaxial phase is connected to <strong>the</strong> glass transition. Accord<strong>in</strong>gly, <strong>the</strong> dynamic <strong>of</strong> <strong>the</strong><br />
polymer cha<strong>in</strong>s plays a decisive role <strong>in</strong> <strong>the</strong> formation <strong>of</strong> <strong>the</strong> biaxial phase.<br />
For potential applications, <strong>the</strong> manipulation <strong>of</strong> <strong>the</strong> m<strong>in</strong>or director <strong>by</strong> external fields without affect<strong>in</strong>g<br />
<strong>the</strong> orientation <strong>of</strong> <strong>the</strong> pr<strong>in</strong>cipal axis is crucial. X-ray scatter<strong>in</strong>g experiments and conoscopy measurements<br />
performed under mechanical deformation reveal that <strong>the</strong> reorientation <strong>of</strong> <strong>the</strong> secondary director<br />
occurs before <strong>the</strong> reorientation <strong>of</strong> <strong>the</strong> primary nematic director sets <strong>in</strong>. This proves <strong>the</strong> selective addressability<br />
<strong>of</strong> <strong>the</strong> biaxial director <strong>by</strong> mechanical fields.<br />
5
S<strong>in</strong>gle molecule spectroscopy <strong>of</strong> membrane bound H + -ATPsynthases from E. coli<br />
Markus Burger *, Peter Gräber*<br />
* Institut für Physikalische Chemie, Albert-Ludwigs Universität Freiburg, Germany<br />
Markus.Burger@physchem.uni-freiburg.de<br />
Subunit movements with<strong>in</strong> <strong>the</strong> H + -ATPsynthase from E. coli (EF0F1) are <strong>in</strong>vestigated <strong>by</strong><br />
s<strong>in</strong>gle molecule FRET spectroscopy. The enzyme is covalently labeled at cyste<strong>in</strong>es with<strong>in</strong> <strong>the</strong><br />
γ-subunit (γC106) and <strong>the</strong> ε-subunit (εC114). The label<strong>in</strong>g procedure is carried out with a<br />
mixture <strong>of</strong> both fluorescent dyes. Therefore, <strong>the</strong> b<strong>in</strong>d<strong>in</strong>g at <strong>the</strong> two positions with <strong>the</strong> donorfluorophor<br />
(ATTO532) and <strong>the</strong> acceptor-fluorophor (ATTO610) occurs statistically. The<br />
donor and acceptor labeled EF0F1 is <strong>in</strong>tegrated <strong>in</strong>to liposomes and s<strong>in</strong>gle-pair fluorescence<br />
resonance energy transfer is measured <strong>in</strong> freely diffus<strong>in</strong>g proteoliposomes with a confocal two<br />
channel microscope. Addition <strong>of</strong> AMPPNP (a non-hydrolysable analogue <strong>of</strong> ATP) leads to a<br />
conformational change <strong>of</strong> <strong>the</strong> ε-subunit from an extended to a folded form. Analysis <strong>of</strong><br />
ensemble-FRET and s<strong>in</strong>gle-pair-FRET <strong>in</strong>dicates distance changes <strong>in</strong> <strong>the</strong> range <strong>of</strong> 4 nm. The<br />
fluorescence time traces obta<strong>in</strong>ed with s<strong>in</strong>gle-pair-FRET reveal a time-dependent switch<strong>in</strong>g <strong>of</strong><br />
<strong>the</strong> ε-subunit <strong>in</strong> <strong>the</strong> presence <strong>of</strong> nucleotides.<br />
ε114 UP<br />
γ106C<br />
ε114 DOWN<br />
Fig. 1. The γ (blue) and ε subunits from E.coli. The ε subunit is shown <strong>in</strong> up (cyan-green) and<br />
down (cyan-red) conformation. Labeled positions are marked with spheres.<br />
6<br />
International Bunsen Discussion Meet<strong>in</strong>g – FRET <strong>in</strong> Life Sciences – 27th - 30th March 2011
Jan Caspar<br />
Arbeitskreis Pr<strong>of</strong>. Dr. P. Spiteller<br />
Institut für Organische Chemie und Biochemie<br />
Titel: Verwundungsaktivierte Verteidigung <strong>in</strong> Marasmius oreades durch Cyanwasserst<strong>of</strong>f<br />
Pilze entwickelten wie auch Pflanzen e<strong>in</strong>e Reihe von Abwehrmechanismen, um sich gegen Fraßfe<strong>in</strong>de<br />
und Schädl<strong>in</strong>ge schützen zu können. Bei der verwundungsaktivierten Verteidigung liegen <strong>in</strong>aktive<br />
Vorläuferverb<strong>in</strong>dungen der eigentlichen Wirkst<strong>of</strong>fe vor, welche durch enzymatische Umwandlung im<br />
Verteidigungsfall <strong>in</strong> ihre bioaktive Form überführt werden. Neben komplexeren, biologisch aktiven<br />
Verb<strong>in</strong>dungen werden auch e<strong>in</strong>fachere Tox<strong>in</strong>e zur Verteidigung e<strong>in</strong>gesetzt. So konnte aus<br />
Fruchtkörpern der Art Aleurodiscus amorphus e<strong>in</strong> Cyanhydr<strong>in</strong> isoliert werden, welches durch e<strong>in</strong>en<br />
oxidativen Mechanismus zu HCN und Aleurodiscoester umgesetzt wird. In Fruchtkörpern der Art<br />
Marasmius oreades konnte nach Beschädigung ebenfalls e<strong>in</strong>e Cyanidfreisetzung festgestellt werden.<br />
Die zugehörige Ausgangsverb<strong>in</strong>dung ist jedoch nicht bekannt. Erste Experimente legen nahe, dass es<br />
sich hierbei um e<strong>in</strong> hochpolares, geschütztes Cyanhydr<strong>in</strong> handelt. Die Substanz soll mittels HPLC-<br />
Techniken isoliert und NMR-spektroskopisch und massenspektrometrisch vollständig charakterisiert<br />
werden. Da die Substanz ke<strong>in</strong> Chromophor enthält, muss die Detektion durch e<strong>in</strong>e Abbaureaktion zur<br />
Freisetzung von Cyanid sowie anschließendem Cyanidnachweis durchgeführt werden.<br />
7
Crater morphologies <strong>in</strong> impact experiments <strong>in</strong>to sandstone targets<br />
A.Dufresne, M.H.Poelchau, T.Kenkmann, and <strong>the</strong> MEMIN-Team<br />
Department <strong>of</strong> Geological Sciences – Geology, Albert-Ludwigs Universität Freiburg, Germany<br />
Understand<strong>in</strong>g <strong>the</strong> dynamics <strong>of</strong> a hypervelocity body impact<strong>in</strong>g onto a planetary surface can<br />
give numerous <strong>in</strong>sights <strong>in</strong>to <strong>the</strong> processes that formed <strong>the</strong> solar system. Dur<strong>in</strong>g an impact<br />
process, <strong>the</strong> impact<strong>in</strong>g projectile <strong>in</strong>itially excavates a roughly hemispherical cavity <strong>in</strong> <strong>the</strong><br />
target, known as <strong>the</strong> “transient crater”, which is usually modified <strong>by</strong> late stage processes.<br />
Constra<strong>in</strong><strong>in</strong>g <strong>the</strong> dimensions <strong>of</strong> <strong>the</strong> transient crater and late stage modification features like<br />
spallation lead to a better understand<strong>in</strong>g <strong>of</strong> <strong>the</strong> physical parameters that affect crater<br />
formation. Therefore, impact experiments <strong>in</strong>to sandstone cubes were performed at <strong>the</strong> two-<br />
stage light gas acceleration facilities <strong>of</strong> <strong>the</strong> EMI Freiburg. Spherical projectiles <strong>of</strong> steel,<br />
meteoritic iron and alum<strong>in</strong>ium were accelerated to velocities <strong>of</strong> 2.5-8 km/s and impact<br />
energies from 0.7 to 58 kJ. In a detailed study, <strong>the</strong> processes <strong>in</strong>fluenc<strong>in</strong>g crater volume,<br />
morphology and spall behaviour were <strong>in</strong>vestigated through visual mapp<strong>in</strong>g, 3D digital<br />
modell<strong>in</strong>g and 2D pr<strong>of</strong>ile analyses. All experimental impact craters <strong>in</strong> dry sandstone targets<br />
share four characteristic morphological features: (1) a light-coloured, fragile, highly<br />
fragmented central part, (2) areas <strong>of</strong> “arrested spallation” where <strong>the</strong> target is fractured but <strong>the</strong><br />
outl<strong>in</strong>ed spall fragments rema<strong>in</strong>s with<strong>in</strong> <strong>the</strong> crater context, (3) typically two <strong>in</strong>ner depressions,<br />
and (4) an outer, shallow (10-20°) dipp<strong>in</strong>g area. Constra<strong>in</strong><strong>in</strong>g <strong>the</strong> percentage <strong>of</strong> spallation<br />
with<strong>in</strong> a crater and determ<strong>in</strong><strong>in</strong>g <strong>the</strong> shape and volume <strong>of</strong> <strong>the</strong> transient crater are vital for<br />
comparison with non-brittle materials like metals, with numerical modell<strong>in</strong>g results, and with<br />
natural impact craters. Transient crater morphologies are derived from (i) parabolas fitted to<br />
<strong>the</strong> central depressions <strong>of</strong> crater pr<strong>of</strong>iles, (ii) reconstructed spallation dimensions, (iii) ejecta<br />
impr<strong>in</strong>t diameters on catcher systems, (iv) ejecta cone angle and dynamic crater diameter<br />
from high-speed videos, and (i) <strong>the</strong> distribution <strong>of</strong> ejecta weight fractions. Results show that<br />
spallation, as a late-stage crater<strong>in</strong>g process, <strong>in</strong>creases <strong>the</strong> volume <strong>of</strong> <strong>the</strong> transient crater <strong>by</strong> a<br />
factor <strong>of</strong> 5-10. The ref<strong>in</strong>ed transient crater model will be applied to o<strong>the</strong>r experimental and to<br />
real-size impact craters <strong>in</strong> due course.<br />
8
<strong>in</strong>-vivo localization <strong>of</strong> <strong>the</strong> OXPHOS complexes <strong>in</strong> E. coli<br />
Erhardt H. and Friedrich T.<br />
Institute for Organic Chemistry and Biochemistry,<br />
Albert-Ludwigs-University Freiburg, Germany<br />
Biological membranes show a high level <strong>of</strong> organization <strong>of</strong> <strong>the</strong>ir compounds. Lipids<br />
and membrane prote<strong>in</strong>s are not randomly distributed with<strong>in</strong> <strong>the</strong>se membranes, but<br />
organized <strong>in</strong> dist<strong>in</strong>ct and dynamic clusters and show a huge variety <strong>in</strong> localization<br />
patterns. It has been proposed that prote<strong>in</strong> complexes connected <strong>by</strong> a common<br />
substrate cha<strong>in</strong> form supercomplexes, which are located <strong>in</strong> specific areas with<strong>in</strong> <strong>the</strong><br />
membrane. Such an organization provides advantages such as stabilization <strong>of</strong> <strong>the</strong><br />
<strong>in</strong>dividual complexes or catalytic enhancement.<br />
The aerobic OXPHOS system <strong>of</strong> E. coli consists <strong>of</strong> several multi-subunit enzyme<br />
complexes. To address <strong>the</strong> question whe<strong>the</strong>r <strong>the</strong>se complexes are localized <strong>in</strong><br />
dist<strong>in</strong>ct lipid areas and if <strong>the</strong>y show any dynamic behavior, we decorated <strong>the</strong><br />
membrane prote<strong>in</strong> complexes with different fluorescent prote<strong>in</strong>s and visualized <strong>the</strong>ir<br />
distribution <strong>in</strong> <strong>the</strong> membrane <strong>by</strong> <strong>in</strong>-vivo fluorescence microscopy. FP-decorated<br />
variants <strong>of</strong> <strong>the</strong> NADH:ubiqu<strong>in</strong>one oxidoreductase, <strong>the</strong> succ<strong>in</strong>ate dehydrogenase, <strong>the</strong><br />
cytochrome-bd complex and <strong>the</strong> F0F1-ATP-synthase, were created <strong>by</strong> means <strong>of</strong> λ-<br />
RED mediated mutagenesis. The labeled complexes were catalytically active and<br />
fully assembled. By fluorescence microscopy we observed an uneven distribution <strong>of</strong><br />
<strong>the</strong> OXPHOS complexes <strong>in</strong> <strong>the</strong> cytoplasmatic membrane <strong>of</strong> liv<strong>in</strong>g E. coli cells. From<br />
<strong>the</strong>se data we conclude that <strong>the</strong>y reside <strong>in</strong> dist<strong>in</strong>ct and dynamic membrane doma<strong>in</strong>s,<br />
which might be <strong>of</strong> functional importance.<br />
References:<br />
Pohl, T., Uhlmann, M., Kaufenste<strong>in</strong>, M. & Friedrich, T., Biochemistry 2007, 46,<br />
10694-10702.<br />
Lenn, T., Leake, M. C., Mull<strong>in</strong>eaux C. W., Mol Microbiol. 2008,70,1397-407.<br />
Mileykovskaya, E. & Dowhan, W., J. Bacteriol. 2000, 182, 1172-1175.<br />
Johnson, A. S., van Horck, S. & Lewis P. J., Microbiology 2004, 150, 2815-2824.<br />
9
Factors controll<strong>in</strong>g stress perturbations <strong>in</strong> faulted reservoirs -<br />
<strong>in</strong>sights from numerical parameter studies<br />
Fischer, K. & Henk, A.<br />
Institut für Geowissenschaften – Geologie, Albert-Ludwigs-Universität Freiburg, Germany<br />
karsten.fischer@geologie.uni-freiburg.de<br />
Das tektonische Spannungsfeld bee<strong>in</strong>flusst die optimale Erschließung e<strong>in</strong>er Lagerstätte <strong>in</strong><br />
vielfältiger Weise. So hängen u.a. Bohrlochstabilität, Orientierung von hydraulisch <strong>in</strong>duzierten<br />
Fracs und – <strong>in</strong>sbesondere <strong>in</strong> geklüfteten Reservoiren – Permeabilitätsanisotropien von<br />
den rezenten <strong>in</strong> situ Spannungen ab. Allerd<strong>in</strong>gs kann das regionale Spannungsfeld durch<br />
Störungen und Lithologiewechsel lokal <strong>in</strong> Magnitude und Orientierung variieren. Insbesondere<br />
bei störungskontrollierten Lagerstätten können solche Perturbationen dazu führen, dass<br />
das Spannungsfeld <strong>in</strong>nerhalb e<strong>in</strong>zelner Störungsblöcke deutlich vom regionalen Trend abweicht.<br />
E<strong>in</strong>e belastbare „pre-drill<strong>in</strong>g“ – Prognose von tektonischen Spannungen zur M<strong>in</strong>derung<br />
des Erschließungsrisikos und zur Optimierung der Bohrungen erfordert daher die E<strong>in</strong>beziehung<br />
der spezifischen Lagerstättengeometrie sowie der mechanischen Eigenschaften<br />
der beteiligten Lithologien und Störungen. Als Prognosewerkzeug bieten sich geomechanische<br />
Lagerstättenmodellierungen auf Basis der F<strong>in</strong>ite Elemente (FE) Methode an.<br />
Im Rahmen des DGMK Forschungsprojekts 721 wird das Potential von solchen geomechanischen<br />
Lagerstättenmodellen für die Prognose von Spannungsfeldern und Kluftnetzwerken<br />
am Beispiel des Erdgasfeldes Nord Hannover untersucht. In der ersten Projektphase wurden<br />
im Rahmen von Parameterstudien die wesentlichen Kontrollfaktoren für Spannungsfeldperturbationen<br />
an Störungen bearbeitet. Ausgehend von e<strong>in</strong>em Standardmodell, das sich h<strong>in</strong>sichtlich<br />
der idealisierten Geometrie e<strong>in</strong>es Störungsblocks, der Materialeigenschaften und<br />
Randbed<strong>in</strong>gungen bereits an die Fallstudie Nord Hannover anlehnt, wurden systematisch die<br />
Magnitude und die Orientierung der maximalen regionalen Hauptnormalspannung (σHmax),<br />
die Reibungskoeffizienten der Störungen sowie die mechanischen Eigenschaften der Geste<strong>in</strong>e<br />
variiert. Darüber h<strong>in</strong>aus wurden Modellszenarien mit mehreren Störungsblöcken bzw.<br />
unterschiedlichen Größen der Störungsblöcke untersucht und zwei- und dreidimensionale<br />
numerische Simulationen mite<strong>in</strong>ander verglichen. Für alle Szenarien wurden die Ergebnisse<br />
<strong>in</strong> Form von Isol<strong>in</strong>ien- und Vektorplots für unterschiedliche Ausgabegrößen von Spannung<br />
und Deformation (Magnituden und Orientierungen der Hauptnormalspannungen, von Mises<br />
Vergleichsspannung, mittlere Spannnung, Differentialspannung, elastische und plastische<br />
Deformationsanteile, „fault slip and dilation tendencies“) dokumentiert.<br />
Die Parameterstudien zeigen, dass die Reibungskoeffizienten der Störungen, der W<strong>in</strong>kel<br />
zwischen Störung und σHmax sowie die mechanischen Eigenschaften der ane<strong>in</strong>andergrenzenden<br />
Geste<strong>in</strong>e den relativ größten E<strong>in</strong>fluss auf Spannungsfeldperturbationen haben. Während<br />
das Standardmodell an den Störungen lediglich kle<strong>in</strong>räumige Veränderungen <strong>in</strong> der<br />
Magnitude der Normalspannungen zeigt, haben z.B. deutlich herabgesetzte Reibungseigenschaften<br />
ausgeprägte Veränderungen <strong>in</strong> Magnitude und Orientierung des Spannungsfeldes<br />
zur Folge. Die Ergebnisse dieser Parameterstudien werden <strong>in</strong> der nächsten Projektphase zur<br />
Validierung e<strong>in</strong>es detaillierten Lagerstättenmodells von Nord Hannover genutzt, das die spezifische<br />
Störungsgeometrie und mechanische Geste<strong>in</strong>seigenschaften berücksichtigt.<br />
10
Clon<strong>in</strong>g, Expression, Purification and Crystallization <strong>of</strong> <strong>the</strong> First<br />
Archaeal 4-Hydroxyphenylpyruvate Dioxygenase<br />
Eduard Frick, 1 Thomas Spatzal, 2 Oliver E<strong>in</strong>sle, 2<br />
Michael Müller, 1 and Wolfgang Hüttel 1<br />
1 Institut für Pharmazeutische Wissenschaftten der Albert-Ludwigs-Universität Freiburg,<br />
Lehrstuhl für Pharmazeutische und Mediz<strong>in</strong>ische Chemie, Albertstr. 25, D-79104 Freiburg,<br />
Email: wolfgang.huettel@pharmazie.uni-freiburg.de<br />
2 Institut für Organische und Biochemie der Albert-Ludwigs-Universität Freiburg,<br />
Lehrstuhl für Biochemie, Albertstr. 21, D-79104 Freiburg,<br />
Email: e<strong>in</strong>sle@bio.chemie.uni-freiburg.de<br />
4-Hydroxyphenylpyruvate dioxygenase (Hpd) catalyzes <strong>the</strong> conversion <strong>of</strong><br />
4-hydroxyphenylpyruvate (4-HPP) <strong>in</strong>to 2,5-dihydroxyphenylacetate (homogentisate). This<br />
reaction is part <strong>of</strong> <strong>the</strong> aerobic tyros<strong>in</strong>e catabolism <strong>in</strong> nearly all forms <strong>of</strong> life. Hpd is a valuable<br />
target for agrichemical (development <strong>of</strong> herbicides) and <strong>the</strong>rapeutical (treatment <strong>of</strong> hereditary<br />
tyros<strong>in</strong>aemia) research. [1]<br />
Enzymes <strong>of</strong> extremophilic organisms <strong>of</strong>fer a promis<strong>in</strong>g access to biocatalysts with <strong>in</strong>creased<br />
stability. We focussed on <strong>the</strong> Hpd from <strong>the</strong> acidophilic and meso<strong>the</strong>rmophilic archaeon<br />
Picrophilus torridus. [2] We managed to clone, express, purify, characterize and crystallize <strong>the</strong><br />
prote<strong>in</strong>. The structure <strong>of</strong> P. torridus Hpd was determ<strong>in</strong>ed at 2.3 Å resolution and will provide<br />
fur<strong>the</strong>r <strong>in</strong>sights <strong>in</strong>to <strong>the</strong> reasons for acid stability <strong>of</strong> certa<strong>in</strong> prote<strong>in</strong>s.<br />
References:<br />
[1] G. R. Moran, Arch. Biochem. Biophys. 2005, 433, 117-128.<br />
[2] C. Schleper, et al., J. Bacteriol. 1995, 177, 7050-7059.<br />
11
Tag der Forschung 2011<br />
Laurdan labeled liposomes<br />
– a new approach to quantify drug partition<strong>in</strong>g<br />
Gesche Först, Rolf Schubert<br />
Dept. <strong>of</strong> Pharmaceutical Technology and Biopharmacy, University <strong>of</strong> Freiburg<br />
gesche.foerst.de@pharmazie.uni-freiburg.de<br />
The knowledge <strong>of</strong> <strong>the</strong> partition behaviour <strong>of</strong> a drug between buffer and biological<br />
membranes is important to predict its distribution and accumulation <strong>in</strong> <strong>the</strong> human<br />
organism. Liposomes are common membrane models and ideal systems for <strong>the</strong><br />
characterization <strong>of</strong> drug-membrane <strong>in</strong>teractions [1] .<br />
The aim <strong>of</strong> this project is to establish an <strong>in</strong>direct screen<strong>in</strong>g method to <strong>in</strong>vestigate <strong>the</strong><br />
liposome/buffer partition coefficient <strong>of</strong> drugs us<strong>in</strong>g <strong>the</strong> easily available fluorescence<br />
dye 6-Lauryl-2-dimethylam<strong>in</strong>onaphtalene (Laurdan) [2] .<br />
After <strong>in</strong>vestigat<strong>in</strong>g <strong>the</strong> partition<strong>in</strong>g behaviour <strong>of</strong> bile salts as amphiphilic model drugs<br />
<strong>in</strong> previous studies [3] <strong>the</strong> Laurdan method has also been applied to <strong>the</strong> ß-blockers<br />
as additional substance class and <strong>the</strong> atta<strong>in</strong>ed results are comparable to literature [4] .<br />
Plemper v. Balen G, Marca Mart<strong>in</strong>et C, Caron G, Bouchard G, Reist M, Carrupt PA, Fruttero R, Gasco A, Testa B.<br />
Liposome/water lipophilicity: methods, <strong>in</strong>formation content, and pharmaceutical applications. Med Res Review. 2004; 24(3):<br />
299-324. doi:10.1002/med.10063<br />
[2] Paternostre M, Meyer O, Grabielle-Madelmont C, Lesieur S, Ghanam M, Ollivon M. Partition coefficient <strong>of</strong> a surfactant<br />
between aggregates and solution: application to <strong>the</strong> micelle-vesicle transition <strong>of</strong> Egg-phosphatidylchol<strong>in</strong>e and Octyl-�-Dglucopyranosid.<br />
Biophys J 1995; 69: 2476-2488<br />
[3] Theobald A, Thesis, Laurdan als Fluoreszenzsonde zur Quantifizierung der Wechselwirkung von Gallensalzen mit<br />
Liposomenmembranen 2009; University <strong>of</strong> Freiburg<br />
[4] Liu X., Hefesha H., Scriba G., Fahr A. Retention Behavior <strong>of</strong> Neutral and Positively and Negatively Charged Solutes on<br />
an Immobilized-Artificial-Membrane Stationary Phase Helv. Chim. Acta 2008; 91: 1505 - 1512<br />
12
Mechanistical studies <strong>of</strong> <strong>the</strong> Rhodium catalyzed<br />
Hydr<strong>of</strong>ormylation with Self-Assembl<strong>in</strong>g Ligands<br />
Urs Gellrich, Bernhard Breit*<br />
Institut für Organische Chemie und Biochemie,<br />
Albert-Ludwigs-Universität Freiburg, Albertstr. 21, 79104 Freiburg<br />
Selectivity control <strong>in</strong> homogeneous metal complex catalysis is most frequently<br />
achieved <strong>by</strong> craft<strong>in</strong>g <strong>the</strong> microenvironment <strong>of</strong> <strong>the</strong> catalytically active metal center<br />
upon b<strong>in</strong>d<strong>in</strong>g <strong>of</strong> appropriate ligand architectures to <strong>the</strong> metal center. Among <strong>the</strong><br />
many ligands used, bidentate ligands occupy an important position. Dur<strong>in</strong>g <strong>the</strong> last<br />
years our group was able to apply self-assembl<strong>in</strong>g ligands, <strong>in</strong>teract<strong>in</strong>g via hydrogen<br />
bonds, [1] successfully <strong>in</strong> many catalytic reactions, for example hydr<strong>of</strong>ormylation,<br />
hydrogenation and o<strong>the</strong>rs. [2,3,4] In order to improve catalytic reactions and ligands it is<br />
<strong>by</strong> all means necessary to completely understand <strong>the</strong> nature <strong>of</strong> <strong>the</strong> catalytically active<br />
species. Hence, onl<strong>in</strong>e monitor<strong>in</strong>g <strong>of</strong> progress<strong>in</strong>g catalytic transformations, supported<br />
<strong>by</strong> quantum mechanical calculations can deliver valuable <strong>in</strong>formations on <strong>the</strong><br />
<strong>in</strong>termediates <strong>in</strong>volved <strong>in</strong> catalysis. We here<strong>in</strong> report on our first <strong>in</strong>sights <strong>in</strong>to<br />
hydr<strong>of</strong>ormylation reactions with <strong>the</strong> 6-DPPon ligand us<strong>in</strong>g onl<strong>in</strong>e<br />
ReactIR TM -spectroscopy toge<strong>the</strong>r with DFT calculations.<br />
Figure 1: Direct observation <strong>of</strong> <strong>the</strong> formation <strong>of</strong> an <strong>in</strong>termediat <strong>of</strong> <strong>the</strong> catalytic cycle and <strong>the</strong><br />
correspond<strong>in</strong>g DFT calculated structure (B3P86/def2-SVP).<br />
[1] W. Seiche, B. Breit Angew. Chem., Int. Ed. 2005, 44, 1640.<br />
[2] C. Waloch, J. Wieland, M. Keller, B. Breit, Angew. Chem. 2007, 119, 309, Angew. Chem. Int. Ed.<br />
2007, 46, 3037.<br />
[3] J. Wieland, B. Breit Nature Chemistry 2010, 2, 832.<br />
[4] M. deGreef, B. Breit Angew. Chem., Int. Ed. 2009, 48, 559.<br />
13
Streptomyces calvus – from bald to beautiful<br />
Arne Gessner<br />
Institut für pharmazeutische Biologie und Biotechnologie<br />
Albert-Ludwigs-Universität Freiburg<br />
arne.gessner@pharmazie.uni-freiburg.de<br />
Streptomyces calvus is most prom<strong>in</strong>ently known for produc<strong>in</strong>g <strong>the</strong> fluor<strong>in</strong>ated compound<br />
nucleocid<strong>in</strong>. S. calvus is also a non-sporulat<strong>in</strong>g stra<strong>in</strong>, an unusual property for Streptomycetes, which<br />
is reflected <strong>in</strong> <strong>the</strong> Lat<strong>in</strong> word for bald, ‘calvus’. Exam<strong>in</strong>ation <strong>of</strong> a draft sequence <strong>of</strong> <strong>the</strong> genome <strong>of</strong> this<br />
stra<strong>in</strong> revealed a polyketide-biosyn<strong>the</strong>sis gene cluster that has high sequence similarity to known<br />
polyene-biosyn<strong>the</strong>sis clusters like those for Nystat<strong>in</strong> or Pimaric<strong>in</strong>, which are valuable natural<br />
products that are <strong>the</strong>rapeutically used to treat fungal <strong>in</strong>fections <strong>in</strong> humans and animals.<br />
A leuc<strong>in</strong>e <strong>in</strong> <strong>the</strong> load<strong>in</strong>g doma<strong>in</strong> <strong>of</strong> a PKS <strong>in</strong> this biosyn<strong>the</strong>sis cluster is encoded <strong>by</strong> a TTA-codon. Such<br />
codons are extremely rare <strong>in</strong> <strong>the</strong> GC-rich genomes <strong>of</strong> Streptomycetes. The correspond<strong>in</strong>g Leu-tRNA is<br />
encoded <strong>by</strong> <strong>the</strong> bldA gene. Sequence alignment <strong>of</strong> <strong>the</strong> S. calvus bldA gene with bldA genes from<br />
o<strong>the</strong>r Streptomyces species showed a po<strong>in</strong>t mutation that is predicted to encode a mis-folded and<br />
most likely non-functional Leu-tRNA (TTA). So it was hypo<strong>the</strong>tisized that complementation <strong>of</strong> S.<br />
calvus with a functional copy <strong>of</strong> <strong>the</strong> bldA gene would not only restore sporulation but could also<br />
activate <strong>the</strong> cryptic polyene cluster. The S. calvus bldA(+) stra<strong>in</strong> did <strong>in</strong>deed rega<strong>in</strong> <strong>the</strong> ability to<br />
sporulate. Additionally, LC-DAD-MS analysis <strong>of</strong> an extract from a seven-day production culture<br />
revealed a new peak that showed an UV-spectrum typical for a polyene. The correspond<strong>in</strong>g polyene<br />
‘annimyc<strong>in</strong>’ was purified and <strong>the</strong> structure elucidated <strong>by</strong> 1 H-, 13 C-, HSQC, HMBC and ROESY NMR<br />
spectroscopy. Disruption <strong>of</strong> PKS genes are currently underway to def<strong>in</strong>e <strong>the</strong> biosyn<strong>the</strong>tic cluster for<br />
annimyc<strong>in</strong>.<br />
14
UNRAVELING THE BIOSYNTHESIS OF KOTANIN AND ITS MONOMERIC PRECURSOR 7-<br />
DEMETHYLSIDERIN IN ASPERGILLUS NIGER<br />
C. Gil Girol, W. Hüttel, M. Müller<br />
Institute for Pharmaceutical Sciences, University <strong>of</strong> Freiburg<br />
Email: michael.mueller@pharmazie.uni-freiburg.de<br />
A. niger shows an <strong>in</strong>termolecular stereo- and regioselective oxidative phenol coupl<strong>in</strong>g <strong>of</strong> <strong>the</strong><br />
coumar<strong>in</strong> derivative 7-demethylsider<strong>in</strong> (2), form<strong>in</strong>g orland<strong>in</strong> (5). F<strong>in</strong>al O-methylation leads to kotan<strong>in</strong><br />
(6) (Fig.1). 1 The biosyn<strong>the</strong>sis <strong>of</strong> kotan<strong>in</strong> (6) <strong>in</strong> A. niger and its absolute configuration was elucidated<br />
through feed<strong>in</strong>g experiments and stereoselective syn<strong>the</strong>sis. 1, 2 Up to now <strong>the</strong>re is no evidence for an<br />
alternative biosyn<strong>the</strong>tic route to 6 <strong>by</strong> oxidative coupl<strong>in</strong>g <strong>of</strong> 3,7-dihydroxycoumar<strong>in</strong> (1) lead<strong>in</strong>g to <strong>the</strong><br />
bicoumar<strong>in</strong> 4 and subsequent O-methylation.<br />
HO<br />
HO<br />
HO<br />
CH 3<br />
CH 3<br />
CH 3<br />
OH<br />
13<br />
C<br />
O O<br />
OH<br />
13<br />
C<br />
O O<br />
O 13C O<br />
OH<br />
HO<br />
CH 3<br />
O 13 CH 3<br />
13<br />
C<br />
O O<br />
H 3CO<br />
1 2 3<br />
?<br />
?<br />
HO<br />
HO<br />
CH 3<br />
CH 3<br />
O 13 CH 3<br />
13<br />
C<br />
O O<br />
O 13C O<br />
OCH 3<br />
H 3CO<br />
H 3CO<br />
4 5 6<br />
Figure 1: Biosyn<strong>the</strong>sis <strong>of</strong> kotan<strong>in</strong> (6) elucidated through feed<strong>in</strong>g experiments and stereoselective syn<strong>the</strong>sis.<br />
CH 3<br />
CH 3<br />
CH 3<br />
O 13 CH 3<br />
13C O O<br />
O 13 CH 3<br />
13<br />
C<br />
O O<br />
O 13C O<br />
OCH 3<br />
Here, we report <strong>the</strong> verification <strong>of</strong> this biosyn<strong>the</strong>tical pathway on genomic level <strong>by</strong> a gene target<strong>in</strong>g<br />
approach. In prelim<strong>in</strong>ary feed<strong>in</strong>g experiments <strong>the</strong> polyketidic orign <strong>of</strong> kotan<strong>in</strong> was proven, an <strong>in</strong> silico<br />
analysis <strong>of</strong> <strong>the</strong> A. niger genome 3 revealed putative PKS clusters cod<strong>in</strong>g for <strong>the</strong> monomeric precursor<br />
2. Subsequent disruption <strong>of</strong> <strong>the</strong> genes led to <strong>the</strong> PKS responsible for kotan<strong>in</strong> (6) biosyn<strong>the</strong>sis.<br />
Fur<strong>the</strong>rmore disruption <strong>of</strong> an O-methyl transferase <strong>of</strong> <strong>the</strong> cluster provided evidence for absence <strong>of</strong><br />
an alternative route to 6 via 1 and 4.<br />
1. Hüttel, W. & Müller, M. Regio- and stereoselective <strong>in</strong>termolecular oxidative phenol coupl<strong>in</strong>g<br />
<strong>in</strong> kotan<strong>in</strong> biosyn<strong>the</strong>sis <strong>by</strong> Aspergillus niger. Chembiochem 8, 521-529 (2007).<br />
2. Hüttel, W., Nieger, M. & Müller, M. A short and efficient total syn<strong>the</strong>sis <strong>of</strong> <strong>the</strong> naturally<br />
occurr<strong>in</strong>g coumar<strong>in</strong>s sider<strong>in</strong>, kotan<strong>in</strong>, isokotan<strong>in</strong> A and desertor<strong>in</strong> C. Syn<strong>the</strong>sis, 1803-1808<br />
(2003).<br />
3. Pel, H.J. et al. Genome sequenc<strong>in</strong>g and analysis <strong>of</strong> <strong>the</strong> versatile cell factory Aspergillus niger<br />
CBS 513.88. Nature Biotechnology 25, 221-231 (2007).<br />
15<br />
1, 2
GAP: A Genome Annotation Pipel<strong>in</strong>e<br />
Grün<strong>in</strong>g BA, Erxleben A, Senger C, Flemm<strong>in</strong>g S, Gün<strong>the</strong>r S*<br />
Pharmaceutical Bio<strong>in</strong>formatics, Institute <strong>of</strong> Pharmaceutical Sciences, University <strong>of</strong><br />
Freiburg, Germany<br />
*e-mail: stefan.guen<strong>the</strong>r[at]pharmazie.uni-freiburg.de<br />
Introduction: Process<strong>in</strong>g <strong>the</strong> explod<strong>in</strong>g number <strong>of</strong> new sequence data requires efficient<br />
implementation <strong>of</strong> several specialised tools on a powerful hardware <strong>in</strong>frastructure. Employed<br />
methods <strong>in</strong>clude genome assembly, genome annotation, pathway reconstruction, data<br />
visualisation, and pathway modell<strong>in</strong>g. Galaxy is a workflow management system for data<br />
process<strong>in</strong>g ideally suited to <strong>the</strong> comb<strong>in</strong>ation <strong>of</strong> various s<strong>of</strong>tware tools. Fur<strong>the</strong>rmore, it ensures data<br />
and process reproducibility <strong>in</strong> terms <strong>of</strong> repeatability and traceability. Accessibility via web <strong>in</strong>terface<br />
facilitates <strong>the</strong> <strong>in</strong>tegration <strong>of</strong> Galaxy <strong>in</strong>to genome annotation projects.<br />
Methods and Results: Based on Galaxy, we have developed <strong>the</strong> comprehensive Genome<br />
Annotation Pipel<strong>in</strong>e (GAP) focused on process<strong>in</strong>g newly sequenced bacterial genomes. Currently,<br />
<strong>the</strong> pipel<strong>in</strong>e <strong>in</strong>cludes several publicly available and self-developed tools: Glimmer3 for <strong>the</strong><br />
prediction <strong>of</strong> ORFs, similarity searches <strong>in</strong> NCBI databases and UniProt, InterProScan for <strong>the</strong><br />
functional annotation <strong>of</strong> gene products, SignalP for <strong>the</strong> prediction <strong>of</strong> prote<strong>in</strong> localisation, Aragorn<br />
for tRNA and tmRNA detection with<strong>in</strong> <strong>the</strong> genome and many more. The pipel<strong>in</strong>e is implemented on<br />
a high-performance computer and allows a very fast complete annotation <strong>of</strong> an average microbial<br />
genome. GAP has been successfully applied for <strong>the</strong> annotation <strong>of</strong> Streptomyces TÜ6071. This is a<br />
bacterium which has a highly-active isoprenoid biosyn<strong>the</strong>sis and produces <strong>the</strong> <strong>in</strong>dustrial important<br />
terpene Phenal<strong>in</strong>olactone which has anti-bacterial activity aga<strong>in</strong>st several Gram-positive bacteria.<br />
The annotated genome sequence <strong>of</strong> Streptomyces TÜ6071 is now available under <strong>the</strong> NCBI<br />
accession number AFHJ00000000.<br />
Future Prospects: Additional sequence analysis tools and and <strong>in</strong>terfaces for complex data<br />
visualisation will be implemented. GAP will soon be publicly available for genome annotation and<br />
systems biology for <strong>the</strong> scientific community.<br />
16
Strukturelle Charakterisierung von Br9 - -Anionen<br />
Heike Haller, Mathias Ellwanger, Sebastian Riedel*<br />
Institut für Anorganische und Analytische Chemie<br />
Uni Freiburg<br />
Seit Jahrzehnten ist e<strong>in</strong>e Vielzahl von Polyiodidanionen bekannt und strukturell untersucht.<br />
Im Bereich der niedrigeren Homologen kennt man deutlich weniger Beispiele. So s<strong>in</strong>d bei den<br />
Polybromiden mehrere Kristallstrukturen bekannt wie zum Beispiel Br10 2- , (Br - )2(Br4 2- ) und<br />
e<strong>in</strong> unendliches 2D Polybromidnetzwerk. Anfang diesen Jahres wurde von Br20 2- als bisher<br />
bromreichste bekannte Verb<strong>in</strong>dung berichtet [1] . Jedoch ist im Bereich der<br />
Polybromidmonoanionen lediglich Br3 - seit … als Kristallstruktur bekannt. Zu Br5 - , Br7 - und<br />
Br9 - gibt es ausführliche ramanspektroskopische Untersuchungen [2] , e<strong>in</strong>e genauere<br />
kristallographische Untersuchung gelang jedoch bislang nicht.<br />
Uns gelang erstmals NMe4Br9 sowie NEt4Br9 mittels E<strong>in</strong>kristallstrukturanalyse zu<br />
charakterisieren.<br />
[1] Wolff, M.; Meyer, J.; Feldmann, C.; Angewandte Chemie 2011,123, 5073-5077.<br />
[2] Chen, X.; Rickard, M.; Hull Jr., J.; Zheng, C.; Leugers, A.; Simoncic, P.; Inorganic Chemistry 2010, 49,<br />
8684-8689.<br />
17
Gallium(I) Chemistry and <strong>the</strong> Syn<strong>the</strong>sis <strong>of</strong> Highly Reactive Polyisobutylene<br />
From Fundamentals to Applications<br />
Higel<strong>in</strong>, A. and Lichtenthaler, M. R., Freiburg i. Br. / D<br />
Pr<strong>of</strong>. Dr. Ingo Kross<strong>in</strong>g, Albert-Ludwigs-Universität Freiburg, Alberstraße 21,<br />
79104 Freiburg i. Br. / D<br />
In 2010 a simple route to univalent gallium salts <strong>of</strong><br />
weakly coord<strong>in</strong>at<strong>in</strong>g anions (WCAs) was developed<br />
<strong>by</strong> SLATTERY et al. [1] Here<strong>in</strong> <strong>the</strong> gallium(I)-cations are<br />
solely coord<strong>in</strong>ated <strong>by</strong> aromatic solvent molecules (e.g.<br />
toluene or fluorobenzene) <strong>in</strong> a bent sandwich fashion.<br />
As only one Ga-F contact is shorter than <strong>the</strong> sum <strong>of</strong><br />
<strong>the</strong> van der Waals radii (3.34 Å) <strong>the</strong> <strong>in</strong>teraction<br />
between <strong>the</strong> gallium(I)-cations and <strong>the</strong> WCAs is very<br />
weak (Figure 1).These novel salts are <strong>the</strong>refore ideal<br />
start<strong>in</strong>g materials for fur<strong>the</strong>r gallium(I) chemistry.<br />
The solvent molecules can be replaced <strong>by</strong> a variety <strong>of</strong><br />
σ-donor ligands, like phosphanes and e<strong>the</strong>rs, lead<strong>in</strong>g<br />
to an entirely new class <strong>of</strong> coord<strong>in</strong>ation compounds<br />
Figure 1: Molecular Structure <strong>of</strong><br />
[Ga(C 6H 5Me) 2] + [Al(OC(C(CF 3) 3) 4] – .<br />
which were <strong>in</strong>accessible with previously known Ga(I) start<strong>in</strong>g materials. The study <strong>of</strong> <strong>the</strong>se<br />
excit<strong>in</strong>g new compounds and <strong>the</strong>ir exotic bond<strong>in</strong>g situations, <strong>in</strong> conjunction with quantum<br />
chemical <strong>in</strong>vestigations, gives fundamental <strong>in</strong>sights <strong>in</strong>to <strong>the</strong> nature <strong>of</strong> <strong>the</strong> chemical bond. On <strong>the</strong><br />
applied side, <strong>the</strong> variation <strong>of</strong> <strong>the</strong> ligands <strong>of</strong>fers an elegant method <strong>of</strong> tun<strong>in</strong>g <strong>the</strong> reactivity <strong>of</strong> <strong>the</strong><br />
gallium(I)-cations. The latter namely are a promis<strong>in</strong>g species for a variety <strong>of</strong> applications, e.g.<br />
<strong>in</strong>itiat<strong>in</strong>g or catalyz<strong>in</strong>g <strong>the</strong> syn<strong>the</strong>sis <strong>of</strong> highly reactive Polyisobutylene (HR-PIB).<br />
Depend<strong>in</strong>g on its molecular weight Polyisobutylene (PIB) has been used <strong>in</strong> many versatile<br />
applications. Thus high molecular weight PIB has gradually substituted natural chicle as <strong>the</strong><br />
ma<strong>in</strong> chew<strong>in</strong>g gum base. The application <strong>of</strong> low molecular weight PIB however relies on <strong>the</strong><br />
PIB be<strong>in</strong>g highly reactive. PIB is classified as highly reactive as soon as <strong>the</strong> content <strong>of</strong> term<strong>in</strong>al<br />
double bonds exceeds 60%. Be<strong>in</strong>g sterically exposed, <strong>the</strong> latter can easily be functionalized.<br />
Therefore HR-PIB represents an essential <strong>in</strong>termediate <strong>in</strong> manufactur<strong>in</strong>g additives for lubricants<br />
and fuels. [2] Up to this day most syn<strong>the</strong>ses <strong>of</strong> HR-PIB show a lack <strong>in</strong> at least one po<strong>in</strong>t. May it<br />
be low reaction temperatures down to –100 °C, dichloromethane as solvent or relatively high<br />
concentrations <strong>of</strong> <strong>the</strong> <strong>in</strong>itiat<strong>in</strong>g or catalyz<strong>in</strong>g species – all <strong>the</strong>se factors sum up to a negative<br />
[3], [4]<br />
economic and environmental balance.<br />
Recently a number <strong>of</strong> reactions showed <strong>the</strong> superior quality <strong>of</strong> <strong>the</strong> univalent gallium salts<br />
<strong>in</strong>itiat<strong>in</strong>g or catalyz<strong>in</strong>g <strong>the</strong> polymerization <strong>of</strong> isobutylene. Thus <strong>the</strong> syn<strong>the</strong>sis <strong>of</strong> HR-PIB can be<br />
carried out at relatively high reaction temperatures up to 0 °C, <strong>in</strong> a non carc<strong>in</strong>ogenic and non<br />
water-hazardous solvent, i.e. toluene, as well as us<strong>in</strong>g relatively low concentrations <strong>of</strong> <strong>the</strong><br />
<strong>in</strong>itiat<strong>in</strong>g or catalyz<strong>in</strong>g species, down to 0.007 mol%. The experimental results were backed <strong>by</strong><br />
quantum-chemical calculations giv<strong>in</strong>g a first h<strong>in</strong>t on a coord<strong>in</strong>ative polymerization mechanism.<br />
[1] J. M. Slattery, A. Higel<strong>in</strong>, T. Bayer, I. Kross<strong>in</strong>g, Angew. Chem. Int. Ed., 2010, 49, 3228.<br />
[2] M. Vierle, Y. Zhang, E. Herdtweck, M. Bohnenpoll, O. Nuyken, F. E. Kühn, Angew. Chem. Int Ed. 2003, 42, 1307.<br />
[3] A. Guerrero, K. Kulbaba, M. Bochmann, Macromol. Chem. and Phys. 2008, 209, 1714.<br />
[4] Y. Li, H. Y. Yeong, E. Herdtweck, B. Voit, F. E. Kühn, Eur. J. Inorg. Chem. 2010, 4587.<br />
18
Characterization <strong>of</strong> airborne dust particles <strong>in</strong> <strong>the</strong> coal m<strong>in</strong><strong>in</strong>g area <strong>of</strong> Cam Pha,<br />
nor<strong>the</strong>rn Viet Nam<br />
T.B. HOÀNG-HÒA 1* , R. GIERÉ 1 , V. DIETZE 2 , U. KAMINSKI 2 AND P. STILLE 3<br />
1 Albert-Ludwigs-Universität, D-79104 Freiburg, Germany (*correspondence: hoahtb@gmail.com)<br />
2 German Meteorological Service, Research Center Human Biometeorology, Stefan-Meier-Str. 4, D-79104, Freiburg, Germany<br />
3 École et Observatoire des Sciences de la Terre, Université de Strasbourg, F-67084 Strasbourg, France<br />
Cam Pha, located <strong>in</strong> Quang N<strong>in</strong>h prov<strong>in</strong>ce, is one <strong>of</strong> <strong>the</strong> largest coal m<strong>in</strong><strong>in</strong>g areas <strong>in</strong> Viet Nam<br />
(reserves ~10 Gt). This study focuses on <strong>the</strong> m<strong>in</strong>eralogical and chemical characterization <strong>of</strong><br />
airborne dust. Exposure to dust is a major challenge for <strong>the</strong> population liv<strong>in</strong>g near <strong>the</strong> m<strong>in</strong>es,<br />
especially <strong>the</strong> open-pit m<strong>in</strong>es. Additional major dust sources comprise m<strong>in</strong>e dumps conta<strong>in</strong><strong>in</strong>g <strong>the</strong><br />
overburden, a coal-process<strong>in</strong>g facility, a coal-shipp<strong>in</strong>g harbour, m<strong>in</strong><strong>in</strong>g traffic, and various<br />
<strong>in</strong>dustries, <strong>in</strong>clud<strong>in</strong>g coal-fired power stations.<br />
Coarse particles (dp >2.5 µm) have been collected with <strong>the</strong> passive sampler device Sigma-2 on<br />
transparent adhesive collection plates for subsequent s<strong>in</strong>gle-particle analysis <strong>by</strong> automated optical<br />
microscopy accord<strong>in</strong>g to VDI guidel<strong>in</strong>e 2119 [1]. Select specimens, sampled dur<strong>in</strong>g different<br />
meteorological conditions, were <strong>in</strong>vestigated fur<strong>the</strong>r <strong>by</strong> SEM-EDX s<strong>in</strong>gle-particle analysis and <strong>by</strong><br />
determ<strong>in</strong><strong>in</strong>g <strong>the</strong>ir bulk chemical and isotopic composition (ICP-MS and MC-ICP-MS, respectively).<br />
W<strong>in</strong>d directions <strong>in</strong>dicate that <strong>the</strong> particles are ma<strong>in</strong>ly derived from <strong>the</strong> open-pit m<strong>in</strong>es, consistent<br />
with bulk chemical and isotopic data ( 87 Sr/ 86 Sr = 0.7278 – 0.7427; εNd = -14.6 – -14.9; 206 Pb/ 207 Pb =<br />
1.1789 – 1.1884), which suggest that <strong>the</strong> dust conta<strong>in</strong>s mostly natural materials (coal, silicate<br />
m<strong>in</strong>erals from sedimentary rocks). Relative to <strong>the</strong> coal, <strong>the</strong> bulk airborne dust is enriched <strong>in</strong> Na,<br />
Mg, K, and Ca, but depleted <strong>in</strong> most o<strong>the</strong>r components. Element-ratio plots reveal some systematic<br />
differences between <strong>the</strong> dust samples and specimens <strong>of</strong> coal and overburden, po<strong>in</strong>t<strong>in</strong>g to an<br />
additional, yet unknown particle source. To verify <strong>the</strong> hypo<strong>the</strong>sis <strong>of</strong> an additional dust source and to<br />
provide quantitative data on <strong>the</strong> m<strong>in</strong>eralogical composition <strong>of</strong> <strong>the</strong> dust samples, we are currently<br />
optimiz<strong>in</strong>g <strong>the</strong> automated SEM-EDX s<strong>in</strong>gle-particle analysis technique.<br />
[1] VDI (1997): VDI guidel<strong>in</strong>e 2119, part 4.<br />
19
Towards <strong>the</strong> Total Syn<strong>the</strong>sis <strong>of</strong> β-Lipomyc<strong>in</strong>:<br />
A Highly Convergent and Adaptable Double Stille-Coupl<strong>in</strong>g<br />
Strategy for <strong>the</strong> Syn<strong>the</strong>sis <strong>of</strong> 3-(Polyenoyl)tetramic Acids<br />
Max H<strong>of</strong>ferberth and Re<strong>in</strong>hard Brückner*<br />
Institut für Organische Chemie und Biochemie, Albert-Ludwigs-Universität, Albertstraße 21,<br />
79104 Freiburg, Germany; E-mail: max_h<strong>of</strong>ferberth@web.de<br />
3-(�-Hydroxymethylidene)pyrrolid<strong>in</strong>e-2,4-diones constitute <strong>the</strong> major tautomer <strong>of</strong> structures<br />
commonly referred to as „acyltetramic acids“. Compounds <strong>in</strong> which „acyl“ equals „poly-<br />
enoyl“ def<strong>in</strong>e a class <strong>of</strong> natural products, which cont<strong>in</strong>ues to evoke <strong>in</strong>terest both <strong>in</strong> <strong>the</strong><br />
syn<strong>the</strong>tic [1] and biosyn<strong>the</strong>tic community [2] .<br />
We syn<strong>the</strong>sized a prototypical (polyenoyl)tetramic acid, namely β-lipomyc<strong>in</strong> (9), [3] � or pos-<br />
sibly a stereoisomer <strong>the</strong>re<strong>of</strong>, s<strong>in</strong>ce <strong>the</strong> 3 D structure <strong>of</strong> 9 still awaits elucidation. Employ<strong>in</strong>g a<br />
novel approach, which is variable and convergent <strong>the</strong> naturally occurr<strong>in</strong>g stereoisomer plus<br />
cha<strong>in</strong>-extended or cha<strong>in</strong>-altered derivatives <strong>the</strong>re<strong>of</strong> should become readily accessible.<br />
[1] R. Schobert, A. Schlenk, Bioorg. Med. Chem. 2008, 16, 4203-4221 and literature cited <strong>the</strong>re<strong>in</strong>.<br />
[2] E. g.: C. Bihlmaier, E. Welle, C. H<strong>of</strong>mann, K. Welzel, A. Vente, E. Breitl<strong>in</strong>g, M. Müller, S. Glaser, A.<br />
Bechthold, Antimicrob. Agents Chemo<strong>the</strong>r. 2006, 50, 2113-2121.<br />
[3] B. Kunze, K. Schabacher, H. Zähner, A. Zeeck, Arch. Mikobiol. 1972, 86, 147-174.<br />
[4] First syn<strong>the</strong>sis and applications <strong>of</strong> 6: A. Sorg, R. Brückner, Angew. Chem. 2004, 116, 4623-4626; Angew.<br />
Chem. Int. Ed. 2004, 43, 4523-4526; J. Burghart, R. Brückner, Eur. J. Org. Chem. 2011, 150-165; J.<br />
Burghart, A. Sorg, R. Brückner, Chem. Eur. J. 2011, 17, 6469-6483.<br />
20
Chorismatases: Characteriz<strong>in</strong>g a new group <strong>of</strong> enzymes<br />
F. Hubrich, J. Andexer<br />
Institut für Pharmazeutische Wissenschaften, Pharmazeutische und Mediz<strong>in</strong>ische Chemie, Albertstr.<br />
25, 79104 Freiburg<br />
Chorismatases are enzymes catalyz<strong>in</strong>g <strong>the</strong> hydrolysis <strong>of</strong> chorismic acid 1 to pyruvat and a cyclic<br />
carboxylic acid. The level <strong>of</strong> saturation <strong>of</strong> <strong>the</strong> carboxylic acid depends on <strong>the</strong> type <strong>of</strong> enzyme<br />
catalyz<strong>in</strong>g this hydrolysis. Enzymes <strong>of</strong> <strong>the</strong> FkbO-type produce 1,5-diene-3,4-dihydroxycyclohexanoic<br />
acid (DHCHC) 3, enzymes <strong>of</strong> <strong>the</strong> Hyg5-type produce <strong>the</strong> aromatic 3-hydroxybenzoic acid (3HBA) 2. 1<br />
These products are precursor molecules for <strong>the</strong> syn<strong>the</strong>sis <strong>of</strong> natural products like ascomyc<strong>in</strong> 5 ,<br />
rapamyc<strong>in</strong> 2 , brasilicard<strong>in</strong> 3 and xanthomonad<strong>in</strong> 4 .<br />
FkbO and Hyg5 are k<strong>in</strong>etically characterized for chorismate, but both <strong>the</strong> structure and <strong>the</strong> reaction<br />
mechanism <strong>of</strong> <strong>the</strong>se enzymes are still unknown. Structural model<strong>in</strong>g will be difficult, because <strong>the</strong>re is<br />
no sequence similarity between chorismatases and o<strong>the</strong>r chorismate us<strong>in</strong>g enzymes.<br />
Phylogenetic analysis 1 and sequence alignments (BLAST) show additional genes <strong>in</strong> different<br />
organisms (Streptomyces and Xanthomonas), that are probably encod<strong>in</strong>g for chorismatases. To show<br />
that <strong>the</strong>se genes products are chorismatases, <strong>the</strong> target genes are <strong>in</strong>troduced <strong>in</strong>to E. coli expression<br />
vectors and stra<strong>in</strong>s. The already purified prote<strong>in</strong> FkbO and Hyg5 are used <strong>in</strong> crystallization<br />
experiments to get a crystal structure <strong>of</strong> a chorismatase.<br />
21
Lawsone and Tetrahydroxynaphthalene Reductase<br />
Syed Masood Husa<strong>in</strong>, Michael Schätzle, Michael Richter, Michael Müller<br />
Institut für Pharmazeutische Wissenschaften der Albert-Ludwigs-Universität Freiburg,<br />
Albertstr. 25, D-79104 Freiburg im Breisgau, Email: michael.mueller@pharmazie.uni-freiburg.de<br />
Lawsone (2-hydroxy-1,4-naphthoqu<strong>in</strong>one), is a red-orange dye present <strong>in</strong> <strong>the</strong> leaves <strong>of</strong><br />
<strong>the</strong> henna plant (Lawsonia <strong>in</strong>ermis). Humans have used henna extracts conta<strong>in</strong><strong>in</strong>g<br />
lawsone as hair and sk<strong>in</strong> pigments for more than 5000 years. We have used this natural<br />
product as a substrate, which on reduction catalyzed <strong>by</strong> <strong>the</strong> enzyme called<br />
tetrahydroxynaphthalene reductase (T4HNR) from Magnapro<strong>the</strong> grisea, gave cis-keto-<br />
diol with high yield (95%) and high ee (99%).<br />
HO<br />
O<br />
O<br />
Lawsone<br />
T 4HNR<br />
NADPH<br />
HO<br />
O<br />
OH<br />
cis-keto-diol<br />
Lawsone was also used extensively to study <strong>the</strong> mechanism <strong>of</strong> T4HNR-catalyzed<br />
reduction that has led to <strong>the</strong> formation <strong>of</strong> a cis-keto-diol.<br />
22
Stereoselectivity <strong>of</strong> <strong>the</strong> Ketoreductase Tyl-KR1 from<br />
Streptomyces fradiae — A VCD spectroscopy analysis<br />
Matthias Häckh, Michael Müller, and Steffen Lüdeke<br />
Institut für Pharmazeutische Wissenschaften der Albert-Ludwigs-Universität Freiburg,<br />
Albertstr. 25, D-79104 Freiburg, Germany<br />
Enzyme-catalysed reactions are best-known for <strong>the</strong>ir high stereoselectivity. Bio-<br />
catalysis benefits from <strong>the</strong>se outstand<strong>in</strong>g properties mak<strong>in</strong>g chiral build<strong>in</strong>g blocks<br />
accessible that can be used for drug syn<strong>the</strong>ses. [1] Oxidoreductases play an important<br />
role <strong>in</strong> polyketide chemistry, especially those with rarely observed “anti-Prelog type”<br />
selectivity, s<strong>in</strong>ce oxidoreductase-catalysed reduction <strong>of</strong> ketones generally leads to<br />
products with “Prelog type” configuration. [2] The stereoselectivity <strong>of</strong> many enzymes is<br />
substrate-specific and might be decreased or even converted for non-physiological<br />
substrates. Exact determ<strong>in</strong>ation <strong>of</strong> <strong>the</strong> absolute configuration <strong>of</strong> different products<br />
formed out <strong>of</strong> different substrates is <strong>the</strong>refore very important for <strong>the</strong> useful application<br />
<strong>of</strong> enzymes <strong>in</strong> biocatalysis. A switch between “anti-Prelog type” and “Prelog type”selectivity<br />
seems to be possible.<br />
The ketoreductase Tyl-KR1 from Streptomyces fradiae is known to reduce racemic 2methyl-3-oxopentanoic<br />
acid N-acetylcysteam<strong>in</strong>e thioester (1) enantioselectively to<br />
anti-(2R,3R)-3-hydroxy-2-methylpentanoic acid N-acetylcysteam<strong>in</strong>e thioester (2). [3]<br />
This “anti-Prelog type” anti-configuration is rarely found <strong>in</strong> natural products.<br />
O O<br />
CH 3<br />
S<br />
H<br />
N<br />
TYL KR 1<br />
OH<br />
R<br />
O<br />
S<br />
1<br />
O<br />
CH3 2<br />
anti-R,R<br />
R<br />
"anti-Prelog type"-reduction<br />
Figure 1: “Anti-Prelog type”-reduction catalysed <strong>by</strong> Tyl-KR1.<br />
We used VCD spectroscopy and quantum chemical calculations to determ<strong>in</strong>e <strong>the</strong><br />
absolute configurations <strong>of</strong> four different polyketide analogues reduced <strong>by</strong> Tyl-KR1.<br />
References<br />
[1] M. Müller, Angew. Chem. Int. Ed. 2005, 44, 362.<br />
[2] K. Faber, Biotransformations <strong>in</strong> Organic Chemistry, 5th ed., Spr<strong>in</strong>ger, 2004.<br />
[3] A. P. Siskos, A. Baerga-Ortiz, S. Bali, V. Ste<strong>in</strong>, H. Mamdani, D. Spiteller, B. Popovic,<br />
J. B. Spencer, J. Staunton, K. J. Weissman, P. F. Leadlay, Chem. Biol. 2005, 12,<br />
1145.<br />
23<br />
H<br />
N<br />
O
Eng<strong>in</strong>eer<strong>in</strong>g an aryl-C-glycoside biocatalyst<br />
J. Härle 1 , M. Weber 1 , B. Lau<strong>in</strong>ger 1 , S. Gün<strong>the</strong>r 2 , B. Kammerer 3 , A. Luzhetskyy 1 , A. Bechthold 1 *<br />
(1) Department <strong>of</strong> Pharmaceutical Biology und Biotechnology<br />
(2) Department <strong>of</strong> Pharmaceutical Bio<strong>in</strong>formatics, Institute <strong>of</strong> Pharmaceutical Sciences, Albert-Ludwigs-<br />
Universität Freiburg, 79104 Freiburg, Germany.<br />
(3) Zentrum für Biosystemanalytik, Albert-Ludwigs-Universität Freiburg, 79104 Freiburg, Germany.<br />
*andreas.bechthold@pharmazie.uni-freiburg.de<br />
Before a natural product potentially becomes a <strong>the</strong>rapeutic agent properties <strong>in</strong>fluenc<strong>in</strong>g <strong>the</strong><br />
pharmacological or pharmacok<strong>in</strong>etic qualities might have to be improved. As sugar substituents play an<br />
essentiell role <strong>in</strong> respect to bioactivity and solubility, <strong>the</strong>y represent a valuable address for modification<br />
(Harle and Bechthold, 2009).S<strong>in</strong>ce <strong>the</strong> sugar moieties are primarily O-l<strong>in</strong>ked to <strong>the</strong> natural product core <strong>the</strong><br />
acid sensitivity strictly limits a pr<strong>in</strong>cipal <strong>the</strong>rapeutical application. A practical alternative is to improve <strong>the</strong><br />
molecule utilizability <strong>by</strong> replac<strong>in</strong>g <strong>the</strong> labile O-aryl bond with an enzymatical and chemical stable Cglycosidic<br />
bond. The result<strong>in</strong>g structural difference might not <strong>in</strong>fluence <strong>the</strong> bioactivity but raises<br />
enormously <strong>the</strong> rigidity. The observed flexibility <strong>of</strong> UrdGT2 catalyz<strong>in</strong>g C- and O-glycosidic bonds (Durr et al.,<br />
2004) brought up <strong>the</strong> idea to eng<strong>in</strong>eer LanGT2, a strictly O-glycosyltransferase <strong>in</strong>volved <strong>in</strong> <strong>the</strong> biosyn<strong>the</strong>sis<br />
<strong>of</strong> <strong>the</strong> anticancer agent landomyc<strong>in</strong> A (Luzhetskyy et al., 2005),towards a C-glycosylat<strong>in</strong>g enzyme.<br />
By <strong>in</strong>tegrat<strong>in</strong>g computational assistance with multiple target am<strong>in</strong>o acid replacement experiments, we<br />
<strong>in</strong>terconverted <strong>the</strong> naturally O-glycosylat<strong>in</strong>g enzyme LanGT2 towards a glycosyltransferase evolved to<br />
catalyzes <strong>the</strong> sugar attachment <strong>by</strong> a C-glycosidic bond. Elucidated <strong>by</strong> alan<strong>in</strong> substitutions and visulized <strong>by</strong> <strong>in</strong><br />
silico prote<strong>in</strong> model<strong>in</strong>g we illustrate <strong>the</strong> assumed catalytic mechanism determ<strong>in</strong><strong>in</strong>g <strong>the</strong> C- and Oglycosylation<br />
respectively. The achievement <strong>in</strong> overcom<strong>in</strong>g natural limitations <strong>by</strong> prote<strong>in</strong> eng<strong>in</strong>eer<strong>in</strong>g might<br />
document a general strategy <strong>in</strong> ga<strong>in</strong><strong>in</strong>g fur<strong>the</strong>r chemical diversity <strong>in</strong> <strong>the</strong> field <strong>of</strong> comb<strong>in</strong>atorial biosyn<strong>the</strong>sis.<br />
References:<br />
Durr,C., H<strong>of</strong>fmeister,D., Wohlert,S.E., Ich<strong>in</strong>ose,K., Weber,M., Von Mulert,U., Thorson,J.S., and Bechthold,A.<br />
(2004). The glycosyltransferase UrdGT2 catalyzes both C- and O-glycosidic sugar transfers. Angew. Chem.<br />
Int. Ed Engl. 43, 2962-2965.<br />
Harle,J. and Bechthold,A. (2009). Chapter 12. The power <strong>of</strong> glycosyltransferases to generate bioactive<br />
natural compounds. Methods Enzymol. 458, 309-333.<br />
Luzhetskyy,A., Taguchi,T., Fedoryshyn,M., Durr,C., Wohlert,S.E., Novikov,V., and Bechthold,A. (2005).<br />
LanGT2 Catalyzes <strong>the</strong> First Glycosylation Step dur<strong>in</strong>g landomyc<strong>in</strong> A biosyn<strong>the</strong>sis. Chembiochem. 6, 1406-<br />
1410.<br />
24
Master Thesis: The Influence <strong>of</strong> Mechanical Defects on <strong>the</strong> Lattice <strong>of</strong> silicon<br />
T. Jauss 1 , J. Wittge 1 , A.N. Danilewsky 1 , A. Cröll 1 , J. Garagorri 2 , R. M. Elizalde 2 , D. Allen 3 , P. McNally 3<br />
1<br />
Universität Freiburg, Kristallographie, Geowissenschaftliches Institut, Freiburg, Germany<br />
2<br />
Centro de Estudios e Investigaciones Tecnicas de Gipuzkoa, CEIT and Tecnun (University <strong>of</strong> Navarra), San<br />
Sebastian, Spa<strong>in</strong><br />
3<br />
Dubl<strong>in</strong> City University, The RINCE Institute, Dubl<strong>in</strong>, Ireland<br />
Correspondence e-mail: thomas.jauss@jupiter.uni-freiburg.de<br />
The aim <strong>of</strong> this Master was <strong>the</strong>sis to exam<strong>in</strong>e <strong>the</strong> impact <strong>of</strong> mechanical damage to <strong>the</strong> silicon lattice. For this<br />
reason controlled damage was applied to silicon wafers with a boron doped layer <strong>by</strong> nano<strong>in</strong>dentation [Gar10]. A<br />
diamond Vickers tip on an Agilent Nano<strong>in</strong>denter II has been used for <strong>in</strong>dentation and <strong>the</strong> result<strong>in</strong>g damage has<br />
been characterized <strong>by</strong> differential <strong>in</strong>terference contrast microscopy (NIC) and high resolution X-ray diffraction<br />
(HRXRD). Damage <strong>by</strong> loads <strong>of</strong> 1N – 5N is compared.<br />
Rock<strong>in</strong>g curves (RC) and reciprocal space maps (RSM) were recorded and analyzed with respect to full width at<br />
half maximum and lattice plane spac<strong>in</strong>g <strong>by</strong> <strong>the</strong> method <strong>of</strong> Bond. The volume <strong>of</strong> <strong>the</strong> disturbed crystal material<br />
around <strong>the</strong> <strong>in</strong>dents is very small compared to <strong>the</strong> undisturbed volume. Therefore a l<strong>in</strong>e <strong>of</strong> seven <strong>in</strong>dents with 1N<br />
<strong>in</strong>dentation load is oriented <strong>in</strong> a way that it lies parallel to <strong>the</strong> l<strong>in</strong>e focus <strong>of</strong> <strong>the</strong> X-ray beam <strong>in</strong> order to <strong>in</strong>crease<br />
<strong>the</strong> signal <strong>in</strong>tensity <strong>of</strong> <strong>the</strong> disturbed crystal. The RSM <strong>of</strong> <strong>the</strong> undisturbed crystal volume is regarded as a<br />
reference, <strong>the</strong> sharp RC are confirmed <strong>by</strong> <strong>the</strong> sharp conf<strong>in</strong>ed peaks <strong>in</strong> <strong>the</strong> RSM. Only artefact streaks <strong>of</strong> <strong>the</strong><br />
monochromator and <strong>the</strong> analyzer are also clearly visible, as well as <strong>the</strong> dynamical streak <strong>of</strong> <strong>the</strong> specimen (Fig.<br />
1a). Despite <strong>the</strong> low count rate, a clear anisotropic <strong>in</strong>tensity distribution around <strong>the</strong> layer related peak is visible at<br />
<strong>the</strong> damaged area (Fig. 1b).<br />
Fig. 1: Reciprocal space maps <strong>of</strong> <strong>the</strong> 004 reflection. Two peaks produced <strong>by</strong> <strong>the</strong> lattice mismatch between <strong>the</strong><br />
silicon substrate and <strong>the</strong> boron doped layer can be observed <strong>in</strong> both cases.<br />
a) Undisturbed area: sharp conf<strong>in</strong>ed peaks <strong>of</strong> <strong>the</strong> silicon substrate and <strong>the</strong> B-doped layer, as well as <strong>the</strong> apparatus<br />
<strong>in</strong>duced artefact streaks and <strong>the</strong> dynamical specimen streak.<br />
b) Disturbed area: an anisotropic broaden<strong>in</strong>g <strong>of</strong> <strong>the</strong> peak bases is clearly visible, but ma<strong>in</strong>ly concentrated around<br />
<strong>the</strong> layer related peak.<br />
[Gar10] Garagorri, J., Gorostegui-Col<strong>in</strong>as, E., Elizalde, M.R., Allen, D., McNally, P. (2010) Nano<strong>in</strong>dentation<br />
<strong>in</strong>duced silicon fracture and 3D modell<strong>in</strong>g, Anales de Mecánica de la Fractura, 27, pages 559-564.<br />
25
Biomonitor<strong>in</strong>g <strong>of</strong> atmospheric particles on cypress leaves<br />
- sampled <strong>in</strong> <strong>the</strong> vic<strong>in</strong>ity <strong>of</strong> a nuclear plant-<br />
R. KALTENMEIER 1* , R. GIERÉ 1 AND L. POURCELOT²<br />
1<br />
Albert-Ludwigs-Universität, D-79104 Freiburg, Germany<br />
(*correspondence: giere@uni-freiburg.de)<br />
²Institut de Radioprotection et de Sûreté Nucléaire, Cadarache,<br />
F-13115 St Paul lez Durance, France<br />
Environmental pollution can be assessed and evaluated <strong>by</strong> plants as biological monitors. Their use<br />
can be selected depend<strong>in</strong>g on <strong>the</strong> <strong>in</strong>vestigation objective, <strong>by</strong> <strong>the</strong>ir ability to reflect past and/ or<br />
current environmental conditions as well as temporal changes (Wolterbeek 2002). Coniferous trees<br />
belong to <strong>the</strong> best passive bio<strong>in</strong>dicators for air, soil and water pollution (Saito et al. 2004). For<br />
cypress leaves (Chamaecyparis nootkatensis), sampled <strong>in</strong> <strong>the</strong> vic<strong>in</strong>ity <strong>of</strong> <strong>the</strong> Malvesi unranium<br />
process<strong>in</strong>g facility, SW France, <strong>in</strong>creased activity <strong>of</strong> act<strong>in</strong>ides and some <strong>of</strong> <strong>the</strong>ir decay products has<br />
been reported (Figure 1). Ejection via smokestacks and artificial ponds <strong>in</strong>side <strong>the</strong> facility are <strong>the</strong><br />
source <strong>of</strong> <strong>the</strong>se enhanced activities. Air dried leaf samples were exam<strong>in</strong>ed systematically <strong>by</strong> scann<strong>in</strong>g<br />
electron microscopy (SEM) <strong>in</strong> secondary and backscattered electron mode <strong>in</strong> comb<strong>in</strong>ation with<br />
energydispersive X-ray spectrometry (EDX), <strong>in</strong> order to characterize particulate matter deposited on<br />
leaf surfaces and to verify <strong>the</strong> existence <strong>of</strong> radioactive particles. For <strong>the</strong> <strong>in</strong>terpretation <strong>of</strong> qualitative<br />
EDX spectra <strong>of</strong> <strong>in</strong>dividual particles trapped on <strong>the</strong> leaf surfaces, signals result<strong>in</strong>g from C or AU coat<strong>in</strong>g<br />
as well as <strong>the</strong> background spectra <strong>of</strong> <strong>the</strong> cypress leaves <strong>the</strong>mselves were taken <strong>in</strong>to account (Figure<br />
2). Particle sizes rang<strong>in</strong>g from
Carbamates as a new prodrug concept for HDACs<br />
Keller, K. [a] , Stolfa, D.A., [a,d] Schlimme, S., [b] Hauser, A-T., [a] Carafa, V., [c] He<strong>in</strong>ke, R.,<br />
[b] Kannan, S., [b] Cellamare, S., [d] Carotti, A., [d] Altucci, L., [c] Jung, M., * [a] and Sippl,<br />
W., * [b]<br />
[a] Albert-Ludwigs-Universität Freiburg, Albertstr. 25, 79104 Freiburg<br />
[b] Mart<strong>in</strong>-Lu<strong>the</strong>r-Universität, Wolfgang-Langenbeck-Str. 4, 06120 Halle/Saale<br />
[c] Seconda Università di Napoli, Vico L. De Crecchio 7, 80138 Napoli<br />
[d] Università degli Studi “Aldo Moro”, Via Orabona 4, 7012 Bari<br />
Histone acetyltransferases (HATs) and histone deacetylases (HDACs) are<br />
responsible for ma<strong>in</strong>ta<strong>in</strong><strong>in</strong>g <strong>the</strong> acetylation equilibrium. HDACs are amidohydrolases<br />
that cleave <strong>the</strong> acetamide bond <strong>by</strong> a z<strong>in</strong>c-dependent mechanism. Inhibitors <strong>of</strong><br />
HDACs have shown great promise <strong>in</strong> <strong>the</strong> development as new anticancer agents.<br />
Hence, a prodrug pr<strong>in</strong>ciple for HDAC <strong>in</strong>hibitors is a valuable concept to optimize<br />
cellular activity.<br />
Our experimental results validate <strong>the</strong> general applicability <strong>of</strong> <strong>the</strong> carbamate group as<br />
a prodrug for hydroxamates, as compound 2 is <strong>in</strong>duc<strong>in</strong>g cellular hyperacetylation <strong>in</strong> a<br />
similar range <strong>of</strong> concentration as compared to <strong>the</strong> parent hydroxamate 1. These<br />
results demonstrate that <strong>the</strong> <strong>in</strong>troduction <strong>of</strong> a carbamate group is a valuable concept<br />
for <strong>the</strong> design <strong>of</strong> new HDAC <strong>in</strong>hibitors with improved pharmacok<strong>in</strong>etic properties.<br />
Introduc<strong>in</strong>g a carbamate group on <strong>the</strong> hydroxamic acid function <strong>of</strong> SAHA<br />
(suberoylanilide hydroxamic acid), a drug already approved as a HDAC <strong>in</strong>hibitor, led<br />
to fur<strong>the</strong>r prodrugs, such as compound 3. We syn<strong>the</strong>sized fur<strong>the</strong>r carbamates from<br />
SAHA with different moieties to fur<strong>the</strong>r <strong>in</strong>vestigate stability, cellular activity and to<br />
study <strong>the</strong> impact <strong>of</strong> different substitution patterns <strong>by</strong> consider<strong>in</strong>g pharmacok<strong>in</strong>etic<br />
aspects like solubility.<br />
Literatur:<br />
S. Schlimme et al., ChemMedChem 2011, 6 (4)<br />
27<br />
31
STABILIZATION OF DIALKYL METAL COMPOUNDS OF GROUP 13 (R2E)2B12Cl12<br />
(E = Al, Ga, In) USING THE WEAKLY COORDINATING DIANION [B12Cl12] 2-<br />
Mathias Keßler, Ardiana Zogaj, Carsten Knapp<br />
Institut für Anorganische und Analytische Chemie Albert-Ludwigs-Universität Freiburg,<br />
79104 Freiburg i. Br., Germany; mathias.kessler@ac.uni-freiburg.de<br />
The perchlor<strong>in</strong>ated closo-dodecaborate [B12Cl12] 2- is a chemically very robust weakly<br />
coord<strong>in</strong>at<strong>in</strong>g dianion, for which improved syn<strong>the</strong>sis have been published recently.[1] It<br />
was used for <strong>the</strong> syn<strong>the</strong>ses <strong>of</strong> trialkyl silylium compounds (R3Si)B12Cl12 and <strong>the</strong><br />
methylat<strong>in</strong>g agent Me2B12Cl12 which <strong>in</strong>dicate its potential to stabilize electrophilic<br />
cations.[2]<br />
The stabilization <strong>of</strong> ion-like dialkyl metal compounds <strong>of</strong> group 13 [R2E] + (E = Al, Ga,<br />
In) is a challeng<strong>in</strong>g task. Bochmann et al. showed that <strong>the</strong><br />
tetrakis(pentafluorophenyl)borate [B(C6F5)4] – anion is not suitable as counterion<br />
because degradation occurs dur<strong>in</strong>g <strong>the</strong> reaction.[3] In 2002 Reed et al. succeeded <strong>in</strong><br />
<strong>the</strong> characterization <strong>of</strong> (Et2Al)(HCB11H5X6) (X = Cl, Br) us<strong>in</strong>g partly halogenated<br />
carborane anions.[4]<br />
The reactions <strong>of</strong> <strong>the</strong> trityl salt [CPh3]2[B12Cl12] with trialkyl metal compounds R3E <strong>of</strong><br />
group 13 (E = Al, In) and Et3Ga·(OEt2) lead<strong>in</strong>g to (R2E)2B12Cl12 (E = Al, In; R = Me,<br />
Et) and [Et2Ga(OEt2)2]2[B12Cl12] will be discussed. Characterization <strong>of</strong> <strong>the</strong> compounds<br />
us<strong>in</strong>g NMR- and Raman spectroscopy and X-Ray diffraction will be presented.[5]<br />
Fig. 1 Part <strong>of</strong> <strong>the</strong> crystal structure <strong>of</strong> (Me2Al)2B12Cl12.<br />
References:<br />
[1] a) Knapp, C.; Guttsche, K.; Geis, V.; Scherer, H.; Uzun, R. Dalton Trans. 2009, 2687-2694. b) Gu,<br />
W.; Ozerov, O. V. Inorg.Chem. 2011, 50, 2726-2728.<br />
[2] a) Kessler, M.; Knapp, C.; Sagawe, V.; Scherer, H.; Uzun, R. Inorg. Chem. 2010, 49, 5223-5230. b)<br />
Bolli, C.; Derendorf, J.; Keßler, M.; Knapp, C.; Scherer, H.; Schulz, C.; Warneke, J. Angew. Chem.<br />
2010, 122, 3616-3619; Angew. Chem. Int. Ed. 2010, 49, 3536-3538.<br />
[3] Bochmann, M.; Sarsfield, M. J. Organometallics 1998, 17, 5908-5912.<br />
[4] Kim, K. C.; Reed, C. A.; Long, G. S.; Sen, A. J. Am. Chem. Soc. 2002, 124, 7662-7663.<br />
[5] Kessler, M.; Knapp, C.; Zogaj, A. Organometallics 2011, accepted.<br />
28
Untersuchung des E<strong>in</strong>flusses von Nan<strong>of</strong>üllst<strong>of</strong>fen auf die Trocknung und<br />
die Eigenschaften von Polymerfilmen mittels forcierter Rayleighstreuung<br />
Kiessl<strong>in</strong>g, Andy, Andy.Kiessl<strong>in</strong>g@fmf.uni-freiburg.de<br />
Bartsch, Eckhard, Eckhard.Bartsch@physchem.uni-freiburg.de<br />
Beschichtungen, welche auf der Verwendung von leicht flüchtigen organischen Substanzen<br />
basieren, tragen u.a. zum bodennahen Ozon bei. Dieses verursacht e<strong>in</strong>e Reizung des<br />
respiratorischen Systems, welches bestehende Atemwegserkrankungen wie Asthma<br />
verschlimmert. Im Gegensatz dazu entstehen bei der Herstellung von Beschichtungen aus<br />
wässrigen Polymerdispersionen ke<strong>in</strong>e schädlichen Emissionen.<br />
Trotz ihrer breiten Anwendung ist die Filmbildung aus wässrigen Polymerdispersionen<br />
mechanistisch nicht <strong>in</strong> allen Details verstanden. E<strong>in</strong>er der wichtigsten Schritte dieser<br />
Filmbildung ist die Teilchenkoaleszenz. Dabei lösen sich durch Interdiffusion der<br />
Polymerketten die Teilchengrenzen auf und e<strong>in</strong> mechanisch stabiler Film entsteht. Dieser<br />
Prozess wird <strong>in</strong> unserer Gruppe mit der Methode der forcierten Rayleighstreuung (FRS) auf<br />
molekularer Ebene verfolgt. Hierbei wird die Diffusion e<strong>in</strong>es Tracers über verschiedene<br />
Weglängen beobachtet, <strong>in</strong>dem durch die Überlagerung zweier Laserstrahlen e<strong>in</strong><br />
holographisches Gitter e<strong>in</strong>gebleicht wird. Dessen Zerfall durch Farbst<strong>of</strong>fdiffusion wird über<br />
das zeitliche Abkl<strong>in</strong>gen e<strong>in</strong>es optischen Braggstreusignals detektiert. Aus der Interpretation<br />
und Modellierung des Signals gew<strong>in</strong>nt man Informationen über die Mobilität des Tracers <strong>in</strong><br />
den verschiedenen Filmkompartimenten, welche durch Diffusionslängen und Diffusionskoeffizienten<br />
charakterisiert wird.<br />
Aktuell wird der E<strong>in</strong>fluss <strong>in</strong>nerer und äußerer Partikel-Grenzflächen anhand von Kern-Schale-<br />
Systemen untersucht. [1] Diese Untersuchungen sollen Aufschluss über den molekularen<br />
Ursprung der makroskopisch beobachteten besonderen Eigenschaften wie Kratzfestigkeit<br />
geben, die bei Beschichtungen aus Materialien komplementärer Eigenschaften auftreten.<br />
Dafür werden die Schalendicke, die Kerngröße, die Art des Kerns und der Schale variiert.<br />
Neben der Natur des Systems haben die Filmbildungsbed<strong>in</strong>gungen (Temperatur,<br />
Tensidkonzentration, Trocknungsgeschw<strong>in</strong>digkeit) enormen E<strong>in</strong>fluss auf die Eigenschaften<br />
der gebildeten Beschichtung. Daher s<strong>in</strong>d die Filmbildungsbed<strong>in</strong>gungen e<strong>in</strong> weiterer wichtiger<br />
Teil der Untersuchungen.<br />
L<strong>in</strong>ks: Stadien der Filmbildung e<strong>in</strong>er Latex-Dispersion, Mitte: elektronenmikroskopische Aufnahme der Teilchenkoaleszenz,<br />
[2] Rechts: schematische Darstellung des FRS-Experiments.<br />
[1] K. Suresh, E. Bartsch, J. Polym. Sci. B. 2007, 45, 2823-2834.<br />
[2] Kolloide – Vorstoß <strong>in</strong> die Nanowelt, Topics <strong>in</strong> Chemistry, BASF AG, Ludwigshafen.<br />
29
In vivo conversion <strong>of</strong> trans-3-hydroxy-L-prol<strong>in</strong>e <strong>by</strong> heterologously<br />
overexpressed microbial trans-4-prol<strong>in</strong>ehydroxylase<br />
Christian Kle<strong>in</strong>, Michael Müller and Wolfgang Hüttel<br />
Lehrstuhl für Pharmazeutische und Mediz<strong>in</strong>ische Chemie, Albert-Ludwigs-Universität<br />
Freiburg, Germany<br />
christian.kle<strong>in</strong>@pharmazie.uni-freiburg.de<br />
Heterologously overexpressed prol<strong>in</strong>e hydroxylases provide <strong>the</strong> possibilities <strong>of</strong> enzymatic<br />
conversions <strong>of</strong> L-prol<strong>in</strong>e for <strong>the</strong> regio- and stereospecific generation <strong>of</strong> hydroxyprol<strong>in</strong>es,<br />
useful chiral synthons for chemical syn<strong>the</strong>sis <strong>of</strong> pharmaceuticals. [1,2] It was also found that<br />
prol<strong>in</strong>e hydroxylases are not strictly substrate specific but accept a set <strong>of</strong> prol<strong>in</strong>e<br />
congeners. [1,3]<br />
Among several so far undiscovered reactions, prol<strong>in</strong>e-4-hydroxylase showed activity with<br />
trans-3-hydroxy-L-prol<strong>in</strong>e result<strong>in</strong>g <strong>in</strong> <strong>the</strong> trans-2,3-cis-3,4-dihydroxyprol<strong>in</strong>e isomer<br />
exclusively. S<strong>in</strong>ce <strong>in</strong> vitro conversion with <strong>the</strong> enzyme suffers from several drawbacks, an <strong>in</strong> vivo<br />
approach was established. Product concentrations <strong>of</strong> up to 4 mM were obta<strong>in</strong>ed <strong>in</strong> <strong>the</strong><br />
fermentation broth <strong>of</strong> simple shake flask cultures and <strong>the</strong> product was isolated via ionexchange<br />
chromatography <strong>in</strong> >90% yield.<br />
References:<br />
[1] C. Kle<strong>in</strong>, W. Hüttel, Adv. Synth. Catal. 2011, 353, 1375-1383.<br />
[2] P. Remuzon, Tetrahedron 1996, 52, 13803-13835.<br />
[3] T. Shibasaki, W. Sakuri, A. Hasegawa, Y. Uosaki, H. Mori, M. Yoshida, A. Ozaki,<br />
Tetrahedron Lett. 1999, 40, 5227-5230.<br />
30
Optical excitations and <strong>in</strong>itial charge transfer phenomena <strong>in</strong><br />
biopolymers and solar cell model compounds: a <strong>the</strong>oretical<br />
approach<br />
S. Kruse, S. Krapf, B. Lampe, T. Koslowski<br />
Institut für Physikalische Chemie, Universität Freiburg, Albertstrasse 23a, D-79104 Freiburg<br />
im Breisgau, Germany<br />
We approach <strong>the</strong> problem <strong>of</strong> optical excitations <strong>in</strong> molecular aggregates <strong>in</strong> complex<br />
biochemical and man-made environments from a computational, all-atom perspective. The<br />
system is divided <strong>in</strong>to a pi orbital part described <strong>by</strong> a Pariser-Parr-Pople model, which is<br />
coupled to <strong>the</strong> charge degrees <strong>of</strong> freedom <strong>of</strong> a classical force field. Strategies for a highaccuracy<br />
reparameterization and an efficient computational solution are presented.<br />
For gamma-D-crystall<strong>in</strong>, a band edge consist<strong>in</strong>g <strong>of</strong> charge-transfer states emerges for a<br />
coupled molecular aggregate compared to <strong>the</strong> uncoupled residues. The energies <strong>of</strong> some<br />
charge-transfer states strongly depend on <strong>the</strong> dielectric properties <strong>of</strong> <strong>the</strong> model, giv<strong>in</strong>g a first<br />
<strong>in</strong>sight <strong>in</strong>to <strong>the</strong> potential temporal evolution <strong>of</strong> <strong>the</strong>se excitations. Biochemical implications<br />
and extensions <strong>of</strong> <strong>the</strong> model to solar cell model compounds are discussed.<br />
31
Stabilized lipid disks<br />
Lena Koehler 1 , Michael Müller 2 , Katar<strong>in</strong>a Edwards 1,3 and Ulrich Mass<strong>in</strong>g 4<br />
1 FRIAS, School <strong>of</strong> S<strong>of</strong>t Matter Research, Freiburg, Germany<br />
2 Institute <strong>of</strong> Pharmaceutical Sciences, Albert-Ludwigs-Universität Freiburg, Germany<br />
3 Physical and Analytical Chemistry, University Uppsala, Sweden<br />
4 Tumor Biology Center, Freiburg, Germany<br />
Background:<br />
A novel type <strong>of</strong> membrane disks with promis<strong>in</strong>g characteristics have been found <strong>in</strong> lipid<br />
mixtures conta<strong>in</strong><strong>in</strong>g polyethylene glycol (PEG)-lipids 1,2 . The lipid disks show planar and<br />
circular shape. The PEG-lipids favor <strong>the</strong> rim <strong>of</strong> <strong>the</strong> disks 3 and <strong>of</strong>fer steric protection aga<strong>in</strong>st<br />
fusion and self-closure. Lipid disks can be produced us<strong>in</strong>g <strong>the</strong> same techniques as for <strong>the</strong><br />
preparation <strong>of</strong> liposomes; such techniques like sonication or several freeze/thaw/vortexcycles<br />
followed <strong>by</strong> extrusion give a very homogenous size distribution.<br />
Stabilized lipid disks:<br />
In order to realize <strong>the</strong> full potential <strong>of</strong> <strong>the</strong> disks <strong>in</strong>creas<strong>in</strong>g <strong>the</strong> robustness <strong>of</strong> <strong>the</strong> disks is<br />
desirable. Therefore <strong>the</strong> formation <strong>of</strong> a stabiliz<strong>in</strong>g shell is envisioned. To meet this goal<br />
modified PEG-lipids bear<strong>in</strong>g a T-L<strong>in</strong>ker between <strong>the</strong> lipid- and <strong>the</strong> PEG-moiety should be<br />
syn<strong>the</strong>sized. These modified PEG-lipids should be employed <strong>in</strong> a cross-l<strong>in</strong>k<strong>in</strong>g reaction<br />
between each o<strong>the</strong>r on <strong>the</strong> rim <strong>of</strong> <strong>the</strong> disks to enhance <strong>the</strong> cohesion and <strong>the</strong>refore <strong>the</strong><br />
stability <strong>of</strong> <strong>the</strong> discs.<br />
References:<br />
1. K. Edwards, M. Johnsson, G. Karlsson, M. Silvander, Biophys J 73 (1997), 258-266<br />
2. M. Johnsson, K. Edwards, Biophys J 85 (2003) 3839–3847<br />
3. P. Wessman, A. Rennie, K. Edwards, BBA Biomembranes 1778 (2008), 2210-2216A.<br />
32
Implications <strong>of</strong> magnetite-mediated ROS formation on genotoxic<br />
effects and signall<strong>in</strong>g pathways <strong>in</strong> A549 cells<br />
Mathias Könczöl 1 , Richard Gm<strong>in</strong>ski 1 , Sandra Ebel<strong>in</strong>g 2 , Ella Goldenberg 3 , Reto Gieré 3 , Bernard<br />
Grobéty 4 , Barbara Ro<strong>the</strong>n-Rutishauser 5 , Irmgard Merfort 2 , Volker Mersch-Sundermann 1<br />
1 Institut für Umweltmediz<strong>in</strong> und Krankenhaushygiene, Universitätskl<strong>in</strong>ikum Freiburg<br />
2 Lehrstuhl für Pharmazeutische Biologie und Biotechnologie, Universität Freiburg<br />
3 Institut für Geowissenschaften, Universität Freiburg<br />
4 Institut für Geowissenschaften, Universität Fribourg, Schweiz<br />
5 Institut für Anatomie, Universität Berne, Schweiz<br />
Magnetite, a ferromagnetic iron oxide, has been characterized as a major component<br />
<strong>of</strong> particulate matter sampled at underground stations, along railway l<strong>in</strong>es and at<br />
weld<strong>in</strong>g workplaces. The abundance <strong>of</strong> magnetite <strong>in</strong> <strong>the</strong>se samples and <strong>the</strong> lack <strong>of</strong><br />
consistent data demand more systematic <strong>in</strong>vestigation, specifically <strong>in</strong>to <strong>the</strong> genotoxic<br />
potential <strong>of</strong> magnetite nano- and micrometer particles and <strong>the</strong>ir underly<strong>in</strong>g<br />
mechanisms. In <strong>the</strong> present study, we studied magnetite for its toxic potential <strong>in</strong><br />
cultured human lung cells (A549). Cell viability (WST-1, NR assay), oxidative stress<br />
(DCFH-DA, lipidperoxidation (LPO)) and genotoxicity (comet assay; CB-MNvit test)<br />
were determ<strong>in</strong>ed as biological endpo<strong>in</strong>ts <strong>of</strong> particle exposure. Additionally, effects on<br />
NF-κB and JNK activation were evaluated <strong>by</strong> EMSA and western blots.<br />
TEM images showed <strong>in</strong>corporation <strong>of</strong> magnetite bulk and nanoparticles <strong>in</strong>to A549<br />
cells. No cytotoxic effects were observed after exposure <strong>of</strong> <strong>the</strong> cells to <strong>the</strong> various<br />
samples. We observed concentration-dependent ROS formation and as a<br />
consecutive reaction high amounts <strong>of</strong> LPO for all samples. Pre<strong>in</strong>cuation with <strong>the</strong> ROS<br />
scavengers butylated hydroxyanisole (BHA) and apocyn<strong>in</strong>, known to <strong>in</strong>hibit NADPH-<br />
oxidase (NOX) and to <strong>in</strong>crease GSH-levels, lead to decreased ROS production. We<br />
detected size and concentration-dependent <strong>in</strong>duction <strong>of</strong> micronuclei (MN), as well as<br />
DNA-damag<strong>in</strong>g effects. These genotoxic effects could be reduced <strong>by</strong> <strong>the</strong> ROS<br />
scavengers N-acetylcyste<strong>in</strong>e and BHA. Moreover, an activation <strong>of</strong> JNK without<br />
<strong>in</strong>creased NF-κB-b<strong>in</strong>d<strong>in</strong>g activity was observed. However, activation <strong>of</strong> JNK seems to<br />
be not ROS dependent. Our experiments suggest that after endocytosis <strong>of</strong> magnetite<br />
particles NOX-activation leads to ROS formation and consecutive LPO. These effects<br />
may play an important role <strong>in</strong> <strong>the</strong> genotoxicity <strong>of</strong> magnetite <strong>in</strong> A549 cells.<br />
33
Gemischte Thiooxowolframate A2[W VI SxO4−x]:<br />
A. Lehner, M. Braitsch, Z. Deng, C. Röhr<br />
Institut f. Anorg. u. Analyt. Chemie, Universität Freiburg<br />
anna@limonite.chemie.uni-freiburg.de<br />
Gemischte Thiooxoorthowolframate(VI) [WSxO4−x] 2− wurden erstmals im 19. Jahrhundert<br />
dargestellt [1,2]. Die spektroskopischen Eigenschaften dieser Ionen wurden <strong>in</strong>tensiv<br />
untersucht [3], während kaum detaillierte Studien der Kristallstrukturen ihrer Salze vorliegen<br />
[4]. Wir präsentieren hier die erste systematische Untersuchung der Kristallchemie<br />
von Alkalimetall- und Ammoniumthiooxowolframaten anhand von Röntgendiffraktometrie<br />
und Bandstrukturrechnungen. Die Strukturchemie und B<strong>in</strong>dungssituation wird dabei im<br />
Vergleich mit den Ergebnissen unserer Untersuchung analoger Thiooxomolybdate [5,6] vorgestellt.<br />
Die Syn<strong>the</strong>sen der Salze A2[WSxO4−x] (A = NH4: x = 2; A = K, Rb: x = 1,2,3; A = Cs:<br />
x = 2,3) erfolgte durch E<strong>in</strong>leiten von H2S-Gas <strong>in</strong> alkalische Lösungen der Oxowolframate.<br />
Aus den gelben Thiooxowolframatlösungen ließen sich die Salze phasenre<strong>in</strong> und e<strong>in</strong>kristall<strong>in</strong><br />
erhalten. Die Verb<strong>in</strong>dungen weisen weit mehr polymorphe Formen auf als bisher erwartet<br />
wurde: zusätzlich zu drei bekannten A2BX4-Typen wurden vier neue Strukturtypen erhalten.<br />
Die Monothiowolframate von K + und Rb + kristallisieren als Hydrate. Während alle Verb<strong>in</strong>dungen<br />
mit [WS2O2] 2− -Anionen isotype monokl<strong>in</strong>e Strukturen ((NH4)2[WS2O2]-Typ, C2/c,<br />
z.B. K2[WS2O2]: a = 1126.5(4), b = 708.2(2), c = 971.5(4) pm, β = 121.86(3) o ) bilden, s<strong>in</strong>d<br />
die K + - und Cs + -Salze dimorph und kristallisieren zusätzlich <strong>in</strong> den bisher unbekannten<br />
Strukturtypen (P21/c bzw. Pbcn). Die Trithiowolframate von Rb + und Cs + kristallisieren<br />
im orthorhomischen β-K2[SO4]-Typ (Pnma), der auch von den Tetrathiowolframaten gebildet<br />
wird. Am Beispiel der Cs-Dithiooxowolframate wurde der <strong>the</strong>rmisch <strong>in</strong>duzierte Phasenübergang<br />
(bei ca. 100 o C) von der orthorhombischen Struktur (Pbcn, VFormele<strong>in</strong>heit =<br />
805 10 6 pm 3 ) <strong>in</strong> die monokl<strong>in</strong>e Form ((NH4)2[WS2O2]-Typ, C2/c, VFormele<strong>in</strong>heit = 797<br />
10 6 pm 3 ) <strong>the</strong>rmoanalytisch untersucht. Bei ca. 260 o C tritt unter Volumenzunahme e<strong>in</strong>e<br />
Umwandlung <strong>in</strong> e<strong>in</strong>e neue Phase im β-K2[SO4]-Typ (Pnma) e<strong>in</strong>. In den wasserfreien Salzen<br />
s<strong>in</strong>d die [WSxO4−x] 2− -Anionen so gepackt, dass die Oxo-Liganden vier- bis fünffach<br />
durch Kationen koord<strong>in</strong>iert s<strong>in</strong>d, während die Thio-Liganden Koord<strong>in</strong>ationszahlen von 6<br />
bis 7 aufweisen. Die Anordnung der Wolframattetraederanionen lässt sich auf e<strong>in</strong>fache Metallpackungen<br />
zurückführen. Die Strukturen der Salzhydrate und Ammoniumsalze werden<br />
h<strong>in</strong>sichtlich ihrer Wasserst<strong>of</strong>fbrückenstruktur diskutiert. Die B<strong>in</strong>dungssituation wird anhand<br />
der Zustandsdichten aus FP-LAPW-Bandstrukturrechnungen auf Basis der experimentellen<br />
(W-S)-/(W-O)- Abstände analysiert.<br />
[1] E. Corleis, Liebigs Ann. Chem. 1886, 232, 244-270. [2] G. Krüss, Liebigs Ann. Chem.<br />
1884, 225, 1. [3] K. H. Schmidt, A. Müller Coord. Chem. Rev. 1974, 14, 115. [4] A. Müller,<br />
W. Sievert, Z. Anorg. Allg. Chem. 1974, 403, 251. [5] A. Lehner, K. Kraut, C. Röhr, Acta<br />
Crystallogr. 2010, A66, 186. [6] A. Lehner, C. Röhr, Z. Anorg. Allg. Chem. (<strong>in</strong> Vorbereitung).<br />
34
Multiple partial melt<strong>in</strong>g events <strong>in</strong> <strong>the</strong> contact aureole <strong>of</strong> <strong>the</strong> Re<strong>in</strong>fjord ultramafic complex,<br />
Seiland Igneous Prov<strong>in</strong>ce, Nor<strong>the</strong>rn Norway<br />
Xiaoyan Li, Kurt Bucher<br />
Re<strong>in</strong>fjord ultramafic complex, one <strong>of</strong> <strong>the</strong> largest layered peridotite and gabbro plutons <strong>in</strong> <strong>the</strong><br />
Seiland Igneous Prov<strong>in</strong>ce, emplaced <strong>in</strong>to <strong>the</strong> lower part <strong>of</strong> <strong>the</strong> Sørøy Succession <strong>of</strong> Kalak nappe<br />
complex, which developed a contact aureole dur<strong>in</strong>g <strong>the</strong> prograde metamorphism. The protolith <strong>of</strong><br />
material contribut<strong>in</strong>g <strong>the</strong> contact aureole consists <strong>of</strong> psammite and semipelite, shallow-mar<strong>in</strong>e sediment<br />
that deposited on <strong>the</strong> Baltica cont<strong>in</strong>ental marg<strong>in</strong> <strong>of</strong> <strong>the</strong> Iapetus Ocean. Based on available researches <strong>in</strong><br />
<strong>the</strong> area, we found that multiple <strong>the</strong>rmal events could have changed <strong>the</strong> lithological unit extensively,<br />
with vary<strong>in</strong>g significances. In order to decipher <strong>the</strong> complicated geological process, we dedicated<br />
abundant field and laboratory works.<br />
Several assemblages have been observed and used as <strong>in</strong>dicators <strong>of</strong> multiple <strong>the</strong>rmal events:<br />
(1) Sil-Kfs-Ab: layered texture, likely product <strong>of</strong> breakdown <strong>of</strong> muscovite.<br />
(2) Bt-Sil-Kfs-Pl-Qtz: different subsets as <strong>in</strong>clusions <strong>in</strong> garnets, act as <strong>the</strong> reactant <strong>of</strong> biotitebreakdown<br />
<strong>in</strong>congruent melt<strong>in</strong>g reaction, form<strong>in</strong>g peritectic garnet.<br />
(3) Opx-Grt-Sil-Bt: occur<strong>in</strong>g locally, biotite partial melted.<br />
(4) Hc-Crn with or without Sil: as bone-like crystals <strong>in</strong> <strong>the</strong> muscovite matrix with <strong>the</strong> whole patch<br />
enclosed <strong>by</strong> garnet and biotite, and hercynite encloses sillimanite, likely resulted from back<br />
reaction between m<strong>in</strong>erals and melts.<br />
(5) Ms or Bt: replac<strong>in</strong>g <strong>the</strong> cordierite and garnet respectively, <strong>the</strong> new hydrous m<strong>in</strong>erals that formed<br />
<strong>by</strong> residual melt or fluid react<strong>in</strong>g with <strong>the</strong> anhydrous m<strong>in</strong>erals.<br />
35
Redox-Neutral Atom-Economic Rhodium-Catalyzed<br />
Coupl<strong>in</strong>g <strong>of</strong> Term<strong>in</strong>al Alkynes with Carboxylic Acids<br />
Toward Branched Allylic Esters<br />
Alexandre LUMBROSO, Philipp KOSCHKER, Bernhard BREIT*<br />
Institut fűr Organische Chemie und Biochemie, Freiburg Institute for Advanced Studies (FRIAS),<br />
Albert-Ludwigs-Universität Freiburg, Albertstrasse 21, 79104 Freiburg im Breisgau, Germany<br />
Branched allylic alcohols and <strong>the</strong>ir ester derivatives are important build<strong>in</strong>g blocks <strong>in</strong><br />
organic syn<strong>the</strong>sis. Numerous approaches for <strong>the</strong>ir preparation are known, <strong>in</strong>clud<strong>in</strong>g<br />
asymmetric versions. [1] More recently, formation <strong>of</strong> allylic esters via CH bond oxidation has<br />
attracted considerable <strong>in</strong>terest and is emerg<strong>in</strong>g as a new tool <strong>in</strong> <strong>the</strong> syn<strong>the</strong>sis <strong>of</strong> complex<br />
molecules. [2] A drawback <strong>of</strong> <strong>the</strong>se reactions with regard to atom economy is <strong>the</strong> requirement<br />
<strong>of</strong> stoichiometric amounts <strong>of</strong> an oxidant. In this work, we have found that us<strong>in</strong>g <strong>the</strong><br />
diphosph<strong>in</strong>e ligand DPEphos L and [Rh(COD)Cl]2, <strong>the</strong> addition <strong>of</strong> carboxylic acids to<br />
term<strong>in</strong>al alkynes lead to <strong>the</strong> branched allylic esters B with high chemoselectivity (Scheme<br />
1). [3] The reaction is a redox-neutral formal propargylic CH oxidation with concomitant<br />
hydride shift where <strong>the</strong> alkyne serves as an <strong>in</strong>ternal oxidant to be reduced simultaneously<br />
toward <strong>the</strong> alkene. This orig<strong>in</strong>al atom-economic process is applicable to a broad substrate<br />
scope and displays a wide functional group tolerance.<br />
Scheme 1. Addition <strong>of</strong> carboxylic acids to term<strong>in</strong>al alkynes via a redox-neutral<br />
atom-economic process<br />
[1] D. M. Hodgson, P. G. Humphreys, In Science <strong>of</strong> Syn<strong>the</strong>sis: Houben-Weyl Methods <strong>of</strong> Molecular<br />
Transformations; Clayden, J. P., Ed.; Thieme: Stuttgart, Germany, 2007; Vol. 36, pp 583.<br />
[2] M. S. Chen, N. Prabagaran, N. A. Labenz, M. C. White, J. Am. Chem. Soc. 2005, 127, 6970.<br />
[3] A. Lumbroso, P. Koschker, N. R. Vautravers, B. Breit, J. Am. Chem. Soc. 2011, 133, 2386.<br />
36
Do photosyn<strong>the</strong>tic bacteria have a protective mechanism<br />
aga<strong>in</strong>st carbonate precipitation at <strong>the</strong>ir surfaces?<br />
Raul E. Mart<strong>in</strong>ez, Insitute für Geochemie, Universität Freiburg, Albertstr. 23b, D-79104<br />
Freiburg, raul.mart<strong>in</strong>ez@m<strong>in</strong>pet.uni-freiburg.de<br />
Closed reactor k<strong>in</strong>etic experiments, SEM and TEM imag<strong>in</strong>g, EDX analyses, and<br />
zeta potential measurements were used to assess <strong>the</strong> existence <strong>of</strong> metabolic process<br />
protect<strong>in</strong>g cyanobacteria aga<strong>in</strong>st carbonate m<strong>in</strong>eralization on <strong>the</strong>ir surfaces. Carbonate<br />
precipitation rates measured at pH <strong>of</strong> ~8.2 and 23 °C <strong>in</strong> <strong>in</strong>itially supersaturated solutions<br />
<strong>in</strong> <strong>the</strong> presence <strong>of</strong> active Synechococcus sp. and Planktothrix sp. correspond closely to<br />
those measured <strong>in</strong> analogous <strong>in</strong>organic control experiments. TEM imag<strong>in</strong>g and EDX<br />
analysis <strong>in</strong>dicates <strong>the</strong> absence <strong>of</strong> Ca 2+ on active Synechococcus sp. and Planktothrix sp.<br />
surfaces. Electrophoretic measurements <strong>of</strong> active cyanobacteria surfaces demonstrate<br />
development <strong>of</strong> a positive surface potential on active Synechococcus sp. and Planktothrix<br />
sp. cyanobacteria at pH 8–10. This positive charge was suppressed <strong>by</strong> <strong>the</strong> presence <strong>of</strong> 1<br />
mM HCO3 - but enhanced <strong>by</strong> <strong>in</strong>creas<strong>in</strong>g aqueous Ca 2+ concentration <strong>in</strong> <strong>the</strong> fluid phase.<br />
These observations suggest <strong>the</strong> existence <strong>of</strong> a mechanism, based on <strong>the</strong> metabolic<br />
ma<strong>in</strong>tenance <strong>of</strong> a positive surface charge at alkal<strong>in</strong>e pH, protect<strong>in</strong>g active cyanobacteria<br />
aga<strong>in</strong>st Ca 2+ adsorption and subsequent carbonate precipitation on <strong>the</strong>ir surfaces. [1]<br />
References:<br />
[1] Mart<strong>in</strong>ez, R. E., Gardes, E., Pokrovsky O. S., Schott, J., Oelkers, E. H., Geochim.<br />
Cosmochim. Acta. 2010, 74, 1329.<br />
37
Zn-Indide der Alkalimetalle mit der Anionenstruktur des β-rhomboedrischen<br />
Bors<br />
V. Mihajlov 1 and C. Röhr 1<br />
1 : Institut f. Anorg. u. Analyt. Chemie, Universität Freiburg, Deutschland<br />
viktoria@goethite.chemie.uni-freiburg.de<br />
β-rhomboedrisches Bor, β-B105 (hR, R3m, Z=3), ist e<strong>in</strong>e der allotropen Modifikationen<br />
des elementaren Bors. Die Kristallstruktur besteht aus zwei kristallographisch unterschiedlichen<br />
B12-Ikosaedern im Verhältnis 1:3 und zwei verbrückten kondensierten<br />
Dreifachikosaedern B28, die über exo-B<strong>in</strong>dungen mite<strong>in</strong>ander verknüpft s<strong>in</strong>d (Abb. 1).<br />
Sie stellt e<strong>in</strong>e hierarchische Variante des MgCu2-Typs dar, bei der die Ikosaeder die<br />
Cu- und die Dreifachikosaeder die Mg-Plätze e<strong>in</strong>nehmen ([B28]2B12[B12)3]). Bereits die<br />
ersten E<strong>in</strong>kristallstrukturdaten [1] zeigen, dass entsprechend der Wade-Regeln nicht<br />
alle Borlagen der B28-E<strong>in</strong>heit voll besetzt s<strong>in</strong>d.<br />
Analoge Gallide und Indide existieren aus sterischen Gründen nur mit Alkalimetallkationen<br />
A + <strong>in</strong> den Lücken zwischen den Clustern, z.B. Na26K8Ga99.1 [2] oder K14Na20In96.3<br />
[3]. Die Elektronenbilanz wird entsprechend durch stärker ausgedehnte Defekte im Anionenteilgitter<br />
wiederhergestellt [4].<br />
E<strong>in</strong> Ausgleich des durch die zusätzlichen A-Atomen e<strong>in</strong>gebrachten Elektronenüberschusses<br />
kann durch den E<strong>in</strong>bau von elektronenärmeren Elementen der späten Übergangsmetallreihe<br />
erreicht werden, z.B. <strong>in</strong> K34Zn18.8In86.2 [5]. Ausgehend von dieser<br />
defektfreien Phase wurde der sterische E<strong>in</strong>fluss der Kationengröße auf die Struktur<br />
systematisch durch Austausch der Alkalimetalle röntgenographisch anhand von E<strong>in</strong>kristalldaten<br />
untersucht. Das Zn/In-Verhältnis bleibt dabei annähernd gleich, die Valenzelektronenkonzentration<br />
(vec) pro M beträgt 3.16 und liegt damit im engen Bereich<br />
zwischen 3.10 und 3.38, <strong>in</strong> den alle Verb<strong>in</strong>dungen mit diesem Strukturtyp auftreten.<br />
Berechnungen der elektronischen Strukturen mittels FP-LAPW-DFT-Methoden zeigen<br />
<strong>in</strong> Übere<strong>in</strong>stimmung hiermit e<strong>in</strong> deutlich ausgeprägtes M<strong>in</strong>imum <strong>in</strong> der totalen<br />
Zustandsdichte am Ferm<strong>in</strong>iveau.<br />
Die sechs Kationenpositionen können <strong>in</strong> zwei Gruppen unterteilt werden. Die drei Positionen<br />
A(1,2,3) (<strong>in</strong>sgesamt 14 Atome) weisen e<strong>in</strong>e kle<strong>in</strong>ere Koord<strong>in</strong>ationssphäre mit<br />
der Koord<strong>in</strong>ationszahl (CN) 12 (gekappte Tetraeder) und die Positionen A(4,5,6) (<strong>in</strong>sgesamt<br />
20 Atome) e<strong>in</strong>e größere mit e<strong>in</strong>er CN von 15 bzw. 16 auf.<br />
Ausgehend von K34Zn18.8In86.3 führte der sukzessive Austausch von K + durch das kle<strong>in</strong>ere<br />
Kation Na + zu der Grenzzusammensetzung K14Na20Zn16.7In88.3 (a = 1771.44(5),<br />
c = 3890.7(2) pm), bei der alle A(1,2,3)-Lagen mit Na + und A(4,5,6)-Lagen mit K +<br />
besetzt s<strong>in</strong>d.<br />
Umgekehrt erfolgte der Austausch von K + auf den Lagen A(4,5,6) durch größere Kationen<br />
ebenfalls nahezu vollständig, so dass Rb13.3K20.7Zn18.9In86.1 und Cs12.3K21.7Zn19.2In85.8<br />
(a = 1838.30(5)/1846.27(9), c = 4017.97(2)/4039.7(2) pm) die Randverb<strong>in</strong>dungen darstellen.<br />
Als Bildungsvoraussetzung für den vorgestellten Strukturtyp können somit zwei limitierende<br />
Größen genommen werden: Zum e<strong>in</strong>en der bereits erwähnte enge vec-Bereich<br />
und zum anderen e<strong>in</strong> Kationen/Anionen-Radienverhältnis von rA/rM = 0.97 - 1.14 für<br />
die Lagen A(1) bis A(3) bzw. rA/rM = 1.14 - 1.31 für A(4) bis A(6).<br />
[1] J. L. Hoard, D. B. Sullenger, C. H. L. Kennard, R. E. Hughes J. Solid State Chem. 1, 268-277 (1970).<br />
[2] C. Bel<strong>in</strong>, M. Charbonnel, J. Solid State Chem. 64, 57-66 (1986).<br />
[3] B. Li, J. D. Corbett, J. Am. Chem. Soc. 127, 926-932 (2005).<br />
[4] C. Bel<strong>in</strong>, M. Charbonnel, Coord. Chem. Rev. 178-180, 529-564 (1998).<br />
[5] B. Li, J. D. Corbett, Inorg. Chem. 45, 8958-8964 (2006).<br />
38
C<br />
B<br />
A<br />
c<br />
O<br />
a<br />
b<br />
Abbildung 1:<br />
39<br />
2<br />
Zn<br />
In
Liposomal encapsulation <strong>of</strong> arsenic trioxide for effective treatment <strong>of</strong><br />
neuroblastoma cancer<br />
Müller, I. 1 , Schleicher, S.², Schubert, R. 1<br />
1<br />
Lehrstuhl für Pharmazeutische Technologie und Biopharmazie,<br />
Universität Freiburg<br />
²Kl<strong>in</strong>ik für K<strong>in</strong>der und Jugendmediz<strong>in</strong> (Hämotologie und Onkologie),<br />
Universitätskl<strong>in</strong>ikum Tüb<strong>in</strong>gen<br />
Arsenic trioxide (ATO) is effectively used as an ant<strong>in</strong>eoplastic drug 1 for <strong>the</strong> treatment<br />
<strong>of</strong> various cancer diseases, for example acute promyelotic leukemia and certa<strong>in</strong><br />
neuroblastoma forms. Due to side effects like renal and heart failure that might occur<br />
<strong>in</strong> oral application, a liposomal encapsulation <strong>of</strong> ATO for parenteral adm<strong>in</strong>istration is<br />
recommended.<br />
This allows for <strong>in</strong>creased accumulation <strong>of</strong> ATO <strong>in</strong> <strong>the</strong> tumor environment because <strong>of</strong><br />
<strong>the</strong> <strong>EPR</strong> effect (enhanced permeability and retention) <strong>in</strong> tumor-support<strong>in</strong>g blood<br />
vessels carry<strong>in</strong>g gap junctions. The accumulation effect could be stimulated <strong>by</strong> an<br />
active target<strong>in</strong>g <strong>of</strong> <strong>the</strong>se liposomes towards specific antigens be<strong>in</strong>g expressed on<br />
cancer cells’ surfaces. This may be achieved via <strong>the</strong> comb<strong>in</strong>ation <strong>of</strong> a sterol based<br />
anchor <strong>in</strong>tegrated <strong>in</strong> <strong>the</strong> liposomal bilayer and correspond<strong>in</strong>g antibodies covalently<br />
attached to this anchor. It was already shown that a sole <strong>in</strong>corporation <strong>of</strong> ATO<br />
solution <strong>in</strong> liposomes did not result <strong>in</strong> a stable formulation (Kall<strong>in</strong>tieri et al 2004) 2 .<br />
Therefore a remote load<strong>in</strong>g mechanism us<strong>in</strong>g nickel acetate (Chen et al. 2006) 3 was<br />
adapted that enabled a precipitation and thus entrapment <strong>of</strong> ATO <strong>in</strong>side <strong>of</strong> <strong>the</strong><br />
hydrophilic liposomal core.<br />
Here prelim<strong>in</strong>ary results with regard to cell viability <strong>of</strong> certa<strong>in</strong> neuroblastoma cell<br />
l<strong>in</strong>es are shown. Cell viability results upon treatment with ATO-solution and<br />
liposomal entrapped ATO, respectively, are compared to solely nickel-loaded or<br />
unloaded control liposomes, as well as non-treated control cells.<br />
1 Ferrara et al. : Acute promyelocytic leukemia: what are <strong>the</strong> treatment options?<br />
Expert Op<strong>in</strong> Pharmaco<strong>the</strong>r. 2010 Mar;11(4):587-96<br />
2 Kall<strong>in</strong>tieri et al.: Arsenic trioxide liposomes: encapsulation efficiency and <strong>in</strong> vitro stability, J Liposome Res. 2004; 14(1-2):27-<br />
38.<br />
3 Chen et al. : Lipid encapsulation <strong>of</strong> arsenic trioxide attenuates cytotoxicity and allows for controlled anticancer<br />
drug release, J. Am. Chem. Soc., 2006, 128 (41), 13348-13349<br />
40
CONVERTING AN AROMATIC TO A POLYKETIDE:<br />
APPLICATION TO THE SYNTHESIS OF THE<br />
C 1 -C 11 FRAGMENT OF RIMOCIDIN<br />
Luc Nachbauer and Re<strong>in</strong>hard Brückner*<br />
Institut für Organische Chemie und Biochemie, Albert-Ludwigs-Universität,<br />
Albertstraße 21, D-79104 Freiburg; lucnachbauer@hotmail.com<br />
Rimocid<strong>in</strong> (1) is a member <strong>of</strong> <strong>the</strong> polyol/polyene family <strong>of</strong> macrolide antibiotics, which consists<br />
<strong>of</strong> ~200 naturally occurr<strong>in</strong>g molecules. We have developed a novel syn<strong>the</strong>sis <strong>of</strong> <strong>the</strong> syn<strong>the</strong>tic<br />
equivalent 2 <strong>of</strong> <strong>the</strong> nor<strong>the</strong>rn moiety <strong>of</strong> rimocid<strong>in</strong> (1). [1] Our route is based on a straightforward<br />
generation <strong>of</strong> <strong>the</strong> polyketide cha<strong>in</strong> <strong>by</strong> <strong>the</strong> oxidative cleavage <strong>of</strong> an ortho-bridged dihydroaromatic<br />
system. [2]<br />
HO<br />
HOOC<br />
14<br />
HO<br />
O<br />
11<br />
18<br />
OH<br />
O-D-mycosam<strong>in</strong><br />
1<br />
O OH<br />
nPr<br />
1<br />
O<br />
O<br />
S<br />
S<br />
11<br />
OTBS<br />
S S<br />
Specifically, we syn<strong>the</strong>sized <strong>the</strong> non-racemic <strong>in</strong>dane 4 from commercially available 5-hydroxy<strong>in</strong>dane<br />
(3) <strong>in</strong> 5 steps. A tandem reaction consist<strong>in</strong>g <strong>of</strong> a Birch reduction and ozonolysis led to triketo-ester<br />
5. S,S-acetal formations and stereoselective reductions gave <strong>the</strong> desired C 1 -C 11 fragment 2.<br />
References:<br />
[1] Isolation: J. W. Davisson, F. W. Tanner, Jr., A. C. F<strong>in</strong>lay, I. A. Solomons, Antibiot. Chemo<strong>the</strong>r. 1951, 1,<br />
289-290.� Total syn<strong>the</strong>ses: G. K. Packard, Y. Hu, A. Vescovi, S. D. Rychnovsky, Angew. Chem. 2004, 116,<br />
2882-2886; Angew. Chem., Int. Ed. 2004, 43, 2822-2826; A. B. Smith III, M. A. Foley, S. Dong, A. Orb<strong>in</strong>, J.<br />
Org. Chem. 2009, 74, 5987-6001.<br />
[2] Ozonolysis <strong>of</strong> bridged hydroaromatics: A. J. Birch, F. Fitton, D. C. C. Smith, D. E. Steere, S. R. Stelfox, J.<br />
Chem. Soc. 1963, 2209-2216.� C. L. Kirkemo, J. D. White, J. Org. Chem. 1985, 50, 1316-1319.<br />
41<br />
2<br />
Et<br />
OTBS<br />
1<br />
OTBS
Noxa/Mcl-1 balance decides <strong>the</strong> effect <strong>of</strong> <strong>the</strong> proteasome<br />
<strong>in</strong>hibitor MG-132 <strong>in</strong> comb<strong>in</strong>ation with anticancer agents <strong>in</strong><br />
pancreatic cancer cell l<strong>in</strong>es<br />
Katr<strong>in</strong> Naumann a , Kathr<strong>in</strong> Schmich a , Christoph Jaeger a , Felix Kratz b , Irmgard Merfort a<br />
a Department <strong>of</strong> Pharmaceutical Biology and Biotechnology, University <strong>of</strong> Freiburg, Stefan-<br />
Meier-Str. 19, D-79104 Freiburg, Germany<br />
b Tumor Biology Center, Cl<strong>in</strong>ical Research, Breisacher Str. 117, D-79106 Freiburg, Germany<br />
Abstract<br />
Pancreatic cancer progresses aggressively and due to chemoresistance responds poorly to<br />
chemo<strong>the</strong>rapy. Thus, <strong>the</strong>re is an urgent need to understand <strong>the</strong> mechanisms <strong>of</strong> cancer cell<br />
resistance <strong>in</strong> order to generate effective strategies to circumvent <strong>in</strong>tr<strong>in</strong>sic chemoresistance <strong>in</strong><br />
this tumor <strong>in</strong>dication. In this work, three pancreatic cancer cell l<strong>in</strong>es, MIA PaCa-2, MDA<br />
Panc-3, and AsPC-1, were treated with <strong>the</strong> proteasome <strong>in</strong>hibitor MG-132 toge<strong>the</strong>r with<br />
campto<strong>the</strong>c<strong>in</strong>, doxorubic<strong>in</strong> or paclitaxel. The comb<strong>in</strong>ation <strong>of</strong> MG-132 and campto<strong>the</strong>c<strong>in</strong> <strong>in</strong> a<br />
ratio <strong>of</strong> 5:1 gave <strong>the</strong> most promis<strong>in</strong>g results and enhanced cytotoxicity compared to <strong>the</strong> s<strong>in</strong>gle<br />
compounds <strong>in</strong> MIA PaCa-2 cells. The <strong>in</strong>crease is shown to be due to an enhanced caspase-3<br />
activity result<strong>in</strong>g <strong>in</strong> apoptosis. Moreover, this comb<strong>in</strong>ation upregulated <strong>the</strong> levels <strong>of</strong> <strong>the</strong> pro-<br />
apoptotic prote<strong>in</strong> Noxa and reduced <strong>the</strong> levels <strong>of</strong> <strong>the</strong> anti-apoptotic prote<strong>in</strong> Mcl-1. In contrast,<br />
<strong>the</strong> comb<strong>in</strong>ation <strong>of</strong> MG-132 with doxorubic<strong>in</strong> only resulted <strong>in</strong> an <strong>in</strong>creased cytotoxic, but also<br />
<strong>in</strong> a decreased apoptotic effect. The lack <strong>of</strong> <strong>the</strong> enhanced apoptosis <strong>in</strong>duction could be<br />
correlated with high levels <strong>of</strong> Mcl-1 <strong>in</strong> response to <strong>the</strong> comb<strong>in</strong>ed treatment with MG-132 and<br />
doxorubic<strong>in</strong>.<br />
Thus, <strong>the</strong> results <strong>in</strong>dicate that regulation <strong>of</strong> <strong>the</strong> anti- and pro-apoptotic Bcl-2 family members<br />
Noxa and Mcl-1 predicts <strong>the</strong> effectiveness <strong>of</strong> <strong>the</strong> comb<strong>in</strong>ation <strong>of</strong> MG-132 with different<br />
anticancer agents on apoptosis <strong>in</strong>duction <strong>in</strong> pancreatic cancer cells.<br />
42
Incorporation <strong>of</strong> <strong>the</strong> H + -ATPsynthase from E. coli <strong>in</strong>to a planar lipid bilayer and<br />
observation <strong>by</strong> spFRET<br />
P. Oswald * and P.Gräber *<br />
* Albert-Ludwigs-University <strong>of</strong> Freiburg, Department <strong>of</strong> Physical Chemistry, Albertstr. 23a,<br />
79104 Freiburg, Germany<br />
peter.oswald@physchem.uni-freiburg.de<br />
H + -ATPsynthases catalyze <strong>the</strong> syn<strong>the</strong>sis <strong>of</strong> ATP from ADP and <strong>in</strong>organic phosphate <strong>by</strong> a<br />
rotational mechanism. Transmembrane proton translocation leads to a rotational movement <strong>of</strong><br />
<strong>the</strong> <strong>in</strong>tramembrane c10-subunits and this movement <strong>in</strong>duces conformational changes at <strong>the</strong><br />
catalytic sites which f<strong>in</strong>ally lead to ATP syn<strong>the</strong>sis. This rotation has been observed <strong>by</strong> s<strong>in</strong>gle<br />
pair Fluorescence Resonance Energy Transfer (spFRET) us<strong>in</strong>g double labeled enzymes, which<br />
were <strong>in</strong>tegrated <strong>in</strong>to <strong>the</strong> membrane <strong>of</strong> liposomes [1]. Catalysis was <strong>in</strong>itiated <strong>by</strong> an acid-based<br />
transition which generates a transmembrane electrochemical potential difference <strong>of</strong> protons. In<br />
this work we try to <strong>in</strong>corporate a double labeled H + -ATPsynthase <strong>in</strong>to a planar lipid bilayer<br />
and we ask whe<strong>the</strong>r it is possible to <strong>in</strong>itiate rotation <strong>by</strong> apply<strong>in</strong>g an electric potential<br />
difference across this bilayer. First <strong>the</strong> double labeled enzyme was reconstituted <strong>in</strong>to<br />
liposomes which conta<strong>in</strong> nystat<strong>in</strong> and ergosterol. Fusion <strong>of</strong> <strong>the</strong> liposome with <strong>the</strong> bilayer can<br />
be observed <strong>by</strong> a current which was due <strong>the</strong> formation <strong>of</strong> K + -ion channels <strong>by</strong> nystat<strong>in</strong> and<br />
ergosterol <strong>in</strong> <strong>the</strong> bilayer.<br />
[1] Diez M., Zimmermann B., Börsch M., Schwe<strong>in</strong>berger E., Steigmiller S., Reuter R.,<br />
Felekyan S., Kudryavtsev V., Seidel C., Gräber P., Proton-powered subunit rotation <strong>in</strong> s<strong>in</strong>gle<br />
membrane-bound F0F1-ATP synthase, Nat. Struct. Mol. Biol., 11, 135-141, (2004).<br />
43
Chas<strong>in</strong>g electrons – Time-resolved spectroscopy on blue-light<br />
sensitive flavoprote<strong>in</strong>s<br />
Bernd Paulus, Erik Schleicher and Stefan Weber<br />
Albert‐Ludwigs‐Universität Freiburg, Institut für Physikalische Chemie<br />
Albertstraße 21, 79104 Freiburg / Germany<br />
Prote<strong>in</strong>s <strong>of</strong> <strong>the</strong> photolyase/cryptochrome‐family use blue light to perform important tasks, such as<br />
entra<strong>in</strong>ment <strong>of</strong> circadian rhythms, DNA repair, regulation <strong>of</strong> plant growth and possibly<br />
magnetoreception <strong>in</strong> migratory birds [1‐3]. To harvest <strong>the</strong> light and use it for prote<strong>in</strong> function, <strong>the</strong>y<br />
carry a flav<strong>in</strong> c<strong>of</strong>actor, e.g. flav<strong>in</strong> aden<strong>in</strong>e d<strong>in</strong>ucleotide (FAD). The c<strong>of</strong>actor has to be <strong>in</strong> <strong>the</strong> proper<br />
redox state. This is obta<strong>in</strong>ed via photoactivation, a process <strong>in</strong>volv<strong>in</strong>g <strong>the</strong> transient formation <strong>of</strong><br />
radical pairs [4]. Us<strong>in</strong>g time‐resolved optical and <strong>EPR</strong>‐spectroscopy we study <strong>the</strong> dynamics <strong>of</strong> this<br />
process as well as <strong>the</strong> <strong>in</strong>fluence <strong>of</strong> <strong>the</strong> surround<strong>in</strong>g prote<strong>in</strong> environment <strong>in</strong> different prote<strong>in</strong>s <strong>of</strong> <strong>the</strong><br />
photolyase/cryptochrome‐family.<br />
The work presented has been performed <strong>in</strong> collaboration with<br />
K. Hitomi and E.D. Getz<strong>of</strong>f (Scribbs Research Institute, La Jolla, CA), P.J. Hore, A. Rob<strong>in</strong>son, K. Henbest,<br />
K. Maeda and C. Timmel (Oxford University), and A.R. Marion and J.R. Norris (University <strong>of</strong> Chigaco).<br />
[1] T. Biskup, E. Schleicher, A. Okafuji, G. L<strong>in</strong>k, K. Hitomi, E.D. Getz<strong>of</strong>f, S. Weber, Angew. Chem. Int.<br />
Ed. 48 (2009) 404‐407.<br />
[2] S. Weber, T. Biskup, A. Okafuji, A.R. Mar<strong>in</strong>o, T. Berthold, G. L<strong>in</strong>k, K. Hitomi, E.D. Getz<strong>of</strong>f, E.<br />
Schleicher, J.R. Norris, J. Phys. Chem. B 114 (2010) 14745‐14754.<br />
[3] K. Henbest, K. Maeda, P.J. Hore, M. Joshi, A. Bacher, R. Bittl, S. Weber, C. Timmel, E. Schleicher,<br />
Proc. Natl. Acad. Sci. USA 105 (2008) 14395–14399.<br />
[4] S. Weber, Biochim. Biophys. Acta 1707 (2005) 1–23.<br />
44
The Deslongchamps Reaction as an Access to <strong>the</strong><br />
Dihydroagar<strong>of</strong>uran Sesquiterpenoids<br />
Denis Petrović and Re<strong>in</strong>hard Brückner*<br />
Institut für Organische Chemie und Biochemie, Albert-Ludwigs-Universität, Albertstr. 21,<br />
79104 Freiburg, Germany; E-mail: denispetrovic@gmx.de<br />
The 1300 plants <strong>of</strong> <strong>the</strong> Celastraceae family 1 are <strong>in</strong>digenous to <strong>the</strong> tropical and subtropical<br />
regions <strong>of</strong> <strong>the</strong> world. Their biological effects are noteworthy and can be traced back to a<br />
number <strong>of</strong> unusually highly oxygenated sesquiterpenoids. 2 Two <strong>of</strong> <strong>the</strong>m are <strong>the</strong><br />
dihydroagar<strong>of</strong>urans 1 and 2.<br />
Consider<strong>in</strong>g 1 and 2 as syn<strong>the</strong>tic targets we plan to derive <strong>the</strong>m from <strong>the</strong> cis-configured<br />
hexal<strong>in</strong>dione 6 via <strong>the</strong> densely functionalized octal<strong>in</strong>dione 3. Hexal<strong>in</strong>dione 6 should be<br />
accessible <strong>by</strong> a DESLONGCHAMPS annulation 3 <strong>of</strong> <strong>the</strong> acceptor-substituted enolate 5 � which<br />
results from an <strong>in</strong>-situ deprotonation <strong>of</strong> a so-called NAZAROV reagent 4 � to <strong>the</strong> estersubstituted<br />
cyclohexenone 4. The latter is an activated qu<strong>in</strong>onemonoketal 4. Engag<strong>in</strong>g this<br />
particular functionality <strong>in</strong> DESLONGCHAMPS annulations represents one <strong>of</strong> several extensions<br />
<strong>of</strong> <strong>the</strong> scope <strong>of</strong> this versatile reaction. 5 In <strong>the</strong> case at hand, choos<strong>in</strong>g 4 furnishes allows to<br />
obta<strong>in</strong> a hexal<strong>in</strong>dione (6) ra<strong>the</strong>r than an octal<strong>in</strong>dione, which would result o<strong>the</strong>rwise. The<br />
latter, <strong>of</strong> course, would lack one <strong>of</strong> <strong>the</strong> C=C bonds and <strong>the</strong>re<strong>by</strong> a site poised for fur<strong>the</strong>r<br />
elaboration.<br />
1 R. Brün<strong>in</strong>g and H. Wagner, Phytochemistry 1978, 17, 1821-1858.<br />
2 A. C. Spivey, M. Weston, S. Woodhead, Chem. Soc. Rev. 2002, 31, 43-59.<br />
3 P.-Y. Caron.; P. Deslongchamps Org. Lett.2010, 12, 508-511 and literature cited <strong>the</strong>re<strong>in</strong>.<br />
4 I. N. Nazarov and S. I. Zauyalou. Zh. Obshch. Khim. 1953, 23, 1703-1706.<br />
5 J. F. Lavalée, P. Deslongchamps, Tetrahedron Lett. 1988, 29, 5117-5118. For our earliest contribution see T.<br />
Tricotet, R. Brückner, Eur. J. Org. Chem. 2007, 1069-1074.<br />
45
Inter- and Intramolecular Hydroacylation <strong>of</strong> Alkenes<br />
employ<strong>in</strong>g a Novel Bifunctionnal Catalyst System<br />
Damien Regent, Nicolas Vautravers, Bernhard Breit*<br />
Institut für Organische Chemie und Biochemie, Freiburg Institute for Advanced Study (FRIAS),<br />
Albert-Ludwigs-Universität Freiburg, Albertstr. 21, 79104 Freiburg, Germany<br />
e-mail: bernhard.breit@chemie.uni-freiburg.de<br />
The hydroacylation <strong>of</strong> alkenes is an attractive atom-economic [1] syn<strong>the</strong>tic method for <strong>the</strong> preparation <strong>of</strong><br />
ketones from aldehydes via transition metal-catalyzed C-H bond activation. [2] One drawback <strong>of</strong> this<br />
reaction is <strong>the</strong> decarbonylation <strong>of</strong> <strong>the</strong> <strong>in</strong>termediate acyl-metal hydride complex (Scheme 1), which<br />
lead to a side product. Many methods have thus been developed <strong>in</strong> order to suppress it. [3] Inspired <strong>by</strong><br />
<strong>the</strong> work <strong>of</strong> Jun et al [4] , we optimized a new bifunctional ligand L comb<strong>in</strong><strong>in</strong>g an am<strong>in</strong>opicol<strong>in</strong>e moiety<br />
and a phosph<strong>in</strong>e function (Scheme 1). [5]<br />
R 1<br />
O<br />
+ H R2<br />
metal b<strong>in</strong>d<strong>in</strong>g<br />
substrate b<strong>in</strong>d<strong>in</strong>g<br />
The am<strong>in</strong>opicol<strong>in</strong>e moiety would act as a reversibly<br />
bound direct<strong>in</strong>g group allow<strong>in</strong>g for facile C-H activation<br />
while simultaneously prevent<strong>in</strong>g <strong>the</strong> undesired<br />
decarbonylation through formation <strong>of</strong> an aldim<strong>in</strong>e.<br />
Integration <strong>of</strong> <strong>the</strong> phosph<strong>in</strong>e function would not only<br />
form an active hydroacylation catalyst, but also enhance<br />
<strong>the</strong> b<strong>in</strong>d<strong>in</strong>g constant <strong>of</strong> <strong>the</strong> direct<strong>in</strong>g group to <strong>the</strong><br />
catalytically active rhodium center.<br />
We applied successfully our bifunctionnal ligand to <strong>the</strong> <strong>in</strong>ter- and <strong>in</strong>tramolecular hydroacylation <strong>of</strong><br />
alkenes [5] , with yields up to 83% <strong>in</strong> <strong>the</strong> <strong>in</strong>termolecular hydroacylation <strong>of</strong> substituted benzaldehydes 1<br />
and diverses alkenes 2 (Scheme 2).<br />
Intermolecular Hydroacylation <strong>of</strong> Alkenes<br />
O<br />
[Rh(COD) 2]BF4 (5 mol%)<br />
FGR1 H<br />
+ R FG<br />
2<br />
ligand L (5 mol%)<br />
toluene (c = 1.1 M)<br />
150 °C, 1 h<br />
1<br />
FG R1<br />
2 3<br />
Scheme 2<br />
9 exemples<br />
up to 93% yield<br />
Similarly, excellent yields up to 98% were also reached <strong>in</strong> <strong>the</strong> <strong>in</strong>tramolecular reaction <strong>of</strong> substituted ov<strong>in</strong>ylbenzaldehyde<br />
4 yield<strong>in</strong>g valuable 1-<strong>in</strong>danone derivatives 5 (Scheme 3).<br />
References:<br />
Ph 2P<br />
ligand L<br />
N N<br />
Rh<br />
H R2 R 1<br />
#<br />
R 1<br />
Intramolecular Hydroacylation <strong>of</strong> Alkenes<br />
[Rh(COD) 2]BF4 (5 mol%)<br />
FGR H<br />
ligand L (5 mol%)<br />
O<br />
toluene (c = 1.1 M)<br />
150 °C, 1 h<br />
O<br />
Scheme 1<br />
R 2<br />
4 5<br />
Scheme 3<br />
10 exemples<br />
up to 98% yield<br />
[1] B. M. Trost, Science 1991, 254, 1471-1477; B. M. Trost, Acc. Chem. Res. 2002, 35, 695-705.<br />
[2] M. C. Willis, Chem. Rev. 2010, 110, 725-748.<br />
[3] T. B. Marder, D. C. Roe, D. Milste<strong>in</strong>, Organometallics 1988, 7, 1451-1453; T. Kondo, N. Hiraishi, Y.<br />
Morisaki, K. Wada, Y. Watanabe, T. Mitsudo, Organometallics 1998, 17, 2131-2134; A. A. Roy, C. P.<br />
Lenges, M. Brookhart, J. Am. Chem. Soc. 2007, 129, 2082-2093.<br />
[4] Y. J. Park, J.-W. Park, C.-H. Jun, Acc. Chem. Res. 2008, 41, 222-234; C.-H. Jun, E.-A. Jo, Eur. J.<br />
Org. Chem. 2006, 2504-2507<br />
[5] N. R. Vautravers, D. D. Regent, B. Breit, Chem. Comm. 2011, ASAP<br />
46<br />
FG R<br />
O<br />
O<br />
R 2 FG
SYNTHESIS AND TESTING OF 2,7-NAPHTHYRIDONES AS A NOVEL<br />
CLASS OF SIRTUIN INHIBITORS<br />
Schiedel, M. 1 , Schemies, J. 1 , Jung, M. 1<br />
1 Institute <strong>of</strong> Pharmaceutical Sciences, Albert-Ludwigs-Universität Freiburg,<br />
Germany, http://jungm.de<br />
Sirtu<strong>in</strong>s represent a specific NAD + -dependent class <strong>of</strong> histone deacetylases<br />
(HDACs). By us<strong>in</strong>g NAD + as a c<strong>of</strong>actor, <strong>the</strong>se enzymes cleave <strong>of</strong>f <strong>the</strong> acetyl<br />
groups from <strong>the</strong> ε-am<strong>in</strong>o group <strong>of</strong> lys<strong>in</strong>es <strong>in</strong> histones and o<strong>the</strong>r prote<strong>in</strong>s, e.g. p53,<br />
FOXO prote<strong>in</strong>s, p300 or HIV tat. The human family <strong>of</strong> sirtu<strong>in</strong>s consists <strong>of</strong> seven<br />
members, which are distributed to different cell compartments and <strong>in</strong>volved <strong>in</strong> <strong>the</strong><br />
regulation <strong>of</strong> various physiological processes like apoptosis, cell differentiation,<br />
metabolism, DNA recomb<strong>in</strong>ation and HIV tat transactivation. Thus, sirtu<strong>in</strong><br />
<strong>in</strong>hibitors are <strong>in</strong>terest<strong>in</strong>g potential drugs for drug discovery. [1]<br />
Besides <strong>the</strong> deacetylated lys<strong>in</strong>es, <strong>the</strong> products <strong>of</strong> <strong>the</strong> catalytic reaction are 2`-Oacetyl-adenos<strong>in</strong>e<br />
diphosphate ribose and nicot<strong>in</strong>amide, which itself acts as a<br />
physiological sirtu<strong>in</strong> <strong>in</strong>hibitor. Thus, we chose nicot<strong>in</strong>amide as a lead structure for<br />
syn<strong>the</strong>sis <strong>of</strong> new potential sirtu<strong>in</strong> <strong>in</strong>hibitors.<br />
Syn<strong>the</strong>sis <strong>of</strong> 2,7-naphthyridones [2]<br />
Quaternary N 1 -alkylnicot<strong>in</strong>amides were treated with an alkal<strong>in</strong>e solution <strong>of</strong><br />
methylketones. After a nucleophilic addition <strong>of</strong> <strong>the</strong> deprotonated methylketone<br />
compound to <strong>the</strong> pyrid<strong>in</strong>ium salts <strong>in</strong> <strong>the</strong> 4-position, <strong>the</strong> 2,7-naphthyridones were<br />
formed <strong>by</strong> dehydrogenation. As an oxidant formic acid was used. The highly<br />
fluorescent products were ei<strong>the</strong>r isolated as a dipolar ion out <strong>of</strong> <strong>the</strong> aqueous layer<br />
or its hydrochloride out <strong>of</strong> an ethanolic phase.<br />
N +<br />
R<br />
O<br />
NH 2<br />
+<br />
C<br />
H 3<br />
O<br />
R'<br />
KOH<br />
0°C<br />
O<br />
R'<br />
N<br />
R<br />
O<br />
NH 2<br />
N<br />
HCOOH<br />
100°C OH - H +<br />
In-vitro test<strong>in</strong>g<br />
To discover new sirtu<strong>in</strong> <strong>in</strong>hibitors among <strong>the</strong> syn<strong>the</strong>sized 2,7-naphthyridones, we<br />
have used a screen<strong>in</strong>g assay, which is based on fluorescence polarisation. [3] As <strong>the</strong><br />
most potent derivative, we could identify 2-benzyl-8-hydroxy-6-(naphthalene-2yl)-2,7-naphthyrid<strong>in</strong>-2-iumchloride<br />
(structure shown below) with 52% <strong>in</strong>hibition <strong>of</strong><br />
Sirt2 at a concentration <strong>of</strong> 25 µM.<br />
N +<br />
Cl<br />
References:<br />
[1] Trapp J., Jung M. (2006) Curr Drug Targets, 7, 1553-1560<br />
[2] Palfreyman M.N., Wooldridge K.R.H. (1974) J. Chem. Soc., Perk<strong>in</strong> Trans, 1,<br />
57-61<br />
[3] Milne J.C., Lambert P.D. et al. (2007) Nature, 450, 712-716<br />
47<br />
OH<br />
N<br />
N +<br />
R<br />
R'<br />
+ H +<br />
N +<br />
R<br />
R'<br />
N<br />
O
Quantenchemische und Matrixisolationsuntersuchungen<br />
zu Eisentetrafluorid<br />
T. Schlöder, S. Riedel*<br />
Institut für Anorganische und Analytische Chemie, Albert-Ludwigs Universität Freiburg, Albertstr. 21, D-79104<br />
Freiburg i. Br. (Germany)<br />
tobias.schloeder@cpg.uni-freiburg.de, sriedel@psichem.de<br />
Eisendi- und trifluorid s<strong>in</strong>d stabile Verb<strong>in</strong>dungen, welche sowohl experimentell 1, 2 als auch<br />
<strong>the</strong>oretisch 3, 4 gut untersucht s<strong>in</strong>d. Das nächst höhere Eisenfluorid FeF4 h<strong>in</strong>gegen ist schwerer<br />
zu greifen. E<strong>in</strong>e neue IR Bande <strong>in</strong> e<strong>in</strong>em Matrixisolationsexperiment wurde diesem Molekül<br />
zugeordnet, nachdem FeF3 mit dem Fluoratomdonor CeF4 zur Reaktion gebracht wurde;<br />
jedoch konnten im Massenspektrum ke<strong>in</strong> entsprechendes Signal gefunden werden 5 .<br />
Hier möchten wir über quantenchemische Rechnungen zu Strukturen und<br />
Schw<strong>in</strong>gungsspektren der Eisenfluoride auf bis zu CCSD(T) Niveau berichten. Weiterh<strong>in</strong><br />
wurde e<strong>in</strong>e <strong>the</strong>rmochemische Stabilitätsanalyse unternommen, bei der die Elim<strong>in</strong>ation von<br />
Fluoratomen und -molekülen (uni- und bimolekular) <strong>in</strong> Betracht gezogen wurde. Nachdem<br />
das Ergebnis zeigte, dass FeF4 <strong>in</strong> der Gasphase stabil ist, wurden Matrixislationsexperimente<br />
zur Syn<strong>the</strong>se durchgeführt. Dabei wurde e<strong>in</strong>e Mischung aus CeF4 und FeF3 erhitzt und die<br />
gasförmigen Spezies über der Probe zusammen mit den Wirtsgasen Neon oder Argon<br />
e<strong>in</strong>gefroren. Die IR Spektren zeigen zusätzlich zu den Banden von FeF2 und FeF3 e<strong>in</strong>e weitere<br />
Absorption, welche basierend auf den quantenchemischen Rechnungen dem Eisentetrafluorid<br />
zugeordnet werden kann.<br />
1. Vogt, N., Journal <strong>of</strong> Molecular Structure 2001, 570, (1-3), 189-95.<br />
2. Hargittai, M., The Journal <strong>of</strong> Chemical Physics 2005, 123, (19), 196101-2.<br />
3. Solomonik, V. G.; Stanton, J. F.; Boggs, J. E., The Journal <strong>of</strong> Chemical Physics 2005,<br />
122, (9), 094322-12.<br />
4. Solomonik, V. G.; Stanton, J. F.; Boggs, J. E., The Journal <strong>of</strong> Chemical Physics 2008,<br />
128, (24), 244104-9.<br />
5. Rau, J. V.; Nunziante, C. S.; Chil<strong>in</strong>garov, N. S.; Leskiv, M. S.; Balducci, G.; Sidorov,<br />
L. N., Inorg. Chem. Commun. 2003, 6, (6), 643-5.<br />
48
Hochkonzentrierte, fließfähige Kolloiddispersionen<br />
Schneider, René, rene.schneider@physchem.uni-freiburg.de<br />
Wiemann, Malte, malte.wiemann@uni-freiburg.de<br />
Bartsch, Eckhard, eckhard.bartsch@physchem.uni-freiburg.de<br />
Hochkonzentrierte, fließfähige kolloidale Dispersionen besitzen e<strong>in</strong> breites Anwendungs<br />
spektrum <strong>in</strong> Industrie und Alltag. Es konnte an e<strong>in</strong>em Modellsystem harter Kugeln,<br />
bestehend aus Polystyrol(PS)Mikrogelkolloiden <strong>in</strong> e<strong>in</strong>em organischen Lösemittel,<br />
mittels Lichtstreuung gezeigt werden, dass durch Zugabe von freiem, nicht<br />
adsorbierendem Polymer der Bereich der Fließfähigkeit zu höherem Volumenanteil der<br />
Kolloide verschoben werden kann [1,2]. Die Wahl des Modellsystems war durch die<br />
Anforderung der Lichtstreuung bestimmt, dass die hochkonzentrierten Dispersionen<br />
transparent se<strong>in</strong> müssen (Isorefraktivität, d.h. Brechungs<strong>in</strong>dex Kolloid ≅ Brechungs<br />
<strong>in</strong>dex Medium). Um zu erkennen, ob die erhaltenen Ergebnisse allgeme<strong>in</strong> gelten, wurde<br />
e<strong>in</strong> neues System syn<strong>the</strong>tisiert, welches dem Modell harter Kugeln näher kommt. Des<br />
weiteren stellte sich die Frage, ob dieser Effekt auch für die <strong>in</strong> der Anwendung wichtige<br />
Materialklasse der wässrigen Polymerdispersionen auftritt und genutzt werden kann.<br />
Daher war die Entwicklung e<strong>in</strong>es weiteren Modellsystems erforderlich, das<br />
Isorefraktivität <strong>in</strong> Wasser gewährleistet. Dazu wurden selbst syn<strong>the</strong>tisierte Per<br />
fluoroacrylatPartikel charakterisiert, die über e<strong>in</strong>e Schicht aus Polyethylenoxidketten <strong>in</strong><br />
Wasser sterisch stabilisiert werden.<br />
L<strong>in</strong>ks: Phasendiagramm<br />
e<strong>in</strong>er PS-Mikrogel/PS-<br />
Polymer-Mischung [2]. C P<br />
gibt die Polymerkonzentration<br />
an, ϕ den<br />
Kolloidvolumenbruch. fluid<br />
= fließfähige Dispersionen.<br />
Rechts: Hochkonzentrierte<br />
Fluoracrylat-Dispersion <strong>in</strong><br />
Wasser (n D =1.332; rechts)<br />
ϕ<br />
und <strong>in</strong> Wasser/Glyzer<strong>in</strong>-<br />
Mischung (n 1.37; l<strong>in</strong>ks)<br />
D<br />
Referenzen:<br />
[1] Eckert, T. und Bartsch, Phys. Rev. Lett. 89, 1257 (2002)<br />
[2] Eckert, T. und Bartsch, J. Phys.: Condens. Matter 16, S4937 (2004)<br />
49
Albert-Ludwigs-Universität Freiburg<br />
Specific siRNA delivery to Alveolar Rhabdomyosarcoma <strong>by</strong><br />
co-modified liposomes<br />
Doris Sehi, Reg<strong>in</strong>e Süss<br />
Department <strong>of</strong> Pharmaceutical Technology and Biopharmacy, University <strong>of</strong> Freiburg,<br />
Germany<br />
Tel: +49 761 203 6329, email: doris.sehi@pharmazie.uni-freiburg.de<br />
Rhabdomyosarcoma is <strong>the</strong> most frequent malignant s<strong>of</strong>t tissue tumor <strong>in</strong><br />
childhood. Especially <strong>the</strong> more aggressive subtype named Alveolar RMS<br />
(ARMS) is highly resistant to all forms <strong>of</strong> <strong>the</strong>rapeutical treatment that are<br />
currently available [1]. RNA <strong>in</strong>terference <strong>of</strong>fers a new promis<strong>in</strong>g approach for<br />
<strong>the</strong> treatment <strong>of</strong> ARMS but lacks an effective way to deliver <strong>the</strong> siRNA <strong>in</strong>to<br />
<strong>the</strong> cancer cells. This study aims to develop a liposomal formulation which is<br />
able to protect <strong>the</strong> siRNA from degradation on its way to <strong>the</strong> tumor and to<br />
achieve specific <strong>in</strong>teraction and successful uptake when hav<strong>in</strong>g arrived <strong>the</strong>re.<br />
Therefore, surface-modified SPC/Cholesterol liposomes were designed<br />
carry<strong>in</strong>g <strong>the</strong> RGD peptide to mediate selective <strong>in</strong>teraction with<br />
rhabdomyosarcoma cells and <strong>the</strong> TAT peptide to promote effective uptake. In<br />
vitro experiments with <strong>the</strong> co-modified liposomes show promis<strong>in</strong>g results.<br />
1. Parham, D., Ellison, D., (2006) Rhabdomyosarcomas <strong>in</strong> Adults and Children, An<br />
Update. Arch Phathol Lab Med 130, 1454-1465<br />
2. Ko, YT. et al (2009) Gene delivery <strong>in</strong>to ischemic myocardium <strong>by</strong> double-targeted<br />
lipoplexes with anti-myos<strong>in</strong> antibody and TAT-peptide Gene Therapy 16, 52-59<br />
50
CoRS Curator: A Text Curation and Annotation Tool<br />
Senger C*, Erxleben A, Grün<strong>in</strong>g BA, Gün<strong>the</strong>r S<br />
Pharmaceutical Bio<strong>in</strong>formatics,<br />
Institute <strong>of</strong> Pharmaceutical Sciences, University <strong>of</strong> Freiburg, Germany<br />
*e-mail: christian.senger[at]pharmazie.uni-freiburg.de<br />
Introduction: Compound related <strong>in</strong>formation is usually published <strong>in</strong> text-form, which is not very<br />
well structured. Fur<strong>the</strong>rmore, associated <strong>in</strong>formation is usually scattered over a great number <strong>of</strong><br />
articles. The Compound Research System (CoRS) is a DFG supported project for establish<strong>in</strong>g an<br />
automated molecule research system and associated modules to extract and connect relevant<br />
<strong>in</strong>formation. CoRS Curator is a s<strong>of</strong>tware tool for facilitat<strong>in</strong>g selection, care, and presentation <strong>of</strong><br />
objects <strong>in</strong> texts and for transferr<strong>in</strong>g this <strong>in</strong>formation to databases <strong>in</strong> a standardised form.<br />
Methods: CoRS Curator uses publicly available text-m<strong>in</strong><strong>in</strong>g and <strong>in</strong>formation retrieval s<strong>of</strong>tware to<br />
preselect texts and highlights found entities. Users are enabled to annotate pieces <strong>of</strong> texts with<br />
predef<strong>in</strong>ed entities via text-highlight<strong>in</strong>g, allow<strong>in</strong>g for a high degree <strong>of</strong> traceability to <strong>the</strong><br />
<strong>in</strong>formation's orig<strong>in</strong>.<br />
In a test case, researchers used <strong>the</strong> tool to collect <strong>in</strong>formation on a set <strong>of</strong> more than 6000 abstracts<br />
related to <strong>the</strong> compound class <strong>of</strong> sesquiterpene lactones, enabl<strong>in</strong>g an iterative fur<strong>the</strong>r development<br />
and evaluation <strong>of</strong> <strong>the</strong> s<strong>of</strong>tware <strong>in</strong> terms <strong>of</strong> <strong>in</strong>terrater agreement (agreement <strong>of</strong> curators on<br />
annotations <strong>of</strong> text pieces), curation speed, and annotation outcome.<br />
Results: The curators needed ~90 man-hours to curate 6650 abstracts for 530 compounds. About<br />
78% <strong>of</strong> <strong>the</strong> abstracts conta<strong>in</strong>ed redundant <strong>in</strong>formation. Interrater agreement was 72% on a test set.<br />
More than 2800 pieces <strong>of</strong> text were annotated with entities (e.g. “source organism“, “assay“, or<br />
“<strong>the</strong>rapeutic action“) provid<strong>in</strong>g important <strong>in</strong>formation <strong>of</strong> <strong>the</strong> analysed compounds.<br />
51
Tag der Forschung - 05.07.2011<br />
Improv<strong>in</strong>g transfection efficiencies <strong>of</strong> lipid-mediated gene delivery<br />
A. Ste<strong>in</strong>bach, R. Süss<br />
- Influence <strong>of</strong> <strong>the</strong> lipid/DNA complex preparation technique -<br />
Institute <strong>of</strong> Pharmaceutical Sciences, Department <strong>of</strong> Pharmaceutical Technology and Biopharmacy,<br />
Sonnenstr. 5, D-79104 Freiburg;<br />
annette.ste<strong>in</strong>bach@pharmazie.uni-freiburg.de<br />
Lipid/DNA complexes are commonly used non-viral gene delivery systems bear<strong>in</strong>g great<br />
potential due to <strong>the</strong>ir low host-immunogenicity. To date <strong>the</strong>ir cl<strong>in</strong>ical use is restricted <strong>by</strong><br />
limited transfection efficiencies caused <strong>by</strong> cellular barriers [1].<br />
To expla<strong>in</strong> and improve low transfection efficiencies <strong>of</strong> DC 30/DNA complexes, different<br />
stages <strong>of</strong> <strong>the</strong> transfection process were addressed as potential transfection barriers <strong>in</strong> <strong>the</strong><br />
two previously characterized cellular models A-10 SMC and MDCK II [2]. Particular<br />
attention was paid to complex <strong>in</strong>ternalisation, <strong>in</strong>tracellular process<strong>in</strong>g, nuclear uptake <strong>of</strong><br />
complex-released pDNA as well as transgene expression.<br />
DC 30/DNA complexes derived from two preparation protocols <strong>in</strong>volv<strong>in</strong>g treatment <strong>of</strong><br />
starter liposomes with a sonication tip before complexation with pDNA compared to an<br />
untreated preparation were <strong>in</strong>cluded <strong>in</strong> our studies.<br />
Transfection deficiency <strong>of</strong> DC 30/DNA complexes <strong>in</strong> MDCK II cells could not be related to<br />
one specific step <strong>in</strong> <strong>the</strong> cellular transfection process. The entirety and <strong>in</strong>terplay <strong>of</strong> all<br />
cellular processes from <strong>in</strong>ternalisation to transgene expression need to be considered to<br />
contribute to <strong>the</strong> low transfection efficiency <strong>in</strong> some cellular systems. The preparation<br />
protocol <strong>of</strong> DC 30/DNA complexes was found to have an <strong>in</strong>fluence on cellular uptake and<br />
cell l<strong>in</strong>e dependently on <strong>the</strong> transfection efficiency, but no impact on <strong>in</strong>tracellular<br />
process<strong>in</strong>g or nuclear uptake <strong>of</strong> complex released pDNA could be observed.<br />
References:<br />
1. Hama et al., Quantitative and mechanism-based <strong>in</strong>vestigation <strong>of</strong> post-nuclear delivery events between<br />
adenovirus and lipoplex. Nucleic Acids Research, 2007. 35(5): p. 1533-1543<br />
2. Schneider S., Dissertation University <strong>of</strong> Freiburg, 2010<br />
52
MEGABLOCKS IN THE RIES IMPACT CRATER, GERMANY: NEW DISCOVERIES AND<br />
STATISTICAL ANALYSIS OF DISTRIBUTION AND LITHOLOGIES.<br />
S. Sturm1, W. Willmes1, H. Hies<strong>in</strong>ger1, T. Kenkmann2 and G. Pösges³, 1Institut für Planetologie, Westfälische<br />
Wilhelms-Universität Münster, Germany (s.sturm@uni-muenster.de), 2 Institut für Geowissenschaften,<br />
Universität Freiburg, Germany, 3Rieskrater-Museum Nördl<strong>in</strong>gen, Germany.<br />
The Ries crater is a well-preserved complex crater, 25 km <strong>in</strong> diameter, located <strong>in</strong> Bavaria, sou<strong>the</strong>rn Germany [1<br />
and references <strong>the</strong>re<strong>in</strong>]. The impact occurred <strong>in</strong> a heterogeneous target that consisted <strong>of</strong> ~600 m sedimentary<br />
cover (Triassic to Jurassic) rest<strong>in</strong>g over a crystall<strong>in</strong>e basement [2]. It consists <strong>of</strong> (i) a 10-12 km diameter <strong>in</strong>ner<br />
crater bas<strong>in</strong> filled with suevite and post-impact lake deposits, (ii) a collar zone <strong>of</strong> upturned and overturned highly<br />
faulted and shock metamorphosed material called <strong>the</strong> “<strong>in</strong>ner r<strong>in</strong>g” and (iii) a zone <strong>of</strong> large blocks (“megablock<br />
zone”) that were displaced dur<strong>in</strong>g <strong>the</strong> crater formation process [e.g., 3]. The Ries has been <strong>in</strong>tensively studied,<br />
however <strong>the</strong> megablock zone still poses questions regard<strong>in</strong>g crater formation mechanics. Here we present new<br />
data <strong>of</strong> <strong>the</strong> megablock zone us<strong>in</strong>g a comb<strong>in</strong>ed approach <strong>of</strong> remote sens<strong>in</strong>g analysis and shallow drill<strong>in</strong>gs.<br />
Formation <strong>of</strong> megablocks is caused <strong>by</strong> two processes: (i) They were ei<strong>the</strong>r thrown outwards dur<strong>in</strong>g <strong>the</strong> crater<br />
excavation stage and were deposited simultaneously with <strong>the</strong> cont<strong>in</strong>uous ejecta blanket (Bunte breccia) and/or<br />
(ii) slumped <strong>in</strong>wards dur<strong>in</strong>g <strong>the</strong> modification stage, lead<strong>in</strong>g to a complex juxtaposition <strong>of</strong> allochthonous<br />
crystall<strong>in</strong>e and allochthonous and parautochthonous sedimentary megablocks. Their abundance and distribution<br />
is a tribute to <strong>the</strong> pre-impact geology, <strong>the</strong> impact process and post-impact erosion. The megablock zone exhibits<br />
a hummocky morphology that is dom<strong>in</strong>ated <strong>by</strong> large Malmian and crystall<strong>in</strong>e megablocks easily detectable at <strong>the</strong><br />
surface [2, 4]. Mega-blocks are also present <strong>in</strong> <strong>the</strong> subsurface as <strong>the</strong>y have been buried <strong>by</strong> Suevite, Bunte Breccia<br />
or post-impact sediment deposition [5]. These subsurface megablocks have only rarely been detected so far and<br />
are not shown <strong>in</strong> <strong>the</strong> present geological map <strong>of</strong> <strong>the</strong> Ries [6].<br />
References: [1] Stöffler, D. (1977) Geologica Bavaria, 75, 443-458. [2] Pohl et al. (1977) Impact and Explosion Crater<strong>in</strong>g,<br />
Flagstaff. [3] Wünnemann, K. et al. (2005) In: Large Meteorite Impacts III, Geological Society <strong>of</strong> America, Boulder. [4]<br />
Hüttner and Schmidt-Kaler (1999) Geologica Bavarica, 104, 7-76. [5] Coll<strong>in</strong>s et al. (2008), Meteoritics and Planetary<br />
Science, 43, 12, 1955-1977. [6] Geologische Karte des Rieses 1:50000 (2005) Bayerisches Geologisches Landesamt,<br />
München.<br />
53
Overproduction <strong>of</strong> Aquifex aeolicus complex I <strong>in</strong> E. coli.<br />
M. Vranas, and T. Friedrich<br />
Institute for Organic Chemistry and Biochemistry,<br />
Albert-Ludwigs-University Freiburg, Germany<br />
The NADH:ubiqu<strong>in</strong>one oxidoreductase, also called complex I, is <strong>the</strong> ma<strong>in</strong> entrance<br />
po<strong>in</strong>t <strong>of</strong> electrons <strong>in</strong>to <strong>the</strong> respiratory cha<strong>in</strong>s. With<strong>in</strong> <strong>the</strong> complex electrons are<br />
transferred from NADH to <strong>Ubiqu<strong>in</strong>one</strong> (Q) coupled with a proton translocation across<br />
<strong>the</strong> membrane accord<strong>in</strong>g to:<br />
NADH + Q + 5H + → NAD + + QH2 + 4H +<br />
The enzyme comprises a noncovalently bound flav<strong>in</strong> mononucleotide and several ironsulfur<br />
clusters as co-factors. In Aquifex aeolicus, a hyper<strong>the</strong>rmophilic bacterium with an<br />
optimal growth temperature <strong>of</strong> 95ºC, <strong>the</strong> 13 nuo-genes, cod<strong>in</strong>g for NuoA-N subunits <strong>of</strong><br />
complex I, are organized <strong>in</strong> three different loci [1, 2].<br />
Heterologous production <strong>of</strong> A. aeolicus complex I is attempted <strong>in</strong> different Escherichia<br />
coli stra<strong>in</strong>s. In some stra<strong>in</strong>s <strong>the</strong> chromosomal nuo-operon was deleted. The deletion <strong>of</strong><br />
<strong>the</strong> 16kb nuo-operon was performed <strong>by</strong> Lambda-Red Recomb<strong>in</strong>eer<strong>in</strong>g technique, a<br />
PCR mediated gene replacement method [3, 4].<br />
Expression plasmids were constructed conta<strong>in</strong><strong>in</strong>g various groups <strong>of</strong> <strong>the</strong> A. aeolicus<br />
nuo-genes, <strong>in</strong>clud<strong>in</strong>g <strong>the</strong> entire nuo-operon from <strong>the</strong> three loci mentioned above. The<br />
stra<strong>in</strong>s were transformed with <strong>the</strong> expression plasmids and <strong>the</strong> heterologous<br />
overproduced prote<strong>in</strong>s were isolated and characterized.<br />
1. Kohlstadt, M. et al., Heterologous Production, Isolation, Characterization and Crystallization<br />
<strong>of</strong> a Soluble Fragment <strong>of</strong> <strong>the</strong> NADH:<strong>Ubiqu<strong>in</strong>one</strong> Oxidoreductase (<strong>Complex</strong> I) from Aquifex<br />
aeolicus. Biochemistry, 2008. 47, 13036–13045<br />
2. Scheide, D., R. Huber, and T. Friedrich, The proton-pump<strong>in</strong>g NADH:ubiqu<strong>in</strong>one<br />
oxidoreductase (complex I) <strong>of</strong> Aquifex aeolicus. FEBS Lett., 2002. 512, 80-4.<br />
3. Datsenko, K.A. and B.L. Wanner, One-step <strong>in</strong>activation <strong>of</strong> chromosomal genes <strong>in</strong><br />
Escherichia coli K-12 us<strong>in</strong>g PCR products. Proc. Natl. Acad. Sci., 2000. 97, 6640-5.<br />
4. Pohl, T. et al., Lambda Red-mediated mutagenesis and efficient large scale aff<strong>in</strong>ity<br />
purification <strong>of</strong> <strong>the</strong> Escherichia coli NADH:ubiqu<strong>in</strong>one oxidoreductase (complex I).<br />
Biochemistry, 2007. 46, 10694-702.<br />
54
Structural Deformations <strong>in</strong> <strong>the</strong> Central Pit <strong>of</strong> a Mart<strong>in</strong><br />
Crater as an Indicator for Impact Direction<br />
G. Wulf, M. H. Poelchau and T. Kenkmann,<br />
Institute <strong>of</strong> Geosciences – Geology, Albert-Ludwigs-University Freiburg, Germany (gerw<strong>in</strong>.wulf@geologie.uni-freiburg.de)<br />
The majority <strong>of</strong> impacts on planetary bodies occur at an oblique impact angle to <strong>the</strong> target<br />
surface because <strong>the</strong> <strong>in</strong>cidence angle follows a Gaussian probability distribution with a mean<br />
value <strong>of</strong> 45° [1]. The ejecta blanket is <strong>the</strong> most dist<strong>in</strong>ctive <strong>in</strong>dicator for <strong>the</strong> impact direction <strong>in</strong><br />
oblique impacts, show<strong>in</strong>g <strong>the</strong> formation <strong>of</strong> “forbidden” zones and “butterfly” patterns [2,3].<br />
The position <strong>of</strong> <strong>the</strong> central uplift relative to <strong>the</strong> crater center was proposed as ano<strong>the</strong>r diagnostic<br />
feature [4], but its statistical relevance could not be verified thus far [5] as <strong>the</strong> position<br />
might be caused <strong>by</strong> heterogeneous target structures [6]. Ano<strong>the</strong>r promis<strong>in</strong>g <strong>in</strong>dicator for<br />
obliquity is provided <strong>by</strong> <strong>the</strong> <strong>in</strong>ternal structure <strong>of</strong> central uplifts. Some terrestrial craters show a<br />
preferred stack<strong>in</strong>g <strong>of</strong> layered bedrock <strong>in</strong> <strong>the</strong> central uplift [7,8] with bedd<strong>in</strong>g strik<strong>in</strong>g perpendicularily<br />
to <strong>the</strong> long axis <strong>of</strong> <strong>the</strong> crater ellipse [8]. The <strong>in</strong>terpretation that this imbrication is<br />
caused <strong>by</strong> remnant horizontal momentum transferred from <strong>the</strong> impact<strong>in</strong>g projectile to <strong>the</strong> target<br />
dur<strong>in</strong>g an oblique impact [8] is supported <strong>by</strong> three-dimensional numerical simulations [9].<br />
Fur<strong>the</strong>r analysis <strong>of</strong> Martian craters [10,11] provide evidence that preferred strike orientation<br />
<strong>in</strong> <strong>the</strong> central uplift could be <strong>in</strong>dicative for an impact direction. Here we present new results <strong>of</strong><br />
<strong>the</strong> mapp<strong>in</strong>g <strong>of</strong> an unnamed Martian crater extend<strong>in</strong>g and confirm<strong>in</strong>g <strong>the</strong>se results for a central<br />
pit structure <strong>of</strong> an oblique impact crater.<br />
[1] Shoemaker, E. M. (1962) Phys. And Astron. <strong>of</strong> <strong>the</strong> Moon, p. 283-359.<br />
[2] Gault D. E. and Wedek<strong>in</strong>d J. A. (1978) Proc. Lunar Planet. Sci. Conf. 9: 3843-75.<br />
[3] Anderson, J. L. B. et al. (2004) Meteoritics & Planet. Sci., 39, 303–320.<br />
[4] Schultz, P. H. and Anderson, R. R. (1996) Geol. Soc. <strong>of</strong> Amer. Spec. Paper, 302, 397-417.<br />
[5] Ekholm, G. A. and Melosh, H. J. (2001) Geophy. Res. Let., 28, 623-626.<br />
[6] Coll<strong>in</strong>s, G. S. et al. (2008) Earth & Planet. Sci. Let., 270, 221–230.<br />
[7] Scherler, D. et al. (2006) Earth & Planet. Sci. Let. 248, 43-53.<br />
[8] Kenkmann, T. and Poelchau, M. H. (2009) Geology, 37, 459-462.<br />
[9] Shuvalov, V. V. et al. (2004) Meteoritics & Planet. Sci., 39, 467-479.<br />
[10] Wulf, G. et al. (2011) LPSC, 42, #1440.<br />
[11] Poelchau, M. H. et al. (2009) LPSC, 40, #1796.<br />
55