Synthesen von NAD+-Analoga und ihre Anwendungen

202 Pages·2015·9.37 MB·German

by  Yan Wang

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Synthesen von NAD -Analoga und
ihre Anwendungen
Dissertation zur Erlangung des akademischen Grades
eines Doktors der Naturwissenschaften
(Dr. rer. nat.)
vorgelegt von
Dipl.-Chem. Yan Wang
an der
Mathematisch-Naturwissenschaftliche Sektion
Fachbereich Chemie
Tag der mu¨ndlichen Pru¨fung: 25. Juni 2015
1. Referent/Referentin: Prof. Dr. Andreas Marx
2. Referent/Referentin: Prof. Dr. Valentin Wittmann
Konstanzer Online-Publikations-System (KOPS)
URL: http://nbn-resolving.de/urn:nbn:de:bsz:352-0-296783

Teile dieser Arbeit sind ver¨offentlicht
Y. Wang, D. R¨osner, M. Grzywa, A. Marx: Kettenterminierende und durch Click-
Chemie modifizierbare NAD+-Analoga zur Markierung von Zielproteinen der ADP-
Ribosyltransferasen, Angew. Chem., 2014, 126, 8298-8301; Chain-terminating and
clickableNAD+ analoguesforlabelingthetargetproteinsofADP-ribosyltransferases,
Angew. Chem. Int. Ed., 2014, 53, 8159-8162.

Danksagung
DievorliegendeArbeitentstandinderZeitvonJuli2010bisJuni2014imArbeitskreis
von Herrn Prof. Andreas Marx am Lehrstuhl fu¨r Organische und Zellul¨are Chemie
im Fachbereich Chemie der Universit¨at Konstanz.
Mein besonderer Dank gilt Herrn Prof. Dr. Andreas Marx fu¨r die Aufnahme in seinen
Arbeitskreis, die U¨berlassung des sehr vielseitigen und interessanten Promotionsthe-
mas, die hervorragende wissenschaftliche Betreuung in jeder Phase der Arbeit sowie
fu¨r die zahlreichen sachkundigen und richtungsweisenden Anregungen.
Bedanken m¨ochte ich mich auch bei Herrn Prof. Dr. Valentin Wittmann fu¨r die U¨ber-
nahme des Zweitgutachtens und Herrn Prof. Dr. Gerhard Mu¨ller fu¨r die U¨bernahme
des Pru¨fungsvorsitzes.
An dieser Stelle m¨ochte ich mich bei allen jetzigen und fru¨heren Mitgliedern der
Arbeitsgruppe fu¨r die unterhaltsame Zeit, die gute Arbeitsatmosph¨are und die Hilfs-
bereitschaft bedanken. In diesem Zusammenhang m¨ochte ich besonders meinen La-
borkollegen Anna-Lena Steck, Meike Liebmann, Meng Zheng fu¨r die unkomplizierte
Zusammenarbeit, die sch¨one und lustige Zeit im Labor und die vielen wissenschaft-
lichen Gespr¨ache danken, Xiaohui Zhao und Sarah Wallrodt fu¨r die sehr anregenden
Gespr¨ache u¨ber Forschung und Science Fiction.
Außerdem m¨ochte ich Daniel R¨osner fu¨r die Unterstu¨tzung bei meinen biochemischen
Experimenten danken, Anna-Lena Steck und Holger Bußkamp fu¨r die Aufnahme der
hochau߬osenden Massenspektren. Mein Dank gilt weiterhin Karin Lanz-Schwarz, To-
bias Strittmatter und Sarah Wallrodt fu¨r das Korrekturlesen dieser Dissertation.
Anke Friemel und Ulrich Haunz danke ich fu¨r die Aufnahme zahlreicher NMR-
Spektren.
All meinen Freunden und Bekannten im Fachbereich Chemie danke ich fu¨r viele un-
vergessliche und lustige Momente.
Ich m¨ochte mich ganz besonders bei meinen Eltern bedanken. Ohne ihre Unterstu¨tz-
ung und den bedingungslosen Familienru¨ckhalt w¨are es nicht m¨oglich gewesen, diese
Arbeit zu beenden.
Nicht zuletzt m¨ochte ich mich bei meiner Ehefrau Jingjing Han bedanken, die in jeder
Situation Frustration und Glu¨ck mit mir teilt.
Abstract
Based on its diverse functions, nicotinamide adenine dinucleotide (NAD+) is well
known to participate in a variety of cellular processes, such as redox metabolism,
signaling pathways, and post-translational modifications. In 1963 Chambon et. al.
reported for the first time about the formation of a nucleic acid like polymer derived
from NAD+, now referring to us as poly(ADP-ribose) (PAR). ADP-ribosylation is a
reversible post-translational modification of proteins, catalyzed by a family of enzy-
mes termed ADP-ribosyltransferases (ARTs), formerly known as poly(ADP-ribose)
polymerases (PARPs). ARTs play important roles in a wide range of biological pro-
cesses, including DNA repair, maintenance of genomic stability and transcriptional
regulation. ADP-ribosylation comprises the transfer of a single or multiple ADP-
ribose moieties from NAD+ to a specific amino acid residues on a target protein,
referring to us as mono(ADP-ribosyl)ation or poly(ADP-ribosyl)ation respectively.
In this process, the covalent transfer onto glutamic acid, aspartic acid (forming an
ester bond) or lysine residues (forming a Schiff base) of target proteins is described.
To further investigate the functions of ADP-ribosylation and determine the crosstalk
with other post-translational modifications, the identification of ADP-ribose acceptor
site is crucial. Mass spectrometry based approaches for the identification of acceptor
sites turned at to be a challenging task, because of the complex structure of attached
PAR.
Encouraged by current research results, the toolbox of NAD+ analogues were ex-
panded by designing and synthesizing NAD+ analogues. New NAD+ analogues are
described in this thesis that are efficiently incorporated by wild-type ARTs and a)
bear an affinity tag that will allow subsequent manipulations such as labeling and
sample enrichment and b) lead to PAR chain termination owing to a lack of the re-
quired hydroxyl group. Since little is known about the substrate scope of ARTs, the
five novel NAD+ analogues 1-5 were successful synthesized and applied in enzymatic
1
studies, and were found to be efficient substrates for ARTD1 and were used to label
ADP-ribosylated ARTD1 and histone H1.2.
The synthesis of the analogues 1-5 was conducted by first synthesizing the modified
adenosine cores 10, 15, 39, 41 and 49, which bear an iodine atom at the nucleoba-
se. The synthesis of these compounds started from commercially available starting
material 6, 11, 28, 33 and 42 respectively. The alkyne function was then introdu-
ced with the Sonogashira reaction to yield 17, 18, 50-52, which were subsequently
converted into the monophosphates 19, 20, 53-55. The respective monophosphates
weresubsequentlyconvertedintotheNAD+ analogues1-5bycouplingwithactivated
β-nicotinamide mononucleotide 25. In addition, the fluorescent dye 27, which was
required in the following biochemical experiment, was synthesized from sulfo-Cy5-
NHS-ester 26 and 3-azido-1-propanamine with good yields. The required affinity tag
58 was synthesized from D-(+)-biotin and an azido linker.
The obtained results of the biochemical experiments showed that the modified NAD+
1 had been accepted by the ARTD1 much better than the 2 in the trans(ADP-
ribos)ylation of histone H1.2 as well as in the auto(ADP-ribos)ylation of ARTD1. By
the systematic modification of the hydroxy groups of adenosine, it was that explored
the substrate scope of ARTD1 in terms of its dependence of the presence of a 2(cid:48)(cid:48)- and
3(cid:48)(cid:48)-OH group in the assembly of PAR. Whereas 1 led to the formation of long PAR,
4 and 5 act as chain terminators when applied in trans- or auto(ADP-ribos)ylation
reactions. This confirms the need for the 2(cid:48)(cid:48)-OH for PAR formation and shows that
the absence of this group cannot be rescued by the presence of a 3(cid:48)(cid:48)-OH. Furthermore
it was found that the 3(cid:48)(cid:48)-OH group participates in the ADP-ribosylation catalysed by
ARTD1 since the employment of 3 leads to less efficient PAR formation as compared
to the reactions where an analogue with both 2(cid:48)(cid:48)- and 3(cid:48)(cid:48)-OH groups (1) was employ-
ed. It was further found that the modifications carried within 5 turn this component
into an efficient chain terminator of PAR synthesis that competes with natural NAD+
2