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Chemical exchange saturation transfer (CEST) and magnetization transfer techniques provide unique and potentially quantitative contrast mechanisms in multiple MRI applications. However, the in vivo implementation of these techniques has been limited by the relatively slow MRI acquisition techniques, especially on high-field MRI scanners. A new, rapid CEST-fast imaging with steady-state free precession technique was developed to provide sensitive CEST contrast in ∼20 sec. In this study at 7 T with in vitro bovine glycogen samples and initial in vivo results in a rat liver, the CEST-fast imaging with steady-state free precession technique was shown to provide equivalent CEST sensitivity in comparison to a conventional CEST-spin echo acquisition with a 50-fold reduction in acquisition time. The sensitivity of the CEST-fast imaging with steady-state free precession technique was also shown to be dependent on k-space encoding with centric k-space encoding providing a 30-40% increase in CEST sensitivity relative to linear encoding for 256 or more k-space lines. Overall, the CEST-fast imaging with steady-state free precession acquisition technique provides a rapid and sensitive imaging platform with the potential to provide quantitative CEST and magnetization transfer imaging data.

Responsive magnetic resonance imaging (MRI) contrast agents can change MR image contrast in response to a molecular biomarker. Quantitative detection of the biomarker requires an accounting of the other effects that may alter MR image contrast, such as a change in the agent’s concentration, magnetic field variations, and hardware sensitivity profiles. A second unresponsive MRI contrast agent may serve as an “internal control” to isolate the detection of the molecular biomarker. Chemical exchange saturation transfer (CEST) MRI contrast agents can be selectively detected, providing the opportunity to combine a responsive CEST agent and an unresponsive CEST agent during the same MRI scan session. When two CEST MRI contrast agents are used for molecular imaging applications, the CEST agents should be designed to maximize accurate quantification of the concentrations of the two agents. From a chemical perspective, CEST agents behave like enzymes that catalyze the conversion of an unsaturated water “substrate” ...

Responsive magnetic resonance imaging (MRI) contrast agents can change MR image contrast in response to a molecular biomarker. Quantitative detection of the biomarker requires an accounting of the other effects that may alter MR image contrast, such as a change in the agent's concentration, magnetic field variations, and hardware sensitivity profiles. A second unresponsive MRI contrast agent may serve as an "internal control" to isolate the detection of the molecular biomarker. Chemical exchange saturation transfer (CEST) MRI contrast agents can be selectively detected, providing the opportunity to combine a responsive CEST agent and an unresponsive CEST agent during the same MRI scan session. When two CEST MRI contrast agents are used for molecular imaging applications, the CEST agents should be designed to maximize accurate quantification of the concentrations of the two agents. From a chemical perspective, CEST agents behave like enzymes that catalyze the conversion of an unsaturated water "substrate" into a saturated water "product". The analysis of CEST agent kinetics parallels the Michaelis-Menten analysis of enzyme kinetics, which can be used to correlate the CEST effect with the concentration of the agent in solution. If the concentration of water "substrate" that is available to the CEST agent is unknown, which may be likely for in vivo MRI studies, then only a ratio of concentrations of the two CEST agents can be measured. In both cases, CEST agents should be designed with minimal T(1) relaxivity to improve concentration quantifications. CEST agents can also be designed to maximize sensitivity. This may be accomplished by incorporating many CEST agents within nanoparticles to create a large number of exchangeable protons per nanoparticle. Finally, CEST agents can be designed with rapid detection in mind. This may be accomplished by minimizing T(1) relaxivity of the CEST agent so that MRI acquisition methods have time to collect many MRI signals following a single selective saturation period. In this Account, we provide an example that shows the sensitive and rapid detection of two CEST agents in an in vivo MRI study of a mouse model of mammary carcinoma. The ratio of the concentrations of the two CEST agents was quantified with analysis methods that parallel Michaelis-Menten enzyme kinetic analysis. This example demonstrates current limitations of the method that require additional research, but it also shows that two CEST MRI contrast agents can be detected and quantitatively assessed during in vivo molecular imaging studies.

PARAmagnetic Chemical Exchange Saturation Transfer (PARACEST) is a novel contrast mechanism for MRI. A PARACEST MRI methodology with high temporal resolution is highly desired for in vivo MRI applications of molecular imaging. To address this need, a strategy has been developed that includes a long selective saturation period before each repetition of a Rapid Acquisition with Relaxation Enhancement (RARE) pulse sequence. This strategy is suitable for the application of PARACEST contrast agents to environments with long T1 relaxation times. An alternative strategy uses short selective saturation periods before the acquisition of each k-space trajectory to maintain steady state conditions, which can be implemented with a Fast Low Angle Shot (FLASH) pulse sequence. These short saturation periods lengthen the total scan time as compared to the first approach but compensate for the loss in PARACEST contrast related to T1 relaxation. Both approaches have been demonstrated in vitro and in vivo with significantly improved temporal resolutions as compared to a conventional gradient-echo PARACEST method without sacrificing CNR efficiency. These demonstrations also adopted a strategy for measuring the PARACEST effect that only requires selective saturation at a single MR frequency, which further improves temporal resolution for PARACEST detection.

A noninvasive assay that tracks the relative in vivo pharmacokinetics of two nanoparticles may accelerate the development of nanoparticles for biomedical applications, and may provide a method to select personalized nanomedicines for individual patients. To develop an in vivo competitive assay, two MRI contrast agents that could be selectively detected through paramagnetic chemical exchange saturation transfer (PARACEST) were conjugated to a second generation and fifth generation polyamidoamine (PAMAM) dendrimer. The CEST effects of each agent was calibrated relative to concentration. The effects of T(1) relaxivities of these dendritic PARACEST magnetic resonance imaging (MRI) contrast agents were found to be negligible relative to their CEST effects with respect to changes in image contrast, which facilitated the measurement of the ratios of their chemical exchange lifetimes. Injection of both contrast agents into a mouse model of mammary carcinoma resulted in a temporal increase in the CEST effect from each agent in the flank tumor. Although the in vivo CEST effects could not be used to determine the absolute concentrations of each agent within the tumor, the ratio of the in vivo CEST effects was used to measure the ratio of the concentrations of the agents. This result demonstrated that the relative in vivo pharmacokinetics of two nanoparticles may be evaluated using PARACEST MRI.

The use of 2D homonuclear J-resolved 1H NMR spectroscopy in the examination of the methine region of poly(cyclohexene oxide) formed by polymerization of cyclohexene oxide employing the Union Carbide calcium−amide−alkoxide catalyst reveals that the majority of the ring opening occurs by backside attack.

Purpose To evaluate magnetic resonance imaging (MRI) in assessing lung inflammation longitudinally in genetic mouse models of cystic fibrosis (CF). MRI is used to view soft tissues noninvasively, but the lung is challenging to image. Materials and Methods Cftr+/+ (wildtype) and Cftr−/− (CF) mice were inoculated with agarose beads laden with Pseudomonas aeruginosa. Longitudinal MR lung images were acquired with cardiac gating. The effects of echo time and respiration gating were evaluated to improve the detection of lung inflammation. Results Cardiac gating and signal averaging sufficiently suppressed motion artifacts without requiring respiration gating. MRI detected moderate to severe inflammation in infected mice, which was confirmed by histology results. Conclusion In vivo longitudinal MRI methods can assess lung inflammation in P. aeruginosa-infected mice, which obviates serial sacrifice. MRI was able to detect inflammation in the absence of other physiological symptoms. J. Magn. Reson. Imaging 2008;28:527–532. © 2008 Wiley-Liss, Inc.

Irreversible responsive PARAmagnetic Chemical Exchange Saturation Transfer (PARACEST) MRI contrast agents constitute a new type of agent for molecular imaging. To investigate the utility of this approach, a novel PARACEST MRI contrast agent, Yb(III)-(1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid)-orthoaminoanilide (Yb-DO3A-oAA), was developed that detects nitric oxide (NO). The agent exhibited two CEST effects at -11 ppm and +8 ppm, which were assigned to chemical exchange from amide and amine functional groups, respectively. The effects of pH, temperature, and concentration were investigated to characterize the complex and to optimize PARACEST detection. This responsive PARACEST MRI contrast agent incurred an irreversible covalent change in the presence of NO and O(2), which caused an irreversible disappearance of both PARACEST effects from MR images. The NO-dependent response of a relaxivity-based MRI contrast agent, Gd-DO3A-oAA, was investigated for comparison. This report highlights the advantages of irreversible MRI contrast agents, demonstrates that large changes in PARACEST can be used to create a highly responsive agent, and indicates challenges that must be overcome to apply this type of contrast agent to in vivo biomedical applications in molecular imaging.

A versatile method is disclosed for solid phase peptide synthesis (SPPS) of molecular imaging contrast agents. A DO3A moiety was derivatized to introduce a CBZ-protected amino group and then coupled to a polymeric support. CBZ cleavage with Et2AlCl/thioanisole was optimized for SPPS. Amino acids were then coupled to the aminoDOTA loaded resin using conventional step-wise Fmoc SPPS to create a product with DOTA coupled to the C-terminus of the peptide. In a second study, the DO3A moiety was coupled to a glycine-loaded polymeric support, and amino acids were then coupled to the amino-DOTA-peptide loaded resin using SPPS, to incorporate DOTA within the peptide sequence. The peptide-(Tm3+-DOTA) amide showed a PARAmagnetic Chemical Exchange Saturation Transfer (PARACEST) effect, which demonstrated the utility of this contrast agent for molecular imaging. These results demonstrate the advantages of exploiting SPPS methodologies through the development of unique DOTA derivatives to create peptide-based molecular imaging contrast agents.

Two relatively new types of exogenous magnetic resonance imaging contrast agents may provide greater impact for molecular imaging by providing greater specificity for detecting molecular imaging biomarkers. Exogenous chemical exchange saturation transfer (CEST) agents rely on the selective saturation of the magnetization of a proton on an agent, followed by chemical exchange of a proton from the agent to water. The selective detection of a biomarker-responsive CEST signal and an unresponsive CEST signal, followed by the ratiometric comparison of these signals, can improve biomarker specificity. We refer to this improvement as a "double-agent" approach to molecular imaging. Exogenous T2-exchange agents also rely on chemical exchange of protons between the agent and water, especially with an intermediate rate that lies between the slow exchange rates of CEST agents and the fast exchange rates of traditional T1 and T2 agents. Because of this intermediate exchange rate, these agents have been relatively unknown and have acted as "secret agents" in the contrast agent research field. This review exposes these secret agents and describes the merits of double agents through examples of exogenous agents that detect enzyme activity, nucleic acids and gene expression, metabolites, ions, redox state, temperature, and pH. Future directions are also provided for improving both types of contrast agents for improved molecular imaging and clinical translation. Therefore, this review provides an overview of two new types of exogenous contrast agents that are becoming useful tools within the armamentarium of molecular imaging.

Introduction: Breast cancer is one of the most common malignancies affecting women in the United States and Europe. Approximately three out of every four women with breast cancer develop metastases in bone which, in turn, diminishes quality of life. The αvβ3 integrin has previously been implicated in multiple aspects of tumor progression, metastasis and osteoclast bone resorption. Therefore, we hypothesized that the αvβ3-selective inhibitor, S247, would decrease the development of osteolytic breast cancer metastases. Materials and methods: Cells were treated in vitro with S247 and assessed for viability and adhesion to matrix components. Athymic mice received intracardiac (left ventricle) injections of human MDA-MB-435 breast carcinoma cells expressing enhanced green-fluorescent protein. Mice were treated with vehicle (saline) or S247 (1, 10, or 100 mg/kg/d) using osmotic pumps beginning either one week before or one week after tumor cell inoculation. Bones were removed and examined by fluorescence microscopy and histology. The location and size of metastases were recorded. Results and conclusions: IC50 for S247 adhesion to αvβ3 or αIIBβ3a substrates was 0.2 nM vs. 244 nM, respectively. Likewise, S247 was not toxic at doses up to 1000 μM. However, osteoclast cultures treated with S247 exhibited marked morphological changes and impaired formation of the actin sealing zone. When S247 was administered prior to tumor cells, there was a significant, dose-dependent reduction (25–50% of vehicle-only-treated mice; P=0.002) in osseous metastasis. Mice receiving S247 after tumor cell inoculation also developed fewer bone metastases, but the difference was not statistically significant. These data suggest that, in the MDA-MB-435 model, the αvβ3 integrin plays an important role in early events (e.g., arrest of tumor cells) in bone metastasis. Furthermore, the data suggest that αvβ3 inhibitors may be useful in the treatment and/or prevention of breast cancer metastases in bone.

Previous work has demonstrated that selective cyclooxygenase-2 (COX-2) inhibitors can act synergistically with radiotherapy to improve tumor debulking and control in preclinical models. The underlying mechanism of this remarkable activity has not yet been determined. Here, we report that radiation can elevate intratumoral levels of COX-2 protein and its products, particularly prostaglandin E2 (PGE2). Furthermore, inhibition of COX-2 activity or neutralization of PGE2 activity enhances radiotherapy even in tumors where COX-2 expression is restricted to the tumor neovasculature. Direct assessment of vascular function by direct contrast enhancement-magnetic resonance imaging showed that the combination of radiation and celecoxib lead to enhanced vascular permeability. These observations suggest that an important mechanism of celecoxib-induced radiosensitization involves inhibition of COX-2-derived PGE2, thus removing a survival factor for the tumor and its vasculature.

Introduction: The purpose of the present study is to examine whether tumor extracellular pH (pHe) is a sensitive and specific biomarker for measuring immediate, short term drug response to an mTOR inhibiting agent (RAD001, everolimus) by a novel, non-invasive MRI-based pH imaging method (acidoCEST). Methods: Two groups of SCID mice with Granta 519 human mantle cell lymphoma flank xenograft models were used in the study. First, to investigate if RAD001 has anti-tumor activity in the Granta 519 model, mice were injected IP with 5 mg/kg per day with RAD001 or vehicle (DMSO) (N=12 per group) every day until tumors reached 2000 mm3. Survival analysis was conducted using GraphPad software. In a separate correlative imaging study mice with tumors averaging 400 mm3 (N=8) were scanned with acidoCEST MRI pretreatment, one day post initial treatment, and one week after continuous treatment. To prepare for each acidoCEST MRI scan session, a mouse was anesthetized with 1.5% isoflurane, respiration rate and body temperature were monitored, and body temperature was maintained at 37.0᠑C with warm air. A bolus of 200 μL of 370 mgI/mL iopamidol was injected IV prior to scanning, followed by continuous infusion of iopamidol at 150 uL/hour during scanning. A CEST-FISP MRI protocol was performed with a Bruker Biospec 7T MRI scanner with a 72 mm volume coil using previously published procedures. The results were analyzed using Matlab, following previously published procedures. The average pHe was determined for each tumor. Pixels which registered a pHe of 6.00 to 6.99 were grouped together as the “acidic fraction,” and those pixels with pH ≥7 were collectively grouped together as the “neutral fraction.” The percent uptake of the contrast agent in the tumor area was measured to assess vascular perfusion in the tumor microenvironment. Results: RAD001 treatment results in a significant survival advantage in Granta 519 flank xenograft tumors. AcidoCEST imaging of tumors showed pretreatment an average pHe value of 6.82, demonstrating that the tumor model was moderately acidic. The standard deviation of pixelwise pHe values was 0.24, indicating moderate heterogeneity of pHe in the measured tumor area. Post initial treatment, the pHe had a statistically significant increase to 6.92 with a standard deviation of 0.14, indicating a decrease in average acidosis and a decrease in heterogeneity of acidosis. After continuous treatment, the pHe dropped to 6.74 with a standard deviation of 0.26, suggesting a return to a more normal metabolic rate. In keeping with the trend, the acidic fraction of the tumor decreased from 33.1% to 19.6%, before increasing to 49% during this study. The tumor growth study showed a temporary growth delay before returning to a more normal growth rate, which paralleled the acidoCEST MRI results. Conclusion: Tumor pHe measured with acidoCEST MRI can detect early response to RAD001. Additional pre-clinical studies are warranted to determine if this biomarker is a robust measure of early therapeutic response. In addition, translation of acidoCEST MRI to the clinic should be pursued to provide this biomarker for clinical studies. Citation Format: Paul J. Akhenblit, Christine M. Howison, Scott W. Malm, Liu Qi Chen, Amanda F. Baker, Mark D. Pagel. Monitoring early therapeutic response by measuring extracellular pH in a tumor model with acidoCEST MRI. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-161. doi:10.1158/1538-7445.AM2014-LB-161

Dynamic MR biomarkers (T2*-weighted or susceptibility-based and T1-weighted or relaxivity-enhanced) have been applied to assess tumor perfusion and its response to therapies. A significant challenge in the development of reliable biomarkers is a rigorous assessment and optimization of reproducibility. The purpose of this study was to determine the measurement reproducibility of T1-weighted dynamic contrast-enhanced (DCE)-MRI and T2*-weighted dynamic susceptibility contrast (DSC)-MRI with two contrast agents (CA) of different molecular weight (MW): gadopentetate (Gd-DTPA, 0.5 kDa) and Gadomelitol (P792, 6.5 kDa). Each contrast agent was tested with eight mice that had subcutaneous MDA-MB-231 breast xenograft tumors. Each mouse was imaged with a combined DSC-DCE protocol three times within one week to achieve measures of reproducibility. DSC-MRI results were evaluated with a contrast to noise ratio (CNR) efficiency threshold. There was a clear signal drop (>95% probability threshold) in the DSC of normal tissue, while signal changes were minimal or non-existent (

Perbenzoylated sialooligosaccharides were found to be stable derivatives, giving intense signals during the matrix-assisted laser desorption/ionization (MALDI) mass spectrometric analysis in the positive-ion mode. Terminal Neu5NAc alpha 2--3 and alpha 2--6Gal units of oligosaccharides undergo characteristic structural changes during benzoylation, yielding easily recognizable mass spectral patterns. Subpicomole carbohydrate samples were successfully benzoylated and analyzed through MALDI mass spectrometry.

The interactions between the mouse major urinary protein isoform MUP-I and the pheromone 2-sec-butyl-4,5-dihydrothiazole have been characterized in solution. (15)N-labeled and (15)N, (13)C-doubly-labeled recombinant MUP-I were produced in a bacterial expression system and purified to homogeneity. Racemic 2-sec-butyl-4, 5-dihydrothiazole was produced synthetically. An equilibrium diffusion assay and NMR titration revealed that both enantiomers of the pheromone bind to the recombinant protein with a stoichiometry of 1 equiv of protein to 1 equiv of racemic pheromone. A micromolar dissociation constant and slow-exchange regime dissociation kinetics were determined for the pheromone-protein complex. (1)H, (15)N, and (13)C chemical shifts of MUP-I were assigned using triple resonance and (15)N-correlated 3D NMR experiments. Changes in protein (1)H(N) and (15)N(H) chemical shifts upon addition of pheromone were used to identify the ligand binding site. Several amide signals, corresponding to residues on one side of the binding site, were split into two peaks in the saturated protein-ligand complex. Similarly, two overlapping ligand spin systems were present in isotope-filtered NMR spectra of labeled protein bound to unlabeled pheromone. The two sets of peaks were attributed to the two possible chiralities of the pheromone. Intermolecular NOEs indicated that the orientation of the pheromone in the MUP-I binding cavity is opposite to that modeled in a previous X-ray structure.

A "smart" PARACEST MRI contrast agent was synthesized to detect caspase-3, which is an important biomarker in apoptosis. The well-known caspase-3 substrate, DEVD (Asp-Glu-Val-Asp), was elongated using the amino group on one sidearm of lanthanide ligand anchored on the polymer support. The amide of DEVD-(Tm-DOTA) showed a PARACEST effect with MR saturation at -51 ppm. DEVD-(Tm-DOTA) amide was successfully cleaved by caspase-3, exposing the free amine group, which showed PARACEST with saturation at +8 ppm. The enzymatic activity of caspase-3 can be detected by the change in PARACEST effect caused by this biotransformation.

The three dimensional structure of several peptides were determined by NMR spectroscopy and distance geometry calculations. Each peptide formed a predictable, rigid structure, consisting of an {alpha}-helix, a {open_quotes}scaffold{close_quotes} region which packed along one face of the helix, and two disulfide bridges which covalently connect the helix and scaffold regions. The peptide Apa-M5 was designed to constrain the M5 peptide from MLCK in a helical geometry using the apamin disulfide scaffold. This scaffold constrains the N- terminal end of the helix with two disulfide bridges and a reverse turn. Like the M5 peptide, Apa-M5 was found to bind calmodulin in a Ca{sup 2+}-dependent 1:1 stoichiometry. However, the dissociation constant of the (Apa-M5)-calmodulin complex, 107 nM, was 100-fold higher than the dissociation constant of the M5-calmodulin complex. This difference was due to a putative steric overlap between the Apa-M5 scaffold and calmodulin. The peptide Apa-Cro was designed to replace the large structural protein matrix of {lambda} Cro with the apamin disulfide scaffold. However, Apa-Cro did not bind the consensus DNA operator half-site of {lambda} Cro, probably due to a steric overlap between the Apa-Cro disulfide framework and the DNA. The amino acid sequence of the scaffold-disulfide bridge arrangement of the peptidemore » Max was derived from the core sequence of scyllatoxin, which contains an {alpha}-helix constrained at the C-terminal end by two disulfide bridges and a two-stranded {beta}sheet scaffold. Max was shown to fold with >84% yield to form a predictable, stable structure that is similar to scyllatoxin. The folding and stability properties of Max make this scaffold and disulfide bridge arrangement an ideal candidate for the development of hybrid sequence peptides. The dynamics of a fraying C-terminal end of the helix of the peptide Apa-AlaN was determined by analysis of {sup 15}N NMR relaxation properties.« less

Proton and deuterium NMR relaxation methods were used to characterize water compartmentalization and hydration in work-free wheat flour doughs. Transverse (spin-spin) relaxation measurements define three motionally unique water compartments in the work-free dough preparations. The apparent occupancy fraction and relative mobility of each water domain are found to be functions of moisture content, temperature, and flour type. Additionally, the number of relaxation-resolved water compartments and their characteristic relaxation rate constants are found to depend critically on both moisture content and the interpulse-delay employed for the multi-pulse relaxation experiments. Under controlled experimental conditions, dynamics between the three water compartments can be observed to be consistent with the onset of flour hydration. The most notable observation during the initial period of hydration is a loss of “free” or “loosely bound” water to environments characterized by less mobility. Freezing studies show that hard wheat doughs have slightly less amorphous, non-freezable water than do soft wheat flour doughs prepared under similar conditions.

This review focuses on MR contrast agents that are responsive to a change in physiological environment. The "response" mechanisms are dependent on 6 physicochemical phenomena, including the accessibility of water to the agent, rotational tumbling time, proton exchange rate, electron spin state, MR frequency, or local field inhomogenieties caused by the agent. These phenomena can be affected by the physiological environment, including changes in concentrations or activities of proteins, enzymes, nucleic acids, metabolites, oxygen and metal ions, and changes in pH and temperature. A total of 52 examples are presented, which demonstrate the variety and creativity of different approaches used to create responsive MRI contrast agents.

Department of Chemistry, Indiana UniVersity Bloomington, Indiana 47405 ReceiVed March 3, 1997 Interest in producing gas-phase nucleic acid ions1a-g has been stimulated by recent mass spectrometry (MS) based sequencing strategies for biomolecules.2a-h Recently mass spectra for plasmid and linear DNAs having molecular weights of 106108 daltons have been recorded.3a-c In contrast to the wealth of structural data for nucleic acids in solution, there is no information about conformations of the gas-phase ions. The conformations of protein ions can influence fragment formation;4 thus, the issue of conformation is important for sequencing nucleic acids by MS. Here, we report the first study of the conformations of a deprotonated DNA oligomer in the gas phase: oligothymidine comprised of ten thymine nucleotides. Using electrospray ionization5 and ion mobility techniques,6 we have measured collision cross sections for the -2 to -6 states, (T10-nH), where n is the deprotonation state, and deprotonated states of oligothymidine with sodium ions attached (NaT10nH)(n-1)-, where n ) 5-7. A sharp structural transition is observed when four or more protons are removed; lower charge states favor compact globular conformers and higher ones favor elongated forms. Addition of a single sodium ion to deprotonated states stabilizes more compact conformers. Conformations that were derived using molecular modeling techniques provide complementary information that allows many important structural features to be delineated. Our experimental apparatus and procedures have been described previously.6 Negatively-charged (deprotonated) oligothymidine ions were formed by electrospraying a ∼3 × 10-5 M oligothymidine7 solution in 49.9:49.9:0.2 water:acetonitrile: ammonium hydroxide. Sodium adducts are readily formed, although no sodium was added. Ions were extracted into a highvacuum region and 30 microsecond pulses were injected into a drift tube containing ∼3.0 Torr of helium in order to record ion mobility distributions. The drift times depend on the ions’ conformations; compact conformers drift through faster than extended forms. Ions that exit the drift tube are focussed into a quadrupole mass spectrometer that transmits only the ion of interest. As ions enter the drift tube they are heated by collisions with the buffer gas. Further collisions cool the ions to the buffer gas temperature. Ion mobility distributions at varying injection energies (∼150-450 eV) yield identical drift times; thus, we expect these data correspond to relatively stable conformations. Figure 1 shows charge normalized ion mobility distributions for the -2 to -6 charge states of (T10-nH). Each distribution shows a single peak. The -2 and -3 states have similar normalized drift times, ∼5.2 ms, suggesting similar conformations. A shift to 6.2 ms is observed for the -4 charge state. The -5 and -6 ions have drift times of 7.2 and 7.4 ms, respectively. These ions favor more open conformations. Ion mobility distributions for oligothymidine ions containing sodium, (NaT10-5H), (NaT10-6H), and (NaT10-7H), are also shown in Figure 1. The average drift time of (NaT10-7H) is similar to that for (T10-6H). The (NaT10-5H) and (NaT106H)5ions have higher mobilities than the pure DNA ions of the same charge. Binding sodium leads to more compact conformations of these high charge states. Drift times can be converted into experimental collision cross sections as described previously6 and are shown in Figure 2. Additional insight was gained by comparing calculated cross sections for an array of conformers generated by molecular modeling techniques to the experimental data.8 Trial conformers were obtained using the Insight II molecular modeling software with the CVFF forcefield.9 The forcefield used a dielectric constant of 1.0 for the surrounding media; thus, these are in Vacuo conformations. For each charge state, 100 different trial conformers were generated by simulated annealing. The temperature was increased to 1000 K over 2 ps, equilibrated for 2 ps, and then cooled over 1 ps to 300 K. The structures obtained were energy minimized, and the atomic coordinates of conformations that fell within 30 kcal/mol of the lowest energy model conformer were used to calculate average cross (1) (a) Hunt, D. F.; Hignite, C. E.; Biemann, K. Biochem. Biophys. Res. Commun. 1968, 33, 378. (b) McNeal, C. J.; Ogilvie, K. K.; Theriault, N. Y.; Nemer, M. J. J. Am. Chem. Soc. 1982, 104, 976. (c) Grotjahn, L.; Frank, R.; Blocker, H. Nucleic Acids Res. 1982, 10, 4671. (d) Cerney, R. L.; Gross M. L.; Grotjahn, L. C. Anal. Biochem. 1986, 156, 424. (e) Covey, T. R.; Bonner, R. F.; Shushan, B. I.; Henion, J. D. Rapid Commun. Mass Spectrom. 1988, 2, 249. (f) Hillenkamp, F.; Karas, M.; Ingendoh, A.; Stahal, B. In Biological Mass Spectrometry; Burlingame, A. L., McCloskey, J. A., Eds.; Elsevier: Amsterdam, 1990. (g) Spengler, B.; Pan, Y.; Cotter, R. Rapid Commun. Mass Spectrom. 1990, 4, 99. (2) (a) Crain, P. F. Mass Spectrom. ReV. 1990, 9, 505. (b) McLuckey, S. A.; Van Berkel, G. J.; Glish, G. L. J. Am. Soc. Mass Spectrom. 1992, 3, 60. (c) McLuckey, S. A.; Habibi-Goudzrzi, S. J. Am. Chem. Soc. 1993 115, 12085. (d) Little, D. P.; Speir, J. P.; Senko, M. W.; O’Connor, P. B.; McLafferty, F. W. Anal. Chem. 1994, 66, 2809. (e) Little, D. P.; Chorush, R. A.; Speir, J. P.; Senko, M. W.; Kelleher, N. L.; McLafferty, F. W. J. Am. Chem. Soc. 1994, 116, 4893. (f) Little, D. P.; McLafferty, F. W. J. Am. Chem. Soc. 1995, 117, 6783. (g) Christian, N. P.; Colby, S. M.; Giver, L.; Houston, C. T.; Arnold, R. J.; Ellington, A. D.; Reilly, J. P. Rapid Commun. Mass Spectrom. 1995, 9, 1061. (h) Ni, J.; Pomerantz, S. C.; Rozenski, J.; Zhang, Y.; McCloskey, J. A. Anal. Chem. 1996, 68, 1989. (3) (a) Fuerstenau, S. D.; Benner, W. H. Rapid Commun. Mass Spectrom. 1995, 9, 1528. (b) Chen, R.; Cheng, X.; Mitchell, D. W.; Hofstadler, S. A.; Wu, Q.; Rockwood, A. S.; Sherman, M. G.; Smith, R. D. Anal. Chem. 1995, 67, 1159. (c) Cheng, X.; Camp II, D. G.; Wu, Q.; Bakhtiarl, R.; Springer, D. L.; Morris, B. J.; Bruce, J. E.; Anderson, G. A.; Edmonds, C. G.; Smith, R. D. Nucleic Acids Res. 1996, 24, 2183. (4) Loo, J. A.; Edmonds, C. G.; Smith, R. D. Science 1990, 248, 201. (5) Fenn, J. B.; Mann, M.; Meng, C. K.; Wong, S. F.; Whitehous, C. M. Science 1989, 246, 64. (6) For a recent review, see: Clemmer, D. E.; Jarrold, M. F. J. Mass. Spectrom. 1997, 32, 577 and references therein. (7) Oligothymidine used was synthesized using an Applied Biosystems 391 DNA synthesizer utilizing the phosphoramidite method. The ions studied here are dephosphorylated at the 5′ end. (8) This approach is similar to a method discussed by Wyttenbach, T.; von Helden, G.; Bowers, M. T. J. Am. Chem. Soc. 1996, 118, 8355. (9) Insight II; BIOSYM/MSI: San Diego, CA, 1995. Figure 1. Ion mobility distributions for deprotonated (T10-nH) (solid lines) and (NaT10-nH) (dotted lines) ions formed by electrospray ionization. The distributions are shown on a modified time scale t*z which normalizes for differences in effective drift fields. 9051 J. Am. Chem. Soc. 1997, 119, 9051-9052

HETCOR NMR spectra reveal that previous assignments of the stereochemistry of poly(lactide) derived from rac and meso-lactide are incorrect; an alternative assignment is made at the tetrad level of resolution.