Journal of the Korean Applied Science and Technology (한국응용과학기술학회지)

The Korean Society of Applied Science and Technology (한국응용과학기술학회)
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NMR Spectroscopy and Mass Spectrometry of 1, 2-Hexanediol Galactoside synthesized using Escherichia coli β-Galactosidase

대장균 베타-갈락토시데이즈를 이용하여 합성된 1, 2-Hexanediol Galactoside의 NMR Spectroscopy 및 Mass spectrometry

  • Kim, Yi-Ok (Department of Biotechnology, Korea National University of Transportation) ;
  • Lee, Hyang-Yeol (Department of Biotechnology, Korea National University of Transportation) ;
  • Jung, Kyung-Hwan (Department of Biotechnology, Korea National University of Transportation)
  • 김이옥 (한국교통대학교 생명공학과) ;
  • 이향렬 (한국교통대학교 생명공학과) ;
  • 정경환 (한국교통대학교 생명공학과)
  • Received : 2016.05.16
  • Accepted : 2016.06.16
  • Published : 2016.06.30
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Abstract

1, 2-Hexanediol galactoside (HD-gal) has been synthesized from 1, 2-hexanediol (HD), a cosmetic preservative, using recombinant Escherichia coli ${\beta}$-galactosidase (${\beta}$-gal) at the high lactose concentration (300 g/l). To confirm the molecular structure of synthesized HD-gal, NMR ($^1H$- and $^{13}C$-) spectroscopy and mass spectrometry of HD-gal were conducted. $^1H$ NMR spectrum of HD-gal showed multiple peaks corresponding to the galactocyl group, which is an evidence of galactocylation on HD. Downfield proton peaks at ${\delta}_H$ 4.44 ppm and multiple peaks from ${\delta}_H$3.96~3.58 ppm were indicative of galactocylation on HD. Up field proton peaks at ${\delta}_H$ 1.60~1.35 ppm and 0.92 ppm showed the presence of $CH_2$ and $CH_3$ protons of HD. $^{13}C$ NMR spectrum revealed the presence of 21 carbons suggestive of ${\alpha}$- and ${\beta}$-anomers of HD-gal. Among 12 carbon peaks from each anomers, the 3 peaks at dC 68.6, 60.9 and 13.2 ppm were assigned to be overlapped showing only 21 peaks out of total 24 peaks. The mass value (protonated HD-gal, m/z = 281.1601) from mass spectrometry analysis of HD-gal, and $^1H$ and $^{13}C$ NMR spectral data were in well agreement with the expecting structure of HD-gal. For further study, the minimum inhibitory concentrations (MICs) of HD-gal against bacteria will be investigated, and, in addition, cytotoxicity to human skin cells of HD-gal will be examined. It is expected that it will eventually be able to develop a new cosmetic preservative, which have low cytotoxicity against human skin cell and maintains antimicrobial effect.

화장품용 방부제로 사용하고 있는 1, 2-hexanediol (HD)에 높은 농도의 lactose (300 g/l)를 넣고, 재조합 대장균 ${\beta}$-galactosidase (${\beta}$-gal)를 이용하여 galactose 한 분자를 결합시키는 transgalactosylation 반응을 시켜서, 1, 2-hexanediol galactoside (HD-gal)을 합성하였다. 그리고, 합성된 HD-gal 분자를 확인하기 위하여, HD-gal에 대한 NMR ($^1H$- and $^{13}C$-) 스펙트럼 분석과 mass 스펙트럼 분석을 실시하였다. HD-gal의 $^1H$ NMR 스펙트럼에서 HD에 갈락토실화가 되었음을 보여주는 다양한 피크를 확인하였다. $^1H$ NMR 스펙트럼의 다운필드인 ${\delta}_H$ 4.44 ppm과 ${\delta}_H$ 3.96~3.58 ppm에서 나타나는 다양한 피크들은 HD에 갈락토실화가 되었다는 것을 잘 암시하고 있으며, 또한 $^1H$ NMR 스펙트럼의 업필드에서 나타나는 ${\delta}_H$ 1.60~1.35 ppm과 0.92 ppm의 피크는 HD의 $CH_2$와 $CH_3$ 작용기로부터 나타나는 피크로써 HD가 본 물질에 존재한다는 것을 나타내고 있다. $^{13}C$ NMR 스펙트럼에서는 HD-gal의 알파-아노머와 베타-아노머의 구조에서 기인하는 총 21의 카본피크가 나타났고, 각 아노머마다 12개의 카본이 존재하는데 이중 ${\delta}_C$ 68.6, 60.9 and 13.2 ppm에 보이는 3개의 카본은 겹쳐서 나타나 총 24개의 피크 중 21개가 나타났다. 또한, 질량스펙트럼 분석에서는 protonated HD-gal인 281.1601 (m/z)의 peak를 확인할 수 있었다. 이를 종합하면, NMR ($^1H$- and $^{13}C$-) 스펙트럼 분석 결과와 질량분석 결과들은 우리가 기대했던 HD-gal의 구조와 매우 잘 일치하고 있다는 것을 알 수 있었다. 앞으로 추가적으로, 세균에 대한 minimum inhibitory concentrations (MICs) 조사와 human skin cell에 대한 독성연구를 추가적으로 진행할 예정이며, 이러한 결과를 근거로 항균력을 유지하면서 피부세포에 대한 독성이 감소된 화장품용 방부제의 연구/개발이 계속되기를 기대하고 있다.

Keywords

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