Gradient Temperature Raman Spectroscopy of Fatty Acids with One to Six Double Bonds Identifies Specific Carbons and Provides Systematic Three Dimensional Structures

Specialized pro-resolving mediators provide promising targets for new drugs and natural products. Much work has been accomplished on the structure/ function of the lipoxygenase and cyclooxygenase enzymes but not on the substrates. A better visualization of three-dimensional lipid structures will allow increased refinement of the interactions that produce the pro-resolving mediators, and lead to improvements in synthetic pathways. We present systematic analysis of oleic (18:1n-9), linoleic (18:2n-6), alpha-linolenic (18:3n-3), arachidonic (20:4n-6), docosapentaenoic (22:5n-3), and docosahexaenoic (22:6n-3) acids. Continuous gradient temperature Raman spectroscopy (GTRS) applies the temperature gradients utilized in differential scanning calorimetry to Raman spectroscopy. GTRS can identify and differentiate specific carbon chain sites, finally allowing Raman analysis to explain why the long-chain polyunsaturated fatty acids (LC-PUFA) exhibit such extreme functional differences despite minimal changes in chemical structure. Detailed vibrational analysis of the important frequency ranges 1450 1200 cm (includes CH2 bending and twisting) and 1750 1425 cm (includes C=C stretching and C-C stretching plus H-C in-plane rocking) shows for the first time that each molecule has its own characteristic set of modes with only some redundancy/commonality. The number and frequency of modes correlates with three-dimensional molecular structure, not the degree of unsaturation. The high degree of specificity of lipoxygenase and cyclooxygenase enzymes should be reconsidered in light of the fact that individual sites on the polyunsaturated fatty acid chain are How to cite this paper: Broadhurst, C.L., Schmidt, W.F., Nguyen, J.K., Qin, J.W., Chao, K.L. and Kim, M.S. (2018) Gradient Temperature Raman Spectroscopy of Fatty Acids with One to Six Double Bonds Identifies Specific Carbons and Provides Systematic Three Dimensional Structures. Journal of Biophysical Chemistry, 9, 1-14. https://doi.org/10.4236/jbpc.2018.91001 Received: October 24, 2017 Accepted: January 15, 2018 Published: January 18, 2018 Copyright © 2018 by authors and Scientific Research Publishing Inc. This work is licensed under the Creative Commons Attribution International License (CC BY 4.0). http://creativecommons.org/licenses/by/4.0/


Introduction
Continuous gradient temperature Raman spectroscopy (GTRS) has proven to be a simple, rapid technique for determining the unique structures of the unsaturated fatty acids [1] [2] [3] [4].While conventional Raman analysis can be useful, to first order the long-chain polyunsaturated fatty acids (LC-PUFA) appear nearly identical, with the vibrational modes of most moieties degenerate, redundant or very broad [5] [6] [7] [8] [9].But logically this cannot be the case, because the LC-PUFA are so vastly different biochemically.Collecting spectra in a temperature gradient provides information not readily available in constant temperature measurements, because the dynamic nature of the vibrational structure can be observed in real time as the molecule changes conformation.Thus, GTRS can identify and differentiate specific carbon chain sites, finally allowing Raman analysis to explain why the LC-PUFA exhibit such extreme functional differences.Recently we applied GTRS to oleic acid (OA, 18:1n-9), linoleic acid (LA, 18:2n-6), arachidonic acid (AA, 20:4n-6), N-3 docosapentaenoic acid (N-3DPA, 22:5n-3), N-6 docosapentaenoic acid (N-6DPA, 22:5n-6), docosahexaenoic acid (DHA, 22:6n-3), and AA and DHA stearic acid phosphatidylcholines, resulting in complete vibrational assignments and new three dimensional (3D) structures for each molecule [1] [2] [3] [4].Readers are referred to these published texts for the full datasets, contour plots and vibrational mode assignments for each of these molecules.
The importance of the LC-PUFA to biological and pharmaceutical sciences cannot be overstated.In particular, the emerging field of specialized pro-resolving mediators provides promising and exciting targets for new anti-inflammatory, neuroprotective and anti-infective drugs and natural products [9] [10] [11] [12] [13].Specialized pro-resolving mediators are highly interesting in that metabolites of not just DHA and EPA, but also n-3DPA and n-6DPA can actively resolve inflammation at very low concentrations, proving new insight into the numerous health benefits epidemiologically associated with diets high in fish and/or total LC-PUFA [14] [15] [16] [17].
All five of the PUFA we present are metabolized by cyclooxygenase and lipoxygenase enzymes into an extensive suite of oxylipin metabolites [18]- [24].
While tremendous work has been accomplished on the structure and function of the various enzymes, it remains largely empirical: there is no systematic explanation as to why conversion rates vary at least 10-fold among PUFA species [23].
One reason for this is that the structure of substrates has been taken for granted beyond the obvious distinction of n-6 vs. n-3.We believe that a better understanding and visualization of the 3D lipid structures will allow increased refinement of the substrate and enzyme interactions that produce the pro-resolving mediators, and lead to improvements in synthetic pathways.Herein we present a detailed, comprehensive vibrational analysis of six fatty acids with 1, 2, 3, 4, 5, and 6 double bonds (OA, LA, alpha-linolenic acid (ALA, 18:3n-3), AA, N-3DPA, and DHA.With the improved GTRS spectroscopic technique, second derivatives can be utilized to resolve and identify vibrational modes which previously were not completely assigned.We show that individual sites on the LC-PUFA chain are nonequivalent although they are technically identical moieties.

Materials and Methods
The GTRS system utilizes a 785-nm laser module (I0785MM0350MF-NL, Innovative Photonic Solutions, Monmouth Junction, NJ) as the excitation source.A fiber optic Raman probe (RPB, InPhotonics, Norwood, MA) is used to focus the laser and acquire the Raman signals.A bifurcated fiber bundle with a 16-bit CCD camera (1024 × 256 pixels; Newton DU920N-BR-DD, Andor Technology, South Windsor, CT) delivers the laser radiation to the probe and transmits the Raman signals to the spectrometer at 0.2˚C increments.The spectrometer detects a Raman shift range of 102.2 to 2538.1 cm −1 with a minimum spectral resolution of 3.7 cm −1 .
OA (cis-9 octadecenoic acid), LA (cis-9, 12 octadecadienoic acid), ALA cis-9, 12, 15 octadecatrienoic acid), AA (cis-5, 8, 11, 14 eicosatetraenoic acid) and DHA (cis-4, 7, 10, 13, 16, 19 docosahexaenoic acid) > 99% purity were purchased from Sigma Chemical Co., St. Louis, MO.N-3DPA (cis-7, 10, 13, 16, 19 docosapentaenoic acid) 98% purity was purchased from Santa Cruz Biotechnology Inc., Dallas, TX. Materials were stored sealed in a freezer until immediately prior to use.Samples were placed in aluminum pans which were then placed on large copper heat sinks with a sample platform on the upper surface.The heat sink and sample holder are chilled in a LN 2 bath, but the sample itself does not come in direct contact with LN 2 or atmospheric water vapor condensate.A ceramic hotplate is used for controlled heating of samples and as stable insulating platform for analysis.The copper heat sinks are placed directly on the ceramic heat surface and serve to buffer the rate of temperature increase.Two K-type thermocouple probes (range −200˚C to 1350˚C) are attached to two sides of the sample area and connected to a dual-input thermometer (Easy View EA15, Extech Instruments, Nashua, NH).The sample temperature is defined as the average value of the two probes.The thermocouple calibration based on test resistance provided by the manufacturer was utilized as well as calibration in LN 2 .
The Raman probe, hotplate, and sample materials are placed in a closed black box to avoid ambient light.Raman spectra can be acquired from −180˚C to 320˚C.
The heating gradient was approximately 1˚C•min −1 for these mainly cryogenic investigations.We did not use a set time schedule for spectral acquisition, but rather acquired spectra each time the sample temperature increased either 1˚C or 0.2˚C, depending on the analysis.System software was developed using Lab VIEW (National Instruments, Austin, TX) to fulfil functions such as camera control, data acquisition, temperature measurement, signal/noise threshold and synchronization [25].All Raman samples were run in triplicate and each dataset analyzed independently to ensure reproducibility.Sigma Plot 13 (Systat Software, Inc.) generated three-dimensional contour plots (frequency, temperature, signal intensity).Software counting statistics ensure that only data with 99% certainty of true signal are utilized.First derivative intensity contour plots were calculated using a three-point running average, i.e. the first and last points calculate the slope of the middle point.The contour plots for the second derivative intensity were calculated using the three-point running average data from the first derivative spectra.Relative intensity is normalized to C=C stretch region circa 1650 cm −1 .Maximum to minimum intensity colors are red > orange > yellow > green > blue > black.Each contour data array contains about 20 Mb.

Results
GTRS collects Raman spectra continuously in an applied thermal gradient without the discontinuity in temperature intrinsic in steady state experiments.In the gradient, the contour plot signal is greatest for molecular sites which are the most elastic and respond most quickly to thermal stress.Figure 1    ).An unusual assignment in ALA is 1601 cm −1 , which corresponds to a difference of two H-C in-plane rocking frequencies, whereas 1695 cm −1 equals a difference of four.
The CH 2 bending assignment in OA at C8 (1408 cm −1 ) is similar to the C8 assignment in ALA (1413 cm −1 ).Twisting at C8 in OA and C14 in ALA occurs at the same frequency.This shifts the CH 2 bending assignment from C8 in ALA to C7 in OA; both are at 1410 cm −1 .
LA has four discrete vibrational modes arising from two double bonds and three CH 2 sites adjacent to C=C sites.C8H 2 bending occurs at 1448 cm −1 and is nearly the same frequency as straight chain aliphatic CH 2 bending at 1450 cm −1 .Journal of Biophysical Chemistry The bending torsion from the six other CH 2 groups on the carbonyl ended aliphatic chain (C2-C7) is minimal.Since C11H 2 twisting near 1327 cm −1 is not observed, torsion from adjacent CH 2 groups in the H-C9=C10-H moiety is minimal.Torsion due to the C14H 2 site results in the twisting modes at 1266 and 1237 cm −1 .In the first case, H a -C twisting is relative to the C14-C13=C12 plane.
In For DHA, twisting towards the methyl-ended chain (1266 cm −1 ) occurs at site C14, at the same frequency as C14H a in LA.DHA with six double bonds has two CH 2 twisting/bending modes whereas AA with four double bonds has three.In AA, these are assignable to CH 2 sites adjacent to the beginning (C4), end (C16) and middle (C10) of the double bond region.In DHA, the beginning site (C3) is shifted about 50 cm −1 lower that the corresponding site in AA (C4).The second site (C14) is shifted about 25 cm −1 higher than from that in AA (C16).A vibrational mode at C12H 2 in DHA would be near its center of mass.C14H 2 is however three carbon atoms past its center of mass.
Four double bonds equal one loop from C6 to C18; the geometry of a selfconsistent twist in the same relative direction from C6 to C18 (with no twist at C12) would result in such a loop forming [3].For DHA (labeling each pattern C a =C b ), all C a = sites could be 1653 cm −1 and all =C b sites could be 1484 cm −1 .
This would explain why for DHA, only two vibrational modes in this spectral region (neither three nor one) are observed.
The bending modes correspond for the CH 2 adjacent to the first double bonds Journal of Biophysical Chemistry The backbone geometry in LC-PUFA structures C8-C9=C10-C11-C12=C13-C14 is a non-repeating moiety because C11 does not repeat twice.The critical structural question of such lipids is redundancy versus symmetry.Symmetry in reference to a molecular site means a conformation and its mirror images are identical.For lipids with an even number of double bonds, the CH2 group in the middle of them is reference site.For those with an odd number of double bonds, the middle of the C=C bond is the reference site.Overall symmetry means that C8-C9=C10-C11 is indistinguishable from C11-C12=C13-C14 or C8-C9= C11-C12.
In LA with two double bonds (C9=C10 and C12=C13), only four spectral lines are observed.All frequencies containing the C-C= and =C-C moiety overlap (1655 cm −1 ).C8H 2 bending before C9= is a single vibrational mode whereas C14 torsion after =C13 has two modes.The CH 2 structure at the carbonyl end of LA is symmetrical; the C14 sites towards the methyl end of the lipid are not equally elastic.The eclipsed conformation of the Fisher projection indicates that C8 to C13 are coplanar and C14 twisting above this plane is unequal to C14 twisting below the plane.
In AA with four double bonds (C5=C6, C8=C9, C11=C12, and C14=C15), only six vibrational modes are observed.Despite five CH 2 sites adjacent to a C=C, only three CH2 bending/twisting modes are detected and symmetrical about C10: C4H 2 before C5, C16H 2 after C15 and C10H 2 .The three spectral lines cannot be divided symmetrically among four sets of two double bonds.The Fisher projection resolves this dilemma: the sequence C5=C6-C7-C8 is not planar; C7-C8=C9-C10 is planar; the sequence C9-C10=C11-C12 is not planar and is in a conformation different than the first one.Moreover, C6 to C11 is the same conformation as C9 to C14, and the conformations C3 to C8 and C12 to C17 are similar.Thus, AA is symmetrical about C12H 2 and clearly not symmetrical about either C8=C9 or C11=C12.
In DHA with six double bonds (C4=C5, C7=C8, C10=C11, C13=C14, C16=C17, and C19=C20), the four vibrational modes observed can only occur because of redundancy.The absence (above −6˚C) of a C12H 2 twisting mode precludes the symmetry observed in AA.In the DHA Fisher projection, the identical gauche (left) cis conformations occurs in three locations (C2 to C7, C11 to C16, and C17 to C22) whereas the other three (C5 to C10, C8 to C13, and C14 to C19) occur as trans conformations.The co-addition curvature at three structurally equivalent cis double bond sites plus the symmetrical trans conformation at the remaining three double bond sites results in a planar "C" shaped molecular structure composed from redundancy in two forms of the C=C-C moieties.The Fisher projections for the gauche (right) cis double bonds in AA are also not identical to the gauche (left) double bonds in DHA.Previously we noted that biochemical energy is more than sufficient to not only recognize, but actually induce nonequivalence among CH and CH 2 groups [4].Our improved lipid spectroscopy and 3D structures likely have bearing on the enzymatic oxidation processes which form specialized pro-resolving mediators.For example, at 0˚C soybean 15-lipoxygenase oxidizes C17 to its hydroperoxy derivative with 100% efficiency for n-6 DPA, 85% for n-3DPA and 50% for DHA [9]. Figure 5 gives a view of these LC-PUFA in the critical area.While full analysis is beyond the scope of this spectroscopic contribution, we hope that this visualization will promote further understanding of the enzymatics involved.The high degree of specificity of lipoxygenase and cyclooxygenase enzymes [18]- [24] should be reevaluated in light of the fact individual sites on the polyunsaturated fatty acid chain are nonequivalent, and each LC-PUFA molecule has an individual, specific structure incorporating torsion.
Figure 1.Composite overlay line spectra for ALA detailing how stacked spectra are transformed to generate contour plots.Selected temperatures are shown for clarity.The discontinuity circa −3˚C represents melting.

Figure 3 .
Figure 3. Structures of the double bond regions and frequency assignments for their carbon sites.(a) OA, LA; (b) ALA, AA; (c) n-3DPA, DHA.
the second case, H b -C twisting is relative to the C14-C13=C12 plane.The aliphatic C14-C15-C16-C17-C18 moiety can be twisted to the right or twisted to the left relative to the planar C9=C10-C11-C12=C13 moiety.No net twist from CH 2 sites adjacent to either the C12=C13 or the C9=C10 moiety was found; LA in this temperature range is flat.AA has the identical structural moieties as LA but twice the number of double bonds with five CH 2 sites adjacent to C=C sites.Unexpectedly, six (not three) vibrational modes are detected.The three that are not observed in the LA data can be assigned to a conformational change at C10.The relative broadness of the LA contour plots reflects a more diffusely distributed elasticity over non-localized molecular conformations.In contrast, the sharp peaks at C4, C10, and C16 in AA are that evidence elasticity is localized at these three sites: i.e. at the beginning, middle and end of the repeating five carbon atom moieties.In AA, the three frequencies (1651, 1489, and 1327 cm −1 ) are not independent variables.The middle one is the average of the other two [(1651 cm −1 + 1327 cm −1 )/2 = 1489 cm−1 ].The difference between the first two results in a constant [(1651 cm −1 − 1487 cm −1 ) = 164 cm −1 ].From the three CH 2 between the double bonds, three in plane H-C rocking modes can occur with a frequency shift of 55 cm −1 per each H-C in-plane rocking site between the double bonds.H-C in plane rocking vibrational modes, depending upon how they add or subtract in relationship to each other, can correspondingly increase or decrease C=C and C-C stretching.

Figure 5 .
Figure 5.Comparison of energy minimized 3D structures for n-3DPA, n-6DPA and DHA based on [1] [2] [3].The position of Fe-O-O-H within the C17 oxidation site can be assumed constant, however the Fe-O-O-H site could equally be towards the inside of the C17 site as towards the outside of the lipid chain.N-6DPA has the cleanest fit at C17 of the three lipids (see purple area).Since n-6DPA has no C19=C20 bond, torsion is due only to previous double bond sites-especially C13=C14.For the C18H2 site, the hydrogen inside the U-shape of the lipid (purple) is in a spatially very different conformation as the hydrogen on the outside (white).