DNA strand scission by activated bleomycin group antibiotics

The bleomycins (BLMs) are a structurally related group of antitumor antibiotics used clinically for the treatment of certain malignancies. The mechanism of action of the BLM is believed to involve DNA strand scission, a process that requires O2 and an appropriate metal ion; the therapeutically relevant metal is probably iron or copper. DNA strand scission by activated Fe X BLM involves oxygenation C-4' of deoxyribose and leads to two sets of products. One set results from scission of the C-3'--C-4' bond of deoxyribose, with concomitant cleavage of the DNA chain. The other set of products consists of free bases and an alkali-labile lesion, the latter of which leads to DNA chain cleavage on subsequent treatment with base. The structures of all of these degradation products have now been established by direct comparison with authentic synthetic samples. Also studied was the activation of BLM with (mono)oxygen surrogates such as iodosobenzene. The chemistry of the activated BLM so formed was remarkably similar to that of activated cytochrome P-450 and structurally related metalloporphyrins, which suggests a mechanistic analogy between the two. Remarkably, both Fe X BLM and Cu X BLM were also shown to be activated by NADPH cytochrome P-450 reductase in a transformation that was dependent on metal ion, O2 and NADPH.     

Chemistry of the alkali-labile lesion formed from iron(II) bleomycin and d(CGCTTTAAAGCG)

Two sets of products are formed from DNA upon treatment with Fe(II).bleomycin + O2. One set, which is believed to derive from a C-4' hydroperoxy derivative of the DNA deoxyribose moiety, includes the four possible base propenals, as well as DNA oligomers having deoxynucleoside 3'-(phosphoro-2"-O-glycolates) at their 3'-termini. The other set of products consists of free bases and alkali-labile lesions, the latter of which had not previously been characterized structurally. By use of the self-complementary dodecanucleotide d(CGCTTTAAAGCG) having a site modified by Fe-bleomycin three nucleotides from the 5'-end, it has been possible to characterize the alkali-labile product as a C-4' hydroxyapurinic acid. When the bleomycin-treated dodecanucleotide was treated with agents that effected decomposition of the alkali-labile lesion, products of the form CpGpx were obtained, and these proved useful for structural characterization of the alkali-labile lesion. Treatment with alkali produced CpGpx, where x was 2,4-dihydroxycyclopentenone. Alternatively, treatment with hydrazine provided a pyridazine derivative, and aqueous alkylamines led to formation of CpGp itself. The structures of all dinucleotides produced from the alkali-labile lesion were verified by direct comparison with authentic synthetic samples.     

C-1' hydrogen abstraction of deoxyribose in DNA strand scission by dynemicin A.

Dynemicin A, which is a hybrid antitumor antibiotic containing anthraquinone and enediyne cores, abstracts the C-1' hydrogen of DNA deoxyribose and then the damaged DNA leads to strand breaks with the formation of 5'- and 3'-phosphate termini. The lesions of C-4' hydrogen also occur at 3' side of G.C base pairs (i. e., 5'-CT and 5'-GA), leading to 5'-phosphate and 3'-phosphoglycolate termini or 4'-hydroxylated abasic sites. The C-1' hydrogen abstraction by dynemicin A is distinct from the preferential C-5' hydrogen abstraction of calicheamicin and neocarzinostatin.     

Oxygen transfer from the nitro group of a nitroaromatic radiosensitizer to a DNA sugar damage product

Mechanisms based on one-electron oxidation appear incomplete in explaining cellular radiosensitization by nitroaromatic compounds such as misonidazole. Evidence is presented for a novel mechanism that may be involved in enhancing DNA strand breakage due to a variety of agents, including ionizing radiation, that generate carbon-centered radicals on DNA deoxyribose. Under anaerobic conditions the carbon-centered radical generated selectively at C-5' of deoxyribose of thymidylate residues in DNA by the antitumor antibiotic neocarzinostatin reacts with misonidazole to produce a DNA damage product in the form of 3'-(formyl phosphate)-ended DNA. In an 18O-transfer experiment we find that the carbonyl oxygen of the activated formyl moiety (trapped as formyl-Tris) is derived from the nitro group oxygen of misonidazole. This result strongly supports a mechanism in which a nitroxide radical adduct, formed by the addition of misonidazole to the radical at C-5' of deoxyribose, cleaves between the N and O so as to form an oxy radical precursor of the formyl moiety and a two-electron reduction species of misonidazole.     

NMR investigation of mithramycin A binding to d(ATGCAT)2: a comparative study with chromomycin A3

The binding of mithramycin A to the d(A1T2G3C4A5T6) duplex was investigated by 1H NMR and found to be similar to that of its analogue chromomycin A3. In the presence of Mg2+, mithramycin binds strongly to d(ATGCAT)2. On the basis of the two-dimensional NOESY spectrum, the complex formed possesses C2 symmetry at a stoichiometry of two drugs per duplex (2:1) and is in slow chemical exchange on the NMR time scale. NOESY experiments reveal contacts from the E-pyranose of mithramycin to the terminal and nonterminal adenine H2 proton of DNA and from the drug hydroxyl proton to both G3NH2 protons, C4H1' proton, and A5H1' proton. These data place the drug chromophore and E pyranose on the minor groove side of d(ATGCAT)2. NOE contacts from the A-, B-, C-, and D-pyranoses of mithramycin to several deoxyribose protons suggest that the A- and B-rings are oriented along the sugar-phosphate backbone of G3-C4, while the C- and D-rings are located along the sugar-phosphate backbone of A5-T6. These drug-DNA contacts are very similar to those found for chromomycin binding to d(ATGCAT)2. Unlike chromomycin, the NOESY spectrum of mithramycin at the molar ratio of one drug per duplex reveals several chemical exchange cross-peaks corresponding to the drug-free and drug-bound proton resonances. From the intensity of these cross-peaks and the corresponding diagonal peaks, the off-rate constant was estimated to be 0.4 s-1. These data suggest that the exchange rate of mithramycin binding to d(ATGCAT)2 is faster than that of chromomycin.     

Poly(deoxyadenylic-deoxythymidylic acid) damage by radiolytically activated neocarzinostatin

The anaerobic reaction of poly(deoxyadenylic-deoxythymidylic acid) with neocarzinostatin activated by the carboxyl radical CO2-, an electron donor generated from gamma-ray radiolysis of nitrous oxide saturated formate buffer, has been characterized. DNA damage includes base release and strand breaks. Few strand breaks are formed prior to alkaline treatment; they bear 3'-phosphoryl termini. In contrast, most (66%) of the base release occurs spontaneously. DNA damage is highly (95%) specific for thymidine sites. Neither DNA-drug covalent adduct nor nucleoside 5'-aldehyde, which are major products in the DNA-nicking reaction initiated by mercaptans and oxygen, is formed in this reaction. Data are presented to show that the CO2(-)-activated neocarzinostatin intermediate is a short-lived free radical able to abstract hydrogen atoms from the C-1' and C-5' positions of deoxyribose. Attack occurs mostly (68%) at the C-1' position, producing a lesion whose properties are consistent with those of (oxidized) apyrimidinic sites.     

Mechanism of bleomycin action: in vitro studies

The cytotoxic activity of bleomucin results from DNA cleavage, which is also accomplished $\text{in vitro}$ by reaction mixtures containing Fe(II), drug and O₂. Bleomycin forms a complex with Fe(II) and O₂ in the presence or absence of DNA. The species attacking DNA forms rapidly from this complex. The nature of the attacking species and of the primary lesion(s) to DNA are not yet known, but two major insults to DNA have been characterized. They are the release of free bases from their glycosidic linkages and, at other residues, the cleavage of the polymer backbone at the deoxyribose C3-C4 bond.     

Photo-induced hole transfer from base to sugar in DNA: relationship to primary radiation damage

This work presents the hypothesis that photo-excitation of G.+ in DNA and model systems results in the same electronic states expected from direct ionization of the sugar phosphate backbone and that these states lead to specific sugar radicals on the DNA sugar phosphate backbone. As evidence we show that visible photo-excitation of guanine cation radicals (G.+) in the dinucleoside phosphate TpdG results in high yields (about 85%) of deoxyribose sugar radicals at the C1' and C3' sites. Further, we have calculated transition energies of hole transfer from G.+ in TpdG using time-dependent density functional theory (TD-DFT) at the B3LYP/6-31G(d) level in gas phase as well as in a solvated environment. These calculations clearly predict that visible excitation of G.+ in TpdG causes transitions from only inner-shell filled molecular orbitals (MOs) to the singly occupied molecular orbital (SOMO) that effectively result in hole transfer from guanine either to the sugar phosphate backbone or to the adjacent base, thymine. The hole transfer is followed by rapid deprotonation from the sugar to form C1' and C3' radicals. These experimental and theoretical results are in agreement with our previously published experimental and theoretical results that photo-excitation of G.+ results in high yields of deoxyribose sugar radicals in DNA, guanine deoxyribonucleosides and deoxyribonucleotides. Photo-excitation of G.+ therefore provides a convenient method to produce and study sugar radicals that are expected to be formed in gamma-irradiated DNA systems unencumbered by the many other pathways involved in direct ionization.     

Atypical abasic sites generated by neocarzinostatin at sequence-specific cytidylate residues in oligodeoxynucleotides

Neocarzinostatin chromophore produces alkali-labile, abasic sites at cytidylate residues in AGC sequences in oligonucleotides in their duplex form. Glutathione is the preferred thiol activator of the drug in the formation of these lesions. The phosphodiester linkages on each side of the abasic site are intact, but when treated with alkali, breaks are formed with phosphate moieties at each end. Similar properties are exhibited by the abasic lesions produced at the purine residue to which the C in AGC is base-paired on the complementary strand. The abasic sites at C residues differ from those produced by acid-induced depurination in the much greater lability of the phosphodiester linkages on both sides of the deoxyribose, in the inability of NaBH4 to prevent alkali-induced cleavage, and in the relative resistance to apurinic/apyrimidinic endonucleases. The importance of DNA microstructure in determining attack site specificity in abasic site formation at C residues is shown not only by the requirement for the sequence AGC but also by the findings that substitution of G by I 5' to the C decreases the attack at C, whereas placement of an I opposite the C markedly enhances the reaction. Quantitation of the abstraction of 3H into the drug from C residues in AGC specifically labeled in the deoxyribose at C-5' or C-1',2' suggests that, in contrast to the attack at C-5' in the induction of direct strand breaks at T residues, abasic site formation at C residues may involve attack at C-1'. Each type of lesion may exist on the complementary strands of the same DNA molecule, forming a double-stranded lesion.     

Conformation and dynamics of the deoxyribose rings of a (nogalamycin)2-d (5''-GCATGC)2 complex studied in solution by 1H-n.m.r. spectroscopy.

The conformation and dynamics of the deoxyribose rings of a (nogalamycin)2-d(5'-GCATGC)2 complex have been determined from an analysis of 1H-1H vicinal coupling constants and sums of coupling constants (J1'-2',J1'-2",epsilon 1', epsilon 2' and epsilon 2") measured from one-dimensional n.m.r. spectra and from H-1'-H-2' and H-1'-H-2" cross-peaks in high-resolution phase-sensitive two-dimensional correlation spectroscopy (COSY) and double-quantum-filtered correlation spectroscopy (DQF-COSY) experiments. The value of J3'-4' has also been estimated from the magnitude of H-3'-H-4' cross-peaks in DQF-COSY spectra and H-1'-H-4' coherence transfer cross-peaks in two-dimensional homonuclear Hartman-Hahn spectroscopy (HOHAHA) spectra. The data were analysed, in terms of a dynamic equilibrium between North (C-3'-endo) and South (C-2'-endo) conformers, by using the graphical-analysis methods described by Rinkel & Altona [(1987) J. Biomol. Struct. Dyn. 4,621-649]. The data reveal that the sugars of the 2C-5G and 3A-4T base-pairs, which form the drug-intercalation site, have strikingly different properties. The deoxyribose rings of the 2C-5G base-pair are best described in terms of an equilibrium heavily weighted in favour of the C-2'-endo geometry (greater than 95% 'S'), with a phase angle, P, lying in the range 170-175 degrees and amplitude of pucker between 35 and 40 degrees, as typically found for B-DNA. For the deoxyribose rings of the 3A-4T base-pair, however, the analysis shows that, for 3A, the C-2'-endo and C3'-endo conformers are equally populated, whereas a more limited data set for the 4T nucleotide restricts the equilibrium to within 65-75% C-2'-endo. The deoxyribose rings of the 1G-6C base-pair have populations of 70-80% C-2'-endo, typical of nucleotides at the ends of a duplex. Although drug-base-pair stacking interactions are an important determinant of the enhanced duplex stability of the complex [Searle, Hall, Denny, & Wakelin (1988) Biochemistry 27, 4340-4349], the current findings make it clear that the same interactions can be associated with considerable variations in the degree of local structural dynamics at the level of the sugar puckers.     

Detection of neocarzinostatin chromophore-deoxyribose adducts as exonuclease-resistant sites in defined-sequence DNA

A 5'-end-labeled DNA restriction fragment was treated with the nonprotein chromophore of neocarzinostatin under anoxia in the presence of dithiothreitol, conditions known to maximize formation of chromophore-deoxyribose adducts. Under conditions where unmodified DNA was digested to completion, chromophore-treated DNA was highly resistant to digestion by exonuclease III plus the 3'----5' exonucleolytic activity of T4 DNA polymerase and partially resistant to digestion by exonuclease III plus snake venom exonuclease. The electrophoretic mobilities of the products of exonucleolytic digestion suggested that (i) digestion by exonuclease III or T4 polymerase terminated one nucleotide before the nucleotide containing the adduct, (ii) the remaining nucleotide directly adjacent to the adduct (3' side) could be removed by snake venom phosphodiesterase, but at a slow rate, (iii) the covalently linked chromophore decreased the electrophoretic mobilities of the digestion products by the equivalent of approximately three nucleotides, and (iv) adducts formed under anaerobic conditions occurred at the same nucleotide positions as the strand breaks formed under aerobic conditions (primarily at T and, to a lesser extent, A residues). The close similarity in sequence specificity of adducts and strand breaks suggests that a common form of nascent DNA damage may be a precursor to both lesions. A chromophore-induced free radical on C-5' of deoxyribose, subject to competitive fixation by addition reactions with either oxygen or chromophore, is the most likely candidate for such a precursor. The base specificity of adduct formation does not reflect the reported base specificity of neocarzinostatin-induced mutagenesis, suggesting that lesions other than adducts may be responsible for at least some neocarzinostatin-induced mutations, particularly those occurring at G X C base pairs.     

Structural studies of lipid A from Pseudomonas aeruginosa PAO1: occurrence of 4-amino-4-deoxyarabinose.

Lipid A derived from Pseudomonas aeruginosa PAO1 contains a biphosphorylated 1-6-linked glucosamine disaccharide backbone. The reducing glucosamine has an unsubstituted glycosidically linked phosphate at C-1. The nonreducing glucosamine has an ester-bound phosphate at C-4' which is nonstoichiometrically substituted with 4-amino-4-deoxyarabinose. Induction of 4-amino-4-deoxyarabinose was dependent on cultural conditions. No pyrophosphate groups were detected. Acyloxyacyl diesters are formed by esterification of the amide-bound 3-hydroxydodecanoic acid with dodecanoic acid and 2-hydroxydodecanoic acids in an approximate molar ratio of 2:1. Dodecanoic and 3-hydroxydecanoic acids are esterified to positions C-3 and C-3' in the sugar backbone. All hydroxyl groups of the glucosamine disaccharide except C-4 and C-6' are substituted. Lipopolysaccharide chemical analyses measured glucose, rhamnose, heptose, galactosamine, alanine, phosphate, and glucosamine. The proposed lipid A structure differs from previous models. There are significant differences in acyloxyacyl diesters, and the proposed model includes an aminopentose substituent.     

Determination of the DNA sugar pucker using 13C NMR spectroscopy

Solid-state 13C NMR spectroscopy of a series of crystalline nucleosides and nucleotides allows direct measurement of the effect of the deoxyribose ring conformation on the carbon chemical shift. It is found that 3'-endo conformers have 3' and 5' chemical shifts significantly (5-10 ppm) upfield of comparable 3'-exo and 2'-endo conformers. The latter two conformers may be distinguished by smaller but still significant differences in the carbon chemical shifts at the C-2' and C-4' positions. High-resolution solid-state NMR of three modifications of fibrous calf thymus DNA shows that these trends are maintained in high-molecular-weight DNA and confirms that the major ring pucker in A-DNA is 3'-endo, while both B-DNA and C-DNA are largely 2'-endo. The data show that 13C NMR spectroscopy is a straightforward and useful probe of DNA ring pucker in both solution and the solid state.     

Backbone dynamics and refined solution structure of the N-terminal domain of DNA polymerase beta. Correlation with DNA binding and dRP lyase activity

Mammalian DNA polymerase beta functions in the base excision DNA repair pathway filling in short patches (1-5 nt) in damaged DNA and removing deoxyribose 5'-phosphate from the 5'-side of damaged DNA. The backbone dynamics and the refined solution structure of the N-terminal domain of beta-Pol have been characterized in order to establish the potential contribution(s) of backbone motion to the DNA binding and deoxyribose 5'-phosphate lyase function of this domain. The N-terminal domain is formed from four helices packed as two antiparallel pairs with a 60 degrees crossing between the pairs. The RMSD of the NMR conformers (residues 13-80) is 0.37 A for the backbone heavy atoms and 0.78 A for all heavy atoms. NMR characterization of the binding site(s) for a ssDNA-5mer, ssDNA-8mer, ssDNA-9mer, and dsDNA-12mer shows a consensus surface for the binding of these various DNA oligomers, that surrounds and includes the deoxyribose 5'-phosphate lyase active site region. Connection segments between helices 1 and 2 and between helices 3 and 4 each contribute to DNA binding. Helix-3-turn-helix-4 forms a helix-hairpin-helix motif. The highly conserved hairpin sequence (LPGVG) displays a significant degree of picosecond time-scale motion within the backbone, that is possibly important for DNA binding at the phosphodiester backbone. An Omega-loop connecting helices 1 and 2 and helix-2 itself display significant exchange contributions (R(ex)) at the backbone amides due to apparent conformational type motion on a millisecond time-scale. This motion is likely important in allowing the Omega-loop and helix-2 to shift toward, and productively interact with, gapped DNA. The deoxyribose 5'-phosphate lyase catalytic residues that include K72 which forms the Schiff's base, Y39 which is postulated to promote proton transfer to the aldehyde, and K35 which assists in phosphate elimination, show highly restricted backbone motion. H34, which apparently participates in detection of the abasic site hole and assists in the opening of the hemiacetal, shows conformational exchange.     

Bleomycin mediated degradation of DNA-RNA hybrids does not involve C-1' chemistry.

Incubation of Fe(II) bleomycin and O2 with a number of 'A'-like DNA-RNA hybrid homopolymers at 4 atm O2 results in formation of base propenal and base in a ratio of approximately 1.0:1.0. This ratio differs dramatically from the corresponding ratio of approximately 10:1.0 observed when activated BLM degrades 'B'-like DNA homopolymers. Experiments were undertaken to determine if the shift to enhanced base production observed in the A-like hybrids is the result of C-1' chemistry in addition to the C-4' chemistry normally observed with B-like DNA under identical conditions. Increased accessibility of the 1'-hydrogen might be anticipated due to widening of the minor groove in the A-like conformers. Experiments using poly([1'-3H]dA) poly(rU) and poly([U-14C]dA) poly(rU) indicated that neither 3H2O nor deoxyribonolactone accompanied adenine release. In addition, studies using poly([4'-2H]dA) poly(rU) and poly([1'-2H]dA) poly(rU) unambiguously establish that the altered base to base propenal ratio is not the result of C-1' chemistry, but a direct consequence of C-4' chemistry.     

A role of oxidative DNA sugar damage in mutagenesis by neocarzinostatin and bleomycin

The anti-tumor antibiotics neocarzinostatin and bleomycin specifically oxidize deoxyribose in DNA at the C-5' and C-4' positions, respectively. The resulting DNA lesions include strand breaks and apyrimidinic sites. Both agents are broad specificity mutagens, inducing, in various systems, base substitutions, frameshifts and deletions. Sequencing studies in bacterial systems have suggested that the base substitutions may result primarily from replicative bypass of the oxidized apyrimidinic sites.     

Slow loss of deoxyribose from the N7deoxyguanosine adducts of estradiol-3,4-quinone and hexestrol-3',4'-quinone. Implications for mutagenic activity

A variety of evidence has been obtained that estrogens are weak tumor initiators. A major step in the multi-stage process leading to tumor initiation involves metabolic formation of 4-catechol estrogens from estradiol (E2) and/or estrone and further oxidation of the catechol estrogens to the corresponding catechol estrogen quinones. The electrophilic catechol quinones react with DNA mostly at the N-3 of adenine (Ade) and N-7 of guanine (Gua) by 1,4-Michael addition to form depurinating adducts. The N3Ade adducts depurinate instantaneously, whereas the N7Gua adducts depurinate with a half-life of several hours. Only the apurinic sites generated in the DNA by the rapidly depurinating N3Ade adducts appear to produce mutations by error-prone repair. Analogously to the catechol estrogen-3,4-quinones, the synthetic nonsteroidal estrogen hexestrol-3',4'-quinone (HES-3',4'-Q) reacts with DNA at the N-3 of Ade and N-7 of Gua to form depurinating adducts. We report here an additional similarity between the natural estrogen E2 and the synthetic estrogen HES, namely, the slow loss of deoxyribose from the N7deoxyguanosine (N7dG) adducts formed by reaction of E2-3,4-Q or HES-3',4'-Q with dG. The half-life of the loss of deoxyribose from the N7dG adducts to form the corresponding 4-OHE2-1-N7Gua and 3'-OH-HES-6'-N7Gua is 6 or 8 h, respectively. The slow cleavage of this glycosyl bond in DNA seems to limit the ability of these adducts to induce mutations.     

DNA strand scission by neocarzinostatin: molecular recognition process responsible for site-specificity

A series of hexanucleotides possessing A-T, G-C, inosine (I)-C and 2-aminoadenine (ANH2)-T base pairs at 5'-side of the target thymine were prepared and their selectivity for C-5' and C4' oxidation in the NCS-mediated degradation was investigated. Quantitative product analysis indicated that preferential C5' oxidation of deoxyribose moiety of the target T occurs at -5'-AT- and 5'-IT- sites, whereas C5' and C4' oxidation occurs competitively at T of -5'-GT- and -5'-ANH2T- sites. Based on the experimental results, an intercalation model that permits competitive hydrogen abstraction from C5' and C4' of deoxyribose moiety has been proposed.     

Sugar radicals in DNA: isolation of neutral radicals in gamma-irradiated DNA by hole and electron scavenging

In this investigation of the radical formation and the reaction of radicals in gamma-irradiated DNA, we report the isolation of putative neutral radicals by the scavenging of holes by Fe(CN)6(4-) and of electrons by Fe(CN)6(3-). Experiments are performed under conditions that emphasize direct and quasi-direct effects (collectively called direct-type effects.) Samples containing Fe(CN)6(4-) show effective scavenging of holes and the ESR spectra obtained arise principally from DNA anion radicals and neutral radicals. On the other hand, for samples containing Fe(CN)6(3-), electron scavenging is highly efficient, and the resulting spectra arise principally from guanine cation radicals and neutral radicals. When both Fe(CN)6(4-) and Fe(CN)6(3-) are present, a near complete scavenging of cation radicals and anion radicals is observed at 77 K, and the ESR spectra that result originate predominantly with neutral radicals which are assigned predominantly to radicals on the sugar phosphate backbone. A notable finding is the presence of spectral components that indicate the formation, through the rupture of the C3'-O bond, of a neutral deoxyribose radical; a concurrent strand break must accompany formation of this radical. This radical was previously reported in argon-ion-irradiated DNA and now, for the first time, is reported in DNA irradiated with low-LET radiation.     

Chemical structure of the lipid A component of Plesiomonas shigelloides and its taxonomical significance

Abstract The chemical structure of the lipid A moiety of the lipopolysaccharide of the type strain of Plesiomonas shigelloides was elucidated. It consists of a β-(1 → 6)-linked glucosamine disaccharide carrying phosphate groups at C-1 of the reducing and at C-4' of the non-reducing glucosamine. It contains a total of 6 residues of fatty acids, 2 amide-linked and 4 ester-linked. The amino groups of the backbone disaccharide are N -acylated by substituted 3-hydroxyacyl residues: at the reducing glucosamine by 3-O-(14:0)14:0; and at the non-reducing glucosamine by 3-O-(12:0)14:0.
Two residues of 3-hydroxytetradecanoic acid are linked to C-3 and C-3' of the glucosamine residues; the hydroxy groups of these ester-linked 3-hydroxytetradecanoic acids are unsubstituted. In free lipid A, the hydroxyl groups at C-4 and C-6' are unsubstituted, indicating that the 2-keto-3-deoxyoctonic acid (KDO) is linked to C-6' of the non-reducing glucosamine, as was shown with enterobacterial lipid A. The taxonomical significance of these structural details is discussed.