Novel ester linked glycosyl amino acids: Convenient building blocks for the synthesis of glycopeptide libraries

Article abstract of DOI:10.1016/S0957-4166(99)00013-0

The completely orthogonally protected aspartic acid derivative FmocAsp(OBn)O'Bu is readily synthesized on a large scale. Deprotection of the β-carboxylic acid allows coupling to various sugar derivatives via free hydroxyl groups to produce novel glycosyl amino acids. Subsequent deprotection of either the α-acid or nitrogen is achieved cleanly to allow elaboration into an oligopeptide, whilst selective deprotection of PMB protected sugar hydroxyls is also readily achievable. Such novel glycosyl amino acid building blocks may be useful for the combinatorial synthesis of novel glycopeptide libraries.

Full text of DOI:10.1016/S0957-4166(99)00013-0

TETRAHEDRON:
ASYMMETRY
Pergamon
Tetrahedron: Asymmetry 10 (1999) 391–401
Novel ester linked glycosyl amino acids: convenient building
blocks for the synthesis of glycopeptide libraries
Richard J. Tennant-Eyles and Antony J. Fairbanks ∗
Dyson Perrins Laboratory, Oxford University, South Parks Road, Oxford OX1 3QY, UK
Received 27 November 1998; accepted 11 January 1999
Abstract
The completely orthogonally protected aspartic acid derivative FmocAsp(OBn)OtBu is readily synthesized on a
large scale. Deprotection of the β-carboxylic acid allows coupling to various sugar derivatives via free hydroxyl
groups to produce novel glycosyl amino acids. Subsequent deprotection of either the α-acid or nitrogen is achieved
cleanly to allow elaboration into an oligopeptide, whilst selective deprotection of PMB protected sugar hydroxyls
is also readily achievable. Such novel glycosyl amino acid building blocks may be useful for the combinatorial
synthesis of novel glycopeptide libraries. © 1999 Elsevier Science Ltd. All rights reserved.
1. Introduction
The oligosaccharide chains of naturally occurring glycoproteins are almost invariably either N- or O-
linked to the anomeric position of the terminal sugar residue.1 Thus the sugars of N-linked glycoproteins
are bound to the peptide backbone via an asparagine residue, which is β-linked through nitrogen to C-1
of a terminal N-acetylglucosamine residue. The synthesis of N-linked glycopeptides,2 therefore, usually
requires the availability of a suitably protected aspartic acid residue which is most commonly coupled
to an N-acetylglucosamine residue which possesses a β-nitrogen at the anomeric position. In contrast,
the oligosaccharides of naturally occurring O-linked glycoproteins are usually linked α to the hydroxyl
of a serine or threonine residue. Synthetic approaches to such materials usually commence with an α-
glycosidation of a serine or threonine hydroxyl.3
The biological activity of glycopeptides has aroused considerable synthetic interest, both in the
construction of naturally occurring materials themselves and also in the synthesis of analogues; a
particularly recent trend is the synthesis of C-linked versions.4 Consideration of analogue synthesis by
structural variation reveals myriad potential targets. This molecular diversity, taken hand in hand with
∗
Corresponding author.
0957-4166/99/$ - see front matter © 1999 Elsevier Science Ltd. All rights reserved.
PII: S0957-4166(99)00013-0

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R. J. Tennant-Eyles, A. J. Fairbanks / Tetrahedron: Asymmetry 10 (1999) 391–401
the potential biological activity, makes the synthesis of libraries of novel glycopeptides an attractive
proposition for the combinatorial chemist.
This possibility of structural variation immediately opens up other modes of linking of the oligosac-
charide chain to the peptide backbone. Perhaps the most obvious modification of the naturally occurring
type of structure is that rather than to link the sugar chain to an amino acid via the anomeric position,
that it could be simply linked via a hydroxyl group of one of the sugar units — conveniently via an
ester bond. Since it is well documented that glycosylation can affect both the biological activity5 and
hydrolytic stability6 of peptides it would also be interesting to investigate any effects that such forms of
non-natural glycosylation may have.
Although studies on the acylation of sugar hydroxyls with the free carboxylic acids of amino acids were
first undertaken nearly 40 years ago,7 and the chemical8 or enzymatic9 synthesis of this type of compound
has subsequently been reported many times, there are only a very few reports in the literature where
sugars have been linked via ester bonds to amino acid side chains.10 The synthesis of novel glycopeptides
containing sugar units which are linked through their hydroxyl groups to the peptide backone via ester
bonds therefore requires a suitably protected aspartic or glutamic acid residue as a linker unit. This
paper details the facile and large scale synthesis of a new completely orthogonally protected aspartic acid
residue and details subsequent manipulations which will allow the synthesis of novel types of O-linked
glycopeptides. In particular, selective deprotection of the β-carboxyl allows standard diimide mediated
coupling to free hydroxyl groups of selectively protected sugars to produce novel O-linked glycosyl
amino acids. Subsequent selective deprotection of either the α-carboxylic acid or nitrogen will allow
extension of the peptide chain in either direction. Selective deprotection of the sugar hydroxyl groups by
oxidative means may also be readily accomplished. All of these transformations can be easily performed
without cleavage of the sugar–amino acid linkage. It is envisaged that these building blocks may be used
for the solid phase combinatorial synthesis of libraries of novel glycopeptides, using standard procedures.
2. Results and discussion
2.1. Synthesis of protected amino acids
Although many protected aspartic acid derivatives are commercially available they are generally very
expensive compared to the parent amino acid.11 Since large amounts of orthogonally protected material
were required for use in many later subsequent synthetic routes the synthesis of the protected amino
acid 1 was undertaken directly from L-aspartic acid, following standard procedures. A literature search
rather surprisingly revealed that the aspartic acid derivative 1 has not been previously described, and its
synthesis is therefore detailed as follows. Refluxing L-aspartic acid with benzyl alcohol and tosic acid
in benzene produced the known dibenzyl ester salt 2.12 Selective hydrolysis of the α-ester yielded the
acid 3,13 which was then converted to the t-butyl α-ester 4.14 Finally, simple Fmoc protection of the
nitrogen produced the orthogonally protected material 1 (Scheme 1). Simple catalytic hydrogenation
allows deprotection of the β-carboxyl in very high yields to yield the free acid 5,15 a standard material
used in N-glycopeptide synthesis. It should be noted that hydrogenation of the benzyl ester is fast and no
competitive loss of the Fmoc nitrogen protection was observed during this reaction (vide infra).
2.2. Coupling reactions to sugar hydroxyls
Initial studies were carried out in the manno series. The known16 selectively protected methyl
mannoside 6 was coupled to the β-carboxyl of 5 with DCC and DMAP in CH2Cl2 to yield the glycosyl

R. J. Tennant-Eyles, A. J. Fairbanks / Tetrahedron: Asymmetry 10 (1999) 391–401
393
Scheme 1. Reagents: (i) TsOH, BnOH, benzene, reflux, 67% (ii) CuSO4, NaOH, EtOH, H2O, 32°C then EDTA, 100°C, 63%
(iii) conc.H2SO4, isobutylene dioxan, 53% (iv) FmocCl, Na2CO3, dioxan, H2O, 95% (v) H2, Pd, EtOH, 95%
amino acid 7 in an acceptable 72% yield (Scheme 2). No resort to more expensive water soluble coupling
reagents was necessary as purification of 7 by standard flash chromatography allowed straightforward
removal of any urea impurities. Since it was envisaged that further sugar units may be added by
glycosidation of an anomerically activated glycopeptide a similar coupling reaction was performed on
thioglycoside 8.17 This proceeded directly analogously to give the thioglycosyl amino acid 9 in 75%
yield. Finally, since it was envisaged that selective deprotection of the sugar hydroxyls may be desirable,
and that problems may be encountered during hydrogenation of benzyl protecting groups on sugars linked
to Fmoc protected amino acids (or indeed benzyl groups of thioglycosides), then similar coupling was
undertaken for the PMB protected methyl glycoside 10.18 This again proceeded cleanly to produce the
PMB protected derivative 11 in 72% yield.
Scheme 2. Reagents (i) 5, DCC, DMAP, CH2Cl2, 72% (ii) 5, DCC, DMAP, CH2Cl2, 75% (iii) 5, DCC, DMAP, CH2Cl2, 72%
2.3. Subsequent protecting group manipulations and peptide bond formation
In all cases removal of either the Fmoc or t-butyl ester amino acid protecting groups was achieved
in high yield under standard conditions, with no loss of the sugar unit via hydrolysis of the ester linker.
Thus treatment of 7 with piperidine in DMF gave the free amine 12 in 88% yield. Treatment of 9 with
TFA/water resulted in quantitative hydroylsis of the t-butyl ester to give the free acid 13 (Scheme 3).
Selective deprotection of benzylated sugar hydroxyls by simple hydrogenation proved problematic. In
all cases not only was loss of the Fmoc nitrogen protection competitive but also considerable amounts
of cleavage of the sugar–acid ester bond were observed, even under slightly acidic conditions. However,

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R. J. Tennant-Eyles, A. J. Fairbanks / Tetrahedron: Asymmetry 10 (1999) 391–401
Scheme 3. Reagents (i) piperidine, DMF, 83% (ii) CF3CO2H, CH2Cl2, quantitative (iii) CAN, CH3CN:H2O, 9:1, CH2Cl2, 80%
(iv) piperidine, DMF (v) Fmoc-glycine, EEDQ, benzene, 95% over two steps
oxidative deprotection of PMB groups was found to occur rapidly and cleanly. Thus treatment of 11 with
ceric ammonium nitrate in acetonitrile/water yielded the triol 14 in 80% yield.
Finally, in order to demonstrate that peptide bond formation of such glycosyl amino acids is completely
straightforward, the triol amino acid 14 was coupled with Fmoc protected glycine. The two step coupling
of 14, involving initial piperidine induced Fmoc removal and subsequent EEDQ mediated coupling with
the Fmoc protected glycine, yielded the dipeptide 15 in 95% overall yield (Scheme 3).
3. Summary and conclusion
To summarize, we have synthesized several novel glycosyl amino acids, in which the sugar residue
is linked to the amino acid via an ester bond of one of the sugar hydroxyls; in this particular instance
mannose linked to aspartic acid. Also investigated and detailed are the necessary subsequent selective
protecting group manipulations which will allow further elaboration of these materials in any of three
senses: namely removal of nitrogen or carboxyl protection of the amino acid to allow peptide bond
formation, or removal of the sugar hydroxyl group protection to allow further functionalization and/or
glycosidation. In all cases it is possible to achieve these transformations without cleavage of the
sugar–amino acid linkage. Finally, the two step EEDQ mediated coupling to Fmoc-glycine is found to
proceed in high yield, demonstrating the feasibility of incorporation of these ester-linked glycosyl amino
acids into synthetic oligopeptides.
Details of further investigations, both on the use of such building blocks for the assembly of libraries
of novel glycopeptides, and of other potential applications of these materials, will be published in due
course.

R. J. Tennant-Eyles, A. J. Fairbanks / Tetrahedron: Asymmetry 10 (1999) 391–401
395
4. Experimental
Melting points were recorded on a Kofler hot block. Proton nuclear magnetic resonance (δH) spectra
were recorded on Varian Gemini 200 (200 MHz), Bruker AC 200 (200 MHz), Bruker DPX 400 (400
MHz) or Bruker AMX 500 (500 MHz) spectrometers. Carbon nuclear magnetic resonance (δC) spectra
were recorded on a Varian Gemini 200 (50.3 MHz). Multiplicities were assigned using DEPT sequence.
All chemical shifts are quoted on the δ scale. Infrared spectra were recorded on a Perkin–Elmer
150 Fourier Transform spectrophotometer. Mass spectra were recorded on VG Micromass 30F, ZAB
1F, Masslab20-250, Micromass Platform 1 APCI, or Trio-1 GCMS (DB-5 column) spectrometers,
using desorption chemical ionization (NH3 DCI), electron impact (EI), chemical ionization (NH3 CI),
atmospheric pressure chemical ionization (APCI), and fast atom bombardment (FAB) techniques as
stated. Optical rotations were measured on a Perkin–Elmer 241 polarimeter with a path length of 1
dm. Concentrations are given in g/100 ml. Hydrogenations were run under an atmosphere of hydrogen
gas maintained by inflated balloon. Microanalyses were performed by the microanalytical service of the
Dyson Perrins laboratory. Thin layer chromatography (TLC) was carried out on Merck glass-backed
sheets, pre-coated with 60F254 silica. Plates were developed using 0.2% w/v cerium (IV) sulfate and 5%
ammonium molybdate in 2 M sulfuric acid. Flash chromatography was carried out using Sorbsil C60
40/60 silica. Solvents and available reagents were dried and purified before use according to standard
procedures; methanol was distilled from magnesium methoxide, dichloromethane was distilled from
calcium hydride, pyridine was distilled from calcium hydride and stored over potassium hydroxide, and
tetrahydrofuran was distilled from a solution of sodium benzophenone ketyl immediately before use.
Hexane was distilled at 68°C before use to remove involatile fractions.
4.1. L-Aspartic acid-dibenzyl-ester-p-toluenesulphonate 2
L-Aspartic acid (50 g, 376 mmol), p-toluenesulphonic acid monohydrate (82.2 g, 432 mmol) and
benzyl alcohol (90.2 ml, 872 mmol) were refluxed in benzene (60 ml) with a Stark–Reed condensor.
After 3 h the reaction mixture was allowed to cool to room temperature and diethyl ether (600 ml) was
added. The mixture was kept in a refrigerator overnight. The resulting crystals were filtered and washed
with cold diethyl ether (200 ml) to give the tosylate salt 2 (121.9 g, 67%) as a white waxy solid; m.p.
152–154°C (hexane/ethyl acetate) [lit.12 m.p. 157–158°C]; [α]D22 +7.04 (c, 1.25 in CHCl3) [lit.12 [α]D
20
+0.9 (c, 1.0 in MeOH)]; δH (200 MHz, CDCl3): 2.27 (3H, s, MePhSO2), 3.13 (2H, m, CH2), 4.49 (1H,
m, CH), 4.89–5.01 (4H, m, 2×CH2Ph), 7.01, 7.72 (4H, 2×d, MePhSO2), 7.14–7.37 (10H, m, Ar).
4.2. L-Aspartic acid-β-benzyl-ester 3
The tosylate salt 2 (121.9 g, 251 mmol) and copper sulphate (251 g in 800 ml water, 1.004 mol) were
stirred in ethanol (450 ml). The pH of the reaction mixture was raised to pH 8 by addition of 5 M NaOH,
and then stirred at 32°C for 1 h. 3 M HCl was then added until the mixture reached pH 3. The resulting
precipitate was filtered and washed with distilled water, ethanol and diethyl ether and then dissolved
in distilled water (1000 ml). EDTA (103 g, 277 mmol) was added and the solution boiled for 10 min,
filtered and allowed to cool to room temperature. The precipitated solid was then filtered and washed
with distilled water (100 ml), ethanol (100 ml) and diethyl ether (100 ml) to give the α acid 3 (35.5 g,
63%) as a white solid; m.p. 209°C (water) [lit.13 m.p. 218–219°C]; [α]D +6.9 (c, 0.89 in H2O) [lit.13
22
[α]D20 +7.73 (c, 1.0 in MeCO2H)]; δH (200 MHz, DMSO): 2.47 (2H, s, NH2), 2.58 (1H, dd, JCH ,CH 5.2
2

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R. J. Tennant-Eyles, A. J. Fairbanks / Tetrahedron: Asymmetry 10 (1999) 391–401
7.6 Hz, CH0 ), 3.48 (1H, m, CH), 5.07 (2H, s, CH2Ph),
2
0
0
Hz, JCH ,CH 16.8 Hz, CH2), 2.88 (1H, dd, J
CH2,CH
2
2
7.35 (5H, m, Ar).
4.3. L-Aspartic acid-β-benzyl-α-tert-butyl ester 4
A mixture of the α acid 3 (28.79 g, 124 mmol) and concentrated sulphuric acid (12 ml) in 1,4-dioxane
(120 ml) was stirred in a pressure bottle at −25°C. Isobutylene gas (56 ml, 618 mmol) was then condensed
into the reaction vessel which was immediately stoppered tightly and allowed to equilibrate to room
temperature. After 20 h the reaction vessel was cooled to −25°C and the pressure released. The reaction
mixture was diluted with 1 M sodium hydroxide (600 ml) and diethyl ether (200 ml). The aqueous phase
was extracted with diethyl ether (3×200 ml) and the combined organic extracts dried (MgSO4), filtered
22
and concentrated in vacuo to give the α-tert-butyl ester 4 (18.01 g, 53%) as a clear oil; [α]D +1.4 (c,
0.29 in CHCl3), [lit.14, [α]D22 +7 (c, 1.0 in ethyl acetate)]; δH (200 MHz, CDCl3): 1.44 (9H, s, (CH3)3C),
5.0 Hz, CH0 ), 3.73 (1H, dd,
CH2,CH
2
0
0
2.74 (1H, dd, JCH ,CH 5.0 Hz, J
7.2 Hz, CH2), 2.79 (1H, dd, J
CH2,CH2
2
CH), 5.13 (2H, s, CH2Ph), 7.37 (5H, m, Ar).
4.4. N-(9-fluorenylmethoxycarbonyl)-L-aspartic acid-β-benzyl-α-tert-butyl ester 1
9-Fluorenylmethoxychloroformate (4.437 g, 17 mmol) was added to a stirred solution of the α-tert-
butyl ester 4 (4.785 g, 17 mmol) and sodium carbonate (9.089 g, 86 mmol) in 1,4-dioxane:water (3:5,
80 ml), at 0°C. The reaction mixture was allowed to equilibrate to room temperature. After 16 h, TLC
(hexane:ethyl acetate, 1:1), indicated complete conversion of the starting material (Rf 0.1) to a single
product (Rf 0.8). The reaction mixture was diluted with ethyl acetate (400 ml) and acidified to congo
red with saturated potassium hydrogensulphate solution. The aqueous layer was extracted with ethyl
acetate (200 ml) and the combined organic extracts washed with brine (200 ml) and distilled water (200
ml), then dried (MgSO4), filtered and concentrated in vacuo. The residue was purified by flash column
chromatography (hexane:ethyl acetate, 3:1) to give the fully protected amino acid 1 (8.18 g, 95%) as
26
a white crystalline solid, m.p. 82–85°C (diethyl ether/hexane); [α]D +26.9 (c, 1.0 in CHCl3); νmax
(KBr disk): 3355 (b, NH), 1721 (s, 3×C_O) cm−1; δH (400 MHz, CDCl3): 1.44 (9H, s, (CH3)3C), 2.90
4.6 Hz, CH02), 4.23 (1H, ap-t,
0
0
(1H, dd, JCH ,CH 4.5 Hz, JCH ,CH 17.0 Hz, CH2), 3.05 (1H, dd, J
CH2,CH
2
2
2
JCH[Fmoc],CH [Fmoc] 7.2 Hz, CH[Fmoc]), 4.39 (2H, m, CH2[Fmoc]), 4.56 (1H, m, JCH,NH 8.4 Hz, CH), 5.16
2
(2H, ABq, J 12.2 Hz, CH2Ph), 5.78 (1H, d, NH), 7.27–7.43, 7.60, 7.72 (13H, m, 2×d, CH2Ph, Ar[Fmoc]);
δC (50.3 MHz, CDCl3): 27.8 (q, (CH3)3C), 36.9 (t, CH2), 47.1, 50.9 (2×d, CH, CH[Fmoc]), 66.8, 67.2
(2×t, CH2Ph, CH2[Fmoc]), 120.2, 125.4, 127.4, 127.9, 128.6, 128.9 (6×d, Ar), 135.7, 141.6, 144.0, 144.2
(4×s, Ar), 156.3, 169.9, 171.1 (3×s, 3×C_O); m/z (CI+): 502.2 (M+H+, 15), 224.1 (35), 178.1 (100%).
(Found: C, 71.88; H, 6.24; N, 2.78. C30H31O6N requires C, 71.84; H, 6.23; N, 2.79%.)
4.5. N-(9-fluorenylmethoxycarbonyl)-L-aspartic acid-α-tert-butyl ester 5
A solution of the fully protected amino acid 1 (18.84 g, 37.0 mmol) in ethanol (200 ml) was
stirred under an atmosphere of hydrogen in the presence of palladium black (200 mg). After 8 h, TLC
(hexane:ethyl acetate, 1:3) indicated complete conversion of the starting material (Rf 0.7) to a single
product (Rf 0.6). The reaction mixture was filtered through Celite and concentrated in vacuo to give the
22
25
mono acid 5 (15.225 g, 99%) as a white foam; [α]D +13.8 (c, 1.0 in CHCl3), [lit.15, [α]D −16.9 (c,
0
1.0 in DMF)]; δH (200 MHz, CDCl3): 1.48 (9H, s, (CH3)3C), 2.91 (1H, dd, JCH ,CH 4.4 Hz, JCH ,CH
2
2
2

R. J. Tennant-Eyles, A. J. Fairbanks / Tetrahedron: Asymmetry 10 (1999) 391–401
397
4.2 Hz, CH0 ), 4.26 (1H, d, JCH[Fmoc],CH [Fmoc] 7.6 Hz, CH[Fmoc]),
2
2
0
17.3Hz, CH2), 3.04 (1H, dd, J
CH2,CH
4.39 (2H, m, CH2[Fmoc]), 4.56 (1H, m, CH), 5.32 (1H, d, NH), 7.28–7.45 (8H, m, Ar).
4.6. Methyl-6-O-carboxy-(N-(9-fluorenylmethoxycarbonyl)-L-aspartic acid-α-tert-butyl ester)-2,3,4-
tri-O-benzyl-α-D-manno-pyranoside 7
DMAP (40 mg) and DCCI (775 mg, 3.8 mmol) were added to a stirred solution of the methyl
pyranoside 6 (1.743 g, 3.8 mmol) and the amino acid 5 (1.543 g, 3.8 mmol) in DCM (20 ml) at 0°C.
The reaction mixture was allowed to equilibrate to room temperature. After 17 h, TLC (hexane:ethyl
acetate, 1:1) indicated conversion of starting materials (Rf 0.5, 0.1 respectively) to a major product (Rf
0.7). The reaction mixture was filtered through Celite (eluted with DCM) and the solvent removed in
vacuo. Purification by flash column chromatography (hexane:ethyl acetate, 5:1) gave the ester 7 (2.308
g, 72%) as a white foam; [α]D22 +35.1 (c, 0.91 in CHCl3); νmax (CHCl3, thin-film): 2910 (br, NH), 1731
t
(C_O) cm−1; δH (400 MHz, CDCl3): 1.51 (9H, s, Bu), 2.91 (1H, dd, JCH ,CH 4.5 Hz, JCH ,CH 17.1
0
2
2
2
0
Hz, CH2), 3.09 (1H, dd, J
4.5 Hz, CH02), 3.35 (3H, s, MeO), 3.38 (2H, m, H-2, H-5), 3.94 (2H,
CH2,CH
m, H-3, H-4), 4.25 (1H, apt, JCH[Fmoc],CH [Fmoc] 7.3 Hz, CH[Fmoc]), 4.35 (2H, m, H-6, CH2[Fmoc]), 4.42
2
(1H, dd, JCH [Fmoc],CH[Fmoc] 10.4 Hz, CH2[Fmoc]), 4.48 (1H, dd, J5,6 1.5Hz, J6 ,6 11.7 Hz, H-60), 4.57
0
0
2
(1H, dd, JCH,NH 8.3 Hz, CH), 4.64 (3H, m, PhCH2), 4.76 (3H, m, H-1, PhCH2), 4.99 (1H, d, J 10.8 Hz,
PhCH2), 5.87 (1H, d, NH), 7.30–7.45, 7.62–7.65, 7.80 (23H, 3×m, Ar); δC (50.3 MHz, CDCl3): 27.8
(q, (CH3)3C), 38.7 (t, CH2), 47.1, 50.9 (2×d, CH, CH[Fmoc]), 54.9 (q, MeO), 64.2, 67.2, 72.1, 72.7, 75.2
(5×t, C-6, CH2[Fmoc] 3×CH2Ph), 69.9, 74.4, 74.7, 80.3 (4×d, C-2, C-3, C-4, C-5), 82.7 (s, (CH3)3C),
99.2 (d, C-1), 120.2, 125.4, 127.6, 127.8, 127.9, 128.0, 128.3, 128.6, 129.1, 129.1 (10×d, Ar), 138.4,
138.5, 141.5, 144.1 (4×s, Ar), 156.4, 169.9, 171.2 (3×s, 3×C_O); m/z (APCI+): 636.6 (M+H+–Fmoc,
7), 770 (29), 548 (25), 243 (100%). (Found: C, 71.45; H, 6.62; N, 1.76. C51H55O11N requires C, 71.39;
H, 6.46; N, 1.63%.)
4.7. Ethyl-6-O-carboxy-(N-(9-fluorenylmethoxycarbonyl)-L-aspartic acid-α-tert-butyl ester)-2,3,4-tri-
O-benzyl-1-thio-α-D-manno-pyranoside 9
DMAP (40 mg) and DCCI (2.01 g, 9.7 mmol) were added to a stirred solution of the thioglycoside 8
(2.4 g, 4.9 mmol) and the amino acid 5 (2.6 g, 6.3 mmol) in DCM (40 ml) at 0°C. The reaction mixture
was allowed to equilibrate to room temperature. After 7 h, TLC (hexane:ethyl acetate, 1:1) indicated
complete conversion of the sugar starting material (Rf 0.2) to a major product (Rf 0.4). The reaction
mixture was filtered through Celite (eluting with DCM) and the solvent removed in vacuo. Purification
by flash column chromatography (hexane:ethyl acetate, 4:1) gave the ester 9 (3.24 g, 75%) as a white
foam; [α]D +59.0 (c, 0.4 in CHCl3); νmax (CHCl3, thin-film): 1732 (s, 3×C_O) cm−1; δH (400 MHz,
24
CDCl3): 1.24 (3H, t, J 7.4Hz, MeCH2S), 1.47 (9H, s, tBu), 2.57 (2H, dq, J 7.3 Hz, J 12.9 Hz, MeCH2S),
2.85 (1H, dd, JCH ,CH 4.4 Hz, J
17.2 Hz, CH2), 3.04 (1H, dd, JCH ,CH 4.6 Hz, CH02), 3.84 (2H, m),
0
0
CH2,CH2
2
2
0
3.92 (1H, ap-t, J 9.5 Hz), 4.19 (2H, m), 4.29 (1H, dd, JCH[Fmoc],CH [Fmoc] 7.3 Hz, JCH[Fmoc],CH [Fmoc] 10.4
2
2
Hz, CH[Fmoc]), 4.38 (3H, m, 1H, CH2[Fmoc]), 4.53 (1H, m, CH), 4.59 (3H, m, PhCH2), 4.70 (2H, q, J 12.3
Hz, PhCH2), 4.95 (1H, d, J 10.8 Hz, PhCH2), 5.34 (1H, s, H-1), 5.82 (1H, d, J 8.3 Hz, NH), 7.25–7.42,
7.59, 7.76 (19H, m, 2×d, Ar); δC (50.3 MHz, CDCl3): 14.9 (q, MeCH2S), 25.4 (t, MeCH2S), 27.8 (q,
(CH3)3C), 36.6 (t, CH2), 47.1, 50.9 (2×d, CH, CH[Fmoc]), 64.1, 67.3, 72.1, 75.3 (4×t, C-6, CH2[Fmoc]
3×PhCH2), 70.2, 74.8, 76.1, 80.4, 82.1 (5×d, C-1, C-2, C-3, C-4, C-5), 82.7 (s, (CH3)3C), 120.2, 125.4,
127.1, 127.3, 128.1, 128.3, 128.7 (7×d, Ar), 138.2, 138.3, 141.5, 144.1 (4×s, Ar), 156.3, 169.9, 171.2

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R. J. Tennant-Eyles, A. J. Fairbanks / Tetrahedron: Asymmetry 10 (1999) 391–401
(3×s, 3×C_0); m/z (APCI+): 666 (M+H+–Fmoc, 20), 162 (79), 121 (52%). (Found: C, 70.21; H, 6.50;
N,1.92. C52H57O10NS requires C, 70.33; H, 6.47; N, 1.58%.)
4.8. Methyl-6-O-carboxy-(N-(9-fluorenylmethoxycarbonyl)-L-aspartic acid-α-tert-butyl ester)-2,3,4-
tri-O-4-methoxybenzyl-α-D-manno-pyranoside 11
DMAP (131 mg, 2.15 mmol) and DCCI (4.431 g, 21.47 mmol) were added to a stirred solution of
the PMB protected methyl glycoside 10 (5.948 g, 10.74 mmol) and the amino acid 5 (6.619 g, 16.10
mmol) in DCM (80 ml) at 0°C. The reaction mixture was allowed to equilibrate to room temperature.
After 8 h 30 min, TLC (hexane:ethyl acetate, 1:1) indicated conversion of the sugar starting material (Rf
0.3) to a major product (Rf 0.7). The reaction mixture was filtered through Celite (eluted with DCM)
and the solvent removed in vacuo. Purification by flash column chromatography (hexane:ethyl acetate,
3:1 to 1:1) gave the ester 11 (8.559 g, 84%, 98% based on recovered starting material) as a white foam;
[α]D +28.3 (c, 1.0 in CHCl3); νmax (KBr disk): 3356 (br, NH), 1732 (3×C_O) cm−1; δH (400 MHz,
26
CDCl3): 1.47 (9H, s, tBu), 2.88, (1H, dd, J
17.4 Hz, JCH ,CH 4.5 Hz, CH2), 3.05 (1H, dd, J
CH2,CH
2
0
0
CH2,CH2
4.7 Hz, CH02), 3.28 (3H, s, MeO), 3.74–3.84 (13H, m, H-2, H-3, H-4, H-5, 3×MeOBn), 4.19 (1H, ap-
t, JCH[Fmoc],CH [Fmoc] 7.2 Hz, CH[Fmoc]), 4.27–4.38 (3H, m, H-60, CH2[Fmoc]), 4.40 (1H, dd, J6,6 11.6
0
2
Hz, J6,5 1.8 Hz, H-6), 4.49–4.54 (4H, m, CH, PhCH2), 4.62–4.68 (3H, m, H-1, PhCH2), 4.86 (1H, d, J
10.8 Hz, PhCH2), 5.85 (1H, d, JNH,CH 8.3 Hz, NH), 6.82–6.87, 7.19–7.22, 7.25–7.31, 7.39, 7.58, 7.75
(20H, 3×m, 3×d, Ar); δC (50.3 MHz, CDCl3): 27.9 (q, (CH3)3C), 36.7 (t, CH2), 47.1, 50.9 (2×d, CH,
CH[Fmoc]), 54.8 (q, MeO), 55.2 (q, 3×MeOBn), 64.2, 67.2, 69.9, 71.7, 72.3, 73.9, 74.4, 74.8, 79.8 (4×d,
5×t, C-2, C-3, C-4, C-5, C-6, CH2[Fmoc] 3×MeOPhCH2), 82.5 (s, (CH3)3C), 99.1 (d, C-1), 113.8, 113.8,
119.9, 125.2, 127.1, 127.7, 129.2, 129.5, 129.7, 130.2, 130.4, 130.5 (12×d, Ar), 141.3, 143.9 (2×s, Ar),
156.0, 169.6, 170.8 (3×s, 3×C_O); m/z (APCI+): 726.8, (M+H+–Fmoc, 91), 121.9 (100%). (Found: C,
68.23; H, 6.64; N, 1.62. C54H61O14N requires C, 68.36; H, 6.48; N, 1.48%.)
4.9. Methyl-6-O-carboxy-(L-aspartic acid-α-tert-butyl ester)-2,3,4-tri-O-benzyl-α-D-manno-
pyranoside 12
Piperidine (1 ml) was added dropwise to a stirred solution of the Fmoc protected ester 7 in DMF (6
ml). After 1 h, TLC (ethyl acetate:methanol, 9:1) indicated complete conversion of starting material (Rf
0.7) to a single product (Rf 0.2). The solvents were removed in vacuo (co-evaporation with toluene) and
the residue purified by flash column chromatography (ethyl acetate:methanol, 9:1) to give the free amine
12 (300 mg, 83%) as a clear oil; [α]D22 +17.5 (c, 2.5 in CHCl3); νmax (CHCl3, thin-film): 2914 (br, NH),
t
1732 (C_O) cm−1; δH (400 MHz, CDCl3): 1.49 (9H, s, Bu), 2.68 (1H, dd, JCH ,CH 4.4 Hz, JCH ,CH
0
2
2
2
0
16.3 Hz, CH2), 2.84 (1H, dd, J
8.2 Hz, CH02), 2.93, 3.00 (2H, 2×s, NH2), 3.37 (3H, s, MeO), 3.77
CH2,CH
0
(1H, dd, CH), 3.83 (2H, m, H-2, H-5), 3.95 (2H, m, H-3, H-4), 4.44 (2H, 2×d, J5,6 3.3 Hz, J5,6 5.5 Hz,
H-6, H-60), 4.67 (3H, m, PhCH2), 4.76 (3H, m, H-1, PhCH2), 4.99 (1H, d, J 10.7 Hz, PhCH2), 7.32–7.43
(15H, m, Ar); δC (50.3 MHz, CDCl3): 27.9 (q, (CH3)3C), 39.1 (t, CH2), 51.8 (d, CH), 54.8 (s, MeO),
63.7, 72.1, 72.7, 75.2 (4×t, C-6, 3×CH2Ph), 69.9, 74.3, 74.5, 80.2 (4×d, C-2, C-3, C-4, C-5), 81.7 (s,
(CH3)3C), 99.0 (d, C-1), 127.8, 128.0, 128.1, 128.3, 128.6 (5×d, Ar), 138.4 (s, Ar), 171.5 (s, C_O); m/z
(APCI+): 636.7 (M+H+, 8), 548.2 (100), 181.0 (26%). HRMS calcd. For C36H45NO9 (M+H+) 636.3173.
(Found 636.3192.)

R. J. Tennant-Eyles, A. J. Fairbanks / Tetrahedron: Asymmetry 10 (1999) 391–401
399
4.10. Ethyl-6-O-carboxy-(N-(9-fluorenylmethoxycarbonyl)-L-aspartic acid)-2,3,4-tri-O-benzyl-1-thio-
α-D-manno-pyranoside 13
TFA (5 ml) was added to a stirred solution of the tert-butyl ester 9 (1.171 g, 1.3 mmol) in DCM (5 ml)
at 0°C, then allowed to equilibrate to room temperature. After 3 h 30 min, TLC (ethyl acetate:methanol,
9:1) indicated complete conversion of starting material (Rf 0.9) to a single product (Rf 0.4).The reaction
mixture was diluted with DCM (50 ml), and the solvents removed in vacuo to give the free acid 13 (1.604
g, quantitative yield) as a pale pink hygroscopic foam; [α]D24 +58.4 (c, 1.0 in CHCl3); νmax (KBr disk):
3406 (br, NH), 1684 (s, 3×C_O) cm−1; δH (400 MHz, CDCl3): 1.23 (3H, t, J 6.9 Hz, MeCH2S), 2.54 (2H,
m, MeCH2S), 2.82, (1H, m, CH2), 3.07 (1H, m, CH02), 3.89 (3H, m), 4.07 (1H, m), 4.19 (1H, s), 4.38 (4H,
m), 4.75 (2H, m), 4.92 (1H, d, J 10.8 Hz, PhCH2), 5.29 (1H, s, H-1), 5.94 (1H, s, NH), 7.16–7.59, 7.70,
7.75 (23H, m, 2×d, Ar); δC (50.3 MHz, CDCl3): 15.3 (q, MeCH2S), 25.1 (t, MeCH2S), 36.6 (t, CH2),
47.0, 51.3 (2×d, CH, CH[Fmoc]), 63.8, 66.2, 70.9, 71.5, 74.7 (5×t, C-6, CH2[Fmoc] 3×PhCH2), 70.3,
74.7, 76.1, 79.9, 81.7 (5×d, C-1, C-2, C-3, C-4, C-5), 120.8, 125.9, 127.7, 128.2, 128.3, 128.9 (6×d, Ar),
138.8, 139.1, 141.4, 141.5 (4×d, Ar), 156.6, 171.1 (2×s, 2×C_O); m/z (APCI+): 854.6 (M+Na+, 41),
548.0 (44), 181.0 (87), 121.8 (100%); m/z (FAB+): 854.4 (M+Na+, 10), 179.1 (30), 91.0 (100%).
4.11. Methyl-6-O-carboxy-(N-(9-fluorenylmethoxycarbonyl)-L-aspartic acid-α-tert-butyl ester)-α-D-
manno-pyranoside 14
The PMB protected ester 11 (1.819 g, 1.9 mmol) and CAN (6.74 g, 11.5 mmol) were stirred in
acetonitrile:water (9:1, 18 ml) for 20 min when TLC (hexane:ethyl acetate, 1:1) indicated complete
conversion of starting material (Rf 0.7) to a single product (Rf 0.1). The reaction mixture was diluted
with DCM (250 ml) and washed with saturated sodium bicarbonate solution (150 ml). The aqueous layer
was extracted with DCM (250 ml) and the combined organic layers were dried (MgSO4), filtered and
concentrated in vacuo. The residue was purified by flash column chromatography (hexane:ethyl acetate,
22
5:1) to yield the triol 14 (897 mg, 80%) as a white foam; [α]D +50.1 (c, 0.9 in CHCl3); νmax (CHCl3,
t
thin-film): 3414 (br, OH, NH), 1731 (3×C_O) cm−1; δH (400 MHz, CDCl3): 1.47 (9H, s, Bu), 2.49,
0
0
2.59, 2.87 (3H, 3×s, 3×OH), 2.91 (1H, dd, JCH ,CH 11.9Hz, JCH ,CH 4.8 Hz, CH2), 2.99 (1H, dd, J
CH2,CH
2
2
2
4.9 Hz, CH02), 3.26 (3H, s, MeO), 3.60 (1H, m, H-5), 3.68–3.81 (3H, m, H-2, H-3, H-4), 4.21 (1H, ap-t,
0
JCH[Fmoc],CH [Fmoc] 6.8 Hz, CH[Fmoc]), 4.29 (1H, dd, J6,6 11.9 Hz, J5,6 1.5 Hz, H-6), 4.35 (1H, s, H-1),
2
4.45–4.57 (3H, m, H-60, CH2[Fmoc]), 4.59 (1H, m, CH), 6.13 (1H, d, JNH,CH 8.9 Hz, NH), 7.32, 7.42,
7.61, 7.78 (8H, 4×m, Ar); δC (50.3 MHz, CDCl3): 27.8 (q, (CH3)3C), 37.7 (t, CH2), 47.0, 51.0 (2×d,
CH, CH[Fmoc]), 55.0 (q, MeO), 63.1, 66.7, 67.4, 64.9, 70.6, 71.5 (4×d, 2×t, C-2, C-3, C-4, C-5, C-6,
CH2[Fmoc]), 83.1 (s, (CH3)3C), 100.9 (d, C-1), 120.0, 125.0, 125.0, 127.2, 127.8 (5×d, Ar), 141.2, 143.7,
143.7 (3×s, Ar), 156.2, 170.1, 170.3 (3×s, 3×C_O); m/z (APCI+): 610.3 (M+Na+, 9), 482.1 (57), 366.2
(M+H+–Fmoc, 47), 310.2 (100%). (Found: C, 60.99; H, 6.21; N, 2.12. C30H37O11N requires C, 61.32;
H, 6.35; N, 2.38%.)
4.12. Methyl-6-O-carboxy-(N-(glycine-N-(9-fluorenylmethoxycarbonyl))-L-aspartic acid-α-tert-butyl
ester)-α-D-manno-pyranoside 15
Piperidine (0.5 ml) was added dropwise to a stirred solution of the triol 14 (37 mg, 0.06 mmol) in
DMF (2.5 ml). After 30 min, TLC (hexane:ethyl acetate, 1:9) indicated complete conversion of starting
material (Rf 0.3) to a single product (Rf 0.1). The solvents were removed in vacuo (co-evaporation with
toluene) and the residue dissolved in benzene:ethanol (1:1, 2 ml). EEDQ (31 mg, 0.13 mmol) and N-(9-

400
R. J. Tennant-Eyles, A. J. Fairbanks / Tetrahedron: Asymmetry 10 (1999) 391–401
fluorenylmethoxycarbonyl)-glycine (28 mg, 0.09 mmol) were added and the solution stirred under argon
for 14 h when TLC (hexane:ethyl acetate, 1:9) indicated conversion of starting material (Rf 0.1) to a
major product (Rf 0.2). The solvents were removed in vacuo and the residue purified by flash column
chromatography (hexane:ethyl acetate, 1:9) to give the dipeptide 15 (39 mg, 95%) as a white foam;
24
[α]D +41.2 (c, 1.03 in CHCl3); νmax (CHCl3, thin-film): 3399 (br, OH, CO2H), 1732 (3×C_O), 1529
(s, NC_O, Amide II) cm−1; δH (400 MHz, CDCl3): 1.45 (9H, s, tBu), 2.99 (2H, m, CH2[Asp.]), 3.31 (3H,
s, MeO), 3.65 (1H, d, J 8.5 Hz, H-5), 3.76–3.82 (2H, m, H-3, H-4), 3.90–4.05 (3H, m, H-2, CH2[Gly.]),
4.22 (1H, apt, J 6.8 Hz, CH[Fmoc]), 4.32–4.43 (3H, m, H-6, CH2[Fmoc]), 4.50 (1H, d, J6,6 11.8 Hz, H-60),
0
4.73 (1H, s, H-1), 4.78 (1H, m, CH), 5.97 (1H, s, NH), 7.30, 7.39, 7.60, 7.75 (8H, 2×t, 2×d, Ar); δC (50.3
MHz, CDCl3): 27.7 (q, (CH3)3C), 36.8, 44.0 (2×t, CH2[Asp] CH2[Gly]), 46.9, 49.3 (2×d, CH, CH[Fmoc]),
55.0 (q, MeO), 63.4, 67.4 (2×t, C-6, CH2[Fmoc]), 67.2, 69.8, 70.6, 71.7 (4×d, C-2, C-3, C-4, C-5), 83.2
(s, (CH3)3C), 107.3 (d, C-1), 120.2, 125.4, 127.3, 127.9 (4×d, Ar), 141.4, 144.0 (2×s, Ar), 157.3, 170.0,
170.9 (3×s, 4×C_O); m/z (APCI+): 645.7 (M+H+, 82), 589.7 (100%).
Acknowledgements
We gratefully acknowledge the EPSRC (to RTE) for financial support.
References
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11. For example 1998 Sigma catalogue prices: L-aspartic acid £17.60 for 500 g, β-benzyl-L-aspartate £92.00 for 25 g.

R. J. Tennant-Eyles, A. J. Fairbanks / Tetrahedron: Asymmetry 10 (1999) 391–401
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17. Readily synthesized in four steps from methyl mannopyranoside in an overall yield of 49%. Full experimental details will
be published separately.
18. Again readily synthezised in four steps from methyl mannopyranoside in an overall yield of 50%. Full experimental details
will be published separately.