Application of chemoenzymatic hydrolysis in the synthesis of 2-monoacylglycerols

Article abstract of DOI:10.1016/j.tet.2012.04.101

The selective biocatalyzed synthesis of 2-monoacylglycerols (2-MAGs) through the use of commercially available immobilized Candida antarctica (Novozym435) and Rhizomucor miehei is explored. Reactions at room temperature result in the formation of a 2-MAG and a corresponding ethyl ester of the fatty acid with immobilized C. antarctica within 2 h with yields ranging from 36% to 83%. Similar reaction conditions with immobilized R. miehei yielded exclusively the 2-MAG after 24 h with yields ranging from 37% to 88%. Yields vary on the acyl group at the sn-2 position and choice of enzyme involved.

Full text of DOI:10.1016/j.tet.2012.04.101

Tetrahedron 68 (2012) 5422e5428
Contents lists available at SciVerse ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Application of chemoenzymatic hydrolysis in the synthesis
of 2-monoacylglycerols
*
Kyle M. Whitten, Alexandros Makriyannis, Subramanian K. Vadivel
Center for Drug Discovery, 116 Mugar Hall, 360 Huntington Avenue, Northeastern University, Boston, MA 02115, Unites States
a r t i c l e i n f o
a b s t r a c t
Article history:
The selective biocatalyzed synthesis of 2-monoacylglycerols (2-MAGs) through the use of commercially
available immobilized Candida antarctica (Novozym435) and Rhizomucor miehei is explored. Reactions at
room temperature result in the formation of a 2-MAG and a corresponding ethyl ester of the fatty acid
with immobilized C. antarctica within 2 h with yields ranging from 36% to 83%. Similar reaction condi-
tions with immobilized R. miehei yielded exclusively the 2-MAG after 24 h with yields ranging from 37%
to 88%. Yields vary on the acyl group at the sn-2 position and choice of enzyme involved.
Ó 2012 Elsevier Ltd. All rights reserved.
Received 13 March 2012
Received in revised form 24 April 2012
Accepted 25 April 2012
Available online 5 May 2012
Keywords:
Cannabinoid
Receptor
Endogenous ligand
2-Monoacylglycerol
Biocatalysis
1. Introduction
2-Monoacylglycerols (2-MAGs) exhibit beneficial emulsifying
properties that are utilized in the food industry,1,2 and in the ad-
ministration of pharmaceuticals.3 The polyunsaturated fatty acid
(PUFA) occupying the sn-2 position is important in the influence of
structured triglycerides absorption and digestion.4 The biological
effects of fatty acids released from the metabolism of glycerols and
amides, or through ingestion, have also been studied.5e7 One of the
more intensively studied 2-MAGs, 2-arachidonoylglycerol (2-AG,
10b), is a physiologically important lipid signaling molecule acting
as a receptor ligand in the endocannabinoid system. Pharmaco-
logical properties of 2-AG include hypotension, neuroprotection,
and appetite stimulation.8
2-AG and other 2-MAGs in biological systems are usually inac-
tivated/catabolized by the hydrolyzing enzyme monoacylglycerol
lipase (MAGL) to produce a fatty acid and glycerol.9
The synthesis and study of 2-MAGs is made difficult due to acyl
migration from the sn-2 to the sn-1 or -3 position (Scheme 1).10,11
This migration is facile and occurs in the presence of acid, base,
heat, and protic solvents.12,13 In the case of 2-AG, acyl migration
renders 1-AG, which is incapable of binding to the endocannabinoid
receptors.14 Many reported syntheses of 2-MAGs involve multiple
Scheme 1.
laborious steps with unfavorable reaction conditions and work ups
that may promote the unwanted acyl migration. An earlier 2-MAG
synthesis began with the coupling of the fatty acid to a 1,3-
triisopropylsilyl (TIPS) glycerol. The removal of the silyl protecting
groups required 24 h with the addition of acetic acid and tetrabu-
tylammonium fluoride.15 Another procedure involved coupling of
fatty acid with 1,3-benzylideneglycerol and removal of the benzyli-
dene with phenylboronic acid. The reaction resulted in the formation
of the mixture of the 1,3- and 1,2-phenylboronate esters, which was
separated and cleaved with methanol and water.16 A third technique
utilizes the ring opening of a glycidal ester with trifluoroacetic acid
to produce a triacylglycerol. The 2-MAG was then formed after
treatment of the triacylglycerol methanol and pyridine.17
Application of lipase in the syntheses of 1,3-diacylglycerol and
1(3)-rac-monoacyl glycerol has been extensively studied and
reviewed.18e26 The selectivity and yield are determined by various
factors, which include amount of enzyme, solvent, temperature,
and the type of lipase used.27,28 Even though, the preparation of
selective 1,3-diacylglycerols has been achieved successfully, it has
been a challenging task for the synthesis of 2-acylglycerols mainly
due to over hydrolysis and the acyl migration from sn-2 to sn-1 or
* Corresponding author. Tel.: þ1 617 373 7620; fax: þ1 617 373 7493; e-mail
addresses: s.vadivel@neu.edu, skvadivel@hotmail.com, k.subramanian@neu.edu
(S.K. Vadivel).
0040-4020/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2012.04.101

K.M. Whitten et al. / Tetrahedron 68 (2012) 5422e5428
5423
sn-3 position. Lipase-mediated selective hydrolysis of triglyceride
using 1,3-regiospecific lipases, esterification of fatty acids or
transesterification of fatty esters with glycerol, and the glycerolysis
of triglycerides have been documented in the literature.29,30 Iri-
of anhydrous THF/CH2Cl2 along with a catalytic amount of 4-
dimethylaminopyridine (DMAP) at 0 ꢀC for 4 h. This generated
the structured triglyceride (‘ABA’ type) in 67e99% yield.
For the hydrolysis step, Novozym 435 was added to the tri-
glyceride in a minimal amount of anhydrous ethanol at room
temperature. By TLC analysis, it was observed that within 1 h the
triglyceride had been completely consumed, and a mixture of 2-
MAG, mono-protected 2-MAG, and ethyl butyrate was generated.
There is no formation of ethyl ester of fatty acid observed during
this period. At this point, additional lipase was added to the mix-
ture, which was allowed to stir until all the mono-protected 2-MAG
was consumed (1 h), affording the 2-MAG. Some significant
amount of ethyl ester was observed during this period, and the
formation of ethyl ester largely depended on the type of carboxylic
acid used. Aryl and unsaturated carboxylic acids showed more re-
sistant toward over hydrolysis compared to saturated fatty acids.
The separation of 1-MAG and 2-MAG is generally performed on
boric acid impregnated TLC plates and silica gel columns.36 Non-
impregnated silica TLC plates do not resolve 1- and 2-MAG. This
separation is a necessary step for most 2-MAG syntheses due to the
unfavorable synthetic conditions used, which result in formation of
considerable 1-MAG as well. In contrast, the highly regiospecific
and neutral reaction conditions when using the lipase result in
minimal or no 1-MAG formation. Although silica gel purification
has been reported to be an inevitable cause of acyl migration in 2-
MAG to 1-MAG,37 we did not observe any migration during column
chromatography with untreated silica gel. The only required step
prior to purification was equilibration of silica gel with hexanes.
During chromatography, the highly non-polar ethyl ester byprod-
uct eluted with ethyl butyrate, and the 2-MAG was collected
without any acyl migration. It was observed that the lipase-
catalyzed hydrolysis reactions involving saturated triglycerides
had isolated yields <50%, with the ethyl ester byproduct being the
major product, whereas the unsaturated triglycerides had yields in
the range of 55e75%, and triglycerides containing phenylalkyl
groups had yields >80% (Table 1). It should also be noted that there
was no observable difference in rate of reaction or isolated yield
from the hydrolysis of a 1,3-diacetylglycerol-protected compound
as compared to the 1,3-dibutrylglycerol-protected compound.
We also screened other 1,3-specific lipases to investigate
whether the transformation can be performed in better yield and
selectivity toward the range of substrates and found that lipase
from Rhizomucor miehei showed excellent selectivity toward hy-
drolyzing ‘ABA’ type triglycerides. The reaction proceeded in
a similar fashion where the triglyceride was consumed quickly, but
hydrolysis of diglycerides took 24e48 h. Even though the reaction
proceeded very slowly compared to C. antarctica lipase, the R.
miehei lipase offered a remarkable improvement in selectivity,
mescu et al. reported
a successful synthesis of various 2-
acylglycerols of fatty acids using regiospecific ethanolysis of sym-
metrical triglycerides with immobilized Candida antarctica lipase
(Novozym 435).22,31 Even though C. antarctica is not considered as
a 1,3-regiospecific enzyme, it has been consistently used for the
preparation of 1,3-acylglycerols and ethanolysis of tri-
glycerides.31,32 All existing methods utilize symmetrical (‘AAA’
type) triglycerides resulting in the formation of corresponding ester
as the byproduct that requires exhaustive purification (Fig. 1).
Fig. 1. Types of triglycerides.
Encouraged by this literature, we recently reported a method for
the synthesis 2-AG, which utilizes a structured glyceride, 1,3-
dibutyryl-2-arachidonate (‘ABA’ type), as a substrate, for we rea-
soned that the anticipated byproduct, ethyl butyrate, can be easily
removed.33 Benefits of this procedure include reactions at ambient
temperature, neutral pH, and conservative reaction time. The
method is simple and green, as the lipase can be recycled. Never-
theless, a significant amount of ethyl arachidonate formed due to
over hydrolysis. Since the reaction is selective and proceeded
quickly, it has become a valuable tool for the radiolabelled synthesis
of 2-AG.34 The following work extends our method to the synthesis
of 2-acylglycerols starting from saturated and unsaturated fatty
acids, and alkyl and aryl carboxylic acids.
2. Results and discussion
To test the general practicality of our method (Scheme 2), we
have synthesized 2-MAGs from various commercially available
long-chain carboxylic acids, including those of biological impor-
tance. The synthesis began with the enzymatic 1,3-diacyl pro-
tection of glycerol by the addition of immobilized C. antarctica
(Novozym 435) to glycerol and vinyl butyrate in anhydrous CH2Cl2
at 0 ꢀC, resulting in the protected glycerol in quantitative yield.32,35
The 1,3-diacylglycerol was then coupled to various medium and
long-chain
acids
through
1-(3-dimethyl-aminopropyl)-3-
ethylcarbodiimide hydrochloride (EDCI) coupling in a 1:1 mixture
Scheme 2.

5424
K.M. Whitten et al. / Tetrahedron 68 (2012) 5422e5428
Table 1
Structures and yields of lipase-catalyzed 2-MAGs
Compound no.
Triglyceride (a)
2-MAG (b)
C. antarcticaa yield (%) R. mieheib yield (%)
2
47
49
36
75
84
82
80
83
3
4
5
6
7
44
72
78
83
8
63
55
67
63
77
79
75
76
9
10
11
12
13
40
83
88
40
14
83
37
a
Remaining yield consisted of the ethyl ester of sn-2 acyl group.
Remaining yield consisted of intermediate diglyceride.
b
providing exclusively 2-acylglycerols in excellent yields without
formation of ethyl ester byproduct. The saturated and unsaturated
fatty acid triglycerides were hydrolyzed in good yields (75e88%)
after 24 h. However, in most of the reactions, some unreacted di-
glyceride intermediate remained. Allowing the reaction to proceed
for an additional 24 h or adding more enzyme did not improve the
yield. When unreacted diglyceride that was separated from the 2-
MAG after 24 h was subjected to an additional treatment of R.
miehei lipase the maximal yield once again reached 80% 2-MAG
formation. Surprisingly, in contrast to C. antarctica lipase, R. mie-
hei lipase showed less reactivity toward aryl esters (13 and 14).
3. Conclusion
Synthesis of 2-MAGs is complicated by the propensity of the acyl
group to shift from the sn-2 to the more stable sn-1 or -3 positions,
the acyl migration being promoted by heretofore standard reaction
conditions. We demonstrate herein that chemoenzymatic hydro-
lysis of structured triglycerides is a mild and efficient means to
synthesize 2-MAGs. The ambient temperature, neutral pH, and lack
of caustic work up are conditions, which markedly limit 2-MAG acyl
migration. The ability to synthesize 2-MAGs from ‘ABA’ type tri-
glycerides is an important aspect of this current methodology in

K.M. Whitten et al. / Tetrahedron 68 (2012) 5422e5428
5425
comparison to utilizing ‘AAA’ type triglycerides. An excess of fatty
acid is not required for this method, which is important when the
preparation of the modified fatty acid involves laborious multistep-
synthesis.38e40 This will further enhance the study of structur-
eeactivity relationships of these biologically important lipid sig-
naling molecules.
additional lipase (100 mg) was added until reaction completion
was observed (1 h). The reaction mixture was diluted with CH2Cl2
(3 mL), and the lipase was filtered off. The solvent was removed
under reduced pressure, and the resulting residue was chromato-
graphed on silica gel (10e50% acetone/hexanes) to yield 2b (31 mg,
47%) as
a
white solid. Rf¼0.26 (30% acetone/hexanes).
Mp¼56e57 ꢀC. 1H NMR (400 MHz, chloroform-d)
¼4.93 (quin,
d
4. Experimental
J¼4.76 Hz,1H), 3.84 (br s, 4H), 2.38 (t, J¼7.69 Hz, 2H), 2.08 (br s, 2H),
1.58e1.69 (m, 2H), 1.20e1.37 (m, 16H), 0.88 (t, J¼6.60 Hz, 3H). 13C
4.1. General methods
NMR (100 MHz, chloroform-d)
d¼174.3, 75.3, 62.8 (2C), 34.6, 32.1,
29.8, 29.7, 29.6, 29.5, 29.3, 25.2 (2C), 22.9,14.4. IR (neat, cmꢁ1) 3352,
2922, 2856, 1730, 1464. HRMS for C15H30O4Na (MNaþ) 297.2041.
Calcd 297.2042.
Lipase acrylic resin from C. antarctica and LipozymeÒ, immobi-
lized from R. miehei were purchased from Sigma Aldrich (USA). All
other reagents were used without prior purification. All reactions
were performed under an atmosphere of argon.
4.5. 1,3-Dihydroxypropan-2-yl dodecanoate (2b, utilizing
All byproducts, ethyl arachidonate, ethyl butyrate, and all di-
glycerides were removed during column chromatography. All
glycerols were purified on a Biotage Isolera One using Luknova
prepackaged 12 g columns equilibrated with hexanes.
Compounds 3ae14a were synthesized following the procedure
described for 2a; while compounds 3be14b were synthesized fol-
lowing the C. antarctica and R. miehei procedures described for 2b.
R. miehei)
LipozymeÒ, immobilized from R. miehei (100 mg) was added to
a solution of 2a (100 mg, 0.24 mmol) stirred in anhydrous EtOH
(1 mL). The reaction mixture was stirred for 24 h, diluted with
CH2Cl2 (3 mL), and the lipase was filtered off. The solvent was re-
moved under reduced pressure, and the resulting residue was
chromatographed on silica gel (10e50% acetone/hexanes) to yield
2b (55 mg, 84%) as an oil. All spectral data was consistent with that
obtained using the procedure with C. antarctica.
4.2. 2-Hydroxypropane-1,3-diyl dibutyrate (1)
Immobilized C. antarctica (750 mg) was added to a solution of
glycerol (2.0 g, 21.6 mmol) and vinyl butyrate (6.2 g, 54.0 mmol) in
anhydrous CH2Cl2 (10 mL) at 0 ꢀC. The resulting mixture was stirred
for 3 h under argon atmosphere. Then, additional lipase (400 mg)
was added to the reaction mixture, which was stirred for an addi-
tional 2 h at 0 ꢀC. The lipase was filtered off, the solvent was
evaporated off under reduced pressure, and the residue was chro-
matographed on silica to yield 1 (5.0 g, 99%) as a colorless oil.
Rf¼0.55 (40% ethyl acetate/hexanes). 1H NMR (500 MHz, chloro-
4.6. 2-(Tetradecanoyloxy)propane-1,3-diyl dibutyrate (3a)
Yield 347 mg, 99%; colorless oil. Rf¼0.47 (15% ethyl acetate/
hexanes). 1H NMR (399 MHz, chloroform-d)
d
¼5.32e5.22 (m, 1H),
4.29 (dd, J¼4.4, 11.7 Hz, 2H), 4.15 (dd, J¼5.9, 11.7 Hz, 2H), 2.35e2.25
(m, 6H), 1.70e1.56 (m, 6H), 1.36e1.19 (m, 20H), 0.94 (t, J¼7.3 Hz,
6H), 0.87 (t, J¼6.6 Hz, 3H). 13C NMR (100 MHz, chloroform-d)
d
¼173.4 (2C), 173.2, 69.1, 62.3 (2C), 36.1 (2C), 34.4, 32.2, 29.91, 29.89
form-d)
d
¼4.20 (dd, J¼11.72, 4.39 Hz, 2H), 4.14 (dd, J¼11.72, 5.86 Hz,
(2C), 29.86, 29.7, 29.6, 29.5, 29.3, 25.1, 22.9, 18.6 (2C), 14.4, 13.9 (2C).
IR (neat, cmꢁ1) 2925, 2854, 1741, 1460. HRMS for C25H46O6Na
(MNaþ) 465.3195. Calcd 465.3192.
2H), 4.04e4.12 (m, 1H), 2.41e2.58 (m, 1H), 2.33 (t, J¼7.32 Hz, 4H),
1.67 (sxt, J¼7.32 Hz, 4H), 0.96 (t, J¼7.57 Hz, 4H). The 13C NMR
spectral data (100 MHz, CDCl3) are in agreement with literature
values.32
4.7. 1,3-Dihydroxypropan-2-yl tetradecanoate (3b)
4.3. 2-(Dodecanoyloxy)propane-1,3-diyl dibutyrate (2a)
C. antarctica: 34 mg, 49%; R. miehei: 57 mg, 82%; white solid.
Rf¼0.22 (30% acetone/hexanes). Mp¼57e58 ꢀC. 1H NMR (399 MHz,
EDCI (383 mg, 2.0 mmol), DMAP (19 mg, 0.16 mmol), and 1
(204 mg, 0.88 mmol) were added to a solution of lauric acid
(160 mg, 0.80 mmol) in a 1:1 mixture of anhydrous THF/CH2Cl2
(10 mL) at 0 ꢀC. The reaction mixture was allowed to stir for 4 h. The
reaction mixture was then diluted with CH2Cl2 (15 mL) and H2O
(15 mL). The organic layer was separated, dried over MgSO4, and
the solvent was removed under reduced pressure. The residue was
chromatographed on silica gel (0e15% ethyl acetate/hexanes) to
yield 2a (221 mg, 67%) as a colorless oil. Rf¼0.50 (15% ethyl acetate/
chloroform-d)
d
¼4.93 (quin, J¼4.8 Hz, 1H), 3.89e3.78 (m, 3H), 2.38
(t, J¼7.3 Hz, 2H), 2.17e2.10 (m, 2H),1.70e1.58 (m, 2H), 1.38e1.19 (m,
20H), 0.88 (t, J¼7.3 Hz, 3H). 13C NMR (100 MHz, chloroform-d)
d
¼174.3, 75.2, 62.8 (2C), 34.6, 32.2, 29.91, 29.87 (2C), 29.8, 29.7,
29.6, 29.5, 29.3, 25.2, 22.9, 14.4. IR (neat, cmꢁ1) 3418, 2926, 2855,
1729, 1466. HRMS for C17H34O4Na (MNaþ) 325.2354. Calcd
325.2355.
4.8. 2-(Palmitoyloxy)propane-1,3-diyl dibutyrate (4a)
hexanes). 1H NMR (400 MHz, chloroform-d)
d
¼5.24e5.31 (m, 1H),
4.30 (dd, J¼12.09, 4.03 Hz, 2H), 4.16 (dd, J¼12.46, 5.86 Hz, 2H),
2.27e2.35 (m, 6H),1.57e1.70 (m, 6H),1.21e1.36 (m,16H), 0.92e0.98
(m, 6H), 0.88 (t, J¼6.60 Hz, 3H). 13C NMR (100 MHz, chloroform-d)
Yield 309 mg, 84%; colorless oil. Rf¼0.39 (15% ethyl acetate/
hexanes). 1H NMR (500 MHz, chloroform-d)
d
¼5.30e5.25 (m, 1H),
4.30 (dd, J¼4.2, 12.0 Hz, 2H), 4.16 (dd, J¼5.9, 11.7 Hz, 2H), 2.35e2.27
d
¼173.4 (2C), 173.2, 77.4, 69.1, 62.3 (2C), 36.1 (2C), 34.4, 32.1, 29.8,
(m, 6H), 1.70e1.58 (m, 6H), 1.34e1.21 (m, 24H), 0.98e0.92 (m, 6H),
29.7, 29.6, 29.5, 29.3, 25.1, 22.9, 18.6 (2C), 14.4, 13.9 (2C). IR (neat,
cmꢁ1) 2926, 2855, 1742, 1460. HRMS for C23H42O6Na (MNaþ)
437.2881. Calcd 437.2879.
0.88 (t, J¼6.8 Hz, 3H). 13C NMR (100 MHz, chloroform-d)
¼173.4,
d
173.2 (2C), 69.1, 62.3 (2C), 36.1, 34.4, 32.2, 29.9 (6C), 29.7, 29.6, 29.5,
29.3, 25.1 (2C), 18.6 (3C), 14.4, 13.9 (2C). IR (neat, cmꢁ1) 2924, 1742,
1460. HRMS for C27H50O6Na (MNaþ) 493.3503. Calcd 493.3505.
4.4. 1,3-Dihydroxypropan-2-yl dodecanoate (2b, utilizing
C. antarctica)
4.9. 1,3-Dihydroxypropan-2-yl palmitate (4b)
Immobilized C. antarctica (Novozym 435, 100 mg) was added to
a solution of 2a (100 mg, 0.24 mmol), stirred in anhydrous EtOH
(1 mL). After the full consumption of 2a (1 h, TLC monitoring),
C. antarctica: 25 mg, 36%; R. miehei: 56 mg, 80%; white solid.
Rf¼0.27 (30% acetone/hexanes). Mp¼64e65 ꢀC. 1H NMR (500 MHz,
chloroform-d)
d
¼4.93 (quin, J¼4.8 Hz, 1H), 3.84 (t, J¼4.9 Hz, 4H),

5426
K.M. Whitten et al. / Tetrahedron 68 (2012) 5422e5428
2.38 (t, J¼7.6 Hz, 2H), 2.13e2.05 (m, 2H), 1.69e1.59 (m, 2H),
1.38e1.20 (m, 24H), 0.88 (t, J¼7.3 Hz, 3H). 13C NMR (100 MHz,
29.3, 29.2, 27.5, 27.4, 25.1, 22.9, 18.6 (3C), 14.4, 13.9 (2C). IR (neat,
cmꢁ1) 3007, 2925, 1742, 1460. HRMS for C29H52O6Na (MNaþ)
519.3658. Calcd 519.3662.
chloroform-d)
d
¼174.3, 75.2, 62.8 (2C), 34.6, 32.2, 29.93, 29.92,
29.89, 29.84, 29.7, 29.6, 29.5, 29.3, 25.2 (2C), 23.3, 22.9, 14.4. IR
(neat, cmꢁ1) 3320, 2917, 2850, 1730, 1471. HRMS for C19H38O4Na
(MNaþ) 353.2668. Calcd 353.2668.
4.15. 1,3-Dihydroxypropan-2-yl oleate (7b)
C. antarctica: 48 mg, 67%; R. miehei: 60 mg, 83%; colorless oil.
Rf¼0.30 (30% acetone/hexanes). 1H NMR (399 MHz, chloroform-d)
4.10. (Z)-2-(hexadec-9-enoyloxy)propane-1,3-diyl dibutyrate
(5a)
d
¼5.41e5.31 (m, 2H), 4.92 (quin, J¼4.8 Hz, 1H), 3.88e3.77 (m, 4H),
2.49 (br s, 2H), 2.37 (t, J¼7.7 Hz, 2H), 2.01 (q, J¼6.4 Hz, 4H), 1.63
Yield 291 mg, 79%; colorless oil. Rf¼0.50 (15% ethyl acetate/
(quin, J¼7.3 Hz, 2H), 1.40e1.19 (m, 20H), 0.88 (t, J¼6.6 Hz, 3H). 13C
hexanes). 1H NMR (399 MHz, chloroform-d)
d¼5.38e5.32 (m, 2H),
NMR (100 MHz, chloroform-d)
d
¼174.4, 130.3, 129.9, 75.1, 62.5 (2C),
5.31e5.24 (m, 1H), 4.30 (dd, J¼4.4, 11.7 Hz, 2H), 4.16 (dd, J¼5.9,
12.5 Hz, 2H), 2.38e2.26 (m, 6H), 2.01 (q, J¼6.6 Hz, 4H), 1.72e1.56
(m, 6H), 1.39e1.20 (m, 16H), 0.95 (t, J¼7.3 Hz, 6H), 0.88 (t, J¼7.0 Hz,
34.6, 32.1, 30.0, 29.9, 29.8, 29.6, 29.4, 29.33, 29.31, 27.45, 27.38, 25.2
(2C), 22.9, 14.4. IR (neat, cmꢁ1) 3415, 3008, 2923, 2854, 1735, 1464.
HRMS for C21H40O4Na (MNaþ) 379.2827. Calcd 379.2824.
3H). 13C NMR (100 MHz, chloroform-d)
d
¼173.3 (2C), 173.1, 130.2,
129.9, 69.1, 62.3 (2C), 36.1 (2C), 34.4, 32.0, 30.0, 29.9, 29.4, 29.3,
29.25, 29.21, 27.5, 27.4, 25.1, 22.9, 18.6 (2C), 14.3, 13.8 (2C). IR (neat,
cmꢁ1) 3007, 2928, 2856, 1742, 1459. HRMS for C27H48O6Na (MNaþ)
491.3347. Calcd 491.3349.
4.16. 2-((9Z,12Z)-Octadeca-9,12-dienoyloxy)propane-1,3-diyl
dibutyrate (8a)
Yield 344 mg, 98%; colorless oil. Rf¼0.38 (15% ethyl acetate/
hexanes). 1H NMR (399 MHz, chloroform-d)
d
¼5.43e5.30 (m, 4H),
4.11. (Z)-1,3-Dihydroxypropan-2-yl hexadec-9-enoate (5b)
5.29e5.24 (m, 1H), 4.30 (dd, J¼4.4, 11.7 Hz, 2H), 4.15 (dd, J¼5.9,
11.7 Hz, 2H), 2.77 (t, J¼6.6 Hz, 2H), 2.36e2.26 (m, 6H), 2.05 (q,
J¼6.6 Hz, 4H), 1.72e1.56 (m, 6H), 1.41e1.22 (m, 14H), 0.95 (t,
J¼7.7 Hz, 6H), 0.89 (t, J¼7.0 Hz, 3H). 13C NMR (100 MHz, chloro-
C. antarctica: 46 mg, 66%; R. miehei: 58 mg, 83%; colorless oil.
Rf¼0.25 (30% acetone/hexanes). 1H NMR (399 MHz, chloroform-d)
d
¼5.40e5.31 (m, 2H), 4.92 (quin, J¼4.8 Hz, 1H), 3.86e3.79 (m, 4H),
form-d)
d
¼173.3 (2C), 173.1, 130.5, 130.2, 128.3, 128.1, 69.1, 62.3 (2C),
2.37 (t, J¼7.7 Hz, 2H), 2.33 (br s, 2H), 2.05e1.97 (m, 4H), 1.63 (quin,
36.1 (2C), 34.4, 31.8, 29.8, 29.6, 29.4 (2C), 29.3, 29.2, 27.4, 25.8, 25.1,
22.8, 18.6 (2C), 14.3, 13.9 (2C). IR (neat, cmꢁ1) 3008, 2929, 2856,
1741, 1459. HRMS for C29H50O6Na (MNaþ) 517.3506. Calcd 517.3505.
J¼7.3 Hz, 2H), 1.39e1.23 (m, 16H), 0.88 (t, J¼6.6 Hz, 3H). 13C NMR
(100 MHz, chloroform-d)
d
¼174.3, 130.3, 129.9, 75.2, 62.6 (2C), 34.6,
32.0, 30.0, 29.9, 29.4, 29.32, 29.30, 29.2, 27.4, 27.4, 25.2, 22.9,14.3. IR
(neat, cmꢁ1) 3405, 3008, 2924, 2855, 1736, 1462. HRMS for
C19H36O4Na (MNaþ) 351.2512. Calcd 351.2511.
4.17. (9Z,12Z)-1,3-Dihydroxypropan-2-yl octadeca-9,12-
dienoate (8b)
4.12. 2-(Stearoyloxy)propane-1,3-diyl dibutyrate (6a)
C. antarctica: 45 mg, 63%; R. miehei: 55 mg, 77%; colorless oil.
Rf¼0.37 (30% acetone/hexanes). 1H NMR (399 MHz, chloroform-d)
Yield 300 mg, 85%; colorless oil. Rf¼0.34 (15% ethyl acetate/
d
¼5.44e5.30 (m, 4H), 4.93 (quin, J¼4.8 Hz, 1H), 3.89e3.76 (m, 4H),
hexanes). 1H NMR (500 MHz, chloroform-d)
d
¼5.30e5.24 (m, 1H),
2.77 (t, J¼6.6 Hz, 2H), 2.38 (t, J¼7.3 Hz, 2H), 2.13 (t, J¼6.2 Hz, 2H),
2.05 (q, J¼6.8 Hz, 4H), 1.69e1.59 (m, 2H), 1.41e1.23 (m, 14H), 0.89 (t,
4.30 (dd, J¼4.4, 11.7 Hz, 2H), 4.16 (dd, J¼6.1, 12.0 Hz, 2H), 2.34e2.27
(m, 6H), 1.70e1.59 (m, 6H), 1.35e1.20 (m, 28H), 0.98e0.93 (m, 6H),
J¼6.6 Hz, 3H). 13C NMR (100 MHz, chloroform-d)
¼174.3, 130.5,
d
0.88 (t, J¼6.6 Hz, 3H). 13C NMR (100 MHz, chloroform-d)
d¼173.4
130.2, 128.3, 128.1, 75.2, 62.8 (2C), 34.6, 31.8, 29.8, 29.6, 29.4, 29.33,
29.30, 27.4, 25.9, 25.2 (2C), 22.8, 14.3. IR (neat, cmꢁ1) 3397, 010,
2926, 2855, 1736, 1459. HRMS for C21H38O4Na (MNaþ) 377.2667.
Calcd 377.2668.
(2C),173.2, 69.1, 62.3 (2C), 36.2 (2C), 34.4, 32.2, 29.9 (4C), 29.89 (3C),
29.86, 29.7, 29.6, 29.5, 29.3, 25.1, 22.9, 18.6 (2C), 14.4, 13.9 (2C). IR
(neat, cmꢁ1) 2924, 1742, 1460. HRMS for C29H54O6Na (MNaþ)
521.3813. Calcd 521.3818.
4.18. (Z)-2-(Icos-11-enoyloxy)propane-1,3-diyl dibutyrate (9a)
Yield 328 mg, 88%; colorless oil. Rf¼0.42 (15% ethyl acetate/
4.13. 1,3-Dihydroxypropan-2-yl stearate (6b)
C. antarctica: 32 mg, 44%; R. miehei: 56 mg, 78%; white solid.
Rf¼0.23 (30% acetone/hexanes). Mp¼68e69 ꢀC. 1H NMR (399 MHz,
hexanes). 1H NMR (399 MHz, chloroform-d)
d
¼5.38e5.32 (m, 2H),
5.30e5.24 (m, 1H), 4.30 (dd, J¼4.4, 11.7 Hz, 2H), 4.16 (dd, J¼5.9,
12.5 Hz, 2H), 2.36e2.26 (m, 6H), 2.05e1.97 (m, 4H), 1.71e1.57 (m,
6H), 1.27 (br s, 24H), 0.95 (t, J¼7.3 Hz, 6H), 0.88 (t, J¼6.6 Hz, 3H). 13C
chloroform-d)
d
¼4.93 (quin, J¼4.6 Hz, 1H), 3.88e3.82 (m, 4H), 2.38
(t, J¼7.7 Hz, 2H), 2.04 (t, J¼5.9 Hz, 2H), 1.65 (quin, J¼7.3 Hz, 2H),
1.38e1.19 (m, 28H), 0.88 (t, J¼6.6 Hz, 3H). 13C NMR (100 MHz,
NMR (100 MHz, chloroform-d)
d
¼173.3 (2C), 173.1, 130.2, 130.0,
chloroform-d)
d¼174.3, 75.3, 62.8 (2C), 34.6, 32.2, 29.95 (5C), 29.91
69.0, 62.3 (2C), 36.1 (2C), 34.4, 32.1, 30.0 (2C), 29.8, 29.7, 29.55 (2C),
29.52, 29.51, 29.3, 27.4 (2C), 25.1 (2C), 22.9, 18.6 (2C), 14.4, 13.9
(2C).IR (neat, cmꢁ1) 3008, 2925, 2855, 1742, 1459. HRMS for
C31H56O6Na (MNaþ) 547.3978. Calcd 547.3975.
(2C), 29.8, 29.7, 29.6, 29.5, 29.3, 25.2, 23.0, 13.8. IR (neat, cmꢁ1
)
3313, 2916, 2849, 1730, 1472. HRMS for C21H42O4Na (MNaþ)
381.2982. Calcd 381.2981.
4.14. (Z)-2-(Oleoyloxy)propane-1,3-diyl dibutyrate (7a)
Yield 290 mg, 82%; colorless oil. Rf¼0.43 (15% ethyl acetate/
4.19. (Z)-1,3-Dihydroxypropan-2-yl icos-11-enoate (9b)
C. antarctica: 40 mg, 55%; R. miehei, 58 mg, 79%; white solid.
Rf¼0.24 (30% acetone/hexanes). Mp¼32e33 ꢀC. 1H NMR (399 MHz,
hexanes). 1H NMR (500 MHz, chloroform-d)
d¼5.39e5.30 (m, 2H),
5.30e5.24 (m, 1H), 4.30 (dd, J¼4.4, 11.7 Hz, 2H), 4.15 (dd, J¼6.1,
12.0 Hz, 2H), 2.36e2.25 (m, 6H), 2.01 (q, J¼6.2 Hz, 4H), 1.71e1.55
(m, 6H), 1.38e1.19 (m, 20H), 0.95 (t, J¼7.3 Hz, 3H), 0.88 (t, J¼6.8 Hz,
chloroform-d)
d
¼5.38e5.32 (m, 2H), 4.93 (quin, J¼4.8 Hz, 1H),
3.89e3.78 (m, 4H), 2.38 (t, J¼7.3 Hz, 2H), 2.20e2.12 (m, 2H), 2.01 (q,
J¼6.6 Hz, 4H), 1.71e1.57 (m, 2H), 1.40e1.18 (m, 24H), 0.88 (t,
3H). 13C NMR (100 MHz, chloroform-d)
129.9, 69.1, 62.3, 36.1 (2C), 34.4, 32.1, 30.0, 29.9, 29.8, 29.6 (2C), 29.4,
d
¼173.4 (2C), 173.1, 130.3,
J¼6.6 Hz, 3H). 13C NMR (100 MHz, chloroform-d)
¼174.3, 130.2,
d
130.0, 75.2, 62.8 (2C), 34.6, 32.1, 30.0 (2C), 29.8, 29.7, 29.7, 29.6 (2C),

K.M. Whitten et al. / Tetrahedron 68 (2012) 5422e5428
5427
29.56, 29.51, 29.3, 27.4, 25.2 (2C), 23.0, 14.4. IR (neat, cmꢁ1) 3405,
4.25. 1,3-Dihydroxypropan-2-yl docosanoate (12b)
3008, 2923, 2854, 1737, 1465. HRMS for C23H44O4Na (MNaþ)
407.3142. Calcd 407.3137.
C. antarctica: 32 mg, 40%; R. miehei: 70 mg, 88%; white solid.
Rf¼0.27 (30% acetone/hexanes). Mp¼79e80 ꢀC. 1H NMR (399 MHz,
chloroform-d)
d
¼4.93 (quin, J¼4.6 Hz, 1H), 3.88e3.81 (m, 4H), 2.38
4.20. 2-((5Z,8Z,11Z,14Z)-Icosa-5,8,11,14-tetraenoyloxy)-
propane-1,3-diyl dibutyrate (10a)
(t, J¼7.7 Hz, 2H), 2.08 (s, 2H), 1.69e1.59 (m, 2H), 1.38e1.19 (m, 36H),
0.88 (t, J¼6.2 Hz, 3H). 13C NMR (100 MHz, chloroform-d)
¼174.3,
d
75.2, 62.8 (2C), 34.6, 32.2, 31.8, 29.94 (7C), 29.90 (2C), 29.8, 29.7,
29.6, 29.5, 29.3, 25.2, 22.94, 22.89, 14.4. IR (neat, cmꢁ1) 3313, 297,
2850, 1730, 1472. HRMS for C25H50O4Na (MNaþ) 437.3610. Calcd
437.3607.
Yield 168 mg, 98%; colorless oil. Rf¼0.36 (30% ethyl acetate/
hexanes). The 1H and 13C spectral data (500 and 100 MHz, CDCl3)
are in agreement with literature values.33 IR (neat, cmꢁ1) 3012,
2931, 1741, 1456. HRMS for C31H50O6Na (MNaþ) 541.3502. Calcd
541.3505.
4.26. 2-(3-Phenylpropanoyloxy)propane-1,3-diyl dibutyrate
(13a)
4.21. (5Z,8Z,11Z,14Z)-1,3-Dihydroxypropan-2-yl icosa-
5,8,11,14-tetraenoate (10b)
Yield 280 mg, 98%; colorless oil. Rf¼0.48 (35% ethyl acetate/
hexanes). 1H NMR (399 MHz, chloroform-d)
d
¼7.33e7.25 (m, 2H),
C. antarctica: 48 mg, 67%; R. miehei: 54 mg, 75%; colorless oil.
Rf¼0.30 (30% acetone/hexanes). The 1H and 13C spectral data (500
and 100 MHz, CDCl3) are in agreement with literature values.33 IR
(neat, cmꢁ1) 3420, 2013, 2927, 1736, 1456. HRMS for C23H38O4Na
(MNaþ) 401.2677. Calcd 401.2668.
7.24e7.15 (m, 3H), 5.31e5.23 (m, 1H), 4.28 (dd, J¼4.4, 11.7 Hz, 2H),
4.13 (dd, J¼5.9, 11.7 Hz, 2H), 2.96 (t, J¼7.7 Hz, 2H), 2.66 (t, J¼8.1 Hz,
2H), 2.34e2.24 (m, 6H), 1.64 (sxt, J¼7.3 Hz, 4H), 0.94 (t, J¼7.3 Hz,
6H). 13C NMR (100 MHz, chloroform-d)
128.7 (2), 128.5 (2), 126.6, 69.4, 62.2 (2), 36.1 (2), 35.9, 31.0, 18.6 (2),
13.9 (2C). IR (neat, cmꢁ1) 3027, 2966, 2877, 1737, 1455. HRMS for
C20H28O6Na (MNaþ) 387.1787. Calcd 387.1784.
d
¼173.3 (2), 172.2, 140.4,
4.22. 2-((4Z,7Z,10Z,13Z,16Z,19Z)-Docosa-4,7,10,13,16,19-
hexaenoyloxy)propane-1,3-diyl diacetate (11a)
4.27. 1,3-Dihydroxypropan-2-yl 3-phenylpropanoate (13b)
EDCI (111 mg, 0.58 mmol), DMAP (6 mg, 0.06 mmol), and diac-
etin (44 mg, 0.25 mmol) were added to a solution of docosahex-
aenoic acid (75 mg, 0.23 mmol) in anhydrous CH2Cl2 (5 mL) at 0 ꢀC.
The reaction mixture was allowed to stir for 4 h. Upon completion,
the reaction mixture was diluted with CH2Cl2 and H2O. The organic
layer was separated, dried over MgSO4, and removed under re-
duced pressure. The resulting residue was chromatographed on
silica gel (0e30% ethyl acetate/hexanes) to yield to 11a (111 mg,
99%) as a colorless oil. Rf¼0.55 (30% ethyl acetate/hexanes). 1H NMR
C. antarctica: 38 mg, 83%; R. miehei: 8 mg, 40%; white foam.
Rf¼0.18 (40% acetone/hexanes). 1H NMR (399 MHz, chloroform-d)
d
¼7.36e7.28 (m, 2H), 7.26e7.18 (m, 3H), 4.89 (td, J¼4.5, 9.3 Hz, 1H),
3.79e3.71 (m, 4H), 3.02e2.96 (m, 2H), 2.77e2.70 (m, 2H), 1.91e1.83
(m, 2H). 13C NMR (100 MHz, chloroform-d)
d¼175.2, 140.4, 128.8
(2), 128.5 (2), 126.7, 75.5, 62.6 (2), 36.1, 31.8. IR (neat, cmꢁ1) 3412,
3029, 2935, 2881, 1731, 1454. HRMS for C12H16O4Na (MNaþ)
247.0945. Calcd 247.0946.
(500 MHz, chloroform-d)
d¼5.47e5.32 (m, 12H), 5.30e5.21 (m, 1H),
4.29 (dd, J¼4.4, 11.7 Hz, 2H), 4.16 (dd, J¼5.9, 12.2 Hz, 2H), 2.93e2.77
(m, 10H), 2.40 (d, J¼2.9 Hz, 4H), 2.14e2.02 (m, 6H), 0.98 (t, J¼7.6 Hz,
3H). The 13C spectral data (100 MHz, CDCl3) and IR data are in
agreement with literature values.32 HRMS for C29H42O6Na (MNaþ)
509.2880. Calcd 509.2879.
4.28. 2-(5-Phenylpentanoyloxy)propane-1,3-diyl dibutyrate
(14a)
Yield 298 mg, 99%; colorless oil. Rf¼0.63 (35% ethyl acetate/
hexanes). 1H NMR (500 MHz, chloroform-d)
d
¼7.31e7.24 (m, 2H),
7.21e7.14 (m, 3H), 5.31e5.23 (m, 1H), 4.30 (dd, J¼4.4, 11.7 Hz, 2H),
4.14 (dd, J¼5.9, 11.7 Hz, 2H), 2.63 (t, J¼7.1 Hz, 2H), 2.35 (t, J¼6.8 Hz,
2H), 2.29 (t, J¼6.8 Hz, 4H), 1.71e1.58 (m, 8H), 0.94 (t, J¼7.3 Hz, 6H).
4.23. (4Z,7Z,10Z,13Z,16Z,19Z)-1,3-Dihydroxypropan-2-yl-
docosa-4,7,10,13,16,19-hexaenoate (11b)
13C NMR (100 MHz, chloroform-d)
(2C), 128.6 (2C), 126.0, 69.2, 62.3 (2C), 36.1 (2C), 35.8, 34.2, 31.0,
24.7, 18.6 (2C), 13.8 (2C). IR (neat, cmꢁ1) 3028, 2965, 2876, 1738,
1454. HRMS for C22H32O6Na (MNaþ) 415.2094. Calcd 415.2097.
d
¼173.4 (2C), 172.9, 142.2, 128.6
C. antarctica: 45 mg, 63%; R. miehei: 63 mg, 76%; colorless oil.
Rf¼0.29 (30% acetone/hexanes). 1H NMR (399 MHz, chloroform-d)
d
¼5.52e5.22 (m, 12H), 4.92 (quin, J¼4.6 Hz, 1H), 3.82 (t, J¼5.1 Hz,
4H), 2.91e2.77 (m, 10H), 2.49e2.37 (m, 4H), 2.22 (t, J¼6.2 Hz, 2H),
2.07 (quin, J¼7.5 Hz, 2H), 0.97 (t, J¼7.3 Hz, 3H). 13C NMR (100 MHz,
chloroform-d)
d
¼173.5, 132.3 (2C), 129.8 (2C), 128.8, 128.6, 128.5
4.29. 1,3-Dihydroxypropan-2-yl 5-phenylpentanoate (14b)
(2C), 128.3 (2C), 128.1, 127.9, 75.4, 62.6 (2C), 34.4, 25.8 (5C), 23.0
(2C), 20.8. IR (neat, cmꢁ1) 3401, 3013, 2663, 1736, 1390. HRMS for
C25H38O4Na (MNaþ) 425.2666. Calcd 425.2668.
C. antarctica: 50 mg, 83%; R. miehei: 24 mg, 37%; white foam.
Rf¼0.26 (40% acetone/hexanes). 1H NMR (399 MHz, chloroform-d)
d
¼7.31e7.25 (m, 2H), 7.22e7.13 (m, 3H), 4.92 (td, J¼4.8, 9.5 Hz, 1H),
3.86e3.76 (m, 4H), 2.64 (t, J¼7.0 Hz, 2H), 2.41 (t, J¼7.0 Hz, 2H),
4.24. 2-(Docosanoyloxy)propane-1,3-diyl dibutyrate (12a)
2.17e2.11 (m, 2H), 1.74e1.60 (m, 4H). 13C NMR (100 MHz, chloro-
form-d)
d
¼174.0, 142.2, 128.6 (4C), 126.1, 75.2, 62.7 (2C), 35.8, 34.4,
Yield 201 mg, 56%; colorless oil. Rf¼0.48 (15% ethyl acetate/
hexanes). Mp¼27e28 ꢀC. 1H NMR (399 MHz, chloroform-d)
31.0, 24.7. IR (neat, cmꢁ1) 3414, 3027, 2936, 2882, 1731, 1454. HRMS
for C14H20O4Na (MNaþ) 275.1257. Calcd 275.1259.
d
¼5.32e5.23 (m, 1H), 4.30 (dd, J¼4.4, 11.7 Hz, 2H), 4.16 (dd, J¼5.9,
11.7 Hz, 2H), 2.36e2.26 (m, 6H), 1.71e1.57 (m, 6H), 1.25 (s, 36H),
0.95 (t, J¼7.3 Hz, 6H), 0.88 (t, J¼6.6 Hz, 3H). 13C NMR (100 MHz,
Acknowledgements
chloroform-d)
d
¼173.4 (2C), 173.2, 69.1, 62.3 (2C), 36.0 (2C), 34.4,
32.2, 29.95 (9C), 29.91 (2C), 29.88, 29.7, 29.6, 29.5, 29.3, 25.1, 22.9,
18.6 (2C), 14.4, 13.9 (2C). IR (neat, cmꢁ1) 2923. 2853, 1742, 1462.
HRMS for C33H62O6Na (MNaþ) 577.4446. Calcd 577.4444.
We thank Dr. David Janero for helpful discussions. We thank Dr.
Furong Sun at the School of Chemical Sciences, University of Illinois
at Urbana-Champaign, Urbana, IL for supplying HRMS data. We

5428
K.M. Whitten et al. / Tetrahedron 68 (2012) 5422e5428
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would like to acknowledge the financial support for this research
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Supplementary data
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