University of Leipzig

Faculty of Chemistry and Mineralogy

Institute of Organic Chemistry



Advanced Practical Course
in Organic Chemistry
WS 2012/2013





Isolation of
Shikimic acid from star aniseeds






Student: Marcel Schlegel (A-16) - 2262291
Supervisor: M.Sc. Liang Xia

Leipzig, 5 October 2012


1 Introduction

Shikimic acid ((3R,4S,5R)-3,4,5-Trihydroxy-1-cyclohexene-1-carboxylic acid) 1 has
its origin in the Japanese flower shikimi (Japanese star anise) from which the acid
was first isolated in 1885. Although the molecule 1 seems to be simple in its structure
there is no efficient access for a chemical synthesis. Thus, shikimic acid 1 is obtained
by extraction from star aniseeds. It serves as a precursor for the synthesis of the drug
oseltamivir 2 used in the treatment of both influenza viruses A and B and expected to
be effective also in the treatment of the dangerous H5N1 avian influenza (bird flu).
Furthermore, aromatic rings like the amino acids tyrosine and phenylalanine are
derived from shikimic acid 1.
[1]


1
Fig. 1: Shikimic acid 1


2
Fig. 2: Oseltamivir 2

The purpose of this experiment is the isolation of shikimic acid 1 from star aniseeds
and the characterisation using
1
H-NMR and
13
C-NMR spectroscopy.





2 Isolation
In a 1 L round-bottomed flask, an 74.3 g amount of crushed star aniseeds was
extracted with 550 mL of ethanol in a Soxhlet apparatus for 4 hours. The brown
extract was filtered, the ethanol removed under reduced pressure and finally dried
using an oil pump for 10 min. 400 mL of distilled water was added to the viscous
brown residue and the mixture was warmed to 80 C in a water bath. The cloudy,
pale brown emulsion with a few millilitres of an oily liquid at the surface was extracted
with diethyl ether (2 x 150 mL) and both ethereal phases were discarded. To the
orange-coloured aqueous phase 0.5 mL of 37 % formalin solution was added and the
mixture heated under reflux for 10 min. After cooling to ambient temperature the
cloudy suspension was filtrated by suction through a sintered glass filter funnel
containing a 3 cm pad of Celite.
Subsequently, a glass column (size 30 cm x 3 cm) with a glass filter funnel in front of
the outlet was prepared for ion exchange: 80 g of Amberlite IRA-402 anion-exchange
resin (chloride form) were mixed with 250 mL of distilled water and poured wet into
the column. A 2 molar solution of 500 mL of sodium acetate was passed 20 times
through the resin charge and finally, the column was flushed once with 150 mL of
distilled water. Then, the 400 mL of the clear, orange-coloured filtrate containing the
shikimic acid 1 were passed through the column five times. The orange-brown liquid
remaining was initially stored and later, the water removed in vacuo to give a brown
viscous residue. A solution of 400 mL of 25 % acetic acid was passed through the
column at the rate of 100 mL within 5 min. After flushing the column with 100 mL of
distilled water the eluate and wash water were combined to give a clear, pale orange
solution. Acetic acid and water were first removed under reduced pressure and
finally, the brown oil remained was dried using an oil pump for 5 h. A brown extract
(mass 4.1 g) of crude shikimic acid 1 remained, which formed an amorphous solid.
In a 500 mL round-bottomed flask, the 4.1 g of crude shikimic acid 1 were placed
together with 4.2 g of charcoal powder and 250 mL of ethyl acetate. The mixture was
heated under reflux for 15 min for decolouration and afterwards, the hot suspension
was filtered through a very close-grained glass filter funnel to yield a colourless
solution of shikimic acid. However, the residue at the glass filter funnel still contained
shikimic acid 1. Hence, four further extractions with 250 mL of each hot ethyl acetate
yielded a final crop of shikimic acid 1 in 1.25 L solution. The filtrate was reduced to
dryness in vacuo to give rise to 3.1 g of shikimic acid 1 as a colourless solid.
Finally, the amount of shikimic acid 1 (3.1 g) was purified by adding 10 mL of cold
ethyl acetate to once wash this material. The suspension obtained was filtrated by
suction through a Hirsch filter funnel and the filtrate obtained was used to transfer the
material on the funnel. The residue at the Hirsch filter funnel was dried using an oil
pump overnight. Last traces of ethyl acetate are not easy to remove.
A colourless, microcrystalline powder of shikimic acid 1 was formed with a yield of
2.5 g (3.4 %).

3 Characterisation

Yield: 2.5 g (3.4 %, colourless powder) (Lit.: 1.2 g [1.5 %])
[1]
Mp.: 180-183 C (ethyl acetate) (Lit.: 185-187 C)
[1]

-186.4 (c = 0.0107 g/mL, water) (Lit.:

-184.2; c = 0.0104 g/mL, water)

[1]
TLC: R
f
= 0.1 (CHCl
3
/CH
3
OH 5:1 v/v) (Lit.: R
f
= 0.1; CHCl
3
/CH
3
OH 5:1 v/v)
[1]


Fig. 3:
1
H-NMR spectrum of shikimic acid 1 in D
2
O (EA = residual ethyl acetate)

1
H-NMR (300 MHz, D
2
O): = 2.22 (ddt, 1H,
2
J
HH
=18.1 Hz,
3
J
HH
=6.5 Hz,
4
J
HH
=1.8 Hz,
H-6), 2.74 (ddt, 1H,
2
J
HH
=18.1 Hz,
3
J
HH
=5.2 Hz,
4
J
HH
=1.6 Hz, H-6), 3.77 (dd, 1H,
3
J
HH
=8.2 Hz,
3
J
HH
=4.2 Hz, H-4), 3.98-4.07 (m, 1H, H-5), 4.42-4.47 (m, 1H, H-3), 6.83
(dt, 1H,
3
J
HH
=3.8 Hz,
4
J
HH
=1.8 Hz, H-2).

Fig. 4:
13
C-NMR spectrum of shikimic acid 1 in D
2
O

13
C-NMR (75 MHz, D
2
O): = 30.5 (C-6), 65.9 (C-3), 66.7 (C-5), 71.2 (C-4), 129.9
(C-1), 137.5 (C-2), 170.2 (COOH).

4 Discussion
The isolation of shikimic acid 1 from star aniseeds was a time-consuming but
successful procedure. The amount of pure shikimic acid 1 (3.4 %) was even twice as
much as described in reference [1] (1.5 %). However, TLC of the 400 mL of residual
orange-brown liquid remained after passing through the ion exchange column
showed that it still contained some shikimic acid together with two less polar
components at least.
The advantage of the method explained in [1] is the low but highly selective solubility
of shikimic acid 1 in ethyl acetate. For that reason distillation, chromatography and
recrystallisation were not necessary. Nevertheless, the
1
H-NMR spectrum shows that
the entire removal of ethyl acetate was not complete, even after using an oil pump
overnight. Since shikimic acid 1 is a polar and hydrophilic compound it forms strong
hydrogen bonds to the ester group of ethyl acetate. Thus, the small amount of
residual solvent could be most likely removed using an oil pump and warming in a
water bath for a longer time.
Furthermore, the acetic acid used for yielding the shikimic acid 1 from the ion
exchange column was not fully removed by the oil pump after 5 h. However, the
residual acetic acid was washed out while purifying with the 10 mL of ethyl acetate.
5 Literature
[1] S. Berger, D. Sicker, Classics in Spectroscopy: Isolation and Structure
Elucidation of Natural Products, 1
st
ed., WILEY-VCH Verlag & Co. KGaA,
Weinheim, 2009, 503-512.