THz Spectra of 1,4-Naphthoquinones and its Four Derivatives* Weining Wang1, Hongqi Li2, Xiang Luo1, Xiaoni Zeng1. 1

Beijing Key Lab for Terahertz Spectroscopy and Imaging, Key Laboratory of Terahertz Optoelectronics, Ministry of Education, Department of Physics, Capital Normal University, Beijing, 100037 China Beijing 100037, China. [email protected] 2

Department of Biology, Frostburg State University, Frostburg, MD, USA, 21532. [email protected] Abstract

Recently some naphthoquinone derivatives have been found with anticancer or other therapeutic properties, but also have some negative side effects. Numerous research projects have been conducted to investigate their properties and therapeutic mechanisms. With Terahertz Time-Domain Spectroscopy (THz-TDS), we have successfully obtained THz spectra of 1,4-naphthoquinone and its four derivatives in a series of naphthazarin – juglone – 1,4-naphthoquinone – menadione – plumbagin, in the range between 0.2 and 2.4~2.8 THz. Although these molecules are almost identical to each other, they have very distinctive THz spectra so that they can be identified much more easily than using conventional spectroscopy. We have comparatively analyzed their THz spectra, and found some possible correlations between THz spectra and molecular structures. These THz spectra cannot only be used as spectral fingerprint, but also provide us their conformational properties that can be used in study of their interaction with biomolecules to reveal their pharmaceutical mechanisms. Key words: THz-TDS, THz spectra, 1,4-naphthoquinone, naphthoquinone derivatives, spectral fingerprint, medicine, molecular conformation.

1. Introduction In recent years, some naphthoquinone derivatives (e.g., plumbagin, juglone, menadione, and naphthazarin) have been found with antibacterial, antifungal, and antiviral properties, and they even could be used as anticancer agent because they can provoke cell apoptosis clinics because of their negative side effects, e.g., necrosis

(5)

(1-5)

. However, they have not been used in

. In order to use them as anticancer agents safely,

their molecular properties and chemotherapeutic mechanism have been studied with various conventional spectroscopy

(1-5)

. In FTIR spectra, absorption peaks are assigned to specific functional groups

(4, 6)

. However,

conformational properties of the entire molecules should be further considered because in reality, molecules with different structural and dynamic conformations would be hard to react each other under normal condition or without helps of catalysts, while molecules with the same leveled vibrational and rotational dynamics could be easily react with each other, even without assistances of enzymes. Therefore, approaches that can detect vibrational and rotational transitions of an entire molecule are really needed to further study interaction between molecules. *

This research is supported with a grant (#10390160) from National Natural Science Foundation of China and a grant (KM200710028004) from Beijing Municipal Education Commission. Terahertz Photonics, edited by Cunlin Zhang, Xi-Cheng Zhang, Proc. of SPIE Vol. 6840, 68400O, (2007) · 0277-786X/07/$18 · doi: 10.1117/12.759883

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Terahertz Time-Domain Spectroscopy (THz-TDS) is just one such desired approach. This new technology uses a THz pulse to stimulate biomolecules, detect their molecular vibrational and rotational transitions to generate THz absorption spectra with a high signal-to-noise ratio (SNR). So, a molecule’s absorption peaks present modes of collective rotational and vibrational transitions of the entire molecule, and THz spectrum exhibits conformational information of the entire molecule, and it is not only determined by molecular structures but also by interactions with other molecules

(7-9)

. However, it has been very hard to find

out the correlation between molecular structures and THz spectra. For the first time, we have comparatively studied a series of naphthoquinones and revealed some correlations between their THz spectra and molecular structures, carbonyl, hydroxyl, and methyl. In addition to our comparative studies of FTIR and THz spectra of plumbagin, juglone, and menadione

(6)

, we also have

examined naphthazarin and 1,4-naphthoquinone with THz-TDS, and obtained their THz spectra in the range between 0.2 and 2.4~2.8 THz. We selected these naphthoquinones because they can be arranged in a series, naphthazarin – juglone – 1,4-naphthoquinone – menadione – plumbagin, with just one different functional group between two neighboring molecules. Therefore, comparatively study of their THz spectra and their molecular structures can lead us to find out correlations between molecular structural changes and spectral changes.

2. Material and Experimental Design Polycrystalline powder samples of juglone (5-hydroxy-1,4-naphthoquinone, 97%), naphthazarin (5,8-dihydroxy-1,4-naphthoquinone, 97%), menadione (2-Methyl-1,4-naphthoquinone, 98%), and plumbagin (5-hydroxy-2-Methyl-1,4-naphthoquinone, 98%), and 1,4-naphthoquinone (98%) were all obtained from Sigma-Aldrich Chemical Co. (USA). Naphthazarin and 1,4-naphthoquinone powders are directly used and other samples were mixed with polyethylene powder in a mass ratio of 2:1, to be pressed to thin disks, 1 mm thick and 1.3 mm in diameter, under a pressure of 2000 kg. THz-TDS apparatus (Fig. 1) used in this project consists of two parts: MaiTai Laser System (from Spectra-Physics, USA) used as a pulse generator, and THz-TDS (from Zomega Terahertz Co., USA). The system was set up according to Wang et al. (2005) (11).

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Probe beam

M9

M8 M4

Detector

M10

Stage

A2

M11

L2

M3

M12 P

PBS L3

Chopper PM4

M14

InAs

PM1

Pump beam

L1

M13

QWP

ZnTe

Si

A4

CBS M7

HWP A1

Sample PM3

M1

A3

PM2

M2

M6

M5

MaiTai Laser

Fig. 1. Diagram of the THz-TDS System.

30

70

Naphthazarin

60 Absorbance/a.u.

Absorption/a.u.

25 20

0.93 15

2.70

Juglone

1.57

10

50 40 30 20

5 Sam:PE=2:1, 0.2~2.8

0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 2.6 2.8 Frequency/THz

10

1.67 0.89 Sam:PE=2:1,0.2~2.8

0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 2.6 2.8 Frequency/THz

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80 70

O

Absorption/a.u.

50 40

1.77

O

2.32

12

60

Absorption/a.u.

M enadione

2.70

1,4-naphthoquinone

2.23

30

8

1.67

4

20

0.87

10

0 0.2

Sam:PE, 0.9~2.8

0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 2.6 2.8

1.42

1.18

1.07

Sam :PE=2:1, 0.2~2.4

0.4

0.6

0.8

1.0

1.2

1.4

1.6 1.8

2.0 2.2

2.4

Frequency/THz

Frequency/THz

15

:rCHa

5

1.9

1.20

1.8

1.40

1.7

1.78 0.85

Plumbagin

Absorption/a.u.

Absorption/a.u.

10

2.0

2.42

Plumbagin

1.20 Sam:PE=2:1, 0.2~2.6

0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 2.6

1.6 1.0

1.1

1.2

1.3

1.4

1.5

1.6

Frequency/THz

Frequency/THz

Fig. 2. THz absorption spectra of 1,4-naphthoquinone, juglone, naphthazarin, menadione, and plumbagin.

3. Results and Discussions THz spectra of 1,4-naphthoquinone, plumbagin, juglone, naphthazarin, and menadione are presented in Fig. 2, and frequencies of their absorption peaks are presented in Table 1. Two THz spectral graphs of plumbagin are presented, with the second showing the vertically magnified absorption peak at 1.40 THz clearly. These molecules are almost identical to each other, except for specific functional groups, so their FTIR spectra are all quite similar to each other and it is hard to distinguish them without a careful analysis

(6)

.

However, their THz spectra are obviously very distinctive. So, THz spectra can be used as spectral fingerprint to identify molecules much more easily than conventional spectra, even among serial molecules with tiny different molecular structures. Since there is just one different functional group between two neighboring molecules in the series of naphthazarin – juglone – 1,4-naphthoquinone – menadione – plumbagin, there should be some regular pattern among their THz spectra and their molecular structures. All of these naphthoquinones exhibit an absorption peak between 0.85-0.93 THz, but 1,4-naphthoquinone does not, because the THz-TDS was temporarily unstable so that the yielded spectrum with the section below 0.90 THz is not valid. Although there are two different groups between juglone and menadione, both exhibit an absorption peak at 0.89 and 0.87 THz, respectively. Since

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1,4-naphthoquinone differs from both juglone and menadione with just one side group respectively, we predict it should also exhibit a low frequency peak, possibly around 0.88 THz. This prediction will be tested as soon as possible. Table 1. Absorption peaks of naphthazarin, juglone, 1,4-naphthoquinone, menadione, and plumbagin.

Naphthazarin 0.93

1.57

Juglone 0.89

1,4-naphthoquinone (0.88?)

Menadione 0.87

Plumbagin 0.85

1.09

1.18

1.20

1.67(?) 2.70

1.42

1.40

1.77

1.67

1.78

2.23

2.32

2.42

2.70

Very excitingly, we have found some correlations between THz spectra and molecular structures in the series of naphthazarin – juglone – 1,4-naphthoquinone – menadione – plumbagin. We list these correlations below: 1. The THz spectrum of 1,4-naphthoquinone has two main absorption peaks at 1.77 and 2.70 THz to represent two rotational and vibrational modes that could be mainly affected by the 1- or 4-carbonyl groups respectively. This correlation is based on our analyses of rest naphthoquinones and our preliminary comparative observation on 1,2-naphthoquinone (to be published separately). 2. Hydroxyl (OH) group appears to counterbalance its diagonal carbonyl group’s dynamics (causing the associated absorption peak or peaks to diminish), but increase its neighbor carbonyl group’s dynamics (enlarging the associated absorption peak or peaks). This correlation is based on following three cases: A). Between 1,4-naphthoquinone and its derivative juglone, the addition of 5-hydroxyl group (OH) is associated with a major change between their THz spectra: the 1.77 peak is extremely reduced with a very tiny trace left at 1.67 THz (drifted toward the low frequency by 0.10 THz). This change suggests that the addition of 5-hydroxyl (OH) could possibly counterbalance the dynamic effect of 1-carbonyl (C=O) in the diagonal position, although it seems did not strengthen the 2.70 THz peak that is determined by the 4-carbonyl. B). Similarly, between juglone and naphthazarin the addition of 8-hydroxyl group (OH) appears counteracted the motion of 4-carbonyl (C=O) in the diagonal position, and caused the offsetting of 2.70 THz peak. In the mean while, it strengthened the dynamics of its neighbor 1-carbonyl (C=O) and caused the associated peak amplified and further shifted another 0.10 THz to 1.57 THz, and the energy oscillation enlarged the absorption peak at 0.93 THz very much. C). The 5-hydroxyl (OH) in Plumbagin appears to be responsible for the dramatic reduce of the 1.78 THz peak that is associated with the 4-carbonyl group. 3. The 2-methyl group (CH3) can increase rotational and vibrational modes, i.e., absorption peaks. Between 1,4-naphthoquinone and its derivative menadione, the addition of 2-Methyl group (CH3) did not only make the 2.23 THz peak shifted to 2.32 THz, but cause the occurrence of a new absorption peak at 1.42 THz, as well. This new peak is visible in the plumbagin spectrum at 1.40 THz to, although it is much smaller due to influence of the 5-hydroxyl (OH). The 2-Methyl group (CH3) could also offset the peak that is associated with

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the diagonal 4-carbonyl, but this is not demonstrated in the spectra of menadione and plumbagin because their valid ranges are limited to 2.4 and 2.6 THz respectively. We hope this can be tested out soon. More interestingly, absorption peaks of juglone and menadione can be all matched with those on the THz spectrum of plumbagin, but there is a difference about 0.10 THz between 1.67 and 1.78, and between 2.32 and 2.42 THz peaks.

4. Conclusions In this paper, all THz spectra of 1,4-naphthoquinone, juglone, naphthazarin, menadione, and plumbagin are demonstrated to be very distinctive so they can be used as spectral fingerprint to identify these serial molecules easily. Much more importantly, for the time the correlations between THz spectra and molecular structures (carbonyl, hydroxyl, and methyl) have been revealed based on our comparative study of THz of serial naphthoquinone molecules. When these correlations are further confirmed and more correlations are revealed, THz spectra will be much more useful in future studies of therapeutic mechanisms of naphthoquinones and other medicinal molecules. The significance will be much more profound in study field of physical chemistry and biochemistry.

References 1. P. J. Guan, D. F, Xu and S. S. Li. Advances in the antitumor activities of naphthoquinones. Chinese Journal of Medicinal Chemistry 60, 249–256 (2004). 2. S. Sugie, K. Okamoto, K. M. W. Rahman, T. Tanaka, K. Kawai, J. Yamahara and H. Mori. Inhibitory effects of plumbagin and juglone on azoxymethane-induced intestinal carcinogenesis in rats. Cancer Letters 127, 177–187 (1998). 3. R. Parimala and P. Sachdanandam. Effect of plumbagin on some glucose metabolising enzymes studied in rats in experimental hepatoma. Molecular and Cellular Biochemistry 125, 59–63 (1993). 4. D. Sajan, K. P. Laladhas, I. Hubert Joe and V. S. Jayakumar. Vibrational spectra and density functional theoretical calculations on the antitumor drug, plumbagin. Journal of Raman Spectroscopy 36, 1001-1011 (2005). 5. J. J. Inbaraj and C. F. Chignell. Cytotoxic Action of Juglone and Plumbagin: A Mechanistic Study Using HaCaT Keratinocytes Chem. Res. Toxicol. 17: 55-62 (2004). 6. W. Wang, X Luo, X. Zeng, Y. Zheng and H. Li. Terahertz and Infrared Spectra of Plumbagin, Juglone, and Menadione. Carnivorous Plants Newsletter (special issue, in press). 7. S. P. Mickan, A. Menikh, H. B. Liu, C. A. Mannella, R. MacColl, D. Abbott, J. Munch and X. C. Zhang. Label-free bioaffinity detection using terahertz technology. Phys. Med. Biol. 47, 3789-3795 (2002). 8. S. P. Mickan and X. C. Zhang. T-Ray Sensing and Imaging. International Journal of High Speed Electronics and Systerms 13, 601-676 (2003). 9. A. Markelz, S. Whitmire, J. Hillebrecht and R. Birge. THz time domain spectroscopy of biomolecular conformational modes. Phys. Med. Biol. 47, 3797-3805 (2002). 10. M. Walther, P. Plochocka, B. Fischer, H. Helm and P. U. Jepsen. Collective Vibrational Modes in Biological Molecules Investigated by Terahertz Time-Domain Spectroscopy. Biopolymers 67, 310-313 (2002).

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11. W. N. Wang, H. Yan, W. Yue, G. Zhao, C. Zhang, H. Liu and X. C. Zhang. THz Spectrum of Reduced Glutathione. Science in China. Ser. G Physics, Mechanics & Astronomy 48, 585-592 (2005).

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