Tight binding ligand approach to oligosaccharide-grafted protein

Article abstract of DOI:10.1016/j.bmcl.2004.01.106

A novel type of artificial glycoprotein was developed, by using dihydrofolate reductase (DHFR) and methotrexate (MTX) as a protein-ligand pair. Various oligosaccharides linked to MTX were shown to bind tightly with DHFR and afforded oligosaccharide-grafted protein, which could be isolated easily by lectin beads.

Full text of DOI:10.1016/j.bmcl.2004.01.106

Bioorganic & Medicinal Chemistry Letters 14 (2004) 2285–2289
Tight binding ligand approach to oligosaccharide-grafted protein
Kiichiro Totani,b Ichiro Matsuoa,b and Yukishige Itoa,b,
*
aRIKEN––The Institute of Physical and Chemical Research, 2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan
bCREST, JST, Kawaguchi 332-1102, Japan
Received 12 December 2003; accepted 31 January 2004
Abstract—A novel type of artificial glycoprotein was developed, by using dihydrofolate reductase (DHFR) and methotrexate
(MTX) as a protein–ligand pair. Various oligosaccharides linked to MTX were shown to bind tightly with DHFR and afforded
oligosaccharide-grafted protein, which could be isolated easily by lectin beads.
Ó 2004 Elsevier Ltd. All rights reserved.
Oligosaccharide parts of glycoproteins play important
roles in a variety of biological events.1 In particular, the
functions of asparagine (Asn)-linked oligosaccharides in
glycoprotein quality control process is attracting recent
attention.2 Efforts to correlate the functions of glycan
chains with their structures are often hampered by the
heterogeneity of glycoproteins.3 Glycoproteins usually
exist as mixtures of various ÔglycoformsÕ that differ in the
structures of oligosaccharides. In order to understand
their functions precisely, it is desired that glycoproteins
having homogeneous and structurally defined oligosac-
charide are available. Since the isolation of homoge-
neous glycoprotein is difficult, various approaches to
prepare artificial glycoproteins have been developed.4
These include (1) modification of natural glycoproteins
using glycosidase and/or glycosyltransferase,5 (2) liga-
tion of synthetic glycopeptide with expressed protein,6
and (3) introducing synthetic sugar derivatives using
chemoselective ligation.7 Fully chemical synthesis of
large glycopeptide has met with substantial success.8 We
wish report herein a complementary approach to car-
bohydrate-grafted protein, which utilizes oligosaccha-
ride–ligand conjugates.9
We focused our attention to the combination of dihy-
drofolate reductase (DHFR)10 and methotrexate (MTX,
1) (Fig. 1).11 DHFR is relatively small protein
(MW ꢀ 18 kDa), which is essential in karyokinasis and
proliferation of cells. MTX is a strong inhibitor of
DHFR having KD < 1 nM.12 Since DHFR inhibition is
considered promising in cancer chemotherapy,13 the
mode of DHFR–MTX binding has been analyzed in
detail.14 To our interest, the inhibitory activity of MTX
is insensitive to the modification of the c-carboxy
Figure 1. The MTX–DHFR complexes.
* Corresponding author. Fax: +81-48-462-4680; e-mail: yukito@postman.riken.go.jp
0960-894X/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.01.106

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K. Totani et al. / Bioorg. Med. Chem. Lett. 14 (2004) 2285–2289
group.15 Therefore, we expected that the oligosacchar-
ide–MTX conjugate 3 bind tightly with DHFR to pro-
vide artificial glycoprotein.
turn was coupled with the pteridine unit 817 and
saponification of c-Me ester gave MTX(atBu) 2.
Pentasaccharide 14g, which corresponds to the core
region of asparagine-linked glycoprotein, was chosen as
the prototypical oligosaccharide and synthesized as
shown in Scheme 2, based on the previously reported
procedure18 with slight modification (Scheme 2).
Removal of TBDPS groupfrom 918 was achieved under
high-pressure conditions.19 Stepwise glycosylations for
10 and 12 using 11 as glycosyl donor afforded penta-
saccharide 13 that was deprotected to give 14g.
Preparation of oligosaccharide–MTX hybrid 3 was
conducted by linking oligosaccharide unit with the
MTX component. This convergent approach would be
flexible to provide various oligosaccharide-grafted
DHFR. The MTX unit 216 was synthesized as depicted
in Scheme 1. L-Glutamate derivative 5 was prepared
from 4 and condensed with 7. Subsequent deprotection
of the benzyloxycarbonyl (Z) groupafforded 6, which in
Me
NH
R1OOC
NHR2
H
3), 4)
5), 6)
MeOOC
t BuOOC
N
COOtBu
O
4 : R1=H, R2=Fmoc
5 : R1=Me, R2=H
1), 2)
6
H2N
N
N
NH2
N
NH2
N
O
Me
N
N
N
N
Br
HO
NZ
Me
H
N
HOOC
N
NH2
tBuOOC
O
7
8
2
Scheme 1. Synthesis of MTX unit 2. Reagents and conditions: (1) CsCO3, MeI, DMF; (2) piperidine, DMF, 87%, two steps; (3) 7, WSCIÁHCl,
HOBt, DMF; (4) H2, Pd(OH)2/C, MeOH, 82%, two steps; (5) 8, Me2NAc, 50 °C, then Et3N; (6) Ba(OH)2, EtOH–H2O (1:1), 95%, two steps.
BnO
OAc
O
BnO
OBn
O
OAc
O
NPhth
O
O
BnO
2), 3)
BnO
O
O
OAll
BnO
Cl
RO
O
11
NPhth
BnO
9 : R=TBDPS
10 : R=H
1)
BnO
BnO
OAc
O
OBn
O
OAc
O
NPhth
O
BnO
HO
HO
4)
BnO
O
OAll
Cl
BnO
11
O
O
NPhth
BnO
BnO
BnO
O
12
OAc
BnO
BnO
OAc
O
BnO
BnO
OBn
O
OAc
O
5), 6), 7), 8), 9), 10)
NPhth
O
O
HO
O
BnO
O
OAll
BnO
O
NPhth
BnO
BnO
BnO
O
OAc
13
BnO
HO
OH
O
HO
HO
OH
O
OH
O
NHAc
O
HO
O
O
HO
HO
O
O
OH
NHAc
HO
HO
HO
HO
O
OH
14g
Scheme 2. Synthesis of pentasaccharide 14g. Reagents and conditions: (1) HFÁpyr–DMF (1:10), 1 GPa, 30 °C, 89%; (2) 11, AgOTf, MS4A, toluene,
ClCH2CH2Cl, 87%; (3) p-TsOHÁH2O, CH3CN, 87%; (4) 11, AgOTf, MS4A, toluene, ClCH2CH2Cl, 72%; (5) H2NCH2CH2NH2–n-BuOH (1:6),
80 °C; (6) pyr–Ac2O (2:1), DMAP; (7) 1 M NaOMe, MeOH, 50 °C, 89%, three steps; (8) [Ir(COD)(PMePh2)2]PF6, H2, THF; (9) HgO, HgCl2,
acetone–H2O (10:1); (10) H2, Pd(OH)2/C, MeOH, 85%, three steps.

K. Totani et al. / Bioorg. Med. Chem. Lett. 14 (2004) 2285–2289
2287
O
O
H
4), 5)
1), 2), 3)
HO
HO
N
NH2
OH
14a-g
15a-g
O
H2N
N
N
NH2
N
Me
H
N
O
H
N
H
O
N
HO
N
H
3a-g
O
HOOC
O
O
HO
OH
OH
O
HO
HO
HO
OH
OH
O
O
O
HO
O
HO
O
HO
HO
HO
HO
O
OH
OH
OH
NHAc
OH
a : GlcNAc
b : Lactose
c : Cellobiose
HO
HO
OH
O
HO
HO
HO
HO
O
O
HO
OH
O
HO
OH
O
HO
HO
O
O
HO
O
HO
OH
O
O
HO
HO
HO
HO
O
OH
OH
HO
OH
d : Melibiose
e : Maltose
f : Maltotriose
HO
HO
OH
O
HO
OH
O
OH
NHAc
O
O
HO
O
HO
O
O
HO
O
NHAc
HO
HO
HO
HO
O
OH
g : Man3GlcNAc2
Scheme 3. Synthesis of bifunctional ligands 3a–g. Reagents and conditions: (1) satd aq NH4HCO3, 40 °C; (2) Fmoc-Gly-Cl, NaHCO3, dioxane–H2O
(1:1); (3) 20% piperidine, DMF, ꢀ93%, three steps; (4) 2, TBTU, DMF, )18 to 0 °C; (5) CF3COOH, ꢀ72%, two steps.
Bifunctional ligands 3a–g were prepared as depicted in
Scheme 3. Commercially available saccharides 14a–f
and aforesaid pentasaccharide 14g were converted to
corresponding glycosylamines20 and acylated with
Fmoc-Gly-Cl.21 Deprotection of Fmoc gave glycine-
linked saccharides 15a–g. Subsequent condensation with
MTX(atBu) 2 was effected by TBTU and acidic cleavage
t
of Bu ester provided sugar–MTX hybrids 3a–g.
In order to assess the affinity to DHFR, inhibitory
activities of 3a–g were measured. For this purpose,
crude preparation of DHFR (E. coli) expressed in a cell-
free protein synthesis system derived from wheat
embryos (PROTEIOSe)22 were employed. The inhibi-
tion assays were performed according to the reported
procedure.23 As summarized in Figure 2, inhibitory
Figure 2. DHFR inhibition by MTX–sugar. Dihydrofolate (FH2)
solution [FH2; 0.104 g/L, 2-mercaptoethanol; 6.06 mL/L in buffer A
(0.05 M TrisÁHCl, pH 7.5)] (130 lL), MTX derivatives [0.01–1 lmol/L
in buffer A] (20 lL), and NADPH/DHFR [NADPH; 0.308 g/L, DHFR
activities of sugar–MTX conjugates (3a–g) were com-
parable with that of unsubstituted MTX (1), suggesting
the formation of tight binding complex. As expected,
MTX(atBu) 2 had substantially reduced avidity.
expression mixture; 38.5 mL/L in buffer A] (50 lL) were added to each
well of the 96-well titer-plate. The absorbance was read in the micro-
plate reader at room temperature at wavelength of 340 nm, using the
kinetic mode with a reading interval of 20 s for 18 min.
Further evidence of the formation of the tight binding
complex between 3 and DHFR was obtained from lectin
binding experiment (Fig. 3), which also facilitated the
isolation of the conjugates. Thus, DHFR preparation
mixture (lane 1) was incubated with pentasaccharide–
MTX 3g (lane 2), in a plastic tube equipped with
membrane filter (Millipore: Ultrafree-MC, 0.22 lm), to
form the 3g–DHFR conjugate. Subsequently, it was
captured by treatment with ConA-Aga and separated by
filtration. At this stage, DHFR vanished from flow
through and from washing fractions (lanes 3 and 4),
implying that 3g–DHFR was retained on ConA-Aga.

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K. Totani et al. / Bioorg. Med. Chem. Lett. 14 (2004) 2285–2289
lectins and enzymes. Further studies are in progress
along this line and will be reported in due course.
Acknowledgements
We thank Dr. Teiji Chihara and his staff for elemental
analyses and the operation of the high-pressure equip-
ment, Dr. Hiroyuki Koshino for high-field NMR mea-
surement, and Ms. Akemi Takahashi for technical
assistance. Financial supports from Presidential Fund of
RIKEN, and Ministry of Education, Culture, Sports,
Science and Technology [Grant-in-Aid for Scientific
Research (B) No. 13480191] are acknowledged.
Figure 3. Lectin binding experiment of pentasaccharide containing
DHFR: lane 1, DHFR prep. mix. in 0.05 M TrisÁHCl (pH 7.5) (buffer
A); lane 2, DHFR prep. mix.+3g in buffer A; lane 3, DHFR prep.
mix.+3g+ConA-Aga (flow through fraction); lane 4, washings; lane 5,
elution fraction (eluent; 0.1 M methyl mannoside in buffer A).
The conjugate was then eluted with 0.1 M a-Me-Man
(lane 5). Similarly, DHFR conjugates carrying 3a and 3b
were isolated using WGA-Aga and RCA120-Aga,
respectively.
References and notes
1. Dwek, R. A. Chem. Rev. 1996, 96, 683–720.
2. Parodi, A. J. Ann. Rev. Biochem. 2000, 69, 69.
3. (a) Rush, R. S.; Derby, P. L.; Smith, D. M.; Merry, C.;
Rogers, G.; Rohde, M. F.; Katta, V. Anal. Chem. 1995, 67,
1442; (b) Opdenakker, G.; Rudd, P. M.; Wormald, M.;
Dwek, R.; Van Damme, J. FASEB J. 1995, 9, 453.
4. Grogan, M. L.; Pratt, M. R.; Marcaurelle, L. A.; Bertozzi,
C. R. Ann. Rev. Biochem. 2002, 71, 593.
5. (a) Witte, K.; Sears, P.; Martin, R.; Wong, C.-H. J. Am.
Chem. Soc. 1997, 119, 2114; (b) Miyazaki, T.; Sakakibara,
T.; Sato, H.; Kajihara, Y. J. Am. Chem. Soc. 1999, 121,
1411.
That the conjugate consists of an equimolar amount of
DHFR and 3g was confirmed in a following manner.
Firstly, 3g–DHFR complex containing eluent (Fig. 3,
lane 5) was separated from the excess ligand by ultra-
filtration (Millipore: Centricon YM3). The amount of
DHFR was determined to be 1.37 lM by BCA protein
assay. This value was in agreement with the amount of
the ligand 3g (1.35 lM) that was determined by the
absorbance of the MTX chromophore at 304 nm (Fig.
4).
6. Shin, Y.; Winans, K. A.; Baches, B. J.; Kent, S. B. H.;
Ellman, J. A.; Bertozzi, C. R. J. Am. Chem. Soc. 1999, 121,
11684.
7. (a) Macmillan, D.; Daines, A. M.; Bayrhuber, M.; Flitsch,
S. Org. Lett. 2002, 4, 1467; (b) Liu, H.; Wang, L.; Brock,
A.; Wong, C.-H.; Schultz, P. G. J. Am. Chem. Soc. 2003,
125, 1702.
8. (a) Hojo, H.; Haginoya, E.; Matsumoto, Y.; Nakahara,
Y.; Nabeshima, K.; Toole, B. P.; Watanabe, Y. Tetrahe-
dron Lett. 2003, 44, 2961; (b) Miller, J. S.; Dudkin, V. Y.;
Lyon, G. L.; Zmuir, T. W.; Danishefsky, S. J. Angew.
Chem., Int. Ed. 2003, 42, 431.
In summary, a novel type of glycoprotein mimetic was
created by employing specific binding of MTX and
DHFR. In principle, it has no limitation in oligo-
saccharide structure that can be introduced. Since con-
version of complex glycan chain to corresponding
glycosylamine is well established,24 it is expected that
various types of sugar–MTX conjugates can be prepared
and converted to artificial glycoproteins, which will be
useful molecular probes to analyze interaction with
9. Other types of noncovalent neoglycoproteins: (a) Shao,
M.-C.; Sokolik, C. W.; Wold, F. In Neoglycoconjugates;
Lee, Y. C., Lee, R. T., Eds.; Academic: San Diego, 1994;
p225; (b) Hamachi, I.; Tajiri, Y.; Murakami, H.; Shinkai,
S. Chem. Lett. 1994, 575; (c) Chen, V. J.; Wold, F.
Biochemistry 1986, 25, 939.
10. Blakley, R. L.; MacDougall, B. M. J. Biol. Chem. 1961,
236, 1163.
11. (a) McGuire, J. J.; Coward, J. K. In Folates and Pterins;
Blakley, R. L., Benkovic, S. J., Eds.; John Wiley: New
York, 1984; Vol. 1, p135; (b) Eilers, M.; Schatz, G. Nature
1986, 322, 228.
12. Appleman, J. R.; Howell, E. E.; Kraut, J.; Kuhl, M.;
Blakley, R. L. J. Biol. Chem. 1988, 263, 9187.
13. Zeng, H.; Chen, Z.-S.; Belinsky, M. G.; Rea, P. A.; Kruth,
G. D. Cancer Res. 2001, 61, 7225.
14. Sawaya, M. R.; Kraut, J. Biochemistry 1997, 36, 586.
15. Rosowsky, A.; Yu, C.-S.; Uren, J.; Lazarus, H.; Wick, M.
J. Med. Chem. 1981, 24, 559.
16. Rosowsky, A.; Forsch, R.; Uren, J.; Wick, M. J. Med.
Chem. 1981, 24, 1450.
Figure 4. UV–visible spectra of 3g–DHFR and 3g. Compound 3g
(24 lM) and DHFR (24 lM) in 0.05 M TrisÁHCl (pH 7.5).
17. Piper, J. R.; Montgomery, J. A. J. Org. Chem. 1977, 42,
208.

K. Totani et al. / Bioorg. Med. Chem. Lett. 14 (2004) 2285–2289
2289
18. Matsuo, I.; Wada, M.; Manabe, S.; Yamaguchi, Y.; Otake,
K.; Kato, K.; Ito, Y. J. Am. Chem. Soc. 2003, 125, 3402.
19. Matsuo, I.; Wada, M.; Ito, Y. Tetrahedron Lett. 2002, 43,
3273.
22. Madin, K.; Sawasaki, T.; Ogasawara, T.; Endo, Y. Proc.
Natl. Acad. Sci. U.S.A. 2000, 97, 559.
23. Widemann, B. C.; Balis, F. M.; Adamson, P. C. Clin.
Chem. 1999, 45, 223.
20. Likhosherstov, L. M.; Novikova, O. S.; Derevitskaja, V.
A.; Kochetkov, N. K. Carbohydr. Res. 1986, 146, c1.
21. Carpino, L. A.; Cohen, B. J.; Stephens, K. E., Jr.; Sadat-
Aalaee, Y.; Tien, J.-H.; Langridge, D. C. J. Org. Chem.
1986, 51, 3732.
24. (a) Stubbs, H. J.; Shia, M. A.; Rice, K. G. Anal. Biochem.
1997, 247, 357; (b) Arsequell, G.; Dwek, R. A.; Wong, S.
Y. C. Anal. Biochem. 1994, 216, 165; (c) Meinjohanns, E.;
Meldal, M.; Paulsen, H.; Dwek, R.; Bock, K. J. Chem.
Soc., Perkin Trans. 1 1998, 549.