One-step synthesis of biotinyl photoprobes from unprotected carbohydrates

Article abstract of DOI:10.1021/jo000414a

A simple and versatile approach for the preparation of carbohydrate photoprobes has been developed. By a single-step reaction at 37 °C, a biotinylated carbene-generating unit was introduced to the reducing end of unprotected carbohydrates. Micromole quantities of N-acetyllactosamine, Lewis X trisaccharide, and sialyl Lewis X tetrasaccharide were easily converted to their biotinylated photoreactive analogues, which enabled the nonradioisotopic chemiluminescent detection of the photolabeled products. Thus, a sequence of lectin photoaffinity labeling, from the probe synthesis to the detection of labeled protein, was readily accomplished within one week. Our strategy may be applicable to any aldehyde-bearing ligand.

Full text of DOI:10.1021/jo000414a

J . Org. Chem. 2000, 65, 5639-5643
5639
On e-Step Syn th esis of Biotin yl P h otop r obes fr om Un p r otected
Ca r boh yd r a tes
Yasumaru Hatanaka,* Uwe Kempin, and Park J ong-J ip
Institute of Natural Medicine, Toyama Medical and Pharmaceutical University,
2630 Sugitani, Toyama, 930-0194 J apan
yasu@ms.toyama-mpu.ac.jp
Received March 21, 2000
A simple and versatile approach for the preparation of carbohydrate photoprobes has been developed.
By a single-step reaction at 37 °C, a biotinylated carbene-generating unit was introduced to the
reducing end of unprotected carbohydrates. Micromole quantities of N-acetyllactosamine, Lewis X
trisaccharide, and sialyl Lewis X tetrasaccharide were easily converted to their biotinylated
photoreactive analogues, which enabled the nonradioisotopic chemiluminescent detection of the
photolabeled products. Thus, a sequence of lectin photoaffinity labeling, from the probe synthesis
to the detection of labeled protein, was readily accomplished within one week. Our strategy may
be applicable to any aldehyde-bearing ligand.
In tr od u ction
Resu lts a n d Discu ssion
We have developed a general method for the rapid
preparation of photoreactive carbohydrate probes. Many
biological phenomena are initiated by the recognition of
carbohydrate ligands by endogenous receptor proteins.1
The method of photoaffinity labeling enables the direct
probing of the target protein through a covalent bond
which is introduced between a ligand and its specific
receptor.2 Recently, phenyldiazirine derivatives which
have an attached biotin residue have been developed as
convenient nonradioactive reagents of photoaffinity label-
ing.3 The method has been successfully applied to iden-
tification of the binding-site region of â1,4-galactosyl-
transferase.3b,c The combination of biotinyl photoprobes
with mass spectrometric sequencing has become a pow-
erful tool for the rapid analysis of protein-ligand inter-
actions.4 However, the application of photoaffinity label-
ing to carbohydrate-binding proteins often relies on mul-
tistep syntheses of photoreactive carbohydrate probes
involving protection, activation and deprotection reac-
tions.5 More generally, the modification of oligosaccha-
rides usually has to be performed on a very small scale
due to ligand availability limitations.6 Therefore, the
current problems of natural and synthetic carbohydrate
availability significantly retards their conversion to pho-
toreactive analogues.
Here we report the novel photophore 6 (Scheme 1) for
the one-step introduction of a carbene-generating phen-
yldiazirine into unprotected carbohydrates. The key
feature of 6 is an aminooxy group. This is designed for
the ligation of the photoreactive group to the terminal
aldehyde of carbohydrates and is based on the recently
developed chemoselective method.7 A biotin tag is also
pre-installed for enabling the nonradioactive detection
and affinity isolation of photolabeled targets based on
avidin-biotin technology. This novel photoreactive re-
agent 6 can be readily prepared as a Boc protected form
7 from a readily available aldehyde 1.8
To demonstrate the effectiveness of our new approach,
we coupled 6 to N-acetyllactosamine (LacNAc, 8) under
conventional ligation conditions.7b HPLC analysis re-
vealed the presence of the three major peaks of products
in the ratio of 2:6:1 (Figure 1), which suggests the
formation of E/Z oxime isomers 9 and/or the existence of
cyclic R/â anomers 10 (Scheme 2).8 The isomerization was
slow enough for the HPLC isolation of each product, and
the structures of 9-Z, 9-E, and 10-â were identified from
1
their H NMR spectra. According to the previous assign-
ment of glucose oximes, doublets at 6.85 and 7.47 ppm
were assigned as the oxime CH protons of Z- and E-form
of 9, respectively.9 The doublet at 4.16 ppm was assigned
as the anomeric proton of 10-â based on its J value (9.8
Hz). The structural heterogeneity at the reducing end
might be a synthetic drawback if this chemistry is
considered for the application to stereoselective glycosy-
lation.10 However, many cell surface carbohydrates usu-
* To whom correspondence should be addressed. Fax: (+81)76-434-
5059.
(1) Dwek, R. A. Chem. Rev. 1996, 96, 683-720.
(2) (a) Brunner, J . Annu. Rev. Biochem. 1993, 62, 483-514. (b)
Kotzyba-Hibert, F.; Kapfer, I.; Goeldner, M. Angew. Chem., Int. Ed.
Engl. 1995, 34, 1296-1312.
(3) (a) Hatanaka, Y.; Hashimoto, M.; Kanaoka, Y. J . Am. Chem. Soc.
1998, 120, 453-454. (b) Koumanov, F.; Yang, J .; J ones, A. E.;
Hatanaka, Y.; Holman, G. D. Biochem. J .1998, 330, 1209-1215. (c)
Hashimoto, M.; Hatanaka, Y. Chem. Pharm. Bull.1999, 47, 667-671.
(4) (a) Fang, K.; Hashimoto, M.; J ockusch, S.; Turro, N. J .; Nakan-
ishi, K. J . Am. Chem. Soc. 1998, 120, 8543-8544. (b) Zhao, Y.; Muir,
T. W.; Kent, S. B. H.; Tischer, E.; Scardina, J . M.; Chait, B. T. Proc.
Natl. Acad. Sci. U.S.A. 1996, 93, 4020.
(7) (a) Cervigni, S. E.; Dumy, P.; Mutter, M. Angew. Chem., Int. Ed.
Engl. 1996, 35, 1230-1232. (b) Zhao, Y.; Kent, S. B. H.; Chait, B. T.
Proc. Natl. Acad. Sci. U.S.A. 1997, 94, 1629-1633. (c) Rodriguez, E.
C.; Winans, K. A.; King, D. S.; Bertozzi, C. R. J . Am. Chem. Soc. 1997,
119, 9905-9906.
(8) (a) Hatanaka, Y.; Hashimoto, M.; Kurihara, H.; Nakayama, H.
Kanaoka, Y. J . Org. Chem. 1994, 59, 383-387. (b) Hashimoto, M.;
Kanaoka, Y.; Hatanaka, Y. Heterocycles 1997, 46, 119-122. (c)
Hashimoto, M.; Hatanaka, Y.; Holman, G. D. submitted.
(9) Finch, P.; Merchant, Z. J . Chem. Soc., Perkin Trans. 1 1975,
1682-1686.
(5) (a) Clark, A. E.; Holman, G. D. Biochem. J . 1990, 269, 615-
622. (b) Hatanaka, Y.; Hashimoto, M.; Nishihara, S.; Narimatsu, H.;
Kanaoka, Y. Carbohydr. Res. 1996, 294, 95-108.
(6) Patel, T. P.; Parekh, R. B. Methods Emzymol. 1994, 230, 57-
66.
10.1021/jo000414a CCC: $19.00 © 2000 American Chemical Society
Published on Web 08/09/2000

5640 J . Org. Chem., Vol. 65, No. 18, 2000
Hatanaka et al.
Sch em e 1a
F igu r e 2. Silver-stained 12% SDS-polyacrylamide gel (lane
1) and the chemiluminescent detection result (lanes 2-5) of
RCA lectin photolabeled with the isomeric mixture of LacNAc
probe (lanes 2 and 5) and HPLC purified 9-E (lanes 3 and 4),
respectively. For the evaluation of specific photolabeling, 0.1
M of LacNAc was incorporated as the inhibitor of the photo-
probe binding during the irradiation (lanes 2 and 3).
Sch em e 2
a
Reagents: (a) NaBH4, EtOH; (b) Ph3P, CBr4, CH2Cl2, 0 °C;
(c) N-hydroxyphthalimide, DMSO, K2CO3; (d) (i) TFA, CH2Cl2, 0
°C, (ii) biotin N-hydroxysuccinimide ester, DMF, Et3N; (e) NH2NH2,
MeOH; (f) Boc2O, CHCl3/CH3CN ) 1:1, Et3N.
the separation of each photoprobe isomer may not be
necessary for the application of current method. The
technique of reductive amination is widely accepted in
the glycoconjugate synthesis as the acyclic type deriva-
tization of reducing terminus.12
To examine this expectation, we directly compared the
labeling ability of the LacNAc probe mixture with that
of purified 9-E using Ricinus communis agglutinin (RCA).
RCA proteins are composed of two subunits, 30 kDa A
and 37 kDa B chains (Figure 2, lane 1). A weak band
between A and B is known to be an impurity resulting
from the closely related other lectin.13 Among these RCA
proteins, only the RCA B chain possesses an affinity for
the terminal â-D-galactosyl residues.14 After the photo-
labeling, these bands were electroblotted from polyacryl-
amide gels onto poly(vinylidene difluoride) membranes
for chemiluminescent detection of the photolabeled prod-
ucts (lanes 2-5).5b Both LacNAc probes equally and
F igu r e 1. HPLC profile of LacNAc probe isomers at equilib-
rium. HPLC separation was performed under isocratic condi-
tions with 85% water/acetonitrile at a flow rate of 1 mL/min.
Peaks were monitored at 360 nm.
ally exist as an oligomeric chain structure. The binding
of receptors to these carbohydrates mainly depends on
the sugar structure at the nonreducing terminus,11 and
the configuration at the reducing end is likely to be less
important for the binding of photoprobes. In such a case,
(12) Feizi, T.; Stall, M. S.; Yuen, C.-T.; Chai, W.; Lawson, A. M.
Methods Enzymol. 1994, 230, 484-519.
(13) Nicolson, G. L.; Blaustein, J .; Etzler M. E. Biochemistry 1974,
13, 196-204.
(14) Baenziger, J . U.; Fiete, D. J . Biol. Chem. 1979, 254, 9795-
9799.
(10) Peri, F.; Dumy, P.; Mutter, M. Tetrahedron 1998, 54, 12269-
12278.
(11) Sharon, N.; Lis, H. Science 1989, 246, 227-246.

One-Step Synthesis of Biotinyl Photoprobes
J . Org. Chem., Vol. 65, No. 18, 2000 5641
specifically identified the galactose binding 37 kDa
subunit of RCA lectin (lanes 4 and 5). No detectable
chemiluminescence was observed in the zone correspond-
ing to the band of the A chain. Furthermore, the presence
of a large excess of LacNAc efficiently prevented the
photoincorporation of both probes by blocking the galac-
tose binding-site in the RCA B chain (lanes 2 and 3).
Other biologically very important antigens,15 Lewis X
trisaccharide (Galâ1-4(FucR1-3)GlcNAc, Lex) and sialyl
Lewis X tetrasaccharide (NeuAcR2-3Galâ1-4(FucR1-
3)GlcNAc, sLex), were next examined. By simple incuba-
tion with 6 at 37 °C, these sugars were also easily con-
verted to the desired photoreactive conjugates. After the
HPLC separation of probe peaks, the possible isomers of
Lex probe (11 and 13) and sLex probe (12 and 14) were
not further purified and were directly used for photola-
beling.
F igu r e 3. Pattern of chemiluminescent detection of biotin-
tagged proteins from lotus lectin (lanes 1 and 2) and MAL
lectin (lanes 3 and 4) after photolabeling. For the inhibition
of specific photolabeling, 0.1 M of FucROMe (lane 1) or
NeuNAcR2-3lactose (lane 3) was incorporated, respectively,
during the photolysis.
is introduced into various unprotected oligosaccharides.
The biotinyl group may be changed to other reporter
groups. A sequence of lectin photoaffinity labeling, from
the probe synthesis to the detection of labeled protein,
was readily accomplished within one week. Our strategy
may be applicable to any aldehyde-bearing ligand. The
present approach may extend the potential of photoaf-
finity labeling to become a rapid and more sensitive
means for the elucidation of protein structures and
binding-sites.
Exp er im en ta l Section
Gen er a l Exp er im en ta l P r oced u r es. The diazirine alde-
hyde 1 was prepared from 3-(3-methoxyphenyl)-3-(trifluoro-
methyl)-3H-diazirine.8 Other chemical reagents were com-
mercially available and were used without further purification.
The reaction flasks containing all diazirine derivatives were
protected from light by wrapping with aluminum foil. The fast
atom bombardment (FAB) mass spectra were obtained by
using 3-nitrobenzyl alcohol as the matrix. Silica gel for column
chromatography was Kieselgel 60 (Merck, No. 7734, 70-230
mesh).
2-[2-[2-(2-ter t-Bu t oxyca r b on yla m in oet h oxy)et h oxy]-
eth oxy]-4-[3-(tr iflu or om eth yl)-3H-diazir in -3-yl]ben zyl Al-
coh ol (2). The aldehyde 1 (3.69 g, 8 mmol) was dissolved in
EtOH (40 mL) followed by the addition of NaBH4 (303 mg, 8
mmol) at 0 °C. After stirring at 0 °C for 30 min, the solvent
was removed and the residue was suspended in H2O. The pH
of this mixture was adjusted to 3 with 1 N HCl at 0 °C and
the alcohol 2 was extracted with ethyl acetate. The extract
was washed with H2O, saturated aqueous NaHCO3, and
saturated aqueous NaCl, and dried over MgSO4. The solvent
was removed by evaporation to leave the alcohol as a yellow
oil which was used in the next step without further purifica-
tion: 1H NMR (400 MHz, CDCl3, 25 °C) δ 7.26 (d, J ) 8.0 Hz,
1H), 6.79 (d, J ) 8.0 Hz, 1H), 6.66 (br s, 1H), 6.01 (br, 1H),
5.02 (br, 1H), 4.66 (s, 2H), 4.22 (t, J ) 4.8 Hz, 2H), 3.84 (t, J
) 4.8 Hz, 2H), 3.7 (m, 2H), 3.6 (m, 2H), 3.51 (t, J ) 5.1 Hz,
2H), 3.3 (m, 2H), 1.45 (s, 9H); MS (FAB+) m/z 486 (100) [M +
Na]+, 464 (16) [M + H]+; HRMS calcd for C20H29F3N3O6 [M +
H]+ ) 464.2008, found 464.1997.
The Lex binding lectin from Lotus tetragonolobus16 was
photolabeled with the Lex probe (Figure 3, lanes 1 and
2) and the R2-3-linked sialic acid residue binding
Maackia amurensis leucoagglutinin (MAL)17 was exam-
ined with the sLex probe (lanes 3 and 4), respectively.
Chemiluminescent detection again demonstrated that
these probes clearly labeled the 27 kDa lotus lectin
subunit (Figure 3, lane 2) and the 33 kDa MAL lectin
protein (lane 4), respectively. The label incorporation into
these bands was diminished in the presence of competi-
tive sugar ligands, methyl R-fucopyranoside (lane 1) and
N-acetyl-neuraminylR2-3lactose (lane 3), respectively.
The result again shows that the isomeric mixtures of
photoprobes are sufficiently useful for the identification
of these lectins.
In summary, we have developed a rapid and versatile
method for the microscale preparation of photoaffinity
probes, whereby a biotinylated carbene-generating unit
2-[2-[2-(2-ter t-Bu t oxyca r b on yla m in oet h oxy)et h oxy]-
eth oxy]-4-[3-(tr iflu or om eth yl)-3H-diazir in -3-yl]ben zyl Br o-
m id e (3). The crude alcohol 2 (prepared from 8 mmol of 1),
carbon tetrabromide (3.32 g, 10 mmol), K2CO3 (1.66 g, 12
mmol) and CH2Cl2 (16 mL) were placed in a flask. Triphen-
ylphosphine (3.15 g, 12 mmol) was slowly added to above
(15) Hakomori, S.; Zhang, Y. Chem. Biol. 1997, 4, 97-104.
(16) Yan, L.; Wilkins, P. P.; Alvatez-Manilla, G.; Do S.-I.; Smith, D.
F.; Cummings, R. D. Glycoconjugate J . 1997, 14, 45-55.
(17) Yamamoto, K.; Konami, Y.; Irimura, T. J . Biochem. 1997, 121,
756-761.

5642 J . Org. Chem., Vol. 65, No. 18, 2000
Hatanaka et al.
suspension at 0 °C. After stirring for 30 min at 0 °C, the
reaction mixture was partitioned between CH2Cl2 and H2O.
The organic layer was dried over MgSO4.and the solvent was
removed under reduced pressure. The brown residue was
chromatographed on silica gel (eluting with ethyl acetate/
hexane ) 1:1) to give 3 (3.62 g, 86% from 1) as a yellow oil:
1H NMR (400 MHz, CDCl3, 25 °C) δ 7.35 (d, J ) 8.1 Hz, 1H),
6.77 (d, J ) 8.1 Hz, 1H), 6.65 (br s, 1H), 5.00 (br, 1H), 4.51 (s,
2H), 4.21 (t, J ) 4.8 Hz, 2H), 3.91 (t, J ) 4.8 Hz, 2H), 3.75 (m,
2H), 3.65 (m, 2H), 3.55 (t, J ) 5.1 Hz, 2H), 3.3 (m, 2H), 1.43
(s, 9H); MS (FAB+) m/z 550 (41) [M + Na]+, 548 (40), 528 (12)
[M + H]+, 526 (11); HRMS calcd for (79Br) C20H28BrF3N3O5
[M + H]+ ) 526.1164, found 526.1193.
H]+; HRMS calcd for C30H44F3N6O8S [M + H]+ ) 705.2893,
found 705.2853
Liga tion of 6 to Oligosa cch a r id es. Deprotection of 7 was
performed with TFA and the resulting 6 was incubated with
a half equivalent of oligosaccharides. Typically, 7 (7.0 mg, 10
µmol) was treated with 200 µL of 50% TFA-CH2Cl2 at 0 °C
for 30 min. After evaporation, the residue was dissolved in
80% aq. acetonitrile (250 µL) containing LacNAc 8 (1.9 mg, 5
µmol). After adjusting the pH to 5-6 with diisopropyleth-
ylamine, the mixture was incubated at 37 °C for 40 h in the
dark. Separation on a silica gel HPLC column (Aquasil SS-
1251, 4.6 250 mm, Sensyu Kagaku Co Ltd., J apan) gave the
isomeric mixture of the glyco-conjugates. Products were moni-
tored at 360 nm. The yields were determined from the UV
spectra of MeOH solutions using the ꢀ value of 7 (ꢀ360 ) 400)
as standard.
La cNAc P r obe (9, 10). HPLC solvent: 10-25% water/
acetonitrile in 20 min at a flow rate of 1 mL/min. Peaks eluting
at 19-21 min were collected to give a glyco-conjugate mix-
ture: yield 63%. Reaction at pH 2 and pH 9 decreased the
yields to 20% and 40%, respectively. Further HPLC separation
(isocratic elution with 85% water/acetonitrile at a flow rate of
1 mL/min) gave pure 9-Z, 9-E, and 10-â.
2-[2-[2-(2-ter t-Bu t oxyca r b on yla m in oet h oxy)et h oxy]-
et h oxy]-4-[3-(t r iflu or om et h yl)-3H -d ia zir in -3-yl]b en zyl-
oxyp h th a lim id e (4). A mixture of the bromide 3 (2.63 g, 5
mmol), N-hydroxyphthalimide (0.98 g, 6 mmol), K2CO3 (0.69
g, 5 mmol), and DMSO (10 mL) was stirred for 12 h at room
temperature. Purification on silica gel (hexane/acetone 3:1)
gave 4 as a yellow oil (2.59 g, 85%): 1H NMR (400 MHz, CDCl3,
25 °C) δ 7.82-7.73 (AB, 4H), 7.54 (d, J ) 7.9 Hz, 1H,), 6.81
(d, J ) 7.9 Hz, 1H,), 6.66 (br s, 1H), 5.27 (s, 2H), 5.05 (br, 1H),
4.15 (t, J ) 4.8 Hz, 2H), 3.86 (t, J ) 4.8 Hz, 2H), 3.7 (m, 2H),
3.6 (m, 2H), 3.54 (t, J ) 5.2 Hz, 2H), 3.3 (m, 2H), 1.42 (s, 9H);
MS (FAB+) m/z 631 (100) [M + Na]+, 609 (9) [M + H]+; HRMS
calcd for C28H32F3N4O8 [M + H]+ ) 609.2172, found 609.2164.
2-[2-[2-(2-Biot in yla m in oet h oxy)et h oxy]et h oxy]-4-[3-
(t r iflu or om et h yl)-3H -d ia zir in -3-yl]b en zyloxyp h t h a lim -
id e (5). Deprotection of 4 (304 mg, 0.5 mmol) was performed
with 1 mL of 50% TFA-CH2Cl2 at 0 °C. After stirring at 0 °C
for 1 h, the reaction mixture was concentrated to provide a
yellow oil. The resulting TFA salt of the deprotected amine
was dissolved in DMF (0.5 mL) and the solution was cooled at
0 °C. Triethylamine (174 µL, 1.25 mmol) was added followed
by a solution of d-biotin N-hydroxysuccinimide ester (171 mg,
0.5 mmol) in DMF (2 mL). The reaction flask was wrapped
with aluminum foil and the mixture was stirred at room-
temperature overnight. The mixture was concentrated under
reduced pressure and the residue was dissolved in CH2Cl2/
MeOH. The organic layer was successively washed with 1 N
NaOH, saturated NaCl, 1 N HCl, and saturated NaCl. After
drying over MgSO4, the solvent was evaporated in vacuo.
Chromatography of the residue on silica gel (eluting with
CHCl3/EtOH ) 10:1) gave 5 (320 mg, 87%) as a colorless
solid: 1H NMR (400 MHz, CDCl3, 25 °C) δ 7.83-7.73 (AB, 4H),
7.53 (d, J ) 7.9 Hz, 1H), 6.84 (s, 1H), 6.81 (d, J ) 7.9 Hz, 1H),
6.64 (s, 1H), 6.61 (s, 1H), 5.64 (s, 1H), 5.26 (s, 2H), 4.5 (m,
1H), 4.3 (m, 1H), 4.15 (t, J ) 4.8 Hz, 2H), 3.90 (t, J ) 4.8 Hz,
2H), 3.7 (m, 2H), 3.65 (m, 2H), 3.55 (m, 2H), 3.4 (br m, 2H),
3.1 (m, 1H), 2.9 (m, 1H), 2.72 (d, J ) 12.5 Hz, 1H), 2.19 (t, J
) 7.5 Hz, 2H), 1.8-1.6 (m, 4H), 1.4 (m, 2H); MS (FAB+) m/z
757 (16), [M + Na]+, 735 (62) [M + H]+; HRMS calcd for
9-Z: 1H NMR (400 MHz, D2O, 25 °C) δ 7.35 (d, J ) 8.0 Hz,
1H), 6.93 (d, J ) 8.0 Hz, 1H), 6.85 (d, J ) 5.8 Hz, 1H), 6.76 (s,
1H), 5.13 (s, 2H), 5.07 (dd, J ) 7.6, 5.8 Hz, 1H), 4.43 (m, 1H),
4.23 (m, 1H), 4.13 (br, 2H), 3.96 (d, J ) 7.6 Hz, 1H), 3.95 (m,
1H), 3.81 (m, 2H), 3.75-3.50 (14H), 3.37 (m, 2H), 3.26 (t, J )
5.2 Hz, 2H), 3.10 (m, 1H), 2.83 (m, 1H), 2.63 (d, J ) 13.2, 1H),
2.08 (t, J ) 7.2, 2H), 1.91 (s, 3H), 1.47 (m, 4H), 1.22 (m, 2H);
HRMS calcd for C39H57F3N7O16S [M - H]- 968.3535, found
968.3594.
9-E: 1H NMR (400 MHz, D2O, 25 °C) δ 7.42 (d, J ) 5.6 Hz,
1H), 7.34 (d, J ) 8.0 Hz, 1H), 6.92 (d, J ) 8.0 Hz, 1H), 6.73 (s,
1H), 5.06 (AB, 2H), 4.85 (dd, J ) 9.3, 5.6 Hz, 1H), 4.44 (m,
1H), 4.23 (d, J ) 7.8 Hz, 1H), 4.20 (m, 1H), 4.15 (br, 2H), 3.99
(m, 1H), 3.82 (m, 2H), 3.75-3.50 (14H), 3.41 (m, 2H), 3.26 (t,
J ) 5.2 Hz, 2H), 3.12 (m, 1H), 2.82 (m, 1H), 2.63 (d, J ) 12.7,
1H), 2.08 (t, J ) 7.2, 2H), 1.85 (s, 3H), 1.47 (m, 4H), 1.22 (m,
2H); HRMS found 968.3528.
10-â: 1H NMR (400 MHz, D2O, 25 °C) δ 7.34 (d, J ) 7.8 Hz,
1H), 6.92 (d, J ) 7.8 Hz, 1H), 6.74 (s, 1H), 4.67 (s, 2H), 4.43
(m, 1H), 4.34 (d, J ) 7.8 Hz, 1H), 4.22 (m, 1H), 4.16 (d, J )
9.8 Hz, 1H), 4.14 (br, 2H), 3.81 (m, 3H), 3.75-3.50 (15H), 3.43
(m, 2H), 3.26 (t, J ) 5.2 Hz, 2H), 3.12 (m, 1H), 2.83 (m, 1H),
2.63 (d, J ) 13.2, 1H), 2.08 (t, J ) 7.2, 2H), 1.88 (s, 3H), 1.47
(m, 4H), 1.22 (m, 2H); HRMS found 968.3635.
LeX P r obe (Mixtu r e of 11 a n d 13). These were prepared
from 0.5 mg of Lex (0.94 µmol) and from peaks eluting at 23-
24 min were collected to give a glyco-conjugate mixture: yield
78%; HPLC solvent: 10-30% water/acetonitrile in 15 min at
a flow rate of 1 mL/min: MS (FAB-) m/z 1114 (47) [M - H]-;
HRMS calcd for C45H66F3N7O20S [M - H]- 1114.4114, found
1114.4163.
C
33H38F3N6O8S [M + H]+ ) 735.2424, found 735.2458.
2-[2-[2-(2-Biot in yla m in oet h oxy)et h oxy]et h oxy]-4-[3-
(t r iflu or om et h yl)-3H -d ia zir in -3-yl]b en zyloxyca r b a m ic
Acid ter t-Bu tyl Ester (7). Phthaloyl group of 5 (73 mg, 0.1
mmol) was deprotected with 1 mL of 1 M hydrazine in
methanol at room temperature. After 30 min, the solvent was
evaporated and the residue was dissolved in toluene. The
residual hydrazine was removed by azeotropic distillation in
vacuo to give crude 6 which was dissolved in 1 mL of CHCl3/
CH3CN (1:1). To this solution, triethylamine (70 µL, 0.5
mmol) and Boc2O (109 mg, 0.5 mmol) were added and the
mixture was stirred at room temperature overnight. The
solvent was evaporated and the residue was chromatographed
on silica gel (eluting with CHCl3/EtOH ) 10:1) to give 7 (64
mg, 95%) as a colorless solid: 1H NMR (400 MHz, CDCl3, 25
°C) δ 8.31 (s, 1H), 7.39 (d, J ) 7.9 Hz, 1H), 6.97 (br, 1H), 6.81
(d, J ) 7.9 Hz, 1H), 6.64 (s, 1H), 6.58 (s, 1H), 5.66 (s, 1H),
4.91 (s, 2H), 4.5 (m, 1H), 4.3 (m, 1H), 4.16 (t, J ) 4.5 Hz, 2H,),
3.87 (t, J ) 4.5 Hz, 2H), 3.7 (m, 2H), 3.65 (m, 2H), 3.55 (m,
2H), 3.4 (br m, 2H), 3.1 (m, 1H), 2.9 (m, 1H), 2.70 (d, J ) 12.8
Hz, 1H), 2.17 (t, J ) 7.3 Hz, 2H,), 1.8s1.6 (m, 4H), 1.45 (s,
9H), 1.4 (m, 2H); UV/VIS (MeOH) λmax (ꢀ) ) 360 (400), 283
(2600); MS (FAB+) m/z 727 (100), [M + Na]+, 705 (12) [M +
sLeX P r obe (Mixtu r e of 12 a n d 14). These were prepared
from 1.0 mg of sLex (1.2 µmol) and from peaks eluting at 20-
22 min were collected to give a glyco-conjugate mixture: yield
71%; HPLC solvent: 15-35% water/acetonitrile in 15 min at
a flow rate of 1 mL/min: MS (FAB-) m/z 1405 (29) [M - H]-;
HRMS calcd for C56H84F3N8O28S [M - H]- 1405.5068, found
1405.5143.
P h otola belin g of Lectin s. Irradiation and analysis were
performed following established procedures.5b Thus, 50 µL each
of photoaffinity labeling sample containing photoprobes (0.1
mM) lectin (0.1 mM) in 0.1 M phosphate buffer (pH 7.6) was
also prepared. In the meanwhile, 50 µL each of control sample
containing photoprobes (0.1 mM) lectin (0.1 mM) and an
inhibitor (0.1M) in 0.1 M phosphate buffer (pH 7.6) was
prepared. Carbohydrates used as inhibitors were LacNAc,
FucROMe, and NeuNAcR2-3lactose for RCA, lotus, and MAL
lectins, respectively. The photoaffinity labeling and control
samples prepared as above were incubated in the dark at 25
°C for 30 min. The samples at 0 °C on ice were then irradiated
for 1 h from above with a 30-W long-wavelength UV lamp
(Funakoshi XX-15) at the distance of 5 cm.

One-Step Synthesis of Biotinyl Photoprobes
J . Org. Chem., Vol. 65, No. 18, 2000 5643
Ch em ilu m in escen t Detection of P h otola beled P r od -
u cts. After irradiation, each of the samples was separated by
conventional methods using 12% SDS-polyacrylamide electro-
phoresis. Following SDS-PAGE, protein bands were elec-
trotransferred onto a PVDF membrane (Immobilon P, Milli-
pore) in 192 mM glycine, 25 mM Tris-HCl, 20% methanol, 0.1%
SDS. Running time was 12 h at 12 mA and a constant
temperature of 4 °C was maintained. The transferred mem-
brane was blocked for 1 h at room temperature with phosphate-
buffered saline with Tween (T-PBS: 0.1 M sodium phosphate,
pH 7.3, 0.15 M NaCl, 0.1% Tween-20) containing 2% skimmed
milk and then washed with T-PBS (5 min, two times). After
soaking the membrane in 1,500 times diluted streptavidins
horseradish peroxidase conjugate (Amersham) for 1 h at room
temperature, the membrane was developed using chemilumi-
nescent detection reagents (RENAISSANCE, DuPont NEN)
for 1 min. The membrane was then wrapped in a plastic sheet
and exposed to Hyperfilm-ECL (Amersham) in the dark. The
results are shown in Figures 2 and 3.
Ack n ow led gm en t. We are very grateful to Profes-
sor G. D. Holman (University of Bath, U.K.), Professor
P. Welzel (University of Leipzig, Germany), and Dr. N.
Nakajima (Toyama Prefect. University, J apan) for valu-
able advice throughout the preparation of the manu-
script. We thank the Ministry of Education, Science,
Sports and Culture, J apan (Grants-in-Aid for Scientific
Research 12470504, Grants-in-Aid for Scientific Re-
search on Priority Areas 08281105, 11121214), the
Fugaku Trust for Medicinal Research, The Naito Foun-
dation, and The Research Foundation for Pharmaceuti-
cal Sciences for the support of this work. U.K. is grateful
to the German Academic Exchange Service (DAAD) for
a postdoctoral fellowship.
J O000414A