Preparation of monolignol γ-acetate, γ-p-hydroxycinnamate, and γ-p-hydroxybenzoate conjugates: Selective deacylation of phenolic acetates with hydrazine acetate

Article abstract of DOI:10.1039/c3ra42818d

We report here a reliable and facile synthesis of a range of monolignol γ-p-hydroxycinnamate (including p-coumarate, ferulate, and caffeate), γ-acetate, and γ-p-hydroxybenzoate conjugates, many not previously reported, that are either putative intermediat

Full text of DOI:10.1039/c3ra42818d

RSC Advances
View Article Online
View Journal | View Issue
PAPER
Preparation of monolignol g-acetate, g-p-
hydroxycinnamate, and g-p-hydroxybenzoate
conjugates: selective deacylation of phenolic acetates
with hydrazine acetate†
Cite this: RSC Adv., 2013, 3, 21964
ad
Matthew Regner,ab Fachuang Lu,abc Hoon Kim,abc
*
Yimin Zhu,
Allison Mohammadi,b Timothy J. Pearsonb and John Ralphabc
We report here a reliable and facile synthesis of a range of monolignol g-p-hydroxycinnamate (including p-
coumarate, ferulate, and caffeate), g-acetate, and g-p-hydroxybenzoate conjugates, many not previously
reported, that are either putative intermediates in the biosynthesis of natural lignins or new monomer-
conjugates destined for upcoming designer lignins. The key was the development of a highly selective
deacylation approach for phenolic acetates; i.e., a method that cleaves phenolic acetates while leaving
the sensitive monolignol ester conjugates intact.
Received 7th June 2013
Accepted 17th September 2013
DOI: 10.1039/c3ra42818d
www.rsc.org/advances
The pathways by which monolignol conjugates are bio-
synthesized have started to become more evident only recently.
Introduction
Lignin, an essential polymer for land plants, contributes much
to the recalcitrance of processing lignocellulosic biomass in
various agricultural and industrial processes, such as ruminant
digestibility, conversion of lignocellulosics to biofuels, and pulp
and papermaking.1–4 This complicated biopolymer is generally
considered to derive primarily from the oxidative coupling of
three monolignols, the hydroxycinnamyl alcohols, p-coumaryl,
coniferyl, and sinapyl alcohols.1,5,6 Natural lignins as well as
lignins in transgenic plants and mutants deriving from ‘mono-
mers of incomplete methylation’, caffeyl and 5-hydroxyconiferyl
alcohols, have also been reported.1,7 Natural lignins are oen
found to be partially g-acylated by various groups.1 The acylation
level in natural lignins can be extremely high such as the ꢀ60%
found in kenaf,8 and ꢀ85% in abaca.9 Monolignol g-ester
conjugates, such as acetates 7, p-hydroxybenzoates 5a–c, and p-
coumarates 5d–f (Scheme 1), have long been implicated as
‘monomer’ precursors for these acylated lignins,8,10–16 and the
contention now has particularly compelling proof.10,16,17
Monolignol p-coumarates 5d–f are now implicated in
grasses,8,10–16 where a putative gene has been identied and the
enzymatic transferase activity demonstrated.18,19 The in planta
function of these acylations remains elusive, but will likely be
aided by the identication of transferase genes involved. Among
the conjugates, monolignol p-hydroxybenzoates 5a–c and
p-hydroxycinnamates 5d–f are particularly intriguing because
the phenolic groups in both the monolignol moiety and the
ester moiety are readily oxidized under lignication conditions,
e.g., by peroxidases-H2O2 or laccases-O2.16 For example, mono-
lignol p-coumarates have been hypothesized to act as radical
shuttles in grasses for efficient generation of sinapyl alcohol
and lignin syringyl unit radicals, which otherwise are produced
by peroxidases only slowly.16,20,21 In a related project, our group
has demonstrated that introducing monolignol ferulate conju-
gates 5h–j into lignication produces readily cleavable ester
linkages in the lignin backbone,16,22 providing promising
potential to facilitate lignin depolymerization in pulping or
biomass pretreatments to reduce energy requirements.
Access to a range of monolignol conjugates 5 and 7 is
therefore required for basic studies into their role in lignin
biosynthesis, and for bioengineering lignin for less recalcitrant
biomass. Synthetic strategies for monolignol p-coumarates have
been discussed,23,24 and some monolignol acetates and p-cou-
marates have previously been synthesized in our laboratories
and elsewhere.15,23–25 Specically, these monolignol conjugates
are synthesized by the coupling of phenol-protected mono-
lignols and appropriate (protected) hydroxycinnamoyl or
benzoyl chlorides. Due to the required regioselectivity (onto the
g-position of the monolignol, Scheme 1), the synthesis requires
aDOE Great Lakes Bioenergy Research Center, University of Wisconsin, 1552 University
Ave, Madison, WI, 53726, USA. Fax: +1 814 949 5700; Tel: +1 608 262 9234
bDepartment of Biochemistry, University of Wisconsin, 446 Babcock St, Madison, WI,
53706, USA
cWisconsin Energy Institute, University of Wisconsin, 1552 University Ave, Madison,
WI, 53726, USA
dDepartment of Chemistry, The Pennsylvania State University, Altoona College, 3000
Ivyside Park, Altoona, PA, 16601, USA. E-mail: yuz30@psu.edu; Fax: +1 814 949
5700; Tel: +1 814 949 5478
† Electronic supplementary information (ESI) available: The 1H and 13C NMR
spectra for all compounds. See DOI: 10.1039/c3ra42818d
21964 | RSC Adv., 2013, 3, 21964–21971
This journal is ª The Royal Society of Chemistry 2013

View Article Online
Paper
RSC Advances
Scheme 1 Synthesis of monolignol conjugates.
protecting the phenolic groups. The most successful protecting
groups for the synthesis are 2,4-dinitrophenyl ether or
acetate.15,23,24 Both groups require strong nucleophiles for
removal; obviously strong bases cannot be considered for
acetate removal because of the sensitive ester present in the
conjugates. These monolignol conjugates are also labile and
sensitive to both basic and acidic conditions. As a result, the
yield for a given compound, for example, coniferyl p-coumarate,
varied from low to good even within our laboratory. Some
monolignol conjugates, such as 5g from g-acylated caffeyl
alcohol, could not be prepared by the reported methods.
Here we report a facile and general synthesis of monolignol
g-acetates, g-p-hydroxybenzoates, and g-p-hydroxycinnamates.
The key to the synthesis was the development of mild and
highly selective deacylation (deacetylation) of phenolic acetates.
Results and discussion
Our preparation started from the synthesis of 4-O-acetyl-mono-
lignols 2 (Scheme 1). In the previous synthesis, 4-O-acetyl-coniferyl
and 4-O-acetyl-sinapyl alcohols, 2b and 2c, were prepared from
coniferaldehyde and sinapaldehyde.24 Unfortunately, p-coumar-
aldehyde and caffealdehyde, the corresponding starting materials
for 4-O-acetyl-p-coumaryl alcohol, 2a, and 3,4-di-O-acetyl-caffeyl
alcohol, 2d, are not commercially available. Although these alde-
hydes could be readily produced in two steps from their benzal-
dehydes, we developed a more straightforward approach to
synthesize the desired 4-O-acetylated hydroxycinnamyl alcohols,
2a–d. Peracetylated, commercially available, hydroxycinnamic
acids, 1a–d, were conveniently converted into mixed anhydrides
that, without purication, were reduced by sodium borohydride to
Scheme 2 A decomposition pathway for monolignol conjugates.
This journal is ª The Royal Society of Chemistry 2013
RSC Adv., 2013, 3, 21964–21971 | 21965

View Article Online
RSC Advances
Paper
yield the phenol-protected monolignols 2a–d. Coupling these 4-O- and g-p-hydroxycinnamates required in current research on
acetyl-monolignols with the corresponding acid chlorides 3 acylated lignins. Our protocol involves highly selective deace-
smoothly gave the desired protected conjugates 4 in good to tylation of phenolic acetates with hydrazine acetate. We found
excellent yields.
that this method is effective for preparing quite labile natural
The main challenge was the removal of the phenolic acetates; monolignol g-esters and their analogs to facilitate studies on
selective cleavage of phenolic esters in the presence of alkyl, and the biological processes in which these conjugates are involved.
particularly conjugated, esters is of considerable synthetic interest.
Several approaches have been reported and these methods show
modest to excellent selectivity in cleaving phenolic esters over
isolated alkyl esters.26–31 However, monolignol conjugates 5 pose
Experimental
General
an additional challenge: monolignol conjugates may be decom- Commercial chemicals, including solvents, were of reagent
posed through quinone methide intermediates (Scheme 2). In the grade or better and used without further purication unless
previous synthesis of coniferyl and sinapyl p-coumarates 5e and 5f, otherwise noted. All reactions were carried out in ame-dried
protecting group removal was achieved using neat pyrrolidine.24 glassware under an argon atmosphere, unless otherwise speci-
However, pyrrolidine is also known to cleave alkyl esters, albeit at a ed. Flash chromatography was performed using silica gel
lower rate.28 To study the stability of monolignol conjugates under (230–450 mesh). 1H and 13C nuclear magnetic resonance (NMR)
the deprotection conditions, selective deacylation of 4-O-p-nitro- spectra were obtained at 300 K at 500 MHz and 125 MHz,
benzoyl coniferyl propionate was monitored by NMR. Instead of respectively on a Bruker Biospin (Billerica, MA) Avance 500 MHz
using neat pyrrolidine, 5% pyrrolidine in d7-DMF was applied to spectrometer equipped with a cryogenically cooled 5 mm triple
slow down the reactions. The phenolic ester was completely resonance (TCI) gradient probe with inverse geometry (proton
cleaved in 6 minutes at room temperature but about 5% decom- coils closest to the sample). Chemical shis are reported in
position of the coniferyl propionate had already occurred by this parts per million (ppm). 1H NMR spectra were referenced to the
time. For more sensitive caffeyl alcohol esters, this deprotection central residual proton peak of the solvent [CDCl3, 7.26 ppm;
condition was far too harsh and no desired product was observed acetone-d6 2.05 ppm]. 13C NMR spectra were referenced to the
in the NMR experiment. Attempts to selectively deacetylate per- central carbon peak of the solvent [CDCl3, 77.16 ppm; acetone-
acetylated caffeyl p-coumarate, 4g, with other reported approaches d6 29.84 ppm] or the internal standard peak for spectra in
also failed to give the desired product, 5g.27,29–31
deuterated water [the methyl carbon peak of 3-(trimethylsilyl)-
We sought a new nucleophile that would selectively cleave propionic-2,2,3,3-d4-acid, sodium salt, 0 ppm]. Fully authenti-
phenolic esters in the presence of alkyl (and even conjugated) cated assignments were made by the usual complement of 1D
esters. As monolignol conjugates 5 and 7 are very sensitive to base, and 2D NMR methods.
the reagent of choice should maintain a proper balance between
Typical procedure for 4-acetoxycinnamyl alcohols 2. To a
nucleophilicity and basicity. We envisioned hydrazine acetate to solution of carboxylic acid 1c (1.6 g, 6.0 mmol) in EtOAc
be one such compound; whereas the basicity of hydrazine is (40.0 mL) were added Et3N (1.3 mL, 9.3 mmol) and ethyl
buffered via acetic acid, this reagent remains a good nucleophile. chloroformate (0.70 mL, 7.2 mmol) at 0 ꢁC. Aer stirring for 1 h
Hydrazine acetate has long been a reagent of choice for selective at 0 ꢁC, the mixture was ltered, rinsed with EtOAc (2 ꢂ 10 mL)
cleavage of glycosidic esters;32 however, it had not been reported and the residue was discarded. The ltrate was collected and
as a reagent for selective cleavage of phenolic esters.
cooled to 0 ꢁC, and DMF (0.5 mL) and NaBH4 (681 mg, 18.0
To our delight, the phenolic ester bond of 4-O-p-nitrobenzoyl mmol) were added to the solution. The mixture was stirred
coniferyl propionate was completely cleaved with 3 equivalents overnight and quenched with H2O (30 mL). The organic layer
of hydrazine acetate in d7-DMF in 20 min, as monitored by was washed with 1 M HCl (1 ꢂ 30 mL) and brine (1 ꢂ 30 mL).
NMR, and no decomposition was observed in the NMR tube The organic extract was dried over MgSO4 and concentrated in
reaction at room temperature for 24 h. We next tried to globally vacuo, and the residue was puried by column chromatography
deprotect the peracetylated monolignol conjugates, 4a–k, with (eluent: 40% ethyl acetate in n-hexanes) to yield compound 2c
1
excess hydrazine acetate. All the reactions were completed in (1.35 g, 89%) as a white solid: H NMR (CDCl3) d 6.64 (s, 2H,
less than one hour. Both thin-layer chromatography and NMR 2/5), 6.57 (d, J ¼ 15.8 Hz, 1H, a), 6.32 (dt, J ¼ 15.8, 5.7 Hz, 1H, b),
of the crude reaction products indicated no detectable side 4.33 (broad s, 2H, g), 3.83 (s, 6H, OMe), 2.34 (s, 3H, OAc); 13C
reactions. Monolignol conjugates were successfully obtained in NMR (CDCl3) d 169.0 (OAc), 152.3 (3/5), 135.3 (1), 131.0 (a),
good to excellent yields aer aqueous workup and column 129.1 (b), 128.4 (4), 103.2 (2/6), 63.7 (g), 56.3 (OMe), 20.6 (OAc);
chromatography. Caffeyl p-coumarate, 5g, which was inacces- HRMS (ESI) m/z calcd for C13H16O5 [M + NH4]+ ¼ 270.1336,
sible by the previously reported approaches, was obtained in found 270.1325.
97% yield. To the best of our knowledge, this is the rst
chemical synthesis of a caffeyl g-ester.
Compound 2a. 77%, white solid: 1H NMR (CDCl3) d 7.41–7.37
(m, 2H, 2/6), 7.06–7.03 (m, 2H, 3/5), 6.60 (d, J ¼ 15.9, 1H, a), 6.32
(dt, J ¼ 15.9, 5.7 Hz, 1H, b), 4.33 (td, J ¼ 5.9, 1.4 Hz, 2H, g), 2.30
(s, 3H, OAc), 1.45 (t, J ¼ 5.9, g-OH); 13C NMR (CDCl3) d 169.6
(OAc), 150.2 (4), 134.6 (1), 130.2 (a), 128.9 (b), 127.6 (2/6), 121.9
Conclusions
We have devised an effective and reproducible synthesis of the (3/5), 63.8 (g), 21.3 (OAc); HRMS (ESI) m/z calcd for C11O3H12
range of hydroxycinnamyl g-acetates, g-p-hydroxybenzoates, [M + NH4]+ ¼ 210.1125, found 210.1121.
21966 | RSC Adv., 2013, 3, 21964–21971
This journal is ª The Royal Society of Chemistry 2013

View Article Online
Paper
Compound 2b. 77%, colorless oil: 1H NMR (CDCl3) d 7.01–6.93
RSC Advances
1
Compound 4d. 51%, white solid: H NMR (CDCl3) d 7.70 (d,
(m, 3H, 2/5/6), 6.58 (d, J ¼ 15.9, 1H, a), 6.31 (dt, J ¼ 15.9, 5.7, 1H, J ¼ 16.0 Hz, 1H, 70), 7.57–7.53 (m, 2H, 20/60), 7.44–7.40 (m, 2H,
b), 4.32 (dd, J ¼ 5.7, 1.5 Hz, 2H, g), 3.84 (s, 3H, OMe), 2.31 (s, 3H, 2/6), 7.15–7.11 (m, 2H, 30/50), 7.08–7.04 (m, 2H, 3/5), 6.69 (d, J ¼
OAc); 13C NMR (CDCl3) d 169.3 (OAc), 151.2 (3), 139.4 (4), 135.9 15.9 Hz, 1H, a), 6.43 (d, J ¼ 16.0 Hz, 1H, 80), 6.31 (dt, J ¼ 15.9, 6.4
(1), 130.6 (a), 129.0 (b), 123.0 (5), 119.3 (6), 110.2 (2), 63.7 (g), Hz, 1H, b), 4.86 (dd, J ¼ 6.4, 1.3 Hz, 2H, g), 2.32 (s, 3H, OAc0),
56.0 (OMe), 20.8 (OAc); HRMS (ESI) m/z calcd for C12H14O4 [M + 2.30 (s, 3H, OAc); 13C NMR (CDCl3) d 169.6 (OAc), 169.3 (OAc0),
Na]+ ¼ 245.0785, found 245.0785.
166.7 (90), 152.3 (40), 150.5 (4), 144.2 (70), 134.2 (1), 133.4 (a),
Compound 2d. 41%, colorless oil: 1H NMR (CDCl3) d 7.24 (dd, 132.2 (10), 129.4 (20/60), 127.8 (2/6), 123.6 (b), 122.3 (30/50), 121.9
J ¼ 8.4, 2.0 Hz, 1H, 6), 7.19 (d, J ¼ 2.0 Hz, 1H, 2), 7.13 (d, J ¼ 8.4 Hz, (3/5), 118.1 (80), 65.2 (g), 21.3 (OAc/OAc0); HRMS (ESI) m/z calcd
1H, 5), 6.56 (d, J ¼ 15.9 Hz, 1H, a), 6.30 (dt, J ¼ 15.9, 5.5 Hz, 1H, b), for C22H20O6 [M + NH4]+ ¼ 398.1599, found 398.1606.
4.30 (ddd, J ¼ 5.8, 5.5, 1.2 Hz, 2H, g), 2.29 (s, 3H, OAc), 2.28 (s, 3H,
Compound 4e. 76%, white solid: 1H NMR (CDCl3) d 7.71 (d, J ¼
OAc), 1.57 (t, J ¼ 5.8, –OH); 13C NMR (CDCl3) d 168.5 (OAc), 168.4 16.0 Hz, 1H, 70), 7.57–7.53 (m, 2H, 20/60), 7.15–7.11 (m, 2H, 30/50),
(OAc), 142.3 (3), 141.5 (4), 135.9 (1), 130.0 (b), 129.3 (a), 124.8 (6), 7.01–6.98 (m, 3H, 2/5/6), 6.67 (d, J ¼ 15.9 Hz, 1H, a), 6.44 (d, J ¼
123.6 (2), 121.2 (5), 63.5 (g), 20.8 (2xOAc); HRMS (ESI) m/z calcd for 16.0 Hz, 1H, 80), 6.31 (dt, J ¼ 15.8, 6.4 Hz, 1H, b), 4.86 (dd, J ¼
C13H14O5 [M + NH4]+ ¼ 268.1180, found 268.1170.
6.4, 1.2 Hz, 2H, g), 3.85 (s, 3H, OMe), 2.31 (s, 6H, OAc/OAc0); 13C
Typical procedure for 4-acetoxycinnamyl 4-acetoxybenzoates NMR (CDCl3) d 169.3 (OAc0), 169.2 (OAc), 166.7 (90), 152.3 (40),
and 4-acetoxycinnamates 4. The acid chlorides 3a, 3b, 3d, and 151.3 (3), 144.2 (70), 139.7 (4), 135.4 (1), 133.8 (a), 132.2 (10),
3e were synthesized following the literature procedure33 and 129.4 (20/60), 123.7 (b), 123.0 (5), 122.3 (30/50), 119.6 (6), 118.1 (80),
were used directly in the following step without any further 110.3 (2), 65.2 (g), 56.0 (OMe), 21.3 (OAc0), 20.8 (OAc); HRMS
purication. To a solution of alcohol 2a (192 mg, 1.0 mmol) in (ESI) m/z calcd for C23H22O7 [M + Na]+ ¼ 433.1258, found
dichloromethane (10.0 mL) were added DMAP (31 mg, 433.1252.
0.25 mmol), Et3N (0.42 mL, 3.0 mmol), and acid chloride 3a (298
1
Compound 4f. 70%, white solid: H NMR (CDCl3) d 7.71 (d,
mg, 1.5 mmol) at 0 ꢁC. The mixture was stirred for 1.5 h at 0 ꢁC J ¼ 16.0 Hz, 1H, 70), 7.57–7.53 (m, 2H, 20/60), 7.15–7.11 (m, 2H,
and then warmed to room temperature. The mixture was 30/50), 6.65 (s, 2H, 2/6), 6.65 (d, J ¼ 15.8 Hz, 1H, a), 6.44 (d, J ¼
washed with 1 M HCl (1 ꢂ 10 mL), sat. NH4HCO3 (1 ꢂ 10 mL), 16.0 Hz, 1H, 80), 6.31 (dt, J ¼ 15.8, 6.4 Hz, 1H, b), 4.87 (dd, J ¼
and brine (1 ꢂ 10 mL), and the organic layer was dried over 6.4, 1.2 Hz, 2H, g), 3.84 (s, 6H, OMe), 2.34 (s, 3H, OAc), 2.31 (s,
MgSO4 and concentrated in vacuo. Purication by column 3H, OAc0); 13C NMR (CDCl3) d 169.3 (OAc0), 168.9 (OAc), 166.7
chromatography (eluent: 20% ethyl acetate in n-hexanes) yiel- (90), 152.32 (40), 152.28 (3/5), 144.2 (70), 134.8 (1), 134.2 (a),
ded 4a (256 mg, 72%) as a white solid: 1H NMR (CDCl3) d 8.13– 132.2 (10), 129.4 (20/60), 128.7 (4), 123.8 (b), 122.3 (30/50), 118.1
8.09 (m, 2H, 20/60), 7.44–7.40 (m, 2H, 2/6), 7.20–7.16 (m, 2H, 30/ (80), 103.4 (2/6), 65.1 (g), 56.3 (OMe), 21.3 (OAc0), 20.6 (OAc);
50), 7.08–7.04 (m, 2H, 3/5), 6.71 (d, J ¼ 15.9 Hz, 1H, a), 6.35 (dt, HRMS (ESI) m/z calcd for C24H24O8 [M + NH4]+ ¼ 458.1810,
J ¼ 15.9, 6.4 Hz, 1H, b), 4.97 (dd, J ¼ 6.4, 1.3 Hz, 2H, g), 2.32 (s, found 458.1810.
13
3H, OAc), 2.30 (s, 3H, OAc0); C NMR (CDCl3) d 169.6 (OAc0),
Compound 4g. 84%, white solid: 1H NMR (CDCl3) d 7.70 (d, J ¼
169.0 (OAc), 165.7 (CO), 154.5 (40), 150.6 (4), 134.1 (1), 133.5 (a), 16.0 Hz, 1H, 70), 7.58–7.53 (m, 2H, 20/60), 7.27 (dd, J ¼ 8.4, 2.0 Hz,
131.4 (20/60), 127.8 (10), 127.8 (2/6), 123.5 (b), 121.9 (3/5), 121.8 1H, 6), 7.24 (d, J ¼ 2.0 Hz, 1H, 2), 7.15 (d, J ¼ 8.4 Hz, 1H, 5), 7.14–
(30/50), 65.7 (g), 21.3 (OAc), 21.3 (OAc0); HRMS (ESI) m/z calcd for 7.10 (m, 2H, 30/50), 6.65 (d, J ¼ 15.9 Hz, 1H, a), 6.43 (d, J ¼ 16.0
C
20H18O6 [M + NH4]+ ¼ 372.1442, found 372.1458.
Hz, 1H, 80), 6.30 (dt, J ¼ 15.9, 6.3 Hz, 1H, b), 4.85 (dd, J ¼ 6.3, 1.2
Compound 4b. 74%, colorless oil: 1H NMR (CDCl3) d 8.14–8.09 Hz, 2H, g), 2.31 (s, 3H, OAc0), 2.30 (s, 3H, OAc), 2.29 (s, 3H, OAc);
(m, 2H, 20/60), 7.21–7.16 (m, 2H, 30/50), 7.02–6.97 (m, 3H, 2/5/6), 13C NMR (CDCl3) d 169.3 (OAc0), 168.4 (OAc), 168.4 (OAc), 166.7
6.70 (d, J ¼ 15.9 Hz, 1H, a), 6.34 (dt, J ¼ 15.9, 6.4 Hz, 1H, b), 4.97 (90), 152.3 (40), 144.2 (70), 142.3 (3), 141.8 (4), 135.4 (1), 132.5 (a),
(dd, J ¼ 6.4, 1.3 Hz, 2H, g), 3.85 (s, 3H, OMe), 2.32 (s, 3H, OAc), 132.2 (10), 129.4 (20/60), 125.0 (6), 124.8 (b), 123.7 (5), 122.3 (30/50),
2.31 (s, 3H, OAc0); 13C NMR (CDCl3) d 169.2 (OAc), 169.0 (OAc0), 121.4 (2), 118.1 (80), 64.9 (g), 21.3 (OAc0), 20.80 (OAc), 20.79
165.7 (CO), 154.5 (40), 151.3 (3), 139.8 (4), 135.4 (1), 133.8 (a), (OAc); HRMS (ESI) m/z calcd for C24H22O8 [M + NH4]+
¼
131.4 (20/60), 127.8 (10), 123.6 (b), 123.0 (5), 121.8 (30/50), 119.5 (6), 456.1653, found 456.1653.
1
110.3 (2), 65.6 (g), 56.0 (OMe), 21.3 (OAc), 20.8 (OAc0); HRMS
Compound 4h. 97%, white solid: H NMR (CDCl3) d 7.68 (d,
(ESI) m/z calcd for C21H20O7 [M + NH4]+ ¼ 402.1548, found J ¼ 16.0 Hz, 1H, 70), 7.44–7.40 (m, 2H, 2/6), 7.13 (dd, J ¼ 8.1, 1.8
402.1533.
Hz, 1H, 60), 7.11 (d, J ¼ 1.7 Hz, 1H, 20), 7.08–7.04 (m, 2H, 3/5),
Compound 4c. 50%, colorless oil: 1H NMR (CDCl3) d 8.14–8.10 7.06 (d, J ¼ 8.1 Hz, 1H, 50), 6.69 (d, J ¼ 15.9 Hz, 1H, a), 6.43 (d,
(m, 2H, 20/60), 7.21–7.17 (m, 2H, 30/50), 6.70 (d, J ¼ 15.9 Hz, 1H, J ¼ 16.0 Hz, 1H, 80), 6.31 (dt, J ¼ 15.9, 6.4 Hz, 1H, b), 4.86 (dd, J ¼
a), 6.68 (s, 2H, 2/6), 6.34 (dt, J ¼ 15.9, 6.3 Hz, 1H, b), 4.98 (dd, J ¼ 6.4, 1.2 Hz, 2H, g), 3.86 (s, 3H, OMe0), 2.33 (s, 3H, OAc0), 2.30 (s,
13
6.4, 1.2 Hz, 2H, g), 3.84 (s, 6H, OMe), 2.34 (s, 3H, OAc), 2.33 (s, 3H, OAc); C NMR (CDCl3) d 169.6 (OAc), 168.9 (OAc0), 166.7
13
3H, OAc0); C NMR (CDCl3) d 169.0 (OAc0), 168.9 (OAc), 165.7 (90), 151.5 (30), 150.5 (4), 144.6 (70), 141.6 (40), 134.2 (1), 133.5
(CO), 154.5 (40), 152.3 (3/5), 134.7 (1), 134.2 (a), 131.4 (20/60), (a/10), 127.8 (2/6), 123.6 (b), 123.4 (50), 121.9 (3/5), 121.4 (60),
128.7 (4), 127.8 (10), 123.7 (b), 121.8 (30/50), 103.4 (2/6), 65.5 (g), 118.2 (80), 111.4 (20), 65.3 (g), 56.0 (OMe0), 21.3 (OAc), 20.8
56.3 (OMe), 21.3 (OAc), 20.6 (OAc0); HRMS (ESI) m/z calcd for (OAc0); HRMS (ESI) m/z calcd for C23H22O7 [M + NH4]+
¼
C
22H22O8 [M + NH4]+ ¼ 432.1653, found 432.1666.
428.1704, found 428.1713.
This journal is ª The Royal Society of Chemistry 2013
RSC Adv., 2013, 3, 21964–21971 | 21967

View Article Online
RSC Advances
Paper
21968 | RSC Adv., 2013, 3, 21964–21971
This journal is ª The Royal Society of Chemistry 2013

View Article Online
Paper
RSC Advances
Compound 4i. 67%, white solid: 1H NMR (CDCl3) d 7.69 (d, J ¼
(OAc), 166.4 (90), 150.5 (4), 143.7 (40), 143.3 (70), 142.6 (30), 134.1
(1), 133.4 (10), 133.4 (a), 127.8 (2/6), 126.6 (60), 124.1 (50), 123.5
(b), 122.9 (20), 121.9 (3/5), 119.2 (80), 65.3 (g), 21.3 (OAc0), 20.8
(OAc), 20.7 (OAc0); HRMS (ESI) m/z calcd for C24H22O8 [M +
NH4]+ ¼ 456.1653, found 456.1644.
16.0 Hz, 1H, 70), 7.13 (dd, J ¼ 8.2, 1.8 Hz, 1H, 60), 7.11 (d, J ¼ 1.8
Hz, 1H, 20), 7.06 (d, J ¼ 8.1 Hz, 1H, 50), 7.02–6.97 (m, 3H, 2/5/6),
6.67 (d, J ¼ 15.9 Hz, 1H, a), 6.43 (d, J ¼ 16.0 Hz, 1H, 80), 6.31 (dt,
J ¼ 15.9, 6.4 Hz, 1H, b), 4.87 (dd, J ¼ 6.5, 1.2 Hz, 2H, g), 3.86 (s,
3H, OMe0), 3.85 (s, 3H, OMe), 2.33 (s, 3H, OAc0), 2.32 (s, 3H,
Typical procedure for 4-acetoxycinnamyl acetates 6. To a
solution of alcohol 2a (192 mg, 1.0 mmol) in pyridine (2 mL)
were added DMAP (12 mg, 0.10 mmol) and acetic anhydride
(0.28 mL, 3.0 mmol) at room temperature. The mixture was
stirred for 24 h, diluted with EtOAc, and washed with 1 M HCl
(3 ꢂ 5 mL) and brine (1 ꢂ 10 mL). The organic layer was dried
over MgSO4 and concentrated in vacuo to a white solid,
which, aer purication by column chromatography (eluent:
15% ethyl acetate in n-hexanes), afforded 6a (215 mg, 92%)
as a white solid: 1H NMR (CDCl3) d 7.41–7.38 (m, 2H, 2/6),
7.07–7.03 (m, 2H, 3/5), 6.63 (d, J ¼ 15.9 Hz, 1H, a), 6.24 (dt, J ¼
15.9, 6.4 Hz, 1H, b), 4.72 (dd, J ¼ 6.5, 1.3 Hz, 2H, g), 2.30 (s,
13
OAc); C NMR (CDCl3) d 169.2 (OAc), 169.0 (OAc0), 166.7 (90),
151.5 (30), 151.3 (3), 144.6 (70), 141.6 (40), 139.8 (4), 135.4 (1),
133.8 (a), 133.4 (10), 123.7 (b), 123.4 (50), 123.0 (5), 121.4 (60),
119.6 (6), 118.2 (80), 111.4 (20), 110.3 (2), 65.2 (g), 56.05 (OMe0),
56.01 (OMe), 20.84 (OAc), 20.82 (OAc0); HRMS (ESI) m/z calcd for
C
24H24O8 [M + NH4]+ ¼ 458.1810, found 458.1811.
Compound 4j. 91%, white solid: 1H NMR (CDCl3) d 7.69 (d, J ¼
16.0 Hz, 1H, 70), 7.13 (dd, J ¼ 8.1, 1.8 Hz, 1H, 60), 7.11 (d, J ¼ 1.8
Hz, 1H, 20), 7.06 (d, J ¼ 8.1 Hz, 1H, 50), 6.66 (s, 2H, 2/6), 6.65 (d,
J ¼ 15.8 Hz, 1H, a), 6.43 (d, J ¼ 16.0 Hz, 1H, 80), 6.31 (dt, J ¼ 15.8,
6.4 Hz, 1H, b), 4.87 (dd, J ¼ 6.4, 1.1 Hz, 2H, g), 3.87 (s, 3H,
OMe0), 3.84 (s, 6H, OMe), 2.34 (s, 3H, OAc0), 2.33 (s, 3H, OAc);
13C NMR (CDCl3) d 169.0 (OAc), 168.9 (OAc0), 166.7 (90), 152.3
(3/5), 151.5 (30), 144.6 (70), 141.6 (40), 134.8 (1), 134.2 (a), 133.4
(10), 128.7 (4), 123.8 (b), 123.4 (50), 121.4 (60), 118.1 (80), 111.4 (20),
103.4 (2/6), 65.1 (g), 56.3 (7), 56.0 (70), 20.8 (OAc0), 20.6 (OAc);
HRMS (ESI) m/z calcd for C25H26O9 [M + NH4]+ ¼ 488.1916,
found 488.1916.
13
3H, OAc), 2.10 (s, 3H, OAc0); C NMR (CDCl3) d 171.0 (OAc0),
169.6 (OAc), 150.5 (4), 134.1 (1), 133.3 (a), 127.7 (2/6), 123.6
(b), 121.9 (3/5), 65.1 (g), 21.3 (OAc), 21.2 (OAc0); HRMS (ESI)
m/z calcd for C13H14O4 [M + NH4]+ ¼ 252.1231, found
252.1224.
1
Compound 6b. 92%, colorless oil: H NMR (CDCl3) d 6.99 (d,
J ¼ 8.1 Hz, 1H, 5), 6.98 (d, J ¼ 1.8 Hz, 1H, 2), 6.96 (dd, J ¼ 8.2, 1.7
Hz, 1H, 6), 6.62 (d, J ¼ 15.9 Hz, 1H, a), 6.24 (dt, J ¼ 15.9, 6.5 Hz,
1H, b), 4.72 (dd, J ¼ 6.5, 1.3 Hz, 2H, g); 3.85 (s, 3H, OMe), 2.31 (s,
1
Compound 4k. 67%, white solid: H NMR (CDCl3) d 7.66 (d,
J ¼ 16.0 Hz, 1H, 70), 7.44–7.40 (m, 2H, 2/6), 7.41 (d, J ¼ 8.7 Hz,
1H, 60), 7.37 (d, J ¼ 2.1 Hz, 1H, 20), 7.23 (d, J ¼ 8.4 Hz, 1H, 50),
7.08–7.04 (m, 2H, 3/5), 6.68 (d, J ¼ 15.9 Hz, 1H, a), 6.42 (d, J ¼
16.0 Hz, 1H, 80), 6.30 (dt, J ¼ 15.9, 6.4 Hz, 1H, b), 4.86 (dd, J ¼
6.4, 1.2 Hz, 2H, g), 2.31 (s, 3H, OAc), 2.30 (s, 3H, OAc0), 2.29 (s,
13
3H, OAc), 2.10 (s, 3H, OAc0); C NMR (CDCl3) d 171.0 (OAc0),
169.2 (OAc), 151.2 (3), 139.7 (4), 135.4 (1), 133.7 (a), 123.7 (b),
123.0 (5), 119.5 (6), 110.3 (2), 65.1 (g), 56.0 (OMe), 21.2 (OAc0),
20.8 (OAc); HRMS (ESI) m/z calcd for C14H16O5 [M + NH4]+ ¼
282.1336, found 282.1340.
13
3H, OAc0); C NMR (CDCl3) d 169.6 (OAc0), 168.2 (OAc0), 168.1
Table 2 13C NMR chemical shift data for conjugates 5 and 7 in acetone-d6
Chemical shis (ppm)
Caffeate
p-Hydroxybenzoates
p-Coumarates
Ferulates
Acetates
Carbon
5a
5b
5c
5d
5e
5f
5g
5h
5i
5j
5k
7a
7b
7c
1
2
3
4
5
6
129.0
128.9
116.3
158.4
116.3
128.9
—
134.7
121.5
65.9
122.6
132.5
116.0
162.6
116.0
132.5
—
129.4
110.1
148.5
147.8
115.8
121.3
56.2
135.0
121.7
65.9
122.6
132.5
116.0
162.6
116.0
132.5
—
128.2
105.1
148.8
137.2
148.8
105.1
56.6
135.2
122.0
65.8
122.6
132.5
116.0
162.6
116.0
132.5
—
129.0
128.8
116.3
158.4
116.3
128.8
—
134.6
121.5
65.6
127.0
131.0
116.7
160.6
116.7
131.0
—
145.5
115.5
167.2
—
129.4
110.1
148.5
147.8
115.8
121.3
56.2
134.9
121.8
65.5
127.0
131.0
116.7
160.6
116.7
131.0
—
145.5
115.5
167.2
—
128.2
105.1
148.8
137.1
148.8
105.1
56.6
135.2
122.0
65.5
127.0
131.0
116.7
160.6
116.7
131.0
—
145.5
115.5
167.2
—
129.7
113.9
146.0
146.3
116.1
120.0
—
134.9
121.5
65.5
127.0
131.0
116.6
160.6
116.6
131.0
—
145.5
115.5
167.2
—
129.0
128.8
116.3
158.4
116.3
128.8
—
134.7
121.5
65.5
127.4
111.3
148.7
150.0
116.0
124.0
56.3
145.8
115.8
167.2
—
129.4
110.1
148.5
147.8
115.8
121.2
56.2
135.0
121.8
65.5
127.4
111.3
148.7
150.1
116.0
124.0
56.3
145.8
115.8
167.2
—
128.2
105.1
148.8
137.2
148.8
105.1
56.6
135.2
122.0
65.4
127.4
111.3
148.7
150.1
116.0
124.0
56.3
145.8
115.7
167.2
—
129.0
128.8
116.3
158.4
116.3
128.8
—
134.6
121.5
65.5
127.6
115.2
146.3
148.8
116.3
122.6
—
145.8
115.5
167.2
—
128.9
128.8
116.3
158.4
116.3
128.8
—
134.6
121.3
65.6
—
—
—
—
—
—
—
—
—
129.4
110.1
148.5
147.8
115.8
121.2
56.2
134.9
121.5
65.6
—
—
—
—
—
—
—
—
—
128.1
105.1
148.8
137.1
148.8
105.1
56.6
135.2
121.8
65.6
—
—
—
—
—
—
—
—
—
OMe
a
b
g
10
20
30
40
50
60
OMe0
70
—
—
166.3
—
—
—
166.3
—
—
—
166.3
—
80
CO
CH3CO
170.8
20.8
170.8
20.8
170.8
20.8
This journal is ª The Royal Society of Chemistry 2013
RSC Adv., 2013, 3, 21964–21971 | 21969

View Article Online
RSC Advances
Compound 6c. 82%, colorless oil: H NMR (CDCl3) d 6.63 (s,
Paper
Compound 7b. 95%, light yellow solid: 1H and 13C NMR
1
2H, 2/6), 6.59 (d, J ¼ 15.8 Hz, 1H, a), 6.24 (dt, J ¼ 15.8, 6.5 Hz, b), (acetone-d6), see Tables 1 and 2, consistent with the previously
4.72 (dd, J ¼ 6.5, 1.3 Hz, 2H, g), 3.83 (s, 6H, OMe), 2.33 (s, 3H, reported data;35 HRMS (ESI) m/z calcd for C12H14O4 [M ꢃ H]ꢃ
¼
13
OAc), 2.11 (s, 3H, OAc0); C NMR (CDCl3) d 171.0 (OAc0), 168.9 221.0819, found 221.0824.
(OAc), 152.3 (3/5), 134.8 (1), 134.1 (a), 128.7 (4), 123.8 (b), 103.4
Compound 7c. 83%, colorless solid: 1H and 13C NMR (acetone-
(2/6), 65.0 (g), 56.3 (OMe), 21.2 (OAc0), 20.6 (OAc); HRMS (ESI) m/ d6), see Tables 1 and 2, consistent with the previously reported
z calcd for C15H18O6 [M + NH4]+ ¼ 312.1442, found 312.1451.
data;35 HRMS (ESI) m/z calcd for C13H16O5 [M ꢃ H]ꢃ ¼ 251.0924,
Typical procedure for removal of phenolic acetate protecting found 251.0929.
group(s). To a solution of conjugate 4a (248 mg, 0.70 mmol) in
DMF (3 mL) was added hydrazine acetate (290 mg, 3.15 mmol) at
Acknowledgements
room temperature. The solution was stirred for 1 h at room
temperature, diluted with EtOAc, and washed with H2O (4 ꢂ 5 mL)
and brine (1 ꢂ 10 mL). The organic layer was then dried over
MgSO4 and concentrated in vacuo to yield an organic residue that,
aer purication by column chromatography (eluent: 40% ethyl
acetate in n-hexanes), afforded 5a (159 mg, 84%) as a white solid:
1H and 13C NMR (acetone-d6), see Tables 1 and 2; HRMS (ESI) m/z
calcd for C16H14O4 [M ꢃ H]ꢃ ¼ 269.0819, found 269.0812.
We are grateful for funding from US Department of Energy, the
Office of Science (DE-SC0006930), and to partial funding for
aspects of this work from the DOE Great Lakes Bioenergy
Research Center (DOE Office of Science BER DE-FC02-
07ER64494. We thank Professor Ronald Raines for insightful
discussions on the stability of these conjugates.
Compound 5b. 80%, light yellow solid: 1H and 13C NMR
(acetone-d6), see Tables 1 and 2, consistent with the previously
Notes and references
reported data;23 HRMS (ESI) m/z calcd for C17H16O5 [M ꢃ H]ꢃ
¼
1 J. Ralph, K. Lundquist, G. Brunow, F. Lu, H. Kim,
P. F. Schatz, J. M. Marita, R. D. Hateld, S. A. Ralph,
J. H. Christensen and W. Boerjan, Phytochem. Rev., 2004, 3,
29–60.
2 R. Vanholme, K. Morreel, J. Ralph and W. Boerjan, Curr.
Opin. Plant Biol., 2008, 11, 278–285.
299.0924, found 299.0925.
1
Compound 5c. 75%, white solid: H and 13C NMR (acetone-
d6), see Tables 1 and 2; HRMS (ESI) m/z calcd for C18H18O6
[MꢃH]ꢃ ¼ 329.1030, found 329.1028.
1
Compound 5d. 89%, white solid: H and 13C NMR (acetone-
d6), see Tables 1 and 2, consistent with the previously reported
data;34 HRMS (ESI) m/z calcd for C18H16O4 [MꢃH]ꢃ ¼ 295.0975,
found 295.0970.
3 X. Li, J. K. Weng and C. Chapple, Plant J., 2008, 54, 569–581.
4 F. Chen and R. A. Dixon, Nat. Biotechnol., 2007, 25, 759–761.
5 R. Vanholme, K. Morreel, J. Ralph and W. Boerjan, Plant
Physiol., 2010, 153, 895–905.
6 K. Freudenberg and A. C. Neish, Constitution and
Biosynthesis of Lignin, Springer-Verlag, Berlin-Heidelberg-
New York, 1968.
7 F. Chen, Y. Tobimatsu, D. Havkin-Frenkel, R. A. Dixon and
J. Ralph, Proc. Natl. Acad. Sci. U. S. A., 2012, 109, 1772–1777.
8 J. Ralph, J. Nat. Prod., 1996, 59, 341–342.
9 A. T. Martinez, J. Rencoret, G. Marques, A. Gutierrez,
D. Ibarra, J. Jimenez-Barbero and J. C. del Rio,
Phytochemistry, 2008, 69, 2831–2843.
Compound 5e. 83%, pale yellow oil: 1H and 13C NMR (acetone-
d6), see Tables 1 and 2, consistent with the previously reported
data;24 HRMS (ESI) m/z calcd for C19H18O5 [M ꢃ H]ꢃ ¼ 325.1081,
found 325.1091.
Compound 5f. 74%, light yellow solid: 1H and 13C NMR
(acetone-d6), see Tables 1 and 2, consistent with the previously
reported data;24 HRMS (ESI) m/z calcd for C20H20O6 [M ꢃ H]ꢃ
¼
355.1187, found 355.1184.
Compound 5g. 97%, light yellow solid: 1H and 13C NMR
(acetone-d6), see Tables 1 and 2; HRMS (ESI) m/z calcd for
C
18H16O5 [M ꢃ H]ꢃ ¼ 311.0924, found 311.0918.
10 F. Lu and J. Ralph, Chem. Commun., 2002, 90–91.
11 D. C. C. Smith, J. Chem. Soc., 1955, 2347.
1
Compound 5h. 76%, white solid: H and 13C NMR (acetone-
d6), see Tables 1 and 2; HRMS (ESI) m/z calcd for C19H18O5
[M ꢃ H]ꢃ ¼ 325.1081, found 325.1082.
12 F. Lu and J. Ralph, Plant J., 2003, 35, 535–544.
13 M. Shimada, T. Fukuzuka and T. Higuchi, Tappi, 1971, 54,
72–78.
14 Y. Nakamura and T. Higuchi, Holzforschung, 1976, 30, 187–
191.
15 J. Ralph, R. D. Hateld, S. Quideau, R. F. Helm, J. H. Grabber
and H.-J. G. Jung, J. Am. Chem. Soc., 1994, 116, 9448–9456.
16 J. Ralph, Phytochem. Rev., 2010, 9, 65–83.
17 F. Lu and J. Ralph, Org. Biomol. Chem., 2008, 6, 3681–3694.
18 R. D. Hateld, J. M. Marita, K. Frost, J. H. Grabber, F. Lu,
H. Kim and J. Ralph, Planta, 2009, 229, 1253–1267.
19 S. Withers, F. Lu, H. Kim, Y. Zhu, J. Ralph and
C. G. Wilkerson, J. Biol. Chem., 2012, 287, 8347–8355.
20 U. Takahama, T. Oniki and H. Shimokawa, Plant Cell
Physiol., 1996, 37, 499–504.
Compound 5i. 94%, yellow oil: 1H and 13C NMR (acetone-d6),
see Tables 1 and 2; HRMS (ESI) m/z calcd for C20H20O6
[M ꢃ H]ꢃ ¼ 355.1187, found 355.1172.
1
Compound 5j. 89%, yellow solid: H and 13C NMR (acetone-
d6), see Tables 1 and 2; HRMS (ESI) m/z calcd for C21H22O7
[M ꢃ H]ꢃ ¼ 385.1292, found 385.1288.
1
Compound 5k. 95%, white solid: H and 13C NMR (acetone-
d6), see Tables 1 and 2; HRMS (ESI) m/z calcd for C18H16O5 [M ꢃ
H]ꢃ ¼ 311.0924, found 311.0932.
1
Compound 7a. 98%, yellow solid: H and 13C NMR (acetone-
d6), see Tables 1 and 2, consistent with the previously reported
data;34 HRMS (ESI) m/z calcd for C11H12O3 [M ꢃ H]ꢃ ¼ 191.0713,
found 191.0711.
21970 | RSC Adv., 2013, 3, 21964–21971
This journal is ª The Royal Society of Chemistry 2013

View Article Online
Paper
RSC Advances
21 R. D. Hateld, J. Ralph and J. H. Grabber, Planta, 2008, 228, 29 A. G. Gonzalez, Z. D. Jorge, H. L. Dorta and F. R. Luis,
919–928. Tetrahedron Lett., 1981, 22, 335–336.
22 J. H. Grabber, R. D. Hateld, F. Lu and J. Ralph, 30 B. Das, J. Banerjee, R. Ramu, R. Pal, N. Ravindranath and
Biomacromolecules, 2008, 9, 2510–2516. C. Ramesh, Tetrahedron Lett., 2003, 44, 5465–5468.
23 Y. Nakamura and T. Higuchi, Cellul. Chem. Technol., 1978, 31 C. Ramesh, G. Mahender, N. Ravindranath and B. Das,
12, 199–208. Tetrahedron, 2003, 59, 1049–1054.
24 F. Lu and J. Ralph, J. Agric. Food Chem., 1998, 46, 2911–2913. 32 G. Excoffier, D. Gagnare and J. P. Utille, Carbohydr. Res.,
25 J. H. Grabber, S. Quideau and J. Ralph, Phytochemistry, 1996,
43, 1189–1194.
26 C. Ramesh, G. Mahender, N. Ravindranath and B. Das, Green
Chem., 2003, 5, 68–70.
27 G. Blay, M. Luz Cardona, M. Begona Garcia and J. R. Pedro,
Synthesis, 1989, 438–439.
1975, 39, 368–373.
33 B. Burja, M. Kocevar and S. Polanc, Tetrahedron, 2009, 65,
8690–8696.
34 D. G. Vassao, D. R. Gang, T. Koeduka, B. Jackson,
E. Pichersky, L. B. Davin and N. G. Lewis, Org. Biomol.
Chem., 2006, 4, 2733–2744.
28 P. Mansson, Tetrahedron Lett., 1982, 23, 1845–1846.
35 A. Zanarotti, J. Org. Chem., 1985, 50, 941–945.
This journal is ª The Royal Society of Chemistry 2013
RSC Adv., 2013, 3, 21964–21971 | 21971