In situ process analytical technology for real time viable cell density and cell viability during live-virus vaccine production.

Viable cell density (VCD) and cell viability (CV) are key performance indicators of cell culture processes in biopharmaceutical production of biologics and vaccines. Traditional methods for monitoring VCD and CV involve offline cell counting assays that are both labor intensive and prone to high variability, resulting in sparse sampling and uncertainty in the obtained data. Process analytical technology (PAT) approaches offer a means to address these challenges. Specifically, in situ probe-based measurements of dielectric spectroscopy (also commonly known as capacitance) can characterize VCD and CV continuously in real time throughout an entire process, enabling robust process characterization. In this work, we propose in situ dielectric spectroscopy as a PAT tool for real time analysis of live-virus vaccine (LVV) production. Dielectric spectroscopy was collected across 25 discreet frequencies, offering a thorough evaluation of the proposed technology. Correlation of this PAT methodology to traditional offline cell counting assays was performed, in which VCD and CV were both successfully predicted using dielectric spectroscopy. Both univariate and multivariate data analysis approaches were evaluated for their potential to establish correlation between the in situ dielectric spectroscopy and offline measurements. Univariate analysis strategies are presented for optimal single frequency selection. Multivariate analysis, in the form of partial least squares (PLS) regression, produced significantly higher correlations between dielectric spectroscopy and offline VCD and CV data, as compared to univariate analysis. Specifically, by leveraging multivariate analysis of dielectric information from all 25 spectroscopic frequencies measured, PLS models performed significantly better than univariate models. This is particularly evident during cell death, where tracking VCD and CV have historically presented the greatest challenge. The results of this work demonstrate the potential of both single and multiple frequency dielectric spectroscopy measurements for enabling robust LVV process characterization, suggesting that broader application of in situ dielectric spectroscopy as a PAT tool in LVV processes can provide significantly improved process understanding. To the best of our knowledge, this is the first report of in situ dielectric spectroscopy with multivariate analysis to successfully predict VCD and CV in real time during live virus-based vaccine production.

In situ Raman spectroscopy for real time detection of cysteine.

Cysteine serves a wide range of important biological and chemical functions and may have an association to neurodegenerative disease and cancer. Rapid, accurate analytical methods for cysteine detection are thus highly desirable. In this work, we report an investigation into the utility of in situ Raman spectroscopy as a Process Analytical Technology (PAT) for real time monitoring of cysteine. Cysteine concentrations are tracked in real time using Raman spectroscopy across a range of pharmaceutically-relevant concentrations, demonstrating the capability of Raman spectroscopy detection for in situ cysteine monitoring. The concentration range over which this analytical methodology can be applied is successfully established. As such, the results herein serve as a proof-of-principle investigation to demonstrate and evaluate the capabilities of a real time Raman spectroscopic approach for in situ cysteine detection, thus informing the range of important chemical and biological processes to which this approach can be applied. To the best of our knowledge, this is the first report of in situ Raman spectroscopy for real time monitoring of dynamically changing cysteine process concentrations.

In situ real time monitoring of emulsification and homogenization processes for vaccine adjuvants.

Adjuvants are commonly employed to enhance the efficacy of a vaccine and thereby increase the resulting immune response in a patient. The activity and effectiveness of emulsion-based adjuvants has been heavily studied throughout pharmaceuticals; however, there exists a lack in research which monitors the formation of a stable emulsion in real time. Process analytical technology (PAT) provides a solution to meet this need. PAT involves the collection of in situ data, thereby providing real time information about the monitored process as well as increasing understanding of that process. Here, three separate PAT tools - optical particle imaging, in situ particle analysis, and Raman spectroscopy - were used to monitor two key steps involved in the formation of a stable emulsion product, emulsification and homogenization, as well as perform a stability assessment. The obtained results provided new insights-particle size decreases during emulsification and homogenization, and molecular changes do not occur during either the emulsification or homogenization steps. Further, the stability assessment indicated that the coarse emulsion product obtained from the emulsification step is stable over the course of 24 hours when mixed. To the best of our knowledge, this is the first report of an analytical methodology for in situ, real time analysis of emulsification and homogenization processes for vaccine adjuvants. Using our proposed analytical methodology, an improved understanding of emulsion-based vaccine adjuvants can now be achieved, ultimately impacting the ability to develop and deliver successful pharmaceuticals.

Process monitoring of polysaccharide deketalization for vaccine bioconjugation development using in situ analytical methodology.

Pneumococcal conjugate vaccines (PCVs) are formed by bioconjugation of a carrier protein to the purified capsular polysaccharide (Ps) from multiple serological strains of Streptococcus pneumoniae. The associated bioconjugation chemistry relies on initial selective modifications to the Ps backbone structure. Among these modifications, removal of a ketal functional group, termed deketalization, is one that is important for pharmaceutical PCV production. Herein, we report a process monitoring investigation into the deketalization of a polysaccharide relevant to PCV process development. We have applied process analytical technology (PAT) for in situ process monitoring to study the deketalization reaction in real time. We find that in situ FTIR spectroscopy elucidates multiple classes of reaction kinetics, one of which correlates strongly with the deketalization reaction of interest. This PAT approach to real time reaction monitoring offers the possibility of improved process monitoring in the pharmaceutical production of PCVs. To our knowledge, this report represents the first PAT investigation into Ps deketalization. Our findings suggest that broader application of PAT to the chemical modifications associated with PCV bioconjugation, as well as other pharmaceutically relevant bioconjugation processes, carries the power to enhance process understanding, control, and efficiency through real time process monitoring.

Utilizing in situ spectroscopic tools to monitor ketal deprotection processes.

The use of protection groups to shield a functional group during a synthesis is employed throughout many reactions and organic syntheses. The role of a protection group can be vital to the success of a reaction, as well as increase reaction yield and selectivity. Although much work has been done to investigate the addition of a protection group, the removal of the protection group is just as important - however, there is a lack of methods employed within the literature for monitoring the removal of a protection group in real time. In this work, the process of removing, or deprotecting, a ketal protecting group is investigated. Process analytical technology tools are incorporated for in situ analysis of the deprotection reaction of a small molecule model compound. Specifically, Raman spectroscopy and Fourier transform infrared spectroscopy show that characteristic bands can be used to track the decrease of the reactant and the increase of the expected products over time. To the best of our knowledge, this is the first report of process analytical technology being used to monitor a ketal deprotection reaction in real time. This information can be capitalized on in the future for understanding and optimizing pharmaceutically-relevant deprotection processes and downstream reactions.

A Different hIAPP Polymorph Is Observed in Human Serum Than in Aqueous Buffer: Demonstration of a New Method for Studying Amyloid Fibril Structure Using Infrared Spectroscopy.

There is enormous interest in measuring amyloid fibril structures, but most structural studies measure fibril formation in vitro using aqueous buffer. Ideally, one would like to measure fibril structure and mechanism under more physiological conditions. Toward this end, we have developed a method for studying amyloid fibril structure in human serum. Our approach uses isotope labeling, antibody depletion of the most abundant proteins (albumin and IgG), and infrared spectroscopy to measure aggregation in human serum with reduced protein content. Reducing the nonamyloid protein content enables the measurements by decreasing background signals but retains the full composition of salts, sugars, metal ions, etc. that are naturally present but usually missing from in vitro studies. We demonstrate the method by measuring the two-dimensional infrared (2D IR) spectra of isotopically labeled human islet amyloid polypeptide (hIAPP or amylin). We find that the fibril structure of hIAPP formed in serum differs from that formed via aggregation in aqueous buffer at residues Gly24 and Ala25, which reside in the putative "amyloidogenic core" or FGAIL region of the sequence. The spectra are consistent with extended parallel stacks of strands consistent with ß-sheet-like structure, rather than a partially disordered loop that forms in aqueous buffer. These experiments provide a new method for using infrared spectroscopy to monitor the structure of proteins under physiological conditions and reveal the formation of a significantly different polymorph structure in the most important region of hIAPP.

Monolayer Sensitivity Enables a 2D IR Spectroscopic Immuno-biosensor for Studying Protein Structures: Application to Amyloid Polymorphs.

Immunosensors use antibodies to detect and quantify biomarkers of disease, though the sensors often lack structural information. We create a surface-sensitive two-dimensional infrared (2D IR) spectroscopic immunosensor for studying protein structures. We tether antibodies to a plasmonic surface, flow over a solution of amyloid proteins, and measure the 2D IR spectra. The 2D IR spectra provide a global assessment of antigen structure, and isotopically labeled proteins give residue-specific structural information. We report the 2D IR spectra of fibrils and monomers using a polyclonal antibody that targets human islet amyloid polypeptide (hIAPP). We observe two fibrillar polymorphs differing in their structure at the G24 residue, which supports the hypothesis that hIAPP polymorphs form from a common oligomeric intermediate. This work provides insight into the structure of hIAPP, establishes a new method for studying protein structures using 2D IR spectroscopy, and creates a spectroscopic immunoassay applicable for studying a wide range of biomarkers.

Enhancing the signal strength of surface sensitive 2D IR spectroscopy.

Spectroscopic techniques that are capable of measuring surfaces and interfaces must overcome two technical challenges: one, the low coverage of molecules at the surface, and two, discerning between signals from the bulk and surface. We present surface enhanced attenuated reflection 2D infrared (SEAR 2D IR) spectroscopy, a method that combines localized surface plasmons with a reflection pump-probe geometry to achieve monolayer sensitivity. The method is demonstrated at 6 µm with the amide I band of a model peptide, a cysteine terminated α-helical peptide tethered to a gold surface. Using SEAR 2D IR spectroscopy, the signal from this sample is enhanced 20 000-times over a monolayer on a dielectric surface. Like attenuated total reflection IR spectroscopy, SEAR 2D IR spectroscopy can be applied to strongly absorbing solvents. We demonstrated this capability by solvating a peptide monolayer with H2O, which cannot normally be used when measuring the amide I band. SEAR 2D IR spectroscopy will be advantageous for studying chemical reactions at electrochemical surfaces, interfacial charge transfer in photovoltaics, and structural changes of transmembrane proteins in lipid membranes.

Site-specific detection of protein secondary structure using 2D IR dihedral indexing: a proposed assembly mechanism of oligomeric hIAPP.

Human islet amyloid polypeptide (hIAPP) aggregates into fibrils through oligomers that have been postulated to contain α-helices as well as ß-sheets. We employ a site-specific isotope labeling strategy that is capable of detecting changes in dihedral angles when used in conjunction with 2D IR spectroscopy. The method is analogous to the chemical shift index used in NMR spectroscopy for assigning protein secondary structure. We introduce isotope labels at two neighbouring residues, which results in an increased intensity and positive frequency shift if those residues are α-helical versus a negative frequency shift in ß-sheets and turns. The 2D IR dihedral index approach is demonstrated for hIAPP in micelles for which the polypeptide structure is known, using pairs of 13C18O isotope labels L12A13 and L16V17, along with single labeled control experiments. Applying the approach to aggregation experiments performed in buffer, we show that about 27-38% of hIAPP peptides adopt an α-helix secondary structure in the monomeric state at L12A13, prior to aggregation, but not at L16V17 residues. At L16V17, the kinetics are described solely by the monomer and fiber conformations, but at L12A13 the kinetics exhibit a third state that is created by an oligomeric intermediate. Control experiments performed with a single isotope label at A13 exhibit two-state kinetics, indicating that a previously unknown change in dihedral angle occurs at L12A13 as hIAPP transitions from the intermediate to fiber structures. We propose a mechanism for aggregation, in which helices seed oligomer formation via structures analogous to leucine rich repeat proteins.

Two-Dimensional Spectroscopy Is Being Used to Address Core Scientific Questions in Biology and Materials Science.

Two-dimensional spectroscopy is a powerful tool for extracting structural and dynamic information from a wide range of chemical systems. We provide a brief overview of the ways in which two-dimensional visible and infrared spectroscopies are being applied to elucidate fundamental details of important processes in biological and materials science. The topics covered include amyloid proteins, photosynthetic complexes, ion channels, photovoltaics, batteries, as well as a variety of promising new methods in two-dimensional spectroscopy.

Spectroscopic Signature for Stable ß-Amyloid Fibrils versus ß-Sheet-Rich Oligomers.

We use two-dimensional IR (2D IR) spectroscopy to explore fibril formation for the two predominant isoforms of the ß-amyloid (Aß1-40 and Aß1-42) protein associated with Alzheimer's disease. Two-dimensional IR spectra resolve a transition at 1610 cm-1 in Aß fibrils that does not appear in other Aß aggregates, even those with predominantly ß-sheet-structure-like oligomers. This transition is not resolved in linear IR spectroscopy because it lies under the broad band centered at 1625 cm-1, which is the traditional infrared signature for amyloid fibrils. The feature is prominent in 2D IR spectra because 2D lineshapes are narrower and scale nonlinearly with transition dipole strengths. Transmission electron microscopy measurements demonstrate that the 1610 cm-1 band is a positive identification of amyloid fibrils. Sodium dodecyl sulfate micelles that solubilize and disaggregate preaggregated Aß samples deplete the 1625 cm-1 band but do not affect the 1610 cm-1 band, demonstrating that the 1610 cm-1 band is due to very stable fibrils. We demonstrate that the 1610 cm-1 transition arises from amide I modes by mutating out the only side-chain residue that could give rise to this transition, and we explore the potential structural origins of the transition by simulating 2D IR spectra based on Aß crystal structures. It was not previously possible to distinguish stable Aß fibrils from the less stable ß-sheet-rich oligomers with infrared light. This 2D IR signature will be useful for Alzheimer's research on Aß aggregation, fibril formation, and toxicity.

A Free Energy Barrier Caused by the Refolding of an Oligomeric Intermediate Controls the Lag Time of Amyloid Formation by hIAPP.

Transiently populated oligomers formed en route to amyloid fibrils may constitute the most toxic aggregates associated with many amyloid-associated diseases. Most nucleation theories used to describe amyloid aggregation predict low oligomer concentrations and do not take into account free energy costs that may be associated with structural rearrangements between the oligomer and fiber states. We have used isotope labeling and two-dimensional infrared spectroscopy to spectrally resolve an oligomeric intermediate during the aggregation of the human islet amyloid protein (hIAPP or amylin), the protein associated with type II diabetes. A structural rearrangement includes the F23G24A25I26L27 region of hIAPP, which starts from a random coil structure, evolves into ordered ß-sheet oligomers containing at least 5 strands, and then partially disorders in the fibril structure. The supercritical concentration is measured to be between 150 and 250 µM, which is the thermodynamic parameter that sets the free energy of the oligomers. A 3-state kinetic model fits the experimental data, but only if it includes a concentration independent free energy barrier >3 kcal/mol that represents the free energy cost of refolding the oligomeric intermediate into the structure of the amyloid fibril; i.e., "oligomer activation" is required. The barrier creates a transition state in the free energy landscape that slows fibril formation and creates a stable population of oligomers during the lag phase, even at concentrations below the supercritical concentration. Largely missing in current kinetic models is a link between structure and kinetics. Our experiments and modeling provide evidence that protein structural rearrangements during aggregation impact the populations and kinetics of toxic oligomeric species.

Not All ß-Sheets Are the Same: Amyloid Infrared Spectra, Transition Dipole Strengths, and Couplings Investigated by 2D IR Spectroscopy.

We report the transition dipole strengths and frequencies of the amyloid ß-sheet amide I mode for the aggregated proteins amyloid-ß1-40, calcitonin, α-synuclein, and glucagon. According to standard vibrational coupling models for proteins, the frequencies of canonical ß-sheets are set by their size and structural and environmental disorder, which determines the delocalization length of the vibrational excitons. The larger the delocalization the lower the frequency of the main infrared-allowed transition, A⊥. The models also predict an accompanying increase in transition dipole strength. For the proteins measured here, we find no correlation between transition dipole strengths and amyloid ß-sheet transition frequency. To understand this observation, we have extracted from the protein data bank crystal structures of amyloid peptides from which we calculate the amide I vibrational couplings, and we use these in a model ß-sheet Hamiltonian to simulate amyloid vibrational spectra. We find that the variations in amyloid ß-sheet structures (e.g., dihedral angles, interstrand distances, and orientations) create significant differences in the average values for interstrand and nearest neighbor couplings, and that those variations encompass the variation in measured A⊥ frequencies. We also find that off-diagonal disorder about the average values explains the range of transition dipole strengths observed experimentally. Thus, we conclude that the lack of correlation between transition dipole-strength and frequency is caused by variations in amyloid ß-sheet structure. Taken together, these results indicate that the amide I frequency is very sensitive to amyloid ß-sheet structure, the ß-sheets of these 4 proteins are not identical, and the assumption that frequency of amyloids scales with ß-sheet size cannot be adopted without an accompanying measurement of transition dipole strengths.

Study of Heat Transfer Dynamics from Gold Nanorods to the Environment via Time-Resolved Infrared Spectroscopy.

Studying the local solvent surrounding nanoparticles is important to understanding the energy exchange dynamics between the particles and their environment, and there is a need for spectroscopic methods that can dynamically probe the solvent region that is in nearby contact with the nanoparticles. In this work, we demonstrate the use of time-resolved infrared spectroscopy to track changes in a vibrational mode of local water on the time scale of hundreds of picoseconds, revealing the dynamics of heat transfer from gold nanorods to the local water environment. We applied this probe to a prototypical plasmonic photothermal system consisting of organic CTAB bilayer capped gold nanorods, as well as gold nanorods coated with varying thicknesses of inorganic mesoporous-silica. The heat transfer time constant of CTAB capped gold nanorods is about 350 ps and becomes faster with higher laser excitation power, eventually generating bubbles due to superheating in the local solvent. Silica coating of the nanorods slows down the heat transfer and suppresses the formation of superheated bubbles.

Electron Dynamics and IR Peak Coalescence in Bridged Mixed Valence Dimers Studied by Ultrafast 2D-IR Spectroscopy.

Dynamic IR peak coalescence and simulations based on the optical Bloch equations have been used previously to predict the rates of intramolecular electron transfer in a group of bridged mixed valence dimers of the type [Ru3(O)(OAc)6(CO)L]-BL-[Ru3(O) (OAc)6(CO)L]. However, limitations of the Bloch equations for the analysis of dynamical coalescence in vibrational spectra have been described. We have used ultrafast 2D-IR spectroscopy to investigate the vibrational dynamics of the CO spectator ligands of several dimers in the group. These experiments reveal that no electron site exchange occurs on the time scale required to explain the observed peak coalescence. The high variability in FTIR peak shapes for these mixed valence systems is suggested to be the result of fluctuations in the charge distributions at each metal cluster within a single-well potential energy surface, rather than the previous model of two-site exchange.

Vibrational cooling dynamics of a [FeFe]-hydrogenase mimic probed by time-resolved infrared spectroscopy.

Picosecond time-resolved infrared spectroscopy (TRIR) was performed for the first time on a dithiolate bridged binuclear iron(I) hexacarbonyl complex ([Fe2(µ-bdt)(CO)6], bdt = benzene-1,2-dithiolate) which is a structural mimic of the active site of the [FeFe]-hydrogenase enzyme. As these model active sites are increasingly being studied for their potential in photocatalytic systems for hydrogen production, understanding their excited and ground state dynamics is critical. In n-heptane, absorption of 400 nm light causes carbonyl loss with low quantum yield (<10%), while the majority (ca. 90%) of the parent complex is regenerated with biexponential kinetics (τ1 = 21 ps and τ2 = 134 ps). In order to understand the mechanism of picosecond bleach recovery, a series of UV-pump TRIR experiments were performed in different solvents. The long time decay (τ2) of the transient spectra is seen to change substantially as a function of solvent, from 95 ps in THF to 262 ps in CCl4. Broadband IR-pump TRIR experiments were performed for comparison. The measured vibrational lifetimes (T1(avg)) of the carbonyl stretches were found to be in excellent correspondence to the observed τ2 decays in the UV-pump experiments, signifying that vibrationally excited carbonyl stretches are responsible for the observed longtime decays. The fast spectral evolution (τ1) was determined to be due to vibrational cooling of low frequency modes anharmonically coupled to the carbonyl stretches that were excited after electronic internal conversion. The results show that cooling of both low and high frequency vibrational modes on the electronic ground state give rise to the observed picosecond TRIR transient spectra of this compound, without the need to invoke electronically excited states.

Exploring the potential of fulvalene dimetals as platforms for molecular solar thermal energy storage: computations, syntheses, structures, kinetics, and catalysis.

A study of the scope and limitations of varying the ligand framework around the dinuclear core of FvRu2 in its function as a molecular solar thermal energy storage framework is presented. It includes DFT calculations probing the effect of substituents, other metals, and CO exchange for other ligands on ΔHstorage . Experimentally, the system is shown to be robust in as much as it tolerates a number of variations, except for the identity of the metal and certain substitution patterns. Failures include 1,1',3,3'-tetra-tert-butyl (4), 1,2,2',3'-tetraphenyl (9), diiron (28), diosmium (24), mixed iron-ruthenium (27), dimolybdenum (29), and ditungsten (30) derivatives. An extensive screen of potential catalysts for the thermal reversal identified AgNO3 -SiO2 as a good candidate, although catalyst decomposition remains a challenge.

Ultrafast infrared studies of the role of spin states in organometallic reaction dynamics.

The importance of spin state changes in organometallic reactions is a topic of significant interest, as an increasing number of reaction mechanisms involving changes of spin state are consistently being uncovered. The potential influence of spin state changes on reaction rates can be difficult to predict, and thus this class of reactions remains among the least well understood in organometallic chemistry. Ultrafast time-resolved infrared (TRIR) spectroscopy provides a powerful tool for probing the dynamics of spin state changes in organometallic catalysis, as such processes often occur on the picosecond to nanosecond time scale and can readily be monitored in the infrared via the absorptions of carbonyl reporter ligands. In this Account, we summarize recent work from our group directed toward identifying trends in reactivity that can be used to offer predictive insight into the dynamics of coordinatively unsaturated organometallic reaction intermediates. In general, coordinatively unsaturated 16-electron (16e) singlets are able to coordinate to solvent molecules as token ligands to partially stabilize the coordinatively unsaturated metal center, whereas 16e triplets and 17-electron (17e) doublets are not, allowing them to diffuse more rapidly through solution than their singlet counterparts. Triplet complexes typically (but not always) undergo spin crossover prior to solvent coordination, whereas 17e doublets do not coordinate solvent molecules as token ligands and cannot relax to a lower spin state to do so. 16e triplets are typically able to undergo facile spin crossover to yield a 16e singlet where an associative, exothermic reaction pathway exists. The combination of facile spin crossover with faster diffusion through solution for triplets can actually lead to faster catalytic reactivity than for singlets, despite the forbidden nature of these reactions. We summarize studies on odd-electron complexes in which 17e doublets were found to display varying behavior with regard to their tendency to react with 2-electron donor ligands to form 19-electron (19e) adducts. The ability of 19e adducts to serve as reducing agents in disproportionation reactions depends on whether the excess electron density localized at the metal center or at a ligand site. The reactivity of both 16e and 17e complexes toward a widely used organic nitroxyl radical (TEMPO) are reviewed, and both classes of complexes generally react similarly via an associative mechanism with a low barrier to these reactions. We also describe recent work targeted at unraveling the photoisomerization mechanism of a thermal-solar energy storage complex in which spin state changes were found to play a crucial role. Although a key triplet intermediate was found to be required for this photoisomerization mechanism to proceed, the details of why this triplet is formed in some complexes (those based on ruthenium) and not others (those based on iron, molybdenum, or tungsten) remains uncertain, and further exploration in this area may lead to a better understanding of the factors that influence intramolecular and excited state spin state changes.

Studying the Dynamics of Photochemical Reactions via Ultrafast Time-Resolved Infrared Spectroscopy of the Local Solvent.

Conventional ultrafast spectroscopic studies on the dynamics of chemical reactions in solution directly probe the solute undergoing the reaction. We provide an alternative method for probing reaction dynamics via monitoring of the surrounding solvent. When the reaction exchanges the energy (in form of heat) with the solvent, the absorption cross sections of the solvent's infrared bands are sensitive to the heat transfer, allowing spectral tracking of the reaction dynamics. This spectroscopic technique was demonstrated to be able to distinguish the differing photoisomerization dynamics of the trans and cis isomers of stilbene in acetonitrile solution. We highlight the potential of this spectroscopic approach for studying the dynamics of chemical reactions or other heat transfer processes when probing the solvent is more experimentally feasible than probing the solute directly.

Reactivity of TEMPO toward 16- and 17-electron organometallic reaction intermediates: a time-resolved IR study.

The (2,2,6,6-tetramethylpiperidin-1-yl)oxyl radical (TEMPO) has been employed for an extensive range of chemical applications, ranging from organometallic catalysis to serving as a structural probe in biological systems. As a ligand in an organometallic complex, TEMPO can exhibit several distinct coordination modes. Here we use ultrafast time-resolved infrared spectroscopy to study the reactivity of TEMPO toward coordinatively unsaturated 16- and 17-electron organometallic reaction intermediates. TEMPO coordinates to the metal centers of the 16-electron species CpCo(CO) and Fe(CO)4, and to the 17-electron species CpFe(CO)2 and Mn(CO)5, via an associative mechanism with concomitant oxidation of the metal center. In these adducts, TEMPO thus behaves as an anionic ligand, characterized by a pyramidal geometry about the nitrogen center. Density functional theory calculations are used to facilitate interpretation of the spectra and to further explore the structures of the TEMPO adducts. To our knowledge, this study represents the first direct characterization of the mechanism of the reaction of TEMPO with coordinatively unsaturated organometallic complexes, providing valuable insight into its reactions with commonly encountered reaction intermediates. The similar reactivity of TEMPO toward each of the species studied suggests that these results can be considered representative of TEMPO's reactivity toward all low-valent transition metal complexes.