RIKA NURIA (F1061141017)

1. PHYTOL
Phytol is an acyclic diterpene alcohol that can be used as a precursor for the manufacture
of synthetic forms of vitamin E and vitamin K1. In ruminants, the gut fermentation of ingested
plant materials liberates phytol, a constituent of chlorophyll, which is then converted to phytanic
acid and stored in fats. In shark liver it yields pristane.
Stucture of phytol:

a. Physical and Chemical Properties

IUPAC Name (2E,7R,11R)-3,7,11,15-

tetramethyl-2-hexadecen-1-ol
Chemical formula C20H40O
Molar mass 296.54 g·mol−1
Density 0.850 g cm−3
Boiling point 203 to 204 °C (397 to 399 °F; 476 to
477 K)at 10 mmHg
Appearance Yellow

b. Biosynthesis
 Biosynthesis of phytol by the non-mevalonic pathway in the Cyanobacterium
Synechocystis sp. UTEX 2470† (according to Proteau, 1998).

The discovery of the non-mevalonate pathway (NMP) to terpenes in bacteria and

higher plants has led to a reexamination of terpene biosynthesis in a number of organisms.
This new route is distinguished by the condensation of pyruvate with glyceraldehyde-3-
 phosphate (G3P) to form deoxyxylulose-5-phosphate, which is converted into isopentenyl
diphosphate (IPP) by an unknown number of steps.It has now been demonstrated that a
variety of bacteria, higher plants,green algae, and a red alga can use this alternate route to
terpenes. The operation of this mevalonate independent route in bacteria and in higher plants
suggested that cyanobacteria also should synthesize terpenes via this pathway, because they
are considered to be the evolutionary precursors of chloroplasts in higher plants. This article
describes experiments to confirm that cyanobacteria can utilize the non-mevalonate pathway.

c. Isolation and Elucidation

Analysis of the isolated phytol using deuterium or 13C NMR showed labeling patterns
consistent with incorporation of labeled glucose via the non-mevalonate pathway to terpenes.
Isolation of Phytol.
The lyophilized cell material was extracted three times with 2:1 CHCl3-MeOH to provide
a crude extract that was loaded onto a flash silica column. Stepwise elution with 20%
EtOAchexanes, 40% EtOAc-hexanes, and 100% EtOAc provided a chlorophyll-containing
fraction in the 40% eluent. This fraction was concentrated and treated with 10 mL of 6% w/v
KOH in MeOH overnight at room temperature. The reaction mixture was diluted with 20 mL
H2O and extracted 3 _ 20 mL with hexanes. The combined organic phases were washed with
20 mL saturated ammonium chloride solution, then 20 mL H2O to obtain phytol
contaminated with a minor amount of a yellow-orange pigment. The basic aqueous layer was
acidified to pH 2-3 with 1N HCl, diluted with 20 mL brine, then extracted 3 _ 30 mL with
hexanes. The hexanes layer was washed with 30 mL of saturated NaHCO3 solution, then 30
mL H2O. Although not expected, the hexanes extract from the acidified solution contained
phytol that was not fully extracted from the basic solution, even after repeated extractions of
the basic layer with hexanes. The pigment was removed from the combined phytol-containing
solutions by passing an ether solution of crude phytol through a short activated-charcoal
column and eluting with Me2CO. The purified phytol (2 mg/L avg. yield) was analyzed by
GCMS and NMR to confirm its identity.
2. Menthol
Menthol is an organic compound made synthetically or obtained from corn mint,
peppermint, or other mint oils. It is a waxy, crystalline substance, clear or white in color, which
is solid at room temperature and melts slightly above. The main form of menthol occurring in
nature is (−)-menthol, which is assigned the (1R,2S,5R) configuration. Menthol has local
anesthetic and counterirritant qualities, and it is widely used to relieve minor throat irritation.
Menthol also acts as a weak kappa opioid receptor agonist.
Structure of menthol:

a. Chemical and physical porperties

IUPAC Name 5-Methyl-2-(propan-2-yl)cyclohexan-1-ol

Chemical formula C10H20O
Molar mass 156.27 g·mol−1
Density 0.890 g·cm−3,
Boiling point 212 °C (414 °F; 485 K)
Melting point 36 to 38 °C (97 to 100 °F; 309 to 311 K)
Appearance White or colorless crystalline solid
solubility Slightly soluble

b. Production
As with many widely used natural products, the demand for menthol greatly exceeds the
supply from natural sources. In the case of menthol it is also interesting to note that
 comparative analysis of the total life-cycle costs from a sustainability perspective has shown
that production from natural sources actually results in consumption of more fossil fuel,
produces more carbon dioxide effluent and has more environmental impact than either of the
main synthetic production routes.

c. Biosynthesis
The biosynthesis of menthol has been investigated in M. x piperita and the enzymes
involved in have been identified and characterized.[9] It begins with the synthesis of the
terpene limonene, followed by hydroxylation, and then several reduction and isomerization
steps.More specifically, the biosynthesis of (−)-menthol takes place in the secretory gland
cells of the peppermint plant. Geranyl diphosphate synthase (GPPS), first catalyzes the
reaction of IPP and DMAPP into geranyl diphosphate. Next (−)-limonene synthase (LS)
catalyzes the cyclization of geranyl diphosphate to (−)-limonene. (−)-Limonene-3-
hydroxylase (L3OH), using O2 and NADPH, then catalyzes the allylic hydroxylation of (−)-
limonene at the 3 position to (−)-trans-isopiperitenol. (−)-Trans-isopiperitenol dehydrogenase
(iPD) further oxidizes the hydroxy group on the 3 position using NAD+ to make (−)-
isopiperitenone. (−)-Isopiperitenone reductase (iPR) then reduces the double bond between
carbons 1 and 2 using NADPH to form (+)-cis-isopulegone. (+)-Cis-isopulegone isomerase
(iPI) then isomerizes the remaining double bond to form (+)-pulegone. (+)-Pulegone
reductase (PR) then reduces this double bond using NADPH to form (−)-menthone. (−)-
Menthone reductase (MR) then reduces the carbonyl group using NADPH to form (−)-
menthol.

d. Elucidation
 A cursory inspection of a one-scan 1D 1H NMR spectrum should reveal most of the
spectral features. Although good quality spectra can be obtained even with low sample
concentrations (_5 m), high concentrations (_100 m) can introduce artifacts and should
be avoided. For example, a one-scan 1D 1H NMR spectrum of menthol
(Figure 1) shows a typical and perfectly acceptable result for a dilute solution. This is
required because many 2D NMR spectroscopic experiments require the observation of
13C satellites (relative intensity, 1). If even the parent 12C peaks (relative intensity, 200)
are not visible, further analysis may require excessive spectrometer time. In addition,
compounds that exhibit complex or broad spectra due to impurities or tautomeric equilibria
are typically not suitable for analysis. It is prudent to obtain the molecular formula of the
unknown by elemental analysis, or preferably, high-resolutionmass spectrometry before
any detailed analysis. The 1D 1H NMR spectrum should be inspected to verify that the
number of protons, as obtained by integration, matches the expected number from the
molecular formula.

Figure 2 dilute one-scan 1D 1H NMR spectrum of menthol. Bold numbers identify each
proton. (500 MHz, 0.5 mg in 700 μL CDCl3= 5m).

3. Limonene
Limonene is a colorless liquid hydrocarbon classified as a cyclic terpene. The more
common d-isomer possesses a strong smell of oranges. It is used in chemical synthesis as a
precursor to carvone and as a renewables-based solvent in cleaning products. The less
common l-isomer is found in mint oils and has a piney, turpentine-like odor. Limonene takes
its name from the lemon, as the rind of the lemon, like other citrus fruits, contains
considerable amounts of this compound, which contributes to their odor.
Structure of limonene:
a. Chemical and physical porperties

IUPAC Name 1-Methyl-4-(1-methylethenyl)-cyclohexene

Chemical formula C10H16
Molar mass 136.24 g·mol−1
Density 0.8411 g/cm3
Boiling point 176 °C (349 °F; 449 K)
Melting point −74.35 °C (−101.83 °F; 198.80 K)
Appearance colorless to pale-yellow liquid
solubility chloroform, ether, CS2, and oils
soluble in CCl4

b. biosynthesis

Limonene is formed from geranyl pyrophosphate, via cyclization of a neryl carbocation or

its equivalent as shown.[6] The final step involves loss of a proton from the cation to form
the alkene.

The most widely practiced conversion of limonene is to carvone. The three step reaction
begins with the regioselective addition of nitrosyl chloride across the trisubstituted double
bond. This species is then converted to the oxime with base, and the hydroxylamine is
removed to give the ketone-containing carvone.

c. Isolation and elucidation

Principle
A suitable source for the isolation of (R)-(+)-limonene is Brazilian sweet orange oil,
obtained from squeezing sweet orange peel. It is a byproduct of making orange juice.
(4R)-(+)-Limonene can be separated by two fractionated distillations in vacuo. The goal is
to obtain a small fraction of nearly pure limonene rather than a complete separation of all
limonene contained from other constituents. Furthermore, it was intended to use standard
laboratory glassware skilfully in this case of an already highly enriched crude material
instead of a sophisticated distillation equipment. Although limonene is thermally stable
enough to be distilled at ambient pressure, it is recommended to do the fractionation in
vacuo.
Method
Brazilian sweet orange oil (36 g) containing 90% of (R)-(+)-limonene is subjected to
fractional distillation in vacuo at 1000 Pa in the stream of an electronically regulated heat
 gun with an outlet temperature of 120 °C. A 10 cm Vigreux column is used and the
distillation is run rather slowly. The refractive index of the orange coloured starting
material is 1.4714 at 24 °C. Two cuts are made the mass of which is 27 g in total (see
table below). The distillation is then interrupted for safety reasons although no increase in
the boiling point is observable. The remaining material has nD 1.4745 at 24 °C and a
mass of 9 g. The two fractions obtained are clear and colourless with an odour not as
heavy in its citrus fruit note as that of the starting oil.

Estimation of the purity of fraction II according to 1H NMR data: ca. 98.5%.

Purification
A 17.0 g amount of fraction II from above is subjected to a second very slow distillation
with the same equipment except for a longer Vigreux column (30 cm).

Fig. 3.1 1H NMR spectrum at 400 MHz in CDCl3

The discussion of the 1H NMR spectrum starts with the observation of the two olefi nic signals
with an intensity ratio of 1:2, indicating that even at 400 MHz the signals of the two olefi nic
protons at C-9 are not separated from each other. The two methyl group signals are clearly
separated, although they are attached to a similar double bond. The other proton signals are diffi
cult to assign using only the 1H spectral information.
Fig. 3.2 COSY spectrum
where both methyl group signals show cross peaks via an allylic coupling over four bonds to their
respective olefi nic protons H-2 and H-9. A cross peak between the olefi nic proton H-2 and the
allylic neighbours H-3 assigns their resonance position