Syntheses of ¹⁵N-labeled pre-queuosine nucleobase derivatives

Abstract

Pre-queuosine or queuine (preQ₁) is a guanine derivative that is involved in the biosynthetic pathway of the hypermodified tRNA nucleoside queuosine (Que). The core structure of preQ₁ is represented by 7-(aminomethyl)-7-deazaguanine (preQ₁ base). Here, we report the synthesis of three preQ₁ base derivatives with complementary ¹⁵N-labeling patterns, utilizing [¹⁵N]-KCN, [¹⁵N]-phthalimide, and [¹⁵N₃]-guanidine as cost-affordable ¹⁵N sources. Such derivatives are required to explore the binding process of the preQ₁ base to RNA targets using advanced NMR spectroscopic methods. PreQ₁ base specifically binds to bacterial mRNA domains and thereby regulates genes that are required for queuosine biosynthesis.

Introduction

The small pyrrolo[2,3-d]pyrimidine 7-(aminomethyl)-7-deazaguanine is a natural product, also termed prequeuosine base (preQ₁ base) [1–2]. This guanine derivative is involved in the complex biosynthetic pathway of the hypermodified tRNA nucleoside queuosine [3]. Recently, preQ₁ base has attracted considerable attention because this nucleobase specifically binds to bacterial mRNA domains and regulates genes that are required for queuosine biosynthesis, by a so-called riboswitch mechanism [4–8]. To explore the binding process of preQ₁ base to the RNA and to shed light on the dynamics underpinning this process advanced NMR spectroscopic methods exist for which ¹⁵N-labeled preQ₁ base derivatives would be highly beneficial. Here, we report efficient routes for the synthesis of three derivatives with complementary ¹⁵N-labeling patterns (Scheme 1).

Scheme 1.

The hypermodified nucleoside queuosine (Q) and the synthetic targets of preQ₁ bases 1 to 3 with complementary ¹⁵N labeling patterns for potential NMR spectroscopic applications. Purine and systematic numbering as indicated.

The synthesis of preQ₁ base has been described first in 1979 by Goto and coworkers from 2-methylthio-6-methoxy-7-methyl-7-deazapurine in 13 steps [9]. Another early, but more efficient procedure was reported by Nishimura in 1988 based on the Mannich reaction using dibenzylamine–formaldehyde and 2-acylaminopyrrolo[2,3-d]pyrimidin-4(3H)-one, which resulted in the selective introduction of the dibenzylaminomethyl group [10]. The following amine exchange reaction of the dibenzylamine function in the Mannich base with ammonia resulted in the preQ₁ base. More recently, Carell and coworkers developed a straightforward pathway based on the key reaction of in situ α-brominated 3-phthalimidopropanal with 2,6-diaminopyrimidin-4-one [11], inspired by Grubb’s synthesis of the Q base (queuine) [12]. Alternatively, Klebe and coworkers employed a Michael addition of the same pyrimidinone to the nitroolefin 2-[(2E)-3-nitroprop-2-en-1-yl]-1H-isoindole-1,3(2H)-dione [13], however, this route seemed inconvenient for our purposes because access to the nitroolefin requires several additional steps.

Results and Discussion

Our aim was to develop a robust synthetic pathway to the preQ₁ derivatives with the three complementary ¹⁵N labeling patterns depicted in Scheme 1. For this undertaking we considered Carell’s synthesis [11] of preQ₁ base as a solid foundation that we intended to adapt and modify accordingly, under the premises of efficacy and cost-minimization for ¹⁵N incorporation.

For [¹⁵N1,¹⁵N3,H₂¹⁵N(C2)]-7-(aminomethyl)-7-deazaguanine (1), we started with the reaction of methyl cyanoacetate (4) and [¹⁵N₃]-guanidine hydrochloride (5) under basic conditions to give the corresponding [¹⁵N1,¹⁵N3,H₂¹⁵N(C2)]-2,6-diaminopyrimidin-4-one (6) in high purity after work-up and reversed-phase column chromatography (C18) (Scheme 2) [14]. Then, the α-bromo aldehyde 7 was obtained in two steps from commercially available 3-phthalimidopropan-1-ol that was oxidized using Dess–Martin periodinane. Subsequent in situ bromination of the 3-phthalimidopropan-1-al with CH₃SiBr, described previously by others [11–12], did not work reliable in our hands.

Scheme 2.

Synthesis of [¹⁵N1,¹⁵N3,H₂¹⁵N(C2)]-preQ₁ base (1). a) CH₃ONa (10 equiv) CH₃OH, reflux, 10 h, RP C18 chromatography, 35%. b) NaOAc·3H₂O (2 equiv), acetonitrile/water, 40 °C, 4 h, 63%. c) H₂N-NH_2·H₂O (10 equiv), ethanol, reflux, 14 h, RP C18 chromatography, 75%. Compound 1 was isolated as salt of trifluoroacetic acid (TFA).

Therefore, in anlogy to Grubb [15] and a more detailed protocol by Yamaguchi [16], we applied 5,5-dibromobarbituric acid [17] to obtain the α-bromo aldehyde 7 which was well stable during purification by column chromatography on SiO₂ and isolated in good yields. The pyrrolo[2,3-d]pyrimidine ring system of preQ₁ base was built in good yields via the cyclocondensation reaction between [¹⁵N1,¹⁵N3,H₂¹⁵N(C2)]-2,6-diaminopyrimidin-4-one (6) and the 2-bromo-3-phthalimidopropan-1-al (7). Finally, deprotection was performed with hydrazine hydrate. The previously published route [11] recommended N-Boc functionalization of the preQ₁ base in the crude reaction mixture to enable flash chromatography on SiO₂ followed by cleavage of the auxiliary function, however, although robust in handling, the yields were rather modest. We therefore decided to directly purify the crude product by reversed-phase column chromatography (HPLC) and obtained compound 1 in excellent yield and purity. We mention that compound 1 was isolated as salt of trifluoroacetic acid (TFA) using 1% TFA in the eluent and it was assumed to exist in 1:1 stoichiometry (preQ₁:TFA) based on ¹H NMR spectra and consideration of pK_a values. However, it is noteworthy that a crystal structure of the preQ₁·TFA salt that was crystallized from saturated aqueous solution showed the co-existence of mono- (N(C'7)) and dications (N(C'7), N3) in the crystal [11].

The synthetic track for the [¹⁵N₃]-preQ₁ base (1) was designed with the concept in mind to access the complementary ¹⁵N patterns of [¹⁵N9]-preQ₁ base (2) and [H₂¹⁵N(C7')]-preQ₁ base (3) by employing the same key steps. In this sense, the key intermediate [H₂¹⁵N(C6)]-2,6-diaminopyrimidin-4-one (13) for target 2 was accessible by first synthesizing ethyl [¹⁵N]-2-cyanoacetate (11) from 2-bromoacetic acid (9) and potassium cyanide [¹⁵N]-KCN, followed by esterification (Scheme 3).

Scheme 3.

Synthesis of [¹⁵N9]-preQ₁ base (2). a) [¹⁵N]-KCN (1 equiv), Na₂CO₃, H₂O, pH 9, 80 °C, 3 h, then room temperature, 20 h, 90%. b) Ethanol (5 equiv), H₂SO₄ cat., reflux, 18 h, 92%. c) CH₃ONa (10 equiv) CH₃OH, reflux, 10 h, RP C18 chromatography, 40%. d) NaOAc·3H₂O (2 equiv), acetonitrile/water, 40 °C, 4 h, 52%. e) H₂N-NH_2·H₂O (10 equiv), ethanol, reflux, 14 h, RP C18 chromatography, 92%. Compound 2 was isolated as salt of trifluoroacetic acid (TFA).

All further steps were conducted in direct analogy as described for target 1, namely reaction with guanidine hydrochloride to furnish compound 13, followed by cyclocondensation with 2-bromo-3-phthalimidopropan-1-al (7) to give the protected [¹⁵N9]-preQ₁ base 14 for subsequent deprotection yielding the desired [¹⁵N9]-preQ₁ base (2) (Scheme 3).

Also for the third target, [H₂¹⁵N(C7')]-preQ₁ base (3), our strategy turn out to be highly convenient. First, we prepared the ¹⁵N-labeled aldehyde 18 as the key intermediate (Scheme 4). This was achieved by reaction of 3-chloropropanol (15) with [¹⁵N]-phthalimide 16 to give [¹⁵N]-3-phthalimidopropan-1-ol (17). All further steps were in direct analogy as described for targets 1 and 2, namely reaction with 5,5-dibromobarbituric acid [17] to obtain [¹⁵N]-2-bromo-3-phthalimidopropan-1-al (19), followed by cyclocondensation with commercially available 2,6-diaminopyrimidin-4-one (20) to give the protected [¹⁵N(C7')]-preQ₁ base 21 for subsequent deprotection yielding the desired [¹⁵N(C7')]-preQ₁ base (3) (Scheme 4).

Scheme 4.

Synthesis of [H₂¹⁵N(C7')] preQ₁ base (3). a) K₂CO₃ (1.5 equiv), DMF, 70 °C, 14 h, 47%. b) Dess–Martin periodinane (1.5 equiv), CH₂Cl₂, 3 h, room temperature. c) 5,5-dibromobarbituric acid (0.6 equiv), acetonitrile, reflux, 2 h, 45%. d) NaOAc·3H₂O (2 equiv), acetonitrile/water, 40 °C, 4 h, 58%. e) H₂N-NH_2·H₂O (10 equiv), ethanol, reflux, 14 h, RP C18 chromatography, 82%. Compound 3 was isolated as salt of trifluoroacetic acid (TFA).

Finally, a direct comparison of ¹H NMR spectra of the three ¹⁵N labeled preQ₁ bases synthesized here is provided in Figure 1.

Figure 1.

Comparison of ¹H NMR spectra of the preQ₁ bases 1, 2 and 3 with complementary ¹⁵N labeling patterns. Conditions: c_{preQ1 base} = 1 mM; DMSO-d₆, 298 K.

Conclusion

In this short note, an efficient and cost-minimizing route for ¹⁵N labeled preQ₁ base derivatives has been described. The synthesis of the pyrrolo[2,3-d]pyrimidine ring system is based on the cyclocondensation reaction between α-bromoaldehydes and 2,6-diaminopyrimidin-4-ones and utilizes [¹⁵N]-KCN, [¹⁵N]-phthalimide, and [¹⁵N₃]-guanidine for ¹⁵N sources to achieve three complementary labeling patterns that cover all five nitrogen atoms of preQ₁ base. The new derivatives carry the potential for modern NMR spectroscopic applications to study the recognition process of these small molecules with RNA aptamer domains from the three preQ₁ riboswitch classes known to this date [4–5,18].

Experimental

General.Chemical reagents and solvents were purchased from commercial suppliers (Sigma-Aldrich) and used without further purification. Organic solvents for reactions were dried overnight over freshly activated molecular sieves (4 Å). The reactions were carried out under argon atmosphere. Analytical thin-layer chromatography (TLC) was carried out on Marchery-Nagel Polygram SIL G/UV254 plates. Column chromatography was carried out on silica gel 60 (70–230 mesh). Reversed-phase column chromatography was performed on a GE Healthcare Äktaprime system using a commercial Götec-Labortechnik GmbH 310-25 LiChroprep RP-18 (40–63 µm) column (Merck Lobar compatible). The LC separation was monitored by ultraviolet (UV) detection at 280 nm. Solvent systems were as described below for the individual compounds. ¹H and ¹³C NMR spectra were recorded on Bruker DRX 300 MHz and Bruker Avance II+ 600 MHz instruments. The chemical shifts (δ) are reported relative to tetramethylsilane (TMS) and referenced to the residual proton or carbon signal of the deuterated solvent: CDCl₃ (7.26 ppm), DMSO-d₆ (2.49 ppm), for ¹H NMR spectra; CDCl₃ (77.0 ppm) or DMSO-d₆ (39.5 ppm) for ¹³C NMR spectra. ¹H and ¹³C assignments are based on COSY and HSQC experiments. MS experiments were performed on a Bruker 7T FT-ICR instrument with an electrospray ion source. Samples were analyzed in the positive-ion mode.

[¹⁵N1,¹⁵N3,H₂¹⁵N(C2)]-2,6-Diaminopyrimidin-4(3H)-one (6). To a solution of methyl cyanoacetate (4, 90 µL, 1.02 mmol) and [¹⁵N₃]-guanidine hydrochloride (5, 100 mg, 1.02 mmol) in methanol (6.6 mL) was added dropwise NaOCH₃ (0.53 g, 9.85 mmol) in methanol (4.1 mL). After the addition was complete, the mixture was refluxed for 10 hours and allowed to cool to room temperature. The mixture was filtrated, and the filtrate evaporated to dryness. The residue was redissolved in water (1 mL) at 90 °C. The yellow solution was then acidified to pH 6 by acetic acid. The crude product was purified by reversed-phase (C18) column chromatography (eluent A: water, eluent B: acetonitrile; 0–15% B in 40 min, 5 mL/min) as eluent to yield 46 mg of compound 6 (35%) as a yellow solid. ¹H NMR (300 MHz, DMSO-d₆) δ 4.41 (s, 1H, CH), 5.82 (s, 2H, NH₂), 6.70 (d, J = 89.03 Hz, 2H, ¹⁵NH₂), 7.91 (d, J = 89.51 Hz, 1H, ¹⁵NH) ppm.

[¹⁵N1,¹⁵N3,H₂¹⁵N(C2)]-2-[(2-Amino-4,7-dihydro-4-oxo-1H-pyrrolo[2,3-d]pyrimidin-5-yl)methyl]-1,3-dihydro-2H-isoindole-1,3-dione (8). Compound 6 (46 mg, 0.37 mmol) and α-bromoaldehyde 7 [15] (100 mg, 0.37 mmol) were suspended in a mixture of acetonitrile and water (1.6 mL; 1:1). Sodium acetate trihydrate (97 mg, 0.72 mmol) was added, and the suspension stirred at 40 °C. After 10 minutes, all solids were disolved and a yellow solution was obtained which rapidly turned into a suspension again, indicating that product 8 started to precipitate. The mixture was stirred for 4 hours, then cooled to room temperature and filtered. The residue was dried under reduced pressure to yield 70 mg of compound 8 (63%) as yellow solid. ¹H NMR (300 MHz, DMSO-d₆) δ 4.84 (s, 2H, CH₂-N), 6.16 (d, J = 88.96 Hz, 2H, ¹⁵NH₂-C2), 6.36 (s, 1H, H-C8), 7.85–7.87 (m, 4H, arom H), 10.42 (d, J = 89.21 Hz, 1H, ¹⁵N1-H), 10.81 (s, 1H, NH) ppm.

Trifluoroacetate salt of [¹⁵N1,¹⁵N3,H₂¹⁵N(C2)]-7-(aminomethyl)-7-deazaguanine (1). Compound 8 (70 mg, 0.22 mmol) was added to a solution containing hydrazine hydrate (111 µL, 2.2 mmol) and ethanol (3 mL). The mixture was refluxed overnight and evaporated to dryness. The residue was purified by reversed-phase (C18) column chromatograpy (eluent A: 1% trifluoroacetic acid in H₂O; eluent B: acetonitrile; 0–20% B in 50 min, 4 mL/min) to yield 49 mg of compound 1 (75%; calculated as mono TFA salt) as a light yellow solid. ¹H NMR (300 MHz, DMSO-d₆) δ 4.01 (s, 2H,CH₂), 6.30 (d, J = 89.1 Hz, 2H, ¹⁵NH₂-C2), 6.78 (s, 1H, H-C8), 8.18 (s, 3H, NH₃⁺), 10.82 (d, J = 89.6 Hz, 1H, ¹⁵N1-H), 11.20 (s, 1H, NH) ppm; ¹³C NMR (75 MHz, D₂O) δ 34.57 (CH₂NH₃⁺), 98.32 (C7), 110.29 (C5), 113.88 (C2), 118.55 (C8), 141.05 (C4), 150.60 (C6=O), 159.09 (COO⁻), 162.18 (CF₃) ppm; HRMS–ESI m/z: [M + H]⁺ calcd for C₇H₉N₂¹⁵N₃O, 183.07909; found, 183.07865.

UV spectroscopic analysis of unlabeled 7-(aminomethyl)-7-deazaguanine (preQ₁ base): UV (H₂O) λ_max (ε) = 218 (13630), 258 (7940) nm. For comparison, see UV spectroscopic data and extinction coefficients of 7-deazaguanine, N9-methyl-7-deazaguanine and preQ₁ nucleoside in references [19–21].

Supporting Information

¹H and ¹³C NMR spectra are provided in Supporting Information File 1. Synthetic procedures for the syntheses of compounds 2, 3, 10, 11, 13, 14, 17–19, and 21.

File 1

Synthetic procedures and NMR spectra of the most typical compounds.

This article is part of the Thematic Series "Nucleic acid chemistry".