We demonstrate that natural isotopic abundance 2D heteronuclear correlation (HETCOR) solid-state NMR spectra can be used to significantly reduce or eliminate the broadening of
1
H and
13
C ...solid-state NMR spectra of organic solids due to anisotropic bulk magnetic susceptibility (ABMS). ABMS often manifests in solids with aromatic groups, such as active pharmaceutical ingredients (APIs), and inhomogeneously broadens the NMR peaks of all nuclei in the sample. Inhomogeneous peaks with full widths at half maximum (FWHM) of ∼1 ppm typically result from ABMS broadening and the low spectral resolution impedes the analysis of solid-state NMR spectra. ABMS broadening of solid-state NMR spectra has previously been eliminated using 2D multiple-quantum correlation experiments, or by performing NMR experiments on diluted materials or single crystals. However, these experiments are often infeasible due to their poor sensitivity and/or provide limited gains in resolution. 2D
1
H-
13
C HETCOR experiments have previously been applied to reduce susceptibility broadening in paramagnetic solids and we show that this strategy can significantly reduce ABMS broadening in diamagnetic organic solids. Comparisons of 1D solid-state NMR spectra and
1
H and
13
C solid-state NMR spectra obtained from 2D
1
H-
13
C HETCOR NMR spectra show that the HETCOR spectrum directly increases resolution by a factor of 1.5 to 8. The direct gain in resolution is determined by the ratio of the inhomogeneous
13
C/
1
H linewidth to the homogeneous
1
H linewidth, with the former depending on the magnitude of the ABMS broadening and the strength of the applied field and the latter on the efficiency of homonuclear decoupling. The direct gains in resolution obtained using the 2D HETCOR experiments are better than that obtained by dilution. For solids with long proton longitudinal relaxation times, dynamic nuclear polarization (DNP) was applied to enhance sensitivity and enable the acquisition of 2D
1
H-
13
C HETCOR NMR spectra. 2D
1
H-
13
C HETCOR experiments were applied to resolve and partially assign the NMR signals of the form I and form II polymorphs of aspirin in a sample containing both forms. These findings have important implications for ultra-high field NMR experiments, optimization of decoupling schemes and assessment of the fundamental limits on the resolution of solid-state NMR spectra.
2D HETCOR experiments enhance the resolution of
1
H and
13
C solid-state NMR spectra by reducing anisotropic bulk magnetic susceptibility (ABMS) signal broadening.
Biodegradable drug-delivery systems can be formulated to release drug for hours to years and have been used for the controlled release of medications in animals and humans. An important consideration ...in developing a drug-delivery matrix is knowledge of the long-term stability of the form of the drug and matrix after formulation and any changes that might occur to the drug throughout the delivery process. Solid-state NMR spectroscopy is an effective technique for studying the state of both the drug and the matrix. Two systems that have been studied using solid-state NMR spectroscopy are presented. The first system studied involved bupivacaine, a local anesthetic compound, which was incorporated into microspheres composed of tristearin and encapsulated using a solid protein matrix. Solid-state 13C NMR spectroscopy was used to investigate the solid forms of bupivacaine in their bulk form or as incorporated into the tristearin/protein matrix. Bupivacaine free base and bupivacaine-HCl have very different solid-state NMR spectra, indicating that the molecules of these compounds pack in different crystal forms. In the tristearin matrix, the drug form could be determined at levels as low as 1:100 (w/w), and the form of bupivacaine was identified upon loading into the tristearin/protein matrix. In the second case, the possibility of using solid-state 13C NMR spectroscopy to characterize biomolecules lyophilized within polymer matrices is evaluated by studying uniformly 13C-labeled asparagine (Asn) in 1:250 (w/w) formulations with poly(vinyl pyrrolidone) (PVP) and poly(vinyl alcohol) (PVA). This work shows the capability of solid-state NMR spectroscopy to study interactions between the amino acid and the polymer matrix for synthetic peptides and peptidomimetics containing selective 13C labeling at the Asn residue.
Full text
Available for:
DOBA, EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, IZUM, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UILJ, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
Asparagine (Asn) degradation kinetics in two model peptides, Gly-Gln-Asn-Gly-Gly (GQNGG) and Val-Tyr-Pro-Asn-Gly-Ala (VYPNGA), were studied at 50°C in pH 7 buffer solutions in the presence and ...absence of 5% (w/v) sucrose or mannitol and at 50°C and 30% relative humidity in solid samples lyophilized from these solutions. Solid formulations were characterized using Karl Fischer coulometric titration, thermal gravimetric analysis (TGA), differential scanning calorimetry (DSC), Fourier-transform infrared spectrometry (FTIR), and solid-state nuclear magnetic resonance (NMR) spectroscopy. GQNGG and VYPNGA showed similar pseudo first-order deamidation rates in solution in the absence of sucrose and mannitol. Adding 5% sucrose or mannitol decreased the rates by no more than 17%. The model peptides degraded 2- to 80-fold more slowly in the solid formulations of sucrose and mannitol than in 5% solutions of these carbohydrates. Ratios of deamidation rates of the model peptides depended upon the solid matrix. In the mannitol solid, the ratio of deamidation rates of GQNGG and VYPNGA (GQNGG:VYPNGA) was ∼8, while in the sucrose solid, the model peptides deamidated at similar rates (GQNGG:VYPNGA≅1). DSC showed the mannitol formulations to be largely amorphous immediately after lyophilization with some ordered, crystalline-like structure; the extent of ordered structure increased during storage as shown by FTIR and ssNMR. In contrast, the sucrose formulation was largely amorphous after lyophilization and remained so during storage. Together, the results showed that 5% sucrose or mannitol in solution does not significantly change the rates of Asn deamidation of the model peptides, while sucrose stabilizes the model peptides against deamidation more than mannitol in the solid state.
Full text
Available for:
BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
Solid-state NMR spectroscopy (SSNMR) is an extremely powerful technique for the analysis of pharmaceutical dosage forms. A major limitation of SSNMR is the number of samples that can be analyzed in a ...given period of time. A solid-state magic-angle spinning (MAS) probe that can simultaneously acquire up to seven SSNMR spectra is being developed to increase throughput/signal-to-noise ratios. A prototype probe incorporating two MAS modules has been developed and spectra of ibuprofen and aspirin have been acquired simultaneously. This version is limited to being a two-module probe due to large amounts of space required for the tuning elements located next to the MAS modules. A new probe design incorporating coaxial transmission lines and smaller MAS modules has been constructed. This probe allows for close proximity of the MAS modules (within 3
cm), adequate proton decoupling power (>50
kHz), and the capability of remote tuning and sample changing. Spectra of hexamethylbenzene (HMB) have been acquired and show signal-to-noise ratios comparable to existing SSNMR probes. Adamantane line widths are also comparable to conventional probe technology. Decoupling powers of 70
kHz have been achieved using a MAS module suitable for 3
cm spacing between modules. Remote tuning has also been achieved with this new coaxial transmission line design. This probe design can be easily scaled to incorporate multiple MAS modules, which is a limitation of the previous design. The number of modules that can be incorporated is only limited by the number of transmission lines that will fit in a cross-sectional diameter of the bore and the axial field length of the magnet.
Full text
Available for:
GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
Two studies were conducted to determine whether attenuated strains of
Salmonella typhimurium, currently being investigated as possible vectors for mucosal vaccines, are able to respond to ...norepinephrine (NE). Bacteria were tested for NE responsiveness before and for 1 week after passage through juvenile rhesus monkeys. NE significantly increased the growth of the attenuated bacteria after being shed from the animal, but not before animal infection. Follow-up in vitro tests were performed by passaging the bacteria in Lauria–Bertani (LB) broth with or without selective antibiotic for the attenuation insert and supplementing with NE. NE increased the growth of bacteria passaged in LB broth with no selective antibiotic, but not in bacteria passaged in LB broth with selective antibiotic. These results show that the attenuated bacteria assumed to be safe for use as a vaccine are able to respond to environmental stimuli, such as NE, and change their characteristics. The results suggest that there may be problems with the stability of attenuated bacteria used as vectors for mucosal vaccines.
Full text
Available for:
IJS, IMTLJ, KILJ, KISLJ, NUK, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
We demonstrate that natural isotopic abundance 2D heteronuclear correlation (HETCOR) solid-state NMR spectra can be used to significantly reduce or eliminate the broadening of 1H and 13C solid-state ...NMR spectra of organic solids due to anisotropic bulk magnetic susceptibility (ABMS). ABMS often manifests in solids with aromatic groups, such as active pharmaceutical ingredients (APIs), and inhomogeneously broadens the NMR peaks of all nuclei in the sample. Inhomogeneous peaks with full widths at half maximum (FWHM) of ∼1 ppm typically result from ABMS broadening and the low spectral resolution impedes the analysis of solid-state NMR spectra. ABMS broadening of solid-state NMR spectra has previously been eliminated using 2D multiple-quantum correlation experiments, or by performing NMR experiments on diluted materials or single crystals. However, these experiments are often infeasible due to their poor sensitivity and/or provide limited gains in resolution. 2D 1H-13C HETCOR experiments have previously been applied to reduce susceptibility broadening in paramagnetic solids and we show that this strategy can significantly reduce ABMS broadening in diamagnetic organic solids. Comparisons of 1D solid-state NMR spectra and 1H and 13C solid-state NMR spectra obtained from 2D 1H-13C HETCOR NMR spectra show that the HETCOR spectrum directly increases resolution by a factor of 1.5 to 8. The direct gain in resolution is determined by the ratio of the inhomogeneous 13C/1H linewidth to the homogeneous 1H linewidth, with the former depending on the magnitude of the ABMS broadening and the strength of the applied field and the latter on the efficiency of homonuclear decoupling. The direct gains in resolution obtained using the 2D HETCOR experiments are better than that obtained by dilution. For solids with long proton longitudinal relaxation times, dynamic nuclear polarization (DNP) was applied to enhance sensitivity and enable the acquisition of 2D 1H-13C HETCOR NMR spectra. 2D 1H-13C HETCOR experiments were applied to resolve and partially assign the NMR signals of the form I and form II polymorphs of aspirin in a sample containing both forms. These findings have important implications for ultra-high field NMR experiments, optimization of decoupling schemes and assessment of the fundamental limits on the resolution of solid-state NMR spectra.
Cellular immune responses were evaluated in 35 infant rhesus monkeys generated from two types of pregnancy conditions. Pregnant females were administered either saline or adrenocorticotropic hormone ...(ACTH) for 2 weeks between Days 120 and 133 postconception, approximately 1 month before parturition. After birth, lymphocytes obtained from infants in the ACTH condition failed to respond as readily to allogeneic cells in mixed lymphocyte cultures, proliferated less to Con A, exhibited lower suppressor function following stimulation with Con A, and showed lower cytolytic activity against target cells. For some measures, the prenatal effect was observed more consistently in male infants. Differences were evident with thesein vitroimmune assays through 6 months of age, indicating that acute disturbance during the prenatal period can have lingering effects on postnatal immunity.
Full text
Available for:
IJS, IMTLJ, KILJ, KISLJ, NUK, SBCE, SBJE, UL, UM, UPCLJ, UPUK
PDF
