N-Dithiasuccinoyl-Protected DeriVatiVes of Glucosamine
J. Am. Chem. Soc., Vol. 118, No. 13, 1996 3149
D-GlcpNAc glycosylation sites occur in protein regions that are
rich in Ser and Thr, and often include a proline residue one to
three positions from the attachment site.7e
Alternatively, in the “active ester” approach, NR-Fmoc-amino
acid pentafluorophenyl esters (Fmoc-AA-OPfp’s) can be gly-
cosylated directly to provide building blocks for SPPS.16 Pfp
esters survive exposure to strong Lewis acids (equimolar) in
organic solvents, are reasonably stable toward oxygen nucleo-
philes under weakly acidic and neutral conditions, and can be
purified by silica gel chromatography with dry organic solvents16a
or reversed-phase HPLC with water-acetonitrile mixtures;16e
these properties make Pfp a suitable CR-carboxyl protecting
group for the glycosylation step.17 Subsequently, Pfp esters are
efficient acylating agents, especially in the presence of an
auxiliary nucleophile such as 1-hydroxybenzotriazole (HOBt)
or 3-hydroxy-2,3-dihydro-4-oxobenzotriazine. Thus, Pfp serves
a dual role of protection and activation, and saves protecting
group manipulation steps.
Stereocontrol in the syntheses of 1,2-trans-glycosides of
carbohydrates by electrophilic activation of an anomeric (C-1)
leaving group is usually accomplished through participation of
the C-2 substituent.18,19 For 2-(acylamino)-2-deoxyhexoses
(e.g., 1), such electrophilic activation forms a reactive oxocar-
benium ion (2) intermediate, which often collapses rapidly to
an oxazolinium ion (3) intermediate (Scheme 1, mechanism A).
Intermediate 3 is expected to undergo nucleophilic attack at C-1
selectively from the â-face of the glycosyl donor to yield only
1,2-trans-glycosides [in those cases where an R/â mixture is
observed, this is often attributed to reactions of 2]. However,
with the 2-amino group acylated, e.g., N-acetyl, the intermediate
3 can be stabilized further through abstraction of the amide
proton to form a relatively stable oxazoline (4).18c Protected
Ser derivatives (e.g., NR-protection, benzyloxycarbonyl, Boc,
Fmoc; CR-protection, Bn, 4-nitrobenzyl, methyl) react with
2-methyl-(1,2-dideoxy-R-D-glycopyrano)[2.1-d]oxazoline (4, R
) Me; prepared intentionally or assumed to be the true
intermediate derived from 1 with L ) Cl, R ) Me) at elevated
temperatures, typically g70 °C, to provide the corresponding
2-acetamido-2-deoxy-â-D-glucopyranosides of these Ser deriva-
tives in marginal to adequate yields.20 Alternatively, saccharides
Many naturally occurring glycoproteins show microhetero-
geneity in the glycan moiety, and isolation of pure glycopeptides
is difficult. Reliable and convenient methods for the chemical
synthesis of glycopeptides8 are of interest since they will make
available materials of well-defined structure in the amounts
required for biological testing and spectroscopic studies of
conformations. Synthetic glycopeptides may also have im-
proved bioavailability characteristics and attenuated in ViVo
clearance, as well as increased stability toward proteases and
reduced immunogenicity.9 The synthesis of Ser and Thr
glycosylated peptides is made challenging by their lability to
strong acids, e.g., hydrogen fluoride,10a and their susceptibility
to base-catalyzed â-elimination of the glycan.10b However, it
has been demonstrated11 that O-glycopeptides are stable to those
bases, e.g., morpholine, piperidine, and 1,8-diazabicyclo[5.4.0]-
undec-7-ene (DBU), that are commonly used to remove the NR-
(9-fluorenylmethyloxycarbonyl) (Fmoc) protecting group for
solid phase peptide synthesis (SPPS), and that acetyl (Ac) and
benzoyl ester groups protecting the glycan hydroxyls can be
cleaved from O-glycopeptides by mild base-catalyzed transes-
terification without significant â-elimination.8 Furthermore,
O-glycosidic linkages appear to be entirely stable to treatment
with concentrated trifluoroacetic acid in the absence or presence
of carbocation scavengers, particularly while the saccharide
retains ester protecting groups.12 Consequently, solid phase
methods using NR-Fmoc-protected glycosylated amino acid
building blocks are emerging as potentially the most flexible
and optimal way to prepare glycopeptides.8
Glycosylation of amino acids, with rare exceptions,13 requires
that both the NR-amino and the CR-carboxyl functions are
blocked. Given NR-amino protection with Fmoc, the CR-
carboxyl is generally protected during the glycosylation step
with a group such as tert-butyl (tBu), allyl (Al), or benzyl (Bn),
orthogonal removal of which provides a building block suitable
for activation and coupling in the context of Fmoc SPPS.14,15
(15) For recent examples of strategies that rely on NR-protection other
than Fmoc, see: (a) Bardaji, E.; Torres, J. L.; Clapes, P.; Albericio, F.;
Barany, G.; Rodriguez, R. E.; Sacristan, M. P.; Valencia, G. J. Chem. Soc.,
Perkin Trans. 1 1991, 1755-1759. (b) Polt, R.; Szabo, L.; Treiberg, J.; Li,
Y.; Hruby, V. J. J. Am. Chem. Soc. 1992, 114, 10249-10258.
(16) (a) Meldal, M.; Jensen, K. J. J. Chem. Soc., Chem. Commun. 1990,
483-485. (b) Meldal, M.; Bock, K. Tetrahedron Lett. 1990, 31, 6987-
6990. (c) Jansson, A. M.; Meldal, M.; Bock, K. Tetrahedron Lett. 1990,
31, 6991-6994. (d) Peters, S.; Bielfeldt, T.; Meldal, M.; Bock, K.; Paulsen,
H. Tetrahedron Lett. 1992, 33, 6445-6448. (e) Jensen, K. J.; Meldal, M.;
Bock, K. J. Chem. Soc., Perkin Trans.1 1993, 2119-2129.
(8) Reviews on glycopeptide synthesis: (a) Paulsen, H. Angew. Chem.,
Int. Ed. Engl. 1990, 29, 823-838. (b) Garg, H. G.; von dem Bruch, K.;
Kunz, H. AdV. Carbohydr. Chem. Biochem. 1994, 50, 277-310. (c) Meldal,
M. In Neoglycoconjugates: Preparation and Applications; Lee, Y. C., Lee,
R. T., Eds.; Academic Press: San Diego, 1994; pp 145-198. (d) Norberg,
T.; Lu¨ning, B.; Tejbrant, J. Methods Enzymol. 1994, 247, 87-106.
(9) (a) Fischer, J. F.; Harrison, A. W.; Bundy, G. L.; Wilkinson, K. F.;
Rush, B. D.; Ruwart, M. J. J. Med. Chem. 1991, 111, 3140-3143. (b)
Powell, M. F.; Stewart, T.; Otvos, L.; Urge, L.; Gaeta, F. C. A.; Sette, A.;
Arrhenius, T.; Thomson, D.; Soda, K.; Colon, S. M. Pharm. Res. 1993, 10,
1268-1273. (c) Neoglycoconjugates: Preparation and Applications; Lee,
Y. C., Lee, R. T., Eds.; Academic Press: San Diego, 1994. (d) Kihlberg,
J.; Åhmann, J.; Walse, B.; Drakenberg, T.; Nilsson, A.; So¨derberg-Ahlm,
C.; Bengtsson, B.; Olsson, H. J. Med. Chem. 1995, 38, 161-169.
(10) (a) Mort, A. J.; Lamport, D. T. A. Anal. Biochem. 1977, 82, 289-
309. (b) Wakabayashi, K; Pigman, W. Carbohydr. Res. 1974, 35, 3-14.
(11) (a) Kihlberg, J.; Vuljanic, T. Tetrahedron Lett. 1993, 34, 6135-
6138. (b) Meldal, M.; Bielfeldt, T.; Peters, S.; Jensen, K. J.; Paulsen, H.;
Bock, K. Int. J. Peptide Protein Res. 1994, 43, 529-536.
(17) The rate of glycosylation of NR-Fmoc-Ser-OPfp was shown to be
slower than that of NR-Fmoc-Ser-OAl, which has a less electron-withdrawing
carboxyl protecting group (ref 16a). Nevertheless, overall yields and purities
are excellent also with Pfp protection. The preceding comments apply as
well to NR-Fmoc-Thr-OPfp (ref 16c).
(18) For reviews see: (a) Paulsen, H. Angew. Chem., Int. Ed. Engl. 1982,
21, 155-173. (b) Schmidt, R. R. In ComprehensiVe Organic Synthesis;
Trost, B. M.; Fleming, I., Eds.; Pergamon Press: Oxford, 1991; pp 33-
64. For an overview of glycosylation with 2-amino-2-deoxy sugars see:
(c) Banoub, J.; Boullanger, P.; Lafont, D. Chem. ReV. 1992, 92, 1167-
1195.
(19) For the synthesis of 2-amino-2-deoxy-1,2-cis-hexosides, Lemieux
and co-workers pioneered the use of an azido group at the 2-position; the
azide is then reduced subsequent to glycosylation (reviewed in refs 8a and
18a). For recent examples see refs 11b, 14b, 14c, and 16d, as well as the
following: (a) Andreotti, A. H.; Kahne, D. J. Am. Chem. Soc. 1993, 115,
3352-3353. (b) Liang R. L.; Andreotti, A. H.; Kahne, D. J. Am. Chem.
Soc. 1995, 117, 10395-10396.
(20) (a) Jacquinet, J.-C.; Zurabyan, S. E.; Khorlin, A. Y. Carbohydr.
Res. 1974, 32, 137-143. (b) Garg, H. G.; Jeanloz, R. W. Carbohydr. Res.
1976, 49, 482-488. (c) Garg, H. G.; Jeanloz, R. W. Carbohydr. Res. 1976,
52, 246-250. (d) Vafina, M. G.; Kim, A.; Molodtsov, N. V. Carbohydr.
Res. 1978, 64, 334-338. (e) Lavielle, S.; Ling, N. C.; Saltman, R.;
Guillemin, R. C. Carbohydr. Res. 1981, 89, 229-236. (f) Arsequell, G.;
Krippner, L.; Dwek, R. A.; Wong, S. Y. C. J. Chem. Soc., Chem. Commun.
1994, 2383-2384.
(12) (a) Paulsen, H.; Merz, G.; Weichert, U. Angew. Chem., Int. Ed.
Engl. 1988, 27, 1365-1367. (b) Kunz, H.; Unverzagt, C. Angew. Chem.,
Int. Ed. Engl. 1988, 27, 1697-1699.
(13) When glycosylations are catalyzed by Lewis acids (SnCl4 and
BF3‚Et2O) strong enough to promote transglycosylation from carboxyl to
hydroxyl, the CR-carboxyl can be left unprotected. This approach requires
the glycosyl donor to be the limiting reagent; see: Elofson, M.; Walse, B.;
Kihlberg, J. Tetrahedron Lett. 1991, 32, 7613-7616.
(14) For strategies that rely on NR-Fmoc protection in combination with
a temporary CR-protecting group see: (a) Schultheiss-Reimann, P.; Kunz,
H. Angew. Chem., Int. Ed. Engl. 1983, 22, 62-63. (b) Paulsen, H.;
Adermann, K.; Merz, G.; Schultz, M.; Weichert, U. Starch 1988, 40, 465-
472. (b) Lu¨ning, B.; Norberg, T.; Tejbrant, J. J. Chem. Soc., Chem. Commun.
1989, 1267-1268. (c) Lu¨ning, B.; Norberg, T.; Tejbrant, J. Glycoconjugate
J. 1989, 5-19. (d) de la Torre, B. G.; Torres, J. L.; Bardaji, E.; Clapes, P.;
Xaus, N.; Jorba, X.; Calvet, S.; Albericio, F.; Valencia, G. J. Chem. Soc.,
Chem. Commun. 1990, 965-967.