Synthesis of exemestane labelled with (13)C

The synthesis of exemestane Aromasin, an irreversible steroidal aromatase inhibitor, specifically labelled with (13)C is reported. The preparation of [(13)C(3)]exemestane was achieved according to an eight-step procedure starting from the commercially available testosterone.     

Insights into the fate of a 13C labelled phenol pulse for stable isotope probing (SIP) experiments

Stable isotope probing (SIP) using DNA or RNA as a biomarker has proven to be a useful method for attributing substrate utilisation to specific microbial taxa. In this study we followed the transfer of a (13)C(6)-phenol pulse in an activated sludge micro-reactor to examine the resulting distribution of labelled carbon in the context of SIP. Most of the added phenol was metabolically converted within the first 100 min after (13)C(6)-phenol addition, with 49% incorporated into microbial biomass and 6% respired as CO(2). Less than 1% of the total (13)C labelled carbon supplied was incorporated into microbial RNA and DNA, with RNA labelling 6.5 times faster than DNA. The remainder of the added (13)C was adsorbed and/or complexed to suspended solids within the sludge. The (13)C content of nucleic acids increased beyond the initial consumption of the (13)C-phenol pulse. This study confirms that RNA labels more efficiently than DNA and reveals that only a small proportion of a pulse is incorporated into nucleic acids. Evidence of continued (13)C incorporation into nucleic acids suggests that cross-feeding of the SIP substrate was rapid. This highlights both the benefits of using a biomarker that is rapidly labelled and the importance of sampling within appropriate timescales to avoid or capture the effects of cross-feeding, depending on the goal of the study.     

Carbon-13 nuclear magnetic resonance study of spin labelled cholesteryl ester in model membranes

Carbon-13 NMR longitudinal relaxation times for unilamellar vesicles of egg phosphatidyl-choline (PC) in aqueous dispersion have been measured following the incorporation of spin labelled cholesteryl palmitate. The spin label induced relaxation rates. 1/T_1.5L, for fatty acyl chain carbons show that the C5 segment of the cholesteryl ester acyl chain is located near the C1 and C2 segments of the phospholipid acyl chains. A greater spin label induced enhancement of relaxation rate was observed for the inner vesicle layer than for the outer, and is attributed to a higher ester incorporation and/or tighter lipid packing in the inner layer.     

Automated sequencing of fluorescently labelled DNA by chemical degradation

A new general method for sequencing fluorescently labelled DNA by chemical degradation has been developed. It is based on the observation that fluorescein attached via a mercaptopropyl or aminopropyl linker arm to the 5'-phosphate of an oligonucleotide is stable during the reactions commonly used in chemical cleavage procedures. DNA to be degraded is first enzymatically synthesized in vitro by annealing and extending a fluorescently labelled primer thereby introducing the fluorescent label at the 5'-end of the fragment. The newly synthesized fluorescently labelled DNA is then chemically degraded using: (a) a set of four different cleavage reactions; or (b) only one reaction comprising methylation of G-residues followed by a partial cleavage with piperidine in the presence of sodium chloride. The fluorescent degradation products are loaded on either four lanes or one lane of the gel, respectively, and the emitted fluorescence detected online during electrophoresis. In the 'four reactions/four lanes' method 200-350 bp (base pairs) can be read from the labelled end. The 'one reaction/one lane' method, in which the nucleotide sequence is determined by measuring different signal intensities following the rule G greater than A greater than C greater than T, currently yields around 100-200 bp of sequence per sample.     

Intra-erythrocyte microviscosity and diffusion of specifically labelled [glycyl-alpha-13C]glutathione by using 13C n.m.r.

[alpha-13C]Glycine was incubated with suspensions of human erythrocytes under special buffer conditions to enrich specifically intracellular glutathione with 13C. The metabolically active cells were then subjected to 13C n.m.r. spectroscopy in which the longitudinal relaxation time(s) (T1) and nuclear Overhauser enhancement(s) of the free glycine and glutathione were measured. With the appropriate analysis, assuming the molecules to be isotropic rotors, intracellular rotational correlation times were calculated. Using these data together with the Stokes-Einstein equation, viscosity and translational diffusion coefficients were calculated. The results were compared with those from cell lysates and extracts. The cytosolic microviscosity probed by glutathione was only 1.9 +/- 0.3 times that of saline, suggesting, therefore, that most enzyme reactions involving this solute are not likely to be diffusion-controlled inside the erythrocyte.     

Structural analysis of the complex of a distamycin analogue with the Dickerson dodecamer 13C labeled at 5'-carbons using NMR spectroscopy.

Structural analysis of the complex of a distamycin analogue (Tallimustine) with the Dickerson dodecamer d(C*G*C*G*A*A*T*T*C*G*C*G) [N*:[5'-(13)C]nucleotide] was performed by NMR spectroscopy and the results will be described in detail.     

13C nuclear magnetic resonance studies of egg phosphatidylcholine.

Spin lattice relaxation times (T1) and apparent spin-spin relaxation times (T2) derived from linewidth have been used to investigate model membranes composed of egg yolk phosphatidylcholine. T1 measurements appear to be largely dominated by segmental motion and as a consequence are not very sensitive to small changes in membrane structure. On the contrary, apparent T2 times are shown to be sensitive to such changes in the membrane and are thus suggested as a useful tool for further investigation of membrane structure.     

Importance of polyunsaturated acyl chains in chlorpromazine interaction with phosphatidylserines: a 13C and 31P solid-state NMR study

The polyunsaturated fatty acid docosahexaenoic acid (DHA, c22:6, n-3) is found at a level of about 50% in the phospholipids of neuronal tissue membranes and appears to be crucial to human health. Dipalmitoyl phosphatidylcholine (DPPC, 16:0/16:0 PC), 1-palmitoyl-2-oleoyl phosphatidylserine (POPS) and the DHA containing 1-stearaoyl-2-docosahexenoyl phosphatidylserine (SDPS) were used to make DPPC (60%)/POPS (29%)/SDPS (11%) bilayers with and without 10 mol% chlorpromazine (CPZ), a cationic, amphiphilic phenothiazine. The T1 relaxation measurements make it clear that the saturated acyl chains carbons (palmitic, stearic and most of the oleic chain) and the choline head group are not affected by CPZ addition. The observed increased signal intensity and T1-values of DHA indicate reduced mobility of C4 and C5 due to CPZ binding. 31P NMR spectra confirm that CPZ binding to the phosphatidylserines in the bilayer enhances phospholipid head group mobility.     

New sequential-assignment routes of nucleic acid NMR signals using a [5'-(13)C]-labeled DNA dodecamer

NMR signal assignments for DNA oligomers have been performed by the well-established sequential assignment procedures based on NOESY and COSY. The H4'/H5'/H5' resonance region is congested and difficult to analyze without the use of isotope-labeled DNA oligomers. Here a DNA dodecamer constructed with 2'-deoxy[5'-(13)C]ribonucleotides, 5'-d(*C*G*C*G*A*A*T*T*C*G*CG)-3' (*N = [5'-(13)C]Nucleotide), was prepared in an effort to analyze the H4'/H5'/H5' resonance region by 2D 1H-13C HMQC-NOESY. In the C5' and H1' resonance region, weak and strong cross peaks for C5'(i)-H1'(i) and C5'(i)-H1'(i-1), respectively, were found, thus enabling the sequential assignment within this region. A similar sequential assignment route was found between C5' and H2'. Proton pair distances evaluated from the canonical B-DNA as well as A-DNA indicated that these sequential-assignment routes on a 2D 1H-13C HMQC-NOESY spectrum work for most nucleic acid stem regions.     

Spin-lattice relaxation times for 13C in isotope-enriched glycine accumulated in frog muscle.

Spin-lattice relaxation times (T1's) of 13C-enriched glycine accumulated in frog muscles were determined at 1 degrees C by the inversion-recovery (180 degrees -tau-90 degree pulse sequence) method and compared with the values obtained in free solution. The value of T1 for the alpha-13C nucleus of glycine in the tissue was 50% of that obtained in free solution. The observed value for T1 in the tissue was not concentration-dependent, and no difference in chemical shift was observed between tissue and free solution. Quantification of the area under the glycine peak suggested that the observed signal represents at least 80% of the intracellular glycine. An average nuclear Overhauser enhancement of 2.83 for intracellular glycine indicates that the relaxation mechanism within the cell is predominantly dipolar, as in free solution. The value of T1 for the 13C' nucleus of glycine in the tissue was 67% of that in a solution of similar concentration. A quantitative analysis of the findings suggests that the observed difference in the value of T1 between tissue and free solution results from a difference in viscosity. The data provide no evidence either for special organization of intracellular water or for glycine binding. It is proposed that intracellular diffusion coefficients may be determined from measurements of 13C T1's of 13C-enriched intracellular solutes.     

(13)C cross-polarization/magic angle spinning NMR studies of alpha-elastin preparations show retention of overall structure and reduction of mobility with a decreased number of cross-links

High-resolution solid-state (13)C NMR spectra are presented for samples of alpha-elastin prepared from the aorta of normal and copper-deficient pigs. Chemical shifts of the various peaks indicate that both the normal and undercross-linked peptides have similar overall structures. However, (13)C T(1), (13)C T(1 rho), and (1)H T(1 rho) measurements indicate that the alpha-elastin peptides obtained from the abnormal elastic fibers samples exhibit altered mobilities, particularly in their side chains. Results from spectra taken with a range of contact times and from dipolar dephasing experiments are consistent with conclusions reached with the relaxation measurements. Namely, the loss of function associated with the undercross-linked sample is correlated to a small but measurable difference in relative mobility.     

Distribution of labelled anti-tenascin antibodies and fragments after injection into intact or partly resected C6-gliomas in rats

Introduction: For treatment of malignant glioma, radioimmunotherapy has become a valuable alternative for more than 2 decades. Surprisingly, very little is known about the distribution of intralesionally administered labelled antibodies or fragments. We investigated the migration of labelled antibodies and antibody fragments injected into intact and partly resected C6-glioma in rats at different times after injection. Materials and methods: Nine days after induction of a C6-glioma, 5 l of ¹²⁵I-labelled murine anti-tenascin antibodies (n=31) or ¹²⁵I-labelled fragments of anti-tenascin antibodies (n=32) was injected slowly into the tumour (group I). In group II the tumour was subtotally resected 9 days after induction of the C6-glioma, and 24 h later the labelled antibodies (n=30) or fragments (n=12) were injected into the resection cavity. At 6, 24 or 48 h after the injection, animals were sacrificed, and brains removed. Distribution of labelled antibodies and fragments was determined by superimposing autoradiographs onto frozen sections and HE-stained neighbouring sections using a digital image analysing system. Results: After injection into intact C6-glioma, labelled antibodies covered a maximum distance of 3.2 ± 1.0, 4.1 ± 1.9 and 4.8 ± 0.9 mm after 6, 24 and 48 h, respectively; while labelled fragments were found at a distance of 6.7 mm (±1.1) after 24 h and 5.8 mm (±0.9) after 48 h (significant in univariate analysis). Following partial tumour resection, the respective distances at 24 h were 3 ± 0.4 mm for anti-tenascin antibodies and 3.4 ± 0.3 mm for Fab fragments. Conclusion: After injection into C6-glioma, labelled fragments are able to cover a greater distance than labelled antibodies. Injection of antibodies and fragments 1 day after tumour resection results in reduced velocity of both antibodies and fragments.     

13C NMR studies of D- and L-phenylalanine binding to cobalt(II) carboxypeptidase A

13C NMR T1 and T2 measurements have been performed on cobalt(II) substituted carboxypeptidase A in the presence of carboxylate-13C-enriched L- and D-phenylalanine. Upon binding to the cobalt enzyme, the longitudinal and transverse relaxation rates T1p-1 and T2p-1 of these inhibitors are enhanced significantly compared to the zinc enzyme, allowing both determination of an affinity constant for inhibitor binding, K, and calculation of the metal-13C carboxylate distances. The L-and D- Phe concentration dependence of T2p-1 yields affinity constants of 290 +/- 60M-1 and 670 +/- 90M-1. The distance measurements calculated for Co-13C from T1p-1 are 0.39 +/- 0.04 and 0.42 +/- 0.04 nm for L-Phe and D-Phe. Both values are too great for direct coordination of their carboxylate groups to the metal atom. Upon formation of their respective ternary enzyme.Phe.N3- complexes, the distances are essentially unaltered. In conjunction with electronic absorption studies on these complexes it can be concluded that N3-, but not the amino acid carboxylate, is bound to the metal.     

Recognition of GC base pairs by triplex forming oligonucleotides containing nucleosides derived from 2-aminopyridine.

We have attempted to alleviate the pH dependency of triplex recognition of guanine by using intermolecular triplexes containing 2-amino-5-(2-deoxy-d-ribofuranosyl)pyridine (AP) as an analogue of 2'-deoxycytidine (dC). We find that for the beta-anomer of AP, the complex between (AP)6T6and the target site G6A6*T6C6is stable, generating a clear DNase I footprint at oligonucleotide concentrations as low as 0.25 microM at pH 5.0, in contrast to 50 microM C6T6which has no effect on the cleavage pattern. This complex is still stable at pH 6.5 producing a footprint with 1 microM oligonucleotide. Oligonucleotides containing the alpha-anomer of AP are much less effective than the beta-anomer, though in some instances they are more stable than the unmodified oligonucleotides. The results of molecular dynamics studies on a range of AP-containing triplexes has rationalized the observed stability behaviour in terms of hydrogen-bonding behaviour.     

Slow motion in the CAA*TTG sequence of a DNA decamer duplex studied by NMR

In DNA duplexes, pyrimidine-purine steps are believed to be flexible or conformationally unstable. Indeed, several DNA crystal structures exhibit a multitude of conformations for CpA*TpG steps. The question arises of whether this structural flexibility is accompanied by dynamical flexibility, i.e., a question pertaining to the energy barrier between conformations. Except for TpA steps, slow motions on the microsecond-to-millisecond time scale have not been detected in duplexes until now. In the present study, such slow motion was investigated by 1H, 13C, and 15N NMR relaxation measurements on a DNA decamer d(CATTTGCATC)*d(GATGCAAATG). The DNA decamer was enriched with 15% 13C and 98% 15N isotopes for each adenosine and guanosine residue. Three lines of evidence support the notion of slow motion in the CAA*TTG moiety. Analysis of (15)N relaxation showed that the order parameter, S2, of guanosine imino NH groups was about 0.8, similar to that of CH groups for this oligomer. The strong temperature dependence of guanosine NH S2 in the CAA*TTG sequence indicated the presence of a large-amplitude motion. Signals of adenosine H8 protons in the CAA*TTG sequence were broadened in 2D 1H NOESY spectra, which also suggested the existence of slow motion. As well as being smaller than for other adenine residues, the 1H T2 values exhibited a magnetic field strength dependence for all adenosine H8 signals in the ATTTG*CAAAT region, suggesting slow motions more pronounced at the first adenosine in the CAA*TTG sequence but extending over the CAAAT*ATTTG region. This phenomenon was further examined by the pulse field strength dependence of the 1H, 13C, and 15N T1rho values. 1H and 13C T1rho values showed a pulse field strength dependence, but 15N T1rho did not. Assuming a two-site exchange process, an exchange time constant of 20-300 micros was estimated for the first adenosine in the CAA sequence. The exact nature of this motion remains unknown.     

13C NMR relaxation times of hepatic glycogen in vitro and in vivo

The field dependence of relaxation times of the C-1 carbon of glycogen was studied in vitro by natural-abundance 13C NMR. T1 is strongly field dependent, while T2 does not change significantly with magnetic field. T1 and T2 were also measured for rat hepatic glycogen enriched with [1-13C]glucose in vivo at 4.7 T, and similar relaxation times were observed as those obtained in vitro at the same field. The in vitro values of T1 were 65 +/- 5 ms at 2.1 T, 142 +/- 10 ms at 4.7 T, and 300 +/- 10 ms at 8.4 T, while T2 values were 6.7 +/- 1 ms at 2.1 T, 9.4 +/- 1 ms at 4.7 T, and 9.5 +/- 1 ms at 8.4 T. Calculations based on the rigid-rotor nearest-neighbor model give qualitatively good agreement with the T1 field dependence with a best-fit correlation time of 6.4 X 10(-9) s, which is significantly smaller than tau M, the estimated overall correlation time for the glycogen molecule (ca. 10(-5) s). A more accurate fit of T1 data using a modified Lipari and Szabo approach indicates that internal fast motions dominate the T1 relaxation in glycogen. On the other hand, the T2 relaxation is dominated by the overall correlation time tau M while the internal motions are almost but not completely unrestricted.     

Extension of the range of DNA sequences available for triple helix formation: stabilization of mismatched triplexes by acridine-containing oligonucleotides.

Triple helix formation usually requires an oligopyrimidine*oligopurine sequence in the target DNA. A triple helix is destabilized when the oligopyrimidine*oligopurine target contains one (or two) purine*pyrimidine base pair inversion(s). Such an imperfect target sequence can be recognized by a third strand oligonucleotide containing an internally incorporated acridine intercalator facing the inverted purine*pyrimidine base pair(s). The loss of triplex stability due to the mismatch is partially overcome. The stability of triplexes formed at perfect and imperfect target sequences was investigated by UV thermal denaturation experiments. The stabilization provided by an internally incorporated acridine third strand oligonucleotide depends on the sequences flanking the inverted base pair. For triplexes containing a single mismatch the highest stabilization is observed for an acridine or a propanediol tethered to an acridine on its 3'-side facing an inverted A*T base pair and for a cytosine with an acridine incorporated to its 3'-side or a guanine with an acridine at its 5'-side facing an inverted G*C base pair. Fluorescence studies provided evidence that the acridine was intercalated into the triplex. The target sequences containing a double base pair inversion which form very unstable triplexes can still be recognized by oligonucleotides provided they contain an appropriately incorporated acridine facing the double mismatch sites. Selectivity for an A*T base pair inversion was observed with an oligonucleotide containing an acridine incorporated at the mismatched site when this site is flanked by two T*A*T base triplets. These results show that the range of DNA base sequences available for triplex formation can be extended by using oligonucleotide intercalator conjugates.     

13C-NMR spectroscopy of human serum high density lipoprotein enriched with labelled phospholipids.

Native human serum high density lipoprotein (HDL) (d = 1.063--1.21g x cm-3) was enriched with phosphatidylcholines labelled with 13C in the polar head group ([N-13CH3]choline) and in the fatty acyl chains ([26-13C]cholesterol) and its linoleic acid ester using the previously described exchange method (Stoffel et al. 1978). The properties of the HDL particles with the exchanged lipid classes were the same as those of the native particles (Mr, CD, fluorescence, lipid and apoprotein stoichiometry, electrophoretic mobility). The T1-times were very similar to those obtained previously with recombined apolipoprotein-[13C]lipid complexes and further support our proposals concerning lipid and apoprotein interactions in the HDL particle.     

Synthesis of backbone deuterium labelled [r(CGCGAAUUCGCG)]2 and HPLC purification of synthetic RNA.

The chemical synthesis of backbone deuterium labelled [r(CGCGAAU*U*CGCG)]2 (U* = [5'-2H]U) is described. An efficient purification procedure was developed using a polymeric reverse phase (PRP) HPLC column at 60 degrees C. This procedure provided pure RNA dodecamer in the multi-milligram quantities (39% overall yield) necessary for dynamics studies using solid-state deuterium NMR. The purification method has been effectively applied to other RNA sequences and will assist biophysical studies which require relatively large quantities of RNA oligomers.