Synthesis of N²-modified 7-methylguanosine 5’-monophosphates as nematode translation inhibitors

Abstract

Preparative scale synthesis of fourteen new N²-modified mononucleotide 5’ mRNA cap analogues was achieved. The key step involved use of an S_NAr reaction with protected 2-fluoro inosine and various primary and secondary amines. The derivatives were tested in a parasitic nematode, Ascaris suum, cell-free system as translation inhibitors. The most effective compound with IC₅₀ ~ 0.9 μM was a N²-p-metoxybenzyl-7-methylguanosine-5’-monophosphate 35.

1. Introduction

Methylation of the exocyclic amino group of guanosine is an important biological modification of RNAs. Modified nucleosides occur frequently in RNAs including N²-methyl- (m²G) and N²,N²-dimethylguanosine (m₂^2,2G) within ribosomal (rRNA)¹ and transfer (tRNA)² RNAs, respectively. N²,N²-dimethylG as a part of the RNA cap structure is also found at the 5’ end of small nuclear RNAs (U snRNA) and some messenger RNAs (mRNA). Eukaryotic mRNAs possess a 5’ terminal cap structure consisting of a 7-methylguanosine linked via a 5’,5’-triphosphate bridge to the first transcribed nucleotide (m⁷GpppN, where N=G, A, C or U; MMG cap).³ In nematodes, however, two different mRNAs co-exist in cells. In addition to mRNAs with the common MMG cap, about 70% of the mRNAs possess an atypical, hypermethylated cap. This cap (TMG-cap, m₃^2,2,7GpppG) has the N⁷-methyl as well as N²,N²-dimethyl in its structure and is added along with a 22 nt spliced leader sequence during trans-splicing.⁴

The cap plays an essential role in several processes during gene expression⁵ by interacting with proteins that specifically recognize its structure.⁶ The best-characterized cap-binding protein is the translation initiation factor eIF4E. Recognition of the mRNA cap by eIF4E is the critical, rate limiting step for efficient translation initiation, and it is a major target for translational control.⁷ It has been shown that eIF4E is a potent oncogene and its overexpression is associated with a variety of human cancers.⁸ Consequently, cap analogs as specific inhibitors to counteract elevated eIF4E level in tumor cells have been explored.⁹

Many nematodes, including Ascaris suum, are parasitic, infect over 2 billion people, and remain a significant health problem. The role of trans-splicing as a mechanism of nematode gene expression and the effect of the TMG cap and a spliced leader addition on mRNA metabolism have been intensively investigated.¹⁰ Ascaris cap-binding proteins must deal with two distinct populations of mRNA suggesting these proteins are unique and may be targets for the discovery of novel cap analogs that can specifically block parasite gene expression. We recently prepared and analyzed a guanosine derivative with a benzyl at N² position (bn²m⁷GMP, 32) to examine the intermolecular interaction of MMG/TMG caps with Ascaris eIF4E-3.¹¹ The addition of an N²-benzyl substituent on a monophosphate led to significant translation inhibition in an Ascaris suum embryo cell-free system. Therefore, we chose to extend these analyses to several other N² modified derivatives.

In the present study we designed and synthesized a series of N²-substituted 7-methylguanosine 5’-monophosphates. This type of cap analog has not been widely studied due to difficulties with the development of a good method to efficiently introduce substituents at the N² position of guanosine. To achieve our goal we used a six step strategy starting from guanosine using an S_NAr reaction with fully protected 2-fluoro inosine and various primary and secondary amines. The synthesized mononucleotide TMG cap analogues were tested in a parasitic nematode, Ascaris suum, cell-free system as translation inhibitors.

2. Results and discussion

2.1 Chemistry

In order to synthesize N²-substituted mononucleotide cap analogues, we adopted and modified a method for the preparation of the N²,N²-dimethylguanosine by Eritja et al.¹² The synthesis was carried out in six steps using inexpensive, commercially available guanosine as the starting material. The synthesis began with the triacetylation of guanosine using acetic anhydride (Ac₂O) in the presence of triethylamine (TEA) and N,N-(dimethylamino)pyridine (DMAP) using a procedure modified from Nair et al.¹³ By carrying out the addition of the Ac₂O at 0°C, N-acetylation was suppressed and 2’,3’,5’-tri-O-acetylguanosine (1) was obtained in a near quantitative yield (98%). In the next step, protection of the O⁶ group of guanosine was carried out with a p-nitrophenylethyl (NPE) group as it is believed to be stable under mild acid and base hydrolyses, and it can be easily cleaved by DBU or DBN in a β-elimination reaction. The reaction was carried out via the Mitsunobu reaction with p-nitrophenylethanol, diisopropyl azodicarboxylate (DIAD), and triphenylphosphine (TPP) in anhydrous toluene at room temperature.¹⁴ A long standing problem with the Mitsunobu reaction is the purification of the desired product from the reaction mixture. We tried replacing triphenylphosphine and diisopropyl azodicarboxylate (which are converted during the reaction into triphenylphosphine oxide and related dialkylhydrazinodicarboxylate, respectively) by other reagents to facilitate the isolation and purification processes.¹⁵ Our observations showed that obtaining pure product was possible using standard reagents but the purification required a very long chromatography process. In order to shorten the isolation of the 2’,3’,5’-tri-O-acetyl-O⁶-[2-(4-nitrophenyl)ethyl] guanosine (2), we partially purified the final product and then used 2 with some impurities (mainly triphenylphosphine oxide) in the next step. By-products remaining after the Mitsunobu reaction were easily removed during the purification of 3. This faster procedure allowed us to obtain a pure fluorinated derivative with good yield (85%).

The main route to N²-substituted guanosine analogues is via nucleophilic displacement of a halogen at the 2 position of inosine. Since fluoro derivatives are more reactive toward nucleophilic displacement in a S_NAr reaction than other halogen derivatives,¹⁶ the fully protected 2’,3’,5’-tri-O-acetyl-O⁶-[2-(4-nitrophenyl)ethyl] guanosine (2) was transformed into N²-fluoro-2’,3’,5’-O-triacetyl-O⁶-[2-(4-nitrophenyl)ethyl] inosine (3). We tested three methods for diazotiation and fluorination of nucleosides. The first was to prepare the 2-fluoro derivative using aqueous diazotization of guanosine in the presence of fluoroboric acid.¹⁷ These conditions, however, were too harsh and led to depurination. A second approach was the introduction of the fluorine atom under anhydrous conditions with t-butyl nitrite as the diazotizing agent and HF in pyridine as the fluoride source.¹⁸ The reaction proceeded smoothly in very good yield (85%), and the main product remained fully protected. However, without careful monitoring, this reaction led to depurination or even failure to obtain product unless very high quality HF/pyridine reagent was used and a low temperature was maintained during the course of the reaction. Consequently, we also explored milder fluorination conditions using polyvinylpyridinium polyhydrogenfluoride (PVPHF) reagent developed by Olah et al.¹⁹ The advantages of this method were easy handling and an extremely convenient work-up, however, yields were limiting. Consequently, the most efficient way to prepare N²-fluoro-2’,3’,5’-O-triacetyl-O⁶-[2-(4-nitrophenyl)ethyl]inosine (3) is the procedure that involves using HF/pyridine for the substitution step.

The 2-fluoro intermediate was used directly in a S_NAr reaction by treating it with two-fold excess of primary or secondary, aliphatic or aromatic amines varying in size and steric branching in anhydrous DMSO (Scheme 2). Incorporated amines were chosen to produce several N² substituents (aliphatic, cyclic, or aromatic) in the cap to vary the size and steric branching of aliphatic N² substituents (compounds 11-17) and to influence the electron density of the aromatic ring through the introduction of various substituents, such as metoxy (7), chloro (8), nitro (9), or phenyl (10), into the benzene ring. In addition to the mono substituted analogues, we also used some secondary amines and synthesized compounds bearing additional methyl (5, 11, 13, 16), ethyl (15) or benzyl (6) groups at the N² position to check the effectiveness of these substituents on the cap as translation inhibitors. The nature of the nucleophile is the most critical parameter in the S_NAr reaction. Therefore, for the simple primary amines, the substitution reaction occurred rapidly and the fluorine derivative was used up in less than 30 min at temperature not higher than 50°C. The reaction with sterically hindered and secondary amines such as dibenzylamine required longer time (in some cases even 2 days) and a higher temperature (65°C). After completion of the substitution reaction 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) was added to the reaction mixture to remove the NPE group followed by the addition of a mixture of THF/MeOH/NaOH_aq in order to complete acetyl deprotection. Chromatographic purification on silica gel yielded pure nucleoside derivatives 4-17 in good yield. It is worth mentioning, that in course of the reaction progress, we observed that all protecting groups (acetyl and NPE) were slowly lost if the substitution reaction was carried out under prolonged heating in the presence of excess amine. This observation led us to a simpler purification procedure in which full deprotection took place without any DBU and final N²-substituted guanosine derivatives were easily crystallized from the reaction mixture by the addition of CH₂Cl₂.

Scheme 2.

Preparation of N²-modified guanosine derivatives via a S_NAr reaction; (a) amine, DMSO, 50°C to 65°C.

^a numbers in brackets correspond to compounds on Scheme 3

All the N²-modified guanosine analogues were further 5’ phosphorylated using the Yoshikawa method²⁰ with phosphorus oxide trichloride in trimethyl phosphate at 4°C and subsequently methylated at the N⁷ position of the guanine ring with CH₃I in DMSO at RT²¹ (Scheme 3). The reaction progress of the last step was monitored by HPLC to avoid over methylation. Purification of the analogues was done using ion-exchange chromatography on DEAE–Sephadex A-25 (HCO₃^- form) and the derivatives were subsequently converted into the sodium salt using Dowex 50WX8. The structure and homogeneity of final products were confirmed by HPLC, mass spectrometry, ¹H NMR and ³¹P NMR.

Scheme 3.

Preparation of N²-modified mononucleotide cap analogues (a) POCl₃, trimethyl phosphate, 4°C (b) methyl iodide, DMSO, RT.

^aR corresponds to various substituents at N2 position of guanosine (see Scheme 2, numbers in brackets)

2.2 Biology

One of the synthesized compounds, bz²m⁷GMP (32), has been recently used in Ascaris suum eIF4E-3/m⁷G- and m₃^2,2,7G- cap binding studies.¹¹ It was shown that bz²m⁷GMP binds to the protein with a 10-fold higher affinity compared with m⁷GMP and is a strong inhibitor of either a m⁷G- or m₃^2,2,7G- capped mRNA translation in a cell-free Ascaris suum translation system. To evaluate the ability of newly synthesized N²-modified 7-methylguanosine 5’-monophosphates to inhibit cap-dependent translation in a cell-free translation system where naturally occurring mono- and trimethylated mRNAs are translated, we tested fourteen cap analogues (32-45) in the same manner as previously reported.^10c,11 Two standard cap analogues were used as internal controls in the experiments: m⁷GTP as a positive control as it is an effective translation inhibitor in various cell-free systems (rabbit reticulocyte,^9a wheat germ,²³ Ascaris suum^10c) and ApppG as a negative control that have very little or no inhibitory efficacy due to lack of the positive charge on guanine that is generated by the N⁷ methyl group. As previously shown, the inhibitory potency of cap analogues generally increases with the length of polyphosphate bridge.^9a Therefore, in order to determine the strength of our new mononucleotide cap analogues we also compared them to m⁷GMP which is known as a weak translation inhibitor. The results (Figure 1, Table 1) indicated that any mono substitution at the N² position of guanine (alkyl – aliphatic, cyclic, or aryl) produced inhibitory compounds with IC₅₀ similar to m⁷GTP. All alkyl substituents led to similar levels of inhibition regardless of their size and steric branching. Aryl substituted compounds were more effective as translation inhibitors. The strongest inhibitor, about 7 times stronger than m⁷GTP, was derivative 35 with an IC₅₀ of 0.9 μM. The electron withdrawing properties of para substituents (NO₂ > Cl > Phe > OCH₃) were roughly correlated with the IC₅₀ of the compounds (12.6 μM, 3.8 μM, 4.3 μM, 0.9 μM, respectively). The enhanced π electron density of the benzene ring increases the effectiveness of cap analogues as translational inhibitors, but other factors such as the size of substituent may also play an important role. Recent studies on the co-crystal structure of m₃^2,2,7GTP with Ascaris eIF4E-3¹¹ indicated that the two methyl groups at the N² position of the base are solvent exposed and do not interact directly with the protein. The N² substituents may form additional interactions with eIF4E influencing conformational changes known to form the binding pocket²⁴ or may change the thermodynamic aspects of the protein binding to the 5’ cap.

Figure 1. Translation inhibition assay in A. suum extract.

The synthesized cap analogues were assayed for their ability to inhibit cap-dependent translation of an m⁷GpppG-capped Renilla luciferase mRNA in an Ascaris suum embryo cell-free translation system. The measurements were carried out as previously described and the % translation activity plotted against the inhibitor concentration.^10c All measurements were made in triplicate in several preparations of extracts. Data presented are representative experiments.

Table 1.

Inhibition of translation in A. suum extract by N²-modified cap analogues

	R =	IC50		R =	IC50
32		2.9 μM	39		>50 μM
33		>50 μM	40		3.9 μM
34		>50 μM	41		>50 μM
			42		4.5 μM
35		0.9 μM	43		>50 μM
36		3.8 μM	44		13.9 μM
37		12.6 μM	45		5.7 μM
38		4.3 μM		m7GTP	6.1 μM
				m7GMP	>50μM

IC₅₀ values were extracted directly from the plotted of % translation activity values against compound concentration. IC₅₀ values > 50 μM - no measurable effect.

Our results also showed that the addition of a second substituent at the N² position of guanosine decreased the level of inhibition significantly (e. g compounds 32 versus 33 or 39 versus 40). These data are consistent with a comparison of the crystal structures of Ascaris eIF4E-3 with m⁷GTP or m₃^2,2,7GTP.¹¹ These studies demonstrated that the addition of the second substituent at the N² position of guanosine leads to the loss of one hydrogen bond between the amine group of guanine and the carboxyl group of Glu116. This loss of a hydrogen bond likely influences the effectiveness of analogues as translation inhibitors.

3. Conclusions

We have prepared fourteen novel N² modified trimethylguanosine cap analogues via a six-step synthesis from guanosine. This general strategy provides a new approach to scale-up the synthesis of a large number of modified cap analogues that should be useful in studying eIF4E function and cap-dependent translation. In addition the idea of exploring cap analogues that possess only one phosphate that typically have little inhibitory activity offers an opportunity to explore compounds that are not highly charged and can be good candidates for new drug development.

4. Materials and methods

All reagents were the highest available purity and purchased from Sigma-Aldrich Chemical Co. Triethylammonium bicarbonate (TEAB) buffer was prepared by bubbling CO₂ through an ice-cold aqueous solution of redistilled triethylamine. Intermediate nucleotides were separated by ion-exchange chromatography on a DEAE-Sephadex A-25 (HCO^3- form) using a linear gradient of TEAB buffer, pH 7.6. Fractions containing products were combined and evaporated under reduced pressure with several additions of ethanol and isolated as triethylammonium salts (TEA salts) and subsequently converted into the sodium salt using Dowex 50WX8 (Na⁺ form). Homogeneity of the final analogues was checked by reversed-phase analytical HPLC. HPLC was performed using a Supelcosil LC-18-T RP column (4.6 × 250 mm, flow rate 1.0 mL/min) with: Method A – a linear gradient of methanol from 0 to 50% (v/v) in 0.05 M ammonium acetate (pH 5.9) in 20 minutes, an isocratic elution of 50% methanol (v/v) in 0.05 M ammonium acetate (pH 5.9) till 30 minutes, Method B – a linear gradient of methanol from 0 to 50% (v/v) in 0.05 M ammonium acetate (pH 5.9) in 10 minutes and then an isocratic elution of 50% methanol (v/v) in 0.05 M ammonium acetate (pH 5.9) till 30 minutes, Method C – an isocratic elution of 50% methanol (v/v) in 0.05 M ammonium acetate (pH 5.9), on a Knauer instrument, with UV detection at 254 nm. MS spectra were acquired using Waters Micromass Q-TOF Premier spectrometer with positive electrospray ionization source. ¹H and ¹³C NMR spectra of intermediate derivatives were obtained with a Varian UnityPlus 200 MHz spectrometer. ¹H and ³¹P NMR spectra of the final compounds were recorded on a Varian INOVA 700 MHz spectrometer.

4.1 Experimental

4.1.1 2’,3’,5’-tri-O-acetylguanosine (1)

2.2 mL of acetic anhydride (22 mmol) was added dropwise to a suspension of guanosine (2 g, 7.0 mmol) (dried for 2 days over P₄O₁₀ in high vacuum), triethylamine (7.7 mL, 55.2 mmol) and N,N-(dimethylamino)pyridine (92 mg, 0.75 mmol) in 27 mL of acetonitrile at 0°C. The mixture was stirred until it became homogeneous and kept an additional 3 hr at room temperature. The reaction was quenched with methanol (2.3 mL). The volume was reduced to 1/3 using a rotary evaporator and diethyl ether was added dropwise to induce precipitation of a fine white powder. The product was collected by filtration, washed with diethyl ether, and then stirred for 2 hr with acetone (30 mL) at 50°C. The filtrate produced 2.8 g (98%) of a fine white powder. ¹H NMR (200 MHz, DMSO) δ 10.54 (s, 1H, NH) 7.93 (s, 1H, H-8), 6.34 (br, 2H, NH₂), 5.98 (d, J = 6.1 Hz, 1H, H-1’), 5.79 (t, J = 5.8 Hz, 1H, H-2’), 5.51 – 5.48 (m, 1H, H-3’), 4.40 – 4.37 (m, 1H, H-4’), 4.33 – 4.31 (m, 1H, H-5’), 4.23 – 4.28 (m, 1H, H-5”), 2.11 (s, 3H, CH₃, acetyl), 2.05 (s, 3H, CH₃, acetyl), 2.04 (s, 3H, CH₃, acetyl); ¹³C NMR (200 MHz, CDCl₃) δ 172.1 (C=O), 171.0 (C=O), 169.9 (C=O), 156.3 (C-6), 152.8 (C-2), 151.2 (C-4), 137.6 (C-8), 119.6 (C-5), 86.1 (C-1’), 82.3 (C-4’), 73.6 (C-2’), 72.3 (C-3’), 61.8 (C-5’), 21.1 (C, acetyl), 20.6 (C, acetyl), 20.2 (C, acetyl); m/z: calcd for C₁₈H₂₁N₅O₅ (M + H)⁺: 410.1306, found: 410.1305

4.1.2 2’,3’,5’-tri-O-acetyl-O⁶-[2-(4-nitrophenyl)ethyl]guanosine (2)

A suspension of 2’,3’,5’-tri-O-acetylguanosine (2.37 g, 5.8 mmol), triphenylphosphine (2.28 g, 8.7 mmol) and 2-(4-nitrophenyl)ethanol (1.45 g, 8.7 mmol) in anhydrous toluene was stirred for 30 min and diisopropyl azodicarboxylate (1.4 mL) was added dropwise over a period of 45 min. The reaction mixture was kept for 12 hours at RT. Then the solvent was evaporated and the residual oil was purified by column chromatography on silica gel with chloroform to produce a pure product of yellowish crystals, 2.26 g (70%). ¹H NMR (200 MHz, CDCl₃) δ 8.17 (d, 2H, Ph), 7.72 (s, 1H, H-8), 7.49 (d, 2H, Ph), 6.05 – 5.91 (m, 2H, H-1’, H-2’), 5.87 – 5.75 (m, 1H, H-3’), 4.73 (t, J = 6.7 Hz, 2H, O-CH₂, NPE), 4.50 – 4.36 (m, 3H, H-4’, H-5’, H-5”), 3.28 (t, 6.7 2H, CH₂Ph, NPE), 2.14 (s, 3H, CH₃, acetyl), 2.09 (s, 3H, CH₃, acetyl), 2.08 (s, 3H, CH₃, acetyl); ¹³C NMR (200 MHz, CDCl₃) δ 171.7 (C=O), 171.0 (C=O), 169.7 (C=O), 161.9, 155.5 (C-6), 154.5 (C-2), 148.8, 148.6, 138.9 (C-8), 129.3 (C, Ph), 124.8 (C, Ph), 113.6 (C-5), 86.8 (C-1’), 82.3 (C-4’), 73.4 (C-2’), 70.6 (C-3’), 67.1 (OCH₂, NPE), 62.5 (C-5’), 35.5 (CH₂Ph), 21.2 (C, acetyl), 20.6 (C, acetyl), 20.2 (C, acetyl); m/z: calcd for C₂₄H₂₆N₆O₁₀ (M + H)⁺: 559.1783, found: 559.1784

4.1.3 N²-fluoro-2’,3’,5’-O-triacetyl-O⁶-[2-(4-nitrophenyl)ethyl]inosine (3)

Dry 2’,3’,5’-tri-O-acetyl-O⁶-[2-(4-nitrophenyl)ethyl]guanosine (1 g, 1.79 mmol) in polypropylene tube under nitrogen was dissolved in anhydrous pyridine (6.75 mL, 0.082 mol). The tube was placed in a dry ice/acetonitrile cooling bath (-35 to -45°C) and 70% HF/pyridine solution (12 mL, 0.42 mol) was added dropwise over a period of 5 min to 45% final HF. The reaction mixture was stirred for 15 min and t-butyl nitrite (0.54 mL, 4.5 mmol) added. After 1hr, the reaction was quenched at 0°C by slowly pouring the reaction mixture into an aqueous K₂CO₃ solution (28.5 g in 25 mL of water) and then extracted three times with ethyl acetate. The organic layers were collected, dried over anhydrous Na₂SO₄ and evaporated to dryness. Purification by column chromatography using as eluate 60:1 CH₂Cl₂ : MeOH gave 0.85 g (85%) of product; TLC silica gel, CH₂Cl₂ : MeOH, 60:1 R_F = 0.4; ¹H NMR (700 MHz, CDCl₃) δ 8.21 – 8.15 (m, 2H, Ph), 8.08 (s, 1H, H-8), 7.50 (d, 2H, Ph), 6.13 (d, J = 5.6 Hz, 1H, H-1’), 5.82 (t, J = 4.9 Hz, 1H, H-2’), 5.60 – 5.55 (m, 1H, H-3’), 4.84 (t, J = 6.7 Hz, 2H, O-CH₂, NPE), 4.46 – 4.42 (m, 2H, H-4’, H-5’), 4.38 – 4.36 (m, 1H, H-5”), 3.32 (t, J = 6.7 Hz, 2H, CH₂Ph, NPE), 2.15 (s, 6H, CH₃, acetyl), 2.08 (s, 3H, CH₃, acetyl). ¹³C NMR (200 MHz, CDCl₃) δ 170.2 (C=O), 169.5 (C=O), 169.3 (C=O), 160.3, 159.3 (C-6), 154.2 (C-2), 147.1, 145.1, 140.8, 129.9 (Ph), 123.8 (Ph), 86.4 (C-1’), 80.5 (C-4’), 73.0 (C-2’), 72.4 (C-3’), 67.7 (OCH₂, NPE), 62.8 (C-5’), 34.9 (CH₂Ph), 20.7 (C, acetyl), 20.5 (C, acetyl), 20.3 (C, acetyl); m/z: calcd for C₂₄H₂₄FN₅O₁₀ (M + H)⁺: 562.1579, found: 562.1581

4.1.4 General procedure for the synthesis of N²-substitutiuted derivatives 4-17

N²-fluoro-2’,3’,5’-O-triacetyl-O⁶-[2-(4-nitrophenyl)ethyl]inosine (250 mg, 0.45 mmol) was dissolved in 2 mL of anhydrous dimethylsulfoxide (DMSO) and then an amine (0.9 mmol) was added. The reaction mixture was stirred at 60°C from 30 minutes to 2 days until the fluoronucleoside completely disappeared (based on TLC) followed by addition of 0.5M NaOH in THF/MeOH/H₂O (5/4/2). The solvent was removed under high vacuum and the resulting oily residue was treated with CH₂Cl₂ to induce precipitation of a fine white powder. The product was collected by filtration and washed several times with methanol and dried over P₄O₁₀ to yield:

4.1.4.1 N²-benzylguanosine (4)

134 mg, 66%; ¹H NMR (200 MHz, CDCl₃) δ 7.69 (s, 1H, H-8), 7.38 – 7.19 (m, 5H, Ph), 5.66 (d, J=5.6 Hz, 1H, H-1’), 4.64 – 4.62 (m, 1H, H-2’), 4.55 – 4.47 (m, 2H, CH₂Ph), 4.12 – 4.10 (m, 1H, H-3’), 3.89 – 3.84 (m, 1H, H-4’), 3.69 – 3.57 (m, 2H, H-5’); ¹³C NMR (50 MHz, CDCl₃) δ 157.4 (C-6), 153.8 (C-2), 150.6 (C-4), 140.4 (Ph) 134.6 (C-8), 128.0 (Ph), 127.2 (Ph), 126.2 (Ph), 117.1 (C-5), 87.5 (C-1’), 85.1 (C-4’), 73.0 (C-2’), 70.6 (C-3’), 61.7 (C-5’), 44.2 (N-CH₂Ph); m/z: calcd for C₁₇H₁₉N₅O₅ (M + H)⁺: 374.1458, found: 374.1453;

4.1.4.2 N²-benzyl, N²-methylguanosine (5)

109 mg, 52%; ¹H NMR (200 MHz, CDCl₃) δ 7.66 (s, 1H, H-8), 7.26 – 7.14 (m, 5H, Ph), 5.64 (d, J=5.5 Hz, 1H, H-1’), 4.62 – 4.60 (m, 1H, H-2’), 4.50 – 4.42 (m, 2H, CH₂Ph), 4.13 – 4.11 (m, 1H, H-3’), 3.81 – 3.74 (m, 1H, H-4’), 3.62 – 3.51 (m, 2H, H-5’), 2.88 (s, 3H, N⁷-CH₃); ¹³C NMR (50 MHz, CDCl₃) δ 157.6 (C-6), 151.1 (C-2), 149.4 (C-4), 140.7, 137.2, 128.6 (Ph), 128.5 (Ph), 127.2 (Ph), 119.3 (C-5), 87.9 (C-1’), 84.6 (C-4’), 73.2 (C-2’), 71.9 (C-3’), 62.3 (C-5’), 44.8 (N-CH₂Ph), 37.0 (N-CH₃); m/z: calcd for C₁₈H₂₁N₅O₅ (M + H)⁺: 388.1615, found: 388.1613;

4.1.4.3 N², N²-dibenzylguanosine (6)

103 mg, 41%, ¹H NMR (200 MHz, CDCl₃) δ 7.59 (s, 1H, H-8), 7.36 – 7.29 (m, 10H, Ph), 5.67 (d, J=5.6 Hz, 1H, H-1’), 4.68 (s, 4H, CH₂Ph), 4.61 (t, J=5.0 Hz, 1H, H-2’), 4.16 – 4.13 (m, 1H, H-3’), 3.87 – 3.83 (m, 1H, H-4’), 3.60 – 3.43 (m, 2H, H-5’); ¹³C NMR (50 MHz, CDCl₃) δ 155.8 (C-6), 152.2 (C-2), 150.3 (C-4), 139.8, 137.3, 128.5 (Ph), 128.2 (Ph), 127.1 (Ph), 119.6 (C-5), 88.9 (C-1’), 85.8 (C-4’), 74.4 (C-2’), 72.0 (C-3’), 62.3 (C-5’), 54.3 (N-CH₂Ph). m/z: calcd for C₂₄H₂₅N₅O₅ (M + H)⁺: 464.1928, found: 464.1918;

4.1.4.4 N²-p-metoxybenzylguanosine (7)

140 mg, 64%, ¹H NMR (200 MHz, CDCl₃) δ 8.02 (s, 1H, H-8), 7.27 (d, 2H, Ph), 6,85 (d, 2H, Ph), 5.92 (d, J=5.4 Hz, 1H, H-1’), 4.73 (t, J=4.9 Hz, 1H, H-2’), 4.48 (s, 2H, CH₂Ph) 4.21 - 4.18 (m, 1H, H-3’), 3.93 – 3.90 (m, 1H, H-4’), 3.72 – 3.68 (m, 2H, H-5’), 3,59 (s, 3H, O-CH₃); ¹³C NMR (50 MHz, CDCl₃) δ 158.9 (C-6), 155.9, 154.1, 150.2, 137.8, 132.8, 127.9, 118.9, 113.9, 88.9 (C-1’), 86.1 (C-4’), 74.3 (C-2’), 72.0 (C-3’), 62.7 (C-5’), 56.0 (OCH₃), 44.6 (N-CH₂Ph). m/z: calcd for C₁₈H₂₁N₅O₆ (M + H)⁺: 404.1564, found: 404.1560;

4.1.4.5 N²-p-chlorobenzylguanosine (8)

153 mg, 69%, ¹H NMR (200 MHz, CDCl₃) δ 8.04 (s, 1H, H-8), 7.36 – 7.30 (m, 4H, Ph), 5.90 (d, J=5.3 Hz, 1H, H-1’), 4.65 (t, J=4.8 Hz, 1H, H-2’), 4.35 (s, 2H, CH₂Ph), 4.18 – 4.15 (m, 1H, H-3’), 3.91 – 3.88 (m, 1H, H-4’), 3.70 – 3.67 (m, 2H, H-5’); ¹³C NMR (50 MHz, CDCl₃) δ 157.2 (C-6), 154.8 (C-2), 150.4 (C-4), 137.9, 136.7, 131.3, 129.2 (Ph), 128.3 (Ph), 119.8 (C-5), 88.6 (C-1’), 85.5 (C-4’), 74.3 (C-2’), 71.7 (C-3’), 62.4 (C-5’), 44.8 (N-CH₂Ph). m/z: calcd for C₁₇H₁₈ClN₅O₅ (M + H)⁺: 408.1069, found: 408.1073;

4.1.4.6 N²-p-nitrobenzylguanosine (9)

134 mg, 59%, ¹H NMR (200 MHz, CDCl₃) δ 8.04 (s, 1H, H-8), 8.22 (d, 2H, Ph), 7.63 (d, 2H, Ph), 5.84 (d, J=5.2 Hz, 1H, H-1’), 4.63 (s, 2H, CH₂Ph) 4.66 – 4.64 (m, 1H, H-2’), 4.18 – 4.15 (m, 1H, H-3’), 3.98 – 3.95 (m, 1H, H-4’), 3.75 – 3.71 (m, 2H, H-5’); ¹³C NMR (50 MHz, CDCl₃) δ 156.6 (C-6), 153.7 (C-2), 150.9, 147.8, 146.4, 136.9 (C-8), 128.2 (Ph), 123.9 (Ph), 118.8 (C-5), 88.4 (C-1’), 86.8 (C-4’), 72.7 (C-2’), 71.2 (C-3’), 62.2 (C-5’), 43.8 (N-CH₂Ph). m/z: calcd for C₁₇H₁₈N₆O₇ (M + H)⁺: 419. 1309, found: 419.1312;

4.1.4.7 N²-p-phenylbenzylguanosine (10)

134 mg, 55%, ¹H NMR (200 MHz, CDCl₃) δ 7.98 (s, 1H, H-8), 7.75 – 7.71 (m, 9H, Ph), 5.89 (d, J=5.3 Hz, 1H, H-1’), 4.69 – 4.65 (m, 2H, CH₂Ph), 4.61 (t, J=4.9 Hz, 1H, H-2’), 4.16 – 4.14 (m, 1H, H-3’), 3.89 – 3.81 (m, 1H, H-4’), 3.64 – 3.41 (m, 2H, H-5’); ¹³C NMR (50 MHz, CDCl₃), δ 158.7 (C-6), 153.2 (C-2), 151.8 (C-4), 140.9, 140.8, 138.1, 137.5, 129.1 (Ph), 128.5 (Ph), 127.7 (Ph), 127.6 (Ph), 127.3 (Ph), 117.4 (C-5), 88.3 (C-1’), 87.5 (C-4’), 75.6 (C-2’), 72.8 (C-3’), 63.7 (C-5’), 43.7 (N-CH₂Ph); m/z: calcd for C₂₃H₂₃N₅O₅ (M + H)⁺: 450.1771, found: 450.1773;

4.1.4.8 N²-butyl-N²-methylguanosine (11)

92 mg, 48%, ¹H NMR (200 MHz, CDCl₃) δ 8.11 (s, 1H, H-8), 5.72 (d, J=4.9 Hz, 1H, H-1’), 4.69 (t, J=5.3 Hz, 1H, H-2’), 4.13 (t, J=3.9 Hz, 1H, H-3’), 3.76 – 3.73 (m, 1H, H-4’), 3.63 – 3.61 (m, 2H, N-CH₂), 3.58 – 3.46 (m, 2H, H-5’), 3.09 (s, 3H, N-CH₃), 1.60 – 1.52 (m, 2H, CH₂ butyl), 1.38 – 1.31 (m, 2H, CH₂CH₃ butyl), 0.90 (t, J = 7.3 Hz, 3H, CH₃ butyl); ¹³C NMR (50 MHz, CDCl₃), δ 158.2 (C-6), 152.3 (C-2), 140.3 (C-4), 136.9 (C-8), 118.5 (C-5), 87.9 (C-1’), 85.6 (C-4’), 74.3 (C-2’), 71.4 (C-3’), 62.4 (C-5’), 51.2 (N-CH₂ butyl), 38.5 (N-CH₃), 27.8 (CH₂ butyl), 20.3 (CH₂ butyl), 13.7 (CH₃ butyl). m/z: calcd for C₁₅H₂₃N₅O₅ (M + H)⁺: 354. 1771, found: 354.1768;

4.1.4.9 N²-butylguanosine (12)

144 mg, 78%, ¹H NMR (200 MHz, CDCl₃) δ 8.09 (s, 1H, H-8), 5.68 (d, J=4.8 Hz, 1H, H-1’), 4.64 – 4.59 (m, 1H, H-2’), 4.11 – 4.08 (m, 1H, H-3’), 3.76 – 3.73 (m, 1H, H-4’), 3.63 (t, J=6.2, 2H, N-CH₂), 3.58 – 3.46 (m, 2H, H-5’), 1.63 – 1.54 (m, 2H, CH₂ butyl), 1.28 – 1.25 (m, 2H, CH₂CH₃ butyl), 0.93 (t, J = 7.3 Hz, 3H, CH₃ butyl); ¹³C NMR (50 MHz, CDCl₃) δ 157.6 (C-6), 154.4 (C-2), 150.3 (C-4), 137.8 (C-8), 117.8 (C-5), 86.8 (C-1’), 84.9 (C-4’), 74.0 (C-2’), 72.3 (C-3’), 62.1 (C- 5’), 42.3 (N-CH₂ butyl), 30.7 (CH₂ butyl), 20.4 (CH₂ butyl), 14.3 (CH₃ butyl). m/z: calcd for C₁₄H₂₁N₅O₅ (M + H)⁺: 340. 1615, found: 340.1619;

4.1.4.10 N²-isobutyl-N²-methylguanosine (13)

100 mg, 52%, ¹H NMR (200 MHz, CDCl₃) δ 7.55 (s, 1H, H-8), 5.64 (d, J=5.2 Hz, 1H, H-1’), 4.64 (t, J=5.0 Hz, 1H, H-2’), 4.16 (t, J=3.8 Hz, 1H, H-3’), 3.86 – 3.81 (m, 1H, H-4’), 3.74 – 3.71 (m, 2H, N-CH₂), 3.60 – 3.45 (m, 2H, H-5’), 2.97 (s, 3H, N-CH₃), 1.89 – 1.85 (m, 1H, CH isobutyl), 0.94 (d, J=6.9, 6H, CH₃ isobutyl); ¹³C NMR (50 MHz, CDCl₃) δ 156.8 (C-6), 152.4 (C-2), 141.9 (C-4), 136.7 (C-8), 119.8 (C-5), 89.3 (C-1’), 85.1 (C-4’), 74.0 (C-2’), 72.3 (C-3’), 62.8 (C-5’), 52.5 (N-CH₂ isobutyl), 38.6 (N-CH₃), 25.2 (CH isobutyl), 20.4 (CH₃ isobutyl). m/z: calcd for C₁₅H₂₃N₅O₅ (M + H)⁺: 354. 1771, found: 354.1777;

4.1.4.11 N²-isobutylguanosine (14)

147 mg, 80%,¹H NMR (200 MHz, CDCl₃) δ 7.64 (s, 1H, h-8), 5.61 (d, J=5.5 Hz, 1H, H-1’), 4.64 – 4.61 (m, 1H, H-2’), 4.15 (t, J=3.9 Hz, 1H, H-3’), 3.87 – 3.83 (m, 1H, H-4’), 3.61 – 3.58 (m, 2H, N-CH₂), 3.58 – 3.47 (m, 2H, H-5’), 1.90 – 1.86 (m, 1H, CH isobutyl), 0.92 (d, J=6.5, 6H, CH₃ isobutyl); ¹³C NMR (50 MHz, CDCl₃) δ 155.9 (C-6), 151.9 (C-2), 149.2 (C-4), 137.5 (C-8), 119.8 (C-5), 87.7 (C-1’), 85.6 (C-4’), 74.6 (C-2’), 72.8 (C-3’), 62.5 (C-5’), 50.3 (N-CH₂ isobutyl), 28.7 (CH isobutyl), 19.2 (CH₃ isobutyl). m/z: calcd for C₁₄H₂₁N₅O₅ (M + H)⁺: 340.1615, found: 340.1615;

4.1.4.12 N², N²-diethylguanosine (15)

101 mg, 55%,¹H NMR (200 MHz, CDCl₃) δ 7.58 (s, 1H, H-8), 5.65 (d, J=5.0 Hz, 1H, H-1’), 4.63 (br, 1H, H-2’), 4.15 (br, 1H, H-3’), 3.83 – 3.78 (m, 3H, H-4’, H-5’), 3.69 – 3.57 (m, 4H, CH₂ ethyl), 1.06 (t, 6H, CH₃ ethyl); ¹³C NMR (50 MHz, CDCl₃) δ 156.8 (C-6), 151.8 (C-2), 141.6 (C-4), 136.9 (C-8), 117.3 (C-5), 87.5 (C-1’), 86.2 (C-4’), 74.2 (C-2’), 71.9 (C-3’), 61.8 (C-5’), 42.6 (N-CH₂ ethyl), 13.1 (CH₃ ethyl). m/z: calcd for C₁₄H₂₁N₅O₅ (M + H)⁺: 340.1615, found: 340.1610;

4.1.4.13 N²-ethyl-N²-methylguanosine (16)

100 mg, 56%,¹H NMR (200 MHz, CDCl₃) 7.79 (s, 1H, H-8), 5.67 (d, J=5.1 Hz, 1H, H-1’), 4.72 – 4.69 (t, J=5.2, 1H, H-2’), 4.16 (br, 1H, H-3’), 3.83 – 3.78 (m, 1H, H-4’), 3.69 – 3.57 (m, 2H, H-5’), 3.62 (q, J = 6.8 Hz, 2H, CH₂ ethyl), 3.12 (s, 3H, N-CH₃), 1.22 (t, J = 7.1 Hz, 3H, CH₃ ethyl); δ ¹³C NMR (50 MHz, CDCl₃) δ 157.7 (C-6), 152.4 (C-2), 140.2 (C-4), 137.5 (C-8), 119.2 (C-5), 88.8 (C-1’), 85.9 (C-4’), 75.0 (C-2’), 71.3 (C-3’), 63.4 (C-5’), 44.4 (N-CH₂ ethyl), 37.5 (N-CH₃), 11.9 (CH3 ethyl). m/z: calcd for C₁₃H₁₉N₅O₅ (M + H)⁺: 326.1458, found: 326.1463;

4.1.4.14 N²-cyclohexylmethylguanosine (17)

144 mg, 70%,¹H NMR (200 MHz, CDCl₃) δ 7.82 (s, 1H, H-8),5.68 (d, J = 3.8 Hz, 1H, H-1’), 4.72 (t, J = 4.9 Hz, 1H, H-2’), 4.15 (t, J = 5.7 Hz, 1H, H-3’), 3.85 – 3.80 (m, 1H, H-4’), 3.61 – 3.52 (m, 2H, H-5’), 3.32 – 3.28 (m, 2H, N-CH₂), 1.82 – 0.98 (m, 11H, cyclohexyl); ¹³C NMR (50 MHz, CDCl₃) δ 156.1 (C-6), 153.2 (C-2), 150.4 (C-4), 137.9 (C-8), 120.1 (C-5), 87.9 (C-1’), 86.6 (C-4’), 73.9 (C-2’), 71.9 (C-3’), 62.3 (C-5’), 44.9 (N-CH₂), 35.7 (cyclohexyl), 29.9 (cyclohexyl), 25.8 (cyclohexyl), 25.1 (cyclohexyl). m/z: calcd for C₁₇H₂₅N₅O₅ (M + H)⁺: 380.1928, found: 380.1925;

4.1.5 General procedure for the synthesis of N²-substitutiuted 5’- monophosphates 32-45

Phosphorus oxide trichloride (POCl₃) (3.5 eq) in trimethyl phosphate (0.05 eq) was cooled to 0°C and added to the dried over P₄O₁₀ compound 4-17 (1 eq). The reaction mixture was stirred at 0°C. After 2 hr, 1 M aqueous TEAB was added to neutralize the pH of the reaction mixture. The product was purified by ion-exchange chromatography on a DEAE-Sephadex A-25 column using a linear 0–1 M TEAB gradient to produce products 18-31 as TEA salts. Methyl iodide (7 eq) was added to a suspension of mononucleotide derivative (18-31) in 1.5 mL anhydrous dimethylsulfoxide (DMSO) and stirred at room temperature for 2 hr. The mixture was poured into water and extracted three times with diethyl ether. The aqueous phase was purified on DEAE–Sephadex using a linear 0 - 0.8 M gradient of TEAB.

4.1.5.1 N²-benzyl-7-methylguanosine-5’-monophosphate (32)

129 mg, 65%, sodium salt; ¹H NMR (700 MHz, D₂O) δ 7.46 – 7.32 (m, 5H, Ph), 6.07 (d, J = 3.4 Hz, 1H, H-1’), 4.61 (s, 2H, CH₂Ph), 4.58 (t, J = 4.7 Hz, 1H, H-2’), 4.43 (t, J =5.7 Hz, 1H, H-3’), 4.35 – 4.33 (m, 1H, H-4’), 4.15 – 4.11 (m, 1H, H-5’), 4.09 (s, 3H, N⁷-CH₃), 4.00 – 3.97 (m, 1H, H-5”); ³¹P NMR (283 MHz, D₂O) 3.234; m/z: calcd for C₁₈H₂₃N₅O₈P: 468.1127, found: 468.1131; HPLC (Method B) t_R 9 min.

4.1.5.2 N²-benzyl-N²,7-dimethylguanosine-5’-monophosphate (33)

110 mg, 68%, sodium salt; ¹H NMR (700 MHz, D₂O) δ 7.45 – 7.31 (m, 5H, Ph), 6.11 (d, J = 3.5 Hz, 1H, H-1’), 4.68 (s, 2H,CH₂Ph), 4.62 (t, J = 4.7 Hz, 1H, H-2’), 4.44 (t, J = 5.4 Hz, 1H, H-3’), 4.35 – 4.32 (m, 1H, H-4’), 4.15 – 4.11 (m, 1H, H-5’), 4.10 (s, 3H, N⁷-CH₃), 4.01 – 3.97 (m, 1H, H-5”), 3.17 (s, 3H, N²-CH₃); ³¹P NMR (283 MHz, D₂O) 3.139; m/z: calcd for C₁₉H₂₅N₅O₈P : 482.1284, found: 482.1287; HPLC (Method B) t_R 13 min.

4.1.5.3 N²,N²-dibenzyl-7-dimethylguanosine-5’-monophosphate (34)

84 mg, 58%, sodium salt; ¹H NMR (700 MHz, D₂O) δ 7.42 – 7.28 (m, 10H, 2x Ph), 6.07 (d, J = 3.6 Hz, 1H, H-1’), 4.82 (s, 4H, CH₂Ph), 4.56 (t, J = 4.9 Hz, 1H, H-2’), 4.42 (t, J = 5.6 Hz, 1H, H-3’), 4.31 – 4.28 (m, 1H, H-4’), 4.09 (s, 3H, N⁷-CH₃), 4.08 – 4.06 (m, 1H, H-5’), 3.97 – 3.93 (m, 1H, H-5”); ³¹P NMR (283 MHz, D₂O) 3.085; m/z: calcd for C₂₅H₂₉N₅O₈P: 558.1597, found: 558.1592; HPLC (Method B) t_R 20.5 min.

4.1.5.4 N²-p-metoxybenzyl-7-methylguanosine-5’-monophosphate (35)

134 mg, 66%, sodium salt; ¹H NMR (700 MHz, D₂O) δ 7.39 (d, 2H, Ph), 6.95 (d, 2H, Ph), 6.08 (d, J = 3.4 Hz, 1H, H-1’), 4.60 (t, J = 4.7 Hz, 1H, H-2’), 4.51 (s, 2H, CH₂Ph), 4.44 (t, J = 5.7 Hz, 1H, H-3’), 4.36 – 4.34 (m, 1H, H-4’), 4.17 – 4.13 (m, 1H, H-5’), 4.07 (s, 3H, N⁷-CH₃), 4.02 – 3.98 (m, 1H, H-5”), 3.79 (s, 3H, OCH₃); ³¹P NMR (283 MHz, D₂O) 3.011; m/z: calcd for C₁₉H₂₅N₅O₉P: 498.1233, found: 498.1237; HPLC (Method B) t_R 12.25 min.

4.1.5.5 N²-p-chlorobenzyl-7-methylguanosine-5’-monophosphate (36)

119 mg, 53 %, sodium salt; ¹H NMR (700 MHz, D₂O) δ 7.40 (s, 4H, Ph), 5.90 (d, J=3.6, 1H, H-1’), 4.66 – 4.62 (m, 1H, H-2’), 4.50 (s, 2H, CH₂Ph), 4.35 - 4.32 (m, 1H, H-3’), 4.29 – 4.26 (m, 1H, H-4’), 4.09 (s, 3H, N⁷-CH₃), 4.07 – 3.97 (m, 2H, H-5’, H-5”); ³¹P NMR (283 MHz, D₂O) 2.590; m/z: calcd for C₁₈H₂₂N₅O₈PCl: 502.0894, found: 502.0897; HPLC (Method B) t_R 15 min.

4.1.5.6 N²-p-nitrobenzyl-7-methylguanosine-5’-monophosphate (37)

96 mg, 49%, sodium salt; ¹H NMR (700 MHz, D₂O) δ 8.25 (d, 2H, Ph), 7.62 (d, 2H, Ph), 6.01 (d, J = 3.5 Hz, 1H, H-1’), 4.68 (s, 2H, CH₂Ph), 4.52 – 4.50 (m, 1H, H-2’), 4.41 – 4.39 (m, 1H, H-3’), 4.32 – 4.29 (m, 1H, H-4’), 4.10 – 4.09 (m, 1H, H-5’), 4.07 (s, 3H, N⁷-CH₃), 3.98 – 3.94 (m, 1H, H-5”); ³¹P NMR (283 MHz, D₂O) 3.339; m/z: calcd for C₁₈H₂₂N₆O₁₀P: 513.0978, found: 513.0976; HPLC (Method C) t_R 4.5 min.

4.1.5.7 N²-p-phenylbenzyl-7-methylguanosine-5’-monophosphate (38)

114 mg, 60 %, sodium salt; ¹H NMR (700 MHz, D₂O) δ 7.84 – 7.41 (m, 9H, Ph-Ph), 6.09 (d, J = 3.5 Hz, 1H, H-1’), 4.69 (s, 2H, CH₂Ph), 4.64 (t, J = 4.7 Hz, 1H, H-2’), 4.46 – 4.44 (m, 1H, H-3’), 4.39 – 4.32 (m, 1H, H-4’), 4.19 – 4.16 (m, 1H, H-5’), 4.08 (s, 3H, N⁷-CH₃), 4.05 – 4.01 (m, 1H, H-5”); ³¹P NMR (283 MHz, D₂O) 3.121; m/z: calcd for C₂₄H₂₅N₅O₈P: 542.1440, found: 542.1443; HPLC (Method C) t_R 15.4 min.

4.1.5.8 N²-butyl-N²,7-dimethylguanosine-5’-monophosphate (39)

78 mg, 55%, sodium salt; ¹H NMR (700 MHz, D₂O) δ 6.11 (d, J = 3.2 Hz, 1H, H-1’), 4.69 (t, J = 4.7 Hz, 1H, H-2’), 4.46 (t, J = 5.9 Hz, 1H, H-3’), 4.38 – 4.35 (m, 1H, H-4’), 4.22 – 4.18 (m, 1H, H-5’), 4.09 (s, 3H, N⁷-CH₃), 4.06 – 4.03 (m, 1H, H-5”), 3.68 – 3.60 (m, 2H, CH₂ butyl), 3.15 (s, 3H, N²-CH₃), 1.66 – 1.60 (m, 2H, CH₂ butyl), 1.37 – 1.28 (m, 2H, CH₂CH₃ butyl), 0.92 (t, J = 7.4 Hz, 3H, CH₃ butyl). ³¹P NMR (283 MHz, D₂O)1.548; m/z: calcd for C₁₆H₂₇N₅O₈P : 448.1440, found: 448.1445; HPLC (Method A) t_R 18.7 min.

4.1.5.9 N²-butyl-7-methylguanosine-5’-monophosphate (40)

134 mg, 60%, sodium salt; ¹H NMR (700 MHz, D₂O) δ 6.11 (d, J = 3.0 Hz, 1H, 1H, H-1’), 4.69 (t, J = 4.8 Hz, 1H, H-2’), 4.46 (t, J = 5.8 Hz, 1H, 1H, H-3’), 4.40 – 4.36 (m, 1H, H-4’), 4.24 – 4.20 (m, 1H, H-5’), 4.08 (s, 3H, N⁷-CH₃), 4.07 – 4.05 (m, 1H, H-5”), 3.45 –3.40 (m, 2H, CH₂ butyl), 1.64 – 1.55 (m, 2H, CH₂ butyl), 1.42 – 1.32 (m, 2H, CH₂CH₃ butyl), 0.91 (t, J = 7.4 Hz, 3H, CH₃ butyl); ³¹P NMR (283 MHz, D₂O) 0.770; m/z: calcd for C₁₅H₂₅N₅O₈P: 434.1284, found: 434.1285; HPLC (Method A) t_R 13.2 min.

4.1.5.10 N²-isobutyl-N²,7-dimethylguanosine-5’-monophosphate (41)

78 mg, 51%, sodium salt; ¹H NMR (700 MHz, D₂O) δ 6.11 (d, J = 3.2 Hz, 1H, H-1’), 4.67 (t, J = 4.8 Hz, 1H, H-2’), 4.46 (t, J = 5.9 Hz, 1H, H-3’), 4.37 – 4.35 (m, 1H, H-4’), 4.22 – 4.18 (m, 1H, H-5’), 4.09 (s, 3H, N⁷-CH₃), 4.07 – 4.03 (m, 1H, H-5”), 3.45 – 3.40 (m, 2H, N-CH₂), 3.17 (s, 3H, N²-CH₃), 2.15 – 2.05 (m, 1H, CH isobutyl), 0.92 (d, J = 6.5 Hz, 6H, CH₃ isobutyl); ³¹P NMR (283 MHz, D₂O) 1.646; m/z: calcd for C₁₆H₂₇N₅O₈P: 448.1440 found: 448.1442; HPLC (Method A) t_R 21.5 min.

4.1.5.11 N²-isobutyl-7-methylguanosine-5’-monophosphate (42)

130 mg, 57 %, sodium salt; ¹H NMR (700 MHz, D₂O) δ 6.12 (d, J = 3.3 Hz, 1H, H-1’), 4.67 (t, J = 4.9 Hz, 1H, H-2’), 4.47 (t, J = 5.6 Hz, 1H, H-3’), 4.38 – 4.36 (m, 1H, H-4’), 4.20 – 4.16 (m, 1H, H-5’), 4.09 (s, 3H, N⁷-CH₃), 4.06 – 4.02 (m, 1H, H-5”), 3.32 – 3.20 (m, 2H, N-CH₂), 1.98 – 1.89 (m, 1H, CH isobutyl), 0.93 (d, J = 6.7 Hz, 6H, CH₃ isobutyl); ³¹P NMR (283 MHz, D₂O) 2.061; m/z: calcd for C₁₅H₂₅N₅O₈P: 434.1284, found: 434.1287; HPLC (Method A) t_R 16 min.

4.1.5.12 N²,N²-diethyl-7-methylguanosine-5’-monophosphate (43)

83 mg, 52%, sodium salt; ¹H NMR (700 MHz, D₂O) δ 6.12 (d, J = 3.1 Hz, 1H, H-1’), 4.70 (t, J = 4.9 Hz, 1H, H-2’), 4.46 (t, J = 5.5 Hz, 1H, H-3’), 4.37 – 4.35 (m, 1H, H-4’), 4.22 – 4.18 (m, 1H, H-5’), 4.08 (s, 3H, N⁷-CH₃), 4.07 – 4.04 (m, 1H, H-5”), 3.60 (q, J = 6.1 Hz, 4H, CH₂ ethyl), 1.22 (t, J = 7.1 Hz, 6H, CH₃ ethyl); ³¹P NMR (283 MHz, D₂O) 1.299; m/z: calcd for C₁₅H₂₅N₅O₈P: 434.1284, found: 434.1291; HPLC (Method A) t_R 14.2 min.

4.1.5.13 N²-ethyl-N²,7-dimethylguanosine-5’-monophosphate (44)

109 mg, 70%, sodium salt; ¹H NMR (700 MHz, D₂O) δ 6.12 (d, J = 3.4 Hz, 1H, H-1’), 4.70 (t, J = 4.8 Hz, 1H, H-2’), 4.46 (t, J = 5.8 Hz, 1H, H-3’), 4.38 – 4.35 (m, 1H, H-4’), 4.23 – 4.19 (m, 1H, H-5’), 4.08 (s, 3H, N⁷-CH₃), 4.07 – 4.05 (m, 1H, H-5”), 3.64 (dd, J = 14.3, 7.1 Hz, 2H), 3.15 (s, 3H), 1.20 (t, J = 7.1 Hz, 3H); ³¹P NMR (283 MHz, D₂O) 0.866; m/z: calcd for C₁₄H₂₃N₅O₈P: 420.1127, found: 420.1128; HPLC (Method A) t_R 13.9 min.

4.1.5.14 N²-cyclohexylmethyl-7-methylguanosine-5’-monophosphate (45)

122 mg, 56%, sodium salt; ¹H NMR (700 MHz, D₂O) δ 6.12 (d, J = 3.6 Hz, 1H, H-1’), 4.66 (t, J = 4.8 Hz, 1H, H-2’), 4.47 (t, J = 5.7 Hz, 1H, H-3’), 4.38 – 4.35 (m, 1H, H-4’), 4.16 – 4.12 (m, 1H, H-5’), 4.10 (s, 3H, N⁷-CH₃), 4.02 – 3.98 (m, 1H, H-5”), 3.30 – 3.27 (m, 2H, N-CH₂), 1.79 – 0.95 (m, 11H, cyclohexyl); ³¹P NMR (283 MHz, D₂O) 3.218; m/z: calcd for C₁₈H₂₉N₅O₈P: 474.1597, found: 474.1592; HPLC (Method A) t_R 15.6 min.

Scheme 1.

Preparation of N²-fluoro-2’,3’,5’-O-triacetyl-O⁶-[2-(4-nitrophenyl)ethyl] inosine; (a) acetic anhydride, DMAP, Et₃N, AcCN, 4°C to RT (b) NPE, PPh₃, DIAD, toluene, RT (c) HF/pyridine, tBuONO, pyridine, -40°C.