Highly regio- and stereoselective synthesis of mannose-containing oligosaccharides with acetobromo sugars as the donors and partially protected mannose derivatives as the acceptors via sugar orthoester intermediates

Full text of DOI:10.1002/(SICI)1521-3773(19990503)38:9<1247::AID-ANIE1247>3.0.CO;2-J

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Enzyme preparation: Human Fhit protein was overexpressed in Escher-
ichia coli as described.[3a] Purification from the crude bacterial extract
comprised ammonium sulfate fractionation, ion-exchange chromatography
on DEAE-Sephacel, gel filtration on Sephadex G-100, and immobilized-
metal-affinity chromatography on TALON (Clonetech) resin from which
adsorbed protein was eluted with imidazole (50mm). Homogeneous
dinucleoside triphosphate hydrolase from yellow lupine seeds was obtained
as described previously.[22]
[20] N. M. H. Thorne, S. Hankin, M. C. Wilkinson, C. Nunez, R. Barra-
clough, A. G. McLennan, Biochem. J. 1995, 311, 717 ± 721.
[21] A. Guranowski, E. Starzynska, P. Brown, G. M. Blackburn, Biochem.
J. 1997, 328, 257 ± 262.
[22] A. Guranowski, E. Starzynska, E. Bojarska, J. Stepinski, E. Darzyn-
kiewicz, Protein Expression Purif. 1996, 8, 416 ± 422.
[23] H. Jakubowski, A. Guranowski, J. Biol. Chem. 1983, 258, 9982 ± 9989.
Received: November 12, 1998 [Z12656IE]
German version: Angew. Chem. 1999, 111, 1324 ± 1327
Keywords: bioorganic chemistry
´ enzyme inhibitors ´
molecular recognition ´ nucleotides ´ trisphosphonic acids
Highly Regio- and Stereoselective Synthesis of
Mannose-Containing Oligosaccharides with
Acetobromo Sugars as the Donors and Partially
Protected Mannose Derivatives as the
Acceptors via Sugar Orthoester
[1] A. G. McLennan, P. C. Zamecnik in Ap4A and Other Dinucleoside
Polyphosphates (Ed.: A. G. McLennan), CRC Press, Boca Raton, FL,
1992, pp. 1 ± 7.
[2] a) Z. Siprashvili, G. Sozzi, L. D. Barnes, P. McCue, A. K. Robinson, V.
Eryomin, L. Sard, E. Ragliabue, A. Graco, L. Fusetti, G. Schwartz,
M. A. Pierotti, C. M. Croce, K. Huebner, Proc. Natl. Acad. Sci. USA
1997, 94, 13771 ± 13776; b) L. D. Barnes, P. N. Garrison, Z. Siprashvili,
A. Guranowski, A. K. Robinson, S. W. Ingram, C. M. Croce, M. Ohta,
K. Huebner, Biochemistry 1996, 35, 11529 ± 11535.
Intermediates**
Wei Wang and Fanzuo Kong*
Many biologically important natural products such as
glycoproteins,[1] the ubiquitous components of the cell mem-
brane, contain an oligomannopyranose core, while the cell
wall of yeast contains branched mannans.[2] Mannose-con-
taining oligosaccharides have been synthesized by well-
established methods[3] that involve multistep selective protec-
tion and deprotection procedures. The use of unprotected or
partially protected mannose and acetobromo sugars as raw
materials in glycosylations is very attractive for organic
chemists because the synthetic routes can be substantially
simplified. In previous work[4a] we described a new method for
regio- and stereoselective synthesis of oligosaccharides by an
orthoester[4d-m] formation/rearrangement procedure with un-
protected glucopyranosides as the glycosyl acceptors and
acetobromo sugars as the donors which gave 1 !6-linked
oligosaccharides in satisfactory yields. In addition, 3-selective
glycosylation was achieved with partially protected glucose
acceptors containing unprotected 2,3- or 3,4-hydroxy groups.
It was found, however, that the glycosylation with unprotect-
ed glycosides as the acceptors was rather slow and difficult to
monitor owing to the poor solubility of the acceptors in the
reaction media. We now report a new strategy for highly
regio- and stereoselective synthesis of mannose-containing di-
and oligosaccharides via orthoester intermediates by coupling
acetobromo sugars with partially protected mannose deriva-
tives as the acceptors, in particular naked mannose 1,2-O-
ethylidenate.
[3] a) C. Brenner, H. C. Pace, P. N. Garrison, A. K. Robinson, A. Rösler,
A. Liu, G. M. Blackburn, C. M. Croce, K. Huebner, L. D. Barnes,
Protein Eng. 1997, 10, 1461 ± 1463; b) H. C. Pace, P. N. Garrison, A. K.
Robinson, L. D. Barnes, A. Draganescu, A. Rösler, G. M. Blackburn,
Z. Siprashvili, C. M. Croce, K. Huebner, C. Brenner, Proc. Natl. Acad.
Sci. USA 1998, 95, 5484 ± 5489.
[4] D. Maksel, A. Guranowski, S. C. Ilgoutz, A. Moir, G. M. Blackburn,
K. R. Gayler, Biochem. J. 1998, 329, 313 ± 319, and references therein.
[5] A. Guranowski, P. Brown, P. A. Ashton, G. M. Blackburn, Biochem-
istry 1994, 33, 235 ± 240.
[6] G. M. Blackburn, M.-J. Guo, A. G. McLennan in Ap4A and Other
Dinucleoside Polyphosphates (Ed.: A. G. McLennan), CRC Press,
Boca Raton, FL, 1992, pp. 305 ± 342.
[7] D. M. Williams, D. L. Jakeman, J. S. Vyle, M. P. Williamson, G. M.
Blackburn, Collect. Czech. Chem. Commun. 1996, 31, 88 ± 91; D. M.
Williams, D. L. Jakeman, J. S. Vyle, M. P. Williamson, G. M. Black-
burn, Bioorg. Med. Chem. Lett. 1998, 8, 2603 ± 2608.
[8] G. M. Blackburn, Chem. Ind. (London) 1981 (5), 134 ± 138.
[9] G. E. Lienhard, I. I. Secemski, J. Biol. Chem. 1973, 248, 1121 ± 1127.
[10] R. M. Dixon, G. Lowe, J. Biol. Chem. 1989, 264, 2069 ± 2074.
[11] X. Liu, X.-R. Zhang, G. M. Blackburn, Chem. Commun. 1997, 52, 87 ±
88.
[12] a) C. Brenner, P. Garrison, J. Gilmour, D. Peisach, D. Ringe, G. A.
Petsko, J. M. Lowenstein, Nature Struct. Biol. 1997, 4, 231 ± 238;
b) M. J. Bessman, D. N. Frick, S. F. OꢁHandley, J. Biol. Chem. 1996,
271, 25059 ± 25062; c) G. Sozzi, K. Huebner, C. M. Croce, Adv. Cancer
Res. 1998, 74, 141 ± 166.
[13] H. Gross, B. Costisella, I. Keitel, S. Ozegowski, Phosphorus Sulfur
Silicon 1993, 83, 203 ± 207.
[14] X. Liu, H. Adams, G. M. Blackburn, Chem. Commun. 1998, 2619 ±
2620.
[15] V. J. Davisson, D. R. Davis, V. M. Dixit, C. D. Poulter, J. Org. Chem.
1987, 52, 1794 ± 1801
It is well known that 3,6-branched mannotrisaccharide
Manpa1 !6(Manpa1 !3)Man is present in all asparagine-
[16] J. G. Moffatt, H. G. Khorana, J. Am. Chem. Soc. 1961, 83, 649 ± 658.
[17] See V. Wittmann, C. H. Wong, J. Org. Chem. 1997, 62, 2144 ± 2147.
[18] These tripodal analogues have been fully characterized by NMR
spectroscopy and mass spectrometry along with all other nucleotide
analogues described here. Positive-ion HR-MS: 2: m/z 571.971, calcd
[*] Prof. Dr. F. Kong, Dr. W. Wang
Research Center for Eco-Environmental Sciences
Academia Sinica
‡
for [C11H15N5O12P3Na3‡H] : 571.970; 4: m/z 1080.898, calcd for
P. O. Box 2871, Beijing 100085 (China)
Fax : (‡ 86)10-62923563
‡
[C21H24ClN10O21P5Na6‡H] : 1080.896 (35Cl); 6: m/z 1398.985, calcd
‡
for [C31H38N15O27P6Na6‡Na] : 1398.977; 8: 1296.023, calcd for
E-mail: fzkong@mail.rcees.ac.cn
‡
[C31H37N15O24P5Na5‡H] : 1296.021. Their purity was established as
[**] This work was supported by the Chinese Academy of Sciences
(Project KJ952J1510) and the National Natural Science Foundation of
China (Project 29802009).
98% or greater by HPLC (BioRad MA70 anion exchange column,
‡
50mm !700mm NH4 HCO3 gradient elution).
[19] A. Guranowski, A. Sillero in Ap4A and Other Dinucleoside Poly-
phosphates (Ed.: A. G. McLennan), CRC Press, Boca Raton, FL,
1992, pp. 81 ± 133.
Supporting information for this article is available on the WWW
under http://www.wiley-vch.de/home/angewandte/ or from the author.
Angew. Chem. Int. Ed. 1999, 38, No. 9
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linked oligosaccharides, and it is
the major Con A binding epitope
on oligomannose-type carbohy-
drates.[5] The syntheses of the tri-
saccharide involve the use of dif-
ferent protective groups and
lengthy reaction routes leading to
the product in low yields.[6] With
the newly developed orthoester
formation/rearrangement strategy
Scheme 1. Conditions and reagents: a) AgOTf/2,4-lutidine, CH2Cl2, molecular sieves (4 Š), 3 h; b) Ac2O/
anhydrous pyridine; c) TMSOTf/CH2Cl2, molecular sieves (4 Š), 30oC, 30 min.
the trisaccharide was readily synthesized[7] by coupling 2,3,4,6-
tetra-O-acetyl-a-d-mannopyranosyl bromide (1, 2.4 equiv)
with 1,2-O-ethylidene-b-d-mannopyranose (2, 1 equiv) in
the presence of silver trifluoromethanesulfonate (AgOTf,
2.2 equiv) and 2,4-lutidine (2.4 equiv) followed by rearrange-
ment (Scheme 1).
tion of 3 to afford the 3,6-branched pentosaccharide di-
orthoester 6 in 88% yield, the rearrangement of which gave
the 3,6-branched pentosaccharide 7 in 74% yield (Scheme 2).
These transformations revealed that the regio- (3,6) and
stereosectivity (1,2-trans) was dependent on the acceptor
rather than the donor structure. The structure of 6 was verified
by its 2D 1H NMR spectrum[8] which showed H4 as a low-field
triplet.
To investigate the application of the new strategy to more
general mannose acceptors, a number of partially protected
mannose derivatives was examined (Scheme 3). Thus, with
methyl 2,3-O-isopropylidene-a-d-mannopyranoside (8), a
The formation of trisaccharide diorthoester 3 is highly
regioselective; no 4-substitution was found, and 3 and 4 were
obtained as white crystals. The structure of 3 was unambig-
uously verified by its 1H NMR spectrum,[8] which showed two
characteristic signals at d ˆ 5.54 and 5.50 for H1' and H1''
respectively, and by its acetylation product 4, which exhibited
a new triplet at d ˆ 5.12, a
clear indication[8] of 3,6-
branched glycosylation. Re-
arrangement[4a±c, 9] of 4 in
the presence of trimethylsil-
yl
trifluoromethanesulfo-
nate (TMSOTf) gave the
3,6-branched mannotrisac-
charide 5 in high yield. Its
1
2D H NMR spectrum also
gave a clear indication[8] of
3,6-branched glycosylation.
The orthoester 3 is a very
important intermediate. It
can be modified at the re-
ducing ends to couple sugar
donors at the 2'- and 2''-
positions, and its transforma-
tion to 5 followed by remov-
al of the 1,2-ethylidene
group can lead to suitable
glycosyl donors such as the
Schmidt reagent and phenyl
thioglycoside. Subsequent
coupling with sugar accept-
ors affords a-[3c] and b-
Scheme 2. Conditions and reagents: a) AgOTf/2,4-lutidine, CH2Cl2, molecular sieves (4 Š), 4 h; b) Ac2O/anhydrous
pyridine; c) TMSOTf/CH2Cl2, molecular sieves (4 Š), N2, 30 min.
linked[10]
oligosaccharides
respectively.
Encouraged by the suc-
cess in coupling 2 with 1, we
tried the glycosylation of 2
with disaccharide as the gly-
cosyl donor. Thus, acetobro-
molactose (2.2 equiv) was
coupled with 2 (1 equiv) un-
der the same conditions as
described for the prepara-
Scheme 3. Conditions and reagents: a) AgOTf/2,4-lutidine, CH2Cl2, molecular sieves (4 Š), 2 ± 3 h; b) Ac2O/
anhydrous pyridine; c) TMSOTf/CH2Cl2, molecular sieves (4 Š), N2, 30 ± 40 min.
1248
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Angew. Chem. Int. Ed. 1999, 38, No. 9

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New York, 1996, 125; c) F. Barresi, O. Hindsgaul, J. Carbohydr. Chem.
1995, 14, 1043; d) R. R. Schmidt, W. Kinzy, Adv. Carbohydr. Chem.
Biochem. 1994, 50, 21; e) K. Toshima, K. Tatsuta, Chem. Rev. 1993, 93,
1503. Glycosylsulfoxide donors: L. Yan, D. Kahne, J. Am. Chem. Soc.
1996, 118, 9239. 1-Hydroxyglycosyl donors: B. A. Garcia, J. A. Poole,
D. Y. Gin, J. Am. Chem. Soc. 1997, 119, 7597. Glycal donors: S. J.
Danishefsky, M. T. Bilodeau, Angew. Chem. 1996, 108, 1482; Angew.
Chem. Int. Ed. Engl. 1996, 35, 1380. 1,2-Anhydroglycosyl donors: Y.
Du, F. Kong, Tetrahedron Lett. 1995, 427.
6-linked orthoester was formed as the sole product in almost
quantitative yield. Its acetylation to 9 followed by rearrange-
ment readily furnished 10. With methyl 6-O-benzoyl-a-d-
mannopyranoside[11] (11) as the acceptor, the 3-linked or-
thoester 12 was obtained in a high yield. Its acetylation to 13
followed by rearrangement, or its rearrangement to 14
followed by acetylation produced the same compound 15,
indicating that both orthoester formation and rearrangement
were regio- and stereoselective. The same 3-selectivity was
obtained with 6-O-benzoyl-1,2-O-ethylidene-b-d-mannopyr-
anose (16) as the acceptor, when the 1,3-linked disaccharide
19 was formed in a satisfactory yield.
Since the rearrangement of 12 to 14 (82%) and the
rearrangement of 13 to 15 (85%) afforded the respective
disaccharides in similar yields, and the rearrangement of 4
afforded the trisaccharide 5, also in a high yield, we
rationalized the mechanism of the rearrangement as shown
in Scheme 4. When TMSOTf was added to the solution of 12
in dichloromethane, selective bond cleavage of the orthoester
linkage rather than 2- or 4-OH bond cleavage occurred to give
a closely associated ion pair (form A). When this ion pair
reaches an appropriate six-membered ring geometry (form B)
rearrangement occurs to afford the required disaccharide 14.
We still consider that steric factors are mainly responsible for
the high regioselectivity in orthoester formation.[4]
[4] a) W. Wang, F. Kong, J. Org. Chem. 1998, 63, 5744; b) F. Kong, W.
Wang, Chinese Pat. Appl. 971257788.4; c) F. Kong, W. Wang, Chinese
Pat. Appl. 98103242.7; d) B. Ernst, A. De Mesmaeker, B. Wagner, T.
Winkler, Tetrahedron Lett. 1990, 6167; e) J. Banoub, P. Boullanger, M.
Potier, G. Descotes, Tetrahedron Lett. 1986, 4145; f) B. M. Dahlin, P. J.
Garegg, R. Johansson, B. Samuelsson, U. Orn, Acta Chem. Scand. Ser.
B 1981, 35, 669; g) J. Banoub, D. R. Bundle, Can. J. Chem. 1979, 57,
2091; h) T. Ogawa, M. Matsui, Carbohydr. Res. 1976, 51, C13; i) S. E.
Zurabyan, M. M. Tikhomirov, V. A. Nesmeyanov, A. Y. Khorlin,
Carbohydr. Res. 1973, 26, 117; j) G. Wulff, W. Kruger, Carbohydr. Res.
1971, 19, 139; k) N. K. Kochetkov, A. F. Bochkov, T. A. Sokolovskaya,
V. J. Snyatkova, Carbohydr. Res. 1971, 16, 17; l) N. K. Kochetkov, A. J.
Khorlin, A. F. Bochkov, Tetrahedron 1967, 23, 693.
[5] a) D. K. Mandal, L. Bhattacharyya, S. H. Koenig, R. D. Brown, S.
Oscarson, C. F. Brewer, Biochemistry 1994, 33, 1157; b) D. K. Mandal,
C. F. Brewer, Biochemistry 1992, 32, 5116; c) B. A. Williams, M. C.
Chervenak, E. J. Toone, J. Biol. Chem. 1992, 267, 22907; d) M. C.
Chervenak, E. J. Toone, J. Am. Chem. Soc. 1994, 116, 10533.
[6] a) M. W. Francoise, J. R. Brisson, J. P. Carver, J. J. Krepinsky,
Carbohydr. Res. 1982, 103, 15; b) T. Ogawa, K. Katano, M. Matsui,
Carbohydr. Res. 1978, 64, C3; c) J. Arnarp, J. Lonngren, Acta Chem.
Scand. Ser. B 1978, 32, 696.
[7] Typical conditions for orthoester preparation: To a
stirred solution of 2,3,4,6-tetra-O-acetyl-a-d-mannopyr-
anosyl bromide (1, 1.02 g, 2.5 mmol), 2,4-lutidine
(276 mL, 2.4 mmol), and 1,2-O-ethylidene-(R,S)-b-d-
mannopyranose (2, 206 mg, 1 mmol) in dichlorome-
thane (20 mL) under nitrogen atmosphere was added
AgOTf (565 mg, 2.2 mmol) in a dark room. The reaction
was carried out at room temperature and monitored by
TLC (petroleum ether/ethyl acetate, 1/1.5). After com-
pletion of the reaction the mixture was partitioned
Scheme 4.
between dichloromethane and water, the organic phase
was concentrated, dried, and subjected to column chromatography on
silica gel with petroleum ether/ethyl acetate (1/1) as the eluent to give
the trisaccharide diorthoester 3 (91% yield based on acceptor 2) as the
sole product. The procedure for the preparation of the disaccharide
orthoesters is the same as described above except for the amounts of
the acetoglycobromide donors (ꢀ1.2 equiv) and the promoters
(ꢀ1.2 equiv). Typical rearrangement conditions: To a stirred solution
of sugar-sugar orthoester 4 (580 mg, 0.64 mmol) in dichloromethane
(10 mL) was added TMSOTf (12 mL, 0.1 equiv) under nitrogen
atmosphere and the reaction was monitored by TLC (petroleum
ether/ethyl acetate, 1/1). After completion of the reaction the mixture
was treated with triethylamine (20 mL), filtered, and the filtrate was
washed with CH2Cl2. The combined solution was washed with 1m HCl
(10 mL), saturated aqueous NaHCO3 (10 mL), and aqueous NaCl
(2 Â 10 mL), dried over anhydrous Na2SO4, and concentrated. The
residue was subjected to column chromatography on silica gel with
petroleum ether/ethyl acetate (1/1) as the eluent to give 5 (510 mg,
88%).
In summary, here we present a very effective regio- and
stereoselective glycosylation method with 1,2-O-ethylidenat-
ed mannose or partially protected mannosides as glycosyl
acceptors and simple acetobromo sugars as the glycosyl
donors by an orthoester formation/rearrangement procedure.
A number of 1 !6-, 1 !3-, and 3,6-branched oligosaccharides
with exclusive 1,2-trans linkage were readily synthesized by
the new strategy. This approach, in terms of yields, simplicity,
and efficiency, will be a general one for the synthesis of
mannose-containing oligosaccharides.
Received: November 10, 1998 [Z12648IE]
German version: Angew. Chem. 1999, 111, 1330 ± 1333
Keywords: oligosaccharides ´ protecting groups ´ rearrange-
ments ´ synthetic methods
1
[8] Physical data and H NMR (400 MHz, CDCl3, 258C, TMS) data for
3 ± 6. 3: m.p. 110 ± 1128C; [a]D ˆ ‡5.2 (c ˆ 1.5 trichloromethane);
1H NMR: d ˆ 5.54, 5.50 (2d, 3J (H1'(1''),H2'(2'')) ˆ 2.7 Hz, 2H; H-1', 1''),
5.30 ± 5.15 (m, H-1, 3', 3'', 4', 4'', 6H; CH3CH), 4.75, 4.63 (2dd, 3J
[1] A. Kobata in Biology of Carbohydrates, Vol. 2 (Ed.: V. Ginsburg), IRL
Press, New York, 1980, pp. 87 ± 161.
3
(H2',H3') ˆ 3.8 Hz, J (H2'',H3'') ˆ 3.9 Hz, 2H; H-2', 2''), 4.23 ± 4.13 (m,
3
3
4H; H-6', 6''), 4.06 (dd, J (H1,H2) ˆ 2.4 Hz, J (H2,H3) ˆ 3.6 Hz, 1H;
H-2), 3.84 ± 3.68 (m, 6H; H-3, 4, 5', 5'', 6), 3.40 ± 3.35 (m, 1H; H-5),
2.11, 2.10, 2.07, 2.07, 2.06, 2.05 (6s, 18H; 6 CH3CO), 1.85, 1.74 (2s, 6H;
2 CH3CO), 1.47 (d, 3J (H,H) ˆ 5.0 Hz, 3H; CH3CH). 4: m.p. 135 ±
1388C; [a]D ˆ ‡0.4 (c ˆ 0.1, trichloromethane); 1H NMR: d ˆ 5.49,
5.43 (2d, 3J (H1',H2'), 3J (H1'',H2'') ˆ 2.8 Hz, 2.6 Hz, 2H; H-1', 1''),
5.30 ± 5.16 (m, 6H; H-1, 3', 3'', 4', 4'', CH3CH), 5.12 (t, 3J (H,H) ˆ
[2] a) T. Kanbe, J. E. Cutler, Infect. Immun. 1994, 62, 1662; b) M.
Stratford, Yeast 1992, 8, 635; c) R. D. Nelson, N. Shibata, R. P.
Podzorski, M. J. Herron, Clin. Microbiol. Rev. 1991, 4, 1; d) R. P.
Podzorski, G. R. Gray, R. D. Nelson, J. Immunol. 1990, 144, 707.
[3] General reviews: a) S. Hanessian, Preparative Carbohydrate Chem-
istry, Marcel Dekker, New York, 1997, Chapters 12 ± 22; b) S. H. Khan,
R. A. O'Neil, Modern Methods in Carbohydrate Synthesis, Harwood,
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9.6 Hz, 1H; H-4), 4.66 (dd, 3J (H1'',H2'') ˆ 2.9 Hz, 3J (H2'',H3'') ˆ 3.9 Hz,
1H; H-2''), 4.58 (dd, 3J (H1',H2') ˆ 2.8 Hz, 3J (H2'H3') ˆ 4.0 Hz, 1H;
H-2'), 4.24 ± 4.08 (m, 5H; H-2, 6', 6''), 4.01 (dd, 3J (H2,H3) ˆ 4.6 Hz, 3J
(H3,H4) ˆ 9.6 Hz, 1H; H-3), 3.75 ± 3.65 (m, 2H; H-5', 5''), 3.60 (d, 2J
(H6a,H6b) ˆ 4.5 Hz, 2H; H-6), 3.55 ± 3.50 (m, 1H, H-5), 2.12, 2.10, 2.07,
2.06, 2.06, 2.05, 2.04 (7s, 21H; 7 CH3CO), 1.77, 1.71 (2s, 6H; 2
CH3CO3), 1.49 (d, 3J (H,H) ˆ 4.9 Hz, 3H; CH3CH). 5: m.p. 177 ±
1788C; [a]D ˆ ‡16.9 (c ˆ 0.15, trichloromethane); 1H NMR: d ˆ
5.36 ± 5.21 (m, 8H; H-1, 2', 3', 3'', 4, 4', 4'', CH3CH), 5.14 (dd, 3J
(H1'',H2'') ˆ 1.9 Hz, 3J (H2'',H3'') ˆ 3.1 Hz, 1H; H-2''), 4.98 (d, 3J
(H1'',H2'') ˆ 1.9 Hz, 1H; H-1''), 4.78 (d, 3J (H1',H2') ˆ 1.7 Hz, 1H;
H-1'), 4.34 ± 4.27 (m, 4H; H-2, 5', 6''), 4.10 ± 4.01 (m, 3H; H-5'', 6'), 3.91
(dd, 3J (H2,H3) ˆ 3.8 Hz, 3J (H3,H4) ˆ 9.7 Hz, 1H; H-3), 3.80 (dd, 3J
(H5,H6a) ˆ 6.3 Hz, 2J (H6a,H6b) ˆ 10.4 Hz, 1H; H-6a), 3.62 ± 3.56 (m,
2H; H-5, 6b), 2.16, 2.15, 2.15, 2.11, 2.09, 2.06, 2.06, 2.00, 1.98 (9s, 27H; 9
CH3CO), 1.53 (d, 3J (H,H) ˆ 5.0 Hz, 3H; CH3CH). 6: [a]D ˆ ‡2.4 (c ˆ
transactivation domainÐis flanked by several phosphoryla-
tion sites (Figure 1). These findings suggest that the correct
orchestration of SRF phosphorylation and glycosylation as
well as the controlled removal ot these covalent protein
modifications appear to be paramount to the regulation of
gene transcription under the control of SRF.
ribosomal
S6 kinase
DNA-protein
kinase
374D S S T D L T Q T S383 S S G T V T L P391
glycosylation
sites
1
3
0.15, trichloromethane); H NMR: d ˆ 5.69 (d, J (H1B,H2B) ˆ 5.1 Hz,
1H; H-1B), 5.65 (d, 3J (H1B',H2B') ˆ 5.2 Hz, 1H; H-1B'), 5.50 (dd, 3J
(H2B,H3B) ˆ 2.4 Hz, 3J (H3B,H4B) ˆ 1.2 Hz, 1H; H-3B), 5.42 (t, 3J
casein kinase II
Figure 1. Phosphorylation and glycosylation sites in the transactivation
domain of the serum response factor (underlined amino acid symbols). A
kinase responsible for the phosphorylation of a certain amino acid is shown.
The phosphorylated and glycosylated heptapeptide 17 synthesized in the
context of this work is highlighted in the box.
3
(H,H) ˆ 1.9 Hz, 1H; H-3B'), 5.38 (2d, J (H,H) ˆ 3.3 Hz, 2H; H-4C,
4C'), 5.26 (d, 3J (H1A,H2A) ˆ 1.6 Hz, 1H; H-1A), 5.26 (q, 3J (H,H) ˆ
5.0 Hz, 1H; CH3CH), 5.16 (2 dd, 3J (H1C(C'),H2C(C')) ˆ 8.0 Hz, 3J
(H2C(C'),H3C(C')) ˆ 9.6 Hz, 2H; H-2C, 2C'), 5.10 (t, 3J (H,H) ˆ 9.5 Hz,
1H; H-4A), 5.00 (2dd, 2H; H-3C, 3C'), 4.60, 4.55 (2d, 3J
(H1C(C'),H2C(C')) ˆ 8.0 Hz, 2H; H-1C, 1C'), 4.41 (dd, 1H; H-2B), 4.35
(dd, 1H; H-2B'), 4.30 ± 4.20 (m, 3H; H-2A, 6Ba, 6B'a), 4.13 ± 4.05 (m,
6H; H-6Bb, 6B'b, 6C, 6C'), 3.98 ± 3.88 (m, 3H; H-3A, 5C, 5C'), 3.85 ±
3.78 (m, 2H; H-5B, 5B'), 3.64 (2t, 3J (H,H) ˆ 9.5 Hz, 2H; H-4B, 4B'),
3.60 ± 3.58 (m, 3H; H-5A, 6A), 2.17 ± 1.98 (13s, 39H; 13 CH3CO), 1.74,
1.68 (2s, 6H; 2 CH3CO3), 1.47 (d, 3J (H,H) ˆ 5.0 Hz, 3H; CH3CH).
[9] a) J. Gass, M. Strobl, P. Kosma, Carbohydr. Res. 1993, 244, 69; b) M. L.
Sznaidman, S. C. Johnson, C. Crasto, S. M. Hecht, J. Org. Chem. 1995,
60, 3942; c) T. Ogawa, K. Beppu, S. Nakabayashi, Carbohydr. Res.
1981, 93, C6; d) W. Wang, F. Kong, Tetrahedron Lett. 1998, 1937.
[10] D. Crich, S. Sun, J. Am. Chem. Soc. 1998, 120, 435.
Peptide conjugates embodying the characteristic structural
elements of the naturally occuring protein conjugates (name-
ly, phosphates and O-glycosides) may be efficient tools for the
study of such biological processes in molecular detail.[5, 6]
However, glycopeptides are very sensitive to acid and base,
and phosphopeptides are even more base labile, with carbo-
hydrate and phosphotriester groups being lost in a b-
elimination reaction at pH values greater than 8 ± 9.[5] In the
synthesis of glycophoshopeptides these problems potentiate
each other and consequently protecting groups have to be
used that can be removed with complete selectivity under the
mildest conditions, preferably at pH 6 ± 8 and room temper-
ature.[5] We now report that such labile peptide conjugates can
successfully be built up by employing the enzymatic removal
of the choline ester and the p-phenylacetoxybenzyloxycar-
bonyl (PhacOZ) group as the key steps.
[11] J. Xia, Y. Hui, Synth. Commun. 1995, 25, 2235.
The choline ester was previously investigated as a possible
blocking group for glycopeptide synthesis, but under the
conditions required for its cleavage (pH 10 ± 11) the sensitive
O-glycosylated peptides were destroyed.[7] This problem can,
however, be solved efficiently by employing the enzyme
butyrylcholine esterase from horse serum for the removal of
the protecting group. To investigate the suitability of the
enzymatic choline ester cleavage for glycopeptide synthesis
O-GlcNAc-modified serine/threonine building blocks 1[8]
were coupled with amino acid choline esters 2[7] to give
glycopeptides 3 in high yield. Glycodipeptide esters 3 were
subsequently treated with butyrylcholine esterase in phos-
phate buffer at pH 6.5 (Scheme 1). In the ensuing enzymatic
reaction the C-terminal choline esters were saponified
exclusively. An attack on the acetate or the N-terminal
urethane groups and an anomerization or b-elimination of the
carbohydrate did not occur. The enzyme accepts different
amino acid combinations and also tolerates sterically de-
manding residues such as phenylalanine at the C-terminus.
The desired, selectively unmasked, glycodipeptides 4 were
isolated in yields of 70 ± 80%. These results clearly indicate
that the enzymatic removal of choline esters can be applied
advantageously to glycopeptide chemistry. Particularly re-
Chemoenzymatic Synthesis of a Characteristic
Glycophosphopeptide from the Transactivation
Domain of the Serum Response Factor**
Jörg Sander and Herbert Waldmann*
Phosphorylation[1] and O-glycosidic attachment of N-ace-
tylglucosamine (GlcNAc)[2] to serine and threonine residues
are key events in the cellular transduction of mitogenic
signals. For instance, the serum response factor (SRF), a
widely found transcription factor, is phosphorylated at various
serine and threonine residues in response to extracellular
stimuli. It then translocates to the nucleus, binds to the serum
response element (SRE, which occurs in the promoters of
numerous genes), and induces gene transcription.[3] In addi-
tion, SRF is glycosylated at four different sites,[4] where the
major glycosylation siteÐmost probably serine383 in the
[*] Prof. Dr. H. Waldmann, Dr. J. Sander
Institut für Organische Chemie der Universität
Richard-Willstätter-Allee 2, D-76128 Karlsruhe (Germany)
Fax: (‡49)721-608-4825
E-mail: waldmann@ochhades.chemie.uni-karlsruhe.de
[**] This research was supported by the Fonds der Chemischen Industrie.
1250
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Angew. Chem. Int. Ed. 1999, 38, No. 9