Cooperative learning : High resolution 13 C and 1H FT-NMR and 2D 1H-13C HSQC of Curcumin:

Mohammed Izmika, Kassandra Dorce, Mohammed Sherwani, and Samira Izmika

By Dr. Robert D. Craig,Ph.D

In this paper, student projects are given as an example on how to introduce FT –NMR into the undergraduate curriculum.

We will incorporate NMR experiments that illustrate the application of high resolution NMR spectroscopy 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) 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

Introduction

This paper will focus on Cooperative learning in science education.

What is Cooperative learning? Cooperative Learning is an instructional strategy that incorporates academic and social skill learning. As science educators, we understand the critical importance for our intermediate learners to be engaged in their learning while developing socially acceptable communication skills

Curcumin is know for it ability to fight amyloid plaque and well as having positive results in treating cervical and brain cancer . However, it is also know for it’s poor bioavailabity.

Natural cancer agents such as Taxol and Curcumin are isloated via ethanol extraction procedure. They are futher purified by size exclusion chromotography and HPLC

Their structure is confirmed by various solvents in FT-NMR experiments and Chemdraw NMR software.

2D -HSQC FT-NMR Utilizes bond coupling between H and C. and is extremely useful in determining structure of organic compounds. It also Eliminates all the H containing C’s so this eliminates many C’s assignments (why carboxyl group CH2 do not appear!)

This leaves only on-H containing C’s to assign.

There are several ways in which phase sensitive data can be recorded. Phase sensitive will be what will be discussed below.

Student became familiar with SpinWorks. This software must know how the data were recorded in order to process it correctly. It is also possible to adjust the timings in a pulse sequence so that there is no required F1 phase correction. This

approach is seen in most Varian NMR data. For Varian data, SpinWorks guesses the F1 detection mode from the name of the pulse sequence. Most phase sensitive Varian pulse sequences are either hypercomplex (States) detection (e.g. HSQC, TOCSY, NOESY) or echo-antiecho (e.g. gHSQC)

Experimental

For the Varian 600mHz, 5mm NMR Sample tubes were used. The NMR sample tubes were“L” Series 5mm NMR tubes (4.960 ± 0.006mm OD; 0.40mm nominal wall; 0.0025mm roundness). Spectra taken by the Varian 600 Mhz spectrometer used TMS as an internal standard. 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, was created by Kirk Marat. It provides us with

excellentdata analysis options andtabular results for ppm shifts for both spectra. Curcumin is a red powder after extraction is performed. This red powder was successively subjected to 1H NMR, 13C NMR, and 2D -HSQC FT-NMR analysis for structuredetermination (compound 1). See image below

Results and discussion

Figure 1 and 2 are the 1H and - 13C of curcumin. The proton spectra give rises to 14 peaks. The carbon spectra displays 13 peaks

Figure 1: the proton spectra of curcumin acquired by the 600 mHz varian spectrometer

For Curcumin, the proton (1H spectrum) shifts are as follows. 2 similar protons on the aromatic groups give rise to shifts at 5.55ppm (aromatic C-OH). 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 (chemdrawNMR was used to confirm this spectral assignment

Table 1 : Varian measurements of curcumin generated by spinworks

curcumin proton from Spin works curcumin carbon 13 from Spin works

Peak shift feq(ppm) Peak shift feq(ppm)

1 7.616 Aromatic C-H 1 182.2

Carboxylic 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

12 5.859Aromatic C-OH 12 76.78 vinylic groups (R2C=C-R

13 5.812Aromatic C-OH 13 55.99 Alkyl 2o and 3o carbon

14 3.962methoxy OCH3 *

missing ref xx 84.4 quartenary to Oxygen

*

missing ref xx 72.9 quartenary to Oxygen

Liu, Sun, and Huang

Students also became familiar with the software Chemdraw NMR. Data from this software was highly beneficial to assign and confirm spectra acquired by the varian

Table 2 : proton spectra of curcumin generated by chemdraw NMR

curcumin proton from chemdraw

shift (ppm) atom index coupling partnerconstant and vectorDelta H

5.35 75.35 257.16 24

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

20 7.5H-C*C*-H

7.16 620 20 1.5 H-C*C*C*-H

6.79

21 21 7.5H-C*C*-H

24 24 1.5 H-C*C*C*-H6.99 3

4 4 7.5H-C*C*-H

6.79 4

3 3 7.5H-C*C*-H

6 6 1.5 H-C*C*C*-H3.83 103.83 274.59 13

7.6 28

30 15.1H>C=C<H

7.6 29

31 15.1H>C=C<H

6.91 30

28 15.1H>C=C<H

6.91 31

29 15.1H>C=C<H

Table 3: proton assignment using chemdraw NMR

Curcumin chemcraw

OH=5.35 5.5 5.00.Aromatic C-OH

0.35 General correction

OH=5.35 5Aromatic 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=C0.52 General correction

CH=6.99 6.99 7.26 1-Benzene-0.11 General correction

-0.53Aromatic 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=C0.52 General correction

CH=6.79 6.79 7.26 1-Benzene-0.44 General correction

-0.17Aromatic 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

-0.17Aromatic C-OH

-0.04 General correction

Analysis of 13 Carbon for Curcumin:

In some important ways 13C spectra are usually less complex and easier to interpret than the 1H NMR spectra.

The interpretation is greatly simplified because each unique carbon atom only produces one 13C peak.

The low natural abundance of 13C nuclei and its inherently low sensitivity also have the effect that this spectra can only be obtained on pulse FT NMR spectrometers. The Varian 600 mHz being highly suitable for this purpose.

Where as carbon-carbon splitting does not occur in 13C NMR spectra, hydrogen atoms attached to carbon can split 13C NMR signals into multiple peaks. It is possible to eliminate signal splitting by 1H -13C coupling by altering instrument parameters to do so.

Students found the concept of 13C chemical shifts highly intriging. Relatively higher electron density around an atom shields the atom from the magnetic field and causes

the 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 atoms are 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 13C NMR spectra

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 mHz varian 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 from The 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.

Analysis of -2D 1H - 13C HSQC spectra Carbon for curcumin:

Below is Figure 3 and 4 they are the 2D 1H - 13C HSQC spectra of Curcumin

Figure 3 the 2D 1H - 13C HSQC spectra of Curcumin

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 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

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

We will shortly find this of high interest.

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 referenced 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.

With peak 1 and 2 (on diagonal) (7.1427 ppm 1H , and 7.1290 ppm 1H ) probably refers to These hydrogens that are H-C=C-H on a benzene ring. 123.8 ppm is reminiscient of

the conjagated ring as well. For a 13C signal in the spectra at 101 ppm, the coupling with With 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.