Enzymatic and chemical syntheses of vacor analogs of nicotinamide riboside, nmn and nad

Article abstract of DOI:10.3390/biom11071044

It has recently been demonstrated that the rat poison vacor interferes with mammalian NAD metabolism, because it acts as a nicotinamide analog and is converted by enzymes of the NAD salvage pathway. Thereby, vacor is transformed into the NAD analog vacor adenine dinucleotide (VAD), a molecule that causes cell toxicity. Therefore, vacor may potentially be exploited to kill cancer cells. In this study, we have developed efficient enzymatic and chemical procedures to produce vacor analogs of NAD and nicotinamide riboside (NR). VAD was readily generated by a base-exchange reaction, replacing the nicotinamide moiety of NAD by vacor, catalyzed by Aplysia californica ADP ribosyl cyclase. Additionally, we present the chemical synthesis of the nucleoside version of vacor, vacor riboside (VR). Similar to the physiological NAD precursor, NR, VR was converted to the corresponding mononucleotide (VMN) by nicotinamide riboside kinases (NRKs). This conversion is quantitative and very efficient. Consequently, phosphorylation of VR by NRKs represents a valuable alternative to produce the vacor analog of NMN, compared to its generation from vacor by nicotinamide phosphoribosyltransferase (NamPT).

Full text of DOI:10.3390/biom11071044

biomolecules
Article
Enzymatic and Chemical Syntheses of Vacor Analogs of
Nicotinamide Riboside, NMN and NAD
Lars Jansen Sverkeli 1,2, Faisal Hayat 3, Marie E. Migaud 3 and Mathias Ziegler 2,
*
1
Department of Biological Sciences, University of Bergen, 5007 Bergen, Norway; lars.sverkeli@uib.no
Department of Biomedicine, University of Bergen, 5009 Bergen, Norway
Mitchell Cancer Institute, University of South Alabama, Mobile, AL 36604, USA;
fhayat@health.southalabama.edu (F.H.); mmigaud@southalabama.edu (M.E.M.)
Correspondence: mathias.ziegler@uib.no
2
3
*
Abstract: It has recently been demonstrated that the rat poison vacor interferes with mammalian
NAD metabolism, because it acts as a nicotinamide analog and is converted by enzymes of the NAD
salvage pathway. Thereby, vacor is transformed into the NAD analog vacor adenine dinucleotide
(VAD), a molecule that causes cell toxicity. Therefore, vacor may potentially be exploited to kill cancer
cells. In this study, we have developed efficient enzymatic and chemical procedures to produce vacor
analogs of NAD and nicotinamide riboside (NR). VAD was readily generated by a base-exchange
reaction, replacing the nicotinamide moiety of NAD by vacor, catalyzed by Aplysia californica ADP
ribosyl cyclase. Additionally, we present the chemical synthesis of the nucleoside version of vacor,
vacor riboside (VR). Similar to the physiological NAD precursor, NR, VR was converted to the
corresponding mononucleotide (VMN) by nicotinamide riboside kinases (NRKs). This conversion
is quantitative and very efficient. Consequently, phosphorylation of VR by NRKs represents a
valuable alternative to produce the vacor analog of NMN, compared to its generation from vacor by
nicotinamide phosphoribosyltransferase (NamPT).
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Citation: Sverkeli, L.J.; Hayat, F.;
Migaud, M.E.; Ziegler, M. Enzymatic
and Chemical Syntheses of Vacor
Analogs of Nicotinamide Riboside,
NMN and NAD. Biomolecules 2021, 11,
1044. https://doi.org/10.3390/
biom11071044
Keywords: NAD+; vacor; base exchange; Vorbruggen chemistry
1. Introduction
Academic Editor: Jesús Fernandez
Since its discovery in the early 1900s [
has been considered a vital cofactor and signaling molecule for all living organisms. Orig-
inally identified as a cofactor for redox reactions [ ], it has since then been shown to
serve as a substrate for several important cellular functions, many of which lead to the
degradation of NAD [ ]. As NAD is constantly consumed, the cell is dependent on a
1], nicotinamide adenine dinucleotide (NAD)
Lucas
1–3
Received: 1 June 2021
Accepted: 7 July 2021
Published: 16 July 2021
4–9
continuous supply of NAD. In mammalian cells, most NAD is synthesized in a two-step
process called the nicotinamide (Nam) salvage pathway (Figure 1). In this process, Nam
is converted to nicotinamide mononucleotide (NMN) by nicotinamide phosphoribosyl
transferase (NamPT) in a rate-limiting reaction [10]. This reaction requires phosphoribosyl
pyrophosphate (PRPP) as co-substrate. NMN is then converted to NAD using ATP in a
reaction catalyzed by nicotinamide mononucleotide adenylyl transferases (NMNATs), of
which there are three isoforms with different subcellular localization and tissue-specific
expression patterns [11]. As Nam is the product of most reactions consuming NAD, the
salvage pathway lets the cell recycle Nam as its main NAD precursor. Given the impor-
tance of NAD, the ability to control the cell’s NAD pool can be a very valuable asset
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from a therapeutic perspective [
promising strategy for modulating the cell’s NAD levels is interfering with NAD produc-
tion [15 20]. Consequently, the NAD biosynthetic pathways have emerged as a topic of
significant research.
5,12–15]. As the NAD pool is constantly replenished, one
–
Biomolecules 2021, 11, 1044. https://doi.org/10.3390/biom11071044
https://www.mdpi.com/journal/biomolecules

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NRKs
Figure 1. Vacor acts as an analogue to nicotinamide: Overview of the salvage pathway of NAD biosynthesis (left).
Nicotinamide is converted to nicotinamide mononucleotide (NMN) in a rate-limiting reaction catalyzed by nicotinamide
phosphoribosyl transferase (NamPT). NMN is then converted to NAD in a reaction catalyzed by nicotinamide mononu-
cleotide adenylyl transferases (NMNATs). The alternative NAD precursor nicotinamide riboside (NR) enters the pathway
by conversion to NMN catalyzed by nicotinamide riboside kinases (NRKs). Vacor is converted to an NAD analogue through
the same pathway, being converted to vacor mononucleotide (VMN) by NamPT and then further converted to vacor adenine
dinucleotide (VAD) by NMNAT2. Possible alternative pathways for generating or consuming VAD that are addressed in
the present study are indicated with question marks.
One compound that has recently seen a certain resurgence in attention is vacor. Origi-
nally developed as a rat poison [21], without a known mechanism of action, recent research
has shown that vacor supplementation leads to accumulation of vacor mononucleotide
(VMN) and vacor adenine dinucleotide (VAD), that is, vacor-containing analogues of
NMN and NAD, respectively [20
NAT2 [20 22], showing that vacor indeed enters the salvage pathway of the NAD biosyn-
thesis (Figure 1). Interestingly, vacor has been shown to act as a potent cytotoxic agent,
leading to rapid NAD depletion and subsequent cell death [20 22]. Furthermore, vacor-
induced cytotoxicity appears to be selective, affecting primarily neuronal and pancreatic
tissue [20 22 23], making vacor an interesting toxin not only from an academic viewpoint,
,22]. This conversion is catalyzed by NamPT and NM-
,
,
,
,
but also as a potential chemotherapeutic agent. The molecular mechanisms of vacor-
induced cytotoxicity have remained unknown. However, it is clear that the accumulation
of vacor-derived NAD analogs are crucial elements [20,22]. Understanding these mecha-
nisms could identify novel biological processes and eventually new therapeutic targets.
Critically, current methods to produce vacor analogs of NAD and its biosynthetic interme-
diates are rather complex and time consuming, as they involve expression and purification
of the enzymes of the salvage pathway of the NAD biosynthesis [20,22]. In this study,
we developed an easy, one-step method for synthesizing VAD (Figure 1), exploiting the
catalytic properties of commercially available ADP-ribosyl cyclases [24,25].

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In addition, while the salvage pathway is the main source of NAD for the cell, alterna-
tive NAD precursors can be converted to NAD through different pathways. Nicotinamide
riboside (NR), in particular, has been extensively studied recently due to its ability to
boost systemic NAD levels when taken as a food supplement [15
the biosynthetic pathway through nicotinamide riboside kinases (NRKs), which convert
NR to NMN in an ATP-dependent reaction [29 30], bypassing the rate-limiting step of the
,18,26–28]. NR enters
,
salvage pathway (Figure 1). Here, we present the chemical synthesis of vacor riboside
(VR) and demonstrate that it acts as an authentic NR analogue, as it is indeed converted to
VMN by NRKs. Thereby, VR could be a useful alternative to vacor, as a tool to study the
mechanisms of vacor toxicity. Taken together, we have developed and validated efficient
methods to produce vacor analogs of NAD and its biosynthetic intermediates that will be
useful to explore the mechanisms of vacor toxicity.
2. Materials and Methods
Unless stated otherwise, all reagents were purchased from Sigma-Aldrich (Oslo,
NO, USA).
2.1. Synthesis of VAD by A. californica ADP-Ribosyl Cyclase
A reaction mix containing 250
NAD+ or cADPr in 10 mM Na-phosphate, pH 7.0, was prepared in a total volume of
500 L. The reaction was started by addition of 0.1 U A. californica ADP-ribosyl cyclase
(Sigma-Aldrich) and incubated at room temperature for 30 min. The reaction mix was then
µM vacor (greyhound chromatography) and 50 µM
µ
filtered using a 10 kDa-cutoff spin column (Amicon) at 14,000
×
g, at 4 ◦C for 10 min, and
the flow-through stored at −80 ◦C for further analysis.
2.2. VR Synthesis
In a 50 mL round bottom flask equipped with a stir bar, 3-picolylamine (1.0 eqv,
5 mmol) was dissolved in 50 mL anhydrous chloroform. The round bottom flask was
evacuated and filled with argon, then a solution of 4-nitrophenyl isocyanate (1.0 eqv,
5 mmol) in 5 mL anhydrous chloroform was added. The resulting mixture was stirred
at RT and the formation of a yellow precipitate was observed immediately. The reaction
mixture was stirred at RT overnight, after which time, the yellow precipitate was filtered
off, washed with chloroform, and dried under reduced pressure to afford vacor as a yellow
powder. Yield 92%, 1H NMR (400 MHz; DMSO-d6)
δ, ppm: 9.43 (s, 1H, NH), 8.55 (brs, 1 H,
NH), 8.46 (d, J = 4.76 Hz, 1 H, Ar-H), 8.14 (d, J = 9.32 Hz, 2 H, Ar-H), 7.72 (d, J = 7.04 Hz,
1 H, Ar-H), 7.64 (d, J = 8.6 Hz, 2 H, Ar-H), 7.37 (q, J = 4.8 Hz, 1 H, Ar-H), 7.02 (t, J = 5.9 Hz,
1 H, Ar-H), 4.35 (d, J = 5.88 Hz, 2 H, CH2NH). 13C NMR (100 MHz; DMSO-d6)
δ, ppm:
154.98 (CO), 149.19, 148.56, 147.47, 140.98, 135.81, 135.47, 125.53, 123.90, 117.44 (Ar-C), 40.38
(NH-CH2); HRMS calcd for C13H13N4O3[M + H]+ 273.09876 found 273.09971.
To a solution of vacor (1 eqv, 1.8 mmol) in anhydrous CH3CN (5 mL), N,O-
bis(trimethylsilyl)-acetamide (BSA) (2.2 eqv, 4.0 mmol), trimethylsilyl chloride (TMSCl)
(1 eqv, 1.8 mmol) and a catalytic amount of ammonium sulfate were added. The resulting
mixture was stirred at room temperature for 3 to 4 h under argon, and then concentrated
under reduced pressure to afford the crude silylated vacor.
Trimethyl silyl triflate (TMSOTf) (3.9 eqv, 7.3 mmol) was added to a mixture of crude
silylated vacor (1 eqv, 1.8 mmol) and D-ribose tetraacetate (1 eqv, 1.8 mmol) in anhydrous
1,2-dichloroethane (15 mL). The reaction mixture was stirred at RT for 3 h. Once the reaction
was complete, the mixture was concentrated under reduced pressure and the residue was
dissolved in 20 mL reagent-grade methanol. The solution was basified with a saturated
aqueous solution of NaHCO3 at 0 ◦C. Once the aqueous methanolic solution reached a
pH between 5 and 6, the addition of NaHCO3 was stopped and the resulting mixture was
concentrated on the rotatory evaporator with no heating. The crude product was washed
with diethyl ether 2–3 times and dried under reduced pressure to afford VR triacetate in
a solid form. Yield 68%, 1H NMR (400 MHz; MeOD)
δ, ppm: 9.03 (s, 1 H, Ar-H), 8.96 (d,

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J = 6.2 Hz, 1 H, Ar-H), 8.58 (d, J = 8.0 Hz, 1 H, Ar-H), 8.09 (t, J = 6.7 Hz, 1 H, Ar-H), 8.04 (d,
J = 7.34 Hz, 2 H, Ar-H), 7.51 (d, J = 8.47 Hz, 2 H, Ar-H), 6.41 (d, J = 4.24 Hz, 1 H, H-1), 5.42
(t, J =4.94 Hz, 1 H, H-2), 5.32 (t, J = 5.24 Hz, 1 H, H-3), 4.68 (q, J = 3.85 Hz, 1 H, H-4), 4.55
(d, J = 3.16 Hz, 2 H, CH2NH), 4.50 and 4.36 (AB part of ABX system, 2 H, JAB = 12.8 Hz,
JAX = 4.0 Hz, JBX = 2.7 Hz, H50), 2.03 (s, 6 H, Ac), 2.00 (s, 3 H, Ac). 13C NMR (100 MHz;
MeOD) δ, ppm: 170.51 (CO), 169.97 (CO), 169.75 (CO), 155.71 (NHCONH), 146.57, 145.96,
142.14, 141.93, 139.64, 139.19, 127.86, 124.46, 117.76 (Ar-C), 97.39 (C-1), 83.05 (C-4), 76.20
(C-2), 69.09 (C-3), 62.36 (C-5), 40.57 (CH2NH), 20.63 (Me), 19.32 (Me), 18.87 (Me).
The crude vacor riboside triacetate (1 eqv, 1.8 mmol) was dissolved in 15 mL an-
hydrous methanol. After 15 min of stirring at RT, 10 mL of an NH3 solution (0.5N) in
1,4-dioxane was added and the resulting mixture was stirred at RT overnight. The reac-
tion progress was monitored by TLC. Upon completion of the reaction, the solvent was
removed under reduced pressure and the crude product was purified by flash column silica
chromatography through gradient elution by using hexane: ethylacetate (1:1), ethylacetate:
MeOH (1:1) and water: MeOH (1:1) as eluents. Yield 62%, 1H NMR (400 MHz; D2O)
δ, ppm:
9.00 (s, 1 H, Ar-H), 8.91 (d, J = 6.12 Hz, 1 H, Ar-H), 8.45 (d, J = 7.92 Hz, 1 H, Ar-H), 8.34 (s,
1 H, Ar-H), 8.04–7.99 (m, 2 H, Ar-H), 7.33 (d, J = 9.2 Hz, 1 H, Ar-H), 6.06 (d, J = 4.6 Hz, 1 H,
H-1), 4.55 (s, 2 H, CH2NH), 4.38–4.35 (m, 1 H, H-3), 4.22 (t, J = 4.52 Hz, 1 H, H-2), 3.88–3.76
(AB part of ABX system, 2 H, JAB = 12.9 Hz, JAX = 2.92 Hz, JBX = 3.8 Hz, H50). 13C NMR
(100 MHz; D2O) δ, ppm: 170.83 (Ar-C), 156.62 (CO), 145.81, 145.29, 141.84, 141.22, 139.08,
138.42, 127.92, 125.17, 118.25 (Ar-C), 99.52 (C-1), 87.59 (C-4), 77.50 (C-2), 69.98 (C-3), 60.53
(C-5), 40.59 (CH2NH); HRMS calcd for C18H21N4O7[M + H]+ 405.14102 found 405.14214.
2.3. Cultivation, Vacor/VR Treatment and Nucleotide Extraction of 293 Cells
293 cells were cultivated in Dulbecco’s modified Eagle’s medium (Gibco) supple-
mented with 10% (v/v) fetal bovine serum (FBS), 2 mM glutamine and penicillin (100 U/mL)/
◦
streptomycin (100
µ
g/mL) (normal medium). The cells were cultured at 37 C in a humidi-
fied atmosphere of 5% CO2. Prior to vacor/VR treatment, the cells were seeded in 12-well
plates (200,000 cells/well) in 1 mL normal medium. The day after seeding, the cells were
washed once with PBS and the medium was changed to the normal medium supplemented
with 100
µM vacor, 100 µM VR or no supplement (control). The cells were then incubated
for 24 h before extraction.
For nucleotide extraction, the cells were placed on ice, the growth medium was
removed, and the cells were washed twice with 0.5 mL ice-cold PBS. Then, 0.5 mL ice-cold
80% (v/v) LC-MS-grade methanol was added to each well and the cells were incubated at
◦
4 C on a shaker for 20 min. After incubation, the cells were detached from the wells using
a cell scraper and the samples were transferred to 1.5 mL tubes. To ensure full transfer,
the wells were washed with 0.3 mL of ice-cold 80% (v/v) LC-MS-grade methanol. The
samples were subsequently frozen at
−
80 ◦C. On the day of LC-MS analysis, the samples
were thawed on a rotating wheel at 4 ◦C before centrifugation at 16,000
×
g for 20 min at
4 ◦C. After centrifugation, 0.6 mL of the supernatant was transferred to a new tube and
0.6 mL acetonitrile was added to each sample.
2.4. LC-MS Analysis
Separation of the nucleotides by liquid chromatography was done using a SeQuant
ZIC-cHILC column (100
×
2.1 mm, 3 µm; Merck) in a Dionex UltiMate 3000 liquid chro-
matographer coupled to a QExactive mass spectrometer (Thermo Scientific, Oslo, NO,
USA). The column compartment was kept at 30 ◦C during the run. The injection volume
for all samples was 10 µL and the flow rate was kept at 0.3 mL/min.
The mobile phase consisted of 20 mM ammonium acetate, pH 6.8 (Buffer A), and
acetonitrile (Buffer B). The gradient was set as follows: Runs were started with 80% Buffer
B for 1 min (flowthrough). The concentration of Buffer B was further decreased to 70%
over 11 min for separation before being brought to 5% over 1 min for washout. After
3.5 min at 5% Buffer B, the concentration of Buffer B was returned to 80% over 1 min

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for equilibration. The column was equilibrated at 80% B for 3.5 min before the next run.
Electrospray ionization was performed using the positive ion polarity mode and a spray
voltage of 3.5 kV. The sheath flow gas flow rate was 48 units, with an auxiliary gas flow
rate of 11 units and a sweep gas flow rate of 2 units.
Mass spectra were recorded using targeted single-ion monitoring at positive polarity,
at a resolution of 70,000. Fragmentation spectra were recorded using MS/MS in parallel
reaction monitoring at a resolution of 70,000, with the normalized collision energy set to
18 units. Data analysis was conducted in the Thermo Xcalibur Qual Browser (Thermo
Scientific, Oslo, NO, USA).
2.5. Recombinant Expression and Purification of NRKs
Pellets from bacterial expression cultures were resuspended in 10 mL resuspension
buffer (20 mM Na-PO4 pH 8.0, 300 mM NaCl) and lysed by 6
×
10 s sonication on ice.
The cell lysate was then centrifuged for 20 min at 13,000 rpm. During this centrifugation,
1 mL Ni-NTA matrix (Qiagen) was transferred to a 15 mL tube and equilibrated with
resuspension buffer by 5 min incubation with 10 mL buffer on a rotating wheel, followed
by 3 min centrifugation at 2000
the supernatant was added to the Ni-NTA matrix and was incubated for 1 h at 4 C
×
g, repeated twice. After centrifugation of the cell lysate,
◦
while rotating to allow binding of the His-tagged protein to the Ni-NTA matrix. After
incubation, the tube was centrifuged for 2 min at 2000
and the matrix was washed by incubation with 10 mL washing buffer (20 mM Na-PO4
×
g. The supernatant was removed,
pH 8.0, 300 mM NaCl, 20 mM Imidazole) for 5 min while rotating, followed by 2 min
centrifugation at 2000
×
g, repeated twice. After washing, the matrix was resuspended
in 2 mL washing buffer and transferred to a column equilibrated with washing buffer.
The Ni-NTA matrix was allowed to pack on top of the column by gravity. Elution was
performed in three fractions by the addition of firstly 300 µL, and then twice 500 µL of the
elution buffer (20 mM Na-PO4 pH 8.0, 300 mM NaCl, 500 mM Imidazole). To assess the
stepwise purification of the protein, samples were collected of the pellet and supernatant
after lysis, after binding to the column and after washing, and analyzed using SDS-PAGE.
2.6. Synthesis of NMN/VMN by NRKs
A reaction mix containing 100
pH 7.0, was prepared in a total volume of 500
g NRK1/NRK2 and incubated at room temperature for 60 min. The reaction mix was
µM VR or NR with 500
µM ATP in 10 mM Na-phosphate,
µL. The reaction was started by addition of
5
µ
then filtered using a 10 kDa-cutoff spin column (amicon) at 14,000
×
g, at 4 ◦C for 10 min,
and the flow-through stored at −80 ◦C for further analysis.
2.7. Cell Proliferation Assay
293 cells were seeded, 10,000 cells/well, in a 96-well plate in a high-glucose DMEM
medium (Gibco) supplemented with 10% FBS. At 24 h after seeding, the medium was
exchanged with a medium containing 100 µM vacor, 100 µM VR or no supplement (con-
trol). The cells were then transferred to an IncuCyte Zoom live cell imager. Growth rate
was assessed by monitoring cell confluency using phase-contrast microscopy at 2 h time
intervals for 48 h.
2.8. Cleavage of VAD/NAD Using A. fumigatus NADase
The reaction mix was prepared in 500 µL reaction buffer containing 50 mM Tris-HCl,
pH 8.0, 500 µM CaCl2 and 150 mM NaCl, supplemented with 10 µM VAD/NAD. The
reaction was started by the addition of 10 ng A. fumigatus NADase [31] and incubated
at room temperature for 30 min. The reactions were then filtered using a 10 kDa-cutoff
spin column at 14,000
further analysis.
×
g, at 4 ◦C for 10 min, and the flow-through stored at
−
80 ◦C for

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3. Results
Vacor is commercially available and can therefore be used as a starting material to
produce NAD analogs. As it will be described below, while VAD can be generated in a
single enzymatic step, VR needs to be chemically synthesized.
3.1. Single-Step Generation of VAD Using Aplysia californica ADP Ribosyl Cyclase
The enzymatic synthesis of VAD described so far requires at least two enzymes
that need to be recombinantly produced. Moreover, this two-step enzymatic synthesis
requires the co-substrates PRPP and ATP (Figure 1). We reasoned that the well-known base-
exchange reaction [24,25,32], which is an exceptional feature of ADP-ribosyl cyclases, could
be exploited for a simplified route to produce VAD. Among others, these cyclases mediate
the substitution of the nicotinamide moiety in NAD by nicotinic acid (the other vitamin B3
form of the pyridine base), thereby generating NAAD, a physiological intermediate of the
Preiss–Handler NAD biosynthetic pathway [33].
We tested this possibility using commercially available Aplysia californica ADP-ribosyl
cyclase in a one-step reaction with vacor and NAD as substrates (Figure 2A, top). The
reaction mix was set up as described in Materials and Methods with a 5
×
excess of vacor to
NAD. Following incubation for 10 min, the reaction mix was analyzed by LC-MS (Figure 2A
bottom), showing that NAD was completely converted to VAD. This reaction yields Nam
as a side product as the base on NAD is exchanged with vacor. While the base exchange
activity of Aplysia californica ADP-ribosyl cyclase was proven efficient in producing VAD,
the same enzyme is also capable of catalyzing the reverse cyclase reaction, adding a base
onto cyclic ADP-ribose (cADPr) [24]. We performed this reaction in a similar manner to
the base-exchange reaction, using cADPr as substrate in the presence of a 5-fold excess of
vacor. This reaction yielded a complete conversion of cADPr to VAD, with no side product
produced (Figure 2B).
The ms spectrum of the product formed matched the expected m/z and natural isotope
distribution of VAD (Figure 2C). To further confirm the identity of the reaction product,
we subjected the product to fragmentation analysis using LC-MS-MS, using NAD+ as
control. The fragmentation spectrum for the reaction product (Figure 2D right) is highly
similar to that of NAD (Figure 2D left), with both spectra containing major fragments at
m/z = 136 and m/z = 428, indicating the presence of an adenosine moiety and an ADP-
moiety. Additionally, the reaction product has a fragment at m/z = 272, corresponding to
the expected mass of vacor. Taken together, this demonstrates that the reaction product has
incorporated vacor as part of its structure, confirming that it is indeed VAD.
Having successfully produced VAD, we wanted to investigate whether VAD can serve
as a substrate for NAD-consuming enzymes. To do this, we used the newly discovered
fungal NADase from Aspergillus fumigatus [31]—an enzyme shown to possess pure NADase
activity, cleaving NAD into ADPr and Nam—in a reaction containing our synthesized VAD,
using NAD as a control. The reaction was analyzed using LC-MS (Figure 2E), revealing
that the NADase is capable of using both VAD and NAD as substrate, yielding ADP-ribose
and the corresponding base as products.

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Figure 2. Synthesis of VAD by Aplysia californica ADP-ribosyl cyclase through base exchange. (A) Scheme of VAD production
utilizing A. californica ADPr-cyclase activity with vacor and NAD as substrates. Chromatograms show the reaction and
control using 50
M NAD+ with 5-fold excess of vacor followed by 10 min incubation using 0.1 U enzyme, quantified by
LC-MS. ( ) Scheme of VAD production utilizing A. californica ADPr-cyclase activity with vacor and cADPr as substrates.
Chromatograms show the reaction and control using 50 excess of vacor followed by 10 min incubation
M NAD+ with 5
using 0.1 U enzyme, detected by LC-MS. ( ) MS spectrum of VAD produced using the A. californica ADPr-cyclase-catalyzed
reaction shown in 2B. ( ) Fragmentation spectra of NAD control (left) and VAD (right) produced using the A. californica
ADPr-cyclase-catalyzed reaction shown in 2B. ( ) Cleavage of NAD and VAD by A. fumigatus NADase. Chromatograms
µ
B
µ
×
C
D
E
show the reactions and controls of 10 µM substrate incubated for 30 min using 10 ng enzyme, detected by LC-MS.
3.2. VR Is Converted to VMN by NRKs
Next, we wanted to investigate whether vacor derivatives can be generated through
alternative entry points in the NAD biosynthetic pathway. In order to do this, we synthe-
sized a vacor analogue of the alternative NAD precursor NR, VR, as described in Materials
and Methods. The synthesis of vacor was performed according to the literature [34] and
its identity confirmed by NMR and MS spectrometry. The ribosylated derivative of vacor
was obtained via Vorbruggen chemistry (Figure 3A) for which vacor was silylated using
N,O-bis(trimethylsilyl)acetamide and trimethylsilyl chloride with a catalytic amount of
ammonium sulfate. Any other silylation methods proved mediocre and led to poor out-
comes in the generation of the triacetylated nucleoside. Crucially, the ribosylation of the
silylated vacor occurred with complete stereoselectivity and in reproducible good yields
using trimethylsilyltriflate as a Lewis acid. The beta-stereochemistry at the C10 position
of the nucleosidic linkage was confirmed by the chemical shift and the multiplicity of the

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hydrogen at the C10 position (6.41, d, J = 4.24 Hz, 1 H, H-1), which matches that observed
in nicotinamide riboside triacetate [35]. The subsequent removal of the three acetyl groups
was achieved under mild reaction conditions that, in addition to the saponification reaction,
also led to the partial hydrolysis of the glycolytic bond and release of vacor. The crude
mixture was therefore purified by silica chromatography. Overall, the fully deprotected VR
was obtained at a 39% yield from 3-picolylamine. The identity and purity of the final VR
product was confirmed using NMR and mass spectroscopy (Figure 3B,C).
Figure 3. Chemical synthesis of VR. (
A) Scheme of the synthesis of VR from vacor via Vorbruggen chemistry. (B) NMR
spectrum of the synthesized VR. (C) Mass spectrum of VR.
In NAD biosynthesis, NRK1 and 2 phosphorylate NR to NMN. To test whether NRKs
are also capable of phosphorylating VR to VMN, we overexpressed both isoforms in E. coli
and purified the enzymes using immobilized metal ion chromatography (Figure 4A). An
enzymatic reaction using the purified NRKs and our synthesized VR was performed as
described in Materials and Methods, with a 5
were analyzed by LC-MS (Figure 4B), showing that both NRK1 and NRK2 were able to
completely convert VR to VMN. It should be noted that due to the inclusion of a negatively
×
excess of ATP. The reaction products

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charged phosphate, the nucleotide products are less efficiently ionized than the nucleoside
substrates. This, in turn, means that the reaction products in this case yield a far lower signal
compared to the substrates. To verify the identity of the reaction product, we performed a
fragmentation analysis using LC-MS-MS (Figure 4C). The major fragmentation ion present
is at m/z = 273, which is the expected m/z of a vacor moiety. Additionally, a major fragment
is observed at m/z 485, corresponding to the expected mass of the full VMN molecule,
indicating that the product formed is indeed VMN.
Figure 4. VR is converted to the NMN analogue, VMN, by both NRK 1 and 2. (A) Purification of NRK1/2 using immobilized
metal ion chromatography, analyzed by SDS-PAGE. B.I.—before induction; A.I.—after induction; Ins.—insoluble fraction;
C.L.—clear lysate; W1—wash 1; W2—wash 2; M—marker; 1—elution fraction 1; 2—elution fraction 2; 3—elution fraction 2.
(B
) Scheme of VAD/NAD production utilizing the NRK activity with VR/NR and ATP as substrates. Chromatograms show
the reaction (VR) and control (NR) using 100 M VR or NR, respectively, with 5-fold excess of ATP followed by 60 min
incubation using 10 g enzyme, analyzed by LC-MS. ( ) Fragmentation spectrum of VMN synthesized from VR by NRKs.
) Abundance of VMN (left) and VAD (right) in 293 cells after 24 h treatment with 100 M vacor or VR. Metabolites were
extracted in methanol and analyzed using LC-MS. All values normalized to the average concentration found in cells treated
with vacor. ( ) Growth curve of the 293 cells in high-glucose DMEM supplemented with 100 M vacor, 100 M VR or
µ
µ
C
(
D
µ
E
µ
µ
control (no supplement). The graph shows the confluence measured for 48 h after supplementation, using IncuCyte Zoom
live cell imaging.

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Having shown that VR can be converted to a product by enzymes of the NAD biosyn-
thetic pathway, we next treated the 293 cells, which are known to express both NRK
and NamPT activity, with 100 µM VR or vacor for 24 h. Following extraction in ice-cold
methanol, the presence or absence of vacor products in the samples was analyzed using
LC-MS (Figure 4D). Interestingly, we observed an accumulation of VMN in cells treated
both with VR and vacor, indicating that NRKs are able to convert VR to VMN also in a
cellular system.
However, VAD—the downstream product generated by NMNAT2—was only ob-
served when the cells were treated with vacor. Following up on this, we wanted to
investigate whether this difference in accumulation of vacor products affected the toxic
effect of the treatment. We therefore performed a growth curve experiment over 48 h using
293 cells treated with 100 µM vacor or VR. The cells were monitored using IncuCyte, and
cell confluency was determined every 2 h (Figure 4E). As expected, cells treated with vacor
showed impaired growth and cell death, with a confluency of 27% compared to the control
at 24 h, decreasing to 19% at 48 h. Cells treated with VR exhibited no signs of growth
impairment compared to the control cells.
4. Discussion
As vacor has gained attention due to its potential to enter the NAD biosynthetic
pathway, with downstream vacor products acting as toxins, a potential use case for vacor
products has emerged. In this paper, we demonstrate an alternative method for produc-
ing VAD, using Aplysia californica ADP ribosyl cyclase in a one-step reaction using only
commercially available materials. Compared to the method previously used to produce
VAD [20,22], the approach presented here is cost-effective and less complex (fewer enzymes,
substrates and products), making the final purification of VAD also easier.
Further, we show that not only vacor, but a vacor analogue of the alternative NAD
precursor NR, can be converted into a vacor product by NRKs, indicating that VR is
also converted in the NAD biosynthetic pathway. The phosphorylation of VR to VMN
was confirmed in cell culture experiments, demonstrating that vacor analogues of NAD
precursors can enter the NAD biosynthetic pathway from several different entry points.
While both NamPT and NRKs are ubiquitously expressed, expression levels across tissues
vary. While Vacor has been shown to mainly affect neuronal and pancreatic cells [20],
different Vacor analogues might exhibit different cytotoxic profiles. In particular, the
fact that VR conversion to VMN is not dependent on NamPT activity can be important.
NamPT is the rate-limiting enzyme of the salvage pathway [10] and a major contributor in
maintaining cellular NAD levels. NamPT is also subject to negative feedback inhibition by
NAD [36], unlike the NRKs. As both NRKs are capable of synthesizing VMN, using VR as
an entry point for vacor toxification could potentially prove efficient in a wide array of cell
lines and tissues.
In this context, it is very interesting to observe that both vacor and VR cause accumula-
tion of VMN in 293 cells. However, accumulation of VAD, with subsequent cell death, was
only observed in the vacor-treated cells, indicating that accumulation of VAD is necessary
for vacor-induced cell death. Interestingly, we observed that VAD can act as a substrate
for NAD-consuming enzymes, as both A. californica ADP-ribosyl cyclase and A. fumigatus
NADase accept it as a substrate. This raises the question of whether vacor/VR or their
products might enter other biological pathways, opening up the possibility that VMN/VAD
might be further metabolized in the cell. As the exact mechanisms behind vacor-induced
cytotoxicity are not well understood, with different explanations proposed [20
highly interesting topic that warrants further investigation.
,22], this is a
Author Contributions: Conceptualization, all authors; experimental work, L.J.S.; chemical syntheses,
F.H.; supervision, M.Z. and M.E.M.; writing—original draft preparation, L.J.S.; review and editing,
all authors. All authors have read and agreed to the published version of the manuscript.

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Funding: This research was funded by the Research Council of Norway (302314) and the MESI-
STRAT project (grant agreement 754688), which received funding from the European Union Horizon
2020 Research and Innovation Program (to MZ). LS has received support from the Meltzer foundation.
Institutional Review Board Statement: Not applicable.
Data Availability Statement: All relevant data of this study are presented. Additional data will be
provided upon request.
Conflicts of Interest: The authors declare no conflict of interest.
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