Detecting Subtle Sequence Signals : . A Gibbs Sampling Strategy for Multiple Alignment

1. 0. Diels and K. Alder, Justus Liebigs Ann. Chem. 460, 98 (1928); 0. Diels, J. H. Blom, W. Koll, ibid. 443, 242 (1925); B. M. Trost, I. Fleming, L. A. Paquette, Comprehensive Organic Synthesis (Pergamon, Oxford, 1991), vol. 5, pp. 316; F. Friguelli and A. Tatichi, Dienes in the Diels-Alder Reaction (Wiley, New York, 1990), and references therein; W. Carruthers, Cycloaddition Reactions in Organic Synthesis (Pergamon, Oxford, 1990), and references therein; G. Desimoni, G. Tacconi, A. Barco, G. P. Pollini, ACS Monograph No. 180 (American Chemical Society, Washington, DC, 1983), and references cited therein. 2. For recent reviews, see U. Pindur, G. Lutz, C. Otto, Chem. Rev. 93, 741 (1993); H. B. Kagan and 0. Riant, ibid. 92, 1007 (1992), and references therein. 3. D. Hilvert, K. W. Hill, K. D. Nared, M-T. M. Auditor, J. Am Chem. Soc. 111, 9261 (1989). 4. A. C. Braisted and P. G. Schultz, ibid. 112, 7430 (1 990). 5. C. J. Suckling, M. C. Tedford, L. M. Bence, J. I. Irvine, W. H. Stimson, Biorg. Med. Chem. Lett. 2, 49 (1992). 6. K. D. Janda, C. G. Sheviin, R. A. Lerner, Science 259, 490 (1993). 7. L. E. Overman, G. F. Taylor, C. B. Petty, P. J. Jessup, J. Org. Chem. 43, 2164 (1978). 8. For example: d-pumiliotoxin, C. L. E. Overman and P. J. Jessup, Tetrahedron Lett. 1253 (1977); J. Am. Chem. Soc. 100, 5179 (1978); d-perhydrogephyrotoxin, L. E. Overman and C. Fukaya, ibid. 102, 1454 (1980); dIisogabaculine, S. Danishefsky and F. M. Hershenson, J. Org. Chem. 44, 1180 (1979); tylidine, L. E. Overman, C. B. Petty, R. J. Doedens, ibid., p. 4183; amaryllidaceae alkaloids, L. E. Overman et al., J. Am. Chem. Soc. 103, 2816 (1981); gephirotoxine, L. E. Overman, D. Lesuisse, M. Hashimoto, ibid. 105, 16, 5373 (1983). 9. Diene 1b was prepared by neutralizing the corresponding carboxylic acid suspended in PBS (sodium phosphate buffer) with one equivalent of NaOH. 10. All new adducts were characterized by NMR and mass spectrometry. 11. In the crude mixture of the cycloaddition, we could not detect any trace of the meta regioisomer by 1H NMR (>98% ortho regioselective). 12. M. J. Frisch et al., Gaussian 92 (Gaussian, Pittsburgh, PA, 1992). 13. R. J. Loncharich, F. K. Brown, K. N. Houk, J. Org. Chem. 54, 1129 (1989). All structures were fully optimized with the 3-21G basis set. Reactants were also fully optimized with the 6-31G* basis set. Vibrational frequencies for all transition structures were calculated at the RHF/3-21 G level and shown to have one imaginary frequency. In addition, the energies were evaluated by single-point calculations with the 6-31G* basis set on 3-21G geometries. 14. K. N. Houk, R. J. Loncharich, J. Blake, W. Jorgensen, J. Am. Chem. Soc. 111, 9172 (1989). 15. L. E. Overman, G. F. Taylor, K. N. Houk, I. N. Domelsmith, ibid. 100, 3182 (1978). 16. M. I. Page and W. P. Jencks, Proc. Nati. Acad. Sci. U.S.A. 68, 1678 (1971). 17. F. Bernardi et al., J. Am. Chem. Soc. 110, 3050 (1989); F. K. Brown and K. N. Houk, Tetrahedron Lett. 25, 4609 (1984). 18. This strategy to avoid product inhibition was first described by Braisted and Schultz in (4). 19. The stereochemistry was established on the basis of 'H NMR analysis. For example: 5-exo, BH6 = 2.65 ppm and 4J6 7a= 3.5 Hz; 5-endo, &H6 = 3.12 ppm, and 6 7=a 0 Hz. 20. G. Kohler and C. Milstein, Nature 256, 495 (1975); the KLH conjugate was prepared by slowly adding 2 mg of the hapten dissolved in 100 &l of dimethylformamide to 2 mg of KLH in 900 gl of 0.01 M sodium phosphate buffer, pH 7.4. Four 8-week-old 129GIX+ mice each received 2 intraperitoneal (IP) injections of 100 gg of the hapten conjugated to KLH and RIBI adjuvant (MPL and TDM emulsion) 2 weeks apart. A 50 >g IP