Exam 6: Organic Chemistry

Which separation technique is optimal for purification of small sample sizes and employs a stationary phase, a solvent mobile phase (under pressure), and a detector to separate compound mixtures based on polarity?

A carboxylic acid is reacted with NaOH in an organic solvent followed by a reaction with an acid chloride.  What is the general structure of the carboxylic acid derivative formed in this reaction?

Passage Students perform a synthesis of a carboxylic acid derivative.They mix a 10.00-mL sample of hexanoic acid, 2.33 mL of ethanol, and 0.64 mL of concentrated sulfuric acid in a flask with a water condenser fitted to its neck.  The mixture is boiled for one hour and then poured into water when the reaction is complete.  The aqueous and organic layers are separated, and the organic layer is washed once with NaHCO₃ and twice with water.  Solid Na₂SO₄ is added to the organic layer to remove any water present.  The organic layer and Na₂SO₄ are separated by filtration.  The filtrate is purified by fractional distillation to yield the carboxylic acid derivative, a colorless liquid. -If the carboxylic acid derivative that formed in the passage is reacted with an excess of 3-methylbutan-1-ol and an acid catalyst, which of the following compounds will be the product?

Compounds 1 and 2 can be described as:

The (R)-enantiomer of the antiasthma drug albuterol is the active isomer.  If a researcher wants to separate a racemic mixture of albuterol, which of the following methods will most likely separate the enantiomers?

The molecule (3R,4S)-3-Bromo-4-chlorocyclohexanone is shown below: Which of the following structures depicts the same molecule?

Passage Solid-phase peptide synthesis is normally carried out on a resin bathed multiple times in dimethylformamide (DMF)-a toxic, polar, aprotic solvent-which poses an environmental problem because a large amount of toxic solvent waste is created.  Cyclic carbonates such as propylene carbonate (PC) and ethylene carbonate (EC) are nontoxic, polar, aprotic solvents that could be used as an alternative to DMF.Compounds 1 and 2 are modified L-amino acids.  They were coupled to form Compound 3, a dipeptide, which was then deprotected to yield Compound 4 (Figure 1). Figure 1  Synthesis of Compound 4To determine the effect that solvent type and temperature have on the synthesis of this peptide, the nontoxic solvents PC and EC as well as the toxic solvent DMF were each used in separate syntheses of Compound 4 at different temperatures.  The reaction solvent was also used to rinse the resin.  Table 1 shows the results of these syntheses.Table 1  Results from the Synthesis of Compound 4 The successful synthesis of Compound 4 using nontoxic solvents encouraged researchers to pursue the synthesis of a larger, biologically active peptide.  Bradykinin, a vasodilator containing nine amino acids (Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg), was synthesized using the same solid-state peptide synthesis protocol used to synthesize Compound 4.  Separate syntheses were done using either DMF or PC, and the purity of bradykinin from each synthesis was analyzed by high-performance liquid chromatography (HPLC) (Figure 2).In each chromatogram, peak 1 has the same retention time as commercially obtained bradykinin, and the other peaks correspond to impurities.  In chromatogram A, the impurity has a similar retention time to the product peak whereas the impurity peaks in chromatogram B have distinct retention times compared to the product peak. Figure 2  HPLC of synthetic bradykinin: (A) synthesis in DMF; (B) synthesis in PC Adapted from S. B. Lawrenson et al., "The greening of peptide synthesis."  Green Chem. ©2017 The Royal Society of Chemistry. -The L designation of the amino acids used in peptide synthesis is based on the absolute configuration of L-glyceraldehyde.  What is the structure of L-glyceraldehyde?

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. -Consider the cyclic form of the sugar from which Compound 1 is derived.  Can this sugar be classified as a pyranose and a hemiacetal?

Which of the following characterize gas-liquid chromatography?Gas mobile phaseLiquid stationary phaseSeparation based on polarityRoom-temperature column conditions

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. -Prior to adduct formation, DNA was isolated from a cellular mixture.  Membrane lipids were first extracted using an organic solvent, which was then evaporated, and DNA was precipitated from the aqueous layer using cold ethanol and sodium acetate.  Which of the following mechanisms allowed for DNA precipitation?

The above chromatogram shows the separation of a mixture containing an ester, a carboxylic acid, an alcohol, and an alkane.  If normal-phase high-performance liquid chromatography is used to separate the mixture components, which peak corresponds to the alcohol?

Two separate reactions are conducted in which a compound containing a ketone, an ester, and a carboxylic acid is reacted with borane (BH₃) in THF in one reaction and with NaBH₄ in methanol in the other.  Which of the following explains why different products are observed?

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. -Why does the bond between glycerol and the ethylhexyl group tend to form at glycerol's C1 hydroxyl group instead of the C2 hydroxyl group?

Suppose that a 0.2 g/mL solution of (S)-ibuprofen in a 2 dm long cell rotates plane-polarized light +10°.  What is the specific rotation for (S)-ibuprofen?

Passage The β-lactam scaffold is an important feature in a class of broad-spectrum antibiotics that includes the penicillins, cephalosporins, and monobactams.  These antibiotics are used to treat a variety of diseases caused by bacteria.  The penicillins work by inhibiting a step in the synthesis of peptidoglycan.  However, the development of antibiotic resistance in the form of enzymes such as β-lactamase is an ongoing problem.An aromatic ring linked to the β-lactam ring has been reported to participate in hydrophobic interactions with β-lactamase active sites, and an aromatic ring bonded to the nitrogen of the β-lactam has shown to be beneficial to biological activity.  With this in mind, researchers synthesized a group of penicillanic acid analogues with substituted aromatic rings bonded to the β-lactam ring in an effort to overcome the challenge of antibiotic resistance.  The substituents on the aromatic rings were varied, including -OCH₃ and -NO₂, to make several analogues.  A key step in the synthesis of these analogues was the coupling of Compounds 1 and 2 to form a compound with two β-lactam rings (Reaction 1). Reaction 1The researchers studied the analogues' structure-activity relationship and antimicrobial activity against the gram-negative bacteria Escherichia coli and the gram-positive bacteria Staphylococcus aureus.  Ampicillin, a penicillin derivative used to treat illnesses brought about by gram-positive and gram-negative bacteria, was used as a control.  The minimal inhibitory concentrations are shown in Table 1.Table 1  Minimal Inhibitory Concentration (MIC) The penicillanic acid analogues were tested for their effect on mammalian cell viability because toxicity to cells is also an important factor to consider for potential drug candidates.  The general acceptable level of cell viability for a drug candidate is 70%.  These results are shown in Figure 1. Figure 1  Cell viability assay results Adapted from De rosa M, Vigliotta G, Palma G, Saturnino C, Soriente A. Novel Penicillin-Type Analogues Bearing a Variable Substituted 2-Azetidinone Ring at Position 6: Synthesis and Biological Evaluation. Molecules. 2015. -The precursor to Compounds 3-6 is shown below with carbonyl carbon atoms labeled. Which of the carbonyl carbon atoms is most likely to undergo hydrolysis when treated with a weak acid at room temperature?

Which term can be used to classify the relationship between two isomers that have the same connectivity but specific rotations of +40° and −25°, respectively?

Passage Fluorescent amino acids are useful for labeling proteins because they can be visualized spectroscopically, facilitating the study of protein structure and interactions.  The noncanonical amino acid dansylalanine, a fluorescent amino acid that is sensitive to the hydrophobicity of its environment, is a good candidate to be incorporated into proteins because of its small size and fluorescent capabilities.  Dansyl chloride and Compound 1 were used to synthesize dansylalanine, as shown in Reaction 1.  Dansylalanine absorbs maximally at 340 nm and emits at approximately 540 nm. Reaction 1  Synthesis of dansylalanineA leucyl-tRNA synthetase from Escherichia coli was engineered to aminoacylate bacterial tRNAs containing a CUA anticodon with dansylalanine.  After verifying that the tRNA/synthetase pair does not interact with endogenous amino acids or tRNAs, it was transfected into the yeast Saccharomyces cerevisiae along with a plasmid encoding a mutated human superoxide dismutase (hSOD).  Using this system, dansylalanine was genetically incorporated into hSOD in place of either Gln-16 or Trp-33, and expressed in S. cerevisiae.Position 16 is located on the surface of hSOD at the N-terminus of a β-strand, and position 33 lies in the center of a strand within a β-barrel.  To probe changes in the structure of hSOD, researchers exposed the purified protein to guanidinium chloride (GdmCl) to denature the protein at concentrations ranging 0-4.5 M.  The experiment was conducted in a 35 mM sodium phosphate buffer at pH 7.2.  The fluorescence of hSOD with dansylalanine at either position 16 or 33 was monitored at each GdmCl concentration, and the results of the unfolding experiment are shown in Figure 1. Figure 1  Plot of dansylalanine fluorescence at different concentrations of GdmCl Adapted from Summerer D, Chen S, Wu N, Deiters A, Chin JW, Schultz PG. A genetically encoded fluorescent amino acid. Proc Natl Acad Sci USA. 2006. -Is the absolute configuration of dansylalanine shown in Reaction 1 different from most naturally occurring amino acids?

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. -Based on the structure of Compound 1 provided in Figure 1, which stretch gives a narrow absorption of weak intensity at approximately 3100 cm⁻¹ in the infrared spectrum?

Compound 1 is shown below. Which of the following conformations of Compound 1 has intramolecular hydrogen bonding?

A solution of Compound 1, shown below, absorbs light maximally at 448 nm in the absence of copper(II) ions but shifts to a 623 nm absorption maximum upon the addition of Cu²⁺.  Which of the following best describes this process?