Using fluorogenic probes for the investigation of selective biomass degradation by fungi

Article abstract of DOI:10.1039/c4gc01659a

A library of fifteen commercially purchased and synthetic fluorogenic probes was employed for the investigation of biomass degradation using extracts of white-rot fungi. These probes were selected or designed to mimic the dominant linkages in celluloses, hemicelluloses, and lignin, the three most abundant polymers found in biomass. The results show that white-rot fungi display a high preference for cleaving mannose- and glucose-based probes, which mimic hemicelluloses. Low degrees of cleavages were noted for xylose- and cellobiose-based probes. No cleavages were observed for probes that mimic the linkages in lignin. Overall, these discoveries prove that it is possible to employ fungi for selective degradation or release of hemicelluloses from biomass.

Full text of DOI:10.1039/c4gc01659a

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ARTICLE TYPE
Using Fluorogenic Probes for the Investigation of Selective Biomass Degradation by Fungi
†
†
‡
‡
†
Qian Zhang, Xinrui Peng, Michelle Grilley, Jon Y. Takemoto, and ChengꢀWei Tom Chang ,*
Department of Chemistry and Biochemistry Utah State University
5
Received (in XXX, XXX) Xth XXXXXXXXX 20XX, Accepted Xth XXXXXXXXX 20XX
DOI: 10.1039/b000000x
OH
HO
O
O
OH
HO
O
O
HO
O
O
A library of fifteen commercially purchased and synthetic
fluorogenic probes were employed for the investigation of
biomass degradation using extract of whiteꢀrot fungi. These
probes were selected or designed to mimic the dominant
linkages in celluloses, hemicelluloses and lignin, the three
most abundant polymers found in biomass. The results show
that whiteꢀrot fungi display a high preference for cleaving
mannoseꢀ and glucoseꢀbased probes, which mimic
hemicelluloses. Low degree of cleavages were noted for
xyloseꢀ and cellobioseꢀbased probes. No cleavages were
observed for probes that mimic the linkages in lignin.
Overall, these discoveries prove that it is possible to employ
fungi for selective degradation or release of hemicelluloses
from biomass.
OH
O
n
Xylan
R
O
MeO
HO
-
R = CH2OH orCO2
O
O
HO
O
1
1
2
0
5
0
O
OH
HO
O
O
OAc
HO
O
MeO
HO
O
OH
O
O
R'O
O
O
R' = Ac,
or
OH
OH
O
n
HO
OH
Arabinoxylan
OH
OH
O
HO
O
O
HO
HO
O
O
OH
OH
O
OH
OH
n
Mannan
OR
OH
OH
HO
HO
O
OH
O
O
O
HO
HO
O
O
O
O
R = H or HO
HO
OH
Glucomannan
n
OH
HO
Figure 2. Representative structure of hemicellulose
is an ongoing effort in searching for "green" processes for
CH OH
2
Introduction
CH
2
CH
HC
CH
2
OH
CH
2
Celluloses, hemicelluloses (commonly present in forms, such
as xylan, arabinoxylan, mannan and glucomannan) and lignin
are the three most abundant polymers found in biomass
(Figures 1ꢀ3). Nonꢀedible biomass is a superior source for
CH
CH
2
OH
HO
HO
2
CH
3
CH
H
3
CO
CH
HC
2
3
3
4
5
0
5
0
OH
OH
CH
HC
CH
2
OH
OCH3
nd
alternative energy in the category of 2 generation biofuels.
OCH
3
O
CH
CH
CH
2
OH
Unlike solar or wind energy, the supply of feedstock biomass
is immense and low cost. Using fungi for biodegradation of
biomass and release of carbohydrates is an effective and
environmentally friendly way of providing feedstock for
biofuel production. However, for many years, the main issue
for utilizing nonꢀedible biomass has been the difficulties in
developing lowꢀcost and continuous separation methods for
extracting fermentable carbohydrates (cellulose or
O
H
3
CO
2
O
CH
HC
CHOH
2
OH
OCH3
O
CH
CH
2
OH
3
H CO
O
2
CH OH
CHOH
HC
CHO
O
OCH
3
O
3
H CO
1
hemicellulose) from the biomass. The common practices for
OH
removing hemicellulose from biomass, especially those
employed by the paperꢀmaking industry during wood pulping,
involve the uses of caustic chemicals (acids, alkaline,
ammonia, ozone) and/or harsh mechanical conditions
(pyrolysis, steam explosion). These practices have the
shortcoming of producing chemical wastes that require further
HOHC
CH
CH
Figure 3. Representative structure of lignin
2
OH
2
hemicellulose isolation, and microbes for costꢀeffective
3
4
5
celluloseꢀbased biofuel production.
treatments or immense energy investments. As a result, there
OH
OH
OH
O
OH
O
HO
HO
O
O
O
HO
HO
O
O
OH
O
Due to the capability of fast rotting of wood, fungi have been the
focus for biodegradation of biomass. This process can be free of
OH
OH
OH
50
Figure 1. Representative structure of cellulose
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3a,4
hazardous chemicals and requires little energy investment.
also differentiate the possible mechanisms that are involved in the
Extensive investigations have been devoted to the search for
fungi, studies of fungal enzymes and the genetic modification of
4
c
microbes. Nevertheless, little progress has been noted. The
major challenges are the complexity of enzymes employed by
various fungi: not all the targeted fungi can degrade wood with
satisfactory efficiency. Another obstacle is that the degradation of
lignin will generate phenols or quinones, which are toxic to the
microbes, commonly yeasts, that will be used for the
5
0
5
0
5
0
5
5
1
1
2
2
3
3
fermentation of the carbohydrates produced from biomass. Over
degradation of lignin will also lead to the formation of excess tar
6
during the gasification of biomass. Therefore, chemical
treatments are required to remove these phenols or quinones
which further complicates the process of ethanol or biofuel
production. We believe that an ideal solution is to identify strains
of fungi that can selectively degrade or release hemicellulose
from biomass without significant degradation of lignin.
breakage of polymers in biomass. All of the examined probes are
shown in Figure 6.
Rationale of Designs
Class I
Hemicelluloses are thought to link to lignin via covalent bonds.
Although the exact linkages have not been fully characterized,
they most likely involve anomeric carbon in the form of
HO
HO
HO
O
O
O
HO
O
HO
O
O
OH
OH
Probe 18
glycosidic bond or hydroxy groups in the form of ether bond
7
(
Figure 4). For achieving the goal of selective degradation or
Class IIa
releasing hemicellulose, a library of synthetic and commerciallyꢀ
available fluorogenic probes were screened to show their
usefulness in identifying and characterizing such linkages. These
probes were designed to contains linkages that mimic those found
in biomass, including: a) the first type (class I) with a
chromophore attached to the anomeric position of cellobiose to
mimic cellulose, b) the second type with chromophores attached
to the anomeric position of xylose, mannose and glucose (class
IIa) to mimic the anomeric linkages between hemicelluloses or
with chromophores attached to the hydroxy group of
mannopyranose and xylofuranose to mimic the ether linkages
between hemicelluloses and lignin (class IIb), and c) the third
HO
O
HO
O
HO
HO
O
O
O
O
HO
HO
O
HO
O
O
HO
O
O
HO
OH
O
OH
Probe 7
Probe 9
Probe 8
HO
O
HO
HO
HO
HO
HO
O
OMe
O
HO
O
OH
HO
HO
OMe
O
O
Probe 14
OH
Probe 19
Probe 16
O
O
Class IIb
HO
O
O
O
O
O
O
HO
O
O
O
HO
OH
MeO
HO
O
HO
OMe
HO
HO
Probe 1
Probe 3
OMe
OH
OH
Probe 4
O
OH
ether bond
MeO
O
lignin
O
OH
HO
O
OH
O
HO
Probe 5
O
HO
lignin
O
O
O
OH
O
Class III
OMe
glycosidic bond
O
O
Figure 4. Possible linkages between hemicelluloses and lignin
O
O
O
O
type (class III) that are designed to mimic various linkages found
in lignin.
O
MeO
MeO
Probe 11
OMe
Probe 12
O
OMe
MeO
Probe 10
OMe
Figure 6. Structures of employed probes
40
45
50
Two
types
of chromophores
were
employed.
4ꢀ
methylumbelliferone (4MU), 1 will be used in the designs that
mimic the phenolic link. 6ꢀmethoxynaphthalenecarbonyl (6MN)
motif, 2 was used in the designs that mimic linkages between
hemicellulose and lignin at benzylic position (Figure 5). These
molecules show no fluorescence when linked to carbohydrates or
55
Synthesis of Probes
All of the probes used with 4MU at the anomeric position are
commercially available. The synthesis of probe 17 began with
other structural moieties. However, upon degradation or breaking 60 converting the perꢀacetylated cellobiose into the glycosyl
of the designed chemical bonds, fluorescent molecules are
released, and are thus termed fluorogenic. In addition, 4MU
undergoes hydrolytic cleavage whereas 6MN fluorescence only
occurs through oxidative cleavage. Thus, these two probes can
bromide followed by glycosylation using (6ꢀMethoxyꢀ2ꢀ
naphthyl)methanol, 7 as the glycosyl acceptor. Deacetylation of 8
afforded the desired probe 17. Similar process was used for the
syntheses of probes 14 and 16 (Scheme 1).
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20
Probe 10 can be synthesized by alkylation of the hydroxy group
of 19 with 17 (Scheme 4). Probe 11 can be prepared by
bromination of the αꢀcarbon of 20 followed by nucleophilic
substitution of Br using 1 as the nucleophile. Probe 12 was
9
Probe 1 was synthesized with alkylation of methyl 2,3,4,ꢀtriꢀOꢀ 25 synthesized as reported in the literature.
benzoylꢀαꢀDꢀmannoside, 11 using 6MN derivative, (6ꢀ
methoxynaphthalenꢀ2ꢀyl)methylꢀ2,2,2ꢀtrichloroacetimidate, 12
5
a
followed by the deprotection of the benzoyl groups (Scheme 2).
Results and Discussion
White rot fungus, Phanerochaete chrysosporium (ATCC24725),
3
3
4
4
5
5
6
0
5
0
5
0
5
0
which uses cellulases and peroxidases to degrade cellulose, lignin
4
d,8
and hemicellulose, has been studied for many years. All of the
probes were tested using crude extract of P. chrysosporium. The
fluorescence intensities of the inoculated solutions were observed
every ten minutes using excitation at 360 nm and emission at 460
nm. Relative fluorescence intensity (RFI) values were calculated
as the difference in fluorescence intensity between the inoculated
solutions and controls (no crude extract of P. chrysosporium
added). The RFI value for each probes was determined 6ꢀ9 times
and the averaged RFI values for all probes are summarized in
Figures 7ꢀ10.
Probe 3 was prepared from methyl mannopyranoside, 14 via a
Mitsunobu reaction using 4MU as the nucleophile (Scheme 2).
Figure 7. RFI values of class I probes
1
0
Similar to the synthesis of probe 3, probe 4 was prepared from
1
,2ꢀOꢀisopropylideneꢀaꢀDꢀxylofuranose via a Mitsunobu reaction
followed by the deprotection of the isopropylidene group
(Scheme 3). In a separate route, the 3ꢀOH of compound 15 was
alkylated with 6MN. Following the deprotection of both trityl and
1
5
time
isopropylidene groups, probe 5 can be obtained which could exist
as an equilibrium of furanose and pyranose forms (Scheme 3).
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The data show that only two of the class IIa probes (probes 7 and
) displayed significant fluorescence increase showing a timeꢀ
8
dependent increase in RFI. Probe 9 also manifested a timeꢀ
60 dependent but rather small increase in RFI. All three probes have
4MU attached to the anomeric position, whereas similar probes
with 6MN attached at the anomeric position (probes 14 and 16)
showed no obvious fluorescence increase. Since 6MN can be
fluorescent only via oxidative cleavage, this result suggests that
Figure 8. RFI values of class IIa probes
5
0
5
0
5
0
5
0
5
0
5
6
7
7
8
8
9
9
5
0
5
0
5
0
5
the activity of glycosidases, not peroxidases, are responsible for
the degradation. However, it is worth mentioning that the lack of
fluorescence from probes 14 and 16 cannot be viewed as the lack
of fungal enzyme activity. In fact, we believe that fugal
glycosidases can degrade these two probes. Nevertheless, the
adducts from such degradation were not fluorescent.
1
1
2
2
3
3
4
4
5
5
The lack of fluorescence increase from class IIb probes attached
with either 4MU or 6MN also implies that peroxidases or
etherases cannot degrade xyloseꢀ or mannoseꢀbased
hemicelluloses, which are connected to lignin via ether linkages.
Therefore, it is possible to release or degrade glucoseꢀ, xyloseꢀ or
mannoseꢀbased hemicelluloses when these are connected to
lignin via glycosidic bonds.
time
The lack of fluorescence increases from class III probes and very
little fluorescence increase from class I probes support the idea of
using fungi to selectively degrade or release hemicelluloses
without breaking down celluloses or lignin from biomass. To
confirm that the aromatic compounds generated from the
degradation of class III probes (probes 10, 11 and 12) will not
cause fluorescence quenching, we have measured the
fluorescence of preꢀmixed aromatic compounds and 4MU. The
Figure 9. RFI values of class IIb probes
10
results showed no fluorescence quenching. Nevertheless, there
is a slight and timeꢀdependent fluorescence increase for probe 18.
It is possible that celluloses may still be degraded but at a much
slower rate than that of the hemicelluloses. In contrast to probe 8
(mannose with βꢀlinked 4MU), probe 19 (mannose with αꢀlinked
time
4
MU) did not exhibit an increase in RFI. Plant mannans are
mannoseꢀbased polysaccharide with mannose linked with β(1ꢀ4)
linkages while yeasts and mammalian glycoproteins have
mannoseꢀbased oligosaccharides carrying mannose linked with
α(1ꢀ6) as the backbone, and α(1ꢀ2) and α(1ꢀ3) linked at the
branches. The lack of RFI increase from probe 19 strongly
indicates that the fungal enzymatic degradation is very selective
Figure 10. RFI values of class III probes
1
1
1
00 toward the plant mannans.
Finally, based on the timeꢀdependent but small increase in RFI of
probe 18, the possibility that the rate of enzymatic
degradation/release on hemicelluloses may be slower for
05 polymers consisting of xylose, mannose or glucose cannot be
ruled out. It is difficult to design or envision a fluorogenic probe
that can mimic the glycosidic bond between two carbohydrate
units. However, since the cellobioseꢀbased probe (probe 18) and,
likely, probe 17 were degraded by the fungal enzymes, it is
10 possible that the glycosidic bonds between xylose, mannose or
glucoseꢀcontaining hemicelluloses and lignin can also be broken
by fungal enzymes albeit, probably, at a slower rate as compared
time
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to the monosaccharideꢀbased probes.
from 100/0 to 4/1). The yields for deacetylation were usually
between 90 – 100%.
Conclusion
60 Methylꢀ6ꢀOꢀ(6ꢀMethoxyꢀ2ꢀnaphthalenylmethyl)ꢀ
5
We have screened a library of fifteen commercially purchased
and synthetic fluorogenic probes to investigate the feasibility of
selective degradation of plant biomass using crude extracts of
whiteꢀrot fungi. These probes contain linkages that mimic the
mannopyronoside (probe 1)
1
H NMR (300 MHz, METHANOLꢀD3) δ 7.7 (m, 3H), 7.43 (dd, J
= 9.42, 1.74 Hz,1H ), 7.19 (d, J = 2.43 Hz, 1H), 7.1 (dd, J =
8.91, 2.73 Hz), 4.7 (s, 3H), 4.62 (d, J = 1.74, 1H, H1), 3.87 (s,
dominant linkages in celluloses, lignin, and the possible linkages 65 3H), 3.81 (t, J =10.32 Hz, 1H), 3.63ꢀ3.76 (m, 5H), 3.36 (s, 3H);
1
3
1
1
2
2
3
3
4
4
5
5
0
5
0
5
0
5
0
5
0
5
between hemicelluloses and lignin. Our results show that fungal
enzymes are very selective in breaking down the βꢀlinked
glycosidic bonds of glucose and mannose. Much slower cleavage
rate of the βꢀlinked glycosidic of cellobiose and xylose was also
noted. No degradation of lignin linkages was observed. Finally,
no cleavage of the αꢀlinked glycosidic bond of mannose suggests
that fungal enzymes are selectively active toward plant mannans,
not mammalian mannans. Fungi are capable of degrading
biomass, including celluloses, hemicelluloses and lignin.
C NMR (100 MHz, METHANOLꢀD3) δ 158.3, 134.7, 133.7,
129.3, 129.2, 127.1, 126.7, 126.6, 118.8, 105.7, 101.8, 73.6, 72.4,
71.6, 71.0, 70.1, 67.7, 54.7, 54.3; ESI/APCI calcd for
C19H24O7 ([M] ) m/z 364.1522; measured m/z 364.1499.
+
+
70
Methylꢀ6ꢀ(4ꢀmethylꢀumbelliferyl)ꢀOꢀαꢀDꢀmannopyranoside
(probe 3).
1
H NMR (300 MHz, METHANOLꢀD3) δ 7.66 (d, J = 8.94Hz,
1H), 7.0 (dd, J = 8.94, 2.4 Hz, 1H), 6.94 (d, J = 2.4 Hz, 1H), 6.14
However, the process often take weeks or months. In our studies, 75 (d, J = 1.02 Hz, 1H), 4.63 (d, J = 1.38 Hz, 1 H), 4.37 (dd, J =
we have shown that the degradation of hemicelluloseꢀbased
probes occurs much faster (within 6 hrs) than that of the ligninꢀ
based probes, which shows no sign of degradation during the
incubation period. The observed rate of degradation is following:
10.65, 1.71 Hz, 1H), 4.26 (dd, J = 10.65, 5.85 Hz, 1H), 3.8 (m,
2H), 3.73 (t, J = 8.91 Hz, 1 H), 3.69 (m, 1H), 3.36 (s, 3H), 2.42
(d, J = 1.35Hz, 3H). C NMR (100 MHz, METHANOLꢀD3) δ
13
162.6, 162.3, 155.2, 154.5, 126.1, 113.7, 112.9, 110.9, 101.7,
hemicelluloseꢀbased probes >> celluloseꢀbased probes > ligninꢀ 80 101.3, 71.4, 71.3, 70.7, 68.3, 62.3, 54.0, 17.4. ESI/APCI calcd for
+
+
based probes. This findings suggests that by controlling the time
of incubating fungal enzymes and biomass, it is possible to
selectively release and separate hemicelluloses while keeping
cellulose and lignin intact. In summary, this study support the use
of fungal enzymes as a tool for developing effective and green
process for isolating and utilizing hemicelluloses from biomass.
C H O ([M] ) m/z 352.1158; measured m/z 352.1152.
17 20 8
6ꢀ(4ꢀMethylꢀumbelliferyl)ꢀOꢀDꢀxylofuranose (probe 4).
0.2g (0.57 mmol) 1,2ꢀOꢀisopropylideneꢀ6ꢀ(4ꢀmethylꢀ
85 umbelliferyl)ꢀOꢀαꢀDꢀxylofuranose was dissolved into a 5mL
solution (HOAc:H O:TFA = 80:20:1) in a 10mL round bottom
2
Experimental section
flask. The solution was heated at 60 °C under reduced pressure
for 2 hours. The completion of the reaction was confirmed by
TLC (eluted with EtOAc/hexane = 3/7). Then the solvent was
General procedure for the Mitsunobu reaction using primary
alcohol and 4ꢀmethylumbelliferone (probe 3 and 16). To a 90 removed under reduced pressure. 5 mL dichloromethane was
solution of starting material (0.5 g) and 4ꢀmethylumbelliferone (1
equiv.) in 20 mL of anhydrous THF, triphenylphosphine (1.5
equiv.) and DIAD (1.5 equiv.) was added. The reaction was
heated at 50 °C overnight. After completion of the reaction
added and the solvent was removed under reduced pressure, this
was repeated 3 times in order to remove AcOH. Then the product
was pumped dry by oil pump for 24 hours. Pure product was
obtained after chromatography (Hexane/EtOAc = 100/10 to
(confirmed by TLC, eluted with EtOAc), the solvent was 95 0/100, yield 110 mg, 0.35 mmol, 62%).
1
removed and the product was purified by column
chromatography (hexane/EtOAc from 80/20 to 0/100). The
obtained product was further purified with recrystallization in a
mixture of EtOAc/MeOH.
H NMR (300 MHz, CDCl ) δ 7.66 (dd, J = 8.9, 1.7 Hz), 7.0 ꢀ
3
6.9 (m, 1H), 6.9 – 6.8 (m, 1H), 6.14 (d, J =1.1 Hz), 5.39 (d, J =
4.1 Hz, 0.4 H), 5.13 (s, 0.4H, this compound is a 1:1 mixture of α
and β), 4.5 – 4.4 (m, 1H), 4.4 – 4.3 (m, 1H), 4.3 – 4.2 (m, 2H),
1
3
100 4.2 – 4.1 (m, 1H), 4.1 – 4.0 (m, 1H), 2.42 (d, J =1.1 Hz, 1H );
C
General procedure for the deprotection of acetyl groups using
NaOMe/MeOH. To a solution of peracetylated sugars (0.1 g) in
anhydrous MeOH (10 mL), a catalytic amount of NaOMe (ca. 1
M in MeOH) was added. The solution was stirred at room
NMR (100 MHz, CDCl ) for the sugar ring there are two sets of
3
peaks since it is a 1:1 mixture of α and β. 162.5, 162.3, 155.1,
154.1, 126.1, 113.7, 112.8, 110.9, 103.3, 101.3, 96.8, 81.1, 80.2,
77.0, 76.9, 75.9, 75.8, 68.6, 67.9, 17.4; ESI/APCI calcd for
+
+
temperature for 6 hours. After completion of the reaction 105 C H O ([M+H] ) m/z 309.0947; measured m/z 309.0959.
15
17
7
(
confirmed by TLC, eluted with EtOAc/MeOH = 9/1), the
+
reaction was quenched by adding Amberlite 120 (H ) resin. The
reaction mixture was filtered through a short column packed with
Celite, and the filtrate was collected and concentrated. The
3ꢀ(6ꢀMethoxyꢀ2ꢀnaphthalenylmethyl)ꢀOꢀDꢀxylofurnose (probe
5). This compound was prepared using the similar procedure for
preparing probe 4 with the isolated yield of 55 %.
1
product usually would be in good quality but can be purified by a 110 H NMR (300 MHz, MeOH) δ 7.79 (s, 1H), 7.72 (d, J =1.7 Hz,
gradient column chromatography (eluted with CH Cl /MeOH
1H), 7.69 (d, J = 2.4 Hz), 7.52 (dd, J = 8.6, 1.7 Hz, 1H), 7.18 (d,
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J = 2.7 Hz, 1H), 7.08 (dd, J = 8.9, 1.7 Hz, 1H), 5.01 (d, J = 3.4
Hz, 0.6 Hz, α ), 4.96 (d, 2H), 4.42 (d, J = 7.2 Hz, β ), 3.87 (s,
yellowish solid. It was confirmed as the right product by H NMR
(Yield 0.78 g, 2.2 mmol, 82%).
1
3
1
3
H), 3.8ꢀ3.5 (m, 5H), 3.3 – 3.2 (m, 1H); C NMR (100 MHz,
H NMR (300 MHz, CDCl ) δ 7.62 (dd, J = 8.25, 2.04 Hz, 1H),
3
MeOH) for the sugar ring there are two sets of peaks since it is a
7.5 ꢀ 7.4 (m, 2H), 7.0 ꢀ 6.9 (m, 2H), 6.79 (d, J = 2.76 Hz, 1H),
5
0
5
0
5
0
5
0
5
0
5
mixture of α and β.157.9, 134.5, 134.4, 129.1, 128.9, 126.8, 60 6.14 (d, J = 1.38 Hz, 1H), 5.33 (s, 2H), 3.97 (s, 3H), 3.93 (s, 3H),
13
1
7
26.6, 126.4, 125.3, 118.5, 105.5, 97.8, 93.1, 84.8, 81.9, 74.9,
4.9, 74.8, 72.5, 70.1, 70.0, 65.9, 61.9, 54.5; ESI/APCI calcd for
2.4 (d, 1.38 Hz); C NMR (100 MHz, CDCl ) δ 191.9, 161.3,
3
161.2, 155.2, 154.5, 152.7, 149.7, 127.5, 125.9, 122.8, 114.5,
112.9, 112.6, 110.4, 110.4, 102.1, 70.7, 56.4, 56.3, 18.7;
+
+
C H O ([M] ) m/z 320.1260; measured m/z 320.1263.
1
7
20
6
+
+
ESI/APCI calcd for C H O ([M] ) m/z 354.1103; measured
20
18
6
1
1
2
2
3
3
4
4
5
5
2ꢀ((1ꢀ(3,4ꢀdimethoxyphenyl)ethoxy)methyl)ꢀ6ꢀ
65 m/z 354.1091.
methoxynaphthalene (probe 10). 1ꢀ(3,4ꢀdimethoxyphenyl)ꢀ
ethanol was prepared by reducing 3,4ꢀdimethoxyacetophenone
(6ꢀMethoxyꢀ2ꢀnaphthalenylmethyl)ꢀβꢀDꢀglucopyranoside
using NaBH in MeOH. 2ꢀbromomethylꢀ6ꢀmethoxyꢀnaphthalene
was prepared by treating 6ꢀmethoxynaphthylꢀmethanol with PBr3
(probe 14).
4
1
H NMR (300 MHz, METHANOLꢀD3) δ 7.8 – 7.7 (m, 3H), 7.48
in Et O for 5 hours. The reaction was quenched by adding sat. 70 (dd, J = 8.5, 1.7 Hz, 1H), 7.20 (d, J = 2.7 Hz, 1H ), 7.10 (dd, J =
2
NaHCO3 to the reaction solution until no bubbling. Then the
8.9, 2.4 Hz), 5.0 – 4.7 (dd, J = 75.9, 11.7 Hz), 4.32 (d, J = 7.5
organic layer was washed by Sat. NaHCO for two times and
Hz, Hꢀ1), 3.9 – 3.8 (m, 1H), 3.88 (s, 3H), 3.7 – 3.6 (m, 1H), 3.4 –
3.2 (m, 3H); C NMR (100 MHz, METHANOLꢀD3) δ 158.1,
134.6, 132.9, 129.2, 128.9, 126.7, 126.6, 118.6, 105.5, 102.0,
3
13
Brine for one time. The organic layer was dried over Na SO and
2
4
the solvent was removed under reduced pressure. The crude
product was use directly for next step. (0.5 g, 2.7 mmol) 1ꢀ(3,4ꢀ 75 76.9, 76.85 (2C), 73.9, 70.7, 70.5, 61.6, 54.5; ESI/APCI calcd for
+
+
dimethoxyphenyl)ethanol and 1eq. 2ꢀbromomethylꢀ6ꢀmethoxyꢀ
naphthalene was dissolved in THF, then 10 equiv. of NaH was
added. After 6 hours, the reaction solution was poured to a flask
with ice to quench the reaction. 300 mL of EtOAc was added to
C H O Na ([M+Na] ) m/z 373.1263; measured m/z 373.1273.
18 22 7
(6ꢀMethoxyꢀ2ꢀnaphthalenylmethyl)ꢀβꢀDꢀxylopyranoside
(probe 16).
1
extract the product. The organic layer was washed by 100 mL 80 H NMR (300 MHz, METHANOLꢀD3) δ 7.8 – 7.7 (m, 3H), 7.46
distilled water, 100 mL 1N HCl and Brine, then dried over
Na SO . Filtration and removal of the solvent followed by
(dd, J = 8.6, 1.7 Hz, 1H), 7.19 (d, J = 2.4 Hz, 1H ), 7.09 (dd, J =
8.9, 2.4 Hz), 4.9 – 4.7 (dd, J = 67.3, 11.7 Hz), 4.32 (d, J = 7.2
Hz, Hꢀ1), 3.9 – 3.8 (m, 1H), 3.88 (s, 3H), 3.6 – 3.4 (m, 2H), 3.3 –
2
4
purification
Hexane/EtOAc = 100:0 to 40/60) afforded desired product
(Yield 0.69g, 1.97mmol, 73%).
with
gradient
column
chromatography
13
(
3.1 (m, 2H); C NMR (100 MHz, METHANOLꢀD3) 134.6,
85 132.9, 129.1, 128.9, 126.8, 126.6, 126.57, 118.6, 1015.5, 102.9,
76.6, 73.8, 70.8, 70.1, 65.8, 54.5; ESI/APCI calcd for
1
H NMR (300 MHz, CDCl ) δ 7.7 – 7.6 (m, 3H), 7.40 (dd, J =
3
+
+
8
.4, 1.8 Hz, 1H), 7.1 – 7.0 (m, 2H), 6.9 – 6.8 (m, 3H), 4.58 – 4.38
C H O Na ([M+Na] ) m/z 343.1158; measured m/z 343.1167.
18 22 7
(
3
dd, J = 46, 11.67 Hz, 2H), 4.47 (q, J = 6.54, 1H), 3.92 (s, 3H),
.90 (s, 3H), 3.89 (s, 3H), 1.49 (d, J = 6.5 Hz, 3H); C NMR
13
(6ꢀMethoxyꢀ2ꢀnaphthalenylmethyl)ꢀβꢀDꢀcellubioside (probe
(100 MHz, CDCl ,) δ 157.8, 149.4, 148.6, 136.5, 134.3, 133.9, 90 17).
3
1
1
7
(
29.5, 128.9, 127.2, 126.8, 126.7, 119.0, 111.1, 109.3, 105.9,
0.5, 56.1, 56.1, 55.5, 29.9, 24.4; ESI/APCI calcd for C H O
7
[M] ) m/z 352.1673; measured m/z 352.1675.
H NMR (300 MHz, METHANOLꢀD3) δ 7.8 – 7.7 (m, 3H), 7.48
(dd, J = 8.5, 1.7 Hz, 1H), 7.20 (d, J = 2.7 Hz, 1H), 7.10 (dd, J =
8.9, 2.4 Hz), 5.0 – 4.7 (dd, J = 71.8, 11.7 Hz), 4. (s, 2H), 4.40
+
22
24
+
(dd, J = 7.5, 3.1 Hz, 2H), 3.9 – 3.8 (m, 3H), 3.88 (s, 3H), 3.7 –
1ꢀ(3,4ꢀdimethoxyphenyl)ꢀ2ꢀ(4ꢀmethylꢀumbelliferoxy)ꢀ
95 3.4 (m, 4H), 3.4 – 3.3 (m, 2H), 3.20 (t, J = 8.6 Hz,1H ); 13C
NMR (100 MHz, METHANOLꢀD3) δ 158.1, 134.6, 132.9, 129.2,
128.9, 126.7, 126.6, 118.6, 105.5, 103.4, 101.9, 79.5, 76.9, 76.6,
75.4, 75.2, 73.7, 70.8, 70.2, 61.2, 60.7, 54.5. ESI/APCI calcd for
ethanone (probe 11). To a cooled (0 Celsius) solution of 3,4ꢀ
dimethoxyacetophenone (0.5 g, 2.7 mmol) in diethyl ether (50
mL), bromine (0.266 g, 3.32 mmol) dissolved in ether ( 20 mL )
was added slowly. After 10 min, the reaction mixture was worked
up, and the organic layer was wash with water and brine. Then 100
organic layer was dried over sodium sulfate. Filtration and
evaporation of the solvent gave a crude product which was used
directly for the next step. The crude product from last step,
potassium carbonate (0.56 g, 4.05 mmol) and 4ꢀ
+
+
C H O Na ([M+Na] ) m/z 535.1791; measured m/z 535.1787.
2
4
32 12
(6ꢀMethoxyꢀ2ꢀnaphthalenylmethyl)ꢀ2,3,4,6ꢀtetraꢀOꢀacetylꢀβꢀ
Dꢀglcucopyranoside (8). Bromoꢀ2,3,4,6ꢀacetylꢀOꢀαꢀDꢀ
glucopyranoside was prepared by treating 1,2,3,4,6ꢀpentaꢀOꢀ
acetylꢀDꢀglucose (0.5 g, 1.28 mmol) with HBr (3mL, 33% in
methylumbelliferone (0.47 g, 2.7 mmol) was dissolved into 50 105 AcOH) in a 20 mL flask with 3 mL anhydrous dichloromethane
mL of acetone. The solution was heated under reflux for
overnight. Upon completion of the reaction (check by TLC,
eluted with EtOAc/hexane = 1/9), the solvent was removed by
compress air. Add 100 ml EtOAc to the crude yellowish solid,
as solvent for 2 hours. The reaction mixture was extracted by 100
mL EtOAc and the organic layer was washed by distilled water,
Sat. NaHCO for three times and Brine for one time. Then the
3
organic layer was dried over anhydrous Na SO4 and filtered. The
2
there was some insoluble yellowish solid. Filtration to get the 110 bromoꢀ2,3,4,6ꢀacetylꢀOꢀαꢀDꢀglucopyranoside was obtained by
removing the solvent under reduced pressure and used directly
6
|
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DOI: 10.1039/C4GC01659A
for next step. Bromoꢀ2,3,4,6ꢀacetylꢀOꢀαꢀDꢀglucopyranoside (1.28
mmol) and (6ꢀMethoxyꢀ2ꢀnaphthyl)methanol (0.34 g, 1.8 mmol)
and 4 Å MS was stirred in 20 mL of anhydrous dichloromethane
1H, H1), 4.71 (dd, J = 26.1, 11.67 Hz, 2H), 4.27 (dt, J = 9.63,
3.45 Hz, 1H, H5), 3.91 (s, 3H), 3.79 (d, J = 3.75 Hz, 2H, H6),
13
3.54 (s, 3H); C NMR (CDCl , 75 MHz) δ 165.8, 165.7, 163.6,
3
for 15 minutes before adding in Ag CO (0.49 g, 1.8 mmol ). The 60 157.8, 134.3, 133.6, 133.4, 133.3, 133.2(2C), 130.1(2C),
2
3
5
mixture was stirred for overnight before filtered through a syringe
packed with Celite and silica gel. Then the solvent was removed
and the crude product was loaded to a column and purified with
gradient (Hexane/EtOAc = 100/10 to 50/50). Pure product was
130.0(3C), 129.9, 129.6, 129.6, 129.44, 129.4, 128.7(2C),
128.5(2C), 128.5(2C), 127.1, 126.80, 126.7, 118.92, 105.9, 98.8,
74.1, 70.7, 70.5, 70.3, 69.1, 67.5, 55.7, 55.5. ESI/APCI calcd for
+
+
C H O ([M] ) m/z 676.2308; measured m/z 676.2300.
40
36 10
obtained (0.41g, 0.79 mmol, 65 % yield over two steps).
65
1
1
1
2
2
3
3
4
4
5
5
0
5
0
5
0
5
0
5
0
5
H NMR (300 MHz, CDCl ) δ 7.8 – 7.7 (m, 3H), 7.37 (dd, J =
1,2ꢀOꢀisopropylideneꢀ6ꢀ(4ꢀmethylꢀumbelliferyl)ꢀOꢀαꢀDꢀ
3
8
.3, 1.4 Hz), 7.2 – 7.1 (m, 2H), 5.70 (d, J = 5.1 Hz, Hꢀ1 ),5.22 (t,
xylofuranose (16).
1
J = 2.8 Hz, Hꢀ2), 4.91 (dd, J = 9.3, 2.1 Hz, Hꢀ3), 4.67 (s, 2H),
4
9
(100 MHz, CDCl ) δ 170.9, 169.9, 169.4, 157.9, 134.3, 132.8,
1
7
H NMR (300 MHz, CDCl ) δ 7.45 (d, J = 8.9 Hz, 1H), 6.88 (dd,
3
.35 (dd, J = 4.5, 3.1 Hz, Hꢀ4), 4.2 – 4.1 (m, 2H), 3.97 (ddd, J =
J = 8.9, 2.4 Hz, 1H ), 6.84 (d, J = 2.4 Hz, 1H), 6.1 (d, 1H), 6.01 (
.6, 5.5, 3.5 Hz, Hꢀ5), 3.9 (s, 3H), 2.1 – 1.8 (m, 12H); C NMR 70 d, J = 3.5 Hz, 1H, Hꢀ1), 4.59 (d, J = 3.8 Hz, 1H, Hꢀ2), 4.55 (ddd,
13
J = 11.3, 5.5, 2.7 Hz, 1H. Hꢀ4), 4.40 (d, J = 2.7 Hz, 1H, Hꢀ3),
4.32 (ddd, J = 16.5, 10.3, 6.2 Hz, 2H, Hꢀ6), 2.77 (s, 1H, OH),
2.37 (d, 3H), 1.52 (s, 3H), 1.33 (s, 3H); 13C NMR (100 MHz,
3
29.6 128.9, 127.3, 126.6, 126.5, 121.7, 119.2, 105.9, 97.22,
3.4, 70.3, 68.4, 67.2, 66.3, 63.3, 55.5, 21.2, 21.0 (3C); ESI/APCI
+
+
calcd for C H O Na ([M+Na] ) m/z 541.1686; measured m/z
5
CDCl ) 161.7, 155.2, 152.9, 125.8, 114.1, 112.9, 112.3, 105.2,
26
30 11
3
41.1677.
75 101.8, 85.5, 78.4, 75.3, 66.4, 29.9, 27.0, 26.4, 22.1, 18.9;
+
+
ESI/APCI calcd for C H O ([M+H] ) m/z 349.1287; measured
18
20
7
(6ꢀMethoxyꢀ2ꢀnaphthalenylmethyl)ꢀ2,3,4,ꢀtriꢀOꢀacetylꢀDꢀ
m/z 349.1295.
xylopyranoside (10). This compound was prepared using the
similar procedure for preparing compound 8 with the isolated
1
1
General procedure for assay of fluorogenic probes. White rot
80 basidiomycete, Phanerochaete chrysosporium ATCC24725 was
grown on potato dextrose agar (PDA) plates and incubated
around 30°C for one week before the fungus was transferred to
liquid culture medium. The culture medium contained, per liter: 2
g KH PO , 0.5 g MgSO •7H O, 0.1 g CaCl •2H O, 1% glucose,
yield of 45 %.
1
H NMR (300 MHz, CDCl ) The compound was obtained as a
3
mixture of alpha and beta conformation (α/β = 1.3/1) δ 7.8 – 7.7
(m, 4.6 H), 7.5 – 7.4 (m, 1.6 H), 7.2 – 7.1 (m, 3.1 H), 5.7 (d, J =
6
2
.9 Hz,1H, H αꢀ1), 5.58 (d, J = 4.5 Hz, 1.3H, H βꢀ1), 5.28 (t, J =
2
4
4
2
2
2
.8 Hz, 1.3H), 5.20 (t, J = 8.3, 1H), 5.1 – 4.8 (m, 4H), 4.68 (s, 85 10 mM pH 4.2 2,2ꢀdimethyl succinate, 1 mg thiaminꢀHCl stock
2H), 4.3 – 4.2 (m, 1.4H), 4.15 (dd, J = 12.0, 5.2 Hz, 1H), 4.0 –
3
3
solution, 0.2 g ammonium tartrate, and 10 mL of trace element
solution (see below). The thiamin and KH PO stock solutions
.9 (m, 1H), 3.90 (s, 5.1H), 3.71 (dd, J = 12.4, 6.9 Hz, 1.4H),
2
4
13
.51 (dd, J = 12.0, 8.6 Hz, 1 H ), 2.1 – 2.0 (m, 21H); C NMR
were filter sterilized; the others are autoclaved as separate
solutions. The stock solution of trace elements is made by first
(100 MHz, CDCl ) δ 170.1, 170.0, 169.5, 169.3, 169.2, 157.9,
3
1
34.3, 132.8, 129.6, 128.9, 127.3, 126.7, 126.6, 126.1, 125.7, 90 dissolving 1.5 g of nitrilotriacetic acid in 800 mL water and the
pH of the solution was adjusted to approximately 6.5 with KOH.
The following were then added with dissolution: 0.5
MnSO ·H O, 1.0 g NaCl, 0.1 g FeSO ·7H O, 0.1 g CoSO , 0.1 g
122.6, 119.1, 105.9, 96.8, 92.3, 74.5, 71.2, 69.7, 69.6, 68.8, 68.5,
7.6, 65.6, 63.0, 59.9, 55.5, 22.9 (2C), 21.0, 20.9, 20.87, 20.81;
6
g
+
+
ESI/APCI calcd for C H O Na ([M+Na] ) m/z 469.1475;
measured m/z 469.1468.
2
3
26
9
4
2
4
2
4
ZnSO ·7H O, 10 mg CuSO ·5H O, 10 mg A1K(SO ) ·12H O, 10
4 2 4 2 4 2 2
95
mg H BO , and 10 mg Na MoO ·2H O. Sterile distilled water
3 3 2 4 2
Methylꢀ2,3,4ꢀTriꢀbenzoylꢀOꢀ6ꢀ(6ꢀMethoxyꢀ2ꢀ
naphthalenylmethyl)ꢀαꢀDꢀmannopyranoside (13).
6
was added to make the total volume of one liter. The medium was
inoculated with a 100 mL suspension of fungal culture containing
spores (absorbance of 0.5 at 650 nm).
ꢀmethoxyꢀ2ꢀnaphthalenylmethyl trichloroacetimidate (0.33 g, 1
mmol) and methyl 2,3,4ꢀtriꢀOꢀbenzoylꢀαꢀDꢀmannopyranoside
0.35 g, 0.7 mmol), 4 Å molecular sieve in anhydrous 100 For fungal growth, 10 ml portions of liquid culture in the growth
(
dichloromethane (20 mL) was stirred for 30 minutes and then
cooled to ꢀ78 °C. After addition of several drops of BF .Et O, the
reaction mixture was stirred overnight. After completion of the
reaction (check by TLC, eluted with EtOAc/hexane = 1/9), the
medium were incubated in 125ꢀml Erlenmeyer flasks capped with
rubberꢀstoppers at 30ꢀ39°C. Enzyme production was stimulated
by including 0.4 mM veratryl alcohol final concentration and
trace elements (7 times the above concentration) at the time of
3
2
reaction mixture was filtered through a short column packed with 105 inoculation. After one week growth, the 10 mL culture aliquots
Celite and silica gel. After removal of most of the solvent, the
crude product was purified with gradient column
chromatography (hexance/EtOAc = 100/0 to 75/25) to furnish the
desired product (0.48 g, 68%). H NMR (CDCl , 300 MHz) δ
8
were centrifuged at 1500 RPM, 5°C for 15 minutes, and the
supernatant fluids were collected and directly used for enzymatic
assays. In a 96ꢀwell microtiter plate, each well contained 100 ꢀL
125 mM sodium tartrate (pH 3), 50 ꢀL 10 mM probes, 50 ꢀL 2
a
1
3
.1ꢀ8.0 (m, 2H), 7.82ꢀ7.9 (m, 4H), 7.7ꢀ7.2 (m, 10H), 7.0ꢀ7.1 (m, 110 mM H O , 50 ꢀL culture supernatant. For the control, each well
2 2
2H), 6.02 (t, J = 9.69 Hz, 1H, H4), 5.85 (dd, J = 9.96, 3.09 Hz,
1
contains 100 ꢀL 125 mM sodium tartrate (pH 3), 50 ꢀL 10 mM
probes, 50 ꢀL 2 mM H O (freshly prepared), and 50 ꢀL H O.
H, H3), 5.7 (dd, J = 3.45, 1.71 Hz, 1H, H2), 5.0 (d, J = 1.71 Hz,
2
2
2
This journal is © The Royal Society of Chemistry [year]
Journal Name, [year], [vol], 00–00 |
7

Green Chemistry
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View Article Online
DOI: 10.1039/C4GC01659A
Fluorescence was measured by using excitation 360/40, emission
60/40 at 37°C. Two duplicated experiments were conducted in
Chem. Rev. 2001, 101, 3397ꢀ3413. (d) Carlile, M. J.; Watkinson, S.
C. The Fungi 1994, Academic Press.
4
55
parallel. Each probes was assayed at least 6ꢀ9 times.
5
6
(a) Lebo, S. E. Jr.; Gargulak, J. D.; McNally, T. J. "Lignin". Kirk‑
Othmer Encyclopedia of Chemical Technology. 2001, John Wiley &
Sons, Inc. (b) Zakzeski, J.; Bruijnincx, P. C. A.; Jongerius, A. L.;
Weckhuysen, B. M. “The Catalytic Valorization of Lignin for the
Production of Renewable Chemicals.” Chem. Rev. 2010, 110, 3552ꢀ
5
Acknowledgment
We acknowledge support from the Sun Grant Western Regional
Center/Department of Transportation.
60
3
599.
Notes and references
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Steam Gasification of Biomass: Catalysts, Thermodynamics and
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J.; Abatzoglou, N. NREL/TPꢀ570ꢀ25357; National Renewable
Energy Laboratories: Golden, CO, 1998. (c) Blasi, C. D.; Branca. C.
1
(a) Keller, R. "Biofuel from Algae Has Problems" Ag Professional,
June 11, 2011. (b) Corma, A.; Iborra, S.; Velty, A. "Chemical Routes
for the Transformation of Biomass into Chemicals." Chem. Rev.
10
15
20
25
30
35
40
45
50
6
7
7
8
8
9
5
0
5
0
5
0
2
007, 107, 2411ꢀ2502. (c) Wright, J. D. "Ethanol from biomass by
“
Kinetics of Primary Product Formation from Wood Pyrolysis”. Ind.
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Cadoche, L.; Lopez, G. D. "Assessment of size reduction as a
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Eng. Chem. Res. 2001, 40, 5547ꢀ5556.
7
8
Laine, C. "Structures of Hemicelluloses and Pectins in Wood and
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2
(a) MäkiꢀArvela, P.; Salmi, T.; Holmbom, B.; Willför, S.; Murzin, D.
U. "Synthesis of Sugars by Hydrolysis of Hemicellulosesꢀ A
Review." Chem. Rev. 2011, 111, 5638ꢀ5666. (b)Aguilar, R; Ramirez,
J. A.; Garrote, G.; Vazquez, M. “Kinetic study of the acid hydrolysis
of sugar cane bagasse.” J. Food Eng. 2002, 55, 309ꢀ318. (c)
(a) Cameron, M. D.; Post, Z. D.; Stahl, J. D.; Haselbach, J.; Aust, S.
D. "Cellobiose dehydrogenaseꢀdependent biodegradation of
polyacrylate polyers by Phanerochaete chrysosporium." Enviorn.
Sci. & Pollut. Res. 2000, 7, 130ꢀ134. (b) Cameron, M. D.; Aust, S. D.
"Degradation of Chemicals by Reactive Radicals Produced by
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Cellobiose Dehydrogenase from Phanerochaete chrysosporium."
Biochemistry and Biophysics 1999, 367, 115ꢀ121.
Future Directions." Kamm, B., Gruber, P. R., Kamm, M., Eds.;
WileyꢀVCH: Weinheim, 2006; Vol. 1. (d) Bjerre, A. B.; Olesen, A.
B.; Fernqvist, T. "Pretreatment of wheat straw using combined wet
oxidation and alkaline hydrolysis resulting in convertible cellulose
and hemicellulose." Biotechnol. Bioeng. 1996, 49, 568–577. (e)
Azzam, A.M. " Pretreatment of cane bagasse with alkaline hydrogen
peroxide for enzymatic hydrolysis of cellulose and ethanol
fermentation." J. Environ. Sci. Health. B. 1989, 24, 421–433. (f)
Dale, B.E., Henk, L.L., Shiang, M., "Fermentation of lignocellulosic
materials treated by ammonia freezeꢀexplosion." Dev. Ind. Microbiol.
9
1
Kajikawa, H., Kudo, H., Kondo, T., Jodai, K., Honda, Y., Kuwahara,
M., & Watanabe, T. "Degradation of benzyl ether bonds of lignin by
ruminal microbes." FEMS microbiology letters, 2000, 187, 15ꢀ20.
0. We selected three aromatic adducts from the possible degradation of
class III probes for this investigation: 3,4ꢀdimethoxyacetophenone,
3
,4ꢀdimethoxybenzyl alcohol and 1ꢀ(3,4ꢀdimethoxylphenyl)ethanol.
There was only a slight decrease in fluorescence when 4MU was
mixed with 3,4ꢀdimethoxyacetopheone and no changes on the other
two combinations. Therefore, we were certain that there was no
degradation of class III probes by fungal enzymes. Please refer to
Supporting Information for more details.
1
984, 26, 223–233.
3
(a) Sanchez, O. J.; Cardona, C. A. "Trends in Biotechnological
Production of Fuel Ethanol from Different Feedstocks." Bioresource
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Page 9 of 9
Green Chemistry
View Article Online
DOI: 10.1039/C4GC01659A
A library of fifteen fluorogenic probes was designed to mimic the dominant linkages in celluloses,
hemicelluloses and lignin, and screened against crude extracts from white-rot fungi. The results show
that fungal enzymes display a high preference for cleaving mannose- and glucose-based probes, which
mimic hemicelluloses. This finding may enable the uses of fungal enzymes as a tool for developing an
effective process for isolating and utilizing hemicelluloses from biomass.