REGIOSELECTIVE CARBON-NITROGEN BOND FORMATION VIA OXIDATIVE TRANSITION METAL CATALYSIS

The synthesis of carbon-nitrogen (C-N) bonds is an area of research that has garnered significant interest due to the ubiquity of C-N bonds in pharmaceuticals and natural products. Various methods exist for forming C-N bonds, most recognizably Buchwald-Hartwig amination, but these reactions typically require functionalization of either the C-H or N-H bond resulting in the formation of harmful byproducts. As a result, the need to explore alternative means for synthesizing C-N bonds merits consideration. An alternative means for achieving C-N bond formation lies in the oxidative cross coupling of carbon-hydrogen (C-H) and nitrogen-hydrogen (N-H) bonds. While this approach eliminates the necessity for the prefunctionalization of CH and N-H bonds, reactions employing oxidative cross coupling can be plagued with poor regioselectivity. As a result, the use of directing groups and transition metal catalysis is typically employed to circumvent this issue. The primary focus of this dissertation is the discussion of newly developed methodologies that enhance the regioselective control of oxidative C-N bond formation. Chapters one and two detail the use of hypervalent iodine in conjunction with a transition metal catalyst to help govern the site of amination on various electron-rich, and moderately deactivated, arene systems. Chapters three and four will deviate from C—N bond forming methodologies and instead discuss synthetic routes for the construction of a molecular probe targeting the D2 receptor and the development of an amino acid coupling protocol to be utilized in the organic teaching laboratory.

either the C-H or N-H bond resulting in the formation of harmful byproducts. As a result, the need to explore alternative means for synthesizing C-N bonds merits consideration. An alternative means for achieving C-N bond formation lies in the oxidative cross coupling of carbon-hydrogen (C-H) and nitrogen-hydrogen (N-H) bonds. While this approach eliminates the necessity for the prefunctionalization of C-H and N-H bonds, reactions employing oxidative cross coupling can be plagued with poor regioselectivity. As a result, the use of directing groups and transition metal catalysis is typically employed to circumvent this issue.
The primary focus of this dissertation is the discussion of newly developed methodologies that enhance the regioselective control of oxidative C-N bond formation. Chapters one and two detail the use of hypervalent iodine in conjunction with a transition metal catalyst to help govern the site of amination on various electron-rich, and moderately deactivated, arene systems. Chapters three and four will deviate from C-N bond forming methodologies and instead discuss synthetic routes for the construction of a molecular probe targeting the D 2 receptor and the development of an amino acid coupling protocol to be utilized in the organic teaching laboratory.
iii ACKNOWLEDGMENTS "The journey of a thousand miles begins with a single step" -Lao Tzu Every journey begins the same way; establish a goal then take that first step. I knew that the path to obtain science's highest degree would not be an easy one, but as and Joseph Brown whose comradery helped alleviate some of the stresses associated with graduate school.
iv I wish to dedicate this dissertation to two individuals: To my loving wife, Tanna Rose, for her unwavering support, unconditional love, and large heart. You will always make me smile and I look forward to a lifetime of happiness.

Introduction
The field of carbon-nitrogen (C-N) bond formation has generated significant attention due to the ubiquity of C-N bonds in a verity of biologically active compounds. Reactions seeking to achieve C-N bond formation can be done in one of two ways: either intramolecularly, resulting in the formation of a heterocyclic compound; or intermolecularly, wherein a nitrogen-containing compound is independently coupled to some carbon species. 1 C-N bond formation can be achieved in a myriad of ways. However, from an environmental perspective, the best way to achieve C-N bond formation is through the oxidative cross coupling of carbonhydrogen (C-H) and nitrogen-hydrogen (N-H) bonds, as this would eliminate the need for prefunctionalization of the C-H bond and would, in principle, increase the atom economy of the overall synthesis. While the use of transition metal catalysts are not always necessary, regioselectivity often becomes an issue when attempting to perform a reaction intermolecularly using oxidative conditions. [2][3][4] As a result, contemporary methods employed for the oxidative construction of C-N bonds often utilize directing groups or late transition metals in order to garner control over the site of amination. Herein, a discussion highlighting contemporary methods for the construction of C-N bonds utilizing different transition metal catalysts will be presented with an emphasis on C-H prefunctionalization alternatives when applicable.

Palladium Catalysis
Perhaps the most recognized amination protocol is the one that is now taught to students in sophomore organic chemistry, the Buchwald-Hartwig reaction. In 1995 Steven Buchwald reported that the C-N bond of an N-arylamine could be formed by reacting aryl bromides with secondary amines in the presence of a base and catalytic amounts of palladium (Scheme 1). 5 While examples of C-N bond formation certainly exist before 1995, the preceding example remains one of the most widely utilized procedures today.

Scheme 1. Buchwald amination reaction 5
The field of oxidative C-H amination employing a palladium catalyst is markedly less developed when compared to reactions yielding carbon-carbon (C-C) bonds. 1 Moreover, the intramolecular reaction has been more studied in comparison to its intermolecular counterpart. The syntheses of carbazoles and indoles have been extensively studied and, in the case of carbazole synthesis, demonstrated good functional group tolerance. 1 By taking advantage of the thermodynamically favored 5membered palladacycle intermediate, the need for C-H bond functionalization is circumvented in both instances through the additional use of stoichiometric amounts of copper 6,7 or hypervalent iodine 8 (Scheme 2).
In addition to carbazoles and indoles, the synthesis of indolines and lactams have also shown to be quite amenable to palladium catalyzed reactions. 7,9 Much like the synthesis of carbazoles, reactions producing indoline derivatives also exhibited excellent functional group tolerance with considerable success being achieved across a  1 Lactam synthesis also proved to be fairly fruitful as high yields were achieved in reactions producing β-, γ-, and δ-derivatives; however, the major drawback to this work was the necessity for a high degree of substitution α to the carbonyl of the amide functional group. 1,7 Scheme 2. Intramolecular synthesis of carbazoles and indazoles 1,[7][8] Few examples of intermolecular palladium catalyzed amination reactions exist via C-H activation. In 2006 Che and associates reported the successful amination of pyridine and oxime ether derivatives. 10 Che demonstrated that catalytic amounts of palladium in the presence of excess K 2 S 2 O 8 were effective in transferring an amide moiety. 1 The electronics of the amide derivative proved to be inconsequential as electron deficient systems were demonstrated to be amenable to Che's protocol (Scheme 3). Most recently, Hartwig and coworkers developed a method for regioselectively coupling phthalimide to sterically encumbered arenes using catalytic amounts of palladium and hypervalent iodine. 11 While Hartwig's findings provided high yielding substrates, his methodology was limited in that regioselectivity was governed by sterics, and owing to the fact that the proposed mechanism proceeded through concerted metalation-deprotonation (CMD), little selectivity between para and meta substituted isomers were observed.

Copper Catalysis
C-N bond formation is substantially more developed with regards to copper catalysis, and an excellent review exists highlighting major accomplishments in the field. 12 For example, one of the better-known copper catalyzed amination reactions involves the arylation of arylamines, the Ullmann coupling reaction. In this reaction, aryl-halides may be coupled to aniline derivatives, or N-containing heterocycles, in the presence of a copper catalyst to yield the corresponding aryl/diarylamines ( Figure 1). 12 N O

Figure 1. Arylation of N-heterocycles 12
Ullmann coupling is typically not amenable to aliphatic systems, i.e.
alkylamines, with the exception of chelating amines such as amino acids. 12 Alternatively, propargyl amines may be synthesized from gold salts, such as AuBr 3 and AuCl, by coupling an alkyne, aldehyde, and amine by C-H activation (Scheme 7). This method does however possess minor drawbacks, as gold salts have a tendency to rapidly reduce into an inert metal species upon olefin activation. 16

Introduction
The synthesis of carbon-nitrogen (C-N) bonds through the oxidative crosscoupling of carbon-hydrogen (C-H) and nitrogen-hydrogen (N-H) bonds is an area that has generated significant interest due to the ubiquity of C-N bonds in a variety of pharmaceuticals and natural products. The utility of late-stage transition metals, more specifically Pd, Rh, Ru and Cu, for C-H bond activation has been extensively studied and the research field has been reviewed several times. 1 Over the course of the last two decades, gold-catalyzed reactions have played a significant role in carbon-carbon

Results and Discussion
We hypothesized that transition metal catalysts that do not metallate arenes by the CMD mechanism would further enhance the regioselectivity of our original findings, and we quickly discovered that heating a solution of chloro(triphenylphosphine)gold(I), phthalimide (1), and PIDA in o-xylene resulted in a 56% conversion of 1 to the desired phthalimide-protected aniline derivatives, 2a and 2b, in a 7:93 ratio, as determined by gas chromatography. Excited by this new lead, we began the optimization process by probing the role of PIDA in the reaction. By conducting control reactions and varying the loading of PIDA, several important trends were observed (Table 1).
First, the necessity for hypervalent iodine in the reaction was illustrated when PIDA was completely omitted from the reaction (Entry 1). Additionally, we observed a steady increase in the conversion of 1 to the desired phthalimide-protected aniline derivative (2) until the reaction plateaued at 4 equiv of PIDA (Entries 2-7). hypothesized that two competing reaction pathways were taking place in these reactions, with metalation of the arene by the gold catalyst being favored over the noncatalyzed amination that we previously described. Other hypervalent iodine sources such as phenyliodine(III) bis(trifluoroacetate) (PIFA), and 2-iodoxybenzoicacid (IBX) were not amenable to this amination; nor were other metal-based oxidants, such as silver acetate.
Attempting to further enhance starting material conversion, we sought to determine if the reaction was stalling as a result of catalyst decomposition or oxidant consumption. A phosphorus NMR of the crude reaction mixture showed that the original gold catalyst still remained in solution. Drawing inspiration from Hartwig's Pd-catalyzed amination, we began adding additional equivalents of oxidant throughout the course of the reaction. 6 It was ultimately determined that by adding an additional 4 equiv of PIDA to the reaction after 12 hrs, a moderate increase in yield could be obtained without impacting regioselectivity. Both the gold-catalyzed reactions that are described herein and the previously described palladium catalyzed and metal-free aminations, also form biaryl side products, thus at least partially accounting for the need for additional equivalents of the oxidant.
Upon the completion of the oxidant screen, we sought to further enhance the conversion of starting material and the regioselectivity by probing the effects of different phosphine ligands ( Table 2). Various gold-phosphine complexes were synthesized according to known precedures 7 and then subjected to the optimized reaction conditions. Bulky biaryl-containing ligands exhibited a drop-off in conversion, while trialkylphosphine ligands provided enhanced conversion of the starting material and simultaneously preserved the lead reaction's regioselectivity. It was ultimately decided to continue the investigation with tricyclohexylphosphine due to its ease of handling. It is also worth mentioning that decreasing catalyst loading severely reduced the reaction rate, while an increase in catalyst loading only modestly impacted regioselectivity.  Subsequently, a variety of simple arenes with various functionalities were subjected to the optimized reaction conditions. These experiments indicated that our protocol appeared to be most amenable to electron-rich systems. As a result, our substrate scope illustrates the effects of the gold-catalyzed reaction on various halogenated arenes and arenes possessing electron-donating groups (EDG) ( Table 3).
The benefit of the gold catalyzed reaction lies in its significantly enhanced regioselectivity. While conducting a similar reaction with other transition metals, such as palladium, may allow for amination of more electron-deficient systems, 6 the goldcatalyzed reaction provides significantly enhanced regioselectivities favoring parasubstituted isomers. We hypothesize that this is the result of an alternate mechanism that differs from our previously reported metal-free radical initiated pathway, and Hartwig's palladium-catalyzed CMD pathway. 5,6 The substitution patterns observed in the gold-catalyzed reaction appear to be governed by the same set of constraints observed in electrophilic aromatic substitution. Moreover, the predominant paraselectivity can be attributed to the large gold atom's preference to avoid positioning itself ortho to substituents.
Perhaps the most significant argument that can be made regarding whether or not the reaction is more heavily influenced by electronics or sterics is best illustrated by 10. In the amination of m-xylene, a clear preference for amination to occur para with respect to either of the methyl groups is observed, rather than aminating at the less sterically encumbered position.
Minor meta-substituted products were also observed in reactions producing 4-8, 10 and 11, all of which are derived from less electron-rich arene substrates. The exception to this rule is the amination of chlorobenzene (6), which surprisingly provided exclusive para-amination. We hypothesize that the meta-substituted products originate from a competing mechanism, the metal-free, radical-mediated reaction pathway. The meta-isomers are more often observed in less electron-rich systems, where electrophilic aromatic metalation (EAM) should be much slower. The inverse of this phenomenon is also illustrated in the reaction of the more electron-rich anisole substrate (3), which exhibits no meta-substitution, presumably because EAM is the dominant reaction pathway. a Regioselectivities determined via GC/MS against standards.

Table 3. Arene Substrate Scope
Having successfully established the substrate scope, we sought to further elucidate the reaction mechanism. To do this we first probed the kinetic isotope effect by performing a competition reaction using an equimolar solution of benzene/benzene-d 6 . A KIE value of 1.04 was obtained, which rules out the possibility 1) 4 equiv PhI(OAc) 2 10 mol % Cy 3 P-Au-Cl Arene (solvent), 100 o C, 24 h 2) 4 equiv PhI(OAc) 2  demonstrates that a CMD pathway is unlikely. In order to substantiate our claim that this reaction proceeds via EAM, additional internal competition reactions were performed ( Table 4). By carrying out the amination procedure in an equimolar mixture of an electron-rich arene with a comparatively electron-deficient arene we observed that amination of the more electron-rich system was dramatically favored in both instances. These findings support the hypothesis that the observed regioselectivity patterns were likely the result of EAM and lead to the proposed mechanism detailed in Scheme 2.  consistent with other Au-catalyzed halogenation, oxygenation and arylation reactions that have been previously reported. 3c-e The Au(III) catalyst could then metalate either the ortho-or para-positions, with the para-position being presumably more favored.
The metallated arene then proceeds to interact with an in situ-generated iodane species (14) via transmetalation. Once the imide reagent has been incorporated onto the gold species, the complex undergoes reductive elimination to afford the desired N-coupled product while regenerating the gold (I) catalyst. Future studies will be directed towards isolating N,O-iodanes, like 14, and studying their reactivities.

Conclusion
In conclusion, a regioselective gold-catalyzed protocol for the amination of arenes has been developed. As phthalimides can be easily converted into free amines, a direct route for regioselectively synthesizing aniline derivatives has been achieved.

Reagents
Substrates, including chloro(tricyclohexylphosphine)gold(I), phthalimide, and all arenes, were purchased from Sigma-Aldrich or Fisher Scientific. Iodobenzene diacetate was purchased from Acros Chemicals. Flash chromatography was performed using a Teledyne-Isco CombiFlash Rf with Redisep Gold silica cartridges or using a Biotage Isolera with SNAP Ultra silica cartridges. All reagents were stored under an inert atmosphere before use.

Instrumentation
GC/MS analyses were performed on an Agilent Technologies 6890 GC system with a 5973 mass selective detector. NMR spectra were obtained using a Bruker Avance 300 MHz spectrometer and a Varian Inova 500 MHz spectrometer.

General Reaction Conditions
A solution of phthalimide (15 mg, 0.102 mmol), iodobenzene diacetate (0.408 mmol), and chloro(tricyclohexylphosphine)gold(I) (0.01 mmol) in 3 mL of arene was assembled in a nitrogen-filled glove box. The reaction vial was then heated on an aluminum well-plate and allowed to magnetically stir for 12 hours at 100 °C. After 12 hours, the reaction was allowed to cool to room temperature and was brought back into the nitrogen-filled glove box where an additional 0.408 mmol of iodobenzene diacetate was added to the reaction vessel. The reaction was then allowed to stir for an additional 12 hours at 100 °C. Excess solvent was removed at reduced pressure and the crude reaction mixture was purified via flash chromatography with ethyl acetate and hexanes. The purified compound was allowed to dry in a preweighed vial overnight under high vacuum in order to determine product yield. Regioselectivities were assessed by NMR and GC/MS. In instances where NMR did not allow for definitive product identification, regioselectivities were determined via GC/MS against a set of standards.

Substrates 4, 8, 10, and 11
Regioselectivities were determined via comparison of GCMS retention times against independently synthesized standards as a result of overlapping signals in 1 H NMR.
Regioselectivity was obtained by integrating the area under each product's respective signal in order to obtain an isomeric ratio. (Table 1 and Table 2)

Results and Discussion
During the course of our previous investigations, we also became interested in the synthesis of new iodane and iodonium reagents containing I-N bonds that could be used as C-H aminating agents. Recently, Muñiz reported several novel hypervalent iodine(III) reagents for metal-free intermolecular allylic amination and diamination of alkenes. 12 Furthermore, diaryliodonium salts had been used by the Sanford group to arylate C-H bonds in the presence of catalytic palladium 13 and by the Gaunt group to arylate C-H bonds in the presence of catalytic copper. 14 Consequently, we synthesized phenyl(diphthalimido)-l 3 -iodane (3) by ligand exchange from iodobenzenebis(trifluoroacetate), PIFA, using a relatively simple ligand exchange reaction. 15 We also successfully synthesized a saccharin-derived iodane (4) and the phthalimide-iodonium salt (5) by modifying a route for synthesizing diaryliodonium salts from in situ-generated iodosobenzene (Scheme 2). 16 The new iodanes and the iodonium salt were subjected to a variety of reaction conditions mediated by palladium or copper with 2-arylpyridine substrates. We hypothesized that the I(III) species would react as an electrophilic amine, and that regioselective metalation of the ortho C aryl -H would create a nucleophilic carbon.
Though this initial hypothesis proved to be incorrect, we discovered a novel method to selectively aminate 2-phenylpyridines using iodanes such as 3, the results of which are presented herein.

Scheme 2 Synthesis of imide-substituted iodanes and an iodonium salt
As a control, we began by heating a solution of phthalimide, 2-phenylpyridine (6), and iodobenzene diacetate (PIDA) in acetonitrile using microwave irradiation and determined that PIDA was not able to directly aminate the pyridine derivative. As a result, the addition of catalytic palladium, along with PIDA or PIFA, was also explored. While modest amination was observed, palladium catalysis did not facilitate amination with acceptable yields. Interestingly, the use of the iodane oxidant (3) provided the desired amination product (7), while the iodonium oxidant (5) exclusively provided the arylated product (8). Consequently, we concluded that the iodane structure apparently favoured C-N bond formation, while the iodonium favoured C-C bond formation. The reason for this trend is not readily apparent and will be the subject of future studies. As a result, we elected to screen copper catalysts and discovered that copper(II) triflate showed higher amounts of amination products The reactions were sluggish, even at 145 °C, so as a compromise, we elected to increase the catalyst loading to 1 equiv. Such a consideration is only possible when dealing with affordable and non-toxic metals such as copper, as opposed to precious metals, like palladium.

Scheme 3 Palladium catalyzed reactions of 3 and 5
Further optimization indicated that Cu(OTf) 2 was the preferred copper salt and that dichlorethane (DCE) was the preferred solvent (See supporting information).
Cu(OAc) 2 and CuCl 2 provided low yeilds of 10 along with acetylated and chlorinated by-products (Table 1, entries 8 and 9). When optimizing the stoichiometry necessary for the iodane, an interesting pattern was observed. Since the I(III) species acted as both the nitrogen source and the oxidant in reactions containing substoichiometric Cu(OTf) 2 , 3 had to be used in excess. However, when reactions containing stoichiometric Cu(OTf) 2 , were run with 1 equiv of 3, lower yields were also observed (entry 6), so the excess iodane reagent was determined to be optimal.     The kinetic isotope effect was also studied using an intramolecular competition  (Table 2), and the competition studies (Table 3) indicate that the reaction favors electron rich arenes, which could be easily oxidized. Thus, we propose a pathway mediated by a radical cation (23) to explain the data (Scheme 4). 2b 82 from an aryl ring containing an electron withdrawing group. We propose that SET is the rate-limiting step in this reaction.

Conclusion
In conclusion, we have developed a novel, useful and economical process for the direct amination of 2-phenylpyridine derivatives. This process requires cheap and commercially available copper triflate and works for a variety of different 2phenylpyridine derivatives. Additionally, the process also works with a bis-saccharin iodane. Future endeavors aim to synthesize additional novel iodanes that contain I-N bonds in order to further develop this and other amination processes. were stored under an inert atmosphere before use.

General procedure for Substrate Library
To a solution of the appropriate 2-arylpyridine (1 equiv) in 1,2-dichloroethane (4 mL) was added the appropriate iodane (2.5 equiv) and Cu(OTf) 2 (1 equiv). The reaction was stirred for the 48 h at 80 o C in an oil bath before dilution with DCM (30 mL) and washing with saturated sodium bicarbonate solution (30 mL). The aqueous phase was

Synthesis of 20
Substrate 4 was subjected to the general procedure as described

Kinetic Isotope Effect
To

Concentration of product, ppm
Area under the curve, GC spectra

Introduction
The D 2 dopamine receptor is a membrane-bound protein that binds the neurotransmitter, dopamine, in the synaptic cleft. In addition to its normal functions that are associated with cognitive reward responses, the D 2 receptor is believed to be associated with a number of neurobehavioral disorders. 1 Degradation or inhibition of any of the D 1 -D 5 receptor subunits of dopamine have been linked to Parkinson's disease, certain motor and hyperactivity disorders, schizophrenia, and substance abuse.
As such, medicinal chemists have exhausted a significant amount of resources protein; and (4) a trifunctional scaffold must be identified that can incorporate the previously mentioned functionalities (Scheme 1).

Scheme 1: Construction of Serine Scaffold
The investigation began with the identification of an amino acid scaffold that could incorporate the three functionalities. As a result, serine was ultimately deemed the most appropriate, as the hydroxyl residue provided an excellent pathway for biotin conjugation via an ester linkage. In order to couple Boc-(L)-Ser-OH (1) to biotin, the serine had to first be protected on the carboxyl terminus. By subjecting 1 so a solution of allylbromide and Na 2 CO 3 in DMF, 2 was produced with an excellent yield (87%) and flash chromatography was unnecessary after extracting the product with EtOAc. EtOAc while the amine, 5, remained in the aqueous layer. 13 C NMR indicated that 5 was epimerized as a result of the deprotection procedure, presumably at serine's chiral center, but the diastereomeric mixture was carried on without resolution. In order to add an additional site of functionalization, 5 was introduced to a stirred solution of succinic anhydride in order to afford 6. The tethered carboxylic acid will serve as a site of attachment for the conjugated spiperone linker 11, which was synthesized according to a known procedure (scheme 2). 3 Introduction of the spiperone moiety has proven to be the most challenging aspect of the proposed synthesis. Prior attempts to directly alkylate spiperone to the amino acid scaffold proved to be ineffective as conditions necessary to carryout the substitution reaction readily hydrolyzed the linker from the serine scaffold or resulted in the corresponding olefin due to the elimination of bromine (Scheme 4). As a result, efforts were directed towards the orthogonal construction of 11 so that the resulting aniline functionality may be coupled directly to 6. However, aniline has proven to be too weak of a nucleophile and this step cannot be achieved through traditional peptide coupling procedures. group, for covalent tethering. The necessity for covalent linkage is due to the likelihood of protein denaturation during probe manipulation. In order to effectively remove the dopamine receptor from the cell membrane, the lipids will need to be dissolved through the use of detergents. This process will potentially liberate spiperone from the D-2 receptor, as a conformational change will alter binding affinity, however, a covalent linkage will allow the probe to remain tethered to the target molecule The desired epoxide moiety can be added to 13 via esterification with glycidol, affording the target molecule, 14. 7

Conclusion
In summation, this manuscript details the efforts made towards the synthesis of a spiperone-based probe and a proposed synthetic route for its successful completion.
Future work will focus on optimization and isolation of the target molecule with significant emphasis placed on the preservation of stereochemistry and synthesis of additional stereoisomers.

Synthesis of 2 1
Boc-L-Ser (1.00g, 4.87 mmol) is dissolved in 25 mL of DMF. Na 2 CO 3 (1.03g, 9.75 mmol) allyl bromide (0.46 mL, 5.37 mmol), and 0.7 mL of water is then added and stirred overnight. The reaction mixture is extracted into EtOAc and carried on to the next step without further purification (yield = 0.103 g, 89%).

Synthesis of 5
To a solution of 4 in EtOAc at 0 o C, 3M HCl was added drop wise. The reaction was monitored by TLC until consumption of the starting material was observed. The reaction was quenched with water and the biotin impurity that could not be removed during the synthesis of 4 was extracted into EtOAc. 5 was present as a colorless oil in the aqueous layer. This protocol led to epimerization of the chiral center.

INTRODUCTION
The second-year organic laboratory course is responsible for training a multitude of students across a wide variety of fields. Students enrolled in this course are not just those with an interest in chemistry, but rather the bulk of the course's population is often composed of students majoring in other scientific disciplines such as biological science, engineering, microbiology, or pharmaceutical sciences. As a result, the implementation of a curriculum that not only appeals to each student, but is also applicable to their chosen professions, is essential for maintaining an enthusiastic and engaged environment. Most students have been exposed to the topic of peptide coupling in their introductory life science courses and will continue to learn about the chemistry of amino acids, peptides and proteins as they progress toward their undergraduate science degrees. Several laboratory exercises about the synthesis of peptides have been described in this Journal, but, with two exception, they all rely on solid phase peptide synthesis techniques. [1][2][3][4]  amino acids, or make use of procedures that do not reflect modern solution-phase peptide chemistry, particularly with respect to stereochemistry preservation. [5][6][7][8][9] In our opinion, the most straightforward method for solution-phase peptide synthesis that has been described as a laboratory exercise was published by 1989, but it involves the use of a rare coupling agent and chiral TLC. 8 Herein, we report a multi-step synthesis of dipeptides that has been performed by over 400 students over the past three years.
The pedagogy of this laboratory exercise allows students to combine multiple techniques that they have learned throughout a typical second-year organic laboratory course.

PEDAGOGY
In the beginning of any organic laboratory course students are introduced to the basic techniques associated with organic synthesis. This typically includes techniques such as separatory extraction, pH manipulation, vacuum filtration, rotary evaporation etc. As they progress through the course the students are expected to become more confident with these skills. By the end of the one or two-semester laboratory course the students should be able to successfully synthesize, purify, and analyze moderately complex small molecules. The procedure detailed herein combines the skills students have learned throughout a typical organic laboratory course and concludes with the isolation and characterization of a bioorganic molecule made from starting materials that the students have developed themselves.
This experiment was carried out in the University of Rhode Island's second semester organic chemistry laboratory. The overarching goal of this experiment was to have the students perform a synthesis that utilized a majority of the skills and techniques covered throughout the semester. Students were graded based upon a predetermined rubric with substantial emphasis placed on the overall yield and purity of the desired product (See notes to the instructor for the complete rubric).
Unfortunately, time typically does not allow for individual inspection of the flasks containing the student's product, and as a result, reported yields are graded based on the honor system. Purity is judged solely on the student's proton NMR where substantial points are deducted for non-solvent impurities.
This experiment has been conducted, and subsequently improved upon, over the course of six semesters. The average class size per semester is approximately 90 students. In the most recent semester yields ranging from 5% -75% were reported for students who were successfully able to complete the experiment. It should be noted that each semester is typically met with several individuals who are unable to obtain product, however, in most cases it was determined that this was the result of an error made during the purification process (See notes to the instructor). In instances where product is not obtained the student is given a standard NMR of their target molecule and is awarded no credit for product yield and purity. Representative student spectra for each peptide can be found in the supporting information.

EXPERIMENTAL
The experiment began with the synthesis of N-acetyl-L-phenylalanine.
Students were instructed to measure 0.20 g of (L)-phenylalanine and synthesize the Nprotected amino acid according to a literature procedure. 1 After suspending Lphenylalanine in 1.0 M NaOH acetic anhydride is slowly, and carefully, added to the reaction vessel and allowed to stir uninterrupted for approximately 30 minutes. The product was extracted in ethyl acetate after acidifying the crude reaction mixture and was rotavapped to dryness. The reaction was very robust, producing yields >85%, with no evidence of unreacted starting material being observed after extraction. After the students had successfully isolated their product, the course instructor divided the class into three groups and each group was given a stock bottle containing a second amino acid that was protected at the carboxyl terminus as a methyl ester. These three amino acids were (L)-leucine methyl ester, (L)-valine methyl ester, and (L)phenylalanine methyl ester. Different amino acids were chosen in order to add variability to the purification and analysis processes in addition to the overall cost effectiveness of each reagent. This protocol should be amenable to any other naturally occurring amino acid, however, only the aforementioned reagents were tested. By completing the table below students determined the necessary amounts of each of the reagents needed in order to construct their dipeptide Table 1. All of the reagents shown in Table 1 were dissolved in 30 mL of DMF and were placed in the students' assigned lab drawers where they remained until their next scheduled lab period, approximately 48 hours later. (Note: Reactions were not stirred during this time.) After the correct solvent system was determined by trial and error, students prepared flash chromatography apparati resembling the image displayed in Figure 1.
The benefits this flash chromatography system, in addition to instructions detailing how to assemble it, are reported in a prior communications. 2 The crude reaction mixture was dissolved in a minimal amount of CH 2 Cl 2 and loaded onto a disposable silica column using a 1 mL syringe. The fractions containing the desired dipeptide were placed into a preweighed flask and concentrated under reduced pressure. The resulting powders were left to dry until the next scheduled lab period, when yields and samples for NMR analysis were obtained.

Figure 1. Flash Column Apparatus 10
Over the course of three years an excess of 400 students have completed this lab.
This experiment has served as the final evaluation of the skills student's have obtained throughout a typical synthesis laboratory. When the dipeptide was appropriately isolated a typical yield within the 50% range was reported. The most common contaminants observed in the final NMRs were the student's respective methyl ester and residual solvent peaks. Due to the fact this experiment was serving as a final exam, communication with the instructor was limited as students were encouraged to perform the experiment as independently as possible. As a result, we hypothesize greater yields and purities may be achieved with more instructor interaction.

CONCLUSION
In summation, the experiment described herein serves as an excellent final assessment of the skills obtained in a second-year organic synthesis laboratory.
Peptide coupling is a topic of significant importance and should appeal to a wide array of students across multiple disciplines while providing a hands-on opportunity to explore one of the more fundamental concepts covered in both introductory and advanced life science courses.

Materials and Methods
All reagents were purchased from either Fisher Scientific or Sigma Aldrich and were used without further purification. NMR spectra were obtained using a Bruker 300 MHz spectrometer in d 6 -DMSO.

Techniques in an Organic Laboratory Course
Throughout your organic chemistry tenure you have been taught the underlying principles necessary to construct simple organic molecules in the laboratory. In this lab you will see how the different branches of science can overlap as you strive to make one of life's most important structures, a peptide.
Thinking back to general biology you will recall that amino acids serve as the building blocks for life. Understanding how amino acid form peptides and proteins is pivotal for the development of vaccines and modern biologic drugs such as Enfuvirtide. The general features of an amino acid is a central carbon atom that is covalently bound to an amino group, a carboxyl group, a hydrogen atom, and an R-group; and with the exception of glycine, R is defined as any alkyl chain that may contain heteroatoms (Figure 1). It is also important to note that stereochemistry plays an important role in peptide synthesis.
You should take note that the central carbon atom present in naturally occurring amino   Place a small amount of the solid into a vial to be used in the future for TLC analysis.

Synthesis Of The Dipeptide:
Your instructor will give you a second amino acid that is already protected on the carboxyl terminus; this is the "R-OMe" section of your data table. Ask your instructor for the molecular weight of this amino acid. When you are determining your molar equivalents, you are going to use your newly synthesized N-acyl-Lphenylalanine as your limiting reagent. EDC is the peptide coupling agent and HOBt Your crude reaction mixture should now be a dry white powder. In order to perform TLC analysis, dissolve your product in 10 mL of CH 2 Cl 2 and find an appropriate solvent system for flash purification by testing a variety of mixture of hexanes and EtOAc. You should observe 2 or 3 spots, and it will be up to you to determine which spot corresponds to your peptide. Start with a 1:1 of Hex:EtOAc (10 mL total) and observe where your spots travel. Remember to co-spot your TLC plate with your acylated amino acid, methylated amino acid, and your extracted product.
Adjust your solvent system accordingly by increasing or decreasing your EtOAc concentration while remembering to keep the overall volume of the solvent system at 10 mL (i.e. if you decrease EtOAc by 1.0 mL be sure to add an additional 1.0 mL of hexanes). Once the appropriate solvent system has been determined (rf of desired product should be 0.3), show your instructor your TLC plate so he/she may confirm.
Transfer your solution into a pre-weighed round bottom and rotavap off the excess CH 2 Cl 2 . Once your round bottom containing the crude product has been dried, dissolve the product in a minimal amount of CH 2 Cl 2 (remember to use as little solvent as possible for this step). Load your sample into your flash column (remembering to saturate the column in the solvent system that you just determine by TLC prior to sample loading) and isolate your desired spot.
Combine all of the test tubes that contain your isolated dipeptide into a dry, preweighed round bottom flask and rotavap off the solvent. Allow your sample to dry overnight and prepare a sample for NMR analysis using deuterated DMSO during the next class.

Inst. 1
The addition of acetic anhydride to the solutions of (L)-phenylalanine in 1 M NaOH is noticeably exothermic. Ensure that students are not holding their flask during addition.

Inst. 2
When acidifying the reaction solution to a pH of 1-2, crystals will begin to crash out of solution. The crystals are pure N-acetyl-L-phenylalanine; however, much of the product still remains in solution. It is easiest to perform an extraction using EtOAc without isolating the crystals by filtration. The crystals will readily dissolve in the organic layer. The crystals can be coaxed out of solution with vigorous shaking if it does crystallization does not spontaneousl occur. This is a good indicator or whether or not the appropriate pH has been reached.

Inst. 3
If no crystals are observed, we have found that this typically is the result of over-acidification. This problem is easily remedied using the 1 M NaOH solution used to carryout the acylation procedure. Over-acidification resulted in severely reduced recovery of the acylated product after extraction, as it remained in the aqueous layer.

Inst. 4
If pH manipulation was performed correctly there was typically no evidence of unreacted (L)-phenylalanine present within the organic layer (TLC = 4% MeOH in CH 2 Cl 2 ).

Inst. 5
N-Acetyl-L-phenylalanine will present as a yellow oil if not completely dried. 10 minutes under high vacuum typically allowed for white powder development. Continuing the experiment using the oil did not inhibit the reaction.

Inst. 6
When distributing the second amino acid be mindful as to whether or not it is the hydrochloride salt derivative. Let the students know the correct molecular weight to put into their data tables.

Inst. 7
EDC was chosen due to the extreme sensitivity some individuals experienced with DCC (i.e. skin irritation and severe rash). Also, DCC was found to still be present in the reaction after extraction, whereas no evidence of EDC was observed.

Inst. 9
The dipeptides were liquid loaded onto the columns using CH 2 Cl 2 because they often did not fully dissolve in the Hex:EtOAc mixture necessary to perform column chromatography. Liquid loading volume depends on column size but in general do not exceed 2 mL of CH 2 Cl 2 when using a 10 g column. See reference 10 of the manuscript for details on the flash column apparatus system.

Inst. 10
Purification by adsorbing the crude reaction material onto silica provides the best separation. This method is slightly more costly, but may serve as a viable option for smaller classes. Biotage SNAP 10 g columns (Biotage HP-Sphere 25 um) provide a means for loading dry silica on top of a disposable silica cartage that can then be attached to the flash chromatography apparatus described.

Inst. 11
Purification difficulties arose from overloading the flash columns. Using a standard 10 g column no more than 2 mL of CH 2 Cl 2 should be used to load the crude reaction material onto the column. If not all of the material can be dissolved in 2 mL then run the column twice.

Inst. 12
It was found that students who did not obtain any product often did not perform the flash chromatography procedure correctly. Rather than spotting test tubes in order to determine if their product had successfully eluted from the column, several students simply stopped after 10 full test tubes had been collected (which was the outcome of a previous lab) and threw away their flash column.

Inst. 13
Rotomers present in final NMR of valine containing product.