Aritalab:Lecture/Biochem/Extraction

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Arita Laboratory

Natural product chemistry starts with "extraction" and "separation　/ purification."

Metabolite Chemistry

Metabolites show huge chemical diversity demonstrating different characteristics.

• molecular weight and size
• polarity and volatility
• solubility and pK_a (acid dissociation constant: [A-][H+]/[HA])

Polarity

highly apolar ←———————————→ highly polar

lipids (fatty acids waxes terpenes)

carotenoids chlorophylls steroids flavonoids

phenolics alcohols

amino acids organic acids organic amines alkaloids nucleosides

sugars nucleotides phsphates metals salts

E.g. cyclohexane < benzene < chloroform < ether < acetone < ethanol < methanol < H2O

Regulation

• substrate-level: cooperativity, feedback or feedforward control
• coenzymes: ATP, NAD, NADP, CoA, FMN, FAD, biotin, THF etc.
• allosteric: homoallostery (regulation by substrate), heteroallostery
• compartmentalization: source → sink flow, plastid, etc.
• hormone: growth factor, neurotransmitter, pheromone, etc.
• channeling: metabolon or multienzyme system

Turn-over rate

Metabolite	Turnover rate mM/s	species	Reference
glucose	1.0	S. cerevisiae aerobic on glucose	De Koning & van Dam 1992
glucose	0.3	Isolated adipocytes treated with insulin	Marshall et al. 2004
ATP	1.5	S. cerevisiae aerobic on glucose (D=0.1/h)	Rizzi et al. 1997
ADP	2.0

Extraction

Quenching

代謝物を計測するには、酵素を失活させ、代謝物の分解を防がなくてはなりません。 光で分解する代表的物質は S-アデノシル-L-メチオニン、酸化しやすい物質はリン酸化合物です。

バクテリア

バクテリアの細胞膜は破れやすく、過塩素酸、熱エタノール、熱水、液体窒素はおろか冷メタノールでも破れてしまいます。 通常は、細胞膜を破壊して細胞内外の代謝物を一緒に計測したものから、培地だけを測ったものを差し引いて細胞内の濃度を算出します。

酵母

冷メタノールを用いるのが一般的です。メタノール失活させる時間はできるだけ短くします。

糸状菌

冷メタノールや液体窒素が一般的です。

植物、動物

組織から目的の細胞を抽出する作業が最も大変です。通常は液体窒素を用いて凍らせ、すり鉢で粉砕します。

Like dissolves like

Solvent for extraction is chosen to share similar polarity with the target compound.

• terpenoid, steroid (alicyclics and aromatics)
  → low polarity solvent (e.g. hexane)
• saccharide, glycoside, amino acid
  → high polarity solvent (e.g. water)
• both
  → methanol

Separation & Purification

Chromatography separates target compounds using their physico-chemical difference in adsorption or partition between stationary and mobile phases.

normal phase

• polarity of stationary phase > polarity of mobile phase
• silica gel + organic solvent
• cyclohexane, benzene, chloroform, ether, acetone, ethanol, methanol

reverse phase

• polarity of stationary phase < polarity of mobile phase
• coated silica beads + water
• water + acetonitrile, water + methanol, water + tetrahydrofuran (THF)

	(High Performance) Liquid Chromatography	Gas Chromatography
Adsorption-type 吸着型	Liquid-solid type (LSC) silica gel, alumina, or porous polymers are used as adsorbent. hydrophobic: polystyrene gel, polymethyl methacrylate (PMMA) hydrophilic: polyhydroxy ethylmethacrylate gel, polyvinyl alcohol gel	Gas-solid type (GSC) Packed column has 2∼4 mm diameter and 30∼60 cm length, and is filled with diatomite, silica and silicon oil.
Partition-type 分配型	Liquid-liquid type (LLC) uses coated-silica beads. Hydrophobic octadecylsil (ODS), phenylmethyl, dichlorophenyl, octyl Hydrophilic aminopropyl, nitrophenol, acrylamido, propylalcohol	Gas-liquid type (GLC) Capillary column has 0.2 mm diameter and > 25 m length, whose inside part is coated with silicon oil.

シリカへの吸着度合い

-COOH ＞ -OH > -NH2 > -C=O > -OCH3 > -Cl