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An unidentified white powder collected as evidence in an intelligence investigation was characterized exclusively by nuclear magnetic resonance (NMR) analysis. A small fraction of the powder dissolved in D 2 O was subjected to a series of one- and two-dimensional techniques which were used to elucidate the molecular structure of the powder's major component and positively identify it as the scopolamine biotoxin. Quantitative one-dimensional experiments identified individual proton and carbon atom sites, and conventional 14 N spectroscopy detected a single nitrogen atom site. Heteronuclear single quantum coherence data correlated all protons to their directly bonded carbon atom, and together with the quantitative spectra, were used to determine the number of protons directly bonded to each carbon atom. The presence of a methyl, carboxyl, and a benzyl group was also identified from these data. Correlation spectroscopy detected a three proton and a nine proton J H,H network, representing a CH 2 CH moiety and seven carbon atom ring, respectively. These five elements were assembled into an almost complete molecular structure by using long-range, J-coupled, 'H- 13 C pairs detected by heteronuclear multiple bond correlation (HMBC) spectroscopy and 1 H- 1 H dipolar-coupled pairs found from nuclear Overhauser effect spectroscopy (NOESY) data. Additional oxygen atom sites were inferred from 1 H- 13 C correlation intensities in the HMBC spectra along with 'H and 13 C chemical shift values, or directly from NOESY correlations. Only a single oxygen atom site could not be inferred from NMR data, but its presence was inferred from comparisons to target analyte structures to complete the structure of the scopolamine molecule. To confirm these results, an ethanol/H 2 O solution of the powder was analyzed by direct infusion into an ion trap mass spectrometer. A prominent base signal was observed at m/z 304.1 amu, corresponding to the protonated molecular ion of scopolamine. Subsequently, the ion was selected and subjected to collision-induced dissociation, producing characteristic major MS/MS fragments at m/z 138.1 and 156.1. Comparisons of 1 H and 13 C chemical shift values and J H,H values measured from our NMR data were found to agree very favorably with previously reported values for scopolamine in D 2 O.

Factor VIII functions as a cofactor for Factor IXa in a membrane-bound enzyme complex. Membrane binding accelerates the activity of the Factor VIIIa–Factor IXa complex approx. 100000-fold, and the major phospholipid-binding motif of Factor VIII is thought to be on the C2 domain. In the present study, we prepared an fVIII-C2 (Factor VIII C2 domain) construct from Escherichia coli, and confirmed its structural integrity through binding of three distinct monoclonal antibodies. Solution-phase assays, performed with flow cytometry and FRET (fluorescence resonance energy transfer), revealed that fVIII-C2 membrane affinity was approx. 40-fold lower than intact Factor VIII. In contrast with the similarly structured C2 domain of lactadherin, fVIII-C2 membrane binding was inhibited by physiological NaCl. fVIII-C2 binding was also not specific for phosphatidylserine over other negatively charged phospholipids, whereas a Factor VIII construct lacking the C2 domain retained phosphatidyl-L-serine specificity. fVIII-C2 slightly enhanced the cleavage of Factor X by Factor IXa, but did not compete with Factor VIII for membrane-binding sites or inhibit the Factor Xase complex. Our results indicate that the C2 domain in isolation does not recapitulate the characteristic membrane binding of Factor VIII, emphasizing that its role is co-operative with other domains of the intact Factor VIII molecule.

Two-dimensional H1-C13 heteronuclear single quantum correlation (HSQC) and fast-heteronuclear multiple quantum correlation (HMQC) pulse sequences were implemented using a cryogenic probehead for detecting Chemical Weapons Convention compounds in complex mixtures. The H1-C13 correlations of target analytes at < or = 25 micrograms/mL were easily detected in a sample where the H1 solvent signal was ~58,000 fold more intense than the analyte H1 signals. The problem of overlapping signals typically observed in conventional H1 spectroscopy was essentially eliminated, while H1 and C13 chemical shift information could be derived quickly and simultaneously from the resulting spectra. The fast-HMQC pulse sequence generated magnitude mode spectra suitable for detailed analysis in ~4.5 h, and can be used to efficiently screen a large number of samples. The HSQC pulse sequence on the other hand, required roughly twice the acquisition time to produce suitable spectra. These spectra, however, were phase sensitive, contained considerably more resolution, and proved to be superior for detecting analyte H1-C13 correlations. Use of the pulse sequences considerably improves the performance of NMR spectroscopy as a complementary technique for the screening, identification, and validation of chemical warfare agents and other small molecule analytes in complex mixtures and environmental samples.

Binding of factor VIII to membranes containing phosphatidyl-L-serine (Ptd-L-Ser) is mediated, in part, by a motif localized to the C2 domain. We evaluated a putative membrane-binding role of the C1 domain using an anti-C1 antibody fragment, KM33scFv, and factor VIII mutants with an altered KM33 epitope. We prepared a dual mutant Lys2092/Phe2093 → Ala/Ala (fVIIIYFP 2092/93) and 2 single mutants Lys2092 → Ala and Phe2093 → Ala. KM33scFv inhibited binding of fluorescein-labeled factor VIII to synthetic membranes and inhibited at least 95% of factor Xase activity. fVIIIYFP 2092/93 had 3-fold lower affinity for membranes containing 15% Ptd-L-Ser but more than 10-fold reduction in affinity for membranes with 4% Ptd-L-Ser. In a microtiter plate, KM33scFv was additive with an anti-C2 antibody for blocking binding to vesicles of 15% Ptd-L-Ser, whereas either antibody blocked binding to vesicles of 4% Ptd-L-Ser. KM33scFv inhibited binding to platelets and fVIIIYFP 2092/93 had reduced binding to A23187-stimulated platelets. fVIIIYFP 2092 exhibited normal activity at various Ptd-L-Ser concentrations, whereas fVIIIYFP 2093 showed a reduction of activity with Ptd-L-Ser less than 12%. fVIIIYFP 2092/93 had a greater reduction of activity than either single mutant. These results indicate that Lys 2092 and Phe 2093 are elements of a membrane-binding motif on the factor VIII C1 domain.

A quantitative 13C NMR method has been developed for characterizing hydrogen cyanide, cyanogen chloride, and phosgene, three Chemical Weapons Convention Schedule 3 compounds in common use today. Towards this end, the 13C spin-lattice relaxation behavior (T1) of the compounds has been assessed at 75 and 126 MMz for temperatures between 5-15 deg C, holding them in their liquid states to dramatically improve detection sensitivity. The derived single exponential T1 values were used to derive relaxation delays for collecting quantitative 13C data sets yielding a signal-to-noise ratio (S/N) exceeding that necessary for certifying the compounds at greater or equal 95 carbon atom % and 99% confidence. At 126 MHz, only a single data acquisition with a high-sensitivity cryogenic probe head exceeded the certifying S/N; however, for analysis at 75 MHz with a conventional probe head, greater or equal 5 acquisitions were necessary for phosgene, and greater or equal 12 acquisitions were necessary for the other two compounds. In terms of accuracy and execution time, the resulting NMR method rivals typical chromatographic methods.

Hydrogen cyanide, cyanogen chloride and phosgene are produced in tremendously large quantities today by the chemical industry. The compounds are also particularly attractive to foreign states and terrorists seeking an inexpensive mass-destruction capability. Along with contemporary warfare agents, therefore, the US Army evaluates protective equipment used by warfighters and domestic emergency responders against the compounds, and requires their certification at ≥ 95 carbon atom % before use. We have investigated the 13C spin-lattice relaxation behavior of the compounds to develop a quantitative NMR method for characterizing chemical lots supplied to the Army. Behavior was assessed at 75 and 126 MHz for temperatures between 5 and 15 °C to hold the compounds in their liquid states, dramatically improving detection sensitivity. T1 values for cyanogen chloride and phosgene were somewhat comparable, ranging between 20 and 31 s. Hydrogen cyanide values were significantly shorter at 10–18 s, most likely because of a 1H13C dipolar contribution to relaxation not possible for the other compounds. The T1 measurements were used to derive relaxation delays for collecting the quantitative 13C data sets. At 126 MHz, only a single data acquisition with a cryogenic probehead gave a signal-to-noise ratio exceeding that necessary for certifying the compounds at ≥ 95 carbon atom % and 99% confidence. Data acquired at 75 MHz with a conventional probehead, however, required ≥ 5 acquisitions to reach this certifying signal-to-noise ratio for phosgene, and ≥ 12 acquisitions were required for the other compounds under these same conditions. In terms of accuracy and execution time, the NMR method rivals typical chromatographic methods. Published in 2007 by John Wiley & Sons, Ltd.