Guided inquiry analysis the use of ft nmr of curcumin
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Guided inquiry analysis the use of ft nmr of curcumin

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Cooperative learning in science education is addressed in this article. How students use a very relevant topic of anti-cancer agents, and the novel technique of (Heteronuclear single Quantum ...

Cooperative learning in science education is addressed in this article. How students use a very relevant topic of anti-cancer agents, and the novel technique of (Heteronuclear single Quantum Correllation Spectroscopy )2D -HSQC FT-NMR to organize spectra data is shown. Here, undergraduates become familiar with making plots of 1H FT-NMR and 13C FT-NMR , learning FT-NMR data processing (spinworks) and also use Chemdraw NMR to present data take with a Varian 600 MHz FT-NMR spectrometer.

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    Guided inquiry analysis the use of ft nmr of curcumin Guided inquiry analysis the use of ft nmr of curcumin Document Transcript

    • Cooperative learning using FT-NMR :Students: Mohammed Izmikna, Kasandra Dorce, MohammedSherwani, Samira Izmikna,By Dr. Robert Craig, Ph.D.Cooperative learning in science education is addressed in thisarticle. How students use a very relevant topic of anti-canceragents, and the novel technique of (Heteronuclear singleQuantum Correllation Spectroscopy )2D -HSQC FT-NMR toorganize spectra data is shown. Here, undergraduates becomefamiliar with making plots of 1H FT-NMR and 13C FT-NMR ,learning FT-NMR data processing (spinworks) and also useChemdraw NMR to present data take with a Varian 600 MHzFT-NMR spectrometer.We can make a tentative spectulationthat For the side chain methyl ester, the coupling constant JH2-H3 changes from 2 Hz to 5 Hz. This confirms a shift inconformational equilibria, after addition of this group. The“deshielding effect” of a carboxylate group on the monomer ofcurcumin is shown by 2D -HSQC FT-NMR data and theChemdrawNMR software.Cooperative learning using FT-NMR : Abstract
    • Cooperative learning in science education is addressed in thisarticle. How students use a very relevant topic of anti-canceragents, and the novel technique of HSQC FT-NMR to organizespectra data is shown. Here, undergraduates become familiarwith making plots of 1H NMR and 13C NMR , learning FT-NMRdata processing (spinworks) and also use Chemdraw NMR topresent data take with a Varian 600 MHz FT-NMRspectrometer. For the side chain methyl ester, the couplingconstant JH2-H3 changes from 2 Hz to 5 Hz. This confirms ashift in conformational equilibria. • The “deshielding effect” of a carboxylate monomer of curcumin is shown by 2D -HSQC FT-NMR data and the ChemdrawNMR software.In this paper, student projects are given as an example on howto introduce FT –NMR into the undergraduate curriculum.The deshielding of the antimitotic agent curcumin and it sidechain methyl ester have been studied by Nmr spectroscopyand by molecular modeling using ChemDrawNMR .Upon carboxylation, some proton shifts change. Studentsproficient in ChemDrawNMR show this in their analysis.
    • For the side chain methyl ester, the coupling constant JH2-H3changes from 2 Hz to 5 Hz. This confirms a shift inconformational equilibria.We will incorporate NMR experiments that illustrate theapplication of high resolution NMR spectroscopy to thestructure determination of Anti-Cancer agents.Adding NMR spectroscopy to a students repertoire of skills willgreatly enhance the laboratory learning experience and enableus to adopt experiments that have been successful elsewhereat utilizing NMR spectroscopy as an essential teaching tool.This paper will also examine the benefits of students involve inperforming NMR experiments at the undergraduate levelAs far as Curcumin is concerned, The effect of the carboxylgroup on the proton spectra(deshielding effect) is clearly shownwith chemdrawNMR software platform.current guidelines of the Committee on Professional Training1(CPT) of the American Chemical Society (ACS) are very clear on
    • its expectation for inclusion of instrumentation, and inparticular NMR spectroscopy, in the chemistry curriculum. Anexcerpt from this document states, “A department should haveseveral major pieces of sophisticated equipment suitable forundergraduate instruction as well as for research. One of thesemust be an NMR spectrometer.”1 Later in this same documentit is noted that the instrument should be an FT-NMR. Inaddition, the most current proposed revision to this documentstates, “The laboratory experience should include synthesis ofmolecules, measurement of chemical properties andphenomena, “hands-on” experience with moderninstrumentation, applications to real-world problems, andcomputational data analysis and modeling.”2
    • Curcumin’s broad spectrum of anti-oxidant, anti-carcinogenic,anti-mutagenic, and anti-inflammatory properties makes itparticularly interesting for the development of pharmaceuticalcompounds. Due to curcumin’s various effects on the functionof numerous unrelated membrane proteins, it has beensuggested that it affects the properties of the bilayer itself.Despite intense interest in the physiological effects ofcurcumin, a general mechanism for its action has not beenidentified.Figure 1Keto-enol form of curcumin, the dominant tatuomer ofcurcumin. The keto-enol from is stabilized by an intramolecularhydrogen bond, shown here by a dashed red line.Here is a mechanism of interest to many.
    • MATERIALS AND METHODSCurcumin (>94%) was purchased from Sigma and used withoutfurther purificationThis red powder was successively subjected to 1H NMR, 13CNMR, and 2D -HSQC FT-NMR analysis for structureFor the Varian 600mHz, 5mm NMR Sample tubes were usedfrom NewEra, inc
    • . The NMR sample tubes were“L” Series 5mm NMR tubes(4.960 ± 0.006mm OD; 0.40mm nominal wall; 0.0025mmroundness).All spectra was processed from the Varian using spinworksplatform.The specta was subsequently confirmed usingChemdraw NMR. It was convinent to use Spinworks to analyzespectra. The Spinworks software, created by Kirk Marat. alsoprovides us with excellent ppm shifts for both spectra.FT NMRAll of the experiments were performed on a Varian Infinity 600MHz solid-state NMR spectrometer. Each sample wasequilibrated for at least 30 minutes before starting theexperiment. 2D 13C-1H correlated (HECTOR) NMR spectra wereobtained using a spin-echo pulse sequence (90°-τ–180°-τ-acquisition; τ = 125 μs) with a 90° pulse length of 5 μs under a30 kHz continuous-wave proton decoupling.
    • Chemical shifts were referenced by setting the isotropicchemical shift peak of TMS to 0 ppm. 2D 13C-1H correlated(HECTOR) quadrupole coupling spectra were recorded using aquadrupolar echo pulse sequence (90°-τ–90°-τ-Acquisition; τ =80 μs) without proton decoupling.RESULTS AND DISCUSSIONSince curcumin affects such a large array of unrelatedmembrane proteins at approximately similar concentrations, ithas been proposed that curcumin can regulate the action ofmembrane proteins indirectly by changing the physicalproperties of the membrane rather than by the direct bindingof curcumin to the protein.High resolution 13 C and 1H NMR , 2D 13C-1H correlated(HECTOR), and 2D 1H-1H correlated (COSY) spectroscopytechniques will be used for elucidating skeletal arrangement ofmonomer units.Applications that also use the 2D 1H-13C HSQC experiment aregaining more interest as a result of the growing feasibility ofacquiring these spectra routinely. The 2D HSQC experiment
    • contains additional information (i.e. 13C chemical shift) as wellas easier identification of labile and diastereotopic protons2D NMR specra may be obtained that indicate couplingbetween hydrogens and carbons to which they are attached. Inthis case it is called heteronuclear correlation spectroscopy(HECTOR, HSQC, or C-H HECTOR).When ambiguities are present in one-dimensional 1H and 13CNMR spectra, a HECTOR or HSQC spectrum can be very usefulfor assigning preciscely which hydrogens and carbons areproducing their respective peaks.In a HSQC spectrum a 13 C spectrum is presented along oneaxis and a 1H spectrum is shown along the other. Cross peaksrelating the two types in a HSC spectrum indicate whichhydrogens are attached to which carbons in a molecule, or viceversa.These cross peaks correlations are the result of instrumentalparameters specified on the NMR spectrometer. If imaginary
    • lines are drawn from a given cross peak in the x-y field to eachrespective axis,The cross peak indicates to the hydrogen giving rise to thecorresponding 1H NMr signal on one axis and is coupled orattached to the carbon that gives rise to corresponding 13CNMR signal on the other axis.Thus, it is readily apparent which hydorgens are attached towhich carbonsFIG 1:The effect of the carboxylated curcumin on proton signal
    • The effect of the carboxylated curcumin on proton signalReferring to the chemdrawNMR data below curcumin from proton chemdraw shift atom index coupling partner constant and vector 5.35 7 delta (ppm) 5.35 25 7.16 24 20 20 1.5 H-C*C*C*-H 6.99 21 20 7.5 H-C*C*-H 7.16 6 20 20 1.5 H-C*C*C*-H 6.79 21 21 7.5 H-C*C*-H 24 24 1.5 H-C*C*C*-H 6.99 3
    • 4 4 7.5 H-C*C*-H 6.79 4 3 3 7.5 H-C*C*-H 6 6 1.5 H-C*C*C*-H 3.83 10 3.83 27 4.59 13 7.6 28 30 15.1 H>C=C<H 7.6 29 31 15.1 H>C=C<H 6.91 30 28 15.1 H>C=C<H 6.91 31 29 15.1 H>C=C<Hcurcumin fromproton chemdrawcarboxylatedshift atom index coupling partner constant and vector 5.35 25 11 35 7.16 24 20 20 1.5 H-C*C*C*-H 6.99 21 H-C*C*- 20 7.5 H 7.3 @@ 6 4 4 1.5 H-C*C*C*-H 6.79 H-C*C*- 21 21 7.5 H 24 24 1.5 H-C*C*C*-H 7.13 3 H-C*C*- 4 4 7.5 H 7.26 @@ 4 H-C*C*- 3 3 7.5 H 6 6 1.5 H-C*C*C*-H 3.83 10 3.83 27 2.3 29
    • 31 31 7.1 H-CH-CH-H 2.3 32 31 31 7.1 H-CH-CH-H4.59 132.01 31 29 29 7.1 H-CH-CH-H 7.6 36 38 38 15.1 H>C=C<H6.91 38 36 36 15.1 H>C=C<H6.91 39 37 37 15.1 H>C=C<H• above is Figure 2:13C spectra of Curcumin
    • above is Figure 3: the 2D1H -13C HSQC spectra of Curcumin• Let’s dive right in, as the research students have provided the spectra and determine the HSQC for Curcumin, with the aid of the ChemdrawNMR software, and previous scan of curcumin (proton and 13C). It is beneficial to keep these spectra on hand. The Spinworks software, created by Kirk Marat.also provides us with excellent ppm shifts for both spectra Working from top down, and left to right, the HSQC for curcumin reads as such. The first peak evident in the spectra is 13C at 55.934 ppm, And crossed with
    • Proton(designed 14) at 3.9620 ppm. The next peak is with Proton(designed 12) at 5.8592 and a 13 C at 101 ppm• This hydrogen must be attached to the OH group , or might be the hydrogen in between the carbonyls on the hexadione bridge.• The carbon 13 peak at 109.3 cross with several protons. Referenced with the spinworks data table for curcumin proton data taken the Varian 600 MHz we have for Peak 3 in the HSQC specta with Peak 12 at 5.8592 ppm And Peak 13 at 5.8124 ppm in the proton spectra. Please refer to table one for the spinworks data.• The carbon 13 peak at 109.3 ppm coupled with a hydrogen (peak 5 at 7.1427 ppm) is an aromatic hydrogen. This hydrogen resides on a benzene ring, and is obviously confirmed by coupling with an aromatic 13C at 109.3 ppm• It is this hydrogen that will be effected in the carboxyalated form of curcumin. The carbon peaks at 122.6 and 123.8 ppm with peak 3 and 4 (off diagonal) of the proton data gives some modest peaks in the specta.Also evident are Peak 3 (122.6 ppm 13C with (6.473 ppm 1H, 6.503 ppm 1H )And, With peak 7 and 8 (shown in the off diagonal) coupling 123.8 ppm 13C with (6.473 ppm 1 H , 6.503 ppm 1H). These hydrogens are on aromatic ring next to hydroxyl groups. A Carbon of 114 ppm is
    • appropriate to be adjacent to these hydrogens. As reference by the ChemdrawNMR softwareplatfom”The benzene CH of which there are 3, give rise to 7.16 ppm, 6.99 ppm and 6.79 ppm. “On the hexadienone bridge, between the two benzene rings (aromatic rings) are 2 pairs of equivalent protons, (see table 2). The software also allows for shift corrections • It is this hydrogen that will be effected in the carboxyalated form of curcumin. The carbon peaks at 122.6 and 123.8 ppm with peak 3 and 4 (off diagonal) of the proton data gives some modest peaks in the specta.Also evident are Peak 3 (122.6 ppm 13C with (6.473 ppm 1H, 6.503 ppm 1H )And, With peak 7 and 8 (shown in the off diagonal) coupling 123.8 ppm 13C with (6.473 ppm 1 H , 6.503 ppm 1H). These hydrogens are on aromatic ring next to hydroxyl groups. A Carbon of 114 ppm is appropriate to be adjacent to these hydrogens. As reference by the ChemdrawNMR softwareplatfom”The benzene CH of which there are 3, give rise to 7.16 ppm, 6.99 ppm and 6.79 ppm. “On the hexadienone bridge, between the two benzene rings (aromatic rings) are 2 pairs of equivalent protons, (see table 2). The software also allows for shift correctionsMono Carboxyated is here
    • Figure 4, 5 and 6 are the 2D1H -13C HSQC spectra of monocarboxylated CurcuminAssign ment of theMONO-CARBOXYLATED CURCUMIN HSQC-ON THEWALL
    • The peak at 6.953 ppm for hydrogen (and 6.94 ppm) corresponds to aC=C-OH on the right benzene ring of the mono carboxylated form.The carbon associated with this Hydrogen is the signal at 114 ppm. Thecarbon experiencing a more electronegative environment is next to thisCarbon at 123.8 ppm . this carbon at 123.8 ppm gives a signal with aproton at 7.063 ppm. This 7.063 also spin couples with a carbonAt 109 ppm. This is associated with a similar fragment,C=C-OCH3 onthe top of rightsided benzene ring. The carbon at 109 ppm couplestwiceWith protons at 7.129 ppm and the one just mentioned thereafter.The proton at 7.143 ppm we can designate Ha , which resides on top ofthe rightsided benzene ring couples with 3 carbons at 109 ppm, 111.8ppmAnd 122.0 ppm. This fragment is the “Ha-‘C=C-OCH3”.The signal at 7.15 ppm we can designate Hb , which resides on top ofthe rightsided benzene ring, as well, and couples with 3 carbons at 109ppm,111.8 ppm And 122.0 ppm. This fragment is the “Hb-‘C=C-OCH3”.a C-C=C-OH makes up the final piece in this portion of the spectra. It isthe bottom of the benzene ring on the right sideof the carboylated curcumin, unaffected by this substituent group. TheHc-C-C=C-OH is responsible for the peak at 7.19 ppm,120ppm
    • and 7.19 ppm,120 ppmThis cluster of peaks resides in the bottom right portion of the HSQC spectramono carboxylated curcumin molecule.The carbon at 140.5 ppm (Ca)signals with a hydrogen residing on the hexadionebrigde (H-Ca=C=H)The carbon at 139.5 ppm (Cb)does the same for this fragment (H-C=Cb=H)(5)Figure 4, 5 and 6 are the 2D1H -13C HSQC spectra of monocarboxylated Curcumin
    • U(6)The one carbon peak at 5.35 ppm sweeps the proton signals with 3.855 ppm and 3.82 ppm.This is the C-O-CH3 groupconclusionHeteronuclear Single Quantum Coherence (HSQC)Plots 1H NMR on x-axis and13 C NMR on y-axis and Utilizes 1 bond coupling between H and C, Eliminating allthe H containing C’s eliminates many C’s assignments. Leaves only on-Hcontaining C’s to assign.Using information from 1H NMR data alone is not a new concept. However, applications thatalso use the 2D 1H-13C HSQC experiment are gaining more interest as a result of the growingfeasibility of acquiring these spectra routinely. The 2D HSQC experiment contains additionalinformation (i.e. 13C chemical shift) as well as easier identification of labile and diastereotopic
    • protons. I would like to thank the students and staff at the college of Staten Island, CUNY formaking this work possible. I find cooperative learning to be very important because it is crucialfor our students to learn to work in groups. This not only helps develop their social skills, butalso enhances their ability to develop the skills necessary to work collaboratively when theyenter graduate school.REFERENCES • Sherman, L.W, “ COOPERATIVE LEARNING IN POST SECONDARY EDUCATION: IMPLICATIONS FROM SOCIAL PSYCHOLOGY FOR ACTIVE LEARNING EXPERIENCES, A presentation to the annual meetings of the American Educational Research Association”, Chicago, IL, April 3-7, 1991. [revised, 20 January, 1996] • Implementation of FT-NMR Across the Chemistry Curriculum , Committee on Professional Training (CPT) of the American Chemical Society, CCCE Dunal Admin – October 16, 2009 to October 16, 2009. • Greenbowe. T.J. and Meltzer, D.E. “Student learning of thermochemical concepts in the context of solution calorimetry.” (2003). International Journal of Science Education, 25(7), 779-800. • Payton F. , Sandusky, P., Alworth, W.L., NMR study of the solution structure of curcumin • Phoung, Thu Ha, Thi Minh, Nguyet Tran, Hong Duong, Pahm, Quan Huan, Nguyen, and Xuan Phuc, Nguyen, The synthesis of poly (lactide)-vitamin E TPGS (PLA-TPGS) copolymer and its utilization to formulater a curcumin nanocarrier,xxxxxxxxxxxx • Phyllis Langone* ‡, Priya Ranjan Debata‡, Sukanta Dolaifl, Gina Marie Curcio, Joseph Del Rosario Inigo, Krishnaswami Raja§, and Probal Banerjee, Coupling to A Cancer Cell-Specific Antibody Potentiates Tumoricidal Properties of Curcumin, International Journal of Cancer, 2008:289:199,1-24 • Kunnumakkara AB, Anand, P., Aggarwal, B.B. Curcumin inhibits proliferation,invasion, angiogenesis and metastasis of different cancers through interaction with multiple cell signaling proteins. Cancer Letters 2008;269:199- 225. • Lopez-Lazaro M. Anticancer and carcinogenic properties of curcumin: considerations for its clinical development as a cancer chemopreventive and chemotherapeutic agent. MolecularNutrition & Food Research 2008;53:S103- S27. • Steward WP, and Gescher, A.J. Curcumin in cancer management: Recent results of analyogue design and clinical studies and desirable future research. Mol. Nutr. Food Res.2008;52:1005-9.