Synthesis of D-galactopyranosides of trans-4-hydroxy-L-proline utilizing the sulfoxide glycosylation method

Article abstract of DOI:10.1071/CH01156

Phenyl 1-thio-β-D-galactopyranoside (1) was converted into two sulfoxide glycosyl donors: phenyl (2,3,4,6-tetra-O-benzyl)-1-thio-β-D-galactopyranoside S-oxide (3) and phenyl (2,3,4,6-tetra-O-pivaloyl)-1-thio-β-D-galactopyranoside S-oxide (5). These glycosyl donors were then each reacted with Nα-fluorenylmethoxycarbonyl-trans-4-hydroxy-L-proline allyl ester (7). The glycosylation reactions were conducted at -70°C with triflic anhydride as promotor, in the presence of 2,6-di-tert-butyl-4-methylpyridine. In the case of the perbenzylated sulfoxide donor (3), the major product was Nα-fluorenylmethoxycarbonyl-trans-4-hydroxy-4-O-[(2,3,4,6-tetra-O-benzyl)- α-D-galactopyranosyl]-L-proline allyl ester (8α). In dichloromethane, the α-to-β ratio was 3:1 and in toluene this improved to 5:1, with a combined yield of 41%. In the case of the perpivaloylated sulfoxide donor (5), Nα-fluorenylmethoxycarbonyl-trans-4-hydroxy-4-O-[(2,3,4,6-tetra-O-pivaloyl )-β-D-galactopyranosyl]-L-proline allyl ester (9) was obtained as the sole glycoside product in 46% yield.

Full text of DOI:10.1071/CH01156

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Aust. J. Chem. 2002, 55, 135–140
Synthesis of D-Galactopyranosides of trans-4-Hydroxy-L-proline
Utilizing the Sulfoxide Glycosylation Method
Carol M. Taylor,A,B Claudette A. WeirC and Charlotte G. JørgensenC,D
A Institute of Fundamental Sciences, Massey University, Private Bag 11-222,
Palmerston North, New Zealand.
B Author to whom correspondence should be addressed (e-mail: C.M.Taylor@massey.ac.nz).
C Department of Chemistry, University of Auckland, Private Bag 92019, Auckland, New Zealand.
D Exchange student from the Technical University of Denmark, Copenhagen, Denmark.
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Phenyl 1-thio-β-D-galactopyranoside (1) was converted into two sulfoxide glycosyl donors: phenyl
(2,3,4,6-tetra-O-benzyl)-1-thio-β-D-galactopyranoside S-oxide (3) and phenyl (2,3,4,6-tetra-O-pivaloyl)-
1-thio-β-D-galactopyranoside S-oxide (5). These glycosyl donors were then each reacted with Nα-
fluorenylmethoxycarbonyl-trans-4-hydroxy-L-proline allyl ester (7). The glycosylation reactions were conducted at
–70°C with triflic anhydride as promotor, in the presence of 2,6-di-tert-butyl-4-methylpyridine. In the case of the
perbenzylated sulfoxide donor (3), the major product was Nα-fluorenylmethoxycarbonyl-trans-4-hydroxy-
4-O-[(2,3,4,6-tetra-O-benzyl)-α-D-galactopyranosyl]-L-proline allyl ester (8α). In dichloromethane, the α-to-β
ratio was 3:1 and in toluene this improved to 5:1, with a combined yield of 41%. In the case of the perpivaloylated
sulfoxide donor (5), Nα-fluorenylmethoxycarbonyl-trans-4-hydroxy-4-O-[(2,3,4,6-tetra-O-pivaloyl)-β-D-
galactopyranosyl]-L-proline allyl ester (9) was obtained as the sole glycoside product in 46% yield.
Manuscript received: 19 October 2001.
Final version: 9 January 2002.
Introduction
O
N
Glycosides of trans-4-hydroxy-L-proline (Hyp) are
widespread in the plant kingdom (Fig. 1).[1] In 1969,
Lamport proposed that the role of Hyp in extensin was to
provide a site for a covalent linkage between the protein and
the cell wall.[2] Hydroxyproline-rich glycoproteins (HRGPs)
are glycosylated by saccharides ranging from a single
arabinose or galactose residue, up to a 75-residue
arabinogalactan.[3] A β-D-xyloside was isolated from the
leaves of sugar beet, Beta vulgaris.[4] Two discoveries of Hyp
glycosides have been made outside the plant kingdom: a
β-D-galactoside from an algae, Chlamydomonas
reinhardtii,[5] and a novel, linear hexasaccharide, linked via
an N-acetylglucosamine (GlcNAc) residue, was recently
identified in SKP1, a glycoprotein of the slime mould
Dictyostelium discoideum.[6]
O
R
HO OH
HO
HO
O
O
HO
HO
OH
OH
β-D-Galp-
β-L-Araf-
OH
HO
HO
O
O
HO
OH
β-D-Xylp-
NHAc
D-GlcNAc
Fig. 1. Glycosides of trans-4-hydroxy-L-proline.
The synthesis of glycosides of Hyp has attracted consid-
erable attention. This is due to the potential of peptide glyco-
sylation to modulate biological activity rather than because
of their occurrence in nature. For example,
H–Tyr–D–Met–Gly–Phe–(β-D-Galp–Hyp)–NH2 is a partic-
ularly potent enkephalin analogue.[7]
We chose to focus on the synthesis of galactosides. Such
Hyp derivatives have been synthesized enzymatically using
β-galactosidases from E. coli and Achatina achatina, but the
chemical yields were less than 30%.[8,9] Several chemical
methods have been employed for the synthesis of Hyp
© CSIRO 2002
10.1071/CH01156
0004-9425/02/010135

136
C. M. Taylor, C. A. Weir and C. G. Jørgensen
glycosides: the Helferich reaction,[10–17] the Koenigs–Knorr
reaction,[12,17–21] the use of anomeric triflates,[22] and the
trichloroacetimidate method.[17,23] Glycosides have rarely
been obtained in greater than 60% yield.
The sulfoxide glycosylation method was introduced by
Kahne and coworkers in 1989.[24,25] A sulfoxide glycosyl
donor is activated with triflic anhydride, in the presence of
2,6-di-tert-butyl-4-methylpyridine (DTBMP), to give a
species which is highly reactive at low temperatures.[26,27]
The method has found wide application in the construction
of glycosidic linkages to sterically hindered alcohols.[28]
Glycosides of Thr have been synthesized and the resulting
Kunz and coworkers.[35] The allyl ester (7) was synthesized
from Fmoc–Hyp–OH (6) according to Scheme 2.
Fmoc
N
Fmoc
N
(i) Cs2CO3, MeOH
(ii) CH2=CHCH2Br, DMF
(86%)
COOH
COOCH2CHCH2
(7)
HO
HO
(6)
Scheme 2
The glycosylation reactions are depicted in Scheme 3.
Sulfoxide (3) has been reported previously and employed in
the synthesis of oligosaccharides containing an α-D-
building
blocks
incorporated
successfully
into
galactopyranosyl group.[32] In the absence of
a
glycopeptides.[29,30] We sought to apply the sulfoxide
glycosylation method to the synthesis of galactosides of
Hyp. We felt that the high reactivity of the glycosylating
species, coupled with the ability to control the
stereochemistry of the glycosidic linkage by an appropriate
choice of protecting groups, boded well for the synthesis of
both α- and β-galactosides. The synthesis of both is
ultimately important for diversity when studying
structure–activity relationships (SARs).
‘neighbouring group’ at C2, the anomeric effect is normally
the dominant force in determining the stereochemistry of the
glycosidic linkage. A mixture of anomers of (8) was
obtained, favouring the α-glycoside. When the reaction was
conducted in dichloromethane a ratio of 3:1 was obtained; in
toluene this improved to 5:1. The glycosides each existed as
mixtures of two rotamers about the C–N bonds of their
carbamate functionality, and most signals in the nuclear
magnetic resonance (NMR) spectra were doubled up. In the
case of the α-glycoside, a pair of signals was observed at δ
96.9 and 97.7 in the 13C NMR spectrum, which was assigned
to the anomeric carbon. These chemical shifts are consistent
with an α-galactoside.[22] The minor glycoside product had a
13C signal at δ 103.3, supporting the presence of a
β-glycosidic linkage. Increasing the amount of donor (3) or
acceptor (7) did not improve the yield; in fact the resulting
increase in by-products made it more difficult to purify the
desired products.
Results and Discussion
Phenyl 1-thio-β-D-galactopyranoside (1) used to be
commercially available at reasonable cost; this is no longer
the case. This starting material was therefore prepared in
[31]*
two steps from 1,2,3,4,6-penta-O-acetyl-D-galactose.
Sulfoxides (3)[32] and (5),[33] intended to lead to α- and
β-glycosides, respectively (see above), were both prepared in
two steps from compound (1) (Scheme 1).
Sulfoxide (5), bearing a pivaloate protecting group at C2,
has been used by Kahne and coworkers to generate
β-galactosides.[33,36,37] The high stereoselectivity observed
in these reactions is attributed to neighbouring group
participation by the bulky pivaloate ester,[38] which
discourages approach of the glycosyl acceptor from the
α-face. The steric bulk of the pivaloyl group can also impede
glycosylation reactions, perhaps accounting for the modest
yield of β-glycoside (9) in the present case. A single
stereoisomer was obtained, which appeared as a mixture of
two rotamers about the carbamate C–N bond in the NMR
spectra. Assignment of the stereochemistry of the glycosidic
BnO OBn
O
PhCH2Br,
NaH, DMF
BnO
X
OBn
(67%)
(2) X = SPh
m-CPBA,
CH2Cl2
HO OH
(87%)
(3) X = SOPh
O
HO
SPh
OH
PivO OPiv
PivO
Me3COCl,
DMAP, py
(1)
O
(74%)
X
OPiv
linkage was based on literature precedent,[33,36,37] and the 13
C
(4) X = SPh
m-CPBA,
CH2Cl2
NMR spectrum, in which two signals were observed at δ
100.3 and 100.5. These signals were attributed to the
anomeric carbon and are consistent with a β-galactoside.
(84%)
(5) X = SOPh
Scheme 1
Conclusion
Our choice of
a trans-4-hydroxy-L-proline (Hyp)
In summary, glycosides (8) and (9) have been synthesized
using the sulfoxide glycosylation method in yields of 41 and
46%, respectively. By comparison, Andreotti and Kahne
glycosyl acceptor involved temporary Nα-protection by the
fluorenylmethyl carbamate (Fmoc) group and subsequent
masking of the carboxylic acid as its allyl ester. This choice
was influenced by protecting groups employed in other
projects ongoing in our laboratory,[34] and the experience of
reported
a
70% yield for the glycosylation of
Fmoc–L-Thr–OBn with phenyl 3,4,6-tri-O-acetyl-2-azido-
*1,2,3,4,6-Penta-O-acetyl-galactopyranoside can be prepared according to this reference. It is also commercially available from Aldrich (Catalogue
No. 13,403-1).

Synthesis of D-Galactopyranosides of trans-4-Hydroxy-L-proline
137
Fmoc
N
O
OCH2CHCH2
Fmoc
N
O
Fmoc
N
BnO OBn
BnO
O
Tf2O, DTBMP
4 Å molecular sieves
BnO OBn
BnO
O
O
O
OCH2CHCH2
OBn
OCH2CHCH2
+
toluene, –70˚C, 2 h
(41%)
+
O
SPh
OBn
OBn
O
HO
O
OBn
(3)
(7)
BnO OBn
(8α)
(8β)
(8α)-to-(8β) ratio 5.0 : 1.0
Fmoc
O
N
PivO OPiv
PivO
PivO OPiv
PivO
(i) Tf2O, DTBMP
OCH2CHCH2
O
CH2Cl2, –70˚C, 10 min
O
O
SPh
O
(ii) (7), CH2Cl2, –70˚C, 3 h
(46%)
OPiv
OPiv
(5)
(9)
Scheme 3
Scheme 3
(E-Merck) and spots were visualized by ultraviolet (UV) light or by
staining with anisaldehyde. Flash chromatography was conducted on
silica gel 60 (230–400 mesh) from Scharlau. High-performance liquid
2-deoxy-1-thio-galactopyranosyl S-oxide (1:1 ratio of
anomers).[29] Similar results were reported for the
attachment of a disaccharide.[30] Their lack of stereocontrol
is a consequence of the azido substitutent at C2. The lower
chemical yield of our reactions may be attributable to steric
hinderance associated with the Hyp–OH glycosyl acceptor,
and complications arising from the conformation of the
pyrrolidine ring.
It may be possible to enhance the nucleophilicity of the
hydroxy group by altering the conformation of the
pyrrolidine ring, although the attempts of Burger et al. were
clearly a move in the wrong direction.[17] It may also be
possible to increase the reactivity of the glycosyl donor in the
formation of β-glycosides by utilizing electron-donating
p-methoxybenzyl (PMB) ether protecting groups, instead of
bulky, electron-withdrawing pivaloate esters. PMB ethers
have been demonstrated to lead to β-glycosides with good
stereoselectivity,[28a,39] although the mechanistic basis for
this is unclear.
chromatography (HPLC) was conducted on
a
Waters 600
semi-preparative system, equipped with a Waters 2487 dual wavelength
UV detector. Methanol was dried and distilled from magnesium
turnings and stored over
4
Å
molecular sieves. N,N-
Dimethylformamide (DMF) was dried and distilled from BaO and
stored over 4 Å molecular sieves. Dichloromethane was freshly distilled
from CaH2. Pyridine was dried and distilled from CaH2 and stored over
KOH pellets. Toluene was freshly distilled from Na. Allyl bromide and
pivaloyl chloride were obtained from Acros. Thiophenol was purchased
from Riedel-de-Häen. Sodium methoxide, Amberlite-IR H+ resin,
sodium hydride (as
a 60% dispersion in mineral oil) and
1,6-di-tert-butyl-4-methylpyridine (DTBMP) were purchased from
Aldrich. Benzyl bromide and N,N-dimethylaminopyridine (DMAP)
were obtained from Merck. m-Chloroperbenzoic acid (m-CPBA) and
caesium carbonate were obtained from Janssen. trans-
4-Hydroxyproline (Hyp) was purchased from Lancaster. All other
reagents were used without further purification.
Phenyl 1-Thio-β-D-galactopyranoside (1)
BF OEt (13.08 mL, 15.09 g, 0.106 mol, 5 equiv.) was added dropwise
·
3
2
While we will seek to improve the efficiency with which
we can produce Hyp glycosides, we already have sufficient
quantities of these glycosylated hydroxyproline building
blocks to incorporate them into peptides and to investigate
the effect of glycosylation on structure and function, relative
to the parent peptides.
to a solution of 1,2,3,4,6-penta-O-acetyl-D-galactose[31] (8.30 g, 0.021
mol, 1.0 equiv.) and thiophenol (2.62 mL, 2.81 g, 0.026 mol, 1.2 equiv.)
in CH2Cl2 (85 mL) at 0°C. The mixture was warmed to room
temperature and then heated at reflux for 5.5 h. The mixture was cooled,
quenched with water and diluted with CH2Cl2 (100 mL), washed with
water (200 mL), sat. aq. NaHCO3 (200 mL), water (200 mL) and brine
(200 mL). The organic layer was dried (MgSO4), filtered and
concentrated. The residue was subjected to flash chromatography, with
3:1 to 1:1 hexanes/EtOAc as eluent, to give mixed fractions of phenyl
Experimental
General
2,3,4,6-tetra-O-acetyl-1-thio-α-D-galactopyranoside
and
phenyl
2,3,4,6-tetra-O-acetyl-1-thio-β-D-galactopyranoside (4.59 g, 49%),
and pure phenyl 2,3,4,6-tetra-O-acetyl-1-thio-β-D-galactopyranoside
(4.62 g, 49%). Total yield 98%.
Optical rotations were determined on either a Perkin Elmer 341 or an
Optical Activity Ltd AA-10 polarimeter. NMR spectra were recorded
on one of the following NMR spectrometers: Bruker DRX-400 (400
MHz for 1H and 100 MHz for 13C), Bruker Avance 400 (400 MHz for
Phenyl
2,3,4,6-tetra-O-acetyl-1-thio-β-D-galactopyranoside:
(Found [HRMS–chemical ionization (CI)]: [M+NH4]+, 458.1485. Calc.
for C20H28NO9S: [M+NH4]+, 458.1484). RF 0.53 (1:1 hexanes/EtOAc).
[α]D20 +5.3° (c, 1.35 in CHCl3). 1H NMR (CDCl3, 200 MHz) δ 1.98, s,
3H, Ac; 2.05, s, 3H, Ac; 2.10, s, 3H, Ac; 2.12, s, 3H, Ac; 3.94, app. t, J
6.5 Hz, 1H, H5; 4.11, dd, J 11.3, 6.1 Hz, 1H, H6a; 4.20, dd, J 11.3, 7.2
Hz, 1H, H6b; 4.72, d, J 9.9 Hz, 1H, H1; 5.05, dd, J 9.9, 3.2 Hz, 1H, H3;
1
1H and 100 MHz for 13C), Bruker AM-200 (200 MHz for H and 50
MHz for 13C) or JEOL 270 JNM-GX270W (270 MHz for 1H and 67.5
MHz for 13C). NMR data for compounds (2), (3A,B), (4), (5A,B), (7),
(8α,β) and (9) are available as supplementary material (website:
http://www.publish.csiro.au/journals/ajc/AccessMat.cfm). Thin-layer
chromatography (TLC) was performed on Kieselgel 60 F254

138
C. M. Taylor, C. A. Weir and C. G. Jørgensen
5.25, app. t, J 9.9 Hz, 1H, H2; 5.42, d, J 3.2 Hz, 1H, H4; 7.30–7.41, m,
3H, ArH; 7.49–7.54, m, 2H, ArH. 13C NMR (CDCl3, 50 MHz) δ 20.1,
20.4, 61.3, 66.9 (2C), 71.5, 74.0, 85.9, 127.7, 128.5, 132.1, 168.9,
169.5, 169.7, 169.8.
Sodium methoxide (518 mg, 9.12 mmol, 3.3 equiv.) was added to a
solution of phenyl 2,3,4,6-tetra-O-acetyl-1-thio-β-D-galactopyranoside
(1.217 g, 2.76 mmol, 1.0 equiv.) in dry methanol (25 mL). The solution
Second-eluting diastereoisomer (3B). RF 0.22 (2:1 hexanes/
EtOAc). H NMR (CDCl3, 400 MHz) δ 3.51, dd, J 8.9, 7.3 Hz, 1H,
1
H6a; 3.61, dd, J 8.9, 5.5 Hz, 1H, H6b; 3.65–3.70, m, 2H, H3, H5; 3.92,
d, J 1.3 Hz, 1H, H4; 4.03, app. t, J 9.0 Hz, 1H, H2; 4.38–4.87, m, 9H,
CH2Ph, H1; 7.07–7.12, m, 2H, ArH; 7.24–7.36, m, 21H, ArH;
7.56–7.59, m, 2H, ArH. 13C NMR (CDCl3, 50 MHz) δ 68.1, 72.3, 72.6,
73.5, 73.6, 74.0, 74.4, 77.4, 83.8, 95.3, 125.9, 127.1, 127.6, 127.8,
128.1, 128.2, 128.4, 130.9, 137.7, 137.9, 138.5, 140.2.
was stirred at room temperature, under nitrogen for
2 h.
Amberlite-IR120 H+ resin (4 g) was added and the suspension stirred
for 15 min. The resin was removed by filtration and washed well with
methanol. The filtrate was concentrated and the residue purified by
flash chromatography, with 9:1 to 17:3 CH2Cl2/MeOH as eluent, to
give (1) as a colourless solid (681 mg, 91%) (Found [HRMS–electron
impact (EI)]: M+, 272.0713. Calc. for C12H16O5S: M+, 272.0718). RF
0.11 (9:1 CH2Cl2/MeOH). 1H NMR (CD3OD, 200 MHz) δ 3.35–3.77,
m, 5H, H2, H3, H5, H6a, H6b; 3.95, d, J 2.6 Hz, 1H, H4; 4.62, d, J 9.2
Hz, 1H, H1; 7.17–7.33, m, 3H, ArH; 7.53–7.57, m, 2H, ArH. 13C NMR
(CD3OD, 50 MHz) δ 62.5, 70.4, 70.9, 76.1, 80.3, 90.1, 128.0, 129.9,
132.0, 135.8.
Phenyl (2,3,4,6-Tetra-O-pivaloyl)-1-thio-β-D-galactopyranoside (4)
Pivaloyl chloride (2.10 mL, 2.05 g, 17.1 mmol, 8 equiv.) and
4-dimethylaminopyridine (131.5 mg, 1.08 mmol, 0.5 equiv.) were
added to a solution of phenyl 1-thio-β-D-galactopyranoside (1) (580.6
mg, 2.13 mmol, 1.0 equiv.) in pyridine (10 mL). The solution was
heated at 100°C overnight, cooled, diluted with CH2Cl2 (120 mL),
washed with water (120 mL) and brine (120 mL). The organic layer was
dried (MgSO4), filtered and concentrated. The residue was purified by
flash column chromatography, with 9:1 hexanes/EtOAc as eluent, to
afford phenyl (2,3,4,6-tetra-O-pivaloyl)-1-thio-β-D-galactopyranoside
(4) (1.09 g, 84%) (Found (HRMS–CI): [M+NH4]+, 626.3377.
C32H52NO9S requires [M+NH4]+, 626.3362). RF 0.55 (5:1
hexanes/EtOAc). 1H NMR (CDCl3, 200 MHz) δ 1.09, s, 9H, Piv; 1.17,
s, 9H, Piv; 1.19, s, 9H, Piv; 1.22, s, 9H, Piv; 3.97–4.21, m, 2H, H5, H6a;
4.19, dd, J 13.7, 9.6 Hz, 1H, H6b; 4.73, d, J 9.6 Hz, 1H, H1; 5.13, dd, J
9.6, 2.8 Hz, 1H, H3; 5.22, app. t, J 9.6 Hz, 1H, H2; 5.42, d, J 2.8 Hz,
1H, H4; 7.29–7.34, m, 3H, ArH; 7.50–7.54, m, 2H, ArH. 13C NMR
(CDCl3, 50 MHz) δ 26.9, 27.0, 38.6, 38.8, 61.3, 66.5, 66.8, 72.0, 74.6,
85.7, 128.2, 128.7, 131.2, 133.5, 176.2, 176.6, 177.0, 177.7.
Phenyl (2,3,4,6-Tetra-O-benzyl)-1-thio-β-D-galactopyranoside (2)
Benzyl bromide (1.93 mL, 2.78 g, 16.3 mmol, 6.5 equiv.) was added to
a solution of phenyl 1-thio-β-D-galactopyranoside (1) (681 mg, 2.50
mmol, 1.0 equiv.) in DMF (7.0 mL) at 0°C. Sodium hydride (651 mg,
16.3 mmol, 6.5 equiv.) was added over 1.75 h. The mixture was warmed
at 40°C for 5 h, and then left to stir at room temperature overnight. The
reaction mixture was diluted with CH2Cl2 (75 mL) and washed with
water (75 mL). The aqueous layer was further extracted with CH2Cl2
(75 mL). The organic extracts were combined and washed with sat. aq.
NaHCO3 (150 mL), water (150 mL) and brine (150 mL), dried
(MgSO4), filtered and concentrated. The residue was purified by flash
column chromatography, with 5:1 hexanes/EtOAc as eluent, to afford
phenyl (2,3,4,6-tetra-O-benzyl)-1-thio-β-D-galactopyranoside (2) as a
colourless solid (1.37 g, 87%) (Found [HRMS–fast atom bombardment
(FAB)]: [M+H]+, 633.2655. C40H41O5S requires [M+H]+, 633.2674).
RF 0.51 (2:1 hexanes/EtOAc). 1H NMR (CDCl3, 200 MHz) δ
3.56–3.67, m, 4H, H3, H5, H6a, H6b; 3.91, app. t, J 9.4 Hz, 1H, H2;
3.97, d, J 2.7 Hz, 1H, H4; 4.37–4.95, m, 9H, CH2Ph, H1; 7.15–7.39, m,
23H, ArH; 7.53–7.58, m, 2H, ArH. 13C NMR (CDCl3, 50 MHz) δ 68.7,
72.7, 73.5 (2C), 74.4, 75.6, 77.3 (2C), 84.1, 87.7, 126.9, 127.4, 127.5,
127.7, 127.8, 127.9, 128.1, 128.3, 128.4, 128.7, 129.3, 129.7, 129.9,
131.4, 131.9, 134.1, 137.8, 138.1, 138.2, 138.7.
Phenyl (2,3,4,6-Tetra-O-pivaloyl)-1-thio-β-D-galactopyranoside
S-Oxide (5)
m-Chloroperbenzoic acid (180 mg, 67% w/w, 0.69 mmol, 1.2 equiv.)
was added to
a
solution of phenyl (2,3,4,6-tetra-O-pival-
oyl)-1-thio-β-D-galactopyranoside (4) (354 mg, 0.58 mmol, 1.0 equiv.)
in CH2Cl2 (10 mL) at –30°C. After stirring for 30 min at –30°C under
N2, the reaction mixture was quenched by the addition of dimethyl
sulfide (3 drops), and warmed to room temperature. The solution was
diluted with CH2Cl2 (50 mL), washed with water (50 mL), sat. aq.
NaHCO3 (50 mL), and water again (50 mL). The organic layer was
dried (MgSO4), filtered and concentrated. The residue was purified by
flash column chromatography, with 5:1 then 1:1 hexanes/EtOAc as
eluent, to afford (5A) as a colourless solid (106 mg), fractions contain-
ing a mixture of (5A) and (5B) (32 mg), and (5B) as a colourless solid
(168 mg). Total yield: 307 mg (84%) (Found (HRMS–DEI, of mixture):
M+, 624.2959. Calc. for C32H48O10S: M+, 624.2968).
Phenyl (2,3,4,6-Tetra-O-benzyl)-1-thio-β-D-galactopyranoside
S-Oxide (3)
First-eluting diastereoisomer (5A). RF 0.38 (3:1 hexanes/EtOAc).
1H NMR (CDCl3, 200 MHz) δ 1.08, s, 18H, Piv; 1.12, s, 9H, Piv; 1.21,
s, 9H, Piv; 3.87–4.09, m, 3H, H5, H6a, H6b; 4.42, d, J 9.8 Hz, 1H, H1;
5.14, dd, J 9.8, 3.0 Hz, 1H, H3; 5.34, d, J 3.0 Hz, 1H, H4; 5.52, app. t,
m-Chloroperbenzoic acid (166 mg, 0.65 mmol, 67% w/w, 1.2 equiv.)
was added to
a
solution of (2,3,4,6-tetra-O-benzyl)-1-phe-
nylthio-β-D-galactoside (2) (340 mg, 0.54 mmol, 1.0 equiv.) in CH2Cl2
(10 mL) at –30°C. After stirring for 20 min at –30°C under N2, the reac-
tion mixture was quenched by the addition of dimethyl sulfide (3
drops), and warmed to room temperature. The solution was diluted with
CH2Cl2 (40 mL), washed with water (40 mL), sat. aq. NaHCO3 (40
mL), and water again (40 mL). The organic layer was dried (MgSO4),
filtered and concentrated. The residue was purified by flash column
chromatography, with 5:1 then 1:1 hexanes/EtOAc as eluent, to afford
the first-eluting diastereoisomer (3A) as a colourless solid (76 mg),
fractions containing a mixture of (3A) and the second-eluting diastere-
oisomer (3B) (129 mg), and (3B) as a colourless solid (100 mg). Total
yield: 305 mg (87%) (Found (HRMS–FAB, of mixture): [M+H]+,
649.2642. Calc. for C40H41O6S: [M+H]+, 649.2623).
J 9.8 Hz, 1H, H2; 7.46–7.55, m, 3H, ArH; 7.59–7.65, m, 2H, ArH. 13
C
NMR (CDCl3, 50 MHz) δ 26.9, 38.6, 38.7, 38.8, 38.9, 61.0, 64.4, 66.5,
72.1, 75.6, 89.5, 125.8, 128.8, 131.5, 138.8, 176.0, 176.7, 177.3, 177.7.
Second-eluting diastereoisomer (5B). RF 0.25 (3:1 hexanes/
1
EtOAc). H NMR (CDCl3, 200 MHz) δ 0.95, s, 9H, Piv; 1.09, s, 9H,
Piv; 1.16, s, 9H, Piv; 1.25, s, 9H, Piv; 3.67–3.80, m, 1H, H5; 4.00–4.14,
m, 2H, H6a, H6b; 4.65, dd, J 8.8, 2.0 Hz, 1H, H3; 5.04–5.19, m, 2H,
H1, H2; 5.31, d, J 2.0 Hz, 1H, H4; 7.50–7.57, m, 3H, ArH; 7.74–7.81,
m, 2H, ArH. 13C NMR (CDCl3, 50 MHz) δ 26.8, 26.9, 38.6, 38.9, 60.2,
64.8, 65.9, 71.7, 74.9, 92.2, 127.1, 128.5, 131.9, 137.1, 176.2, 176.8,
176.9, 177.6.
First-eluting diastereoisomer (3A). RF 0.29 (2:1 hexanes/EtOAc).
1H NMR [(D6)acetone, 200 MHz] δ 3.32, dd, J 9.9, 5.9 Hz, 1H, H6a;
3.55, dd, J 9.9, 5.9 Hz, 1H, H6b; 3.66, td, J 5.9, 1.1 Hz, 1H, H5; 3.96,
dd, J 9.6, 2.9 Hz, 1H, H3; 4.14, dd, J 2.9, 1.1 Hz, 1H, H4; 4.23, d, J 9.6
Hz, 1H, H1; 4.38, app. t, J 9.6 Hz, 1H, H2; 4.61–5.08, m, 8H, CH2Ph;
7.14–7.24, m, 2H, ArH; 7.27–7.39, m, 14H, ArH; 7.43–7.54, m, 7H,
ArH; 7.64–7.69, m, 2, ArH. 13C NMR [(D6)acetone, 50 MHz] δ 69.8,
72.9, 73.6, 74.9, 75.2, 75.5, 76.1, 79.6, 84.9, 94.4, 126.0, 128.2, 128.4,
128.9, 129.2, 129.5, 131.2, 139.6.
Nα-Fluorenylmethoxycarbonyl-trans-4-hydroxy-L-proline
Allyl Ester (7)
Caesium carbonate (82 mg, 0.25 mmol, 0.5 equiv.) was added to a
suspension of N-fluorenylmethoxycarbonyl-trans-4-hydroxy-L-proline
(6) (179 mg, 0.51 mmol, 1.0 equiv.) (prepared according to the general
procedure of Carpino and Han[40]) in dry methanol (2.5 mL). The
homogeneous reaction mixture was left to stir at room temperature
under N2 for 2 h. The methanol was removed under reduced pressure

Synthesis of D-Galactopyranosides of trans-4-Hydroxy-L-proline
139
and the colourless foam was dissolved in dry DMF (2.5 mL) and treated
with allyl bromide (53 µL, 0.61 mmol, 1.2 equiv.). The resulting colour-
less suspension was stirred at room temperature overnight. The reaction
mixture was diluted with EtOAc (30 mL), washed with water (30 mL),
brine (30 mL), dried (MgSO4), filtered and concentrated. The residue
was purified by flash column chromatography, with 1:1
hexanes/EtOAc as eluent, to obtain Nα-fluorenylmethoxycarb-
onyl-trans-4-hydroxy-L-proline allyl ester (7) as a colourless oil (199
mg, 86%) (Found (HRMS–CI): [M+H]+, 394.1646. C23H24NO5
CH=CHB; 5.85–5.99, m, 1H, CH=CH2; 7.21–7.39, m, 24H, ArH;
7.52–7.61, m, 2H, ArH; 7.73–7.76, m, 2H, ArH. 13C NMR
[(D6)acetone, 67.5 MHz] δ 36.1 and 37.3, 47.8 and 48.0, 53.9 and 54.3,
58.3 and 58.7, 65.8 and 66.0, 67.9, 69.5, 73.1, 73.7, 73.9, 75.0, 75.1,
75.3, 75.4, 75.5, 76.9 and 77.7, 80.0 and 80.1, 83.0, 103.3, 117.8 and
118.2, 125.8, 125.9, 126.0, 127.8, 127.9, 128.0, 128.1, 128.2, 128.3,
128.4, 128.5, 128.6, 128.8, 128.9. 133.1, 139.3, 139.7, 139.9, 140.0,
141.8, 144.7, 154.6 and 155.0, 172.3 and 172.5.
1
requires [M+H]+, 394.1654). RF 0.34 (2:1 hexanes/EtOAc). H NMR
Nα-Fluorenylmethoxycarbonyl-trans-4-hydroxy-4-O-[(2,3,4,6-tetra-
|O-pivaloyl)-β-D-galactopyranosyl]-L-proline Allyl Ester (9)
(CDCl3, 200 MHz) (mixture of rotamers) δ 2.10, ddd, J 12.7, 7.6, 5.0
Hz, 1H, Hβ; 2.28–2.45, m, 1H, Hβ´; 3.54, d, J 11.4 Hz, Hδ; 3.68, s, 1H,
OH; 3.75, dd, J 11.4, 4.4 Hz, 1H, Hδ´; 4.13–4.66, m, 5H, Fmoc CH and
CH2, CH2CH=CH2, Hα, Hγ; 5.14–5.35, m, 2H, CH=CH2; 5.75–5.96,
m, 1H, CH=CH2; 7.28–7.41, m, 4H, ArH; 7.51–7.61, m, 2H, ArH; 7.73,
d, J 7.4 Hz, 2H, ArH. 13C NMR (CDCl3, 50 MHz) (mixture of rotamers)
δ 38.4 and 39.3, 47.1, 54.6 and 55.3, 57.6 and 57.9, 65.8, 67.6 and 67.8,
69.2, 70.0, 118.5 and 118.9, 119.9, 124.9, 125.1, 127.0, 127.6, 131.5
and 131.6, 141.2, 143.5, 143.7, 143.9, 154.4 and 154.9, 172.2.
Phenyl (2,3,4,6-tetra-O-pivaloyl)-1-thio-β-D-galactopyranoside S-
oxide (5) (64 mg, 0.10 mmol, 1.0 equiv.) and DTBMP (42 mg, 0.20
mmol, 2.0 equiv.) were azeotroped with toluene three times, dissolved
in dry CH2Cl2 (3 mL) and cooled to –70°C. Triflic anhydride (35 mg,
21 µL, 0.12 mmol, 1.2 equiv.) was added and the solution was left to stir
for 10 min at –70°C. N-Fluorenylmethoxycarbonyl-L-trans-4-hydroxy-
proline allyl ester (7) (87 mg, 0.22 mmol, 2.2 equiv.) was azeotroped
with toluene three times, dissolved in dry CH2Cl2 (2 mL) and added
dropwise to the glycosyl donor mixture. The resulting mixture was
stirred at –70°C under N2 for 3 h. The reaction mixture was quenched
by the addition of sat. aq. NaHCO3 (0.5 mL) and left to warm to room
temperature. The mixture was diluted with EtOAc (35 mL) and washed
with water (15 mL). The organic layer was dried (MgSO4), filtered and
concentrated. The residue was purified by flash column
chromatography, with 5:1 then 3:1 hexanes/EtOAc as eluent. This
afforded Nα-fluorenylmethoxycarbonyl-trans-4-hydroxy-4-O-[(2,3,4,
6-tetra-O-pivaloyl)-β-D-galactopyranosyl]-L-proline allyl ester (9) as a
colourless oil (42 mg, 46%) (Found (HRMS–FAB+): [M+H]+,
892.4496. C49H66NO14 requires [M+H]+, 892.4483). RF 0.21 (3:1
4-O-[(2,3,4,6-Tetra-O-benzyl)-Nα-fluorenylmethoxycarbonyl-D-
galactopyranosyl]-trans-4-hydroxy-L-proline Allyl Ester (8)
Molecular sieves (1 g) were added to
a
solution of
(2,3,4,6-tetra-O-benzyl)-1-thio-β-D-galactopyranoside S-oxide (3B)
(204.5 mg, 0.315 mmol, 1.2 equiv.), N-fluorenylmethoxycarbo-
nyl-trans-4-hydroxyproline allyl ester (7) (103.8 mg, 0.264 mmol, 1.0
equiv.) and DTBMP (233 mg, 1.13 mmol, 3.6 equiv.) in toluene
(15 mL). The suspension was stirred at room temperature under N2 for
1 h and then cooled to –70°C. Triflic anhydride (42 µL, 71 mg, 0.252
mmol, 0.8 equiv.) was added and the mixture was stirred at –70°C for
2 h. Sat. aq. NaHCO3 (5 mL) was added, the suspension warmed to
room temperature and then filtered through a pad of Celite, washing
well with EtOAc. The filtrate was washed with sat. aq. NaHCO3 (50
mL), water (50 mL), brine (50 mL), and then dried (MgSO4), filtered
and concentrated. The residue was partially purified by flash chroma-
tography, with 3:1 to 1:1 hexanes/EtOAc as eluent, to give the
α-anomer (8α) (74.9 mg) and mixed fractions (63.8 mg). The latter was
subjected to HPLC to give more α-anomer (8α) (7.4 mg, for a total of
82.3 mg, 34%) and the β-anomer (8β) (17.8 mg, 7%).
Nα-Fluorenylmethoxycarbonyl-trans-4-hydroxy-4-O-[(2,3,4,6-tetra-
O-benzyl)-α-D-galactopyranosyl]-L-proline allyl ester (8α). (Found
(HRMS–FAB): [M+H]+, 916.4036. Calc. for C57H58NO10: [M+H]+,
916.4060). RF 0.35 (2:1 hexanes/EtOAc). HPLC: Rt 21.9 min (3:1
hexanes/EtOAc; 0.6 mL min–1, 4.6 by 250 mm silica column). 1H NMR
(CDCl3, 400 MHz) δ 2.13–2.22, m, 1H, Hyp Hβ; 2.47–2.55, m, 1H,
Hyp Hβ´; 3.44–3.56, m, 3H, Gal H5, H6a, H6b; 3.68–3.76, m, 1H, Hyp
Hδ; 3.80, dd, J 11.2, 5.2 Hz, 1H, Hyp Hδ´; 3.89, dd, J 10.1, 2.8 Hz, 1H,
Gal H3; 3.91–3.97, m, 2H, Gal H2, H4; 4.02–4.05, m, 1H, Hyp Hα/Hγ;
4.15, dd, J 13.0, 6.3 Hz, 1H, Hyp Hα/Hγ; 4.21–4.63, m, 8H, Gal H1,
Fmoc CH, CH2, CH2CH=CH2, CH2Ph; 4.69–4.95, m, 6H, CH2Ph;
5.15–5.35, m, 2H, CH=CH2; 5.76–5.92, m, 1H, CH=CH2; 7.21–7.44,
m, 24H, ArH; 7.51–7.63, m, 2H, Fmoc ArH; 7.75, d, J 7.5 Hz, 2H,
Fmoc ArH. 13C NMR [(D6)acetone, 67.5 MHz] (mixture of rotamers) δ
37.7 and 38.2, 47.7 and 47.9, 52.7 and 52.8, 56.7 and 59.2, 65.7 and
66.0, 67.9 and 68.0, 70.1, 70.8, 72.8, 73.1, 73.2, 73.6, 75.3, 75.5, 76.4,
77.0, 79.3, 97.4 and 97.9, 117.8 and 118.2, 120.6, 125.8, 125.9, 127.6,
127.7, 127.8, 127.9, 128.0, 128.1, 128.2, 128.3, 128.6, 128.7, 128.8,
133.0 and 133.1, 129.3, 139.8, 129.9, 141.8, 144.6, 144.7, 144.8, 154.7
and 154.9, 172.3 and 172.6.
hexanes/EtOAc). [α]D20 –23.3° (c, 0.70 in CHCl3). H NMR (CDCl3,
1
400 MHz) (mixture of rotamers) δ [0.95, s, 3H; 1.09, s, 3H; 1.11, s, 3H;
1.12, s, 3H; 1.16, s, 6H; 1.17, s, 3H; 1.18, s, 3H; 1.19, s, 3H; 1.25, s,
3H; 1.26, s, 3H; 1.28, s, 3H] total 36H, 4 × Piv; 2.13–2.19, m, 1H, Hyp
Hβ; 2.30–2.41, m, 1H, Hyp Hβ´; 3.68–3.88, m, 1H, Hyp Hδ, Hδ´;
3.94–4.11, m, 2H, Gal H5, H6a; 4.15, dd J 10.8, 6.7 Hz, 1H, H6b;
4.25–4.67, m, 7H, Fmoc CH and CH2, CH2CH=CH2, Gal H1, Hyp Hα,
Hγ; 5.08–5.40, m, 5H, CH2CH=CH2, Gal H2, H3, H4; 5.78–5.96, m,
1H, CH2CH=CH2; 7.28–7.34, m, 2H, ArH; 7.39, t, J 7.4 Hz, 2H, ArH;
7.52–7.55, m, 2H, ArH; 7.59, t, J 6.8 Hz, 2H, ArH; 7.74–7.79, m, 2H.
13C NMR (CDCl3, 100 MHz) (mixture of rotamers) δ 26.8, 27.0 (2C),
27.1, 35.2 and 36.6, 38.7 (2C), 38.9 and 39.1, 47.1 and 47.2, 52.5 and
52.9, 57.4 and 57.7, 60.2 and 61.0, 64.9 and 65.9, 66.0 and 66.6, 67.7,
68.5 and 68.6, 70.9, 71.2 and 71.8, 75.0 and 76.1, 92.2, 100.3 and 100.5,
118.6 and 118.9, 119.9, 124.9 and 125.1, 127.1 and 125.1, 127.1 and
127.2, 127.7, 128.6, 131.4 and 131.6, 132.0, 141.3 (2C), 143.6 and
143.8, 143.9 and 144.1, 154.1 and 154.7, 171.8 and 172.0, 176.6, 176.8,
177.3, 177.8.
Acknowledgments
We thank the Marsden Fund (Grants 96-UOA-617 and
00-MAU-018), administered by the Royal Society of New
Zealand, for financial support. For work conducted at the
University of Auckland, we thank Michael F. Walker for
running mass spectrometry experiments and for
maintenance of the NMR facility. For work conducted at
Massey University, we thank the mass spectrometry facility
at Hort Research and Dr P. J. B. Edwards for maintenance of
the NMR facility.
4-O-[(2,3,4,6-Tetra-O-benzyl)-β-D-galactopyranosyl]-Nα-
fluorenylmethoxycarbonyl-trans-4-hydroxy-L-proline allyl ester (8β).
(Found (HRMS–FAB): [M+H]+, 916.4072. Calc. for C57H58NO10:
[M+H]+, 916.4060). RF 0.20 (2:1 hexanes/EtOAc). HPLC: Rt 30.9 min
(3:1 hexanes/EtOAc; 0.6 mL min–1, 4.6 by 250 mm silica column). 1H
NMR [(D6)acetone, 270 MHz] δ 2.09–2.28, m, 1H, Hyp Hβ;
2.47–2.60, m, 1H, Hyp Hβ´; 3.60–3.82, m, 7H, Hyp Hδ, Hδ´, Gal H2,
H3, H5, H6, H6´; 4.07–4.64, m, 11H, Hyp Hα, Hγ, Fmoc CH, CH2,
OCH2CH=CH2, Gal H1, H4, CH2Ph; 4.73–5.00, m, 6H, CH2Ph; 5.17,
d, J 10.6 Hz, 1H, CH=CHA; 5.35, ddd, J 17.4, 6.6, 1.5 Hz, 1H,
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