Synthesis of 1,1-Linked Galactosyl Mannosides Carrying a Thiazine Ring as Mimetics of Sialyl Lewis X Antigen: Investigation of the Effect of Carboxyl Group Orientation on P-Selectin Inhibition

Article abstract of DOI:10.1021/jo991556b

This paper describes the synthesis of 1,1-linked galactosyl mannosides as sialyl Lewis X mimetics that contain a spiro-ring to position the carboxylate group in a well-defined orientation. It was found that compound 4 is more active as a P-selectin inhibitor (IC₅₀ = 19 μM) than the parent disaccharide 2, which contains a flexible carboxyl group (IC₅₀ = 193 μM). This result is consistent with that observed in the previous NMR study of sialyl Lewis X bound to P-selectin. The chemistry described here should be useful for the development of selective inhibitors of E-, P-, and L-selectins.

Full text of DOI:10.1021/jo991556b

J . Org. Chem. 2000, 65, 2393-2398
2393
Syn th esis of 1,1-Lin k ed Ga la ctosyl Ma n n osid es Ca r r yin g a
Th ia zin e Rin g a s Mim etics of Sia lyl Lew is X An tigen :
In vestiga tion of th e Effect of Ca r boxyl Gr ou p Or ien ta tion on
P -Selectin In h ibition
Kayoko Shibata,† Kazumi Hiruma,†,‡ Osamu Kanie,*,† and Chi-Huey Wong*,†,§
Frontier Research Program, The Institute of Physical and Chemical Research (RIKEN), 2-1 Hirosawa,
Wako-shi, Saitama 351-01 J apan, and Department of Chemistry, The Scripps Research Institute,
10550 North Torrey Pines Road, La J olla, California 92037
Received October 6, 1999
This paper describes the synthesis of 1,1-linked galactosyl mannosides as sialyl Lewis X mimetics
that contain a spiro-ring to position the carboxylate group in a well-defined orientation. It was
found that compound 4 is more active as a P-selectin inhibitor (IC50 ) 19 µM) than the parent
disaccharide 2, which contains a flexible carboxyl group (IC50 ) 193 µM). This result is consistent
with that observed in the previous NMR study of sialyl Lewis X bound to P-selectin. The chemistry
described here should be useful for the development of selective inhibitors of E-, P-, and L-selectins.
Development of small molecules as structural and
functional mimics of complex carbohydrates is a subject
of current interest.1 An intensive effort in this regard is
the development of simple and active compounds as
mimics of the sialyl Lewis X (sLeX) tetrasaccharide (1),2-5
a common ligand for E-, P-, and L-selectins involved in
inflammatory reactions and metastasis.6 Among such
mimetics, â-D-galactopyranosyl-(1,1)-R-D-mannopyrano-
side with a flexible carboxymethyl group (2) is more
active than sLeX in binding to E-, P-, and L-selectins.3
We describe here the synthesis of related structures with
a fixed orientation of the carboxyl group found in the
bound form.
Despite some success in the synthesis of sLeX mimetics
based on the functional group requirement for selectin
binding (see Figure 1),7 the bound conformations of sLeX
remain unclear. A recent NMR study suggests that the
φ and ψ angles around the sialyl glycoside are different
between the solution conformer and the selectin-bound
F igu r e 1. Functional groups of sialyl Lewis X recognized by
E-, P-, and L-selectins.
conformers.8a-c Both E- and P-selectins recognize a
similar conformer that is different from the predominant
solution conformer, and L-selectin recognizes the confor-
* To whom correspondence should be addressed. (O.K.) E-mail:
kokanee@postman.riken.go.jp. Fax: 48-467-9620. (C.H.W.) E-mail:
wong@scripps.edu. Fax: 858-789-2409.
† The Institute of Physical and Chemical Research (RIKEN).
‡ Special Postdoctoral Researcher.
(5) (a) Nigel, M. A.; Alan, H. D.; Fionna, M. M. Tetrahedron Lett.
1993, 34, 3945-3948. (b) Rao, B. N. N.; Anderson, M. B.; Musser, J .
H.; Gilbert, J . H.; Schaefer, M. E.; Foxall, C.; Brandley, B. K. J . Biol.
Chem. 1994, 269, 19663-19666. (c) Hanessian, S.; Prabhanjan, H.
Synlett 1994, 868-870. (d) Kogan, T. P.; Dupre´, B.; Keller, K. M.; Scott,
I. L.; Bui, H.; Market, R. V.; Beck, P. J .; Voytus, J . A.; Revelle, B. M.;
Scott, D. J . Med. Chem. 1995, 38, 4976-4984. (e) Prodger, J . C.;
Bamford, M. J .; Gore, P. M.; Holms, D. S.; Saez, V.; Ward, P.
Tetrahedron Lett. 1995, 36, 2339-2342. (f) Eisele, T.; Toepfer, A.;
Kretzschmar, G.; Schmidt, R. R. Tetrahedron Lett. 1996, 37, 1389-
1392. (g) Liu, A.; Dillon, K.; Campbell, R. M.; Cox, D. C.; Huryn, D. M.
Tetrahedron Lett. 1996, 37, 3785-3788. (h) Kretzschmar, G.; Toepher,
A.; Hu¨ls, C.; Krause, M. Tetrahedron 1997, 53, 2485-2494. (i) Du, M.;
Hindsgaul, O. Synlett 1997, 395-397. (j) Tsukida, T.; Hiramatsu, Y.;
Tsujishita, H.; Kiyoi, T.; Yoshida, M.; Kurosawa, K.; Moriyama, H.;
Ohmoto, H.; Wada, Y.; Saito, T.; Kondo, H. J . Med. Chem. 1997, 40,
3534-3541. (k) Toepher, A.; Kretzschmar, G. Bioorg. Med. Chem. 1997,
7, 1311-1316. (l) Toepher, A.; Kretzschmar, G.; Schuth, S.; Sonnentag,
M. ibid. 1997, 7, 1317-1322. (m) For a review on the subject, see
Simanek, E. E.; McGarvey, G. J .; J ablonowsky, J . A.; Wong, C.-H.
Chem. Rev. 1998, 98, 833-862 and Dasgapta, F.; Rao, B. N. N. 1994,
3, 709-724.
§ The Scripps Research Institute.
(1) For a recent review on the subject, see: Sears, P.; Wong, C.-H.
Angew. Chem., Int. Ed. Engl. 1999, 38, 2300-2324.
(2) (a) Uchiyama, T.; Vassilev, V. P.; Kajimoto, T.; Wong, W.; Lin,
C.-C.; Huang, H.; Wong, C.-H. J . Am. Chem. Soc. 1995, 117, 5395-
5396. (b) Huang, H.; Wong, C.-H. J . Org. Chem. 1995, 60, 3100-3106.
(c) Wang, R.; Wong, C.-H. Tetrahedron Lett. 1996, 37, 5427-5430. (d)
Wu, S.-H.; Shimazaki, M.; Lin, C.-C.; Moree, W. J .; Weitz-Schmidt,
G.; Wong, C.-H. Angw. Chem. Int. Ed. Engl. 1996, 35, 88-90. (e)
Uchiyama, T.; Woltering, T. J .; Wong, W.; Lin, C.-C.; Kajimoto, T.;
Takebayashi, M.; Weitz-Schmidt, G.; Asakura, T.; Noda, M.; Wong,
C.-H. Bioorg. Med. Chem. 1996, 4, 1149-1165. (f) Marron, T. G.;
Woltering, T. J .; Weits-Schmidt, G.; Wong, C.-H. Tetrahedron Lett.
1996, 37, 9037-9040. (g) Lin, C.-C.; Kimura, T.; Wu, S.-H.; Weits-
Schmidt, G.; Wong, C.-H. Bioorg. Med. Chem. Lett. 1996, 6, 2755-
2760. (h) Wong, C.-H.; Moris-Varas, F.; Hung, S.-C.; Marron, T.-G.;
Lin, C.-C.; Gong, K. W.; Weits-Schmidt, G. J . Am. Chem. Soc. 1997,
119, 8152-8158. (i) Cappi, M. W.; Moree, W. J .; Qiao, L.; Marron, T.
G.; Weits-Schmidt, G.; Wong, C.-H. Bioorg. Med. Chem. 1997, 5, 283-
296.
(3) Hiruma, K.; Kajimoto, T.; Weitz-Schmidt, G.; Ollmann, I.; Wong,
C.-H. J . Am. Chem. Soc. 1996, 118, 9265-9270.
(4) Ikeda, T.; Kajimoto, T.; Kondo, H.; Wong, C.-H. Bioorg. Med.
Chem. Lett. 1997, 7, 2485-2490.
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Singhal, A. K.; Hakomori, S.; Paulson, J . C. Science 1990, 250, 1130-
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Berhend, T. L.; Marks, R. M. Cell 1990, 63, 475-484.
10.1021/jo991556b CCC: $19.00 © 2000 American Chemical Society
Published on Web 03/28/2000

2394 J . Org. Chem., Vol. 65, No. 8, 2000
Shibata et al.
F igu r e 3. Structures of sLeX mimetic 1,1-linked galactosyl
mannoside 2 and its derivatives 3 and 4 with fixed carboxyl
group orientation.
Sch em e 1a
F igu r e 2. Schematic representation of rotamers around the
glycosidic bond between sialic acid and galactose of sialyl Lewis
X tetrasaccharide 1. 1-a: free and L-selectin-bound conforma-
tion determined by NMR. 1-b: E- and P-selectin-bound
conformation determined by transferred NOE.
mation similar to the predominant conformer in solution.8d
(Figure 2).
a
Reagents and conditions: (a) (COCl)2-DMSO-Et3N/CH2Cl2;
(b) (i) L-cysteine-pTsOH‚H2O/DMF; ii. CH2N2/MeOH; (c) (i) D-cys-
teine-pTsOH‚H2O/DMF; (ii) CH2N2/MeOH.
Although an investigation of the orientation of the
carboxyl group has been addressed and the synthetic
mimetic found to be inactive,9a the result may be due to
the presence of a large group close to the hydroxyl groups
that are important for binding. An approach to overcome
this problem is the introduction of a tether to support
the carboxyl group in the required orientation from an
unrecognized side of the galactose moiety. Attachment
of a substituent to the R-position of the carboxymethyl
group of the sialic acid mimic in order to restrict the
orientation of the carboxyl group was found to produce
effective E-selectin inhibitors.9b,c To introduce additional
constraints, we decided to synthesize compounds 3 and
4 with the orientation of carboxyl group fixed by a spiro
ring to mimic the conformer bound to L- and E/P-
selectins, respectively (Figure 3).
L-cysteine in the presence of catalytic amounts of p-TsOH
afforded the desired spiro compound 7 in high yield.
However, reaction of 6 with D-cysteine gave, on the other
hand, the undesired product 8 exclusively. The configu-
rations of both compounds were evident from the NOE
analyses as shown in Scheme 1. Further attempts to
improve the yield of the desired compound failed. The
reaction of disaccharide 10 derived from compound 9 was
then attempted (Scheme 2), and compound 11 containing
a fused L-cysteine residue and a 1:2 mixture of 12 and
13 containing a fused D-cysteine residue were obtained,
respectively. The configuration of each compound was
also supported by the NOE analysis. The preferred R-
and S-configurations at the galactose C-3 position for the
respective reactions with L- and D-cysteine were perhaps
due to the steric interaction between the group of L- or
D-cysteine and the 2-OH or the 2-OBn group of the
iminium intermediate as shown in Figure 4, respectively.
In any case, the observation that a single diastereomer
is formed with both enantiomers of cysteine in the
presence of TsOH is remarkable, given that under acidic
deprotection conditions a mixturespresumably of stereo-
isomerssis formed. Additionally, formation of the thia-
zine without an acid catalyst also yields mixtures of
stereoisomers. It is possible that under some of the
conditions described the product distribution is controlled
kinetically while under others a thermodynamic mixture
is generated. Alternatively, the precise reaction condi-
tions may determine the position of a thermodynamic
equilibrium.
Our initial attempt was to introduce a thiazine ring
at C-3 of galactose using the protected 3-ulo compound
6 derived from 510 by Swern oxidation followed by
reaction with L- and D-cysteine. Reaction of 6 with
(7) Kogan, T. P.; Revelle, B. M.; Tapp, S.; Scott, D.; Beck, P. J . A. J .
Biol. Chem. 1995, 270, 14047-14055. Brandley, B. K.; Kiso, M.; Abbas,
S.; Nikrad, P.; Strivastava, O.; Foxall, C.; Oda, Y.; Hasegawa, A.
Glycobiology 1993, 3, 633.
(8) (a) Scheffler, K.; Ernst, B.; Katopodis, A.; Magnani, J . L.; Wang,
W. T.; Weisemann, R.; Peters, T. Angew. Chem., Int. Ed. Engl, 1995,
34, 1841-1844. (b) Cooke, R. M.; Hale, R. S.; Lister, S. G.; Shah, G.;
Weir, M. P. Biochemistry 1994, 33, 10591-10596. (c) Poppe, L.; Brown,
G. S.; Nikrad, P. V.; Shah, B. H. J . Am. Chem. Soc. 1997, 119, 1727-
1736. (d) Ichikawa, Y.; Lin, Y.-C.; Dumas, D. P.; Shen, G.-J .; Garcia-
J unceda, E.; Williams, M. A.; Bayer, R.; Ketcham, C.; Walker, L. E.;
Paulson, J . C.; Wong, C.-H. J . Am. Chem. Soc. 1992, 114, 9283-9298.
(9) (a) Thoma, G.; Schwarzenbach, F.; Duthaler, R. O. J . Org. Chem.
1996, 61, 514-524. (b) Musser, J . H.; Rao, N.; Nashed, M.; Dasgupta,
F.; Abbas, S.; Nematalla, A.; Date, V.; Foxall, C.; Asa, D.; J ames, P.;
Tyrell, D.; Brandley, B. K. Pharmacochem. Libr. 1993, 20, 33-40. (c)
Banteli, R.; Ernst, B. Tetrahedron Lett. 1997, 38, 4059-4062.
(10) Kanie, O.; Ito, Y.; Ogawa, T. Tetrahedron Lett. 1996, 37, 4551-
4554.
Deprotection of the benzyl groups in 11-13 was
unsuccessful under various hydrogenolysis conditions.
Although deprotected materials were obtained when TM-

Synthesis of 1,1-Linked Galactosyl Mannosides
J . Org. Chem., Vol. 65, No. 8, 2000 2395
F igu r e 4. Synthesis of sLex mimics containing a thiazine ring. Other unfavorable rotamers around the N-Ca bond are omitted.
Only preferred conformers are shown as the products.
Sch em e 2a
hydrogenolysis under neutral conditions was used with-
out further purification for the coupling with L- or
D-cysteine in the absence of an acid catalyst. The reaction
proceeded smoothly and yielded spiro compound 3 and
approximately a 1:2 mixture of compounds 4 and 16,
which were isolable by silica gel column chromatography.
It was found, however, that a slow equilibrium exists
between 4 and 16 in water at room temperature (pH 7.4).
Compounds 2, 3, and 4 were then tested as inhibitors
of P-selectin,2 and the IC50 values were 193, 31, and 19
µM, respectively. Attachment of a long chain hydrophobic
group to the mannose 6-position of 2 (see 2a ),13 however,
significantly improves the potency (IC50 ) 3 µM for 2a ),
indicating the importance of the hydrophobic effect on
sugar affinity. While both 3 and 4 direct the carboxylate
“down”, that is, away from the C4′ hydroxyl of the
galactose moiety, the two compounds direct the carboxy-
late in opposite directions. This observation would imply
that fixing one of the two dihedrals of the parent
compound was vital (“up” versus “down”) but fixing the
other was essentially unimportant (“left” versus “right”).
Note that the flexible dihedrals of the parent compound
are interchangeable in this regard. If this is indeed true
then the enhancement in activity seems too large to be
accounted for by a reduction in conformational entropy.
The magnitude of such an enhancement would be no
greater that 0.5 kcal mol-1, while the enhancements for
both compounds 3 and 4 are greater than 1 kcal mol-1
(assuming IC50 values are in fact measures of binding).
Alternatively, if both dihedrals are important to affinity,
then the difference in activity between 3 and 4 is too
small, since one should presumably direct the carboxylate
in the wrong direction for appropriate interaction with
the protein. A more likely basis for the enhancement in
affinity observed for both compounds is the increased
hydrophobicity of 3 and 4 compared to the reference
disaccharide, a concept apparently reinforced by our
experiments.
a
Reagents and conditions: Identical to those described in
Scheme 1.
SOTf and thioanisole were used in TFA,11 an equilibrium
of the acetal through an imine intermediate occurred
during the reaction to give a complex mixture leading to
low yields.12 Interestingly, the acylation reaction of the
amine in order to stabilize the spiro acetal ring did not
proceed at all in our hands.
Finally, we used an unprotected ulo-disaccharide (14)
to avoid the difficulty of deprotections in the presence of
thiazine ring (Scheme 3). Compound 14 obtained by
In summary, we have developed a method for the
preparation of sLeX mimetics with well-defined carboxyl
group orientation for use to address the relationship
between the binding activity and the glycosidic torsion
angles of the two sugar groups. The strategy may find
use in the development of selective inhibitors of selectins.
(11) Fujii, N.; Ikemura, O.; Funakoshi, S.; Matsuo, H.; Segawa, T.;
Nakata, Y.; Inoue, A.; Yajima, H. Chem. Pharm. Bull. 1987, 35, 1076-
1084.
(12) Pera, M. H.; Hamri, A.; Ho, L.; Fillion, H. Sulfur Lett. 1990,
10, 217-222.
(13) Hiruma, K.; Kanie, O.; Wong, C.-H. Tetrahedron. 1998, 54,
15781-15792.

2396 J . Org. Chem., Vol. 65, No. 8, 2000
Shibata et al.
Sch em e 3a
a
Reagents and conditions: (a) Pd(OH)2-C-H2/CHCl3-MeOH; (b) L-cysteine/DMF; (c) D-cysteine/DMF. Inset: The course of equilibrium
between 4 and 16 is shown. The percentage of each compound was estimated by integration of 1H NMR. 0: equilibrium initiated with
100% 4. O: equilibrium initiated with 100% 16. Although several minor signals were detectable in 1H NMR spectra, the total amount of
4 and 16 was determined to be 100%.
(TsOH, 8.2 mg, 0.043 mmol), and molecular sieves (MS AW-
300, 200 mg) in DMF (10 mL) was heated at 60 °C for 3 h,
and then pyridine (Pyr, 5 mL) was added to the mixture. The
mixture was filtered through a Celite pad, washed with MeOH,
and concentrated in vacuo. To a solution of the residue in
MeOH (5 mL) was added a solution of diazomethane in ether.
The mixture was kept until the starting material was con-
sumed, as measured by TLC, and was concentrated after
addition of AcOH and purified on a column of silica gel using
6:1 hexanes-EtOAc to give 7 (280.0 mg, 98.4%): [R]D -22.1°
Exp er im en ta l Section
Gen er a l Meth od s. Dried solvents were used for all reac-
tions. Solutions were evaporated under diminished pressure
at a bath temperature not exceeding 40 °C. Column chroma-
tography was performed on silica gel. Gel permeation chro-
matography was performed using Bio Gel P-2. Optical rota-
1
tions were measured in a 1.0 dm tube at 23 ( 1 °C. H NMR
spectra were recorded in CDCl3 or D2O using Me4Si (δ 0.00)
as the internal standard for solutions in CDCl3 or DOH (δ 4.80
at 25 °C for 1H NMR). 13C NMR spectra were recorded in
CDCl3 or D2O as indicated. Signals for aromatic carbons were
omitted in 13C NMR assignment. The electrospray mass
spectra were recorded in MeOH-H2O (1:1).
1
(c 1.28, CHCl3); H NMR (270 MHz, CDCl3) δ 7.60-7.15 (m,
20H), 5.06 and 4.65 (AB, 2H, J ) 11.2 Hz,), 4.93 and 4.51 (AB,
2H, J ) 11.4 Hz), 4.82 (d, 1H, J ) 9.8 Hz), 4.42 and 4.47 (AB,
2H, J ) 11.2 Hz), 4.20 (d, 1H, J ) 9.8 Hz), 4.02 (br t, 1H, J )
6.3 Hz), 3.76 (ddd, 1H, J ) 5.4, 10.4, 12.8 Hz), 3.75 (s, 3H),
3.65 (dd, 1H, J ) 9.6, 5.0 Hz), 3.58 (dd, 1H, J ) 9.6, 6.9 Hz),
3.62 (s, 1H), 3.17 (dd, 1H, J ) 5.4, 10.4 Hz), 2.82 (d, 1H, J )
12.8 Hz), and 2.68 (t, 1H, J ) 10.4 Hz); 13C NMR (68 MHz,
CDCl3) δ 170.64, 88.93, 86.96, 78.33, 78.02, 77.76, 77.22, 75.78,
75.46, 75.15, 74.81, 73.46, 69.22, 63.60, 52.45, and 39.12.
P h en yl 2,4,6-Tr i-O-ben zyl-1-th io-â-D-xyloh exopyr an osid-
3-u lose (6). To a solution of oxalyl chloride [(COCl)2, 0.84 mL,
9.59 mmol] in dichloromethane (CH2Cl2, 50 mL) was added
dimethyl sulfoxide (DMSO, 1.02 mL, 14.39 mmol) dropwise
at -78 °C. After the mixture was stirred for 30 min, a solution
of compound 5 (1.3 g, 2.40 mmol) dissolved in CH2Cl2 (30 mL)
was added dropwise and the mixture was stirred for 1 h at
the temperature, then triethylamine (Et3N, 2.67 mL, 19.19
mmol) was added and the mixture stirred stirred for 1 h. The
mixture was poured into saturated NH4Cl which was extracted
with CHCl3. The organic layer was washed with brine, dried
over MgSO4, and concentrated in vacuo. Silica gel column
chromatography of the residue using 4:1 hexanes-EtOAc gave
Anal. Calcd for C37H39NO6S2: C, 67.57; H, 5.98; N, 2.13; S,
9.75. Found: C, 67.04; H, 5.93; N, 2.14; S, 9.79.
Met h yl 2′(R)-[P h en yl 3(S)-2,4,6-t r i-O-b en zyl-1-t h io-â-
D-xylop yr a n osid -3,5′-sp ir o[1,4]th ia zin ]-2′-ca r boxyla te (8).
Compound 8 was synthesized as described for the synthesis
of 7 using 6 (51.5 mg, 0.095 mmol), d-cysteine (34.7 mg, 0.286
mmol), TsOH (catalytic amount), MS AW-300 in DMF (2 mL)
for acetalization, and diazomethane/MeOH for methylation:
column 6:1 hexanes-EtOAc; yield 57.6 mg, 92%; [R]D +18.9°
1
6 (1.28 g, 98.7%): [R]D +20.5° (c 1.91, CHCl3); H NMR (270
MHz, CDCl3) δ 7.60-7.09 (m, 20H), 4.75 (d, 1H, J ) 9.8 Hz),
4.54 and 4.40 (AB, 2H, J ) 11.4 Hz), 4.53 and 4.47 (AB, 2H,
J ) 11.9 Hz), 4.32 and 4.29 (AB, 2H, J ) 11.9 Hz, 4.33 (d, 1H,
J ) 9.8 Hz), 3.93 (s, 1H, H-4), and 3.77 (s, 3H, H-5); 13C NMR
(68 MHz, CDCl3) δ 204.74, 88.82, 81.87, 79.43, 77.22, 73.50,
73.35, 72.33, and 67.71.
1
(c 1.26, CHCl3); H NMR (270 MHz, CDCl3) δ 7.64-7.13 (m,
20H), 4.98 and 4.58 (AB, 2H, J ) 10.7 Hz), 4.53 and 4.49 (AB,
2H, J ) 11.9 Hz), 4.88 (d, 1H, J ) 9.9 Hz), 4.86 (s, 2H), 4.34
(t, 1H, J ) 7.0 Hz), 4.05 (d, 1H, J ) 9.9 Hz), 3.89 (ddd, 1H, J
) 5.1, 10.6, 13.4 Hz), 3.76 (s, 3H), 3.67 (d, 2H, J ) 7.0 Hz),
3.52 (s, 1H), 3.28 (d, 1H, J ) 13.4 Hz), 3.20 (dd, 1H, J ) 5.1,
10.6 Hz), and 2.66 (t, 1H, J ) 10.6 Hz); 13C NMR (68 MHz,
CDCl3) δ 170.80, 87.19, 86.54, 85.55, 80.81, 77.20, 76.75, 76.14,
75.69, 75.60, 73.51, 70.64, 68.59, 65.66, 54.41, 52.44, 47.59,
and 39.34.
Anal. Calcd for C33H32O5S: C, 73.32; H, 5.97; S, 5.93.
Found: C, 73.34; H, 5.96; S, 6.05.
Met h yl 2′(S)-[P h en yl 3(R)-2,4,6-t r i-O-b en zyl-1-t h io-â-
D-xylop yr a n osid -3,5′-sp ir o[1,4]th ia zin ]-2′-ca r boxyla te (7).
A mixture of compound 6 (233.9 mg, 0.433 mmol), L-cysteine
(157.4 mg, 1.299 mmol), p-toluenesulfonic acid monohydrate

Synthesis of 1,1-Linked Galactosyl Mannosides
J . Org. Chem., Vol. 65, No. 8, 2000 2397
Anal. Calcd for C37H39NO6S2: C, 67.57; H, 5.98; N, 2.13; S,
9.75. Found: C, 67.68; H, 6.00; N, 2.10; S, 9.57.
2H, J ) 11.3 Hz), 4.83 and 4.65 (AB, 2H, J ) 11.4 Hz), 4.74
(d, 1H, J ) 9.0 Hz), 4.61 and 4.40 (AB, 2H, J ) 12.2 Hz), 4.58
and 4.49 (AB, 2H, J ) 11.6 Hz), 4.53 (s, 2H), 4.37 (s, 2H), 4.21
(dd, 1H, J ) 5.9, 8.4 Hz), 4.09 (ddd, 1H, J ) 1.5, 4.0, 10.0 Hz),
4.00 (t, 1H, J ) 9.6 Hz), 3.89 (dd, 1H, J ) 3.2, 9.3 Hz), 4.85
(ddd, 1H, J ) 5.2, 10.7, 13.1 Hz), 3.79 (s, 3H), 3.77 (d, 1H, J
) 9.0 Hz), 3.72 (dd, 1H, J ) 9.1, 4.3 Hz), 3.61 (dd, 1H, J )
9.1, 2.0 Hz), 3.59 (d, 1H, J ) 1.5 Hz), 3.56 (dd, 1H, J ) 9.0,
8.4 Hz), 3.48 (dd, 1H, J ) 9.0, 5.9 Hz), 3.39 (s, 1H), 3.18 (dd,
1H, J ) 5.6, 10.5 Hz), 3.18 (d, 1H, J ) 13.1 Hz), and 2.66 (t,
1H, J ) 10.5, 4.4 Hz); 13C NMR (100 MHz, CDCl3) δ 170.94,
101.40, 99.20, 85.31, 80.59, 79.80, 78.84, 76.23, 75.86, 75.07,
74.96, 74.84, 73.36, 73.13, 73.02, 72.38, 72.33, 72.29, 69.00,
68.46, 65.65, 52.43, and 39.30.
2,3,4,6-Tetr a-O-ben zyl-r-D-m an n opyr an osyl-(1-1)-2,4,6-
tr i-O-ben zyl-â-D-xylop yr a n osid -3-u lose (10). Compound 10
was synthesized as described for the synthesis of 6 using 9
(412.8 mg, 0.424 mmol), (COCl)2 (0.11 mL, 1.274 mmol), DMSO
(0.15 mL, 2.123 mmol), and Et3N (0.3 mL, 2.123 mmol) in CH2-
Cl2 (20 mL): column 4:1 hexanes-EtOAc; yield 410 mg, quant;
[R]D -5.3° (c 0.66, CHCl3); 1H NMR (400 MHz, CDCl3) δ 7.34-
7.15 (m, 35H), 5.15 (d, 1H, J ) 1.8 Hz), 4.68 (s, 2H), 4.88 and
4.49 (AB, 2H, J ) 11.0 Hz), 4.59 (d, 1H, J ) 7.8 Hz), 4.30 (d,
1H, J ) 7.8 Hz), 4.68-4.32 (m, 10H), 4.08 (brd d, 1H, J ) 2.6,
9.3 Hz), 4.04 (t, 1H, J ) 9.3 Hz), 3.91 (dd, 1H, J ) 3.0, 9.3
Hz), 3.85 (s, 1H), 3.74 (dd, 1H, J ) 1.8, 3.0, 2.9 Hz), 3.73 and
3.58 (m, 5H); 13C NMR (100 MHz, CDCl3) δ 203.85, 102.86,
99.47, 82.40, 80.79, 79.74, 74.95, 74.70, 74.21, 73.65, 73.42,
73.34, 73.20, 72.65, 72.48, 72.42, 68.86, and 67.13.
Anal. Calcd for C65H69NO12S: C, 71.74; H, 6.39; N, 1.29; S,
2.95. Found: C, 71.82; H, 6.48; N, 1.20; S, 2.85.
2′(S )-[r-D -M a n n o p y r a n o s y l-(1-1)-3(R )-â-D -x y lo -
p yr a n osid -3,5′-sp ir o[1,4]th ia zin ]-2′-ca r boxylic Acid (3).
Compound 10 (82.7 mg, 0.085 mmol) was treated with 20%
Pd(OH)2-C (ca. 20 mg) in a 4:1 mixture of MeOH-CHCl3 (1
mL) under H2 atmosphere for 2 h. The reaction mixture was
filtered and the filtrate containing 14 was directly poured into
a mixture of L-cysteine (30.9 mg, 0.256 mmol) and MS AW
300 (ca. 50 mg) in DMF (1 mL), and the resulting mixture was
stirred at 60 °C for 3 h. A residue obtained after filtration and
concentration in vacuo was purified on a column of Bio gel P2
eluted with water to afford 3 (27 mg, 71.5 mg): 1H NMR (500
MHz, D2O) δ 5.15 (d, 1H, J ) 1.4 Hz), 4.58 (d, 1H, J ) 8.3
Hz), 4.04 (dd, 1H, J ) 1.4, 3.2 Hz), 3.98 (dd, 1H, J ) 5.0, 6.9
Hz), 3.93 (ddd, 1H, J ) 2.3, 6.4, 10.1 Hz), 3.89 (d, 1H, J ) 8.3
Hz), 3.87 (dd, 1H, J ) 3.2, 9.6 Hz), 3.83 (s, 1H), 3.77 (dd, 1H,
J ) 6.4, 10.1 Hz), 3.66 (t, 1H, J ) 10.1 Hz), 3.34 (dd, 1H, J )
6.0, 10.1 Hz), and 2.77 (t, 1H, J ) 10.1 Hz); 13C NMR (125
MHz, D2O) δ 178.36, 104.13, 102.22, 86.12, 75.59, 74.59, 72.18,
71.24, 70.17, 69.78, 67.69, 66.16, 62.44, 61.92, 40.57; ESI MS
m/z calcd for C15H25NO12S 443.1, negative mode:442.1 [M -
H]-, positive mode 466.1 [M + Na]+.
Anal. Calcd for C61H62O11: C, 75.44; H, 6.43. Found: C,
75.47; H, 6.64.
Meth yl 2′(S)-[2,3,4,6-Tetr a -O-ben zyl-r-D-m a n n op yr a n -
osyl-(1-1)-3(R )-2,4,6-t r i-O-b e n zyl-â-D-xylop yr a n osid -
3,5′-sp ir o[1,4]t h ia zin ]-2′-ca r b oxyla t e (11). Compound 11
was synthesized as described for the synthesis of 7 using 10
(202.0 mg, 0.208 mmol), L-cysteine (75.7 mg, 0.625 mmol),
TsOH (4 mg), and MS AW-300 (200 mg) in DMF (5 mL) for
acetalization and diazomethane/MeOH for methylation: col-
umn 5:1 hexanes-EtOAc; yield 206.7 mg, 91.3%; [R]D +7.2°
1
(c 0.92, CHCl3); H NMR (400 MHz, CDCl3) δ 7.38-7.16 (m,
35H), 5.21 (d, 1H, J ) 1.8 Hz), 4.89 and 4.51 (AB, 2H, J )
11.4 Hz), 4.82 and 4.46 (AB, 2H, J ) 11.3 Hz), 4.67 (d, 1H, J
) 7.9 Hz), 4.72-4.29 (m, 10H), 4.11 (brd, 1H), 4.07 (t, 1H, J )
9.4 Hz), 3.96 (d, 1H, J ) 6.3 Hz), 3.95 (dd, 1H, J ) ca. 2.0, 9.4
Hz), 3.90 (d, 1H, J ) 7.9 Hz), 3.75 (overlapped 4H), 3.71 (dd,
1H, J ) 10.9, 3.5 Hz), 3.68 (dd, 1H, J ) 1.8, 3.1 Hz), 3.59 (dd,
1H, J ) 10.9, 1.5 Hz), 3.50 (s, 1H), 3.44 (d, 2H, J ) 6.3 Hz),
3.20 (dd, 1H, J ) 5.5, 10.1 Hz), 2.84 (d, 1H, J ) 12.8 Hz), and
2.69 (t, 1H, J ) 10.1 Hz); 13C NMR (100 MHz, CDCl3) δ 170.72,
104.36, 99.64, 85.72, 79.48, 77.90, 76.19, 75.06, 74.89, 74.84,
74.70, 73.20, 73.13, 72.48, 72.43, 72.28, 68.77, 63.24, 52.42,
and 39.47.
2′(R)-[r-D-Ma n n op yr a n osyl-(1-1)-3(R)-â-D-xylop yr a -
n osid -3,5′-sp ir o[1,4]th ia zin ]-2′-ca r boxylic Acid (4) a n d 2′-
(R)-[r-D-Ma n n op yr a n osyl-(1-1)-3(S)-â-D-xylop yr a n osid -
3,5′-sp ir o[1,4]th ia zin ]-2′-ca r boxylic Acid (16). Compounds
4 and 16 were obtained under the conditions described for the
synthesis of 3 using 10 (92.3 mg, 0.095 mmol), Pd(OH)2 (ca.
20 mg), D-cysteine (34.6 mg, 0.285 mmol), and MS AW-300. A
mixture of diastereomers obtained after column chromatog-
raphy on Bio gel P2 (elution: water) was further purified using
Iatro Beads (elution: 2:2:1 EtOAc-EtOH-H2O) to afford 4 (21
mg, 49.9%) and 16 (10 mg, 23.7%).
Anal. Calcd for C65H69NO12S: C, 71.74; H, 6.39; N, 1.29; S,
2.95. Found: C, 71.70; H, 6.41; N, 1.20; S, 2.79.
Meth yl 2′(R)-[2,3,4,6-Tetr a -O-ben zyl-r-D-m a n n op yr a n -
osyl-(1-1)-3(R )-2,4,6-t r i-O-b e n zyl-â-D-xylop yr a n osid -
3,5′-sp ir o[1,4]th ia zin ]-2′-ca r boxyla te (12) a n d Meth yl 2′-
(R)-[2,3,4,6-Tet r a -O-b en zyl-r-D-m a n n op yr a n osyl-(1-1)-
3(S )-2,4,6-t r i-O -b e n zy l-â-D -x y lo p y r a n o s id -3,5′-s p ir o -
[1,4]th ia zin ]-2′-ca r boxyla te (13). Compounds 12 and 13
were obtained under the conditions described for the synthesis
of 7 using 10 (514.3 mg, 0.530 mmol), D-cysteine (192.5 mg,
1.589 mmol), TsOH (10 mg), MS AW-300 (500 mg) in DMF
(15 mL) for acetalization, and diazomethane/MeOH for me-
thylation: column gradient 8:1 f 6:1 hexanes-EtOAc; yield
13 (174.3 mg, 30.2%) and 12 (365.8 mg, 63.5%).
1
Data for compound 4: H NMR (500 MHz, D2O) δ 5.18 (s,
1H), 4.80 (signal overlapped with DOH), 4.13 (ddd, 1H, J )
1.0, 3.4, 6.8 Hz), 4.05 (dd, 1H, J ) 1.8, 3.2 Hz), 3.93 (ddd, 1H,
J ) 1.9, 5.9, 10.0 Hz), 3.87 (d, J ) 9.3 Hz), 3.85 (dd, 1H, J )
3.3, 9.6 Hz), 3.76 (dd, 1H, J ) 5.8, 10.3 Hz), 3.61 (s, 1H), 3.24
(dd, 1H, J ) 5.8, 10.3 Hz), and 2.73 (t, 1H, J ) 10.3 Hz); 13C
NMR (125 MHz, D2O) δ 177.53, 101.76, 101.34, 85.90, 75.58,
73.65, 72.08, 70.24, 69.94, 69.89, 68.45, 66.86, 61.68, 61.11,
and 39.02.
Data for compound 12: [R]D +56.9° (c 1.25, CHCl3); 1H NMR
(400 MHz, CDCl3) δ 7.34-7.16 (m, 35H), 5.20 (d, 1H, J ) 1.7
Hz), 4.97 and 4.66 (AB, 2H, J ) 11.6 Hz), 4.87 and 4.50 (AB,
2H, J ) 10.5 Hz), 4.65 and 4.47 (AB, 2H, J ) 11.6 Hz), 4.67
(d, 1H, J ) 7.9 Hz), 4.62 and 4.52 (AB, 2H, J ) 11.4 Hz), 4.60
and 4.36 (AB, 2H, J ) 11.6 Hz), 4.36 and 4.32 (AB, 2H, J )
11.7 Hz), 4.13 (dd, 1H, J ) 4.4, 6.3 Hz), 4.09 (brd, 1H), 4.05 (t,
1H, J ) 9.3 Hz), 3.93 (dd, 1H, J ) 3.2, 9.3 Hz), 3.77 (t, 1H, J
) 6.6 Hz), 3.74 (s, 1H), 3.72 (dd, 1H, J ) 10.5, 2.3 Hz), 3.70
(d, 1H, J ) 6.1 Hz), 3.69 (s, 3H), 3.64 (dd, 1H, J ) 1.7, 3.2
Hz), 3.60 (dd, 1H, J ) 10.5, 1.5 Hz), 3.47 (dd, 1H, J ) 9.9, 7.0
Hz), 3.44 (dd, 1H, J ) 9.9, 6.0 Hz), 3.18 (dd, 1H, J ) 6.3, 10.5
Hz), 3.08 (dd, 1H, J ) 10.5, 4.4 Hz), and 2.34 (brd, 1H); 13C
NMR (100 MHz, CDCl3) δ 172.83, 103.77, 99.51, 83.01, 82.20,
80.62, 79.63, 75.14, 75.07, 74.98, 74.89, 74.38, 73.18, 73.13,
72.41, 72.26, 68.86, 68.76, 64.63, 52.30, and 37.59.
Data for compound 16: 1H NMR (500 MHz, D2O) δ 5.12 (d,
1H, J ) 1.8 Hz), 4.54 (d, 1H, J ) 8.2 Hz), 4.17 (t, 1H, J ) 6.8
Hz), 4.03 (dd, 1H, J ) 1.8, 3.3 Hz), 3.82 (s, 1H), 3.65 (t, 1H, J
) 9.9 Hz), 3.28 (dd, 1H, J ) 6.8, 10.5 Hz), and 3.14 (dd, 1H, J
) 6.8, 10.5 Hz); 13C NMR (125 MHz, D2O) δ 179.04, 103.09,
101.49, 84.52, 76.18, 73.71, 71.67, 70.41, 69.88, 67.21, 66.85,
61.45, 61.10, and 38.51; ESI MS m/z calcd for C15H25NO12
S
443.1, negative mode 442.1 [M - H]-, positive mode 466.1 [M
+ Na]+.
Ack n ow led gm en t. We thank members of the NMR
laboratory at RIKEN. We also thank Dr. Sadamu
Kurono for electrospray mass spectral measurements.
We are grateful to Dr. Yoshitaka Nagai, Director of the
Glycobiology Research Group, and Dr. Tomoya Ogawa,
Coordinator of the group, Frontier Research Program
Anal. Calcd for C65H69NO12S: C, 71.74; H, 6.39; N, 1.29; S,
2.95. Found: C, 71.81; H, 6.43; N, 1.19; S, 3.14.
Data for compound 13: [R]D +32.4° (c 0.61, CHCl3); 1H NMR
(400 MHz, CDCl3) δ 7.36-7.15 (m, 35H), 5.12 (d, 1H, J ) 1.5
Hz), 4.92 and 4.54 (AB, 2H, J ) 10.4 Hz), 4.86 and 4.48 (AB,

2398 J . Org. Chem., Vol. 65, No. 8, 2000
Shibata et al.
Su p p or tin g In for m a tion Ava ila ble: Copies of the 1H
NMR for compounds 2-4 and 16. This material is available
free of charge via the Internet at http://pubs.acs.org.
of RIKEN, for their continued support and encourage-
ment of our research. This research was supported by
the Science and Technology Agency of the J apanese
Government.
J O991556B