ALBERT

Notes: Why Pentose- and Not Hexose-Nucleic Acids? Purine-Purine Pairing in homo-DNA: Guanine,Isoguanine, 2,6-Diaminopurine, and XanthineThis paper concludes the series of reports in this journal [1-4] on the chemistry of homo-DNA, the constitutionally simplifie dmodel system of hexopyranosyl-(6′ → 4′)-oligonucleotide systems stidued in our laboratory as potentially natural-nucleic-acid alternatives in the context of a chemical aetiology of nucleic-acid structure. The report describes the synthesis and pairing properties of homo-DNA oligonucleotides which contain as nucleobases exclusively purines, and gives, together with part III of the series [3], a survey of what we know today about purine-purine pairingin homo-DNA. In addition, the paper discusses those aspects of the chemistry of homo-DNA which, we think, influence the way how some of the structural features of DNA (and RNA) are to be interpreted on a qualitative level.Purine-purine pairing occurs in the homo-DNA domain in great variety. Most prominent is a novel tridentate Watson-Crick pair between guanine and isoguanine, as well as one between 2,6-diaminopurine and xanthinone, both giving rise to very stable duplexes containing the all-purine strands in antiparallel orientation. For the guanine-isoguanine pair, constitutional assignment is based on temperature-dependent UV and CD spectroscopy of various guanine- and isoguanine-containg duplexes in comparison with duplexes known to be paired in the reverse guanine is replaced by 7-carbauguanine. Isoguanine and 2,6-diaminopurine also have the capability of self-pariring in the reverse-Hoogsteen mode, as previously observed for adenine and guanine [3]. In this type of pairing, the interchangeably. Fig. 36 provides an overall survey of the relative strength of pairing in all possible purine-purine combinations.Watson-Crick pairing of isoguanine with guanine demands the former to participate in its 3H-tautomeric form; hitherto this specific tautomer had not been considered in the pairing chemistry of isoguanine. Whereas (cumulative) purine-purine pairing in DNA (reverse-Hoogsten or Hoogsteen) seems to occur in triplexes and tetrapalexes only, its occurrence in duplexes in a characteristic feature of homo-DNA chemistry. The occurrence of purine-purine Watson-Crick base pairs is probably a consequence of homo-DNA's quasi-linear ladder structure [1][4]. In a double helix, the distance between the two sugar C-atoms, on which a base pair is anchored, is expected to be constrained by the dimensions of the helix; in a linear duplex, however, there would be no restrictions with regard to base-pair length. Homo-DNA's ladder-like model also allows one to recognize one of the reasons why nucleic-acid duplexes prefer to pair in antiparallel, rather than parallel strand orientation: in homo-DNA duplexes, (averaged) backbone and base pair axes are strongly inclined toward one another [4]; the stronger this inclination, the higher the preference for antiparallel strand orientation is expected to be (Fig. 16).In retrospect, homo-DNA turns out to be one of the first artificial oligonucleotide systems (cf. Footnote 65) to demonstrate in a comprehensive way that informational base pairing involving purines and pyrimidines is not a capability unique to ribofuranosyl systems. Stability and helical shape of pairing complexes are not necessary conditions of one another; it is the potential for extensive conformational cooperativity of hte backbone structure with respect to the constellational demands of base pairing and base stacking that determines whether or nor a given type of base-carrying backbone structure is an informational pairing system. From the viewpoint of the chemical aetiology of nucleic-acid structure, which inspired our investigations on hexopyranosyl-(6′ → 4′)-oligonucleotide systems in the first place, the work on homo-DNA is only an extensive model study, because homo-DNA is not to be considered a potential natural-nucleic-acid altenratie. In retrospect, it seems fortunate that the model study was carried out, because without it we could hardly have comprehended the pairing behavior of the proper nucleic-acid alternatives which we have studied later and which will be discussed in Part VI of this series.The English footnotes to Fig. 1-49 provide an extension of this summary.

Notes: The synthesis of the enantiomerically pure, bridgehead-functionalized bicyclo[3.2.1]octanes 11 and 16, containing a conformationally fixed trihydroxypropyl (aminodihydroxypropyl) unit, as well as the X-ray structure of 11 are described. These compounds are of interest as sugar surrogates in the preparation of DNA analogs. Compounds 11 and 16 became available in 10 and 12 steps, respectively, and in an overall yield of 11 and 4% from D-arabinose via a highly stereoselective pinacol coupling as the key step.

Notes: We describe the synthesis of 2′-deoxy-3′,5′-ethano-D-ribonucleosides 1-8 (= (5′,8′-dihydroxy-2′-oxabicyclo-[3.3.0]oct-3′-yl)purines or -pyrimidines) of the nucleobases adenine, thymine, cytosine, and guanine. They differ from natural 2′-deoxyribonucleosides only by an additional ethylene bridge between the centers C(3′) and C(5′). The configuration at these centers (3S,5′R) was chosen as to match the geometry of a repeating nucleoside unit in duplex DNA as close as possible. These nucleosides were designed to confer, as constituents of an oligonucleotide chain, a higher degree of preorganization of a single strand for duplex formation with respect to natural DNA, thus leading to an entropic advantage for the pairing process. The synthesis of these ‘bicyclonucleosides’ was achieved by construction of an enantiomerically pure carbohydrate precursor 18/19 (Schemes 1), which was then converted to the corresponding nucleosides by known methods in nucleoside synthesis (Schemes 2 and 3). In all cases, both anomeric forms of the nucleosides were obtained in pure crystalline form, the relative configuration of which was established by 1H-NMR-NOE spectroscopy. A conformational analysis of the nucleosides with β-configuration at the anomeric center by means of X-ray and 1H-NMR (including NOE) spectroscopy show the furanose part of the molecules to adopt uniformly a 1′exo-conformation with the base substituents preferentially in the anti-range in the pyrimidine nucleosides (anti/syn ca. 2:1) distribution in the purine nucleosides (in solution).

Notes: We present here a nine-step synthesis of the thymine-containing amino ester 1, starting from commercially available methyl N-[(tert-butoxy)carbonyl]-L-serinate. Amino ester 1 is considered as a building block for the preparation of a new nucleic-acid analog with a chiral, flexible polyamide backbone. Key steps in the synthesis are the vitamin-B12-catalyzed addition of 3-bromo-N-[(tert-butoxy)carbonyl]-L-alaninate 2 to ethyl acrylate and the homologation of the corresponding N-protected α-amino acid 4 into the β-amino ester 6 by Arndt-Eistert chemistry. The latter was found to proceed with 10% inversion of configuration at the asymmetric center in 6. Resolution to enantiomerically pure material, however, was easily achieved by simple crystallization of 1.

Notes: Chemistry of α-Aminonitriles. Regioselective Synthesis and Crystal Structure of Uroporphyrinogen (Type I) OctanitrileA regioselective synthesis of uroporphyrinogen-octanitrile (type I) based on the strategy of multiple use of (dimethylmethylidene)ammonium iodide for stepwise regioselective functionalization of the pyrrole nucleus is described. This uroporphyrinogen derivative is remarkably stable and beautifully crystallizes in space group P1 with one molecule per unit cell. The crystal structure of the compound shows interesting conformational characteristics which are interpreted to be caused by subtle stereoelectronic effects. The English Footnotes to Schemes 1-3 and Figs. 1-12 provide an extension of this summary.

Notes: The synthesis of the Fmoc-protected amino acid 2 is presented. First attempts of amide-bond formation to the homodimer 4 in solution showed only poor coupling yields indicative for the low reactivity of the amino and carboxy groups in the building blocks 1 and 2, respectively (Scheme 1). Best coupling yields were found using dicyclohexylcarbodiimide (DCC) without any additive. The oligomerization of building block 2 adopting the Fmoc ((9H-fluoren-9-ylmethoxy)carbonyl) solid-phase synthesis yielded a mixture of N-terminal-modified distamycin-NA derivatives. By combined HPLC and MALDI-TOF-MS analysis, the N-terminal functional groups could be identified as acetamide and N,N-dimethylformamidine functions, arising from coupling of the N-terminus of the growing chain with residual AcOH or DCC-activated solvent DMF. An improved preparation of building block 2 and coupling protocol led to the prevention of the N-terminal acetylation. However, ‘amidination’ could not be circumvented. A thus isolated tetramer of 2, containing a lysine unit at the C-terminus and a N,N-dimethylformamidine-modified N-terminus, not unexpectedly, showed no complementary base pairing to DNA and RNA, as determined by standard UV-melting-curve analysis.

Notes: In three steps, 2-deoxy-D-ribose has been converted into a phosphoramidite building block bearing a (t-Bu)Me2Si protecting group at the OH function of the anomeric centre of the furanose ring. This building block was subsequently incorporated into DNA oligomers of various base sequences using the standard phosphoramidite protocol for automated DNA synthesis. The resulting silyl-oligomers have been purified by HPLC and selectively desilylated to the corresponding free apurinic DNA sequences. The hexamer d (A-A-A-A-X-A) (X representing the apurinic site) which was prepared in this way was characterized by 1H- and 31P-NMR spectroscopy. The other sequences as well as their fragments, which formed upon treatment with alkali base, were analyzed by polyacrylamide gel electrophoresis.

Notes: Why Pentose- and Not Hexose-Nucleid Acids? Part IV . ‘Homo-DNA’: 1H-, 13C-, 31P-, and 15N-NMR-Spectroscopic Investigation of ddGlc(A-A-A-A-A-T-T-T-T-T) in Aqueous SolutionFrom a comprehensive NMR structure analysis, it is concluded that the ‘homo-DNA’ oligonucleotide ddGlc(A-A-A-A-A-T-T-T-T-T) in 3 mM D2O solution (100 mM NaCl, 50 mM phosphate buffer, pH 7.0, T = 50°) forms a duplex of C2-symmetry, with its self-complementary oligonucleotide strands in antiparallel orientation. The 2′,3′-dideoxy-β-D-glucopyranosyl rings are in their most stable chair conformation, with all three substituents equatorial and with the adenine as well as the thymine bases in the anti-conformation. The base pairing is of the Watson-Crick type; this pairing mode (as opposed to the reverse-Hoogsteen mode) was deduced from the observation of inter strand NOEs between the adenine protons H—C(2) and the pyranose protons Hα-C(2′) of the sequentially succeeding thymidine nucleotides of the opposite strand, a correlation which discriminates between the Watson-Crick and the reverse-Hoogsteen pairing mode. The NOEs of the NH protons with either the adenine protons H—C(2) or H—C(8), that are normally used to identify the pairing mode in DNA duplexes, cannot be observed here, because the NH signals are very broad. This line broadening is primarily due to the fact that the exchange of the imino protons with the solvent is faster than for corresponding DNA duplexes.Computer-assisted modeling of the [ddGlc(A5-T5)]2 duplex with the program CONFOR [23], using the linear (idealized) homo-DNA single-strand conformation (α = -60°, β = 180°, γ = 60°, δ = 60°, ∊ = 180°, ζ = -60°, see [1] [3]) as the starting structure, resulted in two duplex models A and B (see Figs. 27-32, Scheme 9, and Table 4) which both contain quasi-linear double strands with the base-pairing axis inclined relative to the strand axes by ca. 60° and 45°, respectively, and with base-pair stacking distances of ca. 4.5 Å. While neither of the two models, taken separately, can satisfy all of the NMR constraints, the NMR data can be rationalized by the assumption that the observed duplex structure represents a dynamic equilibrium among conformers which relate to models A and B as their limiting structure. The required rapid equilibrium appears feasible, since the models A and B are interconvertible by two complementary 120° counter rotations around the α-axis and the γ-axis, respectively, of the phosphodiester backbone. The models A and B correspond to the two types of linear (idealized) single-strand backbone conformation derived previously by qualitative conformational analysis without and with allowance for gauche-trans-phosphodiester conformations, respectively [1] [3]. Refinement of the models A and B with the use of the program AMBER [27] by energy minimization in a water bath and molecular-dynamics simulations (2 ps, 300° K) resulted in two dynamic structures (Figs. 33 and 34, Table 4). These have roughly the same energy, closely resemble the starting structures A and B, and satisfy - as an ensemble - all of the NMR constraints without violating any van der Waals distances by more than 0.2 Å. Extensive fluctuations in base-pair distance and deviations from base-pair coplanarity, as well as the presence of water molecules in the cavities between some of the base pairs, were observed in both dynamic structures A and B, which, on the other hand, did not mutually interconvert within the short simulation time period used. These model properties, together with the conjectured equilibrium between the two structure types A and B, lead to the hypothesis of a homo-DNA duplex containing a ‘partially molten’ pairing core. This proposal could qualitatively account for a high rate of the NH exchange, as well as for part of the previously established [3] deficits in both enthalpic stabilization and entropic destabilization of homo-DNA duplexes relative to corresponding DNA duplexes. The phenomenon of the higher overall stability of homo-DNA duplexes vs. DNA duplexes (e.g, [ddGlc(A5-T5)]2, Tm = 59° vs. [d(A5-T5)]2, Tm = 33°, both at c ≈ 50 μM [3]) can then be seen as the result not only of a higher degree of conformational preorganization of the homo-DNA single strand toward the conformation of the duplex backbone [1] [3], but also of the entropic benefit of greater disorder in the central pairing zone of the homo-DNA duplex. This view of the structure of a homo-DNA duplex relates its characteristic properties to a central structural feature: the average base-pair distance in the models of homo-DNA is too large for regular base stacking (ca. 4.5 Å vs. ca. 3.5 Å in DNA). This difference in the distances between adjacent base pairs is a direct consequence of the quasi-linearity of the homo-DNA double strand as opposed to the right-handed twist of the helical DNA duplexes [1] [3], which is directly related to the specific conformational properties of pyranose rings as opposed to furanose rings [1]. Thus, the structural hypothesis derived from the NMR analysis of [ddGlc(A5-T5)]2 relates the conformational differences between homo-DNA and DNA directly to the sugar ring size, which is the essential constitutional difference between the two types of structure.The English footnotes to Figs. 1-34, Schemes 1-9, and Tables 1-4 provide an extension of this summary.