The Quaternization Reaction of 5-O-Sulfonates of Methyl 2,3-o-Isopropylidene-β-D-Ribofuranoside with Selected Heterocyclic and Aliphatic Amines

The synthesis of N-((methyl 5-deoxy-2,3-O-isopropylidene-β-D-ribofuranoside)-5-yl)ammonium salts are presented. To determine the effect of the nucleophile type and outgoing group on the quaternization reaction, selected aliphatic and heterocyclic aromatic amines reacted with: methyl 2,3-O-isopropylidene-5-O-tosyl-β-D-ribofuranoside or methyl 2,3-O-isopropylidene-5-O-mesyl-β-D-ribofuranoside or methyl 2,3-O-isopropylidene-5-O-triflyl-β-D-ribofuranoside were performed on a micro scale. High-resolution 1H- and 13C-NMR spectral data for all new compounds were recorded. Additionally, the single-crystal X-ray diffraction analysis for methyl 2,3-O-isopropylidene-5-O-mesyl-β-D-ribofuranoside and selected in silico interaction models are reported.


Introduction
Preparation of sulfate esters is one of the very useful reactions in organic chemistry. It was first described by Adolph Strecker in 1868 [1]. Additionally, in the synthesis of carbohydrates, the synthesis of sulfonates has been widely described. In 1953, Tipson described exhaustively the preparation of such derivatives using sulfonyl halides in pyridine [2]. This method is one of the most commonly used methods until now. Another described in the literature method of obtaining esters of p-toluenesulfonic acids is based on the use of an anhydride of this acid [3,4]. Another remarkable method is the use of silver methanesulfonate as the sulfonating agent [5,6]. The interest in sulfonating sugars results from the biological activity of this type of derivatives. Among the glycosaminoglycans with high medicine and industry usage potential, there should be mentioned heparan sulfate, keratan sulfate, dermatan sulfate and chondroitin sulfate. The last one [7] is widely used in the treatment of osteoarthritis resulting in improvements in physical function, quality of life and reductions in pain and disease progression. Another naturally occurring sulfated polysaccharide is heparin. It was discovered accidentally in 1959 by McLean [8] and was initially used clinically as a drug in the field of treatment thrombosis and hemostasis in the 1930s and 1940s. Since it has been shown that the low-molecular heparin is highly active, attempts have been made to develop methods for its synthesis [9,10]. The presence of large amounts of simple sugars in the diet of a modern man is supposed to result in a possible disruption of their metabolic pathways. For example, disturbances in the hexosamine biosynthetic pathway may be associated with type 2 diabetes, Alzheimer's and cancer [11,12]. Hyperactivity in the pentose pathway and glycolysis may result in cancer [13][14][15][16]. An important role in regulating this type of abnormality is associated with naturally occurring derivatives of monosaccharides such as glucose-6-sulphate [17]. For some time, researchers have been interested in sulfate analogues of ribose phosphate (see Figure 1) [18][19][20][21]. Its simple synthesis from methyl 5-deoxy-5-iodo-2,3-O-isopropylidene-D-riboside was described by Musicki and Widlanski [22].
We describe the synthesis of similar sulphate derivatives, which we then quaternized to obtain the appropriate quaternary ammonium salts (QAS) with different counterion. Studies show that different counterions can result in different biological activities of ammonium salts [23,24].

Results and Discussion
This work is an extension of our previously published results of calculations regarding the conformations and mechanisms of formation of quaternary ammonium salts with the results of experimental work. Both types of data show very high convergence, which further increases their value [25,26].
To confirm our assumptions, we used a series of tertiary amines for reactions, ranging from aliphatic to heterocyclic with two rings.
After 14 days of the reaction at 70 • C, the expected product was obtained with a yield of 78%. An analogous reaction with 2-methylpyridine allowed to receive 4d only with a yield of 31%. This result clearly indicates the effect of steric hindrance of the methyl substituent at the C-2 position of the amine on the substitution of the sulfonic group on the terminal group of the sugar derivative 3. After the reaction was completed, the substrate 3 was present in the reaction mixture, next to the expected product 4d, and the prolongation of the reaction time did not improve its yield.
Since 4-(N,N-dimethylamine)pyridine (DMAP) is a solid, the reaction with 3 was initially carried out in acetonitrile solution at 70 • C for a month, which lead to the desired salt with surprisingly low yield. After the chromatographic purification of the post-reaction mixture, the expected salt was obtained in a yield of 37%. In addition, we found the unreacted substrate in the mixture. Since DMAP is both a stronger base and a nucleophile than pyridine, which is the result of the presence of the dimethylammonium residue in the para position, we expected a greater or at least similar quaternization efficiency as for pyridine.
The obtained efficiency came to us as a surprise and prompted us to attempt a synthesis as in the case of pyridine and 2-methylpyridine, so without the use of a solvent. In the next synthesis, we decided to use the melting method developed and published by us for other quaternary ammonium salts. Usually, it leads to the expected products with good yields. The substrate 3 and DMAP were heated in a screw cap ampoule at 100 • C for 48 h. This time, after purification, we obtained the product N-((methyl 2,3-O-isopropylidene-β-D-ribofuranoside)-5-yl)-4-(N,N-dimethylamine)pyridine tosylate (4e) as the pale yellow oil with a yield of 70%. We did not find any substrate in the post-reaction mixture. The result obtained by this method coincided with our expectations and matched logically to a number of previously obtained results.
In addition to heterocyclic monocyclic amines, we used two heterocyclic bicyclic amines in subsequent syntheses.
This result can be explained by the less basicity of quinoline (pK a 4.90 [29]) and greater steric hindrance of the nitrogen atom.
We also carried out the reactions of the tosylate 3 with the following amines: imidazole, 4,4-bipyridyl, 2,2-bipyridyl and 3-carbamoylpyridine. Reactions were carried out in both acetonitrile solutions at 70 • C and solvent-free at 100 • C, and in all cases, we observed a lack of products and only unreacted tosylate 3.
Summing up the reaction of the methyl 2,3-O-isopropylidene-5-O-tosyl-β-D-ribofuranoside (3) with selected amines, we can say that we were able to obtain and characterize six new quaternary ammonium salts. Yields for obtaining N-((methyl 2,3-O-isopropylidene-β-D-ribofuranoside)-5-yl)ammonium tosylates ranged from 28% to 78%. The highest yield was obtained for the synthesis of It came as no surprise to us that the lowest performance was obtained in the case of N-((methyl 2,3-O-isopropylidene-β-D-ribofuranoside)-5-yl)triethylammonium tosylate (4b) (Table 1) [30]. Such results are expected to be caused by the large steric hindrance of the nitrogen atom in the amine molecule and, thus, the difficult substitution of the terminal O-tosyl group.
To compare the effect of the leaving group on the quaternization reactions, we conducted reactions of selected amines with the mesyl analogue of compound 3. Another reason was to study the effect of counterion on the biological properties of the salts obtained.
Compared to the reaction of compound 3, in the case of mesylate 5, we extended the reaction time with triethylamine to 30 days, also leading it at 70 • C, and yet, we obtained the product 6b in only 19% yield. The lower yield despite the longer reaction time was undoubtedly the result of steric hindrance caused by three ethyl groups attached to the amine nitrogen atom.
Due to the case of the reaction of tosylate 3 with DMAP, we obtained much better reactions results using a procedure reaction in a closed ampoule without solvent; a similar procedure was used for compound 5.
In the quaternization reactions we carried out with selected amines, the triflyl derivative proved to be not very useful. It could possibly be the result of the side reactions that this very reactive derivative underwent.

Molecular Modeling
The difference in the reactivity of isoquinoline and quinoline was explained with semi-empirical calculation using the PM6 Hamiltonian [31] in mopac2016 [32]. The tosylate 3 with quinoline were geometry optimized and subjected to the reaction path calculations in mopac2016. After finding, confirming and optimizing the transitional state (TS) complexes, we have calculated the reaction path in two directions: from TS towards the products (4f and tosyl or 4g and tosyl, respectively) and from TS towards the substrates (tosylate 3 and isoquinoline or tosylate 3 and quinoline, respectively). In both cases, the total energy of simulated complexes was measured and visualized in Figure 2.
The resulting energies of both substrates and products of synthesis of 4g are higher than corresponding energies calculated for 4f (~1.5 kcal/mol and~4.6 kcal/mol for substrates and products, respectively). Moreover, the energetic barrier between substrates and TS complexes is over 1 kcal/mol higher in the case of 4g than in the case of 4f. Energy gained after the transition from TS into products side is almost 5 kcal/mol higher for the 4f set of products comparing to the 4g mixture. All 3D models (Protein Data Bank format) of 4f and 4g discussed here (TS, products and substrates of both) are available in the Supplementary Materials.

X-ray Diffraction Analysis of 5
The crystallographic structure of methyl 2,3-O-isopropylidene-5-O-mesyl-β-D-ribofuranoside (5), which was a substrate for the preparation of quaternary ammonium salts, was determined and is shown in Figure 3. With high probability, it can be assumed, which is confirmed by the aforementioned calculation results, that this structure corresponds to that in the solution. Since the quaternization reaction follows the S N 2 mechanism, it could be expected that the steric factor would have a significant impact on the course of the reaction next to the electronic factors.

Materials and Methods
Commercial D-ribose (Merck, New Jersey, NJ, USA) was used. All reactions were monitored by thin-layer chromatography (TLC) on Kieselgel 60 F254 Silica Gel plates (E. Merck, 0.20 mm thickness) using eluent system (v/v) 3:1 CHCl3-MeOH (Merck). The spots were detected by spraying with 5% ethanolic H2SO4 and charring. Other reagents, such as C2H5OH, H2SO4, 2-butanon, tertiary amines, CH3CN, CH2Cl2, Tf2O were obtained from Merck. 1H-NMR and 13C-NMR spectra were recorded at 25 • C with a Varian Mercury (Agilent, Santa Clara, CA, USA) spectrometer at 400.49 and 100.70 MHz, respectively, with Me4Si as the internal standard; positive-ion mode MALDITOF mass spectra was done on a Bruker Biflex III spectrometer (Billerica, Massachusetts, MA, USA).    it is outside the sugar ring, the nucleophile attack is hindered and occurs from above the furan ring, which makes the more difficult the attack the larger the nucleophile is. It seems that the results of the synthesis of 1,4-anhydo-2,3-O-isopropylidene-5-O-tosyl-D,L-ribitol (previously published [31]) contained in Table 2 provide good confirmation of this thesis. The lack of the O-methyl substituent at the sugar ring reduces the steric hindrance and facilitates the attack from the sugar ring side, resulting in better quaternization efficiency with the same amines. Table 2. Crystal data, data collection and refinement for methyl 2,3-O-isopropylidene-5-O-mesyl-β-D -ribofuranoside (5), deposition number CCDC 253950.