Nicotinic acid adenine dinucleotide phosphate analogues substituted on the nicotinic acid and adenine ribosides. Effects on receptormediated Ca2+ release

Article

pubs.acs.org/jmc

Nicotinic Acid Adenine Dinucleotide Phosphate Analogues

Substituted on the Nicotinic Acid and Adenine Ribosides. Eﬀects on

ReceptorMediated Ca²⁺Release

Christopher J. Trabbic,^†Fan Zhang,^‡Timothy F. Walseth,*^,‡and James T. Slama*^,†

^†Department of Medicinal and Biological Chemistry, College of Pharmacy and Pharmaceutical Sciences, University of Toledo, 3000

Arlington Avenue, Toledo Ohio 43614, United States

^‡Department of Pharmacology, University of Minnesota Medical School, 321 Church Street SE, Minneapolis, Minnesota 55455,

United States

S

* Supporting Information

ABSTRACT: Nicotinic acid adenine dinucleotide phosphate

(NAADP) is a Ca²⁺releasing intracellular second messenger in

both mammals and echinoderms. We report that large

functionalized substituents introduced at the nicotinic acid 5-

position are recognized by the sea urchin receptor, albeit with

a 20−500-fold loss in agonist potency. 5-(3-Azidopropyl)-

NAADP was shown to release Ca²⁺with an EC₅₀of 31 μM

and to compete with NAADP for receptor binding with an

IC₅₀of 56 nM. Attachment of charged groups to the nicotinic

acid of NAADP is associated with loss of activity, suggesting

that the nicotinate riboside moiety is recognized as a neutral

zwitterion. Substituents (Br− and N₃−) can be introduced at

the 8-adenosyl position of NAADP while preserving high

potency and agonist eﬃcacy and an NAADP derivative substituted at both the 5-position of the nicotinic acid and at the 8-

adenosyl position was also recognized although the agonist potency was signiﬁcantly reduced.

structural features of NAADP important for its recognition is

incomplete. The lack of information is, in part, due to the

complexity of its structure which prohibits facile synthesis and

testing of structurally modiﬁed analogues. Various reports have

deﬁned the importance of charge, location, and size of

functional groups necessary for NAADP recognition by its

receptor.²⁵⁻²⁷The 3-nicotinyl carboxylic acid, 2′-phosphate,

and the 6-adenosyl amine were all recognized as required for

activity. Removal of these functionalities, as well as changing

their location, resulted in diminished or in complete loss of

activity. More recently, the eﬀect of substitution at the 4- and 5-

nicotinic acid positions of 1 was evaluated.²⁸When nicotinic

acid modiﬁed NAADP analogues were tested using the sea

urchin NAADP receptor, small chemical substitutions present

at the 5-nicotinyl position (amino, methyl, ethyl, azido) were

found to be well tolerated, whereas substitution at the 4-

nicotinyl position (amino, methyl, butyl, phenyl) were shown

to be low potency agonists.

INTRODUCTION

■

Stimulation of Ca²⁺release from intracellular stores is

accomplished through increases in the intracellular concen-

trations of second messengers such as ^D-myo-inositol tri-

sphosphate (IP3), cyclic ADP-ribose (cADPR), and nicotinic

acid adenine dinucleotide phosphate (NAADP, 1), which

interact with speciﬁc receptors to regulate Ca²⁺selective ion

channels. In response to extracellular stimuli, these messenger

molecules are released, bind to their speciﬁc receptors, and

initiate complex changes in the intracellular Ca²⁺concentration,

ultimately converting extracellular stimulation into a highly

regulated spatiotemporal physiological signal.¹⁻³Of the Ca²⁺

releasing second messengers, NAADP is the most potent (EC₅₀

= 20 nM in sea urchin egg homogenates) yet it remains the

least characterized. Originally discovered as a contaminant in

commercial preparations of NADP,⁴NAADP regulates

receptor-mediated Ca²⁺release from intracellular Ca²⁺stores

distinct from those regulated by either IP3 or cADPR.⁵

NAADP-mediated Ca²⁺signaling is best deﬁned in sea urchin

eggs but has been demonstrated in a variety of mammalian and

human systems including pancreatic cells,^2,6,7brain,⁸heart,⁹⁻¹²

kidney cells,¹³lymphocytes,¹⁴⁻¹⁶and the human cell line

Jurkat^17,18among others.¹⁹⁻²²Although NAADP has been

known for two decades,^4,23,24and despite its importance in a

variety of cell signaling pathways, information concerning

The receptor which binds NAADP and controls Ca²⁺release

has not been identiﬁed. The two-pore channel (TPC) was

proposed to be a speciﬁc Ca²⁺channel regulated by NAADP,²⁹

but this has recently been challenged.^30,31Photoaﬃnity studies

Received: February 16, 2015

Published: March 31, 2015

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Figure 1. Enzyme catalyzed base-exchange reaction.

Table 1. 5-Substituted Pyridine-3-carboxylic Acids (2a−f) Obtained and Evaluated as Potential Substrates for the

Chemoenzymatic Synthesis Designed to Produce Novel Pyridinium Substituted NAADP Derivatives

using [³²P]-5-N₃−NAADP have identiﬁed 45, 40, and 35 kDa

proteins in sea urchin homogenates and 21/22 kDa proteins in

cultured human cells that are photolabeled speciﬁcally. These

NAADP binding proteins may be important in the regulation of

Ca²⁺release.^32,33

Figure 1, the enzyme catalyzed base-exchange reaction was

instrumental in producing the substituted NAADP derivatives

which were used in this study. In the presence of NADP (or an

8-substituted NADP) and a high concentration of 5-substituted

nicotinic acid (R₁), the Aplysia ADP-ribosyl cyclase catalyzes

the base exchange of nicotinamide for the 5-substituted

nicotinic acid. The chemoenzymatic synthesis, although

powerful, is subject to the requirement that the pyridine base

be an eﬃcient substrate for the ADP-ribosyl cyclase. This is

usually the case, but there are pyridine bases that fail to

exchange.

Synthesis of 5-Substituted Nicotinic Acids. Simple 5-

substituted nicotinic acids such as 5-hydroxynicotinic acid (2a)

and 5-bromonicotinic acid (2b) were available commercially.

Others were produced in a single step from a commercially

available compound. For example, 5-acetamidonicotinic acid

(2c) was made by treatment of 5-aminonicotinic acid with

acetic anhydride. 5-Thiomethyl nicotinic acid (2d) was derived

from 2b by nucleophilic aromatic displacement with sodium

thiomethoxide.³⁷In total, 10 nicotinic acid derivatives were

obtained and evaluated as base-exchange substrates with the

expectation of producing the corresponding NAADP analogues

(Table 1).

The development of chemical probes that can be used to

identify the NAADP binding proteins requires the production

of more highly functionalized NAADP analogues than those

reported in our previous publication.²⁸Receptor identiﬁcation

will require high potency analogues containing functional

groups suitable for ligand−aﬃnity chromatography or bifunc-

tional derivatives suitable for covalent labeling of the receptor,

followed by aﬃnity isolation of the derivatized protein using

“click chemistry”. This work describes an extension of our study

of the structure−activity relationships (SAR) of NAADP

derivatives to 11 additional NAADP analogues, ﬁve of which

contain large substituents containing functional groups at the

nicotinic acid 5-position. We also evaluate the eﬀect of

substitution on the 8-adenosyl position and for the ﬁrst time

report the activity of a disubstituted NAADP derivative

modiﬁed at both the 5-nicotinyl and 8-adenosyl positions.

RESULTS

■

A majority of the functionalized 5-substituted nicotinic acid

analogues were synthesized using the Sonogashira reaction,^38,39

which eﬀectively coupled a convenient starting material (5-

bromonicotinic acid ethyl ester, (2e) with various terminal

alkynes. The resulting alkynyl nicotinic acid derivative was

Chemistry. Substituted NAADP analogues that would

otherwise be diﬃcult to prepare using purely synthetic

methodologies can be produced from a synthetic pyridine

base and NADP using the Aplysia californica ADP-ribosyl

cyclase³⁴(a version of NAD glycohydrolase).^35,36Illustrated in

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a

Scheme 1. Synthesis of 5-(2-Phenylethyl)-3-pyridinecarboxylic Acid (2f)

a

Reagents and conditions: (a) phenylacetylene, Pd(OAc)₂, CuI, DIPEA, triphenylphosphine, acetonitrile, reﬂux 16 h, stirring under N₂, 82%; (b) H₂,

Pd on C, EtOAc/methanol 10 h, 82%; (c) (i) NaOH, CH₃OH, 18 h, (ii) HCl, 71%.

a

Scheme 2. Synthesis of 5-(3-Acetamidopropyl)nicotinic Acid (6)

a

Reagents and conditions: (a) 3, Pd(PPh₃)₂Cl₂, CuI, DIPEA, CH₃CN, reﬂux, 18 h, 60%; (b) H_2,Pd/C, CH₃OH, 8 h, 91%; (c) (i) NaOH, CH₃OH,

3.5 h, (ii) HCl, 89%.

a

Scheme 3. Synthesis of Compounds 11 and 12

a

Reagents and conditions: (a) 7, Pd(PPh₃)₂Cl₂, CuI, DIPEA, CH₃CN, reﬂux, 6 h, 61%; (b) H₂, Pd/C, CH₃OH, 6 h, 93%; (c) TFA, CH₃OH, 37 °C,

2 h; (d) NaOH, CH₃OH, 8 h, 89%; (e) (i) NaOH, CH₃OH, 37 °C, 4 h, (ii) HCl, 83%.

reduced by hydrogenation, further derivatized, and then

hydrolyzed providing a series of 5-substituted nicotinic acid

analogues. The synthesis of 2f (Scheme 1) illustrates typical

methodologies in the synthesis of 5-substituted pyridine

carboxylic acids. First, Sonogashira reaction of 5-bromonico-

tinic acid ethyl ester with phenylacetylene in the presence of

Pd(OAc)₂, triphenylphosphine, and CuI yielded 5-(2-phenyl-

ethynyl)-3-pyridinecarboxylic acid ethyl ester. This alkyne was

reduced to alkane by catalytic hydrogenation, and hydrolysis

with NaOH provided carboxylic acid (2f).

base providing acetamide 6. Synthesis of 5-(3-aminopropyl)-

nicotinic acid (12) was accomplished by coupling t-Boc-N

protected propargyl amine (7),⁴¹yielding alkyne 8 (Scheme 3).

Standard hydrogenation conditions provided reduced com-

pound 9. Removal of the t-Boc protecting group with TFA

yielded 10 followed by alkaline treatment to produce desired

aliphatic amine 12. Compound 9 was alternatively hydrolyzed,

yielding 11.

Similarly, palladium catalyzed couplings with propargyl

alcohol and 2e (Scheme 4) provided a route to nicotinic acid

derivatives 15 and 18.

Synthesis of NAADP Analogues Containing 8-Sub-

stituted Adenosyl Derivatives. To determine the eﬀect of

substitution within the purine ring of NAADP, we needed to

The synthesis of 5-(3-acetamidopropyl)-nicotinic acid (6) is

illustrated in Scheme 2. Alkyne 3⁴⁰was coupled to 2e under

Sonogashira coupling conditions. The product was then

reduced by hydrogenation (5) and saponiﬁed with aqueous

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a

Scheme 4. Synthesis of Compound 18

a

Reagents and conditions: (a) Propargyl alcohol, Pd(PPh₃)₂Cl₂, CuI, DIPEA, CH₃CN, reﬂux, 16 h, 71%; (b) H₂and Pd/C, CH₃OH, 10 h, 89%; (c)

(i) NaOH, CH₃OH, 37 °C, 4 h, (ii) HCl, 96%; (d) p-methylbenzenesulfonyl chloride, TEA, CH₂Cl₂, reﬂux 28 h, 51%; (e) NaN₃, DMF, 60 °C, 18 h,

73%; (f) (i) NaOH, CH₃OH, 37 °C, 3 h, (ii) HCl, 91%.

a

Scheme 5. Synthesis of 8-N₃-NADP (23)

a

Reagents and conditions: (a) anydrous hydrazine, CH₃CH₂OH, reﬂux; (b) (i) iso-amylnitrite, 1 N HCl, (ii) 1 N NaOH; (c) (i) POCl₃,

trimethylphosphate, (ii) 1 N NaOH, pH = 7; (d) nicotinamide mononucleotide, diisopropylcarbidiimide, pyridine, H₂O; (e) NAD kinase and ATP.

a

Scheme 6. Bromination of NADP to Produce 24

a

Reagents and conditions: (a) Br₂, 0.5 N sodium acetate, pH = 4.

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Table 2. Mono- and Disubstituted NAADP Analogues Obtained Using Our Chemoenzymatic Synthesis

Figure 2. Spectrophotometric titration of 5-hydroxy-NAADP (26). (A) Electronic absorption spectra of 26 determined at varying pH values. (B)

A_{335 nm}versus pH.

produce 8-adenosyl substituted NAD analogues, convert these

to NADP derivatives, and next transform these into NAADP

derivatives using the pyridine base exchange reaction as

depicted in Figure 1. 8-N₃-adenosyl-NADP (22) was

conveniently synthesized starting with 8-bromoadenosine⁴²

according to Scheme 5. 8-Bromoadenosine was converted to 8-

N₃-adenosine (20) in two steps,^43,44and 20 was selectively

phosphorylated producing 21.⁴⁵The pyrophosphate bond was

formed by coupling 21 with commercially available nicotina-

mide mononucleotide, yielding 8-azido-NAD (22).⁴⁶Selective

phosphorylation at the 2′-adenosyl position of 22 was

accomplished enzymatically on a small scale with recombinant

human NAD kinase and ATP, providing 8-azido NADP (23).

8-Bromo-NADP (24) was synthesized directly from NADP

by bromination in buﬀered water (Scheme 6). Conditions for

the synthesis of 8-bromo-NAD^47,48and 8-bromo-AMP⁴⁹have

been previously reported, and NADP was similarly converted to

24.⁵⁰

and either NADP or an 8-substituted NADP derivative as

illustrated in Figure 1. Pyridine bases 2b and 2f failed as

substrates in the enzymatic base exchange, and NAADP

derivatives derived from these were therefore not synthesized.

This illustrates a well know limitation of this chemoenzymatic

synthesis, wherein electron deﬁcient or water insoluble bases

fail to exchange.

Spectroscopic Properties of NAADP Analogues. The

electronic absorption spectrum of 5-hydroxy-NAADP (26) at

low pH exhibits maxima at 256 and at 298 nm (Figure 2A).

This 298 nm band is not observed in the spectrum of NAADP

and is due to the conjugation of the 5-oxosubstituent with the

pyridinium ring. As the pH of the solution was increased, the

long wavelength band shifts to longer wavelength and exhibits a

λ_maxat 333 at high pH. An analysis of the change in the

absorption spectra of 26 as a function of pH (Figure 2B)

enabled us to determine that the pK_afor the ionization of the 5-

OH of 26 was 5.85. The previously characterized 5-amino-

NAADP also exhibits a long wavelength absorption maximum

at 346 nm, but this band does not shift between pH 2.5 and

NAADP Analogues. NAADP analogues 25−35 (Table 2)

were synthesized using the enzyme catalyzed pyridine−base

exchange reaction between a 5-substituted nicotinic acid (R₁)

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Table 3. EC₅₀Values for Ca²⁺Release Induced by NAADP (1) and NAADP Analogues (25−35) Measured Fluorometrically

from Ca²⁺Loaded Sea Urchin Egg Homogenates

compd

EC₅₀SEM, nM (n)

fold increase in EC₅₀

NAADP (1)

25.8 5.7 (19)

1932.3 440.1 (3)

626.2 46.1 (7)

151.3 22.8 (3)

4433.0 578.7 (7)

30749 5654 (8)

160021 54720 (9)

>1000000 (3)

1

5-hydroxy-NAADP (26)

74.9

5-acetamido-NAADP (25)

24.3

5-thiomethyl-NAADP (32)

5.9

5-(3-hydroxypropyl)-NAADP (30)

5-(3-azidopropyl)-NAADP (31)

5-(3-t-Boc-aminopropyl)-NAADP (28)

5-(3-aminopropyl)-NAADP (29)

5-(3-acetamidopropyl)-NAADP (27)

8-bromo-NAADP (33)

171.8

1191.8

6202.4

>33333

1617

27.5

41718 7097 (6)

709.1 63.6 (3)

47.7 1.6 (3)

8-azido-NAADP (34)

1.8

5-azido-8-bromo-NAADP (35)

1363000 145600 (3)

52829

11.5, indicating that the less acidic NH₂group does not ionize

below pH 11.5.

determined by liquid scintillation. Seven concentrations of each

analogue were tested to determine IC₅₀values.

Biological Activity. NAADP analogues were evaluated

using Strongylocentrotus purpuratus (sea urchin) egg homoge-

nates, which serve as a model for NAADP mediated activity in

mammalian cells and were used for much of the character-

ization of the cADPR and NAADP mediated Ca²⁺release. The

ability of NAADP analogues (25−35) to elicit Ca²⁺release

from sea urchin egg homogenates was tested using a

ﬂuorometric assay. Microsomes were loaded with Ca²⁺in the

presence of ATP and 3 μM ﬂuorescent indicator ﬂuo-3. Upon

addition of an NAADP analogue, Ca²⁺was released and its

binding to the Ca²⁺indicator results in an enhanced ﬂuorescent

signal. The increase in ﬂuorescence was proportional to the

released Ca²⁺and can be measured using a ﬂuorescence plate

reader (excitation 490 nm and emission 535 nm). These

experiments were performed in triplicate at a variety of agonist

concentrations, and EC₅₀values were determined (Table 3).

Competition ligand binding studies measured the ability of

analogues to compete with radiolabeled [³²P]NAADP for

receptor binding (Table 4). Binding studies were performed

using 96-well ﬁlter plates. A constant concentration of

[³²P]NAADP (0.2 nM) and varying concentrations of the

test ligand and were incubated simultaneously (90 min) with

sea urchin egg homogenates. After incubation, the homogenate

was ﬁltered, washed, and radioactivity remaining on the ﬁlter

The sea urchin NAADP receptor was shown to be

desensitized upon pretreatment with subthreshold concen-

trations of NAADP. Subsequent challenge with high concen-

trations of NAADP failed to release Ca²⁺from intracellular

stores. A ﬁnal assay determines the ability of subthreshold

concentrations of each analogue to induce receptor desensitiza-

tion. A 7 min pretreatment with varying concentrations of an

NAADP analogue (25−35) was followed by addition of

saturating NAADP (1 μM) and ﬂuorometric measurement of

Ca²⁺release. The response was reported as the IC₅₀value for

inhibition of Ca²⁺release (Table 5). Experiments were

performed in triplicate.

Figure 3 illustrates typical concentration response relation-

ships for NAADP and 32 for the above assays. Both

compounds demonstrate high potency for the sea urchin

NAADP receptor. The IC₅₀for competition ligand binding and

the EC₅₀for subthreshold desensitization were roughly equal,

and both of these values were approximately 100-fold lower

than the EC₅₀for Ca²⁺release. This is in accord with previous

studies that propose that the sea urchin NAADP receptor

contains a high-aﬃnity inhibitory and a low-aﬃnity stimulatory

binding sites.⁵¹5-Thiomethyl-NAADP (32) was shown to be a

full agonist.

Figure 4 depicts the results of bioassay of 5-(3-azidopropyl)-

NAADP (31). Analogue 31 represents an NAADP derivative

containing a large functionalized substituent. It released Ca²⁺as

a partial agonist at μM concentrations, approximately 1000-fold

higher than 1, but was recognized in the nM range in the

competition ligand binding assay and in assays of subthreshold

desensitization.

Table 4. IC₅₀Values Determined for Competition Ligand

Binding between [³²P]NAADP and NAADP Analogues (25−

35)

fold

increase in

IC₅₀

IC₅₀SEM,

nM (n)

compound (structure no.)

NAADP (1)

DISCUSSION

■

0.37 0.03 (16)

18.9 4.9 (3)

15.0 3.0 (4)

6.8 3.3 (3)

49.0 15.2 (4)

56.3 12.8 (4)

3270 1720 (4)

>1 mM (4)

1

51

5-hydroxy-NAADP (26)

Chemoenzymatic synthesis enabled us to eﬃciently produce 11

new NAADP analogues, several of which extend our previously

reported series in that they contained large, functionalized

groups attached to the nicotinic acid ring.²⁸Limitations of the

chemoenzymatic synthesis were nonetheless evident because

2b and 2f could not be converted into the corresponding

NAADP derivatives. Failure of the enzymatic pyridine−base

exchange reaction could result from poor solubility of the

pyridine base, low nucleophilicity of the pyridine nitrogen, or

inability of the enzyme active site to accommodate the structure

of the modiﬁed base.

5-acetamido-NAADP (25)

40.5

5-thiomethyl-NAADP (32)

18.4

5-(3-hydroxypropyl)-NAADP (30)

5-(3-azidopropyl)-NAADP (31)

5-(3-t-Boc-aminopropyl)-NAADP (28)

5-(3-aminopropyl)-NAADP (29)

5-(3-acetamidopropyl)-NAADP (27)

8-bromo-NAADP (33)

132.4

152.2

8840

>2 × 10⁶

4878

42.4

1800 967 (3)

15.7 2.1 (3)

0.7 0.2 (4)

372 (1)

8-azido-NAADP (34)

1.9

5-azido-8-bromo-NAADP (35)

1005

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Table 5. IC₅₀Values Determined for Desensitization of Calcium Release Induced by 1μM NAADP after a 7 min Preincubation

with Varying Concentrations of NAADP Analogues (25−35)

compd (structure no.)

NAADP (1)

IC₅₀SEM, nM (n)

fold increase in IC₅₀

0.47 0.08 (6)

23.1 7.7 (3)

21.5 7.9 (3)

4.2 0.6 (3)

1

49.1

5-hydroxy-NAADP (26)

5-acetamido-NAADP (25)

45.7

5-thiomethyl-NAADP (32)

8.9

5-(3-hydroxypropyl)-NAADP (30)

5-(3-azidopropyl)-NAADP (31)

5-(3-t-Boc-aminopropyl)-NAADP (28)

5-(3-aminopropyl)-NAADP (29)

5-(3-acetamidopropyl)-NAADP (27)

8-bromo-NAADP (33)

122.0 21.8 (3)

45.6 3.8 (3)

942 359 (7)

674000 (1)

259.6

97

2004

1434042

22618.3

32.6

1230.6 174.6 (6)

15.3 5.4 (3)

0.6 0.2 (2)

8-azido-NAADP (34)

1.3

5-azido-8-bromo-NAADP (35)

575.5 57.4 (3)

1224.5

Figure 3. Typical concentration response curves for NAADP (1) (A) and analogue 5-thiomethyl-NAADP (32) (B). The experiments were

performed in sea urchin egg homogenates. The black line represents Ca²⁺release as measured by the increase in ﬂuo-3 ﬂuorescence (left axis). The

blue line corresponds to speciﬁc binding in [³²P]NAADP competition binding experiments. The red line depicts Ca²⁺release after desensitization.

This data is illustrative of potent agonists to the NAADP receptor.

base exchange. The use of NAD kinase enables us to extend the

scope of the chemoenzymatic synthesis and utilize some of the

available purine substituted NAD analogues as precursors to

NAADP derivatives.

All of the NAADP analogues were tested for their ability to

release Ca²⁺ion, for their ability to compete with [³²P]NAADP

in competition ligand binding, and for their ability to induce

subthreshold receptor desensitization. The IC₅₀values

measured for competition ligand binding and for subthreshold

desensitization agree well with each other. These values are

consistently 1−2 orders of magnitude lower than the EC₅₀

values for Ca²⁺release. Previous characterization of the

NAADP receptor in sea urchin egg extracts concluded that

the receptor contained a high aﬃnity inhibitory binding site and

a low aﬃnity stimulatory binding site.⁵¹Our observations are

Figure 4. Concentration response curves for 5-(3-azidopropyl)-

NAADP (31). The experiments were performed in sea urchin egg

homogenates. The black line represents Ca²⁺release. The blue line

corresponds to speciﬁc binding in [³²P]NAADP competition binding

experiments. The red line is measuring Ca²⁺release after

desensitization. This data is illustrative of a partial agonist to the

NAADP receptor.

consistent with this model, assuming that the IC₅₀values

measured using either competition ligand binding or receptor

desensitization reﬂect the strength of interactions with the high

aﬃnity site whereas the Ca²⁺release assay detects the low

aﬃnity stimulatory site.

In our previous publication,²⁸NAADP analogues containing

small substituents (e.g., amino- and methyl-) at the 5-position

of the nicotinic acid ring of NAADP (1) were shown to be

agonists which were approximately equipotent with NAADP. In

this study, 5-hydroxy-NAADP (26) demonstrated unexpected

loss of potency relative to its bioisosteric analogues 5-methyl-

NAADP and 5-amino-NAADP.²⁸Introduction of the phenolic

substituent (26) resulted in a 74.9-fold loss of potency

In this work, the scope of the chemoenzymatic synthesis was

extended through the use of recombinant human NAD kinase

for catalysis of the speciﬁc phosphorylation of the 2′-adenosine

hydroxyl in an NAD derivative. Thus, 8-N₃-NAD (22) was

converted to the NADP analogue 8-N₃-NADP (23), which was

further converted to 8-N₃-NAADP (34) by enzyme catalyzed

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compared to NAADP, whereas 5-amino-NAADP exhibited

only a 1.5-fold decrease in potency. This curious diﬀerence was

unexpected and can best be attributed to the ionization of the

phenolic group at the pH used for bioassay. Bioassay for Ca²⁺

release was performed at pH 7.2, and with a phenolic pK_aof

5.85 compound 26 can be calculated to be 96% ionized. If un-

ionized 26 is the form recognized at the receptor, at the

observed EC₅₀of 1932 nM, the concentration of un-ionized 26

will be 77 nM or 3-fold higher than NAADP (1). This suggests

that the receptor requires the pyridinium functionality to be

zwitterionic and to possess no net charge. This proposal was

further supported by inactivity of 5-carboxy-NAADP²⁸and the

inactivity of 29 containing an ammonium group. The primary

amine of 29 will be protonated at physiological pH, aﬀording a

net +1 charge on the pyridinium ring explaining the loss of

potency when compared with similar analogues 27−31. Upon

the basis of these results, we conclude that the pyridinium

moiety must be zwitterionic and possess no net charge for

receptor-mediated events to occur.

Thiomethyl compound (32) was a full agonist with an EC₅₀

for Ca²⁺release 6-fold higher relative to NAADP (Table 3,

Figure 3) and with IC₅₀values of 18.4- and 8.9-fold increases

for competitive ligand binding and desensitization, respectively

(Tables 4, 5). Additional small substitutions at the 5 position,

exempliﬁed by acetamido (25), were also shown to be high

potency agonists. The slight loss of activity in 25 (EC₅₀= 24.9-

fold increase), as well as its binding (IC₅₀= 42.9-fold increase

for competitive binding, IC₅₀= 51.2-fold increase for

desensitization), could be explained by the increased steric

bulk.

A goal of this study was to develop functionalized NAADP

analogues that retained high aﬃnity for the receptor.

Derivatives with a 3-carbon arm between the pyridine ring

and various functional groups (27−31) were synthesized and

evaluated. Both 27 and 28 are branched, and in the case of t-

Boc derivative (28), contain a sterically large and hydrophobic

group. The smallest member of this series, 30, exhibited about a

2 orders of magnitude decrease in potency in all of our assays

relative to NAADP. The largest member of the series, 28, was

found to be a low-potency partial agonist. This derivative

demonstrated a 3−4 orders of magnitude loss of potency. The

“clickable” azidopropyl analogue (31) displayed interesting

activity in that it was a partial agonist (Figure 4) active in the 30

μM range in inducing Ca²⁺release. Binding assays however

(Tables 4 and 5) suggest that 31 binds with high potency to the

inhibitory binding site with an EC₅₀of approximately 50 nM.

In this work, we additionally reported that substituents could

be introduced at the 8-adenosyl position while retaining agonist

potency. The data in Tables 3−5 show that NAADP analogues

with substituents at the 8-adenosyl position were recognized as

high potency agonists. We found that 8-azido-NAADP (34)

was a high potency agonist, as both the EC₅₀and IC₅₀values

were no more than 2-fold higher than those measured for

NAADP. 8-Bromo-NAADP (33) was shown to be less potent,

with an EC₅₀27.5-fold-higher than that of NAADP. Binding

studies measured IC₅₀values to be 42.4-fold increase for

competitive-ligand binding experiments and 32.6-fold higher

than NAADP for desensitization. Together, the 8-adenosyl

position has been identiﬁed as a position tolerant to

substitution. This is signiﬁcant because previously reported

modiﬁcation of the adenine ring was associated with loss of

activity.²⁶

A disubstituted NAADP derivative has been synthesized and

tested in the sea urchin egg system (Tables 3−5). 5-Azido-8-

bromo-NAADP (35) was a low potency partial agonist,

exhibiting IC₅₀and EC₅₀values 1000-fold higher than

NAADP. Analogue 35 was less potent than predicted by the

activities of either of the monosubstituted analogues, indicating

that the presence of substituents at both the 8-adenosyl and the

5-nicotinic acid interfere with binding. Nonetheless, disub-

stituted 35 was bound at suﬃciently low concentration (370−

576 nM) to suggest that derivatives with substituents on both

the nicotinic acid 5-position and on the 8-adenosyl position can

be developed and applied to receptor identiﬁcation.

We recently reported the results of a study of the activities of

several NAADP analogues in the SKBR3 human cell line.³⁷We

found that the SAR for nicotinic acid substitution diﬀers

signiﬁcantly from the relation we report here based on sea

urchin egg homogenate. In SKBR3 cells, 5-thiomethyl-NAADP

(32) loses much of its eﬃcacy, and in the cases tested, the 4-

substituted analogues are better tolerated than are the 5-

substituted analogues. This indicated that the details of the SAR

for recognition of NAADP analogues will diﬀer across species.

CONCLUSIONS

■

We found that 5-substituted nicotinic acids containing large

and functionalized substituents were excellent substrates for the

enzyme catalyzed base-exchange reaction. Similarly, we ﬁnd

that 8-adenosyl substituted NADP derivatives can be prepared

from NAD analogues and serve as substrates for base-exchange.

This signiﬁcantly extends the scope of our chemo-enzymatic

method. The corresponding NAADP derivatives were evaluated

in sea urchin egg homogenates for their binding, for their ability

to induce subthreshold desensitization, and for Ca²⁺release

activity. The results conﬁrm that small substitutions, as seen

with analogues 25, 26, and 32, were high potency agonists.

Hydroxypropyl (30) and azidopropyl (31) NAADP analogues

demonstrated relatively high activity. The loss of potency

associated with the t-Boc derivative 28 was informative,

indicating the upper limit of steric bulk permitted on the 5-

nicotinyl position. We found that compounds containing

substitution on the 8-adenosyl position were potent agonists,

in particular azide 34, which was found to be almost equipotent

to NAADP itself. Another novel observation was the ability of a

disubstituted NAADP derivative to elicit a receptor-mediated

response at low concentrations. This ﬁnding will prove to be

extremely valuable for the development of NAADP derivatives

for the puriﬁcation and the identiﬁcation of the unknown

NAADP receptor using photoaﬃnity labeling and “click

chemistry” aﬃnity isolation. In addition to further elucidation

of the SAR, we have also conﬁrmed the observation that the

pyridinium moiety on NAADP must be zwitterionic and

possess no net charge on this functionality to be recognized by

the NAADP receptor.

EXPERIMENTAL SECTION

■

Materials. Reagents were obtained from either Acros Organics or

Sigma-Aldrich and puriﬁed when necessary according to instructions

in Perrin’s Puriﬁcation of Laboratory Chemicals.⁵²Anhydrous solvents

were purchased from Sigma-Aldrich (St. Louis, MO) or puriﬁed by

distillation over a drying agent as described in the individual

experimental description. All reagent grade solvents (acetone, DCM,

methanol, ethyl acetate (EtOAc), and hexanes) were purchased from

Pharmaco-Aaper or EMD Chemicals. 5-Aminonicotinic acid was

purchased from AK Scientiﬁc (Union City, CA), 5-hydroxynicotinic

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acid was purchased from TCI America (Portland, OR), NAD was

purchased from Roche Diagnostics (Indianapolis, IN), and NADP

monosodium salt, tetra-hydrate was purchased from Research

Products International (Mt. Prospect, IL). 5-Thiomethylpyridine-3-

carboxylic acid (5-thiomethylnicotinic acid, 2d) was synthesized

according to the procedure described by Ali et al.³⁷

Recombinant Aplysia californica ADP-ribosyl cyclase was produced

by the procedure of H. C. Lee et al.⁵⁰Concentrated solutions of the

puriﬁed enzyme were stored at −80 °C in aliquots of 100 μL at a

concentration of 10 mg/mL. As the catalyst was needed for synthesis,

a single 100 μL aliquot was removed from the freezer and thawed to 0

°C in an ice bucket. This concentrate was diluted to a ﬁnal working

concentration of 0.2 mg protein/mL by diluting it into 4.9 mL of 20

mM 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES)

buﬀer (pH = 7.5). The sample was mixed thoroughly and divided

into 60 μL aliquots, each containing 12 μg of protein at a

concentration of 200 μg/mL. Normally, 3 aliquots (36 μg of protein)

were used for base exchange of 18 μmol of NADP. Diluted samples are

stored at −80 °C and are warmed to 0 °C in an ice bath immediately

prior to use.

General Procedures. Reactions were performed in washed and

oven-dried glassware (≥110 °C) under an inert atmosphere of dry

nitrogen gas. Reactions were stirred using a magnetic stirring apparatus

with Teﬂon-coated stir bars. Microwave heating of reactions was

performed using a Biotage Initiator 2.5 system, and the conditions

were reported by describing the instrument’s adjustable settings: time,

temperature, prestirring, vial type, absorption level, and ﬁxed hold

time. The vials, caps, and stir bars for this system were purchased from

Biotage. Determination of pH was made using a PHM82 standard pH

meter (Radiometer America, Cleveland, OH).

Evaporation of volatile solvents was done under reduced pressure

using a Heidolph rotary evaporator and a pressure regulator connected

to either a water aspirator or a polytetraﬂuoroethylene (PTFE)

diaphragm vacuum pump. In experimental descriptions, reduced

pressures in mbar are often stated. For less volatile solvents, a

traditional two-stage oil-driven vacuum pump connected to a Buchi

rotary evaporator and ﬁtted with a heating bath was used.

chromatography was performed according to the procedure of Still et

al.⁵³using Fisher Silica Gel 60, 200−425 mesh (40−60 mm), weighing

about 50 times the weight of the crude mixture to be puriﬁed.

Alternatively, ﬂash chromatography could be accomplished using a

Combiﬂash Rf system (Teledyne-Isco, Lincoln, NE). Prepacked

columns containing normal phase silica columns (12 or 40 g) and

reversed-phase C₁₈-columns (15.5 g) were purchased from Teledyne-

Isco for this apparatus. Linear gradients could be more reproducibly

produced by the Combiﬂash Rf system and often provides a superior

result.

Reverse phase C18 columns for the Teledyne-Isco Combiﬂash

(Lincoln, NE) and were used to further purify the substituted nicotinic

acids and as a desalting procedure. The method consisted of applying

the dinucleotide as a solution in water followed by applying a gradient

formed from 0 to 80% methanol in water. The product generally elutes

before the gradient starts and the rest of the method can be considered

a wash and storage procedure.

High Pressure Liquid Chromatography (HPLC). HPLC

puriﬁcations were performed on a Bio-Rad BioLogic Duo Flow

system equipped with a 254 nm UV detector. Analytical anion-

exchange columns were purchased from Bio-Rad (Uno-Q, 7 mm × 35

mm, 1.3 mL). Alternately, both analytical and preparative columns

could be packed from BioRex MP1-X2 analytical anion exchange resin.

The procedure was adapted from the volatile water−TFA system

described by Alexson et al. (1981).⁵⁴

Preparation of Macroporous Anion Exchange Resin. Bio-Rad AG-

MP1 anion exchange resin (30 g) (Bio-Rad Laboratories, Hercules,

CA USA) was added to a stirred solution of 4 N NaOH (200 mL) and

thoroughly mixed for 6 h. Stirring was stopped, and the resin was

allowed to settle overnight, at which time, excess liquid was decanted

and a second portion of 2 N NaOH (200 mL) was added and stirred

for 3 h. Once the resin settled, excess liquid was decanted and then

resuspended and stirred (30 min) in water (250 mL). After decanting,

the resin was washed twice more with water as above for a total of

three water washes. We needed to monitor the presence of chloride

anion during the washes with hydroxide anion because it is the

hydroxide anion that displaces the chloride anion. A portion of resin

free supernatant (1 mL) was added to a test tube, acidiﬁed with

concentrated aqueous nitric acid (7 drops), and then assayed with

silver nitrate. If chloride ion is present, insoluble silver chloride

precipitates out of solution. The ﬁrst two washes with NaOH indicated

Cl⁻was present, while the third was Cl⁻free, indicating that

displacement of Cl⁻with OH⁻was complete. Washings with water

were then conducted until supernatants were silver chloride negative

and the pH of the water wash was neutral. If silver chloride continues

to precipitate out of solution, the procedure must be repeated to

appropriately convert the resin to the ⁻OH form. Upon testing

negative for silver chloride, the resin was decanted and resuspended in

2 M TFA (150 mL). This ﬁnal mixture was stirred for 30 min before

storage at 4 °C and was ready for use.

Method 1: Analytical Method. A Bio-Rad Uno-Q column (7 mm ×

35 mm, 1.3 mL of anion-exchange resin) was ﬁtted to a Bio-Rad

BioLogic Duoﬂow HPLC apparatus, equipped with a 250 μL injection

loop. The ﬂow rate was 3 mL/min throughout the separation. The

injection loop was completely ﬁlled with solution of the NAADP

derivative (1 mg/mL) and the chromatography begun by applying 1

mL of d·H₂O through the injection loop. The separation was

developed by (1) applying 10 mL of d·H₂O, (2) forming a linear

gradient between d·H₂O and 50 mM aqueous TFA over a total volume

of 60 mL, and (3) applying 11 mL of 100 mM aqueous TFA. Finally,

the column was re-equilibrated with d·H₂O (11 mL) in preparation for

the next injection.

Melting points were determined in duplicate on a MEL-TEMP II

apparatus and are uncorrected. Elemental analyses were performed by

Atlantic Microlabs (Norcross, GA) and were regarded as being

acceptable when the result was within 0.4% of the theoretical values.

Compounds reported as solvates are denoted on an individual basis,

and the reported empirical formula was calculated by incorporating the

minimum amount of the designated solvent that the compound

encountered during puriﬁcation in order to be within the acceptable

range ( 0.4%).

Proton (¹H) NMR and carbon (¹³C) NMR were determined using

either a Unity-400 spectrophotometer (400 MHz), a Varian Inova-600

spectrophotometer (600 MHz), or a Bruker Avance Cryprobe (600

MHz). Chemical shifts are reported in ppm (δ) and were referenced to

the residual proton signal of the deuterated solvent. When TMS was

present (CDCl₃and deuterated dimethyl sulfoxide (DMSO-d₆)), the

TMS peak at zero ppm was used as the reference. ³¹P NMR spectra

were recorded on a Unity-400 spectrophotometer (162 MHz) and

referenced externally to 85% H₃PO₄. The chemical shifts for ¹H NMR

were reported to the second decimal place, while ¹³C chemical shifts

were reported to the ﬁrst decimal place. The following abbreviations

are used to describe spin multiplicity: s = singlet, d = doublet, t =

triplet, q= quartet, m = multiplet, dd = doublet of doublets. Coupling

constants (J) were reported in hertz (Hz).

Mass spectral analysis of substituted nicotinic acid derivatives and

dinucleotides were performed at The Ohio State University Mass

Spectrometry and Proteomics Facility. Accurate mass analysis was

performed using ESI ionization in the positive ion mode (ESI⁺), and

dinucleotides were measured using matrix-assisted laser desorption/

ionization-time-of-ﬂight (MALDI-TOF) mass spectrometry.

Method 2: Analytical Method. The conditions were identical to

those described under method 1, except that an injection loop and an

injection volume of 50 μL was used and the chromatography begun by

applying 0.8 mL of d·H₂O through the injection loop. Thereafter, the

chromatography was developed by (1) applying 9 mL of d·H₂O, (2)

forming a linear gradient between d·H₂O and 15 mM aqueous TFA

over a total volume of 120 mL, and (3) applying 9 mL of 100 mM

Analytical thin layer chromatography (TLC) was performed on

Baker-ﬂex TLC plates (2.5 cm × 7.5 cm) with a 254 nm ﬂuorescent

indicator (IB-F). Plates were developed in a covered chamber, usually

with 5−10 mL of mobile phase, and visualized by UV-light. Flash

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aqueous TFA. Finally, the column was re-equilibrated with d·H₂O (12

mL) in preparation for the next injection.

automatically with the aid of a fraction collector (Teledyne Isco,

Retreiver 500, 100 drops/tube) and the dinucleotide detected by its

absorption at 254 nm. Repeated lyophilization of the sample from

water ensured complete removal of volatile NH₄HCO₃, leaving pure,

salt-free dinucleotide.

Method 3: Prep-scale HPLC (0−50 mM TFA). A glass column

(Omni, 1.5 cm × 11.5 cm) ﬁlled with Bio-Rad AG-MP1 ion-exchange

resin (triﬂuoroacetate form, see resin preparations) was connected to a

Bio-Rad BioLogic Duoﬂow HPLC apparatus ﬁtted with a 5 mL

injection loop. The ﬂow rate throughout was 5 mL/min. The injection

loop was completely ﬁlled with a solution of the NAADP derivative

(1−3 mg/mL), and the chromatography begun by applying 7 mL of d·

H₂O through the injection loop. The preparative separation was

developed by (1) applying 20 mL of H₂O, (2) forming a linear

gradient between H₂O and 50 mM aqueous TFA, formed over a total

volume of 160 mL, and (3) applying 25 mL of 100 mM aqueous TFA.

Finally, the column was re-equilibrated with d·H₂O (35 mL). Peaks

absorbing at 254 nm were combined, and excess TFA was removed by

extraction of the aqueous solution with DCM (3×). Following the

extraction, residual DCM and TFA were removed by treatment of the

aqueous solution under vacuum for 10 min at 25 mbar.

Method 4: Prep-Scale HPLC (0−20 mM Aqueous TFA). The

procedure was identical to that described under method 3, except that

the chromatography was begun by applying 9 mL of d·H₂O through

the injection loop and developed by (1) applying 15 mL of d·H₂O, (2)

forming a linear gradient between d·H₂O and 20 mM aqueous TFA

over a total volume of 225 mL, and (3) applying 40 mL of 100 mM

aqueous TFA. Finally, the column was re-equilibrated with d·H₂O (40

mL) in preparation for the next injection.

Method 5: Prep-Scale HPLC (0−8 mM aqueous TFA). The

procedure was identical to that described under method 3, except that

the chromatography was begun by applying 9 mL of d·H₂O through

the injection loop and the separation developed by (1) applying 15 mL

of d·H₂O, (2) forming a linear gradient between d·H₂O and 8 mM

aqueous TFA over a total volume of 190 mL, and (3) applying 40 mL

of 100 mM aqueous TFA. Finally, the column was re-equilibrated with

d·H₂O (40 mL) in preparation for the next injection.

Open Column Anion-Exchange Chromatography. Open-

column anion exchange chromatography for the puriﬁcation of

dinucleotides was performed using diethylaminoethyl (DEAE)

substituted cellulose (DE53, product no. 4058200; Whatman Inc.,

Florham Park, NJ, USA). To prepare the commercially obtained

material for use, 50 g of DEAE cellulose was added to a stirred solution

of 1.5 M NH₄HCO₃(300 mL) and thoroughly mixed for 4 h. Stirring

was stopped, and the resin was allowed to settle overnight, at which

time, excess liquid was decanted and a second portion of 1.0 M

NH₄HCO₃(250 mL) was added and stirred for 2 h. Once the resin

settled, excess liquid was decanted and the resin resuspended and

stirred (30 min) in water (300 mL). After decanting, the resin was

washed twice more with water as above for a total of three water

washes. At this point, the pH of a sample of the supernatant should be

close to neutral, even slightly acidic as measured using a wide-range

pH test paper. Repeated washes with water were continued until the

pH was neutral. Unused resin that was not to be used within 2 weeks

was stored at 4 °C in 100 mM NH₄HCO₃.

Method 7: Preparative Anion Exchange Chromatography on

Columns of DEAE Cellulose. NAADP derivatives can be separated

from NADP, ADP-ribose phosphate, and other nucleotides by anion

exchange chromatography on DEAE cellulose. DE-52 cellulose was

slurry packed into glass Bio-Rad Econo-Columns (2.5 cm × 50 cm) to

form a resin bed approximately 42 cm high. Water (100 mL) was

passed through the column to ensure the pH of the eﬄuent from the

column was neutral. The dinucleotide mixture was applied to the

column as a dilute solution at pH 7.5 and washed into the bed with

10−20 mL of distilled water. The separation was developed by the

application of a linear gradient formed between water (350 mL) and

0.6 M NH₄HCO₃(350 mL). A ﬂow rate of about 1−2 mL/min was

achieved using a peristaltic pump (Teledyne-Isco, TRIS) to produce a

slight positive pressure. The separation allowed puriﬁcation of

compounds based on charge, and NAADP derivatives usually eluted

into the middle of the gradient into approximately 320 mM

NH₄HCO₃. Fractions were collected using an automatic fraction

collector (Teledyne-Isco, Retreiver 500, 200 drops/tube) and the

dinucleotides detected by their UV absorbance (ISCO UA-6 UV/vis

detector). UV absorbing peaks were combined, frozen, and lyophilized.

Repeated lyophilization of the sample from water ensured complete

removal of volatile NH₄HCO₃, leaving pure, salt-free dinucleotide.

5-(Acetamido)nicotinic Acid (2c). 5-Aminonicotinic acid (0.250

g, 1.81 mmol), acetic anhydride (1.11 g, 11 mmol), and pyridine (0.72

g, 10 mmol) were added to a round-bottom ﬂask and reﬂuxed for 22 h.

According to the original procedure, a precipitate should form and this

was recrystallized from ethanol. This did not work in our hands.

Instead, solvents were removed by distillation in vacuo. The sample

was dissolved in DMSO and puriﬁed by column chromatography on

silica gel in butanol−acetic acid−water (5:2:3). This gave a mixture of

two compounds. The sample was further puriﬁed by column

chromatography on silica gel (90:9:1 DCM−methanol−acetic acid).

Removal of solvents by distillation under reduced pressure aﬀorded a

dark-yellow solid (126 mg, 39%): mp 277−279 °C (lit.⁵⁵282−284

°C). TLC R_f0.73 in butanol−acetic acid−water (5:2:3). TLC R_f0.36

in DCM−methanol−acetic acid (90:9:1; the R_fvalue was recorded

1

following 2 developments of the TLC plate). H NMR (400 MHz,

DMSO-d₆) δ 10.37 (bs, 1H), 8.88 (s, 1H), 8.74 (s, 1H), 8.58 (s, 1H),

2.11 (s, 3H).

5-Bromonicotinic Acid Ethyl Ester (2e). To a solution of 5-

bromonicotinic acid (5 g, 28.4 mmol) in absolute ethanol (50 mL) was

added 5 N HCl in dioxane (10 mL). The reaction mixture was reﬂuxed

for 48 h. Solvents were removed in vacuo then water (75 mL) was

added and neutralized with 5 N NaOH. Upon neutralization, the

solution was extracted 3× with EtOAc. EtOAc was distilled under

reduced pressure to give an oﬀ-white solid which was recrystallized

twice from water, aﬀording a white solid (5.7 g, 88%); mp 39−40 °C

(lit.⁵⁶38−39 °C). TLC R_f0.52 in 20% EtOAc−hexanes. H NMR

1

Method 6: Desalting of NAADP Derivatives on DEAE Cellulose.

Pyridine dinucleotides 25−35 were desalted and obtained in the

ammonium form using a simple gradient in a volatile NH₄HCO₃

buﬀer followed by repeated lyophilization. A Bio-Rad Econo-Column

(2.5 cm × 8 cm) was ﬁlled with a slurry of DE53 cellulose and allowed

to form a packed column of absorbent 3 cm high. Water (25 mL) was

passed through the column until the eﬄuent pH was neutral. The

sample of dinucleotide was applied to the column as a dilute solution

at pH ≥ 7.5 and properly adsorbed. The separation was developed by

applying a linear gradient formed between water (150 mL) and 0.5 M

NH₄HCO₃(150 mL). Application of a gradient (0−0.5 M

NH₄HCO₃) allowed proper binding of the dinucleotide to the

column. A ﬂow rate of about 1−2 mL/min was achieved using a

peristaltic pump to produce a slight positive pressure. Contaminating

salts such as NaCl, ammonium chloride, or sodium triﬂuoroacetate

eluted into the low ionic strength buﬀer ahead of the dinulcleotide.

NAADP derivatives generally eluted into the mobile phase at

concentrations of NH₄HCO₃> 200 mM. Fractions were collected

(600 MHz, CDCl₃) δ 9.13 (s, 1H), 8.84 (s, 1H), 8.43 (s, 1H), 4.43 (q,

2H, J = 7.2 Hz), 1.42 (t, 3H, J = 7.2 Hz). ¹³C NMR (101 MHz,

CDCl₃) δ 164.0, 154.4, 148.8, 139.5, 127.6, 120.6, 62.0, 14.2.

5-(2-Phenylethyl)-3-pyridinecarboxylic Acid (2f). 2f was

synthesized in three steps starting from 2e.

5-(2-Phenylethynyl)nicotinic Acid Ethyl Ester. To a solution of

2e (250 mg, 1.09 mmol), Pd(OAc)₂(12 mg, 0.054 mmol), and

DIPEA (423 mg, 3.27 mmol) and triphenylphosphine (14 mg, 0.054

mmol) in acetonitrile (7.5 mL) was added phenylacetylene (134 mg,

1.31 mmol) and CuI (14.5 mg, 0.0763 mmol) while stirring under N₂.

After the addition was complete, the reaction mixture was reﬂuxed for

16 h. At this time, TLC indicated that the starting material was

consumed. Solvents were removed in vacuo and the residue

partitioned between water and chloroform. The phases were separated,

and chloroform was concentrated in vacuo. The resulting oil was

puriﬁed by column chromatography on silica gel (20% EtOAc−

hexanes) to yield a brown oil (224 mg, 82%). TLC R_f0.48 in 20%

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EtOAc−hexanes. H NMR (600 MHz, CDCl₃) δ 9.14 (d, 1H, J = 2

5-(3-Acetamidopropyl)nicotinic Acid Ethyl Ester (5). Com-

pound 4 (260 mg, 1.06 mmol) was dissolved in methanol (7 mL) and

transferred to a Pyrex hydrogenation ﬂask and cooled in dry ice−

acetone for 5 min. Pd/C (52 mg, 20% w/w) was added, then the ﬂask

was attached to a PARR hydrogenator and shaken for 8 h at 30 PSI of

H₂. Upon completion, the mixture was ﬁltered through Celite and

solvent was distilled in vacuo and the residue then partitioned between

water and chloroform. The phases were separated, and the chloroform

was distilled in vacuo. This residue was puriﬁed by column

chromatography on silica gel (0−10% methanol−DCM) to yield a

white powder (240 mg, 91%); mp 50−52 °C. TLC R_f0.55 in 10%

methanol−DCM. ¹H NMR (400 MHz, CD₃OD) δ 8.92 (s, 1H), 8.61

(s, 1H), 8.23 (s, 1H), 4.40 (q, 2H, J = 7.2 Hz), 3.21 (t, 2H, J = 6.8

Hz), 2.75 (t, 2H, J = 8 Hz), 1.94 (s, 3H), 1.84 (m, 2H), 1.40 (t, 3H, J

= 7.2 Hz). ¹³C NMR (101 MHz, CD₃OD) δ 173.3, 166.4, 154.2,

148.8, 139.5, 138.6, 128.0, 62.8, 40.0, 31.8, 31.0, 22.7, 14.7. HRMS

calcd for C₁₃H₁₈N₂O₃: 273.1215 (M + Na). Found m/z: 273.1215 (M

+ Na).

Hz), 8.90 (d, 1H, J = 2 Hz). 8.41 (t, 1H, J = 2 Hz), 7.56 (m, 2H), 7.39

(m, 3H) 4.44 (q, 2H, J = 7.14 Hz), 1.43 (t, 3H, J = 7.14 Hz). ¹³C

NMR (150 MHz, CDCl₃) δ 164.7, 155.3, 149.3, 139.3, 131.8, 129.11,

128.52, 125.9, 122.1, 120.5, 93.7, 85.0, 61.7, 14.3. Anal. (C₁₆H₁₃NO₂)

C, H, N.

5-(2-Phenylethyl)nicotinic Acid Ethyl Ester. 5-(2-

Phenylethynyl)nicotinic acid ethyl ester (209 mg, 0.832 mmol) was

dissolved in EtOAc (12 mL) and methanol (4 mL) and transferred to

a glass hydrogenation ﬂask and cooled in dry ice−acetone for 5 min.

Pd/C (42 mg, 20% w/w) was added, and the ﬂask was attached to a

PARR hydrogenator and shaken for 10 h at 30 PSI of H₂. Upon

completion, the mixture was ﬁltered through Celite and solvents were

removed from the ﬁltrate in vacuo. The resulting residue partitioned

between water and chloroform. The chloroform was concentrated

under reduced pressure, and the product was puriﬁed by column

chromatography on silica gel (50% EtOAc−hexanes) to yield a yellow

1

oil (175 mg, 82%). TLC R_f0.54 in 50% EtOAc−hexanes. H NMR

(600 MHz, CDCl₃) δ 9.06 (d, 1H, J = 2 Hz), 8.53 (d, 1H, J = 2.2 Hz),

8.10 (t, 1H, J = 2 Hz), 7.29 (m, 2H), 7.22 (m, 1H), 7.15 (m, 2H) 4.41

(q, 2H, J = 7.14 Hz), 2.98 (m, 4H), 1.41 (t, 3H, J = 7.14 Hz). ¹³C

NMR (150 MHz, CDCl₃) δ 165.4, 153.3, 148.4, 140.3 137.08, 136.80,

128.57, 128.46, 126.39, 126.06, 61.5, 37.2, 34.7, 14.3. Anal.

(C₁₆H₁₇NO₂) C, H, N.

5-(3-Acetamidopropyl)nicotinic Acid (6). Compound 5 (104

mg, 0.42 mmol) was dissolved in methanol (2 mL) and 4N NaOH (2

mL). This mixture was stirred at rt for 3.5 h. The solvent was removed

under reduced pressure and the residue extracted 5 × with hot

methanol (10 mL portions). The resulting 50 mL extract was ﬁltered

through Celite and the solvent removed from the ﬁltrate by distillation

in vacuo. The yellow powder was puriﬁed by reverse phase

chromatography by using a C₁₈cartridge (15.5 g, isocratic elution

with water) using a Combiﬂash Rf system. The product eluted shortly

after the void volume (13.5 mL), which upon removal of solvent in

vacuo aﬀorded an oily white solid (83 mg, 89%); mp 268−270 °C.

5-(2-Phenylethyl)-3-pyridinecarboxylic Acid (2f). 5-(2-

Phenylethyl)nicotinic acid ethyl ester (127 mg, 0.497 mmol) was

dissolved in methanol (3 mL) and 1 N NaOH (3 mL) and stirred at rt

for 18 h. After this time, the methanol was evaporated in vacuo and the

remaining solution was neutralized using 1 N HCl. A cream-colored

precipitate formed, which was collected and washed with ice-cold

water (20 mL). The solid was dried overnight in a vacuum desiccator

at 40 °C equipped to an oil-driven vacuum pump to yield a cream-

colored solid (80 mg, 71%); mp 212−214 °C. TLC R_f0.71 in DCM−

1

TLC R_f0.53 in butanol−acetic acid−water (5:2:3). H NMR (400

MHz, CD₃OD) δ 8.89 (s, 1H), 8.42 (s, 1H), 8.18 (s, 1H), 3.22 (t, 2H,

J = 6 Hz), 2.72 (t, 2H, J = 7.6 Hz), 1.94 (s, 3H), 1.85 (m, 2H). ¹³C

NMR (150 MHz, CD₃OD) δ 173.4, 172.8, 151.1, 149.0, 138.69,

138.58, 135.0, 40.0, 31.8, 31.1, 22.7. HRMS calcd for C₁₁H₁₄N₂O₃:

245.0902 (M + Na). Found m/z: 245.0893 (M + Na).

1

methanol−acetic acid (94:5:1). H NMR (600 MHz, DMSO-d₆) δ

13.37 (s, 1H), 8.89 (d, 1H, J = 2 Hz), 8.62 (d, 1H, J = 2.2 Hz), 8.12 (t,

1H, J = 2 Hz), 7.27 (m, 2H), 7.23 (m, 2H), 7.18 (m, 1H), 3.12 (m,

4H). ¹³C NMR (150 MHz, DMSO-d₆) δ 166.3, 153.4, 147.8, 140.7,

136.82, 136.51, 128.38, 128.17, 126.02, 125.89, 36.3, 33.5. Anal.

(C₁₄H₁₃NO₂) C, H, N.

tert-Butyl-prop-2-ynylcarbamate (7). To a solution of prop-

argylamine (750 mg, 14.0 mmol) in DCM (5 mL) at 0 °C was added a

solution of di-tert-butyldicarbonate (3.36 g, 15.4 mmol) in DCM (6

mL) over a period of 20 min. After this 20 min period, the solution

was allowed to warm to rt and stirred for an additional 48 h. Solvent

was distilled in vacuo, and the resulting yellow oil was stored at 4 °C

for 16 h. A yellowish-white precipitate formed, which was collected,

dried under reduced pressure, and recrystallized from ether−DCM,

aﬀording white crystals (1.5 g, 56%); mp 37−39 °C (lit.⁴⁰34−36 °C).

¹H NMR (400 MHz, CDCl₃) δ 4.79 (s, 1H), 3.93 (s, 2H), 2.22 (s,

3-Acetamidoprop-1-yne (3). To a solution of propargylamine

(500 mg, 9.1 mmol) and TEA (1.37 g, 13.7 mmol) in DCM (5 mL) at

0 °C was added acetic anhydride (1.11 g, 11 mmol). The mixture was

stirred for 1 h at 0 °C then left stirring for an additional 23 h at rt.

Solvent was distilled in vacuo, and then the product was puriﬁed by

bulb-to-bulb distillation (0.3 mmHg, 110−130 °C), aﬀording a clear

oil which was precipitated with hexanes, the precipitate collected, and

recrystallized from ether−DCM to aﬀord white crystals (510 mg,

58%); mp 77−78 °C (Lit.⁴⁰83−85 °C). ¹H NMR (400 MHz, CDCl₃)

δ 5.91 (s, 1H), 4.05 (m, 2H), 2.24 (s, 1H), 2.02 (s, 3H). ¹³C NMR

(101 MHz, CDCl₃) δ 169.7, 79.5, 71.6, 29.2, 23.0.

1H), 1.46 (s, 9H). ¹³C NMR (101 MHz, CDCl₃) δ 155.4, 80.27,

80.05, 71.3, 30.4, 28.4.

5-[3-[[(tert-Butyloxy)carbonyl]amino]propyn-1-yl)nicotinic

Acid Ethyl Ester (8). To a solution of 2e (250 mg, 1.09 mmol),

Pd(PPh₃)₂Cl₂(38 mg, 0.0545 mmol), and anhydrous DIPEA (423 mg,

3.27 mmol) in acetonitrile was added 14 (203 mg, 1.31 mmol) and

CuI (15 mg, 0.0763 mmol) while stirring under N₂. After the addition

was complete, the reaction mixture was reﬂuxed for 6 h. After this

time, TLC indicated complete consumption of starting material. The

solvent was removed by distillation in vacuo and the residue

partitioned between DCM and water. The phases were separated

and the organic phase distilled under reduced pressure. The resulting

residue was puriﬁed by column chromatography on silica gel (0−33%

EtOAc−hexanes), aﬀording a yellow oil which upon freezing at −20

°C for 16 h produced a yellowish-white solid (202 mg, 61%); mp 54−

5-(3-Acetamidoprop-1-yne)nicotinic Acid Ethyl Ester (4). To

a solution of 2e (250 mg, 1.09 mmol), Pd(PPh₃)₂Cl₂(38 mg, 0.0545

mmol), and DIPEA (423 mg, 3.27 mmol) in anhydrous acetonitrile (5

mL) were added 13 (127 mg, 1.31 mmol) and CuI (15 mg, 0.0763

mmol) while stirring under N₂. After the addition was complete, the

reaction mixture was reﬂuxed for 10 h. At the end of this time TLC

indicated that the starting material was consumed. Solvents were

removed in vacuo and the residue partitioned between water and

chloroform. The chloroform was distilled in vacuo, and the residue was

puriﬁed by column chromatography on silica gel (0−5% DCM−

methanol), aﬀording a yellow solid (161 mg, 60%). To obtain

analytically pure samples, the yellow solid was recrystallized from

ether−DCM, aﬀording a white powder; mp 82−84 °C. TLC R_f0.63 in

1

56 °C. TLC R_f0.52 in 50% EtOAc−hexanes. H NMR (400 MHz,

CDCl₃) δ 9.12 (s, 1H), 8.79 (s, 1H), 8.30 (s, 1H), 5.52 (s, 1H) 4.42

(q, 2H, J = 7.2 Hz), 4.21 (s, 2H), 1.48 (s, 9H), 1.41 (t, 3H, J = 7.2

Hz). ¹³C NMR (101 MHz, CDCl₃) δ 164.5, 155.4, 149.4, 139.5, 125.8,

119.9, 90.5, 80.0, 78.6, 61.7, 31.0, 28.4, 28.0. Anal. (C₁₆H₂₀N₂O₄·

0.1CH₂Cl₂) C, H, N. HRMS calcd for C₁₆H₂₀N₂O₄: 327.1321 (M +

Na). Found m/z: 327.1315 (M + Na).

1

5% methanol−DCM. H NMR (400 MHz, CDCl₃) δ 9.11 (s, 1H),

8.78 (s, 1H), 8.29 (s, 1H), 6.82 (s, 1H), 4.42 (q, 2H, J = 7.2 Hz), 4.32

(d, 2H, J = 5.2 Hz), 2.08 (s, 3H), 1.41 (t, 3H, J = 7.2 Hz). ¹³C NMR

(101 MHz, CDCl₃) δ 170.0, 164.5, 155.3, 149.4, 139.6, 125.9, 119.8,

90.0, 78.7, 61.8, 29.8, 23.0, 14.2. Anal. (C₁₃H₁₄N₂O₃) C, H, N. HRMS

calcd for C₁₃H₁₄N₂O₃: 269.0902 (M + Na). Found m/z: 269.0891 (M

+ Na).

5-[3-[(tert-Butyloxy)carbonyl]aminopropyl]nicotinic Acid

Ethyl Ester (9). Compound 8 (100 mg, 0.346 mmol) was dissolved

in methanol (5 mL) and transferred to a Pyrex hydrogenation ﬂask,

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which was capped and cooled in dry ice−acetone for 5 min. Pd/C (20

mg, 20% w/w) was added and mixed, and the ﬂask was connected to a

PARR hydrogenator and shaken for 6 h at 35 PSI of H₂. Upon

completion, the mixture was ﬁltered through Celite. The solvent was

removed by distillation in vacuo and the residue partitioned between

water and DCM. The phases were separated, and the DCM was

concentrated in vacuo. The resulting residue was puriﬁed by column

chromatography on silica gel (50% EtOAc−hexanes), which upon

distillation of the solvents in vacuo aﬀorded a yellowish-white solid (99

was pure by NMR. The product was further puriﬁed by recrystallizing

from ether−DCM to produce white crystals; mp 87−88 °C. ¹H NMR

(400 MHz, CDCl₃) δ 9.13 (s, 1H), 8.86 (s, 1H), 8.33 (s, 1H), 4.53 (d,

2H, J = 6 Hz), 4.42 (q, 2H, J = 7.2 Hz), 1.41 (t, 3H, J = 7.2 Hz). ¹³C

NMR (101 MHz, CDCl₃) δ164.5, 155.3, 149.4, 140.0, 126.0, 120.0,

92.5, 81.0, 61.8, 51.2, 14.2. Anal. (C₁₁H₁₁NO₃) C, H, N. HRMS calcd

for C₁₁H₁₁NO₃: 228.0637 (M + Na). Found m/z: 228.0634 (M + Na).

5-(3-Hydroxypropyl)nicotinic Acid Ethyl Ester (14). Com-

pound 13 (270 mg, 1.32 mmol) was dissolved in methanol (8 mL)

and transferred to a Pyrex hydrogenation ﬂask and cooled in dry ice−

acetone for 5 min. Pd/C (54 mg, 20% w/w) was added, and the ﬂask

was attached to a PARR hydrogenator and shaken for 10 h at 30 PSI of

H₂. Upon completion, the mixture was ﬁltered through Celite and

solvents were removed in vacuo. The resulting residue was partitioned

between water and chloroform. The chloroform was concentrated

under reduced pressure and was puriﬁed by column chromatography

on silica gel (5% methanol−DCM), aﬀording a yellow oil (245 mg,

1

mg, 93%). TLC R_f0.31 (50% EtOAc−hexanes); mp 59−60 °C. H

NMR (400 MHz, CDCl₃) δ 9.06 (s, 1H), 8.61 (s, 1H), 8.13 (s, 1H),

5.07 (s, 1H), 4.42 (q, 2H, J = 7.2 Hz), 3.21 (q, 2H, J = 6.4 Hz), 2.73 (t,

2H, J = 8 Hz), 1.87 (m, 2H), 1.45 (s, 9H), 1.42 (t, 3H, J = 7.2 Hz). ¹³C

NMR (101 MHz, CDCl₃) δ 165.4, 156.0, 153.4, 148.5, 136.8, 136.6,

126.0, 79.2, 61.4, 40.0, 31.4, 30.0, 28.4, 14.3. HRMS calcd for

C₁₆H₂₄N₂O₄: 331.1634 (M + Na). Found m/z: 331.1646 (M + Na).

5-(3-Aminopropyl)nicotinic Acid Ethyl Ester (10). Compound

9 (97.5 mg, 0.316 mmol) was dissolved in water (1 mL) and TFA (1

mL) and stirred for 2 h at 37 °C. The solvent was removed in vacuo,

forming a yellow colored oil. TLC R_f0.67 in butanol−acetic acid−

1

89%). TLC R_f0.37 in 5% methanol−DCM. H NMR (400 MHz,

CDCl₃) δ 9.03 (s, 1H), 8.61 (s, 1H), 8.17 (s, 1H), 4.41 (q, 2H, J = 7.2

Hz), 3.71 (t, 2H, J = 6.4 Hz), 3.62 (s, 1H), 2.83 (t, 2H, J = 7.6 Hz),

1.93 (m, 2H), 1.42 (t, 3H, J = 7.2 Hz). ¹³C NMR (101 MHz, CDCl₃)

δ 165.4, 153.3, 148.2, 137.4, 137.0, 126.1, 61.5, 61.1, 33.7, 29.0, 14.4.

HRMS calcd for C₁₁H₁₅NO₃: 232.0950 (M + Na). Found m/z:

232.0946 (M + Na).

1

water (5:2:3); R_f0.12 in DCM−methanol−acetic acid (90:9:1). H

NMR (400 MHz, CD₃OD) δ 9.17 (s, 1H), 8.90 (s, 1H), 8.77 (s, 1H),

4.47 (q, 2H, J = 7.2), 3.05 (t, 2H, J = 7.2 Hz), 2.99 (t, 2H, J = 8 Hz),

2.08 (m, 2H), 1.42 (t, 3H, J = 7.2 Hz). ¹³C NMR (101 MHz, CD₃OD)

δ 164.3, 148.5, 144.7, 144.4, 141.7, 130.5, 63.7, 40.0, 30.3, 29.5, 14.6.

The compound was immediately taken to the next step in the

formation of 12.

5-(3-Hydroxypropyl)nicotinic Acid (15). Compound 14 (156

mg, 0.746 mmol) was dissolved in methanol (2 mL) and 4 N NaOH

(1 mL) and stirred at 37 °C for 4 h. After this time, the solvent was

evaporated in vacuo, the residue dissolved in water, and the alkali

neutralized using 1 N HCl. After the water was removed by

lyophilization, the residue was applied to a Combiﬂash C18 cartridge

(15.5 g) and eluted isocratically with water. The mixture was

concentrated in vacuo and applied to a 35 mL (bed volume) column

of Amberchrom resin (CG71M, Dow Chemical Company, product no.

10235577; Copenhagen, Denmark), which suﬃciently desalted the

mixture (isocratic, water). Upon drying, a yellowish-white solid was

obtained (129 mg, 96%); mp 216−218 °C. TLC R_f0.16 in DCM−

methanol−acetic acid (90:9:1). TLC R_f0.69 in butanol−acetic acid−

5-[3-[(tert-Butyloxy)carbonyl]aminopropyl]nicotinic Acid

(11). Compound 9 (100 mg, 0.324 mmol) was dissolved in methanol

(2 mL) and 4 N NaOH (1 mL) and stirred at 37 °C for 4 h. After this

time, the solvent was removed and the product was dried in vacuo

then neutralized by dissolving in 1 N HCl. The water was removed by

lyophilization, then the product was immediately puriﬁed by column

chromatography on silica gel (90:9:1 DCM−methanol−acetic acid),

aﬀording a yellowish-white solid (81 mg, 89%); mp 225−227 °C. TLC

R_f0.47 in DCM−methanol−acetic acid (90:9:1); R_f0.79 in butanol−

1

acetic acid−water (5:2:3). H NMR (400 MHz, CD₃OD) δ 8.89 (s,

1

1H), 8.41 (s, 1H), 8.17 (s, 1H), 3.10 (t, 2H, J = 6.4 Hz), 2.71 (t, 2H, J

= 8 Hz), 1.82 (m, 2H), 1.43 (s, 9H). ¹³C NMR (150 MHz, CD₃OD) δ

172.8, 158.6, 151.1, 148.9, 138.8, 138.7, 134.9, 80.0, 40.9, 32.5, 31.0,

28.9. Anal. (C₁₄H₂₀N₂O₄·0.5DCM) C, H, N.

water (5:2:3). H NMR (400 MHz, CD₃OD) δ 8.90 (s, 1H), 8.42 (s,

1H), 8.19 (s, 1H), 3.59 (t, 2H, J = 6.4 Hz), 2.77 (t, 2H, J = 8 Hz), 1.87

(m, 2H). ¹³C NMR (150 MHz, CD₃OD) δ 173.0, 151.2, 148.9, 139.0,

138.7, 135.0, 61.9, 35.0, 30.1. HRMS calcd for C₉H₁₁NO₃: 182.0817

(M + H). Found m/z: 182.0821 (M + H).

5-(3-Aminopropyl)nicotinic Acid (12). Compound 9 (100 mg,

0.324 mmol) was dissolved in water (2 mL) and TFA (1 mL) and was

stirred at rt for 8 h. The solvents were evaporated in vacuo. The crude

residue was examined, and NMR veriﬁed the removal of the t-Boc

group, while the ethyl ester was intact. The sample was then dissolved

in methanol (4 mL) and 4 N NaOH (2 mL) and the mixture allowed

to stir at rt for 8 h. The reaction mixture was neutralized with 1 N

HCl, and solvents were removed in vacuo. The relatively pure mixture

which resulted was further puriﬁed by reverse phase chromatography

on a C₁₈cartridge (15.5 g, 13.5 mL void volume) using a Combiﬂash

Rf system. The product eluted between column volumes 3 and 4 in an

isocratic (water) system. Removal of water by lyophilization yielded a

white solid (48 mg, 83%); mp 270−272 °C. TLC R_f0.33 in butanol−

acetic acid−water (5:2:3). ¹H NMR (600 MHz, D₂O) δ 8.59 (s, 1H),

8.23 (s, 1H), 7.87 (s, 1H), 2.9 (t, 2H, J = 6.4 Hz), 2.59 (t, 2H, J = 8

Hz), 1.85 (m, 2H). ¹³C NMR (150 MHz, D₂O) δ 172.9, 150.2, 147.0,

137.2, 136.3, 132.0, 38.9, 28.8, 28.0. HRMS calcd for C₉H₁₂N₂O₂:

181.0977 (M + H). Found m/z: 181.0981 (M + H).

5-(3-Hydroxy-1-propynyl)nicotinic Acid Ethyl Ester (13). To a

solution of 2e (250 mg, 1.09 mmol), Pd(PPh₃)₂Cl₂(38 mg, 0.0545

mmol), and DIPEA (423 mg, 3.27 mmol) in acetonitrile (5 mL) was

added propargyl alcohol (73 mg, 1.31 mmol) and CuI (15 mg, 0.0763

mmol) while stirring under N₂. After the addition was complete, the

reaction mixture was reﬂuxed for 16 h. At this time, TLC indicated

that the starting material was consumed. Solvents were removed in

vacuo and the residue partitioned between water and chloroform. The

phases were separated, and chloroform was concentrated in vacuo.

The resulting oil was puriﬁed by column chromatography on silica gel

(50% EtOAc−hexanes), aﬀording a yellow solid (160 mg, 71%) that

5-[3-(p-Methylbenzenesulfonyl)propyl]nicotinic Acid Ethyl

Ester (16). Compound 14 (107 mg, 0.51 mmol) was dissolved in dry

DCM (5 mL). To this solution was added p-methylbenzenesulfonyl

chloride (292 mg, 1.5 mmol) and TEA (114 mg, 1.12 mmol). The

mixture was reﬂuxed for 16 h. Upon TLC analysis in 50% EtOAc−

hexanes, starting material was still present. Additional p-toluenesul-

fonyl chloride (50 mg, 0.26 mmol) and TEA (15 mg, 0.15 mmol) were

added to the mixture and allowed to stir at reﬂux for an additional 12

h. This pushed the reaction to completion, and the product was

isolated by removal of solvents by distillation and the residue

partitioned between saturated sodium bicarbonate (50 mL) and DCM

(50 mL). The organic layer was further washed with brine (3×). The

phases were separated, and the DCM was evaporated in vacuo. The

crude material was puriﬁed by chromatography on silica gel in 50%

EtOAc−hexanes to obtain a colorless oil (94 mg, 51%). TLC R_f0.27

1

50% EtOAc−hexanes. H NMR (400 MHz, CDCl₃) δ 9.06 (s, 1H),

8.54 (s, 1H). 8.07 (s, 1H), 7.79 (d, 2H), 7.35 (d, 2H), 4.41 (q, 2H, J =

7.2 Hz), 4.06 (t, 2H, J = 6 Hz), 2.75 (t, 2H, J = 8 Hz), 2.46 (s, 3H),

2.00 (m, 2H), 1.42 (t, 3H, J = 7.2 Hz). ¹³C NMR (101 MHz, CDCl₃)

δ165.4, 153.6, 149.0, 145.2, 136.8, 135.8, 133.0, 130.11, 129.92, 128.0,

126.3, 125.3, 69.2, 61.7, 30.2, 28.7, 21.8, 14.4.

5-(3-Azidopropyl)nicotinic Acid Ethyl Ester (17). To a solution

of 16 (47 mg, 0.13 mmol) in dry DMF (2 mL) was added sodium

azide (17 mg, 0.26 mmol). The reaction mixture was stirred and

heated for 18 h at 60 °C. Upon completion, solvents were distilled

under reduced pressure and the residue puriﬁed by column

chromatography on silica gel in 50% EtOAc−hexanes, yielding a

yellow oil (22 mg, 73%). TLC R_f0.49 in 50% EtOAc−hexanes; R_f0.77

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in DCM−methanol−acetic acid (95:4:1). ¹H NMR (400 MHz,

CDCl₃) δ 9.08 (s, 1H), 8.62 (s, 1H), 8.13 (s, 1H), 4.42 (q, 2H, J = 7.2

Hz), 3.35 (t, 2H, J = 6.4 Hz), 2.79 (t, 2H, J = 8 Hz), 1.95 (m, 2H),

1.42 (t, 3H, J = 7.2 Hz). ¹³C NMR (101 MHz, CDCl₃) δ165.4, 153.5,

148.8, 136.7, 136.1, 126.1, 61.5, 50.4, 30.05, 29.69, 14.3. HRMS calcd

for C₁₁H₁₄N₄O₂: 257.1014 (M + Na). Found m/z: 257.1011 (M +

Na).

5-(3-Azidopropyl)nicotinic Acid (18). Compound 17 (50 mg,

0.21 mmol) was dissolved in methanol (1 mL) and 4 N NaOH (0.5

mL) and stirred for 3 h at 37 °C. Upon completion, solvents were

removed in vacuo and the residue was neutralized with 1 N HCl. The

sample was concentrated to a volume of 0.5 mL then applied to a C₁₈

cartridge (15.5 g), which was then attached to a Combiﬂash Rf system.

The product eluted into the second CV of an isocratic water system.

Water was removed by lyophilization to yield a white solid (39 mg,

91%); mp 77−78 °C. TLC R_f0.40 in DCM−methanol−acetic acid

(95:4:1); R_f0.81 in butanol−acetic acid−water (5:2:3). ¹H NMR (400

MHz, CD₃OD) δ 8.97 (s, 1H), 8.62 (s, 1H), 8.28 (s, 1H), 3.37 (t, 2H

J = 6.4 Hz), 2.83 (t, 2H, J = 7.6 Hz), 1.94 (m, 2H). ¹³C NMR (101

MHz, CD₃OD) δ 168.0, 153.8, 149.1, 139.1, 128.7, 51.8, 31.3, 30.7.

HRMS calcd for C₉H₁₀N₄O₂: 207.0882 (M + H). Found m/z:

207.0881 (M + H).

General Procedure for the Base-Exchange Reaction.

Substituted nicotinic acid (10−20 equiv) was dissolved in water (6

mL) and stirred for 30 min at 37 °C. After this time period, the pH

was adjusted to 4 with 0.5 M NaOH and stirred for 30 min. The pH

was measured again and readjusted to pH = 4 with 0.5 M NaOH, if

necessary. This procedure was repeated until the pH stabilized. After

the pH stabilized, NADP was added and stirred for 5 min at 37 °C.

Next, Aplysia californica ADP-ribosyl cyclase (180 μL, 36 μg) was

added and the reaction was stirred at 37 °C for 2 h. Upon time

completion, HPLC conﬁrmed consumption of starting material

(HPLC method 1: NADP t = 8.96 min, 13 mM TFA). The sample

was then injected onto the preparative HPLC column, and the product

was puriﬁed by anion-exchange chromatography according to method

3. After the product was collected, the combined fractions were added

to a 125 mL separatory funnel and extracted 3× with DCM (35 mL

portions) to remove TFA. The phases were separated, and the

aqueous layer was then distilled in vacuo at 25 mbar for 10 min to

remove any residual DCM and TFA. The pH of this resulting solution

was adjusted between 7.2 and 7.4 with 1 M NaOH. The aqueous

solution was allowed to freeze at −80 °C then lyophilized. This

substance was then dissolved in water (2 mL) and applied to a 3 mL

column of DEAE cellulose according to method 6. The DEAE

cellulose chromatography was developed by applying a 0−500 mM

NH₄HCO₃gradient and the product eluted into ca. 225 mM

NH₄HCO₃. The product showed UV absorbance at 254 nm; fractions

absorbing at this wavelength were collected and combined. The

combined fractions were then lyophilized until a foamy white residue

appeared. The sample was redissolved in water (2 mL) and

lyophilized. This process was repeated twice more, aﬀording pure

substituted NAADP derivative.

Preparation of NAADP Analogues. NAADP (1). Nicotinic acid

(40 mg, 0.325 mmol) was suspended in water (6 mL) and stirred for

30 min at 37 °C. After this time, the pH was adjusted to 4 with 0.5 M

NaOH and stirred for 30 min. The pH was measured again and

readjusted to pH = 4 with 0.5 N NaOH, if necessary. This procedure

was repeated until the pH stabilized. After the pH stabilized, NADP

(20 mg, 0.024 mmol) was added and stirred for 5 min at 37 °C. Next,

Aplysia ADP-ribosyl cyclase (180 μL, 36 μg) was added and the

reaction was stirred at 37 °C for 2 h. After 2 h, analysis by HPLC

conﬁrmed consumption of starting material (NADP). The sample was

then injected onto the preparative HPLC column, and the product was

puriﬁed by anion exchange chromatography in water−TFA gradients

according to HPLC method 3. NAADP eluted between 22.85 and

28.40 min (27−36%). The combined aqueous fractions were added to

a 125 mL separatory funnel and extracted 3 times with DCM (35 mL

portions). The phases were separated and the aqueous layer was

distilled in vacuo at 25 mbar for 10 min to remove residual DCM and

TFA. The pH of the resulting solution was adjusted to between 7.2

and 7.4 with 1 M NaOH. The aqueous solution was frozen then

lyophilized, producing a white, amorphous solid. The sample was

dissolved in water (2 mL) and applied to a 3 mL column of DEAE

cellulose according to method 6. The product 1 eluted into 225 mM

NH₄HCO₃. The fractions showing UV absorption at 254 nm were

combined and lyophilized. The sample was redissolved in water (2

mL) and again lyophilized, aﬀording pure NAADP as a white powder.

¹H NMR (400 MHz, D₂O) δ 9.12 (s, 1H), 9.01 (s, 1H), 8.75 (s, 1H),

8.44 (s, 1H), 8.14 (s, 1H), 8.07 (s, 1H), 6.18 (s, 1H), 6.04 (s, 1H),

5.12 (s, 1H), 4.64 (s, 1H), 4.5 (s, 1H), 4.41 (s, 3H), 4.32- 4.21 (m,

+

4H). MALDI-TOF MS calcd for C₂₁H₂₈N₆O₁₈P₃: 745.067. Found m/

z: 744.897 (M+). HPLC method 1: t = 12.80, 23 mM TFA. Method 7:

product eluted into 300 mM NH₄HCO₃.

8-N₃-NAD (22). NMN (15 mg, 0.045 mmol) and 21 (63 mg, 0.13

mmol) were dissolved in a mixed solvent system of H₂O (1 mL) and

pyridine (5 mL). Diisopropylcarbodiimide (DIC; 0.23 mL × 5) was

added in ﬁve portions over 5 d. The mixture was stirred at rt. Twenty-

four h after the last addition of DIC, the mixture was poured into H₂O

(25 mL) and the mixture was stirred for 2 h. Precipitates were ﬁltered

oﬀ, and the ﬁltrate was lyophilized. The solid residue was dissolved in

H₂O (20 mL) and was injected onto the preparative HPLC column,

and the product was puriﬁed by anion exchange chromatography in

H₂O−TFA gradients according to HPLC method 5. 22 eluted

between 15.76 and 17.23 min (2% 100 mM TFA). The combined

aqueous fractions were added to a 125 mL separatory funnel and

extracted 3 times with DCM (35 mL portions). The phases were

separated, and the aqueous layer was distilled in vacuo at 25 mbar for

10 min to remove residual DCM and TFA. The pH of the resulting

solution was adjusted to between 7.2 and 7.4 with 1 M NaOH. The

aqueous solution was frozen and lyophilized, producing a white,

amorphous solid. The amorphous solid was dissolved in H₂O (2 mL)

and applied to a 3 mL column of DEAE cellulose and puriﬁed

according to method 6. The product 22 eluted into 50 mM

NH₄HCO₃. The fractions showing UV absorption at 254 nm were

combined and lyophilized. The sample was redissolved and again

lyophilized, aﬀording pure 22 as a cream-colored powder (3.6 mg,

11%). ¹H NMR (400 MHz, D₂O) δ 9.11 (s, 1H), 8.91 (d, 1H, J = 6.4

Hz), 8.63 (d, 1H, J = 8 Hz), 8.00 (t, 1H, J = 8 Hz), 5.91 (d, 1H, J = 4.4

Hz), 5.67 (d, 1H, J = 5.2 Hz), 4.80−4.08 (m, 10H). ³¹P NMR (162

MHz, D₂O) δ −10.70. HPLC method 2: t = 8.69 min, 2 mM TFA.

8-N₃-NADP (23). 8-N₃-NAD (22) was converted to 8-N₃-NADP

(23) enzymatically using NAD kinase. 8-N₃-NAD (22) (13.7 mg, 19.5

μmol) was dissolved in 14 mL of 20 mM HEPES, pH 7.3, 3 mM ATP,

5 mM MgCl₂, 2.5 mM creatine phosphate, and 0.15 mg/mL creatine

kinase. The reaction was initiated by addition of 20 μL of 1 mg/mL

human NAD kinase (Axxora) and was allowed to proceed at rt in the

dark. After 1 week, the reaction had proceeded to 69% completion.

The resulting 8-N₃-NADP was puriﬁed by chromatography on a 1.5

cm × 15 cm AG MP-1 column using an HPLC system consisting of a

Beckman 128 solvent module and a 166 UV detector. The 8-N₃-

NADP was eluted at a ﬂow rate of 3 mL/min with a concave upward

gradient of TFA from 1.5 to 150 mM over 50 min, 8-N₃-NAD eluted

from 15 to 19 min, and 8-N₃-NADP from 31 to 35 min. Then 3 mL of

the reaction was injected each time and the 8-N₃-NADP peak was

pooled from each run. The TFA was extracted from the puriﬁed 8-N₃-

NADP by treating the pooled fractions (40 mL) with 10 mL of a 3:1

mixture of 1,1,2-trichlorotriﬂuoroethane/tri-N-octylamine.⁵⁷The

resulting aqueous layer was dried in a Savant Speed-Vac concentrator

as 10 mL aliquots. A total of 6.4 mg (4.3 μmol) of pure 23 as the

octylamine salt was isolated, representing a 22% yield. The 8-N₃-NAD

peak was also collected and processed in a similar manner and the

NAD kinase reaction repeated as described above to isolate more 8-

N₃-NADP. ¹H NMR (600 MHz, D₂O) δ 9.34 (s, 1H), 9.18 (d, 1H, J =

6.3 Hz), 8.87 (d, 1H, J = 6.7 Hz), 8.23 (m, 2H), 6.09 (d, 1H, J = 5.52

Hz), 6.02 (d, 1H, J = 5.16 Hz), 5.34 (m, 1H), 4.66 (t, 1H, J = 5.22

Hz), 4.54 (m, 1H), 4.48 (t, 1H, J = 5.22 Hz), 4.43 (m, 1H), 4.35 (m,

1H), 4.29−4.19 (m, 4H). ³¹P NMR (162 MHz, D₂O) δ 0.61, −10.78.

+

HRMS calcd for C₂₁H₂₈N₁₀O₁₇P₃: 785.084. Found m/z: 785.142 (M

+).

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8-Br-NADP (24). To a stirred solution of NADP (100 mg, 0.12

mmol) in 0.5 M sodium acetate (1.5 mL, pH = 4.75) was added Br₂

(0.2 mL, 4 mmol). The reaction was stirred vigorously using a

magnetic stirrer for 3 h while wrapped in aluminum foil to protect

from light. Upon completion, the mixture was diluted with water (8.5

mL) then DCM (20 mL) was added. The reaction mixture was

transferred to a separatory funnel, and the organic layer was discarded.

The aqueous layer was further extracted 5 times with DCM (20 mL).

The pH was adjusted to 7 with 4 M NaOH and was concentrated in

vacuo until the total volume was ca. 10 mL. The crude product was

puriﬁed by anion exchange chromatography according to HPLC

method 4 in 5 mL portions. 8-Br-NADP eluted between 29.44 and

37.39 min (11−15 mM TFA). Fractions exhibiting UV absorption at

254 nm were combined and extracted 3 times with DCM (10 mL).

The aqueous layer was distilled in vacuo at 25 mbar for 10 min to

remove residual DCM and TFA. The resulting solution was

neutralized with 0.5 M NaOH then lyophilized. The amorphous

solid was dissolved in water (2 mL) and applied to a 3 mL column of

DEAE cellulose according to method 6. The product 30 eluted into

175 mM NH₄HCO₃. The fractions showing UV absorption at 254 nm

were combined and lyophilized. The sample was redissolved and again

lyophilized, aﬀording pure 8-Br-NADP as a yellowish-white solid (56

puriﬁed by anion exchange chromatography in water−TFA gradients

according to HPLC method 3. 5-Hydroxy-NAADP eluted between

25.60 and 29.84 min (32−38 mM TFA). The combined aqueous

fractions were added to a 125 mL separatory funnel and extracted 3

times with DCM (35 mL portions). The phases separated, and the

aqueous layer was distilled in vacuo at 25 mbar for 10 min to remove

residual DCM and TFA. The pH of this solution was adjusted to

between 7.2 and 7.4 with 1 M NaOH. The aqueous solution was

frozen and lyophilized, producing a white, amorphous solid. The

amorphous solid was dissolved in water (2 mL) and applied to a 3 mL

column of DEAE cellulose and puriﬁed according to method 6. The

product 26 eluted into 240 mM NH₄HCO₃. The fractions showing

UV absorption at 254 nm were combined and lyophilized. The sample

was redissolved and again lyophilized, aﬀording pure 5-hydroxy-

1

NAADP as a white powder (9.55 mg, 63%). H NMR (400 MHz,

D₂O) δ 8.43−8.39 (d, 3H), 8.13 (s, 1H), 7.96 (s, 1H), 6.12 (d, 1H, J =

4.8 Hz), 5.82 (s, 1H, J = 4.8 Hz), 5.01 (m, 1H), 4.58 (t, 1H), 4.39 (s,

1H), 4.34−4.15 (6H). ³¹P NMR (162 MHz, D₂O) δ 0.91, −10.8.

+

MALDI-TOF MS calcd for C₂₁H₂₈N₆O₁₉P₃: 761.062. Found m/z:

761.153 (M+). HPLC method 1: t = 14.13 min, 26 mM TFA. Method

7: 350 mM NH₄HCO₃.

5-(3-Acetamidopropyl)-NAADP (27). 5-(3-Acetamidopropyl)-

nicotinic acid (6) (37.5 mg, 0.169 mmol) was suspended in water

(6 mL) and stirred for 30 min at 37 °C. The pH initially was 8.4 and

was adjusted to 4 with 1 N HCl and stirred for 30 min. The pH was

measured again and readjusted to pH = 4 with 1 N HCl. After this

time, the pH stabilized. NADP (15 mg, 0.018 mmol) was added and

stirred for 5 min at 37 °C. Next, Aplysia ADP-ribosyl cyclase (180 μL,

36 μg) was added and the reaction was stirred at 37 °C for 5 h. At the

end of 5 h, analysis by HPLC conﬁrmed that the starting material

(NADP) had been consumed. The sample was then injected onto the

preparative HPLC column, and the product was puriﬁed by anion

exchange chromatography in water−TFA gradients according to

HPLC method 3. 5-(3-Acetamidoaminopropyl)-NAADP eluted

between 21.87 and 26.24 min (26−33 mM TFA). The combined

aqueous fractions were added to a 125 mL separatory funnel and

extracted 3 times with DCM (35 mL portions). The phases separated,

and the aqueous layer was distilled in vacuo at 25 mbar for 10 min to

remove residual DCM and TFA. The pH of the resulting solution was

then adjusted to between 7.2 and 7.4 with 1 M NaOH. The aqueous

solution was frozen and lyophilized, producing a white, amorphous

solid. The amorphous solid was dissolved in water (2 mL) and applied

to a 3 mL column of DEAE cellulose and puriﬁed according to method

6. The product 27 eluted into 225 mM NH₄HCO₃. The fractions

showing UV absorption at 254 nm were combined and lyophilized.

The sample was redissolved and again lyophilized, aﬀording pure 5-(3-

1

mg, 51%). H NMR (400 MHz, D₂O) δ 9.37 (s, 1H), 9.18 (s, 1H),

8.94 (d, 1H, J = 7.6 Hz), 8.31 (s, 1H), 8.20 (s, 1H), 6.21 (s, 1H), 6.11

(s, 1H), 5.53 (s, 1H), 4.5−4.22 (9H). ³¹P NMR (162 MHz, D₂O)

+

2.09, −10.79. MALDI-TOF MS calcd for C₂₁H₂₇BrN₆O₁₈P₃: 821.994.

Found m/z: 821.992 (M+). HPLC method 2: t = 22.72 min, 7 mM

TFA.

5-(Acetamido)-NAADP (25). 5-(Acetamido)nicotinic acid (2c) (35

mg, 0.194 mmol) was suspended in water (6 mL) and stirred for 30

min at 37 °C. After this time, the pH was adjusted to 4 with 0.5 M

NaOH and stirred for 30 min. The pH was measured again and

readjusted to pH = 4 with 0.5 M NaOH, if necessary. This procedure

was repeated until the pH has stabilized. After the pH stabilized,

NADP (15 mg, 0.018 mmol) was added and stirred for 5 min at 37 °C.

Next, Aplysia ADP-ribosyl cyclase (180 μL, 36 μg) was added and the

reaction was stirred at 37 °C for 4 h. At the end of 4 h, analysis by

HPLC conﬁrmed that the starting material (NADP) had been

consumed. The sample was injected onto the preparative HPLC

column, and the product was puriﬁed by anion exchange

chromatography in water−TFA gradients according to HPLC method

3. 5-(Acetamido)-NAADP eluted between 24.12 and 27.91 min (31−

37 mM TFA). The combined aqueous fractions were added to a 125

mL separatory funnel and extracted 3 times with DCM (35 mL

portions). The phases separated, and the aqueous layer was distilled in

vacuo at 25 mbar for 10 min to remove residual DCM and TFA. The

pH of the resulting solution was adjusted to between 7.2 and 7.4 with

1 M NaOH. The aqueous solution was frozen and lyophilized,

producing a white, amorphous solid. The amorphous solid was

dissolved in water (2 mL) and applied to a 3 mL column of DEAE

cellulose and puriﬁed according to method 6. The product 25 eluted

into 225 mM NH₄HCO₃. The fractions showing UV absorption at 254

nm were combined and lyophilized. The sample was redissolved and

again lyophilized, aﬀording pure 5-(acetamido)-NAADP as a white

1

acetamidopropyl)-NAADP as an oﬀ-white powder (9.7 mg, 58%). H

NMR (400 MHz, D₂O) δ 9.28 (s, 1H), 9.12 (s, 1H), 8.89 (s, 1H), 8.58

(s, 1H), 8.40 (s, 1H), 6.26 (d, 1H, J = 4.8 Hz), 6.11 (d, 1H, J = 4.8

Hz), 5.09 (m, 2H), 4.63−4.23 (9H), 3.18 (t, 2H, J = 6.4 Hz), 2.97 (t,

2H, J = 7.6 Hz), 1.93 (s, 5H). ³¹P NMR (162 MHz, D₂O) δ 0.39,

+

−10.68. MALDI-TOF MS calcd for C₂₆H₃₇N₇O₁₉P₃: 844.136. Found

m/z: 844.217 (M+). HPLC method 1: t = 12.45 min, 22 mM TFA.

5-(3-t-Boc-Aminopropyl)-NAADP (28). 5-(3-t-Boc-aminopropyl)-

nicotinic acid (11) (35 mg, 0.125 mmol) was suspended in water (5

mL) and DMSO (1 mL) and stirred for 30 min at 37 °C. After this

time, the pH was adjusted to 4 with 0.5 M NaOH and stirred for 30

min. The pH was measured again and readjusted to pH = 4 with 0.5 M

NaOH, if necessary. This procedure was repeated until the pH

stabilized. After the pH stabilized, NADP (15 mg, 0.018 mmol) was

added and stirred for 5 min at 37 °C. Next, Aplysia ADP-ribosyl

cyclase (180 μL, 36 μg) was added and the reaction was stirred at 37

°C for 6 h. At the end of 6 h, analysis by HPLC conﬁrmed that the

starting material (NADP) had been consumed. The sample was then

injected onto the preparative HPLC column, and the product was

puriﬁed by anion exchange chromatography in water−TFA gradients

according to HPLC method 3. 5-(3-t-Boc-aminopropyl)-NAADP

eluted between 22.57 and 27.75 min (27−34 mM TFA). The

combined aqueous fractions were added to a 125 mL separatory funnel

and extracted 3 times with DCM (35 mL portions). The phases

1

powder (7.45 mg, 47%). H NMR (400 MHz, D₂O) δ 9.24 (s, 1H),

9.16 (s, 1H), 8.88 (s, 1H), 8.47 (s, 1H), 8.36 (s, 1H), 6.18 (s,1H), 6.12

(s, 1H), 5.06 (s, 1H), 4.59 (s, 1H), 4.52 (s, 1H), 4.42−4.23 (7H), 2.18

(s, 3H). ³¹P NMR (162 MHz, D₂O) δ 0.35, −10.56. HPLC method 1:

t = 13.68 min, 25 mM TFA. Method 7: 340 mM NH₄HCO₃.

5-Hydroxy-NAADP (26). 5-Hydroxynicotinic acid (2a)(35 mg,

0.252 mmol) was suspended in water (6 mL) and stirred for 30 min

at 37 °C. After this time, the pH was adjusted to 4 with 0.5 M NaOH

and stirred for 30 min. The pH was measured again and readjusted to

pH = 4 with 0.5 M NaOH. This procedure was repeated until the pH

stabilized. After the pH stabilized, NADP (15 mg, 0.018 mmol) was

added and stirred for 5 min at 37 °C. Next, Aplysia ADP-ribosyl

cyclase (180 μL, 36 μg) was added and the reaction was stirred at 37

°C for 2 h. At the end of 2 h, analysis by HPLC conﬁrmed that the

starting material (NADP) had been consumed. The sample was then

injected onto the preparative HPLC column, and the product was

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Article

separated, and the aqueous layer was distilled in vacuo at 25 mbar for

10 min to remove residual DCM and TFA. The pH of the resulting

solution was adjusted to between 7.2 and 7.4 with 1 M NaOH. The

aqueous solution was frozen and lyophilized, producing a white,

amorphous solid. The amorphous solid was dissolved in water (2 mL)

and applied to a 3 mL column of DEAE cellulose and puriﬁed

according to method 6. The product 28 eluted into 215 mM

NH₄HCO₃. The fractions showing UV absorption at 254 nm were

combined and lyophilized. The sample was redissolved and again

lyophilized, aﬀording pure 5-(3-t-Boc-aminopropyl)-NAADP as a

white solid (7.6, 42%). ¹H NMR (400 MHz, D₂O) δ 8.96 (s, 1H), 8.79

(s, 1H), 8.62 (s, 1H), 8.42 (s, 1H), 8.09 (s, 1H), 6.11 (s, 1H), 5.96 (s,

1H), 4.60−4.10 (10H), 3.06 (s, 2H), 2.79 (s, 2H), 1.79 (s, 2H), 1.34

(s, 9H). ³¹P NMR (162 MHz, D₂O) δ 0.44, −10.39. MALDI-TOF MS

dissolved in water (2 mL) and applied to a 3 mL column of DEAE

cellulose and puriﬁed according to method 6. The product (30) eluted

into 225 mM NH₄HCO₃. The fractions showing UV absorption at 254

nm were combined and lyophilized. The sample was redissolved and

again lyophilized, aﬀording pure 5-(3-hydroxypropyl)-NAADP as a

1

white powder (9.5 mg, 59%). H NMR (400 MHz, D₂O) δ 8.96 (s,

1H), 8.82 (s, 1H), 8.65 (s, 1H), 8.42 (s, 1H), 8.14 (s, 1H), 6.13 (d,

1H, J = 5.6 Hz), 5.96 (d, 1H, J = 4.4 Hz), 5.03 (m, 2H), 4.6 (m, 1H),

4.45 (s, 1H), 4.38−4.18 (m, 6H), 3.59 (t, 2H, J = 6.4 Hz), 2.86 (t, 2H,

J = 8.4 Hz), 1.86 (m, 2H). ³¹P NMR (162 MHz, D₂O) δ 0.55,

+

−10.64). MALDI-TOF MS calcd for C₂₄H₃₄N₆O₁₉P₃: 803.109.

Found m/z 803.214 (M+). HPLC method 1: t = 12.70 min, 23

mM aqTFA. Method 7: 310 mM NH₄HCO₃.

5-(3-Azidopropyl)-NAADP (31). 5-(3-Azidopropyl)nicotinic acid

(18) (35 mg, 0.170 mmol) was suspended in water (5 mL) and

DMSO (1 mL) and stirred for 30 min at 37 °C. After this time, the pH

was adjusted to 4 with 0.5 M NaOH and stirred for 30 min. The pH

was measured again and readjusted to pH = 4 with 0.5 M NaOH, if

necessary. This procedure was repeated until the pH stabilized. After

the pH stabilized, NADP (15 mg, 0.018 mmol) was added and stirred

for 5 min at 37 °C. Next, Aplysia ADP-ribosyl cyclase (120 μL, 12 μg)

was added and the reaction was stirred at 37 °C for 5 h. At the end of

5 h, analysis by HPLC conﬁrmed that the starting material (NADP)

had been consumed. The sample was injected onto the preparative

HPLC column, and the product was puriﬁed by anion exchange

chromatography in water−TFA gradients according to HPLC method

3. 5-(3-Azidopropyl)-NAADP eluted between 23.46 and 27.03 min

(30−35 mM TFA). The combined aqueous fractions were added to a

125 mL separatory funnel and extracted 3 times with DCM (35 mL

portions). The phases separated, and the aqueous layer was distilled in

vacuo at 25 mbar for 10 min to remove residual DCM and TFA. The

pH of the resulting solution was adjusted to between 7.2 and 7.4 with

1 M NaOH. The aqueous solution was frozen and lyophilized,

producing a white, amorphous solid. The amorphous solid was

dissolved in water (2 mL) and applied to a 3 mL column of DEAE

cellulose and puriﬁed according to method 6. The product 31 eluted

into 225 mM NH₄HCO₃. The fractions showing UV absorption at 254

nm were combined and lyophilized. The sample was redissolved and

again lyophilized, aﬀording pure 5-(3-azidopropyl)-NAADP as a white

+

calcd for C₂₉H₄₃N₇O₂₀P₃: 902.178. Found m/z: 902.278 (M+).

HPLC method 1: t = 12.75 min, 23 mM TFA.

5-(3-Aminopropyl)-NAADP (29). 5-(3-Aminopropyl)nicotinic acid

(12) (35 mg, 0.194 mmol) was dissolved in water (6 mL) and stirred

for 30 min at 37 °C. After this time, the pH was adjusted to 4 with 1 M

HCl and stirred for 30 min. The pH was measured again and

readjusted to pH = 4 with 1 M HCl. This procedure was repeated until

the pH stabilized. After the pH stabilized, NADP (15 mg, 0.018

mmol) was added and stirred for 5 min at 37 °C. Next, Aplysia ADP-

ribosyl cyclase (180 μL, 36 μg) was added and the reaction was stirred

at 37 °C for 6 h. At the end of 6 h, analysis by HPLC conﬁrmed that

the starting material (NADP) had been consumed. The sample was

then injected onto the preparative HPLC column, and the product was

puriﬁed by anion exchange chromatography in water−TFA gradients

according to HPLC method 3. 5-(3-Aminopropyl)-NAADP eluted

between 12.83 and 15.44 min (12−16 mM TFA). The combined

aqueous fractions were added to a 125 mL separatory funnel and

extracted 3 times with DCM (35 mL portions). The phases separated,

and the aqueous layer was distilled in vacuo at 25 mbar for 10 min to

remove residual DCM and TFA. The pH of the resulting solution was

adjusted to between 7.2 and 7.4 with 1 M NaOH. The aqueous

solution was frozen and lyophilized, producing a white, amorphous

solid. The amorphous solid was dissolved in water (2 mL) and applied

to a 3 mL column of DEAE cellulose and puriﬁed according to method

6. The product 29 eluted into 140 mM NH₄HCO₃. The fractions

showing UV absorption at 254 nm were combined and lyophilized.

The sample was redissolved and again lyophilized, aﬀording pure 5-(3-

1

powder (8.5 mg, 52%). H NMR (400 MHz, D₂O) δ 9.30 (s, 1H),

9.12 (s, 1H), 8.91 (s, 1H), 8.57 (s,1H), 8.38 (s, 1H), 6.25 (d, 1H, J =

5.6 Hz), 6.10 (d, 1H, J = 5.2 Hz), 5.08 (s, 1H), 4.62 (s, 1H), 4.56 (s,

1H), 4.53−4.23 (m, 7H), 3.38 (t, 2H, J = 6.4 Hz), 3.02 (t, 2H, J = 7.2

Hz), 1.98 (m, 2H). ³¹P NMR (162 MHz, D₂O) δ 0.39, −10.40.

1

aminopropyl)-NAADP as a white powder (10.5 mg, 66%). H NMR

(400 MHz, D₂O) δ 8.94 (s, 1H), 8.80 (s, 1H), 8.60 (s, 1H), 8.34 (s,

1H), 8.06 (s, 1H), 6.06 (d, 1H, J = 4.8 Hz), 5.96 (d, 1H, J = 4 Hz),

4.89 (partially buried in HDO peak, 1H), 4.54 (m, 1H), 4.44 (s, 1H),

4.32−4.13 (6H), 3.02 (t, 2H, J = 6.8 Hz), 2.84 (t, 2H, J = 7.6 Hz), 1.97

(m, 2H). ³¹P NMR (162 MHz, D₂O) δ 4.22, −10.82. MALDI-TOF

+

MALDI-TOF MS calcd for C₂₄H₃₃N₉O₁₈P₃: 828.116. Found m/z:

828.214 (M+). HPLC method 1: t = 13.17 min, 24 mM TFA.

5-Thiomethyl-NAADP (32). 5-Thiomethylnicotinic acid (2d) (35

mg, 0.207 mmol) was suspended in water (5 mL) and DMSO (1 mL)

and stirred for 30 min at 37 °C. After this time, the pH was adjusted to

4 with 0.5 M NaOH and stirred for 30 min. The pH was measured

again and readjusted to pH = 4 with 0.5 M NaOH, if necessary. This

procedure was repeated until the pH stabilized. After the pH stabilized,

NADP (15 mg, 0.018 mmol) was added and stirred for 5 min at 37 °C.

Next, Aplysia ADP-ribosyl cyclase (120 μL, 24 μg) was added and the

reaction was stirred at 37 °C for 2 h. At the end of 2 h, analysis by

HPLC conﬁrmed that the starting material (NADP) had been

consumed. The sample was then injected onto the preparative HPLC

column, and the product was puriﬁed by anion exchange

chromatography in water−TFA gradients according to HPLC method

3. 5-Thiomethyl-NAADP eluted between 23.60 and 27.89 min (29−35

mM TFA). The combined aqueous fractions were added to a 125 mL

separatory funnel and extracted 3 times with DCM (35 mL portions).

The phases separated, and the aqueous layer was distilled in vacuo at

25 mbar for 10 min to remove residual DCM and TFA. The pH of the

resulting solution was adjusted to between 7.2 and 7.4 with 1 M

NaOH. The aqueous solution was frozen and lyophilized, producing a

white, amorphous solid. The amorphous solid was dissolved in water

(2 mL) and applied to a 3 mL column of DEAE cellulose and puriﬁed

according to method 6. The product 32 eluted into 225 mM

NH₄HCO₃. The fractions showing UV absorption at 254 nm were

+

MS calcd for C₂₄H₃₅N₇O₁₈P₃: 802.125. Found m/z 802.182 (M+).

HPLC method 1: t = 6.93 min, 11 mM aq TFA.

5-(3-Hydroxypropyl)-NAADP (30). 5-(3-Hydroxypropyl)nicotinic

acid (15) (35 mg, 0.193 mmol) was suspended in water (6 mL)

and stirred for 30 min at 37 °C. After this time, the pH was adjusted to

4 with 0.5 M NaOH and stirred for 30 min. The pH was measured

again and readjusted to pH = 4 with 0.5 M NaOH, if necessary. This

procedure was repeated until the pH stabilized. After the pH stabilized,

NADP (15 mg, 0.018 mmol) was added and stirred for 5 min at 37 °C.

Next, Aplysia ADP-ribosyl cyclase (180 μL, 36 μg) was added and the

reaction was stirred at 37 °C for 4 h. At the end of 4 h, analysis by

HPLC conﬁrmed that the starting material (NADP) had been

consumed. The sample was then injected onto the preparative HPLC

column, and the product was puriﬁed by anion exchange

chromatography in water−TFA gradients according to HPLC method

3. 5-(3-Hydroxypropyl)-NAADP eluted between 21.73 and 26.53 min

(26−34 mM aqTFA). The combined aqueous fractions were added to

a 125 mL separatory funnel and extracted 3 times with DCM (35 mL

portions). The phases separated, and the aqueous layer was distilled in

vacuo at 25 mbar for 10 min to remove residual DCM and TFA. The

pH of the resulting solution was adjusted to between 7.2 and 7.4 with

1 M NaOH. The aqueous solution was frozen and lyophilized,

producing a white, amorphous solid. The amorphous solid was

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combined and lyophilized. The sample was redissolved and again

lyophilized, aﬀording pure 5-thiomethoxy-NAADP as a white powder

(8 mg, 51%). ¹H NMR (400 MHz, D₂O) δ 9.08 (s, 1H), 8.88 (s, 1H),

8.72 (s, 1H), 8.57 (s, 1H), 8.40 (s, 1H), 6.25 (s, 1H), 6.07 (s, 1H),

5.08 (s, 1H), 4.62−4.20 (9H), 2.65 (s, 3H). ³¹P NMR (162 MHz,

D₂O) δ 0.31, −10.72. MALDI-TOF MS calcd for C₂₂H₃₀N₆O₁₈P₃S⁺:

791.055. Found m/z: 791.139 (M+). HPLC method 1: t = 13.44 min,

25 mM TFA.

was then injected onto the preparative HPLC column, and the product

was puriﬁed by anion exchange chromatography in H₂O−TFA

gradients according to HPLC method 3. 35 eluted between 31.04

and 35.76 min (30−37 mM TFA). The combined aqueous fractions

were added to a 125 mL separatory funnel and extracted 3 times with

DCM (35 mL portions). The phases separated, and the aqueous layer

was distilled in vacuo at 25 mbar for 10 min to remove residual DCM

and TFA. The pH of the resulting solution was adjusted to between

7.2 and 7.4 with 1 M NaOH. The aqueous solution was frozen and

lyophilized, producing a white, amorphous solid. The amorphous solid

was dissolved in water (2 mL) and applied to a 3 mL column of DEAE

cellulose and puriﬁed according to method 6. The product 35 eluted

into 225 mM NH₄HCO₃. The fractions showing UV absorption at 254

nm were combined and lyophilized. The sample was redissolved and

again lyophilized, aﬀording pure 35 as a light-yellow powder (5 mg,

35%). ¹H NMR (600 MHz, D₂O) δ 8.88 (s, 1H), 8.64 (s, 1H), 8.57 (s,

1H), 8.15 (s, 1H), 6.13 (d, 1H, J = 4.4 Hz), 5.89 (d, 1H, J = 4.2 Hz),

5.39 (m, 1H), 4.41−4.19 (m, 9H). ³¹P NMR (162 MHz, D₂O) δ 5.00,

8-Bromo-NAADP (33). Nicotinic acid (35 mg, 0.207 mmol) was

dissolved in water (5 mL) and DMSO (1 mL) and stirred for 30 min

at 37 °C. After this time, the pH was adjusted to 4 with 0.5 M NaOH

and stirred for 30 min. The pH was measured again and readjusted to

pH 4 with 0.5 M NaOH, if necessary. This procedure was repeated

until the pH has stabilized. After the pH has stabilized, 8-Br-NADP

(15 mg, 0.018 mmol) was added and stirred for 5 min at 37 °C. Next,

Aplysia ADP-ribosyl cyclase (180 μL, 36 μg) was added and the

reaction was stirred at 37 °C for 2 h. At the end of 2 h, analysis by

HPLC conﬁrmed that the starting material 24 had been consumed.

The sample was then injected onto the preparative HPLC column, and

the product was puriﬁed by anion exchange chromatography in water−

TFA gradients according to HPLC method 3. 8-Br-NAADP eluted

between 23.00 and 27.97 min (28−35 mM TFA). The combined

aqueous fractions were added to a 125 mL separatory funnel and

extracted 3 times with DCM (35 mL portions). The phases separated,

and the aqueous layer was distilled in vacuo at 25 mbar for 10 min to

remove residual DCM and TFA. The pH of the resulting solution was

adjusted to between 7.2 and 7.4 with 1 M NaOH. The aqueous

solution was frozen and lyophilized, producing a white, amorphous

solid. The amorphous solid was dissolved in water (2 mL) and applied

to a 3 mL column of DEAE cellulose and puriﬁed according to method

6. The product 33 eluted into 220 mM NH₄HCO₃. The fractions

showing UV absorption at 254 nm were combined and lyophilized.

The sample was redissolved and again lyophilized, aﬀording pure 8-Br-

+

−10.39. MALDI-TOF MS calcd for C₂₁H₂₆BrN₉O₁₈P₃: 863.979.

Found m/z: 863.973 (M+). HPLC method 1: t = 15.04 min, 29 mM

TFA. Method 7: 300 mM NH₄HCO₃.

Biological Assays. All compounds tested (1, 25−35) were

repuriﬁed by preparative anion exchange chromatography immediately

prior to their bioassay and the purity of the collected fractions veriﬁed

by analytical anion exchange HPLC (AG MP-1). The purity of the

tested compounds was in all cases ≥95% (see Supporting

Information).

Ca²⁺Release. Testing of NAADP derivatives for Ca²⁺release on

cell free receptor systems were performed on homogenates (1.25% v/

v) prepared from sea urchin eggs (Strongylocentrotus purpuratus)

diluted with intracellular medium containing 250 mM potassium

gluconate buﬀer (pH 7.2), 0.5 mM ATP, 4 mM creatinine phosphate,

creatinine kinase, and 3 μM ﬂuorescent indicator, ﬂuo-3. The dilutions

and all experiments were conducted at 17 °C. Fluo-3 is a calcium

chelating indicator which is nonﬂuorescent in absence of Ca²⁺ions but

after binding Ca²⁺it emits ﬂuorescence. This ﬂuorescence can be

measured using a ﬂuorescence plate reader (excitation 490 nm and

emission 535 nm, suitable to avoid interference from reduced pyridine

nucleotide).⁵⁸

1

NAADP as a yellowish-white powder (8 mg, 51%). H NMR (400

MHz, D₂O) δ 9.22 (s, 1H), 9.01 (d, 1H, J = 6.4 Hz), 8.89 (d, 1H, J = 8

Hz), 8.25 (s, 1H), 8.18 (m, 1H), 6.21 (d, 1H, J = 4 Hz), 6.04 (d, 1H, J

= 5.2 Hz), 5.49 (m, 1H), 4.91 (m,1H), 4.49−4.31 (8H). ³¹P NMR

(162 MHz, D₂O) δ 5.17, −9.91. MALDI-TOF MS calcd for

+

C₂₁H₂₇BrN₆O₁₈P₃: 822.978. Found m/z: 823.208 (M+). HPLC

Receptor Desensitization. Subthreshold concentrations of tested

analogues were added to a standard Ca²⁺release assay and incubated

for the speciﬁed time before the addition of saturating concentrations

of NAADP.

Method 1: t = 13.47 min, 25 mM TFA.

8-Azido-NAADP (34). First, 2.6 mg (1.74 μmol) of 8-N₃-NADP

(23) trioctylammonium salt was dissolved in 2.2 mL water. The

solution was adjusted to 100 mM sodium acetate by adding 0.8 mL of

500 mM sodium acetate, pH 4.0. Nicotinic acid (1 mL of 200 mM)

was added, and the base-exchange reaction was started by adding 5 μL

of 200 μg/mL Aplysia ADP-ribosyl cyclase and allowed to incubate at

room temperature in the dark. The reaction was monitored by

analyzing 50 μL of reaction on the analytical chromatography system.

The reaction was complete at 6 h as judged by the disappearance of 8-

N₃-NADP. The 8-N₃-NAADP was puriﬁed using the same system as

described for 8-N₃-NADP (23) after adjusting the pH of the reaction

to 6 by adding 200 μL of 2 M Tris-base. 8-N₃-NAADP eluted between

41 and 45 min. The puriﬁed 8-N₃-NAADP was processed as described

for 8-N₃-NADP (23), resulting in the isolation of 1.2 mg (0.65 μmol;

37% yield) of 34 isolated as the trioctylammonium salt. ¹H NMR (600

MHz, D₂O) δ 8.99 (s, 1H), 8.93 (d, 1H, J = 6.6 Hz), 8.63 (d, 1H, J =

8.16 Hz) 8.06 (s, 1H), 7.99 (t, 1H, J = 7.02 Hz), 5.93 (d, 1H, J = 4.98

Hz), 5.90 (d, 1H, J = 5.34 Hz), 5.23 (m, 1H), 4.29−4.12 (m, 9H). ³¹P

NMR (162 MHz, D₂O) δ 0.59, −10.76. MALDI-TOF MS calcd for

Receptor Binding. The competitive binding studies were performed

according to our previously published procedures.⁵⁸The binding

assays were done in triplicate in 96-well ﬁlter plates containing the cell

free sea urchin egg homogenate and constant concentration of

radioligand [³²P]NAADP (0.2 nM). Seven concentrations of each

compound were tested to determine their IC₅₀. The competitor and

[³²P]NAADP were incubated simultaneously with the sea urchin egg

homogenate for 90 min at 4 °C. The homogenate was ﬁltered and

washed, and the radioactivity retained on the ﬁlter was determined by

liquid scintillation. Nonspeciﬁc binding was deﬁned as radioactivity

not displaced by 1 μM NAADP and represented between 0.05 and 2%

of total binding.

ASSOCIATED CONTENT

■

S

* Supporting Information

+

¹H NMR spectra, results from combustion analysis, HPLC

results for tested dinucleotides, and UV absorption spectra for

tested dinucleotides. This material is available free of charge via

the Internet at http://pubs.acs.org.

C₂₁H₂₇N₉O₁₈P₃: 786.068. Found m/z: 786.068 (M+).

8-Bromoadenosyl-5-azido-NAADP (35). 5-Azido-nicotinic acid (33

mg, 0.201 mmol) was dissolved in H₂O (5 mL) and DMSO (1 mL)

and stirred for 30 min at 37 °C. After this time period, the pH was

adjusted to 4 with 0.5 M NaOH and stirred for 30 min. The pH was

measured again and readjusted to pH = 4 with 0.5 M NaOH, if

necessary. This procedure was repeated until the pH stabilized. After

the pH stabilized, 8-Br-NADP (15 mg, 0.018 mmol) was added and

stirred for 5 min at 37 °C. Next, Aplysia ADP-ribosyl cyclase (180 μL,

36 μg) was added and the reaction was stirred at 37 °C for 5 h. HPLC

conﬁrmed that the starting material had been consumed. The sample

AUTHOR INFORMATION

■

Corresponding Authors

*For T.F.W.: phone, (1)-612-625-2627; fax, (1)-612-625-8408;

E-mail, walse001@umn.edu.

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(12) Brailoiu, G. C.; Gurzu, B.; Gao, X.; Parkesh, R.; Aley, P. K.;

Trifa, D. I.; Galione, A.; Dun, N. J.; Madesh, M.; Patel, S.; Churchill,

G. C.; Brailoiu, E. Acidic NAADP-sensitive calcium stores in the

endothelium: agonist-specific recruitment and role in regulating blood

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*For J.T.S.: phone, (1)-419-383-1925; fax, (1)-419-383-1909;

E-mail, james.slama@utoledo.edu.

Author Contributions

The manuscript was written through contributions of all

authors. All authors have given approval to the ﬁnal version of

the manuscript. Taken in part from the doctoral dissertation

submitted by Christopher J. Trabbic to the University of

Toledo College of Graduate Studies for the Ph.D. degree in

Medicinal Chemistry, May 2012.

Notes

The authors declare no competing ﬁnancial interest.

ACKNOWLEDGMENTS

■

This work was supported by a University of Toledo

Interdisciplinary Research Initiation Program grant and by

NIH grant no. GM 100444.

ABBREVIATIONS USED

■

cADPR, cyclic adenosine diphosphate ribose; CV, column

volume in reference to column chromatography; DEAE,

diethylaminoethyl group; DIPEA, diisopropylethylamine;

EtOAc, ethyl acetate; HEPES, 4-(2-hydroxyethyl)piperazine-1-

ethanesulfonic acid buﬀer; IP3, ^D-myo-inositol trisphosphate;

NAADP, nicotinic acid adenine dinucleotide phosphate; PTFE,

polytetaﬂuoroethylene; t-Boc, tert-butyloxycarbonyl protecting

group; TEA, triethylamine; TPC, two-pore channel

(19) Coxon, C. H.; Lewis, A. M.; Sadler, A. J.; Vasudevan, S. R.;

Thomas, A.; Dundas, K. A.; Taylor, L.; Campbell, R. D.; Gibbins, J. M.;

Churchill, G. C.; Tucker, K. L. NAADP regulates human platelet

function. Biochem. J. 2012, 441, 435−442.

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Bincoletto, C.; Han, S. W.; Stilhano, R. S.; Ureshino, R. P.; Bloor-

Young, D.; Churchill, G.; Piacentini, M.; Patel, S.; Smaili, S. S.

Nicotinic acid adenine dinucleotide phosphate (NAADP) regulates

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DOI: 10.1021/acs.jmedchem.5b00279

J. Med. Chem. 2015, 58, 3593−3610

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Nicotinic acid adenine dinucleotide phosphate analogues substituted on the nicotinic acid and adenine ribosides. Effects on receptormediated Ca2+ release

DOI: 10.1021/acs.jmedchem.5b00279

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