Combined chemical and enzymatic synthesis of a C-glycopeptide and its inhibitory activity toward glycoamidases

Article abstract of DOI:10.1021/ja9712027

A novel chemoenzymatic approach to synthesizing high-mannose-type N-glycopeptide and its C-linked glycopeptide analog is described, The synthesis consists of two steps: a chemical synthesis of GlcNAc-containing peptides and an enzymatic glycosyl transfer

Full text of DOI:10.1021/ja9712027

VOLUME 119, NUMBER 46
NOVEMBER 19, 1997
© Copyright 1997 by the
American Chemical Society
Combined Chemical and Enzymatic Synthesis of a
C-Glycopeptide and Its Inhibitory Activity toward
Glycoamidases
Lai-Xi Wang,† Mei Tang,† Tadashi Suzuki,‡ Ken Kitajima,‡ Yasuo Inoue,‡,§
Sadako Inoue,‡,§ Jian-Qiang Fan,† and Yuan C. Lee*,†
Contribution from the Department of Biology, Johns Hopkins UniVersity,
Baltimore, Maryland 21218, and Department of Biophysics and Biochemistry,
UniVersity of Tokyo, Tokyo 113, Japan
ReceiVed April 15, 1997X
Abstract: A novel chemoenzymatic approach to synthesizing high-mannose-type N-glycopeptide and its C-linked
glycopeptide analog is described. The synthesis consists of two steps: a chemical synthesis of GlcNAc-containing
peptides and an enzymatic glycosyl transfer of Man9GlcNAc to the terminal GlcNAc in the peptides in an aqueous
medium containing organic solvents. The essential enzyme used is an endo-â-N-acetyl-glucosaminidase from
Arthrobacter protophormiae (Endo-A). This approach should be generally applicable to the synthesis of both natural
and designed high-mannose-type glycopeptides. It has been found that, while the natural high-mannose-type
N-glycopeptide 2 can be rapidly hydrolyzed by glycoamidases [commonly called N-glycanase or, systematically,
peptide-N4-(N-acetyl-â-D-glucosaminylasparagine amidase], the synthetic C-glycopeptide 1 with an insertion of a
methylene group at the crucial asparagine-GlcNAc linkage is resistant to the enzyme-catalyzed hydrolysis and shows
apparent inhibitory activity toward glycoamidases of plant, bacterial, and animal origin, with the Ki values ranging
from 1 to 160 µM for different enzymes. The C-glycopeptide 1 is the first, broad spectrum inhibitor for glycoamidases,
which is expected to be a useful tool in the study of the mechanism and biological functions of the enzymes.
Introduction
matrix interactions.2-4 Although it is well-known that removal
of the carbohydrate moieties from certain glycoproteins could
cause structural and physiological changes in their core proteins,
thus affecting the properties of the proteins, little is known about
the functional importance of physiological protein “de-glyco-
sylation”. However, recent findings have indicated that protein
de-glycosylation mediated by glycoamidase [peptide-N4-(N-
acetyl-â-D-glucosaminyl)asparagine amidase] may be an im-
portant posttranslational modification of proteins for regulating
their biological functions in living organisms.5,6
Protein glycosylation is one of the most common co- and
posttranslational modifications of proteins, in which sugar chains
are added to specific Asn or Ser/Thr residues to form the N-
and O-linked glycoproteins, respectively.1 Protein glycosylation
plays a crucial role in the expression of most cell-surface and
secreted proteins and is frequently required for the correct
folding, stability, antigenicity, and biological functions of
proteins, and as recognition markers for cell-cell and cell-
Glycoamidase (also called N-glycanase, peptide N-glycanase,
§ Present address: Institute of Biological Chemistry, Academia Sinica,
Nankang, Taipei, Taiwan.
(1) (a) Kornfeld, R.; Kornfeld, S. Annu. ReV. Biochem. 1985, 54, 631.
(b) Ivatt, R. J. The Biology of Glycoproteins; Plenum Press, New York,
1984. (c) Hart, G. W. Curr. Opin. Cell. Biol. 1992, 4, 1017. (d) Carraway,
K. L.; Hull, S. R. Glycobiology 1991, 1, 131. (e) Ruoslahti, E. Annu. ReV.
Cell Biol. 1988, 4, 229. (f) Fukuda, M. Cell Surface Carbohydrates and
Cell DeVelopment; CRC Press: Boca Raton, 1992.
† Johns Hopkins University.
‡ University of Tokyo.
* To whom all correspondence should be addressed. Tel: 410-516-
7041. Fax: 410-516-8716. E-mail: yclee@jhu.edu.
X Abstract published in AdVance ACS Abstracts, November 1, 1997.
S0002-7863(97)01202-X CCC: $14.00 © 1997 American Chemical Society

11138 J. Am. Chem. Soc., Vol. 119, No. 46, 1997
Wang et al.
subsequently isolated from different plant sources.11 Further-
more, other glycoamidases have been found recently in animal
organs and cultured cells, e.g., in early embryos of Medaka fish
(Oryzias latipes),12 in organs and tissures of mice,13 and in
cultured animal cells.14,15 More recently, it has been shown
that glycoamidase activity are present in human cytomegalovirus
infected cells16 and in human tumor cell lines,17 in which the
enzymes are assumed to be involved in the destruction of newly
synthesized major histocompatibility (MHC) class I molecules
and in the processing of the tumor antigen presented by MHC
class I molecules.16,17 The wide occurrence of glycoamidase
strongly suggests that protein N-de-glycosylation mediated by
the enzyme may be a universal feature in living organisms as
a functionally important mechanism of regulation.5,6,16,17
To study the mechanism and biological functions of glyco-
amidases, we are interested in various glycopeptide substrates
and their analogs which can serve as useful probes. Among
others, substrate analogs with a modification at the crucial
carbohydrate-peptide linkage in the natural glycopeptides is
of great interest. For this purpose, we have chosen a high-
mannose-type N-glycopeptide analog, a C-glycopentapeptide 1
as our first target molecule (Figure 2). The C-glycopeptide 1
contains all the structural features of the natural N-glycopeptide
2 except the inserted methylene group at the crucial asparagine-
GlcNAc linkage. However, in comparison with peptide syn-
thesis, glycopeptide synthesis involves many more functional
groups and is a more challenging task when a complex
oligosaccharide chain is attached.18 Several approaches are
available for N-glycopeptide synthesis, including solid phase
synthesis using glycosylated amino acid building blocks,19 the
convergent coupling of Asp-containing peptide with protected
or unprotected glycosylamine,20 and combined chemical and
enzymatic synthesis using peptidase and/or glycosyltrans-
ferases.21 However, these approaches are not quite suitable for
the synthesis of the target molecule 1 because they are either
Figure 1. Schematic depiction of glycoamidase-catalyzed hydrolysis
of N-glycopeptides.
and glycopeptidase) is an enzyme that releases the intact
oligosaccharides from N-glycopeptides or N-glycoproteins by
cleaving the â-aspartylglucosylamine amide linkage. Upon
hydrolysis, the oligosaccharide is released in the form of
glycosylamine which will be spontaneously hydrolyzed to
reducing oligosaccharide, and the asparagine residue is converted
to aspartic acid (Figure 1).7,8 Two glycoamidases which have
been well characterized and widely used for glycoprotein studies
are the plant glycoamidase A from almond emulsin7 and the
bacterial glycoamidase F from FlaVobacterium meningosepti-
cum.8 Both glycoamidases A and F can liberate N-glycans of
high-mannose-, complex-, and hybrid-type in N-glycopeptides
from vertebrates, given that both the amino and carboxyl groups
of the glycosylated asparagine (Asn) residue are present in
peptide linkage.7,8 However, a striking difference in substrate
specificity exists between the two enzymes. For example, the
bacterial enzyme is unable to release N-glycans bearing a (1,3)-
fucose residue on the Asn-linked N-acetylglucosamine (GlcNAc),
often found in glycoproteins from insects and plants.9,10 In
addition to glycoamidase A, more glycoamidases have been
(10) (a) Tretter, V.; Altmann, F.; Marz, I. Eur. J. Biochem. 1991, 199,
647. (b) Altmann, F.; Schweiszer, S.; Weber, C. Glycoconjugate J. 1995,
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1996, 96, 683. (b) Varki, A. Glycobiology 1993, 3, 97. (c) Rasmussen, J.
R. Curr. Opin. Struct. Biol. 1992, 2, 682. (d) Kobata, A. Eur. J. Biochem.
1992, 209, 483. (e) Cumming, D. A. Glycobiology 1991, 1, 115. (f) Elbein,
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(18) For a comprehensive review on glycopeptide synthesis, see: Meldal,
M. In Neoglycoconjugates: Preparation and Applications; Lee, Y. C., Lee,
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C-Glycopeptide and Glycoamidases
J. Am. Chem. Soc., Vol. 119, No. 46, 1997 11139
Figure 2. Structure of a high-mannose-type natural N-glycopeptide and the designed C-glycopeptide.
limited to the synthesis of glycopeptides bearing simple sugar
moiety or restricted to the synthesis of glycopeptides with natural
linkages.
group strategy for preparing the key C-(2-acetamido-2-deoxy-
glucosyl)methylamine as the building block, and the synthesis
of the GlcNAc-CH2-pentapeptide 14, is summarized in Scheme
1. Reduction of the â-glycosyl cyanide 328 via catalytic
hydrogenation followed by N-protection of the resulting gly-
cosylmethylamine gave 4. A large J1,2-value (9.8 Hz) indicated
that 4 was in the desired 1,2-trans-configuration. The N-
phthaloyl derivative 4 was successfully converted into the
N-acetyl derivative 5 by treatment of 4 with hydrazine hydrate
followed by acetylation. De-N-Boc-protection of 5, followed
by its condensation with Boc-Asp-OBn, provided the fully
protected N4-(2-acetamido-2-deoxy-â-D-glucopyranosylmethyl)-
asparagine 7.29 This compound can serve as a building block
for synthesizing various neo-glycopeptides containing the
GlcNAc-CH2-Asn structural unit. For the preparation of
GlcNAc-CH2-pentapeptide 14, a stepwise solution-phase syn-
thetic approach was used. De-O-benzylation of 7 followed by
coupling with dipeptide H-Ala-Ser-OMe (9) gave the tripeptide
derivative 10, which was then elongated at the N-terminus to
pentapeptide derivative 13 by coupling with the dipeptide Boc-
Tyr-Ile-OH 12. Deprotection of 13 afforded the GlcNAc-CH2-
pentapeptide 14, which was purified by reversed phase high-
performance liquid chromatography (RP-HPLC). The overall
yield from the glycosyl cyanide 3 is 23% (Scheme 1).
We describe in this paper a novel strategy for the synthesis
of the C-glycopeptide 122 and the natural high-mannose N-
glycopeptide 2, as well as their biological activity toward various
glycoamidases. The synthetic strategy consists of two key
steps: a chemical synthesis of a GlcNAc containing glycopep-
tide or neoglycopeptide and an enzymatic transfer of a Mann-
GlcNAc moiety (n ) 5-9) to the terminal GlcNAc residue in
the peptide. The essential enzyme used is endo-â-N-acetylglu-
cosaminidase from Arthrobacter protophormiae (Endo-A),23
which hydrolyzes the glycosidic bond in the N,N′-diacetylchi-
tobiose moiety of high-mannose-type N-glycans. Endo-A was
also reported to have a transglycosylation activity, being able
to transfer a Man5-9GlcNAc residue to the 4-OH of a terminal
GlcNAc residue, although the transglycosylation activity is very
low compared with its hydrolytic activity.24 Our strategy was
based on our finding that the transglycosylation yield of Endo-A
could be substantially enhanced by performing the enzymatic
reaction in aqueous organic solvents.25 The synthetic C-
glycopeptide 1 has been found to be a competitive inhibitor for
all glycoamidases of plant, bacterial, and animal origins we have
tested.
A Convergent Approach to the GlcNAc-CH2-Pentapeptide
14. Compound 14 was also synthesized by a convergent
approach, as shown in Scheme 2. First, a pentapeptide 18 with
a free â-carboxyl group in the Asp residue was prepared by a
stepwise solution peptide synthesis. Then 18 was condensed
with the glycosylmethylamine derivative 6 using 2-(1H-benzo-
triazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate
(HBTU)20,30 as the coupling reagent, giving 13 in 65% yield.
Deprotection of 13 followed by RP-HPLC purification provided
the C-glycopentapeptide 14 (Scheme 2). In comparison, the
convergent approach is superior to the stepwise approach in
both the length of the synthetic scheme and the overall yield
for the preparation of 14.
Results and Discussion
Stepwise Synthesis of a GlcNAc-CH2-Pentapeptide (14).
There has been an increasing interest in C-glycosides and related
compounds in recent years, because of their mimetic effects
and resistance to glycosidase-catalyzed hydrolysis.26 However,
the synthesis of C-glycosides of 2-acetamido-2-deoxy sugars
has proven to be a difficult task because the common Lewis
acid-catalyzed C-glycosylation and nucleophilic substitution
reaction usually lead to the formation of oxazolines when applied
to the 2-acetamido-2-deoxy sugars.27 We chose a protecting
(22) Part of the synthesis of the C-glycopeptide was communicated,
see: Wang, L. X.; Fan, J. Q.; Lee, Y. C. Tetrahedron Lett. 1996, 37, 1975.
(23) Takegawa, K.; Nakoshi, M.; Iwahara, S.; Yamamoto, K.; Tochikura,
T. Appl. EnViron. Microbiol. 1989, 55, 3107.
(24) (a) Takegawa, K.; Yamaguchi, S.; Kondo, A.; Iwamoto, H.; Nakoshi,
M.; Kato, M.; Iwahara, M. Biochem. Int. 1991, 24, 849. (b) Takegawa, K.;
Yamaguchi, S.; Kondo, A.; Kato, I.; Iwahara, S., Biochem. Int. 1991, 25,
829. (c) Takegawa, K.; Tabuchi, M.; Yamaguchi, S.; Kondo, A.; Kato, I.;
Iwahara, S. J. Biol. Chem. 1995, 270, 3094.
(25) Fan, J. Q.; Takegawa, K.; Iwahara, S.; Kondo, A.; Kato, I.;
Abeygunawardana, C.; Lee, Y. C. J. Biol.Chem. 1995, 270, 17723.
(26) For a comprehensive review on the synthesis of C-glycosides and
related compounds, see: Bertozzi C. R.; Bednarski, M. D. In Modern
Methods in Carbohydrate Synthesis; Khan, S. H., O’Neill, R. A., Eds.;
Howard Academic Publishers: 1996; pp 316-351.
(27) For recent examples on the synthesis of C-glycosides of 2-acetamido-
2-deoxy sugars, see: (a) Burkhart, F.; Hoffmann, M.; Kessler, H. Angew.
Chem., Int. Ed. Engl. 1997, 36, 1191. (b) Wei, A.; Haudrechy, A.; Audin,
C.; Jun, H. S.; Haudrechy-Bretel, N.; Kishi, Y. J. Org. Chem. 1995, 60,
Enzymatic Transfer of Man9GlcNAc to the GlcNAc-CH2-
Pentapeptide. To prepare the high-mannose-type C-glycopep-
tide 1 by an Endo-A-catalyzed glycosyl transfer reaction, a
2160. (c) Kim, K. I.; Hollingsworth, R. I. Tetrahedron Lett. 1994, 35, 1031.
(d) Hoffmann, M.; Kessler, H. Tetrahedron Lett. 1994, 35, 6067. (e) Giannis,
A.; Munster, P.; Sandhoff, K.; Steglich, W. Tetrahedron 1988, 44, 7177.
(f) Bertozzi, C. R.; Bednarski, M. D. Tetrahedron Lett. 1992, 33, 3109. (g)
Grondin, R.; Leblanc, C.; Hoogsteen, K. Tetrahedron Lett. 1991, 32, 5021.
(28) Myers, R. W.; Lee, Y. C. Carbohydr. Res. 1986, 154, 145.
(29) In this paper, we prefer to name and number the compounds as
C-glycosyl derivatives because the systematic heptitol nomenclature may
not invoke synthetically and biochemically relevant analogies. Moreover,
the heptitol nomenclature will be very tedious especially when the
substituents become large and complex.
(30) Knorr, R.; Trzeciak, A.; Bannwarth, W.; Gillessen, D. Tetrahedron
Lett. 1989, 30, 1927.

11140 J. Am. Chem. Soc., Vol. 119, No. 46, 1997
Wang et al.
Scheme 1
Scheme 2
Scheme 3
medium prevented the use of a higher concentration of the
acceptor substrate. Under an optimal condition (see Experi-
mental Section), the transglycosylation product, H-Tyr-Ile-Asn-
(Man9GlcNAc2CH2)-Ala-Ser-NH2 (1), was obtained in 26%
yield after RP-HPLC purification, and the excess acceptor could
be effectively recovered and recycled. It is worthy to note that,
in contrast to using a host of glycosyltransferases,21 the present
approach allows the installation of a complex oligosaccharide
chain into glycoconjugates in one step using only one enzyme.
Although Endo-A is specific for hydrolyzing and transfering
high-mannose-type N-glycans, the approach described here
should be readily extendable to complex- and hybrid-type
N-glycopeptides and their analogs, when a suitable endoenzyme
becomes available.32
suitable high-mannose N-glycan donor is required. We chose
Man9GlcNAc2Asn as the donor substrate because Man9GlcNAc2-
Asn can be conveniently prepared from soybean agglutinin
(isolated from soybean flour) by exhaustive protease digestion
and subsequent gel filtration separation.31 The transglycosyl-
ation was performed in acetate buffer (pH 6.0) containing 35%
acetone with 4-fold excess of the acceptor 14 (50 mM) (Scheme
3). Unfortunately, the low solubility of 14 in the reaction
(32) Recently, an endo-â-N-acetylglucosaminidase from Mucor hiemalis
(called Endo-M) was shown to be able to transfer disialo complex-type
oligosaccharide to the terminal GlcNAc residue in glycopeptides, see: (a)
Haneda, K.; Inazu, T.; Yamamoto, K.; Kumagai, H.; Nakahara, Y.; Kobata,
A. Carbohydr. Res. 1996, 292, 61. (b) Yamamoto, K.; Kadowaki, S.;
Watanabe, J.; Kumagai, H. Biochem. Biophys. Res. Commun. 1994, 203,
244.
(31) (a) Lis, H.; Sharon, N. J. Biol. Chem. 1978, 253, 3468. (b) Dorland,
L., van Halbeek, H.; Vliegenthart, J. F. G.; Lis, H.; Sharon, N.; J. Biol.
Chem., 1981, 256, 7708. (c) Fan, J. Q.; Kondo, A.; Kato, I.; Lee, Y. C.
Anal. Biochem. 1994, 219, 224.

C-Glycopeptide and Glycoamidases
J. Am. Chem. Soc., Vol. 119, No. 46, 1997 11141
Scheme 4
Figure 3. RP-HPLC patterns of the N-glycopeptides 2 and 21 and of
the corresponding C-glycopeptides 1 and 14. The RP-HPLC was
performed on a Microsorb MW ODS column (4.6 × 250 mm, Rainin,
Ridgefield, NJ) (mobile phase, 9% aqueous MeCN containing 0.05%
trifluoroacetic acid; flow rate, 1.0 mL/min).
1H-NMR of 1 showed eight H-1 R-Man signals at δ 5.400-
4.894 and one H-1 â-Man signal at δ 4.783. The H-1 GlcNAc-2
signal appeared as a distorted doublet at δ 4.618 with a relatively
large J-value (cat. 7.5 Hz), indicating a â-anomeric configuration
for the newly formed glycosidic linkage. Amino acid and sugar
composition analysis of 1 gave satisfactory results. Moreover,
the structure of 1 was confirmed by high-resolution FAB-MS.
Synthesis of a Natural High-Mannose N-Glycopeptide 2.
To demonstrate the general applicability of the synthetic
strategy, a natural high-mannose N-glycopeptide 2 was also
synthesized. First, 2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-â-
D-glucopyranosylamine (19)33 was condensed with the pen-
tapeptide 18 using HBTU as the coupling reagent to give 20,
which was then converted into the GlcNAc-N-pentapeptide 21
Via sequential deprotections (41% overall yield from 18).
Finally, an enzymatic transfer of a Man9GlcNAc to 21, as in
the preparation of the C-glycopeptide 1, provided a 25% yield
of the desired high-mannose N-glycopeptide 2 after RP-HPLC
purification (Scheme 4).
incubated with glycoamidases A, F, Sm, and HO, respectively,
under the optimal conditions for 18 h. The reaction mixture
was analyzed by RP-HPLC. In all cases, we observed no
change in the C-glycopeptide after enzymatic incubation, nor
was the formation of any detectable hydrolytic products. In
contrast, under the same conditions, the synthetic natural
N-glycopeptide 2 was rapidly hydrolyzed (data not shown).
Therefore, the C-glycopeptide is not a substrate for all the
glycoamidases so far tested, although the only structural
difference between the C- and N-glycopeptides is the presence
of a CH2 group between the anomeric carbon and the amide
nitrogen. The results suggest a strict structural requirement by
the glycoamidases at the cleaving site of the substrate. Alter-
natively, the nature of the potential leaving group of the
enzymatic hydrolysis (glycosylmethylamine in the C-glycopep-
tide versus glycosylamine in the N-glycopeptide) may also
account for the lack of hydrolytic activity of the C-glycopeptide,
considering that the pKa of glycosylmethylamine is usually 2-3
units higher than that of the corresponding glycosylamine.35
(2) Inhibition of Glycoamidase-Catalyzed Hydrolysis by the
C-Glycopeptide. The effects of the C-glycopeptide 1 on the
glycoamidase-catalyzed hydrolysis were studied. The 14C-
labeled asialofetuin glycopeptide I (asialo-fetGP I), Leu-
Asn(Man3Gal3GlcNAc5)-Asp-Ser-Arg, which was prepared from
fetal calf serum fetuin with a triantennary complex-type glycan
chain,14 was used as the substrate for glycoamidases Sm and
HO. In contrast, the synthetic N-glycopeptide 2 was used as
the substrate for glycoamidases A and F. The Km of the asialo-
fetGP I was determined to be 170 and 110 µM for glycoami-
dases Sm and HO, respectively; and the Km of 2 for glycoam-
idases A and F was determined to be 8 and 80 µM, respectively.
As can be seen in Figure 4, the C-glycopeptide 1 shows apparent
A comparison of the elution position of the C-glycopeptide
with that of the N-glycopeptide in RP-HPLC reveals an
interesting feature (Figure 3). Under the same HPLC conditions,
the C-glycopeptide 1 is actually eluted earlier (tR ) 5.62 min)
than the corresponding N-glycopeptide 2 (tR ) 6.15 min).
Similarly, the GlcNAc-CH2-pentapeptide 14 was eluted earlier
(tR ) 10.72 min) than the GlcNAc-N-pentapeptide 21 (tR
)
13.45 min). It seems that an insertion of the hydrophobic
methylene group, rather than enhancing its interaction with the
hydrophobic stationary phase of the column, has a “negative”
effect.
Biological Activity of the C-Glycopeptide toward Glyco-
amidases. The potential biological activity of the C-glycopep-
tide was tested with several typical glycoamidases originated
from plant, bacteria, and animals. Glycoamidase A from almond
emulsin and glycoamidase F from FlaVobacterium meningosep-
ticum are well-known and are commercially available.7,8,34
Moreover, we isolated two new glycoamidases: a bacterial
glycoamidase Sm from Sphingobacterium multiVorum and an
animal glycoamidase HO from hen oviduct. These enzymes
were purified to homogeneity, completely devoid of protease
and exo- and endoglycosidase activities and were used for
studying the biological activity of the C-glycopeptide.
(1) Hydrolysis of the synthetic C-glycopeptide 1 by Various
glycoamidases. To examine whether the C-glycopeptide can
be hydrolyzed by the glycoamidases, the C-glycopeptide was
(33) Marks, G. S.; Neuberger, A. J. Chem. Soc. 1961, 4872.
(34) (a) Tarentino, A. L.; Plummer, T. H., Jr. Biochem. Biophys. Res.
Commun. 1993, 197, 179. (b) Kunkel, T. A.; Bebenek, K.; McClary, J.
Methods Enzymol. 1991, 204, 125. (c) Tarentino, A. L.; Plummer, T. H.,
Jr. Methods Enzymol. 1994, 230, 44.
(35) (a) BeMiller, J. N.; Gilson, R. J.; Myers, R. W.; Santoro, M. M.;
Yadav, M. P. Carbohydr. Res. 1993, 250, 93. (b) Isbell, H. S.; Frush, H. L.
J. Res. Nat. Bur. Stand. 1951, 46, 132. (c) Isbell, H. S.; Frush, H. L. J.
Org. Chem. 1958, 23, 1309.

11142 J. Am. Chem. Soc., Vol. 119, No. 46, 1997
Wang et al.
Figure 4. Inhibition of various glycoamidases by the C-glycopeptide 1. The N-glycopeptide 2 was used as the substrate for glycoamidases A and
F; in contrast, a radiolabeled glycopeptide asialo-[14C]fetGP I was used as the substrate for glycoamidases Sm and HO. The substrate concentration
was fixed at 11 µM. The IC50 were obtained by using nonlinear regression (logistics equation) with the Graphpad Prism program (GraphPad
Software, Inc., Grand Junction, CO).
as previously described.14 The glycopeptide obtained was 14C-labeled
inhibitory activity toward all the glycoamidases tested. Anaylsis
at the amino terminal residue by reductive methylation13 at the
by the Lineweaver-Burk plot (1/V vs 1/[S]) in the presence of
Radioisotope Centre, University of Tokyo. Specific radioactivity for
1 at various concentrations reveals that the inhibition of the
asialo-[14C]fetGP I was determined to be 1.1 × 105 dpm/nmol.
C-glycopeptide 1 against the glycoamidases is competitive in
Glycoamidase Sm was purified from Sphingobacterium multiVorum
to homogeneity, completely devoid of protease and exo- and endogly-
cosidase activities (details will be reported elsewhere). The molecular
weight of the enzyme was estimated to be 86 000 by Sephacryl S-200
gel filtration.
nature (data not shown), and the enzyme-inhibitor dessociation
constant Ki of 1 for glycoamidases A, F, Sm, and HO was
determined to be 1, 43, 152, and 157 µM, respectively. This is
the first, broad-spectrum inhibitor for glycoamidases.
Glycoamidase HO was purified from hen oviduct as follows: 270
g of hen oviduct was homogenized in a Waring blender in 2 volumes
(w/v) of 50 mM HEPES (N-(2-hydroxyethyl)piperazine-N′-2-ethane-
sulfonic acid) buffer (pH 7.5) containing 2 mM DTT (dithiothreitol)
and various protease inhibitors, i.e., leupeptin, 0.5 µg/ml; aprotinin,
1.0 µg/mL; pepstatin, 0.7 µg/mL; soybean trypsin inhibitor, 0.5 µg/
mL; phenylmethanesulfonyl fluoride, 100 µM (all the purification
procedures were carried out at 4 °C or on ice). The homogenate was
centrifuged at 10 000g for 10 min, and the precipitate was washed by
suspension with an appropriate volume of the homogenization buffer.
NaCl was then added to the combined supernatant to a final concentra-
tion of 2.4 M, and the solution was applied on a TSK butyl-Toyopearl
650M column (Tosoh; 5.5 × 10 cm) equilibrated with 50 mM HEPES
buffer (pH 7.5) containing 2.4 M NaCl and 2 mM DTT. The column
was washed first with 500 mL of equilibration buffer, followed by
elution with 700 mL of 50 mM HEPES buffer (pH 7.5) containing 0.4
M ammonium sulfate and 2 mM DTT. Glycoamidase-positive fraction
was then eluted with 500 mL of 50 mM HEPES buffer (pH 7.5)
containing 2 mM DTT, and the fraction obtained was concentrated by
ultrafiltration with YM-30 membrane (Amicon) to 100 mL. Am-
monium sulfate was then added to the fraction up to 0.4 M, and the
resulting solution was applied to a butyl-Toyopearl 650 M column (1.7
× 9 cm) equilibrated with 50 mM HEPES buffer (pH 7.5) containing
0.4 M ammonium sulfate and 2 mM DTT and was washed with 100
mL of equilibration buffer. Glycoamidase-positive fraction was then
eluted with 50 mM HEPES buffer (pH 7.5) containing 2 mM DTT,
concentrated by YM-30 membrane, and applied to a Sephacryl S-300
column (2.5 × 155 cm) equilibrated and eluted with 50 mM Tris-HCl
buffer (pH 7.5) containing 0.25 M sucrose and 4 mM DTT, collecting
5.6 mL fractions. The glycoamidase-active fractions (41-45) were
pooled and concentrated by ultrafiltration with a YM-30 membrane to
Conclusion
We have described a novel strategy for the synthesis of high-
mannose-type N-glycopeptide and its C-linked glycopeptide
analog, which allows the installation of a complex oligosac-
charide moiety into glycoconjugate in one single step using one
enzyme. This strategy should be generally applicable to the
preparation of many desirable high-mannose-type glycopeptides
and their analogs and can be extended to the hybrid- and
complex-types, when a suitable endoenzyme becomes available.
The synthetic C-glycopeptide 1 with a CH2 between the â-amide
and the anomeric carbon demonstrates an apparent inhibitory
activity toward various glycoamidases of plant, bacterial, and
animal origin, which comprises the first and broad-spectrum
inhibitor for the enzymes and should have considerable potential
as a tool in the study of the mechanism and biological functions
of diverse glycoamidases.
Experimental Section
Materials and Methods. Man9GlcNAc2Asn was prepared from
soybean agglutinin by exhaustive pronase digestion, followed by gel
filtration on Sephadex G-50.31 Endo-N-acetyl-â-glucosaminidase from
Arthrobacter protophormiae (Endo-A) is a gift from Takara Shuzo Co.
(Ohtsu, Japan). Glycoamidase A (from almond) and glycoamidase F
(from FlaVobacterium) were purchased from Seikagaku America, Inc.
(Rockville, MD) and Glyko, Inc. (Novato, CA), respectively.
Asialofetuin glycopeptide I (asialo-fetGP I) having a triantennary
complex-type glycan chain, Leu-Asn(Man3Gal3GlcNAc5)-Asp-Ser-Arg,
was prepared from fetal calf serum fetuin (Nacalai Tesque, Co., Japan)

C-Glycopeptide and Glycoamidases
J. Am. Chem. Soc., Vol. 119, No. 46, 1997 11143
15 mL and the mixture was applied to a DEAE-Memsep 1010 column
(Millipore). The flow-through fraction containing glycoamidase activity
was concentrated by absorption on a Gigapite (Hydroxyapatite) column
(Seikagaku Kogyo Co., Tokyo; 1.2 × 3 cm) equilibrated with 50 mM
Tris-HCl buffer, pH 7.5, containing 2 mM DTT, and eluted with 0.1
M sodium phosphate buffer (pH 7.0) containing 50 mM HEPES buffer
(pH 7.5) and 2 mM DTT. The enzyme fraction was purified 87-fold
to 5.2 milliunit/mg protein in a 2.0% yield from the supernatant of
10 000g centrifugation. This fraction is devoid of endo-â-N-acetyl-
glucosaminidase, â-N-acetylglucosaminidase, â-galactosidase, and R-
mannosidase activities under the glycoamidase assay conditions.
HPLC was performed on a Gilson HPLC system equipped with a
UV detector (ISCO, Lincoln, NE), a Rheodyne 7125 injector, and a
Fiatron CH-30 column heater. High-performance anion exchange
chromatography (HPAEC) was performed on a Bio-LC [(Dionex Corp.,
Sunnyvale, CA) HPAEC system], equipped with a pulsed amperometric
detector (PAD-II), and was managed with AI-450 chromatography
software (Dionex).
Melting points were determined with a Fisher-Johns apparatus and
uncorrected. Elemental analysis was performed by Galbraith Labora-
tories, Knoxville, TN. 1H-NMR spectra were recorded with a Bruker
AMX-300 or a Varian Unity Plus 500 NMR spectrometer at 25 °C
unless otherwise specified. Multiplicities are abbreviated as follows:
singlet (s), doublet (d), triplet (t), quartet (q), and multiplet (m). HR-
FABMS was recorded with a VG ProSpec mass spectrometer, and the
ES-MS was obtained with a Hewlett-Packard Model 5989 B electro-
spray ionization mass spectrometer. Thin layer chromatography (TLC)
hydrazine was removed by coevaporation with toluene (3 × 10 mL).
The residue was then treated with acetic anhydride (2 mL) and pyridine
(4 mL) at 20 °C for 6 h. The solution was poured into ice water (15
mL), stirred at 20 °C for 3 h, and evaporated. The residue was dissolved
in CHCl3 (50 mL), washed with 0.1 N HCl, saturated NaHCO3, and
water; dried (Na2SO4); and filtered. The filtrate was evaporated and
the residue was chromatographed on silica gel with 35:1 CHCl3-EtOH
1
as the eluent to afford 5 (345 mg, 76%): mp 171-172 °C; H-NMR
(300 MHz, CDCl3) δ 5.740 (d, 1 H, J ) 9.3 Hz, NH), 5.124-5.044
(m, 3 H, H-3,4 and NH), 4.248 (dd, 1 H, J ) 4.5, 12.1 Hz, H-6a),
4.108 (dd, 1 H, J ) 1.9, 12.1 Hz, H-6b), 3.952 (q, 1 H, J ) 9.4 Hz,
H-2), 3.650-3.525 (m, 2 H, H-1,5), 3.462 and 3.051 (m, each 1 H,
CH2N), 2.095, 2.038, 2.031, and 1.965 (s, each 3 H, 4 Ac), 1.445 (s,
9 H, Boc). Anal. Calcd for C20H32N2O10: C, 52.16; H, 7.00; N, 6.08.
Found: C, 52.15; H, 7.17; N, 5.98.
N4-[C-(2-Acetamido-3,4,6-tri-O-acetyl-2-deoxy-â-D-glucopyrano-
syl)methyl]-N2-(tert-butoxycarbonyl)-L-asparagine Benzyl Ester [Boc-
Asn(tri-O-acetyl-â-GlcNAc-CH2)-OBn] (7). To a solution of 5 (168
mg, 0.365 mmol) in CH2Cl2 (6 mL) was added a solution of HCl in
anhydrous dioxane (4 M, 2.0 mL), and the mixture was stirred at 20
°C for 30 min when TLC (9:1 CHCl3-EtOH) showed the disappearance
of starting material. The reaction mixture was evaporated and the
residue was coevaporated with toluene (3 × 6 mL). Trituration of the
residue with ether gave 6 as its hydrochloride salt (146 mg, quantitative),
which was used in the next step without further purification. The solid
was suspended in CH2Cl2 (6 mL) and THF (2 mL), to which were
added triethylamine (55.8 mL, 0.402 mmol), HOBt (59.2 mg, 0.438
mmol), and Boc-Asp-OBn (142 mg, 0.438 mmol). The solution was
cooled to 0 °C, and DCC (107 mg, 0.438 mmol) was added. After
stirring at 0 °C for 30 min and at 20 °C for 10 h, the white suspension
was filtered and the precipitate was washed with CH2Cl2 (2 × 3 mL).
The filtrate and washings were combined and evaporated. The residue
was chromatographed on silica gel with 30:1 CHCl3-EtOH as the
was carried out on precoated plates of silica gel (E. Merck, 60F254
,
0.25 mm layer thickness). Column chromatography was performed
on silica gel (E. Merck). Ratios of solvents for TLC and column
chromatography were expressed in volume.
General Procedure for Peptide Synthesis. An O-protected amino
acid hydrochloride (1.0 equiv) and an N-protected amino acid (1.0
equiv) were dissolved in CH2Cl2 or DMF at 0 °C. To the solution
were sequentially added triethylamine (1.1 equiv), HOBt (1.0 equiv),
and DCC (1.1 equiv). The mixture was stirred at 0 °C for 30 min,
then at room temperature for 10-20 h (monitored by TLC). White
precipitate was removed by filtration, and the filtrate was evaporated
in Vacuo to dryness. The desired peptide was finally purified by column
chromatography on silical gel.
Deblocking of N-Boc Group in Peptide Synthesis. The N-Boc-
protected compound was dissolved in 4 M HCl in anhydrous dioxane
at 0 °C and the mixture was stirred at the same temperature. After
completion of the reaction (monitored by TLC), the reaction mixture
was evaporated and the residue was coevaporated with toluene in Vacuo.
Trituration of the residue with ether gave the peptide as its hydrochlo-
ride.
N-tert-Butoxycarbonyl-C-(3,4,6-tri-O-acetyl-2-deoxy-2-phthalimido-
â-D-glucopyranosyl)methylamine (4). 3,4,6-Tri-O-acetyl-2-deoxy-2-
phthalimido-â-D-glucopyranosyl cyanide (3)28 (955 mg, 2.15 mmol)
was dissolved in THF-EtOH (9:1, v/v, 20 mL), to which 10%
palladium on charcoal (110 mg) was added, and the mixture was stirred
under hydrogen atmosphere for 16 h. Then the reaction mixture was
filtered. The filtrate was evaporated to give a white foam, which was
treated with di-tert-butyl dicarbonate (1.41 g, 6.45 mmol) and triethyl-
amine (2 mL) in THF (15 mL) at 30 °C for 10 h. After concentration,
the reaction mixture was diluted with CH2Cl2 (60 mL), washed with
saturated NaHCO3, brine, and water, dried (Na2SO4), and filtered. The
filtrate was evaporated and the residue was chromatographed on silica
gel, eluting with 35:1 CHCl3-EtOH to provide 4 (1.02 g, 86%): 1H-
NMR (300 MHz, CDCl3) δ 7.787-7.666 (m, 4 H, phthaloyl), 5.719
(t, 1 H, J ) 9.8 Hz, H-3), 4.104 (t, 1 H, J ) 10.0 Hz, H-4), 4.847 (t,
1 H, J ) 5.9 Hz, NH), 4.490 (dt, 1 H, J ) 1.2 and 10.3 Hz, H-1),
4.300 (t, 1 H, J ) 9.8 Hz, H-2), 4.298 (dd, 1 H, J ) 4.6, 12.3 Hz,
H-6a), 4.085 (dd, 1 H, J ) 2.1, 12.3 Hz, H-6b), 3.799 (dd, 1 H, J )
2.1, 4.6, 9.8 Hz, H-5), 3.272 (m, 2 H, CH2N), 2.074, 1.977, and 1.780
(s, each 3 H, 3 Ac), 1.335 (s, 9 H, Boc). Anal. Calcd for C26H32N2O11
H2O: C, 55.12; H, 6.05; N, 4.94. Found: C, 54.82; H, 5.91; N, 4.68.
N-tert-Butoxycarbonyl-C-(2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-
â-D-glucopyranosyl)methylamine (5). A mixture of 4 (540 mg, 0.984
mmol) in ethanol (10 mL) containing hydrazine monohydrate (1.5 mL)
was heated at 80 °C for 2 h. The mixture was evaporated and trace
1
eluent to yield 7 (209 mg, 86%): mp 190-192 °C; H-NMR (300
MHz, DMSO-d6) δ 7.962 (d, 1 H, J ) 9.3 Hz, NH), 7.900 (t, 1 H, J
) 7.2 Hz, CH2NH), 7.344 (s, 5 H, phenyl), 7.154 (d, 1 H, J ) 7.1 Hz,
NH), 5.093 (s, 2 H, PhCH2), 5.030 (t, 1 H, J ) 9.7 Hz, H-3), 4.839 (t,
1 H, J )9.7 Hz, H-4), 4.351 (m, 1 H, R-CH Asp), 4.187 (dd, 1 H, J
)4.6, 12.1 Hz, H-6a), 3.963 (dd, 1 H, J ) 1.8, 12.2 Hz, H-6b), 3.795
(q, 1 H, J ) 9.8 Hz, H-2), 3.685 (ddd, 1 H, J ) 1.8, 4.6, 9.7, H-5),
3.529 (m, 1 H, H-1), 3.352 and 2.948 (m, each 1 H, CH2N), 2.595 and
2.465 (each dd, each 1 H, J ) 5.9, 13 Hz, â-CH2 Asp), 1.997, 1.960,
1.908, and 1.776 (s, each 3 H, 4 Ac), 1.358 (s, 9 H, Boc). Anal. Calcd
for C31H43N3O13: C, 55.93; H, 6.51; N, 6.31. Found: C, 56.04; H,
6.41; N, 5.96.
N4-[C-(2-Acetamido-3,4,6-tri-O-acetyl-2-deoxy-â-D-glucopyrano-
syl)methyl]-N2-(tert-butoxycarbonyl)-L-asparagine [Boc-Asn(tri-O-
acetyl-â-GlcNAc-CH2)-OH] (8). Compound 7 (195 mg, 0.293 mmol)
was hydrogenated in THF-EtOH (1:2, v/v, 9 mL) in the presence of
10% palladium on charcoal (20 mg) under hydrogen atmosphere. After
10 h, the mixture was filtered through a Celite pad and the filtrate was
evaporated in Vacuo to provide 8 (169 mg, quantitative): mp 192-
1
194 °C; H-NMR (300 MHz, DMSO-d6) δ 12.445 (s, 1 H, CO2H),
7.981 (d, 1 H, J ) 9.4 Hz, NH), 7.858 (t, 1 H, J ) 6.5 Hz, CH2NH),
6.898 (d, 1 H, J ) 8.1 Hz, NH), 5.030 (t, 1 H, J ) 9.5 Hz, H-3), 4.840
(t, 1 H, J ) 9.5 Hz, H-4), 4.312-4.206 (m, 2 H, H-6a and R-CH Asp),
3.973 (dd, 1 H, J ) 1.8, 12.1 Hz, H-6b), 3.782 (q, 1 H, J ) 9.5 Hz,
H-2), 3.703 (m, 1 H, H-5), 3.450 (m, 1 H, H-1), 3.418 and 2.904 (m,
each 1 H, CH2N), 2.563-3.390 (m, 2 H, â-CH2 Asp), 2.005, 1.954,
1.904, and 1.778 (s, each 3 H, 4 Ac), 1.362 (s, 9 H, Boc). Anal. Calcd
for C24H37N3O13: C, 50.08; H, 6.48; N, 7.30. Found: C, 49.83; H,
6.79; N, 6.90.
L-Alanyl-L-serine Methyl Ester (H-Ala-Ser-OMe) (9). H-Ser-
OMe hydrochloride (1.56 g, 10 mmol) and Boc-Ala-OH (1.89 g, 10
mmol) were coupled in CH2Cl2 (30 mL) according to the standard
procedure. The product was purified by chromatography on silica gel
using 25:1 CHCl3-EtOH as the eluent, giving tert-butoxycarbonyl-L-
alanyl-L-serine methyl ester (Boc-Ala-Ser-OMe) (2.76 g, 95%) as a
colorless syrup: 1H-NMR (300 MHz, DMSO-d6) δ 8.006 and 6.943
(each d, each 1 H, J ) 7.8 Hz, 2 NH), 5.061 (t, 1 H, J ) 5.6 Hz, OH
Ser), 4.325 and 4.056 (m, each 1 H, R-CH Ser and Ala), 3.760-3.558
(m, 2 H, â-CH2 Ser), 3.617 (s, 3 H, OMe), 1.370 (s, 9 H, Boc), 1.168

11144 J. Am. Chem. Soc., Vol. 119, No. 46, 1997
Wang et al.
(d, 3 H, J ) 7.1 Hz, Me Ala). Anal. Calcd for C12H22N2O6: C, 49.64;
H, 7.64; N, 9.65. Found: C, 49.50; H, 7.71; N, 9.58. Cleavage of the
N-Boc protecting group in Boc-Ala-Ser-OMe using the standard
procedure afforded 9 in quantitative yield as its hydrochloride, which
was used for peptide synthesis without purification.
H-4), 4.460-4.025 (m, 6 H, H-6a and 5 R-CH), 3.985 (dd, 1 H, J )
1.8, 11.2 Hz, H-6b), 3.820 (q, 1 H, J ) 8.5 Hz, H-2), 3.790-3.598
(m, 3 H, H-5 and â-CH2 Ser), 3.610 (s, 3 H, OMe), 3.510 (m, 1 H,
H-1), 3.348 and 2.982 (m, each 1 H, CH2N), 2.910-2.520 (m, 4 H,
â-CH2 Asn and Tyr), 2.005, 1.954, 1.902, and 1.766 (s, each 3 H, 4
Ac), 1.715, 1.390, and 1.260 (m, 2 H, â-CH and g-CH2 Ile), 1.297 (s,
9 H, Boc), 1.181 (d, 1 H, J ) 7.0 Hz, Me Ala), 0.803 (m, 6 H, 2 Me
Ile). Anal. Calcd for C46H69N7O19 H2O: C, 53.01; H, 6.87; N, 9.41.
Found: C, 52.83; H, 7.06; N, 9.20.
N4-[C-(2-Acetamido-3,4,6-tri-O-acetyl-2-deoxy-â-D-glucopyrano-
syl)methyl]-N2-(tert-butoxycarbonyl)-L-asparaginyl-L-alanyl-L-
serine Methyl Ester [Boc-Asn(tri-O-acetyl-â-GlcNAc-CH2)-Ala-Ser-
OMe] (10). Compound 8 (158 mg, 0.274 mmol) and the hydrochloride
salt of H-Ala-Ser-OMe 9 (98 mg, 0.431 mmol) were coupled in DMF
(5 mL) according to the standard procedure. The product was purified
by chromatography on silica gel using 30:1 to 15:1 CHCl3-EtOH as
the eluent, giving 10 (172 mg, 84%): mp 177-179 °C; 1H-NMR (300
MHz, DMSO-d6) δ 8.256 (d, 1 H, J ) 7.6 Hz, NH), 7.960 (d, 1 H, J
) 9.5 Hz, NH), 7.851 (m, 2 H, 2 NH), 6.962 (d, 1 H, J ) 7.5 Hz,
NH), 5.056 (m, 2 H, H-3 and OH Ser), 4.850 (t, 1 H, J ) 9.6 Hz,
H-4), 4.401-4.285 (m, 2 H, R-CH Ser and Asn), 4.250-4.105 (m, 2
H, H-6a and R-CH Ala), 3.983 (dd, 1 H, J ) 1.9, 10.8 Hz, H-6b),
3.816 (q, 1 H, J ) 8.8 Hz, H-2), 3.792-3.650 (m, 3 H, H-5 and â-CH2
Ser), 3.618 (s, 3 H, OMe), 3.507 (m, 1 H, H-1), 3.350 and 2.985 (m,
each 1 H, CH2N), 2.585-2.360 (m, 2 H, â-CH2 Asn), 2.013, 1.962,
1.911, and 1.779 (s, each 3 H, 4 Ac), 1.366 (s, 9 H, Boc), 1.202 (d, 3
H, J ) 7.0 Hz, Me Ala). Anal. Calcd for C31H49N5O16 H2O: C, 48.62;
H, 6.71; N, 9.14. Found: C, 48.45; H, 6.50; N, 9.22.
N-tert-Butoxycarbonyl-L-tyrosyl-L-isoleucine (Boc-Tyr-Ile-OH)
(12). Boc-Tyr-OH (563 mg, 2 mmol) and H-Ile-OBn p-toluenesulfonate
(787 mg, 2 mmol) were coupled by the standard procedure to give,
after chromatographic purification, N-tert-butoxycarbonyl-L-tyrosyl-L-
isoleucine benzyl ester (Boc-Tyr-Ile-OBn) (737 mg, 76%): 1H-NMR
(300 MHz, CDCl3) δ 7.998 (s 1 H, OH Tyr), 7.327 (s, 5 H, Ph), 6.997
and 6.735 (d, each 2 H, 4 aromatic H Tyr), 6.468 (d, 1 H, J ) 8.5 Hz,
NH), 5.114 (s, 2 H, PhCH2), 5.080 (d, 1 H, J ) 8.1 Hz, NH), 4.522
(dd, 1 H, J )5.1, 8.4 Hz, R-CH Ile), 4.265 (m, 1 H, R-CH Tyr), 2.945-
2.870 (m, 2 H, â-CH2 Tyr), 1.820--1.050 (m, 4 H, â-CH2 and γ-CH2
Ile), 1.398 (s, 9 H, Boc), 0.844-0.777 (m, 6 H, 2 Me Ile). Anal. Calcd
for C27H36N2O6: C, 66.92; H, 7.49; N, 5.78. Found: C, 66.78; H,
7.82; N, 5.65.
Hydrogenation of Boc-Tyr-Ile-OBn (680 mg, 1.40 mmol) in THF-
EtOH (1:1, 15 mL) was performed in the presence of 10% Pd/C (100
mg) under hydrogen atmosphere at 20 °C. After 12 h, the catalyst
was removed by filtration and the filtrate was evaporated. The product
was purified by chromatography on silica gel using 7:1 CHCl3-MeOH
as the eluent to provide 12 (492 mg, 89%) as a colorless syrup. 1H-
NMR (300 MHz, CDCl3): δ 8.013 (s, 1 H, OH Tyr), 6.978 and 6.708
(d, each 2 H, 4 aromatic H Tyr), 6.519 (d, 1 H, J ) 8.1 Hz, NH),
5.378 (d, 1 H, J ) 7.9 Hz, NH), 4.456 and 4.310 (m, each 1 H, R-CH
Tyr and Ile), 2.980-2.820 (m, 2 H, â-CH2 Tyr), 1.825-1.170 (m, 3
H, â-CH and g-CH2 Ile), 1.427 (s, 9 H, Boc), 0.899-0.862 (m, 6 H, 2
Me Ile). Anal. Calcd for C20H30N2O6: C, 60.89; H, 7.67; N, 7.10.
Found: C, 60.80; H, 7.92; N, 6.90.
N4-[C-(2-Acetamido-2-deoxy-â-D-glucopyranosyl)methyl]-N2-(L-
tyrosyl-L-isoleucyl)-L-asparaginyl-L-alanyl-L-serine amide [H-Tyr-
Ile-Asn(GlcNAc-CH2)-Ala-Ser-NH2] (14). A solution of 13 (42 mg,
41 µmol) in methanol (30 mL) was saturated with ammonia at -5 °C
for 30 min. The solution was kept at 20 °C for 3 days, then evaporated
to dryness. Trituration of the residue with 2-propanol-water gave a
white solid (35 mg), which was then treated with aqueous 3 M HCl at
20 °C for 1 h. The clear solution was evaporated to dryness and the
product was purified by RP-HPLC on a Microsorb MW ODS column
(4.6 × 250 mm, Rainin, Ridgefield, NJ) with a UV detector (double
detection at 215 and 280 nm) (mobile phase, 9% aqueous MeCN
containing 0.05% trifluoroacetic acid; flow rate, 1.0 mL/min). The
product (tR ) 10.72 min) was collected and lyophilized to provide the
GlcNAc-CH2-pentapeptide 14 (25.7 mg, 80%): mp 226--229 °C; 1H-
NMR (300 MHz, D2O) δ 7.046 and 6.807 (each d, each 2 H, J ) 8.3
Hz, 4 aromatic H Tyr), 4.590 (t, 1 H, J ) 6.8 Hz, R-CH Tyr), 4.350
(t, 1 H, J ) 4.9 Hz, R-CH Ser), 4.265 (q, 1 H, J ) 7.3 Hz, R-CH Ala),
4.115-4.020 (m, 2 H, H-6a and R-CH Asn), 3.905-3.834 (m, 3 H,
R-CH Ile and â-CH2 Ser), 3.720-3.620 (m, 2 H, H-2,6b), 3.503-3.335
1
(m, 5 H, H-1,3,4,5 and /2 CH2N), 3.192 (dd, 1 H, J ) 7.5, 12.5 Hz,
1/2 CH2N), 2.994 (m, 2 H, â-CH2 Asn), 2.845 and 2.682 (dd, each 1
H, J ) 6.8, 13.0 Hz, â-CH2 Tyr), 1.959 (s, 3 H, N-Ac), 1.710 (m, 1 H,
â-CH Ile), 1.412 and 1.065 (m, each 1 H, g-CH2 Ile), 1.358 (d, 1 H, J
) 7.2 Hz, Me Ala), 0.805-0.764 (m, 6 H, 2 Me Ile); HR-FABMS
calcd for C34H54N8O13 + H+ 783.3928, found: 783.3902.
N-tert-Butoxycarbonyl-â-benzyl-L-aspartyl-L-alanyl-L-serine Meth-
yl Ester [Boc-Asp(OBn)-Ala-Ser-OMe] (15). The hydrochloride of
H-Ala-Ser-OMe (9) (795 mg, 3.5 mmol) was dissolved in DMF (10
mL). Triethylamine (0.73 mL, 5.25 mmol) and p-nitrophenyl N-tert-
butoxycarbonyl-â-benzyl-L-aspartate [Boc-Asp(OBn)-OPNP] (3.11 g,
7.0 mmol) were added, and the mixture was stirred at 20 °C for 16 h.
After evaporation of the solvent, the residue was dissolved in CHCl3
(60 mL), washed with saturated NaHCO3 and water, dried (Na2SO4),
and evaporated. The product was purified by chromatography on silica
gel using 30:1 CHCl3-EtOH as the eluent to give 15 (1.58 g, 91%):
1H-NMR (300 MHz, DMSO-d6) δ 8.260, 7.862, and 7.232 (each d,
each 1 H, J ) 7.6 Hz, 3 NH), 7.356 (s, 5 H, Ph), 5.096-5.038 (m, 3
H, PhCH2 and OH Ser), 4.362-4.302 (m, 3 H, 3 R-CH), 3.741-3.564
(m, 2 H, â-CH2 Ser), 3.634 (s, 3 H, OMe), 2.778 (dd, 1 H, J ) 5.1, 16
1
1
Hz, /2 â-CH2 Asp), 2.574 (dd, 1 H, J ) 9.1, 16 Hz, /2 â-CH2 Asp),
1.369 (s, 9 H, Boc), 1.195 (d, 2 H, J ) 7.0 Hz, Me Ala).
N2-(N-tert-butoxycarbonyl-L-tyrosyl-L-isoleucyl)-â-benzyl-L-as-
partyl-L-alanyl-L-serine Methyl Ester [Boc-Tyr-Ile-Asp(OBn)-Ala-
Ser-OMe] (17). De-N-Boc-protection of Boc-Asp(OBn)-Ala-Ser-OMe
(15) (495 mg, 1.0 mmol) with 4 M HCl in anhydrous dioxane (4 mL)
was performed by the standard procedure to obtain H-Asp(OBn)-Ala-
Ser-OMe (16) (432 mg, quantitative) as its hydrochloride salt. Then
16 and Boc-Tyr-Ile-OH (12) (395 mg, 1.0 mmol) were coupled in DMF
(8 mL) by the standard procedure, and the product was purified by
chromatography on silica gel using 9:1 CHCl3-EtOH as the eluent to
N4-[C-(2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-â-D-glucopyrano-
syl)methyl]-N2-(N-tert-butoxycarbonyl-L-tyrosyl-L-isoleucyl)-L-as-
paraginyl-L-alanyl-L-serine Methyl Ester [Boc-Tyr-Ile-Asn(tri-O-
acetyl-â-GlcNAc-CH2)-Ala-Ser-OMe] (13). A solution of 10 (150
mg, 0.201 mmol) in CH2Cl2 (6 mL) was treated with 4M HCl in
anhydrous dioxane (2 mL) at 20 °C for 2 h, the reaction mixture was
evaporated in Vacuo, and coevaporated with toluene (3 × 6 mL).
Trituration of the residue with diethyl ether gave the hydrochloride
salt of 11 (139 mg, quantitative) as a white solid, which was used
immediately without characterization. The white solid was suspended
in DMF (5 mL), followed by the sequential addition of triethylamine
(33.4 mL, 0.24 mmol), Boc-Tyr-Ile-OH (12) (103 mg, 0.26 mmol),
HOBt (35 mg, 0.26 mmol), and DCC (62 mg, 0.30 mmol). The mixture
was stirred at 0 °C for 30 min and at 20 °C for 16 h. After water was
added (0.3 mL), the resulting suspension was filtered and the filtrate
was evaporated in Vacuo. The residue was chromatographed on silica
gel using 20:1 to 9:1 CHCl3-EtOH as the eluent to give 13 (126 mg,
61%): 1H-NMR (300 MHz, DMSO-d6) δ 9.141 (s, 1 H, OH Tyr), 8.214
(d, 2 H, J )7.6 Hz, 2 NH), 7.943 (m, 2 H, 2 NH), 7.839 (d, 1 H, J )
7.6 Hz, NH), 7.712 (d, 1 H, J )8.1 Hz, NH), 7.023 and 6.628 (d, each
2 H, J ) 8.1 Hz, 4 aromatic H Tyr), 6.939 (d, 1 H, J ) 7.8 Hz, NH),
5.060-4.979 (m, 2 H, H-3 and OH Ser), 4.885 (t, 1 H, J ) 9.6 Hz,
1
give the pentapeptide 17 (638 mg, 81%): mp 196-198 °C; H-NMR
(300 MHz, DMSO-d6) δ 9.149 (s, 1H, OH Tyr), 8.414, 8.220, 7.808,
7.730, and 6.930 (each d, each 1 H, J ) 7.5-7.8 Hz, 5 NH), 7.345 (s,
5 H, Ph), 7.014 and 6.630 (each d, each 2 H, J ) 8.3 Hz, 4 aromatic
H Tyr), 5.067 (m, 3 H, PhCH2 and OH Ser), 4.715-3.400 (m, 5 H, 5
R-CH), 3.692 (m, 2 H, R-CH2 Ser), 3.618 (s, 3 H, OMe), 2.910-2.520
(m, 4 H, R-CH2 Tyr and Asp), 1.705 (m, 1 H, â-CH Ile), 1.460 and
1.085 (m, each 1 H, r-CH2 Ile), 1.297 (s, 9 H, Boc), 1.181 (d, 3 H, J
) 7.0 Hz, Me Ala), 0.828-0.795 (m, 6 H, 2 Me Ile).
N2-(N-tert-butoxycarbonyl-L-tyrosyl-L-isoleucyl)-L-aspartyl-L-ala-
nyl-L-serine Methyl Ester [Boc-Tyr-Ile-Asp(OH)-Ala-Ser-OMe]
(18). Compound 17 (394 mg, 0.5 mmol) was dissolved in THF-
EtOH-MeOH (1:1:1, 18 mL) and subjected to hydrogenation in the

C-Glycopeptide and Glycoamidases
J. Am. Chem. Soc., Vol. 119, No. 46, 1997 11145
presence of 10% Pd/C (80 mg) under hydrogen atmosphere. After 16
h, the catalyst was removed by filtration through a Celite pad, and the
filtrate was evaporated and chromatographed on silica gel using 5:1 to
2:1 CHCl3-MeOH as the eluent to give 18 (220 mg, 63%): mp 170
84.7 µmol), diisopropylethylamine (12 mL, 85 µmol), HOBt (13.5 mg,
100 µmol), and HBTU (160 mg, 84.7 µmol) were mixed in DMF (2.5
mL). After 48 h, DMF was evaporated in Vacuo and the residue was
chromatographed on a column (2 × 45 cm) of Sephadex LH-20 in 1:1
CHCl3-95% EtOH to give a crude product, which was crystallized
from EtOH to afford 20 (43.5 mg, 50%): 1H-NMR (300 MHz, DMSO-
d6) δ 9.165 (s, 1 H, OH Tyr), 8.697, 8.370, 8.212, 7.925, 7.750, 7.665,
and 6.920 (each d, each 1 H, J ) 7.0-8.0 Hz, 7 NH), 7.024 and 6.636
(each d, each 2 H, J ) 8.4 Hz, 4 aromatic H Tyr), 5.170-4.950 (m, 3
H, H-1,3, and OH Ser), 4.830 (t, 1 H, J ) 9.5 Hz, H-4), 4.550-3.665
(m, 11 H, H-2,5,6a,6b,5 R-CH and â-CH2 Ser), 3.619 (s, 3 H, OMe),
2.890-2.285 (m, 4 H, â-CH2 Asn and Tyr), 1.993, 1.963, 1.909, and
1.745 (s, each 3 H, 4 Ac), 1.690, 1.405, and 1.060 (m, 3 H, â-CH and
r-CH2 Ile), 1.307 (s, 9 H, Boc), 1.178 (d, 3 H, J ) 7.0 Hz, Me Ala),
0.799 (m, 6 H, 2 Me Ile).
1
°C (dec); H-NMR (300 MHz, DMSO-d6) δ 9.210 (s, 1H, OH Tyr),
8.670, 8.192, 8.038, 7.703, and 6.950 (each d, each 1 H, J ) 5.8-8.0
Hz, 5 NH), 7.345 (s, 5 H, Ph), 7.016 and 6.632 (each d, each 2 H, J )
8.1 Hz, 4 aromatic H Tyr), 4.525-4.080 (m, 6 H, 5 R-CH and OH
Ser), 3.710 (m, 2 H, â-CH2 Ser), 3.612 (s, 3 H, OMe), 2.880-2.215
(m, 4 H, â-CH2 Tyr and Asp), 1.710 (m, 1 H, â-CH Ile), 1.415 and
1.065 (m, each 1 H, r-CH2 Ile), 1.308 (s, 9 H, Boc), 1.190 (d, 3 H, J
) 7.1 Hz, Me Ala), 0.850-0.710 (m, 6 H, 2 Me Ile).
Condensation of Pentapeptide (18) with Glycosylamine (6). A
Convergent Approach to the Synthesis of 14. Compound 18 (50
mg, 71.6 µmol) and the hydrochloride salt of 6 (43 mg, 107 µmol)
were dissolved in DMF (2 mL), then N,N′-diisopropylethylamine (38
µL, 215 µmol) , HOBt (14.5 mg, 107.5 µmol), and HBTU (82 mg,
215 µmol) were added sequentially. The mixture was stirred at 0 °C
for 2 h and at 20 °C for 40 h. DMF was evaporated and the residue
was applied to a column (2 × 45 cm) of Sephadex LH-20. The column
was preequilibrated and eluted with 1:1 CHCl3-95% EtOH to give a
crude product (65 mg), which was further purified by column
chromatography on silica gel eluted with 9:1 CHCl3-EtOH to afford
13 (48 mg, 65%), which was identical to the product obtained by the
stepwise synthesis. Removal of the protecting groups in 13 by
sequential treatment with ammonia-saturated methanol and 3 M HCl
as described gave 14 after RP-HPLC purification.
N4-(2-Acetamido-2-deoxy-â-D-glucopyranosyl)-N2-(L-tyrosyl-L-
isoleucyl)-L-asparaginyl-L-alanyl-L-serine Amide [H-Tyr-Ile-Asn-
(GlcNAc)-Ala-Ser-NH2] (21). Compound 20 (35 mg, 34 µmol) was
treated with ammonia-saturated MeOH (30 mL) for 2 days, then the
solution was evaporated to dryness. Trituration of the residue with
EtOH gave a white solid. The solid was treated with aqueous 3 M
HCl at 20 °C for 1 h and evaporated to dryness. The product was
purified by RP-HPLC on a Microsorb MW ODS column (4.6 × 250
mm, Rainin, Ridgefield, NJ) with a UV detector (double detection at
215 and 280 nm) (mobile phase: 9% aqueous MeCN containing 0.05%
trifluoroacetic acid; flow rate: 1.0 mL/min). The product, eluted at
13.45 min, was collected and lyophilized to obtain the GlcNAc-
pentapeptide (21) (22 mg, 81%): mp 223-225 °C; 1H-NMR (300 MHz,
D2O) δ 7.077 and 6.809 (each d, each 2 H, J ) 8.3 Hz, 4 aromatic H
Tyr), 5.019 (d, 1 H, J ) 9.7 Hz, H-1), 4.643 (t, 1 H, J ) 6.8 Hz, R-CH
Tyr), 4.359 (t, 1 H, J ) 5.3 Hz, R-CH Ser), 4.283 (q, 1 H, J ) 7.3 Hz,
R-CH Ala), 4.203 (t, 1 H, J ) 7.1 Hz, R-CH Asn), 4.074 (d, 1 H, J )
8.3 Hz, R-CH Ile), 3.859-3.421 (m, 8 H, H-2,3,4,5,6a,6b, and â-CH2
Ser), 3.150-2.980 (m, 2 H, â-CH2 Asn), 2.895 (dd, 1 H, J ) 7.7, 16
Transfer of Man9GlcNAc to the GlcNAc-CH2-Pentapeptide Using
Endo-A. Synthesis of the C-Glycopeptide 1. A mixture consisting
of Man9GlcNAc2Asn (2.4 µmol), the GlcNAc-CH2-pentapeptide 14 (12
µmol), and enzyme (100 mU) in 25 mM NH4OAc buffer (240 mL, pH
6.0) containing 35% acetone was incubated at 37 °C for 20 min. The
reaction was stopped by boiling in a 100 °C water bath (3 min). The
product was purified by RP-HPLC on a Microsorb MW ODS column
(4.6 × 250 mm, Rainin, Ridgefield, NJ) with a UV detector (double
detection at 215 and 280 nm) (mobile phase, 9% aqueous MeCN
containing 0.05% trifluoroacetic acid; flow rate, 1.0 mL/min). The
transglycosylation product eluted at 5.62 min was collected and
lyophilized to obtain 1 (1.56 mg, 0.634 µmol, 26%). The excess
GlcNAc-CH2-pentapeptide (acceptor) was eluted after 10 min under
the conditions and was efficiently recovered: 1H-NMR (500 MHz, 60
°C in D2O, set the DHO signal at δ 4.441) of 1 δ 7.112 and 6.855
(each d, 4 H, J ) 8.3Hz, 4 aromatic H Tyr), 5.400, 5.344, 5.312, 5.143,
5.094, 5.083, 5.078, and 4.894 (each br. s, each 1 H, 8 H-1 R-Man),
4.783 (s, 1 H, H-1 â-Man), 4.687 (t, 1 H, J ) 6.8 Hz, R-CH Tyr),
4.618 (distorted d, J ) 7.5 Hz, H-1 GlcNAc-2), 4.373-3.448 (m,
multiple protons), 3.174 (dd, 1 H, J ) 7.5, 14 Hz, 1/2 CH2N), 2.889
(d, 2 H, J ) 6.4 Hz, â-CH2 Asn), 2.865 and 2.714 (each dd, 2 H, J )
6.9, 14.5 Hz, â-CH2 Tyr), 2.086 and 2.044 (s, each 3 H, 2 NAc), 1.765
(m, 1 H, â-CH Ile), 1.426 (d, 3 H, J ) 7.3 Hz, Me Ala), 1.314 and
1.100 (m, 2 H, g-CH2 Ile), 0.868-0.838 (m, 6 H, 2 Me Ile);
HR-FABMS calculated for C96H157N9O63 + H+ 2444.9436; Found
2444.9462.
1
1
Hz, /2 â-CH2 Tyr), 2.719 (dd, 1 H, J ) 5.8, 16 Hz, /2 â-CH2 Tyr),
1.947 (s, 3 H, Ac), 1.706 (m, 1 H, â-CH Ile), 1.378 and 1.086 (m,
each 1 H, r-CH2 Ile); 1.367 (d, 3 H, J ) 7.2 Hz, Me Ala), 0.827-
0.789 (m, 6 H, 2 Me Ile). HR-FABMS calcd for C33H52N8O13 + H+
769.3732, Found 769.3734.
Transfer of Man9GlcNAc to the GlcNAc-Pentapeptide by Endo-
A. Synthesis of the Natural High-Mannose N-Glycopeptide (2). A
mixture consisting of Man9GlcNAc2Asn (2 µmol), the GlcNAc-
pentapeptide 21 (10 µmol), and enzyme (70 mU) in 25 mM NH4OAc
buffer (100 µL, pH 6.0) containing 35% acetone was incubated at 37
°C for 20 min. The reaction was stoped by boiling at 100 °C for 3
min, and the transglycosylation product was purified by RP-HPLC on
a Microsorb MW ODS column (4.6 × 250 mm, Rainin, Ridgefield,
NJ) detected at 215 and 280 nm (mobile phase, 9% aqueous MeCN
containing 0.05% TFA; flow rate, 1 mL/min). The transglycosylation
product, eluted at 6.15 min, was collected and lyophilized to give 2
(1.25 mg, 0.51 µmol, 25%). The excess GlcNAc-pentapeptide acceptor
21, which was eluted much later, was efficiently recovered: 1H-NMR
(500 MHz, 60 °C in D2O, set the DHO signal at δ 4.441) of 2 δ 7.136
and 6.874 (each d, each 2 H, J ) 8.4Hz, 4 aromatic H Tyr), 5.402,
5.349, 5.315, 5.146, 5.099, 5.084, 5.078, and 4.901 (each br. s, each 1
H, 8 H-1 R-Man), 5.048 (d, 1 H, J ) 9.5 Hz, H-1 GlcNAc-1), 4.790
(s, 1 H, H-1 â-Man), 4.711 (t, 1 H, J ) 6.6 Hz, R-CH Tyr), 4.639
(distorted d, 1 H, J ) 6.6 Hz, H-1 GlcNAc-2), 4.484-3.539 (m,
multiple protons), 2.959 (d, 2 H, J ) 6.6 Hz, â-CH2 Asn), 2.927 and
2.750 (each dd, each 1 H, J ) 6.6, 16 Hz, â-CH2 Tyr), 2.092 and
2.028 (s, each 3 H, 2 NAc), 1.790 (m, 1 H, â-CH Ile), 1.423 (d, 3 H,
J ) 7.0 Hz, Me Ala), 1.395 and 1.150 (m, 2 H, g-CH2 Ile), 0.868-
0.835 (m, 6 H, 2 Me Ile); ESI-MS calcd for C95H155N9O63 (M) 2431
(average mass), and 2429.9 (exact mass); Found 1217 [(M + 2 H)2+],
1228 [(M + H + Na)2+], and 1239 [(M + 2 Na)2+].
Compositional Analysis of 1. The monosaccharide analysis was
carried out by the established HPAEC-PAD method.36 In brief, a
sample was hydrolyzed with 4N HCl at 100 °C for 6 h (for GlcN) or
with 2M trifluoroacetic acid at 100 °C for 4 h (for Man), then
evaporated by speed-vac and diluted to a suitable concentration for
HPAEC-PAD analysis. The ratio between Man and GlcNAc was found
to be 9:1. The released GlcNAc-CH2NH2 was detected without
quantitative analysis. For amino acid analysis, the sample was
hydrolyzed using 4 N HCl at 110 °C for 20 h. TLC (5:5:1:3 EtOAc-
pyridine-AcOH-H2O) of the hydrolysate clearly showed the presence
of Tyr, Ile, Asp, Ala, Ser, and GlcN-CH2NH2.
N4-(2-Acetamido-3,4,6-tri-O-acetyl-2-deoxy-â-D-glucopyranosyl)-
N2-(N-tert-butoxycarbonyl-L-tyrosyl-L-isoleucyl)-L-asparaginyl-L-
alanyl-L-serine methyl ester [Boc-Tyr-Ile-Asn(tri-O-acetyl-â-GlcNAc)-
Ala-Ser-OMe] (20). 2-Acetamido-3,4,6-tri-O-acetyl-2-deoxy-â-D-
glucopyranosylamine (19)33 (88 mg, 254 µmol), compound 18 (59 mg,
Assay of the Activity of Glycoamidases. The activity of glyco-
amidases HO and Sm was assayed using asialo-[14C]fetGP I as the
substrate as previously described.14 Briefly, the reaction mixture
containing the glycoamidase in a total volume of 5 µL of 50 mM
HEPES buffer (pH 7.5) and 2 mM DTT together with 11 µM of asialo-
[14C]fetGP I (6,100 dpm) was incubated in a polypropylene microtube
at 37 °C for 3 h. For bacterial glycoamidase Sm, the substrate was
(36) Hardy, M.; Townsend, R. R.; Lee, Y. C. Anal. Biochem. 1988, 170,
54.

11146 J. Am. Chem. Soc., Vol. 119, No. 46, 1997
Wang et al.
incubated at 25 °C for 20 min in 5 µL of 20 mM Tris-HCl buffer (pH
7.1). Reaction products were separated by paper chromatography
followed by measurement of the radioactivities by the Bio-Imaging
analyzer (Fujix BAS 2000).
A and F, the enzymatic reactions were performed at various substrate
concentrations (0.1-1000 µM) when the inhibitor was present at various
concentrations (10, 50, and 100 µM). The Lineweaver-Burk double-
reciprocal plots (1/V vs 1/[S]) were created, and the slops of each of
these lines were then plotted against [I], which was fitted to a straight
line. The intercept at the [I] axes is equal to -Ki. By this method,
the Ki of the C-glycopeptide 1 for glycoamidases A and F was
determined to be 1 and 43 µM, respectively. The Lineweaver-Burk
plots at different [I] also revealed that the inhibition by the C-
glycopeptide 1 is competitive in nature. For glycoamidases Sm andHO,
the Ki values were calculated by the equation of Cheng and Prusoff,37
Ki ) IC50 / (1 + [S]/Km), taking the above determined data of IC50 ([S]
) 11 µM) and Km.
Hydrolysis of the C-Glycopeptide by Glycoamidases. To examine
whether C-glycopeptide 1 would be hydrolyzed by the glycoamidases,
10 nmol of the C-glycopeptide was incubated with the respective
glycoamidase under the conditions described for assaying enzymatic
activity for 18 h. The reaction mixture was then evaporated by a
SpeedVac concentrator (Savant Instruments, Inc., Farmingdale, NY).
RP-HPLC analysis showed neither the change of the C-glycopeptide
nor the presence of any hydrolytic products.
The activity of glycoamidases A and F was assayed using the
synthetic N-glycopeptide 2 [H-Tyr-Ile-Asn(Man9GlcNAc2)-Ala-Ser-
NH2] as substrate. Briefly, the reaction mixture containing the
glycoamidase and the substrate was incubated in a total volume of 50
µL of 40 mM ammonium acetate buffer (pH 5 for glycoamidase A
and pH 8 for glycoamidase F) for a predetermined time. The enzymatic
reactions were stopped by boiling in a water bath for 3 min and the
hydrolytic product, the pentapeptide H-Tyr-Ile-Asp-Ala-Ser-NH2, was
analyzed by RP-HPLC, which was quantitated by intergrating the peak
observed at 210 and/or 280 nm.
Inhibition of Glycoamidases by the C-Glycopeptide (1). For
practical reasons, the synthetic N-glycopeptide 2 was used as the
substrate for glycoamidases A and F, but a radiolabeled glycopeptide
asialo-[14C]fetGP I was used as the substrate for glycoamidases Sm
and HO in the inhibitory experiments. In all the cases, enzymes were
used at the amount that would give less than 10% hydrolysis of the
respective substrate. The initial rates of the enzymatic reactions were
determined by quantitating the peptide released. The Km values of the
two substrates for the respective enzymes were obtained by Lin-
eweaver-Burk double-reciprocal plots (1/V vs. 1/[S]). The inhibitory
activity of 1 against glycoamidases was examined by performing the
enzymatic reaction at a fixed substrate concentration ([S] ) 11 µM)
for a pre-determined time when the inhibitor was present at various
concentrations (0.1-1000 µM). The IC50 values were obtained by using
nonlinear regression (logistics equation) with the Graphpad Prism
program (GraphPad Software, Inc., Grand Junction, CO) and the results
were shown in Figure 4. To determine the Ki values for glycoamidases
Acknowledgment. We thank Takara Shuzo Co. (Ohtsu,
Japan) for providing Endo-A, Drs. Yuanda Zhang and R. B.
van Breemen (University of Illinois, Chicago) for recording the
mass spectra, and Dr. Charles A. Long (Johns Hopkins
University) for recording the 500 MHz NMR. This work was
supported by NIH Grant DK09970 (to Y.C.L).
JA9712027
(37) Cheng Y.; Prusoff, W. H. Biochem. Pharmacol. 1973, 22, 3099.