Branching of poly(ADP-ribose): Synthesis of the Core Motif

Article abstract of DOI:10.1021/acs.orglett.5b02143

The synthesis of the core motif of branched poly(adenosine diphosphate ribose) (poly(ADPr)) is described, and structural analysis reasserted the proposed stereochemistry for branching. For the synthesis, a ribose trisaccharide was first constructed with only α-O-glycosidic linkages. Finally, the adenine nucleobase was introduced via a Vorbrüggen-type glycosylation reaction. The orthogonality of the selected protecting groups was demonstrated, allowing for the construction of branched poly(ADPr) oligomers in the near future.

Full text of DOI:10.1021/acs.orglett.5b02143

Letter
pubs.acs.org/OrgLett
Branching of poly(ADP-ribose): Synthesis of the Core Motif
Hans A. V. Kistemaker, Herman S. Overkleeft, Gijsbert A. van der Marel,* and Dmitri V. Filippov*
Leiden Institute of Chemistry, Leiden University, P.O. Box 9502, 2300 RA Leiden, The Netherlands
S
* Supporting Information
ABSTRACT: The synthesis of the core motif of branched poly(adenosine diphosphate
ribose) (poly(ADPr)) is described, and structural analysis reasserted the proposed
stereochemistry for branching. For the synthesis, a ribose trisaccharide was first
constructed with only α-O-glycosidic linkages. Finally, the adenine nucleobase was
introduced via a Vorbruggen-type glycosylation reaction. The orthogonality of the
̈
selected protecting groups was demonstrated, allowing for the construction of branched
poly(ADPr) oligomers in the near future.
oly(adenosine diphosphate ribose) (poly(ADPr)) is an
intriguing biopolymer which is synthesized by poly(ADPr)
(ADPr) fragments has not been reported yet because of the
P
limited number and the random positions of branching points
in ADPr chains. In addition, enzymatic preparation followed by
multiple chromatographic purifications produced nonhomoge-
nous poly(ADPr) fragments.2d,5 Recently, the first chemical
syntheses of well-defined ADPr-related model compounds were
reported.4,6 Apart from the synthesis of mono-ADPribosylated
oligopeptides,7 we reported the synthesis of dimeric and
trimeric ADPr-fragments in milligram amounts, suitable for
detailed molecular studies.6 As part of a program toward the
synthesis and evaluation of structurally defined poly(ADPr)
fragments, we here present the first synthesis of 2′-O-((2″-O-
(α-D-ribofuranosyl)-α-D-ribofuranosyl)-β-D-ribofuranosyladeno-
sine (10), the branching point in poly(ADPr) (Scheme 1).
Target 10 can be considered as a hyperglycosylated
nucleoside, a structural element that is also found in a variety
of biomolecules such as initiator t-RNA, complex nucleoside
antibiotics, and neuroactive spider toxins.8 The synthesis of this
class of molecules presents a challenge as O-glycosylation of a
protected nucleoside is accompanied by nearly unavoidable side
reactions with nucleophilic positions in the nucleobase. In
addition, en route to 10, α-ribosyl linkages need to be installed.
Two general routes for the synthesis of glycosylated nucleo-
sides can be considered. In the first route, a suitably protected
nucleoside is O-glycosylated, while in the second route
introduction of the heterocyclic nucleobase is the final
glycosylation event. Most of the reported syntheses to α-2′-
O-ribosylated adenosine rely on the ribosylation of a suitably
protected adenosine derivative.4,8a,9 The approach to α-2′-O-
ribosylated adenosine we reported proved to be inefficient
during the scaling up procedure, in particular, for the O-
glycosylation of adenosine. Therefore, we devised a new route
polymerases (PARPs) for modifying various proteins and
enzymes. This reversible post-translational modification (PTM)
is associated with a wide variety of biological processes like
DNA damage repair, mitosis, apoptosis, and transcription.1 The
first ADPr moiety was enzymatically transferred to an acceptor
protein by a nucleophilic attack of the side chain of a specific
amino acid (Glu, Asp, Asn, Arg) on nicotinamide adenine
dinucleotide (NAD+), thereby releasing nicotinamide. The
resulting mono-ADPribosylated protein is the starting point for
PARP enzymes to form poly-ADPr chains. PARP enzymes
elongate the chains to over 200 units in size and also branch the
polymer after every 20−50 elongation reactions (Figure 1).2 In
the linear part the characteristic pyrophosphates in the
poly(ADPr) chain link the 5′-OH of adenosine with the 5′-
OH of ribose, while the 2′-OH of adenosine is connected via an
unique α O-glycosidic bond with ribose. At the branching point,
the ribose moiety in the chain becomes trifunctionalized by
linking its 2′-OH via an α-O-glycosidic linkage with another
ribose.2a,h
Although the involvement of ADPribosylation in many
cellular processes has become clear over the past decades, the
mechanism of action and mode of interaction at a molecular
level remains elusive. So far, the crystal structures of relevant
enzymes have been studied with the use of enzymatically
prepared linear ADPr fragments3 and, recently, the first
example of a synthetic ADPr dimer.4 On the contrary,
information on the manner of binding of branched ADPr is
scarce. However, on the basis of the information obtained from
crystal structure studies with poly(ADPribose) glycohydrolase
(PARG) and fractionated poly(ADPr), it is suggested that
branched ADPr cannot bind with the enzymes that recognize
linear ADPr.3b Therefore, it is not excluded that there are
unknown enzymes that specifically recognize these branched
ADPr structures. Isolation of well-defined branched poly-
Received: July 24, 2015
© XXXX American Chemical Society
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Figure 1. Schematic representation of branched poly(ADPr) with the core motif of branching depicted in red.
Scheme 1. Synthesis of 2′-O-((2″-O-(α-D-Ribofuranosyl)-α-D-ribofuranosyl)-β-D-ribofuranosyladenosine (10)
by initially forming a ribose disaccharide and introducing the
adenine base at the anomeric center in a later stage of the
synthesis.6 This new route afforded α-2′-O-ribosylated
adenosine in sufficient quantities and ultimately allowed the
synthesis of linear ADPr-oligomers.
Guided by these results, we set out to assemble target
compound 10 by postponing the crucial introduction of the
adenine base at the trisaccharide stage of the synthesis (Scheme
1). The required ribose trisaccharide 7 is orthogonally
protected and possesses only cis-glycosidic linkages. Although
α-configured C-ribofuranoside linkages can be installed stereo-
selectively, introduction of similar O-glycosidic linkages usually
require optimization experiments to attain acceptable stereo-
selectivity. First, α-configured ribose disaccharide 4 was
obtained by TMSOTf-mediated condensation of commercially
available tribenzoylribose 1 with known N-phenyltrifluoroace-
timidate donor 2.9a The glycosylation reaction was extremely
fast (<1 min) with just a minimal amount of TMSOTf (1 mol
%) and high yielding (84%), while the corresponding β-
disaccharide could not be detected. In order to stereoselectively
introduce the next ribose moiety at the 2″-OH in dimer 4, the
benzyl ethers had to be removed first. Unexpectedly,
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hydrogenolysis of the benzyl groups in 4 using catalytic
amounts of Pd/C was accompanied by a significant degree of
glycosidic bond cleavage. Apparently, besides the formation of
the debenzylated dimer, an intramolecular side reaction
occurred that resulted in a mixture of 1,5-anhydroribofuranose
and starting compound 1 (Scheme 2). Treatment of the crude
selective manner and in a yield of 63%. UV−vis spectroscopic
analysis of the main product confirmed that adenine was
correctly linked via its N9 position to the anomeric center,
while the minor byproducts could be assigned to adenosine
derivatives with incorrect N1, N3, and/or N7 linkages.12
Compound 8 is orthogonally protected to allow complete
control over the elongation reactions in future poly(ADPr)
syntheses. To demonstrate the orthogonality, we selectively
cleaved the TBDPS group without affecting the TIPS group to
give compound 9 with a free hydroxyl group. Finally, all the
protecting groups in 8 were removed by sequential treatment
with TBAF and NH4OH to afford target 10, the core motif of
branched ADPr.
Scheme 2. Products Formed during Hydrogenolysis of 4
Compound 10 was carefully analyzed with all relevant 1D
and 2D NMR spectroscopic methods, and all the H and C
atoms could be assigned (Figure 2, 6.20 (H1′), 5.25 (H1″),
4.96 (H1‴)). The anomeric configuration of the ribosyl
residues was ascertained by measuring the value of JH1C2
using HSQC-HECADE experiments for compounds 6 and 8
(see the Supporting Information).13 Having thus obtained the
deprotected branched nucleoside of ADPr, we felt that a
comparison of the spectroscopic data of our synthetic material
with a compound isolated from natural sources was in order.
The only available spectroscopic information on branched
ADPr has been reported by Miwa and co-workers.2a They
performed an enzymatic synthesis to generate poly(ADPr),
purified the material, treated it with snake venom phospho-
diesterase to hydrolyze all the pyrophosphate linkages, and
finally fractionated the mixture using a weak anion-exchange
column.2h By doing so, they isolated 2′-ribosyladenosine-5′,5″-
bis-phosphate (Ado(P)-Rib-P) as the major product and a
unknown minor byproduct which was later identified to be 2′-
O-((2″-O-(α-D-ribofuranosyl)-α-D-ribofuranosyl)-β-D-ribofura-
nosyladenosine 5′,5″,5‴-O-tri-phosphate (Ado(P)-Rib-(P)-Rib-
mixture with 1,3-dichloro-1,1,3,3-tetraisopropyldisiloxane
(TIPDS) chloride yielded a sufficient amount of ribose
disaccharide 5 (2 mmol). Alternatively, disaccharide 5 could
uneventfully be obtained by our previously reported con-
densation of triisopropylsilyl (TIPS)-protected donor 3 with
acceptor 1.6 Subsequent hydrogenation kept the TIPS group in
place while glycosidic bond cleavage could not be detected.
Potentially, removal of the TIPS group and installment of the
TIPDS moiety should give 5 in higher yields.
The next glycosylation event, in which disaccharide acceptor
5 was reacted with TIPS donor 3, proceeded in a complete α-
selective manner to give trisaccharide 6 in 57% yield. At this
stage, the removal of the benzyl-protecting groups was
undertaken as we previously experienced that the presence of
an adenine moiety complicates hydrogenolysis. Subjection of
trisaccharide 6 to Pd/C-catalyzed hydrogenation not only
removed the benzyl ethers but was also accompanied by partial
opening of the TIPDS ring at the primary position. This
drawback was negated by treatment of the crude mixture with
HF·pyridine to completely remove the TIPDS while leaving the
TIPS intact. Introduction of a TBDPS at the 5″-OH and finally
acetylation of the secondary hydroxyl functionalities gave
trisaccharide 7. With building block 7 available, the crucial
1
P). The observed H NMR shifts for the anomeric protons
(6.41 (H1′), 5.45 (H1″), and 5.21 (H1‴)) by Miwa and co-
workers show the same relative shifts as we observed for
1
compound 10. The H NMR shifts for compound 10 are,
however, uniformly shifted approximately 0.2 ppm upfield
because of the lack of phosphate monoesters at the primary
hydroxyls.
In conclusion, we have successfully synthesized the core
structure of branched poly(ADPr) with two ribose residues
having α-glycosidic linkages via the preparation of an
introduction of the adenine base by a Vorbruggen glycosylation
̈
reaction was undertaken. Heating a mixture of trisaccharide 7
with N6-benzoyladenine, HClO4−SiO2,10,11 and N,O-bis-
(trimethylsilyl)trifluoroacetamide (BSTFA) in acetonitrile
gave fully protected2′-O-((2″-O-(α-D-ribofuranosyl)-α-D-ribo-
furanosyl)-β-D-ribofuranosyladenosine derivative (8) in a β-
1
Figure 2. H NMR spectrum compound 10 (500 MHz, D2O).
C
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orthogonally protected trisaccharide and ensuing a Vorbruggen
̈
glycosylation reaction for the regio- and stereoselective
introduction of the adenine base. Spectroscopic analysis of
core structure 10 supports the reported structure of the isolated
naturally occurring poly(ADPr). The obtained result in
combination with previously reported synthesis for linear
ADPr oligomers is a basis to undertake the synthesis of
branched poly(ADPr) oligomers. In order to obtain the
required building block for the synthesis of branched poly-
ADPr oligomers, the previously reported protecting group
strategy will be applied to compound 8 as part of ongoing
research.6 The presented methodology is also a valuable asset
for future syntheses of other glycosylated nucleosides, such as
complex nucleoside antibiotics.
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Marel, G. A.; Filippov, D. V. Org. Lett. 2011, 13, 2920−2923.
(b) Knapp, S.; Gore, V. K. J. Org. Chem. 1996, 61, 6744−6747.
(10) Kistemaker, H. A. V.; van der Heden van Noort, G. J.;
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15, 2306−2309.
(11) Agarwal, A.; Rani, S.; Vankar, Y. D. J. Org. Chem. 2004, 69,
6137−6140. Immobilized perchloric acid on silica was prepared by
adding HClO4 (2 mmol, as a 70% aqueous solution) to a slurry of
silica gel (5 g, 200 mesh) in Et2O (15 mL) and stirring for 1 h at room
temperature. The solvent was removed under reduced pressure, and
the resulting powder was dried at 110 °C for 2 h under reduced
pressure to obtain HClO4−SiO2 (0.4 mmol H+/g), which was used
directly.
ASSOCIATED CONTENT
■
S
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.or-
glett.5b02143.
1
Experimental procedures, H, 13C, and 2D NMR spectra
and characterization of all compounds (PDF)
AUTHOR INFORMATION
■
Corresponding Authors
*E-mail: marel_g@chem.leidenuniv.nl.
*E-mail: filippov@chem.leidenuniv.nl.
Notes
(12) (a) Acton, E. M.; Ryan, K. J.; Goodman, L. J. Org. Chem. 1971,
36, 2646−2657. (b) Framski, G.; Gdaniec, Z.; Gdaniec, M.; Boryski, J.
Tetrahedron 2006, 62, 10123−10129. (c) Richert, C.; Roughton, A. L.;
Benner, S. A. J. Am. Chem. Soc. 1996, 118, 4518−4531.
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
(13) Napolitano, J. G.; Gavín, J. A.; García, C.; Norte, M.; Fernan
́
dez,
The Netherlands Organization for Scientific Research (NWO)
is acknowledged for financial support.
J. J.; Hernandez Daranas, A. Chem. - Eur. J. 2011, 17, 6338−6347.
́
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