New approach for the efficient synthesis of carbamate-tethered glycosylated amino acids and their insertion into peptides

Article abstract of DOI:10.1055/s-2008-1032087

An efficient two-step route for the synthesis of carbamate-tethered glycosylated amino acids by coupling of oxycarbonyl chlorides derived from side-chain hydroxyl group of N^α-Fmoc-Ser, Thr, Tyr and homoserine (Hser) with appropriately protected

Full text of DOI:10.1055/s-2008-1032087

496
LETTER
New Approach for the Efficient Synthesis of Carbamate-Tethered
Glycosylated Amino Acids and Their Insertion into Peptides
S
ynthesis of Carba
.
mate-Tether
P
edGlycosylate
.
d
A
minoA
c
H
ids emantha, Vommina V. Sureshbabu*
Peptide Research Laboratory, Department of Studies in Chemistry, Central College Campus, Dr. B. R. Ambedkar Veedhi, Bangalore Uni-
versity, Bangalore 560 001, India
Fax +91(80)22292848; E-mail: hariccb@rediffmail.com
Received 29 October 2007
nooximes,8 amides,9 thioamides,10 phosphorus linkages,11
Abstract: An efficient two-step route for the synthesis of carba-
triazoles,12 and carbamates.13–17 The present communica-
tion describes a simpler and efficient synthesis of the car-
bamate-tethered glycosylated amino acids and their
mate-tethered glycosylated amino acids by coupling of oxycarbonyl
chlorides derived from side-chain hydroxyl group of Na-Fmoc-Ser,
Thr, Tyr and homoserine (Hser) with appropriately protected sugar-
1-amine has been described. The utility of these neoglycoamino ac- inclusion into peptides.
ids as building blocks for the synthesis of neoglycopeptides pos-
sessing the carbamate moiety has been demonstrated.
Out of the two reports on the synthesis of carbamate-teth-
ered glycosylated amino acids, Ichikawa et al. used a cou-
pling reaction between a sugar isocyanate and the side-
chain hydroxyl group of N-protected Ser.18 This protocol
involves separation of isomers of the sugar isocyanates
and the establishment of stereochemistry at the anomeric
carbon of the sugar moiety. Alternatively, the one-pot
synthesis of glycopeptidyl carbamates was reported start-
ing from sugar-1-acid.19 The sugar-1-acyl azide, generat-
ed using DPPA, was subjected to the Curtius
rearrangement to obtain the corresponding isocyanate,
and then coupled with N-protected amino alcohol. The
retrosynthetic analysis for the insertion of the carbamate
group between the sugar and the hydroxyl group of an
amino acid leads to yet another approach (Scheme 1).
Thus, the reaction of an oxycarbonyl chloride with an
amine also furnishes a carbamate bond.
Key words: neoglycopeptides, oxycarbonyl chlorides, carbamates,
carbamate-tethered glycosylated amino acids, carbonates
Glycopeptides1 have gained much synthetic focus owing
to their roles in a range of diverse biochemical processes
such as cell recognition, adhesion, and signaling.2 Glyco-
sylation also enhances the bioavailability of peptides
through increasing the solubility in biological systems.3 A
result of these studies has been the development of glyco-
peptide mimetics called neoglycopeptides.4 They serve as
useful probes for understanding biorecognition processes
carried out by the carbohydrate-binding protein receptor.5
Naturally occurring O-linked glycopeptides contain a
sugar attached to peptide back bone through the hydroxyl
group of Ser/Thr.6 Recently, much attention has been paid
to glycopeptide mimics where the natural glycosydic link-
age is replaced by non-native bonds such as ureas,7 ami-
Herein we report a simple approach for the synthesis of
carbamate-tethered glycosylated amino acids. The strate-
R
H
O
O
N
R
R
R
O
COOMe
FmocHN
c
a
b
R
R
R
O
O
O
NCO
COOH
NH2
R
R
R
R
R
R
R
R
R
+
+
+
Cl
OH
OH
O
O
COOMe
COOMe
COOMe
FmocHN
FmocHN
FmocHN
Scheme 1 Synthesis of carbamate-tethered glycosylated amino acids: (a) Ichikawa’s approach; (b) Ikegami’s one-pot approach using DPPA;
(c) present strategy
SYNLETT 2008, No. 4, pp 0496–0500
x
x.
x
x.
2
0
0
8
Advanced online publication: 12.02.2008
DOI: 10.1055/s-2008-1032087; Art ID: D35107ST
© Georg Thieme Verlag Stuttgart · New York

LETTER
Synthesis of Carbamate-Tethered Glycosylated Amino Acids
497
Table 1 List of Na-Fmoc-aminooxy Carbonyl Chloride Esters and Corresponding Pentafluorophenyl Carbonates
Entry Fmoc-aminooxycarbonyl
Yield Mp
HRMS
Pentafluorophenyl-carbonate 3
Yield Mp
HRMS
chloride ester 1
(%)
(°C)
[M + Na]
Obsd/(Calcd)
(%)
(°C)
[M + Na]
Obsd/(Calcd)
O
Cl
O
O
426.8014
(426.8028)
F5
574.4051
(574.4054)
O
O
1
2
92
89
97
108
COOMe
COOMe
FmocHN
FmocHN
1a
3a
O
O
O
Cl
502.8981
(502.8988)
650.5007
(650.5014)
F5
88
85
80
78
96
94
95
110
137
121
O
O
COOBz
COOBz
FmocHN
FmocHN
3b
1b
O
O
Cl
O
440.8285
(440.8294)
588.4324
(588.4320)
F5
3
4
113
111
O
O
COOMe
COOMe
FmocHN
FmocHN
1c
3c
O
O
O
Cl
516.9260
(516.9253)
664.5270
(664.5279)
F5
O
O
COOBz
COOBz
FmocHN
FmocHN
1d
3d
O
O
O
Cl
O
F5
502.8993
(502.8988)
650.5021
(650.5014)
O
5
6
82
85
86
92
91
114
140
FmocHN
FmocHN
COOMe
COOMe
1e
3e
O
O
F5
Cl
O
O
O
440.8299
(440.8294)
588.4316
(588.4320)
118
COOMe
FmocHN
COOMe
FmocHN
1f
3f
gy involves the reaction between protected sugar-1-amine peratures. On the other hand, the required sugar-1-amines
and oxycarbonyl chlorides derived from Na-Fmoc-Ser, were prepared by following the established procedures.22
Thr, Tyr, and HSer carboxyl esters.
The 2,3,4,6-tetra-O-acetyl/benzoyl-b-D-glycosyl-1-azide
was prepared via the 1-bromo intermediate23 and then re-
duced to the corresponding amine using the catalytic hy-
drogenation.
Starting with serine, the Fmoc group was selected for N-
protection due to the varied advantages associated with it.
The a-COOH was converted into either the methyl ester
or the benzyl ester. The Na-Fmoc-Ser-OMe was treated In the next stage, 1 was coupled with sugar-1-amine in the
with triphosgene20 in the presence of pyridine at –20 °C in presence of N-methylmorpholine (NMM). In a typical re-
a well-ventilated hood (Scheme 2). The conversion of the action, Na-Fmoc-Ser(OCOCl)OMe (1a, 1 mmol) was re-
hydroxyl group into its oxycarbonyl chloride was com- acted with tetra-acetyl-D-glucosyl-1-amine in presence of
plete in about two hours. The IR analysis of the reaction NMM at 0 °C (Scheme 2). The coupling was complete in
mixture had a peak at 1770 cm–1 corresponding to the new 30 minutes and a simple workup led to the isolation of
carbonyl group. After a simple workup, the crude product neoglycocarbamate conjugate 2 in quantitative yield. A
was recrystallized as an off-white solid to obtain Na- single recrystallization with ethyl acetate–hexane elevat-
Fmoc-Ser(OCOCl)OMe in excellent yield (94%) as an ed the product purity to above 99% as judged by HPLC.
analytically pure sample.21 The procedure was successful- No much significant change either in the reaction time or
ly utilized for the synthesis and isolation of oxycarbonyl the product yield was observed when the above reaction
chloride derivatives of Thr, Tyr, and HSer as solid prod- was repeated with other bases such as pyridine, Et3N, and
ucts in satisfactory yield and purity (Table 1). Scaling up DMAP. Further, the coupling of 1 with three other sugar
of the protocol up to 25 mmol per batch went smoothly. amines as well as that of sugar-1-amines with oxycarbon-
These synthons were found to be stable without any no- yl chlorides derived from Fmoc-Thr, Tyr, and HSer esters
ticeable decomposition when stored under subzero tem- was accomplished (2a–i).24
Synlett 2008, No. 4, 496–500 © Thieme Stuttgart · New York

498
H. P. Hemantha, V. V. Sureshbabu
LETTER
OH
This is done by the synthesis of active carbonates by treat-
ing with a phenol. When Na-Fmoc-Ser(OCOCl)OMe (1a)
was treated with pentafluorophenol in the presence of
NMM, the corresponding carbonate 3a precipitated out as
a solid which was collected by filtration (Scheme 2). Sim-
ilarly, the pentafluorophenyl carbonates 3b–f of other
amino acids were also made (Table 1).25 In the following
step, the pentafluorophenyl carbonates were also coupled
with sugar-1-amine in the presence of NMM which yield-
ed the carbamate-linked glycosylated amino esters 2a–i in
quantitative yield in about an hour after a simple workup.
FmocHN
COOMe
triphosgene,
pyridine
OH
Cl
O
O
O
F5
F5
O
O
NMM
Finally, the glycosylated amino acid carbamates were uti-
lized for peptide-chain elongation along both N- as well as
C-terminals. In the first series, the Fmoc group was re-
moved from 2a using 20% diethyl amine (DEA) in
CH2Cl2 and the resulting free amine was directly coupled
with Fmoc-Ala-OH via the mixed anhydride method. The
reaction was complete in an hour and the resulting Na-
Fmoc-Ala-Ser(OCONH-2,3,4,6-tetra-O-acetyl-b-D-glu-
copyranosyl)OMe (4a) was isolated as a pure solid by
flash chromatography using 40% ethyl acetate in hexane.
Consistent results in both yield and purity were recorded
when the above procedure was repeated to obtain another
two glycosylated peptidyl carbamates 4b,c extended on
N-side. In the following work, Na-Fmoc-Thr(OCONH-
FmocHN
COOMe
FmocHN
COOMe
3
1
R
O
R
R
NMM, r.t.
85–90%
H2N
R
R
H
O
R
R
N
O
R
O
FmocHN
COOMe
2
R = OAc, OBz
2,3,4,6-tetra-O-acetyl-b-D-galactopyranosyl)OBz
(2e)
was subjected to catalytic hydrogenation using Pd/C and
the resulting free acid was coupled to H-Phe-OBz to ex-
tend the peptide chain on C-terminal of the carbamate-
tethered glycosylated amino acid. The product Na-Fmoc-
Thr(OCONH-2,3,4,6-tetra-O-acetyl-b-D-galactopyrano-
syl)Phe-OBz (5a) was isolated after a simple workup fol-
lowed by flash chromatography in good yield and purity
(Figure 1). The same was true when the protocol was re-
peated to synthesize two more peptides in this manner
(5b,c). All the products 4a–c and 5a–c were fully charac-
terized by 1H NMR, 13C NMR, and MS.
Scheme 2 Synthesis of Na-Fmoc-Ser(OCOCl)OMe, pentafluoro-
phenyl carbonate, and their coupling with sugar amine
All the carbamate-tethered glycosylated amino acid esters
were isolated either through recrystallization or using col-
umn chromatography in quantitative yield and purity
(Table 2).
As the oxycarbonyl chlorides need to be stored at lower
temperature, it is desirable to convert them into stable
compounds while retaining the reactivity.
Table 2 List of Carbamate-Tethered Glycosylated Amino Acid Esters 2
Entry Compound 2
Yield Mp
HRMS [M + Na]
(%)
(°C)
Calcd
Obsd
1
2
3
4
5
6
7
8
9
Na-Fmoc-Ser(O-CO-NH-2,3,4,6-tetra-O-acetyl-b-D-glucopyranosyl)OMe (2a)
Na-Fmoc-Ser(O-CO-NH-2,3,4,6-tetra-O-benzoyl-b-D-glucopyranosyl)OMe (2b)
Na-Fmoc-Ser(O-CO-NH-2,3,4,6-tetra-O-acetyl-b-D-galactopyranosyl)OBn (2c)
Na-Fmoc-Thr(O-CO-NH-2,3,4,6-tetra-O-acetyl-b-D-glucopyranosyl)OMe (2d)
Na-Fmoc-Thr(O-CO-NH-2,3,4,6-tetra-O-acetyl-b-D-galactopyranosyl)OBn (2e)
88
80
87
82
80
122
131
147
177
163
197
129
118
190
737.6597 737.6604
985.9372 985.9379
813.7557 813.7550
751.6863 751.6870
827.7822 827.7818
1039.9369 1039.9375
813.7552 813.7648
1102.0063 1102.0075
751.6863 751.6851
Na-Fmoc-Thr(O-CO-NH-2,2¢,3,3¢,4¢,6,6¢-hepta-O-acetyl-b-D-maltopyranosyl)OMe (2f) 84
Na-Fmoc-Tyr(O-CO-NH-2,3,4,6-tetra-O-acetyl-b-D-glucopyranosyl)OMe (2g)
85
Na-Fmoc-Tyr(O-CO-NH-2,2¢,3,3¢,4¢,6,6¢-hepta-O-acetyl-b-D-maltopyranosyl)OMe (2h) 81
Na-Fmoc-Hser(O-CO-NH-2,3,4,6-tetra-O-acetyl-b-D-galactopyranosyl)OMe (2i)
80
Synlett 2008, No. 4, 496–500 © Thieme Stuttgart · New York

LETTER
Synthesis of Carbamate-Tethered Glycosylated Amino Acids
499
(12) Kuijpers, B. H. M.; Groothuys, S.; Keereweer, A. R.;
Quaedflieg, P. J. L. M.; Blaauw, R. H.; Delft, F. L. V.;
Rutjes, F. P. J. T. Org. Lett. 2004, 6, 3123.
(13) Houben-Weyl: Synthesis of Peptides and Peptidomimetics,
Vol. E22c; Goodman, M.; Felix, A.; Moroder, L.; Toniolo,
C., Eds.; Thieme: Stuttgart, New York, 2003.
OAc
H
O
OAc
OAc
N
O
R1
O
OAc
O
FmocHN
COOMe
N
H
R2
(14) Wo, Y.; Kohn, J. J. Am. Chem. Soc. 1991, 113, 687.
(15) Moran, E. J.; Wilson, T. E.; Cho, C. Y.; Cherry, S. R.;
Schultz, P. G. Biopolymers (Pept. Sci.) 1995, 37, 213.
(16) Cho, C. Y.; Moran, E. J.; Cherry, S. R.; Stephans, J. C.;
Fodor, S. P. A.; Adams, C. L.; Sundaram, A.; Jacobs, J. W.;
Schultz, P. G. Science 1993, 261, 1303.
(17) Cho, C. Y.; Youngquist, R. S.; Paikoff, S. J.; Beresini, M. H.;
Hebert, A. R.; Berleau, L. T.; Liu, C. W.; Wemmer, D. E.;
Keough, T.; Schultz, P. G. J. Am. Chem. Soc. 1998, 120,
7706.
4
4a: R1 = H, R2 = Me
4b: R1 = H, R2 = CHMe2
4c: R1 = Me, R2 = H
OAc
O
AcO
AcO
H
N
O
R1
H
AcO
O
N
COOBz
FmocHN
(18) Ichikawa, Y.; Ohara, F.; Kotsuki, H.; Nakano, K. Org. Lett.
2006, 8, 5009.
O
R2
(19) Sawada, D.; Sasayama, S.; Takahashi, H.; Ikegami, S.
Tetrahedron Lett. 2006, 47, 7219.
5
5a: R1 = Me, R2 = Bn
5b: R1 = R2 = Me
5c: R1 = H, R2 = Me
(20) (a) Eckert, H.; Forster, B. Angew Chem., Int. Ed. Engl. 1987,
26, 894. (b) Alternatively, the reaction was also carried out
by purging phosgene in to a solution of Fmoc-Ser-OMe (1.1
mmol) and the resulting Fmoc-Ser(OCOCl)OMe (1a) was
isolated by the removal of solvent. But considering the
health hazards and handling difficulties associated with
phosgene, the use of triphosgene is preferred.
Figure 1 Peptide-chain extension on either terminal of 2
In conclusion, we have developed a simple route for the
synthesis of carbamate-tethered glycosylated amino acids
2 starting from oxycarbonyl chlorides derived from
Fmoc-Ser, Thr, Tyr, and Hser methyl/benzyl esters. Shelf-
stable carbonates 3 were also synthesized on treatment of
oxycarbonyl chlorides 1 with pentafluorophenol which
showed equal reactivity as that of their precursor. Further,
the neoglycosylated amino acids were employed as build-
ing blocks in the preparation of carbamate-tethered
neoglycopeptides.
(21) Procedure for the Synthesis of Na-Fmoc-
Ser(OCOCl)OMe (1a)
To a stirred solution of Na-Fmoc-Ser-OMe (1 mmol) in
anhyd CH2Cl2 (15 mL) at 0 °C was added triphosgene (0.4
mmol) and pyridine (1.5 mmol) and stirring was continued
for 30 min or till the completion of reaction (TLC analysis).
Then it was washed with sat. sodium meta bisulfate solution
(2 × 10 mL), H2O (2 × 10 mL) and dried over anhyd
Na2SO4. The solvent was evaporated under reduced pressure
and the residue was precipitated in EtOAc–hexane in to a
pure solid powder which was filtered and dried.
Selected Spectral Data of Fmoc-Ser(OCOCl)OMe (1a)
Yield 92%; mp 108 °C. 1H NMR (300 MHz, CDCl3): d =
2.40 (m, 1 H), 3.20 (s, 3 H), 3.70 (t, 2 H), 3.90 (d, 2 H), 4.28
(m, 1 H), 5.30 (m, 1 H), 7.10–7.50 (m, 8 H). 13C NMR (300
MHz, CDCl3): d = 43.1, 50.2, 55.4, 67.1, 70.8, 119.0, 122.2,
125.3, 127.0, 141.0, 149.0, 152.0, 156.0, 170.5. IR (KBr):
1698, 1770 cm–1. HRMS: m/z calcd for [M + Na]: 426.8028;
found :426.8014.
Acknowledgment
We thank the Council of Scientific and Industrial research (CSIR)
New Delhi, India, for financial support, Sophisticated Instrument
Facility and Molecular Biophysics Unit at IISc, Bangalore, for ana-
lytical data.
(22) Furniss, B. S.; Hannaford, A. J.; Smith, P. W. G.; Tatchell,
A. R. Vogel’s Textbook of Practical Organic Chemistry;
Addison Wesley Longman Ltd.: London, 1989.
(23) Deng, S.; Gangadharmath, U.; Chang, C. T. J. Org. Chem.
2006, 71, 5179.
References and Notes
(1) Varki, A. Glycobiology 1993, 3, 97.
(2) Dwek, R. A. Chem. Rev. 1996, 96, 683.
(3) (a) Selvador, L. A.; Eloffson, M.; Kihlberg, J. Tetrahedron
1995, 51, 5643. (b) Taylor, C. M. Tetrahedron 1998, 54,
11317. (c) Davis, B. G. Chem. Rev. 2002, 102, 579.
(4) (a) Carbohydrate Chemistry; Boons, G.-J., Ed.; Chapman
and Hall: London, 1998. (b) Dondoni, A.; Marra, A. Chem.
Rev. 2000, 100, 4395.
(24) Preparation of Carbamate-Tethered Glycosylated
Amino Acid Ester 2
To a solution 1 or 3 (1 mmol) in THF (15 mL) was added
glycosyl-1-amine at 0 °C followed by NMM (1.5 mmol) and
the reaction mixture was stirred till the completion of
reaction. The solvent was evaporated and the residue was
taken in CH2Cl2 (15 mL). To this, 10% citric acid solution
(15 mL) was added and the resulting layers were separated.
The organic phase was washed with 10% Na2CO3 solution
(2 × 10 mL), H2O (2 × 10 mL), and brine (15 mL). The
residue, after the removal of solvent, was triturated with
hexane to get the product as white crystalline solid.
Spectral Data of Na-Fmoc-Thr(OCONH-2,3,4,6-tetra-O-
acetyl-b-d-glucopyranosyl)OMe (2d)
(5) Arya, P.; Kuttere, K. M. K.; Barkley, A. J. Comb. Chem.
2000, 2, 120.
(6) Kunz, H. Angew. Chem., Int. Ed. Engl. 1987, 26, 294.
(7) Ichikawa, Y.; Nishiyama, T.; Isobe, M. Synlett 2000, 1253.
(8) Cassarco, M. R.; Brown, R. T. J. Org. Chem. 2003, 68, 8853.
(9) McDonald, F. E.; Danishefsky, S. J. J. Org. Chem. 1992, 57,
7001.
(10) Khorlin, A. Y.; Zurabyan, S. E.; Macharadze, R. G.
Carbohydr. Res. 1980, 85, 201.
(11) Gustafson, G. L.; Gander, J. E. Methods Enzymol. 1984, 107,
172.
Yield 82%. 1H NMR (300 MHz, CDCl3): d = 1.2 (d, 3 H),
1.8 (m, 1 H), 2.0 (s, 12 H), 2.31 (m, 1 H), 3.75 (s, 3 H), 4.12
Synlett 2008, No. 4, 496–500 © Thieme Stuttgart · New York

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H. P. Hemantha, V. V. Sureshbabu
LETTER
(25) Preparation of Fmoc-Ser(OCOOPfp)OMe (3a)
(d, 2 H), 4.23 (m, 1 H), 4.45 (m, 5 H), 4.98 (d, 2 H), 5.4 (m,
2 H), 7.40–7.63 (m, 8 H). 13C NMR (300 MHz, CDCl3): d =
17.1, 21.3, 21.8, 22.1, 45.0, 55.7, 62.2, 62.5, 67.0, 68.2, 68.9,
71.1, 71.5, 72.8, 90.5, 118.8, 123.2, 125.9, 127.3, 141.5,
149.0, 152.0, 157.2, 166.0, 168.2, 169.3, 178.0. IR (KBr):
1695, 1762 cm–1. HRMS: m/z calcd for [M + Na]: 751.6863;
found: 751.6870.
Pentafluorophenol (1.1 mmol) was added to a stirred
solution of Fmoc-Ser(OCOCl)OMe (1 mmol) in anhyd
CH2Cl2 (15 mL) at 0 °C followed by NMM (1.5 mmol).
After 30 min the precipitated carbonate was filtered and the
residue was washed with H2O, Et2O, and dried under suction
to afford the product as white solid powder.
Synlett 2008, No. 4, 496–500 © Thieme Stuttgart · New York

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