R3COCI
pyr.
C H20C O R2
I
C H O C O R3
I
C Hp C O R1
t riac i dTG
s y m m et r i c a l d i a c i dTG
44
The ketal blocking group was removed by borate replacement ; the acyl acetone glycerol was reacted with boric acid ( 2 . 4 mol ) in 400ml
0
of 2-methoxyethanol for 1 .5 h at 1 00 C . The react ion was carried out with careful exclusion of wat er in a 21 flask fitt e d with an air con denser , a silica gel drying t ube and a magnetic st irrer bar . At the complet ion of the react ion the flask cont ents were allowe d t o cool and were then filt ered. The boric aci d precipitate was washed repeatedly with a t ot al of 21 of diethyl ether-chloroform (3 : 1 ) and the c ombined filt rat es were extract ed with wat er ( 4 x 1 1 ) and dried with anhydrous Na2so4• �1ost
0
of the solvent was removed under reduced pressure at 30-35 C and the
crude 1 -MG was then crystallised two or three t imes from 1 0- 1 5 vol . of diethyl ether-hexane ( 6 : 4 ) at 20°C for the saturat e d 1 -HGs and -20°C for 1 -oleoylglycerol . Examinat ion of the recrystallised products by TLC showed that only traces of the 2-isomer were present . Furthermore , IR spec troscopy of the methyl est er prepared from 1 -oleoylglyc erol demonst rat ed that no det ectable isomerisat ion of the double bond had occurred.
�- 1 -pabnit oylglyc erol m . p .
yield
�-1 -st earoylglycerol m . p .
yield
�-1 -oleoylglyc erol yield
2 . 3 . 3
( a ) Monoacid
observed 75-76°C
lit erature 76.5°C ( Lut t on , 1 971 b ) 73% based on fatty ac id
observed 80-81 °C
lit erature 81.6°C ( Lutton , 1 971 b ) 71%
66x;
The preparat ion of monoaci d 1 , 3-diacylglycerols ( 1 , 3-DGs ) was the first st ep in the synthesis of symmetrical diacid TGs ( Figs . 2-2 and 2-3 scheme 2 ) .
1 , 3-Dipalmitoyl- and 1 , 3-dist earoyl-glycerol were prepared by the direct esterification of glycerol in an homogeneous reaction system
(Hartman , 1957). Anhydrous glycerol ( 0 . 05 mol ) was dissolved in
� ,� ' -
.
dimethylformamide ( 1 0ml ) , chloroform ( 75ml ) and pyridine ( 25ml ) in a dry
stoppered flask. St earoyl or palmitoyl chloride ( 0 . 1 1 mol ) in chloroform ( 150ml ) was then added dropwise to the magnet ically st irred solut ion.
If nec essary , more pyridine or dimethylformamide was added at this stage to ensure that the flask contents were homogeneous . After 4- 1 6h ,
45 0
clear solution was then evaporat ed t o dryness at 30-35 C . Examination of the crude react ion product by TLC showed that the chi e f impurit ies were methyl ester , TG , 1 , 2-DG and 1 -HG . The oily solid was then crystallised successively from 300ml of absolut e ethanol and 250ml of
0
hexane at 20 c . This procedure removed pyridine , dimethylformamide and most lipid contaminants except for TG . The latt er was removed by t wo
()
further recrystallisat ions from 1 0 vol . of hexane at 35 C . TLC of the final product showed no detectable impurit ies.
0
m.p. observed 73-5-74.0 c
1 , 3-dipalmitoylglycerol
lit erature 73.5-74. 0°C (Hartman ,
1 957 ) yield 51% based on glycerol
1 , 3-dist earoylglyc erol m.p. observed 79-80°C
literature 80°C ( Hartman , 1 957 ) yield 56%
()
The combined 35 C hexane filtrates from the 1 , 3-DG puri ficat ion
were evaporat ed to dryness and the resultant solid was recrystallised
twice from absolute ethanol-hexane ( 1 :
1 )
and then twice from chloroformhexane ( 1 : 5 ) . TLC of the recrystallised TG showed no detectable impurit ies.
t ripalmitoylglycerol m . p . observed
lit erature
yield 1 5% based
trist earoylglycerol rn.p. observed
literat ure
yield 6% ( c ) Diac id
on
65.8-66.2°C
66.4°C ( Lutton and Fehl,
1 970 )
glycerol 0
71.5-72.5 c
0
73-5 c ( Lutton and Fehl ,
1 970 )
Diacid �- 1 , 3-diacylglycerols were int ermediat es in the synthe sis of all t riacid TGs _and the diac id TGs , BPP , BSS , OOP and OOS (Figs.
2-2 and 2-3 scheme 1 ) .
( i ) Diacid of Holecular
Rac- 1 -oleoyl-3-stearoylglyc erol and �-1 -oleoyl-3-palmitoylglycerol were prepared by the est erificat ion of �-1 -oleoylglycerol ( 1 00g/l
in chloroform ) with an equimolar quant ity of stearoyl or palmitoyl
standing overnight , methanol was added to react with any remaining acyl chloride and t he solut ion was evaporat ed under reduced pressure at
0
30-35 C . The resultant solid �t�as t aken up in ether and the solut ion was then extract ed four t imes with wat er t o remove pyridine and evaporated
()
t o dryness at 30-35 C . Finally , the crude 1 , 3-diacylglycerol was
46
crystallised from 95% ethanol ( 1 0 vol ; 0°C ), followed by two recrystall-
o
isat ions from hexane ( 1 0 vol. ; 0 C ) . No impurities could be detected in
the products by TLC. Based on 1 -l'·\G , the yields of 1 , 3-00 were 70 and
64%
for �- 1 -oleoyl-3-st earoylglycerol and 1 -oleoyl-3-palmit oylglycerol respect ively .1 -Palmitoyl-3-st earoylglycerol was recovered as a by-product from the preparat ion of the TG PPS. 1 -St earoylglycerol ( 0 . 05 mol ) was
incomplet ely est erified by react ion with palmit oyl chloride (0.10 mol )
for 24h and the TG recovered by 4 crystallisat ions of the crude react ion mixture from acetone-chloroform-hexane ( 4 : 1 : 1 ) . The first two filtrat es were then c ombined and recrystallised t hree times from hexane ( 1 0- 1 5 vol . ) at 35°C t o yield
9�6
(based on 1 -hG ) of the pure 1 , 3-00.( ii ) Diacid 1 of Low 1-lolecular
Honobutyryl 1 , 3-00s were prepared from palmitoyl or st earoyl 1 -NGs because of the difficulty of synthesising pure 1-butyrylglycerol.
Rac-1 -st earoylglycerol ( 1 00g/l in chloroform ) was esterified with butyryl chloride ( 1 : 1.05 molar rC:J.t io ) in the presence o f excess pyridine. Aft er leaving overnight , the solut ion was evaporat ed in vacuo at 30-35°C t o remove chloroform , pyridine and any remaining butyryl chloride . Examinat ion of the crude product by TLC showed that the main impurit ies were pyridine hydrochloride , unreacted 1 -MG , 1 , 2-DG and TG .
Pyridine hydrochloride was removed by dissolving the crude solid in warm hexane (5 vol . ) , cooling t o 20°C and filt ering through a Milli pore membrane filter under posit ive nitrogen pressure . ( Pyridine hydro chloride could not readily be removed by extract ion with water because of the very stable emulsions formed when organic solutions of the crude 00 were shaken with wat er. Presumably DGs containing short and long
ch_ain fatty acids are effective emulsif iers ). The clear hexane solut ion was
0
evaporat ed t o dryness at 30-35 C and t he resultant solid was chromatographed on a c olumn of aluminium oxide-boric aci d to remove 1 -NG and remaining
traces of pyridine hydrochloride ( Sect ion 2 . 2. 2 ) . Finally , to remove TG and 1 , 2-DG , the product was crystallised twice from 1 5 vol . of hexane at
-5°C. The overall yield of �- 1 -butyryl-3-stearoylglyc erol was 61% ( base d
47 Rac- 1 -butyryl-3-palmitoylglycerol was prepared in a similar manner , although the overall yield was much lovJer (29";6 base d on 1 -HG ) because of t he higher molar rat io of acyl chloride t o 1 -MG ( 1 .5 : 1 ) and the loss of mat erial during repeat ed at t empt s to remove 1 -Iv!G by extrac t i on , crystallisat ion and , finally , c olumn chromatography. Because of the large excess of acyl chloride , the TG BBP acc ount ed for about half
of the crude react ion produc t . It was recovered from the final two hexane filtrates and purified by c olumn chromat ography on alumina
( Jensen et 1966). The yield of pure BBP was 32% based on 1 -l'iG.
an intermediat e in the preparat ion of the TGs ESS and PSS , was synthesise d according t o the scheme shown in Fig . 2-4.
Glycerol- 1 -tetrahydropyranyl
(
TlfP)
ether (Barry and Crai g , 1 955 ) was est erified with stearoyl chloride ( 1 0/o molar excess ) in a one phasesyst em containing chloroform , E ,� ' -dimethylformamide and excess pyridine.
Aft er 1 0 days , methanol was added and the solut ion was evaporat ed
0
in vacuo at 35-40 C . The resultant slurry was taken up in ether , and the solut ion was washed with wat er ( t o remove pyri dine and ,li ,Ji' - dirnethyl formamide ) , dried with anhydrous Na2soL� and evaporat e d to dryness in vacuo .
The THP group was removed from the crude �- 1 ,2-dist earoyl-3-
( tetrrulydropyranyl )-glyceryl ether by a modificat ion of the proce dure of
Gigg and Gigg ( 1 967 ) which utilised the cleavage and prot ect ive proper t ies of boric acid ( compare the removal of the isopropylidene group in the preparat ion of rac-1-l'iGs ). Boric ac i d (0. 51+ mol ) and �- 1 ,2-
dist earoyl-3- ( t etrahydropyranyl )-glyceryl ether ( 0.0189 mol ) in 1 40ml of
2-methoxyethrulol were plac ed in a round-bot tom flask f it t e d with an air condenser , a silica gel drying tube and a magnetic st irrer bar . The flask was transferred to a boiling water bath and the content s were
0
vigorously stirred for 1 . 5h. After cooling to 20 C , the slurry was
dilut ed with dichloromethane and filt ered t o remove boric ac i d . The clear filtrat e was washed with wat er and then evaporated to dryness at 35-40°C . Examinat i on of the crude react ion product by TLC showed that
the main impurit ies were : 1 -MG , 1 - ( mono )st earoyl-3- ( t et rahydropyranyl )
glyc eryl ether , st earic ac i d and ( running together ) methyl st earate and
unchanged 1 ,2-distearoyl-3- ( t etrahydropyranyl )glyceryl ether. Only a
trac e ( < 1 /o) of 1 , 3-00 was present with the 1 12-00 , which forme d the
majority of the react ion product . The 1 ,2-DG was purified by cryst allis at ion of the crude product from 20 vol . of acetone-methanol ( 1 : 1 ) at