Facile synthesis of chiral N-glycosylated amino acids

Article abstract of DOI:10.1007/s11164-010-0133-6

Five designed chiral glycosylated amino acids have been synthesized for the first time by coupling of 1,3,4,6-tetra-O-acetyl-β-D-glucosamine sulfate (2), previously prepared by direct acetylation of D-glucosamine hydrochloride with acetic anhydride, with

Full text of DOI:10.1007/s11164-010-0133-6

Res Chem Intermed (2010) 36:237–243
DOI 10.1007/s11164-010-0133-6
Facile synthesis of chiral N-glycosylated amino acids
•
•
•
Lin Fa Wang Ling Qiang Kong Li Fan
Da Cheng Yang
Received: 27 September 2009 / Accepted: 12 January 2010 / Published online: 11 March 2010
Ó Springer Science+Business Media B.V. 2010
Abstract Five designed chiral glycosylated amino acids have been synthesized for
the first time by coupling of 1,3,4,6-tetra-O-acetyl-b-D-glucosamine sulfate (2),
previously prepared by direct acetylation of D-glucosamine hydrochloride with
acetic anhydride, with chiral Fmoc-protected amino acids and DIC, HOBt, and
DIEA under mild conditions. The structures of these new compounds were char-
1
acterized by IR, H NMR, and 13C NMR spectroscopy and ESI MS.
Keywords N-Glycosylated amino acid Á Fmoc-protected amino acid Á
Glucosamine Á Chiral
Introduction
Most of the proteins occurring in nature are glycosylated with O and N-linked
oligosaccharides [1]. Carbohydrate moieties in glycoproteins play a crucial role in a
number of biological processes including cell recognition, cell adhesion, infection,
and tumor metastasis [2]. Glycosylation of peptides has also been shown to increase
proteolytic stability and to promote blood brain barrier (BBB) permeability [3, 4].
Glycosylation-enhanced crossing of the blood–brain barrier has been observed with
nonnatural glycosylation of enkephalin peptides [5, 6]. However, despite the
biological relevance of glycopeptide and, more generally, glycoconjugate structures,
the preparation of such compounds by chemical synthesis is still a challenging and
laborious task.
Glucosamine, a natural component of glycoproteins found in connective tissues
and gastrointestinal mucosal membranes, has therapeutic potential for the treatment
of a variety of diseases, including arthritis, inflammatory bowel disease, and general
L. F. Wang Á L. Q. Kong Á L. Fan Á D. C. Yang (&)
College of Chemistry and Chemical Engineering, Southwest University, Chongqing 400715, China
e-mail: hxydc@swu.edu.cn
123

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L. F. Wang et al.
OH
OAc
OAc
O
(1)
O
(2)
OAc
O
HO
HO
AcO
AcO
AcO
AcO
OH
OAc
NH2.HCl
NH
R
NH2.H2SO4
3
1
2
O
3a: R=CH3, 3b: R= CH(CH3)2, 3c: R= CH2CH(CH3)2, 3d: R=CH(CH3)CH2CH3
3e: R=CH2C6H5
H
FmocHN
Scheme 1 The synthetic route to the target compounds. Reagents (1) Ac2O, H2SO4, (2) Fmoc-protected
amino acid, Et3N, DIC, HOBt, DIEA
inflammatory damage [7, 8]. 1,3,4,6-tetra-O-acetyl-b-D-glucosamine, a key inter-
mediate in the synthesis of derivatives of D-glucosamine, is obtained by selective
protection of 1,3,4,6-hydroxyl groups of D-glucosamine. Various methods have
been reported for synthesis of the intermediate:
(1) all the groups of D-glucosamine were protected with acetic anhydride, and the
amino protection group was then removed selectively [9];
(2) different protecting groups were used to protect the amino group and four
hydroxyl group, and then the amino-protecting group was removed [10–13];
and
(3) one-pot synthesis of 1,3,4,6-tetra-O-acetyl-b-D-glucosamine sulfate with
acetic anhydride under acidic conditions[14, 15].
Of these three methods, the conditions used for the first method are harsh. The
second method is inconvenient because of relatively long reaction steps. Compar-
atively, the third method has convenient operation, low cost, and good yield.
In our study the third method was further improved to obtain 2; this was then
coupled with protected amino acids to produce the designed new target derivatives
3, which will be used as synthetic building blocks of glycopeptides. The synthetic
route is shown in Scheme 1.
Experimental
Synthesis of 1,3,4,6-tetraacetyl-b-D-glucosamine sulfate (2)
To a stirred solution of acetic anhydride (11.3 mL), cooled in an ice-bath, was
added D-glucosamine hydrochloride (2.6 g, 12 mmol). After stirring for 10 min,
sulfuric acid (1 mL) was added dropwise to the ice-cold reaction mixture and
stirring at room temperature was continued for 1 day. Much white solid appeared
after dropwise addition of ethanol (5 mL) to the reaction mixture at 0 °C. The
reaction mixture was filtered, then the residue was washed with ethyl acetate until
the smell of acetic anhydride disappeared. The white product obtained was dried in
vacuo to yield 74.6%. The compound was characterized by determination of melting
point, IR spectroscopy, and MS. 1,3,4,6-Tetra-O-acetyl-b-D-glucosamine sulfate
(II): IR (cm-1): 3459, 3108 (mN–H), 2992, 2942 (mC–H), 1754 (mC=O), 1622 (dN–H),
123

Facile synthesis of chiral N-glycosylated amino acids
239
1238 (s, br, mC–O–C), 1036 (s, mC–O–C); ESI MS: 370.2 (M? ? 23); 371.2
(M? ? 23 ? 1).
A general method for synthesis of 3
A solution of protected amino acid (1.3 mmol), DIC (204 mg, 1.62 mmol), HOBt
(220 mg, 1.62 mmol), and DIEA (168 mg, 1.3 mmol) in THF (5 mL) was stirred in
a round-bottomed flask at 0 °C for 1 h. A solution of D-glucosamine sulfate
(579 mg, 1.3 mmol in 5 mL THF) containing triethylamine (263 mg, 2.6 mmol)
was then added. After stirring for 12 h, the solvent was removed under reduced
pressure. The solid residue was dissolved in 20 mL ethyl acetate and washed with
0.1 M hydrochloric acid (2 9 10 mL) and 5% potassium bicarbonate solution
(2 9 15 mL). The organic layer was dried over Na2SO4 and the volume was
reduced to 4 mL and purified via flash chromatography (SiO2) with ethyl acetate
and petroleum ether as eluent. The chemical structures of these products were
identified by IR and NMR spectroscopy and MS.
Results and discussion
Considering the esterification of all the hydroxyl groups of D-glucosamine and
protection of amino group, our initial efforts focused on the amount of acetic
anhydride (Table 1) and sulfuric acid used (Table 2). The experimental results
showed that the optimum molar ratio of 1/Ac2O/H2SO4 was 1:10:1.6. It was found
that when the molar ratio of Ac2O/1 was 6:1 a lower yield was obtained. This was
because the least amount of acetic anhydride was unfavorable to dissolution of
D-glucosamine hydrochloride, and less white product was precipitated when ethanol
was added dropwise to the reaction mixture. When the ratio was 12:1, not only was
more ethyl acetate needed to wash the residue, but the yield also decreased. The
effect of temperature on the reaction was then investigated (Table 3). Although the
yield was almost same at 25 or 40 °C, the reaction at 40 °C was faster than that at
25 °C. In a series of exploratory experiments the highest yield was 91.5%,
validating the optimum reaction condition.
A comparative study of the coupling reaction between glucosamine and protected
amino acids was performed using different solvents (Table 4) and use of different
coupling reagents for amide bond formation, for example TBTU, HATU, DIC, and
Table 1 Effect on experimental results of the amount of acetic anhydride used
Entry
1/Ac2O (mol/mol)
Yield (%)
m.p. (°C)
1
2
3
4
1:6
38
64
75
71
168.0–170.1
168.2–169.6
167.8–169.5
167.6–170.0
1:8
1:10
1:12
r.t., 1/H2SO4 = 1:1.6 (molar ratio)
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L. F. Wang et al.
Table 2 Effect on experimental results of the amount of sulfuric acid used
Entry
1/H2SO4 (mol/mol)
Yield (%)
m.p. (°C)
1
2
3
4
1:1.1
1:1.3
1:1.6
1:1.8
64.1
68.4
74.6
73.3
168.2–171.2
167.5–170.9
167.6–169.3
167.5–172.1
r.t., 1/Ac2O = 1:10 (molar ratio)
Table 3 Effect on experimental results of reaction temperature
Entry
T (°C)
Yield (%)
m.p. (°C)
1
2
3
4
10
25
40
60
71.1
75.4
76.3
72.1
167.6–169.7
167.9–170.1
168.2–170.3
161.2–169.4
1/Ac2O/H2SO4 = 1:10:1.6 (molar ratio)
Table 4 The effect of different solvents on the coupling reaction of glucosamine with Fmoc-Ala-OH
Entry
Solvent
Yield (%)
1
2
3
4
DCM
THF
46
61
54
32
CH3CN
DMF
Reagents: Fmoc-Ala-OH (0.5–1.5 mmol), GlcAc4-NH2ÁH2SO4 (1 equiv), TBTU/HOBt (1 equiv), Et3N
(2 equiv), DIEA (1 equiv)
Table 5 The effect of different coupling reagents on the reaction of glucosamine with Fmoc-Ala-OH
Entry
Coupling reagent
Yield (%)
1
2
3
4
DCC
31
74
59
47
DIC
TBTU
PyBOP
Reagents: Fmoc-Ala-OH (0.5–1.5 mmol), GlcAc4-NH2ÁH2SO4 (1 equiv), THF (5 mL), HOBt (1 equiv),
Et3N (2 equiv), DIEA (1 equiv)
PyBOP (Table 5). The experimental results showed that the coupling reaction of
Fmoc-Ala-OH with tetraacetyl-protected glucosamine worked well using HOBt and
DIC as coupling reagents in THF. During selection of the solvents it was found
experimentally that for fewer by-product solvents with low dielectric constant
should be used as far as possible to dissolve the reactants. Using the same reaction
condition, four other N-glycosylated amino acids target compounds were obtained
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Facile synthesis of chiral N-glycosylated amino acids
241
7
8
OCOCH3
H
19
6
4
18
17
H
5
O
7
8
7
8
3a, R= CH3;
20
H3COCO
H3COCO
2
OCOCH3
3
1
3b, R= CH(CH3)2;
3c, R= CH2CH(CH3)2;
3d, R= CH(CH3)CH2CH3;
3e, R=CH2C6H5;
21
21
7
O
16
H
8
H
N
12
20
19
O
13
11
H
NH
15
10
9
14
16
17
R
O
18
Scheme 2 The structures of the target compounds
by coupling 2 with four other protected amino acids (Fmoc-Leu-OH, Fmoc-IIe-OH,
Fmoc-Val-OH, and Fmoc-Phe-OH) (Scheme 2); the yields were 71, 78, 58, and
83% respectively.
3a: Fmoc-Ala-NH-Glucose (Ac): m.p. 198.6–202.3 °C; 1H NMR (300 MHz,
DMSO-d6, d ppm): 7.94 (d, 1H, J = 8.3 Hz, H-9), 7.89 (d, 2H, J = 7.4 Hz, H-20),
7.72 (d, 2H, J = 7.3 Hz, H-17), 7.48 (d, 1H, J = 8.0 Hz, H-12), 7.42 (t, 2H,
J = 7.4 Hz, H-19), 7.33 (t, 2H, J = 7.3 Hz, H-18), 5.92 (d, 1H, J = 3.0 Hz, H-1),
5.20 (t, 1H, J = 10.1 Hz, H-3), 5.02 (t, 1H, J = 9.5 Hz, H-4), 4.26–4.01 (m, 8H,
H-2,5,6,11,14,15), 2.19, 2.02, 1.99, 1.93 (s, each, 3H, H-8), 1.5 (d, 3H, J = 6.8 Hz,
CH3); 13C NMR (75 MHz, DMSO-d6, d ppm): 173.4 (C-10), 170.1, 169.9, 169.2,
169.1 (C-7), 155.6 (C-13), 144.0, 143.8 (C-16), 140.7 (C-21), 127.7 (C-17), 127.1
(C-20), 125.3 (C-18), 120.1 (C-19), 89.7 (C-1), 70.1 (C-3), 69.1 (C-5), 67.9 (C-4),
65.6 (C-14), 61.4 (C-6), 49.9 (C-2), 49.7 (C-11), 46.7 (C-15), 20.9, 20.5, 20.4 (C-8),
18.3 (CH3); IR (cm-1): 3341 (mN–H), 3068 (m=C–H), 2986, 2908 (mC–H), 1754 (mC=O),
1676 (mO=C–N), 1604, 1451 (mC=C), 1236 (s, br, mC–O–C), 1044 (s, mC–O–C); ESI MS:
663.3 ([M ? Na]?), 679.2 ([M ? K]?).
3b: Fmoc-Val-NH-Glucose (Ac): m.p. 199.6–204.8 °C; 1H NMR (300 MHz,
DMSO-d6, d ppm): 7.96 (d, 1H, J = 8.2 Hz, H-9), 7.88 (d, 2H, J = 7.4 Hz, H-20),
7.73 (d, 2H, J = 7.3 Hz, H-17), 7.50 (d, 1H, J = 8.0 Hz, H-12), 7.42 (t, 2H,
J = 7.4 Hz, H-19), 7.33 (t, 2H, J = 7.3 Hz, H-18), 6.01 (d, 1H, J = 3.1 Hz, H-1),
5.17 (t, 1H, J = 10.1 Hz, H-3), 4.97 (t, 1H, J = 9.5 Hz, H-4), 4.37–4.03 (m, 8H,
H-2,5,6,11,14,15), 2.21–2.32 (m, 1H, CH (CH3)2), 2.16, 2.01, 1.96, 1.94 (s, each,
3H, H-8), 0.92 (d, 6H, J = 5.93 Hz, CH (CH3)2); 13C NMR (75 MHz, DMSO-d6, d
ppm): 173.3 (C-10), 170.0, 169.8, 169.2, 169.1 (C-7), 155.7 (C-13), 144.0, 143.7
(C-16), 140.8 (C-21), 127.7 (C-17), 127.1 (C-20), 125.3 (C-18), 120.1 (C-19), 89.5
(C-1), 70.0 (C-3), 69.1 (C-5), 68.0 (C-4), 65.6 (C-14), 61.4 (C-6), 51.4 (C-2), 51.1
(C-11), 46.7 (C-15), 31.6 (CH), 20.9, 20.5, 20.4 (C-8), 19.0, 17.8 (CH3); IR (cm-1):
3334 (mN–H), 3072 (m=C–H), 2989, 2904 (mC–H), 1753 (mC=O), 1674 (mO=C–N), 1601,
1454 (mC=C), 1237 (s, br, mC–O–C), 1036 (s, mC–O–C); ESI MS: 691.3 ([M ? Na]?),
707.3 ([M ? K]?).
3c: Fmoc-Leu-NH-Glucose (Ac): m.p. 211.5–215.2 °C; 1H NMR (300 MHz,
DMSO-d6, d ppm): 8.01 (d, 1H, J = 8.3 Hz, H-9), 7.89 (d, 2H, J = 7.4 Hz, H-20),
7.72 (d, 2H, J = 7.3 Hz, H-17), 7.48 (d, 1H, J = 8.0 Hz, H-12), 7.42 (t, 2H,
J = 7.3 Hz, H-19), 7.33 (t, 2H, J = 7.2 Hz, H-18), 5.94 (d, 1H, J = 3.3 Hz, H-1),
123

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L. F. Wang et al.
5.19 (t, 1H, J = 10.2 Hz, H-3), 5.02 (t, 1H, J = 9.6 Hz, H-4), 4.28–4.02 (m, 8H,
H-2,5,6,11,14,15), 2.19, 2.03, 1.98, 1.91 (s, each, 3H, H-8), 1.39–1.57 (m, 3H,
CH2CH), 0.87–0.92 (m, 6H, CH3); 13C NMR (75 MHz, DMSO-d6, d ppm):173.2
(C-10), 170.0, 169.7, 169.2, 169.0 (C-7), 155.8 (C-13), 144.1, 143.7 (C-16), 140.7
(C-21), 127.6 (C-17), 127.1 (C-20), 125.3 (C-18), 120.1 (C-19), 89.5 (C-1), 69.8
(C-3), 69.1 (C-5), 68.1 (C-4), 65.5 (C-14), 61.4 (C-6), 52.2 (C-2), 50.1 (C-11), 46.7
(C-15), 40.6 (CH2), 22.8, 21.6 (CH3), 21.13 (CH), 20.9, 20.5, 20.4 (C-8); IR (cm-1):
3322 (mN–H), 2960, 2901 (mC–H), 1754 (mC=O), 1602, 1468 (mC=C), 1236 (s, br,
m
C–O–C), 1032 (s, mC–O–C); ESI MS: 705.3 ([M ? Na]?), 721.2 ([M ? K]?).
3d: Fmoc-IIe-NH-Glucose (Ac): m.p. 205.4–208.3 °C; 1H NMR (300 MHz,
DMSO-d6, d ppm): 7.98 (d, 1H, J = 8.2 Hz, H-9), 7.88 (d, 2H, J = 7.4 Hz, H-20),
7.72 (d, 2H, J = 7.3 Hz, H-17), 7.49 (d, 1H, J = 8.0 Hz, H-12), 7.42 (t, 2H,
J = 7.3 Hz, H-19), 7.33 (t, 2H, J = 7.3 Hz, H-18), 5.96 (d, 1H, J = 3.2 Hz, H-1),
5.18 (t, 1H, J = 10.1 Hz, H-3), 5.01 (t, 1H, J = 9.5 Hz, H-4), 4.35–4.03 (m, 8H, H-
2,5,6,11,14,15), 2.18, 2.10 (s, each, 3H, H-8), 1.97–2.01 (m, 7H, H-8 and CH),
1.18–1.46 (m, 2H, CH2), 0.89–0.95 (m, 6H, CH3); 13C NMR (75 MHz, DMSO-d6, d
ppm):173.3 (C-10), 170.0, 169.8, 169.2, 169.1 (C-7), 155.7 (C-13), 144.0, 143.7
(C-16), 140.7 (C-21), 127.7 (C-17), 127.1 (C-20), 125.2 (C-18), 120.1 (C-19), 89.5
(C-1), 69.9 (C-3), 69.1 (C-5), 68.1 (C-4), 65.6 (C-14), 61.4 (C-6), 53.5 (C-2), 50.0
(C-11), 46.7 (C-19), 38.5 (CH), 23.6 (CH2CH3), 20.8, 20.5, 20.4 (C-8), 15.7
(CHCH3), 11.7 (CH2CH3); IR (cm-1): 3342 (mN–H), 2962, 2925 (mC–H), 1756 (mC=O),
1608, 1475 (mC=C), 1234 (s, br, mC–O–C), 1038 (s, mC–O–C); ESI MS: 705.3
([M ? Na]?).
3e: Fmoc-phe-NH-Glucose (Ac): m.p. 207.5–209.1 °C; 1H NMR (300 MHz,
DMSO-d6, d ppm): 8.17 (d, 1H, J = 8.7 Hz, H-9), 7.88 (d, 2H, J = 7.50 Hz, H-20),
7.65–7.61 (m, 2H, ArH), 7.43–7.19 (m, 10H, H-12 and ArH), 5.98 (d, 1H,
J = 3.3 Hz, H-1), 5.24 (t, 1H, J = 10.1 Hz, H-3), 5.03 (t, 1H, J = 9.8 Hz, H-4),
4.29–3.99 (m, 8H, H-2,5,6,11,14,15), 2.89–2.84 (m, 1H, CH2), 2.78–2.51 (m, 1H,
CH2), 2.21, 2.03, 1.99, 1.91 (s, each, 3H, H-8); 13C NMR (75 MHz, DMSO-d6, d
ppm): 173.4 (C-10), 170.0, 169.8, 169.2, 169.1 (C-7), 155.7 (C-13), 143.8, 143.6
(C-16), 140.6 (C-21), 137.7, 129.1, 128.0, 126.3 (C6H5), 127.6 (C-17), 127.0 (C-20),
125.2 (C-18), 120.0 (C-19), 89.5 (C-1), 69.9 (C-3), 69.1 (C-5), 68.0 (C-4), 65.6
(C-14), 61.3 (C-6), 55.6 (C-11), 50.1 (C-2), 46.5 (C-15), 20.8, 20.5, 20.4 (C-8); IR
(cm-1): 3341 (mN–H), 3071 (m=C–H), 2984, 2914 (mC–H), 1756 (mC=O), 1676 (mO=C–N),
1608, 1457 (mC=C), 1238 (s, br, mC–O–C), 1045 (s, mC–O–C); ESI MS: 739.3
([M ? Na]?), 755.3 ([M ? K]?).
Conclusion
Five designed chiral glycosylated amino acids were firstly synthesized in the yield
of 58–83% under mild conditions. One practical synthesis of 2 was developed with
promising application industrially for its convenient operation, low cost and stable
high yield. All of these will lay foundation to a certain extent for the scale synthesis
of glycoproteins.
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243
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