Synthesis, insertion, and characterization of SARS-CoV-2 membrane protein within lipid bilayers.

Throughout history, coronaviruses have posed challenges to both public health and the global economy; nevertheless, methods to combat them remain rudimentary, primarily due to the absence of experiments to understand the function of various viral components. Among these, membrane (M) proteins are one of the most elusive because of their small size and challenges with expression. Here, we report the development of an expression system to produce tens to hundreds of milligrams of M protein per liter of Escherichia coli culture. These large yields render many previously inaccessible structural and biophysical experiments feasible. Using cryo-electron microscopy and atomic force microscopy, we image and characterize individual membrane-incorporated M protein dimers and discover membrane thinning in the vicinity, which we validated with molecular dynamics simulations. Our results suggest that the resulting line tension, along with predicted induction of local membrane curvature, could ultimately drive viral assembly and budding.

Interhelical E@g-N@a interactions modulate coiled coil stability within a de novo set of orthogonal peptide heterodimers.

The designability of orthogonal coiled coil (CC) dimers, which draw on well-established design rules, plays a pivotal role in fueling the development of CCs as synthetically versatile assembly-directing motifs for the fabrication of bionanomaterials. Here, we aim to expand the synthetic CC toolkit through establishing a "minimalistic" set of orthogonal, de novo CC peptides that comprise 3.5 heptads in length and a single buried Asn to prescribe dimer formation. The designed sequences display excellent partner fidelity, confirmed via circular dichroism (CD) spectroscopy and Ni-NTA binding assays, and are corroborated in silico using molecular dynamics (MD) simulation. Detailed analysis of the MD conformational data highlights the importance of interhelical E@g-N@a interactions in coordinating an extensive 6-residue hydrogen bonding network that "locks" the interchain Asn-Asn' contact in place. The enhanced stability imparted to the Asn-Asn' bond elicits an increase in thermal stability of CCs up to ~15°C and accounts for significant differences in stability within the collection of similarly designed orthogonal CC pairs. The presented work underlines the utility of MD simulation as a tool for constructing de novo, orthogonal CCs, and presents an alternative handle for modulating the stability of orthogonal CCs via tuning the number of interhelical E@g-N@a contacts. Expansion of CC design rules is a key ingredient for guiding the design and assembly of more complex, intricate CC-based architectures for tackling a variety of challenges within the fields of nanomedicine and bionanotechnology.

Supplemental habitat is reservoir dependent: Identifying optimal planting decision using Bayesian Decision Networks.

Environmental management often requires making decisions despite system uncertainty. One such example is mudflat mediation in flood control reservoirs. Reservoir mudflats limit development of diverse fish assemblages due to the lack of structural habitat provided by plants. Seeding mudflats with agricultural plants may mimic floodplain wetlands once inundated and provide fish habitat and achieve habitat management objectives. However, planting success is uncertain because of unpredictable water level fluctuations that affect plant survival and growth. Decision support tools can account for uncertainty that influences decision outcomes and reduce the risk in reservoir mudflat planting decisions. We used Bayesian decision networks and sensitivity analyses to quantify uncertainty surrounding mudflat plantings as supplemental fish habitat in four northwest Mississippi reservoirs. When averaged across all uncertainty, planting was the optimal decision only in Enid Lake. Response profiles indicated planting decisions depended on elevation contours within Enid, Sardis, and Grenada reservoirs. No planting was optimal at all elevations for Arkabutla Lake. These results provide a quantified basis for establishing best management practices and identify key system states that influence decision outcomes. The process used in this study to evaluate planting decisions can be applied to any reservoir by modifying reservoir dependent inputs to evaluate planting decisions to provide supplemental fish habitat.

The binding and specificity of chemokine binding proteins, through the lens of experiment and computation.

Chemokines are small proteins that are critical for immune function, being primarily responsible for the activation and chemotaxis of leukocytes. As such, many viruses, as well as parasitic arthropods, have evolved systems to counteract chemokine function in order to maintain virulence, such as binding chemokines, mimicking chemokines, or producing analogs of transmembrane chemokine receptors that strongly bind their targets. The focus of this review is the large group of chemokine binding proteins (CBP) with an emphasis on those produced by mammalian viruses. Because many chemokines mediate inflammation, these CBP could possibly be used pharmaceutically as anti-inflammatory agents. In this review, we summarize the structural properties of a diverse set of CBP and describe in detail the chemokine binding properties of the poxvirus-encoded CBP called vCCI (viral CC Chemokine Inhibitor). Finally, we describe the current and emerging capabilities of combining computational simulation, structural analysis, and biochemical/biophysical experimentation to understand, and possibly re-engineer, protein-protein interactions.

A generalized approach for sperm cryopreservation in the genus Pomoxis: Sperm cryopreservation and fertilization efficiency of black-stripe black crappie, Pomoxis nigromaculatus.

Approaches for white crappie, Pomoxis annularis sperm cryopreservation have led to interest in applying similar methods to black-stripe black crappie, Pomoxis nigromaculatus. Their rarity in wild populations makes them a preferred phenotype for hatchery use. Sperm cryopreservation procedures were compared between black-stripe black crappie and white crappie for sperm motility and egg fertilization rate. There was no difference in black-stripe black crappie sperm motility after thawing between 5% dimethyl sulfoxide (DMSO, 45% motility) and 10% methanol (50% motility). However, fertilization rates were higher (p < .001) for sperm cryoprotected with 5% DMSO (38 ± 8%) than 10% methanol (22 ± 7%). Hatchery use requires sperm-to-egg ratios and fertilizing potential of single doses (i.e., 0.5 ml straw). Using black-stripe black crappie sperm (2.5 × 108 sperm/ml; 5% DMSO), the highest fertilization (27%) was found using single straws with 785 eggs (0.25 ml); total sperm:egg ratio: 159,000:1; motile sperm:egg ratio: 71,700:1. Therefore, sperm of two Pomoxis species could be cryopreserved using 350 mOsmol/kg Hanks' balanced salt solution as an extender, 5% DMSO as a cryoprotectant, cooling at 40°C/min, and thawing for 8 s at 40°C to maintain sperm motility and fertility. Basic protocols can be generalized within a genus if variables such as sperm concentration, process timing, and sample volumes are controlled.

Discerning the dietary habits of the smooth butterfly ray Gymnura lessae using two distinct methods, otolith identification and metagenetics.

Two different methods, metagenetics and free-otolith identification, were used to identify prey in the stomach contents of 531 Gymnura lessae captured by trawling in Mobile Bay, Alabama 2016-2018. Both methods were found to produce analogous results and were therefore combined into a single complete dataset. All prey were teleosts; the families Sciaenidae and Engraulidae were the most important prey (prey specie index of relative importance 89.3% IPSRI ). Multivariate analyses indicated that the diet of G. lessae varied with sex and seasonality. Specifically, variability was probably due to morphologically larger females consuming larger teleost prey species compared with males, whereas seasonal variability was probably due to changes in the available prey community composition. The findings indicate that both metagenetics and free otolith identification, used independently or complementarily, offer robust means of characterising dietary habits for teleost-specialised species such as G. lessae, which may play an important role in the structure and maintenance of coastal food webs such as those in Mobile Bay.

Biophysical and Computational Studies of the vCCI:vMIP-II Complex.

Certain viruses have the ability to subvert the mammalian immune response, including interference in the chemokine system. Poxviruses produce the chemokine binding protein vCCI (viral CC chemokine inhibitor; also called 35K), which tightly binds to CC chemokines. To facilitate the study of vCCI, we first provide a protocol to produce folded vCCI from Escherichia coli (E. coli.) It is shown here that vCCI binds with unusually high affinity to viral Macrophage Inflammatory Protein-II (vMIP-II), a chemokine analog produced by the virus, human herpesvirus 8 (HHV-8). Fluorescence anisotropy was used to investigate the vCCI:vMIP-II complex and shows that vCCI binds to vMIP-II with a higher affinity than most other chemokines, having a Kd of 0.06 ± 0.006 nM. Nuclear magnetic resonance (NMR) chemical shift perturbation experiments indicate that key amino acids used for binding in the complex are similar to those found in previous work. Molecular dynamics were then used to compare the vCCI:vMIP-II complex with the known vCCI:Macrophage Inflammatory Protein-1ß/CC-Chemokine Ligand 4 (MIP-1ß/CCL4) complex. The simulations show key interactions, such as those between E143 and D75 in vCCI/35K and R18 in vMIP-II. Further, in a comparison of 1 µs molecular dynamics (MD) trajectories, vMIP-II shows more overall surface binding to vCCI than does the chemokine MIP-1ß. vMIP-II maintains unique contacts at its N-terminus to vCCI that are not made by MIP-1ß, and vMIP-II also makes more contacts with the vCCI flexible acidic loop (located between the second and third beta strands) than does MIP-1ß. These studies provide evidence for the basis of the tight vCCI:vMIP-II interaction while elucidating the vCCI:MIP-1ß interaction, and allow insight into the structure of proteins that are capable of broadly subverting the mammalian immune system.

Innate and adaptive immune responses in migrating spring-run adult chinook salmon, Oncorhynchus tshawytscha.

Adult Chinook salmon (Oncorhynchus tshawytscha) migrate from salt water to freshwater streams to spawn. Immune responses in migrating adult salmon are thought to diminish in the run up to spawning, though the exact mechanisms for diminished immune responses remain unknown. Here we examine both adaptive and innate immune responses as well as pathogen burdens in migrating adult Chinook salmon in the Upper Willamette River basin. Messenger RNA transcripts encoding antibody heavy chain molecules slightly diminish as a function of time, but are still present even after fish have successfully spawned. In contrast, the innate anti-bacterial effector proteins present in fish plasma rapidly decrease as spawning approaches. Fish also were examined for the presence and severity of eight different pathogens in different organs. While pathogen burden tended to increase during the migration, no specific pathogen signature was associated with diminished immune responses. Transcript levels of the immunosuppressive cytokines IL-10 and TGF beta were measured and did not change during the migration. These results suggest that loss of immune functions in adult migrating salmon are not due to pathogen infection or cytokine-mediated immune suppression, but is rather part of the life history of Chinook salmon likely induced by diminished energy reserves or hormonal changes which accompany spawning.

Occupancy modeling for improved accuracy and understanding of pathogen prevalence and dynamics.

Most pathogen detection tests are imperfect, with a sensitivity < 100%, thereby resulting in the potential for a false negative, where a pathogen is present but not detected. False negatives in a sample inflate the number of non-detections, negatively biasing estimates of pathogen prevalence. Histological examination of tissues as a diagnostic test can be advantageous as multiple pathogens can be examined and providing important information on associated pathological changes to the host. However, it is usually less sensitive than molecular or microbiological tests for specific pathogens. Our study objectives were to 1) develop a hierarchical occupancy model to examine pathogen prevalence in spring Chinook salmon Oncorhynchus tshawytscha and their distribution among host tissues 2) use the model to estimate pathogen-specific test sensitivities and infection rates, and 3) illustrate the effect of using replicate within host sampling on sample sizes required to detect a pathogen. We examined histological sections of replicate tissue samples from spring Chinook salmon O. tshawytscha collected after spawning for common pathogens seen in this population: Apophallus/echinostome metacercariae, Parvicapsula minibicornis, Nanophyetus salmincola/ metacercariae, and Renibacterium salmoninarum. A hierarchical occupancy model was developed to estimate pathogen and tissue-specific test sensitivities and unbiased estimation of host- and organ-level infection rates. Model estimated sensitivities and host- and organ-level infections rates varied among pathogens and model estimated infection rate was higher than prevalence unadjusted for test sensitivity, confirming that prevalence unadjusted for test sensitivity was negatively biased. The modeling approach provided an analytical approach for using hierarchically structured pathogen detection data from lower sensitivity diagnostic tests, such as histology, to obtain unbiased pathogen prevalence estimates with associated uncertainties. Accounting for test sensitivity using within host replicate samples also required fewer individual fish to be sampled. This approach is useful for evaluating pathogen or microbe community dynamics when test sensitivity is <100%.

Tolerance and efficacy of emamectin benzoate and ivermectin for the treatment of Pseudocapillaria tomentosa in laboratory zebrafish (Danio rerio).

Tolerance of adult zebrafish and efficacy of emamectin benzoate and ivermectin in eliminating Pseudocapillaria tomentosa infection were evaluated. In the tolerance study, behavioral changes, fecundity, histopathology, and mortality were evaluated for in-feed administration of emamectin (0.05, 0.10, and 0.25 mg/kg) and ivermectin (0.05 and 0.10 mg/kg). All doses of emamectin were well tolerated. Ivermectin 0.05 mg/kg administration resulted in mild behavioral changes and a transient decrease in fecundity. Ivermectin 0.10 mg/kg administration resulted in severe behavioral changes and some mortality. In the efficacy study, emamectin (0.05 and 0.25 mg/kg) and ivermectin (0.05 mg/kg) were evaluated for their efficacy in eliminating P. tomentosa infection. Emamectin reduced parasite burden in infected zebrafish, and ivermectin eliminated intestinal nematode infections. Despite a small margin of safety, ivermectin 0.05 mg/kg was effective at eliminating P. tomentosa infection in adult zebrafish. Higher doses or a longer course of treatment may be needed for complete elimination of P. tomentosa infection using emamectin. In this study, we propose two possible treatments for intestinal nematode infections in zebrafish.

Design of High-Specificity Nanocarriers by Exploiting Non-Equilibrium Effects in Cancer Cell Targeting.

Although targeting of cancer cells using drug-delivering nanocarriers holds promise for improving therapeutic agent specificity, the strategy of maximizing ligand affinity for receptors overexpressed on cancer cells is suboptimal. To determine design principles that maximize nanocarrier specificity for cancer cells, we studied a generalized kinetics-based theoretical model of nanocarriers with one or more ligands that specifically bind these overexpressed receptors. We show that kinetics inherent to the system play an important role in determining specificity and can in fact be exploited to attain orders of magnitude improvement in specificity. In contrast to the current trend of therapeutic design, we show that these specificity increases can generally be achieved by a combination of low rates of endocytosis and nanocarriers with multiple low-affinity ligands. These results are broadly robust across endocytosis mechanisms and drug-delivery protocols, suggesting the need for a paradigm shift in receptor-targeted drug-delivery design.

Electric field-driven water dipoles: nanoscale architecture of electroporation.

Electroporation is the formation of permeabilizing structures in the cell membrane under the influence of an externally imposed electric field. The resulting increased permeability of the membrane enables a wide range of biological applications, including the delivery of normally excluded substances into cells. While electroporation is used extensively in biology, biotechnology, and medicine, its molecular mechanism is not well understood. This lack of knowledge limits the ability to control and fine-tune the process. In this article we propose a novel molecular mechanism for the electroporation of a lipid bilayer based on energetics analysis. Using molecular dynamics simulations we demonstrate that pore formation is driven by the reorganization of the interfacial water molecules. Our energetics analysis and comparisons of simulations with and without the lipid bilayer show that the process of poration is driven by field-induced reorganization of water dipoles at the water-lipid or water-vacuum interfaces into more energetically favorable configurations, with their molecular dipoles oriented in the external field. Although the contributing role of water in electroporation has been noted previously, here we propose that interfacial water molecules are the main players in the process, its initiators and drivers. The role of the lipid layer, to a first-order approximation, is then reduced to a relatively passive barrier. This new view of electroporation simplifies the study of the problem, and opens up new opportunities in both theoretical modeling of the process and experimental research to better control or to use it in new, innovative ways.

Validating clustering of molecular dynamics simulations using polymer models.

BACKGROUND: Molecular dynamics (MD) simulation is a powerful technique for sampling the meta-stable and transitional conformations of proteins and other biomolecules. Computational data clustering has emerged as a useful, automated technique for extracting conformational states from MD simulation data. Despite extensive application, relatively little work has been done to determine if the clustering algorithms are actually extracting useful information. A primary goal of this paper therefore is to provide such an understanding through a detailed analysis of data clustering applied to a series of increasingly complex biopolymer models. RESULTS: We develop a novel series of models using basic polymer theory that have intuitive, clearly-defined dynamics and exhibit the essential properties that we are seeking to identify in MD simulations of real biomolecules. We then apply spectral clustering, an algorithm particularly well-suited for clustering polymer structures, to our models and MD simulations of several intrinsically disordered proteins. Clustering results for the polymer models provide clear evidence that the meta-stable and transitional conformations are detected by the algorithm. The results for the polymer models also help guide the analysis of the disordered protein simulations by comparing and contrasting the statistical properties of the extracted clusters. CONCLUSIONS: We have developed a framework for validating the performance and utility of clustering algorithms for studying molecular biopolymer simulations that utilizes several analytic and dynamic polymer models which exhibit well-behaved dynamics including: meta-stable states, transition states, helical structures, and stochastic dynamics. We show that spectral clustering is robust to anomalies introduced by structural alignment and that different structural classes of intrinsically disordered proteins can be reliably discriminated from the clustering results. To our knowledge, our framework is the first to utilize model polymers to rigorously test the utility of clustering algorithms for studying biopolymers.

A bimodal distribution of two distinct categories of intrinsically disordered structures with separate functions in FG nucleoporins.

Nuclear pore complexes (NPCs) gate the only conduits for nucleocytoplasmic transport in eukaryotes. Their gate is formed by nucleoporins containing large intrinsically disordered domains with multiple phenylalanine-glycine repeats (FG domains). In combination, these are hypothesized to form a structurally and chemically homogeneous network of random coils at the NPC center, which sorts macromolecules by size and hydrophobicity. Instead, we found that FG domains are structurally and chemically heterogeneous. They adopt distinct categories of intrinsically disordered structures in non-random distributions. Some adopt globular, collapsed coil configurations and are characterized by a low charge content. Others are highly charged and adopt more dynamic, extended coil conformations. Interestingly, several FG nucleoporins feature both types of structures in a bimodal distribution along their polypeptide chain. This distribution functionally correlates with the attractive or repulsive character of their interactions with collapsed coil FG domains displaying cohesion toward one another and extended coil FG domains displaying repulsion. Topologically, these bipartite FG domains may resemble sticky molten globules connected to the tip of relaxed or extended coils. Within the NPC, the crowding of FG nucleoporins and the segregation of their disordered structures based on their topology, dimensions, and cohesive character could force the FG domains to form a tubular gate structure or transporter at the NPC center featuring two separate zones of traffic with distinct physicochemical properties.

Intramolecular cohesion of coils mediated by phenylalanine--glycine motifs in the natively unfolded domain of a nucleoporin.

The nuclear pore complex (NPC) provides the sole aqueous conduit for macromolecular exchange between the nucleus and the cytoplasm of cells. Its diffusion conduit contains a size-selective gate formed by a family of NPC proteins that feature large, natively unfolded domains with phenylalanine-glycine repeats (FG domains). These domains of nucleoporins play key roles in establishing the NPC permeability barrier, but little is known about their dynamic structure. Here we used molecular modeling and biophysical techniques to characterize the dynamic ensemble of structures of a representative FG domain from the yeast nucleoporin Nup116. The results showed that its FG motifs function as intramolecular cohesion elements that impart order to the FG domain and compact its ensemble of structures into native premolten globular configurations. At the NPC, the FG motifs of nucleoporins may exert this cohesive effect intermolecularly as well as intramolecularly to form a malleable yet cohesive quaternary structure composed of highly flexible polypeptide chains. Dynamic shifts in the equilibrium or competition between intra- and intermolecular FG motif interactions could facilitate the rapid and reversible structural transitions at the NPC conduit needed to accommodate passing karyopherin-cargo complexes of various shapes and sizes while simultaneously maintaining a size-selective gate against protein diffusion.

Influence of sulfur oxidation state and steric bulk upon trifluoromethyl ketone (TFK) binding kinetics to carboxylesterases and fatty acid amide hydrolase (FAAH).

Carboxylesterases metabolize numerous exogenous and endogenous ester-containing compounds including the chemotherapeutic agent CPT-11, anti-influenza viral agent oseltamivir, and many agrochemicals. Trifluoromethyl ketone (TFK)-containing compounds with a sulfur atom beta to the ketone moiety are some of the most potent carboxylesterase and amidase inhibitors identified to date. This study examined the effects of alkyl chain length (i.e., steric effects) and sulfur oxidation state upon TFK inhibitor potency (IC50) and binding kinetics (k(i)). The selective carboxylesterase inhibitor benzil was used as a non-TFK containing control. These effects were examined using two commercial esterases (porcine and rabbit liver esterase) and two human recombinant esterases (hCE-1 and hCE-2) as well as human recombinant fatty acid amide hydrolase (FAAH). In addition, the inhibition mechanism was examined using a combination of 1H NMR, X-ray crystallography, and ab initio calculations. Overall, the data show that while sulfur oxidation state profoundly affects both inhibitor potency and binding kinetics, the steric effects dominate and override the contributions of sulfur oxidation. In addition, the data suggest that inclusion of a sulfur atom beta to the ketone contributes an increase (approximately 5-fold) in inhibitor potency due to effects upon ketone hydration and/or intramolecular hydrogen bond formation. These results provide further information on the nature of the TFK binding interaction and will be useful in increasing our understanding of this basic biochemical process.

Mutagenic potency of food-derived heterocyclic amines.

The understanding of mutagenic potency has been primarily approached using "quantitative structure-activity relationships" (QSAR). Often this method allows the prediction of mutagenic potency of the compound based on its structure. But it does not give the underlying reason why the mutagenic activities differ. We have taken a set of heterocyclic amine structures and used molecular dynamic calculations to dock these molecules into the active site of a computational model of the cytochrome P4501A2 enzyme. The calculated binding strength using Boltzman distribution constants was then compared to the QSAR value (HF/6-31G* optimized structures) and the Ames/Salmonella mutagenic potency. Further understanding will only come from knowing the complete set of mutagenic determinants. These include the nitrenium ion half-life, DNA adduct half-life, efficiency of repair of the adduct, and ultimately fixation of the mutation through cellular processes. For two isomers, PhIP and 3-Me-PhIP, we showed that for the 100-fold difference in the mutagenic potency a 5-fold difference can be accounted for by differences in the P450 oxidation. The other factor of 20 is not clearly understood but is downstream from the oxidation step. The application of QSAR (chemical characteristics) to biological principles related to mutagenesis is explored in this report.

Environmental effects on the structure of metal ion-DOTA complexes: an ab initio study of radiopharmaceutical metals.

Quantum chemical calculations were performed to study the differences between the important radiopharmaceutical metals yttrium (Y) and indium (In) bound by DOTA and modified DOTA molecules. Energies were calculated at the MP2/6-31+G(d)//HF/6-31G(d) levels, using effective core potentials on the Y and In ions. Although the minimum energy structures obtained are similar for both metal ion-DOTA complexes, changes in coordination and local environment significantly affect the geometries and energies of these complexes. Coordination by a single water molecule causes a change in the coordination number and a change in the position of the metal ion in In-DOTA, but Y-DOTA is hardly affected by water coordination. When one of the DOTA carboxylates is replaced by an amide, the resulting structures show a large variation between the Y and In ions. A six-residue model of the active site containing metal ion-DOTA showed that the Y-DOTA structure optimized to a structure similar to the crystal structure but that the water molecule in In-DOTA disrupts the salt bridge between Arg98B and a carboxylate side chain of DOTA. These observed differences could in part explain the differential binding constants for Y-DOTA and In-DOTA to the antibody 2D12.5.

Dynamic force spectroscopy of parallel individual Mucin1-antibody bonds.

We used atomic force microscopy to measure the binding forces between Mucin1 (MUC1) peptide and a single-chain variable fragment (scFv) antibody selected from a scFv library screened against MUC1. This binding interaction is central to the design of molecules used for targeted delivery of radioimmunotherapeutic agents for prostate and breast cancer treatment. Our experiments separated the specific binding interaction from nonspecific interactions by tethering the antibody and MUC1 molecules to the atomic force microscope tip and sample surface with flexible polymer spacers. Rupture force magnitude and elastic characteristics of the spacers allowed identification of the rupture events corresponding to different numbers of interacting proteins. We used dynamic force spectroscopy to estimate the intermolecular potential widths and equivalent thermodynamic off rates for monovalent, bivalent, and trivalent interactions. Measured interaction potential parameters agree with the results of molecular docking simulation. Our results demonstrate that an increase of the interaction valency leads to a precipitous decline in the dissociation rate. Binding forces measured for monovalent and multivalent interactions match the predictions of a Markovian model for the strength of multiple uncorrelated bonds in a parallel configuration. Our approach is promising for comparison of the specific effects of molecular modifications as well as for determination of the best configuration of antibody-based multivalent targeting agents.

1,3- vs 1,5-intramolecular alkylation reactions in isophosphoramide and phosphoramide mustards.

It is well-established that at pH 7.4, intramolecular 1,3-N-alkylation reactions in isophosphoramide mustard (IPM) and phosphoramide mustard (PM) produce electrophilic alkylating agents with aziridinyl moieties. To investigate the role of 1,5-intramolecular cyclizations in the chemistry of IPM and PM, the five-membered ring phospholidine products of these reactions were independently synthesized and characterized by (31)P NMR. In 0.33 M BisTris, pH 7.4, 37 degrees C, the intramolecular O-alkylation product of IPM [2-(2-chloroethylamino)-2-tetrahydro-2H-1,3,2-oxazaphospholidine-2-oxide (11)] had a chemical shift of delta 33.0 and a half-life of 3.3 h. The O-alkylation product of PM [2-amino-3-(2-chloroethyl)tetrahydro-2H-1,3,2-oxazaphospholidine-2-oxide (12)] displayed a chemical shift of delta 30.6 and a half-life of 26.9 h. For both IPM and PM, 1,5-N-alkylation provides the same product [1-(2-chloroethyl)-2-hydroxy-tetrahydro-2H-1,3,2-diazaphospholidine-2-oxide (13)]. Because of its instability, 13 was generated in situ and was not isolated; however, the chemical shift (delta 33.0) and reactivity (half-life 0.3 h at 25 degrees C) of the species attributed to 13 were consistent with the assigned structure. Resonances with (31)P NMR chemical shifts indicative of 11 or 12 did not appear in reaction solutions of IPM or PM. The compound assigned as 13 gave hydrolysis products that were not found in reaction solutions of IPM or PM. The collective data supported the conclusion that intramolecular 1,5-alkylations do not contribute to the chemistry of IPM or PM in aqueous solutions at pH 7.4, 37 degrees C. Conversely, 11 and 12 were found to be the major if not exclusive products formed in DMSO solutions of the respective cyclohexylammonium salts of IPM and PM. Both 11 and 12 were relatively noncytotoxic against a series of cell lines, but there were differences in mutagenicities. Chinese hamster ovary cells were exposed to 11 or 12 for one half-life of each compound; 11 was nonmutagenic up to 500 microM, while 12 (500 microM) was mutagenic with 246 mutant colonies/10(6) surviving cells.