Cooperative learning using FT-NMR :

Students: Mohammed Izmikna, Kasandra Dorce, Mohammed Sherwani, Samira Izmikna,

By Dr. Robert Craig, Ph.D.

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. We can make a tentative spectulation that For the side chain methyl ester, the coupling constant JH2-H3 changes from 2 Hz to 5 Hz. This confirms a shift in conformational equilibria, after addition of this group. The “deshielding effect” of a carboxylate group on the monomer of curcumin is shown by 2D -HSQC FT-NMR data and the ChemdrawNMR software.

Cooperative learning using FT-NMR :

Abstract

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 HSQC FT-NMR to organize spectra data is shown. Here, undergraduates become familiar with making plots of 1H NMR and 13C NMR , learning FT-NMR data processing (spinworks) and also use Chemdraw NMR to present data take with a Varian 600 MHz FT-NMR spectrometer. For the side chain methyl ester, the coupling constant JH2-H3 changes from 2 Hz to 5 Hz. This confirms a shift 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 how to introduce FT –NMR into the undergraduate curriculum.

The deshielding of the antimitotic agent curcumin and it side chain methyl ester have been studied by Nmr spectroscopy and by molecular modeling using ChemDrawNMR .

Upon carboxylation, some proton shifts change. Students proficient in ChemDrawNMR show this in their analysis.

For the side chain methyl ester, the coupling constant JH2-H3 changes from 2 Hz to 5 Hz. This confirms a shift in conformational equilibria.

We will incorporate NMR experiments that illustrate the application of high resolution NMR spectroscopy to the structure determination of Anti-Cancer agents.

Adding NMR spectroscopy to a students repertoire of skills will greatly enhance the laboratory learning experience and enable us to adopt experiments that have been successful elsewhere at utilizing NMR spectroscopy as an essential teaching tool.

This paper will also examine the benefits of students involve in

performing NMR experiments at the undergraduate level

As far as Curcumin is concerned, The effect of the carboxyl

group on the proton spectra(deshielding effect) is clearly shown

with 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 in

particular NMR spectroscopy, in the chemistry curriculum. An

excerpt from this document states, “A department should have

several major pieces of sophisticated equipment suitable for

undergraduate instruction as well as for research. One of these

must be an NMR spectrometer.”1 Later in this same document

it is noted that the instrument should be an FT-NMR. In

addition, the most current proposed revision to this document

states, “The laboratory experience should include synthesis of

molecules, measurement of chemical properties and

phenomena, “hands-on” experience with modern

instrumentation, applications to real-world problems, and

computational data analysis and modeling.”2

Curcumin’s broad spectrum of anti-oxidant, anti-carcinogenic,

anti-mutagenic, and anti-inflammatory properties makes it

particularly interesting for the development of pharmaceutical

compounds. Due to curcumin’s various effects on the function

of numerous unrelated membrane proteins, it has been

suggested that it affects the properties of the bilayer itself.

Despite intense interest in the physiological effects of curcumin, a general mechanism for its action has not been identified.

Figure 1

Keto-enol form of curcumin, the dominant tatuomer of curcumin. The keto-enol from is stabilized by an intramolecular hydrogen bond, shown here by a dashed red line.

Here is a mechanism of interest to many.

MATERIALS AND METHODS

Curcumin (>94%) was purchased from Sigma and used without

further purification

This red powder was successively subjected to 1H NMR, 13C

NMR, and 2D -HSQC FT-NMR analysis for structure

For the Varian 600mHz, 5mm NMR Sample tubes were used

from NewEra, inc

. The NMR sample tubes were“L” Series 5mm NMR tubes

(4.960 ± 0.006mm OD; 0.40mm nominal wall; 0.0025mm

roundness).

All spectra was processed from the Varian using spinworks

platform.The specta was subsequently confirmed using

Chemdraw NMR. It was convinent to use Spinworks to analyze

spectra. The Spinworks software, created by Kirk Marat. also

provides us with excellent ppm shifts for both spectra.

FT NMR

All of the experiments were performed on a Varian Infinity 600 MHz solid-state NMR spectrometer. Each sample was equilibrated for at least 30 minutes before starting the experiment. 2D 13C-1H correlated (HECTOR) NMR spectra were obtained using a spin-echo pulse sequence (90°-τ–180°-τ-acquisition; τ = 125 μs) with a 90° pulse length of 5 μs under a 30 kHz continuous-wave proton decoupling.

Chemical shifts were referenced by setting the isotropic chemical shift peak of TMS to 0 ppm. 2D 13C-1H correlated (HECTOR) quadrupole coupling spectra were recorded using a quadrupolar echo pulse sequence (90°-τ–90°-τ-Acquisition; τ = 80 μs) without proton decoupling.

RESULTS AND DISCUSSION

Since curcumin affects such a large array of unrelated membrane proteins at approximately similar concentrations, it has been proposed that curcumin can regulate the action of membrane proteins indirectly by changing the physical properties of the membrane rather than by the direct binding of curcumin to the protein.

High resolution 13 C and 1H NMR , 2D 13C-1H correlated (HECTOR), and 2D 1H-1H correlated (COSY) spectroscopy techniques 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 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

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).

When ambiguities are present in one-dimensional 1H and 13C NMR spectra, a HECTOR or HSQC spectrum can be very useful for assigning preciscely which hydrogens and carbons are producing their respective peaks.

In a HSQC spectrum a 13 C spectrum is presented along one axis and a 1H spectrum is shown along the other. Cross peaks relating the two types in a HSC spectrum indicate which hydrogens are attached to which carbons in a molecule, or vice versa.

These cross peaks correlations are the result of instrumental parameters specified on the NMR spectrometer. If imaginary

lines are drawn from a given cross peak in the x-y field to each respective axis,

The cross peak indicates to the hydrogen giving rise to the corresponding 1H NMr signal on one axis and is coupled or attached to the carbon that gives rise to corresponding 13C NMR signal on the other axis.

Thus, it is readily apparent which hydorgens are attached to which carbons

FIG 1: The effect of the carboxylated curcumin on proton signal

The effect of the carboxylated curcumin on proton signal

Referring to the chemdrawNMR data below

curcumin proton

from chemdraw

shift atom index coupling partner constant and vector

5.35 7 delta (ppm)5.35 257.16 24

20 20 1.5 H-C*C*C*-H6.99 21

20 7.5 H-C*C*-H7.16 6

20 20 1.5 H-C*C*C*-H6.79

21 21 7.5 H-C*C*-H24 24 1.5 H-C*C*C*-H

6.99 3

4 4 7.5 H-C*C*-H6.79 4

3 3 7.5 H-C*C*-H6 6 1.5 H-C*C*C*-H

3.83 103.83 274.59 13

7.6 2830 15.1 H>C=C<H

7.6 2931 15.1 H>C=C<H

6.91 3028 15.1 H>C=C<H

6.91 3129 15.1 H>C=C<H

curcumin proton

from chemdraw

carboxylated

shift atom index coupling partnerconstant and vector

5.35 2511 35

7.16 2420 20 1.5 H-C*C*C*-H

6.99 21

20 7.5H-C*C*-H

7.3 @@ 64 4 1.5 H-C*C*C*-H

6.79

21 21 7.5H-C*C*-H

24 24 1.5 H-C*C*C*-H7.13 3

4 4 7.5H-C*C*-H

7.26 '@@ 4

3 3 7.5H-C*C*-H

6 6 1.5 H-C*C*C*-H3.83 103.83 27

2.3 2931 31 7.1 H-CH-CH-H

2.3 3231 31 7.1 H-CH-CH-H

4.59 132.01 31

29 29 7.1 H-CH-CH-H7.6 36

38 38 15.1H>C=C<H

6.91 38

36 36 15.1H>C=C<H

6.91 39

37 37 15.1H>C=C<H

•

above is Figure 2: 13C spectra of Curcumin

above is Figure 3: the 2D 1H - 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 1H , 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 1H , 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

Mono Carboxyated is here

Figure 4, 5 and 6 are the 2D 1H - 13C HSQC spectra of mono carboxylated Curcumin

Assign ment of theMONO-CARBOXYLATED CURCUMIN HSQC-ON THE WALL

The peak at 6.953 ppm for hydrogen (and 6.94 ppm) corresponds to a C=C-OH on the right benzene ring of the mono carboxylated form.

The carbon associated with this Hydrogen is the signal at 114 ppm. The carbon experiencing a more electronegative environment is next to this

Carbon at 123.8 ppm . this carbon at 123.8 ppm gives a signal with a proton at 7.063 ppm. This 7.063 also spin couples with a carbon

At 109 ppm. This is associated with a similar fragment, C=C-OCH3 on the top of rightsided benzene ring. The carbon at 109 ppm couples twice

With 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 of the rightsided benzene ring couples with 3 carbons at 109 ppm, 111.8 ppm

And 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 of the rightsided benzene ring, as well, and couples with 3 carbons at 109 ppm,

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 is the bottom of the benzene ring on the right side

of the carboylated curcumin, unaffected by this substituent group. The Hc- C-C=C-OH is responsible for the peak at 7.19 ppm,120ppm

and 7.19 ppm,120 ppm

This cluster of peaks resides in the bottom right portion of the HSQC spectra mono carboxylated curcumin molecule.

The carbon at 140.5 ppm (Ca)signals with a hydrogen residing on the hexadione brigde (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 2D 1H - 13C HSQC spectra of mono carboxylated 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 group

conclusionHeteronuclear Single Quantum Coherence (HSQC)Plots 1H NMR on x-axis and 13C

NMR on y-axis and Utilizes 1 bond coupling between H and C, Eliminating all the

H containing C’s eliminates many C’s assignments. Leaves only on-H containing

C’s to assign.

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 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

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