Exam 6: Organic Chemistry

Passage Ethanol is an important source of energy that is frequently used as a supplement to gasoline.  It can be obtained from renewable resources such as the cellulose found in plant matter.  When subjected to a process called gasification, the plant mass is converted to a mixture called syngas, composed predominantly of carbon monoxide and molecular hydrogen.  Syngas can be passed over a rhodium catalyst to generate ethanol and other products.  After using this method, a group of researchers analyzed the resulting products by gas chromatography using helium as a carrier gas.  The results are shown in Figure 1. Figure 1  Gas chromatograph of syngas catalysis productsMass spectrometry revealed that the process yielded similar amounts of ethanol, acetic acid, and acetaldehyde, with trace amounts of ethane.  In addition, the ethanol peak was found to be contaminated with ethyl acetate.  Another group of researchers in the same laboratory had recently produced Compound 1 as a by-product in a separate reaction.  They combined Compound 1 with purified ethyl acetate to form megestrol acetate, a drug used in the treatment of certain cancers.  This reaction also produced ethanol, as shown in Scheme 1. Scheme 1 Adapted from Lopez L, Velasco J, Montes V, Marinas A, Cabrera S, Boutonnet M, Järås S. Catalysts  2015. -Ethyl acetate could be better separated from ethanol by doing which of the following?

Researchers separate a mixture by both simple distillation and vacuum distillation.  Which expression accurately describes the relationship of the mixture components' boiling points (bp) in each distillation?

Passage Chloramphenicol (Compound 1) is an antibiotic that acts against gram-positive and gram-negative bacteria by blocking peptidyl transfer, which inhibits protein synthesis by bacterial ribosomes.  However, it tastes bitter, so most patients cannot tolerate it.  As a result, synthesis of hydroxyl-substituted chloramphenicol analogues has become of interest because ester chloramphenicol analogues taste better than chloramphenicol.  Substituents on the primary hydroxyl group of chloramphenicol analogues are typically hydrolyzed quickly in vivo to regenerate Compound 1.  Compound 3, a chloramphenicol analogue, was synthesized by the transesterification of Compound 1 and vinyl propionate (Compound 2), catalyzed by the enzyme Lip_BA lipase (Figure 1). Figure 1  Transesterification of chloramphenicol catalyzed by Lip_BAAlthough enzyme catalysis allows for efficient regiospecific and stereospecific bond formation, the reaction conditions for the conversion efficiency of Compound 1 to Compound 3, as well as the purity of Compound 3, must also be optimized.  The reaction solvent, temperature, and time were studied to find the conditions that gave the highest conversion rate and purity.  Each parameter was altered individually while all others were kept constant, and the enzyme concentration was 4.5 g/L in each trial.  The results of this study are shown in Table 1.  The purity of Compound 3 was found to be over 90% when 1,4-dioxane was used and over 70% when dichloromethane was used.Table 1  Transesterification Conditions Optimization The transesterification reaction was monitored by thin-layer chromatography (Figure 2), and the product was confirmed by ¹H NMR.  The ¹H NMR spectra of Compounds 1 and 3 can be distinguished by the shift of H_e and H_f due to the addition of the electronegative ester functional group. Figure 2  Thin-layer chromatography of reaction mixture Adapted from F. Dong et al., "Transesterification Synthesis of Chloramphenicol." Molecules. ©2017 MDPI. -How does the addition of the ester to Compound 1 affect the shift of H_e and H_f?  The signals for H_e and H_f will move:

Passage Aldehydes are organic compounds encountered in everyday life and used as intermediates in metabolism.  Common aldehydes include formaldehyde, which is used in the fixation of tissues, and cinnamaldehyde, which provides the flavor and smell of cinnamon (Figure 1). Figure 1  Common aldehydesThe aldol condensation shown in Figure 2 is an important carbon-carbon bond-forming reaction that has many applications in the synthesis of bioactive molecules.  It also takes place in some metabolic processes, such as the citric acid cycle and gluconeogenesis.  The aldol condensation begins with the aldol reaction, which can be acid- or base-catalyzed and requires nucleophilic addition to a carbonyl to provide the aldol product, which can be a β-hydroxy ketone or aldehyde.  The final step of the aldol condensation consists of an acid- or base-catalyzed dehydration of the aldol product to yield an α,β-unsaturated ketone or aldehyde. Figure 2  General aldol reaction and condensationThe reverse of an aldol reaction or condensation is known as a retro-aldol, which is important in metabolism, most notably in glycolysis.  This reaction breaks a carbon-carbon bond to form aldehydes and/or ketones.  In glycolysis, fructose 1,6-bisphosphate is broken down into an aldehyde and a ketone, glyceraldehyde 3-phosphate (GAP) and dihydroxyacetone phosphate (DHAP), respectively (Figure 3). Figure 3  Retro-aldol in glycolysis -In some synthesis reactions, the carbonyl functional group needs to be protected from nucleophilic attack.  The reaction below of cinnamaldehyde with ethylene glycol shows the formation of which carbonyl-protecting group?

What functional group forms in an acid-catalyzed reaction between a secondary amine and a ketone?

Passage The protozoan Euglena gracilis expresses glycosyltransferases (enzymes that add carbohydrates to molecules) and glycosidases (enzymes that remove carbohydrates) within its membrane.  These enzymes were previously studied using radiolabeled sugars.  To avoid the use of radioactive materials, fluorescence-based assays were investigated.  Derivatives of the fluorescent molecule coumarin were attached to carbohydrates (Figure 1) for detection in fluorescence assays.Compound 1, a carbohydrate derivative with hydroxyl groups protected by benzoate (Bz), reacted with Compound 2 to form Compound 3.  ¹³C NMR confirmed the formation of Compound 3 by observing a distinct C-H coupling at the anomeric carbon.  Hydroxyl deprotection and addition of Compound 4 (a coumarin analogue) gave Compound 5, a fluorescent coumarin derivative.  Compound 6, another coumarin derivative, was made following a similar sequence except an additional sugar was attached before the addition of the coumarin group. Figure 1  Synthesis of fluorescent coumarin derivatives (Note: HCT denotes the fluorescent butyl hydroxycoumarin triazole group.)Small vesicles called microsomes were isolated from Euglena gracilis membranes, and microsomal glycosyltransferase activities were investigated using a hexose donor substrate and either Compound 5 (Figure 2) or Compound 6 (Figure 3) as the acceptor substrate.  Thin-layer chromatography (TLC) analysis indicated the formation of a new product in each assay.  These products were purified and characterized by liquid chromatography-mass spectrometry (LC-MS).  The molecular ion for each product was subjected to another round of MS, in which individual hexose units (MW) = 162) were cleaved from the coumarin derivatives that were formed during the assay.  The MW of hydroxycoumarin triazole (HCT) is 301. Figure 2  Analysis of Compound 5 as the acceptor substrate: (A) TLC analysis of fluorescent products; (B) Mass spectrum of product A fragmented molecular ion peak Figure 3  Analysis of Compound 6 as the acceptor substrate: (A) TLC analysis of fluorescent products; (B) Mass spectrum of product B fragmented molecular ion peak Adapted from: I. M. Ivanova et al., "Fluorescent mannosides serve as acceptor substrates for glycosyltransferase and sugar-1-phosphate transferase activities in Euglena gracilis membranes." Carbohydrate Research. © 2017 Elsevier. -What is the configuration of the sugar in the coumarin derivatives shown in Figure 1?

Passage Chloramphenicol (Compound 1) is an antibiotic that acts against gram-positive and gram-negative bacteria by blocking peptidyl transfer, which inhibits protein synthesis by bacterial ribosomes.  However, it tastes bitter, so most patients cannot tolerate it.  As a result, synthesis of hydroxyl-substituted chloramphenicol analogues has become of interest because ester chloramphenicol analogues taste better than chloramphenicol.  Substituents on the primary hydroxyl group of chloramphenicol analogues are typically hydrolyzed quickly in vivo to regenerate Compound 1.  Compound 3, a chloramphenicol analogue, was synthesized by the transesterification of Compound 1 and vinyl propionate (Compound 2), catalyzed by the enzyme Lip_BA lipase (Figure 1). Figure 1  Transesterification of chloramphenicol catalyzed by Lip_BAAlthough enzyme catalysis allows for efficient regiospecific and stereospecific bond formation, the reaction conditions for the conversion efficiency of Compound 1 to Compound 3, as well as the purity of Compound 3, must also be optimized.  The reaction solvent, temperature, and time were studied to find the conditions that gave the highest conversion rate and purity.  Each parameter was altered individually while all others were kept constant, and the enzyme concentration was 4.5 g/L in each trial.  The results of this study are shown in Table 1.  The purity of Compound 3 was found to be over 90% when 1,4-dioxane was used and over 70% when dichloromethane was used.Table 1  Transesterification Conditions Optimization The transesterification reaction was monitored by thin-layer chromatography (Figure 2), and the product was confirmed by ¹H NMR.  The ¹H NMR spectra of Compounds 1 and 3 can be distinguished by the shift of H_e and H_f due to the addition of the electronegative ester functional group. Figure 2  Thin-layer chromatography of reaction mixture Adapted from F. Dong et al., "Transesterification Synthesis of Chloramphenicol." Molecules. ©2017 MDPI. -What is the number of signals corresponding to aromatic hydrogen atoms present in the ¹H NMR spectrum of Compound 3?

Passage Two key ingredients found in many soaps are PEG 150 distearate and ethylhexyl glycerin.  Both can be synthesized from glyceryl tristearate (Figure 1). Figure 1  Formation of PEG 150 distearate and ethylhexyl glycerin from glyceryl tristearateThe first step in this process requires the saponification of glyceryl tristearate.  The resulting stearate molecules can then react with PEG 150 in an acid-catalyzed Fischer esterification to form PEG 150 distearate.  The glycerol produced during saponification is used as a nucleophile to form ethylhexyl glycerin through a base-catalyzed S_N2 reaction with an ethylhexyl halide.Studies have shown that hand washing with soap can substantially reduce the number of germs present on hands and that washing hands before preparing food is particularly effective in reducing illness.  Researchers examined the effect of washing for 20 seconds with different amounts of soap containing PEG 150 distearate and ethylhexyl glycerin and found an inverse correlation between the volume of soap used and the amount of bacteria recovered from the hands.  The data are shown in Figure 2. Figure 2  Bacteria recovered from hands after washing with varying amounts of soapThe researchers then investigated the effect of hand washing with different kinds of soap on the transfer of bacteria from hands to objects.  Volunteers washed with a fixed volume of soap for 30 seconds, then handled sterilized plastic spheres for 30 seconds.  Bacteria were then recovered from the spheres.  The results are shown in Table 1.Table 1 Bacteria Recovered from Objects Adapted from  Fuls JL, Rodgers ND, Fischler GE, et al. Alternative hand contamination technique to compare the activities of antimicrobial and nonantimicrobial soaps under different test conditions. Appl Environ Microbiol. 2008. -Chemically equivalent protons are present in each of the following compounds EXCEPT:

Passage Aldehydes are organic compounds encountered in everyday life and used as intermediates in metabolism.  Common aldehydes include formaldehyde, which is used in the fixation of tissues, and cinnamaldehyde, which provides the flavor and smell of cinnamon (Figure 1). Figure 1  Common aldehydesThe aldol condensation shown in Figure 2 is an important carbon-carbon bond-forming reaction that has many applications in the synthesis of bioactive molecules.  It also takes place in some metabolic processes, such as the citric acid cycle and gluconeogenesis.  The aldol condensation begins with the aldol reaction, which can be acid- or base-catalyzed and requires nucleophilic addition to a carbonyl to provide the aldol product, which can be a β-hydroxy ketone or aldehyde.  The final step of the aldol condensation consists of an acid- or base-catalyzed dehydration of the aldol product to yield an α,β-unsaturated ketone or aldehyde. Figure 2  General aldol reaction and condensationThe reverse of an aldol reaction or condensation is known as a retro-aldol, which is important in metabolism, most notably in glycolysis.  This reaction breaks a carbon-carbon bond to form aldehydes and/or ketones.  In glycolysis, fructose 1,6-bisphosphate is broken down into an aldehyde and a ketone, glyceraldehyde 3-phosphate (GAP) and dihydroxyacetone phosphate (DHAP), respectively (Figure 3). Figure 3  Retro-aldol in glycolysis -Do aldehydes and ketones tend to have higher or lower boiling points than their corresponding alcohols?

Can α-D-fructose and β-D-fructose be classified as anomers?

If an alcohol were to undergo a substitution reaction, which of the following modifications would improve the alcohol's leaving group ability?

Citrulline is an important component in the urea cycle and in the cellular production of nitric oxide (NO).  When L-arginine is oxidized via a nitric oxide synthase (NOS) enzyme, it results in the formation of L-citrulline and the release of NO(g).  Which structure most likely corresponds to citrulline?

Passage Electrospray ionization mass spectrometry (ESI-MS) employs a soft ionization technique that, unlike traditional ionization via electron bombardment, results in little to no fragmentation of target molecules.  During electron bombardment, electrons collide with the analyte and cause the molecule to lose an electron whereas during ESI-MS, ions are produced by passing the analyte solution through an electrospray needle that has a potential difference applied with respect to a counterelectrode.  This leads to the formation of charged droplets, which are repelled from the needle toward the counterelectrode and mass spectrometer, as shown in Figure 1. Figure 1  ESI-MS ionization mechanismDuring the droplet's flight, the solvent is evaporated off analyte molecules.  Ionization occurs through the protonation of basic sites on target molecules.  Reaction 1 shows the number of protons n added to molecule M.M → (M + nH)ⁿ⁺Reaction 1Researchers used ESI-MS to quantify the formation of four pyridyloxobutyl-DNA (POB-DNA) adducts, shown in Figure 2.  POB-DNA adduct formation involves highly mutagenic pyridyloxobutylating agents, which are formed from cytochrome P450-mediated hydroxylation of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), an abundant N-nitrosamine carcinogen found in cigarette smoke.  Adducts were produced in vitro by treating samples of calf thymus DNA with NNK.  The DNA adducts were then separated by reversed-phase high-performance liquid chromatography (RP-HPLC) and analyzed via ESI-MS. Figure 2  Molecular structure of POB-DNA adducts Adapted from Lao Y, Villalta PW, Sturla SJ, Wang M, Hecht SS. Quantitation of pyridyloxobutyl DNA adducts of tobacco-specific nitrosamines in rat tissue DNA by high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry. Chem Res Toxicol. 2006. -Which of the following changes could be made to this experiment to increase the signal intensity of peaks that correspond to smaller mass-to-charge (m/z) values relative to larger m/z values?

Passage Students used three classification tests to study the reactivity of alcohol, aldehyde, and ketone functional groups in organic compounds.  The organic compounds provided to the students are given in Table 1.Table 1  Organic Compounds Tested First, the students performed the Lucas test by mixing a few drops of each compound with 2 mL of the Lucas reagent (ZnCl₂ and HCl).  This reagent is used to test for secondary and tertiary alcohols.  A positive test result is indicated by a cloudy reaction mixture upon addition of the Lucas reagent due to the formation of an insoluble compound by an S_N1 reaction with a secondary or tertiary alcohol (Reaction 1). Reaction 1Next, a test for alcohols and aldehydes was performed by the addition of a drop of chromic acid reagent (CrO₃ dissolved in aqueous H₂SO₄) into samples of the organic compounds.  The disappearance of the orange color from chromic acid and formation of a blue-green suspension is indicative of a positive result for this test.  Compound A was found to be an example of a positive result, as shown in Reaction 2. Reaction 2Finally, samples of the compounds were also dissolved in water, followed by the addition of NaOH and iodine.  This test is known as the iodoform test, which is used to identify methyl ketones and secondary alcohols.  A positive result is indicated by the formation of iodoform (CHI₃), a yellow precipitate (Reaction 3). 3Table 2  Selected Observations from the Classification Tests -Based on the results presented in Table 2, which compounds would give a positive result for the iodoform test?CH₃CH₂CHOCH₃(CH₂)₄OHCH₃(CH₂)₂C(O)CH₃CH₃(CH₂)₂CH(OH)CH₃

Passage Hemophilia B is a blood clotting disorder caused by a factor IX (FIX) deficiency.  FIX is a 57-kDa, vitamin K-dependent protease that activates factor X, leading to the conversion of prothrombin to thrombin for propagation of the clotting cascade.  Activated FIX has two major domains:  a γ-carboxyglutamic acid domain and a serine protease domain.  The γ-carboxyglutamic acid domain participates in the oxidation of vitamin K using metallic cofactors, as shown in Figure 1. Figure 1  Oxidation of vitamin KTo further analyze the γ-carboxyglutamic acid-rich domain of FIX, an analogous synthetic peptide composed of matching residues 1 through 49 on FIX was evaluated by proton nuclear magnetic resonance (NMR) spectroscopy.  Analysis of the proton chemical shift before the addition of metal ions suggested that the synthetic peptide contained normal structural elements.  Large chemical shifts were observed after the addition of calcium and beryllium, as shown in Figure 2. Figure 2  Results of NMR spectroscopy (tetramethylsilane [TMS] peak has been removed)The synthetic analog was then placed in solution with vitamin K hydroquinone and cofactors required for vitamin K oxidation.  The oxidation products of vitamin K in Reactions 1 and 2 were collected and evaluated under high-performance liquid chromatography (HPLC) using hexane as the mobile phase.  Analysis demonstrated cis and trans isomers of vitamin K₁ and a trans-epoxy vitamin K₁, as shown in Figure 3. Figure 3  Results of HPLC separation Adapted from Freedman SJ, Furie BC, Furie B, Baleja JD. Structure of the metal-free gamma-carboxyglutamic acid-rich membrane binding region of factor IX by two-dimensional NMR spectroscopy. J Biol Chem. 1995. -During the oxidation of vitamin K in Reaction 1, an amino acid residue is carboxylated and results in the following structure: What was the most likely initial amino acid residue?

Passage A group of students studied the reactivity of four alkyl halides (R−X) in two nucleophilic substitution reactions.  Nucleophilic substitution reactions can go through one of two mechanisms.  The S_N2 reaction requires substitution of a leaving group with a nucleophile in a single step (Reaction 1), and the S_N1 reaction requires elimination of the leaving group and formation of an intermediate before nucleophilic substitution can occur, resulting in a two-step process (Reaction 2).R−X + NaI → R−I + NaX↓Reaction 1R−X + AgNO₃ → R⁺ + AgX↓ + HOCH₂CH₃ → R−OCH₂CH₃ + HNO₃Reaction 2Experiment 1The students performed the S_N2 reaction (Reaction 1) by the addition of a few drops of each alkyl halide into a separate test tube containing 1 mL of 15% solution of NaI in acetone at 25°C.  After mixing, they monitored the reaction for the formation of a precipitate (sodium halide salt).  If no precipitate formed after 5 minutes, they raised the temperature of the reaction to 50°C and again checked for the formation of a precipitate.  Formation of a precipitate indicated the substrate underwent nucleophilic substitution.  The results observed for the Finkelstein reaction are shown in Table 1.Table 1  Results of the S_N2 Reaction Experiment 2The students repeated the procedure followed in Experiment 1 except they added a few drops of each alkyl halide into a separate test tube containing 1 mL of 1% solution of AgNO₃ in ethanol at 25°C.  The results observed for this reaction are shown in Table 2.Table 2  Results of the S_N1 Reaction -If 3-bromo-2,4-dimethylpentane (shown below) was used as a substrate in the experiments described in the passage, what would the observation be for reaction with NaI at 25°C?

Thin-layer chromatography was used to determine the best solvent system for purification of a mixture by column chromatography.  One spot was observed near the origin using hexanes, one spot near the solvent front using ethyl acetate, and two spots separated using 1:1 hexanes/ethyl acetate.  Which statement accurately describes the observations?

When a carboxylic acid reacts with phosphorus tribromide and bromine and then water, an α-bromoacid is formed.  What is the structure of the product of this reaction if a three-carbon carboxylic acid is used?

Passage Ethanol is an important source of energy that is frequently used as a supplement to gasoline.  It can be obtained from renewable resources such as the cellulose found in plant matter.  When subjected to a process called gasification, the plant mass is converted to a mixture called syngas, composed predominantly of carbon monoxide and molecular hydrogen.  Syngas can be passed over a rhodium catalyst to generate ethanol and other products.  After using this method, a group of researchers analyzed the resulting products by gas chromatography using helium as a carrier gas.  The results are shown in Figure 1. Figure 1  Gas chromatograph of syngas catalysis productsMass spectrometry revealed that the process yielded similar amounts of ethanol, acetic acid, and acetaldehyde, with trace amounts of ethane.  In addition, the ethanol peak was found to be contaminated with ethyl acetate.  Another group of researchers in the same laboratory had recently produced Compound 1 as a by-product in a separate reaction.  They combined Compound 1 with purified ethyl acetate to form megestrol acetate, a drug used in the treatment of certain cancers.  This reaction also produced ethanol, as shown in Scheme 1. Scheme 1 Adapted from Lopez L, Velasco J, Montes V, Marinas A, Cabrera S, Boutonnet M, Järås S. Catalysts  2015. - What atom indicated would be most readily deprotonated if Compound 2 reacts with NaOH?

Passage The N-terminus of an amino acid must be protected with 9-fluorenylmethoxycarbonyl (Fmoc) during solid state peptide synthesis to avoid side reactions (Scheme I). Scheme IThe Fmoc protecting group is typically removed with piperidine (Scheme II), a heterocyclic secondary amine. Scheme IIBecause the use of piperidine is restricted, structurally similar amines were examined as possible replacements.  To determine whether piperidine analogues were suitable alternatives for Fmoc removal, deprotection of Compound 2 was carried out with several amine bases (Scheme III). Scheme IIICompound 2 was prepared from L-tryptophan and Fmoc-Cl, and the reaction was monitored by thin-layer chromatography (TLC) and visualized by ultraviolet light.  The deprotections were done with piperidine and each analogue using a microwave set to 155 watts at 75 °C for 15 seconds.  The products of the deprotections were analyzed by high-performance liquid chromatography (HPLC) and mass spectrometry; the retention times of the products were compared to tryptophan and Compound 2 standards. Adapted from O.P. Luna et al, "Deprotection Reagents in Fmoc Solid Phase Peptide Synthesis: Moving Away from Piperidine?." Molecules. ©2016 MDPI. -Based on information in the passage, which amine was most likely proposed as a replacement for piperidine in Scheme III?