Preparation and characterization of 4-methoxy cinnamoyl glycerol

Article abstract of DOI:10.1007/s11746-008-1197-y

Glycerol was reacted with 4-methoxy cinnamic acid to prepare the corresponding 4-methoxy cinnamoyl glycerol. The reaction proceeded in toluene under reflux conditions with p-toluenesulfonic acid catalyst. Reaction of equimolar amounts of reactants produced the monoester in 20% yield after 2 h. The product was isolated and characterized by FTIR, mass spectrometry, and NMR techniques. Results of mass spectrometry and NMR experiments showed that the ester linkage formed between the carboxylic acid and the primary hydroxyl of glycerol. The ester displayed strong absorbance between 250 and 350 nm and shows potential as a UV filter in formulations where hydrophilic compounds are advantageous. AOCS 2008.

Full text of DOI:10.1007/s11746-008-1197-y

J Am Oil Chem Soc (2008) 85:347–351
DOI 10.1007/s11746-008-1197-y
ORIGINAL PAPER
Preparation and Characterization of 4-Methoxy Cinnamoyl
Glycerol
R. A. Holser Æ T. R. Mitchell Æ R. E. Harry-O’kuru Æ
S. F. Vaughn Æ E. Walter Æ D. Himmelsbach
Received: 13 March 2007 / Revised: 7 January 2008 / Accepted: 10 January 2008 / Published online: 6 February 2008
Ó AOCS 2008
Abstract Glycerol was reacted with 4-methoxy cinnamic
acid to prepare the corresponding 4-methoxy cinnamoyl
glycerol. The reaction proceeded in toluene under reflux
conditions with p-toluenesulfonic acid catalyst. Reaction of
equimolar amounts of reactants produced the monoester in
20% yield after 2 h. The product was isolated and char-
acterized by FTIR, mass spectrometry, and NMR
techniques. Results of mass spectrometry and NMR
experiments showed that the ester linkage formed between
the carboxylic acid and the primary hydroxyl of glycerol.
The ester displayed strong absorbance between 250 and
350 nm and shows potential as a UV filter in formulations
where hydrophilic compounds are advantageous.
Introduction
Derivatives of cinnamic acid such as octyl methoxycin-
namate (OMC) and 2-ethylhexyl-p-methoxycinnamate find
use as organic UV filters in sunscreens and cosmetic
formulations [1, 2]. These cinnamates and the related
esters formed by combining p-methoxy cinnamic acid or
ferulic acids with long chain acylglycerides exhibit pre-
dominantly lipophilic characteristics [3–5]. However, for
certain applications it would be advantageous to have a
UV filter with more hydrophilic character, e.g., to limit the
penetration of a topical formulation into the skin [6–8].
This can be achieved by appropriate selection of the
alcohol used to form the ester. For example, reducing the
length of the carbon chain or introducing polar groups will
increase the hydrophilic properties of the resulting prod-
uct. Glycerol is a particularly attractive substrate for this
reaction due to its small size, three carbons, and three
hydroxyl groups. The additional hydroxyl groups intro-
duce significant polarity to the structure and provide sites
for further derivatization.
Keywords Glycerol Á 4-methoxy cinnamic acid Á
Ester
Disclaimer The use of trade, firm, or corporation names in this
publication is for the information and convenience of the reader.
Such use does not constitute an official endorsement or approval by
the United States Department of Agriculture or the Agricultural
Research Service of any product or service to the exclusion of others
that may be suitable.
The current supply of glycerol is increasing as biodiesel
production expands in response to the demand for renew-
able fuels which has made the co-product glycerol widely
available and relatively inexpensive. Glycerol is also
obtained from plant and animal triglycerides during the
production of soaps, fatty acids, and fatty esters for nonfuel
applications [9].
R. A. Holser (&) Á T. R. Mitchell Á D. Himmelsbach
Richard Russell Agricultural Research Center,
Agricultural Research Service,
United States Department of Agriculture,
950 College Station Road,
Athens, GA 30605, USA
The monoester of glycerol and 4-methoxy cinnamic acid
was prepared directly in one step to produce an organic UV
filter with more hydrophilic character than related com-
pounds in the cinnamate class [10]. The development of
such new products can provide additional outlets for this
surplus glycerol. Applications for these compounds include
polymer additives and personal care formulations where
e-mail: Ronald.Holser@ars.usda.gov
R. E. Harry-O’kuru Á S. F. Vaughn Á E. Walter
National Center for Agriculture Utilization Research,
Agricultural Research Service,
United States Department of Agriculture,
1815 North University Street,
Peoria, IL 61604, USA
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J Am Oil Chem Soc (2008) 85:347–351
Infrared Spectroscopy (IR)
the UV absorbing properties may limit damage from out-
door exposure [11, 12].
Infrared spectra were obtained with a Thermo Nicolet,
Nexus FT-IR 470 spectrometer, using the ZnSe ATR
accessory. Samples, 10–20 mg, were dissolved in diethyl
ether (2 mL) and a drop was placed onto the ZnSe crystal.
The solvent was allowed to evaporate before the spectra
were collected. Spectral data were collected over the range
600–4,000 cm-1 and processed by Omnilab software.
Strong absorptions measured in the 3,200–3,600 cm-1
region were observed for hydroxyl groups and near
1,700 cm-1 for the ester linkage.
Experimental Procedures
Materials
Glycerol, 4-methoxy cinnamic acid, toluene, and diethyl
ether were purchased from Sigma-Aldrich Chemicals,
(St. Louis, MO, USA). The p-toluenesulfonic acid catalyst
was purchased from Fisher Scientific Co. (Pittsburgh, PA,
USA). BSTFA reagent was purchased from Pierce Chem-
icals (Rockford, IL, USA). All materials were used as
received.
GC–MS
Mass spectra of silylated samples were collected using the
Agilent 6890N gas chromatograph equipped with the 5973
mass selective detector operated in EI mode. Separations
were achieved on the HP-5 ms column, 30 m 9 0.25 mm
ID 9 0.25 micron film thickness. Helium was used as the
carrier gas with a linear velocity of 35 cm/sec. The oven
temperature was programmed from 120 to 240 °C at
10 °C/min with an initial 2-min hold and a final ten minute
hold. The inlet was heated to 230 °C and set for splitless
injections with a one microliter injection volume. The
detector source was heated to 230 °C and the detector
quadrapole was heated to 150 °C. Data were collected and
processed via Chemstation software.
Esterification
Reactions were performed in 250-mL glass vessels fitted
with a condenser and Dean Stark trap. Equimolar reac-
tions used 2 g of 4-methoxy cinnamic acid (8 mMol) and
0.73 g glycerol (8 mMol). The reactants were placed into
the reactor with 100 mL toluene and 100 mg of p-tolu-
enesulfonic acid catalyst (6 mol%, relative to glycerol).
The mixture was heated to reflux, 110 °C, on a magnet-
ically stirred hot-plate. Separation and recovery of the
monoester from the reactants was achieved by extraction
of the reaction mixture with distilled water, 2:1 volume
ratio, followed by extraction with diethyl ether, 1:1 vol-
ume ratio. The product was obtained as a clear viscous
liquid.
HPLC–MS–MS
Samples were analyzed with a tandem MS system using a
methanol/water gradient flowing at 0.2 mL/min. Each
solvent contained 1 vol% formic acid with the gradient
starting at 50% methanol and increasing to 100% over
50 min with a ten minute hold. Injections were made using
a Finnigan Spectra System AS3000 autosampler onto a
Beckman Ultrasphere C18 column measuring 4.6 mm 9
250 mm. The eluent was monitored at 280 nm by the
Finnigan Spectra System UV 6000 LP before entering the
MS.
HPLC Analysis
Reaction samples were analyzed by HPLC on an Agilent
1100 system equipped with DAD-ELSD-MS detectors. The
strong UV absorbance of 4-methoxy cinnamic acid and the
glycerol ester products provided a sensitive method of
detection. The absorbance of column effluents was moni-
tored at 192, 210, 215, 273, and 288 nm using the diode
array detector with UV spectra collected under the peaks
by scanning from 190 to 400 nm. The 4-methoxy cinnamic
acid and the esters strongly absorb from 250 to 350 nm
with maxima near 290 nm. Glycerol does not absorb in this
region but was detected by ELSD. The MS was not rou-
tinely used for quantitative analysis. Compounds were
separated on an Alltech C18AQ column (Grace, Deerfield,
IL, USA) measuring 150 mm 9 4.6 mm with 5 lm
packing. Isocratic methanol at a flow rate of 0.5 mL/min
was used to elute compounds. Data were collected and
processed via Chemstation software.
Mass spectrometry was performed using the Finnigan
LCQ Duo with electrospray ionization in positive ion
mode. The MS parameters were optimized for the acid and
the instrument scanned from 100 to 1,000 mass units with
the most intense ions fragmented at 27% normalized col-
lision energy. Typical settings were 4.5 kV source voltage,
80 lA source current, 56 V capillary voltage, and 95 °C
capillary temperature. All recorded spectra were the aver-
age of three microscans.
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J Am Oil Chem Soc (2008) 85:347–351
1D NMR
349
Results and Discussion
Spectra were collected at 300.0 K with the Varian Mercury
Plus 400 Spectrometer using the 5-mm Mercury probe.
Samples were dissolved in CD3OD and spectra were
acquired at 400.15 MHz for1H NMR and 100.63 MHz for
13C NMR. Chemical shifts (d) for 1H NMR are reported as
ppm from 3.3 ppm for CD3OD and from 47.9 ppm for
CD3OD for 13C NMR.
The esterification of glycerol with 4-methoxy cinnamic
acid proceeded in toluene at reflux conditions using p-tol-
uenesulfonic acid catalyst to form the monoester product
(Fig. 1). The disappearance of the acid and the formation
of the ester were followed over the course of the reaction
by HPLC with UV detection (Fig. 2). While the absorbance
spectrum of the 4-methoxy cinnamic acid was unchanged
by the esterification with glycerol the polarity of the ester
product was significantly increased compared to the free
acid. This is indicated by the elution order obtained with
the C18 HPLC column. Also, as shown in a related work
this esterification reaction can proceed to form the diester
at longer reaction times [13]. However, the condensation of
glycerol with two molecules of 4-methoxy cinnamic acid to
produce such a diester results in a product that is less polar
than the free acid. The objective to obtain a more hydro-
philic UV filter was satisfied by synthesis of the monoester
rather than the diester. This was achieved at shorter reac-
tion times to avoid the sequential reaction of monoester to
diester [13].
1H NMR
7.7 (d, J = 16.0 Hz, 1H), 7.5 (d, J = 8.8 Hz, 2H), 7.0
(d, J = 8.9 Hz, 2H), 6.4 (d, J = 16.0, 1H), 4.3
(dd, J = 4.2, 11.4, 2H), 4.2 (m , 1H), 3.8 (s, 3H), 3.6
(d, J = 1.9, 2H).
13C NMR
168.0 C10, 162.0 C4, 145.3 C8, 130.0 C2, 130.0 C6, 127.1
C1, 114.7 C9, 114.3 C3, 114.3 C5, 70.1 C12, 65.5 C11,
62.9 C13, 54.8 C7.
The 4-methoxy cinnamoyl glycerol product (monoester)
was recovered from the reaction mixture by extraction with
deionized water followed by extraction of the resulting
aqueous phase with diethyl ether. The first extraction
separated the product from unreacted 4-methoxy cinnamic
acid and the second extraction separated the product from
unreacted glycerol. The product was obtained in greater
DEPT Experiments
Spectra were collected at 300.0 K with the Varian Mercury
Plus 400 Spectrometer using the 5-mm Mercury probe.
Samples were dissolved in CD3OD and spectra were
acquired at 400.15 MHz for 1H NMR and 100.63 MHz for
13C NMR. Samples were scanned 64 times.
HSQC Experiments
Spectra were collected at 300.0 K with the Varian Mercury
Plus 400 Spectrometer using the 5-mm Mercury probe.
Samples were dissolved in CD3OD and spectra were
acquired at 400.15 MHz for 1H NMR and 100.63 MHz for
13C NMR. Samples were scanned 64 times.
HMBC Experiments
Spectra were collected on the Varian Inova 500 Spec-
trometer equipped with the 5 mm gradient inverse probe
operated at a temperature of 298 K with a frequency of
499.80 Hz and 125.67 Hz for 1H NMR and 13C NMR,
respectively. Samples were dissolved in CD3OD and
scanned 16 times.
Fig. 1 Esterification of glycerol and 4-methoxy cinnamic acid to
form the monoester
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J Am Oil Chem Soc (2008) 85:347–351
The fragmentation pattern of such a derivatized compound
provides evidence of the location of the ester linkage.
Different ion fragments are generated depending on whe-
ther the ester formed at the primary or secondary hydroxyl
of glycerol. This approach has been applied to determine
the positional isomers for structures such as monoacyl-
glycerides, mono-alkyl glycerides, and in particular feru-
loyl glycerol [14, 15]. The latter compound is relevant
because of the structural similarity between ferulic acid, 4-
methoxy cinnamic acid, and the esters these compounds
form with glycerol. Mass spectra obtained by GC-MS
analysis of the TMS derivative of the isolated product, 4-
methoxy cinnamoyl glycerol, are presented in Fig. 3. The
molecular ion (M) is observed at m/z 396 with the char-
acteristic ion m/z 293 (m/z M-103) produced by cleavage
between carbons 12 and 13. This ion, m/z M-103, is
observed with 1-substituted glycerols but not 2-substituted
glycerols and provides evidence that the ester linkage
formed at the primary hydroxyl of glycerol. Other peak
assignments are shown for the ions generated by cleavage
at the bonds indicated in Fig. 3 and include m/z 161, 133,
179, and 191.
Fig. 2 Chromatogram of an equimolar reaction mixture showing the
elution of the product 4-methoxy cinnamoyl glycerol (1) before 4-
methoxy cinnamic acid (2). Separation obtained with methanol
mobile phase on a C18 column and detection by UV absorption
than 98% purity after evaporation of the ether. The yield of
product was nearly 20% after a two hour reaction with
complete selectivity for the monoester.
The 4-methoxy cinnamoyl glycerol product was further
characterized by FTIR, LC–MS, GC–MS, and NMR. The
infrared spectrum showed the presence of a broad band in
the 3,200–3,600 cm-1 region typical of the O–H stretching
mode of the hydroxyl group. This was attributed to the
unreacted hydroxyl groups of the glycerol portion of the
molecule following formation of the ester linkage. A signal
at 1,701 cm-1 indicated the presence of the ester function
shifted from the typical 1,740 cm-1 location by the double
bond conjugated with the aromatic ring structure. A pair of
strong sharp bands was observed at 1,172 and 1,116 cm-1
consistent with the C–O stretching mode.
The results of DEPT and HMBC experiments provided
additional evidence that the ester linkage formed between
the primary hydroxyl of glycerol and 4-methoxy cinnamic
acid. The DEPT spectra (not shown) displayed all carbons
directly bonded to hydrogens. The carbons comprising the
glycerol moiety (C11, C12, and C13) were of particular
interest and shown to have two protons attached to C11,
one proton attached to C12, and two protons attached to
C13. Moreover, C11 and C13 displayed different shifts and
were therefore not equivalent. If the ester had formed at the
C12 hydroxyl group then C11 and C13 would be equiva-
lent. The connectivity across multiple bonds was
investigated through HMBC experiments that examined
the long-range coupling between carbons and protons.
Figure 4 shows results obtained for such an experiment
optimized for an 8-Hz coupling. The relevant peaks from
Analysis by LC–MS identified the molecular ion of the
ester with m/z 252.8. The location of the ester linkage on
the glycerol moiety was determined from the mass spec-
trum of the corresponding trimethyl silyl derivative (TMS).
Fig. 3 Mass spectrum of TMS
derivatized 4-methoxy
cinnamoyl glycerol
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J Am Oil Chem Soc (2008) 85:347–351
351
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Fig. 4 HMBC spectrum of 4-methoxy cinnamoyl glycerol
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the sides of the corresponding axes. The cross peaks for
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the resonant carbon and proton nuclei, respectively. The
long range correlations obtained from this experiment,
especially between C10 and H11, show the connectivity
through the ester linkage and are consistent with a product
formed at the primary hydroxyl site of glycerol.
Acknowledgments The authors are grateful to Gregory P. Wylie,
University of Georgia, Department of Chemistry for performing the
NMR experiments.
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