2328 J . Org. Chem., Vol. 66, No. 7, 2001
Mitchell et al.
peptide with a disialyl biantennary complex-type sac-
charide moiety N-linked to asparagine.15 Glycoconjugate
vaccines against certain types of cancer have been
synthesized.16 Glycopeptides17 and neoglycopeptides18 can
now be assembled once the required amino acid glycoside
is in hand. Despite these advances, the pharmacology and
biology of glycopeptides is still far less developed than
peptides, largely because glycopeptides are considerably
more difficult to synthesize.
Peptides have been discounted as drug candidates due
to a perceived instability to hydrolytic enzymes and pH
extremes and due to the blood-brain barrier (BBB), which
blocks their entry into the central nervous system.19
Glycosylation has been used as a method to penetrate
the BBB, and enkephalin analogues glycosylated at or
near the C-terminus were shown to elicit prolonged and
profound analgesia in mice.20 Similar results have been
obtained with glycosylated vasopressin analogues21 and
with deltorphin and dermorphin glycopeptide ana-
logues.22
transport rates, as shown by physicochemical data, do
not correlate with lipophilicity,25 which reinforces the
premise that another transport mechanism is at work.
This transport phenomenon may be related to the
observation that glycosylation of peptides has been shown
to improve absorption rates from the intestinal tract.26
Design Cr iter ia . The “message sequence,” or phar-
macophore, used in these studies was borrowed from the
work of Hruby et al.,27 the δ-selective enkephalin parent
sequence D-Pen2,D-Pen5-enkephalin (DPDPE).28 The Tyr-
DCys-Gly-Phe-DCys “message” was chosen as a relatively
unselective ligand (µ vs δ) to maximize the possibility of
interaction with a receptor within the brain, and to
observe any influences of the glycoside moiety on receptor
selectivity.29 In addition, it was hoped that this would
help establish a “pharmacological baseline” with which
to compare the effectiveness of transport in vivo. Since
D-amino acids have been used in several peptides to
circumvent degradation by proteases and hydrolases,30
D-serine and D-threonine were also used as glycoside-
bearing amino acids. Surprisingly, these O-linked D-
amino acid glycosides do not appear to have been
examined previously in any context.
At this point, the precise role of the glycoside in peptide
and protein transport processes remains enigmatic. Most
O-linked glycoproteins found in nature have a core glycan
structure initiated with a serine- or threonine-linked
R-GalNAc, and it is believed that the glycan moiety plays
a decisive role in intracellular transport (protein traf-
ficking).31 Thus, related glycopeptide structures may
provide new insights on protein trafficking. Mannose-6-
phosphate binding proteins are involved in the transport
of glycoproteins to lysosomal compartments for enzymatic
digestion.32 Also, due to the importance of â-GlcNAc-
Ser/Thr glycosides in both the â-amyloid precursor
protein (APP)33 and nuclear pore proteins,34 â-GlcNAc
was included as one of the glycosyl moieties. The disac-
charides were incorporated to examine the effect of a
larger glycan moiety on the pharmacology of these
glycopeptides, and the xyloside (present in gel-forming
mucins)35 was included to see whether a pentose glycoside
Originally, it was envisaged that the glucose trans-
porter GLUT-1 found at the BBB would transport the
opiate message sequences to the targeted receptors
within the brain. Subsequent work by Davis et al.23 with
14C-labeled glycopeptides showed that this mode of
transport is unlikely and led to the conclusion that other
mechanisms were responsible for the BBB permeability
of these glycopeptides.24 Paradoxically, the observed
(15) Mizuno, M,; Haneda, K.; Iguchi, R.; Muramoto, I.; Kawakami,
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J eanloz, R. W. Adv. Carbohydr. Chem. Biochem. 1985, 43, 135-201.
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Kunz, H. Angew. Chem., Int. Ed. Engl. 1987, 26, 294-308. (e) Gigg,
J .; Gigg, R. Top. Curr. Chem. 1990, 154, 77. (f) Paulsen, H. Angew.
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Barany, G. J . Pept. Res. 2000, 55, 81-91. For examples of enzymatic
synthesis see, (m) Wong, C. H.; Schuster, M.; Wang, P.; Sears, P. J .
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