An expeditious route to GlcNAc-Cbz-Asn by chemo-enzymatic synthesis

Article abstract of DOI:10.1055/s-2002-19355

A short-step route to GlcNAc-Cbz-Asn was developed. Treatment of GlcNAc in sat. aq. NH₄HCO₃ solution and subsequent electorodialytic desalting provided ammonia-free glycosylamine in large quantity. The product was coupled with Cbz-As

Full text of DOI:10.1055/s-2002-19355

LETTER
57
An Expeditious Route to GlcNAc-Cbz-Asn by Chemo-enzymatic Synthesis
An
Expeditious Ro
o
ute to GlcN
A
c-Cbz-A
h
sn by Chemo
i
-enzym
h
atic Synthes
i
is de Tokuda,a Yoichi Takahashi,a Kaori Matoishi,a Yukishige Ito,b Takeshi Sugai*a
a
Department of Chemistry, Keio University, 3-14-1, Hiyoshi, Yokohama 223-8522, Japan
E-mail: sugai@chem.keio.ac.jp
b
RIKEN (The Institute of Physical and Chemical Research), 2-1, Hirosawa, Wako-shi, 351-0198, Japan
Received 10 October 2001
Abstract: A short-step route to GlcNAc-Cbz-Asn was developed.
Treatment of GlcNAc in sat. aq. NH4HCO3 solution and subsequent
electorodialytic desalting provided ammonia-free glycosylamine in
large quantity. The product was coupled with Cbz-Asn -isobutyl
ester -fluoride, and finally, the isobutyl ester was deprotected by
enzyme-catalyzed hydrolysis under mild conditions.
OH
OH
NH4HCO3
/ H2O
O
HO
HO
O
HO
HO
OH
NH2
NHAc
NHAc
2
3
2
electoro-
dialytic
desalting
Key words: amino acids, coupling, enzymes, glycosides, hydroly-
ses
3
lyophilization
H2O
(54%)
+
NH4HCO3
H2O
2 (9%)
NH4HCO3
Increasing demand on N-acetylglucosaminylasparagine
(1a) in the synthesis of glycoproteins and glycopeptides,1
especially endo- -N-acetlylglucosaminidase-catalyzed
chemo-enzymatic synthesis,2 has prompted the develop-
ment of expeditious routes to 1a itself as well as the pro-
tected forms. Here we present a chemo-enzymatic
approach to N-Cbz derivative 1b (Figure 1), which does
not depend on the so-far developed “glycosyl azide” inter-
mediate.3–7 Our scheme has the following two features: 1)
as few synthetic steps as possible; 2) intermediates carry-
ing the least protecting groups.
Scheme 1 Synthesis of Ammonia-free Glycosylamine 3.
This problem was cleanly solved by means of electrodia-
lytic desalting. The concentration of NH4HCO3 was mon-
itored by the measurement of electroconductivity, which
lies in linear relationship as depicted in Figure 2.
10
8
OH
O
H
HO
HO
N
CO2H
NHR
6
AcHN
O
1a : R = H
1b : R = Cbz
4
Figure 1 GlcNAc-Asn and Cbz derivative.
First, glycosylamine 3 was prepared according to Kochet-
kov’s report8 (Scheme 1). There is a great advantage in
Kochetkov’s procedure in that the desired compound 3 is
obtained in only one step from 2. Indeed, treatment of 2 in
a sat. aq. NH4HCO3 solution at 35 °C for 4 days reached
an equilibrium of 3 and 2 in 86:14 ratio. This method of
preparation is very simple; however, there remained a del-
eterious effect of the co-existing NH4HCO3 and H2O,
which show considerable nucleophilicity toward the ami-
no acid acyl donor. Our initial attempts using vacuum
pump-dried workup9 only resulted in a very low yield of
the subsequent coupling step.
2
40
60
0
20
120
80
100
concentration of NH4HCO3 (mmol/L)
Figure 2 Calibration of NH4HCO3 in aq. solution.
The electrodialysis was carried out with a membrane Ac-
220-550 (Asahi Chemical Co.), which allows all ionized
components with less than MW 220 to go through. Typi-
cal example of time course on the decrease of NH4HCO3
is shown in Figure 3. In the preparative-scale experi-
ment,10 99.5% of the initial NH4HCO3 was removed. The
subsequent lyophilization provided a mixture of 3 (54%)
Synlett 2002, No. 1, 28 12 2001. Article Identifier:
1437-2096,E;2002,0,01,0057,0060,ftx,en;Y20901ST.pdf.
© Georg Thieme Verlag Stuttgart · New York
ISSN 0936-5214

58
Y. Tokuda et al.
LETTER
nucleophile,13 glycosylamine 3, since this amine is a free
sugar and, moreover, contaminated with GlcNAc (2, ca.
9%). The acids 5a and 6a were treated with cyanuric
fluoride14 to give stable fluorides 5b (92%)15 and 6b
(90%), respectively.
150
100
50
1500
1000
The coupling reactions were examined in the presence of
NaHCO3, as listed in Table. The complete dissolution of
the glycosylamine 3 in a polar solvent such as DMF was
essential for the efficient progress of the reaction, and the
yield reached as high as 84%.16 The great advantage of
acyl fluoride indeed is the cleanness of the reaction prod-
uct. In contrast, an attempted reaction between 3 and 5a
with a conventional N-hydroxysuccinimide protocol8 pro-
vided the desired product; however, we faced many diffi-
culties throughout the purification of such a polar
polyhydroxy compound from the debris of the coupling
reagents.
500
0
0
0
100
200
time (min)
300
400
Figure 3 Electrodialytic Desalting of NH4HCO3 in the Reaction
Mixture.
Table Coupling Reactions Between 3 and Acyl Fluorides.
Substrate
6b
Solvent
Time (h)
Yield (5)
63
and 2 (9%). Through this step, no interchange between
amine 3 and GlcNAc 2 was observed.
H2O–dioxane (1:5)
CH3CN
2.5
24
1
Next, a selectively protected ( -ester) form 5a11 of N-Cbz-
L-aspartate was prepared according to the reported proce-
dure via a cyclic oxazolidinone 6a.12 Activation of the
acyl donor was accomplished by the conversion to acyl
fluoride,6 expecting enhanced affinity toward the nitrogen
6b
ND
65
6b
DMF
5b
H2O–dioxane (1:3)
dioxane
2.5
24
1
21
5b
ND
84
5b
DMF
O
(HCHO)
CO2H
NHCbz
X
n
Finally, enzyme-catalyzed hydrolysis17 under mild condi-
tions as neutral pH was applied to deprotection of the -
ester, to avoid the decomposition of glycosyl amide and/
or epimerization at the -position of the asparagine moi-
ety. Bacillus licheniformis protease (subsilisin, Sigma)
showed very low activity; in turn, papain-catalyzed
hydrolysis18 worked well on the isobutyl ester to give 1b
in 67% isolated yield (Scheme 2).19 The cyclic ester 1d,
however, was a poor substrate toward either enzyme.
HOOC
O
/ benzene
reflux, 2 h
O CbzN
4
6a : X = OH
6b : X = F
7
pyridine
/ CH2Cl2
i-BuONa
/ i-BuOH
F
X
COOi-Bu
NHCbz
6a
N
N
O
F
N
F
5a : X = OH
5b : X = F
In conclusion, a chemo-enzymatic short-step route to
GlcNAc-Cbz-Asn 1b was established, involving ammo-
nia-free formation of N-acetylglucosaminylamine (3) in a
preparative scale, followed by the coupling with acyl flu-
oride 5b, and papain-catalyzed hydrolysis of the ester pro-
tective group of -carboxyl group at the final step.
7
7
pyridine
/ CH2Cl2
OH
O
O
NaHCO3
DMF
H
HO
HO
3
3
+
6b
5b
N
O
AcHN
O
CbzN
Acknowledgement
1d
This work was accomplished as „Science and Technology Program
on Molecules, Supra-Molecules and Supra-Structured Materials“ of
an Academic Frontier Promotional Project, by Japan’s Ministry of
Education, Culture, Sports, Science and Technology, the Monbu-
kagakusho. This was also supported by a Grant-in-Aid for Scientific
Research, which is acknowledged with thanks.
OH
O
H
HO
HO
+
N
CO2R
NHCbz
AcHN
O
1c : R = i-Bu
1b : R = H
papain, L-cysteine
/ DMF-buffer
Scheme 2 Synthesis of 1b.
Synlett 2002, No. 1, 57–60 ISSN 0936-5214 © Thieme Stuttgart · New York

LETTER
An Expeditious Route to GlcNAc-Cbz-Asn by Chemo-enzymatic Synthesis
59
(dd, 1 H, J = 3.9, 17.1 Hz), 2.85 (dd, 1 H, J = 4.4, 17.1 Hz),
1.86 (m, 1 H), 0.83 (d, 6 H, J = 6.3 Hz); 13C NMR (100 MHz,
CDCl3) 175.83, 170.36, 155.89, 135.84, 128.40, 128.01,
127.96, 71.98, 67.23, 50.24, 36.46, 27.63, 19.06; IR (KBr)
3350, 3127, 2954, 1761, 1727, 1692, 1280, 1224, 1063
cm–1. Its NMR spectrum was in good accordance with that
reported previously.12 No -ester was detected. In contrast,
the ring-opening reaction20 of a cyclic anhydride of N-Cbz-
aspartate with isobutyl alcohol gave a mixture of -ester and
-ester (ca. 10:1, 3.92 for -ester and 3.86 for -ester).
Attempts for enzyme-catalyzed selective hydrolysis21 of
diethyl N-Cbz-aspartate or ethyl N-Cbz-asparagine gave no
fruitful results, such as the formation of a diacid, or entirely
no reaction.
References
(1) (a) Kunz, H. Angew. Chem. Int. Ed. Engl. 1987, 26, 294.
(b) Taylor, C. M. Tetrahedron 1998, 54, 11317. (c) Seitz,
O. CHEMBIOCHEM 2000, 1, 214.
(2) (a) Yamamoto, K.; Kadowaki, S.; Watanabe, J.; Kumagai,
H. Biochem. Biophys. Res. Commun. 1994, 203, 244.
(b) Haneda, K.; Inazu, T.; Yamamoto, K.; Kumagai, H.;
Nakahara, Y.; Kobata, A. Carbohydr. Res. 1996, 292, 61.
(c) Mizuno, M.; Haneda, K.; Iguchi, R.; Muramoto, I.;
Kawakami, T.; Aimoto, S.; Yamamoto, K.; Inazu, T. J. Am.
Chem. Soc. 1999, 121, 284.
(3) Cowley, D. E.; Hough, L.; Peach, C. M. Carbohydr. Res.
1971, 19, 231.
(4) (a) Bolton, C. H.; Jeanloz, R. W. J. Org. Chem. 1963, 28,
3228. (b) Bolton, C. H.; Hough, L.; Khan, M. Y. Biochem. J.
1966, 101, 184.
(12) Yuki, H.; Okamoto, Y.; Taketani, Y.; Tsubota, T.;
Murabayashi, Y. J. Polymer Sci. 1978, 16, 2237.
(13) (a) Carpino, L. A.; Beyermann, M.; Wenschuh, H.; Bienert,
M. Acc. Chem. Res. 1996, 29, 268. (b) Manabe, S.; Ito, Y. J.
Am. Chem. Soc. 1999, 121, 9754.
(5) Matsuo, I.; Isomura, M.; Ajisaka, K. J. Carbohydr. Chem.
1999, 18, 841.
(6) Ito, Y.; Gerz, M.; Nakahara, Y. Tetrahedron Lett. 2000, 41,
1039.
(14) (a) Carpino, L. A.; Sadat-Aalaee, D.; Chao, H. G.; DeSelms,
R. H. J. Am. Chem. Soc. 1990, 112, 9651. (b) Carpino,
L. A.; Mansour, E. M. E. J. Org. Chem. 1992, 57, 6371.
(15) Mp 37.5-40.0 °C (colorless fine needles from CH2Cl2–
hexane); 1H NMR (400 MHz, CDCl3) 7.37 (m, 5 H), 5.72
(d, 1 H, J = 7.3 Hz), 5.14 (d, 1 H, J = 12.2 Hz), 5.10 (d, 1 H,
J = 12.2 Hz), 4.67 (ddd, 1 H, J = 2.4, 4.8, 7.3 Hz), 3.98 (d, 2
H, J = 6.8 Hz), 3.23 (dd, 1 H, J = 2.4, 18.1 Hz), 3.13 (dd, 1
H, J = 4.8, 18.1 Hz), 1.95 (m, 1 H), 0.92 (d, 6 H, J = 6.8 Hz);
13C NMR (100 MHz, CDCl3) 160.90 (d, J = 357 Hz),
169.21, 155.65, 135.67, 128.44, 128.20, 128.01, 72.43,
67.38, 49.98, 35.14 (d, J = 52 Hz), 27.64, 18.93; IR (KBr)
3341, 2961, 1840, 1727, 1691, 1533, 1294, 1269, 1096 cm–1.
(16) To a solution of 3 (782.9 mg, 2.42 mmol) in anhydrous DMF
(15 mL) was added NaHCO3 (383 mg, 4.56 mmol) and
fluoride (5b, 872.8 mg, 2.94 mmol) at room temperature.
After stirring for 2 h, the reaction mixture was filtered
through a Celite pad and washed with 1,4-dioxane. The
filtrate was evaporated and purified by silica gel column
chromatography. Elution with CHCl3–MeOH (5:1) afforded
1c (1.07 g, 84%). Mp 195.0–196.5 °C; [ ]D25 +6.9 (c 0.81,
MeOH); 1H NMR (400 MHz, CD3OD) 7.28 (m, 5 H), 5.09
(d, 1 H, J = 12.2 Hz), 5.05 (d, 1 H, J = 12.2 Hz), 4.93 (d, 1
H, J = 9.8 Hz), 4.58 (dd, 1 H, J = 4.9, 7.3 Hz), 3.88 (d, 2 H,
J = 6.8 Hz), 3.81 (dd, 1 H, J = 1.5, 12.2 Hz), 3.73 (t, 1 H, J
= 9.8 Hz), 3.64 (ddd, 1 H, J = 0.9, 3.4, 12.2 Hz), 3.44 (m, 1
H), 3.30 (m, 2 H), 2.79 (dd, 1 H, J = 4.9, 16.0 Hz), 2.68 (dd,
1 H, J = 7.3, 16.0 Hz), 1.90 (s, 3 H), 1.89 (m, 1 H), 0.90 (d,
6 H, J = 6.8 Hz); 13C NMR (100 MHz, CD3OD) 175.45,
172.73, 172.26, 158.96, 137.83, 129.31, 128.92, 128.82,
80.24, 79.67, 76.24, 72.50, 71.76, 67.74, 62.62, 56.06,
52.14, 38.49, 28.97, 22.92, 19.40; IR (KBr) 3295, 3091,
2957, 1749, 1703, 1654, 1546, 1296, 1271 cm–1. Anal.
Calcd. For C24H35N3O10: C, 54.85; H, 6.71; N, 8.00. Found:
C, 54.59; H, 6.84; N, 7.56.
(7) Inazu, T.; Kobayashi, K. Synlett 1993, 869.
(8) (a) Likhosherstov, L. M.; Novikova, O. S.; Derevitskaja, V.
A.; Kochetkov, N. K. Carbohydr. Res. 1986, 146, C1.
(b) Campa, C.; Donati, I.; Vetere, A.; Gamini, A.; Paoletti,
S. J. Carbohydr. Chem. 2001, 20, 263.
(9) Otvos, L. Jr.; Urge, L.; Hollosi, M.; Rwoblewski, K.;
Gradzyk, G.; Fasman, G. D.; Thurin, J. Tetrahedron Lett.
1990, 31, 5889.
(10) GlcNAc (2, 40.4 g, 182.9 mmol) was dissolved in H2O (220
mL) and saturated with NH4HCO3. The conversion of
GlcNAc into glucosylamine 3 was determined by 1H NMR
spectroscopy by comparing anomeric protons. After stirring
for 4 days at 35 °C, the conversion reached 86%, and the
solution was diluted with H2O (150 mL). The mixture was
desalted by AC-220-550 on Asahi Chemical Micro Acylyzer
S3. At the initial stage, the conductivity was 87.8 mS and
after the desaltation at the 15 V (1.07 A), it reached 0.6 mS.
NH4HCO3 was estimated to be 5.6 mmol/L based on the
calibration linear as described in Figure 2. The yields of 3
and 2 were quantitatively estimated to be 54% and 9%,
respectively, by 1H NMR using methyl -D-glucopyranoside
as internal standard. The mixture was frozen and lyophilized
to give a highly hygroscopic white powder (30.1 g). Again at
this stage, the amounts of 3 and 2 in the solid were estimated
to be 22.3 g (101 mmol) and 3.0 g (14 mmol), respectively.
No decomposition of 3 was observed into 2 and ammonia
through the lyophilization. 1H NMR (400 MHz, D2O) 4.16
(d, 1 H, J = 9.3 Hz), 3.85 (dd, 1 H, J = 2.1, 12.4 Hz), 3.69
(ddd, 1 H, J = 2.1, 4.6, 12.4 Hz), 3.62 (t, 1 H, J = 9.3 Hz),
3.52 (m, 1 H), 3.55-3.40 (m, 2 H), 2.07 (s, 3 H); 13C NMR
(100 MHz, D2O) 175.26, 85.01, 77.63, 75.36, 70.90, 61.71,
57.21, 23.21; IR (KBr) 3352, 1652, 1558, 1375, 1047 cm–1.
(11) Ester 5a was obtained from oxazolidinone 6a12 with sodium
isobutoxide. The alkoxide solution prepared from isobutyl
alcohol (37.8 mL) and sodium (0.83 g, 36.2 mmol) was
added dropwise into the solution of 6a (10.1 g, 36.2 mmol)
in isobutyl alcohol (55 mL) at 65 °C with stirring. After the
stirring was continued for 3 h, AcOH (10 g) was added to the
reaction mixture. Conventional work up procedure and
subsequent silica gel column chromatography of the residue
[CHCl3–MeOH–AcOH (90:3:2)] gave 5a (5.5 g, 47% yield).
Recrystallization from n-hexane–Et2O afforded fine
colorless needles. Mp 65.0–66.0 °C [lit.12 mp 68–69 °C];
[ ]D22 -4.4 (c 1.3, AcOH); [ ]43522 -10.3 (c 1.3, AcOH) [lit.12
[ ]42722 -11.8 (c 1.3, AcOH)]; 1H NMR (400 MHz, CDCl3)
7.28 (m, 5 H), 5.71 (d, 1 H, J = 8.3 Hz), 5.06 (m, 2 H), 4.58
(ddd, 1 H, J = 3.9, 4.4, 8.3 Hz), 3.92 (d, 2 H, J = 6.8 Hz), 3.04
(17) (a) Waldmann, H.; Sebastian, D. Chem. Rev. 1994, 94, 911.
(b) Braun, P.; Waldmann, H.; Kunz, H. Synlett 1992, 39.
(18) (a) Shin, C.; Takahashi, N.; Seki, M. Bull. Chem. Soc. Jpn.
1991, 64, 3575. (b) Hallinan, K. O.; Crout, D. H. G.;
Errington, W. J. Chem. Soc., Perkin 1 1994, 3537.
(c) Miyazawa, T.; Iwanaga, H.; Yamada, T.; Kuwata, S.
Biotechnol. Lett. 1994, 16, 373. (d) Eberling, J.; Braun, P.;
Kowalszyk, D.; Schultz, M.; Kunz, H. J. Org. Chem. 1996,
61, 2638.
(19) The coupling product (1c, 200.9 mg, 0.38 mmol) was
dissolved in a mixture of DMF (2.0 mL) and 0.2 M
phosphate buffer (pH 7.0, 2.0 mL). The pH was adjusted to
7.0 with 2 mol/L HCl, and papain (Sigma, P3375, 56.4 mg,
Synlett 2002, No. 1, 57–60 ISSN 0936-5214 © Thieme Stuttgart · New York

60
Y. Tokuda et al.
LETTER
107 units) and L-cysteine (11.0 mg, 0.09 mmol) were added.
Then the mixture was stirred at 35 °C with a controlled
addition of 0.5 mol/L NaOH to maintain the pH at 7.0. After
stirring for 7 h, the reaction mixture was brought to pH 8.0
with 0.5 mol/L NaOH and then filtered through a Celite pad
and washed with H2O. The filtrate was adjusted to be pH 4.7
by the addition of 2 mol/L HCl, concentrated in vacuo, and
the residue was purified by silica gel column
136.89, 129.48, 129.13, 128.49, 79.09, 78.41, 74.96, 70.31,
68.02, 61.35, 55.17, 51.82, 38.31, 22.91; IR (KBr) 3292,
3073, 1716, 1689, 1655, 1542, 1374, 1256, 1065 cm–1. Anal.
Calcd. For C20H27N3O10: C, 51.17; H, 5.80; N, 8.95. Found:
C, 51.00; H, 5.90; N, 8.75. The NMR spectrum of fully
deprotected form(1a) was identical with that reported
previously.22
(20) (a) Bergmann, M.; Zervas, L.; Salzmann, L. Chem. Ber.
1933, 66, 1288. (b) Le Quesne, W. J.; Young, G. T. J. Chem.
Soc. 1952, 24.
(21) (a) Miyazawa, T.; Minowa, H.; Miyamoto, T.; Imagawa, I.;
Yanagihara, R.; Yamada, T. Tetrahedron: Asymmetry 1997,
8, 367. (b) Matoishi, K.; Sano, A.; Imai, N.; Yamazaki, T.;
Yokoyama, M.; Sugai, T.; Ohta, H. Tetrahedron:
Asymmetry 1998, 9, 1097.
chromatography. Elution with EtOAc–MeOH–H2O (7:2:1)
afforded 1b (119.2 mg, 67%). Analytical sample (colorless
fine needles from acetone–H2O); mp 174.0–176.0 °C(dec)
[lit.4 mp 172–174 °C]; [ ]D20 +8.1 (c 1.00, H2O) [lit.4 [ ]D
25.5
+5.2 (c 1, H2O)]; 1H NMR (400 MHz, D2O) 7.45 (m, 5 H),
5.17 (m, 2 H), 5.09 (d, 1 H, J = 9.8 Hz), 4.57 (dd, 1 H, J =
4.3, 7.8 Hz), 3.89 (dd, 1 H, J = 2.0, 12.2 Hz), 3.82 (dd, 1 H,
J = 9.8, 10.3 Hz), 3.76 (dd, 1 H, J = 4.9, 12.2 Hz), 3.63 (dd,
1 H, J = 8.3, 10.3 Hz), 3.52 (m, 2 H), 2.89 (dd, 1 H, J = 4.4,
(22) Dorland, L.; Schut, B. L.; Vliegenthart, J. F. G.; Strecker, G.;
Foournet, B.; Spik, G.; Montreuil, J. Eur. J. Biochem. 1977,
73, 93.
16.1 Hz), 2.79 (dd, 1 H, J = 7.8, 16.1 Hz), 1.96 (s, 3 H); 13
C
NMR (100 MHz, D2O) 175.39, 173.42, 172.19, 158.47,
Synlett 2002, No. 1, 57–60 ISSN 0936-5214 © Thieme Stuttgart · New York