Factor F430 from Methanogenic Bacteria: On the Nature of the Isolation Artefacts of F430, a Contribution to the Chemistry of F430 and the Conformational Stereochemistry of the Ligand Periphery of Hydroporphinoid Nickel(I1) ComplexesFactor F430 (l), a coeiizyme from methanogenic bacteria, when heated in aqueous solution isomerizes to 12,13-di-epi-F430 (5) via 13-epi-F430 (3). The equilibrium mixture of the three F430 isomers in aqueous phosphate buffer solution (pH 7,100")contains 88 % of 5,8 % of 3, and 4% of 1 (Scheme I ) . The structural assignment for the F430 isomers rests on FAB-MS-, UVjVIS-, 'H-and I3C-NMR spectra of their pentamethyl esters. Chemical proof for the double epimerization at the two chiral centers of F430s ring C was provided by ozonolytic degradation of the di-epimer to give a ring-C-derived succinimide derivative that was shown to be the enantiomer of the one previously obtained by ozonolysis of F430M (see Scheme 2). The two F430 ring-C epiiners 3 and 5 are the isolation artefacts described in the earlier F430 literature. F430 is susceptible to autoxidation in air and the product, that absorbs at 560 nm, was shown to be the 12,13-didehydro derivative 8 of F430 by spectroscopic characterization of its pcntamethyl ester 9. The dehydrogenation product 8 can be diastereoselectively reduced with Zn in AcOH to give natural F430 as the main product rather than the thermodynamically more stable F430-di-epimer (Scheme 3 ) . In the double epimerization of F430, the two ring-C side chains change from a trans-quasi-diaxial arrangement to the (locally) enantiomorphic position in which the same side chains are again in a trans-quasi-diaxial arrangement. This equilibrium paradox as well as the kinetic diastereoselectivity of the reduction of 12,13-didehydro-F430 (8) are rationalized to be consequences of the general phenomenon documented earlier (see the preceding paper) according to which hydroporphinoid Ni(I1) complexes all show a characteristic conformational ruffling of their ligand system due to the tendency of the (small) Ni(lI) ion to contract thc size of the ligand's central coordination hole (see Fig.5 and 6

Ziagen, (1S,cis)-4-[2-amino-6-(cyclopropylamino)-9H-purin-9-yl]- 2-cyclopentene-1-methanol, was synthesized from (1S,4R)-azabicyclo[2.2.1]hept-5-en-3-one by efficient processes which bypass problematic steps in earlier routes. 2-Amino-4,6-dichloro-5-formamidopyrimidine is a key intermediate which makes possible an efficient construction of the purine from a chiral cyclopentenyl precursor.

A discrete tetravalent conjugate, 7a (LJP 394), consisting of four oligonucleotides attached to a common carrier or platform was prepared. Single-stranded oligonucleotide 20-mers consisting of alternating deoxycytidine-deoxyadenosine nucleotides, (CA)10, were attached to a tetrabromoacetylated platform by displacement with sulfhydryl-terminated linkers. The tetrabromoacetylated platform 3a was synthesized in three steps using triethylene glycol bis-(chloroformate). The single-stranded conjugate was characterized by polyacrylamide gel electrophoresis, DNA sequencing, phosphate analysis, carbon and nitrogen combustion analysis, and correlation of stoichiometry to conversion in the conjugation process. HPLC and capillary electrophoretic methods were developed to evaluate purity. The tetrakis, single-stranded conjugate was annealed with a stoichiometric amount of a complementary single-stranded oligonucleotide 20-mer consisting of alternating thymidine-deoxyguanosine nucleotides, (TG)10. The double-stranded conjugate LJP 394 was characterized by melt temperature and hyperchromicity, phosphate analysis, and carbon and nitrogen combustion analysis. LJP 394 inhibits binding of DNA to anti-double-stranded oligonucleotide antibodies and reduces anti-oligonucleotide-specific plaque (antibody)-forming cells in an immunized mouse model by a proposed mechanism involving cross-linking B cell surface immunoglobins.

Factor F430, the porphinoid nickel-containing coenzyme of the methylcoenzyme-M reductase of methanogenic bacteria is shown to be the 33,83, 122,133, 18*-pentaacid derivative of the pentamethylester F430M, the structure of which had been determined previously (see structural formulae 1 and 2). The structure assignment rests on chromatographic, UVjVIS-, CD-, IR-, and '3C-NMR-spectroscopic as well as FAB-mass spectral comparison of F430 with F430M and the pentaacid prepared by acid-catalyzed hydrolysis of F430M.in the cells of Methanobacterium thermoautotrophicum, factor F430 is present in a 'bound' and also, depending on the growth conditions, in a 'free' form, the latter being defined as the part of total F430 that can be extracted from the cells under extremely mild conditions (80% EtOH at O d ) . From the (protein)-'bound' form, F430 is extracted by subsequently treating the cells at 0-4" with 80% EtOH containing (e.g.1 2 M LiC1.From both sources, the extracted factor is the same pentaacid, and there is no indication for the existence of a protein-free F430 species that would contain additional (covalently bound) structural elements.in unserer vorangegangenen Mitteilung [l] zur Kenntnis des Faktors F430 [2], eines Bestandteils der Methylcoenzym-M-Reduktase aus methanogenen Bakterien [3], haben wir uber die Strukturermittlung von F430M mittels biosynthetischer, chemischer und instrumentalanalytischer Methoden berichtet. F430M ist das .Perchlorat eines durch siiurekatalysierte Methanolyse von F430-Isolaten [4] gewonnenen, chromatographisch und spektroskopisch einheitlichen Ni(I1)-Komplexes, fur dein wir die Struktur 1 (X = C10,)') abgeleitet haben [ 11. Seine charakteristischen UVjVIS-und CD-spektroskopischen Daten unterscheiden sich kaum von jenen, welche bislang der spektroskopischen Definition [2], Erkennung und Gehaltsbestimmung [2] [4,] des naturlichen Faktors in F430-isolaten aus methanogenen Bakterien gedient haben. Deshalb nahmen wir ') Die Zuordnung der Chiralitat von 1 im Bereiche der Ringe A und B Cjedoch nicht des Rings C) sowie der Kontiguration an C(17) sind bislang experimentell nicht direkt belegt [l].