Synthesis of mannosyl and oligomannosyl threonine building blocks found on the mycobacterium tuberculosis 45 kDa MPT 32 glycoprotein

Article abstract of DOI:10.1071/CH01204

Mycobacterium tuberculosis secretes a 45 kDa glycoprotein (MPT 32) carrying mannosylated threonine residues. A general strategy was developed to couple peracetylated mannose, α-(1,2)-linked mannobiose or mannotriose to Fmoc-threonine-benzyl ester. Activation of mannosyl acetates or fluorides by borontrifluoride etherate gave the desired Fmoc-glycosyl amino acid building blocks in good yields. The synthesis leads to stable compounds and can easily be scaled up.

Full text of DOI:10.1071/CH01204

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Aust. J. Chem. 2002, 55, 161–165
Synthesis of Mannosyl and Oligomannosyl Threonine Building Blocks
Found on the Mycobacterium tuberculosis 45 kDa MPT 32 Glycoprotein*
Daniel Varon ,A,B Eduardo Lioy,A Manuel E. Patarroyo,A Xaver SchrattB and Carlo UnverzagtB,C
A Sección de Química, Fundación Instituto de Inmunología de Colombia, Cra 50 No 26-00, Bogotá, Colombia.
B Bioorganische Chemie, Universität Bayreuth, Gebäude NW 1, 95440 Bayreuth, Germany.
C Author to whom correspondence should be addressed (e-mail: Carlo.Unverzagt@uni-bayreuth.de).
Mycobacterium tuberculosis secretes a 45 kDa glycoprotein (MPT 32) carrying mannosylated threonine residues.
A general strategy was developed to couple peracetylated mannose, α-(1,2)-linked mannobiose or mannotriose to
Fmoc-threonine-benzyl ester. Activation of mannosyl acetates or fluorides by borontrifluoride etherate gave the
desired Fmoc-glycosyl amino acid building blocks in good yields. The synthesis leads to stable compounds and can
easily be scaled up.
Manuscript received: 18 December 2001.
Final version: 11 April 2002.
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Introduction
AcO
AcO
AcO
OAc
O
During the invasion of phagocytic cells by Mycobacterium
tuberculosis, the bacterial surface carbohydrates, especially
mannose residues, play an important role in the receptor-
mediated binding and internalization process.[1] Mannose
has been found to be a component of various molecules
generated by mycobacteria, the most important being
lipoarabinomannan (LAM) and secreted glycoproteins.[2]
The only M. tuberculosis glycoprotein whose glycosylation
pattern has been completely elucidated up to now is the 45
KDa MPT32.[3] In this glycoprotein, four threonine residues
are O-glycosidically modified by α-D-mannose, an α-(1,2)-
linked dimannoside, or the corresponding trimannoside. The
structures of these oligomannosides are similar to the
capping motifs reported for lipoarabinomannan.[4] Pure
carbohydrates have long been known not to induce T cell
responses, however, carbohydrates conjugated to lipids[5] or
AcO
AcO
AcO
OAc
O
AcO
AcO
AcO
O
O
AcO
AcO
AcO
AcO
AcO
O
O
OAc
O
O
O
AcO
AcO
AcO
AcO
O
O
O
Fmoc Thr OH
Fmoc
Thr OH
Fmoc
Thr OH
A
B
C
Fig. 1. Glycosyl amino acid building blocks A–C derived form the
oligomannoside structures found on Mycobacterium tuberculosis
MPT32 glycoprotein.
particular, the difficult purification of larger quantities of C-
terminally unprotected threonine derivatives[10] or the latent
instability of threonine pentafluorophenyl esters[11]
prompted us to develop a more robust strategy that could be
conveniently scaled up. Our interest turned to Fmoc-
threonine-benzyl ester (3)[12] which had been successfully
glycosylated using glycosyl halides and silver triflate.[13] To
avoid unstable glycosyl halides and silver salts, the
mannosylation of (3) was investigated using the
mannosylperacetate[14] (1) or the fluoride[14] (2). When
activated with BF3 Et2O both donors gave the protected α-
mannosyl threonine (4)[13,17] in yields of 51 and 75%,
respectively. This compound is stable and can easily be
purified in large quantities. In a previous report an α-
carboxy unprotected threonine was reacted with the
peracetate (1) under similar conditions[10] with nearly
identical yields, suggesting that the benzyl ester moiety of
peptides[6] can stimulate
T
cells. Thus, synthetic
M. tuberculosis glycopeptides appear to be good candidates
for the development of molecules capable of stimulating T
cell response, which is required for protection against
tuberculosis.[7] In order to assemble M. tuberculosis MPT32
glycopeptides in a stepwise manner on a solid support,[8] we
developed a general strategy for the synthesis of the
appropriately protected glycosyl amino acid building blocks
A–C (Fig. 1).
*†
.
Results and Discussion
There are several reports on the synthesis of O-mannosylated
amino acids,[9] however, our attempts to utilize the
previously disclosed strategies revealed some drawbacks. In
*
Dedicated to Professor Peter Köll on the occasion of his 60th birthday.
For activation of glycosyl fluorides using BF3 Et2O see reference 15. For activation of glycosyl acetates using BF3 Et2O see reference 16.
†
.
.
© CSIRO 2002
10.1071/CH01204
0004-9425/02/010161

162
D. Varon et al.
compound (3) was not influencing the course of the reaction.
The mannosyl fluoride (2) turned out to be a far more
reactive donor, which possibly explains the significantly
higher yields. Catalytic hydrogenation[13] of the benzyl ester
(4) furnished the monosaccharide–threonine building block
A[13] in high yield (Fig. 2).
peracetylmannose (1). After 24 hours of reaction time, a
55% yield of the disaccharide–threonine derivative (11) was
obtained. Prolonged reaction time (36 hours) led to a
maximum yield of 61%. Using the dimannosyl fluoride (8)
as an alternative donor improved the yield in the
glycosylation step to 70%. Despite the lack of neighboring
group participation in donors (7) and (8), only α-linked
product was obtained.
Surprisingly, the reaction of Fmoc-Thr-OBzl (3) with the
peracetylated trimannoside (9) could not be accomplished.
However, the trimannosylated threonine building block (12)
was obtained in 61% yield using the more reactive
trisaccharide fluoride (10) (Fig. 4). Liberation of the free
carboxylic acid was effected by catalytic hydrogenation
affording the target oligomannosyl–threonines B and C in
high yields.
AcO
AcO
AcO
AcO
AcO
OAc
O
OAc
O
OAc
O
AcO
AcO
a
b
AcO
AcO
OAc
O
O
O
c
(1)
O
O
O
O
O
N
O
N
H
H
AcO
OAc
O
OH
AcO
AcO
F
A
(2)
(4)
Fig. 2. Glycosylation of Fmoc-Thr-OBzl (3). (a) Fmoc-Thr-OBzl
.
(3), BF3 Et2O, absolute CH2Cl2, 24 h, (52%). (b) Fmoc-Thr-OBzl (3),
.
BF3 Et2O, absolute CH2Cl2, 2 h, (75%). (c) Pd/C 10%, H2, MeOH, 2 h
(93%).
AcO
OAc
O
AcO
AcO
AcO
AcO
AcO
AcO
AcO
AcO
OAc
O
O
O
To obtain the oligosaccharide–amino acid building blocks
B and C, containing α-(1,2)-linked mannobiose and
mannotriose, the peracetylated oligomannosides were
synthesized initially (Fig. 3). Glycosylation of 1,3,4,6-tetra-
O-acetyl-β-D-mannopyranose (6)[18] with the thiomannoside
(5)[14] using N-iodosuccinimide (NIS) and triflic acid
(TfOH) as activants[14] gave the dimannoside (7)[19] in 67%
yield. Subsequently, the disaccharide (7) was converted into
the corresponding glycosyl fluoride (8) by reaction with the
HF–pyridine complex.[20] The disaccharide fluoride (8)
proved to be a valuable donor in the glycosylation of (6),
furnishing the trimannoside (9) in 60% yield. Subsequent
acidolysis of the trisaccharide (9) with HF–pyridine led to
the trisaccharide fluoride (10).
a
b
(7)
(8)
AcO
AcO
AcO
AcO
AcO
AcO
O
O
O
O
d
O
O
(10)
Fmoc
Thr OR
Fmoc
Thr OR
(11) R = Bzl
R = H
(12) R = Bzl
R =H
c
e
B
C
Fig. 4. Glycosylation of Fmoc-Thr-OBzl (3) with oligomannosyl
donors. (a) Fmoc-Thr-OBzl (3), BF3 Et2O, absolute CH2Cl2, 36 h,
(61%). (b) Fmoc-Thr-OBzl (3), 4 Å molecular sieves, BF3 Et2O,
absolute CH2Cl2, 2 h, (70%). (c) Pd/C 10%, H2, MeOH, 2 h (95%).
(d) BF3 Et2O, absolute CH2Cl2, 3 h, (61%). (e) Pd/C 10%, H2,
MeOH, 2 h (91%).
.
.
.
With the four oligomannosyl building blocks (7)–(10) in
hand, the glycosylation of Fmoc-Thr-OBzl (3) was
investigated. Reaction of peracetyldimannoside (7) with
Fmoc-Thr-OBzl (3) was performed as elaborated for
Conclusion
In conclusion we have devized a general strategy to obtain
mannosylated threonines as valuable building blocks in
solid-phase glycopeptide synthesis. Mannose and
oligomannosides can conveniently be coupled to Fmoc-Thr-
OBzl (3) by use of borontrifluoride etherate activating either
a mannosyl acetate or fluoride. The reactions are useful for
large-scale synthesis and provide stable compounds that can
be easily purified.
AcO
OAc
AcO
AcO
AcO
AcO
OAc
O
OAc
O
O
AcO
AcO
AcO
AcO
(5)
+
AcO
SEt
OH
b
a
AcO
AcO
AcO
AcO
AcO
O
O
O
O
O
OAc
AcO
AcO
AcO
OAc
F
Experimental
(8)
(7)
(6)
AcO
AcO
AcO
AcO
OAc
O
OAc
O
Materials and Methods
AcO
AcO
Reagents for synthesis were obtained from Fluka, Aldrich, Sigma or
Acros. Thin-layer chromatography (TLC) was carried out on Merck
silica gel 60 F254 plates; compounds were visualized by heating with a
1 : 1 mixture of 0.2% 3-methoxyphenol in ethanol and 1 M H2SO4.
Flash chromatography was carried out using Merck silica gel 60
(0.040–0.063 mm). Nuclear magnetic resonance (NMR) spectra (1H,
13C, H–H correlation spectroscopy (COSY), heteronuclear multiple
quantum coherence (HMQC), HMQC-COSY, and total correlation
spectroscopy (TOCSY)) were recorded on a Bruker DRX-500
spectrometer. The chemical shifts are expressed in δ (ppm) relative to
tetramethylsilane. Electrospray-ionization time-of-flight (ESI-TOF)
mass spectra (MS) were recorded on a Micromass LCT spectrometer.
c
d
AcO
AcO
AcO
AcO
AcO
AcO
O
O
O
O
(8)+(6)
AcO
AcO
AcO
AcO
O
O
O
O
OAc
AcO
AcO
F
(9)
(10)
Fig. 3. Synthesis of oligomannosyl donors. (a) NIS, TfOH, 4 Å
molecular sieves, 0°C, 30 min (67%). (b) HF–pyridine, CH2Cl2, 2 h
(70%). (c) BF3 Et2O, 4 Å molecular sieves, CH2Cl2, 3 h (60%). (d)
HF–Pyridine, CH2Cl2, 3 h (55%).
.

Synthesis of Mannosyl and Oligomannosyl Threonine Building Blocks
163
1
N-(9-Fluorenylmethoxycarbonyl)-L-threonine Benzyl Ester (3)[12]
671.22. H NMR (500 MHz, (D6)DMSO) δ 7.90, d, J 7 Hz, 2H, Ar-
Fmoc; 7.77, d, J 7 Hz, 2H, Ar-Fmoc; 7.48–7.30, m, 5H, Ar-Fmoc and
NH; 5.30, dd, J 10 Hz, 1H, H4; 5.12–4.92, m, 3H, H1, H2, and H3;
4.32–4.00, m, 8H, H5, H6a, H6b, CH2-Fmoc, CH-Fmoc, Hα, and Hβ;
2.12–1.88, 4 × s, 12H, Ac; 1.22, d, J 6 Hz, 3H, H3γ. 13C NMR (125
MHz, (D6)DMSO) δ 170.6–169.9, C=O; 157.0, C=O; 128.1–120.6, Ar-
Fmoc; 98.5, C1; 76.5, Cβ; 69.0, C2; 68.5, C5; 66.8, C3; 66.4, C4; 66.3,
CH2-Fmoc; 62.6, C6; 60.3, Cα; 47.2, CH-Fmoc; 21.1–20.9, Ac; 18.5,
Cγ.
To a stirred solution of 8.31 g (32.7 mmol) of H-Thr-OBzl hemioxalate
in 120 mL of dry dichloromethane were added 12.5 g (36 mmol) of 9-
fluorenylmethyloxycarbonyl-N-hydroxysuccinimide (Fmoc-OSu) and
5.5 mL (40 mmol) of triethylamine. After TLC (AcOEt/cyclohexane,
1 : 1) indicated the reaction was complete, the organic phase was
washed twice with 1 N HCl and 2 M KHCO3. The organic phase was
dried over MgSO4 and concentrated under reduced pressure. The
remainder was purified by flash chromatography using AcOEt/
cyclohexane (1 : 1) as eluent. Yield: 12.83 g (28.7 mmol, 84%) of (3) as
a white solid. [α]D20 –6.94 (c, 0.5 in AcOEt) (lit.[12a] [α]D20 +6.65 in
AcOEt; lit.[12b] [α]D20 –4.0 (c, 1.0 in CHCl3)); RF 0.56 (AcOEt/
cyclohexane, 1 : 1); ESI-TOF MS: Found 432.3 [M + 1]+. Calc. for
C26H25NO5: 431.17. 1H NMR (500 MHz, (D6)DMSO) δ 7.90, d, J 7.5
Hz, 2H, Ar-Fmoc; 7.88, d, J 7 Hz, 2H, Ar-Fmoc; 7.46–7.28, m, 10H,
Ar-Fmoc, Ar-Bzl, and NH; 5.16, s, 2H, CH2-Bzl; 4.87, d, J 7 Hz, 1H,
OH; 4.35–4.12, m, 5H, H9-Fmoc, CH2-Fmoc, Hα, Hβ; 1.11, d, J 6 Hz,
3H, Hγ. 13C NMR (125 MHz, (D6)DMSO) δ 170.5–169.1, C=O; 157.1,
C=O; 156.7, C=O; 128.9–120.8, Ar-Fmoc and Ar-Bzl; 66.5, Cβ; 66.1,
CH2-Bzl and CH2-Fmoc; 59.7, Cα; 45.4, CH-Fmoc; 16.5, Cγ.
Acetyl-O-(2,3,4,6-tetra-O-acetyl-α-D-mannopyranosyl)-(1→2)-3,4,6-
tri-O-acetyl-β-D-mannopyranose (7)[19]
A 5 g portion of powdered and activated 4 Å molecular sieves was
added to a solution of thioglycoside (5) (12.1 g, 30.9 mmol) and
tetraacetyl mannose (6) (9.75 g, 28 mmol) in 150 mL of absolute
dichloromethane. After stirring the suspension at 0ºC for 15 min, N-
iodosuccinimide (15.77 g, 70.11 mmol) was added. The reaction was
started by the dropwise addition of triflic acid (1.95 mL, 22.4 mmol).
When the reaction was terminated (TLC; AcOEt/cyclohexane, 2 : 1),
the reaction mixture was diluted with 200 mL of dichloromethane,
filtered over Celite and washed with 2 M KHCO3, a 10% solution of
Na2S2O3 (× 3) and water. The organic phase was dried over MgSO4 and
concentrated. Flash chromatography (AcOEt/cyclohexane, 2 : 1) of the
remainder yielded 12.7 g (18.8 mmol, 67%) of (7). [α]D20 +5.0 (c, 0.5 in
CHCl3); (lit.[19a] [α]D20 +1.2 (c, 1.0 in CHCl3); lit.[19b] [α]D20 +5.7 (c,
0.78 in CHCl3); lit.[19c] [α]D20 +0.7 (c, 0.6 in CHCl3); lit.[19d] [α]D20 +9.0
(c, 0.25 in CHCl3)); RF 0.42 (AcOEt/cyclohexane, 2 : 1); ESI-TOF MS:
Found: 701.2 [M +Na]+. Calc. for C28H38O19: 678.20. 1H NMR
(500 MHz, (D6)DMSO) δ 6.15, d, J < 1 Hz, 1H, H1; 5.31, dd, 1H, J 10
Hz, H4; 5.26–5.14, m, 4H, H3, H1´, H2´, and H3´; 5.07, dd, J 10 Hz,
1H, H4´; 4.19, m, 1H, H2; 4.16–3.98, m, 6H, H5, H6a, H6b, H5′, H6a´,
and H6b´; 2.15–1.95, 8 × s, 24H, Ac. 13C NMR (125 MHz, (D6)DMSO)
δ 98.1, C1′; 90.5, C1; 75.4, C2; 69.7, C5; 68.9, C3; 68.8, C2´; 68.7, C5′;
68.0, C3´; 65.5, C4´; 64.7, C4; 62.0, C6′; 61.2, C6; 20.8–20.3, Ac.
N-(9-Fluorenylmethoxycarbonyl)-O-(2,3,4,6-tetra-O-acetyl-α-D-
mannopyranosyl)-L-threonine Benzyl Ester (4)[13,17]
(a) From pentaacetate (1). Fmoc-Thr-OBzl (3) (3.83 g, 8.90
mmol) was added to a solution of pentaacetylmannose (1) (3.81 g, 9.90
mmol) in 180 mL of absolute dichloromethane. The atmosphere in the
.
flask was replaced by argon and 10.1 mL (80.1 mmol) of BF3 Et2O was
slowly added. When maximum conversion was observed (TLC, AcOEt/
cyclohexane, 1 : 1; 24 h) the mixture was diluted with 180 mL of
dichloromethane and washed with 1 M KHCO3. The organic phase was
dried over MgSO4 and concentrated. Purification of the remainder by
flash chromatography (AcOEt/cyclohexane, 1 : 1) gave (4) (4.02 g, 5.17
mmol, 51% yield) as a white solid. [α]D20 +31.7 (c, 0.1 in CHCl3) (lit.[17]
[α]D20 +34.0 (c, 1.1 in CHCl3)); RF 0.58 (AcOEt/cyclohexane, 1 : 1).
(b) From fluoride (2). A suspension of fluoride (2) (150 mg,
0.43 mmol), Fmoc-Thr-OBzl (3) (220 mg, 0.50 mmol) and freshly
ground and heated 4 Å molecular sieves (400 mg) in 5 mL of absolute
dichloromethane was stirred for 30 min at ambient temperature. After
O-(2,3,4,6-Tetra-O-acetyl-α-D-mannopyranosyl)-(1→2)-3,4,6-tri-O-
acetyl-α-D-mannopyranosyl Fluoride (8)
To a stirred solution of disaccharide (7) (5.20 g, 7.7 mmol) in 20 mL of
dry dichloromethane was added 20 mL of HF–pyridine (65% HF) at
0°C. Once the reaction was complete (3 h, TLC; AcOEt/cyclohexane,
2 : 1), the mixture was diluted with 150 mL of dichloromethane and
poured onto a mixture of cold water and ice in a Teflon separatory
funnel. The organic phase was washed three times with 1 N HCl and
2 M KHCO3, dried over MgSO4 and concentrated. After flash
chromatography (AcOEt/cyclohexane, 2 : 1) 3.4 g of fluoride (8) was
obtained (5.32 mmol, 70%). [α]D20 +13.7 (c, 0.4 in CH2Cl2); RF 0.48
(AcOEt/cyclohexane, 2 : 1); ESI-TOF MS: Found: 661.3 [M + Na]+.
Calc. for C26H35FO17: 638.19. 1H NMR (500 MHz, (D6)DMSO) δ 5.92,
dd, J 49 Hz, 1H, H1; 5.30, dd, J 10 Hz, 1H, H4; 5.22–5.15, m, 4H, H3,
H1´, H2´, and H3´; 5.12, dd, J 10 Hz, 1H, H4´; 4.34, m, 1H, H2; 4.19–
4.05, m, 6H, H5, H6a, H6b, H5´, H6a´, and H6b´; 2.12–1.96, 7 × s, 21H,
Ac. 13C NMR (125 MHz, (D6)DMSO) δ 105.5, d, C1; 98.1, C1´; 74.0,
d, C2; 70.3, C5; 68.6, C3; 68.6, C2´; 68.3, C5´; 68.0, C3´; 65.3, C4´;
64.3, C4; 61.8, C6´; 61.1, C6; 2.12–1.96, Ac.
.
addition of BF3 Et2O (55 µL, 0.43 mmol), TLC analysis (AcOEt/
cyclohexane, 1 : 1) revealed that the reaction was complete within 1 h.
The molecular sieves were removed by filtration over celite and the
diluted filtrate was washed with 1 N HCl and 2 M KHCO3. The organic
phase was dried over MgSO4, concentrated and purified by flash
chromatography (AcOEt/cyclohexane, 1 : 1.2). Yield: 240 mg (0.32
mmol) 75% of compound (4). ESI-TOF MS: Found: 762.5 [M + 1]+.
Calc. for C40H43NO14: 761.27. 1H NMR (500 MHz, (D6)DMSO) δ
8.20, d, J 6 Hz, 1H, NH; 7.89, d, J 7 Hz, 2H, Ar-Fmoc; 7.82, t, J 7 Hz,
2H, Ar-Fmoc; 7.50–7.32, m, 9H, Ar-Fmoc and Ar-Bzl; 5.20, dd, J 10
Hz, 1H, H4; 5.15–5.00, m, 4H, H2, H3, and PhCH2; 4.91, d, J < 1 Hz,
1H, H1; 4.30–4.00, m, 8H, H5, H6a, H6b, CH2-Fmoc, CH-Fmoc, Hα,
and Hβ; 2.05–1.92, 4 × s, 12H, Ac; 1.20, d, J 6 Hz, 3H, Hγ. 13C NMR
(125 MHz, (D6)DMSO) δ 170.9–169.0, C=O. 157.6, C=O; 146.7, C=O;
128.3–120.1, Ar-Fmoc and Ar-Bzl; 97.8, C1; 76.0, Cβ; 68.9, C2; 68.3,
C5; 68.0, C3; 66.2, CH2-Bzl; 65.9, CH2-Fmoc; 65.8, C4; 61.5, C6; 58.8,
Cα; 46.5, CH-Fmoc; 20.6–20.3, 4 × C; 17.4, Cγ.
Acetyl-O-(2,3,4,6-tetra-O-acetyl-α-D-mannopyranosyl)-(1→2)-O-
(3,4,6-tri-O-acetyl-α-D-mannopyranosyl)-(1→2)-3,4,6-tri-O-acetyl-β-
D-mannopyranose (9)
N-(9-Fluorenylmethoxycarbonyl)-O-(2,3,4,6-tetra-O-acetyl-α-D-
mannopyranosyl)-L-threonine A[13]
A suspension of fluoride (8) (1.6 g, 2.5 mmol), tetraacetyl mannose (6)
(830 mg, 2.38 mmol) and powdered and activated 4 Å molecular sieves
(3.2 g) in 50 mL of absolute dichloromethane was stirred for 30 min.
The reaction was started by slow addition of BF3Et2O (750 µL,
5.96 mmol). After 3 h complete reaction was observed (TLC; AcOEt/
cyclohexane, 1.5 : 1). The mixture was diluted with 100 mL of
dichloromethane and the molecular sieves were filtered off over celite.
The combined organic phases were extracted with 2 M KHCO3, dried
over MgSO4 and concentrated. Flash chromatography of the remainder
using AcOEt/cyclohexane (1.5 : 1) gave 1.45 g (1.50 mmol, 60%) of
trisaccharide (9). [α]D20 +17.9 (c, 0.5 in CH2Cl2); RF 0.29 (AcOEt/
Compound (4) (3.5 g, 4.5 mmol) was dissolved in 300 mL of absolute
MeOH. After addition of 620 mg of palladium on activated carbon (5%
Pd) the reaction was stirred under a hydrogen atmosphere for 90 min.
The removal of the benzyl group was monitored by TLC
(dichloromethane/MeOH, 10 : 1). Following complete deprotection, the
catalyst was filtered off and washed repeatedly. The organic phase was
concentrated and purified by flash chromatography using
dichloromethane/MeOH (10 : 1). Yield: 2.80 g (4.01 mmol, 93%).
[α]D20 +30.4 (c, 0.3 in CH2Cl3); RF 0.53 (dichloromethane/MeOH,
10 : 1); ESI-TOF MS: Found: 672.4 [M + 1]+. Calc. for C33H37NO14:

164
D. Varon et al.
cyclohexane, 1.5 : 1); ESI-TOF MS: Found: 989.2 [M + Na]+. Calc. for
C40H54O27: 966.29. H NMR (500 MHz, (D6)DMSO) δ 6.15, d, J 1.5
N-(9-Fluorenylmethoxycarbonyl)-O-[(2,3,4,6-tetra-O-acetyl-α-D-
1
mannopyranosyl)-(1→2)-(3,4,6-tri-O-acetyl-α-D-mannopyranosyl)]-
L-threonine B
Hz, 1H, H1; 5.32, dd, J 10 Hz, 1H, H4; 5.25–5.02, m, 9H, H3, H1´, H2´,
H3´, H4, H1´´, H2´´, H3´´, and H4´´; 4.20, m, 1H, H2; 4.15–4.01, m,
9H, H5, H6a, H6b, H5´, H6a´, H6b´, H5´´, H6a´´, and H6b´´; 2.16–1.95,
33H, Ac. 13C NMR (125 MHz, (D6)DMSO) δ 170.6–169.1, C=O; 98.6,
C1´; 92.6, C1´´; 90.5, C1; 75.4, C2; 69.7, C5; 68.9, C3; 68.8, C5´´; 68.5,
2 × C, C2´´, and C3´; 68.4, 2 × C, C2´, and C3´´; 68.0, C5´; 65.5, C4´´;
65.4, C4´; 68.4, C4; 62.0, C6´´; 61.6, C6´; 61.2, C6; 20.4–19.0, Ac.
To a solution of benzyl ester (11) (5.23 g, 4.98 mmol) in 150 mL
absolute MeOH was added 44 mg of palladium on activated carbon
(10%). The suspension was stirred under a hydrogen atmosphere for 3
h (TLC; acetone/cyclohexane, 2 : 1). Removal of the catalyst by
filtration and evaporation of the solvent gave a crude product, which
was purified by flash chromatography using acetone/cyclohexane
(2 : 1). Yield 4.87 g (4.98 mmol, 95%) of compound B. [α]D20 +48.4 (c,
0.4 in CH2Cl2); Rf 0.33 (acetone/cyclohexane, 2 : 1); ESI-TOF MS:
(2,3,4,6-Tetra-O-acetyl-α-D-mannopyranosyl)-(1→2)-O-(3,4,6-tri-O-
acetyl-α-D-mannopyranosyl)-(1→2)-3,4,6-tri-O-acetyl-α-D-
mannopyranosyl Fluoride (10)
1
Found: 960.4 [M + 1]+. Calc. for C45H53NO22: 959.31. H NMR (500
MHz, (D6)DMSO) δ 7.85, d, J 7 Hz, 2H, Ar-Fmoc; 7.75, d, J 9 Hz, 1H,
NH; 7.70, d, J 7 Hz, 2H, Ar-Fmoc; 7.38, t, J 7 Hz, 2H, Ar-Fmoc; 5.22,
dd, J2,3 3, J3,4 10 Hz, 1H, H3; 5.18, dd, J2´,3´ 3, J3´,4´ 10 Hz, 1H, H3´;
5.14–5.06, m, 4H, H1, H4, H2´, and H4´; 4.99, d, J < 1 Hz, 1H, H1´;
4.23–3.98 m, 11H, H5, H6a, H6b, H5´, H6a´, H6b´, Hα, Hβ, CH2-
Fmoc, and CH-Fmoc; 3.90, s, 1H, H2; 2.07–1.91, 7 × s, 21H, Ac; 1.21,
d, J 6 Hz, 3H, Hγ. 13C NMR (125 MHz, (D6)DMSO) δ 171.2–170.5,
C=O; 157.6, C=O; 128.1–126.4, Ar-Fmoc; 120.2, Ar-Fmoc; 99.6, C1;
98.8, C1´; 76.9, C2; 76.7, Cβ; 70.2, C3; 69.6, C2´; 69.2, C5; 68.9, C5´;
68.8, C3´; 66.8, C4´; 66.7, CH2-Fmoc; 66.0, C4; 62.6, C6´; 62.5, C6;
59.6, Cα; 47.4, CH-Fmoc; 21.4–21.0, Ac; 18.8, Cγ.
To a stirred solution of trisaccharide (9) (1.1 g, 1.14 mmol) in 10 mL of
absolute dichloromethane at 0°C were added 10 mL of HF–pyridine
(65%). The biphasic reaction was complete after 3 h (TLC; acetone/
cyclohexane, 1.5 : 1). The mixture was diluted with 100 mL of
dichloromethane and added to a mixture of ice and water in a Teflon
separatory funnel. The organic phase was washed with 1 N HCl (× 3),
2 M KHCO3, dried over MgSO4 and concentrated. The crude product
was purified by flash chromatography using AcOEt/cyclohexane (2 : 1)
to obtain 0.580 g (0.63 mmol, 55%) of a white solid. [α]D20 +13.7 (c, 0.4
in CH2Cl2); RF 0.56 (acetone/cyclohexane, 1.5 : 1). ESI-TOF MS:
Found: 949.2 [M + Na]+. Calc. for C38H51FO25: 926.27. 1H NMR (500
MHz, (D6)DMSO) δ 5.90, dd, J 49 Hz, 1H, H1; 5.31, dd, J 10 Hz, 1H,
H4; 5.23–5.15, m, 5H, H3, H3´, H3´´, H2´, and H1´; 5.13, dd, J 10 Hz,
1H, H4´; 5.09, dd, J 10 Hz, 1H, H4´´; 5.07–5.00, m, 2H, H1´´ and H2´´;
4.34, m, 1H, H2; 4.18–3.99, m, 9H, H5, H6a, H6b, H5´, H6a´, H6b´,
H5´´, H6a´´, and H6b´´; 2.13–1.93, 30H, Ac. 13C NMR (125 MHz,
(D6)DMSO) δ 170.2–169.1, C=O; 106.4, d, C1; 98.0, C1´; 91.0, C1´´;
73.8, d, C2; 70.3, C5; 70.1, C2´; 68.7, C5´; 68.6, C2´´; 68.6, C3; 68.4,
C5´´; 68.3, C3´; 68.0, C3´´; 65.8, C4´; 65.3, C4´´; 64.3, C4; 62.4, C6´´;
62.8, C6´; 61.1, C6; 20.8–20.3, Ac.
N-(9-Fluorenylmethoxycarbonyl)-O-[O-(2,3,4,6-tetra-O-acetyl-α-D-
mannopyranosyl)-(1→2)-O-(3,4,6-tri-O-acetyl-α-D-
mannopyranosyl)-(1→2)-(3,4,6-tri-O-acetyl-α-D-mannopyranosyl)]-
L-threonine Benzyl Ester (12)
A suspension of fluoride (10) (460 mg, 0.45 mmol), Fmoc-Thr-OBzl
(1) (220 mg, 0.50 mmol) and 1 g of powdered and activated 4 Å
molecular sieves in 10 mL of absolute dichloromethane was stirred for
.
20 min. Subsequently, BF3 Et2O (110 µL, 0.9 mmol) was added. After
4 h the reaction was complete (TLC; acetone/cyclohexane, 1 : 1.5). The
mixture was diluted with 25 mL of dichloromethane and the molecular
sieves were filtered off over Celite. The organic phase was extracted
with 2 M KHCO3, dried over MgSO4 and concentrated. Flash
chromatography of the remainder using acetone/cyclohexane (1 : 4)
yielded 370 mg (0.28 mmol, 61%) of compound (12). [α]D20 +27.0 (c,
1.0 in CH2Cl2); RF 0.61 (acetone/cyclohexane, 1 : 1.5); ESI-TOF MS:
Found: 1338.4 [M + 1]+. Calc. for C64H75NO30: 1337.44. 1H NMR (500
MHz, (D6)DMSO) δ 8.12, d, J 9 Hz, 1H, NH; 7.89, d, J 7.5 Hz, 2H, Ar-
Fmoc; 7.70, t, J 7 Hz, 2H, Ar-Fmoc; 7.45–7.25, m, 9H, Ar-Fmoc and
Ar-Bzl; 5.29–5.07, m, 12H, H1, H3, H4, H1´, H2´, H3´, H4´, H2´´,
H3´´, H4´´, and H2-Bzl; 4.92, d, J < 1 Hz, 1H, H1´´; 4.37–3.98, m, 14H,
H5, H6a, H6b, H5´, H6a´, H6b´, H5´´, H6a´´, H6b´´, CH-Fmoc, CH2-
Fmoc, Hα, and Hβ; 3.81, br s, 1H, H2; 2.14–1.93, 30H, Ac; 1.24, d, J
6 Hz, 3H, Hγ. 13C NMR (125 MHz, (D6)DMSO) δ 170.4–169.2, C=O;
154.2, C=O; 100.1, C1; 98.9, C1´´; 91.8, C1´; 77.3, C2; 77.1, Cβ; 70.8,
C3; 70.4, C2´; 69.7, C5; 69.5, C3´; 69.4, C2´; 69.2, 2 × C, C5´ and C3´´;
69.0, C5´´; 67.6, CH2-Bzl; 67.2, C4´´; 66.9, CH2-Fmoc; 66.4, 2 × C,
C4´ and C4; 62.9, C6´´; 62.5, C6´; 62.3, C6; 58.6, Cα; 47.6, CH-Fmoc;
20.6–19.7, Ac; 18.6, Cγ.
N-(9-Fluorenylmethoxycarbonyl)-O-[(2,3,4,6-tetra-O-acetyl-α-D-
mannopyranosyl)-(1→2)-(3,4,6-tri-O-acetyl-α-D-mannopyranosyl)]-
L-threonine Benzyl Ester (11)
(a) From acetate (7). A portion of disaccharide (7) (9.42 g,
13.88 mmol) and amino acid (2) (5.17 g, 11.55 mmol) were dissolved
in 150 mL of absolute dichloromethane. The flask was flushed with
.
argon and BF3 Et2O complex (5.3 mL, 41.7 mmol) was added dropwise.
After 24 h the reaction mixture was diluted and washed with 2 M
KHCO3. The organic phase was dried over MgSO4, concentrated and
purified by flash chromatography with acetone/cyclohexane (1 : 1.2) to
obtain 6.76 g (6.5 mmol, 55%) of glycosyl amino acid (11). RF 0.52
(acetone/cyclohexane, 1 : 1.2).
(b) From fluoride (8). A suspension of fluoride (8) (200 mg,
0.32 mmol), Fmoc-Thr-OBzl (3) (150 mg, 0.34 mmol) and 400 mg of
ground and preactivated 4 Å molecular sieves in 6 mL of absolute
.
dichloromethane was stirred for 20 min. Subsequently, BF3 Et2O
(80 µL, 0.63 mmol) was added. After 2 h (TLC; acetone/cyclohexane,
1 : 1.2) the reaction was completed and the solids were removed by
filtration over Celite. The diluted filtrate was washed with 2 M KHCO3,
dried over MgSO4 and concentrated. The remainder was subjected to
flash chromatography (acetone/cyclohexane 1 : 1), yielding 235 mg
(0.22 mmol, 70%) of compound (11). [α]D20 +22.4 (c, 0.4 in CH2Cl2);
RF 0.52 (acetone/cyclohexane, 1 : 1.2). ESI-TOF MS: Found: 1050.7
[M + 1]+. Calc. for C52H59NO22: 1049.35. 1H NMR (500 MHz,
(D6)DMSO) δ 8.1, d, J 9 Hz, 1H, NH; 7.82, d, J 7 Hz, 2H, Ar-Fmoc;
7.67, t, J 7 Hz, 2H, Ar-Fmoc; 7.38–7.25, m, 9H, Ar-Fmoc and Ar-Bzl;
5.23–5.04, m, 8H, H1, H3, H4, H2´, H3´, H4´ and H2-Bzl; 4.89, s, 1H,
H1´; 4.32–4.20, m, 5H, CH2-Fmoc, CH-Fmoc, Hα, and Hβ; 4.10–3.97,
m, 6H, H5, H6a, H6b, H5´, H6a´, H6b´; 3.77, s, 1H, H2; 2.09–1.92, 7
× s, 21H, Ac; 1.20, d, J 6 Hz, 3H, Hγ. 13C NMR (125 MHz, (D6)DMSO)
δ 170.80–170.1, C=O; 157.4, C=O; 141.5, C=O; 129.1–126.0, Ar-
Fmoc; 99.5, C1; 98.8, C1´; 77.0, C2; 76.8, Cβ; 70.1, C3; 69.6, C2´;
69.2, C5; 68.8, C3´; 67.4, CH2-Bzl; 67.0, C4´; 66.8, CH2-Fmoc; 66.6,
C4; 62.7, C6´; 62.4, C6; 59.6, Cα; 47.4, CH-Fmoc; 21.2–21.1, Ac;
18.3, Cγ.
N-(9-Fluorenylmethoxycarbonyl)-O-[O-(2,3,4,6-tetra-O-acetyl-α-D-
mannopyranosyl)-(1→2)-O-(3,4,6-tri-O-acetyl-α-D-mannopyrano-
syl)-(1→2)-(3,4,6-tri-O-acetyl-α-D-mannopyranosyl)]-L-threonine C
A suspension of compound (12) (0.35 g, 0.26 mmol) and 40 mg of
palladium on carbon (10% Pd) in 5 mL of absolute MeOH was stirred
under a hydrogen atmosphere. After 3 h (TLC; acetone/cyclohexane,
2 : 1) the catalyst was removed by filtration. The combined washings
were evaporated and purified by flash chromatography using acetone/
cyclohexane (2 : 1). Yield 0.30 g (0.24 mmol, 91%) of glycosyl amino
acid C. [α]D20 +41.4 (c, 0.5 in CH2Cl3); RF 0.25 (acetone/cyclohexane,
3 : 1). ESI-TOF MS: Found: 1248.5 [M + 1]+. Calc. for C57H69NO30:
1247.39. 1H NMR (500 MHz, (D6)DMSO) δ 7.91, d, J 7.5 Hz, 2H, Ar-
Fmoc; 7.88, d, J 9 Hz, 1H, NH; 7.80, t, J 7 Hz, 2H, Ar-Fmoc; 7.77, d, J
7 Hz, 2H, Ar-Fmoc; 7.45, t, J 7.5 Hz, 2H, Ar-Fmoc; 5.30–5.08, m, 10H,
H1, H3, H4, H1´, H2´, H3´, H2´´, H3´´, and H4´´; 4.88, d, J < 1 Hz, 1H,
H1´´; 4.28–3.96, m, 14H, H5, H6a, H6b, H5´, H6a´, H6b´, H5´´, H6a´´,

Synthesis of Mannosyl and Oligomannosyl Threonine Building Blocks
165
H6b´´, Hα, Hβ, CH-Fmoc, and CH2-Fmoc; 3.88, s, 1H, H2; 2.12–1.94,
30H, Ac; 1.23, d, J 6 Hz, 3H, Hγ. 13C NMR (125 MHz, (D6)DMSO) δ
99.9, C1; 99.0, C1´´; 92.0, C1´; 77.0, C2; 76.9, Cβ; 70.9, C3; 70.4, C2´;
69.8, C5; 69.7, C3´; 69.3, C2´´; 69.2, C5´; 69.0, C3´´; 68.9, C5´´; 67.5,
C4´´; 67.0, CH2-Fmoc; 66.8, C4´; 66.6, C4; 62.8, C6´´; 62.4, C6´; 62.3,
C6; 57.9, Cα; 47.5, CH-Fmoc; 20.2–19.1, Ac; 19.0, Cγ.
[8] H. Herzner, T. Reipen, M. Schultz, H. Kunz, Chem. Rev. 2000,
100, 4495.
[9] G. Arsequell, G. Valencia, Tetrahedron: Asymmetry 1997, 8,
2839.
[10] L. A. Salvador, M. Elofsson, J. Kihlberg, Tetrahedron 1995, 51,
5643.
[11] A. M. Jansson, M. Meldal, K. Bock, J. Chem. Soc., Perkin Trans.
1 1992, 1699.
Acknowledgments
[12] (a) C.-D. Chang, M. Waki, M. Ahmad, J. Meienhofer, J. D.
Lundell Haug, Int. J. Pept. Protein Res. 1980, 15, 59; (b) J. M.
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[13] H. Vegad, C. J. Gray, P. J. Somers, A. S. Dutta, J. Chem. Soc.,
Perkin Trans. 1 1997, 1429.
This research has been supported by the President
of Colombia’s office and the Fonds der Deutschen
Chemischen Industrie. We also thank Dr Fanny Guzman for
her support.
[14] K. Toshima, K. Tatsuta, Chem. Rev. 1993, 93, 1503.
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J. Chem. Soc., Chem. Commun. 1984, 1155; (b) S. Hashimoto,
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