Introduction

1. CONFORMATIONAL ANALYSIS OF DEOXYFLUORO SUGARS AND NUCLEOSIDES: PSEUROT PROGRAM INCORPORATING COMPUTER ANALYSIS OF THE BOND ANGLE CORRECTION TERM Ivan V. ANISHENKO,1 Cornelis ALTONA,2 and Igor A. MIKHAILOPULO1* 1Institute of Bioorganic Chemistry, National Academy of Sciences, Acad. Kuprevicha 5/2, 220141 Minsk, Belarus 2Gorlaeus Laboratories, Leiden Institute of Chemistry, Leiden University, NL-2300 RA Leiden, The Netherlands Abstract: An extension of the PSEUROT program 6.3 consisting in computer optimization of rmsd values by means of the Geff variations within the calculated by Chattopadhyayaet al. (J. Org. Chem. 1998, 63, 4967) limits. An application of this approach to the search for the correct output pseudorotation parameters resulting from a new generalized Karplus-type equation gives rise to the low rmsd and ΔJ(H,H) and ΔJ(H,F) values employing scale factors for 3J(H,H) and 3J(H,F) equal to 1.0. In continuation of this study, we applied the extended FLUOROT analysis for related 2'-fluorinated arabinosides, viz., 6-amino-3-bromo-1-(2-deoxy-2-fluoro--D-arabinofuranosyl)-1H-pyrazolo[3,4-d]pyrimidin-4-one (1; FAra-PP) [5] and 1-(2-deoxy-2-fluoro--D-arabinofuranosyl)-6-azauracil (2; FAra-6NU) [1b,e]. Both compounds showed the remarkably rigid N-conformation of the pentofuranose rings in solution. In the present work, we applied for both nucleosides the A and B parameters of Barchi et al. [2] and Lowary et al. [6] calculated for -D-arabinofuranose. The resulted pseudorotation parameters are shown in Table 1 . Introduction In our previous papers [1,2] we have analyzed the conformational peculiarities of deoxyfluoro sugars and nucleosides using the PSEUROT 6.3 program [3] (the FLUOROT version [2]), which based on a new generalized Karplus-type equation relating vicinal proton-fluorine coupling constants to H-C-C-F torsion angles [4]. The last correction term of the generalized Karplus-type equation (term F) describes the H-C-C (HCC) and F-C-C (FCC) bond angle changes from the tetrahedral values. The bond angles  were determined for a number of deoxyfluoro nucleosides from 3-21G ab initio calculations and the parameters A-G were optimized [4]. In our calculations, we used slightly changed the bond angle correction term, which involves the determination of the range of best pseudorotational parameters on the basis of 3J(H,H) and 3J(H,F) and then fixing the con-formers found to manually fit ΔGeff along the coupled path in question [1a,f;2]. Results and Discussion The vicinal proton-fluorine coupling constants are expressed through the Karplus-type equation (1) The last summand in (1) describes the dependence of 3J(H,F) on the F-C-C and H-C-C bond angle changes. Eventually both bond angles can be reduced to one parameter Geff = 3.72[(FCC+HCC)/2 – 110] affecting the magnitude of 3J(H,F). In case the whole fluorinated pentofuranose system is considered, several F-C-C-H coupled paths should be taken into account with the corresponding set of Geff values which are incorporated in the FLUOROT program as fixed parameters. Since the bond angle values HCC and FCC can undergo slight changes [4] leading to the variations in the magnitude of proton-fluorine coupling constants 3J(H,F) they consequently influence the quality of the fit during FLUOROT calculations. Hereby we introduce an automated procedure making it possible to optimize the rmsd values by varying the Geff parameters within their tolerable limits. This approach was realized in a computer program which is hereinafter referred to as the extended FLUOROT program. The new methodology is based on the Altona’s original PSEUROT 6.3 (FLUOROT) program whereby the best fit of experimental 3J(H,H) and 3J(H,F) coupling constants to the conformational parameters of a pentofuranose ring is performed under the assumption of the two-state N/S equilibrium. The original FLUOROT routine is employed in an unmodified fashion each time when the new set of Geff values is tested in the process of rmsd optimization. It was observed that for some pentofuranose systems the variation of the HCC and FCC bond angles within the permissible limits leads to the smooth (or approximately smooth) rmsd dependence on Geff parameters obtained from FLUOROT calculations. In this case the search for the Geff parameters resulting in the lowest rmsd values can be successfully performed by the Fletcher-Reeves conjugate gradient algorithm. For the other compounds the very same rmsd profile is rather rigid and it may be moreover complexed with the value areas at which the FLUOROT routines doesn’t converge and rmsd is not defined. For the latter case a more computationally expensive simulated annealing protocol is applied. The program also provides for the possibility of the multiple optimizations of Geff parameters starting each time from the random point in the permissible region in the Geff space. It allows one (i) to attain a more complete coverage of the search space and (ii) to estimate the possible deviations in the calculated pseudorotation parameters and the values of the N/S population. The extension to the FLUOROT program was written in C++ and the input format didn’t change from the original FLUOROT program. The calculations started with no fixed parameters; scale factors for both [H,H] and [H,F] couplings equal to 1.0 were used throughout, and the calculated A and B parameters [2]. Barchi et al. have scrupulously investigated the stereochemistry of 1-(2,3-dideoxy-2,3-difluoro--D-arabinofuranosyl)uracil (FFAra-U), and 1-(2,3-dideoxy-2,3-difluoro--D-xylofuranosyl)-uracil (FFXylo-U) and -6-azauracil (FFXylo-6NU) using an totality of spectral and theoretical methods [2]. Conclusion Refinement of the pseudorotation parameters employing extended FLUOROT program led usually to the very low rms deviations and ΔJ(H,H) and ΔJ(H,F) values compared to those previously obtained by manual fitting of ΔGeff along the coupled path under consideration. The earlier FLUOROT calculations [2] as well as presented here clearly evidence in favour of the use of the scale factors equal to 1.0 for both [H,H] and [H,F] couplings. Acknowledgements: Financial support by the International Science and Technology Centre (project #B-1640) is gratefully acknowledged. IAM is thankful to Dr. Joe J. Barchi, Jr., (Laboratory of Medicinal Chemistry, NCI, Frederick, Maryland 21702) for numerous stimulating discussions and to the A. von Humboldt-Stiftung (Bonn – Bad-Godesberg, Germany) for partial financial support of this study. References 1. (a) Mikhailopulo, I.A., Pricota, T.I., Sivets, G.G., Altona, C. J. Org. Chem., 2003, 68, 5897-5908; (b) Seela, F., Peng, X., Li, H., Chittepu, P., Shaikh, K.I., He, J.; He, Y., Mikhailopulo, I. Collection Symp. Series, 2005, 7, 1-20; (c) Mikhailopulo, I.A., Sivets, G.G. Helv. Chim. Acta, 1999, 82, 2052-2065; (d) Krawiec, K., Kierdaszuk, B., Kalinichenko, E.N., Rubinova, E.B., Mikhailopulo, I.A., Eriksson, S., Munch-Petersen, B., Shugar, D. Nucleosides, Nucleotides & Nucl. Acids, 2003, 22, 153-173; (e) Mikhailopulo, I.A., Sokolov, Y.A., He, J., Chittepu, P., Rosemeyer, H., Seela, F. Nucleosides Nucleotides & Nucl. Acids, 2005, 24, 701-705. (f) Sivets, G.G., Kalinichenko, E.N., Mikhailopulo, I.A. Helv. Chim. Acta, 2007, 90, 1818-1836. 2. 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