Synthesis of Glycosidic (β-1′′→6, 3′ and 4′) Site Isomers of Neomycin B and Their Effect on RNA and DNA Triplex Stability

Glycosidic (β-1′′→6, 3′ and 4′) site isomers of neomycin B (i.e., neobiosamine (β-1′′→6, 3′ and 4′) neamines) have been synthesized in a straightforward manner. Peracetylated neomycin azide was used as a common starting material to obtain neobiosamine glycosyl donor and 6, 3′,4′-tri-O-acetyl neamine azide that after simple protecting group manipulation was converted to three different glycosyl acceptors (i.e., 5,6,4′-, 5,3′,4′- and 5,6,3′-tri-O-acetyl neamine azide). Glycosylation between the neobiosamine glycosyl donor and the neamine-derived acceptors gave the protected pseudo-tetrasaccharides, which were converted, via global deprotection (deacetylation and reduction of the azide groups), to the desired site isomers of neomycin. The effect of these aminoglycosides on the RNA and DNA triplex stability was studied by UV-melting profile analysis.


Introduction
Among the small molecular ligands that target nucleic acids, aminoglycosides (AGs) deserve special attention [1].Their binding to a variety of nucleic acid targets has been extensively studied.Binding to the ribosomal decoding site is the basis of AGs' bactericidal effect [2][3][4][5].The continuous increase of antibiotic resistant infectious diseases maintains the interest around this RNA target, and a significant effort has been paid to optimization and modification of existing AG-based lead compounds to provide new potential antibacterial drugs [6,7].Relatively high binding affinities have also been reported for other structurally resembling binding sites in ribozymes [7,8] and important regions of HIV RNAs (Trans Activation Response element (TAR), Reverse Response Element (RRE) and Dimerization Initiation Site (DIS)) [9][10][11][12]).In these RNA targets, the binding sites are bulges and internal loops, which, in contrast to canonical double helices, are able to form appropriate hydrogen bonds and electrostatic interactions with AGs.AGs can also act as groove binders that stabilize DNA-and RNA-triple helices and their hybrids [13][14][15][16][17].Among the AGs studied for the triplex recognition, neomycin B has shown to be the most effective groove binder.The binding occurs to the Watson-Hoogsteen groove, in which favorable contacts with rings II and IV (cf.Scheme 1), together with the appropriate shape complementarity, may take place [14,17].In each case, however, electrostatic interactions dominate AGs' affinity to nucleic acids, which makes the binding promiscuous and may disturb detailed characterization of the binding motifs.Conformational adaption of the nucleic acid targets (in particular RNA) disturbs modelling and discovery of specific ligands even further [1].Chemically synthesized structural analogs of the known AG-ligands are hence important tools that may be used for the exclusion analysis of the binding requirements.
In the present study, a straightforward synthesis of glycosidic (β-1 →6, 3 and 4 ) site isomers of neomycin B is described (Note: C6-, C3 -and C4 -substituted neamines are not neomycin class AGs, i.e., 4,5-disubstituted deoxystreptamines, and the correct names for these compounds are neobiosamine(β-C1 →C6, 3 and 4 )neamines).The key steps of the synthesis (Scheme 1) were (1) acid-catalyzed thiolysis of neomycin azide [18], which gave useful intermediates for both glycosyl donor and the acceptors, and (2) selective diacetylation of neamine azide, which after simple protecting group manipulation afforded the glycosyl acceptors.Glycosylation between the neobiosamine donor and triacetylated neamine acceptors resulted in the azide masked pseudo-tetrasaccharides, which, via global deprotection, were converted to the desired site isomers of neomycin B (Scheme 2).In order to evaluate how the changed glycosidic connection (β-1 →3 ,4 or 6) between the neamine and neobiosamine cores affects the groove binding, the stability of RNA and DNA triple helix models in the presence of these structural analogs (and neomycin B) was studied by UV-melting profile analysis.
The anomeric mixture of phenylthio 2,5,3 4 -tetra-O-acetyl 2 ,6 -diazidoneobiosamine (4) may be used as such as a glycosyl donor, but the mixture of the donor makes the control and monitoring of the reaction (cf.Scheme 2) complex.Therefore, the phenylthioglycoside (4) was hydrolyzed and the resulted hemiacetal converted to pure α-anomer of tricloroacetimidate 6.Overall, this leaving group conversion (from 4 to 6) could be carried out in 86% yield (iv and v/Scheme 1).

Glycosylation and Global Deprotection
A standard procedure using TMSOTf as a catalyst was applied for the glycosylation between acceptors 14-16 and donor 6 (1.5 equiv.).Each reaction was performed at −20 °C (for 2 h) in dry dichloromethane under nitrogen.For the purification reasons (the fully protected pseudotetrasaccharides 17-19 remained contaminated by traces of donor 6, despite a laborious chromatographic purification), the glycosylation was followed by subsequent deacetylation (0.1 mol L −1 NaOMe in MeOH) and the neomycin azides 20-22 could be isolated in acceptable yields (49-69%).The purity of the products (20)(21)(22) was confirmed by reversed phase-high performance liquid chromatography (RP HPLC) analysis (Figure 1).Finally, the azide masks were removed by Staudinger reaction using trimethylphosphine and aqueous ammonia, followed by elution through

The Effect of 23-25 on DNA and RNA Triplex Stability
The effect of the glycosidic site isomers of neomycin B (23)(24)(25) and neomycin (1) on the stability of simple DNA and RNA triple helices was studied by UV-melting profile experiments (Table 1).The DNA triple helix is consisted of a purine rich region of c-Myc promoter 1 [21], used as a model in several previous studies [22][23][24][25][26][27].The formation of this triple helix, consisting of CH + •G-C-triplets, requires slightly acidic pH.The intramolecular RNA triplex is also a known model (in fact, this model may exist as a mixture with its dimer [28]).The measurements were carried out using 2 µmol L −1 of the oligonucleotides in a mixture of 10 mmol L −1 sodium cacodylate and 0.1 mol L −1 NaCl at pH 6.0 (both RNA and DNA model) and at pH 7.0 (RNA model only) in the presence of 5 and 10 eq. of the AGs (1 and 23-25).The temperature was changed at a rate of 0.2  1), the C1 →C4 (25), C1 →C3 (23) and C1 →C6 (24) site isomers, respectively.On the RNA triplex, the effect with the C1 →C3 (23) and C1 →C6 (24) site isomers was, in fact, slightly higher compared to that with neomycin B (1) (∆T m 3 = +11.3and +10.6 Probably, the carbohydrate core and spatial orientation of the amino groups on it do not play a marked enough role to result in discrimination between the affinities of the glycosidic site isomers (1,(23)(24)(25).This may be an unexpected observation as, e.g., previous studies with kanamycin class aminoglycosides (kanamycin = a 4,6-disubstituted deoxystreptamine consisted of neamine and α-D-glucos-3-amine at C6) show a modest or no effect on the poly U-A-U DNA triplex [14].The effect of 24 (a 4,6-disubstituted deoxystreptamine) on the DNA triplex was the smallest (∆T m 3 = +13.9ΔTm-values in parentheses.Error limits for each Tm-value (an average of three temperature ramps) were less than 1°C.Tm 3 and Tm 2 correspond to melting values of the triplex and duplex, respectively.

RNA Triplex Model
∆T m -values in parentheses.Error limits for each T m -value (an average of three temperature ramps) were less than 1 • C. T m 3 and T m 2 correspond to melting values of the triplex and duplex, respectively.

General Remarks
Pyridine, methanol and dichloromethane were dried over 3Å molecular sieves.Nuclear magnetic resonance (NMR) spectra were recorded using a 500 MHz instrument.The chemical shifts for 1 H and 13 C-NMR resonances are given in parts of million from the residual signal of the deuterated solvents (CD 3 OD and CD 3 Cl 3 ).Mass spectra were recorded using electrospray ionization (ESI-TOF).The NMR spectral data for all new compounds are showed in the Supplementary Materials.