High throughput screening of O-glycosylation conditions

Article abstract of DOI:10.1016/j.tetlet.2005.03.066

We report a novel methodology for rapid and quantitative screening of O-glycosylation reactions of application to the analysis of parallel reaction systems. Our system exploits perdeuterated benzyl (Bn-d₇) ether, and stereoselectivity and yield

Full text of DOI:10.1016/j.tetlet.2005.03.066

Tetrahedron
Letters
Tetrahedron Letters 46 (2005) 3521–3524
High throughput screening of O-glycosylation conditions
Akihiro Ishiwataa,b and Yukishige Itoa,b,
*
aRIKEN (The Institute of Physical and Chemical Research), 2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan
bCREST, JST, Kawaguchi, Saitama 332-1102, Japan
Received 3 February 2005; revised 10 March 2005; accepted 14 March 2005
Available online 6 April 2005
Abstract—We report a novel methodology for rapid and quantitative screening of O-glycosylation reactions of application to the
analysis of parallel reaction systems. Our system exploits perdeuterated benzyl (Bn-d7) ether, and stereoselectivity and yield are eval-
1
uated by H NMR and MALDI-TOF MS, respectively. This paper summarizes over 240 screenings of 1 ! 3 linkage formation
between glucose residues targeting the a-isomer in high yield.
ꢀ 2005 Elsevier Ltd. All rights reserved.
High-throughput
screening
(RO)
Optimization of reaction conditions is essential in multi-
step organic synthesis. In oligosaccharide synthesis, too,
the efficiency of glycosylation is of primary importance.1
Ideally, glycosylation should proceed in high yield and
with complete stereoselectivity. Although there are a
number of factors (i.e., solvent, temperature, molar
ratio, leaving group, promoter, additives, concentration,
etc.) that may affect the results, their effects are hardly
predictable. To be meaningful, screening of reaction
conditions should be conducted with each result evalu-
ated quantitatively in terms of both yield and selectivity.
In a conventional procedure, >0.05 mmol of substrates
are typically required for each trial and results are eval-
uated after work-up, chromatographic isolation and
spectroscopic analysis of products. The optimization
processes, as a whole, tends to be time and material con-
suming. We report herein a novel methodology for rapid
and quantitative screening of O-glycosylation reactions,
which can be applied to the analyses of parallel reaction
systems. Our system exploits perdeuterated benzyl (Bn-
d7) ether, and stereoselectivity and yield are evaluated
by 1H NMR and MALDI-TOF MS, respectively
(Scheme 1).2,3
n
O
H
(RO)
1
m
O
(OR)
O
n
HO
O
(RO)
m
+
O
O
O
A
X
α-(A-D) β-(A-D)
R = Bn-d
+
7
D
1
H NMR
stereoselectivity
R = Bn-d
MALDI-TOF Mass
yield
7
∆
MW (Bn-d
7 – Bn)
A-D
D
= + 7
A
R = Bn-d
7
α
β
R = Bn
(standards)
6.0
5.0
∆MW =
7n
7m
7(m+n)
H-1
Scheme 1. Quantitative screening of O-glycosylation reactions by 1H
NMR and MALDI-TOF MS.
metallic conditions, and can be removed by catalytic
hydrogenolysis under mild conditions. However, direct
1H NMR analysis of oligosaccharides with multiple O-
Bn groups is problematic. Benzylic methylene signals
appear at 4–5 ppm as AB-quartets, obscuring the signals
derived from anomeric protons. By employing Bn-d7,6
the benzylic methylene signals disappear and the iso-
meric ratio of glycosylated products can be determined
easily by the relative integration of the anomeric signals.
On the other hand, addition of Bn-d7 increases the
molecular weight (M.W.) by +7 Da as compared to
Bn. For the yield, inspection of the MS spectrum of a
reaction mixture supplemented with a defined amount
of non-labeled substrate (donor or acceptor) or of prod-
uct should provide a quantitative estimate7 of the prod-
uct yield and substrate recovery. Thus by combining
MALDI-TOF MS and high-field NMR, reactions per-
formed on micromolar scales can be analyzed rapidly.
Bn ether4 is one of the most widely used protective
groups in carbohydrate chemistry.5 It is stable under a
variety of acidic, basic, oxidative, reductive, and organo-
Keywords: Stereoselective glycosylation; Rapid and quantitative scre-
ening; Deuterium-labeled compound; MALDI-TOF MS; 1H NM R.
*
Corresponding author. Tel.: +81 48 462 1111; fax: +81 48 462
4680; e-mail: yukito@riken.jp
0040-4039/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2005.03.066

3522
A. Ishiwata, Y. Ito / Tetrahedron Letters 46 (2005) 3521–3524
O
O
O
O
O
O
OMP
O
O
RO
O
O
RO
RO
OMP
HO
RO
1H, 1D
3H-β, 3D-β
MeOTf
DTBMP
O
O
O
+
RO
+
MS4A
RO
O
H : R = Bn
or
D : R = Bn-d
O
O
SMe
O
O
RO
O
RO
7
O
OMP
2H, 2D
RO
3H-α, 3D-α
Scheme 2. Glycosylation of 1 with 2.
Figure 1. (a) MALDI-TOF MS spectrum of product 3D (3H-a was used for the standard); (b)1H NMR spectra of the crude mixture in C6D6. The
mixture includes the products (3D-a and 3D-b) concomitant with unreacted substrates (1D and 2D).
As an initial demonstration of the strategy, the reaction
depicted in Scheme 2 was examined with the aim of
optimizing the glycosylation to form a1 ! 3 linkage
between glucose (Glc) residues.8 This structure corre-
sponds to the sub-terminal region of high-mannose type
tetradecasaccharide Glc3Man9GlcNAc2, a glycan that is
transferred from dolichol diphosphosphate (Dol-PP) to
nascent polypeptide as the precursor of all types of
asparagine (Asn)-linked glycoproteins.9,10 As monosac-
charide substrates, the thioglycoside 2 and the acceptor
1 were prepared in their Bn-d7 and Bn protected forms
from b-D-glucose pentaacetate.11 To begin with, non-
labeled substrates 1H and 2H (1.2 equiv) were reacted
in the presence of 1.2 equiv of methyl trifluoromethane-
sulfonate (MeOTf),12 2,6-di-tert-butyl-4-methylpyridine
labeled and non-labeled standard samples widely sepa-
rated with almost same ionizing properties (Fig. 1a).
The H NMR spectrum of 3D-a is simpler than that
of 3H-a, especially in the anomeric region (4.5–
6.0 ppm) as mentioned above (Fig. 1b).
1
As a next step, systematic screening was conducted in a
parallel setting; reactions were performed with ꢀ2 mg
(ꢀ5 lmol) of each substrate. MALDI-TOF MS spectra
of the crude mixtures were measured using stock solu-
tions of 1H, 2H, and 3H (1.0 mMeach in CH 3CN)
and yields were calculated from peak heights relative
to the standards.15 Anomeric ratios were estimated from
relative integrations of H-1 signals (in C6D6) of a- (d
5.89 ppm, J 3.6 Hz) and b- (d 5.26 ppm, J 7.2 Hz)
isomers.15
˚
(DTBMP), and molecular sieves (MS) 4 A in (CH2Cl)2
in order to obtain the mixture of the products and
substrates. Under these conditions, an 80% yield of
disaccharide 3H13 was obtained as a 5.0:1 mixture of
a- and b-isomers with 33% and 20% recovery of donor
(2H) and acceptor (1H), respectively. With a standard
sample of the disaccharide 3H in hand, the same glyco-
sylation was conducted with deuterium-labeled com-
pounds 1D and 2D in various solvents. In (CH2Cl)2,
product 3D13 was obtained in identical yield and
selectivity as 3H. It was confirmed that the ionizing
properties are nearly identical between deuterated/non-
deuterated (3H vs 3D) and a- and b-isomers (3H-a vs
3D-b).14 For easy quantitative estimation of the yield
by MS analysis, it is essential to have the ion peaks of
In total, 86 solvents were tested at ambient temperature
(ꢀ23 ꢁC) and 49 at 50 ꢁC. The reactions were carried out
in the presence of 4.2 equiv of MeOTf, DTBMP, and
MS4A. Some of these results are summarized in Table
1, which displays the following features: (1) In contrast
to general perception, the degrees of stereoselectivity
are quite variable among halogenated hydrocarbons
(entries 1–5). Among them, chloroform proved to be
most effective, orienting the reaction to a-selectivity
(a:b = 10.9:1, entry 3). (2) Compared to benzene, tolu-
ene, and p-xylene, substantially higher selectivity was
observed for aromatics with electron-withdrawing sub-
stituents (entries 9–13). (3) Among ethereal solvents (en-

A. Ishiwata, Y. Ito / Tetrahedron Letters 46 (2005) 3521–3524
3523
Table 1. Selected results of the effect of the solvent on the glycosyl-
ation of 1D and 2D at room temperature and 50 ꢁCa
Mixed solvents systems (105 in total) were also screened;
CHCl3, toluene, c-C5H9OMe, n-BuCN, and EtOAc were
selected as the principal solvents, each of which was
mixed with 21 solvents in 1:1 ratio. Only 45 results are
shown in Table 2. Overall, among the 240 reaction con-
ditions screened, it was concluded that the optimum
conditions were for entries 1–3 (3–1) in Table 2
(CHCl3–c-C5H9OMe, 1:1, rt) in terms of both yield
(quantitative) and selectivity (a:b = 11.4:1).
Solvent entry
Yield/% (a:b) at rt
Yield/% (a:b) at 50 ꢁC
1. CH2Cl2
2. (CH2Cl)2
3. CHCl3
61 (5.89:1)
96 (3.68:1)
60 (10.9:1)
25 (2.02:1)
73 (1.86:1)
65 (1.86:1)
66 (1.46:1)
68 (1.31:1)
69 (3.65:1)
83 (3.13:1)
55 (3.11:1)
68 (4.33:1)
94 (3.13:1)
76 (4.31:1)
78 (6.91:1)
57 (5.50:1)
65 (3.33:1)
87 (4.11:1)
43 (3.78:1)
77 (2.72:1)
0 (—)
—
97 (4.18:1)
100 (4.95:1)
85 (1.87:1)
58 (2.54:1)
100 (2.12:1)
91 (1.84:1)
97 (4.18:1)
100 (1.64:1)
99 (4.85:1)
96 (3.13:1)
98 (3.47:1)
100 (2.34:1)
—
4. CCl4
5. C2Br2F4
6. Benzene
7. Toluene
8. Xylene
Our system enables the qualitative and facile screening
of glycosylation reactions performed in parallel. Further
refinements are possible to enhance the throughput.
Firstly, MALDI-TOF MS may be used for the initial
screening. The relatively time-consuming NMR analyses
could be limited to high-yielding entries. Faster process-
ing should be possible by automation and scaling-down
with state-of-the-art instruments such as nano-probe
NMR17 and microreactors.18
9. PhF
10. PhCl
11. PhOMe
12. PhCO2Et
13. o-C6H4Cl2
14. Et2O
15. c-C5H9OMe
16. t-BuOMe
17. THP
42 (17.6:1)
1.4 (—)
8.9 (4.18:1)
94 (1.60:1)
90 (3.23:1)
96 (3.38:1)
—
18. Dioxane
19. DME
20. EtOAc
21. Acetone
22. DMP
Acknowledgments
0 (—)
—
This work was partly supported by ꢀEcomolecular Sci-
enceꢁ and ꢀChemical Biologyꢁ programs in RIKEN and
a Grant-in-Aid for Scientific Research from the Japan
Society for the Promotion of Science (Grant No.
16390011). We thank Dr. T. Chihara and his staff for
elementary analyses, and Ms. A. Takahashi for technical
assistance.
23. DEC
53 (2.80:1)
7 (1:2.15)
28 (1:1.06)
99 (1.06:1)
93 (2.82:1)
30 (1:3.42)
0 (—)
98 (2.31:1)
33 (1:2.11)
32 (1:1.48)
60 (1.30:1)
100 (2.63:1)
27 (1:1.96)
—
24. MeCN
25. EtCN
26. n-BuCN
27. CCl3CN
28. DTP
29. DMF
30. DMSO
0 (—)
—
a Abbreviations: THP: tetrahydropyrane, DME: dimethoxyethane,
DMP: 2,2-dimethoxypropane, DEC: diethyl carbonate, DTP: 2,6-di-
References and notes
tert-butylpyridine,
dimethylsulfoxide.
DMF:
dimethylformamide,
DMSO:
1. (a) Carbohydrates in Chemistry and Biology; Ernst, B.,
Hart, G. W., Sinay, P., Eds.; Wiley-VHC: Weinheim,
¨
1999; Vols.1 and 2; (b) Glycoscience, I–III; Fraser-Reid,
B., Tatsuta, K., Thiem, J., Eds.; Springer: Berlin, 2001.
2. Ac-d3 protection for the screening of enantioselective
catalyst using ESI-Mass Reetz, M. T.; Becker, M. H.;
Klein, H.-W.; Sto¨ckigt, D. Angew. Chem. Int. Ed. 1999, 38,
1758–1761.
3. Isotope labeling for the analysis of solid phase chemistry
using UV–vis and ESI-Mass Congreve, M. S.; Ley, S. V.;
Scicinski, J. J. Chem. Eur. J. 2002, 8, 1769–1776.
4. (a) Greene, T. W.; Wuts, P. G. M. Protective Groups in
Organic Synthesis, 3rd ed.; Wiley: New York, 1999; (b)
Kocienski, P. Protecting Groups; Thieme: Stuttgart, 2003.
tries 14–18), cyclopentyl methyl ether gave the highest
selectivity. (4) A number of liquids that are rarely used
for glycosylation (e.g., EtOAc, CCl3CN, o-C6H4Cl2,
c-C5H9OMe, DEC, . . .) gave high yields of products,
while dipolar (DMSO, DMF, . . .), ketonic, and acetalic
liquids proved to be unsuitable for O-glycosylation. (5)
Although the yield was low, the substantial b-selectivity
observed for 2,6-di-tert-butylpyridine (entry 28) may be
indicative of the reverse anomeric effect.16
Table 2. Selected results of the effect of the solvent mixture (1:1) on the glycosylation of 1D and 2D at room temperature
Principal gradient
Solvent (1:1)
(1) CHCl3
(2) Toluene
(3) c-C5H9OMe
(4) n-BuCN
(5) EtOAc
Entry Y./% a:b
Entry Y./% a:b
Entry Y./% a:b
Entry Y./% a:b
Entry Y./% a:b
1. CHCl3
1–1
1–2
1–3
1–4
1–5
1–6
1–7
1–8
1–9
1–10
60
78
10.9:1 2–1
78
66
24
76
55
49
38
76
51
80
3.64:1 3–1
100
35
78
47
15
98
80
69
84
89
11.4:1 4–1
51
66
47
99
95
97
72
54
42
75
1.69:1 5–1
42
54
15
95
77
49
92
50
92
99
3.65:1
2. Toluene
3. c-C5H9OMe
4. n-BuCN
5. EtOAc
3.64:1 2–2
11.4:1 2–3
1.69:1 2–4
3.65:1 2–5
11.3:1 2–6
3.67:1 2–7
7.28:1 2–8
3.23:1 2–9
3.36:1 2–10
1.46:1 3–2
4.11:1 3–3
2.51:1 3–4
3.48:1 3–5
4.79:1 3–6
1.81:1 3–7
8.72:1 3–8
4.09:1 3–9
1.16:1 3–10
5.33:1 4–2
6.91:1 4–3
2.15:1 4–4
4.26:1 4–5
8.26:1 4–6
3.16:1 4–7
4.95:1 4–8
4.73:1 4–9
3.67:1 4–10
1.70:1 5–2
2.15:1 5–3
1.06:1 5–4
1.19:1 5–5
1.77:1 5–6
1.66:1 5–7
2.03:1 5–8
1.95:1 5–9
2.18:1 5–10
3.69:1
4.26:1
1.19:1
2.72:1
3.67:1
3.08:1
3.80:1
3.56:1
4.25:1
100
51
42
81
6. CH2Cl2
7. Decaline
8. Dioxane
9. DME
77
55
100
85
10. DEC

3524
A. Ishiwata, Y. Ito / Tetrahedron Letters 46 (2005) 3521–3524
5. (a) Roy, R.; Andersson, O.; Letellier, M. Tetrahedron Lett.
Glc1C2H, Glc1C4H, Glc2C4H, Glc2C6H), 4.05 (1H, dd,
J = 10.8, 5.6 Hz, Glc2C6H), 4.14 (1H, t, J = 9.2 Hz,
Glc1C3H), 4.29 (1H, t, J = 9.2 Hz, Glc2C3H), 4.57–4.65
(1H, m, Glc2C5H), 4.87 (1H, d, J = 8.0 Hz, Glc1C1H),
5.89 (1H, d, J = 3.6 Hz, Glc2C1H), 6.69 (2H, d,
J = 9.2 Hz, MPM), 6.98 (2H, d, J = 9.2 Hz, MPM); 3D-
1992, 33, 6053–6056; (b) Zhang, Z.; Ollmann, I. R.; Ye,
X.-S.; Wischnat, R. F.; Baasov, T.; Wong, C.-H. J. Am.
Chem. Soc. 1999, 121, 734–753.
6. Meddour, A.; Courtieu, J. Tetrahedron: Asymmetry 2000,
11, 3635–3644.
7. Recent methods for quantitative analysis (a) using deute-
rium-labeled amino acid Nakanishi, T.; Iguchi, K.;
Shimizu, A. Clin. Chem. 2003, 49, 829–831; and using
labeled reagent for pre-treatment before MS analysis (b)
Kuyama, H.; Watanabe, M.; Toda, C.; Ando, E.; Tanaka,
K.; Nishimura, O. Rapid Commun. Mass Spectrom. 2003,
17, 1642–1650.
8. Recent review for the synthesis of 1,2-cis glycoside, see
Demchenko, A. V. Synlett 2003, 1225–1240.
9. Essentials in Glycobiology; Varki, A., Cummings, R.,
Esko, J., Freeze, H., Hart, G., Marth, J., Eds.; Cold
Spring Harbor Laboratory: New York, 1999.
b: 1H NM R (CD6, 400 MHz) d 0.90–1.95 (10H, m,
6
cyclohexyl · 2), 3.10–3.20 (1H, m, Glc1C5H), 3.19–3.27
(1H, m, Glc2C5H), 3.27 (1H, s, MeO), 3.68–3.74 (4H, m,
Glc1C6H, Glc2C2H, Glc2C3H, Glc2C6H), 3.78–3.85 (3H,
m, Glc1C42H, Glc1C6H, Glc2C4H), 3.89 (1H, t,
J = 8.0 Hz, Glc1C2H), 3.94 (1H, dd, J = 9.2, 5.2 Hz,
Glc2C6H), 4.17 (1H, t, J = 8.8 Hz, Glc1C3H), 4.91 (1H,
d, J = 7.6 Hz, Glc1C1H), 5.26 (1H, d, J = 7.2 Hz,
Glc2C1H), 6.69 (2H, d, J = 9.2 Hz, MPM), 7.00 (2H, d,
J = 9.2 Hz, MPM).
14. We checked the ionization ratio of labeled to non-labeled
compound by comparing the apex values (mV) of the
MALDI-TOF MS peaks; for (3H-a)/(3D-a), y = 1.04·; for
(3H-a)/(3D-b), y = 0.967·.
10. Dwek, R. A. Chem. Rev. 1996, 96, 683–720.
11. 1H was synthesized from pentaacetyl-b-D-glucose as
follows: (1) p-(MeO)C6H4OH, BF3ÆOEt2, (2) NaOMe, (3)
1,1-dimethoxycyclo-hexanone, CSA, 90% in three steps,
(4) TIPSCl, imidazole, (5) BnBr, NaH, and (6) TBAF,
59% in three steps. 2H was synthesized from pentaacetyl-
b-D-glucose as follows: (1) TMSSMe, TMSOTf, 66%
(a:b = 1:5), (2) b-isomer, NaOMe, (3) 1,1-dimethoxycyclo-
hexanone, CSA, 77% in two steps, (3) BnBr, NaH, 99%.
1D and 2D were synthesized according to the procedure
for 1H and 2H, respectively, except benzyl-d7 bromide was
used instead of BnBr. Bn-d7 was synthesized from toluene-
d8 following the procedure in Ref. 6.
12. Lo¨nn, H. Carbohydr. Res. 1985, 139, 105–113.
13. Spectroscopic data of alpha isomer of 3H-a: 1H NM R
(C6D6, 400 MHz) d 0.90–2.10 (10H, m, cyclohexyl · 2),
3.08 (1H, td, J = 10.0, 5.2 Hz, Glc1C5H), 3.27 (1H, s,
MeO), 3.56 (1H, t, J = 10.8 Hz, Glc1C6H), 3.68–3.74 (2H,
m, Glc1C6H, Glc2C2H), 3.76–3.89 (4H, m, Glc1C2H,
Glc1C4H, Glc2C4H, Glc2C6H), 4.04 (1H, dd, J = 10.4,
5.2 Hz, Glc2C6H), 4.14 (1H, t, J = 9.2 Hz, Glc1C3H), 4.29
(1H, t, J = 8.8 Hz, Glc2C3H), 4.56–4.65 (1H, m,
Glc2C5H), 4.86 (2H, s, Bn), 4.88 (1H, d, J = 8.0 Hz,
Glc1C1H), 5.01 (1H, d, J = 11.6 Hz, Bn), 5.06 (1H, d,
J = 10.8 Hz, Bn), 5.10 (1H, d, J = 10.8 Hz, Bn), 5.19 (1H,
d, J = 11.6 Hz, Bn), 5.89 (1H, d, J = 3.6 Hz, Glc2C1H),
15. General procedure for the small scale screening: each
100 lL of solution of acceptor 1D (0.0966 g, 0.208 mmol),
donor 2D (0.1212 g, 0.250 mmol) and DTBMP (0.0768 g,
0.374 mmol) in 6.0 mL of CH2Cl2 were pipetted into
multiplicate tubes, and the mixtures were evaporated by
flashing with N2 gas. In each tube, 3.47 lmol of acceptor,
4.17 lmol of donor and 6.23 lmol of DTBMP were
prepared for the reaction. After MS4A (25 mg) and each
solvent (200 lL) were added to the mixture, methyl
trifluoromethanesulfonate (2.0 lL, 18 lmol) was added
to each tube. The mixtures were magnetically stirred at
room temperature for 24 h, and the reactions quenched by
triethylamine. The mixtures were filtered through Celite,
washed with aqueous satd NaHCO3 solution, dried over
Na2SO4, and evaporated by flashing with N2 to give crude
mixtures.
Determination of the stereoselectivity by 1H NMR: the
anomeric ratios of crude mixtures were estimated from the
relative intensities of H-1 signals (in C6D6) of a- (d
5.89 ppm, J 3.6 Hz) and b- (d 5.26 ppm, J 7.2 Hz) isomers.
Quantitative MALDI-TOF MASS analysis: the crude
mixtures were diluted with 700 lL of CH3CN. A 4.0 lL
measure of 1.0 mMstandard solution of each of the three
non-labeled compounds was pre-mixed with 2.0 lL of the
crude solutions for MS analysis. The resulting solutions
were measured by MALDI-TOF MASS using the RAS-
TER function. The molar ratio of labeled to non-labeled
compound was obtained from the ratio of each value (mV)
at the apex of the ion peak of [M+Na]+.
6.69 (2H, d, J = 9.2 Hz, MPM), 6.98 (2H, d, J = 9.2 Hz,
1
MPM), 7.10–7.75 (15H, m, Ar); 3H-b: H NM R (CD6,
6
400 MHz) d 0.90–1.95 (10H, m, cyclohexyl · 2), 3.12–3.22
(1H, m, Glc1C5H), 3.20–3.28 (1H, m, Glc2C5H), 3.28
(1H, s, MeO), 3.62–3.77 (4H, m, Glc1C6H, Glc2C2H,
Glc2C3H, Glc2C6H), 3.78–3.85 (3H, m, Glc1C42H,
Glc1C6H, Glc2C4H), 3.89 (1H, t, J = 8.0 Hz, Glc1C2H),
3.95 (1H, dd, J = 9.2, 5.2 Hz, Glc2C6H), 4.17 (1H, t,
J = 8.8 Hz, Glc1C3H), 4.88 (1H, d, J = 11.6 Hz, Bn), 4.91
(1H, d, J = 7.6 Hz, Glc1C1H), 4.93 (1H, d, J = 10.0 Hz,
Bn), 4.96 (1H, d, J = 12.0 Hz, Bn), 5.07 (1H, d,
J = 12.0 Hz, Bn), 5.10 (1H, d, J = 10.0 Hz, Bn), 5.11
(1H, d, J = 11.6 Hz, Bn), 5.26 (1H, d, J = 7.2 Hz,
Glc2C1H), 6.69 (2H, d, J = 9.2 Hz, MPM), 7.00 (2H, d,
J = 9.2 Hz, MPM), 7.10–7.50 (15H, m, Ar); 3D-a: 1H
NMR (C6D6, 400 MHz) d 0.90–2.10 (10H, m, cyclo-
hexyl · 2), 3.08 (1H, td, J = 10.0, 5.2 Hz, Glc1C5H), 3.27
(1H, s, MeO), 3.55 (1H, t, J = 10.4 Hz, Glc1C6H), 3.68–
3.74 (2H, m, Glc1C6H, Glc2C2H), 3.76–3.89 (4H, m,
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