A data file was created containing internal coordinates and derived parameters for 178~-D-furanoside fragments, occurring in X-ray structures of ribo-, 2'-deoxyriboand arabinonucleoside derivatives. The variability in bond angles and torsion angles can be described by four conformational parameters; the kind and degree of ring puckering as expressed in phase angle of pseudorotation P and puckering amplitude " m , the conformation of the side-chain 0 and the glycosidic torsion angle X. Comparison between out-of-plane and torsion angle methods for calculation of pseudorotation parameters revealed that the latter method is better suited to describe the internal coordinates of the non-equilateral five-membered ring. Inspection of the distribution of phase angles and puckering amplitudes lends additional support to the hypothesis that conformational interconversion of the familiar Nand S type ring conformers is a process of hindered pseudorotation, proceeding through the 04'endo conformation (P = 90°) and maintaining a constant puckering amplitude. Both syn-anti distribution and preferred ranges of X are correlated with furanose conformation in ribonucleosides; the distribution is also affected by the type of base, purines showing higher syn/anti ratios than pyrimidines. Three modes are observed for the backbone torsion angle 0(C4'-C5'); the most abundant rotamer is 0., followed by 0•. The O. rotamer has so far been found only in S-type furanosides. Regressions based on existing or modified equations were used to express variations in internal coordinates in terms of the four parameters involved. As a consequence of its anomeric character, the glycosidic bond length can be described by a cyclic function of X. Endocyclic bond angles' and torsion angles show small but systematic deviations from the pseudorotation equations derived for equilateral rings. Based on observed correlations with the four parameters, reasonably accurate coordinates for exocyclic atoms, including hydrogen atoms, could also be obtained. Coordinates for model {3-D-furanoside fragments with preferred X and 0 orientations and variable pseudorotation parameters, covering a part of the N;:= S interconversion pathway, are presented. Their usefulness in future theoretical and experimental studies on nucleic acid conformations, both in the solid state and in solution, is discussed.• This paper is part XIII in the series Nucleic Acid Constituents from this laboratory. For part XII sec Nature, 281, 235 (1<)79).

SynopsisThe relationship between published vicinal proton-proton coupling constants and the pseudorotation properties of the pyrrolidine ring in L-proline, 4-hydroxy-~-proline, 4-fluoro-L-proline, and several linear and cyclic model proline peptides is investigated. Compared to earlier studies, several important improvements are incorporated (1) a new empirical generalization of the classical Karplus equation is utilized, which allows a valid correction for the effects of electronegativity and orientation of substituents on 35HH; (2) an empirical correlation between proton-proton torsion angles and the pseudorotational parameters P and T, is derived; and (3) the best fit of the conformational parameters to the experimental coupling constants is obtained by means of a computerized iterative least-squares procedure. Two pseudorotation ranges were considered, classified as type N ( x 2 positive sign) and type S (XZ negative sign). The conformational equilibrium is fully described in terms of four geometrical parameters (PN, TN, Ps, 7s) and the equilibrium constant K . The present results indicate that, in general, the geometrical properties found in x-ray studies of proline and hydroxyproline residues are well preserved in solution. Several novel features are encountered, however. It is demonstrated that the proline ring occurs in a practically 1:l conformational equilibrium between well-defined N-and S-type forms. Introduction of an amide group at the C-terminal end has no observable effect on this equilibrium, but the formation of a peptide bond a t the imino nitrogen site results in a pronounced, but not exclusive, preference for an S-type form which is roughly 1.1 kcal/mol more stable than its N-type counterpart. The hydroxyproline ring system in neutral or acidic medium displays a pure N-type state, but N-acetylation results in the appearance of a minor (S-type) conformation. Cyclic proline dipeptides similarly exist in a biased conformational equilibrium. The major form (77-88%) corresponds to the N-type conformer observed in the solid state; the minor S-form has not been observed before. In contrast, cyclic hydroxyproline dipeptides display complete conformational purity. Ranges of endocyclic torsion angles deduced for the various classes of pyrrolidine derivatives in solution are presented. Each torsion appears confined to a surprisingly narrow range, comprising about 4'-8" in most cases. In all, the proline ring is far less "floppy" than hitherto assumed.

The relationship between vicinal N M R proton-proton coupling constants and the pseudorotational properties of the sugar ring in nucleosides and nucleotides is reinvestigated. Compared with our earlier study several important improvements are introduced: first, a new empirical generalization of the classical Karplus equation is utilized, which allows an accurate correction for the effects of electronegativity and orientation of substituents on 3J(EIH); second, empirical correlations between the parameters governing the conformation of p-D-furanosides (taken from an analysis of 178 crystal stmctores) were used to define proton-proton torsion angles as a fnnctiou of the pseudorotation parameters P and a,,,; and, third an iterative least-squares computer program was devised to obtain the best fit of the conformational parameters to the experimental coupling constants. N M R data for the sugar ring in the following compounds were taken from the literature and analysed: 3',5'-cy&c nudeotides, a base-stacked nionucleotide, 2'-~ydroarabinonucleosides, or-~-2',2-0-cyclouridine, 2'-and 3'-aminosubstituted ribonucleosides, 2'-and 3'-deoxyribonucleosides. The present results confirm that the conformational properties found in the solid state are, on the whole, preserved in solution.

The rates of deacylation of 5'-U-acetyl-, -methoxyacetyl-, -phenoxyacetyl-, -formyl-, and -chloroacetyl-uridines (10a-e. respectively) in aqueous and methanolic ammonia have been measured. From these data, a procedure has been developed for the general preparation of 2'-0-(methoxytetrahydropyranyl)-3'-O-acyl ribonucleosides (9) by selective deacylation of suitably designed 2'-0-(methoxytetrahydropyranyl) -3',5'-di-O-acyl derivatives (8) * Two series of 2'-acetal 3'-esters (9), designed as building blocks for oligoribonucleotide synthesis, have been prepared from each of the four main ribonucleosides : one series (derived from uridine, 4-N-benzoylcytidine, adenosine, and 2-N-benzoylguanosine) consists of 3'-acetates or -benzoates and the other series (derived from uridine, 4-N-p-anisoylcytidine, 6-N-p-anisoyladenosine, and 2-N-benzoylguanosine) consists of 3'-methoxyacetates. All these 2'-U-(methoxytetrahydropyranyl) -3'-U-acyl ribonucleosides (9) have been obtained in satisfactory yields and all except one have been isolated as pure crystalline solids.IN our approach to oligoribonucleotide 2 9 3 synthesis it is desirable to have four building blocks derived from each ribonucleoside: a terminal 2',3'-, a terminal 2',5'-, a non-terminal 2',3'-, and a non-terminal 2',5'-derivative [( 1)-(4), respectively; see Scheme 11. The 2'-hydroxyfunctions are always blocked by acid-labile protecting groups; in the terminal building blocks (1) and (2), the 3'and 6'-hydroxy-functions, respectively, are also

Leu-Val -Thr-Leu-OH, p -LPH -( 70-77). 390 mg (0.43 mmol) of H-70-77-0tBu were treated with 90% aqueous TFA and purified in the Same way as was described for 7(t76. Yield 244 mg; [a]~? -121.3' (C = 0.5, 10% HoAc). Homogeneous on TLC: R, 0.48 (e). Amino acid analysis: Thr 1.94, Ser 0.98, Glu 1.04, Pro 1.04, Val 1.04, Leu 1.96. Peptide content 93.6 %. LAO digestion: no racemization was found. HPLC: a linear gradient of 20-70% B was run in 25 min; main component: 95.3%. W. A . A . J. B$ et al. / Synthesis of fragments of human 0-lipotropin, p,-LPH. Part I Acknowledgements The valuable technical assistance of Miss T. Nillesen and of A. Hendrix, H. polderdijk and M . UM Tilborg is greatfully acknowledged. We would like to thank the Biochem. R & D Lab B-4 Of Organon In'. (head Ir. B. ' . for performing the amino acid analyses, LAO digestions and HPLC experiments.

Proton NMR studies at 360 MHz are reported on the adenine dinucleoside monophosphates N6-dimethyladenyly(3'-5')-N6-dimethyladenosine (m(6)(2)Apm(6)(2)A), ApA, rApdA, dAprA and on the methyl phosphate esters of the monomers m(6)(2)Ap, pm(6)(2)A, Ap and pA. Complete 1H-NMR spectral assignments are given. The dimers were also investigated by means of circular dichroism to obtain accurate thermodynamic parameters of the stacking equilibrium. With the aid of the thermodynamic data NMR coupling constants are extrapolated to values appropriate to the stacked conformers. A modernized version of pseudorotation analysis is used to delineate the conformational behaviour of the ribose and 2'-deoxyribose rings. It is shown that the unmethylated dimers can be arranged in two groups (dApdA/dAprA vs ApA/rApdA) according to their melting temperatures. ApA and the fully N6-methylated dimer m(6)(2)Apm(6)(2)A prefer to adopt the classical right-handed N-N stacked conformation. Both dimers with a 2'-deoxyribose ring at the 5'-OH end (dApdA and dAprA) behave similarly and occur in solution as a 75:25 mixture of S-S and S-N stacked states. The fully stacked hybrid dimer rApdA displays an unexpectedly large amount of S conformers (greater than 40%) in both sugar rings. This finding is rationalized by the postulation of a right-handed helical S-S stacked state on the basis of NMR and circular dichroic data.

Crystals of the title compound are orthorhombic, space group P2, 2, 2, .with Z = 4 in a unit cell of dimensions: a = 16.48(1), b = 4.752(4), and c = 15.94(1) A. The structure was determined from three-dimensional diffractometer data by direct methods, and refined by least-squares methods to R 4.04%. The dioxolan and ribofuranose rings are cis-connected about the C(2')-C(3') bond, The relatively flattened ribose ring 23") has the C(2')endo S-conformation, the glycosidic torsion angle lies in the normal anti range (xCN 56.5') and the orientation of the C(5')-O(5') side-chain is gauche-gauche. The crystal packing is governed by O(2) N(3) (2.696 A) and O(4) * * * O(5') (2.907 A) hydrogen bonds. lH n.m.r. spectroscopy reveals the existence of various conformational equilibria in solution. Vicinal coupling constants between ribose protons are consistent with an N [C(3')-endo] == S [C(2')-endo] equilibrium with a slightly more puckered ribose ring than occurs in the crystal structure. The C(5')-0(5') side-chain shows almost equal populations of gauche-gauche-and gauche-trans-rotamers with a minor contribution of the transgauche-conformation. The c.d. spectrum and the chemical shifts of the base protons H(5) and H(6) indicate a preference for the anti-conformation about the glycosidic bond.KNOWLEDGE of conformation and geometry of fragments of nucleic acids or their analogues may be helpful for the interpretation of physical data (n.m.r., c.d.), for correlation with tRNA structures and, more generally, helical structures of nucleic acids. 0 endo R1=H, R2=OMe (S) ex0 R1=OMe, Ra=H (R)

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