Methanolysis of ethyl esters of N-acetyl amino acids catalyzed by cyclosophoraoses isolated from Rhizobium meliloti

Article abstract of DOI:10.1016/j.carres.2007.10.033

Methanolysis of four ethyl esters, N-acetyl-l-phenylalanine ethyl ester, N-acetyl-l-tyrosine ethyl ester, N-acetyl-l-tryptophan ethyl ester, and ethyl phenylacetate was catalyzed by a mixture of microbial cyclooligosaccharides termed cyclosophoraoses isolated from Rhizobium meliloti. Cyclosophoraoses [cyclic-(1→2)-β-d-glucans, collectively 'Cys'] are a mixture of large-ring molecules consisting of various numbers of glucose residues (17-27) linked by β-(1→2)-glycosidic bonds. Cys as a catalytic carbohydrate enhanced the methanolysis about 233-fold for N-acetyl-l-tyrosine ethyl ester in comparison with a control. The effect of dry organic solvents on the methanolysis of N-acetyl-l-tyrosine ethyl ester was investigated by high-performance liquid chromatography (HPLC), and it was found that the rate enhancement correlated closely with the hydrophobicity of the solvent.

Full text of DOI:10.1016/j.carres.2007.10.033

Available online at www.sciencedirect.com
Carbohydrate Research 343 (2008) 274–281
Methanolysis of ethyl esters of N-acetyl amino acids catalyzed
by cyclosophoraoses isolated from Rhizobium meliloti
Heylin Parka and Seunho Junga,b,
*
aDepartment of Advanced Technology Fusion, Konkuk University, Seoul 143-701, Republic of Korea
bDepartment of Bioscience and Biotechnology, Bio/Molecular Informatics Center, Konkuk University,
Seoul 143-701, Republic of Korea
Received 4 August 2007; received in revised form 19 October 2007; accepted 30 October 2007
Available online 5 November 2007
Abstract—Methanolysis of four ethyl esters, N-acetyl-L-phenylalanine ethyl ester, N-acetyl-L-tyrosine ethyl ester, N-acetyl-L-trypto-
phan ethyl ester, and ethyl phenylacetate was catalyzed by a mixture of microbial cyclooligosaccharides termed cyclosophoraoses
isolated from Rhizobium meliloti. Cyclosophoraoses [cyclic-(1!2)-b-D-glucans, collectively ‘Cys’] are a mixture of large-ring mole-
cules consisting of various numbers of glucose residues (17–27) linked by b-(1!2)-glycosidic bonds. Cys as a catalytic carbohydrate
enhanced the methanolysis about 233-fold for N-acetyl-L-tyrosine ethyl ester in comparison with a control. The effect of dry organic
solvents on the methanolysis of N-acetyl-L-tyrosine ethyl ester was investigated by high-performance liquid chromatography
(HPLC), and it was found that the rate enhancement correlated closely with the hydrophobicity of the solvent.
ꢀ 2007 Elsevier Ltd. All rights reserved.
Keywords: N-Acetyl amino acids; Ethyl esters; Methanolysis; Rhizobium meliloti; Cyclosophoraoses; Catalytic oligosaccharides
1. Introduction
molecule for various inclusion complexation technolo-
gies, primarily owing to its characteristic scaffold
dictated by the b-(1!2)-glycosidic linkage (Fig. 1A).
Cyclosophoraoses (collectively ‘Cys’) are a family of
unbranched cyclic-(1!2)-b-D-glucans that are found
almost exclusively in fast-growing soil bacteria of the
Agrobacterium and Rhizobium species as intra- and
extra-oligosaccharides.1,2 Cys is produced as a mixture
of large-ring molecules, each of which has various
degrees of polymerization (DPs).3,4 Cys is synthesized
in the cytosol and is transported to the periplasmic space
where it plays an important role in regulating the
osmolarity in response to external osmotic shocks.5 In
addition, Cys is involved in the initial stage of the
root-nodule formation of the Rhizobium species in nitro-
gen fixation.6,7 Cys is suspected to be involved in the
complexation with various plant flavonoids throughout
the process of root-nodule formation.8 Several reports
have shown that Cys has good potential as a host
Degree of polymerization 17-27
A
HO
OH
HO
O
O
O
HO
O
β(1→ 2)
HO
B
*
Figure 1. Chemical structure of Cys (degree of polymerization 17–27)
Corresponding author. Tel.: +82 2 450 3520; fax: +82 2 452 3611;
(A) and stereoview of the proposed molecular model of Cys (B).
e-mail: shjung@konkuk.ac.kr
0008-6215/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2007.10.033

H. Park, S. Jung / Carbohydrate Research 343 (2008) 274–281
275
Examples include application as a solubility enhancer of
2. Results and discussion
poorly soluble guest molecules,9–11 a chiral NMR sol-
vating agent12 a chiral additive in capillary electrophore-
sis (CE),13 and a catalyst for methanolysis.14 Even if the
exact three-dimensional structure of Cys is not clearly
identified, nuclear magnetic resonance (NMR)15 and
conformational studies15,16 suggest that Cys has flexible
backbone structures and narrower cavity sizes than
expected (Fig. 1B).
2.1. Identification of neutral cyclosophoraoses
The isolation, purification, and structural analyses of
Cys were carried out as described previously.9–11 Puri-
fied Cys was separated with a Rf value of 0.13 on
TLC. We confirmed that Cys is composed of
unbranched cyclic b-(1!2)-D-glucans with various sizes
from 17 to 27 in DP by NMR spectroscopy and matrix-
assisted laser desorption/ionization-time of flight
(MALDI-TOF) mass spectometry. Based on MALDI-
TOF mass spectrometry, the number-average molecular
weight Mn of Cys was determined as 3568.6.10
In general, protein enzymes, ribozymes,17 DNA-
zymes,18 and catalytic antibodies19 as typical biocata-
lysts have been studied by many researchers in various
fields; however, catalytic carbohydrates derived from
living organisms are the exception. Cyclomaltooligosac-
charides [cyclodextrins, cyclic-(1!4)-a-D-glucans] as
nonnatural carbohydrates have been known to show
catalytic functions.20,21 Recently, it has been reported
that a few microbial carbohydrates, Cys,14 a-cyclosop-
horohexadecaose [a cyclic-(1!2)-b-D-glucan with one
a-(1!6)-linkage],22 succinoglycan,23 and Zoogloeal
zooglans24 function as novel biological catalysts for
the methanolysis of ester compounds. Among them,
Cys enhanced the rates of the methanolysis reactions
of both oxazolone compounds and phospholipids. The
characteristic scaffold induced by b-(1!2)-glycosidic
linkages of Cys would provide an appropriate space
for the binding of substrates, thus affording the possibil-
ity of a catalytic reaction.14
Herein, we investigated catalytic ability of Cys for the
methanolysis of three ethyl esters of N-acetyl amino
acids and ethyl phenylacetate. The esters of N-acetyl
amino acids have been used as low-molecular-weight
substrates for transesterification in organic solvents or
hydrolysis in water by nucleophilic catalysts such as ser-
ine proteases.25–27 The effect of nonaqueous solvents on
the methanolysis was also investigated.
2.2. Methanolysis of ethyl esters catalyzed by Cys
The methanolysis (Fig. 2) of four ethyl esters, N-acetyl-
L-phenylalanine ethyl ester (APEE), N-acetyl-L-tyrosine
ethyl ester (AYEE), N-acetyl-L-tryptophan ethyl ester
(AWEE), and ethyl phenylacetate (EPA), catalyzed by
1
Cys was monitored by H NMR spectroscopy. Figure
1
3 shows the H NMR spectra of APEE (substrate) and
N-acetyl-L-phenylalanine methyl ester (APME, product)
in the presence of 0.1 equiv of Cys at different periods of
time. The proton peak of the exchanged methyl group of
the product appeared at 30 min and increased with reac-
tion time. After 11 h, methanolysis of APEE by Cys was
almost completed (P96%), but in the absence of Cys the
reaction proceeded slowly (66%). The Cys-induced
methanolysis of AYEE, AWEE, and EPA was also
monitored by in similar manner of APEE (data not
shown). The results in Figure 4 and Table 1 show that
the reactions were enhanced approximately 53-, 233-,
38-, and 19-fold for APEE, AYEE, AWEE, and EPA,
respectively, in the presence of 0.1 equiv of Cys in
O
O
A
Cys
MeOH, 60 ºC
R1
R1
O CH3
O CH2CH3
R2
R2
O
O
B
O
CH CH
2 3
O
CH CH
2 3
NHAc
NHAc
HO
APEE
AYEE
O
O
O
CH CH
2 3
NHAc
O
CH CH
2 3
N
H
AWEE
EPA
Figure 2. Scheme of methanolysis (A) and chemical structures (B) of ethyl esters.

276
H. Park, S. Jung / Carbohydrate Research 343 (2008) 274–281
O
O
11
8
5
11'
8'
5'
12 13
12'
CH3
Cys
2
2'
HN
10
9
10'
9'
O
CH2CH3
O
CH3CH2OH
+
HN
MeOH, 60 ºC
7
7'
O
O
H3C
4
H3C
4'
14 h
8 h
5 h
3 h
2 h
1 h
12’
0.5 h
5
12
2
13
4
0 h
8
6
4
2
Chemical Shift (ppm)
Figure 3. 1H NMR spectra of APEE (substrate), and N-acetyl-L-phenylalanine methyl ester (APME, product) in the presence of 0.1 equiv of Cys at
different periods of time.
100
75
50
25
0
MeOH. Depending on the chemical functional group
linked to ethyl esters, the extent of the rate enhancement
for the methanolysis of esters by Cys was different. Cys
showed the most effective catalytic activity for AYEE
among the four ethyl esters. Although the uncatalyzed
methanolysis of esters of N-acetyl amino acids (APEE,
AYEE, and AWEE) was slower than that of EPA
(Table 1), the extent of the rate enhancement for
methanolysis of esters of N-acetyl amino acids by Cys
was higher. The esters containing the N-acetylated
amino group were found to be more suitable substrates
than EPA for methanolysis by Cys. Glucose, linear-
(1!2)-b-D-glucan, and amylose were also tested as cat-
alysts to identify the effect of noncyclic carbohydrates
for the methanolysis of AYEE, which had the highest
value of kcat_Cys/kuncat. The reactions were monitored
by HPLC. Linear-(1!2)-b-D-glucans were prepared by
acid hydrolysis of Cys, and then their structural analysis
0
5
10
15
20
25
Time (h)
Figure 4. Time-course of methanolysis of ethyl esters in the absence or
presence of 0.1 equiv of Cys at 60 ꢁC. The data were fitted to a single
exponential to obtain kcat_Cys and kuncat for each substrate. Symbols:
APEE (h), AYEE (e), AWEE (n), and EPA (s) in the absence of
Cys, and APEE (j), AYEE (r), AWEE (m), and EPA(d) in the
presence of Cys.

H. Park, S. Jung / Carbohydrate Research 343 (2008) 274–281
Table 1. Methanolysis of ethyl esters catalyzed by Cys
277
about 180- and 16-fold, respectively. These results can
be caused by the structural differences of carbohydrates
used in the reaction. Glucose and amylose may lack
proper space for the binding of substrate, and linear-
(1!2)-b-D-glucan could not completely form the suit-
able structure to accept the substrate. From the results,
we suggest that both of the characteristic scaffolds
induced by b-(1!2)-glycosidic linkages and the cyclic
structures would provide an appropriate space for the
binding of substrates, thus affording the possibility of
a catalytic reaction. Additionally, to confirm the cata-
lytic properties of Cys species with different DPs, each
cyclosophoraose (from 17 to 27) was fractionated by
preparative HPLC. Then the catalytic activity of each
cyclosophoraose fraction with DP 19, DP 22, a mixture
kuncat (minꢀ1
)
kcat_Cys (minꢀ1
)
kcat_Cys/kuncat
APEE
AWEE
AYEE
EPA
0.319
0.240
0.028
0.516
17.034
9.230
6.610
9.702
53.40
37.96
233.36
18.81
was investigated by NMR spectroscopy (Fig. 5A) and
MALDI-TOF mass spectrometry (data not shown),
indicating that the oligosaccharides are b-(1!2)-linked
linear glucans with various DP values from 4 to 22.
Figure 5B shows the comparison of catalysis by Cys
and noncyclic carbohydrates. Glucose and amylose
showed no catalytic effect on the methanolysis. Cys
and linear-(1!2)-b-D-glucan enhanced the reaction
OH
OH
A
O
1
O
OH
HO
5
6
O
2
4
O
Ha
O
Hb
3
HO
HO
OH
O
HO
OH
OH
O
Hc
HO
HO
OH
OH
H3,6’ H5,4
H2
H6
H1
Hc
Ha
Hb
5.5
5.0
4.5
Chemical Shift (ppm)
4.0
3.5
B
100
75
50
25
0
0
10
20
30
40
Time(h)
Figure 5. 1H NMR spectra of Cys and linear (1!2)-b-D-glucan (A) and time-course of methanolysis of AYEE (B) in the absence (d) or presence of
carbohydrates used as catalysts: Cys (j), linear-(1!2)-b-D-glucan (r), amylose (.), and glucose (m).

278
H. Park, S. Jung / Carbohydrate Research 343 (2008) 274–281
of DP 19 and 22, and DP 17–27 was investigated for the
methanolysis of AYEE by HPLC. We determined that
Cys species with different degrees of polymerization pos-
sess almost the same catalytic properties (data not
shown). The results can be explained by the fact that
Cys has a flexible structure owing to the b-(1!2)-glyco-
sidic linkages and a great water solubility.
tion of intermediates that are produced from the release
of the ethyl group of the substrate. To assess whether
Cys was incorporated into the intermediate, a MAL-
DI-TOF mass spectrometric analysis of the reaction
mixture was performed. In the mass spectrum of the
reaction mixture for the methanolysis of APEE by
Cys, peaks at m/z 2969, 3131, 3293, 3455, 3617, 3779,
3941, 4103, and 4266 were newly detected (Fig. 7A).
These molecular ions correspond to the results from a
m/z shift of 189, which could be attributed to the acyl
moiety (N-acetyl-L-phenylalaninyl) of APEE, as com-
pared to the native Cys with DP 17–25. Through the
mass analysis, these sequential peaks were proposed to
be acyl intermediates formed on Cys (Fig. 7A). New
peaks for the potential acyl intermediates of AWEE
(Fig. 7B) and AYEE (data not shown) were also de-
tected, showing m/z shift of 228 and 205, respectively.
In conclusion, we have described that Cys, as a cyclic
catalytic carbohydrate, isolated from a soil microorgan-
ism, Rhizobium meliloti, catalyzed the methanolysis of
four ethyl esters at 60 ꢁC. Among the tested substrates,
Cys enhanced the methanolysis of AYEE with the high-
est value of kcat_Cys/kuncat. A comparison of the esters of
N-acetyl amino acids and EPA indicated that Cys
showed a catalytic effect with preference for the sub-
strates containing an N-acetamido group. We also inves-
tigated the effect of nonaqueous organic solvents on the
methanolysis of AYEE. The best solvent was the most
hydrophobic solvent, hexadecane. In addition, possible
acyl intermediates formed on Cys during reaction were
revealed by MALDI-TOF mass spectrometric analysis,
suggesting that Cys-catalyzed methanolysis likely pro-
ceeds through a nucleophilic pathway with an intermedi-
ate formation step.
2.3. Effect of nonaqueous media on the methanolysis of
ethyl esters catalyzed by Cys
The effects of eight organic solvents on the methanolysis
of AYEE which had the highest value of kcat_Cys/kuncat
were investigated by HPLC. As a control experiment,
MeOH was used as both a substrate and an organic sol-
vent
(kuncat = 0.034 minꢀ1
,
kcat_Cys = 5.55 minꢀ1).
HPLC analysis revealed that the reaction progress in
each organic solvent was different. Cys was catalytically
active (kcat_Cys = 6.30 minꢀ1) and enhanced the methan-
olysis of AYEE about 180-fold (kcat_Cys/kuncat) in dry
hexadecane. Similar kinetic analyses were carried out
for the Cys-catalyzed methanolysis of AYEE in other
organic solvents. The effect of each organic solvent
was analyzed by comparing the values of krel, which is
the ratio of kcat_Cys in each organic solvent to kcat_Cys
in MeOH. The values of krel broadly correlated with
the logarithm of the octanol–water (logP) partition
coefficient for a given solvent as shown in Figure 6. This
plot shows that Cys-catalyzed methanolysis of AYEE is
dependent on the nature of the solvent, and that there is
a correlation with the hydrophobicity of the organic sol-
vent. The best solvents were water-immiscible hydro-
phobic solvents, while the worst were hydrophilic
solvents.
2.4. Potential acyl intermediates formed on Cys during the
methanolysis
3. Experimental
3.1. Materials
The methanolysis of the ethyl esters by a nucleophile can
occur through a nucleophilic pathway with the forma-
N-Acetyl-L-phenylalanine ethyl ester (APEE) and N-
acetyl-L-tyrosine ethyl ester (AYEE) were obtained from
Sigma Chemical Co. (St. Louis, MO, USA) and Fluka
Chemical Co. (St. Gallen, Switzerland), respectively.
N-Acetyl-L-tryptophan ethyl ester (AWEE), ethyl phen-
ylacetate (EPA), D2O (99.9 atom % D), and CDCl3
(99.9 atom % D) were purchased from Aldrich Chemical
Co. (Milwaukee, WI, USA). All the other chemicals
containing anhydrous glucose, amylose, and anhydrous
organic solvents were purchased from Sigma Chemical
Co. (St. Louis, MO, USA).
1.3
1
1
0.7
2
3
4
0.4
6
7
0.1
5
8
-0.2
-2
0
2
4
6
8
10
log P
3.2. General experimental procedures
Figure 6. The effects of organic solvents on the methanolysis of AYEE
(1, hexadecane; 2, octane; 3, carbon tetrachloride; 4, toluene; 5,
benzene; 6, tetrahydrofuran; 7, acetonitrile; 8, dioxane).
NMR spectra were recorded in D2O or CD3Cl with a
Bruker AMX spectrometer (operated at 500 MHz for

H. Park, S. Jung / Carbohydrate Research 343 (2008) 274–281
279
A
O
O
O
NHAc
HO
HO
Acyl-Cys 1
[Cys + 2H + Na]+
DP 22, m/z 3589
100
75
50
25
0
[Acyl-Cys 1 + 3H + Na]+
DP22, m/z 3779
DP 21
DP 23
*
DP 20
DP 25
DP 24
*
DP 26
*
*
*
DP 18 DP 19
*
DP 17
*
*
3000
3500
4000
4500
Mass (m/z)
[Cys + 2H + Na]+
B
100
75
50
25
0
DP 22, m/z 3589
[Acyl-Cys 2 + 2H + Na]+
DP22, m/z 3817
*
*
*
*
*
*
*
3000
3500
4000
4500
Mass (m/z)
Figure 7. MALDI-TOF mass spectra of reaction mixtures containing Cys and potential acyl intermediates (Acyl-Cys 1 for APEE and Acyl-Cys 2 for
AWEE) of the methanolysis of APEE (A) and AWEE (B). Each signal at m/z 2778, 2941, 3103, 3265, 3427, 3589, 3752, 3914, 4076, and 4238
corresponds to the DP of 17–26 Cys cationized with one sodium ion, respectively. The asterisks indicate the sequential peaks, which were proposed to
be Acyl-Cys 1 (A) containing an N-acetyl phenylalanine group (m/z 2969, 3131, 3293, 3455, 3617, 3779, 3941, 4103, and 4266) and Acyl-Cys 2 (B)
containing an N-acetyl tryptophan group (m/z 3170, 3332, 3494, 3656, 3817, 3980, 4143, and 4303). The chemical structure of a possible Acyl-Cys 1 is
shown in the square box.
1H and 125 MHz for 13C) at 25 ꢁC. All NMR measure-
ments were performed with 0.7-mL samples in 5-mm
NMR tubes. Tetramethylsilane (TMS, Me4Si) was used
as an external reference, and chemical shifts were cali-
brated with an accuracy of 0.05 ppm. A Jupiter C18 col-
umn (5 lM, 250 · 4.60 mm) was used for HPLC
(Shimadzu, Japan) experiments. The analysis was car-
ried out at 30 ꢁC and a flow rate of 1 mL/min with the
mobile phase (70:30 water–acetonitrile). Elution was
monitored at 278 nm for AYEE. MALDI-TOF mass

280
H. Park, S. Jung / Carbohydrate Research 343 (2008) 274–281
spectra were obtained with a MALDI-TOF mass spec-
trometer (Voyager-DETM STR Bio-Spectrometry, Ap-
plied Biosystems, Framingham, MA, USA) in the
positive-ion mode using 2,5-dihydroxybenzoic acid
(DHB) as the matrix. Approximately 0.5 lL of the sam-
ple/matrix mixture was applied to the MALDI probe,
and the solvent was removed by evaporation.
lyzed by NMR spectroscopy or HPLC. The values of
kcat_Cys and kuncat were calculated by integration of the
1
respective H NMR signals, or HPLC peaks were as-
signed to the ethyl ester and methyl ester. To measure
the possible acyl intermediates, each ethyl ester of N-
acetyl amino acid (200 mM) was dissolved in 1 mL of
MeOH, and Cys (14 mg) was then added. At 3 h the
reaction mixture was completely evaporated at room
temperature and subjected to extraction after adding
1 mL of water and chloroform, respectively. The inter-
mediates dissolved in the water layer were then analyzed
by MALDI-TOF mass spectrometry.
3.3. Preparation of cyclosophoraoses (Cys)
R. meliloti were cultured in a 5-L jar fermenter contain-
ing glutamic acid and mannitol salts (GMS) medium to
a late logarithmic phase at 30 ꢁC.28,29 Cells were har-
vested by centrifugation (8000 rpm at 4 ꢁC) and then ex-
tracted with 75% (v/v) ethanol at 70 ꢁC for 30 min. After
centrifugation, the supernatant was concentrated on a
vacuum rotary evaporator. The concentrated sample
was chromatographed on a Sephadex G-50 column
(3 · 130 cm) at a rate of 1 mL/min, and eluant fractions
(7 mL) were assayed for carbohydrates by the phenol–
sulfuric acid method. The fractions containing Cys were
pooled, concentrated, and desalted using a Sephadex G-
10 column (2 · 20 cm). The desalted sample was then
applied to a column (2 · 20 cm) of DEAE-cellulose to
separate neutral and anionic Cys. After the neutral
Cys was desalted using a Sephadex G-10 column
(2 · 20 cm), they were also confirmed on thin-layer chro-
matography (TLC, 5:5:4 BuOH–EtOH–water), NMR
spectroscopy, and MALDI-TOF mass spectrometry.
For further separation, each cyclosophoraose with dif-
ferent DP was fractionated by preparative HPLC (LC-
6AD, Shimadzu, Japan). The column used was a Bond-
clone C8 column (10 lM, 250 · 21.2 mm), and the eluant
was 96:4 water–MeOH with flow rate of 4 mL/min.
Acknowledgment
This research was supported by the National R&D pro-
ject for Biodiscovery in MOST 2004, SDG.
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