Monitoring Poly(ADP-ribosyl)glycohydrolase Activity with a Continuous Fluorescent Substrate

Full text of DOI:10.1016/j.chembiol.2018.09.008

Resource
Monitoring Poly(ADP-ribosyl)glycohydrolase Activity
with a Continuous Fluorescent Substrate
Graphical Abstract
Authors
Bryon S. Drown, Tomohiro Shirai,
Johannes Gregor Matthias Rack,
Ivan Ahel, Paul J. Hergenrother
Fluorescent PARG and ARH3 Substrates
NH2
General Substrate
O
O
OH
N
N
PARG
or
ARH3
N
O
P
O
O
P
O
Correspondence
hergenro@illinois.edu
O
O
O
O
O
N
O
O
O
CF3
+
HO
OH
HO
OH
CF3
TFMU-ADPr
ADP-ribose
In Brief
O
N
ARH3-selective Substrate
Drown et al. describe the design and
synthesis of fluorescent activity probes
for the (ADP-ribosyl)hydrolases PARG
and ARH3. These probes are operational
in biochemical assays or in cell lysate,
and were utilized to identify an
N
N
O
O
OH
NH
O
P
O
O
P
O
ARH3
only
O
O
O
O
O
O
O
O
CF3
HO
OH
HO
OH
+
CF3
TFMU-IDPr
ADP-ribose
Discovery of Endogenous ARH3 Inhibitor
NH2
N
N
endogenous inhibitor of ARH3.
HO
O
O
P
O
O
P
O
H
N
H
N
O
O
N
N
PAR
H2N
O
O
O
ARH3
NH
HO
OH
HO
OH
ADPr-Arg
ADP-ribose
Highlights
d
d
d
d
Synthesis of fluorescent substrates that are enzymatically
cleaved by PARG and ARH3
Synthesis of a fluorescent substrate that is enzymatically
cleaved by ARH3 only
Use of these substrates in cell lysate to measure PARG and
ARH3 activity
Discovery that ARH3 is inhibited by the metabolite ADP-
ribosyl arginine
Drown et al., 2018, Cell Chemical Biology 25, 1–9
December 20, 2018 ª 2018 Elsevier Ltd.
https://doi.org/10.1016/j.chembiol.2018.09.008

Please cite this article in press as: Drown et al., Monitoring Poly(ADP-ribosyl)glycohydrolase Activity with a Continuous Fluorescent Substrate, Cell
Chemical Biology (2018), https://doi.org/10.1016/j.chembiol.2018.09.008
Cell Chemical Biology
Resource
Monitoring Poly(ADP-ribosyl)glycohydrolase
Activity with a Continuous Fluorescent Substrate
Bryon S. Drown, Tomohiro Shirai, Johannes Gregor Matthias Rack, Ivan Ahel, and Paul J. Hergenrother1,3,*
1
1
2
2
1
Department of Chemistry and Institute for Genomic Biology, University of Illinois at Urbana-Champaign, 261 Roger Adams Lab Box 36-5, 600
S. Mathews Avenue, Urbana, IL 61801, USA
2Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OX1 3RE, UK
3Lead Contact
*Correspondence: hergenro@illinois.edu
https://doi.org/10.1016/j.chembiol.2018.09.008
SUMMARY
PARylation as central for proper DNA damage response (An-
drabi et al., 2006; Dantzer et al., 2000; Wang et al., 2016), chro-
The post-translational modification (PTM) and matin maintenance (Becker et al., 2016), cell division (Chang
signaling molecule poly(ADP-ribose) (PAR) has an et al., 2005), and DNA replication (Illuzzi et al., 2014).
In contrast, much is still unknown about the regulation of ADPr
erasure. Several families of enzymes are responsible for removal
and degradation of PARylation. Poly(ADP-ribose) glycohy-
impact on diverse biological processes. This PTM is
regulated by a series of ADP-ribosyl glycohydrolases
(
PARG enzymes) that cleave polymers and/or liberate
0
00
drolase (PARG) hydrolyzes the 2 ,1 glycosidic linkage (red in
Figure 1A) of polymers mainly in an exo manner (Barkauskaite
et al., 2013). PARG modifies PARylated proteins by removing
polymers down to the last unit, which it is incapable of removing
monomers from their protein targets. Existing
methods for monitoring these hydrolases rely on
detection of the natural substrate, PAR, commonly
achieved via radioisotopic labeling. Here we disclose
a general substrate for monitoring PARG activity,
(Slade et al., 2011). ADP-ribosyl hydrolase 3 (ARH3 or ADPRHL2)
is also capable of degrading PAR polymers, albeit at a reduced
TFMU-ADPr, which directly reports on total PAR hy- rate (Mashimo et al., 2014, 2013; Niere et al., 2012; Oka et al.,
drolase activity via release of a fluorophore; this sub- 2006; Ono et al., 2006). Until recently, neither of these proteins
strate has excellent reactivity, generality (processed were believed capable of removing the last ADPr unit directly
bonded to the protein side chain. This hydrolysis is carried
by the major PARG enzymes), stability, and usability.
A second substrate, TFMU-IDPr, selectively reports
on PARG activity only from the enzyme ARH3. Use
of these probes in whole-cell lysate experiments
out by ARH1 or an enzymatic member of the macrodomain
family (terminal ADP-ribosyl protein glycohydrolase (TARG1),
MACROD1, and MACROD2 in mammals) (Barkauskaite et al.,
2015; Feijs et al., 2013; Jankevicius et al., 2013; Mashimo
has revealed a mechanism by which ARH3 is inhibited
by cholera toxin. TFMU-ADPr and TFMU-IDPr are
versatile tools for assessing small-molecule inhibi-
tors in vitro and probing the regulation of ADP-ribosyl
catabolic enzymes.
et al., 2013; Moss et al., 1992; Rosenthal et al., 2013; Sharifi
et al., 2013). Recently ARH3 was shown to be the sole enzyme
capable of hydrolyzing mono-ADP-ribosyl serine (Abplanalp
et al., 2017; Fontana et al., 2017). Beyond substrate recognition,
little is known about how these ADPr erasers are regulated or the
relative importance of different erasers in various biological con-
texts. In particular, study of PAR cleavage has been limited by
the challenge of separating contributions to PAR degradation
by PARG and by ARH3. Several reports suggest that the specific
INTRODUCTION
Many biological processes are regulated by post-translational activity of PARG is significantly greater than ARH3; however, the
modifications (PTMs), with ADP-ribosylation being one of the cellular distribution of these two enzymes differs, as do their reg-
most thoroughly studied nucleotide-containing modifications. ulatory domains (Mashimo et al., 2014). Genetic knockout of
This modification includes a collection of ‘‘writers,’’ ‘‘readers,’’ ARH3 does not affect the lifetime of long polymers but does
and ‘‘erasers.’’ The writers, poly(ADP-ribosyl) polymerases result in longer-lived short polymers (Fontana et al., 2017);
(
PARPs), modify protein substrates with adenine diphosphate thus, much more detailed information on the relative contribution
ribose (ADPr). Nearly all nucleophilic amino acids have been of PAR-degrading enzymes is needed.
shown to be modified: glutamate, aspartate, cysteine, lysine,
The most widely employed enzyme assays for measuring
and serine (Altmeyer et al., 2009; Barkauskaite et al., 2015; Lei- PARG/ARH3 activity rely on radioisotopically labeled and enzy-
decker et al., 2016; Vyas et al., 2014; Zhen et al., 2017). While matically produced PAR (Cortes et al., 2004; Finch et al., 2012;
the majority of PARPs can only transfer a single ADPr unit from M e´ nard and Poirier, 2011; Sharifi et al., 2013). The production
+
NAD (Feijs et al., 2013), the founding member of the family, and isolation of labeled PAR is limited to 0.3–2-mg scale (M e´ nard
PARP1 (but also PARP2 and tankyrases), can synthesize large and Poirier, 2011). After treatment with PARG, reaction mixtures
polymers termed poly(ADP-ribose) (PAR) (Vyas et al., 2014). are separated by TLC or HPLC and quantified by autoradi-
A wealth of literature over several decades has established ography or liquid scintillation counting. This technique, while
Cell Chemical Biology 25, 1–9, December 20, 2018 ª 2018 Elsevier Ltd.
1

Please cite this article in press as: Drown et al., Monitoring Poly(ADP-ribosyl)glycohydrolase Activity with a Continuous Fluorescent Substrate, Cell
Chemical Biology (2018), https://doi.org/10.1016/j.chembiol.2018.09.008
A
NH2
N
N
O
P
O
O
P
O
NH2
N
PARG
or
ARH3
HO
HO
O
O
N
N
O
O
O
N
N
O
P
O
O
P
O
ADP-ribose
O
O
OH
HO
O
N
O
O
O
HO
OH
HO
OH
poly(ADP-ribose) (PAR)
n
1
,2-cis selective
glycosylation
pyrophosphate
NH2
N
B
coupling
N
PARG
or
ARH3
O
P
O
O
P
O
OH
O
O
N
N
O
+
ADP-ribose
= 405 nm
max
O
O
O
O N
2
O N
2
HO
OH
OH
HO
HO
OH
4
-nitrophenol
pNP-ADPr, 1
NH2
N
N
N
PARG
or
ARH3
O
P
O
O
P
O
O
O
OH
+
O
O
O
O
O
N
O
O
O
ADP-ribose
HO
OH
= 384 nm
= 502 nm
CF3
TFMU
ex
em
TFMU-ADPr, 2
CF3
Figure 1. Design of PARG/ARH3 Substrate
(
(
A) PAR is cleaved by PARG and ARH3 via hydrolysis of the glycosyl bond (red).
B) Synthetic PARG/ARH3 substrates mimic ADP-ribose and release a chromophore or fluorophore upon hydrolysis. Synthesis of these compounds requires a
late-stage pyrophosphate formation and 1,2-cis selective glycosylation.
sensitive, is laborious and not scalable. Recent efforts to develop tivity and specificity. Retrosynthetic analysis of pNP-ADPr
more scalable PARG activity assays have been modestly suc- identified two major synthetic hurdles (Figure 1B): a late-stage
cessful; for example, a four-component FRET system to detect pyrophosphate coupling and a 1,2-cis selective glycosylation
the interaction between a PARylated protein and XRCC1 (Kim of a furanoside. Recent work on the synthesis of dimeric PAR
et al., 2015; Stowell et al., 2016). While this approach has been (Lambrecht et al., 2015) overcame similar challenges, and it
implemented in microtiter format and utilized to discover a was envisioned that an analogous strategy could be applied to
first-in-class PARG inhibitor, it suffers from an inability to accu- the synthesis of this simplified PARG substrate.
rately measure enzyme kinetics and cannot operate in cell lysate
(
Glycosylation of electron-deficient phenols such as pNP and
James et al., 2016). There clearly is still an unmet need for a TFMU is often challenging. Most methods rely on anchimeric
facile and continuous activity assay for PAR-degrading enzy- assistance, leading to the undesired 1,2-trans product (Bordoni
matic activity. Herein is described the design, synthesis, and et al., 2010). We found that acceptable a-selectivity could
evaluation of fluorescent probes for PAR hydrolyzing enzymes, be achieved by directly activating a hemi-acetal with bulky pro-
including a compound that is processed by both ARH3 and tecting groups under Mitsunobu conditions. As outlined in Fig-
PARG, and another that is a selective substrate for ARH3. This ure 2, this strategy was employed using orthogonally protected
latter probe has enabled the first direct measurements of ribose 3, prepared in three steps from D-ribonic g-lactone (Fig-
ARH3 activity in cells and was then used to discover the first spe- ure S1A) (Lambrecht et al., 2015); subsequent removal of the
cific means for cellular ARH3 inhibition.
trityl group proceeded smoothly under strictly anhydrous condi-
tions to prevent hydrolysis of the glycoside, producing 4 and 5.
RESULTS AND DISCUSSION
3
Although direct phosphorylation with POCl was possible, we
found phosphoramidite coupling followed by fluorenylmethyl
removal more amenable on scale to give phosphates 6 and 7
Design and Synthesis of PARG Substrates
Reporter substrates for PARG and ARH3, pNP-ADPr and TFMU- (Figures 2, S1B, and S1C). Pyrophosphate formation was
ADPr, were designed, inspired by the natural substrate, PAR. The accomplished via in situ oxidative chlorination of H-phospho-
0
00
2
,1 -glycosidic bond that is cleaved in PAR (highlighted red in nate 8 (Figures 2 and S1D), to produce 9 and 10, followed by
Figure 1A) was replaced with a phenolic glycoside. Initial efforts global desilylation to provide pNP-ADPr and TFMU-ADPr.
focused on utilizing 4-nitrophenol (pNP) as a chromophore re- The late-stage pyrophosphate coupling featured in the synthe-
porter but also extended to the fluorophore 4-(trifluoromethyl) sis of these compounds also facilitates the construction of other
umbelliferone (TFMU) for applications requiring greater sensi- non-natural substrates for PARG. The recently co-crystalized
2
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Please cite this article in press as: Drown et al., Monitoring Poly(ADP-ribosyl)glycohydrolase Activity with a Continuous Fluorescent Substrate, Cell
Chemical Biology (2018), https://doi.org/10.1016/j.chembiol.2018.09.008
For pNP:
a) ADDP, Bu P
3
For TFMU:
O
O
OH
O
RO
O
OH
HO
b) DIAD, Ph P
OH
OTBS
OCPh3
OTBS
3
+
or
c) CF CO H
O N
2
3
2
TBSO
TBSO
(
CF CO) O
CF3
TFMU
3
3 2
4, R = pNP
5, R = TFMU
pNP
R = pNP, 38%
R = TFMU, 24%
d) i-Pr NP(OFm)
2
2
Et NH
3
DCI, CH CN, CH Cl
O
P
O
3
2
2
then: t-BuOOH
RO
O
e) Et N, CH CN
O
OH
3
3
R = pNP, 60%
TBSO
OTBS
R = TFMU, 72%
6
7
, R = pNP
f) NCS, DIPEA
, R = TFMU
CH CN
NH2
NH2
3
g) DBU, THF
N
N
N
N
CN
O
O
P
O
R = pNP, 88%
R = TFMU, 70%
O
N
N
4 steps
N
N
HO
H
O
4
9%
HO
OH
TBSO
OTBS
adenosine
8
NH2
N
N
N
2
X
O
P
O
O
P
O
9, R = pNP, R = TBS, X = DBUH
1
RO
R O
O
O
N
10, R = TFMU, R = TBS, X = DBUH
1
O
O
O
h) Et3
1
2
(pNP-ADPr), R = pNP, R = H, X = NH , 98%
1 4
(TFMU-ADPr), R = TFMU, R = H, X = NH , 96%
OR1
R O
1
OR1
1
4
1
Figure 2. Synthesis of pNP-ADPr and TFMU-ADPr
0
ADDP, 1,1 -(azodicarbonyl)dipiperidine; DBU, 1,8-diazabicycloundec-7-ene; DIAD, diisopropyl azodicarboxylate; Fm, 9H-fluorenylmethyl; DCI, 4,5-dicyanoi-
midazole; NCS, N-chlorosuccinimide; TBS, tert-butyldimethylsilyl.
structure of Latimeria chalumnae ARH3 with ADPr (unpublished) and 4B). A commonly used ortholog of PARG from Tetrahymena
indicates that the binding modes of hPARG and LchARH3 are thermophila (ttPARG) (Barkauskaite et al., 2013; Dunstan et al.,
dramatically different. In particular, the adenine binding site of 2012; Lambrecht et al., 2015) was also characterized and found
LchARH3 is much less organized and more solvent exposed. to effectively hydrolyze pNP-ADPr and TFMU-ADPr (Table 1
M
Molecular docking sugar nucleotides with varied purine bases and Figure S3A). The measured K of ARH3 was similar to
suggests that PARG is highly discriminatory while ARH3 would the enzyme’s reported activity against O-acetyl-ADP-ribose
accommodate other bases (Table S1). Key interactions between (Kasamatsu et al., 2011). The kinetic parameters of pNP-ADPr
ADPr and hPARG Glu727 and Ile726 cannot be recapitulated and TFMU-ADPr were quite similar, likely due to the leaving
with IDPr (inosine diphosphate ribose), and the hPARG binding groups having similar pK
site is too restrictive to allow for additional favorable interactions predicted selectivity of TFMU-IDPr for ARH3 over PARG
Figure 3A). Conversely, the LchARH3 binding site is predicted was confirmed (Figure 4C). Further, both PARG and ARH3 selec-
a
s (7.15 and 7.26). Gratifyingly, the
(
to allow IDPr to shift and make additional interactions with tively hydrolyzed the a anomer of pNP-ADPr over the b anomer,
Lys132 (Figure 3B). We thus hypothesized that replacing the nu- as is consistent with their natural substrates (Figures S3C and
cleobase of phenolic substrates, specifically substitution of S3D). Hydrolysis of substrates depends on catalytically active
adenine for hypoxanthine, would provide a substrate selective protein; inactivating mutations in hPARG (the E756N mutant,
for ARH3. Therefore the compounds TFMU-IDPr and pNP- Lambrecht et al., 2015) and hARH3 (the D77N/D78N mutant,
IDPr were designed and synthesized from inosine (Figures 3C Fontana et al., 2017) abolish substrate processing (Figures S3F
and S2).
and S3G).
In Vitro Processing of Substrates
Use of Substrates to Monitor ARH3 Activity Assay in Cell
Using pNP-ADPr and TFMU-ADPr, the first continuous PARG Culture
and ARH3 activity assays were developed. Enzymatic hydrolysis The ability of TFMU-IDPr to selectively report on ARH3 activity
of phenolic glycosides was easily monitored by an increased within the cellular milieu would be an important application of
absorbance or fluorescence (Figures S3A and S3B). Kinetic these probes. While the experiments in Figure 4 demonstrate
parameters for human PARG and ARH3 were determined by the selectivity of this substrate for ARH3 over PARG in a puri-
non-linear fitting of initial reaction rates (Table 1 and Figures 4A fied enzymatic system, in cells, other hydrolases that recognize
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Please cite this article in press as: Drown et al., Monitoring Poly(ADP-ribosyl)glycohydrolase Activity with a Continuous Fluorescent Substrate, Cell
Chemical Biology (2018), https://doi.org/10.1016/j.chembiol.2018.09.008
A
B
Glu 727
Ile 726
Lys 132
C
O
N
O
N
a) TBSCl, 96%
N
N
N
N
NH b) Cl CCO H, 66%
CN
NH
3
2
O
P
O
c) (PhO) P(O), 83%
2
O
O
d) HOCH CH CN, 73%
2
2
HO
H
O
HO
OH
TBSO
OTBS
O
1
1
inosine
O
N
N
2
NH4
NH
O
O
e) NCS, DIPEA
8%
N
6
O
O
O
O
P
P
O
O
O
7
+
11
O
O
f) Et N-3HF
3
HO
OH
HO
OH
5
3%
CF3
TFMU-IDPr, 12
Figure 3. Molecular Docking Sugar Nucleotides with hPARG and LchARH3 Using Glide XP
(
(
(
A) ADPr (blue) and IDPr (yellow) are docking into hPARG (PDB: 5A7R).
B) ADPr (blue) and IDPr (yellow) are docking into LchARH3 (PDB: 6G1Q).
C) Synthesis of TFMU-IDPr; for pNP-IDPr synthesis please see Figure S2.
ADP-ribose could potentially process this substrate, thus two blot. Thus, it was hypothesized that in this cell line, ARH3 is
experiments were conducted to assess the activity of ARH3 mutated or being affected by an endogenous inhibitor.
and PARG with these substrates in cell lysate. First, the hydroly-
A unique feature of MCF10A cells is the particular additives in
sis of TFMU-IDPr was evaluated in cell lysate from an ARH3 the growth media, and most notable is the inclusion of cholera
knockout isogenic cell line pair derived from U2OS cells (Fontana toxin (CTX), a known ADP-ribosyltransferase (Gill and Meren,
et al., 2017). Knockout of ARH3 resulted in near complete loss of 1978; Wang and Schultz, 2014). Suspecting that the presence
TFMU-IDPr activity (Figure 5A), while TFMU-ADPr hydrolysis of CTX may impact ARH3 activity, MCF10A cells were passaged
was only partially decreased (Figure 5B). Second, substrate pro- five times in the absence of CTX, and ARH3 activity was restored
cessing was evaluated in the presence of the selective PARG (FigureS5A). Thisrecoveryofactivity wasreversed(ina dose-and
inhibitor PDD00017273 (James et al., 2016). Hydrolysis of time-dependent manner) with subsequent addition of CTX,
TFMU-ADPr was completely abolished by PDD00017273 in and this phenotype was reproduced in other cell lines (MCF7
ꢀ
/ꢀ
ARH3
cell lysate, but TFMU-IDPr was unaffected by PARG and MIA PaCa-2) (Figures S5A and S5B). In all these cell lines,
inhibitor. Taken together, these experiments indicate that CTX-mediated ARH3 inhibition occurred rapidly (t1/2 < 60 min,
TFMU-IDPr is selectively cleaved by ARH3 in cell lysate, and Figure S5B). The treatment of cells with CTX appears to be the
TFMU-ADPr is cleaved only by ARH3 and PARG.
first example of selective inhibition ARH3 in cells.
Survey of Cancer Cell Lines
Mechanism of ARH3 Inhibition by ADP-Ribosyl-Arginine
As the activity of ARH3 in various cell types is unknown, experi- Having identified cholera toxin as causing ARH3 inhibition in cell
ments were conducted to assess ARH3 activity in a variety of culture, several experiments were performed to elucidate the
cancer cell lines. Twenty cell lines were analyzed for ARH3 activ- toxin’s mechanism of action. CTX was first found to have no
ity, measured using TFMU-IDPr, and compared with ARH3 direct effect on ARH3 in vitro (Figure S5C). The canonical mech-
abundance (measured by western blot, Figure 5D); in general, anism of CTX involves mono-ADP-ribosylation of an active-site
ARH3 activity and abundance are well correlated (Figure 5E, arginine on Gsa (G-protein alpha subunit); this PTM results in
Table S2). Strikingly, no ARH3 activity is observed with the constitutive activation of adenylyl cyclase (AC), which syn-
MCF10A cells despite the obvious presence of ARH3 by western thesizes cyclic AMP (cAMP) (Figure S6) (Inageda et al., 1991;
4
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Please cite this article in press as: Drown et al., Monitoring Poly(ADP-ribosyl)glycohydrolase Activity with a Continuous Fluorescent Substrate, Cell
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ures 6A and 6B). Interestingly, ADPr-Arg demonstrates markedly
reduced activity against PARG (Figure 6C). This differential inhi-
bition is in contrast to the results for the substrate analogue in-
Table 1. Kinetic Parameters Derived for ADP-Ribosylhydrolase
Substrates
Substrate
Enzyme
K
M
(mM)
Vmax (mmol/min/mg)
hibitor of PARG, the compound ADP-HPD (Koh et al., 2003;
Slama et al., 1995a, 1995b); analysis of ADP-HPD shows that
this compound inhibits both PARG and ARH3 (Figure 6C). Impor-
tantly, ADPr-Arg substantially accumulated in MCF7 cells
following CTX treatment, from concentrations below the limit of
detection in untreated cells to 2.62 ± 0.05 mM in the treated cells
(Figure S5G). Changes in related metabolites were also
observed: cAMP concentration increased as expected for the
a
TFMU-ADPr
hPARG
66.2 ± 15
210 ± 13
6.3 ± 0.2
>1500
0.84 ± 0.05
28.6 ± 0.6
1.61 ± 0.02
ND
b
ttPARG
c
hARH3
hPARG
ttPARG
hARH3
ttPARG
hARH3
ttPARG
hARH3
TFMU-IDPr
>1500
ND
312 ± 30
210 ± 10
3.2 ± 0.6
>1500
1.79 ± 0.06
16.9 ± 0.5
1.7 ± 0.1
ND
pNP-ADPr
pNP-IDPr
+
canonical activity of CTX, NAD concentration decreased (it is
a substrate for ADPr-Arg synthesis), but arginine remained un-
changed, perhaps due to the presence of exogenous arginine
in media. The same trend for these metabolic changes was
also observed in U2OS cells (Figure S5H).
410 ± 20
5.2 ± 0.2
ADPr, adenine diphosphate ribose; ARH3, ADP-ribosyl hydrolase 3; IDPr,
inosine diphosphate ribose; ND, not determined; PARG, poly(ADP-
ribose) glycohydrolase; pNP, 4-nitrophenol; TFMU, fluorophore 4-(tri-
fluoromethyl)umbelliferone.
DISCUSSION
a
Homo sapiens full-length PARG.
b
Tetrahymena thermophila PARG.
This manuscript describes the first continuous substrates for the
glycohydrolases that catabolize PAR. The syntheses of these
substrates are robust and scalable (hundreds of milligrams of
each were prepared), and as such these compounds should
c
H. sapiens full-length ARH3.
Kato et al., 2007). Thus the role of AC and cAMP in CTX-depen- find routine use for the measurement of kinetics, the assessment
dent ARH3 inactivation were investigated. cAMP was found to of inhibitors, and for high-throughput screening applications. In
only weakly inhibit ARH3 (IC50 = 2.96 ± 0.05 mM, Figure S5D). addition, their ability to report on this enzymatic activity in cell
Furthermore, treatment with forskolin, a known activator of AC lysate will enable a variety of experiments where PARG or
(
Florio et al., 1999; Takeda et al., 1983), did not result in ARH3 in- ARH3 activity is the needed readout, and in such experiments
hibition (Figure S5E). Finally, other ADP-ribosyltransferase bac- the ARH3-selective substrate TFMU-IDPr can be used to differ-
terial ecto-toxins that target other amino acid residues do not entiate cellular ARH3 enzymatic activity from PARG activity.
inhibit ARH3 activity (Figure S5F). With these experiments point-
ing upstream of AC, direct interaction with ADP-ribosyl arginine differential contribution of enzyme family members to total enzy-
ADPr-Arg) was considered. To make assessments, ADPr-Arg matic activity. The potential impact such experiments can have
Selective enzyme assays are powerful for assessment of the
(
was synthesized as described previously (Oppenheimer, 1978) is apparent in the widespread use of a variety of compounds
and assessed in the in vitro assay. This compound was found as caspase substrates. For caspases, specificity can be imbued
to potently inhibit ARH3 in vitro, with a K
i
value of 18 ± 2 nM (Fig- through use of peptide sequences known to be specifically
Figure 4. Michaelis-Menton Kinetics of Re-
A
B
1
1
0
.6
.2
.8
0.8
0.6
0.4
0.2
0.0
combinantly Expressed Human PARG and
ARH3
(A) Kinetics of human PARG processing TFMU-
ADPr.
(B) Kinetics of human ARH3 processing TFMU-
ADPr.
H. sapiens PARG
H. sapiens ARH3
0.4
0.0
(C) Selectivity of TFMU-IDPr for processing by hu-
man ARH3 over human PARG. Error bars indicate
SEM, n = 3.
0
50 100 150 200 250 300 350 400
0
20
40
60
80 100 120 140
[TFMU-ADPr] ( M)
[TFMU-ADPr] ( M)
C 1.6
1.2
0.8
0.4
0.0
hARH3
hPARG
0
300
600
900
1200 1500
[TFMU-IDPr] ( M)
Cell Chemical Biology 25, 1–9, December 20, 2018
5

Please cite this article in press as: Drown et al., Monitoring Poly(ADP-ribosyl)glycohydrolase Activity with a Continuous Fluorescent Substrate, Cell
Chemical Biology (2018), https://doi.org/10.1016/j.chembiol.2018.09.008
A
TFMU-IDPr
B
TFMU-ADPr
C
*
*
*
**
ARH3
+/+ -/-
*
*
4
3
2
1
00
00
00
00
0
5000
*
*
*
n.s.
*
**
*
*
4000
3000
2000
PARG
n.s.
*
*
ARH3
actin
n.s.
-/-
1000
0
PARGi (µM)
0
10
0
10
PARGi (µM)
0
10
0
10
ARH3
+/+
ARH3
+/+
-/-
D
E
4
3
2
1
00
00
00
00
0
ARH3
actin
0
.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5
Relative ARH3 Expression
(ARH3/actin)
Figure 5. Measurement of PARG Activity in Cell Lysate and Validation of TFMU-IDPr Selectivity
A) Selectivity for ARH3 by TFMU-IDPr validated by CRISPR-Cas9 knockout of ARH3 in U2OS cells. PARG activity was measured in the presence and absence of
PARG inhibitor PDD00017273 using TFMU-IDPr at 200 mM. Error bars represent SEM, n = 3. Significance levels are given by asterisks: *p < 0.05, **p < 0.01,
**p < 0.001, n.s., p > 0.05.
(
*
(
(
(
B) Same as A but with 200 mM TFMU-ADPr.
C) Validation of ARH3 knockout in U2OS as measured by western blotting.
D) Western blotting to determine ARH3 expression levels in various indicated cell lines; representative blot of two independent replicates shown, see Figure S4
for all replicates.
E) Correlation of ARH3 expression (measured by western blotting) with ARH3 activity (measured by TFMU-IDPr hydrolysis). Data points reflect average values
(
from different mammalian cell lines. Red indicates MCF10A. ARH3 activity was measured with 200 mM TFMU-IDPr.
recognized by different caspase isozymes, and such tool com- toxin (PT) catalyze the mono-ADP-ribosylation of arginine
pounds have been widely employed to understand conditions (CTX and CDT) and cysteine (PT) residues. While the back-
under which specific caspases are activated, and have been crit- ground ADP-ribosylation of arginine by CTX has been
ical to mapping the apoptotic cell death pathway (Berger et al., observed with isolated protein (Oppenheimer, 1978), the activ-
2
006; Por e˛ ba et al., 2013). The lack of analogous reagents for ity of CTX has largely been regarded to be highly selective for
monitoring of PAR processing has necessitated reliance on proteinaceous arginine. We observe and quantify ADP-ribosy-
non-ideal methods, such as isozyme-general processing of ra- lation of free arginine for the first time in cell culture at concen-
diolabeled substrates. In the case of PAR-processing enzymes, trations that rationalize ARH3 inhibition. The absence of ARH3
the development of specific substrates is considerably more inhibition following CDT treatment, despite creating the same
complicated than for a protease (where specificity can be built PTM, may result from differential propensities to produce
in though understanding of the endogenous protein substrates). free and proteinaceous ADPr-arginine. However, the possibility
The recently solved X-ray structures of ARH3 and PARG, and remains that the observed ADPr-arginine results from proteo-
subsequent docking studies, has now allowed for the design of lytic breakdown of ADP-ribosylated G proteins rather than
reporter substrates, one that is general for ARH3 and PARG, direct ADP-ribosylation. In this case, the differential activities
and one that is specific for ARH3.
of bacterial toxins can be attributed to stabilities of their
As a first application, these substrates have been used to respective protein targets. Some of the phenotypes described
discover that CTX suppresses cellular ARH3 activity through upon treatment by bacterial toxins (i.e., loss of tight junctions)
the production of ADPr-arginine, an ARH3-selective inhibitor. has also been described for PAR overproduction (Cuzzocrea
While the effect of bacterial exotoxins on cAMP synthesis and Genovese, 2000; Guichard et al., 2013; Mazzon et al.,
and downstream processes has been extensively described 2002; Nusrat et al., 2001). While the exact mechanism by
(Figure S6), their effects off-pathway are less understood. which these processes operate is yet unknown, inhibition of
CTX, Clostridium difficile binary toxin (CDT), and pertussis ARH3 may assist in the ability of toxins to elevate PAR
6
Cell Chemical Biology 25, 1–9, December 20, 2018

Please cite this article in press as: Drown et al., Monitoring Poly(ADP-ribosyl)glycohydrolase Activity with a Continuous Fluorescent Substrate, Cell
Chemical Biology (2018), https://doi.org/10.1016/j.chembiol.2018.09.008
A
B
6000
4500
3000
1500
0
0.008
0.006
0.004
0.002
[
ADPr-Arg] (nM)
[ADPr-Arg] (nM)
0
5
10
100
50
25
10
5
2
5
50
00
1
0
0
10
20
30
40
50
-0.2 0.0 0.2 0.4 0.6 0.8 1.0
[
TFMU-ADPr] ( M)
/[TFMU-ADPr] ( M-1)
1
C
D
1
20
00
1
NH2
N
N
O
O
80
60
40
20
0
O
P
O
O
P
O
ARH3 - ADP-HPD
H
N
H
N
O
O
N
N
ARH3 - ADPr-arginine H3N
PARG - ADP-HPD
O
O
O
NH2
HO
OH
HO
OH
PARG - ADPr-arginine
ADPr-Arg
-
20
0
.001 0.01
0.1
1
10
100
[
Inhibitor] (µM)
Figure 6. Selective Inhibition of ARH3 by ADP-ribosylated Arginine
(
(
(
(
A) Kinetics of inhibition of ARH3 by ADPr-Arg.
B) Lineweaver-Burke plot of ADPr-Arg inhibition of ARH3.
C) Selectivity of ADPr-Arg for ARH3 over hPARG is indicated by a shift in dose-response curve.
D) Structure of ADPr-Arg.
concentrations. The ready availability of these ARH3 and
PARG substrates will now facilitate additional discoveries
about the regulation of cellular PAR processing.
d KEY RESOURCES TABLE
d CONTACT FOR REAGENT AND RESOURCE SHARING
d EXPERIMENTAL MODEL AND SUBJECT DETAILS
B Cell Lines
d METHOD DETAILS
SIGNIFICANCE
B Buffer Composition
B In Vitro Enzyme Kinetics
ADP-ribosylation has long captured interest due to its prev-
alence in normal biological processes and in disease states,
but study of this PTM has traditionally relied on imprecise
and laborious methods for measuring the activity of ADPr
erasers. As such, the conditions and mechanisms by which
PAR is regulated are poorly understood. Here we introduce a
rapid and precise technology for measuring poly(ADP-
ribose) glycohydrolase activity. Recent advances in pyro-
phosphate couplings have also improved our capability to
construct sugar nucleotides in a modular and scalable
fashion, leading to robust and scalable syntheses of the
target substrates. These substrates are useful for the detec-
tion and quantitation of endogenous cellular PARG and
ARH3 activity, and the utility of this tool is increased with
the discovery that replacement of the nucleobase provides
selectivity for ARH3 over PARG. Together this toolset is ex-
pected to greatly enhance the ability to interrogate PAR-
cleaving enzymes.
B Cell Lysate ARH3 Activity Assay
B ADP-ribosylated Arginine Enzymatic Synthesis
B Metabolic Profiling
B Recombinant Protein Expression and Purification
B Western Blot Analysis
B Molecular Docking
B General Chemical Synthesis
B Synthesis of PARG Substrates
d QUANTIFICATION AND STATISTICAL ANALYSIS
SUPPLEMENTAL INFORMATION
Supplemental Information includes six figures, two tables, and one data file
and can be found with this article online at https://doi.org/10.1016/j.
chembiol.2018.09.008.
ACKNOWLEDGMENTS
We express our thanks to Prof. Hening Lin (Cornell University) for ARH3
plasmid. We thank David Boothman (Indiana University School of Medicine)
for the MIA PaCa cell line, Timothy Fan (University of Illinois, Urbana) for the
CA46 cell line, Navdeep Chandel (Northwestern University) for the MEF cell
line, Gregory Riggins (Johns Hopkins) for the D54 cell line, and Richard Hotch-
kiss (Washington University, St. Louis) for the Jurkat cell line. We are grateful
STAR+METHODS
Detailed methods are provided in the online version of this paper
and include the following:
Cell Chemical Biology 25, 1–9, December 20, 2018
7

Please cite this article in press as: Drown et al., Monitoring Poly(ADP-ribosyl)glycohydrolase Activity with a Continuous Fluorescent Substrate, Cell
Chemical Biology (2018), https://doi.org/10.1016/j.chembiol.2018.09.008
for the University of Illinois and the NIH (R21 CA212732) for funding this work.
The work in I.A.’s laboratory is funded by the Wellcome Trust (grant number
mammalian cells lacking Poly(ADP-ribose) polymerase-1. Biochemistry 39,
7559–7569.
1
01794) and Cancer Research United Kingdom (grant number C35050/
Dunstan, M.S., Barkauskaite, E., Lafite, P., Knezevic, C.E., Brassington, A.,
Ahel, M., Hergenrother, P.J., Leys, D., and Ahel, I. (2012). Structure and mech-
anism of a canonical poly(ADP-ribose) glycohydrolase. Nat. Commun. 3, 878.
A22284).
AUTHOR CONTRIBUTIONS
Feijs, K.L.H., Forst, A.H., Verheugd, P., and L u€ scher, B. (2013). Macrodomain-
containing proteins: regulating new intracellular functions of mono(ADP-ribo-
syl)ation. Nat. Rev. Mol. Cell Biol. 14, 443–453.
B.S.D. performed chemical synthesis of PARG/ARH3 substrates, molecular
docking, cell culture, and enzyme assays. T.S. performed chemical synthesis
of ADP-HPM. J.G.M.R. obtained structure of LchARH3. B.S.D., I.S., J.G.M.R.,
I.A., and P.J.H. designed experiments and analyzed data. B.S.D. and P.J.H.
wrote the manuscript.
Finch, K.E., Knezevic, C.E., Nottbohm, A.C., Partlow, K.C., and Hergenrother,
P.J. (2012). Selective small molecule inhibition of poly(ADP-ribose) glycohy-
drolase (PARG). ACS Chem. Biol. 7, 563–570.
Florio, C., Frausin, F., Vertua, R., and Gaion, R.M. (1999). Amplification of the
cyclic AMP response to forskolin in pheochromocytoma PC12 cells through
adenosine A(2A) purinoceptors. J. Pharmacol. Exp. Ther. 290, 817–824.
DECLARATION OF INTERESTS
The University of Illinois has filed a patent on materials related to this work.
Fontana, P., Bonfiglio, J.J., Palazzo, L., Bartlett, E., Matic, I., and Ahel, I. (2017).
Serine ADP-ribosylation reversal by the hydrolase ARH3. Elife 6, 861.
Received: April 17, 2018
Revised: July 17, 2018
Gill, D.M., and Meren, R. (1978). ADP-ribosylation of membrane proteins cata-
lyzed by cholera toxin: basis of the activation of adenylate cyclase. Proc. Natl.
Acad. Sci. USA 75, 3050–3054.
Accepted: September 12, 2018
Published: October 11, 2018
Guichard, A., Cruz-Moreno, B., Cruz-Moreno, B.C., Aguilar, B., van Sorge,
N.M., Kuang, J., Kurkciyan, A.A., Wang, Z., Hang, S., Pineton de Chambrun,
G.P., et al. (2013). Cholera toxin disrupts barrier function by inhibiting exo-
cyst-mediated trafficking of host proteins to intestinal cell junctions. Cell
Host Microbe 14, 294–305.
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Please cite this article in press as: Drown et al., Monitoring Poly(ADP-ribosyl)glycohydrolase Activity with a Continuous Fluorescent Substrate, Cell
Chemical Biology (2018), https://doi.org/10.1016/j.chembiol.2018.09.008
STAR+METHODS
KEY RESOURCES TABLE
REAGENT or RESOURCE
Antibodies
SOURCE
IDENTIFIER
Mouse anti-ARH3
Rabbit anti-beta-actin
Rabbit anti-PARP1
HRP-conjugated goat anti-rabbit
HRP-conjugated horse anti-mouse
Chemicals, Peptides, and Recombinant Proteins
TFMU-ADPr
Santa Cruz Biotech
Cell Signaling
Cell Signaling
Cell Signaling
Cell Signaling
Cat# sc-374162
Cat# 4970
Cat# 9542
Cat# 7074
Cat# 7076
This paper
n/a
TFMU-IDPr
This paper
n/a
pNP-ADPr
This paper
n/a
pNP-IDPr
This paper
n/a
ADPr-Arg
This paper
n/a
ADP-HPD
Calbiochem
Cat# 118415
Cat# A0752
Cat# 5952
Cat# EN300-05279
Cat# 241326
n/a
ADP-ribose
Sigma-Aldrich
Tocris
PDD 00017273
4
4
2
-(trifluoromethyl)umbelliferone
-nitrophenol
Enamine Building Blocks
Sigma-Aldrich
(Lambrecht et al., 2015)
,3-di-(tert-butyldimethylsilyl)-5-(triphenylmethyl)-D-
ribofuranose
,1’-(azodicarbonyl)dipiperidine
1
Accela ChemBio Inc.
Sigma-Aldrich
Sigma-Aldrich
Sigma-Aldrich
GFS Chemicals
(Lambrecht et al., 2015)
Sigma-Aldrich
Sigma-Aldrich
Oakwood Chemical
Sigma-Aldrich
Sigma-Aldrich
Macherey-Nagel
Sigma-Aldrich
Fisher Scientific
Fisher Scientific
Sigma Aldrich
Fisher Scientific
Fisher Scientific
EMD
Cat# SY001006
Cat# 225541
Cat# T84409
Cat# 90827
Diisopropyl azodicarboxylate
Triphenylphosphine
Tributylphosphine
1
,2-dimethoxyethane
N,N-diisopropyl bis(9-methylfluorenyl)phosphoramidite
,5-Dicyanoimidazole
Cat# 2510
n/a
4
Cat# 554030
Cat# 109681
Cat# 003029
Cat# A3784
N-chlorosuccinimide
Triethylamine trihydrofluoride
L-Arginine
NAD+
Cat# N0632
Silicagel 60M
Cat# 815381.25P
Cat# 217514
Cat# A38-212
Cat# S271-500
Cat# DTT-RO ROCHE
Cat# S311-100
Cat# BP151-100
Cat# SX0715-1
Cat# BDH9266.500
Cat# M33
Dowex 50Wx8
Acetic Acid
Sodium Chloride
DTT
EDTA
Triton-X-100
Sodium phosphate dibasic
Potassium phosphate dibasic
Magnesium chloride
Glycine
VWR Analytical
Fisher Scientific
Bio-Rad
Cat# 161-0718
Cat# M6250
Cat# G33
2-mercaptoethanol
Sigma-Aldrich
Fisher Scientific
Fisher Scientific
Corning
Glycerol
Tween 20
Cat# BP337
0.05% Trypsin
Cat# 25-052-Cl
(Continued on next page)
e1 Cell Chemical Biology 25, 1–9.e1–e19, December 20, 2018

Please cite this article in press as: Drown et al., Monitoring Poly(ADP-ribosyl)glycohydrolase Activity with a Continuous Fluorescent Substrate, Cell
Chemical Biology (2018), https://doi.org/10.1016/j.chembiol.2018.09.008
Continued
REAGENT or RESOURCE
SOURCE
IDENTIFIER
Cat# 539134
Cat# 100-106
Cat# A30075-100.0
Cat# 409-10
Cat# 11058-021
Cat# 100B
Protease Inhibitor Cocktail Set III, EDTA-free
Calbiochem
Fetal Bovine Serum
Gemini
BSA
Research Products International Corp.
Polyplus
INTERFERin Transfection Agent
Opti-MEM Media
ThermoFisher
Cholera Toxin from Vibrio cholerae
List Biological Labs
List Biological Labs
List Biological Labs
List Biological Labs
Qiagen
Binary Toxin from Clostridium difficile, Subunit A
Cat# 157A
Binary Toxin from Clostridium difficile, Subunit B
Cat# 157B
Pertussis Toxin from B. pertussis
Ni-NTA
Cat# 180
Cat# 30230
Cat# 12481C100
Cat# K22000
Cat# BP1760
Cat# C0378
Cat# BP152
Cat# I202
IPTG
Gold Biotechnology
Research Products International
Fisher Scientific
Sigma-Aldrich
Kanamycin
Ampicillin
Chloramphenicol
Tris base
Fisher Scientific
Sigma-Aldrich
Imidazole
Leupeptin
Sigma-Aldrich
Cat# L2884
Cat# P4265
Cat# A1153
Cat# 78830
n/a
Pepstatin A
Aprotinin
Sigma-Aldrich
Sigma-Aldrich
PMSF
Sigma-Aldrich
Human full-length PARG
(Barkauskaite et al., 2013;
Tucker et al., 2012)
T. thermophila PARG protein
(Dunstan et al., 2012)
n/a
n/a
Human ARH3 protein
(Barkauskaite et al., 2013;
Finch et al., 2012)
Human ARH3 D77N/D78N protein
Human PARG catalytic domain wild type protein
Human PARG catalytic domain E756N protein
Critical Commercial Assays
(Fontana et al., 2017)
(Tucker et al., 2012)
(Tucker et al., 2012)
n/a
PARG26
n/a
Pierce BCA Protein Assay Kit
ThermoFisher
ThermoFisher
Lonza
Cat# 23227
SuperSignal West Pico
Cat# 34577
Penicillin/Streptomycin
Cat# 17-602E
Cat# 456-1093
Cat# 161-0732
Cat# 161-0737
Cat# 21059
Mini-PROTEAN TGX 4-20% Precast Gel
SDS (Tris/Glycine) Buffer (10x)
Laemmili Sample Buffer
Bio-Rad
Bio-Rad
Bio-Rad
Restore Western Blot Stripping Buffer
Immobilon-P PVDF Transfer Membrane
Deposited Data
ThermoFisher
EMD Millipore
Cat# IPV00010
Structure of Human PARG
(Lambrecht et al., 2015)
PDB:5A7R
PDB:6G1Q
Structure of Latimeria chalumnae ARH3
Experimental Models: Cell Lines
Human: A549
ATCC
Cat# CCL-185, RRID:CVCL_0023
RRID:CVCL_1101
Human: CA46
From Tim Fan (University of
Illinois, Urbana)
Human: HCT-116
Human: HEK293TN
Human: HeLa
ATCC
Cat# CCL-247, RRID:CVCL_0291
Cat# LV900A-1
System Biosciences
ATCC
Cat# CCL-2, RRID:CVCL_0030
Cat# HB-8065, RRID:CVCL_0027
Human: HepG2
ATCC
(Continued on next page)
Cell Chemical Biology 25, 1–9.e1–e19, December 20, 2018 e2

Please cite this article in press as: Drown et al., Monitoring Poly(ADP-ribosyl)glycohydrolase Activity with a Continuous Fluorescent Substrate, Cell
Chemical Biology (2018), https://doi.org/10.1016/j.chembiol.2018.09.008
Continued
REAGENT or RESOURCE
Mouse: MEF
SOURCE
IDENTIFIER
n/a
From Navdeep Chandel
(Northwestern University)
Human: MIA PaCa-2
From David Boothman (Indiana
University School of Medicine)
RRID:CVCL_0428
Human: U2OS
Human: U2OS ARH3 KO
Human: U87
ATCC
Cat# HTB-96, RRID:CVCL_0042
(Fontana et al., 2017)
ATCC
Cat# HTB-14, RRID:CVCL_0022
Cat# CRL-1593, RRID:CVCL_0007
RRID:CVCL_7185
Human: U937
ATCC
Human: D54
From Gregory Riggins
(Johns Hopkins)
Human: Daudi
Human: HCC1937
Human: HCC38
Human: HFF-1
Human: Hs578t
Human: Jurkat
ATCC
ATCC Cat# CCL-213, RRID:CVCL_0008
Cat# CRL-2336, RRID:CVCL_0290
Cat# CRL-2314, RRID:CVCL_1267
Cat# SCRC-1041, RRID:CVCL_3285
Cat# HTB-126, RRID:CVCL_0332
n/a
ATCC
ATCC
ATCC
ATCC
From Richard Hotchkiss
(Washington University, St. Louis)
Human: MCF7
ATCC
ATCC
ATCC
ATCC
Cat# HTB-22, RRID:CVCL_0031
Cat# CRL-10317, RRID:CVCL_0598
Cat# HTB-70, RRID:CVCL_0527
Cat# CRL-1690, RRID:CVCL_0556
Human: MCF10A
Human: SK-MEL-5
Human: T98G
Experimental Models: Organisms/Strains
E. coli: Rosetta 2 (DE3)
Recombinant DNA
Plasmid: hARH3-pDEST
Plasmid: hPARG-pColdTF
Plasmid: ttPARG-pET28a
Software and Algorithms
Prism 6
Novagen
Cat# 71400
Hening Lin (Cornell University)
Ivan Ahel (Oxford University)
Ivan Ahel (Oxford University)
n/a
n/a
n/a
GraphPad
Molecular Devices
NIH
SoftMax Pro 6.4
Build 204720
ImageJ (FIJI)
http://imagej.net/Fiji/Downloads
Image Lab 4.1
Bio-Rad
PyMOL
PyMOL Molecular Graphics
System (built from source)
https://www.pymol.org/
Version 2018-1
Small Molecule Drug Discovery Suite
Other
Schr o¨ dinger
CombiFlash Rf+
Teledyne Isco
Molecular Devices
Bio-Rad
Cat# 68-5230-022
Model# M3
SpectraMax Multi-mode Microplate Reader
ChemiDoc MP Imaging System
RediSep Rf C18 Gold 5.5g
RediSep Rf C18 Gold 150g
Zorbax Eclipse Plus C18 1.8mm 2.1x50mm
Luna C18 5 mm 21.2x150mm
Digital Sonifier
Cat# 170-8280
Teledyne Isco
Teledyne Isco
Agilent
Cat# 69-2203-328
Cat# 69-2203-338
Cat# 959741-902
Cat# 00F-4252-P0-AX
Model# S-450
Phenomenex
Branson Ultrasonics
CONTACT FOR REAGENT AND RESOURCE SHARING
All requests for reagents and resources should be directed to the lead contact, Paul Hergenrother (hergenro@illinois.edu).
e3 Cell Chemical Biology 25, 1–9.e1–e19, December 20, 2018

Please cite this article in press as: Drown et al., Monitoring Poly(ADP-ribosyl)glycohydrolase Activity with a Continuous Fluorescent Substrate, Cell
Chemical Biology (2018), https://doi.org/10.1016/j.chembiol.2018.09.008
EXPERIMENTAL MODEL AND SUBJECT DETAILS
Cell Lines
A549, D54, HCC1937, HCC38, HCT-116, Jurkat, and U937 cells were grown in RPMI 1640 supplemented with 10% fetal bovine
serum (Gemini) and 1% pen/strep. U2OS, Daudi, HEK293TN, HeLa, Hs578t, MEF, MIA PaCa-2, and SK-MEL-5 cells were
grown in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum and 1% pen/strep. HFF-1 cells were
grown in Dulbecco’s modified Eagle’s medium supplemented with 15% fetal bovine serum and 1% pen/strep. HepG2, MCF7,
T98G, and U87 cells were grown in Eagle’s Minimum Essential Medium supplemented with 10% fetal bovin serum and 1% pen/strep.
MCF10A cells were grown in Dulbecco’s modified Eagle’s medium supplemented with 2% horse serum, 20 ng/mL EGF, 0.5 mg/mL
hydrocortisone, 10 mg/mL insulin, 100 ng/mL cholera toxin, and 1% pen/strep. Phenol red was added to all media except for
ꢁ
MCF10A as a pH indicator. All cells were cultured at 37 C in a 5% CO
2
environment. Media were prepared by the University of
Illinois School of Chemical Sciences Cell Media Facility. Sex of cell lines: A549 (Male, 58 years old), CA46 (Male, age unknown),
D54 (Female, age unknown), Daudi (Male, 16 years old), HCC1937 (Female, 23 years old), HCC38 (Female, 50 years old), HCT-
1
16 (Male, age unknown), HEK293TN (Female, fetus), HeLa (Female, 31 years old), HepG2 (Male, 15 years old), HFF-1 (Male,
newborn), Hs578t (Female, 74 years old), MCF7 (Female, 69 years old), MCF10A (Female, 36 years old), MEF (sex and age unknown),
MIA PaCa-2 (Male, 65 years old), Jurkat (Male, 14 years old), SK-MEL-5 (Female, 24 years old), T98G (Male, 61 years old), U2OS
(
Female, 15 years old), U87 (Male, age unknown), U937 (Male, 37 years old).
METHOD DETAILS
Buffer Composition
PARG Activity Lysis Buffer: 30 mM Tris (pH 7.5), 500 mM NaCl, 20% glycerol, 1% Triton X-100, 1:500 protease inhibitor cocktail.
Lysate Activity Buffer: 50 mM K
T. thermophila PARG reaction buffer: 50 mM K
Human PARG reaction buffer: 50 mM K HPO , 50 mM KCl, 10 mM b-mercaptoethanol, pH 7.40.
Human ARH3 reaction buffer: 50 mM Na HPO , 10 mM MgCl , 5 mM DTT, pH 7.40.
PARG Purification Lysis buffer: 50 mM NaH PO , 300 mM NaCl, 10 mM imidazole, 0.5 mg/mL lysozyme, 1 ug/mL leupeptin, 1 ug/mL
pepstatin A, 2 ug/mL aprotinin, 1 mM PMSF, pH 8.0.
PARG Purification Wash buffer: 50 mM NaH PO , 300 mM NaCl, 10 mM imidazole, pH 8.0.
PARG Purification Elution buffer: 50 mM NaH PO , 300 mM NaCl, 300 mM imidazole, pH 8.0.
PARG Purification Dialysis buffer: 25 mM Tris, 50 mM NaCl, 1 mM DTT, 10% glycerol, pH 7.5.
2
HPO
4
(pH 7.4), 50 mM KCl, 5 mM MgCl
2
, 5 mM DTT.
2
HPO , 50 mM KCl, 10 mM b-mercaptoethanol, pH 7.40.
4
2
4
2
4
2
2
4
2
4
2
4
3
Towbin Transfer Buffer: 20% CH OH, 192 mM glycine, 50 mM Tris, pH 8.3.
In Vitro Enzyme Kinetics
1
2
0X dilutions of substrate were prepared from 10 mM stock solutions in MilliQ H O into reaction buffer. 5 mL 10X substrate was added
to a 384-well plate. 45 mL 2.2 nM enzyme solution in reaction buffer was added to a single column of the 384-well plate via 16-channel
Matrix pipet. Plate was placed into plate reader. After shaking for 5 s, absorbance or fluorescence was recorded at 2 s intervals for
5
min. Initial reaction rates were determined by fitting the linear portions of reaction progress curves using SoftMax Pro 6.4. Initial
rates were plotted against substrate concentration and fit to the Michaelis-Menton equation using a non-linear curve-fitting algorithm
with GraphPad Prism 6. For pNP detection, plate reader was configured: absorbance at 405 nm. For TFMU detection, plate reader
was configured: Excitation 385 nm, Cutoff 495 nm, Emission 502 nm, 6 reads/well, Low Gain.
Cell Lysate ARH3 Activity Assay
Cells were cultured in T75 flasks in appropriate medium. Cells were trypsinized, and scrapped (if necessary), and counted. 1x10 cells
6
were harvested and washed with cold PBS 2x. Cell pellet was lysed by addition of activity lysis buffer (150 mL) and incubated on ice for
ꢁ
3
0 min. Lysate was clarified by centrifugation at 14,300xg at 4 C for 10 min. Supernatant was transferred to chilled, empty 500 mL
tube. Total protein content of lysate was evaluated by BCA assay. ARH3 expression level was determined by western blotting
analysis. 5 mL lysate was added to well of 384-well black plate. 45 mL TFMU-IDPr (200 mM final) in reaction buffer was added to
well. Reaction progress was monitored by fluorescent plate reader (ex 385 nm, cutoff 495, em 502 nm, 6 reads/well, high gain).
ADP-ribosylated Arginine Enzymatic Synthesis
CTX (100 mL, 500mg/mL, 10mg/mL final) was added to 5 mL buffer (400 mM K
2
HPO
4
, 20 mM DTT, pH 7.2) containing 200 mM arginine
ꢁ
and 10 mM NAD. Mixture was incubated at 37 C for 16 h. Protein was removed from reaction mixture by filtration through 3k MWCO
spin filter. Filtrate was direction subjected to ion-pairing preparative HPLC using Luna C18 21.5x150mm column. Solvent A: 20 mM
3
Et N,HOAc (pH 7.2), solvent B: acetonitrile. Gradient (A:B, 20 mL/min): 98:2, 0 min; 98:2, 2 min; 75:25, 16 min; 75:25, 23 min; 40:60,
2
7 min, 40:60, 30 min. ADP-ribosylated arginine eluted at ꢂ5 min as two separate anomers. Individual anomers gradually intercon-
verted in ꢂ1 h, so characterization and inhibition experiments were performed with mixture of anomers. Product-containing fractions
1
were determined by LCMS and lyophilized to yield flocculent white solid (7.8 mg, 21%). H NMR was consistent with previous reports
(
Oppenheimer, 1978).
Cell Chemical Biology 25, 1–9.e1–e19, December 20, 2018 e4

Please cite this article in press as: Drown et al., Monitoring Poly(ADP-ribosyl)glycohydrolase Activity with a Continuous Fluorescent Substrate, Cell
Chemical Biology (2018), https://doi.org/10.1016/j.chembiol.2018.09.008
Metabolic Profiling
Six-well plates were seeded with cells at 3x105 cells/well. When cells were ꢂ80% confluent, cells were treated with 100 ng/mL CTX
for 6 h. Cells were washed with PBS once and detached with trypsin, quenching trypsin with media with 10% FBS. Cell viability was
ꢁ
verified by Trypan Blue exclusion. Cells were centrifuged at 400xg at 4 C for 4 min and washed with PBS twice. Cell pellets were
resuspended with cold 70:30 methanol:water and placed on ice. Cell suspension was sonicated on ice (20%, 10 s on, 10 s off,
ꢁ
3
0 s total) and continued to be incubated on ice for 30 min. Lysate was clarified by centrifugation at 14,300xg at 4 C for 30 min. Super-
ꢁ
natent was transferred to an empty 0.5 mL tube and stored at -80 C until analysis. Samples were analyzed with the 5500 QTRAP
LC/MS/MS system (Sciex, Framingham, MA) in Metabolomics Lab of Roy J. Carver Biotechnology Center, University of Illinois at
Urbana-Champaign. Software Analyst 1.6.2 was used for data acquisition and analysis. The 1200 series HPLC system (Agilent
Technologies, Santa Clara, CA) includes a degasser, an autosampler, and a binary pump. The LC separation was performed on
an Agilent SB-Aq (4.6 x 50mm, 5mm) with mobile phase A (0.1% formic acid in water) and mobile phase B (0.1% formic acid in
acetontrile). The flow rate was 0.3 mL/min. The linear gradient was as follows: 0-3min, 100%A; 5-10min, 2%A; 11-15min, 100%A.
ꢁ
The autosampler was set at 10 C. The injection volume was 5 mL. Mass spectra were acquired under positive electrospray ionization
ꢁ
(
ESI) with the ion spray voltage of +5500 V. The source temperature was 450 C. The curtain gas, ion source gas 1, and ion source
gas 2 were 36, 65, and 60, respectively. Multiple reaction monitoring (MRM) was used for quantitation: Arg m/z 175.1–> m/z 70.1;
ADP-Arg m/z 716.2–> m/z 428.1; cAMP m/z 330.2–> 232.0; ADP-ribose m/z 560.1–> m/z 348.1; NAD m/z 664.2–> m/z 136. Limit
of quantitation (LOQ) for the compounds were: 10 ng/mL ADPr-Arg, 10 ng/mL ADP-ribose, 10 ng/mL arginine, 20 ng/mL cAMP,
+
and 100 ng/mL NAD .
Recombinant Protein Expression and Purification
Human ARH3 was expressed as previously described (Barkauskaite et al., 2013; Finch et al., 2012). Briefly, human ARH3 cloned into
a pDEST vector with an N-terminal His
6
-tag was obtained from Hening Lin (Cornell University, Cornell, NY) was transformed into
Rosetta2 (DE3) E. coli cells. 500 mL culture of LB (100 mg/mL ampicillin and 20 mg/mL chloramphenicol) was inoculated with
ꢁ ꢁ
0 mL of overnight culture. The culture was grown at 37 C to mid-log phase (OD600 = 0.5) and cooled to 18 C. Protein expression
1
was induced with 0.1 mM IPTG for 18 h and harvested. Pellet was resuspended in 13 mL lysis buffer and lysed by sonication. Lysate
ꢁ
was clarified by centrifugation (35,000xg, 30 min, 4 C). Supernatent was purified on Ni-NTA beads by batch protocol. Fractions
ꢁ
containing pure protein were combined, dialyzed, and stored at -80 C. Protein concentration was determined by BCA assay.
T. thermophila PARG enzyme was expressed using a modified protocol a previously reported (Dunstan et al., 2012). Briefly,
T. thermophila PARG2 cloned into a pET28a vector was obtained from Ivan Ahel (University of Oxford, Oxford, UK) and transformed
into Rosetta2 (DE3) E. coli. 500 mL culture of LB (100 mg/mL kanamycin and 20 mg/mL chloramphenicol) was inoculated with 10 mL of
ꢁ
overnight culture. The culture was grown to mid-log phase (OD600 = 0.7). Culture was induced with 0.3 mM IPTG at 30 C for 3 h and
harvested. Pellet was resuspended in 13 mL lysis buffer and lysed by sonication. Lysate was clarified by centrifugation. Protein was
ꢁ
purified on Ni-NTA beads by batch protocol. Fractions containing pure protein were combined, dialyzed, and stored at -80 C. Protein
concentration was determined by BCA assay.
Human PARG cloned into a pColdTF vector was obtained from Ivan Ahel (University of Oxford, Oxford, UK) and transformed into
ꢁ
Rosetta2 (DE3) E. coli. Culture of Terrific Broth supplemented with appropriate antibiotics was grown to an OD600 = 0.6-0.8 at 37 C.
ꢁ
Culture was induced with 0.1 mM IPTG at 18 C for 16 h and harvested. Pellet was resuspended in lysis buffer (40 mM HEPES,
3
00 mM NaCl, 20 mM imidazole, 10% glycerol, 1 mM TCEP, pH 8.0) supplemented with benzonase, lysozyme and protease inhibitor
Roche Complete EDTA-free protease inhibitor tablet) and lysed by freeze/thaw. Lysate was clarified by centrifugation. Protein was
(
purified on Ni-NTA beads by batch and eluted with lysis buffer supplemented with 500 mM imidazole.
Western Blot Analysis
Samples loading was normalized based on total protein content as assessed by BCA assay and subjected to SDS-PAGE. Protein
was transferred to PVDF membranes (Merck Millipore). Membranes were cut along molecular weight markers and then blocked
with TBS-T buffer (25 mM Tris-HCl, 150 mM NaCl, 0.05% Tween 20, pH 7.5) supplemented with either 5% BSA (PARP1) or 5%
ꢁ
non-fat dried milk (ARH3) for 1 h. Membrane strips were incubated overnight with primary antibodies in blocking buffer at 4 C
with gentle rocking. Membranes were washed 3x with TBS-T and incubated with HRP-conjugated secondary antibody in TBS-T
for 1 h. Membranes were washed 3x with TBS-T, developed using SuperSignal West Pico and imaged using a ChemiDoc MP system.
Membrane strip containing ARH3 bands was stripped and reimaged with anti-actin antibody. Dilutions for antibodies were: anti-
PARP1 (1:3000), anti-ARH3 (1:1000), anti-actin (1:3000), anti-rabbit (1:3000), and anti-mouse (1:3000). Band quantification was per-
formed using FIJI.
Molecular Docking
Protein and ligand preparation, docking, and scoring was performed using the Schrodinger Suite 2018-1. Proteins were prepared
using the Protein Prep Wizard using standard settings with pH set to 7.4. Receptor grids were generated based on bound ADP-
ribose. Additional positional constraints were applied so that the nucleobase occupies the adenine binding site and the ribose
ring occupies the active site. Sugar nucleotide protonation state and tautomer form was determined using LigPrep. Docking was
performed with Glide using both standard precision (SP) and extra precision (XP). Docking poses were exported and rendered using
PyMOL.
e5 Cell Chemical Biology 25, 1–9.e1–e19, December 20, 2018

Please cite this article in press as: Drown et al., Monitoring Poly(ADP-ribosyl)glycohydrolase Activity with a Continuous Fluorescent Substrate, Cell
Chemical Biology (2018), https://doi.org/10.1016/j.chembiol.2018.09.008
General Chemical Synthesis
All reactions were run in flame or oven dried glassware under and atmosphere of dry nitrogen unless otherwise noted. Acetonitrile,
tetrahydrofuran, methanol, dimethylformamide, toluene, and methylene chloride using in reactions were obtained from a solvent
ꢁ
˚
dispensing system. 4 A molecular sieves were dried at 150 C on high vacuum overnight. Pyridine, diazabicyclo[5.4.0]undec-7-
˚
2
ene, diisopropylethylamine, and trimethylamine were distilled from CaH and stored on 4 A sieves. All other reagents were of
standard commercial purity and were used as received. Analytical thin-layer chromatography was performed on EMD Merck silica
gel plates with F254 indicator. Plates were visualized with UV light (254 nm) or staining with p-anisaldehyde. Silica gel for column
chromatography was purchased from Macherey-Nagel (40-63 mm particle size).
1
13
19
31
Unless otherwise indicated, H, C, F, and P NMR spectra were recorded at 500, 126, 470, and 202 MHz, respectively.
1
13
H and C NMR spectra were referenced to the residual solvent peak. F NMR spectra were referenced using absolute refer-
19
3
1
2 4
encing based on 1H spectra. P NMR spectra were externally referenced to 85% H PO (0.00 ppm) in water. Chemical shits
are reported in ppm and multiplicities are reported as s (singlet), d (doublet), t (triplet), q (quartet), p (pentet), h (hextet), m (multi-
plet), and br (broad). For annotated NMR spectra see Data S1. Mass spectrometry analysis was performed by the University of
Illinois Mass Spectrometry Center.
Preparative C18 chromatography was performed using a Teledyne Isco Combiflash Rf system with RediSep Gold columns. Silyl
ether protected intermediates were separated using a gradient of H
O:35% CH CN over 6 min, ramping to 100% CH CN over 6 min, and holding 100% CH
separated using a gradient of 10 mM Et N,HOAc (pH 7.2)/CH CN beginning with 100% buffer, ramping to 15% buffer:85% CH
CN over 6 min, and holding 50% buffer:50% CH CN for 4 min.
2
O/CH
3
CN beginning with 95% H
CN for 5 min. Unprotected substrates were
CN
2 3
O:5% CH CN, ramping to 65%
H
2
3
3
3
3
3
3
over 10 min, ramping to 50% buffer:50% CH
3
3
Synthesis of PARG Substrates
5
-O-trityl-D-ribono-1,4-lactone (S1)
Synthesized with modification of previous report (Taylor et al., 2002). Specifically, to a 250 mL round bottom flask was added
D-ribonic acid g-lactone (2.15 g, 14.5 mmol, 1 eq), 4-dimethylaminopyridine (360 mg, 2.9 mmol, 0.2 eq), and pyridine (42 mL).
Once fully dissolved, triphenylmethyl chloride (4.86 g, 17.4 mmol, 1.2 eq) was added as a solid in one portion to the stirring reaction
ꢁ
mixture at room temperature. The solution was stirred at 70 C for 16 h. The cooled reaction mixture was diluted with dichloromethane
3 4
and washed with 1 M HCl twice and satd aq NaHCO once. The organic layer was dried over MgSO , filtered through a pad of celite,
and concentrated in vacuo. The residue was purified by silica gel chromatography, eluting with 1:1 hexanes-EtOAc, yielding
compound S1 as a white solid (5.0 g, 88%).
1
6
H NMR (500 MHz, DMSO-d ) d 7.41 – 7.24 (m, 15H), 5.93 (d, J = 7.6 Hz, 1H), 5.45 (d, J = 4.0 Hz, 1H), 4.54 (dd, J = 7.6, 5.5 Hz, 1H),
4
.39 – 4.34 (m, 1H), 4.02 (ddd, J = 5.3, 4.0, 1.2 Hz, 1H), 3.41 – 3.33 (m, 1H), 3.14 (dd, J = 11.0, 3.8 Hz, 1H).
13
6
C NMR (126 MHz, DMSO-d ) d 176.2, 143.2, 128.2, 128.1, 127.3, 86.7, 83.4, 69.4, 68.6, 63.0.
+
22 5
HRMS (ESI-TOF) m/z: [M+Na] Calcd for C24H O Na 413.1365; Found 413.1362.
2
,3-bis-O-tert-butyldimethylsilyl-5-O-trityl-D-ribono-1,4-lactone (S2)
Synthesized with modification of previous reports (Taylor et al., 2002). Specifically, to a 100 mL round bottom flask was added S1
ꢁ
(
2.0 g, 5.1 mmol, 1 eq), imidazole (1.9 g, 27 mmol, 5 eq), and dimethylformamide (20 mL). The solution was cooled to 0 C. Tert-bu-
tyldimethylsilyl chloride (2.9 g, 19 mmol, 4 eq) was added as a solid in one portion. Solution was allowed to warm to room temperature
and was stirred for 16 h. Reaction mixture was diluted with diethyl ether and quenched with the addition of satd aq NH Cl. Organic
layer was washed with satd aq NaHCO thrice and brine once. Organic layer was dried over Na SO , filtered, and concentrated. Res-
idue was purified by silica gel chromatography, eluting with 5% Et
4
3
2
4
2
O-hexane, to yield compound S2 as a white foam (3.1 g, 97%).
Cell Chemical Biology 25, 1–9.e1–e19, December 20, 2018 e6

Please cite this article in press as: Drown et al., Monitoring Poly(ADP-ribosyl)glycohydrolase Activity with a Continuous Fluorescent Substrate, Cell
Chemical Biology (2018), https://doi.org/10.1016/j.chembiol.2018.09.008
1
3
H NMR (500 MHz, CDCl ) d 7.40 (d, J = 8.4 Hz, 6H), 7.31 (t, J = 7.6 Hz, 6H), 7.25 (t, J = 7.1 Hz, 3H), 4.68 (d, J = 5.2 Hz, 1H), 4.29
(
t, J = 3.0 Hz, 1H), 3.95 (dd, J = 5.2, 1.1 Hz, 1H), 3.62 (dd, J = 11.0, 3.7 Hz, 1H), 3.21 (dd, J = 11.0, 2.8 Hz, 1H), 0.93 (s, 9H), 0.80 (s, 9H),
.18 (s, 3H), 0.11 (s, 3H), 0.01 (s, 3H), -0.06 (s, 3H).
0
1
3
C NMR (126 MHz, CDCl
3.45, -4.47, -4.70, -5.04.
3
) d 175.27, 143.19, 128.63, 128.18, 127.49, 84.70, 77.16, 72.16, 70.44, 62.39, 25.96, 25.75, 18.51, 18.20,
-
+
HRMS (ESI-TOF) m/z: [M+Na] Calcd for C36
H
50
O
5
Si
2
Na 641.3094; Found 641.3093.
2
,3-bis-O-tert-butyldimethylsilyl-5-O-trityl-D-ribose (3)
AlH) in hexane (17.5 mL, 17.5 mmol, 1.5 eq) was added dropwise via addition funnel over 5 min to a stirring
solution of S2 (7.25 g, 11.7 mmol, 1 eq) in CH
quenched with the addition of CH
A 1 M solution of (i-Bu
2
2
ꢁ
ꢁ
2
Cl
2
(120 mL) at -78 C. The resulting solution was stirred at -78 C for 3 h. Reaction was
ꢁ
3
OH (4 mL) added dropwise via addition funnel and allowed to warm to 0 C. After stirring for 30 min,
2
00 mL 0.5 M potassium sodium tartrate was added and mixture was stirred until aluminum salts were fully dissolved. Extracted
mixture with CH Cl three times. The combined organic layers were dried over MgSO , filtered through a pad of celite, and concen-
O-hexanes, to yield an interconverting mixture of
2
2
4
trated. The residue was purified by silica gel chromatography, eluting with 10% Et
2
diastereomers of compound 3 as a white foam (6.5 g, 90%).
1
b Anomer (Major). H NMR (500 MHz CDCl
3
) d 7.50 – 7.43 (m, 6H), 7.35 – 7.30 (m, 6H), 7.29 – 7.24 (m, 3H), 5.13 (dd, J = 11.4,
.3 Hz, 1H), 4.24 (d, J = 11.4 Hz, 1H), 4.16 (t, J = 4.5 Hz, 1H), 3.92 (d, J = 4.6 Hz, 1H), 3.26 (dd, J = 10.4, 4.9 Hz, 1H), 3.11
4
(
dd, J = 10.4, 3.3 Hz, 1H), 0.93 (s, 9H), 0.85 (s, 9H), 0.13 (s, 3H), 0.10 (s, 3H), 0.04 (s, 3H), -0.02 (s, 3H)
1
a Anomer (Minor). H NMR (500 MHz CDCl
3
) d 7.50 – 7.43 (m, 6H), 7.35 – 7.30 (m, 6H), 7.29 – 7.24 (m, 3H), 5.19 (d, J = 4.6 Hz, 1H),
.40 (dd, J = 7.3, 4.0 Hz, 1H), 4.20 (t, J = 4.0 Hz, 1H), 4.11 (ddd, J = 7.0, 3.8, 2.6 Hz, 1H), 3.97 (d, J = 4.1 Hz, 1H), 3.49 (dd, J = 10.3,
.7 Hz, 1H), 3.10 (dd, J = 10.2, 3.9 Hz, 1H), 2.71 (d, J = 4.6 Hz, 1H), 0.91 (s, 9H), 0.75 (s, 9H), 0.11 (s, 3H), 0.09 (s, 3H), -0.03 (s, 3H), -0.19
4
2
(
s, 3H)
Both Anomers.
4.43, 81.78, 77.06, 74.56, 72.58, 71.74, 63.77, 63.31, 26.06, 25.96, 25.92, 25.86, 18.42, 18.26, 18.11, 18.09, -4.08, -4.29, -4.46,
4.49, -4.55, -4.67, -4.90, -4.94.
1
3
3
C NMR (126 MHz, CDCl ) d 143.80, 129.01, 128.77, 128.01, 127.95, 127.26, 127.23, 102.19, 97.90, 87.05, 87.01,
8
-
+
52 5 2
HRMS (ESI-TOF) m/z: [M+Na] Calcd for C36H O NaSi 643.3251; Found 643.3251.
4-nitrophenyl 2,3-bis-O-tert-butyldimethylsilyl-5-O-trityl-D-ribofuranoside (S3)
˚
To a 250 mL round bottom flask, were added 3 (3.80 g, 6.11 mmol, 1 eq), 4-nitrophenol (2.51 g, 18.1 mmol, 3 eq), 4 A molecular sieves
ꢁ
(
200 mg), and toluene (100 mL). Cooled mixture to 0 C. Added n-Bu
3
P (3.80 mL, 0.81 g/mL, 3.1 g, 15 mmol, 2.5 eq) dropwise via
ꢁ
syringe. Added 1,1’-(azodicarbonyl)dipiperidine as a solid in one portion. Mixture was stirred at 0 C for 30 min and then allowed
ꢁ
to warm to room temperature and stirred for an additional 4 h. The reaction mixture was cooled to 0 C, and 30% H
2
O
2
(5 mL)
was added to fully quench phosphine. After stirring for 30 min, the reaction mixture was diluted with hexane (100 mL). The resulting
yellow precipitate was removed by filtration through a pad of celite. The filtrate was washed with satd aq NaHCO five times, dried
over Na SO4, filtered, and concentrated. The residue was purified by silica gel chromatography, eluting with 5% Et O-hexanes, to
yield compounds S3 (a mixture of anomers, a:b 69:31) as a pale yellow foam (2.87 g, 63%).
3
2
2
1
a-anomer. H NMR (500 MHz, CDCl
3
) d 8.21 (d, J = 9.0 Hz, 2H), 7.48 – 7.43 (m, 6H), 7.32 (t, J = 7.7 Hz, 6H), 7.29 – 7.24 (m, 3H), 7.13
d, J = 9.3 Hz, 2H), 5.65 (d, J = 4.1 Hz, 1H), 4.38 (t, J = 4.7 Hz, 1H), 4.18 (q, J = 3.0 Hz, 1H), 4.05 (dd, J = 5.2, 2.2 Hz, 1H), 3.42
dd, J = 10.5, 3.5 Hz, 1H), 3.12 (dd, J = 10.6, 3.1 Hz, 1H), 0.92 (s, 9H), 0.84 (s, 9H), 0.09 (s, 3H), 0.08 (s, 3H), 0.04 (s, 3H), -0.12 (s, 3H).
(
(
1
3
C NMR (126 MHz, CDCl
3.43, 26.00, 25.86, 18.44, 18.17, -4.37, -4.39, -4.40, -4.73.
3
) d 162.98, 143.82, 142.01, 128.79, 128.04, 127.33, 125.94, 116.35, 100.21, 86.98, 86.66, 73.83, 72.23,
6
+
HRMS (ESI-TOF) m/z: [M+Na] Calcd for C42
H
55NO
7
Si
) d 8.20 (d, J = 9.2 Hz, 2H), 7.38 – 7.33 (m, 6H), 7.20 – 7.13 (m, 11H), 5.59 (d, J = 1.4 Hz, 1H), 4.41
dd, J = 6.9, 4.1 Hz, 1H), 4.30 (dd, J = 4.1, 1.4 Hz, 1H), 4.23 (ddd, J = 6.9, 4.1, 2.6 Hz, 1H), 3.38 (dd, J = 10.6, 2.7 Hz, 1H), 3.00
dd, J = 10.6, 4.2 Hz, 1H), 0.92 (s, 9H), 0.76 (s, 9H), 0.13 (s, 3H), 0.13 (s, 3H), 0.02 (s, 3H), -0.16 (s, 3H).
2
Na 764.3415; Found 764.3413.
1
b-anomer. H NMR (500 MHz, CDCl
3
(
(
e7 Cell Chemical Biology 25, 1–9.e1–e19, December 20, 2018

Please cite this article in press as: Drown et al., Monitoring Poly(ADP-ribosyl)glycohydrolase Activity with a Continuous Fluorescent Substrate, Cell
Chemical Biology (2018), https://doi.org/10.1016/j.chembiol.2018.09.008
1
3
H NMR (500 MHz, CDCl ) d 8.20 (d, J = 9.2 Hz, 2H), 7.38 – 7.33 (m, 6H), 7.20 – 7.13 (m, 11H), 5.59 (d, J = 1.4 Hz, 1H), 4.41
(
(
dd, J = 6.9, 4.1 Hz, 1H), 4.30 (dd, J = 4.1, 1.4 Hz, 1H), 4.23 (ddd, J = 6.9, 4.1, 2.6 Hz, 1H), 3.38 (dd, J = 10.6, 2.7 Hz, 1H), 3.00
13
dd, J = 10.6, 4.2 Hz, 1H), 0.92 (s, 9H), 0.76 (s, 9H), 0.13 (s, 3H), 0.13 (s, 3H), 0.02 (s, 3H), -0.16 (s, 3H). C NMR (126 MHz,
) d 162.09, 143.74, 142.27, 128.82, 127.75, 127.09, 125.87, 116.26, 105.06, 86.55, 83.33, 76.60, 71.64, 62.95, 25.92, 25.89,
8.26, 18.11, -3.99, -4.28, -4.38, -4.86.
CDCl
3
1
+
7 2
HRMS (ESI-TOF) m/z: [M+Na] Calcd for C42H55NO Si Na 764.3415; Found 764.3414.
4-(trifluoromethyl)umbellifer-7-yl 2,3-bis-O-tert-butyldimethylsilyl-5-O-trityl-D-ribofuranoside (S4)
A 100 mL round bottom flask was charged with 4-(trifluoromethyl)umbelliferone (1.69 g, 7.36 mmol, 1.5 eq), 2,3-di-(tert-butyldime-
˚
thylsilyl)-5-(triphenylmethyl)-D-ribofuranose (3) (3.05 g, 4.91 mmol, 1 eq), triphenylphosphine (1.93 g, 7.37 mmol, 1.5 eq), and 4 A mo-
lecular sieves (600 mg). Material was dissolved in dimethoxyethane (35 mL) and stirred for 15 min at room temperature. DIAD
(
1.45 mL, 7.36 mmol, 1.5 eq) was added dropwise. Reaction mixture was stirred at room temperature for 2 hours and then poured
into hexane (400 mL). Resulting precipitate was removed by filtration. Filtrate was washed with satd aq NaHCO 4x. Organic phase
was dried over Na SO and evaporated. Residue was purified by silica gel (5% Et O:hexane) to give S4 (2.47 g, 60%) as a mixture of
3
2
4
2
anomers. While anomers were could be separated by silica gel chromatography, the mixture of diastereomers was routinely carried
into the next step as they were more easily separable with the trityl group removed.
1
a-anomer. H NMR (500 MHz, CDCl
3
) d 7.55 (dt, J = 9.1, 1.9 Hz, 1H), 7.39 – 7.34 (m, 6H), 7.27 – 7.21 (m, 6H), 7.20 – 7.15 (m, 3H), 7.03
(
d, J = 2.3 Hz, 1H), 6.95 (dd, J = 9.0, 2.4 Hz, 1H), 6.54 (s, 1H), 5.55 (d, J = 4.2 Hz, 1H), 4.31 (dd, J = 5.3, 4.3 Hz, 1H), 4.08 (q, J = 3.1 Hz,
1
H), 3.96 (dd, J = 5.2, 1.9 Hz, 1H), 3.32 (dd, J = 10.6, 3.4 Hz, 1H), 3.03 (dd, J = 10.6, 3.1 Hz, 1H), 0.83 (s, 9H), 0.75 (s, 9H), -0.00 (s, 6H),
0.05 (s, 3H), -0.21 (s, 3H).
-
13
2
C NMR (126 MHz, CDCl
3
1
) d 161.79, 159.62, 156.25, 143.83, 141.75 (q,
3
J
CF = 32.8 Hz), 128.79, 128.07, 127.34, 126.43
3
(
d,
J
CF = 2.3 Hz), 121.79 (q,
2.25, 63.49, 26.01, 25.85, 18.44, 18.16, -4.37, -4.41, -4.43, -4.72.
JCF = 275.5 Hz), 114.85, 112.50 (q, JCF = 5.7 Hz), 107.61, 104.46, 100.23, 87.03, 86.87, 73.87,
7
1
9
F NMR (471 MHz, CDCl
3
) d -64.68.
b-anomer. H NMR (500 MHz, CDCl ) d 7.50 (dq, J = 8.9, 1.8 Hz, 1H), 7.25 – 7.20 (m, 6H), 7.06 – 7.01 (m, 10H), 6.99 (dd, J = 9.0,
.5 Hz, 1H), 6.48 (s, 1H), 5.45 (d, J = 1.3 Hz, 1H), 4.32 (dd, J = 6.9, 4.0 Hz, 1H), 4.17 (dd, J = 4.1, 1.4 Hz, 1H), 4.09 (dt, J = 6.8,
.3 Hz, 1H), 3.30 (dd, J = 10.6, 2.8 Hz, 1H), 2.86 (dd, J = 10.7, 3.8 Hz, 1H), 0.80 (s, 9H), 0.63 (s, 9H), 0.00 (s, 3H), 0.00 (s, 3H),
1
3
2
3
-
0.11 (s, 3H), -0.28 (s, 3H).
13
2
C NMR (126 MHz, CDCl
3
1
) d 160.98, 159.37, 156.10, 143.77, 141.55 (q,
3
J
CF = 32.7 Hz), 128.85, 127.75, 127.07, 126.43
3
(
q,
J
CF = 2.4 Hz), 121.70 (q,
JCF = 275.6 Hz), 114.14, 112.96 (q, JCF = 5.7 Hz), 107.92, 105.19, 104.79, 86.55, 83.32, 76.62,
7
1.46, 62.51, 25.93, 25.90, 18.27, 18.11, -3.98, -4.28, -4.37, -4.84.
19
F NMR (471 MHz, CDCl
3
) d -64.75.
+
HRMS (ESI-TOF) m/z: [M+Na] Calcd for C46
55 7 3 2
H O F NaSi 855.3336; Found 855.3342.
4
-nitrophenyl 2,3-bis-O-tert-butyldimethylsilyl-D-ribofuranoside (4)
ꢁ
To a stirring solution of compound S3 (5.26 g, 7.09 mmol, 1 eq) in dichloromethane (60 mL) at 0 C was added trifluoroacetic anhydride
4.00 mL, 1.487 g/mL, 5.95 g, 28.3 mmol, 4 eq) as a 2 M solution in CH Cl dropwise via addition funnel. Trifluoroacetic acid (1.6 mL,
.489 g/mL, 2.38 g, 20.9 mmol, 3 eq) was added as a 2 M solution in CH Cl dropwise via addition funnel. Reaction was stirred at
room temperature for another 10 min. Reaction mixture was quenched with the addition of Et N (4 mL, 0.7255 g/mL, 2.9 g,
9 mmol, 4.1 eq) followed by CH OH (30 mL). Mixture was stirred for an additional 15 min at room temperature before being
diluted with H O. Extracted three times with CH Cl . Combined organic layers were dried over Na SO and concentrated in vacuo.
Resulting residue was purified by silica gel chromatography (5:1 Hexane:EtOAc) to yield compound 4a (2.15 g, 61%) and compound
b (0.96 g, 27%).
(
2
2
1
2
2
3
2
3
2
2
2
2
4
4
Cell Chemical Biology 25, 1–9.e1–e19, December 20, 2018 e8

Please cite this article in press as: Drown et al., Monitoring Poly(ADP-ribosyl)glycohydrolase Activity with a Continuous Fluorescent Substrate, Cell
Chemical Biology (2018), https://doi.org/10.1016/j.chembiol.2018.09.008
1
a-anomer (4a). H NMR (500 MHz, CDCl
3
) d 8.17 (d, J = 9.2 Hz, 2H), 7.10 (d, J = 9.3 Hz, 2H), 5.60 (d, J = 3.6 Hz, 1H), 4.20 – 4.15
(
(
m, 3H), 3.81 (ddd, J = 12.2, 4.5, 2.5 Hz, 1H), 3.67 (ddd, J = 12.3, 7.0, 3.1 Hz, 1H), 1.94 (dd, J = 7.4, 4.5 Hz, 1H), 0.91 (s, 18H), 0.12
s, 3H), 0.09 (s, 3H), 0.07 (s, 6H).
1
3
C NMR (126 MHz, CDCl
4.30, -4.39, -4.40, -4.71.
3
) d 162.65, 142.13, 125.91, 116.34, 100.21, 86.99, 77.16, 73.96, 71.45, 62.32, 25.96, 25.90, 18.42, 18.20,
-
+
HRMS (ESI-TOF) m/z: [M+Na] Calcd for C23
H
41NNaO
) d 8.15 (d, J = 9.2 Hz, 2H), 7.07 (d, J = 9.2 Hz, 2H), 5.46 (d, J = 1.3 Hz, 1H), 4.36 (dd, J = 7.0,
.1 Hz, 1H), 4.21 (dd, J = 4.2, 1.3 Hz, 1H), 4.15 (dt, J = 6.7, 3.1 Hz, 1H), 3.80 (ddd, J = 12.5, 4.4, 2.7 Hz, 1H), 3.54 (ddd, J = 12.4, 8.8,
.5 Hz, 1H), 1.68 (dd, J = 8.8, 4.3 Hz, 1H), 0.91 (s, 9H), 0.91 (s, 9H), 0.13 (s, 6H), 0.10 (s, 3H).
7 2
Si 522.2314; Found 522.2297.
1
b-anomer (4b). H NMR (500 MHz, CDCl
3
4
3
1
3
C NMR (126 MHz, CDCl
4.15, -4.45, -4.46, -4.90.
3
) d 161.52, 142.47, 125.92, 116.23, 105.00, 84.24, 77.16, 76.85, 70.72, 61.21, 25.94, 25.85, 18.20, 18.17,
-
-
HRMS (ESI-TOF) m/z: [M-H] Calcd for C23
H
40NO
7
Si
2
498.2343; Found 498.2336.
4-(trifluoromethyl)umbellifer-7-yl 2,3-bis-O-tert-butyldimethylsilyl-D-ribofuranoside (5)
To a 100 mL-RBF equipped with addition funnel was added S4 (2.31 g, 2.77 mmol) and dichloromethane (24 mL). Trifluoroacetic
anhydride (1.56 mL, 11.0 mmol, 4 eq) diluted with 5 mL of dichloromethane was added to stirring solution via addition funnel at
room temperature. Trifluoroacetic acid (0.64 mL, 8.4 mmol, 3 eq) diluted with 5 mL of dichloromethane was added to stirring solution
dropwise. Reaction mixture was stirred at room temperature for 20 min. Reaction mixture was neutralized by addition of neat triethyl-
amine (5.0 mL, 36 mmol, 13 eq) via addition funnel followed by methanol (10 mL). Fuming mixture was stirred for 30 min and poured
into satd aq NH
dried over Na SO
anomers 5a (658 mg, 40%) and 5b (767 mg, 47%) as white solids.
4
Cl. Layers were separated and aqueous layer extracted 3x with dichloromethane. Combined organic layers were
2
4
, filtered, and evaporated. Residue was purified by silica gel chromatography (6:1 hexane:EtOAc) to give separable
1
a-anomer (5a). H NMR (600 MHz, CDCl
3
) d 7.63 (dd, J = 9.0, 1.8 Hz, 1H), 7.09 (d, J = 2.4 Hz, 1H), 7.03 (dd, J = 9.0, 2.4 Hz, 1H), 6.62
s, 1H), 5.60 (d, J = 3.7 Hz, 1H), 4.22 – 4.14 (m, 3H), 3.81 (dd, J = 12.3, 2.7 Hz, 1H), 3.67 (dd, J = 12.2, 3.1 Hz, 1Hf), 1.82 (bs, 1H), 0.920
s, 9H), 0.918 (s, 9H), 0.12 (s, 3H), 0.09 (s, 3H), 0.08 (s, 3H), 0.08 (s, 3H).
(
(
1
3
2
3
C NMR (151 MHz, CDCl
3
) d 161.44, 159.55, 156.17, 141.73 (q,
J
CF = 32.72 Hz), 126.49 (q,
q, JCF = 275.62 Hz), 114.80, 112.68 (q, JCF = 5.68 Hz), 107.80, 104.46, 100.25, 87.04, 74.01, 71.45, 62.34, 25.98, 25.91, 18.44,
8.21, -4.29, -4.36, -4.37, -4.69.
JCF = 2.40 Hz), 121.74
1
3
(
1
1
9
F NMR (564 MHz, CDCl
3
) d -64.74.
+
HRMS (ESI-TOF) m/z: [M+H] Calcd for C27
H
42
O
7
F
3
Si
) d 7.64 (dd, J = 8.9, 1.9 Hz, 1H), 7.06 (d, J = 2.4 Hz, 1H), 7.01 (dd, J = 9.0, 2.4 Hz, 1H), 6.65
s, 1H), 5.47 (d, J = 1.2 Hz, 1H), 4.38 (dd, J = 7.2, 4.1 Hz, 1H), 4.21 (dd, J = 4.1, 1.2 Hz, 1H), 4.17 (dt, J = 7.2, 3.0 Hz, 1H), 3.86 – 3.79 (m,
2
591.2421; Found 591.2437.
1
b-anomer (5b). H NMR (500 MHz, CDCl
3
(
1
H), 3.56 (ddd, J = 12.4, 9.1, 3.3 Hz, 1H), 1.46 (dd, J = 9.1, 4.2 Hz, 1H), 0.93 – 0.92 (s, 18H), 0.14 (s, 9H), 0.12 (s, 3H).
2
1
3
3
C NMR (151 MHz, CDCl
3
) d 160.36, 159.23, 156.13, 141.54 (q,
3
J
CF = 32.82 Hz), 126.71 (q,
JCF = 2.40 Hz), 121.69
1
(
q, JCF = 275.34 Hz), 114.34, 113.23 (q, JCF = 5.66 Hz), 108.27, 105.14, 104.57, 84.23, 76.90, 70.68, 61.22, 25.99, 25.91, 18.28,
1
8.23, -4.08, -4.38, -4.41, -4.83.
1
9
3
F NMR (564 MHz, CDCl ) d -64.77.
4-nitrophenyl 2,3-bis-O-tert-butyldimethylsilyl-a-D-ribose-5-(difluorenylmethyl phosphate) (S5)
To a 20 mL reaction vial, 4a (71.1 mg, 0.142 mmol, 1 eq) and bis-(9H-fluoren-9-ylmethyl)-N,N-diisopropylamidophosphite (Lam-
brecht et al., 2015) (95.5 mg, 0.183 mmol, 1.3 eq) were added. Dissolved material by addition of CH
ꢁ
2
Cl
to 0 C. 4,5-dicyanoimidazole (24.0 mg, 0.203 mmol, 1.4 eq) was added as a solution in acetonitrile (0.25 mL). After stirring at 0 C for
2
(1.25 mL). Cooled solution
ꢁ
15 min, mixture was allowed to warm to room temperature and was stirred for an additional 2 h. Once starting material was consumed
ꢁ
as indicated by TLC (75:25 hexane:EtOAc), mixture was cooled to 0 C and subjected to dropwise addition of tert-butyl hydroperoxide
e9 Cell Chemical Biology 25, 1–9.e1–e19, December 20, 2018

Please cite this article in press as: Drown et al., Monitoring Poly(ADP-ribosyl)glycohydrolase Activity with a Continuous Fluorescent Substrate, Cell
Chemical Biology (2018), https://doi.org/10.1016/j.chembiol.2018.09.008
(
0.14 mL, 0.70 mmol, 5 eq) as a 5 M solution in decane. Mixture was stirred for an additional 1 h and quenched with H
extracted with CH Cl three times. Combined organic layers were dried over Na SO , concentrated, and purified by silica gel chro-
matography eluting with 60:40 hexane:EtOAc to yield compound S5 as a white foam (111.5 mg, 84%).
2
O. Mixture was
2
2
2
4
1
H NMR (500 MHz, CDCl
.44 – 7.34 (m, 4H), 7.32 – 7.24 (m, 4H), 6.91 (d, J = 9.2 Hz, 2H), 5.26 (m, 1H), 4.38 – 4.26 (m, 4H), 4.22 – 4.12 (m, 3H), 4.06
ddd, J = 11.4, 5.8, 3.1 Hz, 1H), 4.04 – 4.00 (m, 2H), 3.91 (ddd, J = 11.1, 7.0, 3.8 Hz, 1H), 0.91 (s, 9H), 0.90 (s, 9H), 0.11 (s, 3H),
3
) d 8.00 (d, J = 9.2 Hz, 2H), 7.77 – 7.70 (m, 4H), 7.57 (dd, J = 18.2, 7.5 Hz, 2H), 7.52 (dd, J = 7.3, 6.3 Hz, 2H),
7
(
0
.05 (s, 3H), 0.03 (s, 6H).
13
3
C NMR (126 MHz, CDCl ) d 162.23, 143.16, 143.10, 142.98, 142.94, 142.10, 141.47, 141.44, 128.10, 128.08, 128.06, 127.27,
1
6
27.22, 125.78, 125.21, 125.17, 125.13, 125.07, 120.19, 120.18, 120.15, 116.09, 99.76, 83.93, 83.87, 77.16, 73.46, 71.13, 69.56,
9.51, 69.48, 69.43, 66.55, 66.51, 48.03, 48.00, 47.97, 47.93, 25.90, 25.86, 18.38, 18.12, -4.25, -4.37, -4.47, -4.80.
31
P NMR (202 MHz, CDCl
3
) d -0.41.
+
HRMS (ESI-TOF) m/z: [M+Na] Calcd for C51
2
H62NO10NaSi P 958.3548; Found 958.3554.
Triethylammonium 4-nitrophenyl 2,3-bis-O-tert-butyldimethylsilyl-a-D-ribose-5-phosphate (6)
0 mL reaction vial was charged with compound S5 (464 mg, 0.50 mmol, 1 eq). Added acetonitrile (5 mL) and triethylamine (freshly
distilled from CaH , 1 mL) successively. Stirred at room temperature for 16 h. Added 1 mL toluene to stirring solution and concen-
2
2
trated in vacuo. Residue was redissolved in methanol (0.5 mL) and purified by C-18 chromatography to yield the triethylammonium
salt of compound 6 as a tan foam (241 mg, 71%).
1
3
H NMR (500 MHz, CD OD) d 8.20 (d, J = 9.3 Hz, 2H), 7.17 (d, J = 9.3 Hz, 2H), 5.71 (d, J = 4.2 Hz, 1H), 4.39 (dd, J = 5.4, 4.2 Hz, 1H),
4
.31 (dd, J = 5.4, 2.3 Hz, 1H), 4.20 (dq, J = 3.9, 1.9 Hz, 1H), 3.99 (ddd, J = 10.1, 4.4, 3.1 Hz, 1H), 3.95 (ddd, J = 9.1, 4.6, 3.4 Hz, 1H), 3.17
q, J = 7.3 Hz, 6H), 1.31 (t, J = 7.3 Hz, 9H), 0.94 (s, 9H), 0.93 (s, 9H), 0.16 (s, 3H), 0.13 (s, 3H), 0.13 (s, 3H), 0.10 (s, 3H).
(
13
C NMR (126 MHz, CD
7.40, 26.49, 26.46, 19.15, 18.96, 9.11, -4.10, -4.31 (2C), -4.43.
3
OD) d 164.14, 143.13, 126.62, 117.36, 101.35, 87.69 (d, J = 8.9 Hz), 74.75, 73.14, 65.98 (d, J = 5.2 Hz),
4
3
1
P NMR (202 MHz, CD
3
OD) d 0.77.
-
HRMS (ESI-TOF) m/z: [M-H] Calcd for C23
H
2
41NO10PSi 578.2012; Found 578.2017.
Triethylammonium 4-(trifluoromethyl)umbellifer-7-yl 2,3-bis-O-tert-butyldimethylsilyl-a-D-ribose-5-phosphate (7)
Method A)
(
ꢁ
To a stirring solution of 5a (507 mg, 0.858 mmol) in THF (8 mL) at 0 C was added triethylamine (1.4 mL, 10 mmol, 12 eq) followed by
ꢁ
phosphorus oxychloride (0.16 mL, 1.7 mmol, 2 eq). Reaction mixture was stirred at 0 C for 40 min then warmed to room temperature
ꢁ
and stirred for an additional 20 min. After cooling to 0 C once again, reaction was quenched with addition of water (1 mL). Mixture was
stirred for an additional 30 min at room temperature then solvent was removed by rotary evaporator. Residue was purified by C18
chromatography to give 7 (225 mg, 34%) as a white solid.
1
3
H NMR (500 MHz, CD OD) d 7.68 (dd, J = 8.8, 2.0 Hz, 1H), 7.12 – 7.06 (m, 2H), 6.71 (s, 1H), 5.72 (d, J = 4.3 Hz, 1H), 4.41 (dd, J = 5.4,
4
.2 Hz, 1H), 4.32 (dd, J = 5.4, 2.2 Hz, 1H), 4.21 (dq, J = 3.9, 1.9 Hz, 1H), 4.01 – 3.91 (m, 2H), 3.19 (q, J = 7.3 Hz, 6H), 1.31 (t, J = 7.3 Hz, 9H),
.95 (s, 9H), 0.93 (s, 9H), 0.16 (s, 3H), 0.14 (s, 6H), 0.11 (s, 3H).
0
13
2
3
C NMR (126 MHz, CD
1
3
OD) d 163.04, 160.83, 157.41, 142.36 (q,
3
J
CF = 32.30 Hz) 127.32 (q,
JCF = 1.72 Hz), 123.22
3
(
(
q, JCF = 274.75 Hz), 115.81, 113.88 (q, JCF = 5.81 Hz), 108.56, 105.05, 101.51, 87.94 (d, JCP = 9.1 Hz), 74.81, 73.20, 65.99
2
d, JCP = 5.2 Hz), 47.56, 26.48f, 26.46, 19.16, 18.99, 9.15, -4.12, -4.34 (2C), -4.43.
1
9
F NMR (470 MHz, CD
P NMR (202 MHz, CD
3
OD) d -66.10.
OD) d 0.83.
31
3
Cell Chemical Biology 25, 1–9.e1–e19, December 20, 2018 e10

Please cite this article in press as: Drown et al., Monitoring Poly(ADP-ribosyl)glycohydrolase Activity with a Continuous Fluorescent Substrate, Cell
Chemical Biology (2018), https://doi.org/10.1016/j.chembiol.2018.09.008
-
HRMS (ESI-TOF) m/z: [M-H] Cald for C27
H
41
F
3
O10PSi
2
669.1933; Found 669.1918.
4
-(trifluoromethyl)umbellifer-7-yl 2,3-bis-O-tert-butyldimethylsilyl-a-D-ribose-5-(difluorenylmethyl phosphate) (S6)
To a stirring solution of compound 5a (373.9 mg, 0.633 mmol) and N,N-diisopropyl bis(9-methylfluorenyl)phosphoramidite (403.8 mg,
.774 mmol) in CH Cl (12 mL). Dicyanoimidazole (151 mg, 1.3 mmol) was added as a solution in acetonitrile (4.8 mL). Stirred at room
temperature for 3 h. Reaction mixture was cooled to 0 C added t-butylperoxide as a 5 M solution in decane (0.6 mL). Stirred at 0 C for
h. Reaction mixture was quenched with water and extracted with CH Cl . Combined organic layers were dried over Na SO and
evaporated. Residue was purified by silica gel chromatography (1:1 hexane:EtOAc) to give compound S6 as a yellow foam
0
2
2
ꢁ
ꢁ
1
2
2
2
4
(
579.5 mg, 90%).
1
3
H NMR (500 MHz, CDCl ) d 7.76 – 7.68 (m, 4H), 7.58 – 7.46 (m, 5H), 7.43 – 7.32 (m, 4H), 7.31 – 7.22 (m, 5H), 6.95 (d, J = 2.4 Hz, 1H),
6
.86 (dd, J = 9.0, 2.4 Hz, 1H), 6.61 (s, 1H), 5.34 – 5.30 (m, 1H), 4.34 (td, J = 6.3, 1.4 Hz), 4.29 (ddt, J = 9.9, 6.8, 3.7 Hz), 4.20 – 4.12
m, 3H), 4.05 – 4.00 (m, 3H), 3.90 (ddd, J = 11.1, 7.1, 3.8 Hz, 1H), 0.90 (s, 9H), 0.89 (s, 9H), 0.10 (s, 3H), 0.04 (s, 3H), 0.02 (s, 3H),
(
0
.02 (s, 3H).
1
3
2
C NMR (126 MHz, CDCl
3
) d 161.13, 159.34, 156.03, 143.16 (d, J = 11.6 Hz), 143.04 (d, J = 9.6 Hz), 141.53 (q, JCF = 32.8 Hz),
3
1
41.52, 141.47, 128.07 (d, J = 1.7 Hz), 127.28 (d, J = 1.2 Hz), 127.26, 126.45 (q, JCF = 2.2 Hz), 125.22 (d, J = 6.8 Hz), 125.16
1
3
(
d, J = 4.2 Hz), 121.72 (q, JCF = 275.5 Hz), 120.19 (d, J = 1.9 Hz), 120.16, 114.20, 112.80 (q, JCF = 5.7 Hz), 107.81, 104.59,
9
9.82, 84.30 (d, J = 7.4 Hz), 73.54, 71.26, 69.54 (t, J = 5.7 Hz), 66.66 (d, J = 5.8 Hz), 48.04 (dd, J = 7.9, 5.7 Hz), 25.94, 25.88,
8.42, 18.15, -4.25, -4.36, -4.42, -4.74.
1
1
9
F NMR (471 MHz, CDCl
P NMR (203 MHz, CDCl
3
) d -64.71.
) d -1.55.
3
1
3
+
62 10 3 2
HRMS (ESI-TOF) m/z: [M+Na] Calcd for C55H O F NaSi P 1049.3469; Found 1049.3500.
Triethylammonium 4-(trifluoromethyl)umbellifer-7-yl 2,3-bis-O-tert-butyldimethylsilyl-a-D-ribose-5-phosphate (7)
Method B)
2
To a stirring solution of compound S6 (1.18 g, 1.00 mmol) in acetonitrile (10 mL) was added Et N (freshly distilled from CaH , 2.5 mL).
(
3
Mixture was stirred at room temperature for 16 h. Reaction mixture was co-evaporated with toluene to dryness. Residue was purified
by C18 reverse phase chromatography to give compound 7 as a white solid (80%).
1
3
H NMR (500 MHz, CDCl ) d 7.57 (dt, J = 9.0, 1.8 Hz, 1H), 7.04 (d, J = 2.4 Hz, 1H), 7.00 (dd, J = 9.0, 2.4 Hz, 1H), 6.56 (s, 1H), 5.54
(
d, J = 4.1 Hz, 1H), 4.25 (dd, J = 5.4, 4.1 Hz, 1H), 4.22 (dd, J = 5.4, 1.9 Hz, 1H), 4.16 (dt, J = 4.0, 2.2 Hz, 1H), 3.98 (ddd, J = 11.3, 5.4,
3
0
.8 Hz, 1H), 3.93 (ddd, J = 11.2, 5.6, 4.1 Hz, 1H), 3.06 (q, J = 7.3 Hz, 6H), 1.30 (t, J = 7.3 Hz, 9H), 0.86 (s, 9H), 0.86 (s, 9H), 0.07 (s, 3H),
.04 (s, 3H), 0.03 (s, 3H), 0.02 (s, 3H).
1
3
2
3
C NMR (126 MHz, CDCl
1
3
) d 161.76, 159.58, 156.06, 141.72 (q,
3
J
CF = 32.6 Hz), 126.33 (q,
J
CF = 1.70 Hz), 121.66
3
(
(
q, JCF = 275.6 Hz), 114.93, 112.20 (q, JCF = 5.7 Hz), 107.32, 104.07, 100.00, 86.72 (d, JCP = 8.6 Hz), 73.52, 71.66, 64.78
2
d, JCP = 4.8 Hz), 45.47, 25.90, 25.84, 18.29, 18.08, 8.62, -4.46, -4.47, -4.49, -4.66.
1
9
F NMR (471 MHz, CDCl
P NMR (203 MHz, CDCl
3
) d -64.74.
) d 1.69.
3
1
3
+
42 3 2
HRMS (ESI-TOF) m/z: [M+Na] Calcd for C27H O10NaPF Si 693.1904; Found 693.1901.
e11 Cell Chemical Biology 25, 1–9.e1–e19, December 20, 2018

Please cite this article in press as: Drown et al., Monitoring Poly(ADP-ribosyl)glycohydrolase Activity with a Continuous Fluorescent Substrate, Cell
Chemical Biology (2018), https://doi.org/10.1016/j.chembiol.2018.09.008
2
To a 100 mL round-bottom flask, adenosine (5.0 g, 19 mmol, 1 eq), imidazole (6.9 g, 102 mmol, 5.4 eq), and dimethylformamide
’,3’,5’-tris-O-(tert-butyldimethylsilyl)-adenosine (S7)
ꢁ
(
40 mL) were added. Once fully dissolved, solution was cooled to 0 C. Tert-butyldimethylsilyl chloride (12.2 g, 81 mmol, 4.2 eq)
was added to the stirring solution. Once fully dissolved, reaction vessel was allowed to warm to room temperature. Stirred at
room temperature for 16 h. Reaction was quenched by the addition of satd aq NH Cl. Extracted three times with CH Cl . Combined
organic layers were dried over MgSO , filtered through a pad of celite, and evaporated. Residue was purified by silica gel chroma-
tography eluting with 50:50 hexane:EtOAc to yield S7 as a white foam (12.5 g, 96%).
4
2
2
4
1
3
H NMR (500 MHz, CDCl ) d 8.32 (s, 1H), 8.15 (s, 1H), 6.11 (bs, 2H), 6.02 (d, J = 5.2 Hz, 1H), 4.68 (t, J = 4.8 Hz, 1H), 4.31 (t, J = 3.9 Hz,
1
H), 4.12 (q, J = 3.5 Hz, 1H), 4.02 (dd, J = 11.4, 4.2 Hz, 1H), 3.77 (dd, J = 11.3, 2.9 Hz, 1H), 0.94 (s, 9H), 0.92 (s, 9H), 0.78 (s, 9H),
.13 (s, 3H), 0.12 (s, 3H), 0.09 (s, 3H), 0.09 (s, 3H), -0.06 (s, 3H), -0.24 (s, 3H).
0
13
C NMR (126 MHz, CDCl
8.22, 17.99, -4.28, -4.57, -4.59, -4.95, -5.24 (2C).
3
) d 155.70, 153.03, 150.05, 139.69, 120.19, 88.41, 85.58, 75.89, 72.11, 62.66, 26.20, 25.98, 25.81, 18.65,
1
+
56 5 4 3
HRMS (ESI-TOF) m/z: [M+H] Calcd for C28H N O Si 610.3640; Found 610.3647
2
’,3’-bis-O-(tert-butyldimethylsilyl)-adenosine (S8)
A 500 mL round-bottom flask was charged with S7 (4.4 g, 7.2 mmol, 1 eq). Compound was dissolved in wet tetrahydrofuran (120 mL).
ꢁ
Solution was cooled to 0 C. To vigorously stirring solution, trichloroacetic acid (56 g, 340 mmol, 47 eq) was added as an ice-cold
ꢁ
solution in H
2
O (25 mL). Stirred reaction mixture at 0 C for 3 h. Quenched reaction by slowly cannulating reaction mixture into ice
cold satd aq NaHCO
3
. Once evolution of gas ceased, extracted three times with EtOAc. Dried organic layer over Na
2
SO
4
, filtered,
2 2 3
and evaporated. Residue was purified by silica gel chromatography eluting with 95:5 CH Cl :CH OH to yield compound S8 as a white
solid (3.3 g, 92%).
1
3
H NMR (500 MHz, 1:1 CDCl :DMSO (v:v)) d 8.24 (s, 1H), 8.11 (s, 1H), 7.29 (s, 2H), 6.08 (dd, J = 9.3, 3.3 Hz, 1H), 5.86 (d, J = 7.1 Hz, 1H),
4
.87 (dd, J = 7.2, 4.5 Hz, 1H), 4.26 (dd, J = 4.4, 1.4 Hz, 1H), 4.01 (q, J = 2.1 Hz, 1H), 3.75 (dt, J = 12.6, 3.2 Hz, 1H), 3.58 (ddd, J = 12.1, 9.4,
.6 Hz, 1H), 0.90 (s, 9H), 0.69 (s, 9H), 0.09 (s, 3H), 0.08 (s, 3H), -0.18 (s, 3H), -0.52 (s, 3H).
2
13
3
C NMR (126 MHz, 1:1 CDCl :DMSO (v:v)) d 156.22, 151.89, 148.40, 140.12, 119.86, 88.45, 87.40, 73.99, 73.06, 61.60, 25.52,
2
5.33, 17.65, 17.36, -4.88, -4.97, -5.07, -6.02.
+
HRMS (ESI-TOF) m/z: [M+H] Calcd for C22
H
42
N
5
O
4
Si
2
496.2775; Found 496.2774.
Triethylammonium 2’,3’-bis-O-(tert-butyldimethylsilyl)-adenosin-5’-yl H-phosphonate (S9)
To a 20 mL reaction vial was added compound S8 (324 mg, 0.654 mmol, 1 eq). Added dry pyridine (6.5 mL). Once fully dissolved,
diphenyl phosphite (0.65 mL, 1.22 g/mL, 795 mg, 3.4 mmol, 5 eq) was added, and reaction mixture was stirred at room temperature
for 1.5 h. Then, water (0.5 mL) was added followed by triethylamine (0.5 mL). After stirring for an additional 30 min, the reaction mixture
was concentrated and purified by C-18 chromatography to yield the triethylammonium salt of compound S9 as a white foam
(
331 mg, 77%).
1
1
3
HNMR(500MHz,CD OD)d8.51(s, 1H),8.22(s, 1H),6.83(d, JHP = 618.5 Hz, 1H), 6.10 (d, J=6.7Hz, 1H), 4.87(dd,J= 6.7, 4.4 Hz, 1H),
4
.41 (dd, J = 4.4, 1.9 Hz, 1H), 4.21 (td, J = 3.9, 2.1 Hz, 1H), 4.14 (ddd, J = 11.0, 6.7, 4.2 Hz, 1H), 4.08(ddd, J = 11.4, 6.7, 3.8 Hz, 1H), 3.15 (q,
J = 7.3 Hz, 6H), 1.27 (t, J = 7.3 Hz, 9H), 0.97 (s, 9H), 0.74 (s, 9H), 0.18 (s, 3H), 0.16 (s, 3H), -0.02 (s, 3H), -0.31 (s, 3H).
13
3
3
C NMR (126 MHz, CD OD) d 157.20, 153.66, 151.00, 141.41, 120.32, 88.64, 86.71 (d, JCP = 8.1 Hz), 77.07, 74.49, 64.22
2
(
d, JCP = 4.5 Hz), 47.61, 26.44, 26.24, 18.90, 18.68, 9.16, -4.17, -4.28 (2C), -5.08.
31
3
P NMR (202 MHz, CD OD) d 4.14.
Cell Chemical Biology 25, 1–9.e1–e19, December 20, 2018 e12

Please cite this article in press as: Drown et al., Monitoring Poly(ADP-ribosyl)glycohydrolase Activity with a Continuous Fluorescent Substrate, Cell
Chemical Biology (2018), https://doi.org/10.1016/j.chembiol.2018.09.008
-
HRMS (ESI-TOF) m/z: [M-H] Calcd for C22
H
41
N
5
O
6
PSi
2
558.2338; Found 558.2323.
2’,3’-bis-O-(tert-butyldimethylsilyl)-adenosin-5’-yl 2-cyanoethyl phosphonate (8)
To a 25 mL round-bottom flask was added compound S9 (238 mg, 0.360 mmol, 1 eq). Dried material by co-azeotroping with dry
ꢁ
acetonitrile three times. Pyridine was added (4 mL) and solution was cooled to 0 C. 3-hydroxypropionitrile (0.070 mL, 1.04 g/mL,
3 mg, 1.0 mmol, 3 eq) was added to stirring solution. Pivaloyl chloride (0.09 mL, 0.98 g/mL, 88 mg, 0.73 mmol, 2 eq) was added
7
dropwise. After stirring at 0 C for 2.5 h, reaction mixture was concentrated, azeotroping with toluene. Resulting residue was redis-
ꢁ
solved in acetonitrile and purified by C-18 chromatography to yield compound 8 as a white solid as a mixture of diastereomers
(
159 mg, 72%).
1
3
H NMR (500 MHz, CDCl ) d 8.30 (s, 1H), 8.30 (s, 1H), 8.00 (s, 1H), 7.98 (s, 1H), 6.91 (d, J = 720.9 Hz, 1H), 6.83 (d, J = 723.5 Hz, 1H)
6
.50 (d, J = 7.8 Hz, 2H), 5.87 (dd, J = 5.8, 4.2 Hz, 2H), 4.89 (q, J = 4.1 Hz, 2H), 4.52 - 4.14 (m, 10H), 2.72 (t, J = 6.3 Hz, 2H), 2.69
(
t, J = 6.2 Hz, 2H), 0.90 (s, 9H), 0.90 (s, 9H), 0.80 (s, 18H), 0.10 (s, 3H), 0.09 (s, 3H), 0.09 (s, 3H), 0.08 (s, 3H), -0.03 (s, 6H), -0.19
s, 3H), -0.19 (s, 3H).
(
1
3
3
C NMR (126 MHz, CDCl ) d 156.00, 155.96, 153.01, 149.52, 149.49, 140.14, 139.98, 120.54, 116.33, 89.94, 89.83, 82.60, 82.57,
8
1
2.55, 82.52, 77.16, 74.42, 74.33, 71.68, 71.60, 64.90, 64.85, 64.77, 64.73, 60.20, 60.16, 60.12, 60.08, 25.87, 25.76, 19.98, 19.93,
8.09, 17.94, -4.30, -4.31, -4.61, -4.63, -4.75, -4.76, -4.83, -4.85.
3
1
P NMR (202 MHz, CDCl
3
) d 8.70, 8.23.
-
HRMS (ESI-TOF) m/z: [M-H] Calcd for C25
44 6 6 2
H N O PSi 611.2604; Found 611.2594.
a-1’’-O-(4-nitrophenyl)-2’,2’’,3’,3’’-O-tetrakis-(tert-butyldimethylsilyl)-ADP-ribose (9)
To a 20 mL reaction vial was added 6 (158.6 mg, 0.233 mmol, 1.0 eq) and 8 (138.3 mg, 0.226 mmol, 1.0 eq). Mixture was dried by co-
azeotroping with dry acetonitrile three times and placing under vacuum over P
2 5
O for 12 h. The mixture was dissolved in acetonitrile
(
as 1 M solutions in acetonitrile and stirred at room temperature for 1 h. 1,8-diazabicycloundec-7-ene (350 mg, 2.3 mmol, 10 eq) was
3.0 mL). Then, (i-Pr NEt (129 mg, 1.00 mmol, 4.4 eq) and N-chlorosuccinimide (90.8 mg, 0.68 mmol, 3 eq) were sequentially added
2 2
)
added as a 1 M solution in acetonitrile. After stirring for 30 min, reaction mixture was evaporated in vacuo and purified by C18 chro-
matography to yield compound 9 as a white foam (287 mg, 88%).
1
3
H NMR (500 MHz, CD OD) d 8.67 (s, 1H), 8.18 (s, 1H), 8.14 (d, J = 9.2 Hz, 2H), 7.11 (d, J = 9.2 Hz, 2H), 6.15 (d, J = 7.5 Hz, 1H),
5
.64 (d, J = 4.3 Hz, 1H), 4.85 (dd, J = 7.5, 4.5 Hz, 1H), 4.46 (d, J = 4.4 Hz, 1H), 4.37 (dd, J = 5.4, 4.2 Hz, 1H), 4.35 - 4.27 (m, 3H), 4.21
ddt, J = 16.3, 4.3, 2.3 Hz, 2H), 4.11 (hept, J = 5.7, 5.0 Hz, 2H), 3.58 - 3.53 (m, 4H), 3.50 (t, J = 6.5, 1.7 Hz, 4H), 3.36 (t, J = 5.6 Hz, 4H),
(
2
0
.73 - 2.67 (m, 4H), 1.99 (pd, J = 5.4, 1.3 Hz, 4H), 1.79 - 1.62 (m, 12H), 1.34 (d, J = 6.5 Hz, 2H), 0.98 (s, 9H), 0.93 (s, 9H), 0.91 (s, 9H),
.70 (s, 9H), 0.20 (s, 3H), 0.17 (s, 3H), 0.15 (s, 3H), 0.13 (s, 3H), 0.11 (s, 3H), 0.08 (s, 3H), -0.01 (s, 3H), -0.38 (s, 3H).
1
3
3
C NMR (126 MHz, CD OD) d 167.42, 164.19, 157.30, 153.85, 151.17, 143.00, 141.66, 126.58, 120.04, 117.32, 101.23, 87.89,
8
2
7.86, 87.82, 87.64, 87.56, 77.60, 74.87, 74.76, 73.28, 66.76, 66.71, 66.66, 55.23, 49.53, 49.00, 39.34, 33.60, 30.01, 27.54, 26.49,
6.44, 26.21, 24.99, 20.43, 19.59, 19.14, 18.97, 18.93, 18.65, -4.07, -4.09, -4.19, -4.26, -4.29, -5.26.
3
1
P NMR (202 MHz, CD
3
OD) d -11.30 (d, J = 21.8 Hz), -11.67 (d, J = 21.6 Hz).
-
HRMS (ESI-TOF) m/z: [M-H] Calcd for C45
81 6 16 2 4
H N O P Si 1135.4267; Found 1135.4273.
e13 Cell Chemical Biology 25, 1–9.e1–e19, December 20, 2018

Please cite this article in press as: Drown et al., Monitoring Poly(ADP-ribosyl)glycohydrolase Activity with a Continuous Fluorescent Substrate, Cell
Chemical Biology (2018), https://doi.org/10.1016/j.chembiol.2018.09.008
a-1’’-O-(4-(trifluoromethyl)umbellifer-7-yl)-2’,2’’,3’,3’’-O-tetrakis-(tert-butyldimethylsilyl)-ADP-ribose (10)
To a stirring solution of 7 (95.8 mg, 0.124 mmol) and 8 (76.3 mg, 0.124 mmol) in dichloromethane (5 mL) was added DIPEA as a 1 M
solution in CH
Mixture was stirred for 30 min at room temperature and then 1,8-diazabicycloundec-7-ene as a 1 M solution in THF (1 mL,
mmol, 8 eq) was added. After stirring for an additional 30 min, solvent was removed by rotavap. Residue was purified by C18 chro-
matography to give 10 (124 mg, 70%) as a white solid.
3 3
CN (0.74 mL, 0.74 mmol, 6 eq) and N-chlorosuccinimide as a 1 M solution in CH CN (0.62 mL, 0.62 mmol, 5 eq).
1
1
H NMR (500 MHz, CD
H), 5.27 (d, J = 4.3 Hz, 1H), 4.42 (dd, J = 7.2, 4.4 Hz, 1H), 4.05 (dd, J = 4.4, 1.3 Hz, 1H), 3.99 (dd, J = 5.4, 4.3 Hz, 1H), 3.95 – 3.87
3
OD) d 8.29 (s, 1H), 7.81 (s, 1H), 7.24 (dd, J = 8.9, 2.0 Hz, 1H), 6.65 (m, 2H), 6.29 (s, 1H), 5.73 (d, J = 7.2 Hz,
1
(
m, 3H), 3.85 – 3.80 (m, 2H), 3.75 – 3.70 (m, 2H), 3.18 – 3.14 (m, 1H), 3.11 (t, J = 6.0 Hz, 2H), 2.96 (dd, J = 10.1, 4.4 Hz, 2H), 2.91
p, J = 1.6 Hz, 1H), 2.80 (q, J = 7.4 Hz, 3H), 2.33 – 2.27 (m, 2H), 1.60 (pd, J = 5.5, 1.1 Hz, 2H), 1.38 – 1.24 (m, 4H), 0.97 (ddt,
J = 14.4, 9.2, 5.2 Hz, 22H), 0.57 (s, 9H), 0.54 (s, 9H), 0.51 (s, 9H), 0.31 (s, 9H), -0.21 (s, 3H), -0.24 (s, 3H), -0.25 (s, 3H), -0.28
(
(
s, 3H), -0.29 (s, 3H), -0.31 (s, 3H), -0.41 (s, 3H), -0.77 (s, 3H).
13
3
C NMR (126 MHz, CD OD) d 163.03, 160.75, 157.36, 153.02, 151.00, 142.21, 141.85, 127.28, 120.00, 115.77, 113.82, 108.45,
1
2
05.03, 101.34, 88.03, 87.46, 87.39, 77.72, 74.77, 73.27, 66.76, 66.63, 55.43, 55.25, 43.49, 39.35, 33.62, 30.00, 27.54, 26.49, 26.48,
6.22, 24.99, 20.43, 19.57, 19.14, 18.98, 18.92, 18.86, 18.65, 17.44, 13.05, 9.12, -4.09, -4.18, -4.20, -4.24, -4.28, -4.31, -5.21.
19
F NMR (470 MHz, CD
P NMR (202 MHz, CD
3
OD) d -66.37.
OD) d -10.38 (d, J = 21.2 Hz), -10.80 (d, J = 21.2 Hz).
31
3
-
81 3 5 16 2 4
HRMS (ESI-TOF) m/z: [M-H] Cald for C49H F N O P Si 1226.4188. Found 1226.4139.
pNP-ADPr (1). To a 25 mL round-bottom flask was added 9 (109.7 mg, 0.0761 mmol, 1 eq) and THF (3.0 mL). Once starting material
ꢁ
was fully dissolve, triethylamine trihydrofluoride (0.5 mL) was added neat dropwise. Reaction vessel was sealed and heated to 65 C.
Reaction was closely monitored by TLC (80:20 2-propanol:0.2% NH
was cooled to room temperature and concentrated in vacuo. Remaining acid was quenched by dropwise addition of satd aq
NaHCO . Aqueous solution of crude product was purified by C18 chromatography utilizing ion-pairing reagent in the mobile phase
10 mM Et N-HOAc, pH 7.0) to yield compound 1 as a triethylammonium salt. The triethylammonium salt was eluted through Dowex
0W-8X (ammonium-form) to give the ammonium salt of compound 1 as a white powder (53.8 mg, 98%).
4
OH(aq)) Once reaction had gone to completion, reaction mixture
3
(
3
5
1
H NMR (500 MHz, D
pNP-2,6), 6.04 (d, J = 5.7 Hz, 1H, Ade-1’), 5.67 (d, J = 4.4 Hz, 1H, Rib-1’’), 4.62 (t, J = 5.7 Hz, 1H, Ade-2’), 4.46 (dd, J = 5.1,
2
O) d 8.36 (s, 1H, Ade-2), 8.03 (s, 1H, Ade-8), 7.92 (d, J = 9.2 Hz, 2H, pNP-3,5), 6.92 (d, J = 9.2 Hz, 2H,
3
4
.6 Hz, 1H, Ade-3’), 4.41 (dd, J = 6.0, 4.6 Hz, 1H, Rib-2’’), 4.36 (m, 2H, Ade-4’, Rib-4’’), 4.27 (dd, J = 6.2, 2.6 Hz, 1H, Rib-3’’),
.23 (m, 2H, Ade-5’), 4.08 (m, 2H, Rib-5’’).
13
C NMR (126 MHz, D
9.7, 65.7, 65.3.
2
O) d 161.8, 155.2, 152.5, 148.7, 141.5, 139.6, 125.6, 118.3, 116.3, 100.4, 86.8, 84.7, 83.8, 74.5, 71.3, 70.4,
6
31
P NMR (202 MHz, D
2
O) d -11.2.
-
HRMS (ESI-TOF) m/z: [M-H] Calcd for 679.0808; Found 679.0805.
TFMU-ADPr (2). To a stirring solution of 10 (67.2 mg, 0.044 mmol) in THF (1 mL) was added triethylamine trihydrofluoride (0.3 mL).
ꢁ
Mixture was heated to 65 C and stirred for 2 h. Once cooled to room temperature, reaction was quenched with addition of satd aq
NaHCO
3
. Aqueous mixture was purified by ion-pairing chromatography (10 mM Et
3
N-HOAc, C18) followed by ion exchange (Dowex
5
0W-8, ammonium form) to provide TFMU-ADPr (2) as a white solid (33.8 mg, 96%).
1
H NMR (500 MHz, D
H), 6.50 (s, 1H), 5.76 (d, J = 5.6 Hz, 1H), 5.56 (d, J = 4.5 Hz, 1H), 4.41 (t, J = 5.3 Hz, 1H), 4.33 – 4.28 (m, 2H), 4.28 – 4.25 (m, 1H), 4.22
2
O) d 8.11 (s, 1H), 7.71 (s, 1H), 7.25 (dd, J = 9.1, 1.8 Hz, 1H), 6.70 (dd, J = 9.0, 2.5 Hz, 1H), 6.58 (d, J = 2.5 Hz,
1
(
q, J = 4.1 Hz, 1H), 4.15 (dd, J = 6.2, 2.8 Hz, 1H), 4.15 – 4.05 (m, 2H), 4.01 (ddd, J = 11.5, 5.0, 3.0 Hz, 1H), 3.94 (dt, J = 11.4, 4.4 Hz, 1H).
Cell Chemical Biology 25, 1–9.e1–e19, December 20, 2018 e14

Please cite this article in press as: Drown et al., Monitoring Poly(ADP-ribosyl)glycohydrolase Activity with a Continuous Fluorescent Substrate, Cell
Chemical Biology (2018), https://doi.org/10.1016/j.chembiol.2018.09.008
1
3
2
2
C NMR (126 MHz, D O) d 161.71, 159.94, 154.33, 154.18, 151.56, 147.93, 141.37 (q, JCF = 32.8 Hz), 139.31, 125.98, 121.09
1
3
3
(
q, JCF = 275.1 Hz), 117.67, 114.65, 112.35 (q, JCF = 4.3 Hz), 107.33, 103.50, 100.16, 86.90, 84.51 (d, JCP = 8.2 Hz), 83.42
3
2
2
(
d, JCP = 8.3 Hz), 74.46, 71.13, 70.10, 69.57, 65.66 (d, JCP = 4.0 Hz), 65.11 (d, JCP = 4.2 Hz).
1
9
F NMR (471 MHz, D
P NMR (203 MHz, D
2
O) d -64.43.
O) d -11.08 (d, J = 21.9 Hz), -11.28 (d, J = 21.5 Hz).
3
1
2
-
25 3 5 16 2
HRMS (ESI-TOF) m/z: [M-H] Cald for C25H F N O P 770.0729; found 770.0723.
2’,3’,5’-tris-O-(tert-butyldimethylsilyl)-inosine (S10)
Intermediate was prepared according to a modified version of a previous report (H o¨ bartner and Silverman, 2005). Briefly, a 100 mL
round-bottom flask was charged with inosine (1.0 g, 3.7 mmol, 1 eq) and imidazole (1.7 g, 25 mmol, 6.7 eq). Material was dissolved in
dimethylformamide (4 mL). Tert-butyldimethylsilyl chloride (2.8 g, 18.7 mmol, 5 eq) was added in one portion. The solution was stirred
at room temperature for 16 h. Reaction mixture was diluted with CH
layer was washed two times with satd aq NaHCO , dried over Na SO
4
2
Cl
2 3
and quenched with the addition of satd aq NaHCO . Organic
3
2
, filtered through a pad of celite, and evaporated to give S10 as
1
a white solid (2.2 g, 96%) which was used in subsequent reactions without further purification. H NMR spectrum was consistent with
literature.
1
3
H NMR (500 MHz, CDCl ) d 12.94 (bs, 1H), 8.24 (s, 1H), 8.10 (s, 1H), 6.02 (d, J = 4.9 Hz, 1H), 4.50 (t, J = 4.6 Hz, 1H), 4.30
(
t, J = 4.0 Hz, 1H), 4.13 (q, J = 3.4 Hz, 1H), 4.00 (dd, J = 11.4, 3.7 Hz, 1H), 3.79 (dd, J = 11.4, 2.5 Hz, 1H), 0.96 (d, J = 0.7 Hz, 9H),
.93 (d, J = 0.7 Hz, 9H), 0.81 (d, J = 0.7 Hz, 9H), 0.15 (s, 3H), 0.14 (s, 3H), 0.10 (s, 3H), 0.09 (s, 3H), -0.02 (s, 3H), -0.18 (s, 3H).
0
2’,3’-bis-O-(tert-butyldimethylsilyl)-inosine (S11)
A 100 mL round-bottom flask was charged with S10 (1.2 g, 2.0 mmol, 1 eq). Compound was dissolved in tetrahydrofuran (31 mL).
ꢁ
Solution was cooled to 0 C. To vigorously stirring solution, trichloroacetic acid (15 g, 92 mmol, 47 eq) was added as an ice-cold so-
ꢁ
lution in H
NaHCO
Residue was purified by silica gel chromatography eluting with 93:7 CH
2
O (6.8 mL). Stirred reaction mixture at 0 C for 3 h. Quenched reaction by slowly cannulating reaction mixture into satd aq
3
. Once evolution of gas ceased, extracted three times with EtOAc. Dried organic layer over Na
2 4
SO , filtered, and evaporated.
2
Cl :CH OH to yield compound S11 as a white solid
2
3
(
641 mg, 66%).
1
3
H NMR (500 MHz, CDCl ) d 13.34 (bs, 1H), 8.46 (s, 1H), 7.95 (s, 1H), 5.80 (d, J = 7.7 Hz, 1H), 5.68 (bs, 1H), 4.87 (dd, J = 7.8, 4.6 Hz,
1
H), 4.31 (d, J = 4.5 Hz, 1H), 4.16 (s, 1H), 3.93 (dd, J = 12.9, 2.1 Hz, 1H), 3.72 (d, J = 13.0 Hz, 1H), 0.94 (d, J = 0.9 Hz, 9H), 0.75
d, J = 0.9 Hz, 9H), 0.12 (s, 3H), 0.11 (s, 3H), -0.12 (s, 3H), -0.52 (s, 3H).
(
1
3
C NMR (126 MHz, CDCl
4.42, -4.46, -4.49, -5.70.
3
) d 158.90, 147.87, 145.79, 141.04, 126.72, 91.05, 89.33, 74.79, 73.84, 62.91, 25.93, 25.80, 18.20, 17.92,
-
+
HRMS (ESI-TOF) m/z: [M+H] Calc for C22
H
40
N
4
O
5
Si
2
497.2610; Found 497.2614.
Triethylammonium 2’,3’-bis-O-(tert-butyldimethylsilyl)-inosin-5’-yl H-phosphonate (S12)
A 100 mL round-bottom flask was charged with S11 (420 mg, 0.85 mmol, 1 eq) and pyridine (8.5 mL). Diphenylphosphite (0.81 mL,
1
2
.223 g/mL, 990 mg, 4.2 mmol, 5 eq) was added dropwise. After stirring reaction mixture at room temperature for 1.5 h, H O (0.5 mL)
and triethylamine (0.5 mL) were added sequentially. Reaction was stirred for 30 min, then concentrated in vacuo. Remaining pyridine
was removed by repeated azeotropic evaporation with toluene. Residue was purified by C18 chromatography to provide compound
S12 as a white foam (464 mg, 83%).
e15 Cell Chemical Biology 25, 1–9.e1–e19, December 20, 2018

Please cite this article in press as: Drown et al., Monitoring Poly(ADP-ribosyl)glycohydrolase Activity with a Continuous Fluorescent Substrate, Cell
Chemical Biology (2018), https://doi.org/10.1016/j.chembiol.2018.09.008
1
3
HNMR(500 MHz, CD OD)d8.48(s,1H), 8.16 (s,1H), 6.84(d, JHP =620.8Hz,1H), 6.07 (d, J=6.6Hz,1H), 4.83(dd, J= 6.5, 4.4Hz,1H),
4
.41(dd, J=4.4, 2.1Hz,1H), 4.24-4.20(m,1H),4.20-4.14 (m, 1H), 4.12- 4.05(m, 1H), 3.23(q, J=7.3Hz,6H), 1.30(t, J=7.3Hz,9H), 0.96
s, 9H), 0.77 (s, 9H), 0.17 (s, 3H), 0.15 (s, 3H), 0.00 (s, 3H), -0.27 (s, 3H).
(
13
C NMR (126 MHz, CD
d, J = 4.4 Hz), 47.49, 26.44, 26.26, 18.88, 18.67, 9.19, -4.18, -4.29, -4.31, -5.10.
3
OD) d 158.78, 150.22, 146.99, 140.81, 125.52, 89.00, 86.60(d, JCP = 8.0 Hz), 77.09, 74.17, 64.05
(
3
1
P NMR (202 MHz, CD
3
OD) d 4.11.
-
HRMS (ESI-TOF) m/z: [M-H] Calc for C22
41 4 7 2
H N O PSi 559.2179; Found 559.2164.
2
’,3’-bis-O-(tert-butyldimethylsilyl)-inosin-5’-yl 2-cyanoethyl phosphonate (11)
ꢁ
To a 20 mL reaction vial was added compound S12 (246 mg, 0.372 mmol, 1 eq) and dry pyridine (4 mL). Solution was cooled to 0 C.
3-hydroxypropionitrile (0.070 mL, 1.04 g/mL, 73 mg, 1.0 mmol, 2.8 eq) was added to stirring solution. Pivaloyl chloride (0.09 mL, 0.98
ꢁ
g/mL, 88 mg, 0.73 mmol, 2 eq) was added dropwise. After stirring at 0 C for 2.5 h, reaction mixture was concentrated, azeotroping
with toluene. Resulting residue was redissolved in acetonitrile and purified by C-18 chromatography to yield compound 11 as a white
solid as a mixture of diastereomers (166 mg, 73%).
1
3
H NMR (500 MHz, CDCl ) d 8.40 (s, 0.5H), 8.36 (s, 0.5H), 8.03 (s, 0.5H), 8.01 (s, 0.5H), 6.98 (d, J = 722.4 Hz, 0.5H), 6.94
(
d, J = 722.1 Hz, 0.5H), 5.90 (dd, J = 4.7, 3.2 Hz, 1H), 4.79 (t, J = 4.5 Hz, 1H), 4.76 (t, J = 4.0 Hz, 1H), 4.52 – 4.23 (m, 7H), 2.87 –
.75 (m, 2H), 0.92 (s, 9H), 0.91 (s, 9H), 0.805 (s, 9H), 0.799 (s, 9H), 0.12 (s, 3H), 0.10 (s, 3H), 0.09 (s, 3H), -0.017 (s, 3 H), -0.022
s, 3H), -0.19 (s, 3H), -0.21 (s, 3H)
2
(
13
3
C NMR (126 MHz, CDCl ) d 159.03, 148.66, 148.64, 145.75, 145.68, 139.73, 139.53, 125.75, 125.69, 116.73, 116.60, 89.71,
8
1
9.64, 83.15, 83.10, 83.03, 82.97, 77.36, 74.94, 74.74, 71.73, 71.66, 64.55, 60.60, 60.56, 60.53, 25.87, 25.75, 20.17, 20.12, 20.08,
8.10, 17.94, -4.30, -4.56, -4.58, -4.69, -4.70, -4.90, -4.93.
31
P NMR (202 MHz, CDCl
3
) d 8.66, 8.41.
-
HRMS (ESI-TOF) m/z: [M-H] Calc for C25
44 5 7 2
H N O PSi 612.2444; Found 612.2448.
a-1’’-O-(4-nitrophenyl)-2’,2’’,3’,3’’-O-tetrakis-(tert-butyldimethylsilyl)-IDP-ribose (S13)
To a 20 mL reaction vial was added 6 (41.1 mg, 0.0604 mmol, 1.0 eq) and 11 (38.7 mg, 0.0630 mmol, 1.04 eq). Mixture was dried by
co-azeotroping with dry acetonitrile three times and placing under vacuum over P
nitrile (1.0 mL). Then, (i-Pr NEt (31 mg, 0.24 mmol, 4.0 eq) and N-chlorosuccinimide (24 mg, 0.18 mmol, 3 eq) were sequentially
added as 1 M solutions in acetonitrile and stirred at room temperature for 1 h. 1,8-diazabicycloundec-7-ene (92 mg, 0.6 mmol,
0 eq) was added as a 1 M solution in acetonitrile. After stirring for 30 min, reaction mixture was evaporated in vacuo and purified
by C18 chromatography to yield compound S13 as a white foam (34.6 mg, 40%).
2 5
O for 12 h. The mixture was dissolved in aceto-
2 2
)
1
1
H NMR (500 MHz, CD
d, J = 4.3 Hz, 1H), 4.46 (d, J = 4.3 Hz, 1H), 4.39 (dd, J = 5.4, 4.3 Hz, 1H), 4.35 – 4.28 (m, 3H), 4.23 (t, J = 4.8 Hz, 1H), 4.19 (dt, J = 4.3,
3
OD) d 8.55 (s, 1H), 8.14 (d, J = 9.2 Hz, 2H), 8.08 (s, 1H), 7.12 (d, J = 9.3 Hz, 2H), 6.08 (d, J = 7.5 Hz, 1H), 5.66
(
2
.6 Hz, 1H), 4.15 – 4.07 (m, 2H), 3.64 – 3.56 (m, 4H), 3.53 (t, J = 6.1 Hz, 4H), 3.36 (t, J = 5.6 Hz, 4H), 2.74 – 2.65 (m, 4H), 2.02 (tdd, J = 6.9,
5
.2, 4.2 Hz, 4H), 1.82 – 1.66 (m, 14H), 0.97 (s, 9H), 0.93 (s, 9H), 0.91 (s, 9H), 0.73 (s, 9H), 0.20 (s, 3H), 0.17 (s, 3H), 0.15 (s, 3H), 0.13
s, 3H), 0.11 (s, 3H), 0.08 (s, 3H), -0.00 (s, 3H), -0.36 (s, 3H).
(
31
P NMR (202 MHz, CD
C NMR (126 MHz CD
8.02 (d, J = 9.2 Hz), 87.70 (d, J = 9.3 Hz), 77.11, 74.81, 73.33, 66.71 (d, J = 5.5 Hz), 66.52 (d, J = 5.4 Hz), 55.33, 49.57, 39.37,
3
OD) d -11.3 (d, J = 21.9 Hz), -11.6 (d, J = 21.8 Hz).
OD) d 167.50, 164.28, 159.27, 150.61, 147.02, 143.00, 141.44, 126.59, 125.58, 117.36, 101.30, 88.57,
13
3
8
3
3.70, 30.01, 27.53, 26.50 (3C), 26.48, 26.22, 24.97, 20.44, 19.15, 18.99, 18.93, 18.68, -4.04, -4.11, -4.20, -4.23, -4.26, -4.30,
-
4.32, -5.32.
Cell Chemical Biology 25, 1–9.e1–e19, December 20, 2018 e16

Please cite this article in press as: Drown et al., Monitoring Poly(ADP-ribosyl)glycohydrolase Activity with a Continuous Fluorescent Substrate, Cell
Chemical Biology (2018), https://doi.org/10.1016/j.chembiol.2018.09.008
-
HRMS (ESI-TOF) m/z: [M-H] Calc for C45
H
80
N
5
O
17
P
2
Si
4
1136.4107; Found 1136.4115.
a-1’’-O-(4-(trifluoromethyl)umbellifer-7-yl)-2’,2’’,3’,3’’-O-tetrakis-(tert-butyldimethylsilyl)-IDP-ribose (S14)
To a stirring solution of 7 (93.6 mg, 0.121 mmol) and 11 (75.5 mg, 0.123 mmol) in dichloromethane (1 mL) was added DIPEA as a 1 M
3 3
solution in CH CN (0.73 mL, 0.73 mmol, 6 eq) and N-chlorosuccinimide as a 1 M solution in CH CN (0.61 mL, 0.61 mmol, 5 eq).
Mixture was stirred for 30 min at room temperature and then DBU as a 1 M solution in THF (1 mL, 1 mmol, 8 eq) was added. After
stirring for an additional 30 min, solvent was removed by rotary evaporator. Residue was purified by C18 chromatography to give S14
(
126 mg, 68%)
1
3
H NMR (500 MHz, CD OD) d 8.56 (s, 1H), 8.09 (s, 1H), 7.65 (dd, J = 9.6, 2.0 Hz, 1H), 7.05 (dd, J = 6.8, 2.4 Hz, 3H), 6.70 (s, 1H), 6.07
(
d, J = 7.4 Hz, 1H), 5.70 (d, J = 4.3 Hz, 1H), 4.89 (dd, J = 7.4, 4.4 Hz, 1H), 4.44 (d, J = 4.5 Hz, 1H), 4.40 (dd, J = 5.4, 4.3 Hz, 1H), 4.35 –
.25 (m, 6H), 4.22 (q, J = 3.9 Hz, 4H), 4.12 (t, J = 4.7 Hz, 3H), 3.72 (hept, J = 6.7 Hz, 2H), 3.58 (m, 2H), 3.52 (t, J = 5.9 Hz, 2H), 3.37
t, J = 5.7 Hz, 2H), 3.20 (q, J = 7.4 Hz, 2H), 2.76 – 2.68 (m, 2H), 2.01 (pd, J = 5.6, 1.3 Hz, 2H), 1.80 – 1.64 (m, 6H), 1.38 (t, J = 7.2 Hz, 14H),
.96 (s, 9H), 0.94 (s, 9H), 0.91 (s, 9H), 0.73 (s, 9H), 0.19 (s, 3H), 0.16 (s, 3H), 0.15 (s, 3H), 0.13 (s, 3H), 0.12 (s, 3H), 0.09 (s, 3H), -0.00
s, 3H), -0.36 (s, 3H).
4
(
0
(
1
3
2
3
C NMR (126 MHz, CD OD) d 167.45, 163.08, 160.79, 158.93, 157.38, 150.54, 146.83, 142.33 (q, JCF = 32.6 Hz), 141.41, 129.48,
1
3
3
1
8
3
27.29, 125.52, 123.23 (q, JCF = 274.9 Hz), 115.77, 113.81 (q, JCF = 6.4 Hz), 108.44, 105.04, 101.36, 88.59, 88.12 (d, JCP = 9.2 Hz),
3
2
2
7.55 (d, JCP = 9.4 Hz), 77.15, 74.79, 74.74, 73.31, 66.79 (d, JCP = 5.5 Hz), 66.54 (d, JCP = 5.4 Hz), 55.40, 55.27, 43.49, 39.36, 33.64,
0.04, 27.56, 26.49, 26.49, 26.47, 26.22, 25.00, 20.44, 19.57 (d, J = 1.7 Hz), 19.15, 19.00, 18.92, 18.67, 13.11, -4.06, -4.10, -4.20,
-
4.22, -4.24, -4.30, -4.32, -5.29.
3
1
P NMR (203 MHz, CD
3
OD) d -11.41 (d, J = 21.3 Hz), -11.81 (d, J = 21.9 Hz).
-
HRMS (ESI-TOF) m/z: [M-H] Cald for C49
80 3 4 17 2 4
H F N O P Si 1227.4028; Found 1227.4056.
pNP-IDPr (S15). To a 7 mL reaction vial was added S13 (17.4 mg, 0.0121 mmol, 1 eq) and THF (1.0 mL). Once starting material was
ꢁ
fully dissolve, triethylamine trihydrofluoride (0.2 mL) was added neat dropwise. Reaction vessel was sealed and heated to 60 C. Re-
action was closely monitored by TLC (80:20 2-propanol:0.2% NH
was cooled to room temperature and concentrated in vacuo. Remaining acid was quenched by dropwise addition of satd aq
NaHCO . Aqueous solution of crude product was purified by C-18 chromatography utilizing ion-pairing reagent in the mobile phase
10 mM Et N-HOAc) to yield compound pNP-IDPr (S15) as a triethylammonium salt. The triethylammonium salt was eluted through
Dowex 50W-8X (ammonium-form) to give the ammonium salt of compound pNP-IDPr (S15) as a white powder (6.2 mg, 75%).
4
OH(aq)). Once reaction had gone to completion, reaction mixture
3
(
3
1
H NMR (500 MHz, D
J = 4.5 Hz, 1H), 4.70 (t, J = 5.5 Hz, 1H), 4.50 (dd, J = 5.2, 3.5 Hz, 1H), 4.45 (dd, J = 6.2, 4.5 Hz, 1H), 4.42 - 4.37 (m, 2H), 4.31 (dd, J = 6.0,
2
O) d 8.37 (s, 1H), 8.09 (s, 1H), 8.06 (d, J = 9.1 Hz, 2H), 7.06 (d, J = 9.0 Hz, 2H), 6.06 (d, J = 5.9 Hz, 1H), 5.79 (d,
2
.5 Hz, 1H), 4.29 – 4.22 (m, 2H), 4.16 - 4.05 (m, 2H)
3
1
P NMR (202 MHz, D
2
O) d -11.14 (d, J = 21.8 Hz, 1P), -11.36 (d, J = 21.6 Hz, 1P)
1
3
1
13
C NMR (from H, C-HMBC, 500 MHz, D O) 162.04, 158.43, 148.37, 146.14, 141.63,139.60, 125.85, 123.59, 116.73, 100.32,
2
8
7.36, 84.78, 83.97, 74.53, 71.21, 70.38, 69.64, 65.57, 64.86.
-
24 5 17 2
HRMS (ESI-TOF) m/z: [M-H] Calc for C21H N O P 680.0648; Found 680.0650.
e17 Cell Chemical Biology 25, 1–9.e1–e19, December 20, 2018

Please cite this article in press as: Drown et al., Monitoring Poly(ADP-ribosyl)glycohydrolase Activity with a Continuous Fluorescent Substrate, Cell
Chemical Biology (2018), https://doi.org/10.1016/j.chembiol.2018.09.008
TFMU-IDPr (12). To a stirring solution of S13 (119.9 mg, 0.78 mmol) in THF (1.0 mL) was added triethylamine trihydrofluoride (0.5 mL,
ꢁ
ꢁ
3
.1mmol, 40 eq). Reactionmixture wassealed withTeflon capand heatedto 60 C. After stirring at 60 C for2 h, solvent wasremovedby
rotavap. ResiduewasneutralizedwithsatdaqNaHCO andaqueousmixturewaspurifiedbyion-pairingchromatography(10mMEt N-
HOAc, C18) followed by ion exchange (Dowex 50W-8, ammonium form) to provide TFMU-IDPr (12) (33.6 mg, 53%) as a white solid.
3
3
1
H NMR (500 MHz, D
.70 (s, 1H), 5.95 (d, J = 5.6 Hz, 1H), 5.77 (d, J = 4.4 Hz, 1H), 4.61 (t, J = 5.4 Hz, 1H), 4.49 – 4.42 (m, 2H), 4.44 – 4.39 (m, 1H), 4.37
q, J = 3.8 Hz, 1H), 4.30 (dd, J = 6.3, 2.8 Hz, 1H), 4.28 – 4.21 (m, 2H), 4.16 (ddd, J = 11.5, 5.1, 3.2 Hz, 1H), 4.09 (dt, J = 11.3, 4.6 Hz, 1H).
2
O) d 8.27 (s, 1H), 7.96 (s, 1H), 7.48 (dd, J = 9.1, 1.9 Hz, 1H), 6.93 (dd, J = 9.0, 2.4 Hz, 1H), 6.85 (d, J = 2.4 Hz, 1H),
6
(
1
3
2
C NMR (126 MHz, D
2
O) d 161.91, 160.21, 157.82, 154.77, 148.22, 145.93, 141.55 (q, JCF = 32.9 Hz), 139.48, 126.22, 123.33,
1
3
3
1
21.31 (q,
3
J
CF = 276.4, 275.8 Hz), 115.03, 112.72 (q, JCF = 8.2 Hz), 107.67, 103.87, 100.30, 87.54, 84.77 (d, JCP = 8.2 Hz),
2
2
8
3.86 (d, JCP = 8.5 Hz), 74.62, 71.32, 70.36, 69.71, 65.81 (d, JCP = 5.1 Hz), 65.29 (d, JCP = 5.3 Hz).
3
1
P NMR (202 MHz, D
F NMR (470 MHz, D
2
O) d -11.09 (d, J = 21.5 Hz), -11.31 (d, J = 21.3 Hz).
O) d -64.55.
19
2
-
HRMS (ESI-TOF) m/z: [M-H] Cald for C25
H
24
F
3
N
4
O
17
P
2
771.0569; Found 771.0566.
4
-nitrophenyl 2,3-bis-O-tert-butyldimethylsilyl-b-D-ribose-5-(difluorenylmethyl phosphate) (S5b)
To a 20 mL reaction vial, 4b (360.4 mg, 0.721 mmol, 1 eq) and bis-(9H-fluoren-9-ylmethyl)-N,N-diisopropylamidophosphite (Lam-
brecht et al., 2015) (488 mg, 0.936 mmol, 1.3 eq) were added. Dissolved material by addition of CH
ꢁ
2
Cl
to 0 C. 4,5-dicyanoimidazole (129 mg, 1.1 mmol, 1.5 eq) was added as a solution in acetonitrile (2 mL). After stirring at 0 C for
2
(11 mL). Cooled solution
ꢁ
15 min, mixture was allowed to warm to room temperature and was stirred for an additional 2 h. Once starting material was consumed
ꢁ
as indicated by TLC (75:25 hexane:EtOAc), mixture was cooled to 0 C and subjected to dropwise addition of tert-butyl hydroperoxide
(
0.5 mL, 2.5 mmol, 3.5 eq) as a 5 M solution in decane. Mixture was stirred for an additional 1 h and quenched with H
extracted with CH Cl three times. Combined organic layers were dried over Na SO , concentrated, and purified by silica gel chro-
matography eluting with 80:20 hexane:EtOAc to yield compound S5b as a white foam (628 mg, 93%).
2
O. Mixture was
2
2
2
4
1
3
H NMR (500 MHz, CDCl ) d 7.80 (d, J = 9.2 Hz, 2H), 7.72 (dd, J = 11.9, 7.5 Hz, 3H), 7.50 – 7.33 (m, 9H), 7.30 – 7.20 (m, 4H), 6.76
(
(
d, J = 9.2 Hz, 2H), 5.31 (m, 1H), 4.28 (dd, J = 7.0, 4.0 Hz, 1H), 4.21 – 3.98 (m, 9H), 3.92 (dt, J = 11.3, 4.0 Hz, 1H), 0.91 (s, 9H), 0.90
s, 9H), 0.11 (s, 3H), 0.10 (s, 3H), 0.08 (s, 3H), 0.06 (s, 3H).
13
3
C NMR (126 MHz, CDCl ) d 161.50, 143.09, 143.07, 143.04, 142.22, 141.43, 141.40, 141.37, 128.07, 128.04, 127.22, 127.20,
1
6
27.18, 125.64, 125.20, 125.14, 125.12, 120.19, 120.16, 120.12, 116.01, 105.07, 81.77, 81.70, 76.53, 70.84, 69.47, 69.45, 69.42,
9.41, 65.74, 65.70, 48.02, 47.98, 47.96, 47.92, 25.95, 25.86, 18.21, 18.14, -4.10, -4.43, -4.44, -4.86.
31
P NMR (202 MHz, CDCl
3
) d -1.68.
+
HRMS (ESI-TOF) m/z: [M+Na] Calcd for C51
2
H62NO10NaSi P 958.3548; Found 958.3540.
Triethylammonium 4-nitrophenyl 2,3-bis-O-tert-butyldimethylsilyl-b-D-ribose-5-phosphate (6b)
0 mL reaction vial was charged with compound S5b (551 mg, 0.59 mmol, 1 eq). Added acetonitrile (6 mL) and triethylamine (1.5 mL)
2
successively. Stirred at room temperature for 16 h. Added 1 mL toluene to stirring solution and concentrated in vacuo. Residue was
redissolved in methanol (0.5 mL) and purified by C-18 chromatography to yield the triethylammonium salt of compound 6b as a tan
foam (322 mg, 80%).
1
3
H NMR (500 MHz, CD OD) d 8.22 (d, J = 9.2 Hz, 2H), 7.19 (d, J = 9.3 Hz, 2H), 5.55 (d, J = 3.0 Hz, 1H), 4.40 (dd, J = 4.3, 3.0 Hz, 1H),
4
.36 (t, J = 4.3 Hz, 1H), 4.23 (dt, J = 5.7, 4.4 Hz, 1H), 4.00 (dt, J = 10.0, 4.7 Hz, 1H), 3.87 – 3.80 (m, 1H), 3.15 (q, J = 7.3 Hz, 6H), 1.28
t, J = 7.3 Hz, 9H), 0.95 (s, 9H), 0.93 (s, 9H), 0.19 (s, 3H), 0.17 (s, 3H), 0.16 (s, 3H), 0.16 (s, 3H).
(
Cell Chemical Biology 25, 1–9.e1–e19, December 20, 2018 e18

Please cite this article in press as: Drown et al., Monitoring Poly(ADP-ribosyl)glycohydrolase Activity with a Continuous Fluorescent Substrate, Cell
Chemical Biology (2018), https://doi.org/10.1016/j.chembiol.2018.09.008
1
3
C NMR (126 MHz, CD
7.53, 26.46, 26.38, 19.05, 18.97, 9.11, -4.09, -4.17, -4.33, -4.52.
3
OD) d 163.55, 143.73, 126.70, 117.67, 106.77, 85.92 (d, J = 9.2 Hz), 78.04, 73.84, 66.50 (d, J = 4.8 Hz),
4
3
1
P NMR (202 MHz, CD
3
OD) d 0.66.
-
HRMS (ESI-TOF) m/z: [M-H] Calcd for C23
H
2
41NO10Si P 578.2007; Found 578.2009.
b-1’’-O-(4-nitrophenyl)-2’,2’’,3’,3’’-O-tetrakis-(tert-butyldimethylsilyl)-ADP-ribose (9b)
To a 20 mL reaction vial was added 6b (113.7 mg, 0.167 mmol, 1.0 eq) and 8 (110.0 mg, 0.179 mmol, 1.1 eq). Mixture was dried by co-
azeotroping with dry acetonitrile three times and placing under vacuum over P
2.0 mL). Then, (i-Pr NEt (64.6 mg, 0.5 mmol, 3 eq) and N-chlorosuccinimide (53.4 mg, 0.4 mmol, 2.4 eq) were sequentially added as
M solutions in acetonitrile and stirred at room temperature for 1 h. 1,8-diazabicycloundec-7-ene (350 mg, 2.3 mmol, 10 eq) was
2 5
O for 12 h. The mixture was dissolved in acetonitrile
(
2 2
)
1
added as a 1 M solution in THF. After stirring for 30 min, reaction mixture was evaporated in vacuo and purified by C18 chromatog-
raphy to yield compound 9b as a white foam (149.6 mg, 60%).
1
3
H NMR (500 MHz, CD OD) d 8.64 (s, 1H), 8.173 (s, 1H), 8.172 (d, J = 9.2 Hz, 2H), 7.15 (d, J = 9.2 Hz, 2H), 6.10 (d, J = 7.5 Hz, 1H),
5
.51 (d, J = 4.1 Hz, 1H), 4.80 (dd, J = 7.5, 4.4 Hz, 1H), 4.45 (t, J = 4.2 Hz, 1H), 4.42 (d, J = 4.5, 1H), 4.38 (dd, J = 4.4, 2.7 Hz, 1H), 4.26
(
(
(
td, J = 5.9, 2.8 Hz, 1H), 4.23 (m, 2H), 4.16 (m, 1H), 4.08 (qt, J = 10.9, 5.5 Hz, 2H), 3.55 (m, 4H), 3.49 (t, J = 5.9 Hz, 4H), 3.35 (m, 9H), 2.68
m, 4H), 1.99 (m, 4H), 1.70 (m, 12H), 0.97 (s, 9H), 0.94 (s, 9H), 0.90 (s, 9H), 0.69 (s, 9H), 0.18 (s, 3H), 0.17 (s, 3H), 0.16 (s, 3H), 0.15
s, 6H), 0.14 (s, 3H), -0.03 (s, 3H), -0.40 (s, 3H)
1
3
C NMR (126 MHz, CD
d, J = 8.6 Hz), 86.75 (d, J = 9.0 Hz), 78.21, 77.67, 74.85, 74.26, 66.86 (d, J = 5.2 Hz), 66.68 (d, J = 5.1 Hz), 55.25, 49.85, 49.54, 39.34,
3.62, 30.02, 27.55, 26.51, 26.48, 26.44, 26.21, 24.98, 20.43, 19.08, 18.99, 18.92, 18.64, -4.08, -4.13, -4.19, -4.21, -4.26, -4.29, -5.29.
3
OD) d 167.45, 163.85, 157.31, 153.80, 151.17, 143.64, 141.65, 126.69, 120.02, 117.69, 107.14, 87.75, 87.63
(
3
3
1
P NMR (202 MHz, CD
3
OD) d -11.44 (d, J = 21.5 Hz), -11.65 (d, J = 21.5 Hz).
-
HRMS (ESI-TOF) m/z: [M-H] Calcd for C45
82 6 16 2 4
H N O P Si 1135.4267; Found 1135.4272.
b-pNP-ADPr (1b). To a 20 mL reaction vial was added 9b (24.1 mg, 0.0167 mmol, 1 eq) and THF (1 mL). Triethylamine trihydrofluor-
ide (0.1 mL, 0.989 g/mL) was added dropwise to stirring solution. Reaction vessel was sealed and heated to 60 C for 4 h. Reaction
was closely monitored by TLC (80:20 2-propanol:0.2% NH
cooled to room temperature and concentrated in vacuo. Remaining acid was quenched by dropwise addition of satd aq NaHCO
Aqueous solution of crude product was purified by C18 chromatography utilizing ion-pairing reagent in the mobile phase (10 mM
Et N-HOAc, pH 7.0) to yield compound 1b as a triethylammonium salt. The triethylammonium salt was eluted through Dowex
0W-8X (ammonium-form) to give the ammonium salt of compound 1b as a white powder (8.5 mg, 71%).
4
OH(aq)) Once reaction had gone to completion, reaction mixture was
3
.
3
5
1
2
H NMR (500 MHz, D O) d 8.33 (s, 1H), 8.11 (s,1H), 7.94 (d, J = 9.3 Hz, 2H), 6.93 (d, J = 9.4 Hz, 2H), 5.99 (d, J = 5.4, 1H),
5
.64 (d, J = 1.0 Hz, 1H), 4.62 (t, J = 5.2 Hz, 1H), 4.46 (m, 2H), 4.34 (m, 2H), 4.29 (td, J = 5.9, 4.1 Hz, 1H), 4.19 (m, 2H), 4.16
q, J = 3.9 Hz, 1H), 4.08 (m, 1H)
(
1
3
C NMR (126 MHz, D
2.78, 74.68, 70.70, 70.32, 66.69, 65.14.
2
O) d 181.63, 161.38, 155.43, 152.74, 148.68, 141.68, 139.53, 125.67, 118.51, 116.45, 105.24, 87.17, 83.80,
8
3
1
P NMR (202 MHz, D
2
O) d -10.19.
-
HRMS (ESI-TOF) m/z: [M-H] Calcd for C21
26 6 16 2
H N O P 679.0808; Found 679.0809.
QUANTIFICATION AND STATISTICAL ANALYSIS
All the data were presented as mean ± standard error of mean from at least three independent trials. All data fitting and statistical
analysis was performed using GraphPad Prism (version 6).
e19 Cell Chemical Biology 25, 1–9.e1–e19, December 20, 2018