IDENTIFICACIÓN DE LOS CRITERIOS DE EVALUACIÓN

were unsuccessful mainly due to difficulties in performing the copper-mediated

C-glycosylation (Chapter 4.2.3). Therefore, a simpler synthetic procedure was

developed for an altered ligand, which contains a similar arrangement of donor atoms.[196]

The troublesome C-glycosylation of a sugar moiety with an (aromatic) ring can be

circumvented if the ring contains a nitrogen atom with a free valence as in the case of the natural DNA bases. Another example is Shionoya’s hydroxypyridone ligand (12).

Likewise, a new ligand based on the hydroxyphenyl-oxazoline system was developed by formally transforming the phenyl ring of structure 43 into the pyrrole ring of

OH N O O OH HO N OH N O O OH HO _O OH HO N O NH O 43 51 52

Figure 33: Formal transformation of the hydroxyphenyl-oxazoline nucleoside 43 via the hypothetic

hydroxypyrrole-oxazoline 51 into the 3-(2-oxazolidinylidene-)indol-2-one nucleoside 52.

Although compound 51 is difficult to synthesize, the derivative 52 is easily accessible

according to published methods for the preparation of compound 53 (Figure 34).[197]

N H

O NH O

Figure 34: Free 3-(2-oxazolidinylidene-)indol-2-one ligand 53. Note that compound 53 appears in the

literature as the tautomer shown here (C=O double bond) whereas the hydroxyphenyl-oxazoline in 43

is believed to exist as depicted in Figure 33 (C=N double bond).[197]

The resulting ligand 52 (Figure 33) is accessible from the commercially available

compound isatin (54), which is an industrial intermediate in the fabrication of indigo.

This strategy of a simple and quick synthetic access from isatin, however, implied that the final ligand 52 contains an additional benzene ring and therefore is sterically

more bulky than the parental system.

Ligands similar to molecule 53 are known to form complexes with a couple of

transition metal ions (Cu2+, Ni2+, Co2+).[198] The synthesis of the DNA building block is summarized in Scheme 11.

O OTol TolO N O Cl Cl O OH DMTO N O NH O O O DMTO N O NH O P O N N O OH HO N O NH O O OTol TolO N O NH O N H O Cl Cl N O Cl Cl O Cl Cl N H O O O OTol TolO Cl a b c d e f g 54 55 56 29 57 58 52 59 60

Scheme 11: Synthesis of the 3-(2-oxazolidinylidene-)indol-2-one nucleoside 52 and the corresponding

phosphoramidite 60. a) CHCl2COCl, NEt3, CHCl3, 20 °C, 1 h, 80 %; b) NaOH(aq), 89 %; c) 29, DBU, dry

MeCN, 17 h, 37 % (18 % β, 19 % α); d) ethanolamine, THF, 35 %; e) K2CO3, MeOH, 54 %; f) DMT-Cl,

pyridine, 65 %; g) CED-Cl, NEt(iPr)2, THF, r.t., 32 h, yield not determined.

In contrast to the three consecutive steps in the synthesis of the free ligand 53, the

order of the steps for the synthesis of the glycosylated ligand 52 was changed. First,

isatin 54 was reacted with a mixture of dichloroacetylchloride and triethylamine in

chloroform (which leads to in situ formation of dichloroketene) to the acylated

dichloromethyleneindolone 55. Besides the unavoidable acylation of the nitrogen

atom, the dichloroketene reacts in a [2+2] cycloaddition with the carbonyl group in 3-position to a spiro-annelated β-lactone, which fragments under cycloreversion and loss of CO2 to compound 55. Afterwards, the unwanted dichloroacetyl group is

removed from N1 by saponification with aqueous NaOH to yield molecule 56. The N-glycosylation using glycosyl donor 29 and DBU was performed with a total yield of

37 % to give nucleoside 57 (19 % α, 18 % β). Side reactions were not examined but

a hydrolysis or polymerization of a fraction of compound 56 is probable. The anomers

could be differentiated by the through space coupling of the hydrogen atoms at C1’,

C2’ and C3’ using 1H-NOESY-NMR spectroscopy. The closure of the oxazoline ring of molecule 58 by treatment of 57 with ethanolamine in THF yielded the protected

ligandoside 58 in 35 % yield. The ring closure was performed after the glycosylation

to avoid the regioselectivity problems that were expected when the ligand 53 would

have been taken for the reaction (due to its two nucleophilic nitrogen atoms). Deprotection of the sugar hydroxyl group yielded nucleoside 52 and subsequent

Interestingly, a protection of the free NH group of molecule 59 (or OH-group of its

tautomer) was not possible with a variety of protecting groups (SEM, TES, TIPS). The DMT group could selectively be introduced onto the sugar’s 5’-position in moderate yields. However, generation of the phosphoramidite 60 was certainly

complicated by the free NH functionality: a mixture of two phosphorylated compounds (each as a mixture of diastereomers) was isolated. Although silica column chromatography of 60 resulted in partial decomposition, it was decided to use the

impure phosphoramidite for DNA synthesis. The coupling of the ligand nucleoside was of medium performance and the raw DNA material consisted of a mixture of the expected product and failure sequences.[199]

One hairpin and two complementary single strands containing nucleoside 52 were

synthesized, purified by RP-HPLC and characterized by high resolution ESI mass spectrometry (Figure 35).

D28-In-a 5’-CACATTAITGTTGTA-3’

D28-In-b 3’-GTGTAATIACAACAT-5’

D29-In 5’-GTAGAITTTTITCTAC-3’

Figure 35: Duplex D28-In-a/b and hairpin D29-In containing the 3-(2-oxazolidinylidene-)indol-2-one

nucleoside 52 prepared in this work.

According to the standard protocol, the melting temperature was measured in the absence and presence of metal ions (not shown). Even without any metal ions, the melting temperature of the duplex was, with 45.2 °C, 4 K higher than the melting temperature of a similar duplex containing the salicylic aldehyde bases 25 (Chapter

4.4.2). This small effect can be explained with the additional π-surface introduced with the two nucleosides 52. Unfortunately, addition of metal ions did not alter the

melting point of the duplex or the hairpin containing nucleoside 52. ESI mass

spectrometric analysis likewise did not furnish any data supporting a coordination of metal ions by the nucleosides 52. Figure 36 shows the ESI spectra of the

Figure 36: a) ESI mass spectrum of a mixture of D28-In-a and D28-In-b. The duplex breaks up into its

single strand components under ESI conditions. Found for [D28-In-a-7H+]7-:660.5391; calculated for [C154H185N50O91P14]7-: 660.5382; found for [D28-In-b-7H+]7-:663.1140; calculated for

[C154H183N56O87P14]7-: 663.1129; b) ESI mass spectrum of hairpin D29-In. Found for [D29-In-8H+]8-:

624.2293; calculated for [C170H202N49O100P15]8-: 624.2281. In both cases, no metal complexation could

be observed in the ESI experiments.

The reason for the incapability of the strands D28-In-a/b and D29-In to coordinate

metal ions might be that the nucleobases 52 exist in a conformation with the benzene

rings pointing away from the sugar moieties into the middle of the DNA duplex. This means that the potentially coordinating parts of the molecules are not facing each other in the DNA duplex and a metal ion cannot be coordinated between the complementary strands.