A new protecting group: 9-fluorenylmethoxycarbonyl (FMOC) in the synthesis of 1,2-diacylglycerols

Article abstract of DOI:10.1016/S0009-3084(97)00034-0

The synthesis of 1,2-L-dipalmitoyl-sn-glycerol, 1,2-L-distearoyl-sn-glycerol and 1,2-L-dioleoyl-sn-glycerol are described here using 9-fluorenylmethoxycarbonyl (FMOC) group for protection of the 3-position of glycerol which can be selectively removed by Et₃N treatment on the overall 60-70% yield based on 1,2-isopropylidene-sn-glycerol. Little or no acyl migration occured during deprotection and purification.

Full text of DOI:10.1016/S0009-3084(97)00034-0

Chemistry and Physics of Lipids
87 (1997) 171–174
Short communication
A new protecting group: 9-fluorenylmethoxycarbonyl (FMOC)
in the synthesis of 1,2-diacylglycerols
´
Agnes Nyilas *
Department of Bioorganic Chemistry, Box 581, Biomedical Center, Uni6ersity of Uppsala, S-75 123 Uppsala, Sweden
Received 10 February 1997; received in revised form 18 April 1997; accepted 7 May 1997
Abstract
The synthesis of 1,2-
L
-dipalmitoyl-sn-glycerol, 1,2-L-distearoyl-sn-glycerol and 1,2-L-dioleoyl-sn-glycerol are de-
scribed here using 9-fluorenylmethoxycarbonyl (FMOC) group for protection of the 3-position of glycerol which can
be selectively removed by Et3N treatment on the overall 60–70% yield based on 1,2-isopropylidene-sn-glycerol. Little
or no acyl migration occured during deprotection and purification. © 1997 Elsevier Science Ireland Ltd.
1. Introduction
ranyl (Lok, 1978). The 2,2,2-trichloroethoxycar-
bonyl (Pfeiffer et al., 1970); trifluoroacetyl (Lok,
1978); levulinate (Fro¨ling et al., 1984);
methoxyethoxymethyl (Duralski et al., 1989); and
t-butyldimetylsilyl (Sonnet, 1991), protecting
groups have also been used in the synthesis of
diacyl-sn-glycerols. Removal of these protecting
groups from diacyl-sn-glycerols generally resulted
in some acyl migration. We suggest using an
9-fluorenylmethoxycarbonyl (FMOC) protecting
group which is removable by non-nucleophylic
tertiary base with i-elimination.
The glyceryl derivatives, especially the phos-
pholipids, are important components of biological
membranes (Jain, 1972; Han et al., 1994). Phos-
pholipid synthesis requires a careful choice of
protecting groups for the different functionalities
and mild deprotection conditions, in order to
avoid acyl group migration. The most frequently
used glycerol protecting groups are: benzyl (Eibl
and Wolley, 1986; Hong et al., 1988); allyl and
trityl (Eibl and Wolley, 1986); and terahydropy-
The FMOC group was first used in peptide
chemistry by Carpino and Han (1970). Later it
was introduced to nucleosides by Heikkila and
Chattopadhyaya (1983).
* Present address: Tisza Lajos krt. 18/D, 6720. Szeged,
Hungary. Tel.: +36 62 487310.
0009-3084/97/$17.00 © 1997 Elsevier Science Ireland Ltd. All rights reserved.
PII S0009-3084(97)00034-0

´
A. Nyilas / Chemistry and Physics of Lipids 87 (1997) 171–174
172
2. Experimental procedures
aromatic of FMOC; 4.0–4.4 (m, 5H) CH2, CH
of FMOC and 3-H2C; 3.8 (m, 1H) 2-HC; 3.6
(m, 2H) 1-H2C; 2.73(bs, 1H) OH, 2.37 (bs, 1H)
OH. 13C-NMR (CDCl3) l: 155.13 (CꢀO);
142.99, 141.04, 127.71, 124.84, 119.86 (aromatic
carbons of FMOC); 69.8, 68.45, 62.98 (glycerol
carbons), 46.51 (CH2 of FMOC).
1H-NMR and 13C-NMR spectra were mea-
sured with a Jeol Fx 90 Q spectrometer using
tetramethylsilane as internal standard in l scale.
Merck Kieselgel G was used for short column
chromatography.
Pyridine was dried by heating under reflux
with calcium hydride for ca. 3 h then distilled at
atmospheric preasure and stored over molecula
sieves (4 A) in a dark bottle. 9-Fluorenyl chloro-
formate (FMOC-Cl), palmitic-, stearic- and oleic
acid were purchased from Sigma.
2.3. 3-FMOC-1,2-dipalmitoyl-sn-glycerol (4a)
Compound 3 (0.3 g, 0.94 mmol) and palmitic
acid (0.48 g, 1.88 mmol) were dissolved in 13 ml
dichloromethane and cooled to 0°C. DCC (1.32
g, 6.4 mmol) and DMAP (24.4 mg, 0.2 mmol)
were dissolved in 10 ml dichloromethane and
added to the reaction mixture. After 2–3 h stir-
ring the reaction mixture was evaporated and
dissolved in 50 ml dichloromethane and the di-
cyclohexylurea was then filtered. The product 4a
was purified on silica gel column and eluted
with 50% hexane–dichloromethane. The proper
fractions were evaporated giving the compound
Elemental analyses were performed in Upp-
sala. Results were within 90.4% of theoretical
values.
2.1. 1,2-Isopropylidene-3-FMOC-sn-glycerol (2)
1,2-Isopropylidene-sn-glycerol (0.66 g,
5
mmol) was dissolved in 50 ml dry pyridine, 9-
fluorenyl chloroformate (1.55 g, 6 mmol) was
added and the reaction mixture was stirred for 1
h at RT. The reaction mixture was divided into
500 ml chloroform and 500 ml saturated ammo-
nium bicarbonate. The organic phase was evapo-
rated to dryness and coevaporated with toluene
several times to remove all traces of pyridine.
The oil residue was dissolved in dichloro-
methane, loaded on silica gel and eluted with
dichloromethane and 5% methanol–dichloro-
methane. The proper fractions were evaporated
and the resulting oil was 1.44 g (81.3%). 1H-
NMR (CDCl3) l: 7.23–8.0 (m, 8H) aromatic of
FMOC; 3.6–4.5 (m, 8H) CH and CH2 of
FMOC, 2-HC, 3-H2C and 1-H2C; 1.45 (s, 3H)
CH3; 1.38 (s, 3H) CH3.
1
4a (0.8 g, 100%). H-NMR (CDCl3) l: 7.23–7.8
(m, 8H) aromatic of FMOC; 5.3 (m, 1H) 2-HC;
4.38 (m, 3H) CH and CH2 of FMOC; 3.2 (m,
3H) 3-H2C and 1-H2C; 2.33 (m, 4H) OCH2;
1.2–2.1 (m, 26H) CH2; 0.87 (bs, 6H) CH3.
2.4. 3-FMOC-1,2-Distearoyl-sn-glycerol (4b)
Compound 4b was prepared in the same way
as compound 4a (0.57 g, 87%). 1H-NMR
(CDCl3) l:7.25–8.0 (m, 8H) aromatic of
FMOC; 5.3 (m, 1H) 2-HC; 4.38 (m, 3H) CH
and CH2 of FMOC; 3.2 (m, 4H) 3-H2C and
1-H2C; 2.33 (m, 4H) 2 OCH2, 1.1–2.2 (m, 30H)
CH2; 0.877 (bs, 6H) CH3.
2.2. 3-FMOC-sn-glycerol (3)
2.5. 3-FMOC-1,2-Dioleoyl-sn-glycerol (4c)
Compound 2 (0.46 g, 1.3 mmol) was dissolved
in 10 ml dioxane and 10 ml of 80% formic acid
was added. The reaction mixture was stirred at
RT for 45 min then evaporated, loaded on silica
gel column and eluted with 2–5% methanol–
dichloromethane. The proper fractions were
evaporated and the resulting oil was 0.37 g
(89%). 1H-NMR (CDCl3) l: 7.1–7.7 (m, 8H)
Compound 4c was prepared in the same way
as compound 4a (0.5 g, 95%). H-NMR (CDCl3)
1
l:7.19–7.91 (m, 8H) aromatic of FMOC; 5.33
(m, 5H) 2-HC and 2CHꢀCH; 4.36 (m, 3H) CH
and CH2 of FMOC; 3.23 (m, 3H) 3-H2C and
1-H2C; 2.33 (m, 4H) 2 OCH2, 1.1–2.19 (m,
26H) CH2; 0.88 (m, 6H) CH3.

´
A. Nyilas / Chemistry and Physics of Lipids 87 (1997) 171–174
173
2.6. 1,2-Dipalmitoyl-sn-glycerol (5a)
erols with Et3N in dry pyridine, resulting in an
80% yield.
What are the similarities and advantages of
FMOC group compared to the other protecting
groups in glycerol chemistry?
1. It is easy to introduce FMOC-Cl after 1 h at
RT.
Compound 4a (0.63 g, 0.79 mmol) was dis-
solved in 8 ml dry pyridine and dry Et3N (0.8 ml,
7.95 mmol) was then added. The reaction mixture
was stirred at RT for 2 h, evaporated and loaded
on silica gel column. The column was eluted with
50% hexane–dichloromethane and the product
(5a) was eluted with dichloromethane. The proper
fractions were poured together and evaporated
2. The products 2,3,4,5 can be quickly purified
on silica gel.
3. In the 1H-NMR and 13C-NMR spectra, the
aromatic part and CH2 of FMOC in com-
pound 3 can be easily seen.
1
giving the compound 5a 0.38 g (84%). H-NMR
(CDCl3+CD3OD) l: 4.99 (m, 1H) 2-HC; 4.19
(m, 2H) 1-H2C; 3.6 (m, 2H) 3-H2C; 2.27 (m, 4H)
2 CH2CO; 1.22–1.73 (m, 26H) 2 (CH2)13; 0.81 (t,
6H) 2 CH3. 13C-NMR (CDCl3 + CD3OD) l:
173.65 (CꢀO); 71.86, 62.16, 60.64 (glycerol car-
bons); 48.51 and 33.88 (CH2–CꢀO); 31,66 (CH2–
CH2–CH3); 29.44 ((CH2)10 both acyl chain); 13.84
(CH3 both acyl chain).
4. The fatty acids can be introduced almost
quantitatively despite the bulkiness of the pro-
tecting group.
5. The deblocking condition, Et3N in dry
pyridine for 2 h, does not cause acyl migra-
tion, as no other product was observed on
TLC.
6. The overall yield of diacyl-sn-glycerols is simi-
lar to using benzyl protection. However, ben-
zyl can not be used in the case of unsaturated
fatty acids, therefore removal with Pd/C,
debenzylation and detrtitylation can be
achieved using bromodimethylborane as
shown by Kodali and Duclos (1992).
2.7. 1,2-Distearoyl-sn-glycerol (5b)
Compound 5b was produced similar to 5a (0.37
1
g, 80%). H-NMR (CDCl3) l: 5.0 (m, 1H) 2-HC;
4.2 (m, 2H) 1-H2C; 3.65 (m, 2H) 3-H2C, 2.3 (m,
4H) 2 CH2CO; 1.5 (m, 30H) CH2; 0.88 (t, 6H) 2
CH3.
1
7. H-NMR and 13C-NMR spectra of compound
5a are similar to that of Kodali and Duclos
(1992).
We will show in our next paper, the use of the
above prepared diacyl-sn-glycerols in the synthe-
sis of 1-i-D-arabinofuranosylcytosine-phospho-
lipid.
2.8. 1,2-Dioleoyl-sn-glycerol (5c)
Compound 5c was produced similar to com-
1
pound 5a (0.25 g, 80%). H-NMR (CDCl3) l: 5.4
(m, 4H) 2 CHꢀCH; 4.26 (m, 2H) 1-H2C; 3.72 (m,
2H) 3-H2C; 2.34 (m, 4H) 2 CH2CO; 1.27 (m, 26H)
CH2; 0.88 (t, 6H) 2 CH3.
Acknowledgements
The author gratefully acknowledges the finan-
cial support from the Swedish Board of Technical
Development and the Swedish Natural Science
Research Council. I thank Professor J. Chatto-
padhyaya for valuable discussion.
3. Results and discussion
In our strategy we have employed the method
used by Eibl (1981) for the synthesis of 1,2-iso-
propylidene-sn-glycerol. We have protected the
3-position with FMOC-Cl in 82% yield, the iso-
propilidene group was removed with formic acid,
and the fatty acids, either saturated or unsatu-
rated, were introduced using the method of Neises
and Steiglich (1978) almost quantitatively. The
FMOC group was removed from diacyl-sn-glyc-
References
Carpino, L.A., Han, G.Y., 1970. The 9-fluorenylmethoxycar-
bonyl function,
a new base-sensitive aminoprotecting
group. J. Am. Chem. Soc. 92, 5748–5750.

´
A. Nyilas / Chemistry and Physics of Lipids 87 (1997) 171–174
174
Duralski, A.A., Spooner, P.J.R., Watts, A., 1989. Synthesis of
optically active polyunsaturated diacylglycerols. Tetrahe-
dron Lett. 30, 3585–3588.
Hong, C.I., An, S.H., Schliselfield, L., Buchheit, D.J.,
Nechaev, A., Kirisits, A.J., West, C.R., 1988. Nucleoside
conjugates. 10. Synthesis and antitumor activity of 1-i-D-
Eibl, H., Wolley, P., 1986. Synthesis of enantiomerically pure
glycerol esters and ethers. 1. Methods employing the pre-
cursor 1,2-isopropilidene-sn-glycerol. Chem. Phys. Lipids
41, 53–63.
arabinofuranosylcytosine 5%-diphosphate-1,2-dipalmitins. J.
Med. Chem. 31, 1793–1798.
Jain, J.K., 1972. The Biomolecular Lipid Membrane: A Sys-
tem, von Nostrand-Reinhold, New York.
Eibl, H., 1981. An improved method for the preparation of
1,2-isopropylidene-sn-glycerol. Chem. Phys. Lipids 28, 1–
6.
Froling, A., Pabon, H.J.J., Ward, J.P., 1984. Synthesis of
long-chain 1-saturated-2-unsaturated diacylglycerols via
levulinoyl protection. Chem. Phys. Lipids 36, 29–38.
Han, G.W., Ruble, J.R., Craven, B.M., 1994. The crystal
structure of 1,2-dipalmitoyl-sn-glycerol at 123 K. Chem.
Phys. Lipids 71, 219–225.
Kodali, D.R., Duclos, R.I., 1992. Debenzylation and detrityla-
tion by bromodimethylborane: Synthesis of mono-acid or
mixed-acid 1,2 or 2,3-diacylglycerols. Chem. Phys. Lipids
61, 169–173.
Lok, C.M., 1978. Versatile methods for the synthesis of mixed-
acid 1,2-diacylglycerols. Chem. Phys. Lipids 22, 323–337.
Neises, B., Steiglich, W., 1978. Simple method for the esterifi-
cation of carboxylic acids. Angew. Chem. Int. Ed. 17 (7),
522–523.
Heikkila, J., Chattopadhyaya, J., 1983. The 9-fluorenyl-
methoxy-carbonyl (FMOC) group for the protection of
amino function of cytidine, adenosine, guanosine and their
2%-deoxysugar derivatives. Acta Chem. Scand. B37 (3),
263–265.
Pfeiffer, F.R., Miao, C.K., Weisbach, J.A., 1970. Glyc-
erolipids. III. Stereospecific synthesis of D-and L-1,2-diglyc-
erides via glycerol carbonates. J. Org. Chem. 35, 221–224.
Sonnet, P.E., 1991. A short highly region-and stereoselective
synthesis of triacylglycerols. Chem. Phys. Lipids 58, 35–39.
.