Synthesis & HyperCEST Testing of CTV Derivatives: A Bowl Shaped Compound That Encapsulates Xenon

The primary focus of this dissertation is the synthesis and testing of cyclotriveratrylenes (CTV) as potential biomedical molecular imaging contrast agents. CTVs are bowl shaped molecules that have a hydrophobic pocket capable of reversibly binding Xenon-129 (Xe) gas, a spin 1⁄2 noble gas, which is diamagnetic and produces a signal when a strong magnetic field is applied to it. These CTVs then create two pools of Xe which produce distinct signals in an NMR scan. The creation of the two pools of Xe is necessary for the application of an imaging technique named HyperCEST, Hyperpolarized Chemical Exchange Saturation Transfer, which increases the sensitivity of the scan by up to 10,000 times. The ease of synthesis for the CTV as well as its ability to be conjugated to biochemical ligands should expedite the synthesis of targeted Xe biosensors. The last chapter deals with, Process oriented guided inquiry learning (POGIL), which is a pedagogical method that has demonstrated improvement in student performance, increases in attendance, and decreases in failing grades and withdrawals. A three-year study was conducted where attendance was mandatory for all students across both the POGIL and traditional lecture formats to measure the effect of POGIL. There was a decrease in the standard deviation of exam grades in the POGIL lecture sections however there was no statistically significant change in grades received by the students. This is in keeping with previous studies that have found a decrease in grades of D, F, and W’s.

x  morphological, which focuses on the entire body, and molecular, which deals with the processes occurring within the cells. Of these six types none are able to accomplish the three major requirements for the ideal medical imaging technique, high spatial resolution, selectivity, and low toxicity in vivo. Selectivity refers to the ability for the imaging technique to detect the molecular process that is indicative of the disease.

LIST OF TABLES
MRI meets two of the criteria for an ideal imaging technique; it has excellent resolution and is benign to the subject. To obtain an image it exposes a subject to a strong magnetic field causing the magnetic nuclei to align and relax at different rates based upon their chemical environment. This produces a signal that the MRI then uses to make a three dimensional image of the scanned area. The most abundant MRI active isotope in the human body is hydrogen-1, which is contained in water, fats, and soft tissues. Unfortunately the widespread distribution of hydrogen-1 does not allow for the desired selectivity. The current method of increasing selectivity for MRI imaging is to use a gadolinium contrast agent, which has severe side effects in some patients, effectively trading an increase in selectivity at the cost of preserving the health of the subject. Alternatively, if an MRI is paired with another medical imaging technique, such as PET scanner, the two images can be overlaid. This is often an uncomfortable process for the subjects, since they must be immobilized for the images, and any shift of location during the imaging procedures would render the images useless as a pair. In addition, the subjects are exposed to radiation as part of the PET scan, again trading possible adverse effects to the subject for an increase in selectivity.
However, 1 H is not the only nucleus that is MRI active and capable of being introduced into the human body. To be an ideal choice the chosen nucleus would need to be capable of being distributed throughout the body, including crossing the blood brain barrier, would have no background signal in the subjects, and be nontoxic to the subject. Xenon-129 ( 129 Xe) meets all three of those requirements, and is not only benign, but also has been previously approved for medical applications. 2 Furthermore, any MRI scanner that is equipped with a broadband coil is capable of detecting the 129 Xe signal. 129 Xe is administered orally and is lipid soluble meaning that it diffuses throughout the body and across the blood brain barrier. 129 Xe has a large chemcial shift range of approximately 2000 ppm, and the shift is largely dependent on the environment surrounding the xenon atom. This gives 129 Xe the potential to act as a  129 Xe created by the molecular host encapsulation of 129 Xe.
selective imaging agent. It has already been applied to detect lung activity in MRI scans where it shows the portions of the lungs that are filled with gas when the patient inhales. 3 The major drawback has been the sensitivity of the MRI, which is typically capable of detecting molecules that are present concentrations greater than 10 -3 to 10 -5 M, while the typical PET or SPECT molecular imaging technique has a detection limit of 10 -9 to 10 -12 M. To overcome this limitation hyperpolarized 129 Xe gas has been used providing approximately a 100,000 increase in sensitivity and putting it on approximately the level of the leading molecular imaging techniques. 4 Conventional NMR/MRI techniques only detect approximately 1.0 ppm of the available nuclei in a given sample. This is because NMR and MRI capitalize on the magnetic moments of certain nuclei, which are caused by the nuclei having odd numbers of protons and/or neutrons. In the case of 129Xe, the nucleus has an odd number of neutrons (75) and an even number of protons (54), and its spin value has been experimentally determined to be ½. This means that when the 129Xe is exposed to a magnetic field, the atoms organize themselves into two energy states -one with the magnetic moments aligned with the external field (lower energy state) and one with the magnetic moments opposed to the external field (higher energy state). The ratio of the states is determined by the energy difference between the two levels, which is determined by the strength of the magnetic field. The Boltzmann distribution predicts that at room temperature the population difference of the two states is on the order of parts per million in the presence of a 7 T magnetic field (a 300 MHz NMR).
Hyperpolarization artificially increases the population of the lower energy state, causing an overall increase in signal.
In a typical spin-echo NMR experiment, a radio frequency pulse is applied causing the magnetic moment of the nuclei to become perpendicular to the external magnetic field. Once the pulse is complete, the transverse magnetization begins to decay as the sample returns to equilibrium. This decay is detected by the receiver, producing a free induction decay pattern, or what is commonly referred to as a FID.
The ability to make 129 Xe a selective biosensor centers on changing the environment surrounding the xenon. To accomplish this it was hypothesized that xenon atoms could be encapsulated in molecular hosts thus causing a unique signal to appear in the 129 Xe NMR spectrum ( Figure 2 forces to form the relationship that a cavity to guest size ratio of 55% ± 9% results in optimal binding, they determined that their cryptophane, with a ratio of 51%, was within this optimal binding ratio. 7 This increase in binding strength resulted in an increased signal intensity from the bound xenon. A three and half times increase in binding constant versus cryptophane-A was observed with the optimal cavity size of cryptophane-1,1,1, which corresponded to an improvement in the signal to noise ratio and a sharpening of the bound xenon peak.  (Figure 3). 4 HyperCEST takes advantage of the ability to selectively presaturate at a specific frequency, with a targeted radio frequency pulse. In this technique an initial reading is conducted giving a baseline signal (Soff) then the scan is repeated with the presatureation pulse frequency on giving a reduced signal (Son). CEST values (C) can then be obtained by using equation 1.
The higher the value of C the more sensor is present in a given location. The key to this technique is the significant change in signal for the bound xenon and the free exchange of the xenon into and out of the host. HyperCEST shifted our focus from the small peak corresponding to the encapsulated xenon to the large peak corresponding to the unbound xenon. The HyperCEST experiment shifts the focus for an ideal xenon imaging probe from one that tightly binds xenon to that rapidly exchanges xenon in and out of its hydrophobic cavity.
The synthesis of cryptophanes has been accomplished following three main routes: (i) the template method, (ii) "two step" synthesis, and (iii) capping method (Scheme 1). 8 All three methods involve the use of electron donating groups in both the meta and para positions to facilitate the Friedel-Crafts reaction. The two substitutions also act as ortho/para directing groups, making it necessary for the meta group to be the stronger electron donating to ensure that the substitution occurs at the correct position for ring formation. Each of the three methods has different advantages and accomplishes the synthesis of various cryptophanes with varying degrees of success (  Current cryptophanes have a long and low yielding synthetic procedure that results in an ether linked macrocycle that can be difficult to functionalize. 9 Functionalization is necessary to make the cryptophane selective and water soluble, critical qualities for a biosensor. Two of the three synthetic strategies go through cyclotriveratrylenes (CTVs) to obtain cryptophanes. CTVs have further been used in the past to bind large hydrophobic molecules called fullerenes. 10 The volume of a fullerene is 450 Å 3 , which is approximately 10 times the volume of xenon. 11 There have been multiple previously CTVs synthesized often with high yielding procedures, most notably the aCTG which has a reported cyclization step yield of 95%. 6 Additionally, much of the work on functionalizing the cryptophanes can be directly applied to CTVs. This will facilitate the rapid synthesis of a number of CTV derivatives that can meet the requirements for an effective biosensor. We hypothesize that CTVs will provide a hydrophobic cavity capable of binding 129 Xe and changing its chemical shift. That combined with its short high-yielding synthesis will make CTVs an ideal candidate to replace cryptophanes as the primary focus of 129 Xe molecular imaging research.

MANUSCRIPT 1 A BOWL-SHAPED MOLECULAR PROBE FOR XENON-129 NMR Abstract
A novel molecular probe for 129 Xe magnetic resonance spectroscopy and imaging is disclosed. Prior work in the field has predominantly relied on the use of cryptophanes, cage-shaped compounds that fully encapsulate xenon atoms, thus giving rise to a unique signal in the 129 Xe NMR spectrum. Herein, we report a cyclotriveratrylene (CTV) compound that is capable of reversibly binding xenon, and that the binding event can be detected as a unique peak in the 129 Xe NMR spectrum.
The ease of synthesis for the CTV as well as its ability to be conjugated to biochemical ligands should expedite the synthesis of targeted 129 Xe biosensors.

Main Text
Hyperpolarized xenon-129 magnetic resonance imaging (HP-Xe MRI) is a promising technology for diagnostic imaging which does not rely on ionizing radiation. The non-toxic noble gas 129 Xe has a nuclear spin of ½ and can be hyperpolarized by optical pumping to increase the magnetic resonance signal intensity 10,000-100,000-fold. 1 Once inhaled, HP-Xe readily enters the blood stream, and because it is lipid soluble, it distributes into nearly all organs of the body, even crossing the blood-brain barrier. The distribution of the HP-Xe can then be imaged in whole bodies using an MRI scanner that is equipped with a broadband coil. 2 Examples of the current applications of this technology include distinguishing between the white and gray matter in the brain and the diagnosis of maladies such as chronic obstructive pulmonary disease and stroke. 3 In light of the extra sensitivity that can be gained via the hyperpolarization process, several research groups have pursued HP-Xe MRI as a method for performing molecular imaging of biochemical receptors via MRI. 4 As first outlined by Pines and Wemmer, a biosensor can be constructed by tethering a ligand for binding a biochemical target to a molecular host that is capable of encapsulating HP-Xe. 5 The most common host molecule used for encapsulating xenon is cryptophane-A, which consists of two bowl-shaped cyclotriveratrylenes (CTVs) joined by three ethylene linkers. 6 The encapsulation of xenon in a cryptophane host creates two pools of 129 Xe spins, which can be detected as two distinct peaks in the 129 Xe NMR spectrum ( Figure   4), as the residence time of xenon in a cryptophane is sufficiently long (tens of milliseconds) to be detected on the NMR time scale. The exchange of the xenon in and out of the host can be exploited to achieve an additional 10,000-fold sensitivity enhancement. 7 This is achieved by detecting the decrease in the signal of the large pool of unbound 129 Xe spins while saturating the signal of the pool of encapsulated 129 Xe spins with a targeted radio frequency pulse.
Since the xenon atoms are reversibly bound by the host, the targeted pulse also causes the partial saturation of the pool of unbound 129  alternative to current molecular imaging techniques that rely on probes containing radionuclides.
The key requirement for the development of a HP-Xe molecular probe is the presence of a molecular or macromolecular species that can bind the 129 Xe atoms and create two distinguishable pools of nuclear spins. Nearly all HP-Xe probes that have been developed to date have used cryptophanes for this purpose. 10,11 One of the biggest impediments to research in this field has been the synthesis of these molecular cages, such as Dmochowski's tri-alkyne, 1 ( Figure 4). The 10-step synthesis is expensive, low yielding and technically difficult. It is the bottleneck in the development of HP-Xe biosensors.
Consequently, we hypothesized that the HyperCEST technique created a paradigm shift in the field, and that there now existed a need to develop new xenon hosts that were optimized for exchange kinetics rather than binding affinity. We predicted that bowl-shaped cavitands such as cyclotriveratrylenes (CTVs) would be superior hosts for HyperCEST imaging due to their more open architectures that should allow for faster chemical exchange. Herein, we disclose the first example of 129 Xe magnetic resonance spectroscopy using such a bowl-shaped contrast agent, indicating that it has the potential to be a scaffold for constructing HyperCEST biosensors.
While most CTVs contain six ether groups around their rim, Collet reported that substituting one of these ethers for an amide, more than tripled the yield of the key Friedel-Crafts trimerization step. 12 As shown in Scheme 1, we followed this strategy to synthesize the amino-CTV (5) in four steps with an excellent yield (average step yield = 83%, overall yield = 47%). In addition to facilitating the Friedel-Crafts step, we hypothesized that the amines in this CTV derivative could serve as functional handles to conjugate ligands for eventual studies with targeted HP-Xe biosensors.
To analyze the size of the cavity of 5 and to compare it with that of a cryptophane cage, X-ray quality crystals were grown in methanol ( Figure 5A). The bond lengths and angles of 5 were virtually identical to the CTV portion of Dmochowski's triallyl cryptophane-A, 13 as demonstrated by the superimposed image shown in Figure 5B.
The longest diameter of Dmochowski's cryptophane is 9.3 Å, while the depth of the bowl-shaped cavity of the CVT, 5, is only 3.3 Å. Thus, when compared to cryptophane A derivatives, 5 has approximately 36% of the volume that can be used to accommodate a xenon atom.
To support our hypothesis that CTVs could be used as 129 Xe molecular probes, a xenon atom was computationally docked in the cavity of the amino-CTV host, 5, and the energy was minimized using a Hartree-Fock 3-21G basis set, yielding a calculated binding energy of -0.52 kcal/mol ( Figure 5C). Though this is a fraction of the free energies of association that have been previously described for xenon-cryptophane complexes, 14 the computed favorable binding energy indicates that the formation of a host-guest complex between xenon and 5 could possibly produce the two spin pools necessary for HyperCEST.  Ultimately, 129 Xe NMR spectroscopy confirmed that the probe 5 was, in fact, capable of serving as a HP-Xe host ( Figure 6). A 50 mM solution of 5 was prepared in DMSO and placed in a syringe containing 1 atm of HP-Xe (polarization ~37%). A 129 Xe magnetic resonance spectrum was then acquired using a Phillips 3T MRI scanner. A small peak corresponding to Xe@5 was observed in the 129 Xe NMR spectrum 51 ppm upfield from the free 129 Xe peak.
The host-guest relationship of the tri-amino CTV, 5, with xenon described herein could represent a significant advance in xenon biosensor development. It allows for the rapid synthesis of CTV-derived HP-Xe molecular probes. Future work in our laboratories will be dedicated toward the further optimization of the bowl-

Supporting Information
Experimental procedures and characterization data for novel compounds, description of the 129 Xe NMR protocol using a clinical MRI scanner, crystal structure data for

Introduction
There has been a strong interest in developing a contrast agent for magnetic resonance imaging (MRI) for diagnostic biomedical applications that will shift MRI from a primarily morphological imaging tool to a molecular imaging tool. While the majority of work in this field has focused on the development of paramagnetic contrast agents that can perturb the T1 relaxation time of protons, the use of xenon-based imaging agents is a viable alternative that could prove to be a superior molecular imaging platform. Xenon-129 ( 129 Xe) is a non-toxic noble gas that has a ½ nuclear spin and is capable of being hyperpolarized via optical pumping. 1 129 Xe is delivered through inhalation and distributes throughout the body including crossing the blood brain barrier and is currently used in medical diagnoses. 2 129 Xe magnetic resonance molecular imaging is currently being pursued by several research groups, and until our recent communication, has primarily been pursued using cryptophane hosts that have been functionalized with ligands for binding to a molecular target. To take advantage of these properties, large macrocyclic compounds named cryptophanes were developed to bind xenon.
Cryptophanes encapsulate 129 Xe reversibly with a residence time of approximately 40 milliseconds. This presents two pools of 129 Xe that are detected by the NMR at unique chemical shifts. This phenomenon can be further exploited for an increase in sensitivity by saturating the signal of the encapsulated 129 Xe with a targeted radio frequency pulse. This effectively "turns off" the signal from these encapsulated 129 Xe atoms. The scan is designed with a mixing time, to allow the depolarized 129 Xe atoms to exchange out of the molecular host's cavity, thus decreasing the intensity of the peak corresponding to unbound 129 Xe. Indirectly detecting the presence of the cryptophane probe by observing the decrease in the large peak is 10,000 more sensitive than trying to directly detect the small peak corresponding to encapsulated 129 Xe in aqueous solution, 3 thus allowing for the detection of quantities of the molecular probe in sub-picomolar concentrations (Figure 7). 4 The technique that Pines developed, and Dmochowski, Stevens and Schröder have since repeated, is called Hyperpolarized Chemical Exchange Saturation Transfer (HyperCEST). Due to its high sensitivity, HyperCEST has the potential to replace the use of radionuclides that current molecular imaging techniques utilize. HyperCEST pulse applied 89% reduction same solution with application of a prepulse at the frequency corresponding to the encapsulated species (red).
With the advent of HyperCEST, we hypothesized that a new xenon host that was optimized for its exchange kinetics, rather than binding affinity, was the next step towards the development 129 Xe molecular probes. Cyclotriveratrylenes (CTVs) were chosen due to their open architecture and their known affinity for hydrophobic guest molecules. They have exhibited a ball-and-socket-style binding of large hydrophobic compounds such as C60 fullerenes. 5 Additionally, they are an intermediate in two of the main synthetic strategies currently used to synthesize cryptophanes, which essentially are two CTVs joined by three diether linkers. CTVs are quickly and easily synthesized from cheap, readily available starting materials. As we recently reported in our communication, the amino functionalized CTV binds xenon gas and that this binding is apparent by 129 Xe NMR, thus demonstrating the two pool system of 129 Xe spins that is necessary for HyperCEST. It has been shown that CTVs, when binding charged substances, can self-assemble with hydrogen bonds to form a dimeric compound encapsulating the guest. 6 In addition, the methoxy and amino groups around the exterior ring of the compound in our earlier publication are within range of each other to have hydrogen bonding interactions ( Figure 8). To explore the effects the functional groups around the outer ring of the CTV have on 129 Xe binding, a set of CTVs was synthesized that varies the number and type of hydrogen bond donors/acceptors present (Figure 9).  HCl/EtOH, 74% yield.

Chemical Synthesis
The amine CTV (5) was synthesized in five steps starting with methyl 4amino-3-methoxybenzoate (2) which was reduced to 4-amino-3-methoxybenzol alcohol using five equivalents of lithium aluminum hydride in THF/ether (1:1). After a Fiezer and Fiezer workup, the crude reaction mixture was vacuum filtered, and the solvent was removed via rotary evaporation. The crude product was loaded onto silica gel and purified via automated flash chromatography using a ISOLERA 1 ® yielding the product (3) in 87% yield. The purified product was immediately taken on to the next reaction due to its quick decomposition.
The diacetate (4) was synthesized from the amino alcohol (3), which was dissolved in pyridine (anhydrous) and acetic anhydride was added drop wise over three minutes at 0 ᵒC under an ambient atmosphere. The reaction mixture was capped, warmed to room temperature and allowed to react for 12 hours. The reaction was then cooled to 0 ᵒC, and ice was added to the reaction mixture. Concentrated hydrochloric acid was added drop wise until the reaction was brought to pH 1. The product precipitated and was vacuum filtered and washed with DI H2O. The product 4 was dried with a V-10 rapid solvent evaporator, giving a 81% yield of a white crystalline powder. The product was of sufficient purity to take directly on to the next step.
The CTV (5) was synthesized from the monomer (4), dissolving it in glacial acetic acid and adding percholoric acid (65%) drop wise over five minutes. The reaction mixture was then stirred overnight. After 12 hours, the volume was doubled by adding DI H2O, and the reaction was stirred for an additional 15 minutes to allow for complete precipitation. Vacuum filtration through a fine sintered glass frit and washing with DI H2O and acetone provided the pure product the triacetal CTV (7) in 91% yield as a white paste.
The deprotected CTV (5)  Compound (8), the trihydroxy CTV, was synthesized from 3-methoxy benzyl alcohol (11) following the previously published procedure. 7 Phosphorous pentoxide was added to a round bottom flask with a stir bar and suspended in dichloromethane.
The alcohol (11) was added drop wise over five minutes and was heated at 40 ᵒC for one hour. The product (8) was obtained by recrystallization in sufficient purity to take on to the next step in 1% yield.

Conclusion
Six CTVs were synthesized for HyperCEST testing to determine if they were capable of reversibly binding xenon. The aim of the HyperCEST testing will be to determine if they form a dimeric compound through hydrogen bonding selfassembling a cryptophane like structure to encapsulate 129 Xe. These CTVs are currently waiting HyperCEST testing by our collaborator. physiology, computer science, engineering, math, and biochemistry. 3 The student-centered active learning focuses on the development of "process skills" as well as teaching the core material. These process skills include information processing, critical thinking, problem solving, teamwork, and communication. 4 The existing body of work on POGIL has demonstrated a variety of results including positive impact on grades, a decrease in D, F, and withdrawal rates, an increase in attendance, and more enjoyment in the classroom. 5 As one study recently pointed out, increased attendance is a challenging outcome to obtain with conventional teaching methods. 6 However, some of those studies have shown that this method does not necessarily result in an increase in grades. In addition, there is some doubt about whether the increase in grades can be attributed to the increase in attendance or due to the pedagogy itself. 7

Aims and Research Questions
This study aims to investigate the impact of adopting POGIL on student performance in a first semester general chemistry course while holding attendance constant. Specifically, we ask if a full implementation of POGIL improves the performance of the students relative to other non-POGIL sections of the general chemistry class, and relative to the previous year's POGIL section taught by the same instructor.

Institution
The United States Coast Guard Academy is a four-year public undergraduate college that has an enrollment of approximately 900 students. It has a stated goal of graduating 130 students from STEM majors each year, which is approximately 60% of each class. 8 General Chemistry is a required course for all students in their freshman year and is made up of students intending to major in science, mathematics, and engineering as well as government and management. Attendance to all class meetings is required.

Students
The students are made up of a diverse body from across the United States. Each incoming class is composed of individuals who were under the age of 26 and who have no dependents on the day that they were required to report. All students are

Course Setup
The lecture portion of General Chemistry I is conducted in seven sections, taught by seven different instructors. The class meets three times per week for 50 minutes in a three-tiered classroom capable of holding 40 students. Class size was approximately 35 students per class. All students are required to attend each class unless they had an approved excusal to miss from their instructor. Generally, the only approved excusals were for varsity athletes who were missing class for a competition.
At the beginning of the semester, chapter-specific objectives for each chapter were handed out to each student (available in supporting information). The objectives were Generally, students were not permitted to withdraw from the course without withdrawing from the institution. As a general rule, students do not withdraw from the institution as a result of the chemistry course but instead for a variety of reasons that lead them to conclude they do not wish to pursue a career in the U.S. Coast Guard. Students who did not complete the semester were excluded from this study.
Written homework was graded on a scale of 1 to 5 for each assignment and collected at the end of each chapter by the assigned lecture instructor. Online homework was due at the same time and was graded for completeness. It was assigned through an online homework system, ChemSkill Builder. As long as a student received a score better than 65% on an individual section, they would receive full credit. The grading was completed by the online system. There were five hourly exams given throughout the semester. All exams were administered during a common testing period, where all students enrolled in the course took the exam at the same time. Each page of an exam was graded by one instructor for all seven sections ensuring that all exams were graded equitably across all sections. Finally, online student surveys were taken on a voluntary basis at the end of the course. The surveys were anonymous and not attributed to specific sections or instructors, therefore they were only used for anecdotal conclusions.

Experimental Design
In the first year of data that was collected, all seven sections were taught by different instructors in a traditional lecture format. In the second and third year, one section was taught using POGIL, by an instructor who attended a weeklong implementation workshop offered by the POGIL project. POGIL was implemented following all of the tenets of a basic POGIL classroom 9 including: • Students worked in groups of 3 or 4.
• The activities follow the learning cycle and were designed using POGIL principles.
• The instructor acted as a facilitator.
• Only short lectures (if any) were used, and the majority of class time was used for student centered learning.
• Students had assigned roles within their groups and rotated through the roles.
• The first time new information was introduced was in class, via POGIL.
• Groups were expected to complete all of the activities together in class, ensuring that all members understood the information.
In lieu of graded quizzes at the beginning of the course, as used in many implementations of POGIL, 1b an anonymous clicker system was used approximately weekly to give the students immediate anonymous feedback on how they were performing relative to the objectives that had been covered in previous classes.

Data Analysis
In year one, all sections (242 students) were taught using traditional methods.
This year was used as a control, comparing the instructor who would later implement POGIL to the remaining sections. In years two and three, one section was taught using a full implementation of POGIL, while the remaining sections continued to be taught using traditional methods. For years two and three, 14% of students enrolled in the General Chemistry course experienced the POGIL implementation, with 241 and 263 students enrolled in the course, respectively.
For all three years, data was analyzed for each of the five exams given during the semester, overall averages on homework assignments and final exam scores. Data analysis was performed using independent two-group t-tests comparing traditional and POGIL implementations, and results were considered statistically significant (i.e., the null hypothesis is rejected) at the p = 0.1 level.
For year one, the instructor who would later implement POGIL had statistically significant higher scores in his section on semester exam averages, t(52) = 1.71, p < 0.10, and final exam score, t(58) = 2.17, p < 0.05. Differences on homework assignment averages did not show a statistically significant difference. Table 3 shows averages for year one. Tables 4 and 5 contain averages for each of these categories for years two and three, respectively. In years two and three, with full POGIL implementation, all but one of the average scores for each category noted above are higher for the POGIL sections when compared to the traditional sections. However, none of the differences in exam scores or final exam scores are statistically significant at the p = 0.1 level of confidence. There is, however, an advantage in the POGIL section on homework averages in year three, t(52) = 2.20, p < 0.05.  Of the ten exams given during the semester over years two and three, averages from the POGIL section outscored the traditional section on eight exams, although the differences were only statistically significant for three of those eight exams: Exam two in year 2, t(60) = 2.01, p < 0.05, Exam three in years three, t(49) = 1.8, p < 0.01, and Exam 5 in year three, t(48) = 2.67, p < 0.05. One of the two exams where the traditional sections scored higher than the POGIL section was also significant: Exam three in year two, t(48) = -2.34, p < 0.05. The averages for each of these exams can be seen in Table 5. However, a paired two-tailed t-test showed that the standard deviation for the exams between the POGIL and traditional classroom was statistically significantly smaller, t(10) = 2.2912, p < 0.05.

Conclusions and Discussion
In this study, we were able to evaluate the impact of a full POGIL implementation in its first two years for a first-year general chemistry classroom while holding attendance constant. Due to experimental design we were able to convey the same amount of information and produce the same level of performance as a traditional lecture. A visual representation of the grades appeared to show an improvement in the letter grades obtained by the students in the POGIL sections (graph 1.) A Χ 2 analysis was conducted to determine if the difference was significant.
It revealed that there was no significant difference between the two groups,

% of Students Earning Each Grade
Letter Grade

Pogil Non-Pogil
Instructor preparation was important for the successful implementation. The weeklong POGIL workshop combined with ongoing discussions from experienced practitioners greatly accelerated the learning curve and decreased any negative impacts on the class. Anecdotally, the classroom was much more enjoyable with an increase in interaction with the students. Further study on the impact of POGIL on process skills is definitely warranted and planned for the future. Previous implementations of POGIL have reported an increase in grades and attendance but were unable to determine the interrelation of these two variables. In this implementation, where we were able to hold attendance constant, we did not see a significant increase in grades for the POGIL classroom but did see a decrease in the standard deviation of the grades between the POGIL and traditional lecture sections.
At institutions where attendance is not compulsory, an increase in attendance alone should be enough of a positive outcome to warrant POGIL's consideration.

Supporting Information
An example syllabus and chapter specific objectives are provided for the course in appendix 3.

Author Information
Corresponding Author MHz spectrometers.

Chemical Synthesis.
Unless otherwise noted all reactions were carried out under normal atmosphere at room temperature.

4-amino-3-methoxy-benzenemethanol (3):
To an oven dried 100 mL RBF with a stir bar lithium aluminum hydride (27.61 mmol) was added under N2(g). The vessel was capped and cooled to 0ᵒC. The LiAlH4 was suspended in anhydrous diethyl ether (37 mL). Methyl-4-amino-3-methoxy-benzoate (5.52 mmol) was dissolved in anhydrous tetrahydrofuran (17 mL) and then added to the RBF across 5 min. The reaction was allowed to warm to room temperature and stirred for approximately 4 hours with the reaction progress being monitored via TLC.
When the reaction appeared complete by TLC it was cooled to 0 ᵒC and quenched using the Fieser method. After stirring for an additional hour the reaction was vacuum filtered and the solid was washed with approximately 30 mL of dichloromethane. The filtrate was concentrated via rotary evaporation and the resulting yellow oil was purified via flash chromatography on a 10g Ultra SNAP column. The fractions containing the desired compound were combined and dried via rotoary evaporation and on a V-10 rapid solvent evaporator. 4-amino-3-methoxy-benzenemethanol (3) was obtained in 87% yield.

Characterization of Compounds
Known compounds were obtained by the procedures above and characterized via NMR spectroscopy; a 1 H NMR spectrum was provided for each compound and the relevant reference was cited. All novel compounds obtained have been characterized with 1 H NMR, 13 C NMR, and either Mass Spectroscopy or Elemental Analysis.
Spectroscopic Data for Compounds in Scheme 1:

Crystallographic Data
A colorless crystal of sample was mounted on a Cryoloop with paratone-N oil and data was collected at 90 K with a Bruker CMOS detector using Mo K alpha radiation generated from a Mo rotating anode. Data were corrected for absorption with SADABS. Structure was solved by direct methods and all non-hydrogen atoms were refined anisotropically by full matrix least squares on F 2 . Hydrogen atom H1N was found from a Fourier difference map and was refined isotropically with N-H distance

Xe Magnetic Resonance Spectroscopy
Representative Procedure: 129 Xe gas was polarized to 39% polarization using a Xemed polarizer utilizing spin exchange optical pumping (SEOP). Polarized 129 Xe was stored in a tetlar bag and kept inside the magnet bore to minimize depolarization. The amino-CTV (5) was dissolved in dimethyl sulfoxide (DMSO) within a 5 mL syringe to create a 50 mM solution. 1 mL of hyperpolarized 129 Xe was introduced into the syringe and vigorously shaken within the magnet bore. All ambient air was removed from the syringe and 129 Xe delivery tubes prior to the introduction of polarized 129 Xe into the syringe. The syringe was placed inside a custom built quadrature birdcage RF coil within the magnet bore.
A Phillips Achieve 3T clinical MRI scanner was used to obtain NMR spectra utilizing the manufacturer's control software. A RF pulse sequence consisting of 10-5 ms pulses followed by 5-5 ms pulses with a 5 ms pulse interval was applied, and an MR spectrum was collected. MR spectra were processed using MATLAB (v. 8 MHz spectrometers.

Chemical Synthesis.
Unless otherwise noted all reactions were carried out under normal atmosphere at room temperature.

4-amino-3-methoxy-benzenemethanol (3):
To an oven dried 100 mL RBF with a stir bar, lithium aluminum hydride (27.61 mmol) was added under N2(g). The vessel was capped and cooled to 0 ᵒC. The LiAlH4 was suspended in anhydrous diethyl ether (37 mL). Methyl-4-amino-3methoxy-benzoate (5.52 mmol) was dissolved in anhydrous tetrahydrofuran (17 mL) and then added to the RBF over 5 min. The reaction was allowed to warm to room temperature and stirred for approximately 4 hours, and the reaction progress was monitored via TLC. When the reaction appeared complete by TLC, it was cooled to 0 ᵒC and quenched using the Fieser method. After stirring for an additional hour, the reaction was vacuum filtered, and the solid was washed with approximately 30 mL of dichloromethane. The filtrate was concentrated via rotary evaporation, and the resulting yellow oil was purified via flash chromatography on a 10 g Ultra SNAP column. The fractions containing the desired compound were combined and dried first via rotary evaporation and subsequently using a V-10 rapid solvent evaporator. (3) was obtained in 87% yield.

Cyclotriveratrylene (6):
Scandium(III) triflate (64 mg) was added to a 5 mL reaction vessel with a magnetic stir bar which was then sealed and purged with dry nitrogen. 3,4-dimethoxybenzyl alcohol (500 mg) was added then added and acetonitrile (3 mL) and it was heated to 60 ᵒC in an oil bath overnight. The reaction was then rotovaped to dryness and redissolved in dichloromethane and extracted with DI-H2O. The dichloromethane was rotovaped to dryness. The product was washed with ethyl acetate yielding the 114 mg (26% yield) of the pure product.

Cyclotricatechylene (9):
Cyclotriveratrylene (6)   Course Description: Chemistry I is the first half of a one-year curriculum in general chemistry. It is a four-credit-hour course that consists of three one-hour lectures and one three-hour laboratory each week. The course presents an introduction to elementary concepts of chemistry, covering topics of matter and measurement, atomic theory and inorganic nomenclature, mass relationships, reactions in aqueous solution, gas laws and reactions, enthalpy, quantum theory, periodic trends in the elements, chemical bonding, and intermolecular forces.

Lecture
Online The grade of "H" (Honors) will be given in lieu of "A" for those students who have distinguished themselves by demonstrating mastery of all four overall course objectives.

Final Exam:
The comprehensive final exam will include material from the entire semester, and will be scheduled in accordance with a SUPTNOTE published during the semester.

Course Notebook
Each cadet will maintain a notebook that contains all administrative handouts, lecture notes, homework assignments, exams, and laboratory documents. This notebook can become an indispensable study aid and self-assessment tool. Further, your returned assignments are the only physical record of your graded effort. In the rare event that a mistake is made while entering your grades, a properlymaintained portfolio of your graded assignments may be the only evidence available to correct the problem. Selected notebooks will be collected and retained for submission to the Accreditation Board for Engineering and Technology (ABET) reviewers when they visit during the Fall 2007 semester.

Chemistry Resources
USCGA Chemistry Blackboard: http://uscg.blackboard.com is the official course web site, and it should be your first source in obtaining course information. All course and laboratory documents will be posted there, along with homework and exam solutions, announcements, student grades, textbook resources, and more. Safeguard your BlackBoard password and visit the site frequently.
Chemistry Faculty: Outside of the classroom and laboratory, any member of the Chemistry Faculty will help you with any aspect of the course. Approach the instructor to whom you are assigned before seeking out other faculty members.

Cadet Academic Assistance Program (CAAP):
During scheduled CAAP sessions, one or more of the Chemistry Faculty will be available in the evening during study hour to assist students as needed, for lecture or lab questions. An official Chemistry CAAP schedule can be found at http://uscg.blackboard.com. Cadet Reading/Writing Center: By appointment, this resource is prepared to help you with your writing (including post-lab questions) and can also help you improve your skills at reading technical textbooks.

Chemistry II
Chemistry I & II are essentially one seamless course. As such, the knowledge you gain from Chemistry I will be important to your understanding of Chemistry II. At the conclusion of this one-year chemistry curriculum you will be administered a standardized, multiple-choice exam, covering both semesters.

Collaboration and Consultation
Collaboration is working together on an assignment with someone who is in the same course and is also responsible for completing the same assignment. Consultation is discussing an assignment with someone who is not currently taking the course but does have the information you need. Consultation with a Chemistry Instructor is always allowed on every assignment. Consultation with anyone outside of the Chemistry Faculty (e.g. an upper-class tutor) is allowed whenever collaboration is allowed. Collaboration is allowed and highly encouraged on ChemSkill Builder, homework problems, and studying for exams.

Individual Effort:
If an assignment is marked as an individual effort assignment, then all work is expected to be done by one individual with the exception of assistance provided by a Chemistry Instructor. No other collaboration or consultation is permitted. Individual effort applies to examinations, quizzes, and when indicated in laboratory reports.

Group Effort:
A group effort assignment applies to laboratory exercises where two or more students are assigned to a laboratory group. Full collaboration is expected within each assigned group, instructors may be consulted for assistance, but two separate groups may not collaborate together in whole or in part. Many lab assignments are broken up into group effort sections and individual effort sections; each section will be clearly labeled in every laboratory exercise.
If you have any doubt concerning the collaboration policy for an assignment, you shall first assume that collaboration is prohibited, and then you should seek clarification from a Chemistry Instructor. 2 Understand the progression of steps in the "scientific method" and how these steps are applied to written problems and laboratory exercises.