Poly(ADP-Ribose) - A unique natural polymer structural features, biological role and approaches to the chemical synthesis

Article abstract of DOI:10.1080/15257770.2014.984073

Poly(ADP-ribose) (PAR) is a natural polymer, taking part in numerous important cellular processes. Several enzymes are involved in biosynthesis and degradation of PAR. One of them, poly(ADP-ribose)polymerase-1 (PARP-1) is considered to be a perspective ta

Full text of DOI:10.1080/15257770.2014.984073

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Poly(ADP-Ribose)—A Unique Natural
Polymer Structural Features, Biological
Role and Approaches to the Chemical
Synthesis
Mikhail S. Drenicheva & Sergey N. Mikhailova
a Engelhardt Institute of Molecular Biology, Russian Academy of
Sciences, Moscow, Russian Federation
Published online: 16 Mar 2015.
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Natural Polymer Structural Features, Biological Role and Approaches to the Chemical Synthesis,
Nucleosides, Nucleotides and Nucleic Acids, 34:4, 258-276, DOI: 10.1080/15257770.2014.984073
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Nucleosides, Nucleotides and Nucleic Acids, 34:258–276, 2015
Copyright ꢀC Taylor and Francis Group, LLC
ISSN: 1525-7770 print / 1532-2335 online
DOI: 10.1080/15257770.2014.984073
POLY(ADP-RIBOSE)—A UNIQUE NATURAL POLYMER STRUCTURAL
FEATURES, BIOLOGICAL ROLE AND APPROACHES
TO THE CHEMICAL SYNTHESIS
Mikhail S. Drenichev and Sergey N. Mikhailov
Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow, Russian
Federation
Poly(ADP-ribose) (PAR) is a natural polymer, taking part in numerous important cellular
2
processes. Several enzymes are involved in biosynthesis and degradation of PAR. One of them,
poly(ADP-ribose)polymerase-1 (PARP-1) is considered to be a perspective target for the design of
new drugs, affecting PAR metabolism. The structure of PAR was established by enzymatic hydrolysis
and further analysis of the products, but total chemical synthesis of PAR hasn’t been described yet.
Several approaches have been developed on the way to chemical synthesis of this unique biopolymer.
Keywords DNA alkylation; nucleotide excision and repair; bioconjugates; nucleic acid
biosynthesis; modified nucleosides; enzyme inhibition
Poly(ADP-ribose) (PAR) participates in numerous cellular processes such as
DNA repair and replication, modulation of chromatin structure, transcrip-
tion, cell differentiation and also in pathogenesis of various diseases such as
cancer, diabetes, ischemia and inflammations.[1–3] PAR is the last one among
important natural biopolymers, which has not been chemically synthesized.
The development of methods for the synthesis of PAR is still a challenge
for chemists. A biological function of PAR may be associated with its unique
structure, bearing the most negative charge per monomeric unit among all
known natural biopolymers. Ionic interactions between negatively charged
PAR and positively charged proteins play an important role in the formation
of nucleoprotein complexes.[20]
In the previous reviews [1–2, 4–9] biochemical, biological, and some medic-
inal aspects of PAR functions were described in details. The present review
Received 23 September 2014; accepted 31 October 2014.
Address correspondence to Sergey N. Mikhailov, Engelhardt Institute of Molecular Biology, Russian
Academy of Sciences, Vavilov str. 32, Moscow 119991, Russian Federation. E-mail: smikh@eimb.ru
Color versions of one or more of the figures in the article can be found online at
www.tandfonline.com/lncn.
258

Poly(ADP-Ribose)—A Unique Natural Polymer
259
summarizes structural peculiarities, physicochemical properties, and per-
spectives in chemical synthesis of PAR.
1. DISCOVERY AND STRUCTURE ELUCIDATION OF PAR
Poly(ADP-ribosylation) is a posttranslational modification of proteins in
eukaryotic cells, catalyzed by poly(ADP-ribose)polymerases (PARPs). These
enzymes promote the conversion of NAD+ to poly(ADP-ribose) (PAR) with
the release of nicotinamide (Scheme 1).[3]
SCHEME 1 Structure and biosynthesis of PAR: a. poly(ADP-ribose) polymerase-1 (PARP). b. (ADP-
ribosyl)proteinlyase. c. poly(ADP-ribose) glycohydrolase (PARG).
PAR is a complex branched biopolymer, in which 2ꢁ-O-α-D-ribofur-
anosyladenosine moieties are bound by pyrophosphate linkages (Scheme 1).
PAR molecules either isolated from natural sources or synthesized in vitro
contain more than 200–300 monomeric units.[1, 3] Linear segments of 20–50
units alternate with branched fragments.[1, 3, 10, 13] The chemical structure
of PAR was determined by enzymatic hydrolysis of the pyrophosphate bonds
by phosphodiesterase with subsequent enzymatic dephosphorylation by al-
kaline phosphatase and further characterization of the products by NMR

260
M. S. Drenichev and S. N. Mikhailov
FIGURE 1 Structural fragments of PAR (1-4) and 2ꢁ-O-(5ꢁꢁ-O-phospho-β-D-ribofuranosyl-1-yl)nucleo-
sides (5-6)—the closest natural analogues of 1.
spectroscopy.[3, 10, 13] Enzymatic hydrolysis of PAR by snake venom phos-
phodiesterase yielded disaccharide nucleoside[11, 10, 13] and trisaccharide
nucleoside[10] diphosphates 1 and 2 (Figure 1). Nucleoside diphosphates
1-2 were hydrolyzed by alkaline phosphatase to corresponding nucleosides
3 and 4.[11]
To date, about a hundred disaccharide nucleoside derivatives have been
isolated from various natural sources.[3, 10] 2ꢁ-O-β-D-ribofuranosyladenosine
Arp (5) and its guanosine derivative—Grp (6) were isolated from tRNA.[12-14]
The greatest difference in 1H-NMR spectrum of monomeric unit 1
in comparison to 2ꢁ-O-(5ꢁꢁ-O-phospho-β-D-ribofuranosyl-1-yl)-adenosine 5,
the closest natural analogue of 1, is observed in coupling constants of the
extra carbohydrate moiety due to the difference in configurations of C-1ꢁꢁ
atoms. Configuration of O-glycosidic bond in isolated nucleosides was un-
ambiguously established by comparative analysis of 13C-NMR spectra of 2
and anomeric methyl-D-ribofuranosides.[10] These compounds contain an
extra carbohydrate residue linked to one of the nucleoside hydroxyl groups
via an O-glycosidic bond. The presence of a disaccharide residue and a het-
erocyclic base makes their properties similar to those of carbohydrates and
nucleosides.[3]
The ability of forming helicoidal secondary structure by long-chain PAR
has much in common with the structure of DNA and RNA.[1] PAR can form
regular helical structures, stabilized by base stacking and hydrogen bonding
interactions.[15–16] Although PAR exists in an extended conformation in low
salt buffers, it may undergo some structural changes in high salt buffers,
showing cooperative helix-coil transitions with hypochromicity up to 40%
for long chain molecule.[15] The rise of ionic strength of solution leads to
more intensive Cotton effects for long chain PAR polymers. Melting curves
monitored by CD-spectra at high salt concentrations have sharp transitions

Poly(ADP-Ribose)—A Unique Natural Polymer
261
with Tm values increasing from 20 to 40◦C upon the rise of ionic strength
from 3 M to 5 M NaCl, respectively.[15]
Recently, an efficient one-pot enzymatic synthesis of 13C, 15N-enriched
PAR with 8% overall yield was performed by Schultheisz and co-workers.[16]
The isotopically labeled PAR was studied by the NMR methods. According
to the NOESY-data adenine base is characterized by anti-conformation. Het-
erocyclic moieties in PAR are accessible both for inter- and intramolecular
interactions, that is in agreement with possibilities of helix-coil transitions, es-
tablished by CD-spectroscopy.[16] Three-dimensional structural information
available by investigations of isotopically labeled PAR provides possibility for
modeling and prediction of PAR complexation with proteins.
In cell, PAR is covalently linked to PARPs, histones, and other nuclear
proteins.[1] Covalent binding of target proteins with PAR is not specific. En-
zymatic modification proceeds either on backbone carboxyl groups of glu-
[17]
tamic/aspartic acid residues (formation of ester linkages)
or on lysine
ε-amino groups.[18–19] The site of poly(ADP-ribosylation) was determined
by enzymatic hydrolysis of long PAR-fragments by ADP-ribosylhydrolase
(ARH3) and further analysis of ADP-ribosylated peptides by electron transfer
dissociation.[18] Modification of proteins by ADP-ribose can also be char-
acterized according to their chemical properties: ADP-ribosylated lysine
residues are stable in 1M hydroxylamine at pH 7, while glutamic and aspartic
acid residues are easily cleaved under such conditions, releasing ADP-ribose
fragments.[20]
2. ENZYMES OF PAR METABOLISM
Several enzymes are involved in biosynthesis and degradation of PAR
(Scheme 1). In cellular nuclei PAR is synthesized by PARPs (scheme 1, path-
way a) from NAD+ as a substrate. At present time about 17 enzymes of PARP
family are known, among them are PARP-1, PARP-2, PARP-3, tankyrases
etc. The properties of the most enzymes from PARP family have not been
thoroughly studied yet.
PARP-1 (EC 2.4.2.30) is responsible for the synthesis of 90% of PAR in
cell[21] and appears to be the major poly(ADP-ribosyl)ating enzyme in higher
eukaryotes after DNA-damage. PARP-1 assists in the repair of single-strand
DNA nicks by base excision repair (BER) pathway in mammals[8] and is
defined as a protein, catalyzing PAR formation in response to genotoxic
stress or mitogenic stimuli.[1, 17] Apart from its role in BER, PARP-1 is involved
in modulation of chromatin structure, DNA-replication, gene transcription,
repair of DNA double strand breaks (DSB), restarting of collapsed replica-
tion forks and genomic maintenance possibly by interaction with p53.[1, 8,
22, 23] PARP-1 consists of three main domain structures: DNA-binding domain
(DBD) with molecular mass 46 kDa, automodification domain (AMD) with

262
M. S. Drenichev and S. N. Mikhailov
molecular mass 22 kDa and catalytic domain with molecular mass 54 kDa
(cat-PARP).[2]
DBD includes nuclear localization signal (NLS)[8] and three zinc finger
motifs FI, FII [1, 8–9] and FIII.[8–9] FI and FII recognize damaged DNA struc-
[1, 6, 9]
ture
and are important for the binding to single- and double-strand
breaks (SSB and DSB).[1, 6, 8] FIII participates in alterations of PARP-1 cat-
alytic activity in response to the coupling with damaged DNA [8–9], as a result
the catalytic activity increases up to 500-fold resulting in PAR-ylation of va-
riety of proteins during 15 to 30 s after damage.[8] AMD, which is necessary
for regulation of PARP-1 catalytic activity, is located in the central part of
PARP and contains acceptor aminoacids for covalent attachment of PAR.[4]
AMD also contains leucine-zipper motif at N-terminal region [1], which is sug-
gested to be involved in the dimerization of PARP and complexation with
other nuclear proteins.[1] Recent studies revealed, that enzymatic activity of
RARP-1 is regulated only by automodification of acceptor lysine residues
(Lys-498, 521, and 524) located in AM-domain.[20]
Cat-PARP is responsible for PAR biosynthesis and implements at least
three enzymatic reactions [1]: (a) initiation (the attachment of the first ADP-
ribose moiety to an acceptor amino acid); (b) elongation of PAR chain by
joining of additional ADP-ribose monomers; (c) generation of branching
points.
Active site of catalytic domain consists of two sub-domains: acceptor sub-
domain for binding adenosine and donor sub-domain for binding nicoti-
namide residue of NAD+ Aminoacid sequence in cat-PARP is conserved
and insignificantly differs for different species of higher eykariotes.[2] The
mechanism of poly(ADP-ribosylation) has not been thoroughly investigated
yet. A multipoint fixation of NAD+ at nicotinamide, pyrophosphate, and
adenosine moieties is supposed to direct 2ꢁ-O-hydroxyl group for nucle-
ophilic attack on C-1ꢁ carbon atom with the following release of nicoti-
namide. It has been shown that base modified NAD-analogues: 1,N 6-etheno-
NAD+(ε-NAD+), nicotiamide guanine dinucleotide (NGD+), nicotinamide
hypoxantine dinucleotide (NHD+) are rather poor substrates for PARP-1 as
compared to NAD+, leading to short-chain modified molecules of PAR.[24]
Nevertheless, modified NAD-analogues open possibilities of fluorescent la-
beling of substrate proteins of PARPs, for example, by «click chemistry»
[25], other NAD-consuming enzymes[26], detection of membrane-associated
proteins and even for visualization of organelles, cells, and tissues.[27]
Ionic interactions with target proteins underlie in functional role of
PAR.[28] When linked to various target proteins negatively charged PAR sig-
nificantly changes their properties.[29] PAR-ylation of PARP-1 itself is a gen-
eral pathway of enzymatic activity regulation.[20] A series of proteins partici-
pating in DNA synthesis and repair, transcription, modulation of chromatin
structure, cell cycle are recognized by PARP-1 as specific targets.[1, 28] A wide
[1, 28]
range of proteins are involved in DNA repair, particularly—histones
,

Poly(ADP-Ribose)—A Unique Natural Polymer
263
SCHEME 2 Formation of repair complex on nicked DNA.
X-ray repair cross-complementing proteins (XRCC) [28], DNA-polymerase
[1, 28], catalytic subunit of DNA-polymerase ε, double strand-break re-
β
pair protein MRE-11, DNA ligases I and III, DNA mismatch repair pro-
tein MSH2, DNA-topoisomerases, DNA-dependent protein kinase catalytic
subunit etc.[28] Lysine and arginine-containing motifs in these proteins are
involved in ionic interactions with PAR.[28] It was shown, that binding of
protein depends on molecular weight of PAR. For example p53 manifests
ability to form complexes with long PAR chains (>40 units), while protein
XPA can bind to short (20–40 units) and long (>40 units) PAR chains.[22]
In solution one protein can form several complexes with PAR of different
molecular weights.[22]
Scheme 2 summarizes literature data on interactions of different
proteins, participating in DNA repair.
Ionizing radiation, free radicals, and alkylating agents cause modi-
fication of heterocyclic bases in DNA. The excision of modified base
by DNA-glycosylases and further hydrolysis of injured DNA site by AP-
endonucleases lead to the nick in the DNA-strand, named single-strand break

264
M. S. Drenichev and S. N. Mikhailov
TABLE 1 Composition of repair complexes under two different BER-mechanisms
Type of repair
Participating proteins
Short patch repair (single strand break)
PARPs-1/2, XRCC-1,
DNA-polymerase-β (POL-β),
Ligase-IIIα (LIG-IIIα), WRN (helicase)
PARPs-1/2, XRCC-1,
Long patch repair (multiple single strand breaks)
DNA-polymerases-δ/ε (POL-δ/ε),
Ligase-I (LIG-I), WRN,
Flap-endonuclease-1 (FEN-1),
Proliferating cell nuclear antigen (PCNA)
(SSB). After activation by SSB PARP-1 homodimer poly(ADP-ribosyl)ates
histones, facilitating uncoiling of nicked DNA from modified histones by
electrostatic repulsion.[29–31] X-ray repair cross-complementing I protein
(XRCC-1) has high affinity to PAR and serves as a scaffold for binding
with the repair enzymes and construction of the repair complex[28, 31]
(Table 1).
Protein repair complexes under short patch repair and long patch re-
pair are quite different.[31] PARPs, XRCC-1, DNA-polymerase-β (POL-β),
and ligase-IIIα participate in short patch repair. Long-patch repair complex
contains DNA-polymerases-δ/ε instead of DNA-polymerase-β, ligase-I instead
of ligase-IIIα and also contains flap-endonuclease-1 and protein PCNA that
acts as a processivity factor for DNA-polymerase-ε. It has been shown that
enzyme PARP-2, attached to PARP-1, accelerates DNA repair. PARP-1 in com-
plex with PARP-2 works more effectively than single PARP-1.[31] In fact the
scheme, described here, is simplified, because PARP-1 is involved in many
cellular processes and can recruit hundreds of proteins bearing specific sites
for binding with PAR.[8]
As PARP-1 is involved in DNA repair mechanism, it is considered as the
perspective enzymatic target for design of new drugs for the treatment of
cancer, ischemia and some other diseases.[2, 32–34] It has been shown that
breast cancer-associated genes BRCA1 and BRCA2 play an important role
in the repair of double strand breaks (DSB) through homologous recom-
bination, suppressing tumor growth.[8, 33] The loss of one gene function
(PARP-1 or BRCA1/2) is not dramatic event for the cell, but the loss of both
genes (PARP-1 and BRCA1/2) is lethal. In BRCA1/2 deficient cells PARP-1
is upregulated. In this case, PARP-1 inhibitors can be used as single agents
for the treatment of BRCA-deficient tumors, such as breast and ovarian
cancer.[8, 33] PARP-1 inhibition is a reliable diagnostic test for identification
of BRCA-deficient forms of cancer and for effectivity of PARP inhibition
therapy.[33] The use of alkylating agents in chemotherapy of cancer leads
to DNA damage both in healthy and cancer cells. Therefore the system of
DNA-repair is being activated in both cases. Inclusion of PARP-1 inhibitors
into the complex chemotherapy is expected to decrease concentration of

Poly(ADP-Ribose)—A Unique Natural Polymer
265
TABLE 2 Current clinical trials of PARP-1 inhibitors∗
Single agent/
combination
Therapeutic
indication
Compound
Rucaparib
Company
Pfizer
Phase (2014)
Single agent
Solid tumors,
pancreatic
cancer
Ovarian cancer
Hematological
tumors
2
3
Single agent
Single agent
BMN-673
BioMartin
1 (terminated)
Combination with
temozolomide
(TMZ)
Solid tumors
2
GPI21016/E7016 MGI-
Pharma/Eisai
Combination with
TMZ
Combination with
TMZ
Combination with
hemcytabine
hydrochloride and
cisplatin
Solid tumors
Melanoma
1 (completed)
2
2
Veliparib
Abbott
Adenocarcinoma
and pancreatic
cancer
Single agent
Single agent
Inotek/Genetech Combination with
TMZ
Solid tumors
Solid tumors
Melanoma
1 (completed)
1
1 (terminated)
Olaparib
INO-1001
AstraZeneca
Niraparib
CEP-8983
Merck
Combination with
TMZ
Single agent
Solid tumors
Solid tumors
1
Cephalon
1-2 (terminated)
∗http://www.clinical trials.gov (a service of the U.S. National Institutes of Health)
alkylating agents and, hence, to decrease the toxicity of the therapy. PARP-
1 inhibitors can be used for the treatment of different forms of cancer
in combination with chemotherapeutic agents and also in monotherapy
(Table 2).
A wide range of natural compounds are known to inhibit PARP-1: nucle-
osides, purines and pyrimidines, vitamins (A, K, F), endocrine substances,
taurine, antibiotics, metal ions (Zn2+, Cd2+, Hg2+, Cu2+, Cu+, Co2+, Ni2+).[35]
Substituted benzamides were the first known synthetic analogues of nicoti-
namide and first inhibitors of PARP-1, competing with NAD+ for binding
with PARP-1.[34] Further experiments have led to the discovery of new classes
of inhibitors based on analogues of 3-aminobenzamide with nanomolar in-
hibitory constants [33–34, 36] (Figure 2).
The most important structural motifs in PARP-1 inhibitors are carboxam-
ide fragment and aromatic moiety.[33, 36] To improve some characteristics
of PARP-1 inhibitors auxiliary pharmacophores, ionogenic groups or hy-
drophobic moieties are attached to the molecule of inhibitor via linker of
variable length. Attachment of such functionalized substituents may increase
solubility in water or selectivity of accumulation in cells or ability to form
stable complexes with target enzyme PARP-1.[33]

266
M. S. Drenichev and S. N. Mikhailov
FIGURE 2 Structural motifs in lead PARP-1 inhibitors.[33–34]
Natural nucleosides and nucleotides are less active toward PARP-1 than
polycyclic analogues of 3-aminobenzamide (IC50 ∼ 200 μM).[35] Their prop-
erties can be improved by chemical modifications. Disaccharide nucleosides
seem to be advantageous class of PARP-1 inhibitors due to their cell pene-
trability and low cytotoxicity.[37]
It has been shown, that many of PARP-1 inhibitors, even the drugs for
clinical trials, possess cross-binding affinity to more than one member of
PARP family, thus demonstrating polypharmacology in PARP-inhibition.[29]
To better understand the results of biochemical and clinical studies and
also to avoid the risks, associated with broad-range PARP-inhibition, novel
selective PARP inhibitors are required. Biological activity of the most known
PARP-1 inhibitors is based on interaction with the target in the nicotinamide
pocket of the enzymatic active site.
Nowadays, several routes to new compounds are proposed: (a) targeting
the putative adenine binding site and (b) targeting the D-loop of catalytic
subunit, which is variable in structure and in sequence.[29]

Poly(ADP-Ribose)—A Unique Natural Polymer
267
Biochemical degradation of PAR is mediated by two enzymes, viz.,
ADP-ribosyl protein lyase (scheme 1, pathway b) and poly(ADPribose)
glycohydrolase (PARG) (scheme 1, route c)[17], playing the major role in
stopping of DNA repair system. O-Glycoside bond between adenosine and
ribofuranose moieties in PAR-polymer is split by PARG. The structure of
PARG and several functions, carried out by PARG in cell, are still not well-
38]
studied because of increased sensitivity of this enzyme to proteolysis.[17
,
PARG exists in two isoforms[38]: full-sized enzymatic form (110 kDa) is ac-
cumulated in nucleus, while short form of enzyme (85 kDa) prevails (85%
from total quantity of PARG) and is localized in cytoplasm, Golgii apparatus
and endoplasmatic reticulum. N -terminal part of enzyme is supposed to reg-
ulate PARG localization in cell compartments.[38] Preferable localization of
PARG in cytoplasm is likely necessary for cell protection, because free PAR
is shown to be toxic and can cause cell death, penetrating from nucleus to
cytoplasm.[39] Besides DNA repair and regulation PARP-1 activity in cell nu-
cleus PARG also participates in DNA replication, carrying out the recovery
of initial structure (chromatin recondensation).[17] It was also hypothesized
that PARG participates in apoptosis.[38]
ADP-ribosyl protein lyase catalyzes the removal of proximal adenin-
ediphosphoribosyl (ADPR) residue from proteins but does not cleave
poly(ADP-ribosyl)ated proteins.[17, 40] ADPR-hydrolases (ARH), enzymes,
splitting single ADPR-moieties and short PAR fragments are also known.[41]
Earlier studies indicated that PARG can also function as lyase and catalyze
cleavage of ADPR residues linked to carboxylate groups of histone H1.[17]
3. NEGATIVELY CHARGED NATURAL BIOPOLYMERS AND PAR
In PAR polymer disaccharide units are joined by pyrophosphate link-
age, that makes PAR the most electronegative biopolymer (in pyrophos-
phate pK1 < 2.0, pK2 = 2.64 [42]). Other biopolymers, such as DNA and
RNA, contain only one phosphate group per monomeric unit and have
only one negative charge at neutral pH. Heparan sulfate and heparin be-
long to the glycosaminoglycan polymers built from repeating disaccharide
units. Their main disaccharide unit is shown in Table 3. The presence of
sulfate and carboxyl groups defines the substantial negative charge of these
biopolymers under neutral conditions. In heparin, such groups reach an av-
erage of 2.7 on the disaccharide unit. The main difference between heparin
and heparan sulfate is the lower degree of sulfonation of the later.[43] Tei-
hoic acids isolated from cell wall of gram-positive bacteria consist of repeat-
ing polyol or glycosylpolyol residues joined together with phosphodiester
linkages.[44]

268
M. S. Drenichev and S. N. Mikhailov
TABLE 3 Some negatively charged biopolymers
Charge per monomeric
unit at neutral pH
Polymer
Structure of monomeric unit
Ionogenic group
Phosphate
DNA or RNA
1
PAR
Pyrophosphate
2
Heparin
Sulfate, carboxyl∗
1.35∗
Glycerol teihoic acid
Ribitol teihoic acid
Phosphate
Phosphate
1∗∗
1
∗2.7 on disaccharide unit; ∗∗2 on diglycerol unit.
4. APPROACHES TO THE CHEMICAL SYNTHESIS OF PAR
The development of methods for the synthesis of PAR is still a chal-
lenging problem for chemists and the first obvious step on this way is the
synthesis of disaccharide monomeric unit of PAR. To Oligonucleotides con-
taining disaccharide nucleosides have been obtained so far.[3] However,
the formation of pyrophosphate internucleoside linkage remains the major
problem. Trisaccharide nucleoside, located in the branching points of PAR,
also hasn’t been obtained by chemical approaches.

Poly(ADP-Ribose)—A Unique Natural Polymer
269
4.1. Synthesis of Disaccharide Nucleosides
Disaccharide nucleosides can be synthesized by one of two routes. These
consist of coupling of a protected disaccharide with a heterocyclic base
derivative or the formation of an O-glycosidic bond between a nucleoside car-
rying one free hydroxy group and an activated monosaccharide. Obviously,
the use of available natural carbohydrates and nucleosides can significantly
shorten the synthetic procedure.[3]
Recently 2ꢁ-O-α-D-ribofuranosyladenosine, a monomeric unit of PAR,
was obtained for the first time.[45] The strategy, based on the inversion
SCHEME 3 Synthesis of 2ꢁ-O-α-D-ribofuranosyladenosine.

270
M. S. Drenichev and S. N. Mikhailov
of configuration at C-2 of additional monosaccharide residue in 2ꢁ-O-α-
D-arabinofuranosyladenosine 14, was chosen (Scheme 3). The initial step
was the condensation of TIPDS-protected adenosine 7, carrying one free
hydroxyl group, with 1-O-acetyl-2,3,5-tri-O-benzoyl-α/β-D-arabinofuranose 9
(Scheme 3, conversion i).
The use of sodium methoxide afforded O-debenzoylation (Scheme 3,
conversion ii) but N 6-benzoyl derivative 12 was isolated with only 47% yield
due to the partial desilylation. The use of alkylamines in ethanol for deacyla-
tion of 3ꢁ,5ꢁ-O-TIPDS-protected nucleosides provided more mild conditions
as compared with sodium methoxide, and helped to avoid partial desilylation
of TIPDS-protecting group, essentially increasing the yield of deprotection
stage.[46]
Inversion of configuration at C-2 of the extra monosaccharide residue
in 14 was conducted in two steps—oxidation of the 2ꢁꢁ-hydroxyl group to a
ketone (not shown) and its reduction with sodium borohydride (Scheme 3,
conversion iv).
The use of partially protected nucleoside 8 (R = H) and methylamine in
methanol allowed the increase of the overall yield of the resulting product
3 from 13 to 21%.[47]
Recently, an approach for the preparation of divergently protected 2ꢁ-
O-α-D-ribofuranosyladenosine for the synthesis of various ADP-ribosylated
peptides and PAR oligomers was developed (Scheme 4).
SCHEME 4 Synthesis of protected 2ꢁ-O-α-D-ribofuranosyladenosine.

Poly(ADP-Ribose)—A Unique Natural Polymer
271
Condensation of adenosine derivative 16 with 1-(N -phenyl)-2,2,2-
trifluoroacetamido-2,3,5-tri-O-benzyl-β-D-ribofuranose (17) in the presence
of TMSOTf (scheme 4, conversion i) proceeded with preferable formation
of α-anomer.[48] Benzyl protecting groups (Bn) were removed by BCl3 at
−78◦C (Scheme 4, conversion ii). Further manipulations with protecting
groups led to disaccharide nucleoside 22. However, the yield of 2ꢁ-O-α-D-
ribofuranosyladenosine (3), obtained by this method (10%), was lower than
in previously described scheme 3.
Recently, the new method for the preparation of 2ꢁ-O-α-D-ribofuran-
osylnucleosides have been reported.[49] The described glycosylation
of 3ꢁ,5ꢁ-O-TIPDS-protected uridine with 1-thiotolyl-2,3,5-tri-O-benzyl-D-
ribofuranoside under AgOTf/N -iodosuccinimide catalysis proceeded with
high yield (82%) of blocked 2ꢁ-O-α-D-ribofuranosyluridine.
SCHEME 5 Pyrophosphate modification of oligodeoxynucleotides.

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M. S. Drenichev and S. N. Mikhailov
4.2. Pyrophosphorylation
To date, different methods for the preparation of oligonucleotides, con-
taining disaccharide nucleosides, joined by phosphodiester linkages, have
been elaborated.[3, 50–53] Introduction of labile pyrophosphate bond requires
more mild conditions. Formation of pyrophosphate linkages by various
chemical methods is widely-known and well-described in the literature.[26,
54–57]
The synthesis of oligomers, consisting of nucleosides joined by py-
rophosphate linkages, was done quiet recently.[58] Amidophosphite method
was used for the preparation of such modified DNA-oligomers (Scheme 5).
SCHEME 6 Solid-phase modification of heptapeptide by ADPR.
Synthesis of modified DNA-oligomers on polymeric carrier didn’t re-
quire protection of functional groups in nucleosides: sterically hindered
diphosphytylating reagent, attached to the polymer, facilitated selectivity of

Poly(ADP-Ribose)—A Unique Natural Polymer
273
the reaction at 5ꢁ-OH group. After the standard synthetic procedures, includ-
ing oxidation of pyrophosphites to pyrophosphates, modified oligodeoxynu-
cleotides were released from the polymeric carrier by acidic hydrolysis.
Oligodeoxynucleotides, containing 3ꢁ-5ꢁ-pyrophosphate internucleotide
linkages, formed stable duplexes with complementary DNA strands and
haven’t been degraded under enzymatic hydrolysis.[58] Expanding the de-
scribed protocol on PAR oligomers requires divergently protected disaccha-
ride nucleosidic «building unit».
4.3. ADP-ribosylation of Proteins
Peptides containing ADP-ribosyl moiety can be considered as substrates
for elongation by PARP-1.[59] Modifications of histones by ADPR may be
valuable instrument for investigation of protein interactions with PAR and
mechanisms of PARP, PARG, and ADP-ribosylproteinlyase action in vitro.
Methods of peptide chemistry can be recruited for such modifications. As an
example, ADP-ribosylated isosterical fragment of histone H2B was obtained
by this way (Scheme 6).[59]
This fragment is located at the N -terminal part of histone and undergoes
PAR-ylation during DNA replication or repair.[59] Glutamine, containing ri-
bose moiety (compound A), was included into heptapeptide by peptide
chemistry. After selective removal of tert-butyldiphenylsilyl (TBDPS) protect-
ing group, followed by phosphorylation, the resulting peptide reacted with
activated nucleotide B. Deprotection and cleavage from the polymeric car-
rier was carried out in NH3/MeOH. Peptides, functionalized by aminooxy
groups, can be also site-specifically conjugated to ADP-ribose.[60]
CONCLUSION
In a summary, poly(ADP-ribose) (PAR) is a unique biopolymer, partic-
ipating in numerous key processes in a cell. Its chemical synthesis remains
a challenging problem, which hasn’t been solved yet. PAR is the most elec-
tronegative biopolymer among all known in nature. This property underlies
its binding affinity to a wide range of proteins, including PARP-1, which is
the major enzyme, catalyzing the synthesis of PAR chains with variable length
to regulate the functions of specific proteins and recruit them depending on
the cellular event. Understanding of PAR functions in cell requires chemical
synthesis of regular PAR oligomers. Some natural sulfated and phosphory-
lated biopolymers may be considered as PAR mimics.
ACKNOWLEDGMENTS
The authors are grateful to Dr. V.I. Tararov and Dr. E.N. Timofeev for
useful discussions.

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M. S. Drenichev and S. N. Mikhailov
FUNDING
This work was supported by the Russian Foundation for Basic Research
(14-04-31514) and Russian Academy of Sciences (Program ‘Molecular and
Cell Biology’).
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