Synthesis of ribosyl-ribosyl-adenosine-5′,5′′,5′′′(triphosphate) - The naturally occurring branched fragment of poly(ADP ribose)

Article abstract of DOI:10.1039/c7cc05755e

Poly-adenosine diphosphate ribose (PAR) is a branched biopolymer that occurs as a result of post-translational modification of proteins. In 1981 Miwa et al. determined the structure of enzymatically prepared branched PAR. We present the first synthesis of the same branched PAR fragment and have shown by NMR that the structure proposed by Miwa is correct.

Full text of DOI:10.1039/c7cc05755e

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Marilyn M. Olmstead, Alan L. Balch, Josep M. Poblet, Luis Echegoyenet al.
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Synthesis of Ribosyl-ribosyl-adenosine-5’,5’’,5’’’(triphosphate)―the
Naturally Occurring Branched fragment of Poly ADP Ribose
Received 00th January 20xx,
Accepted 00th January 20xx
Qiang Liu,a Hans. A. V. Kistemaker,a Herman S. Overkleeft,a Gijsbert A. van der Marel,a and Dmitri
V. Filippov* a
DOI: 10.1039/x0xx00000x
www.rsc.org/
Poly-adenosine diphosphate ribose (PAR) is a branched al2. They performed an enzymatic synthesis using NAD+ and
biopolymer that occurs as a post-translational modification of calf thymus nuclei, to get PAR in vitro. Subsequent hydrolysis
proteins. In 1981 Miwa et al. determined the structure of of all the pyrophosphate linkages in PAR by treatment with
enzymatically prepared branched PAR. We present the first snake venom phosphodiesterase led to the isolation of
synthesis of the same branched PAR fragment and show by branched PAR fragment
1. The configuration of 1 was
NMR that the structure proposed by Miwa is correct. determined by derivatization and with the aid of
Poly ADP ribosylation (PARylation) is an important post- physicochemical techniques including gas chromatography,
translational modification in which negatively charged ADP- mass spectrometry, and 1H-NMR spectroscopy2. Shortly after
11
ribose units are transferred to an acceptor protein using NAD+ the structure elucidation, two different groups10, reported
(nicotinamide adenine dinucleotide) as a donor and PARPs the existence of branched PAR in vivo, indicating that the
(poly ADP ribose polymerases) as the involved enzymes1,
2
branched PAR fragment made from enzymatic synthesis is
(Figure 1). PARylation and the resulting polymers (PARs) are indeed the naturally occurring product. Furthermore,
involved in many biological events such as DNA repair, enzymatic synthesis is widely applied to simulate in vivo
transcriptional regulation and apoptosis3. PAR chains can be conditions and to produce PAR12-14. In this respect, the organic
either linear or branched4. Linear PAR can grow to over 200 synthesis
units in size, with a branching site occurring on average once ribofuranosyl-(1’’
of
O-α-D-ribofuranosyl-(1’’’
→
2’’)-O-α-D-
is
→
2’)-adenosine-5’,5’’,5’’’-tris(phosphate)
1
every 20 to 50 elongation reactions1. While the knowledge on a challenging and valuable goal that can confirm this structure
linear PAR is steadily growing, less progress is made with the elucidation and will support future biological studies. As part
role of branched PAR and its function is still unclear. There are of a program to develop synthetic methodologies towards PAR
a few reports on branched PAR after its discovery by Miwa et related molecules such as ribosylated amino acids15, mono-
al4 at the end of 1970s. For example, the branched and not the ADP ribosylated peptides16,
17
and ADP ribose dimer and
linear PAR chains bind most preferably to histones5 and other trimer18, we here present the synthesis and structural analysis
nuclear proteins6. The branching point is reported not to be of compound
. The synthetic route toward the O-α-D-
the endoglycosidic cleavage site of poly-ADP-ribose ribofuranosyl-(1’’’ 2’’)-O-α-D-ribofuranosyl-(1’’ 2’)-
glycohydrolase (PARG)7 which indicates there might be adenosine-5’,5’’,5’’’-tris(phosphate)
is guided by our earlier
1
→
→
1
undiscovered enzymes that specifically recognize the branched reported synthesis of the core motif of branched PAR19 by
PAR structure8, 9. In 1981 the chemical structure of the adaptation of the protective group strategy and simultaneous
branching point of PAR was established as O-α-D- introduction of three phosphate triester functions on the
ribofuranosyl-(1’’’
→
adenosine-5’,5’’,5’’’-tris(phosphate) (Figure 1, 1) by Miwa et trisaccharide with phosphoramidite chemistry.
2’’)-O-α-D-ribofuranosyl-(1’’
→
2’)-
5’,5’’, 5’’’-primary hydroxyls of a suitably protected branched
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The first stage of the synthetic route comprises the opening or total cleavage of the TIPDS group. This side
introduction of two challenging 1,2 cis-α-glycosidic linkages by reaction could not be prevented by the use of other catalysts
the synthesis of key intermediate 620 (Scheme 1). We suggest such as Pd(OH)2. We presumed that the lability of the TIPDS
to call the parent trisaccharide parotriose (PAR + triose, Figure group on the trisaccharides made the hydrogenolysis
1,
The hydrogenolysis of the benzyl groups in fully protected be attained
parobiose, (Scheme 1) obtained according to our earlier for 24 h to remove all silyl groups. Hydrogenolysis of the thus
reported method18 was followed by removal of obtained compound
using Pd/C in methanol for 24 h
(triisopropylsilyl) TIPS group with HF·Pyridine to give 2’’,3’’,5’’- afforded compound , provided with five hydroxyl functions, in
OH parobiose derivative in good yield. It is of interest that high yield (Scheme 2). Readjusting the protection by
the presence of the TIPS instead of the benzyl group at the 5’- installation of TBDPS groups on the primary hydroxyls of
OH of compound 2, avoided glycosidic bond cleavage during compound and acetylation of the remaining secondary
hydrogenolysis as reported in our previous study19, resulting in hydroxyls set the stage for introduction of N6-benzolyadenine
a significant improved yield and making a large scale synthesis on the reducing end of parotriose . Vorbrüggen type
possible. The 3’’,5’’-OH functions in triol
were selectively glycosylation using HClO4-SiO2 as catalyst and persilylated N6-
masked with the diol protecting TIPDS group by treatment benzolyadenine proceeded completely selective and
with 1,3-dichloro-1,1,3,3-tetraisopropyldisiloxane (TIPDSCl2) in furnished 10 in high yield19. The selective glycosylation on the
pyridine to get alcohol . Coupling of partially protected N-9 position and not on the N-3 or N-7 positions was
parobiose with N-phenyl trifluoroacetimidate donor ascertained by UV-spectroscopy. Before three identical
afforded the fully protected parotriose with complete α- phosphate triesters could be installed on the 5’,5’’, 5’’’-primary
selectivity and improved yield19. Subsequent introduction of hydroxyls, protective group manipulation was required to
adenine base requires number of protective group ensure regioselective phosphorylation. Thus, saponification of
manipulations. The benzyl-protecting groups in should the acetyl and benzoyl esters with aqueous NaOH in
preferably be replaced because the adenine moiety would pyridine/ethanol gave intermediate 11, allowing protection of
complicate hydrogenolysis20. However, we observed that the the remaining free 5’-OH group with
TBDPS group.
removal of benzyl ethers in by Pd/C-catalyzed hydrogenation Surprisingly the reaction of the 5’-OH in 11 with TBDPSCl
B) and the corresponding ribose disaccharide parobiose (
A
). problematic but selective removal of the TIPDS in
6 could not
.
Therefore, we treated with an excess of Et3N·HF
6
2
7
8
3
8
9
3
β
4
4
5
6
a
6
a
6
was tedious (more than 4 days) and was accompanied by ring failed, but fortunately the equally suitable TBS group was
introduced successfully using the more reactive TBSCl in
pyridine. Subsequent acetylation of this intermediate gave
fully protected 12. After removal of the silyl groups by
HF·pyridine all primary hydroxyl functions were released to
give triol 13, amenable to the simultaneous introduction of
three di-tert-butylphosphate triesters. Treatment of 13 with 10
equivalents
di-tert-butyl-N,N–diisopropylphosphoramidite
using 1-methylimidazole and 1-methylimidazolium chloride as
activator combination under strictly anhydrous conditions21
and subsequent oxidation of the intermediate phosphite
Scheme 1. Synthesis of protected parotriose 6 from parobiose 2
2 | J. Name., 2012, 00, 1-3
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triesters gave 14 in moderate yield. The low reactivity of 5’-OH chromatography, followed by column chromatography on
of the adenosine moiety as noticed in the silylation of 11 phosphocellulose, which we could not easily attain. By doing
decreases also the yield of the phosphitylation reaction as the this, Miwa obtained compound
1 as acidified sample (pH=3)
formation of target 14 was accompanied by bis- while our product occurs in neutralized form (pH=7) as
phosphorylated product. In the final stage, the tert-butyl ammonium salt. To get a more accurate comparison, we
groups of the phosphate triester in fully protected 14 were changed the pH of our sample in D2O from 7 to 3 by adding
removed with HCl/HFIP in 1 h followed by ammonolysis of the CD3COOD. Under these conditions the NMR spectrum of our
acyl groups to furnish tris-phosphorylated parotriose
excellent yield.
1
in product and the one of Miwa proved to be virtually identical
(Figure 2A vs Figure 2B). A small difference of multiplicity at
Having target compound
compare the spectroscopic data of our synthetic product with in our measurement. Overall the chemical shifts of all protons
those reported by Miwa2 for the natural product (Figure 2A). In in our compound
are approximately 0.1 ppm upfield from
1 at our disposal, we could R2’’’ and R3’’’ could be attributed to the slightly different field
1
the first instance, we observed significant differences between those of Miwa’s compound mainly because of they used DSS
the 1H-NMR-spectra (see Supporting Information). We (sodium 2,2-dimethyl-2-silapentane-5-sulfonate) as reference
speculated that these differences originate mainly from a pH while we did not. Taken together, we conclude that the
difference of the NMR samples, which in turn may be due to synthetic compound and the isolated compound have the
different isolation procedures. Our procedure involved global same chemical structure.
deprotection by ammonia treatment, followed by purification
In conclusion, for the first time, O-α-D-ribofuranosyl-
2’’)-O-α-D-ribofuranosyl-(1’’ 2’)-adenosine-5’,5’’,5’’’-
by HW-40 gel filtration using 0.15M NH4OAc in H2O as eluent (1’’’
→
→
under essentially neutral conditions. Contrary, Miwa firstly tris(phosphate), a tris-phosphorylated branched PAR fragment
desalted the isolated product by DEAE-cellulose column was obtained by organic synthesis. Comparison of the 1H-NMR
spectra of this fragment and the naturally occurring product
showed the same chemical shifts which means that the
structure of
1 was identical to the naturally occurring
compound2 and that the regio- and stereochemistry of
branching point of PAR was correctly elucidated by Miwa et al.
Importantly, synthetic methodology in this work represents a
next step to the future synthesis towards more complicated
branched PAR fragments. Such fragments will be a valuable
asset for future biological studies toward the biological
function of branched ADPr. As last remark, we propose to give
the disaccharide as occurs in linear PAR-chains parobiose and
the native trisaccharide responsible for the branching points in
PAR – parotriose, as trivial names.
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Acknowledgments
We are grateful for the support Q.L. received from China
Scholarship Council (CSC) and H.A.V.K. received from Netherlands
Organization for Scientific Research (NWO).
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Total synthesis provided the first independent confirmation of the chemical structure of the
branching point in poly-ADP-ribose.