Nicotinamide Benzimidazolide Dinucleotides, Non-Cyclisable Analogues of NAD⁺

Abstract

Benzimidazole-based nucleotides and dinucleotides have been synthesised to increase the range of chemical tools available to probe the NAD⁺ biology space. They were examined for their reactivity in alkylation-type reactions, where they yielded unstable alkylated heteoaromatic adducts, both chemically and enzymatically. While unsuited for NAD⁺ cyclases, these NAD⁺ analogues could be viable substrates for non-adenine modifying NAD⁺-dependent enzyme classes.

Key words

nucleotides - benzimidazoribosyl nucleosides - phosphorylation - cyclic adenosine diphosphoribose - enzymes

• References and Notes

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• 34 5-Phenyl-1-N-(5′-Phosphato-β-d-ribofuranosyl)-benzimidazole (20): A 62% isolated yield of compound 20 was achieved when purification was performed on reverse-phase column (Varian: prepacked C18 column), product elution was carried out using a linear gradient 5–90% of 10 mM AF (adjusted to pH 5) against MeOH. ¹H NMR (300 MHz, D₂O): δ = 8.54–8.66 (m, 1 H), 7.97 (m, 1 H), 7.79–7.87 (m, 1 H), 7.53–7.75 (m, 3 H), 7.41 (t, J = 7.3 Hz, 2 H), 7.31 (t, J = 7.3 Hz, 1 H), 6.08 (d, J = 6.2 Hz, 1 H), 4.55–4.62 (m, 1 H), 4.36–4.48 (m, 1 H), 4.22–4.33 (m, 1 H), 4.06–4.16 (m, 2 H). ³¹P NMR (121 MHz, D₂O): δ = 0.49. HPLC: t _R = 12.88 min. MS (ES): m/z [M – H]^– calcd for C₁₈H₁₈N₂O₇P: 405.0852; found: 405.0869.
• 35 2-(3′-Pyridyl)-1-N-(5′′-Phosphato-β-d-ribofuranosyl)-benzimidazole (21): A 25% yield of compound 21 was achieved when the initial purification was performed on ion-exchange resin (DEAE Sepharose Fast Flow) using gradient miliQ water against 0.1 M triethylamine formate (TEAF) at pH 5 with 10% of MeOH (0–100%) followed by reverse-phase purification (Varian: prepacked C18 column), product elution was carried out using a linear gradient 5–90% of 10 mM AF (adjusted to pH 5) against MeOH. ¹H NMR (300 MHz, D₂O): δ = 8.74 (s, 1 H), 8.64 (d, J = 4.2 Hz, 1 H), 8.08–8.17 (m, 1 H), 7.96 (d, J = 7.6 Hz, 1 H), 7.70 (d, J = 7.3 Hz, 1 H), 7.58 (dd, J = 5.1, 7.9 Hz, 1 H), 7.30–7.43 (m, 2 H), 5.80 (d, J = 7.7 Hz, 1 H), 4.82 (dd, J = 6.3, 7.6 Hz, 1 H), 4.35 (dd, J = 3.1, 6.3 Hz, 1 H), 4.10–4.16 (m, 1 H), 3.99–4.03 (m, 2 H). ¹³C NMR (100 MHz, D₂O): δ = 150.5, 149.0, 142.0, 138.7, 132.6, 125.6, 124.6, 124.4, 124.1, 119.1, 113.9, 89.0, 84.2 (d, J = 8.7 Hz), 70.7, 69.6, 64.1 (d, J = 4.8 Hz). ³¹P NMR (121 MHz, D₂O): δ = 2.24. HPLC: t _R = 15.38 min. MS (ES): m/z [M + H]⁺ calcd for C₁₇H₁₉N₃O₇P: 408.0961; found: 408.0952.
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• 40 General Procedure for the CDI-Mediated Pyrophosphate Bond Formation: Prior to the reaction a stock solution of NMN was prepared by dissolving NMN (100 mg) in anhyd DMF (1 ml) and anhyd formamide (1 mL). CDI (2.5 equiv) and Et₃N (2 equiv) were then added to a solution of nucleotide (1.2 equiv) in anhyd DMF (0.5 mL). The reaction was stirred for 12 h at r.t. The reaction was then quenched with anhyd MeOH and evaporated to dryness. The resulting solid was dissolved in new portion of anhyd DMF (0.5 mL) and a solution of NMN (1 equiv) was added. The reaction was followed by ³¹P NMR and once the imidazolate peak had disappeared, the reaction was diluted with MilliQ water and applied on DEAE sepharose. Isolation of the NAD analogues was performed using ion-exchange resin (DEAE Sepharose Fast Flow) using gradient MilliQ water against 0.1 M ammonium formate buffer (AF) at pH 5 with 10% of MeOH (0–100%).
• 41 Nicotinamide Benzimidazole Dinucleotide (25): The product was isolated after 48 h in 32% of yield. ¹H NMR (400 MHz, D₂O): δ = 9.12 (s, 1 H), 8.98 (d, J = 6.2 Hz, 1 H), 8.61 (d, J = 8.1 Hz, 1 H), 8.38 (s, 1 H), 7.94–8.07 (m, 1 H), 7.58 (d, J = 7.58, 15.6 Hz, 2 H), 7.25–7.34 (m, 3 H), 6.98 (d, J = 7.8 Hz, 1 H), 5.91 (dd, J = 5.9, 9.3 Hz, 2 H), 3.98–4.51 (m, 10 H). ¹³C NMR (100 MHz, D₂O): δ = 180.0, 171.0, 145.4, 142.1, 142.1, 139.7, 133.4, 129.9, 128.5, 124.2, 123.5, 121.6, 119.1, 114.9, 111.5, 99.9, 88.2 (d), 83.7 (d), 77.5, 72.9, 70.4, 70.10, 65.59 (d), 64.81 (d). ³¹P NMR (162 MHz, D₂O): δ = –10.95 to –12.08 (m, P–O–P). MS (ES): m/z [M – H]^– calcd for C₂₃H₂₈N₄O₁₄P₂: 646.1077; found: 646.1078. HPLC: t _R = 7.17 min.
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• 43 Nicotinamide 5-Phenylbenzimidazole Dinucleotide (26): The product was isolated after 48 h in 43% of yield. ¹H NMR (400 MHz, D₂O): δ = 8.86 (s, 1 H), 8.67 (s, 1 H), 8.58 (s, 2 H), 8.09 (s, 1 H), 7.85 (s, 1 H), 7.29–7.68 (m, 9 H), 5.91 (s, 1 H), 5.52 (s, 1 H), 3.94–4.42 (m, 10 H). ³¹P NMR (162 MHz, D₂O): δ = –11.09 to –11.60 (m, P–O–P). HPLC: t _R = 8.87 min. MS (ES): m/z [M]⁺ calcd for C₂₉H₃₃N₄O₁₄P₂ ⁺: 723.1469; found: 723.1436.
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