Cooperative learning of nmr in anticancer agents

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This paper will focus on Cooperative learning in science education.
Curcumin extract is subjected to 1H NMR, 13C NMR, and 2D -HSQC FT-NMR analysis for structure
the 2D NMR specra may be obtained that indicate coupling between hydrogens and carbons to which they are attached. In this case it is called heteronuclear correlation spectroscopy (HECTOR, HSQC, or C-H HECTOR).

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Cooperative learning of nmr in anticancer agents

  1. 1. Cooperative learning :High resolution 13 C and 1HFT-NMR and2D 1H-13C HSQC ofCurcumin:Mohammed Izmika, KassandraDorce, Mohammed Sherwani, and Samira IzmikaBy Dr. Robert D. Craig,Ph.DIn this paper, student projects are given as an example on how to introduce FT –NMR into theundergraduate curriculum.We will incorporate NMR experiments that illustrate the application of high resolution NMRspectroscopy to the structure determination of Anti-Cancer agents.High resolution 13 C and 1H NMR , 13 C –distortionless enhancement by polarization transfer(DEPT) , 2D 13C-1H correlated (HECTOR), and 2D 1H-1H correlated (COSY) spectroscopytechniques will be used for elucidating skeletal arrangement of monomer units.Applications that also use the 2D 1H-13C HSQC experiment are gaining more interest as a resultof the growing feasibility of acquiring these spectra routinely. The 2D HSQC experimentcontains additional information (i.e. 13C chemical shift) as well as easier identification of labileand diastereotopic protons
  2. 2. This leaves only on-H containing C’s to assign.Introduction There are several ways in which phaseThis paper will focus on Cooperative sensitive data can be recorded. Phaselearning in science education. sensitive will be what will be discussed below.What is Cooperative learning? CooperativeLearning is an instructional strategy that Student became familiar with SpinWorks.incorporates academic and social skill This software must know how the datalearning. As science educators, we were recorded in order to process itunderstand the critical importance for our correctly. It is also possible to adjust theintermediate learners to be engaged in timings in a pulse sequence so that there istheir learningwhile developing socially no required F phase correction. This 1acceptable communication skills approach is seen in most Varian NMR data. For Varian data, SpinWorks guesses the F 1 detection mode from the name of the pulseCurcumin is know for it ability to fight sequence. Most phase sensitive Varianamyloid plaque and well as having positive pulse sequences are either hypercomplex (States) detection (e.g. HSQC, TOCSY,results in treating cervical and brain cancer . NOESY) or echo-antiecho (e.g. gHSQC)However, it is also know for it’s poorbioavailabity.Natural cancer agents such as Taxol andCurcumin are isloated via ethanol extractionprocedure. They are futher purified by size Experimentalexclusion chromotography and HPLC For the Varian 600mHz, 5mm NMR SampleTheir structure is confirmed by various tubes were used. The NMR sample tubessolvents in FT-NMR experiments and were“L” Series 5mm NMR tubes (4.960 ±Chemdraw NMR software. 0.006mm OD; 0.40mm nominal wall; 0.0025mm roundness). Spectra taken by2D -HSQC FT-NMR Utilizes bond coupling the Varian 600 Mhz spectrometer used TMSbetween H and C.and is extremely useful in as an internal standard. All spectra wasdetermining structure of organic processed from the Varian usingcompounds. It also Eliminates all the H spinworksplatform.Thespecta wascontaining C’s so this eliminates many C’s subsequently confirmed using Chemdrawassignments (why carboxyl group CH2 do NMR. It was convinent to use Spinworks tonot appear!) analyze spectra. The Spinworkssoftware,was created by Kirk
  3. 3. Marat. It provides us with excellentdataanalysis options andtabular results for ppmshifts for both spectra. Curcumin is a redpowder after extraction is performed. Thisred powder was successively subjected to1 H NMR, 13C NMR, and 2D -HSQC FT-NMRanalysis for structuredetermination (compound 1). See imagebelow
  4. 4. Results and discussionFigure 1 and 2 are the 1H and - 13C of curcumin. The proton spectra give rises to 14peaks. The carbon spectra displays 13 peaksFigure 1: the proton spectra of curcumin acquired by the 600 mHzvarian spectrometerFor Curcumin, the proton (1H spectrum) shifts are as follows. 2 similar protons on thearomatic groups give rise to shifts at 5.55ppm (aromatic C-OH). The benzene CH ofwhich there are 3, give rise to 7.16 ppm, 6.99 ppm and 6.79 ppm. On the hexadienonebridge, between the two benzene rings (aromatic rings) are 2 pairs of equivalentprotons(chemdrawNMR was used to confirm this spectral assignment
  5. 5. Table 1 : Varian measurements of curcumin generated by spinworkscurcumin proton from Spin works curcumin carbon 13 from Spin worksPeak shift feq(ppm) Peak shift feq(ppm) Carboxylic 1 7.616 Aromatic C-H 1 182.2 acid peak 2 7.59 Aromatic C-H 2 147.7 Alkene 3 7.271 Aromatic C-H 3 146.6 Alkene 4 7.27 Aromatic C-H 4 140.5 Alkene 5 7.143 Aromatic C-H 5 127.5 aromatic 13C 6 7.129 Aromatic C-H 6 123.8 aromatic 13C 7 7.063 Aromatic C-H 7 114.8 RCH=CH2 and R2C=CH2 8 6.954 1-Benzene 8 109.3 double bonded RCH=CHR 9 6.94 1-Benzene 9 107.6 double bonded RCH=CHR 10 6.502 1-Benzene 10 77.19 vinylic groups (R2C=C-R 11 6.476 1-Benzene 11 76.97 vinylic groups (R2C=C-R Aromatic C- 12 5.859 OH 12 76.78 vinylic groups (R2C=C-R Aromatic C- 13 5.812 OH 13 55.99 Alkyl 2o and 3o carbon methoxy 14 3.962 OCH3 * missing ref xx 84.4 quartenary to Oxygen * missing ref xx 72.9 quartenary to Oxygen Liu, Sun, and HuangStudents also became familiar with the software Chemdraw NMR. Data from thissoftware was highly beneficial to assign and confirm spectra acquired by the varian
  6. 6. Table 2 :proton spectra of curcumin generated bychemdraw NMRcurcumin proton from chemdrawshift (ppm) atom index coupling partner constant and vector Delta H 5.35 7 5.35 25 7.16 24 20 20 1.5 H-C*C*C*-H 6.99 21 H-C*C*- 20 7.5 H 7.16 6 20 20 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 6.99 3 H-C*C*- 4 4 7.5 H 6.79 4 H-C*C*- 3 3 7.5 H 6 6 1.5 H-C*C*C*-H 3.83 10 3.83 27 4.59 13 7.6 28 H>C=C< 30 15.1 H 7.6 29 H>C=C< 31 15.1 H 6.91 30 H>C=C< 28 15.1 H 6.91 31 H>C=C< 29 15.1 H
  7. 7. Table 3: proton assignment using chemdraw NMRCurcumin chemcrawOH=5.35 5.5 5.00. Aromatic C-OH 0.35 General correctionOH=5.35 5 Aromatic C-OH 0.35 General correctionCH=7.16 7.26 7.26 -0.49 1 –O-C -0.17 1 –O -0.04 1 –C=C 0.52 General correctionCH=6.99 6.99 7.26 1-Benzene -0.11 General correction -0.53 Aromatic C-OH -0.55 General correctionCH=7.16 7.16 7.26 -0.49 1 –O-C -0.17 1 –O -0.04 1 –C=C 0.52 General correctionCH=6.79 6.79 7.26 1-Benzene -0.44 General correction -0.17 Aromatic C-OH -0.04 General correctionCH=6.99 6.99 -0.11 1 –O-C -0.53 1 –O -0.05 1 –C=C 0.42 General correctionCH=6.79 6.79 7.26 1-Benzene -0.44 General correction -0.17 Aromatic C-OH -0.04 General correction
  8. 8. Analysis of 13 Carbon for Curcumin:In some important ways 13C spectra are usually less complex and easier to interpretthan the 1H NMR spectra.The interpretation is greatly simplified because each unique carbon atom only producesone 13C peak.The low natural abundance of 13C nuclei and its inherently low sensitivity also have theeffect that this spectra can only be obtained on pulse FT NMR spectrometers. TheVarian 600 mHz being highly suitable for this purpose.Where as carbon-carbon splitting does not occur in 13C NMR spectra, hydrogen atomsattached to carbon can split 13C NMR signals into multiple peaks. It is possible toeliminate signal splitting by 1H -13C coupling by altering instrument parameters to doso.Students found the concept of 13C chemical shifts highly intriging. Relatively higherelectron density around an atom shields the atom from the magnetic field and causesthe signal to occur upfield (lower ppm and to the right) in the NMR spectrum.For example, carbon atoms that are attached only to other carbon and hydrogen atomsare relatively shielded from the magnetic field by the density of electrons around them,and, as a consequence carbon atoms of this type produce peaks which are upfield in 13CNMR spectra
  9. 9. Below is the proton spectra of curcumin acquired by the 600 MHz Varian spectrometer Figure 2: the carbon 13 spectra of curcumin acquired by the 600 mHzvarian spectrometer When analyzing the hector spectra – it might be first beneficial to designate the carbon spectral lines first. Then, sweep these lines across the proton specta. For the 13 spectra of curcumin, it is better we see this before address the Hector spectra some peaks fromThe peak at 55.954 is due to alkyl 2o or alkyl 3o carbon. Alkyl 2o and 3o carbon reside in the 50 ppm region, so obviously the 55.985 ppm corresponds to such a group. There are three peaks at 77.18662, 76.96907 and 76.78016. these belong probably to vinylic groups on the hexadione bridge (R2C=C-R). The peaks from 109 to 107,(109.3 and 107.6) Are probably due to double bonded carbons also on the hexadione bridge. They are for RCH=CHR carbon 13 resonances. Similar RCH=CHR carbon 13 resonances, RCH=CH2 and R2C=CH2 from the 127.5 ppm, 123.8 ppm, 122.6 ppm and 114.8 ppm. this will also encompus the peaks at 147.7 ppm, 146.6 ppm and 140.5 ppm. Lastly , aromatic 13C are from 120 to 135 ppm.
  10. 10. Analysis of -2D 1H - 13C HSQC spectra Carbon for curcumin:Below is Figure 3 and 4 they are the 2D1H - 13C HSQC spectra ofCurcuminFigure 3 the 2D 1H - 13C HSQC spectra ofCurcumin
  11. 11. Figure 4 (zoomed at 200%) are the 2D 1H - 13C HSQC spectra of Curcumin.The 2D NMR specra may be obtained that indicate coupling between hydrogens andcarbons to which they are attached. In this case it is called heteronuclear correlationspectroscopy (HECTOR, HSQC, or C-H HECTOR).When ambiguities are present in one-dimensional 1H and 13C NMR spectra, a HECTORor HSQC spectrum can be very useful for assigning preciscely which hydrogens andcarbons are producing their respective peaks. In a HSQC spectrum a 13 C spectrum ispresented along one axis and a 1H spectrum is shown along the other. Cross peaks
  12. 12. relating the two types in a HSC spectrum indicate which hydrogens are attached towhich carbons in a molecule, or vice versa.These cross peaks correlations are the result of instrumental parameters specified onthe NMR spectrometer. If imaginary lines are drawn from a given cross peak in the x-yfield to each respective axis,. The cross peak indicates to the hydrogen giving rise to thecorresponding 1H NMr signal on one axis and is coupled or attached to the carbon thatgives rise to corresponding 13C NMR signal on the other axis. Thus, it is readilyapparent which hydorgens are attached to which carbonsLet’s dive right in, as the research students have provided the spectra and determinethe HSQC for Curcumin, with the aid of the ChemdrawNMR software, and previous scanof curcumin (proton and 13C). It is beneficial to keep these spectra on hand. TheSpinworks software, created by Kirk Marat.also provides us with excellent ppm shifts forboth spectraWorking from top down, and left to right, the HSQC for curcumin reads as such. Thefirst peak evident in the spectra is 13C at 55.934 ppm, And crossed withProton(designed 14) at 3.9620 ppm. The next peak is with Proton(designed 12) at5.8592 and a 13 C at 101 ppmThis hydrogen must be attached to the OH group , or might be the hydrogen in betweenthe carbonyls on the hexadione bridge.The carbon 13 peak at 109.3 cross with several protons. Referenced with the spinworksdata table for curcumin proton data taken the Varian 600 MHz we have for Peak 3 in theHSQC specta with Peak 12 at 5.8592 ppm And Peak 13 at 5.8124 ppm in the protonspectra. 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 anaromatic hydrogen. This hydrogen resides on a benzene ring, and is obviouslyconfirmed by coupling with an aromatic 13C at 109.3 ppmWe will shortly find this of high interest.It is this hydrogen that will be effected in the carboxyalated form of curcumin. Thecarbon peaks at 122.6 and 123.8 ppm with peak 3 and 4 (off diagonal) of the protondata gives some modest peaks in the specta. Also evident are Peak 3 (122.6 ppm 13Cwith (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 1H , 6.503 ppm 1H) . These hydrogens are onaromatic ring next to hydroxyl groups. A Carbon of 114 ppm is appropriate to be
  13. 13. adjacent to these hydrogens. As referenced by the ChemdrawNMRsoftwareplatfom”Thebenzene CH of which there are 3, give rise to 7.16 ppm, 6.99 ppm and 6.79 ppm. “Onthe hexadienone bridge, between the two benzene rings (aromatic rings) are 2 pairs ofequivalent protons, (see table 2). The software also allows for shift corrections.With peak 1 and 2 (on diagonal) (7.1427 ppm 1H , and 7.1290 ppm 1H ) probably refersto These hydrogens that are H-C=C-H on a benzene ring. 123.8 ppm is reminiscient ofthe conjagated ring as well. For a 13C signal in the spectra at 101 ppm, thecoupling withWith peak 12 (5.8592 ppm) and 13 (off diagonal) (5.8124ppm 1H ) is rather confusing.Most likely,these 2 similar protons on the aromatic groups give rise to shifts at 5.55ppm(aromatic C-OH).CONCLUSION • Using information from 1H NMR data alone is not a new concept. However, applications that also use the 2D 1H-13C HSQC experiment are gaining more interest as a result of the growing feasibility of acquiring these spectra routinely. The 2D HSQC experiment contains additional information (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 for making this work possible. I find cooperative learning to be very important because it is crucial for our students to learn to work in groups. This not only helps develop their social skills, but also enhances their ability to develop the skills necessary to work collaboratively when they enter the work force.

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