Emissive Synthetic Cofactors: An Isomorphic, Isofunctional, and Responsive NAD+ Analogue

Article abstract of DOI:10.1021/jacs.7b05852

The synthesis, photophysics, and biochemical utility of a fluorescent NAD⁺ analogue based on an isothiazolo[4,3-d]pyrimidine core (N^tzAD⁺) are described. Enzymatic reactions, photophysically monitored in real time, show N^tzAD⁺ and N^tzADH to be substrates for yeast alcohol dehydrogenase and lactate dehydrogenase, respectively, with reaction rates comparable to that of the native cofactors. A drop in fluorescence is seen as N^tzAD⁺ is converted to N^tzADH, reflecting a complementary photophysical behavior to that of the native NAD⁺/NADH. N^tzAD⁺ and N^tzADH serve as substrates for NADase, which selectively cleaves the nicotinamide's glycosidic bond yielding ^tzADP-ribose. N^tzAD⁺ also serves as a substrate for ribosyl transferases, including human adenosine ribosyl transferase 5 (ART5) and Cholera toxin subunit A (CTA), which hydrolyze the nicotinamide and transfer ^tzADP-ribose to an arginine analogue, respectively. These reactions can be monitored by fluorescence spectroscopy, in stark contrast to the corresponding processes with the nonemissive NAD⁺.

Full text of DOI:10.1021/jacs.7b05852

Subscriber access provided by University of Florida | Smathers Libraries
Communication
Emissive synthetic cofactors: An isomorphic,
isofunctional and re-sponsive NAD+ analogue
Alexander R. Rovira, Andrea Fin, and Yitzhak Tor
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.7b05852 • Publication Date (Web): 18 Oct 2017
Downloaded from http://pubs.acs.org on October 18, 2017
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a free service to the research community to expedite the
dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts
appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been
fully peer reviewed, but should not be considered the official version of record. They are accessible to all
readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered
to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published
in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just
Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor
changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers
and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors
or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
Journal of the American Chemical Society is published by the American Chemical
Society. 1155 Sixteenth Street N.W., Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 11
Journal of the American Chemical Society
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 1
94x74mm (600 x 600 DPI)
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 2 of 11
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Scheme 1
1
67x65mm (300 x 300 DPI)
ACS Paragon Plus Environment

Page 3 of 11
Journal of the American Chemical Society
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 2
8
9x65mm (300 x 300 DPI)
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 4 of 11
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 3
89x75mm (300 x 300 DPI)
ACS Paragon Plus Environment

Page 5 of 11
Journal of the American Chemical Society
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 4
3
11x265mm (300 x 300 DPI)
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 6 of 11
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
TOC Figure
8
0x44mm (300 x 300 DPI)
ACS Paragon Plus Environment

Page 7 of 11
Journal of the American Chemical Society
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Emissive synthetic cofactors: An isomorphic, isofunctional and respon-
sive NAD analogue
+
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Alexander R. Rovira, Andrea Fin, Yitzhak Tor*
Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, California 92093-0358, United
States.
Supporting Information Placeholder
ABSTRACT: The synthesis, photophysics, and biochemical util-
+
ity of a fluorescent NAD analogue based on an isothiazolo[4,3-
tz
+
d]pyrimidine core (N AD ) are described. Enzymatic reactions,
tz
+
tz
photophysically monitored in real time, show N AD and N ADH
to be substrates for yeast alcohol dehydrogenase and lactate dehy-
drogenase, respectively, with reaction rates comparable to that of
tz
+
the native cofactors. A drop in fluorescence is seen as N AD is
tz
converted to N ADH, reflecting a complementary photophysical
+
tz
+
tz
behavior to that of the native NAD /NADH. N AD and N ADH
serve as substrates for NADase, which selectively cleaves the nic-
tz
tz
+
otinamide’s glycosidic bond yielding ADP-ribose. N AD also
serves as a substrate for ribosyl transferases, including human
adenosine ribosyl transferase 5 (ART5) and Cholera Toxin Subunit
tz
A (CTA), which hydrolyze the nicotinamide and transfer ADP-
ribose to an arginine analogue, respectively. These reactions can
be monitored by fluorescence spectroscopy, in stark contrast to the
+
corresponding processes with the non-emissive NAD .
+
Figure 1. Comparing the photophysical behavior of native NAD
tz
+
and N AD in reactions involving alcohol dehydrogenase.
+
Modified oligonucleotides represent a fraction of the innova-
tive ways for exploiting fluorescent nucleoside analogues. The
involvement in metabolic and regulatory processes make NAD a
key cofactor and its emissive analogues have been explored for
1
1
1
+
vast biochemistry of nucleosides and nucleotides as coenzymes and
secondary messengers offers unique opportunities for their emis-
sive surrogates as biophysical and mechanistic tools. Pioneered by
giants such as Leonard and Shugar, most early studies were done
decades. One NAD analogue that has been widely employed is
6
+
+
1
,N -etheno NAD (NAD ), originally introduced by Leonard and
12
coworkers. Enzymatic cleavage at the nicotinamide heterocycle
(
forming the corresponding nicotinamide and adenosine diphos-
6
with perturbing emissive nucleoside analogues (e.g., 1,N -ethen-
+
phate riboside) is required to induce a large change in NAD ’s
2
,3
oadenosine) or poorly emissive ones (e.g., 8-azapurines). A key
principle for the universal implementation of such probes is to min-
imize structural and functional perturbations, which are inevitable
consequences of replacing any native residue with a synthetic ana-
logue. We define nucleosides that fulfill such critical constraints as
being isomorphic and isofunctional, respectively. To serve as ef-
fective emissive probes, at least one of the analogue’s photophysi-
cal characteristics must respond to structural and environmental
13,14,15
emission quantum yield.
Other non-isomorphic fluorescent
+
11b
NAD analogues have been developed for similar applications,
11c,16
and a clickable version was recently reported.
Intriguingly,
while the reduced native cofactor, NADH, is emissive (em 460 nm,
1
7
+
), the native NAD is not. We thus sought to develop an
isomorphic and isofunctional redox couple with orthogonal photo-
physical behavior to the native substrate (Figure 1). Such a syn-
thetic cofactor could expand the processes that can be visualized in
4
changes. We describe such an attribute as responsiveness.
1
8
real-time by fluorescence spectroscopy.
NAD+ and NADH (Figure 1), the corresponding reduced
Our lab has developed a family of emissive isomorphic and
isofunctional ribonucleosides based on an isothiazolo[3,4-d] py-
rimidine core. Considering the red-shifted absorption band and
we hypothesized
that by replacing adenosine with A (Figure 1, Scheme 1), our flu-
orescent adenosine analogue, an emissive NAD analogue with dis-
5
form, are key determinants of the cellular redox state. In addition
6
+
to its metabolic roles and extracellular signaling functions, NAD
1
9
is also a substrate for several key enzymes, including poly-ADP-
ribose polymerases (PARP), mono-ADP-ribose transferases
+
17,20
emissive nature of NADH relative to NAD ,
tz
7
,8,9,10
(
ART), sirtuins, cyclases, and DNA ligases.
Its
+
tinct photophysical features and the potential to enhance the spec-
+
troscopic monitoring of NAD -dependent processes will be ob-
tained.
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 8 of 11
tz
a
Scheme 1. Synthesis of N AD
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
a
Reagents and conditions: (a) POCl
3
, Proton Sponge, trimethyl
o
phosphate, 4 C, 2 h, 50%. (b) i. β-Nicotinamide mononucleotide,
CDI, Et
tz
21
3
N, DMF, rt, 6 h; ii. AMP, DMF, rt, 4 days, 20%.
Herein we report the synthesis, photophysics, and enzymatic inter-
tz
+
+
conversions of N AD , an NAD analogue based on our isomor-
phic isothiazolo heterocyclic system that is emissive and isofunc-
+
Figure 2. (a) Enzymatic cycle for NAD consumption and regener-
tz
+
tz
tional. While the N AD /N ADH couple is complementary in its
ation with ADH and LDH. (b) ADH-mediated oxidation of ethanol
photophysical behavior to that of the native cofactors
tz
+
to acetaldehyde using N AD followed by UV and fluorescence
+
tz
+
NAD /NADH, the emissive N AD facilitates the fluorescence-
based monitoring of ADP-ribosylation reactions, which are “fluo-
rescently-silent” with the native cofactor.
tz
+
spectroscopies (ex = 330 nm). (c) As in b, showing N AD (red)
tz
to N ADH (orange) conversion, followed by HPLC (monitored at
330 nm). (d) ADH-mediated oxidation of ethanol to acetaldehyde
followed by LDH-mediated reduction of pyruvic acid to lactic acid
To prepare N AD , the previously synthesized tzA was
tz
+
19
tz
tz
+
+
treated with POCl
3
and trimethyl phosphate to give AMP
with N AD (red/orange) and NAD (grey) followed by real-time
(
Scheme 1), which was coupled to activated β-nicotinamide mon-
emission at 410 nm (ex = 330 nm) and 465 nm (ex = 335 nm),
1
1c,21
onucleotide following published protocols.
The spectroscopic
21
respectively. Dashed lines represent weighted curve fits.
tz
+
tz
properties of N AD closely resemble that of A, the core nucleo-
tz
+
side. The absorption and emission maxima are found to be 336 and
When subjecting N AD to ADH and ethanol in buffer (pH 7.6),
tz
4
11 nm, respectively, with a fluorescence quantum yield of 3.8 ±
0.4% (Table 1).
The biocompatibility and photophysical responsiveness of
the conversion to the corresponding N ADH was effectively mon-
itored via a large decrease in its visible fluorescence intensity (ex
330 nm, em 410 nm) and increase in absorbance at 330 nm (Figure
2
1
tz
+
2b).
N AD , was initially tested with S. cerviseae alcohol dehydrogen-
+
ase (ADH). This dehydrogenase catalyzes the reversible oxidation
of ethanol to acetaldehyde, using NAD as a cofactor (Figure 2a).
Subjecting NAD to the same enzymatic reaction with ADH
under the same conditions yielded a comparable rate with t = 23±3
+
½
+
tz
+
and 21±1 s for NAD and N AD , respectively (Figure S4 and Ta-
ble S2). The reaction was then reversed by adding excess acetalde-
hyde after the initial dehydrogenation was complete. The fluores-
cence signal was restored within seconds (Figure S5). Similarly,
Table 1. Photophysical properties
Stokes
shift

abs ()a

em ()a
a
tz
+
HPLC reaction monitoring showed near-full conversion of N AD
tzAb
338 (7.79)
336 (6.9)
338 (7.2)
336 (10.7)
259 (16.9)
339 (6.22)
410 (0.05)
411 (0.038)
411 (0.044)
412 (0.015)
-
5.23
5.41
5.23
5.49
-
tz
to N ADH after 5 min followed by instantaneous regeneration of
21
the former following the addition of acetaldehyde (Figure 2c).
tz
+c
N AD
+
To further challenge our emissive NAD analogue and assess
tz
+ d
N AD
its biochemical compatibility, it was tested with lactate dehydro-
genase (LDH), a metabolic enzyme catalyzing the interconversion
tz
d e
N ADH
NAD+ b
NADH b
+
22
of pyruvate to lactate and concurrently NADH to NAD . After
tz
+
consumption of N AD with ADH and ethanol, the reaction is
460 (0.02)
3
7.76
treated with pyruvic acid followed by LDH. The reformation of
tz
+
tz
a
–1
–1
N AD from N ADH shows nearly full restoration of fluorescence

abs, , em and Stokes shift are in nm, 10 M cm , nm and
3
–1
intensity (Figure 2d, red/orange). This behavior was complemen-
10 cm , respectively. All values reflect the average of at least
three independent measurements. See Table S1 for experimental
+
tary to that of NAD , essentially mirroring its time course, which
b
c
d
shows enhanced emission at 465 nm, arising from the formation of
NADH, followed by a subsequent decrease in emission after the
addition of LDH (Figure 2d, grey).
errors. See references 17 and 19. Measured in MilliQ water
Measured in Tris buffer pH 7.6. Measured at ADH reaction end,
assuming complete consumption, Tris buffer pH 7.6.
e
2
ACS Paragon Plus Environment

Page 9 of 11
Journal of the American Chemical Society
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
tz
+
tz
+
Figure 3. (a) Enzymatic cycle for N AD consumption by ADH
and NADase. (b) UV-Vis and emission (ex = 330 nm) spectra of
Figure 4. (a) Treatment of N AD with ART5 and CTA to yield
ADPR and ADPR-agmatine, respectively. (b) Steady state emis-
sion spectra following treatment of N AD with ART5 at 0 (blue)
and 18 min (red), as well as NAD at 0 (green) and 18 min (orange),
ex = 335 nm; Inset: Fluorescence based kinetics of aforementioned
reaction (em = 410 nm, ex = 335 nm, blue solid), and normalized
HPLC-monitored product formation from reactions with N AD
(blue, dashed) and native NAD (red, dashed). (c) Steady state emis-
sion spectra ( = 335 nm) following treatment of N AD with
CTA; reaction sampled at 0 (blue), 20 (green), 50 (orange), and
tz
+
tz
+
21
N AD at time 0 (red), after oxidizing ethanol to acetaldehyde with
ADH (orange) and subsequent treatment with NADase (blue). (c)
Real-time emission intensity at 410 nm (ex = 330 nm) of the enzy-
matic oxidation of ethanol to acetaldehyde by ADH with N AD
orange, bottom time scale) followed by cleavage with NADase
blue, top time scale). Inset: Cleavage of N AD with NADase
blue) followed by real-time emission at 410 nm (ex = 330 nm).
+

tz
+
tz
+
(
(
(
tz
+
tz
+
ex
21
tz
+
To shed light on the photophysical behavior of N AD and
9
0 min (pink); Inset: Reactions with CTA following normalized
emission intensity at 410 nm (ex = 335 nm, blue, solid), normal-
ized HPLC-monitored product formation from reactions with
tz
N ADH, their response upon enzymatic cleavage of the nicotina-
mide moiety with porcine brain NADase was evaluated (Figure 3).
+
NADase specifically cleaves NAD at the nicotinamide-ribose
tz
+
+
21
N AD (blue, dashed) and native NAD (red, dashed).
1
2,23
linkage, yielding nicotinamide and ADP-ribose (ADPR).
Treat-
tz
tz
+
ing N ADH (generated from N AD with ADH and ethanol) with
NADase yielded a net 30% emission enhancement above the initial
level before treatment with ADH (Figure 3b and 3c). Upon treat-
ment of N AD with NADase, a 40% increase in emission was ob-
served (Figure 3c, inset). We hypothesize that the diminished emis-
sion observed upon reducing N AD to N ADH could arise from
static quenching or a filtering effect by the reduced nicotinamide
moiety, which in NADH absorbs at nearly the same wavelength as
A (Table 1). The observed emission enhancement upon treatment
with NADase and subsequent conversion to ADP Ribose (Figure
) suggests, however, that the photophysics of these molecules is
influenced by a myriad of intramolecular ground and excited state
interactions between the nicotinamide and A moieties. These may
include a combination of filter effects, photoinduced electron trans-
fer (PET), and additional quenching pathways arising from prox-
imity-driven interactions between the nicotinamide and isothia-
as they facilitate clearer detection of reactivity and biocompatibil-
ity. Two commercially available proteins with reported mono-
ADP-ribosylation activity were used: human ART5, a transferase
originally cloned from Yac-1 lymphoma cells in mice, and Chol-
era toxin subunit A (CTA), derived from Cholera toxin, a protein
5
toxin family. While arginine-specific ARTs operate
in diverse biological systems and are regulated in complex man-
we chose ART5 as it has been identified as a major pro-
ducer of arginine-specific ADP-ribose modification and CTA, as
tz
+
26
27
tz
+
tz
from the AB
25, 28
ners,
tz
29
tz
30
it has been well-studied as an arginine-specific ADPR transferase.
3
Upon treatment with ART5 and agmatine (a commonly used
Arg surrogate in ART assays), both NAD and N AD were
found to primarily undergo hydrolysis, under a variety of condi-
tions, forming ADP Ribose and ADP Ribose, respectively (Figure
). This process, which has been documented for NAD ,
found to occur at the same rate (Figure 4b), as detected via emission
(for N AD ) and HPLC (for both NAD and N AD ). Upon treat-
ment with CTA and agmatine, both NAD and N AD were found
2
9
+
tz
+
tz
tz
+ 26a,29
4
was
+
zolo-pyrimidine core, as reported for NAD and related ana-
tz
+
+
tz
+ 31
13a,17,20,24
logues.
+
tz
+
Finally, to illustrate the unique features of the emissive
tz
to produce primarily ADPR-agmatine and ADPR-agmatine, re-
spectively, as monitored via emission (for N AD ) and HPLC (for
tz
+
+
N AD compared to the non-emissive NAD and take advantage
of the photophysical changes induced upon cleavage of the nicotin-
amide moiety, we have expanded the enzymatic processes moni-
tored to ADP ribosyl transfer reactions. While several enzyme
classes exploit NAD through cleavage of the nicotinamide (e.g.,
tz
+
+
tz
+
both NAD and N AD ) (Figure 4c). These reactions show com-
plete consumption of the corresponding substrates within 90
minutes, and formation of roughly 10% of hydrolyzed ADPR or
2
5
+
tz
tz
+
ADPR as a minor product. N AD seemed to react slightly slower
PARPs, sirtuins), we employed arginine-specific mono-ADP-
ribose transferases (ARTs),
than the native cofactor with CTA. Importantly, however,
ADPR-agmatine was found to have near identical photophysical
properties as A, thus facilitating the fluorescence-based monitor-
ing of the enzyme-mediated ADP-ribosylation.
tz
tz
3
2
3
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 10 of 11
tz
+
6. Garten, A.; Schuster, S.; Penke, M.; Gorski, T.; de Giorgis, T.; Kiess, W., Nat. Rev.
Endocrinol. 2015, 11, 535-546.
While the reactions of N AD with alcohol and lactate dehy-
drogenase exemplify the isofunctionality of our emissive cofactor
within the context of biochemically-relevant redox reactions, its re-
activity with mono ADP-ribose transferases reinforces this notion.
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
7
1
. (a) Chambon, P.; Mandel, P.; Weill, J. D., Biochem. Biophys. Res. Commun. 1963,
1, 39-43; (b) Hayaishi, O.; Ueda, K., Annu. Rev. Biochem 1977, 46, 95-116;
(c) Cervantes-Laurean, D.; Minter, D. E.; Jacobson, E. L.; Jacobson, M. K.,
Biochemistry 1993, 32, 1528-1534; (d) Hassa, P. O.; Haenni, S. S.; Elser, M.; Hottiger,
M. O., Microbiol. Mol. Biol. Rev. 2006, 70, 789-829.
tz
+
In particular, the suitability of N AD as a substrate for ADP ribo-
syl transferases, key enzymes responsible for diverse post-tran-
scriptional modifications of cellular regulatory significance, illus-
trated with CTA-mediated ADP-ribosylation of agmatine, show-
cases the formation of a new glycoside linkage to an amino acid
derivative. Above all and in stark contrast to the non-emissive na-
tive NAD , such processes yield fluorescent ribosylated products
and can be kinetically monitored by enhanced fluorescence signals,
due to the displacement of the quenching nicotinamide moiety.
8
. (a) Boulikas, T., Anticancer Res. 1991, 11, 489-527; (b) Hottiger, M. O., Annu. Rev.
2
5
Biochem. 2015, 84, 227-263.
9. Bonkowski, M. S.; Sinclair, D. A., Nat. Rev. Mol. Cell Biol. 2016, 17, 679-690.
10. Moss, J.; Stanley, S. J., J. Biol. Chem. 1981, 256, 7830-3.
tz
1
1. (a) Pankiewicz, K. W.; Watanabe, K. A.; Lesiak-Watanabe, K.; Goldstein, B. M.;
Jayaram, H. N., Curr. Med. Chem. 2002, 9, 733-741; (b) Pergolizzi, G.; Butt, J. N.;
Bowater, R. P.; Wagner, G. K., Chem. Commun. 2011, 47, 12655-12657; (c) Wang,
Y.; Rosner, D.; Grzywa, M.; Marx, A., Angew. Chem., Int. Ed. 2014, 53, 8159-8162.
+
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2. Barrio, J. R.; Secrist, J. A.; Leonard, N. J., Proc. Natl. Acad. Sci. U. S. A. 1972, 69,
2039-2042.
In summary, the isothiazolo[4,3-d]pyrimidine-based NAD+
analogue displays isofunctionality and complementary photophys-
ical behavior when compared to its native counterpart, with the ox-
13. (a) Gruber, B. A.; Leonard, N. J., Proc. Natl. Acad. Sci. U. S. A. 1975, 72, 3966-
3
969; (b) Luisi, P. L.; Baici, A.; Bonner, F. J.; Aboderin, A. A., Biochemistry 1975,
14, 362-368.
14. Lee, C.-Y.; Everse, J., Arch. Biochem. Biophys. 1973, 157, 83-90.
tz
+
+
idized form (N AD ) being much more emissive than the reduced
15. To our knowledge, the enzymatic conversion of εNAD to εNADH has not been
tz
+
photophysically studied. The cofactors have shown photophysical differences when
bound to GDH: Dieter, H.; Koberstein, R.; Sund, H., FEBS. Lett. 1974, 47, 90-93.
one (N ADH). To our knowledge, no fluorescent NAD analogues
+
with photophysical behavior complementary to the native NAD
1
6. Willner has electrochemically studied surface-bound cofactors; See: (a) Bardea,
and NADH couple have been previously reported. Furthermore,
A.; Katz, E.; Bückmann, A. F.; Willner, I., J. Am. Chem. Soc. 1997, 119, 9114-9119;
(b) Katz, E.; Willner, I., Angew. Chem. Int. Ed. 2004, 43, 6042-6108.
tz
+
N AD serves as faithful substrate for ADP-ribose transferases.
1
9
7. Scott, T. G.; Spencer, R. D.; Leonard, N. J.; Weber, G., J. Am. Chem. Soc. 1970,
2, 687-695.
+
tz
+
Unlike the non-emissive NAD , N AD facilitates the kinetic mon-
itoring of the enzymatic hydrolysis and transferase activity by flu-
orescence spectroscopy and yields visibly fluorescent products.
18. (a) Vranken, C.; Fin, A.; Tufar, P.; Hofkens, J.; Burkart, M. D.; Tor, Y., Org.
Biomol. Chem. 2016, 14, 6189-6192; (b) Deen, J.; Vranken, C.; Leen, V.; Neely, R.
K.; Janssen, K. P. F.; Hofkens, J., Angew. Chem. Int. Ed. 2017, 56, 5182-5200.
19. (a) Rovira, A. R.; Fin, A.; Tor, Y., J. Am. Chem. Soc. 2015, 137, 14602-14605; (b)
Rovira, A. R.; Fin, A.; Tor, Y., Chem. Sci. 2017, 8, 2983-2993.
tz
+
N AD has thus been subjected to five enzymes, which share com-
+
mon mechanistic pathways with most other NAD -utilizing reac-
tions, where the nicotinamide moiety serves as either a redox unit
20. Hull, R. V.; Conger III, P. S.; Hoobler, R. J., Biophys. Chem. 2001, 90, 9-16.
21. See supporting information for experimental details.
tz
+
or as a leaving group. A synthetic cofactor such as N AD , with
unprecedented photophysical responses and biocompatibility,
could therefore enhance and expand the real-time visualization of
cofactor-dependent processes by fluorescence spectroscopy.
2
2. (a) Månsson, M.-O.; Larsson, P.-O.; Mosbach, K., FEBS Lett. 1979, 98, 309-313;
(b) Chenault, H. K.; Whitesides, G. M., Bioorg. Chem. 1989, 17, 400-409; (c) Faber,
K., Biotransformations in organic chemistry. Springer-Verlag: Berlin ; New York,
1992.
2
2
3. Kaplan, N. O.; Colowick, S. P.; Nason, A., J. Biol. Chem. 1951, 191, 473-483.
4. Tanaka, M.; Ohkubo, K.; Fukuzumi, S., J. Phys. Chem. A 2006, 110, 11214-11218.
ASSOCIATED CONTENT
Supporting Information
Synthetic and analytical details, photophysical data, enzymatic pro-
tocols and HPLC traces. This material is available free of charge
via the Internet at http://pubs.acs.org.
25. Endogenous ADP-Ribosylation; Koch-Nolte, F., Ed.; Springer International
Publishing: 2015.
2
6. (a) Weng, B. Y.; Thompson, W. C.; Kim, H. J.; Levine, R. L.; Moss, J., J. Biol.
Chem. 1999, 274, 31797-31803; (b) Okazaki, I. J.; Moss, J., Annu Rev Nutr 1999, 19,
485-509.
2
7. Vanden Broeck, D.; Horvath, C.; De Wolf, M. J., Int. J. Biochem. Cell Biol. 2007,
39, 1771-5.
8. (a) Moss, J.; Stanley, S. J.; Osborne, J. C., Jr., J. Biol. Chem. 1981, 256, 11452-6;
b) Hottiger, M. O.; Hassa, P. O.; Luscher, B.; Schuler, H.; Koch-Nolte, F., Trends
2
(
Biochem. Sci 2010, 35, 208-219.
29. Glowacki, G.; Braren, R.; Firner, K.; Nissen, M.; Kuhl, M.; Reche, P.; Bazan, F.;
Cetkovic-Cvrlje, M.; Leiter, E.; Haag, F.; Koch-Nolte, F., Protein Sci. 2002, 11, 1657-
AUTHOR INFORMATION
Corresponding Author
ytor@ucsd.edu
1
670.
30. (a) Moss, J.; Vaughan, M., J. Biol. Chem. 1977, 252, 2455-2457; (b) Tsai, S. C.;
Noda, M.; Adamik, R.; Moss, J.; Vaughan, M., Proc. Natl. Acad. Sci. U.S.A. 1987, 84,
5
139-42.
31. High levels of glycohydrolase activity versus transferase activity have been
reported for ART5. See reference 29.
Notes
Yitzhak Tor provides consulting services to TriLink Biotechnolo-
gies. The terms of the arrangements have been reviewed and ap-
proved by UCSD in accordance with its conflict of interest policies.
2. ADP-ribose and tzADPR-agmatine have the same photophysical properties as
tz
3
tzA, the parent nucleoside. See SI and Figures S9 and S15.
ACKNOWLEDGMENT
We thank the National Institutes of Health for generous support
(GM 069773) and UCSD’s Chemistry and Biochemistry MS Facil-
ity.
REFERENCES
1
. Sinkeldam, R. W.; Greco, N. J.; Tor, Y., Chem. Rev. 2010, 110, 2579-2619.
2. Leonard, N. J. B., J. R., Crit. Rev. Biochem. 1984, 15, 125-199.
3
2
. Wierzchowski, J.; Antosiewicz, J. M.; Shugar, D., Mol. BioSyst. 2014, 10, 2756-
774.
4. Sinkeldam, R. W.; Tor, Y., Org. Biomol. Chem. 2007, 5, 2523-2528.
. (a) Everse, J.; Anderson, B.; You, K., The Pyridine nucleotide coenzymes. Academic
5
Press: New York, 1982; (b) Ying, W. H., Antioxid. Redox Signaling 2008, 10, 179-
206; (c) Collins, Y.; Chouchani, E. T.; James, A. M.; Menger, K. E.; Cocheme, H. M.;
Murphy, M. P., J. Cell Sci. 2012, 125, 801-806.
4
ACS Paragon Plus Environment

Page 11 of 11
Journal of the American Chemical Society
1
2
3
4
5
6
7
8
9
Insert Table of Contents artwork here
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
5
ACS Paragon Plus Environment