An efficient synthetic route to N-glycosylamino acids using N α-Fmoc-Asp/Glu-5-oxazolidinone as internal protection

Article abstract of DOI:10.1055/s-2007-986645

A new facile synthetic route to prepare N-glycosylamino acids in fewer steps is reported. 5-Oxazolidinone served as an effective protecting moiety for N^α-Fmoc-Asp/Glu and after glycosylation, the ring opening resulted in free α-carboxylic acid, which can be directly used to extend the chain to obtain N-glycopeptides. Both the minimum number of steps as well as circumvention of orthogonal protection strategy leads to a cost-effective route for their synthesis. This protocol can be easily scaled up to prepare N-glycosylated Asn/Gln acids in large quantities in fairly good yields. Georg Thieme Verlag Stuttgart.

Full text of DOI:10.1055/s-2007-986645

2492
LETTER
An Efficient Synthetic Route to N-Glycosylamino Acids Using
Na-Fmoc-Asp/Glu-5-oxazolidinone as Internal Protection
Synthetic Route
t
a
o
N
-Glycos
o
ylaminoAcids Venkataramanarao, Vommina V. Sureshbabu*
Department of Studies in Chemistry, Central College Campus, Bangalore University, Dr. B. R. Ambedkar Veedhi, Bangalore 560 001, India
E-mail: hariccb@rediffmail.com
Received 9 May 2007
Alternatively, the use of allyl ester was also exploited
Abstract: A new facile synthetic route to prepare N-glycosylamino
which involves the deprotection of allyl group using pal-
acids in fewer steps is reported. 5-Oxazolidinone served as an effec-
ladium(0) or Wilkinson catalyst [(PPh3)3Rh(I)Cl].10,11 An
equally important aspect of this kind of orthogonal strate-
tive protecting moiety for Na-Fmoc-Asp/Glu and after glycosyla-
tion, the ring opening resulted in free a-carboxylic acid, which can
be directly used to extend the chain to obtain N-glycopeptides. Both gy is the generation of the free b/g-carboxyl of Fmoc-Asp/
the minimum number of steps as well as circumvention of ortho-
Glu-Ot-Bu/Allyl/Me after deprotecting the concerned
gonal protection strategy leads to a cost-effective route for their
protecting group of the side chain, and its preparation,
synthesis. This protocol can be easily scaled up to prepare N-glyco-
which itself is a multistep process.
sylated Asn/Gln acids in large quantities in fairly good yields.
Consequently, it is desirable to develop both mild and
Key words: 5-oxazolidinone, glycosyl amine, protecting group, N-
efficient protection and easy deprotection strategy to con-
struct N-glycosylated amino acids in a facile way. In this
context, we envisaged the usage of 5-oxazolidinones as
superior to the corresponding orthogonal protecting
groups12 because (1) they are capable of providing com-
plete protection for both Na- and carboxylic functions; (2)
they are easy to prepare; (3) the b/g-carboxyl functions for
direct coupling are readily available; and (4) mild condi-
tions are sufficient for their cleavage. Further, Fmoc-Asp/
Glu-5-oxazolidinone can be obtained in about 20% higher
yields than the corresponding Boc analogues.
The Na-Fmoc-Asp/Glu-OH were converted into their 5-
oxazolidinones using paraformaldehyde and a catalytic
amount of p-toluenesulfonic acid in toluene under micro-
wave irradiation in 95% yield (Scheme 1).13 In these Na-
Fmoc-Asp/Glu-5-oxazolidinones, the b- or g-carboxylic
acid moiety is now readily available for chemoselective
transformation.14 The carbohydrate partner of the glyco-
peptide was synthesized using the literature procedure
(Scheme 2).15,16 The azido functionality was introduced
using penta-O-acetyl glucosyl pyranose via its 1-bromo
compound and then reduced to obtain the required
glycosyl amine.
glycosylamino acid, coupling agent, building-block strategy
N-Linked and O-linked glycoproteins and glycopeptides
are of significant biological interest due to their wide-
spread role in cell recognition, cell adhesion and tumor
metastasis.1 Glycosylation of peptides has been shown to
increase proteolytic stability and promote blood–brain
barrier permeability.2 Glycosylation also enhances
solubility3–5 and may contribute to the stabilization of
peptide structures. Recent developments in the area of
glycopeptide synthesis have been reviewed.6 It is there-
fore desirable that a convenient and high yielding proce-
dure be available for the routine synthesis of N-
glycopeptides.
In the literature, the commonly employed approach for the
synthesis of glycosylated amino acid makes use of Na-
Fmoc-Asp-Ot-Bu as the key intermediate with b-carboxyl
group being activated using pentafluorophenyl ester or
acid chloride or in situ activation with Dhbt followed by
coupling with glycosylamine to obtain Na-Fmoc-Asn(sug-
ar)-Ot-Bu. The resulting N-glycosylated amino acid has
been utilized as a building block for chain extension after
deprotecting tert-butyl group ester group with TFA fol-
lowed by activation of a-carboxylic group.7 However, the
glycosydic bond is labile to the acidic conditions em-
ployed in the process of tert-butyl deprotection and the
molecule is prone to decomposition. This naturally results
in formation of more contaminants leading to laborious
procedure for purification of the final targets.8 A similar
problem is encountered in the solid-phase peptide synthe-
sis, where TFA is used to cleave glycopeptides from
Wang resin.9
O
O
paraformaldehyde
cat. PTSA
Fmoc
N
Fmoc-HN
O
OH
n
n
MW
COOH
COOH
1
n = 1 or 2
Scheme 1
HO
Ac2O, pyridine
AcOH, HBr
AcO
AcO
O
O
HO
HO
HO
NH2
AcO
OH
NaN3, DMF
Pd/C, H2
AcO
SYNLETT 2007, No. 16, pp 2492–2496
0
1
.1
0
.2
0
0
7
2
Advanced online publication: 12.09.2007
DOI: 10.1055/s-2007-986645; Art ID: G10907ST
© Georg Thieme Verlag Stuttgart · New York
Scheme 2

LETTER
Synthetic Route to N-Glycosylamino Acids
2493
The glucosylamines were then coupled with the b- or g- HBTU/DIPEA to furnish the compound 3 in 90% yield.17
carboxylic acid moiety of Na-Fmoc-Asp/Glu-5-oxazolidi- As an extension of this methodology, several other glyco-
none. The coupling between the glycosyl amine and Na- sylamines were prepared and coupled to the Na-Fmoc-
Fmoc-Asp/Glu-5-oxazolidinone was carried out with Asp/Glu-5-oxazolidinones and in all cases these corre-
different coupling agents. Fmoc-Asp/Glu-5-oxazolidino- sponding glycosylated 5-oxazolidinones were obtained in
nes were converted into pentafluorophenyl ester, ONSu 88–90% yield. Finally, the compound 3 was hydrolyzed
ester, acid chloride and were also directly coupled using back to its acid by treating with LiOH (1 equiv, 1 N aq so-
HBTU/DIPEA. The best results were obtained using lution) in tetrahydrofuran for 30 minutes. These results
Table 1 Synthesis of Na-Fmoc-glycosylated Amino Acids
Entry
Amine
Product
Yield (%)
84
Mass (Calcd/Obsd)
707.2064/707.2080
COOH
Fmoc-HN
AcO
AcO
H
O
NH2
O
N
AcO
AcO
AcO
AcO
1
AcO
AcO
O
2a
4a
AcO
AcO
AcO
AcO
Fmoc-HN
COOH
H
O
N
O
2
3
NH2
82
88
707.2064/707.2134
955.26/955.2
AcO
AcO
AcO
AcO
O
2b
4b
Fmoc-HN
H
COOH
BzO
BzO
BzO
BzO
O
O
NH2
O
BzO
N
BzO
BzO
BzO
O
2c
4c
AcO
AcO
AcO
AcO
AcO
AcO
O
Fmoc-HN
COOH
AcO
AcO
AcO
4
5
6
H
75
78
80
995.29/995.4
O
AcO
O
AcO
N
O
NH2
O
AcO
AcO
AcO
O
2d
2a
4d
Fmoc-HN
COOH
AcO
H
N
721.2221/721.2413
721.2221/721.2455
O
OAc
AcO
AcO
O
4e
Fmoc-HN
COOH
AcO
AcO
H
N
2b
O
AcO
OAc
O
4f
Fmoc-HN
COOH
BzO
H
N
7
8
2c
83
77
969.28/969.3
O
OBz
BzO
BzO
O
4g
2d
1009.30/1009.4
COOH
AcO
Fmoc-HN
O
AcO
AcO
AcO
H
O
AcO
N
O
AcO
OAc
O
4h
Synlett 2007, No. 16, 2492–2496 © Thieme Stuttgart · New York

2494
R. Venkataramanarao, V. V. Sureshbabu
LETTER
O
Fmoc-HN
COOH
HBTU, DIPEA
Fmoc
N
O
LiOH
+
1
2
AcO
AcO
n
AcO
AcO
n
O
HN
O
O
HN
AcO
O
AcO
AcO
AcO
4
3
HBTU, DIPEA
O
R1
Fmoc-HN
N
COOMe
H
AcO
AcO
n
O
HN
O
n = 1 or 2
AcO
AcO
5
Scheme 3
are summarized in Table 1. Initial studies on hydrolysis of In conclusion, we have described in this letter a facile ap-
5-oxazolidinone using aqueous LiOH in methanol result- proach to the synthesis of Na-Fmoc-Asn/Gln-glycosylated
ed in partial deprotection of Fmoc group. Finally, the amino acids. The Na-Fmoc-Asn/Gln-glycosylated amino
same reaction was carried in tetrahydrofuran as a solvent acids can be used directly in the synthesis of glycopep-
which gave pure product. The site-selective cleavage of 5- tides by the building-block strategy. We have adopted Na-
oxazolidinone ring can be attributed to higher lability of Fmoc-5-oxazolidinone moiety for protection of a-amino
5-oxazolidinone ring towards base-catalyzed hydrolysis and carboxylic group as an alternative to the conventional
in the presence of Fmoc or acetyl groups (Scheme 3).18 protection and deprotection strategy for Asp/Glu. Further-
The presence of free acid group in 3 enabled the extension more, all four steps, namely preparation of Fmoc-Asp/
on C-terminus by coupling with amino acid ester using Glu, 5-oxazolidinone, glycosylation, and ring opening,
HBTU/DIPEA that resulted in the formation of N-glyco- are high-yielding reactions. Both the intermediates as well
peptides (Table 2). The glycosylated 5-oxazolidinones as the final products are crystalline solids. The entire
derivatives and their amino acids were analyzed by IR, 1H protocol is completely devoid of the use of any acid
NMR, mass and their purities confirmed using RP-HPLC. treatment. As all the reagents used for protection and
The HPLC profile of glycosylated 5-oxazolidinones 3a deprotection are simple, the overall strategy leads to N-
and 3b contained peak at tR = 22.45 and 21.53 minutes glycosylated Asn/Gln by way of a less expensive route.
whereas the glycoyslated acids 4a and 4e showed sharp The scale-up of this protocol is easy.
peaks at tR values 19.24 and 17.72 minutes, respectively.
All the HPLC profiles were recorded using Eclipse XDB-
C18 column 2.5 with the gradient elution method as
water–acetonirile (10–90%) in 30 minutes.
Table 2 Synthesis of Na-Fmoc-glycosylated Amino Acids
Entry
1
Product
Yield (%)
89
Mass (Calcd/Obsd)
792.2592/792.2514
O
Fmoc-HN
AcO
N
COOMe
H
H
O
N
AcO
AcO
OAc
O
5a
O
AcO
AcO
Fmoc-HN
N
COOMe
H
H
2
90
792.2592/792.2552
O
N
5b
AcO
OAc
O
5b
Synlett 2007, No. 16, 2492–2496 © Thieme Stuttgart · New York

LETTER
Synthetic Route to N-Glycosylamino Acids
2495
Table 2 Synthesis of Na-Fmoc-glycosylated Amino Acids (continued)
Entry
Product
Yield (%)
Mass (Calcd/Obsd)
O
Fmoc-HN
H
N
COOMe
BzO
3
H
78
1068.3531/1068.78
O
BzO
BzO
N
OBz
O
5c
O
Fmoc-HN
N
H
COOMe
AcO
4
5
86
88
806.2748/806.2864
806.2748/806.2912
H
N
O
AcO
AcO
OAc
O
5d
O
Fmoc-HN
AcO
AcO
N
COOMe
H
H
N
O
AcO
OAc
O
5e
O
Fmoc-HN
N
H
COOMe
BzO
6
81
1082.3687/1082.46
H
N
O
OBz
BzO
BzO
O
5f
(8) Wong, S. Y.; Guile, G. R.; Rademacher, T. W.; Dwek, R. A.
Glycoconjugate J. 1993, 10, 227.
(9) (a) Meldal, M. Curr. Opin. Struct. Biol. 1994, 4, 710.
(b) Meldal, M.; Bock, K. Glycoconjugate J. 1994, 11, 59.
(10) Anisfeld, S. T.; Lansbury, P. T. Jr. J. Org. Chem. 1990, 55,
5560.
Acknowledgment
We thank the University Grants Commission, Govt. of India for
financial assistance. R.V. thanks the Council of Scientific and
Industrial Research (CSIR), New Delhi, Govt. of India for the
award of a fellowship.
(11) (a) Likhosherstov, L.; Novikova, O.; Derveitskaja, V. A.;
Kochetkov, N. K. Carbohydr. Res. 1986, 146, C1.
(b) Corey, E. J.; Suggs, J. W. J. Org. Chem. 1973, 38, 3234.
(12) For similar applications of oxazolidinones in the synthesis of
2,3-diaminopropionic acid (2,3-Dap) and 2,4-diamino-
butanoic acid (2,4-Dab). See: (a) Teng, H.; He, Y.; Wu, L.;
Su, J.; Feng, X.; Qui, G.; Liang, S.; Hu, X. Synlett 2006,
877. (b) Ramanarao, R. V.; Tantry, S. J.; Sureshbabu, V. V.
Synth. Commun. 2006, 36, 2912. (c) Teng, H.; Jiang, Z.;
Wu, L.; Su, J.; Feng, X.; Qiu, G.; Liang, S.; Hu, X. Synth.
Commun. 2006, 36, 3803.
(13) For an efficient synthesis of Fmoc-amino acid-5-oxa-
zolidinones employing a rapid protocol under microwave
irradiation, see: (a) Tantry, S. J.; Kantharaju; Sureshbabu,
V. V. Tetrahedron Lett. 2002, 43, 9461. (b) For further
optimized reaction conditions using MeCN as a solvent, see:
Govender, T.; Arvidsson, P. I. Tetrahedron Lett. 2006, 47,
1691.
References and Notes
(1) Hojo, H.; Nakahara, Y. Curr. Protein Pept. Sci. 2000, 1, 23.
(2) (a) Polt, R.; Palian, M. M. Drugs Future 2001, 26, 561.
(b) Polt, R.; Porreca, F.; Szabo, L. Z.; Bilsky, E. J.; Davis, P.;
Abbruscato, T. J.; Davis, T. P.; Horvath, R.; Yamamura, H.
I.; Hruby, V. J. Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 7114.
(3) Middagh, C. R.; Litman, G. W. J. Biol. Chem. 1987, 262,
3671.
(4) Nomoto, M.; Yamaoa, K.; Haga, M.; Hayashi, M. J. Pharm.
Sci. 1998, 87, 326.
(5) (a) Andreotti, A. H.; Kahne, D. J. Am. Chem. Soc. 1993, 115,
3352. (b) Laczko, I.; Hollosi, M.; Urge, L.; Urgen, K. E.;
Weiner, D. B.; Mantzsch, H. H.; Thurin, J.; Otvos, L. Jr.
Biochemistry 1992, 31, 4282.
(6) (a) Hojo, H.; Nakahara, Y. Pept. Sci. 2007, 88, 308.
(b) Haase, C.; Seitz, O. Top. Curr. Chem. 2007, 267, 1.
(c) Liu, L.; Bennett, C. S.; Wong, C.-H. Chem. Commun.
2006, 21.
(7) Cohen-Anisfield, S. T.; Lansbury, P. T. Jr. J. Am. Chem. Soc.
1993, 115, 10531.
(14) Scholtz, J. M.; Bartlett, P. A. Synthesis 1989, 131.
(15) Deng, S.; Gangadharmath, U.; Chang, C. W. T. J. Org.
Chem. 2006, 71, 5179.
Synlett 2007, No. 16, 2492–2496 © Thieme Stuttgart · New York

2496
R. Venkataramanarao, V. V. Sureshbabu
LETTER
(16) (a) Thiem, J.; Wiemann, T. Angew. Chem., Int. Ed. Engl.
1990, 29, 80. (b) Ameijde, J.; Albada, H. B.; Liskamp, R. M.
J. J. Chem. Soc., Perkin Trans. 1 2002, 1042.
4.88 (t, 1 H), 5.06 (t, 1 H), 5.12 (s, 2 H), 5.27 (m, 2 H), 5.50
(br d, 1 H), 7. 33 (t, 2 H), 7.41 (t, 2 H), 7.52 (d, 2 H), 7.77 (d,
2 H). HRMS (ES): m/z calcd for C35H38N2NaO14 [M + Na]+:
733.2221; found: 733.2254.
(17) Typical Procedure for the Synthesis of Glycosylated Na-
Fmoc-Asp/Glu-5-oxazolidinones
(18) Typical Procedure for the Preparation of Glycosylated
To a solution of Fmoc-Asp/Glu-5-oxazolidinone (10 mmol)
in 10 mL of THF was added 1-amino b-glucose (10 mmol)
in THF (40 mL), HBTU (10 mmol), and DIPEA (11 mmol),
and stirred at r.t. for about 30 min. The solvent was
evaporated under reduced pressure and the residue was
diluted with H2O (15 mL) and extracted with EtOAc (3 × 20
mL). The combined organic layer was washed with 10%
citric acid (3 × 10 mL), 5% Na2CO3 solution (3 × 15 mL),
brine, and dried over anhyd Na2SO4. The solution was
concentrated and purified by column chromatography using
EtOAc–hexane (2:8).
Na-Fmoc-Asn/Gln-OH
To the glycosylated Na-Fmoc-Asn/Gln-5-oxazolidinone (10
mmol) dissolved in THF was added 1 N LiOH solution (1
equiv) and stirred at r.t. for 30 min. The reaction was
monitored by TLC. After the completion of reaction, the
reaction mixture was acidified with 10% citric acid solution
and extracted with EtOAc. The organic layer was washed
with brine and dried over anhyd Na2SO4. The solvent was
removed in vacuo, and the resulted residue was purified by
column chromatography using CHCl3, MeOH and AcOH
(40:2:1).
Selected Spectral Data
Compound 4a: white solid. IR (KBr): nmax = 1705, 1733
cm–1. 1H NMR (300 MHz, CDCl3): d = 2.03–2.13 (4 s, 12 H),
2.81 (m, 2 H), 3.80 (m, 1 H), 4.03 (t, 1 H), 4.24 (m, 2 H),
4.42–4.52 (m, 2 H), 4.98 (t, 1 H), 5.05 (t, 1 H), 5.21–5.35 (m,
2 H), 5.92 (br d, 1 H), 7. 33 (t, 2 H), 7.41 (t, 2 H), 7.52 (d, 2
H), 7.77 (d, 2 H). HRMS (ES): m/z calcd for C33H36N2NaO14
[M + Na]+: 707.2064; found: 707.2080.
Compound 4e: white solid. IR (KBr): nmax = 1698, 1732
cm–1. 1H NMR (300 MHz, CDCl3): d = 1.86 (m, 2 H), 2.03–
2.13 (4 s, 12 H), 2.22 (m, 2 H), 3.80 (m, 1 H), 4.03 (d, 1 H),
4.21–4.32 (m, 3 H), 4.48 (m, 2 H), 4.88 (t, 1 H), 5.06 (t, 1 H),
5.27 (2 H, m), 5.50 (br d, 1 H), 7. 33 (t, 2 H), 7.41 (t, 2 H),
7.52 (d, 2 H), 7.77 (d, 2 H). HRMS (ES): m/z calcd for
C34H38N2NaO14 [M + Na]+: 721.2221; found: 721.2413.
Compound Fmoc-Asn(2,3,4,6-tetra-O-acetyl-b-D-gluco-
pyranosyl)oxazolidinone (3a: n = 1): white solid. IR (KBr):
n
max = 1700, 1735, 1801 cm–1. 1H NMR (300 MHz, CDCl3):
d = 2.03–2.13 (4 s, 12 H), 2.81 (m, 2 H), 3.80 (m, 1 H), 4.03
(t, 1 H), 4.24 (m, 2 H), 4.42–4.52 (m, 2 H), 4.98 (t, 1 H), 5.05
(t, 1 H), 5.11 (s, 2 H), 5.21–5.35 (m, 2 H), 5.92 (br d, 1 H),
7. 33 (t, 2 H), 7.41 (t, 2 H), 7.52 (d, 2 H), 7.77 (d, 2 H).
HRMS (ES): m/z calcd for C34H36N2NaO14 [M + Na]+:
719.2064; found: 719.2081.
Compound Fmoc-Gln(2,3,4,6-tetra-O-acetyl-b-D-gluco-
pyranosyl)oxazolidinone (3b: n = 2): white solid. IR (KBr):
n
max = 1698, 1730, 1800 cm–1. 1H NMR (300 MHz, CDCl3):
d = 1.86 (m, 2 H), 2.03–2.13 (4 s, 12 H), 2.22 (m, 2 H), 3.80
(m, 1 H), 4.03 (d, 1 H), 4.21–4.32 (m, 3 H), 4.48 (m, 2 H),
Synlett 2007, No. 16, 2492–2496 © Thieme Stuttgart · New York