Welcome and thanks for reading. "A bear is wiser than a man because a man does not know how to live all winter without eating anything." Abenaki (People of the Dawn) saying. This is the third post describing the metabolic and nutritional chemistry of bear hibernation. Click on the following links for Part 1 and Part 2.
Specifically, this post begins discussion about the organic chemistry of the multi-step (4 distinct steps overall), enzyme-dependent degradation (catabolic) pathway known as the β-oxidation of saturated fatty acids, where β is the lower case Greek letter beta. This elegantly tuned, precisely organized, irreversible bond breaking-making catabolic pathway permits a hibernating bear to not die from starvation during the long foodless, calorie deficient winter months. Basically, it permits a bear to "eat" itself, specifically to "eat" its white adipose fat reserves that a bear sufficiently accumulated (hopefully) during the summer and late autumn non-stop, eat-a-thon.
What: The β-oxidation of saturated fatty acids pathway will be discussed in a series of posts, this being the first. Step 1 of 4 is discussed in this post. Each post discusses the chemistry that is taking place (redox, isomerism, and addition-elimination, decarboxylation, regio- and stereospecific reactions, and so on) and includes 'learning assessment questions' (my terminology, hence the single apostrophe marks) to ask students to gauge their understanding of:
1. the intricacies and specifics of the reaction chemistry involved, and
2. the spectroscopic tools to decipher the substrate and/or intermediate structures.
Who- Faculty: The intended audience is for faculty who teach Organic Chemistry 1 or 2 but of course the questions can be adapted to meet the instructional needs of General Chemistry topics as well.
Who- Students: For students to get the most educational 'bang-for-the buck', it is assumed that students have a working and foundational knowledge of the following ochem topics:
- stereochemistry (relative configuration about a chiral center, enantiomeric and disastereomeric relationships, optical rotation and purity, and prochirality)
- carbon hybridization, ideal bond angles, and molecular geometry about a central atom
- computing/determining a molecule's Index of Hydrogen Deficiency (IHD); also referred to as the Degree(s) of Unsaturation (this post uses IHD)
- 1H and broadband proton-decoupled 13C NMR, IR, and DEPT 1H NMR
Let's begin. Remember in Part 2 the relative rapid change in body mass of the brown bears (Ursus arctos) of Katmai National Park and Preserve (KNPP) in Alaska? Free-range bears (= wild) emerge from their winter dens in the spring 15-30% lighter than when they entered their den in late autumn (Lundberg et al., 1976; Nelson, 1980; Nelson et al., 1983; Yellowstone Grizzly Bears, 2017). The photo below of KNPP brown bear 909 shows the distinct change in body mass due to fat accumulation from early summer (photo taken a few weeks after 909 emerged from the den hungry and lean) to early fall (photo taken with about another 3-4 weeks before 909 hunkers down for the winter). The weight gain in fat is obvious.
The net loss in body mass during hibernation is due entirely to the 'burning' of white adipose fat. There is no appreciable net loss of muscle (protein catabolism) or bone mass (Lundberg et al., 1976; Nelson et al., 1975). Bears break down stored white fat reserves while hibernating in order to keep vital and required maintenance of physiological functions like breathing and core body temperature from shutting down.
Summary of β-Oxidation Pathway: A fat molecule (3 moles fatty acid/mole fat; 3 moles ester functional group/mole fat) is first hydrolyzed by lipase enzymes into a molecule of glycerol and three molecules of free fatty acids (see Note below). Before a free fatty acid can be metabolized, it first must be activated with ATP [via phosphorylation (transfer of PO3-) to give an acyl adenylate (fatty acid-AMP where AMP = adenosine monophosphate) + PPi (= P2O7-4)] and then with reaction of the sulfhydryl group of the large carrier molecule coenzyme A (CoASH) to form a fatty acyl-CoA. Synthetase enzymes catalyze this activation. The polar functional group linking the non-polar hydrocarbon fatty acid moiety to CoA is a thioester (O=C-S). Through a series of four, enzyme-dependent steps the fatty acyl-CoA is then degraded with the loss of two carbon atoms per cycle or turn to form acetyl-CoA (CH3COCoA where CO is a carbonyl group, C=O) and a shorter (by two carbons) fatty acyl-CoA. The shorter fatty acyl-CoA then re-enters the pathway for another round of degradation (= loss of two carbons to form acetyl-CoA). The pathway repeats until all the carbons in the original fatty acid are converted into acetyl-CoA. Each acetyl-CoA formed enters into the Tricarboxylic Acid (TCA) cycle, aka Kreb's and Citric Acid cycles, which in turn is coupled to the electron transport chain (the reduced coenzymes FADH2 and NADH) and the eventual production of ATP, the molecular energy currency.
Note: The side chain length and degree of saturation/unsaturation for the free fatty acids vary. The β-oxidation pathway discussed in this post deals with saturated, unbranched, even numbered carbon moieties. And the discussion below begins with the formation of an unbranched, saturated fatty acyl-CoA with a 16 carbon side chain.
The overall multi-step β-oxidation of saturated fatty acids reaction pathway is summarized below. (Students are not given the reaction pathway summary below.)
Let's begin by taking a look at the chemistry involved in STEP 1 of the β-oxidation pathway (shown below). What follows is a series of questions (identified with Q) students answer with respect to STEP 1. You are free to use these questions as is or modify them. Please though practice conspicuous attribution if you use or modify the work that follows. Thanks as always. FYI- I won't tell students that the series of reactions that will occupy consecutive lectures is the β-oxidation of saturated fatty acids pathway. It is not important that students know the name of the reaction pathway. What I'm trying to accomplish is that students understand the chemistry by closely studying the bond making-breaking scenarios without having to look at preliminary information first. Everything they need to know about the chemistry is in the actual chemistry, not the name.
Some Selective 'Learning Assessment Questions':
Done individually or in groups. If the latter, I recommend the Think-Pair-Share-Report Out (TPSRO) strategy to promote increased student engagement (= 'theoretical buy in'). The References section includes information about TPSRO.
Q1: The reaction above (STEP 1) is the first of four in a metabolic pathway used by mammals (including humans) during fasting or when starving. A is a redox prothestic group. Complete STEP 1 Table 1 below based on the STEP 1 reaction scheme described above.
Q2: Determine if the italicized statement below is TRUE or FALSE. If FALSE, then re-write the statement to be TRUE.
In the reaction the fatty acid moiety of the substrate changes from more to less saturated. ΔIHDreaction = -1. The ideal bond angle, hybridization, and structural geometries for both α and β carbons change from 109.5o to 120o, from sp3 to sp2, and from tetrahedral to trigonal pyramidal respectively. And B is the reduced form of A, an oxidizing agent. The STEP 1 reaction is an example of a dehydration reaction that produces an intermediate with trans (or E) geometry.
Q3: If the statement above is FALSE, quantify the percentage of the statement that is incorrect.
Q4: Below is the idealized, theoretical* IR spectrograph (excluding isotope effects) for the fatty acid moiety only of the fatty acyl-CoA substrate. On the blank axes draw the idealized IR spectrograph for intermediate 1. Compare/contrast your idealized spectrograph of intermediate 1 with another student or group. Reconcile any differences in the spectra.# Be prepared to defend your IR spectrum for intermediate 1 if called upon by the instructor.
Instructor: *: See Notes for Q4: 1. below. #: See Notes for Q4: 2. below.
ANSWERS:
Q2: FALSE; The reaction the fatty acid moiety of the substrate changes from more to less saturated (TRUE). ΔIHDreaction = -1 (FALSE; = +1 = (# sites unsaturation in intermediate 1 - # sites of unsaturation in substrate) = 2 - 1). The ideal bond angle, hybridization, and structural geometries for both α and β carbons change from 109.5o to 120o (TRUE), from sp3 to sp2 (TRUE), and from tetrahedral to trigonal pyramidal (FALSE; trigonal planar) respectively. And B is the reduced form (TRUE) of A, an oxidizing agent (TRUE). The STEP 1 reaction is an example of a dehydration reaction (FALSE; is a dehydrogenation reaction) that produces an intermediate with trans (or E) geometry (TRUE).
Commentary about TRUE/FALSE questions: I likely am in the chemistry education community minority in that I like TRUE/FALSE questions. But my liking comes with a distinct qualifier- the TRUE/FALSE question is multi-sentence and each sentence is comprised of at least one correct/incorrect statement. Basically, as I see it (but perhaps not by others and that's cool by me) a one sentence TRUE/FALSE question is useless as a gauge to know if students truly grasp the content. A multi-sentence TRUE/FALSE is more useful (but perhaps still useless overall but maybe that's another post some other time) as it requires a comparative synthesis of the information and a final judgement call as to whether the information is 100% TRUE, 90% TRUE (but 10% FALSE so the statement taken as a whole is FALSE), 80% TRUE (20% FALSE), and so on. Of course, a student still has a 50% chance of guessing it right (or wrong) regardless of the number of sentences and pieces of information but from after-class talks with students over the years many have 'confessed' that they find my multi-sentence TRUE/FALSE questions challenging and hate these question types the most on my quizzes and exams. The fact that students for the most part use the two words 'challenging' and 'hate' in the same context tells me- at least anecdotally- that such an approach to gauge student learning is comparatively useful in engaging their intellect and/or knowledge of the content material under question.
Q3: % FALSE statements = (# FALSE statements/total # statements) x 100 = (3/9) x 100 = 33%
Q4:
Notes for Q4:
1. An "idealized, theoretical" IR spectrograph means that all predicted peaks appear in the expected stretching frequency range and that there is no "mudding up" of absorption peaks from the instrument type (dispersive or grating versus FTIR), solvent in which the analyte is dissolved, liquid vs. gas phase data collection, and so on. Basically, a perfect instrument is used to make a perfect measurement and perfect peaks are produced, seen, and are easily interpretable. It's not reality but my purpose is to get sophomore level ochem students to understand and apply foundational spectroscopic principles to solving molecular structure problems. The purpose is not for ochem students to learn the molecular electronic ground state potential energy surface or quantum selection rules of IR spectroscopy.
2. So what does the following mean, Reconcile any differences in the spectra. First, it's unlikely that students or groups working independently will draw identical IR spectrograph. Rather, students or groups will draw different IR spectra for likely different reasons. As an educator I think it's valuable and necessary for college students to not simply share their ideas but to defend them as well. This in turn may mean that a student(s) is (are) convinced to change their way of thinking by another student or group (perhaps from initially correct to incorrect). To illustrate, a hypothetical situation is below. For the two spectra below, will Student B convince Student A that the moderately strong absorption at ~2300cm-1 is incorrect? Or will Student A successfully counter Student B? Will Student A convince Student B to get rid of the weak absorption at ~3100cm-1, or will Student B hold ground and stick with the original answer? I find this teaching approach useful for two reasons:
a. it promotes (ideally) dialogue, intellectual engagement, and the free-flow of ideas between students and/or groups, and
b. it shows that one can steadfastly defend a position (say, Student A refuses to get rid of the moderately weak absorption at ~2300cm-1, the approximate region where C≡C and C≡N bonds appear) but in the end- and in spite of one's resolute defense and reasons for it- still be wrong.
3. For the intermediate IR spectrograph, I'm just looking for new absorptions in the correct stretching frequency (wavenumber) regions because of the formation of the trans disubstituted alkene functional group. Whether the vinylic hydrogens absorb at 3045 or 3100cm-1 is not of concern to me. As long as students show an absorption peak in the approximately correct region I'm OK with that- ~3100-3000cm-1 (ish) for the vinylic hydrogens and ~1600cm-1 (ish) for the C=C.
4. New absorptions are drawn relative to the strong absorption of the C=O group. In IR some absorption intensities are strong (s), some moderate (m), and others weak (w). I expect students to know that the C=C and =C-H stretching IR absorptions are predicted to be weaker than the C=O stretching IR absorption. But I do not sweat it if students draw the C=C and =C-H stretching frequencies of equal intensity. But if they do, it's another learning/teaching opportunity to have them speculate about what the relative absorption intensities are predicted to be.
5. If students do not show a vinylic (=C-H) absorption at all, perhaps it is because students buried it in the more intense and busy sp3C-H absorption frequencies (specifically the 3000-2950cm-1 range). That's reasonable. But ask students to explain the absence of the absorption, if applicable. Possibilities: a. Either the absorption is buried in the saturated C-H upper range, or b. they forgot about it. If they forgot it, then ask why did they forget it. And I don't sweat it if students only show one =C-H absorption as opposed to two on account of the asymmetry of the disubstituted C=C. The difference is probably so small anyway that overall it looks like only one absorption exists.
6. I'm OK if students draw the intermediate's carbonyl group (C=O) absorption at the same wavenumber (= Stretching frequency value ~1750cm-1) as in the substrate in spite of the conjugation of the C=O with the alkene. The C=O absorption shift is real- with conjugation to the π electrons between the α and β carbons- but small. And besides it is difficult to show the actual magnitude of the shift using the scale of the x-axis. Perhaps as a side question after the activity ask students if the C=O absorption is predicted to shift to a higher or lower wavenumber when in conjugation with the neighboring alkene. If yes, is the magnitude and/or direction of absorption shift dependent on alkene geometry- cis versus trans?
Closing Remarks: The next post (Part 4) will explore the reaction chemistry of Steps 2 and 3.
References
Lundberg, D.A., Nelson, R.A., Wahner, H.W., and Jones, J.D. 1976. Protein metabolism in black bear before and during hibernation. Mayo Clinic Proceedings 51: 716-722.
Nelson, R.A., Jones, J.D., Wahner, H.W., Mcgill, D.B., and Code, C.F. 1975. Nitrogen metabolism in bears: urea metabolism in summer starvation and in winter sleep and role of urinary bladder in water and nitrogen conservation. Mayo Clinic Proceedings 50: 141-146.
Nelson, R.A. 1980. Protein and fat metabolism in hibernating bears. Federation Proceedings 39: 2955-2958.
Nelson, R.A., Folk, Jr., G.E., Pfeiffer, E.W., Craighead, J.J., Jonkel, C.J., and Steiger, D.L. 1983. Behavior, biochemistry, and hibernation in black, grizzly, and polar bears. Ursus 5: 284-290. DOI: 10.2307/3872551
Yellowstone Grizzly Bears: ecology and conservation of an icon of wildness. 2017. Editors: P.J. White, Kerry A. Gunther, and Frank T. van Manen; Contributing authors: Daniel D. Bjornlie [and thirteen others]; Managing editor: Jennifer A. Jerrett. Yellowstone National Park, [Wyoming]: National Park Service, Yellowstone National Park; [Bozeman, Montana]: U.S. Geological Survey, Northern Rocky Mountain Science Center. Downloadable from: https://www.nps.gov/yell/learn/nature/upload/Yellowstone_Grizzlies_Web.pdf
For Think-Pair-Share-Report Out (TPSRO)
Sam McKagan and Daryl McPadden. "Best practices for whiteboarding in the physics classroom." PhysPort (developed by AAPT, American Association of Physics Teachers), October 26, 2017; successfully retrieved November 2019 (https://www.physport.org/recommendations/Entry.cfm?ID=101319)
Heather Macdonald and Rebecca Teed. "How to Give Interactive Lectures" Science Education Research Center (SERC) at Carleton College (in Starting Point: Teaching Entry Level Geoscience); successfully retrieved November 2019 (https://serc.carleton.edu/introgeo/interactive/howto.html)
"Think-Pair-Share" Science Education Research Center (SERC) at Carleton College (in Starting Point: Teaching Entry Level Geoscience); successfully retrieved November 2019 (https://serc.carleton.edu/introgeo/interactive/tpshare.html)