32 research outputs found
Chelators for Radioimmunotherapy: I. NMR and Ab Initio Calculation Studies on 1,4,7,10-Tetra(carboxyethyl)-1,4,7,10-tetraazacyclododecane (DO4Pr) and 1,4,7-Tris(carboxymethyl)-10-(carboxyethyl)-1,4,7,10-tetraazacyclododecane (DO3A1Pr)
This work describes the modification of the chelating agent 1,4,7,10-tetraazacyclododecane-N,N‘,N‘ ‘,N‘ ‘‘-tetraacetic acid (DOTA) to improve the rate of metal loading for radioimmunotherapy applications. Previous ab initio calculations predicted that the compounds 1,4,7,10-tetra(carboxyethyl)-1,4,7,10-tetraazacyclododecane (DO4Pr) and 1,4,7-tris(carboxymethyl)-10-(carboxyethyl)-1,4,7,10-tetraazacyclododecane (DO3A1Pr) have a ca. 2000-fold improvement in yttrium metal loading rates compared to those of DOTA (Jang, Y. H.; Blanco, M.; Dasgupta, S.; Keire, D. A.; Shively, J. E.; Goddard, W. A., III. J. Am. Chem. Soc. 1999, 121, 6142−6151). In this study, we report the synthesis, purification, 1H-NMR chemical shift assignments, pKa values, metal loading rate measurements, and additional ab initio calculations of these two compounds. The yttrium loading rates of DO3A1Pr are approximately twice those of DOTA, at pH 4.6 and 37 °C. The NMR data indicates that the DO4Pr analogue forms a stable type I complex but does not form a type II complex. The new ab initio calculations performed on DO4Pr and DO3A1Pr indicate that the rate-determining step is the deprotonation of the first macrocycle amine proton, not the second proton as assumed in the previous calculations. The new calculations predict an improvement in the rate of metal loading that more closely matches the experimentally observed change in the rate
Chemoenzymatic synthesis and structural characterization of 2-O-sulfated glucuronic acid-containing heparan sulfate hexasaccharides
Heparan sulfate and heparin are highly sulfated polysaccharides that consist of a repeating disaccharide unit of glucosamine and glucuronic or iduronic acid. The 2-O-sulfated iduronic acid (IdoA2S) residue is commonly found in heparan sulfate and heparin; however, 2-O-sulfated glucuronic acid (GlcA2S) is a less abundant monosaccharide (∼<5% of total saccharides). Here, we report the synthesis of three GlcA2S-containing hexasaccharides using a chemoenzymatic approach. For comparison purposes, additional IdoA2S-containing hexasaccharides were synthesized. Nuclear magnetic resonance analyses were performed to obtain full chemical shift assignments for the GlcA2S- and IdoA2S-hexasaccharides. These data show that GlcA2S is a more structurally rigid saccharide residue than IdoA2S. The antithrombin (AT) binding affinities of a GlcA2S- and an IdoA2S-hexasaccharide were determined by affinity co-electrophoresis. In contrast to IdoA2S-hexasaccharides, the GlcA2S-hexasaccharide does not bind to AT, confirming that the presence of IdoA2S is critically important for the anticoagulant activity. The availability of pure synthetic GlcA2S-containing oligosaccharides will allow the investigation of the structure and activity relationships of individual sites in heparin or heparan sulfate
Development of methods for data quantitation of spiked salmon host cell DNA in protamine sulfate by qPCR
Protamine sulfate (PS) is an approximately 4 kDa cationic polypeptide derived from chum salmon used to reverse heparin-induced anticoagulation in patients. Because the presence of residual host cell salmon DNA (resDNA) in PS drug product can pose safety concerns, processing steps during PS manufacturing are designed to target the reduction of these impurities. However, given protamine׳s positively charged structure, isolating and measuring negatively charged residual DNA is challenging. Suitable resDNA methods for PS require the generation of host DNA reference materials, efficient DNA extraction procedures and assay sensitivity and accuracy as high as possible. Here, optimization data are shown for the extraction of DNA present in PS drug products and for the generation of reference standard from protease-digested research grade chum salmon DNA. The lower limit of quantitation (LLOQ) for the reference standard determined from protease-digested DNA (0.0025–156.25 pg/μL) was 0.0025 pg/μL. The extraction procedure LLOQ, determined from DNA (0.01–1.25 pg/μL) spiked into PS samples, was 5 pg DNA per mg PS. The data supporting the LLOQs were evaluated using acceptance criteria of 70–130% recovery with % correlation coefficient (CV) ≤ 25% for DNA concentrations and curve metrics (slopes, R2 and y-intercepts) within 2SD of the mean. The data presented here complement a broader study (Sommers et al., 2018) [1] and are particularly useful for the development of resDNA methods for challenging drug products. Keywords: Protamine sulfate, Real-time polymerase chain reaction, Residual host cell DNA, TaqMan, DNA reference standar
Chemoenzymatic synthesis and structural characterization of 2-O-sulfated glucuronic acid-containing heparan sulfate hexasaccharides
Heparan sulfate and heparin are highly sulfated polysaccharides that consist of a repeating disaccharide unit of glucosamine and glucuronic or iduronic acid. The 2-O-sulfated iduronic acid (IdoA2S) residue is commonly found in heparan sulfate and heparin; however, 2-O-sulfated glucuronic acid (GlcA2S) is a less abundant monosaccharide (∼<5% of total saccharides). Here, we report the synthesis of three GlcA2S-containing hexasaccharides using a chemoenzymatic approach. For comparison purposes, additional IdoA2S-containing hexasaccharides were synthesized. Nuclear magnetic resonance analyses were performed to obtain full chemical shift assignments for the GlcA2S- and IdoA2S-hexasaccharides. These data show that GlcA2S is a more structurally rigid saccharide residue than IdoA2S. The antithrombin (AT) binding affinities of a GlcA2S- and an IdoA2S-hexasaccharide were determined by affinity co-electrophoresis. In contrast to IdoA2S-hexasaccharides, the GlcA2S-hexasaccharide does not bind to AT, confirming that the presence of IdoA2S is critically important for the anticoagulant activity. The availability of pure synthetic GlcA2S-containing oligosaccharides will allow the investigation of the structure and activity relationships of individual sites in heparin or heparan sulfate
High-Throughput Differentiation of Heparin from Other Glycosaminoglycans by Pyrolysis Mass Spectrometry
Sensors
with high chemical specificity and enhanced sample throughput are
vital to screening food products and medical devices for chemical
or biochemical contaminants that may pose a threat to
public health. For example, the rapid detection of oversulfated chondroitin
sulfate (OSCS) in heparin could prevent reoccurrence of heparin adulteration
that caused hundreds of severe adverse events including deaths worldwide
in 2007–2008. Here, rapid pyrolysis is integrated with direct
analysis in real time (DART) mass spectrometry to rapidly screen major
glycosaminoglycans, including heparin, chondroitin sulfate A, dermatan
sulfate, and OSCS. The results demonstrate that, compared to traditional
liquid chromatography-based analyses, pyrolysis mass spectrometry
achieved at least 250-fold higher sample throughput and was compatible
with samples volume-limited to about 300 nL. Pyrolysis yielded an
abundance of fragment ions (<i>e.g.,</i> 150 different <i>m</i>/<i>z</i> species), many of which were specific
to the parent compound. Using multivariate and statistical data analysis
models, these data enabled facile differentiation of the glycosaminoglycans
with high throughput. After method development was completed, authentically
contaminated samples obtained during the heparin crisis by the FDA
were analyzed in a blinded manner for OSCS contamination. The lower
limit of differentiation and detection were 0.1% (w/w) OSCS in heparin
and 100 ng/μL (20 ng) OSCS in water, respectively. For quantitative
purposes the linear dynamic range spanned approximately 3 orders of
magnitude. Moreover, this chemical readout was successfully employed
to find clues in the manufacturing history of the heparin samples
that can be used for surveillance purposes. The presented technology
and data analysis protocols are anticipated to be readily adaptable
to other chemical and biochemical agents and volume-limited samples