Synthesis and evaluation of a photoactive probe with a multivalent carbohydrate for capturing carbohydrate-lectin interactions

Article abstract of DOI:10.1021/bc400306g

Lectins are ubiquitous carbohydrate-binding proteins of nonimmune origin that are characterized by their specific recognition of defined monosaccharide or oligosaccharide structures. However, the use of carbohydrates to study lectin has been restricted by the weak binding affinity and noncovalent character of the interaction between carbohydrates and lectin. In this report, we designed and synthesized a multifunctional photoaffinity reagent composed of a trialkyne chain, a masked latent amine group, and a photoreactive 3-trifluoromethyl-3- phenyl-diazirine group in high overall yield. Two well-defined chemistries, Huisgen-Sharpless click chemistry and amide bond coupling, were the key steps for installing the multivalent character and tag in our designed photoaffinity probe. The photolabeling results demonstrated that the designed probe selectively labeled the target lectin, RCA₁₂₀ (Ricinus communis Agglutinin), in an E. coli lysate and an asialoglycoprotein receptor (ASGP-R) on intact HepG2 cell membranes. Moreover, the probe also enabled the detection of weak protein-protein interactions between RCA₁₂₀ and ovalbumin (OVA).

Full text of DOI:10.1021/bc400306g

Article
pubs.acs.org/bc
Synthesis and Evaluation of a Photoactive Probe with a Multivalent
Carbohydrate for Capturing Carbohydrate−Lectin Interactions
Tsung-Che Chang,† Chian-Hui Lai,† Chih-Wei Chien,†,§ Chien-Fu Liang,† Avijit Kumar Adak,†
Yung-Jen Chuang,‡ Yu-Ju Chen,§ and Chun-Cheng Lin*,†
†Department of Chemistry and
‡Institute of Bioinformatics and Structural Biology, National Tsing Hua University, 101 Sec. 2, Kuang Fu Rd., Hsinchu 30013, Taiwan
§Institute of Chemistry, Academia Sinica, 128 Sec. 2, Academia Rd, Nankang Taipei 115, Taiwan
S
* Supporting Information
ABSTRACT: Lectins are ubiquitous carbohydrate-binding
proteins of nonimmune origin that are characterized by their
specific recognition of defined monosaccharide or oligosacchar-
ide structures. However, the use of carbohydrates to study lectin
has been restricted by the weak binding affinity and noncovalent
character of the interaction between carbohydrates and lectin. In
this report, we designed and synthesized a multifunctional
photoaffinity reagent composed of a trialkyne chain, a masked
latent amine group, and a photoreactive 3-trifluoromethyl-3-
phenyl-diazirine group in high overall yield. Two well-defined
chemistries, Huisgen-Sharpless click chemistry and amide bond
coupling, were the key steps for installing the multivalent
character and tag in our designed photoaffinity probe. The
photolabeling results demonstrated that the designed probe
selectively labeled the target lectin, RCA120 (Ricinus communis Agglutinin), in an E. coli lysate and an asialoglycoprotein receptor
(ASGP-R) on intact HepG2 cell membranes. Moreover, the probe also enabled the detection of weak protein−protein
interactions between RCA120 and ovalbumin (OVA).
monovalent carbohydrate ligand conjugated with a photo-
affinity probe may not be suitable for lectin labeling.
Multivalent carbohydrate structures, which greatly enhance
the weak affinity of individual monoligands to their binding
lectins, is a solution to overcome weak affinity.19−21 For
example, the asialoglycoprotein receptor (ASGP-R) in rat liver
membranes was photoaffinity-labeled by a multivalent YEE-
(ahGalNAc)3-glycoprobe containing three terminal GalNAc
residues.22,23 In addition, Shin and co-workers developed
photoaffinity probes possessing a trivalent fucose or mannose
structure and used them to specifically photolabel spiked
exogenous ConA from bacterial cell lysates of A. aurantia.24
Several photoaffinity functionalities are known and these
functionalities are stable in the absence of ultraviolet (UV)
radiation.25,26 The most notable feature is that these functional
groups can be appropriately incorporated and localized within
the proteins upon UV irradiation. However, native carbohy-
drate−lectin binding interactions may be impeded by the steric
bulk of the proximal photoaffinity groups,27 resulting in a low
efficiency of photolabeling and, consequently, hampering
INTRODUCTION
■
Carbohydrate−lectin interactions are involved in various
biological recognitions and mediate many processes including
cellular recognition,1 adhesion,2 signal transduction,3 glyco-
protein clearance,4 immunomodulation,5,6 inflammation,7 and
host−pathogen recognition.8 To fully understand the biological
implications of carbohydrate−lectin interactions in living
organisms, it is imperative to investigate and profile their
functions under physiological conditions. Moreover, a
quantitative assessment of the variations in lectins expressed
in different cell states and cell types is essential for elucidating
their biological roles in disease development. However, probing
carbohydrate interacting proteins is challenging due to the
properties of the carbohydrate−lectin interaction: it is non-
covalent, reversible, and weak (Kd in the mM range).9
Photoaffinity labeling was introduced in the 1960s10 and has
proven to be a powerful tool to probe proteins by attaching a
photoaffinity reagent to the corresponding small molecule
ligands.11−18 Although these photoaffinity probes worked very
well by forming covalent bonds with target proteins upon UV
irradiation, the success of labeling interacting proteins is highly
reliant on high-affinity (Kd in the nM range) interactions
between the probe and the protein. Thus, for the low affinity
interaction between a carbohydrate ligand and a lectin, a
Received: July 2, 2013
Revised: October 11, 2013
Published: October 23, 2013
© 2013 American Chemical Society
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a
Scheme 1. Convergent Synthesis of the Multifunctional Skeleton Precursor of the Multivalent Photoaffinity Probes
a
Reagents and conditions: (a) (i) TFA, DCM, r.t., 2 h, and then neutralization; (ii) block A, HBTU, DMF, Et3N, r.t., overnight; yield for two steps, n
= 1, 86%; n = 3, 78%; (b) (i) DBU, DCM, r.t., 1 h; (ii) block B, HBTU, DMF, Et3N, r.t., 12 h, yield for two steps, n = 1, 80%; n = 3, 85%; (c) (i)
LiOH, MeOH, H2O, r.t., 1 h, and then neutralization; (ii) block C, HBTU, DMF, Et3N, r.t., overnight, yield for two steps, n = 1, 72%; n = 3, 89%;
(d) (i) LiOH, MeOH, H2O, r.t., 1 h, and then neutralization; (ii) block D, HBTU, DMF, Et3N, r.t., overnight, yield for two steps, n = 1, 73%; n = 3,
81%. HBTU: O-benzotriazole-N,N,N’,N’-tetramethyl-uronium-hexafluoro-phosphate; DBU: 1,8-diazabicycloundec-7-ene.
purification and/or identification. As shown in Scheme 1 (see
the Supporting Information for details), we designed a
multifunctional photoaffinity reagent (10) derived from a
glutamic acid core and carrying a powerful photocross-linker, 3-
trifluoromethyl-3-phenyl-diazirine,25,26 and two well-defined
synthetic handles to enable subsequent conjugations. The
presence of a trialkyne in the probe could be simply and
smoothly used to assemble carbohydrate ligands by Cu(I)-
assisted click chemistry28 for efficient photolabeling. The
important multivalent ligand character of the probe could
strengthen some weak ligand/receptor interactions and
consequently increase the efficiency of covalent bond formation
between the probe and the interacting protein. Subsequently,
the amine of the probe could be ligated29 with a variety of tags
for visualization (e.g., fluorescence) or enrichment (e.g., biotin)
of the labeled proteins. Furthermore, in 10, the photoreactive
group is not close to the multivalent ligand, minimizing
negative effects on the ligand−receptor interaction.
Merck), 2-amino-2-(hydroxymethyl)-1,3-propanediol (Tris,
Acros), 6-aminohexanoic acid (Acros), Fmoc-L-glutamic acid
5-tert-butyl ester (Fmoc-Glu(OtBu)-OH, AnaSpec), suberic
acid bis-N-hydroxysuccinimide ester (DSS, Fluka), and ethanol-
amine (Acros) were used as received. Bovine serum albumin
(BSA, A9418), Ricinus communis agglutinin 120 (RCA120,
L7886), concanavalin A (ConA, C7642), wheat germ agglutinin
(WGA, L9640), and ovalbumin (OVA, A5378) were purchased
from Sigma-Aldrich (St. Louis, MO, USA). The BCA protein
assay kit was obtained from Pierce (Rockford, IL, USA). The P-
2 gel was purchased from Bio-Rad. Monoclonal antibiotin
antibody conjugated with HRP (A0185) was purchased from
Sigma-Aldrich. Polyclonal anti-ASGP1 (asialoglycoprotein
receptor 1) antibody (ARP33805_T100) and anti-ASGP2
(asialoglycoprotein receptor 2) antibody (ARP33820_P050)
were purchased from AVIVA systems biology. Donkey
antirabbit HRP-conjugated antibody (RPN510) was obtained
1
from GE Healthcare. H and 13C NMR spectra were recorded
on either a Bruker AV-400 or an AV-600 MHz NMR
instrument. Chemical shifts are expressed in ppm using residual
CDCl3 (7.24 ppm), CD3OD (3.30 ppm), or D2O (4.67 ppm at
298 K) as an internal standard. Multiplicities are reported using
the following abbreviations: s = singlet, d = doublet, t = triplet,
EXPERIMENTAL PROCEDURES
■
Materials and Methods. Dimethyl formaldehyde (DMF,
Merck), 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetra-methyluronium
hexafluorophosphate (HBTU, Merck), triethylamine (NEt3,
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q = quartet, m = multiplet, br = broad. J = coupling constant
values are expressed in Hertz. Low-resolution and high-
resolution mass spectra were recorded under ESI-TOF mass
spectroscopy conditions. Analytical thin-layer chromatography
(TLC) was performed on precoated plates (silica gel 60). Silica
gel 60 (E. Merck) was employed for all flash chromatography.
All reactions were performed in oven-dried glassware (120 °C)
under an atmosphere of argon unless indicated otherwise. All
solvents were dried and distilled by standard techniques.
Building Block A. The Fmoc-Glu-OMe was prepared by
following the procedures described in ref 30.
3.02 mmol) in dry DCM (6 mL) was added TFA (1.20 mL)
dropwise at 4 °C. After being stirred for 2.5 h at room
temperature, the mixture was then concentrated to dryness in
vacuo by coevaporation with toluene. The residue in MeOH
(10 mL) was neutralized with Dowex resin 550 (OH−) at 4 °C
followed by filtration and then concentration in vacuo to give
building block C as a colorless oil (770 mg, 99%). Rf 0.15
1
(MeOH/DCM = 1/9); H NMR (400 MHz, CD3OD): δ
1.29−1.42 (m, 4H), 1.46−1.53 (m, 2H), 1.57−1.69 (m, 6H),
2.19 (t, J = 7.4 Hz, 2H), 2.32 (t, J = 7.4 Hz, 2H), 2.90 (t, J = 7.6
Hz, 2H), 3.15 (t, J = 7.0 Hz, 2H), 3.64 (s, 3H); 13C NMR (100
MHz, CDCl3): δ 24.4, 25.0, 25.9, 26.3, 29.0, 33.7 (x2), 36.0,
39.1 (x2), 51.4, 173.2, 174.1. HRMS (ESI) m/z Calcd for
C13H27N2O3 [M+H]+ 259.2021; found: 259.2022.
Methyl 6-[(6-{[tert-Butoxycarbonyl]amino}hexanoyl)-
amino]hexanoate. Commercially available 6-[(tert-
butoxycarbonyl)amino]hexanoic acid (1.08 g, 4.67 mmol), N-
hydroxysuccinimide (537 mg, 4.67 mmol), and 1-ethyl-3-(3-
dimethylaminopropyl) carbodiimide hydrochloride (1.07 g,
5.61 mmol) were dissolved in dry dichloromethane (23 mL),
and the resulting mixture was stirred at room temperature
under a nitrogen atmosphere for 3 h, until TLC indicated
complete disappearance of starting material. The mixture was
partitioned between dichloromethane and H2O. The organic
layer was separated, dried over MgSO4, filtered, and
concentrated in vacuo to give the desired activated ester. To a
stirred solution of the activated ester in dry dichloromethane
(25 mL) was added 6-aminohexanoic acid (613 mg, 4.67
mmol) and DIPEA (1.63 mL, 9.34 mmol). After being stirred
at room temperature for 7 h, the mixture was concentrated to
dryness in vacuo. The residue was dissolved in dry DMF (10
mL), and then iodomethane (2.33 mL, 37.36 mmol) and
cesium carbonate (2.28 g, 7.00 mmol) were added at room
temperature. After being stirred at room temperature for 1.5 h,
the mixture was concentrated to dryness in vacuo. The above
residue was dissolved in ethyl acetate and washed with H2O.
The organic layer was dried over MgSO4, filtered, and
concentrated in vacuo. The residue was purified by column
chromatography on silica gel (EtOAc/hexane = 1/1) to give
the title compound (1.22 g, 73% for three steps) as a colorless
N-tert-Butoxycarbonyl-1-[4-[3-(trifluoromethyl)-3H-di-
azirin-3-yl]phenoxy]carbonyl-3-propane-diamine. [4-[3-
(Trifluoromethyl)-3H-diazirin-3-yl]phenoxy]acetic acid31,32
(160 mg, 0.615 mmol), commercially available N-Boc-1,3-
propanediamine (161 mg, 0.923 mmol), and HBTU (280 mg,
0.738 mmol) were dissolved in dry DMF (6 mL) and treated
with triethylamine (129 μL, 0.923 mmol) at room temperature.
After being stirred at room temperature under a nitrogen
atmosphere for 8 h, the mixture was concentrated to dryness in
vacuo, and ethyl acetate was added. The organic layer was
washed with brine, dried over MgSO4, filtered, and
concentrated in vacuo to provide the crude product. The
residue was purified by column chromatography on silica gel
(EtOAc/hexane = 1/1) to give the desired product as a yellow
1
oil (210 mg, 80%). Rf 0.26 (EtOAc/Hexane = 1/1); H NMR
(400 MHz, CDCl3): δ 1.37 (s, 9H), 1.57−1.63 (m, 2H), 3.11
(q, J = 6.1 Hz, 2H), 3.34 (q, J = 6.3 Hz, 2H), 4.45 (s, 2H), 4.88
(br, 1H), 6,93 (d, J = 8.7 Hz, 2H), 7.13(d, J = 8.7 Hz, 2H), 7.35
2
(br, 1H); 13C (100 MHz, CDCl3): δ 28.1 (q, JCF = 32 Hz),
1
28.3, 30.1, 35.3, 36.9, 67.2, 79.4, 115.1, 122.1 (q, JCF = 272
Hz), 122.3, 128.3, 156.6, 158.3, 167.8. HRMS (ESI) m/z Calcd
for C18H23F3N4O4Na [M+Na]+ 439.1569; found: 439.1565.
Building Block D. To a solution of the above coupling
product (210 mg, 0.505 mmol) in dry DCM (4 mL) was added
TFA (1 mL) dropwise at 4 °C. After being stirred at room
temperature for 45 min, the mixture was concentrated to
dryness in vacuo by coevaporation with toluene. The residue in
MeOH (5 mL) was neutralized with Dowex resin 550 (OH−)
at 4 °C and then filtered and concentrated in vacuo to give
building block D as a yellow oil (153 mg, 96%). Rf 0.15
1
oil. Rf 0.3 (EtOAc/Hexane = 1/1); H NMR (400 MHz,
CDCl3): δ 1.24−1.33 (m, 4H), 1.39 (s, 9H), 1.42−1.50 (m,
4H), 1.55−1.63 (m, 4H) 2.11 (t, J = 7.6 Hz, 2H), 2.27 (t, J =
7.4 Hz, 2H), 3.05 (q, J = 6.6 Hz, 2H), 3.19 (q, J = 7.0 Hz, 2H),
3.61 (s, 3H); 13C NMR (100 MHz, CDCl3): δ 24.0, 24.9, 25.8,
25.9, 27.9, 28.7, 29.2, 33.3, 35.8, 38.6, 39.8, 50.9, 78.1, 155.6,
172.7, 173.5. HRMS (ESI) m/z Calcd for C18H35N2O5 [M
+H]+ 359.2546; found: 359.2556.
1
(EtOAc/Hexane = 1/1 containing 10% MeOH); H NMR
Building Block B. To a solution of methyl 6-[(6-{[tert-
butoxycarbonyl]amino}hexanoyl)amine]-hexanate (4.50 g,
12.52 mmol) in MeOH (12.5 mL)/H2O (12.5 mL) was
added LiOH (301.5 mg, 12.57 mmol) at room temperature.
After being stirred at room temperature for 0.5 h, the mixture
was neutralized with Amberlite IR-120 (H+) resin and then
filtered and concentrated in vacuo to give building block B as a
colorless oil (4.29 g, 99%), which was used in the next step
without further purification. Rf 0.65 (MeOH/DCM = 1/9); 1H
NMR (400 MHz, CD3OD): δ 1.29−1.39 (m, 4H), 1.42 (s,
9H), 1.44−1.54 (m, 4H), 1.56−1.65 (m, 4H), 2.16 (t, J = 7.6
Hz, 2H), 2.28 (t, J = 7.4 Hz, 2H), 2.98−3.04 (m, 2H), 3.13−
3.18 (m, 2H); 13C NMR (100 MHz, CD3OD): δ 25.8, 26.6,
27.3, 27.4, 28.8, 30.0, 30.5, 35.4, 36.9, 40.1, 41.1, 79.7, 158.3,
175.8, 178.2. HRMS (ESI) m/z Calcd for C17H33N2O5 [M+H]
(400 MHz, CD3OD): δ 1.83−1.90 (m, 2H), 2.92 (t, J = 7.3 Hz,
2H), 3.37 (t, J = 6.6 Hz, 2H), 4.58 (s, 2H), 7.08 (d, J = 8.8 Hz,
2H), 7.23 (d, J = 8.8 Hz, 2H). 13C NMR (100 MHz, CD3OD):
δ 29.2 (q, 2J CF = 40.2 Hz), 29.6, 37.3, 38.0, 68.1, 116.5, 122.8,
1
123.6 (q, J = 273.0 Hz), 129.4, 160.4, 170.7. HRMS (ESI)
CF
m/z Calcd for C13H16F3N4O2 [M+H]+ 317.1225; found:
317.1234.
1-Azido-3,6-dioxaoct-8-yl 2,3,4,6-tetra-O-acetyl-β-D-
galactopyranoside (β-D-Galactoside 15). The β-D-Galacto-
side 15 was prepared by following the procedure described in
ref 33.
N-(tert-Butyloxycarbonyl)-2-(propargyloxy)-
aminoethane (1). To a solution of ethanolamine (3.05 g, 50
mmol) in dioxane (50 mL) was added (Boc)2O (16.42 g, 75
mmol), and the resulting mixture was stirred at room
temperature overnight. After the complete absence of starting
material (as judged by TLC), the volatiles were removed under
reduced pressure, and the residue was redissolved in 30 mL of
+
345.2389; found: 345.2390.
Building Block C. To a solution of methyl 6-[(6-{[tert-
butoxycarbonyl]amino}hexanoyl)amino]-hexaneate (1.08 g,
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CH2Cl2. The solution was washed successively with 1% HCl
(60 mL), brine (2 × 30 mL), and H2O (30 mL), then dried
over MgSO4, filtered, and concentrated to yield N-(tert-
butoxycarbonyl)ethanolamine, which was used directly in the
subsequent steps. 1H NMR (400 MHz, CDCl3): δ 1.42 (s, 9H),
2.50 (br, 1H), 3.26 (q, J = 5.2 Hz, 2H), 3.67 (q, J = 4.5 Hz,
2H), 4.97 (br, 1H). HRMS (FAB) m/z Calcd for C7H16NO3
[M+H]+ 162.1130; found: 162.1129. A solution of N-(tert-
butoxycarbonyl)ethanolamine (1.60 g, 9.938 mmol) and
propargyl bromide (80 wt % in toluene, 2.13 mL, 19.87
mmol) in dry DMF (10.7 mL) was stirred at 0 °C. Portions of
finely ground KOH (1.11 g, 19.87 mmol) were added over a
period of 15 min. The mixture was then heated to 35 °C and
stirred for 16 h under a nitrogen atmosphere. The mixture was
quenched with H2O and extracted with ethyl acetate. The
organic layer was washed with brine, dried over MgSO4,
filtered, and concentrated in vacuo to give the crude product.
The residue was purified by column chromatography on silica
gel (EtOAc/hexane = 1/10) to give compound 1 (1.23 g, 63%)
as a yellow oil. Rf 0.50 (EtOAc/Hexane = 1/2); 1H NMR (400
MHz, CDCl3): δ 1.42 (s, 9H), 2.42 (t, J = 2.4 Hz, 1H), 3.31 (q,
J = 5.0 Hz, 2H), 3.56 (t, J = 5.0 Hz, 2H), 4.13 (d, J = 2.4 Hz,
2H); 13C NMR (100 MHz, CDCl3): δ 28.7, 41.0, 58.8, 69.6,
75.9, 80.0, 80.5, 158.3. HRMS (FAB) m/z Calcd for
C10H18NO3 [M+H]+ 200.1287; found: 200.1291.
5.69 (d, J = 8.0 Hz, 1H), 5.99 (br, 1H), 7.30 (t, J = 7.4 Hz, 2H),
7.38 (t, J = 7.4 Hz, 2H), 7.59 (d, J = 7.2 Hz, 2H), 7.74 (d, J =
7.4 Hz, 2H); 13C NMR (100 MHz, CDCl3): δ 28.3, 32.8, 47.0,
52.3, 53.2, 58.5, 59.2, 66.8, 68.2, 74.6, 79.4, 119.8, 125.0, 126.9,
127.5, 141.1, 143.5, 143.7, 156.1, 171.8, 172.4. HRMS (ESI) m/
z Calcd for C34H36N2NaO8 [M+Na]+ 623.2369; found:
623.2368.
Compound 5. To a solution of compound 3 (2.79 g, 6.03
mmol) in dry DCM (20 mL) was added 8-diazabicycloundec-7-
ene (1.8 mL, 12.06 mmol) at room temperature. After being
stirred at room temperature for 1 h, the mixture was
concentrated to dryness in vacuo. The residue was purified by
silica gel column chromatography (EtOAc/hexane = 1/1 + 20%
MeOH) to afford the desired product. The above compound,
building block B (2.07 g, 6.03 mmol), and HBTU (2.74 g, 7.24
mmol) were dissolved in dry DMF (30 mL), and triethylamine
(1.26 mL, 9.05 mmol) was added at room temperature. After
being stirred under a nitrogen atmosphere at room temperature
for 12 h, the mixture was concentrated to dryness in vacuo and
extracted with ethyl acetate. The organic layer was washed with
brine, dried over MgSO4, filtered, and concentrated in vacuo to
give the crude product. The residue was purified by column
chromatography on silica gel (EtOAc/hexane = 1/1 + 10%
MeOH) to give compound 5 (2.74 g, 80%) as a white syrup. Rf
1
0.50 (EtOAc/Hexane = 1/1 + 15% MeOH); H NMR (400
MHz, CDCl3): δ 1.25−1.36 (m, 4H), 1.39 (s, 9H), 1.43−1.51
(m, 4H), 1.56−1.65 (m, 4H), 2.11−2.30(m, 7H), 2.43 (t, J =
2.4 Hz, 1H), 3.06 (q, J = 6.4 Hz, 2H), 3.21 (q, J = 6.4 Hz, 2H),
3.36−3.51 (m, 2H), 3.57 (t, J = 5.2 Hz, 2H), 3.70 (s, 3H), 4.12
(d, J = 2.4 Hz, 2H), 4.47−4.52 (m, 1H), 4.66 (br, 1H), 5.87
(br, 1H), 6.49 (br, 1H), 6.88 (br, 1H); 13C NMR (100 MHz,
CDCl3): δ 24.7, 25.1, 25.9, 26.1, 27.1, 28.1, 28.8, 29.3, 29.5,
32.0, 35.6, 36.1, 38.8, 38.9, 40.1, 51.8, 52.1, 57.9, 68.1, 74.7,
78.6, 79.1, 155.9, 172.2, 172.3, 173.0, 173.2. HRMS (ESI) m/z
Calcd for C28H48N4O8Na [M+Na]+ 591.3363; found 591.3370.
Compound 6. The synthesis of compound 6 is similar as
described in the synthesis of compound 5. The crude
compound was purified by column chromatography in silica
gel (Hexane/Acetone = 1/1) to give compound 6 (yield: 85%)
as white syrup Rf 0.3 (EtOAc/Hexane = 1/1 + 10% MeOH);
1H NMR (400 MHz, CDCl3): δ 1.26−1.37 (m, 4H), 1.40 (s,
9H), 1.44−1.52 (m, 4H), 1.89−1.98 (m, 1H), 2.07−2.31 (m,
7H), 2.42 (t, J = 2.4 Hz, 3H), 3.07 (q, J = 6.6 Hz, 2H), 3.22 (q,
J = 6.8 Hz, 2H), 3.71 (s, 3H), 3.81 (dd, J = 9.2, 14.0 Hz, 6H),
4.12 (d, J = 2.4 Hz, 6H), 4.50−4.55 (m, 1H), 4.62 (br, 1H),
5.77 (br, 1H), 6.70 (br, 1H); 13C NMR (100 MHz, CDCl3): δ
24.8, 25.3, 26.1, 26.3, 27.6, 28.3, 29.0, 29.7, 33.0, 35.9, 36.4,
39.0, 40.3, 51.8, 52.4, 58.6, 59.4, 68.3, 74.7, 79.4, 156.0, 172.3,
172.6, 172.9, 173.2. HRMS (ESI) m/z Calcd for
C36H56N4NaO10 [M+Na]+ 727.3894; found: 727.3900.
Compound 7. To a solution of compound 5 (900 mg,
1.583 mmol) in MeOH (10 mL)/H2O (10 mL) was added
LiOH (37.9 mg, 1.583 mmol) at room temperature. After being
stirred at room temperature for 1 h, the mixture was neutralized
with Amberlite IR-120 (H+) resin, then filtered and
concentrated in vacuo to afford the acid as a white solid. The
above acid, building block C (766 mg, 2.058 mmol), and
HBTU (900 mg, 2.374 mmol) were dissolved in dry DMF (7.9
mL) and treated with triethylamine (440 μL, 3.166 mmol) at
room temperature under a nitrogen atmosphere. After being
stirred overnight at room temperature, the reaction was
concentrated to dryness in vacuo, and ethyl acetate was
added. The organic layer was washed with brine, dried over
N-(tert-Butyloxycarbonyl)tris[(propargyloxy)methyl]-
aminomethane (2). The compound 2 was prepared
according to the procedure described in ref 34.
Compound 3. To a solution of compound 1 (1.20 g, 6.03
mmol) in dry DCM (10 mL) was added TFA dropwise (2.5
mL) at 4 °C. After being stirred at room temperature for 2 h,
the mixture was concentrated to dryness in vacuo by
coevaporation with toluene. The residue was dissolved in
MeOH (12 mL) and neutralized with Dowex resin 550 (OH−)
at 4 °C, followed by filtration and concentration in vacuo to give
a yellow oil. To a solution of the above compound (598 mg),
building block A (2.30 g, 6.03 mmol), and HBTU (3.43 g, 9.05
mmol) in dry DMF (6 mL) was added triethylamine (1.68 mL,
12.06 mmol) at room temperature under a nitrogen
atmosphere. After being stirred at room temperature overnight,
the mixture was concentrated to dryness in vacuo, and ethyl
acetate was added. The organic layer was washed with brine,
dried over MgSO4, filtered, and then concentrated in vacuo to
give the crude product. The residue was purified by column
chromatography on silica gel (EtOAc/hexane = 1/2) to give
compound 3 (2.40 g, 86%) as a white syrup. Rf 0.42 (EtOAc/
Hexane = 1/1); 1H NMR (400 MHz, CDCl3): δ 1.92−2.05 (m,
1H), 2.17−2.26 (m, 3H), 2.41 (t, J = 2.4 Hz, 1H), 3.39−3.50
(m, 2H), 3.56 (t, J = 5.0 Hz, 2H), 3.72 (s, 3H), 4.11 (d, J = 2.4
Hz, 2H), 4.19 (t, J = 7.0 Hz, 1H), 4.32−4.40 (m, 3H), 5.79 (d,
J = 8.0 Hz, 1H), 6.16 (br, 1H), 7.29 (t, J = 7.4 Hz, 2H), 7.38 (t,
J = 7.4 Hz, 2H), 7.58 (dd, J = 5.0, 7.0 Hz, 2H), 7.74(d, J = 7.4
Hz, 2H); 13C NMR (100 MHz, CDCl3): δ 28.2, 32.3, 39.2,
47.1, 52.5, 53.4, 58.2, 66.9, 68.5, 74.8, 79.3, 120.0, 125.0, 127.0,
127.6, 141.2, 143.6, 143.8, 156.2, 171.9, 172.4. HRMS (ESI) m/
z Calcd for C26H28N2NaO6 [M+Na]+ 487.1845; found:
487.1836.
Compound 4. The synthesis of compound 4 is similar to
the synthesis of compound 3. Compound 4: White syrup
1
(yield: 78%). Rf 0.40 (EtOAc/Hexane = 1/1); H NMR (400
MHz, CDCl3): δ 1.90−1.97 (m, 1H), 2.16−2.24 (m, 3H), 2.39
(t, J = 2.4 Hz, 3H), 3.73 (s, 3H), 3.79−3.86 (m, 6H), 4.11 (d, J
= 2.4 Hz, 6H), 4.21 (t, J = 6.8 Hz, 1H), 4.34−4.43 (m, 3H),
1898
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Bioconjugate Chemistry
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MgSO4, filtered, and concentrated in vacuo to give the crude
product. The residue was purified by column chromatography
on silica gel (EtOAc/hexane = 1/1 + 15% MeOH) to give
compound 7 (873 mg, 72%) as a white syrup. Rf 0.25 (EtOAc/
Hexane = 1/1 + 20% MeOH); 1H NMR (600 MHz, CD3OD):
δ 1.28−1.36 (m, 8H), 1.41 (s, 9H), 1.44−1.53 (m, 8H), 1.57−
1.65 (m, 8H), 1.84−1.91 (m, 1H), 2.00−2.07 (m, 1H), 2.85 (t,
J = 1.5 Hz, 1H), 3.01 (t, J = 4.7 Hz, 1H), 3.12−3.18 (m, 6H),
3.32−3.43 (m, 2H), 3.54−3.58 (m, 2H), 3.64 (s, 3H), 4.15 (d, J
= 1.5 Hz, 2H), 4.24−4.30 (m, 1H); 13C NMR (150 MHz,
CD3OD): δ 25.6, 26.4, 26.6 (x2), 26.7 27.4, 27.5, 28.8, 29.1,
30.0 (x2), 30.1, 30.6, 33.2, 34.6, 36.6, 36.9, 37.0, 40.1 (x2), 40.2,
41.2, 51.9, 54.3, 58.8, 58.9, 69.2, 76.0, 79.7, 80.5, 158.5,
173.8,174.8,175.7, 175.9 (x2), 176.0. HRMS (ESI) m/z Calcd
for C40H70N6NaO10 [M+Na]+ 817.5051; found: 817.5049.
Compound 8. Compound 8 was prepared similarly as
described in the synthesis of compound 7. The residue was
purified by column chromatography in silica gel (EtOAc/
Hexane = 1/1 + 17% MeOH) to give compound 8 (yield:
89%) as white syrup. Rf 0.55 (EtOAc/Hexane = 1/1 + 20%
Hz, 2H); 13C NMR (150 MHz, CDCl3): δ 24.9, 25.0, 25.1,
25.2, 26.1, 26.2, 27.9 (q, JCF = 40 Hz), 28.1, 28.2, 28.7, 28.9,
2
29.4, 29.5, 33.0, 35.6, 35.7, 35.8, 36.0, 36.1, 38.9, 39.0, 40.2,
52.6, 58.5, 59.3, 67.0, 68.2, 74.8, 78.8, 79.3, 115.0, 121.9 (q, 1JCF
= 273 Hz), 122.0, 128.1, 156.0, 158.1, 167.9, 171.6, 172.6,
173.1, 173.2, 173.4, 173.7. HMRS (ESI) m/z Calcd for
C60H89F3N10NaO13 [M+Na]+ 1237.6460; found: 1237.6461.
Compound 11. To a solution of compound 9 (182 mg,
0.169 mmol) in EtOH/H2O/t-BuOH (5.6 mL; 3:2:5 = v/v/v)
was added 1-azido-3,6-dioxaoct-8-yl 2,3,4,6-tetra-O-acetyl-β-D-
galacto-pyranoside (94 mg, 0.186 mmol) and DIPEA (27 μL,
0.169 mmol) at room temperature. The mixture was stirred for
5 min, and then copper iodide (3 mg, 0.0169 mmol), a
saturated solution of copper(II) sulfate (49 μL), and sodium
ascorbate (6.7 mg, 0.0338 mmol) were added. The mixture was
stirred at 35 °C for another 6 h. The solvent was concentrated
to dryness in vacuo, and the resulting residue was purified by
column chromatography on silica gel (MeOH/DCM = 1/10)
to give compound 11 (193 mg, 72%) as a yellow syrup. Rf 0.45
1
(MeOH/DCM = 1/9); H NMR (400 MHz, CD3OD): δ
1
MeOH); H NMR (400 MHz, CD3OD): δ 1.27−1.37 (m,
1.26−1.34 (m, 8H), 1.41 (s, 9H), 1.44−1.53 (m, 8H), 1.56−
1.71 (m, 10H), 1.84−1.90 (m, 1H), 1.93 (s, 3H), 1.98−2.05
(m, 1H), 2.01 (s, 3H), 2.02 (s, 3H), 2.11 (s, 3H), 2.12−2.20
(m, 6H), 2.22−2.27 (m, 4H), 3.01 (q, J = 6.6 Hz, 2H), 3.12−
3.18 (m, 8H), 3.21−3.28 (m, 2H), 3.35−3.44 (m, 2H), 3.51−
3.58 (m, 8H), 3.67−3.75 (m, 1H), 3.87−3.93 (m, 3H), 4.08−
4.15 (m, 3H), 4.22−4.31 (m, 1H), 4.54 (s, 2H), 4.57−4.61 (m,
4H), 4.68 (d, J = 7.5 Hz, 1H), 5.04−5.14 (m, 2H), 5.37 (d, J =
3.2 Hz, 1H), 7.08 (d, J = 8.8 Hz, 2H), 7.22 (d, J = 8.8 Hz, 2H),
7.94 (s, 1H), 8.04 (s, 1H); 13C NMR (150 MHz, CD3OD): δ
8H), 1.42 (s, 9H), 1.45−1.54 (m, 9H), 1.58−1.65 (m, 8H),
1.83−1.91 (m, 1H), 1.96−2.04 (m, 1H), 2.16 (t, J = 7.5 Hz,
4H), 2.24 (t, J = 7.3 Hz, 4H), 2.32 (t, J = 7.3 Hz, 4H), 2.83 (t, J
= 2.3 Hz, 3H), 2.99−3.04 (m, 2H), 3.12−3.19 (m, 6H), 3.64 (s,
3H), 3.75−3.80 (m, 6H), 4.14 (d, J = 2.3 Hz, 6H), 4.21−4.26
(m, 1H); 13C NMR (100 MHz, CD3OD): δ 25.5, 26.3, 26.6
(x2), 27.3, 27.5, 28.8, 29.1, 29.9, 30.0 (x2), 30.6, 33.6, 34.6,
36.6, 36.9, 40.0, 40.1, 40.2, 41.1, 52.0, 54.2, 59.4, 60.9 (x2),
69.0, 76.0, 79.7, 80.5, 158.3, 173.7, 174.8, 175.6, 175.8 (x3).
HMRS (ESI) m/z Calcd for C48H78N6NaO12 [M+Na]+
953.5575; found: 953.5572.
20.5, 20.6, 20.8, 26.4, 26.6, 26.7, 27.4, 27.5, 28.8, 29.0 (q, 2JCF
=
39 Hz), 29.1, 29.2, 30.0, 30.1, 30.3, 30.6, 33.1, 33.2, 36.7, 37.0,
37.4, 37.5, 40.1, 40.3, 41.2, 43.8, 51.4, 54.2, 54.3, 55.8, 62.5,
64.7, 68.2, 68.8, 69.7, 69.8, 70.3, 70.4, 71.3, 71.4, 71.5, 72.3,
79.7, 102.3, 116.6, 122.8, 123.6 (q, 1JCF = 272 Hz), 125.9, 129.4,
145.6, 158.5, 160.4, 170.5, 171.3, 171.4, 171.9, 172.0, 173.8,
174.7, 175.9 (x3), 176.2. HMRS (ESI) m/z Calcd for
C72H112F3N13NaO23 [M+Na]+ 1606.7844; found: 1606.7844.
Compound 12. Compound 12 was prepared by following
the procedures as described in the synthesis of compound 11.
The residue was purified by column chromatography in silica
gel (MeOH/DCM = 1/8) to give compound 12 (157 mg,
70%) as yellow syrup. Rf 0.43 (MeOH/DCM = 1/9); 1H NMR
(400 MHz, CD3OD): δ 1.28−1.36 (m, 8H), 1.41 (s, 9H),
1.44−1.51 (m, 8H), 1.56−1.69 (m, 10H), 1.77−1.85 (m, 1H),
1.86−1.97 (m, 1H), 1.93 (s, 6H), 1.94 (s, 3H), 2.01 (s, 18H),
2.11−2.24 (m, 19H), 2.99−3.05 (m, 4H), 3.12−3.18 (m, 8H),
3.50−3.71 (m, 26H), 3.75−3.80 (m, 2H), 3.85−4.00 (m, 9H),
4.10−4.21 (m, 9H), 4.24−4.47 (m, 6H), 4.53−4.60 (m, 8H),
4.67−4.70 (m, 3H), 5.04−5.15 (m, 6H), 5.37−5.39 (m, 3H),
7.04 (dd, J = 8.8, 14.2 Hz, 1H), 7.09 (d, J = 8.8 Hz, 1H), 7.23
(d, J = 8.8 Hz, 1H), 7.41 (dd, J = 8.8, 14.2 Hz, 1H), 7.94−8.00
(m, 3H); 13C NMR (150 MHz, CD3OD): δ 20.5, 20.6, 20.7,
Compound 9. Compound 9 was prepared by following the
procedures as described in the synthesis of compound 7. The
residue was purified by column chromatography in silica gel
(MeOH/DCM = 1/12) to give compound 9 (yield: 73%) as
1
lightly yellow syrup. Rf 0.50 (MeOH/DCM = 1/9); H NMR
(400 MHz, CD3OD): δ 1.27−136 (m, 8H), 1.41 (s, 9H),
1.44−1.51 (m, 8H), 1.55−1.69 (m, 10H), 1.83−1.92 (m, 1H),
1.98−2.08 (m, 1H), 2.14−2.20 (m, 6H), 2.22−2.28 (m, 4H),
2.85 (t, J = 2.3 Hz, 1H), 2.98−3.03 (m, 2H), 3.12−3.18 (m,
8H), 3.26−3.40 (m 4H), 3.56 (t, J = 5.5 Hz, 2H), 4.14 (d, J =
2.3 Hz, 2H), 4.24−4.31 (m, 1H), 4.54 (s, 2H), 7.08 (d, J = 8.8
Hz, 2H), 7.22 (d, J = 8.8 Hz, 2H); 13C NMR (150 MHz,
CD3OD): δ 26.3, 26.5, 26.6, 26.7, 27.3, 27.4, 27.5, 28.8, 29.1,
2
29.2 (q, JCF = 40 Hz), 29.9, 30.0, 30.2, 30.5, 33.0, 33.1, 36.6,
36.9, 37.3, 37.4, 40.1, 40.2, 41.1, 49.8, 54.1, 54.2, 58.8, 68.1, 69.0
1
69.1, 76.0, 76.1, 79.6, 80.5, 116.5, 122.6, 123.5 (q, JCF = 272
Hz), 129.3, 158.3, 160.3, 170.3, 173.7, 174.7, 175.8 (x3), 176.0.
HMRS (ESI) m/z Calcd for C52H81F3N10NaO11 [M+Na]+
1101.5936; found: 1101.5945.
Compound 10. Compound 10 was prepared by following
the procedures as described in the synthesis of compound 7.
The residue was purified by column chromatography in silica
gel (EtOAc/Hexane = 1/1 + 15−30% MeOH) to give
compound 10 (yield: 81%) as light yellow syrup. Rf 0.50
2
20.8, 26.4, 26.6, 26.7, 27.4, 27.5, 28.8, 29.0 (q, JCF = 38 Hz),
29.2, 30.0, 30.1, 30.3, 30.6, 30.7, 33.6, 36.7, 37.0, 37.4, 37.5,
40.2, 41.2, 50.5, 51.4, 51.5, 54.4, 62.5, 62.6, 64.1, 64.3, 65.2,
1
(EtOAc/Hexane = 1/1 + 20% MeOH); H NMR (400 MHz,
68.1, 68.2, 68.8, 70.2, 70.3, 70.4, 71.1, 71.2, 71.4, 71.5, 71.8,
CD3OD): δ 1.27−1.36 (m, 8H), 1.41 (s, 9H), 1.45−1.53 (m,
8H), 1.56−1.71 (m, 10H), 1.81−1.91 (m, 1H), 1.95−2.03 (m,
1H), 2.14−2.20 (m, 6H), 2.22−2.27 (m, 4H), 2.83 (t, J = 2.3
Hz, 3H), 3.01 (t, J = 7.0 Hz, 2H), 3.13 (m, 8H), 3.27−3.29 (m,
2H), 3.75−3.80 (m, 6H), 4.13 (d, J = 2.3 Hz, 6H), 4.23−4.26
(m, 1H), 4.54 (s, 2H), 7.08 (d, J = 8.8 Hz, 2H), 7.22 (d, J = 8.8
1
72.3, 79.8, 102.2, 115.7, 115.9, 116.6, 122.8, 123.6 (q, JCF
=
273 Hz), 125.2, 129.4, 130.2, 147.6, 158.5, 160.4, 170.5, 171.3
(x3), 171.5 (x3), 171.9 (x3), 172.0 (x3), 173.7, 174.7, 175.9
(x3), 176.2. HMRS (ESI) m/z Calcd for C120H182F3N19NaO49
[M+Na]+ 2753.2184; found: 2753.2190.
1899
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Bioconjugate Chemistry
Article
Compound 13. To a solution of compound 11 (133 mg,
0.084 mmol) in dry DCM (2 mL) was added TFA (0.6 mL)
dropwise at 4 °C. After being stirred at room temperature for
1.5 h, the mixture was concentrated to dryness in vacuo by
coevaporation with toluene. The residue in MeOH (12 mL)
was neutralized with Dowex resin 550 (OH−) at 4 °C, filtered,
and concentrated in vacuo to give a crude product, which was
used in the next reaction without purification. The above
material and biotin-OSu35 (42.9 mg, 0.126 mmol) were
dissolved in dry DMF (2 mL), and Et3N (17.5 μL, 0.126
mmol) was added. The reaction was stirred at room
temperature for 12 h under a nitrogen atmosphere. After the
reaction was complete (as judged by TLC), the mixture was
concentrated to dryness in vacuo by coevaporation with
toluene. The resulting residue was dissolved in MeOH (4
mL) and stirred with NaOMe (13.6 mg, 0.252 mmol) at room
temperature. After 1 h at room temperature, the reaction
mixture was neutralized with Amberlite IR-120 (H+) resin,
filtered, and concentrated in vacuo to give the crude product.
The residue was purified by Bio-Gel P2 size exclusion
chromatography to give compound 13 as a colorless syrup
(60 mg, 46% overall yield for three steps). Rf 0.22 (MeOH/
DCM = 1/2); 1H NMR (600 MHz, CD3OD): δ 1.29−1.36 (m,
8H), 1.39−1.45 (m, 2H), 1.47−1.52 (m, 10H), 1.57−1.75 (m,
12H), 1.84−1.90 (m, 1H), 1.99−2.05 (m, 1H), 2.14−2.19 (m,
8H), 2.22−2.27 (m, 4H), 2.69 (d, J = 12.7 Hz, 1H), 2.91 (dd, J
= 5.0 Hz, 12.7 Hz, 1H), 3.13−3.21 (m, 12H), 3.28 (dd, J = 9.4
Hz, 16.2 Hz, 1H), 3.37(t, J = 5.4 Hz, 2H), 3.45−3.53 (m, 3H),
3.55−3.58 (m, 2H), 3.60 (s, 4H), 3.62−3.64 (m, 3H), 3.67−
3.76 (m, 3H), 3.82 (d, J = 3.3 Hz, 1H), 3.89 (t, J = 5.0 Hz, 2H),
3.96−3.99 (m, 1H), 4.24−4.30 (m, 3H), 4.48 (dd, J = 5.0, 8.0
Hz, 1H), 4.54 (s, 2H), 4.58 (t, J = 5.0 Hz, 2H), 4.61 (s, 2H),
7.01−7.06 (m, 1H), 7.08 (d, J = 8.8 Hz, 1H), 7.22 (d, J = 8.8
Hz, 1H), 7.38−7.43 (m, 1H), 8.04 (s, 1H); 13C NMR (150
MHz, CD3OD): δ 26.4, 26.6, 26.7, 26.9, 27.5, 27.6, 29.0 (q, 2JCF
= 39 Hz), 29.2, 29.5, 29.7, 30.0, 30.1, 30.3, 30.4, 30.7, 33.0,
33.2, 36.7, 36.8, 37.0, 37.4, 37.5, 40.2, 40.3, 40.4, 41.0, 51.4,
54.4, 57.0, 61.6, 62.5, 63.4, 64.7, 68.2, 69.6, 69.8, 70.3, 71.3,
176.2. HMRS (MALDI-TOF, Matrix: CHCA (10 mg/mL)
0.1% TFA and 50% acetonitrile) m/z Calcd for
C
101H164F3N21NaO37S [M+Na]+ 2375.11; found: 2375.12.
Labeling of RCA120 by the Trivalent (14) Galactose
Photoaffinity Probe. RCA120 (0.1 μM, 500 μL in PBS buffer,
pH 7.4) was incubated with various concentrations of
compound 14 (25, 50, 75, and 100 μM). The mixture was
briefly agitated at 4 °C for 30 min to ensure proper mixing and
then irradiated with a UV lamp (365 nm, 2.76 mW/cm2)
(Panchum, photochemical reactor, PR-2000) at 4 °C for 30
min. Excess trigalactose photoaffinity probe was removed from
the reaction via spin concentration (Microcon centrifugal filter
10,000 MWCO, Millipore, MA). An aliquot of this purified
mixture was treated with SDS-PAGE loading buffer (0.3 M
Tris-Cl, 10% SDS, 30% glycerol, 9.3% DTT, pH 6.8) and
heated at 95 °C for 10 min followed by electrophoresis.
Samples were visualized by Coomassie blue staining and
verified by Western blotting. Negative control experiments
were performed as described above without UV irradiation.
Labeling of RCA120 by Monovalent (13) Galactose
Photoaffinity Probe. RCA120 (0.1 μM, 500 μL in PBS buffer
at pH 7.4) was incubated with various concentrations of
compound 13 (12.5, 25, 50, 100, and 200 μM). The
subsequent procedure was the same as that described above.
Selective Labeling of RCA120 by Trivalent Galactose
Photoaffinity Probe (14) in a Protein Mixture. A 1.7-mL
Eppendorf tube was charged with a solution of protein mixture
I (OVA, BSA, ConA, RCA120, and WGA) or II (BSA, ConA,
RCA120, and WGA) (0.1 μM for each protein, 500 μL in PBS
buffer at pH 7.4) and a solution of compound 14 (final 50 μM).
The subsequent procedure was the same as previously
described. A negative control experiment was performed by
omitting the irradiation step.
Synthesis of Magnetic Nanoparticles. The core Fe3O4
particles (MNPs) and the amine-functionalized MNPs (NH2-
MNPs) were prepared following reported procedures.36
Synthesis of Streptavidin-MNP (Streptavidin-Conju-
gated MNP). NH2-MNPs (10 mg) and DSS (50 mg) were
mixed in DMSO (500 μL), and the resulting mixture was gently
rotated at 37 °C for 6 h. After magnetic separation, the
supernatant was discarded, and the MNPs were washed three
times with DMSO, ddH2O, and PBS (pH 7.4) to give (OSu)-
activated MNPs. To conjugate streptavidin with MNP, the
(OSu)-activated MNPs were dispersed in PBS, and a
streptavidin solution (10 mg in 1.0 mL PBS) was added. The
resulting mixture was stirred at 4 °C overnight. The resulting
MNPs were separated from the supernatant by applying a
magnet and were washed three times with ddH2O. The protein
amount on the MNPs was determined with a BCA Protein
Assay Kit (PIERCE, Rockford, IL). An Agilent spectrometer
8453 was used to measure the absorbance of the dye solutions.
The calibration curve for the quantitation was obtained by
measuring the absorbance at 562 nm of a 1 mL dye solution
containing bovine serum albumin (BSA; 0 μg, 2 μg, 4 μg, 6 μg,
8 μg, and 10 μg). The BCA working reagent was prepared by
mixing 50 parts BCA working reagent A and 1 part BCA
working reagent B (reagent A:reagent B = 50:1 (v/v)). Each
standard protein or unknown sample was pipetted into 1.7 mL
Eppendorf centrifuge tubes. One milliliter of BCA working
reagent was added to each tube and then vortexed to mix well.
After incubation at 60 °C for 30 min, the samples were
separated by applying a magnet. The supernatants were
interrogated with an UV/vis spectrometer at 562 nm. This
1
71.4, 72.5, 74.9, 76.7, 105.1, 115.9, 116.6, 122.8, 123.7 (q, JCF
= 273 Hz), 125.9, 129.4, 130.8, 145.6, 160.5, 166.1, 170.5,
173.8, 174.9, 175.9 (x3), 176.0, 176.2. HMRS (ESI) m/z Calcd
for C69H110F3N15NaO19S [M+Na]+ 1564.7673; found:
1564.7659.
Compound 14. Compound 14 was synthesized by the
same methods as described in the synthesis of compound 13.
The residue was purified by Bio-Gel P2 size exclusion
chromatography to give compound 14 as colorless syrup
1
(43% for three steps). Rf 0.1 (MeOH/DCM = 1/1); H NMR
(600 MHz, CD3OD): δ 1.29−1.36 (m, 8H), 1.41−1.44 (m,
2H), 1.47−1.53 (m, 10H), 1.56−1.74 (m, 12H), 1.78−1.87 (m,
1H), 1.91−2.01 (m, 1H), 2.16−2.19 (m, 8H), 2.23−2.26 (m,
4H), 2.70 (d, J = 12.7 Hz, 1H), 2.91 (dd, J = 4.0, 12.7 Hz, 1H),
2.98−3.05 (m, 1H), 3.12−3.20 (m, 13H), 3.27−3.28 (m, 1H),
3.33−3.35 (m, 1H), 3.47−3.77 (m, 52H), 3.82−3.84 (m, 4H),
3.88−3.91 (m, 5H), 3.93−3.98 (m, 6H), 4.20−4.31 (m, 8H),
4.46−4.58 (m, 16H), 7.01−7.06 (m, 1H), 7.08 (d, J = 8.4 Hz,
1H), 7.22 (d, J = 8.4 Hz, 1H), 7.38−7.43 (m, 1H), 8.00 (s,
3H); 13C NMR (150 MHz, CD3OD): δ 26.4, 26.6, 26.7, 26.9,
2
27.5, 29.0 (q, JCF = 39 Hz), 29.5, 29.7, 30.0, 30.1, 30.3, 36.7,
36.8, 37.0, 37.4, 37.5, 40.2, 41.0, 51.4, 56.9, 61.6, 62.5, 63.3,
65.2, 68.2, 69.6, 70.3, 71.3, 71.4, 72.5, 74.9, 76.7, 105.1, 115.9,
1
116.6, 122.8, 123.6 (q, JCF = 273 Hz), 125.9, 126.0, 129.4,
130.8, 145.6, 160.4, 166.0, 170.5, 173.8, 174.9, 176.0 (x4),
1900
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Bioconjugate Chemistry
Article
a
Scheme 2. Syntheses of Mono- and Tri-Galactoses of Photoaffinity Probes 13 and 14
a
Reagents and conditions: (a) β-D-Galactoside 15a, CuI (catalytic), CuSO4 (catalytic), sodium ascorbate, DIPEA, EtOH:H2O:CH3CN = 3:2:5, r.t. 6
h, n = 1, 80%; n = 3, 82%. (b) (i) TFA, DCM, r.t., 1.5 h, and then neutralization; (ii) D-biotin, HBTU, Et3N, DMF, r.t.,12 h; (iii) NaOMe (catalytic),
MeOH, r.t., 1 h, n = 1, 46%; n = 3, 43% (over three steps). DIPEA: N,N-diisopropylethylamine.
measurement should be completed within 10 min. The
absorbance intensity of the blank standard was subtracted
from the absorbance intensity of the measured sample. The
concentration of the tested sample was determined from the
calibration curve. The amount of streptavidin was calculated to
be 106 μg/mg MNP.
heat-inactivated fetal bovine serum (FBS, Gibco), 100 U mL−1
penicillin (Gibco), 100 μg mL−1 streptomycin (Gibco), and
0.25 μg mL−1 Fungizone (BioSOURCE). Cells were incubated
at 37 °C in an ambient air/5% CO2 atmosphere and
subcultured every 3 days.
Labeling the Membrane Protein ASGP-R (Asialogly-
coprotein receptor) on HepG2 cells with the Trivalent
Galactose Photoaffinity Probe (14) and the Purification
of Labeled Proteins by Streptavidin-MNP. HepG2 cells (1
× 107) were seeded onto sterile glass coverslips, placed in a 100
mm dish, and cultured in 10.0 mL DMEM medium with 10%
FBS overnight. The cells were washed three times with serum-
free DMEM and then serum-starved for 30 min on ice in the
same medium. After 30 min, the cells were removed from their
Petri dish for passage by gently pipetting up and down several
times with 1.0 mL of serum-free DMEM on ice. To the above
solution was added compound 14 (2 mM, in ddH2O (5 μL)),
followed by incubation on ice for 1.5 h. After the binding step,
the cells were irradiated for 30 min with a UV lamp (365 nm,
2.76 mW/cm2) at 4 °C. Then, Streptavidin-MNPs (1.0 mg in
PBS buffer (20 μL) at pH 7.4) were added, and the resulting
mixture was vortexed for 30 min at 4 °C, followed by
centrifugation at 200 g for 2 min to collect the cells. The cells
were then lysed with 1.0 mL lysis buffer (1xRIPA buffer [20
mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM Na2EDTA, 1
mM EGTA, 1% NP-40, 1% sodium deoxycholate, 2.5 mM
sodium pyrophosphate, 1 mM -glycerophosphate, 1 mM
Na3VO4, 1 μg/mL leupeptin], 1 mM PMSF, and protease
inhibitor cocktail (Roche, 04693116001)) for 30 min at 4 °C.
The MNPs in the above mixture were magnetically isolated,
and the resulting particles were washed three times with 500 μL
washing buffer (sodium dodecyl sulfate: PBS = 0.3: 99.7%, v/
Labeling and purification of RCA120 from E. coli Cell
LysateA. A 1.7-mL Eppendorf tube was charged with a
solution of E. coli BL21 (DE3) cell lysate (delivered as 300 μL
of solution in 20 mM Tris-HCl buffer at pH 8.0), RCA120
(delivered as 24 μL of a 56.4 μM stock solution in PBS buffer at
pH 7.4), PBS buffer (delivered as 1.0 mL at pH 7.4), and a
solution of compound 14 (delivered as 50 μL of a 2 mM
solution in PBS buffer at pH 7.4). The mixture was briefly
agitated to ensure proper mixing and then incubated with
agitation at 4 °C for 30 min, followed by subsequent irradiation
with a UV lamp (365 nm, 2.76 mW/cm2) at 4 °C for 30 min.
Excess compound 14 was removed from the reaction via spin
concentration (Microcon centrifugal filter 10,000 MWCO,
Millipore, MA). An aliquot of this purified mixture (50 μL) was
diluted with PBS buffer at pH 7.4 (950 μL) and then incubated
with Streptavidin-MNP (1.5 mg/protein) at 4 °C for 30 min
with vortexing. The RCA120-Streptavidin-MNP complex was
isolated by applying a magnet, and the resulting MNPs were
washed three times with 500 μL washing buffer (sodium
dodecyl sulfate:PBS = 0.3:9.7% (v/v)). The samples were then
treated with SDS-PAGE loading buffer (0.3 M Tris-Cl, 10%
SDS, 30% glycerol, 9.3% DTT, pH 6.8), heated at 95 °C for 10
min, and then analyzed by electrophoresis, visualized by
Coomassie blue staining, and verified by Western blotting.
Cell Culture. HepG2 cells were cultured in T-75 flasks with
Dulbecco’s Modified Eagle’s Medium (DMEM, Gibco), 10%
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Figure 1. (A) Concentration-dependent labeling of RCA120 (0.1 μM) by probe 14 with and without UV irradiation. (B) A partial enlargement of the
Western blot (left), and Coomassie stained (right) SDS-PAGE in part A (marked with an arrow), indicative of cross-linking at A chain of RCA120
.
(C) Comparison of RCA120 (0.1 μM) labeling efficiencies with various concentrations of probe 13 and probe 14 (50 μM). N/P lane: only RCA120
(0.2 μM) as a negative control.
v). The captured proteins were divided into four aliquots and
analyzed by SDS-PAGE with Coomassie blue visualization and
Western blotting with antibodies against biotin and the ASGP-
R subunits (H1 and H2).
length was then elongated by (i) cleavage of Fmoc with 1,8-
diazabicycloundec-7-ene, (ii) coupling with building block B
using HBTU, (iii) hydrolysis of methyl esters under basic
conditions, and (iv) coupling with building block C to furnish
compound 8. Hydrolysis of the methyl ester of 8 was followed
by amide bond formation with photoreactive group D to yield
photoaffinity probe 10 in ten steps with 27% overall yield from
2. Following these same procedures, compound 9 (22% overall
yield from 1) was synthesized.
To demonstrate the photolabeling efficiency of this multi-
valent ligand, Ricinus communis Agglutinin 120 (RCA120) was
chosen as the target for a proof-of-concept study. RCA120 was
isolated from the seed of the castor bean and consists of a
dimeric chain [BAAB] in which the two chains, A−B, are
covalently connected by a disulfide bridage.38,39 RCA120
recognizes nonreducing terminal β-D-galactose (β-Gal) and
shows low avidity for monosaccharide (Ka 7.7 × 103 M−1 for
methyl-β-D-galactoside),40 while that for poly-Gal (more than
37 gal) was enhanced to Ka 1.8 × 108 M−1.41 Thus, the β-D-
galactopyranoside 15 was assembled on skeletons 9 and 10 by
Cu(I)-catalyzed click chemistry to give the mono- and
trigalactosyl precursors 11 and 12, respectively (Scheme 2).
Removal of the Boc group on precursor 11 (or 12) was
followed by coupling with biotin, which was used as a tag to
purify the labeled protein. Finally, global hydrolysis of the acetyl
groups on the galactose was performed with sodium methoxide
to furnish the desired photoaffinity probes 13 and 14 (see the
SI).
Western Blotting. Proteins were transferred onto a PVDF
membrane with a semidry transfer blot apparatus (BioRad).
Samples were resolved on SDS-polyacrylamide gels. The gels
were then rinsed with transfer buffer (50 mM Tris-HCl, pH 8.3,
20% methanol, 40 mM glycine in distilled water). The PVDF
membrane (Millipore) was activated in a bath of methanol and
then equilibrated in transfer buffer. The membranes were
blocked with 5% milk/TBST at room temperature for 1 h
followed by incubation with the corresponding primary
antibody in 5% milk/TBST at 4 °C for 16 h. The membranes
were washed with TBST (4 × 10 min) and then incubated with
an appropriate secondary antibody at room temperature for 1 h.
Finally, the membranes were washed again with TBST before
visualization by chemiluminescence (PerkinElmer,
NEL102001EA).
RESULTS AND DISCUSSION
■
Synthesis of Trigalactose Photoaffinity Probe. To
highlight the effect of the multivalent character of the designed
probe on the photolabeling of target proteins, the monovalent
probe 9 (Scheme 1) was also synthesized for comparison. To
readily access scaffolds with flexible spatial arrangements, we
used commercially available 6-aminohexanoic acid, which is
commonly employed as a heterobifunctional linker in
glycocluster synthesis.37 The multifunctional skeletons 9 and
10 were efficiently constructed using monoalkyne 1 and
trialkyne 2, respectively, via a series of deprotection and amide
bond formations with building blocks A, B, C, and D (see
Supporting Information for detailed syntheses). For example,
the Boc group of 2 was removed with 20% TFA/DCM
followed by coupling with building block A to give 4. The linker
Evaluation and Characterization of Photolabeling
Ability of Probe (14). To assess the photolabeling efficiency
of probes on RCA120, RCA120 (0.1 μM) was incubated with
probe 14 for 30 min at 4 °C and then irradiated with or without
UV (365 nm) at 4 °C for 30 min. The excess labeling reagent in
the resulting solution was removed by ultrafiltration (10 kDa
molecular weight cutoff), and the resulting mixture was
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Figure 2. (A) OVA and RCA120 were labeled simultaneously by probe 14 (50 μM) from a protein pool containing BSA, ConA, RCA120, OVA, and
WGA (lane 1: presence of UV; lane 2: absence of UV). Indicated bands: “a” OVA (lane 1 of Western blot); “b” B chain of RCA120 (lane 1 of
Western blot); “#” excess probe 14. (B) OVA was incubated with probe 14 and then irradiated with UV (lane 1); RCA120 was specifically labeled by
probe 14 (50 μM) from a protein pool containing BSA, ConA, RCA120, and WGA (lane 2: presence of UV; lane 3: absence of UV). The
concentration of each protein was 0.1 μM. BSA: bovine serum albumin; OVA: ovalbumin; ConA: concanavalin A; WGA: wheat germ agglutinin.
Figure 3. RCA120 in E. coli lysate was photolabeled with 14 and purified by streptavidin-MNP. Wash 1 was performed with 0.3% SDS in PBS to
remove nonspecifically adsorbed proteins (lane 7 in Figure 3A); Wash 2 was performed with PBS to remove excess detergent (SDS) (lane 8 in
■
Figure 3A); Indicated bands: , nonspecific adsorption of proteins on streptavidin-MNP (lane 9 in Figure 3A); a, B chain of RCA120 (lane 9 in
Figure 3A, B); b, A chain of RCA120 (lane 9 in Figure 3A, B); c, fragment of streptavidin protein on MNPs (lane 9 in Figure 3A); d, unknown
photolabeled proteins that associated with the galactose of 14 in the E. coli lysate. (C) Comparison of expansion (20−55 kDa) of gel electrophoresis
images (lane 9) of A and B.
analyzed by Western blotting with an antibiotin antibody and
Coomassie blue staining on the SDS-PAGE. As shown in
Figure 1A, probe 14 successfully labeled RCA120 in a dose-
dependent manner after UV irradiation. The effective
concentration of probe 14 for efficient labeling of the B chain
of RCA120 was found to be 25 μM (Figure 1A). It is not
surprising that the major labeling occurred on the B chain
because the B chain (37 kDa) is a lectin moiety that binds β-D-
galactosides, while the A chain (29.5 kDa) is a toxin bearing
rRNA N-glycosidase activity. However, because the diazirine
group in probe 14 is flexible and the carbohydrate binding site
of the B chain is linked with the A chain in RCA120, the A chain
was also labeled (Figure 1B). Increasing the concentration of
14 resulted in increased labeling of the A chain due to the
proximity effect.42 To avoid undesired labeling, 50 μM 14 was
used in the subsequent experiments. The efficiency of RCA120
labeling by the mono- and trigalactose photoaffinity probes 13
and 14 was also evaluated. Therefore, various concentrations of
monovalent probe 13 (200−12.5 μM) were incubated with
RCA120 following the same photolabeling process described in
Figure 1A. As evident from Figure 1C, 13-treated RCA120
displays a considerably decreased band intensity compared to
14-treated ones, directly suggesting the superior photolabeling
efficiency of 14. Conducting a competitive binding experiment
using different concentrations (1−200 μM) of galactoside
(15b) also revealed suppression of cross-linking as the
concentration 15b increased; however, a strong band is still
visible even in the presence of large excess (200 μM) of 15b
(Figure S5). The results clearly demonstrated that photoaffinity
probe 14 containing a multivalent ligand provided much better
labeling efficiency due to the higher affinity of the multivalent
interaction through statistical rebinding mode,19 where a
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Figure 4. Photolabeling of the asialoglycoprotein receptor (ASGP-R) with trigalactose photoaffinity probe 14. Lane 1, HepG2 cells (1 × 107, total
protein was 45 mg) were incubated with probe 14 (10 μM) followed by UV irradiation, incubation with streptavidin-coated magnetic nanoparticles,
cell lysis, purification with a magnet and subjected to SDS-PAGE followed by (A) Coomassie staining; Western blot with an antibody against (B)
biotin, (C) ASGP-R subunit H1, and (D) ASGP-R subunit H2. Lane 2, HepG2 cells (1 × 107, total protein was 45 mg) were lysed, and a portion was
subjected to SDS-PAGE as described for lane 1 as a negative control. Indicated bands: “a” H2 subunit of ASGP-R associated with probe 14; “b” H1
subunit of ASGP-R associated with probe 14; “c” H1 subunit of ASGP-R; “d” H2 subunit of ASGP-R.
nearby ligand quickly replaces the bound ligand due to its
proximity, leading to reduce off-rate.
with detergent (0.3% SDS in PBS buffer), and the resulting
captured complex was divided into two aliquots, analyzed by
SDS-PAGE, and visualized by Coomassie blue staining (Figure
3A) and Western blotting (Figure 3B). The results
demonstrated that probe 14 selectively labeled the B chain of
RCA120 (band a in Figure 3A and B) in a complicated cell
lysate, although trace amounts of unknown photolabeled
proteins were observed (band d in Figure 3B). The visualized
bands a and b in Figure 3A were further subjected to in-gel
digestion followed by LC-MS/MS analysis. RCA120 was indeed
identified by multiple peptides (sequence coverage 37%) with
high confidence (p < 0.05) (see Figure S8 for MS/MS spectra
of all identified peptide sequences).
Photolabeling ASGP-R by Probe 14 on the Membrane
of HepG2 Cells. Encouraged by the above results, we
attempted to evaluate the potential of probe 14 for labeling
cell surface carbohydrate binding proteins. The ASGP-R is
located on hepatocytes and is a Ca2+-dependent carbohydrate-
binding protein.44 ASGP-R is expressed on the plasma
membrane of mammalian liver cells (highly expressed in liver
cancer cells) and maintains serum glycoprotein homeostasis
through endocytosis of asialoglycoproteins (ASGPs), desialy-
lated glycoproteins with terminal galactose or GalNAc residues.
ASGP-R consists of two homologous subunits, designated H1
(46 kDa) and H2 (50 kDa), in human hepatocytes; these
subunits form a noncovalent hetero-oligomeric complex with
an estimated ratio of 2−5:1.45 Previously, we demonstrated that
preassembled trigalactose multivalent ligands provided better
endocytosis efficiency than monogalactose multivalent ligands
by targeting ASGP-R on the HepG2 cell surface.46 Thus, we
anticipate that the trigalactose probe 14 should exhibit high
affinity and specificity for ASGP-R.
Evaluation of Photolabeling Selectivity of Probe (14)
in a Protein Pool. To examine the labeling specificity of probe
14, a mixture of five proteins (BSA, OVA, RCA120, ConA, and
WGA at 0.1 μM) was incubated with probe 14 (50 μM)
followed by UV irradiation (Figures S3 and S4). Unexpectedly,
OVA and the B chain of RCA120 were concurrently labeled
instead of specifically labeling of RCA120 (Figure 2A, lane 1 in
the Western blot). OVA is a glycosylated protein with
nonreducing LacNAc on the protein surface.43 Thus, OVA
may have been labeled due to the interaction of the
carbohydrate moiety of ovalbumin with RCA120 and cross
labeling by probe 14. However, a nonspecific hydrophobic
interaction between ovalbumin and probe 14 could not be
excluded. To further investigate this undesired labeling,
ovalbumin (0.1 μM) was incubated with probe 14 (50 μM)
and then irradiated. However, no labeled band was observed in
the Western blot (Figure 2B, lane 1), indicating the absence of
nonspecific interactions between probe 14 and OVA.
Furthermore, incubation of probe 14 with the above protein
mixture without OVA revealed specific labeling of the B chain
of RCA120 (Figure 2B, lane 2). The labeling of OVA by probe
14 could thus be clearly attributed to the presence of RCA120
.
RCA120 has four carbohydrate binding sites, two of which
exhibit high affinity for β-D-galactopyranoside and are separated
by 11 nm.41 The protein microarray based assay was used to
further confirm the interaction between RCA120 and OVA, as
shown in Figure S6. Thus, probe 14 and OVA may bind the
galactose binding sites of RCA120, with a proximal orientation
of 14 and OVA in the resulting complex. Upon UV irradiation,
the generated carbene can reach and label OVA. Despite the
short lifetime of the carbene derived from 14, the photo-cross-
linking yield of 14 to RCA120 was estimated to be ∼36 5%
(Figure S7). Thus, probe 14 could serve as an efficient probe
for lectin labeling and protein−protein interactions.
To demonstrate the potential applications of 14 in specific
labeling, we employed probe 14 to detect an exogenous lectin
in E. coli lysates. Accordingly, an E. coli lysate (300 μg protein)
spiked with RCA120 (24 μg) was incubated with 14 (100 μM)
and then irradiated, ultrafiltered (cutoff of 10 kDa), and
separated using streptavidin-coated magnetic nanoparticles
(streptavidin-MNPs). The captured proteins were washed
HepG2 cells derived from a human hepatocellular carcinoma
expressing ASGP-R on the cell surface were incubated with
probe 14 (10 μM) for 1.5 h on ice to permit binding of the
probe with the cell surface receptor while preventing
endocytosis. The unbound probe 14 was removed from the
plasma membrane by washing the cells with PBS buffer (pH
7.4) followed by UV irradiation for 30 min at 4 °C to furnish
photocrossing with ASGP-R. Due to interference from
endogenous biotinylated proteins in HepG2 cells during
purification,47 the streptavidin−biotin interaction was per-
formed before cell lysis. Streptavidin-MNPs were applied to
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capture biotinylated ASGP-R on the plasma membrane. The
cells were lysed, and the labeled proteins were separated by
applying a magnet and then washed with detergent (0.3% SDS
in PBS buffer). The resulting captured proteins were divided
into four aliquots and analyzed by SDS-PAGE with Coomassie
blue visualization (Figures 4A and S9) and Western blotting
with antibodies against biotin and the ASGP-R subunits. Two
bands with molecular weights of approximately 46 and 50 kDa
were observed in the Western blot with an antibiotin antibody
(Figure 4B, lane 1, “a” and “b” markers), indicating successful
photolabeling of H1 and H2 of ASGP-R by 14. To confirm the
two observed bands, Western blotting with anti-H1 and H2 of
ASGP-R was performed and the results were consistent with
the results of Western Blotting with the antibiotin antibody
(Figure 4C and D, lane 1). The strong cross-linking at these
two subunits of ASGP-R is in good agreement with the results
obtained from a previous glycoprobe-based photoaffinity
labeling method using terminal GalNAc (N-acetylgalactos-
amine) residues.23 Notably, the bands at 25−37 kDa and 55−
65 kDa in the Western blot with antibiotin antibody (Figure
4B, lane 1) may be the result of an undesired streptavidin−
biotin interaction with endogenous biotinylated proteins.
However, unidentified galactose binding proteins may also
contribute to these bands. Overall, probe 14 was demonstrated
to be an efficient photoaffinity probe for labeling the membrane
protein ASGP-R on HepG2 cells.
ACKNOWLEDGMENTS
■
This work was financially supported by the National Tsing Hua
University, Academia Sinica, and the National Science Council,
Taiwan.
REFERENCES
■
(1) Bertozzi, C. R., and Kiessling, L. L. (2001) Chemical
glycobiology. Science 291, 2357−2364.
(2) Drickamer, K., and Taylor, M. E. (1993) Biology of animal lectin.
Annu. Rev. Cell Biol. 9, 237−264.
(3) Kiessling, L. L., Gestwichi, J. E., and Strong, L. E. (2006)
Synthetic multivalent ligands as probes of signal transduction. Angew.
Chem., Int. Ed. 45, 2348−2368.
(4) Lee, S. J., Evers, S., Roeder, D., Parlow, A. F., Risteli, J., Risteli, L.,
Lee, Y. C., Feizi, T., Langen, H., and Nussenzweig, M. C. (2002)
Mannose receptor-mediated regulation of serum glycoprotein homeo-
stasis. Science 295, 1898−1901.
(5) Sperandio, M., Gleissner, C. A., and Ley, K. (2009) Glycosylation
in immune cell trafficking. Immunol. Rev. 230, 97−113.
(6) Rudd, P. M., Elliott, T., Cresswell, P., Wilson, I. A., and Dwek, R.
A. (2001) Glycosylation and the immune system. Science 291, 2370−
2376.
(7) Dube, D. H., and Bertozzi, C. R. (2005) Glycans in cancer and
inflammation-potential for therapeutics and diagnostics. Nat. Rev. Drug
Discovery 4, 477−488.
(8) Lee, Y. C. (2010) Warfare between pathogens and hosts: the
trickery of sugars. Trends Glycosci. Glycotechnol. 22, 95−106.
(9) Sharon, N., and Lis, H. (1998) Lectins: carbohydrate-specific
proteins that mediate cellular recognition. Chem. Rev. 98, 637−674.
(10) Singh, A., Thorton, E. R., and Westheimer, F. H. (1962) The
photolysis of diazoacetylchymotrypsin. J. Biol. Chem. 237, 3006−3008.
(11) Han, S., Collins, B. E., Bengtson, P., and Paulson, J. C. (2005)
Homomultimeric complexes of CD22 in B cells revealed by protein-
glycan cross-linking. Nat. Chem. Biol. 1, 93−97.
CONCLUSIONS
■
In conclusion, a convergent synthesis of a versatile photoaffinity
scaffold was developed. The desired ligand and tag of the probe
were conveniently conjugated via orthogonal reactions: Cu(I)-
catalyzed click reaction and amide bond formation. The
advantage of the developed synthetic route was that the use
of click chemistry permitted the efficient assembly of various
ligands on the probe to form a multivalent ligand scaffold that
provided greater affinity and thus improved labeling efficiency.
The trigalactose photoaffinity probe was designed to
demonstrate the importance of multivalent ligand binding in
lectin labeling. The results demonstrated selective labeling of
RCA120 in a protein mixture and E. coli lysates, as well as
labeling of ASGP-R on the surface of HepG2 cells.
Furthermore, transient and weak protein−protein interactions
between the LacNAc moiety of OVA and the carbohydrate
binding chain of RCA120 were detected by the use of probe 14.
It is anticipated that the designed photoaffinity scaffold could
be used to quickly assemble diverse affinity ligands and facilitate
the interrogation of unknown protein interactions.
(12) Ballell, L., Alink, K. J., Slijper, M., Versluis, C., Liskamp, R. M. J.,
and Pieters, R. J. (2005) A new chemical probe for proteomics of
carbohydrate-binding proteins. ChemBioChem 6, 291−295.
(13) Walter, M., Wiegand, M., and Lindhorst, T. K. (2006) Synthesis
of cluster mannosides carrying a photolabile diazirine group. Eur. J.
Org. Chem., 719−728.
(14) Wiegand, M., and Lindhorst, T. K. (2006) Synthesis of
photoactive α-mannosides and mannosyl peptides and their evaluation
for lectin labeling. Eur. J. Org. Chem., 4841−4851.
(15) Vodovozova, E. L. (2007) Photoaffinity labeling and its
application in structural biology. Biochemistry (Mosc.) 72, 1−20.
(16) Tanaka, Y., and Kohler, J. J. (2008) Photoactivatable
crosslinking sugars for capturing glycoprotein interactions. J. Am.
Chem. Soc. 130, 3278−3279.
(17) Nakamura, Y., Miyatake, R., and Ueda, M. (2008)
Enantiodifferential approach for the detection of the target membrane
protein of the jasmonate glycoside that controls the leafmovement of
albizzia saman. Angew. Chem., Int. Ed. 47, 7289−7292.
(18) Tamura, T., Tsukiji, S., and Hamachi, I. (2012) Native FKBP12
engineering by ligand-directed tosyl chemistry: labeling properties and
application to photo-cross-linking of protein complexes in vitro and in
living Cells. J. Am. Chem. Soc. 134, 2216−2226.
ASSOCIATED CONTENT
■
S
* Supporting Information
(19) Gestwichi, J. E., Cairo, C. W., Strong, L. E., Oetjen, K. A., and
Kiessling, L. L. (2002) Influencing receptor-ligand binding mecha-
nisms with multivalent ligand architecture. J. Am. Chem. Soc. 124,
14922−14933.
Spectra, photolabeling yields, and additional experimental
details. This material is available free of charge via the Internet
at http://pubs.acs.org.
(20) Jayaraman, N. (2009) Multivalent ligand presentation as a
central concept to study intricate carbohydrate-protein interactions.
Chem. Soc. Rev. 38, 3463−3483.
AUTHOR INFORMATION
■
Corresponding Author
(21) Lahmann, M. (2009) Architectures of multivalent glycomimetics
for probing carbohydrate-lectin interactions. Top. Curr. Chem. 288,
17−65.
*E-mail: cclin66@mx.nthu.edu.tw. Tel: (+) 886-3-5753147.
Notes
(22) Lee, R. T., and Lee, Y. C. (1987) Affinity labeling of the
galactose/N-acetylgalactosamine-specific receptor of rat hepatocytes:
The authors declare no competing financial interest.
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preferential labeling of one of the subunits. Biochemistry 26, 6320−
(42) Vila-Perello′, M., Pratt, M. R., Tulin, F., and Muir, T. W. (2007)
Covalent capture of phospho-dependent protein oligomerization by
site-specific incorporation of a diazirine photo-cross-linker. J. Am.
Chem. Soc. 129, 8068−8069.
6329.
́
(23) Lauc, G., Lee, R. T., Dumic, J., and Lee, Y. C. (2000)
Photoaffinity glycoprobes-a new tool for the identification of lectins.
Glycobiology 10, 357−364.
(43) Yamashita, K., Tachibana, Y., and Kobata, A. (1978) The
structures of the galactose-containing sugar chains of ovalbumin. J.
Biol. Chem. 253, 3862−3869.
(24) Lee, M.-R., Jung, D.-W., Williams, D., and Shin, I. (2005)
Efficient solid-phase synthesis of trifunctional probes and their
application to the detection of carbohydrate-binding proteins. Org.
Lett. 7, 5477−5480.
(44) Ashwell, G., and Harford, J. (1982) Carbohydrate-specific
receptors of the liver. Annu. Rev. Biochem. 51, 531−554.
(45) Weigel, P. H., and Yik, J. H. N. (2002) Glycans as endocytosis
signals: the cases of the asialoglycoprotein and hyaluronan/
chondroitin sulfate receptors. Biochim. Biophys. Acta 1572, 341−363.
(46) Lai, C.-H., Lin, C.-Y., Wu, H.-T., Chan, H.-S., Chuang, Y.-J.,
Chen, C.-T., and Lin, C.-C. (2010) Galactose encapsulated multi-
functional nanoparticle for HepG2 cell internalization. Adv. Funct.
Mater. 20, 3948−3958.
(25) Moss, R. A. (2006) Diazirines: carbene precursors par
excellence. Acc. Chem. Res. 39, 267−272.
(26) Das, J. (2011) Aliphatic diazirines as photoaffinity probes for
proteins: recent developments. Chem. Rev. 111, 4405−4417.
(27) Ingenhorst, G., Bindseil, K. U., Boddien, C., Drose, S., Gaßel,
̈
M., Altendorf, K., and Zeeck, A. (2001) Synthesis of a doubly labelled
concanamycin derivative for ATPase binding studies. Eur. J. Org. Chem.
2001, 4525−4532.
(47) The detail of endogenous biotinylated proteins in HepG2 cells
was viewed in Figure S7.
(28) Becer, C. R., Hoogenboom, R., and Schubert, U. S. (2009) Click
chemistry beyond metal-catalyzed cycloaddition. Angew. Chem., Int. Ed.
48, 4900−4908.
(29) Montalbetti, C. A. G. N., and Falque, V. (2005) Amide bond
formation and peptide coupling. Tetrahedron 61, 10827−10852.
(30) Dumez, E., Snaith, J. S., and Jackson, R. F. W. (2009) Synthesis
of macrocyclic potential protease inhibitors using a generic scaffold. J.
Org. Chem. 67, 4882−4892.
(31) Hatanaka, Y., Hashimoto, M., Nakayama, H., and Kanaoka, Y.
(1994) An entry to chromogenic carbene precursors for photoaffinity
labeling. Chem. Pharm. Bull. 42, 826−831.
(32) Lu, X., Cseh, S., Byun, H.-S., Tigyi, G., and Bittman, R. (2003)
Total synthesis of two photoactivatable analogues of the growth-
factor-like mediator sphingosine 1-phosphate: differential interaction
with protein targets. J. Org. Chem. 68, 7046−7050.
(33) Cecioni, S., Oerthel, V., Iehl, J., Holler, M., Goyard, D., Praly, J.-
P., Imberty, A., Nierengarten, J.-F., and Vidal, S. (2001) Synthesis of
dodecavalent fullerene-based glycoclusters and evaluation of their
binding properties towards a bacterial lectin. Chem.Eur. J. 17, 3252−
3261.
́
(34) Chabre, Y. M., Contino-Pepin, C., Placide, V., Shiao, T. C., and
Roy, R. (2008) Expeditive synthesis of glycodendrimer scaffolds based
on versatile TRIS and mannoside derivatives. J. Org. Chem. 73, 5602−
5605.
(35) He, B., Velaparthi, S., Pieffet, G., Pennington, C., Mahesh, A.,
Holzle, D. L., Brunsteiner, M., Breemen, R. V., Blond, S. Y., and
Petukhov, P. A. (2009) Binding ensemble profiling with photoaffinity
labeling (BEProFL) approach: mapping the binding poses of HDAC8
inhibitors. J. Med. Chem. 52, 7003−7013.
(36) Wu, H.-T., Hsu, C.-C., Tasi, C.-F., Lin, P.-C., Lin, C.-C., and
Chen, Y.-J. (2011) Nanoprobe-based immobilized metal affinity
chromatography for sensitive and complementary enrichment of
multiply phosphorylated peptides. Proteomics 11, 2639−2653.
(37) Tian, X., Pai, J., Baek, K.-H., Ko, S.-K., and Shin, I. (2011)
Fluorophore-labeled, peptide-based glycoclusters: synthesis, binding
properties for lectins, and detection of carbohydrate-binding proteins
in cells. Chem. Asian J. 6, 2107−2113.
(38) Nicolson, G. L., Blaustein, J., and Etzler, M. E. (1974)
Characterization of two plant lectins from ricinus communis and their
quantitative interaction with a murine lymphomat. Biochemistry 13,
196−204.
(39) Sweeney, E. C., Tonevitsky, A. G., Temiakov, D. E., Agapov, I. I.,
Saward, S., and Palmer, R. A. (1997) Preliminary crystallographic
characterization of ricin agglutinin. Proteins: Struct., Funct., Genet. 28,
586−589.
(40) Sharma, S., Bharadwaj, S., Surolia, A., and Podder, S. K. (1998)
Evaluation of the stoichiometry and energetics of carbohydrate binding
to Ricinus communis agglutinin: a calorimetric study. Biochem. J. 333,
539−542.
(41) Spain, S. G., and Cameron, N. R. (2011) The binding of
polyvalent galactosides to the lectin Ricinus communis agglutinin 120
(RCA120): an ITC and SPR study. Polym. Chem. 2, 1552−1560.
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