Chemo-enzymatic synthesis of adenine substituted nicotinic acid adenine dinucleotide phosphate (NAADP) analogs

Abstract

Nicotinamide adenine dinucleotide phosphate (NADP) is an indispensable metabolic co-substrate and nicotinic acid adenine dinucleotide phosphate (NAADP) is an important Ca²⁺ releasing intracellular second messenger. Exploration of the NADP and NAADP interactome often requires the synthesis of NADP derivatives substituted on the adenosine nucleoside. The introduction of the 2′-phosphate of NADP makes the synthesis of substituted NADP derivatives difficult. We have employed recombinant human NAD kinase expressed in E. coli as an enzymatic reagent to convert readily available synthetic NAD derivatives to NADP analogs, which were subsequently transformed into NAADP derivatives using enzyme catalyzed pyridine base exchange. 8-Ethynyl-NADP, 8-ethynyl-NAADP and 5-N₃-8-ethynyl-NAADP were synthesized starting from a protected 8-ethynyladenosine using a combination of chemical and enzymatic steps and the NAADP derivatives shown to be recognized by the sea urchin NAADP receptor at low concentration. Our methodology will enable researchers to produce mono- and bi-substituted NADP and NAADP analogs that can be applied in proteomic studies to identify NADP and NAADP binding proteins.

Graphical Abstract

1. Introduction

Nicotinamide adenine dinucleotide phosphate is an important metabolic cosubstrate and a presumed precursor to nicotinic acid adenine dinucleotide phosphate (NAADP), an endogenous dinucleotide and an intracellular second messenger for Ca²⁺ release in echinoderms,^{1, 2} mammals^{3, 4} and plants⁵. NAADP has been shown to be involved in various cellular functions like fertilization,⁶ smooth muscle contraction,⁷ insulin secretion³, neuronal differentiation⁸, and T cell activation.⁹ Although the physiological importance of NAADP has been recognized, the molecular mechanism of the NAADP-induced Ca²⁺ signaling remains ambiguous. While NAADP mobilizes Ca²⁺ release from acidic lysosome-like organelles,^{10, 11} the identity of the molecular components in the NAADP-evoked calcium release pathway remains to be fully elucidated.¹²

We previously described the structure-activity-relationships (SAR) of a series of NAADP analogs, a majority of which contained substituted nicotinic acids.^{13, 14} These compounds could be produced from NADP and a synthetic nicotinic acid derivative using enzyme catalyzed pyridine base exchange as illustrated in Figure 1. The effect of modification of the purine ring is less well understood because of the difficulty in obtaining the adenine substituted NADP analogs, which we required for NAADP production. Due to the difficulty in introducing the adenosyl 2′-phosphate, the production of NADP by synthesis still remains difficult, and our inability to access purine substituted NADP analogs prevented us from producing many desirable NAADP derivatives. In this study, we report the development of an effective methodology for production of NADP and NAADP analogs from NAD derivatives. NAD derivatives are readily accessible by chemical synthesis through coupling of an adenosine 5′-monophosphate (AMP) analog with nicotinamide mononucleotide (NMN). In this work we show that recombinant human NAD kinase can be used to enzymatically introduce the adenosyl 2′-phosphate and convert simple adenine substituted NAD analogs into NADP derivatives.

Figure 1.

Aplysia ADP-ribosyl cyclase catalyzed pyridine base exchange reactions.

This enzymatic methodology enables us to synthesize mono-functionalized 8-ethynyl-NADP (5), 8-ethynyl-NAADP (8) and determine the effect of 8 on Ca²⁺ release ability and desensitization of the sea urchin NAADP receptor. We further describe the synthesis of photoactive 5-N₃-8-ethynyl-NAADP (9).

2. Results

2.1. Chemo-enzymatic synthesis

5-Substituted NAADP derivatives were produced from substituted nicotinic acid analogs and commercial NADP using the pyridine base exchange reaction catalyzed by Aplysia californica ADP-ribosyl cyclase^{14, 15} (Figure 1).

To introduce a functional group to the 8-adenosyl position, an 8-substituted NAD derivative was synthesized chemically and then selectively phosphorylated using recombinant human NAD kinase¹⁶ to give an 8-substituted NADP derivative (Figure 2).

Figure 2.

Selective phosphorylation of NAD derivative catalyzed by recombinant human NAD kinase

The resulting 8-substituted NADP could be subjected to the pyridine base exchange reaction with nicotinic acid or nicotinic acid analogs to generate a mono- or bi-functional NAADP derivative, respectively.

2.2. Synthesis of 8-ethynyl-NAD (4)

Protected 8-ethynyl adenosine (1) was synthesized in three steps from adenosine by first protecting the three ribosyl hydroxyl groups as tert-butyldimethylsilyl (TBDMS) ethers. Next, the 8-adenosyl was selectively lithylated using n-butyl lithium and the 8-adenosyl anion iodinated with cyanogen iodide.¹⁷ Protected 8-iodoadenosine derivative could be easily converted into 1 using the Sonogashira reaction and trimethylsilylacetylene.¹⁸ Although conversion of 8-bromoadenosine to the protected 8-ethynyladenosyl derivative was reported,¹⁹ the use of the bromoadenosine offered no advantage and in our hands, compound 1 was successfully produced only from protected 8-iodoadenosine.

Difficulty was encountered in our attempt to remove all of the silyl groups of compound 1 using tetrabutylammonium fluoride (TBAF), where we encountered complex reaction mixtures. This result was also observed by others, who suggested that TBAF gave rise to conjugate addition products.^{18, 20} We first successfully removed the acetylinic-trimethylsilyl (TMS) protecting group with K₂CO₃/methanol and next selectively deprotected the 5’-OH using aqueous trifluoroacetic acid (TFA) to yield 8-ethynyl-2’,3’-bis-O-(tert-butyldimethylsilyl)-adenosine (2).²¹ In a preliminary experiment, we discovered that the remaining TBDMS groups were removed in situ during 5′-phosphorylation in a reaction mixture of trimethylphosphate (TMP) and POCl₃, which was developed for the selective phosphorylation of the 5’-OH of nucleosides.²² Silyl groups were reported to be deprotected by a catalytic amount of HCl,²³ so the HCl generated as a co-product during the phosphorylation is likely to be the cause of the in situ deprotection of TBDMS groups.

The free 5’-OH of compound 2 was phosphorylated successfully using POCl₃/TMP, while the 2’- and 3’- hydroxyls were deprotected simultaneously. The resulting 8-ethynyl-AMP (3) was coupled with commercially available β-nicotinamide mononucleotide (NMN) in aqueous solution using a water soluble carbodiimide, EDC, to yield 8-ethynyl-NAD (4) (Scheme 1).

Scheme 1. Synthesis of 8-ethynyl-NAD (4)^a.

^aReagents and conditions: (a) (i) K₂CO₃, MeOH, 1 h, (ii) THF-TFA-H₂O (4: 1: 1), 0 °C, 2 h, 90%; (b) (i) POCl₃, TMP, 0 °C, 2 h, (ii) quenched with water, 62%; (c) β-nicotinamide mononucleotide, MgCl₂, HEPES (pH = 6.8), EDC, 37 °C, 2 h, 16%.

2.3. Synthesis of 8-ethynyl-NADP (5)

The selective phosphorylation of 8-ethynyl-NAD (4) at the adenosyl 2’-OH to yield 8-ethynyl-NADP (5) was accomplished using recombinant human NAD kinase. The enzyme catalyst is available commercially, or it can be produced from an easily constructed E. coli expression vector (see Experimental Procedures section 5.2.1.). Compound 4 with its 8-adenosyl substitution is phosphorylated by the NAD kinase at a rate slower than that for NAD, so to compensate a large amount of the kinase was utilized. Initial concentrations of ATP, the phosphoryl donor for NAD kinase, were maintained by adding phosphocreatine and creatine phosphokinase to the reaction to continuously convert the product ADP back to ATP (Scheme 2).

Scheme 2. Synthesis of 8-ethynyl-NADP (5)^a.

^aReagents and conditions: (a) ATP, MgCl₂, phosphocreatine, creatine phosphokinase, recombinant human NAD kinase, HEPES buffer (pH = 7.3), ambient temperature, 48 h, 70%.

2.4. Synthesis of 8-ethynyl-NAADP (8) and 5-N₃-8-ethynyl-NAADP (9)

The nicotinamide moiety of 8-ethynyl-NADP (5) was exchanged for nicotinic acid (6) to yield 8-ethynyl-NAADP (8), or 5-N₃-nicotinic acid (7)¹³ to give 5-N₃-8-ethynyl-NAADP (9). As ADP-ribosyl cyclase also catalyzes the cyclization and hydrolysis of NADP,²⁴ a high concentration of nicotinic acid facilitates the pyridine base exchange reaction, and inhibits the production of the cyclic and linear ADP-ribose byproducts (Scheme 3). We attribute the lower yield of compound 9 compared to 8 to difficulties in monitoring the conversation of 5 to 9 and subsequently in separating 9 from overlapping peaks.

Scheme 3. Synthesis of 8-ethynyl-NAADP (8) and 5-N₃-8-ethynyl-NAADP (9)^a.

^aReagents and conditions: Aplysia californica ADP-ribosyl cyclase, H₂O, pH = 4, 16h (8) 6h (9), 79% (8) and 40% (9).

2.5. Spectroscopic properties for 8-ethynyl-NAADP (8) and 5-N₃-8-ethynyl-NAADP (9).

The UV/Vis spectra of compound 8 and 9 were different both from typical NADP analogs and from each other. Compound 8 exhibited an absorption maximum at 292 nm which did not change on exposure to short wavelength UV light. Compound 9 exhibited an absorption maximum at 267 nm with a shoulder at ca 290 nm owing to the addition of an azido group to the 5-nicotinic acid position (Figure 3). Upon UV irradiation of 9 using a short wavelength mineral light for 10 minutes, we observed that the absorption maximum shifted to 292 nm. This change is consistent with the photochemical reaction of the 5-azido-nicotinic acid producing a nitrene and nitrene-derived products.

Figure 3.

A. UV/Vis spectrum of 8-ethynyl-NAADP (8). B. UV/Vis spectrum of 5-N₃-8-ethynyl-NAADP (9) before (solid blue line) and after (dotted red line) UV irradiation for 10 minutes. All spectra were obtained in 20 mM HEPES at pH 7.

2.6. Biological activities

NAADP analogs were assayed using sea urchin egg homogenates for their ability to induce the release of Ca²⁺ and for the ability to induce time dependent sub-threshold desensitization of the Ca²⁺ release response.

We first assayed 8-ethynyl-NAADP (8) to determine the effect of 8-adenosyl substitution. Increasing concentrations of NAADP (as a positive control) or analog 8 were added to sea urchin egg homogenates and the Ca²⁺ release measured using the fluorescent Ca²⁺ indicator, Fluo-3. We observed a concentration dependent increase in Ca²⁺ release (Figure 4) and determined that the EC₅₀ values for 8-ethynyl-NAADP (8) were 89-fold greater than that of NAADP.

Figure 4.

Mobilization of Ca²⁺ release (solid line) by increasing concentration of NAADP (red) and 8-ethynyl-NAADP (black), and receptor desensitization to subsequent addition of 1 μM NAADP (dash line). Ca²⁺ concentrations were detected fluorometrically by monitoring the peak fluo-3 fluorescent values.

In sea urchin egg homogenates NAADP displays an atypical concentration-response relationship: on extended incubation (2 – 7 min), a subthreshold concentration of NAADP that does not itself induce Ca²⁺ release will inhibit the Ca²⁺ signaling on subsequent treatment with a supramaximal concentration of NAADP.^{25, 26} Such an atypical characteristic can be explained by a “two-site” hypothesis: low concentration of NAADP binds to a high-affinity allosteric site of NAADP receptor and mediates self-desensitization, while high concentration of NAADP binds to the low affinity orthosteric site and mobilizes Ca²⁺ release.²⁷ For this experiment, the sea urchin homogenates were treated with test compound at a low concentration and after seven minutes incubation, 1 μM NAADP added to all samples and the subsequent Ca²⁺ release was monitored fluorometrically. The response, shown in Figure 4, was reported as the IC₅₀ value. The addition of the 2-carbon substituent to the adenylyl 8-position resulted in an 86-fold decrease in desensitization potency.

The bi-substituted analog 9 was assayed similarly, and the results of both Ca²⁺ release and sub-threshold desensitization were already reported in a companion publication.²⁸ The EC₅₀ and IC₅₀ values of NAADP, 8, 9 and two other related mono-functional NAADP analogs, 5-N₃-NAADP¹³ and 8-N₃-NAADP¹⁴, are summarized in Table 1.

Table 1.

Structure-activity relationships for NAADP, mono- and bi-functional NAADP analogs^a

Structure	Name	R1	R2	EC50 (nM)a	IC50 (nM)a
	NAADP	H	-H	8.0 ± 0.7	0.14 ± 0.01
	8-ethynyl-NAADP (8)	-H	-C≡C-H	709.6 ± 98.9	12.1 ± 1.1
	5-N3-8-ethynyl-NAADPb (9)	-N3	-C≡C-H	2860.0 ± 406.9	99.0 ± 7.7

^aValues are mean ± SEM of three independent assays.

^bSee our companion paper Gunaratne et al., 2018²⁸

3. Discussion

In our previous publications, we have synthesized nicotinic acid substituted NAADP derivatives from commercially available NADP and synthetic 4- or 5-subsitiuted nicotinic acids using the enzyme catalyzed pyridine base-exchange reaction.^{13, 14} Production of adenosyl substituted NAADP analogs is significantly more challenging, since this requires the production of adenosyl substituted NADP derivatives and their use as substrates for base-exchange. Chemical synthesis of NADP as reported by Dowden et al, (2004)²⁹ is not always a satisfactory solution, since the process requires multiple steps sand becomes more difficult when the 8-adenosyl position is substituted. We have applied recombinant human NAD kinase to solve the problem of converting a synthetic NAD into an NADP derivative. Recombinant human NAD kinase is available commercially (Sigma-Aldrich; Creative Enzymes, Shirley, NY;), but we preferred to construct our own bacterial expression plasmid starting from a plasmid containing full length human NADK purchased from Origene. Although 8-ethynyl-NAD is phosphorylated more slowly by the recombinant human NAD kinase than is NAD, the availability of large amounts of the recombinant enzyme enables the phosphorylation of 8-ethynyl-NAD to be accomplished in good yield and in a reasonably short time. We therefore now have developed a method to utilize substituted adenosine 5′-monophosphates, convert them to the NAD derivative chemically, and then convert the NAD analog to the NADP or the NAADP enzymatically.

Compound 8 and 9 were assayed using sea urchin egg homogenates to determine their EC₅₀ and IC₅₀ values. This begins to offer some insight into the effect of substitution at the 8-adenosyl position on NAADP analog activity. Compared to NAADP, 8-ethynyl-NAADP (8) was 90-fold less potent as an agonist and in sub-threshold desensitization.

The bifunctional 5-N₃-8-ethynyl-NAADP (9) was recognized by the sea urchin NAADP receptor less well than the mono-functionalized analog, with an IC₅₀ of approximately 100 nM in the sub-threshold desensitization assay. This loss of potency associated with bi-substitution was previously observed.¹⁴

In summary, we successfully developed a synthetic method which permits us to introduce a “clickable” functional group to the 8-adenosyl position of NADP and NAADP and further to introduce a photoactive group at the 5-nicotinyl position using enzyme catalyzed 2′-phosphorylation and pyridine base exchange reactions. The effects of the functionalized NAADP analogs on binding to the NAADP receptor were evaluated. Both the mono- and bi-functional NAADP analogs were shown to unexpectedly lose potency as agonists for the sea urchin NAADP receptor.

In our published companion paper,²⁸ low concentrations of microinjected 9 was shown to elicit Ca²⁺ release in cultured human cells and to specifically photolabel NAADP 23–24 kDa binding protein(s), further emphasizing differences in the recognition of NAADP analogs between sea urchin homogenates and mammalian cells.

4. Conclusions

Our study offers a method to convert synthetic NAD analogs into both NADP and NAADP derivatives. The access to clickable substituted NADP analogs will be of value in future proteomic studies to define NADP binding proteins. The clickable and photoactive probe 9 will be used to identify NAADP binding proteins important in Ca²⁺ signaling.

5. Experimental procedures

5.1. Chemical methods

5.1.1. General procedures

Reagents and anhydrous solvents were purchased from Sigma-Aldrich or Acros Organics and used without purification unless stated otherwise. NAD kinase used in preliminary experiments was purchased from Alexis/Enzo. Aplysia californica ADP-ribosyl cyclase was produced and purified according to Munshi & Lee.¹⁵ Proton (¹H) NMR spectra were recorded at 600 MHz and proton decoupled carbon (¹³C) NMR spectra were recorded at 150.9 MHz using Bruker Avance Cryprobe. Chemical shifts are reported in parts per million (ppm, δ) and referenced to the residual proton signal of the deuterated solvent (¹H, δ 4.70, D₂O) or internal standard of tetramethylsilane (TMS) when CDCl₃ was used. ³¹P NMR spectra were recorded on a VXRS 400 spectrophotometer (161.9 MHz) with 85% H₃PO₄ as external reference. High-resolution mass spectrometry (HRMS) was performed using nanoESI-TOF mass spectrometry. Final tested compounds were >95% pure as determined by analytical HPLC.

Anion exchange chromatography was adapted from the volatile liquid chromatography system described by Alexson et al.³⁰ and was performed using a Bio-Rad BioLogic DuoFlow™ HPLC system equipped with a 254 nm UV detector. The stationary phase was anion exchange AG MP-1 resin trifluoroacetate form (200–400 mesh). The mobile phase solvent A was deionized water and solvent B was 100 mM aqueous TFA.

Analytical anion exchange HPLC general method. A glass column (5 × 50 mm, 1 mL) packed with AG MP-1 resin and a 2 mL injection loop were fitted to the HPLC system. The flow rate was maintained at 1 mL/min. The chromatography was developed as followed: 1) 5 mL solvent A was applied through the injection loop. 2) A linear step-gradient of solvent B increased from 0% to 1% over a volume of 1 mL, to 2% over a volume of 3 mL, to 4% over a volume of 4 mL, to 8% over a volume of 3 mL, to 16% over a volume of 5 mL, to 32% over a volume of 4 mL, to 64% over a volume of 4 mL, to 100% over a volume of 2 mL, and then decreased linearly from 100% to 1 % solvent B over a volume of 6 mL and maintain at 1% solvent B over a volume of 3 mL.

Preparative HPLC general method. A glass column (15 × 113 mm, 20 mL) packed with AG MP-1 resin and a 5 mL injection loop were fitted to the HPLC system. The flow rate was 6 mL/min throughout the purification. The chromatography was developed as followed: 1) 6 mL solvent A was applied through the injection loop, 2) 25 mL solvent A was applied through the column, 3) a linear gradient of TFA was run over a total volume of 185 – 400 mL (as specified in individual synthesis), 4) the column was cleaned using 40 mL solvent B and re-equilibrated using 40 mL solvent A. The purified products were collected, pooled in a 1000 mL round bottom flask and applied to a rotatory evaporator at 35 °C using a diaphragm pump at ca. 20 mm Hg to remove most of the TFA.

5.1.2. Synthesis and characterization of new compounds

8-(Trimethylsilyl)ethynyl-2’,3’,5’-tris-O-(tert-butyldi-methyl-silyl)-adenosine (1),

was prepared from 2’,3’,5’-tris-O-(tert-butyldimethylsilyl)-8-iodoadenosine¹⁷ according to the procedure of Manfredini et al.,(1995)¹⁸. Product 1 was isolated as a yellow crystalline solid in 82 % yield, Mp 86–87 °C; Reported¹⁸, 85–87 °C. TLC: R_f = 0.45 in 30% EtOAc-hexane. UV/Vis (MeOH) λ_max 299.02 nm. ¹H NMR (600 MHz, CDCl₃) δ 8.30 (s, 1H), 6.27 (br, 2H), 6.16 (d, 1H, J = 6.90), 5.40 (dd, 1H, J = 6.90, 4.30), 4.46 (dd, 1H, J = 4.20, 0.90), 4.19 (dd, 1H, J = 10.44, 9.0), 4.12 – 4.10 (m, 1H), 3.78 (dd, 1H, J = 10.62, 4.98), 0.97 (s, 9H), 0.92 (s, 9H), 0.79 (s, 9H), 0.30 (s, 9H), 0.17 (s, 3H), 0.15 (s, 3H), 0.08 (s, 6H), −0.10 (s, 3H), −0.49 (s, 3H). ¹³C NMR (150.9 MHz, CDCl₃) δ 155.6, 153.4, 149.3, 135.6, 120.1, 103.6, 92.0, 89.4, 86.3, 72.9, 71.8, 62.9, 26.0, 25.9, 25.7, 18.4, 18.1, 17.8, −0.6, −4.5, −4.6, −4.7, −5.3, −5.4, −5.5.

8-Ethynyl-2’,3’-bis-O-(tert-butyldimethylsilyl)-adenosine (2).

To a solution of compound 1¹⁸ (2.0 g, 2.83 mmol) in 20 mL MeOH was added K₂CO₃ (1.95 g, 14.15 mmol). The reaction mixture was stirred at room temperature for 1 hour. The reaction was monitored by TLC (EtOAc-hexane, 3:7). When the reaction was complete, the reaction mixture was filtered, and the solvent was removed from the filtrate under reduced pressure. The residue was dissolved in 400 mL EtOAc, washed with water (2 × 200 mL), dried over anhydrous Na₂SO₄ and the solvent evaporated. The crude product was dissolved in 20 mL THF and cooled to 0 °C in an ice bath, followed by addition of 10 mL of 50% aqueous TFA solution. The resulting solution was stirred at 0 °C for 2 hours (TLC, EtOAc-hexane, 3:7). After the reaction was complete, the reaction mixture was diluted with 400 mL EtOAc, washed with saturated aqueous NaHCO₃ (3 × 150 mL), water (2 × 200 mL) and saturated aqueous NaCl (200 mL). The organic solvent was removed under reduced pressure. The crude product was purified by column chromatography on silica gel (EtOAc-hexane, 30–80%) to give a yellow crystalline solid (1.32 g, 90%). Mp 226–229 °C. [α]^22.5₅₈₉ = −1251° (c=1.01, CH₃OH). TLC: R_f = 0.23 in 50% EtOAc-hexane. UV/Vis (MeOH) λ_max 292.18 nm. ¹H NMR (600 MHz, CDCl₃) δ 8.37 (s, 1H), 6.20 (d, 1H, J = 8.04), 6.03 (br, 2H), 5.07 (dd, 1H, J = 7.98, 4.5), 4.35 (d, 1H, J = 4.5), 4.18 (s, 1H), 3.95 (dd, 1H, J = 13.08, 1.62), 3.73, (d, 1H, J = 12.90), 3.55 (s, 1H), 0.97 (s, 9H), 0.80 (s, 9H), 0.16 (s, 3H), 0.14 (s, 3H), −0.12 (s, 3H), −0.61 (s, 3H). ¹³C NMR (150.9 MHz, CDCl₃) δ 155.7, 153.3, 148.4, 134.5, 120.1, 90.1, 89.6, 84.7, 74.0, 73.9, 71.9, 63.1, 25.8, 25.7, 18.1, 17.7, −4.5, −4.6, −4.7 −5.9. HRMS calcd for C₂₄H₄₁N₅O₄Si₂: 520.2775 (M + H). Found m/z 520.2762 (M + H).

8-Ethynyl-AMP (3).

Compound 2 (30 mg, 0.058 mmol) was suspended in 100 μL trimethylphosphate (distilled and stored under argon) at 0 °C, followed by addition of POCl₃ (distilled and stored under argon, 11 μL, 0.12 mmol). The reaction solution became clear upon addition of POCl₃ and was continuously stirred at 4 °C for 2 hours. To monitor the reaction by analytical HPLC, 5 μL of the solution was removed from the reaction and diluted to 100 μL with water and pH was adjusted to 7 using 1 M NaOH. After 2 hours, the reaction was quenched by adding 15 mL of water and the pH was adjusted to 7 using 1 M NaOH. The crude product was purified using anion-exchange chromatography by preparative HPLC, where 0 – 6 mM TFA was applied in a linear gradient over a total volume of 300 mL. The fraction containing the product was frozen and lyophilized to give an amorphous white solid (13 mg, 62%). Analytical HPLC: t = 14.13 min, 6% solvent B. UV/Vis (H₂O, pH = 7) λ_max 291.3 nm. ¹H NMR (600 MHz, D₂O) δ 8.33 (s, 1H), 6.13 (d, 1H, J = 5.4), 5.06 (t, 1H, J = 5.52), 4.54 (t, 1H, J = 5.16), 4.23 (d, 1H, J = 0.78), 4.20 (q, 1H, J = 4.8), 4. 06 (m, 2H). ¹³C NMR (150.9 MHz, D₂O) δ 149.5, 147.8.0, 145.3, 136.3, 118.3, 89.3, 88.7, 83.9, 72.0, 70.0, 69.8, 64.3. ³¹P NMR (161.9 MHz, D₂O) δ 5.07. HRMS calcd for C₁₂H₁₄N₅O₇P: 372.0709 (M + H). Found m/z 372.0718 (M + H).

8-Ethynyl-NAD (4).

Compound 3 (12 mg, 0.032 mmol) and β-nicotinamide mononucleotide (54 mg, 0.163 mmol, Ark Pharm Inc, AK152018) were dissolved in 1.5 mL 1 M MgCl₂. The resulting solution was evaporated to dryness using a rotatory evaporator equipped with a vacuum pump. The residue was redissolved in 2 mL of 1.5 M HEPES (pH = 6.8). The reaction was initiated by adding EDC (0.5 mL of a 5 M solution), and then incubated at 37 °C for 2 hours. To monitor the reaction by analytical HPLC, 5 μL of solution was removed from the reaction and diluted to 100 μL with water. After 2 hours, the reaction solution was diluted to 10 mL with water and purified using preparative HPLC, where a linear gradient of 0 – 3 mM TFA was applied over a total volume of 300 mL. The fraction containing the product was lyophilized and re-purified if necessary. Compound 4 was isolated as a white solid (3.6 mg, 16%). Analytical HPLC: t = 10.99 min, 3% solvent B. UV/Vis (H₂O, pH = 7) λ_max 291.7 nm with a shoulder at 275.5 nm. ¹H NMR (600 MHz, D₂O) δ 9.20 (s, 1H), 9.02 (d, 1H, J = 6.12), 8.74 (d, 1H, J = 7.98), 8.14 – 8.11 (m, 2H), 5.94 (d, 1H, J = 5.58), 5.89 (d, 1H, J = 4.26), 5.04 (t, 1H, J = 5.94), 4.49 (t, 1H, J = 5.52), 4.32 – 4.12 (m, 9H). ¹³C NMR (150.9 MHz, D₂O) δ 165.5, 149.9, 147.9, 146.0, 145.8, 142.6, 139.8, 136.1, 133.9, 128.8, 118.4, 99.9, 89.2, 88.8, 87.1, 83.8, 77.5, 71.9, 70.7, 70.1, 69.7, 65.2, 64.8. ³²P NMR (161.9 MHz, D₂O) δ −10.12, −10.44. HRMS calcd for C₂₃H₂₈N₇O₁₄P₂⁺: 688.1170 (M⁺). Found m/z 688.1181 (M +).

8-Ethynyl-NADP (5).

A reaction mixture was prepared by adding water (5176 μL), HEPES (1925 μL of a 50 mM solution in water, pH = 7.3), ATP (100 mM stock solution, 154 μL), MgCl₂ (1 M stock solution, 38 μL), phosphocreatine disodium salt hydrate (1 M stock solution, 22 μL, Sigma Aldrich, P7936–1G), followed by 1 mg of creatine phosphokinase (from rabbit muscle, Sigma Aldrich, C3755–35KU). The reaction mixture was gently added to a 20 mL glass vial which contained compound 4 (4.0 mg, 5.8 μmol). The reaction was initiated by adding 600 μL of recombinant human NAD kinase (250 μg/mL stock solution) and monitored by analytical HPLC. After the reaction was stirred at room temperature for 48 hours, analytical HPLC showed that about 80 % of compound 4 converted to product 5. The resulting mixture was purified using preparative HPLC, where a linear gradient of 0 – 20 mM TFA was applied over a total volume of 185 mL. The fraction containing the product was frozen and lyophilized to give an amorphous white powder (3.12 mg, 70%). Analytical HPLC: t = 19.04 min, 13% solvent B. UV/Vis (H₂O, pH = 7) λ_max 291.6 nm with a shoulder at 274.4 nm. ¹H NMR (600 MHz, D₂O) δ 9.29 (s, 1H), 9.11 (d, 1H, J = 6.18), 8.83 (d, 1H, J = 8.04), 8.37 (s, 1H), 8.19 (t, 1H, 7.62), 6.22 (d, 1H, J = 5.28), 6.00 (d, 1H, J = 5.52), 5.42 (m, 1H), 4.64 (t, 1H, J = 5.22), 4.41 – 4.36 (m, 2H), 4.32 (m, 1H), 4.24 (m, 1H), 4.21– 4.12 (m, 4H), 4.12 – 4.07 (m, 1H). ³²P NMR (161.9 MHz, D₂O) δ 0.77, −10.44, −10.74. HRMS calcd for C₂₃H₂₉N₇O₁₇P₃⁺: 768.0833 (M⁺). Found m/z 768.0861 (M⁺).

8-Ethynyl-NAADP (8).

Nicotinic acid (6) (14 mg, 0.117 mmol) was dissolved in 3.9 mL water. The resulting solution was adjusted to pH 4 and then added to a 20 mL glass vial which contained 8-ethynyl-NADP (2.7 mg, 3.5 μmol). The pH of the solution was adjusted to 4 again if necessary. Aplysia californica ADPR cyclase (100 μL, 0.2 mg/mL) was added to initiate the reaction. The reaction was incubated at 37 °C and monitored using analytical HPLC. After 16 hours, analytical HPLC showed consumption of the 8-ethynyl-NADP (7). The reaction mixture was purified using preparative HPLC, where a linear gradient of 0 – 40 mM TFA was applied over a total volume of 185 mL. The fraction containing the product was frozen and lyophilized to give an amorphous white powder (2.14 mg, 79%). Analytical HPLC: t = 23.50 min, 26% solvent B. UV/Vis (20 mM HEPES buffer, pH = 7) λ_max 291.7 nm with a shoulder at 275.2. Molar extinction coefficient: 16.2 × 10³ L mol⁻¹ cm⁻¹ at 291.6 nm. ¹H NMR (600 MHz, D₂O) δ 9.28 (s, 1H), 9.13 (d, 1H, J = 6.30), 8.88 (d, 1H, J =7.98), 8.38 (s, 1H), 8.17 (t, 1H, 7.11), 6.22 (d, 1H, J = 5.40), 6.00 (d, 1H, J = 5.40), 5.44 (m, 1H), 4.63 (t, 1H, J = 5.11), 4.39 – 4.36 (m, 2H), 4.32 (m, 1H), 4.26 – 4.07 (m, 6H). HRMS calcd for C₂₃H₂₈N₆O₁₈P₃⁺: 769.0673 (M⁺). Found m/z 769.0658 (M⁺).

5-N₃-8-Ethynyl-NAADP (9).

A solution of 5-N₃-nicotinic acid¹³ (7) (12.8 mg, 0.078 mmol), in 5.8 mL water was adjusted to pH 4 using 0.1–1 M NaOH. The resulting solution was added to a 20 mL glass vial which contained compound 5 (4.0 mg, 5.2 μmol) and the pH was adjusted to 4 again if necessary. Aplysia californica ADPR cyclase (100 μL, 0.2 mg/mL) was added to initiate the reaction. The reaction was stirred gently at 37 °C and monitored using analytical HPLC. After 6 hours, the reaction mixture was purified using preparative HPLC, where a linear gradient of 0 – 25 mM TFA was applied over a total volume of 400 mL. The fraction containing the product was frozen and lyophilized to give an amorphous yellow powder (1.68 mg, 40%). Analytical HPLC: t = 23.56 min, 26% solvent B. UV/Vis (20 mM HEPES buffer, pH = 7) λ_max 266.7 nm with a shoulder at 292.2 nm. Molar extinction coefficients: 19.9 × 10³ L mol⁻¹ cm⁻¹ at 266.7 nm and 17.9 × 10³ L mol⁻¹ cm⁻¹ at 292.2 nm. ¹H NMR (600 MHz, D₂O) δ 8.73 (s, 1H), 8.51 (s, 1H), 8.43 (s, 1H), 8.06 (s, 1H), 6.04 (d, 1H, J = 5.10), 5.71 (d, 1H, J = 5.22), 5.33 (m, 1H), 4.21 – 4.06 (m, 10H). HRMS calcd for C₂₃H₂₇N₉O₁₈P₃⁺: 810.0687 (M⁺). Found m/z 810.0653 (M⁺).

Determination of extinction coefficients of compound 8 and 9.

Solutions 8-ethynyl-NAADP (8) and 5-N₃-8-ethynyl-NAADP (9) were prepared, the UV absorptions were measured, and the concentrations of the samples determined by quantifying the total phosphate content of 100 μL aliquot according to Ames (1966).³¹

5.2. Biological methods

5.2.1. NAD kinase cloning, expression and purification

To obtain enzymatically active NADK the human cDNA was cloned into a bacterial expression vector, transformed into bacteria, expressed and purified from bacterial lysate. A plasmid containing full length human NADK was purchased from Origene (Cat. # RC00544) and used as a template for polymerase chain reaction amplification followed by Gibson Assembly cloning (InFusionHD kit, Clontech) into the pET15b vector (Novagen cat. # 69661–3). pET15b has an IPTG inducible promoter for bacterial expression and a His-Tag coding sequence for amino terminus tagging and Ni⁺ column purification of the NADK protein. The NADK cDNA was amplified using a forward primer 5’-GTGCCCGCGGCAGCCATATGCACATTCAGGACCCCG CGAGC-3’. This sequence contains the 21 bases complementary to the template strand of the NADK gene starting at base 262 of the cDNA for efficient template binding and PCR amplification (underlined), 6 bases of the NdeI restriction endonuclease recognition sequence and a 15-base sequence from the pET15b vector for efficient Gibson Assembly cloning. The reverse primer 5′-TTGTTAGCAGCAGCCGGATCCTCAGCCCTCCTCCTCCTCCTCCTC-3′ was designed with 21 bases complementary to the coding strand of the cDNA ending with base 1566 (underlined), an antisense stop codon to replace the epitope tag provided by the Origene vector, 6 bases of the BamHI recognition sequence and 15 bases from the pET15b vector for Gibson Assembly cloning. PCR amplification was performed with CloneAmp HiFi PCR mix from the InFusionHD kit for 30 cycles of 10 seconds at 98 °C, 15 seconds at 55 °C and 15 seconds at 72 °C. Agarose gel purified PCR product was combined with gel purified NdeI and BamHI double digest linearized vector along with InFusion reaction mix and incubated for 15 minutes at 50 °C. The Gibson Assembly reaction product was transformed into Stellar (Clontech) competent bacteria by manufacturer’s protocol and positive clones selected with ampicillin. Positive clones were confirmed by diagnostic restriction digest. A single vector clone was chosen to be transformed into Escherichia coli strain BL21(DE3)pLysS by standard methods. BL21(DE3)pLysS provides for IPTG induction with rapid and massive overexpression of the cloned gene. All restriction enzyme analyses were performed with endonucleases from New England Biolabs. All DNA gel purifications were performed by QIAquick Gel Extraction Kit, Qiagen.

The above cloning procedure provides for expression of a truncated NADK protein with a histidine tag. Ohashi et al (2011)³² showed that the truncated protein provided a significantly more stable purified product after Ni⁺ column purification. Methods for purification of NADK published by Lerner et al 2001¹⁶ and Ohashi et al 2011³² were used as guidelines for the following scheme. Briefly, a 2 × 1-liter late log phase culture of BL21(DE3)pLysS was induced to express truncated NADK from the pET15b vector by addition of 2mM IPTG for 3 hours. The cells were harvested, pelleted and lysed by repeated pipetting with 240 mL of 10 mM Tris, pH 8.0 supplemented with PMSF and Complete Protease Inhibitor tablets (Roche). The lysate was sonicated to shear the DNA and clarified by centrifugation at 20,000g for 10 minutes at 4°C. A one-step purification protocol based on Ohashi et al 2011³² was used for purification of the bacterial lysate. All purification steps were performed in a 4 °C cold room. HisBind purification kit (Novagen) was used to prepare a column with a bed volume of 30 mL packed beads according to manufacturer’s protocol and washed with 10 mM Tris, pH 8.0. The lysate was applied to the HisBind beads as a batch and incubated for 1 hour with gentle rocking before applying in aliquots to the empty 60 mL glass column. All eluates were collected for analysis. The column was washed with 5 × 30 mL (1 bed volume) of wash buffer (10 mM Tris, pH 8.0, 30 mM imidazole, 0.3 M NaCl). The column was eluted in one fraction of 2 bed volumes of elution buffer (10 mM Tris, pH 8.0, 150 mM imidazole, 0.3 M NaCl). The eluted fraction was dialyzed against 2 liters of 10 mM Tris, pH 8.0 and 5% glycerol for 20 hours with 2 changes using dialysis cartridges (Pierce) with a 10 kDa molecular weight cut-off. The dialyzed, purified NADK was sterile filtered through a 0.22 μm syringe filter and aliquoted into cryovials and stored at −80°C. Samples were checked for purity by SDS-PAGE.

5.2.2. Biological assays.

Ca²⁺ release and receptor desensitization assays in sea urchin egg homogenates were performed as previously described.¹⁴

Abbreviations

ADPR

adenosine diphosphate ribose

EDC

1-ethyl-3-(3-dimethylaminopropyl)carbodiimide∙HCl

EtOAc

ethylacetate; HEPES buffer, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid

MeOH

methyl alcohol

NAADP

nicotinic acid adenine dinucleotide phosphate

TBDMS

tert-butyldimethylsilyl protecting group

TMP

trimethylphosphate