Two Facile General Methods for the Conjugation of Three Different Molecules

Article abstract of DOI:10.1002/bkcs.12208

In the quest to synthesize compounds that are uniquely optimized for fighting each individual target disease, the importance of developing a new methodology for the bioconjugation of compounds with different functional groups or biological activities cann

Full text of DOI:10.1002/bkcs.12208

Article
DOI: 10.1002/bkcs.12208
BULLETIN OF THE
K. Oh et al.
KOREAN CHEMICAL SOCIETY
Two Facile General Methods for the Conjugation of Three
Different Molecules
†,‡,
Keumrok Oh,† Dong Seok Shin,‡ Hyeong Baik Kim,† Uthaiwan Sirion,§ and Dae Yoon Chi
*
†Department of Chemistry, Sogang University, 35 Baekbeomro Mapogu, Seoul 04107, Korea
‡FutureChem Co., Ltd., 26 Seongsuil-ro 10-gil, Seongdong-gu, Seoul 04782, Korea.
*E-mail: dychi@sogang.ac.kr
§Department of Chemistry, Faculty of Science, Burapha University, Chonburi 20131, Thailand
Received December 1, 2020, Accepted December 21, 2020, Published online January 25, 2021
In the quest to synthesize compounds that are uniquely optimized for fighting each individual target dis-
ease, the importance of developing a new methodology for the bioconjugation of compounds with differ-
ent functional groups or biological activities cannot be overemphasized. This study investigates two novel
methods for click reaction: coupling a molecule with three functional compounds by repetitive copper(I)
catalyzed azide/alkyne 1,3-dipolar cycloadditions. Method A uses three copper(I) catalyzed azide/alkyne
1,3-dipolar cycloadditions in series. Method B uses two copper(I) catalyzed azide/alkyne 1,3-dipolar
cycloadditions and one conjugation reaction of amine and isothiocyanate.
Keywords: Conjugation chemistry, Bioconjugation, Tripodal conjugation, Click reaction
Introduction
integrin protein. Introduction of carbohydrate to RGD
derivative shows around 30% of cell uptake high. However,
as the synthesis of F-18 labeled carbohydrate-RGD requires
very complicated protection and deprotection steps, the
development of this compound is not so easy.12–15 Herein,
we have described the development of a new convenient
method, which can conjugate three different complicated
molecules.16,17
As our understanding of biological functions and processes
increases, so grows the need to facilitate the syntheses of
big biomolecules connected with one or more different
molecules. Commercial automatic synthesizers have made
possible the rapid and convenient preparation of
oligopeptides, oligonucleic acids, and oligosaccharides,
using similar and repeated coupling methods of limited
number of protected monomers.1–4 However, the synthesis
of compounds that have different functional groups remains
an arduous and labor-intensive task.
In 2001, Huisgen azide/alkyne 1,3-dipolar cycloadditions
have been widely applied after these cycloaddition reac-
tions could be proceeded regiospecifically by using
Cu(I) catalyst.5,6 The most attractive merit of these cycload-
dition reactions is that the reactions go in very mild condi-
tions, even in aqueous phase and in rt.7 This cycloaddition
reaction was also applied successfully to the synthesis of
fluorine-18 labeled biomolecules in extremely low concen-
trations, giving various fluorine-18 labeled 1,2,3-triazoles
with high radiochemical yield.8 Conjugation of two differ-
ent big molecules, one with azido group and the other with
a terminal alkyne group, became feasible without compli-
cated protective groups.9–11
Results and Discussion
Method A. Scheme 2 shows the basic concept of this
method to connect three molecules X, Y, and Z having
azide group. It has been found that terminal alkyne goes
well while internal alkynes do not go in Huisgen cycloaddi-
tion. As the pKa of proton of terminal alkyne is around
25, terminal alkynes can be deprotonated and protected by
various silyl groups. We had to find two proper silyl groups
that could be discriminated in the deprotection step. In
acidic media, the relative stabilities of silyl groups are: tri-
methylsilyl (TMS) (1) < triethylsilyl (TES) (64) < tert-
butyldimethylsilyl
(TIPS) (700 000) < tert-butyldiphenylsilyl
(TBS)
(20 000) < triisopropylsilyl
(TBDPS)
(5 000 000). In basic media, the relative stabilities are:
TMS (1) < TES (10−100) < TBS ~ TBDPS (20 000)
< triisopropylsilyl (TIPS) (100 000).18 TMS and TBS were
chosen, tested, and found to be suitable silyl groups.
Among the various methods we tried out to synthesize
6BMH, the route shown in Scheme 1 is the most practical
for mass production. Propargylamine (2H) was selected as
the starting material of this route. Boc-protected
propargylamine 3H was successfully monoalkylated with
Molecular imaging using fluorescent or radioactive label-
ing is a new field to understand the functions of biological
systems. To incorporate label either fluorescent or radioac-
tive group to bioconjugated molecules by Huisgen cycload-
dition reactions, a new facile method that can connect three
different biomolecules is needed. Arg-Gly-Asp (RGD)
derivative was prepared by our group to image αvβ3
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KOREAN CHEMICAL SOCIETY
using 12a, 12b, and 12c. Compound 8 was also used in the
same manner from 6BMH using 12a, 12b, and 12d via
7BM (79%), 7H (98%), 7B (87%), 7H (95%), and
8 (87%). Table 1 shows the yields and the azides used for
the synthesis of the compounds 9, 10, and 11.
Method B. In Table 1, the yield of synthesizing 11 from
11H and 12c is as low as 23%. Except for this reaction, the
rest reaction proceeded generally in high yield. The last
reaction to prepare 11 is depending on the size of azide.
Between center of tripodal amine and triazoles, the number
of carbons will be important. In this case, as the spacer car-
bon from the center of tripodal amine to the triazoles is
only one, the length of the space may be short, resulting in
arising steric hinder after three sequential click reactions. In
the case of a large molecule such as RGDfK, we found that
the yield decreased. The stepwise click reaction of (tri-
propargyl)amines such as 6BMH tends to decrease the
yield due to steric hindrance as the click reaction proceeds.
In the case of introducing a large molecule, we designed 13
as shown in Scheme 3. Compound 13 has only two propa-
rgylic groups that are used, and the other one is connected
to amine using the sulfonamide group. Sulfonamide group
has three methylene as spacer and protected amine by boc
group in ω-position. By using these three methylene
groups, steric hindrance could be reduced. In the case of a
compound with a larger steric hindrance, a longer aliphatic
chain could be used instead of the three methylene spaces.
If boc is used as a protecting agent in amine, it can be
removed relatively easily. When this unprotected amine in
ω-position reacts with a molecule having an isothiocyanate
group, it will be able to conjugate easily by the formation
of thiourea. Scheme 3 shows an example of Method B that
efficiently attaches three very polar compounds, such as
glucose, RGDfK, and DOTA. To obtain the structure–
activity relationships (SAR) of these synthesized tripod
Scheme 1. Synthesis of Trialkyne 6BMH.
TBS mesylate 4B. Then, deprotection of boc group and
alkylation with TMS mesylate 4M provided one of the tar-
get tripodal amine 6BMH. The overall yield to prepare
6BMH from propargylamine (2H) was 70% in four steps.
As shown in Schemes 2 1) click reaction, 2) deprotection
of protection group A, 3) click reaction, 4) deprotection of
protection group B, and 5) click reaction through the reac-
tion until
a
total of five steps was synthesized
trifunctionalized molecule. To selectively deprotect the silyl
group, 6BMH (A = TMS, B = TBS) was selected in accor-
dance with the relative stability of the silyl group in basic
conditions.
Azido compounds to the Cu(I)-catalyzed alkyne/azide
1,3-dipolar cycloadditions were used to 12a-12f, as shown
in Table 1. Using compound 6BMH and the three selected
azides in Table 1, compounds 7, 8, 9, 10, and 11 were syn-
thesized in the order as shown in Scheme 1. As an exam-
ple, compound 7 was prepared in five steps via 7BM
(79%), 7H (98%), 7B (87%), 7H (95%), and 7 (95%)
according to the series of sequence as shown in Scheme 1
Scheme 2. Schematic of tripod compound (Method A). Compound number notation in all schemes: 7BM-11BM, group A = TMS,
B = TBS; 7BH-11BH, Group A = H, B = TBS; 7B 11B, X,Y-clicked, Group B = TBS; 7H 11H, X,Y-clicked, Group B = H; 7–11,
X,Y,Z-clicked. Silyl group notation: B = TBS (t-butyldimethylsilyl); M = TMS (trimethylsilyl); E = TES (triethylsilyl); I = TIPS
(triisopropylsilyl); D = TBDPS (t-butyldiphenylsilyl); P = TPS (triphenylsilyl).
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Table 1. Various azido compounds and reaction yields of each Producta.
Compd
7
8
12a (7BM, 79%; 7BH, 98%)
12a (7BM, 79%; 7BH, 98%)
12ba (7B, 87%; 7H, 95%)
9
12b (7B, 87%, 7H, 95%)
Rhodamine-N3
12e (9, 33%)
12c (10, 90%)
12c (11, 23%)
10
11
12a (7BM, 79%; 7BH, 98%)
12a (7BM, 79%; 7BH, 98%)
12d (10B, 68%; 10H, 71%)
a Product numbers and yields in parenthesis.
Scheme 3. Typical example of MethodꢀB: Synthesis of RGDfK-NOTA-Glucose 20. Reaction conditions: i. CuSO4, NaASC, t-BuOH,
water, rt, 18 h, 87%; ii. TBAF, THF, 0 C, 1 h, 84%; iii. 7 N NH3 in MeOH, rt., 18 h, 95%; iv. CuSO4, NaASC, MeOH, water, rt, 2 h,
71%; v. 50% TFA in CH2Cl2, rt, 2 h; vi. 0.1 M Na2CO3 (aq):DMF = 1:4, rt, 20 h, 51%.
compounds, the number of compounds should be prepared.
For example, after metal chelated compound 20 with diag-
nostic and/or therapeutic radioisotope could be an excellent
candidate of cancer treatment based on protein receptor
radionuclide therapy (PRRT).19 The moiety of carbohydrate
will increase pharmacokinetic by interaction with glycopro-
teins in endothelial cell, and RGD will be binding to
integrin expressed from cancer.12–14
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To reduce the steric effects, the tripod synthesis for the
(2H, 10.0 mg, 181 mmol) dissolved in THF (10 mL),
dropwisely. The reaction mixture was stirred for 2 h at rt
and concentrated to give yellow oil. Purification with silica
column chromatography (EtOAc:hexane = 1:9) gave a pale
white solid (25.6 g, 95%): mp. 37–38 ꢀC; 1H NMR
(500 MHz, CDCl3) δ 1.46 (s, 9H), 2.24 (t, J = 3.0 Hz, 1H),
3.92 (s, 2H), 4.98 (br s, 1H); 13C NMR (125 MHz, CDCl3)
δ 28.3, 30.3, 71.2, 79.9, 80.2, 155.4. HRMS (ESI+): m/z
bioconjugation started with the sulfonamide 13, having one
TBS protected propargylic group and one propargyl group.
Click reaction of the sulfonamide 13 and protected
azidoglucose 12d followed by deprotection of the TBS
ether produced compound 14 in 87% yields. After
deprotection of TBS group and tetraacetate groups, a sec-
ond click reaction of 16 with protected RGDfK 17 pro-
duced 18 in 71% yield. Then, deprotection of the boc, pbf
and t-butoxy groups with 50% trifluoroacetic acid in dic-
hloromethane provided a deprotected amine compound.
The conjugation reaction of primary amine to
1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA) iso-
thiocyanate 19 produced thiourea 20 under basic condition,
purified in 51% yield after isolation by HPLC.
calcd. for C8H13NNaO2 [M + Na]+: 178.0838, found
+
178.0839.
tert-Butyl(3-methoxyprop-1-yn-1-yl)dimethylsilane (4B).
To a solution of t-butyldimethylsilylated propargyl alcohol
ꢀ
(1.7 g, 9.98 mmol) in anhydrous dichloromethane at 0 C
was added triethylamine (1.81 mL, 13.0 mmol). After being
stirred for 15 min at the same temperature, methanesulfonyl
chloride (0.86 mL, 11.0 mmol) was added dropwisely and
stirred for 1 h. The reaction mixture was quenched by addi-
tion of water. The aqueous layer was extracted three times
with dichloromethane, the organic layer was dried over
Na2SO4 and concentrated under reduced pressure to afford
Conclusion
Until now, linking compounds with three different func-
tional groups such as 20 has been a notoriously long and
laborious process. We sought to develop an efficient and
elegant method of synthesis. We first designed Method A
and it proceeds well as we desired. However, as the three
different functional groups become larger, there may be
limitations due to steric hindrance of this method, so we
went back to the drawing table and designed Method
B. The synthesis of the very complicated compound 20
containing carbohydrate, RGD derivative and NOTA dem-
onstrated high yield. Both of these methods can be applied
to synthesize compounds having multimodality in the field
of molecular imaging.
1
the mesylate 4B as a colorless oil (2.38 g, 88%): H NMR
(500 MHz, CDCl3) δ 0.13 (s, 6H), 0.93 (s, 9H), 3.12
(s, 3H), 4.86 (s, 2H); 13C NMR (125 MHz, CDCl3) δ −5.0,
16.4, 25.9, 39.1, 58.3, 94.2, 97.3. HRMS (ESI+): m/z calcd.
for C10H20NaO3SSi+ [M + Na]+: 271.0795, found
271.0797.
(3-Methoxyprop-1-yn-1-yl)trimethylsilane (4M). To a solu-
tion of trimethylsilylated propargyl alcohol (3.57 g,
27.8 mmol) in anhydrous dichloromethane at 0 ꢀC was
added triethylamine (5.04 mL, 36.2 mmol). After being
stirred for 15 min at the same temperature, methanesulfonyl
chloride (2.38 mL, 30.6 mmol) was added dropwisely and
stirred for 1 h. The reaction mixture was quenched by addi-
tion of water. The aqueous layer was extracted three times
with dichloromethane, the organic layer was dried over
Na2SO4 and concentrated under reduced pressure to afford
Experimental
General Information. All commercial reagent and solvents
were used without further purification unless otherwise speci-
fied. Reagents and solvent were commercially purchased form
Sigma-Aldrich (MO, USA), Merck (Darmstadt, Germany),
TCI (Tokyo, Japan), and FutureChem. Reaction progress was
monitored by analytical thin layer chromatography (TLC)
with Merck 60 F-254 silica plates and visualized by UV light
(254 nm) or PMA indicator. Flash column chromatography
was performed on silica gel (Kieselgel 60; 230–400 mesh).
1H and 13C NMR spectra were recorded on 400-MR
(400 MHz-1H, 100 MHz-13C) or Varian Inova-500
(500 MHz-1H, 125 MHz-13C) and chemical shift (δ, ppm) are
reported in parts per million downfield from tetramethylsilane.
The hard copies of NMR spectra of all compounds is avail-
able in Supplementary Information. The melting point was
determined by fisher US/9300 and high-resolution mass
obtained on a 4.7 Tesla IonSpec Electrospray Ionization-Fou-
rier transform mass spectrometry (ESI-FTMS) or Micromass
LCT ESI-Time of Fright (ESI-TOF) mass spectrometer.
1
the mesylate 4M as a colorless oil (5.66 g, 89%): H NMR
(500 MHz, CDCl3) δ 0.18 (s, 9H), 3.11 (s, 3H), 4.82
(s, 2H); 13C NMR (125 MHz, CDCl3) δ −0.6, 39.0, 58.2,
95.7, 96.7. HRMS (ESI+): m/z calcd. for C7H14NaO3SSi+
[M + Na]+: 229.0325, found 229.0327.
tert-Butyl
(3-(tert-butyldimethylsilyl)prop-2-yn-1-yl)
(prop-2-yn-1-yl)carbamate (5BH). To the solution of 3H
(155 mg, 1.0 mmol) in anhydrous THF (3 mL) was added
60% NaH (48 mg, 1.2 mmol) at 0 ꢀC and stirred for
15 min. 4B (238 mg, 0.96 mmol) in anhydrous THF
(4 mL) was dropwise at 0 ꢀC and stirred for 4 h at
rt. Reaction mixture was washed with saturated aqueous
NaHCO3 and then extracted with EtOAc, dried, filtered,
and concentrated. Silica column chromatography provided
1
5BH as colorless oil (304 mg, 92%): H NMR (500 MHz,
CDCl3) δ 0.10 (t, J = 0.5 Hz, 6H), 0.93 (s, 9H), 1.48 (d,
J = 0.5 Hz, 9H), 2.20 (s, 1H), 4.17 (br, 4H); 13C NMR
(125 MHz, CDCl3) δ −4.8, 16.4, 26.0, 28.2, 35.0, 36.3,
71.8, 78.9, 80.8, 86.7, 101.2, 154.2. HRMS (ESI+): m/z
tert-Butyl prop-2-yn-1-ylcarbamate (3H). To a solution
of di-t-butyl dicarbonate (37.7 g, 173 mmol) in THF
(30 mL) at 0 ꢀC was added solution of propargylamine
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calcd. for C17H29NNaO2Si+ [M + Na]+: 330.1860, found
330.1860.
101.0, 122.6, 128.0, 128.6, 129.0, 134.5, 144.9. HRMS
(ESI+): m/z calcd. for C22H30N4NaSi+ [M + Na]+:
401.2132, found 401.2135.
N-((1-Benzyl-1H-1,2,3-triazol-4-yl)methyl)-3-(tert-
butyldimethylsilyl)-N-((1-(4-methoxybenzyl)-1H-
3-(tert-Butyldimethylsilyl)-N-(prop-2-yn-1-yl)-N-(3-(tri-
methylsilyl)prop-2-yn-1-yl)prop-2-yn-1-amine (6BMH).
To the solution of 5BH (5.7 g, 18.5 mmol) in dic-
hloromethane (30 mL) was added TFA (10 mL) and stirred
for 2 h at rt. Reaction mixture was washed with saturated
aqueous NaHCO3 and then extracted with dichloromethane,
dried, filtered, and concentrated to boc-deprotected com-
pound. Deprotected compound was dissolved in acetonitrile
(30 mL), to which was added 4M (4.58 g, 22.2 mmol) in
acetonitrile (10 mL), then stirred for 18 h at 60 ꢀC. The
reaction mixture was washed with saturated aqueous
NaHCO3 and then extracted with ethyl acetate, dried, fil-
tered, and concentrated. Silica column chromatography pro-
1,2,3-triazol-4-yl)methyl)prop-2-yn-1-amine (7B). To a
solution of 7BH (450 mg, 1.19 mmol) in acetonitrile
(16 mL) were added p-methoxybenzyl azide (233 mg,
1.43 mmol), diisopropylethylamine (0.02 mL, 0.12 mol)
and CuI (22 mg, 0.115 mmol). The reaction mixture was
stirred 2 h and concentrated under reduce pressure to afford
crude oil. Silica column chromatography (EtOAc:hexane
=1:1) provided 7B as a yellow oil (561 mg, 87%): 1H
NMR (500 MHz, CDCl3) δ 0.08 (s, 6H), 0.91 (s, 9H), 3.34
(br, 2H), 3.78 (s, 3H), 3.64–3.98 (m, 4H), 5.40 (s, 2H),
5.48 (s, 2H), 6.87 (d, J = 8.5 Hz, 2H), 7.20 (d, J = 8.5 Hz,
2H), 7.24 (d, J = 7.5 Hz, 2H), 7.31–7.37 (m, 3H), 7.46 (s,
1H), 7.47 (s, 1H); 13C NMR (125 MHz, CDCl3) δ −4.5,
−0.1, 16.4, 26.1, 29.6, 43.2, 47.9, 54.6, 54.1, 55.3, 88.8,
114.4, 122.6, 122.9, 126.5, 128.0, 128.7, 128.9, 129.0,
129.6, 134.6, 145.0, 159.8. HRMS (ESI+): m/z calcd for
C30H39N7NaOSi+ [M + Na]+: 564.2878, found 564.2876.
N-((1-Benzyl-1H-1,2,3-triazol-4-yl)methyl)-N-
1
vided 6BMH as an oil (4.71 g, 80%): H NMR (500 MHz,
CDCl3) δ 0.11 (s, 6H), 0.17 (s, 9H), 0.94 (s, 9H), 2.25
(t, J = 2.5 Hz, 1H), 3.46 (s, 2H), 3.47 (d, J = 2.5 Hz, 2H),
3.50 (s, 2H); 13C NMR (125 MHz, CDCl3) δ −4.6, −0.1,
16.5, 26.0, 41.7, 42.9, 42.9, 73.2, 78.6, 88.4, 90.0, 100.6,
100.9. HRMS (ESI+): m/z calcd. for C18H32NOSi2 [M
+
+ H]+: 318.2068, found 318.2068.
N-((1-Benzyl-1H-1,2,3-triazol-4-yl)methyl)-3-(tert-
butyldimethylsilyl)-N-(3-(trimethylsilyl)prop-2-yn-1-yl)
prop-2-yn-1-amine (7BM). To a solution of 6BM (73 mg,
0.23 mmol) in acetonitrile (0.5 mL) were added benzyl
azide (32 μL, 0.25 mmol), solution of CuI (460 μL, 0.1 M
in acetonitrile) and diisopropylethyl amine (460 μL, 0.1 M
in acetonitrile). The reaction mixture was stirred for 3 h
and concentrated under reduced pressure to afford a crude
oil. The organic layer was extracted three times with ethyl
acetate, dried over Na2SO4, and absorbed on silica for col-
umn chromatography (EtOAc:hexane = 1:5). After purifica-
tion, 7BM was obtained as a white solid (82 mg, 79%):
((1-(4-methoxybenzyl)-1H-1,2,3-triazol-4-yl)methyl)
prop-2-yn-1-amine (7H). To the 7B (470 mg, 0.87 mmol)
in THF (25 mL) was added 1 M TBAF (0.9 mL, 9 mmol)
at 0 ꢀC. The reaction was stirred for 30 min and then
washed with saturated aqueous NaHCO3 and extracted with
EtOAc. The organic layers were dried, filtered and concen-
trated. Silica column chromatography (EtOAc:hexane = 1:2)
1
provided the 7H as a yellow oil (343 mg, 95%): H NMR
(500 MHz, CDCl3) δ 2.21 (s, 1H), 3.29 (s, 2H), 3.76 (s,
3H), 3.79 (br, 4H), 5.39 (s, 2H), 5.46 (s, 2H), 6.85 (d,
J = 8.5 Hz, 2H), 7.18 (d, J = 8.5 Hz, 2H), 7.21–7.34 (m,
5H), 7.45 (s, 1H), 7.49 (s, 1H); 13C NMR (125 MHz,
CDCl3) δ 41.9, 47.6, 53.5, 53.9, 55.2, 73.7, 78.2, 114.3,
122.7, 123.0, 126.5, 127.9, 128.5, 128.9, 129.5, 134.6,
144.4, 144.5, 159.7. HRMS (ESI+): m/z calcd. for
C24H25N7NaO+ [M + Na]+: 450.2013, found 450.2016.
1-(1-Benzyl-1H-1,2,3-triazol-4-yl)-N-((1-(2-(2-(2-fluoro-
ethoxy)ethoxy)ethyl)-1H-1,2,3-triazol-4-yl)methyl)-N-
((1-(4-methoxybenzyl)-1H-1,2,3-triazol-4-yl)methyl)
methanamine (7). To the 7H (34 mg, 0.08 mmol) in aceto-
nitrile (2 mL) was added 12c (17 mg, 0.096 mmol), 0.1 M
1
mp. 72–74 ꢀC; H NMR (500 MHz, CDCl3) δ 0.09 (s, 6H),
0.15 (s, 9H), 0.93 (s, 9H), 3.43 (s, 2H), 3.46 (s, 2H), 3.82
(s, 2H), 5.51 (s, 2H), 7.25–7.29 (m, 2H), 7.33–7.40 (m,
3H), 7.42 (s, 1H); 13C NMR (125 MHz, CDCl3) δ −4.6,
−0.1, 16.4, 26.0, 43.0, 43.1, 48.1, 54.1, 88.3, 89.9, 100.8,
101.2, 122.6, 128.1, 128.7, 129.1, 134.5, 145.2. HRMS
(ESI+): m/z calcd. for C25H38N4NaSi2 [M + Na]+:
+
473.2527, found 473.2525.
N-((1-Benzyl-1H-1,2,3-triazol-4-yl)methyl)-3-(tert-
butyldimethylsilyl)-N-(prop-2-yn-1-yl)prop-2-yn-1-amine
(7BH). To a solution of 7BM (596 mg, 1.32 mmol) dis-
solved in methanol (8 mL) was added NaOMe (2.64 mL,
1.32 mmol, 0.5 M solution in methanol) and stirred for 1 h
at rt. Then the mixture was concentrated under reduced
pressure and dissolved again in EtOAc for filtration over
pad of silica. Resulting solution was evaporated to afford a
diisopropylethylamine
in
acetonitrile
(0.16 mL,
0.016 mmol) and 0.1 M CuI in acetonitrile (0.16 mL,
0.016 mmol). After 2 h, reaction mixture was concentrated.
Silica column chromatography (2–8% methanol in dic-
hloromethane) provide 7 as a sticky green oil (46 mg,
1
95%): H NMR (500 MHz, CDCl3) δ 3.61–3.67 (m, 6H),
1
white solid 7BH (490 mg, 98%): mp. 52–53 ꢀC; H NMR
3.71–3.73 (m, 7H), 3.80 (s, 3H), 3.87 (t, J = 8.5 Hz, 2H),
4.46 (t, J = 4.0 Hz, 1H), 4.53 (t, J = 4.5–5.5 Hz, 2H), 4.56
(t, J = 4.0 Hz, 1H), 5.30 (s, 1H), 5.44 (s, 2H), 5.51 (s, 2H),
6.88 (d, J = 8.5 Hz, 2H), 7.22 (d, J = 8.5 Hz, 2H), 7.26 (d,
J = 6.5 Hz, 2H), 7.32–7.38 (m, 3H), 7.64 (s, 1H), 7.69 (s,
1H), 7.86 (s, 1H); 13C NMR (125 MHz, CDCl3) δ 50.5,
(500 MHz, CDCl3) δ 0.07 (s, 6H), 0.90 (s, 9H), 2.20 (t,
J = 2.5 Hz, 1H), 3.40 (d, J = 2.5 Hz, 2H), 3.44 (s, 2H),
3.81 (s, 2H), 5.48 (s, 2H), 7.20–7.25 (m, 2H), 7.29–7.36
(m, 3H), 7.41 (s, 1H); 13C NMR (125 MHz, CDCl3) δ
−4.7, 16.3, 26.0, 41.8, 43.0, 47.9, 54.0, 73.1, 78.6, 88.4,
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55.1, 69.1, 69.9, 70.1, 70.2, 70.2, 70.3, 70.4, 70.5, 70.5,
70.6, 70.6, 71.2, 82.2, 82.3, 83.6, 83.6, 114.2, 126.4,
127.9, 128.5, 128.8, 129.6, 134.4, 159.6. HRMS (ESI+): m/
methyl-N-[3-(t-butyldimethylsilyl)propargyl]-N-
propargylamine (10B). To a solution of 7BH (100 mg,
0.26 mmol) in acetonitrile (2 mL) were added protected
azidoglucose 12d (118 mg, 0.32 mmol), 0.1
z calcd. for C30H37FN10NaO3 [M + Na]+: 627.2926, found
M
+
627.2929.
diisopropylethylamine in acetonitrile (0.26 mL,
(2R,3R,4S,5R,6R)-2-(Acetoxymethyl)-6-(4-((((1-benzyl-
1H-1,2,3-triazol-4-yl)methyl)((1-(4-methoxybenzyl)-1H-
1,2,3-triazol-4-yl)methyl)amino)methyl)-1H-1,2,3-triazol-
1-yl)tetrahydro-2H-pyran-3,4,5-triyl triacetate (8). To
the 7H (122 mg, 0.29 mmol) in acetonitrile (2 mL) was
added protected azidoglucose 12d (118 mg, 0.32 mmol),
0.1 M diisopropylethylamine in acetonitrile (0.31 mL,
0.031 mmol) and 0.1 M CuI in acetonitrile (0.31 mL,
0.031 mmol). After 3 h, reaction mixture was concentrated.
Silica column chromatography (5% methanol in dic-
hloromethane) provide 8 as an oil (200 mg, 87%): 1H
NMR (500 MHz, CDCl3) δ 1.80 (s, 3H), 2.03 (s, 3H), 2.07
(s, 3H), 2.09 (s, 3H), 3.64–3.77 (m, 6H), 3.80 (s, 3H),
3.99–4.01 (m, 1H), 4.16 (d, J = 12.5 Hz, 1H), 4.30 (dd,
J = 12.5, 5.0 Hz, 1H), 5.26 (t, J = 9.0 Hz, 1H), 5.40–5.47
(m, 4H), 5.52 (s, 2H), 5.83 (d, J = 8.5 Hz, 1H), 7.23 (d,
J = 9.0 Hz, 2H), 7.26–7.38 (m, 5H), 7.64 (s, 1H), 7.68 (s,
1H), 8.00 (s, 1H); 13C NMR (125 MHz, CDCl3) δ 20.0,
20.4, 20.5, 20.6, 46.9, 47.0, 53.6, 54.0, 55.2, 61.4, 67.6,
70.4, 72.5, 75.0, 85.7, 114.3, 122.4, 123.4, 123.7, 126.7,
127.9, 128.6, 129.0, 129.5, 134.7, 144.0, 144.1, 144.6,
159.7, 168.7, 169.3, 169.9, 170.5. HRMS (ESI+): m/z
0.026 mmol) and 0.1 M CuI in acetonitrile (0.26 mL,
0.026 mmol). The reaction mixture was stirred 3 h and con-
centrated under reduce pressure to afford a crude oil. Silica
column chromatography (EtOAc:hexane = 1:1) provided
1
10B as a yellow oil (136 mg, 68%): H NMR (500 MHz,
CDCl3) δ 0.05 (s, 6H), 0.88 (s, 9H), 1.95 (s, 3H), 1.99 (s,
3H), 2.00 (s, 3H), 3.30 (br, 2H), 3.76 (br, 2H), 3.78 (br,
2H), 3.97–3.99 (m, 1H), 4.08 (d, J = 12.5 Hz, 1H), 4.24
(dd, J = 12.5, 5.0 Hz, 1H), 5.17 (t, J = 9.5 Hz, 1H),
5.31–5.39 (m, 2H), 5.45 (s, 2H), 5.82 (d, J = 8.0 Hz, 1H),
7.19 (d, J = 6.5 Hz, 2H), 7.25–7.30 (m, 3H), 7.45 (s, 1H),
7.74 (s, 1H); 13C NMR (125 MHz, CDCl3) δ −4.7, 13.9,
16.2, 19.9, 20.3, 20.4, 25.9, 43.0, 47.6, 53.8, 61.3, 67.4,
70.2, 72.3, 74.8, 85.4, 88.7, 100.8, 121.3, 122.8, 127.8,
128.4, 128.8, 134.5, 144.8, 145.2, 168.5, 169.1, 169.6,
170.2. HRMS (ESI) m/z calcd. for C36H49N7NaO9Si [M
+ Na]+ 774.3259, found 774.3260.
N-(1-Benzyl-1,2,3-triazol-4-yl)methyl-N-[1-(O-
tetraacetyl-1-deoxyglucose-1-yl)-1,2,3-triazol-4-yl]
methyl-N-propargylamine (10H). To 10B (136 mg,
0.18 mmol) in THF (9 mL) was added 1 M TBAF
ꢀ
(0.02 mL, 0.2 mmol) at 0 C. The reaction was stirred for
calcd. for C38H44N10NaO10 [M + Na]+: 823.3134, found
2 h and then concentrated. Silica column chromatography
+
823.3136.
(2% methanol in dichloromethane) provided 10H as a yel-
low oil (82 mg, 71%): H NMR (500 MHz, CDCl3) δ 1.76
1
N-(9-(3-((2-(2-(2-(2-(4-((((1-Benzyl-1H-1,2,3-triazol-4-yl)
methyl)((1-(4-methoxybenzyl)-1H-1,2,3-triazol-4-yl)
methyl)amino)methyl)-1H-1,2,3-triazol-1-yl)ethoxy)eth-
oxy)ethoxy)ethyl)carbamoyl)phenyl)-6-(diethylamino)-
(s, 3H), 1.97 (s, 3H), 2.01 (s, 3H), 2.02 (s, 3H), 2.24 (s,
1H), 3.29 (br, 2H), 3.78 (br, 4H), 3.97–4.00 (m, 1H), 4.10
(d, J = 12.5 Hz, 1H), 4.25 (dd, J = 12.5, 5.0 Hz, 1H), 5.19
(t, J = 9.5 Hz, 1H), 5.33–5.40 (m, 2H), 5.47 (s, 2H), 5.83
(d, J = 8.0 Hz, 1H), 7.22 (d, J = 7.5 Hz, 2H), 7.27–7.33
(m, 3H), 7.50 (s, 1H), 7.79 (s, 1H); 13C NMR (125 MHz,
CDCl3) δ 19.9, 20.3, 20.4, 20.5, 41.8, 47.4, 47.6, 53.9,
60.2, 61.3, 67.5, 70.3, 72.3, 73.7, 74.9, 85.5, 121.5, 122.9,
127.9, 128.5, 128.9, 134.6, 144.7, 145.0, 168.7, 169.2,
169.7, 170.3. HRMS (ESI) m/z calcd. for C30H35N7NaO9
[M + Na]+ 660.2394, found 660.2394.
3H-xanthen-3-ylidene)-N-ethylethanaminium
chloride
(9). To azidorhodamine 12e (100 mg, 0.157 mmol) in ace-
tonitrile (2 mL) was added 7H (74 mg, 0.173 mmol),
diisopropylethylamine (5 μL, 0.052 mmol) and CuI (3 mg,
0.016 mmol). After 12 h, the reaction mixture was concen-
trated. Silica column chromatography (5% methanol in dic-
hloromethane) provide 9 as a white solid (60 mg, 33%):
mp. 69–71 ꢀC; 1H NMR (500 MHz, CDCl3) δ 1.12 (t,
J = 7.0 Hz, 12H), 3.12 (t, J = 7.0 Hz, 2H), 3.27–3.32 (m,
13H), 3.41–3.42 (m, 2H), 3.50 (s, 4H), 3.58–3.86 (m,
11H), 4.46 (br, 2H), 6.23 (dd, J = 9.0, 2.0 Hz, 2H), 6.34
(d, J = 2.0 Hz, 2H), 6.38 (s, 1H), 6.40 (s, 1H), 6.84 (d,
J = 7.0 Hz, 2H), 6.88 (d, J = 8.0 Hz, 2H), 7.23 (d,
J = 9.0 Hz, 2H), 7.20 (d, J = 8.0 Hz, 2H), 7.23 (d,
J = 7.0 Hz, 2H), 7.28–7.85 (m, 9H); 13C NMR (125 MHz,
CDCl3) δ 12.4, 39.1, 44.2, 55.2, 64.7, 67.6, 69.2, 69.8,
70.2, 70.3, 97.6, 105.3, 107.9, 114.3, 122.5, 123.6, 126.6,
127.8, 127.9, 128.5, 128.6, 128.9, 129.5, 130.7, 132.3,
134.6, 148.6, 153.1, 153.6, 159.7, 168.1. HRMS (ESI+): m/
(2S,3S,4R,5S,6R)-2-(Acetoxymethyl)-6-(4-((((1-benzyl-
1H-1,2,3-triazol-4-yl)methyl)((1-(2-(2-(2-fluoroethoxy)
ethoxy)ethyl)-1H-1,2,3-triazol-4-yl)methyl)amino)
methyl)-1H-1,2,3-triazol-1-yl)tetrahydro-2H-pyran-
3,4,5-triyl triacetate (10). To 10H (65 mg, 0.10 mmol) in
acetonitrile (2 mL) was added 12c (22 mg, 0.12 mmol),
0.1 M diisopropylethylamine in acetonitrile (0.1 mL,
0.01 mmol) and 0.1 M CuI in acetonitrile (0.1 mL,
0.01 mmol). After 1.5 h, reaction mixture was concentrated.
Silica column chromatography (2–5% methanol in dic-
hloromethane) provide 10 as a yellow solid (75 mg, 90%):
1H NMR (500 MHz, CDCl3) δ 1.79 (s, 3H), 1.99 (s, 3H),
2.03 (s, 3H), 2.05 (s, 3H), 3.60 (br, 6H), 3.69 (br, 2H),
4.01 (br, 3H), 4.14 (d, J = 11.5 Hz, 1H), 4.28 (d,
J = 9.5 Hz, 1H), 4.44 (br, 1H), 4.53 (br, 3H), 5.23 (br, 1H),
z calcd. for C60H72N13NaO6 [M + Na]+: 1093.5615, found
1093.5617.
N-(1-Benzyl-1,2,3-triazol-4-yl)methyl-N-[1-(O-
tetraacetyl-1-deoxyglucose-1-yl)-1,2,3-triazol-4-yl]
+
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5.40 (br, 1H), 5.49 (br, 3H), 5.87 (br, 1H), 7.23–7.32 (m,
5H), 8.04 (br, 3H); 13C NMR (125 MHz, CDCl3) δ 20.1,
20.4, 20.5, 20.6, 29.6, 50.6, 61.4, 67.6, 69.1, 70.0, 70.2,
70.3, 70.4, 70.4, 70.5, 70.6, 70.7, 72.6, 75.1, 82.4, 83.7,
128.1, 128.6, 128.9, 134.5, 168.7, 169.2, 169.8, 170.5.
concentrated. Silica column chromatography (2–5% metha-
nol in dichloromethane) provide 11 as an oil (30 mg, 23%):
1H NMR (400 MHz, CDCl3) δ 1.42 (s, 9H), 1.82–1.84 (m,
1H), 1.99–2.14 (m, 4H), 2.38–2.40 (m, 2H), 3.18 (q,
J = 6.0 Hz, 2H), 3.45 (m, 1H), 3.60–3.64 (m, 6H),
3.70–3.76 (m, 7H), 3.85 (t, J = 5.0 Hz, 2H), 4.39–4.44 (m,
2H), 4.51–4.56 (m, 2H), 5.51 (s, 2H), 7.17 (t, J = 5.6 Hz,
1H), 7.16–7.34 (m, 5H), 7.12 (s, 1H), 7.89 (s, 1H), 7.96 (s,
1H); 13C NMR (100 MHz, CDCl3) δ 10.0, 10.9, 14.1, 22.7,
23.0, 23.7, 28.0, 28.9, 29.3, 29.7, 30.0, 32.8, 36.2, 38.7,
47.4, 47.6, 47.7, 50.2, 53.4, 54.2, 61.0, 69.4, 70.2, 70.4,
70.6, 71.8, 77.2, 81.7, 82.3, 83.9, 123.7, 124.5, 124.8,
128.1, 128.7, 129.1, 134.6, 143.7, 143.9, 144.4, 173.3,
HRMS (ESI+) m/z calcd. for C36H47FN10NaO11 [M
+
+ Na]+: 837.3302, found 837.3301.
N-(1-Benzyl-1,2,3-triazol-4-yl)methyl-N-[(3-(t-
butyldimethylsilyl)propargyl)-1,2,3-triazol-4-yl]methyl-
N-{1-[3-(N-Fmoc-O-t-butylglutamin-N’-yl)propyl]-
1,2,3-triazol-4-yl}methylamine (11B). To a solution of
7BH (71 mg, 0.20 mmol) in acetonitrile (2 mL) were added
12f (148 mg, 0.29 mmol), 0.1 M diisopropylethylamine in
acetonitrile (0.01 mL, 0.01 mmol) and 0.1 M CuI in aceto-
nitrile (0.2 mL, 0.02 mmol). The reaction mixture was
stirred 0.5 h and concentrated under reduce pressure to
afford a crude oil. Silica column chromatography (2%
methanol in dichloromethane) provided 11B as oil
(110 mg, 64%): 1H NMR (400 MHz, CDCl3) δ 0.09 (s,
6H), 0.92 (s, 9H), 1.44 (s, 9H), 1.88–1.94 (m, 1H),
2.05–2.31 (m, 5H), 3.12–3.27 (m, 2H), 3.36 (s, 2H), 3.79
(s, 2H), 3.83 (s, 2H), 4.19–4.24 (m, 2H), 4.34–4.42 (m,
4H), 5.48 (s, 2H), 5.75 (d, J = 8.0 Hz, 1H), 6.63 (m, 1H),
7.23–7.41 (m, 9H), 7.47 (s, 1H), 7.58–7.59 (m, 2H), 7.65
(s, 1H), 7.75 (d, J = 7.6 Hz, 2H); 13C NMR (100 MHz,
CDCl3) δ −4.5, 16.5 26.1, 28.0, 28.9, 30.0, 32.5, 36.3,
43.4, 47.1, 47.5 (2C), 48.2, 54.0, 67.0, 82.4, 88.9, 101.0,
120.0, 123.0, 123.6, 125.1, 125.2, 127.1, 127.7, 128.1,
128.7, 129.1, 134.6, 141.3, 143.7, 143.9, 144.8, 145.0,
156.5, 171.2, 172.6. HRMS (ESI) m/z calcd. for
C49H63N9NaO5Si [M + Na]+ 908.4614, found 908.4616.
N-(1-Benzyl-1,2,3-triazol-4-yl)methyl-N-{1-[3-(N-O-t-
butylglutamin-N’-yl)propyl]-1,2,3-triazol-4-yl}methyl-N-
propargylamine (11H). To 11B (100 mg, 0.11 mmol) in
THF (2 mL) was added 1 M TBAF (0.033 mL, 0.33 mmol)
at rt. The reaction was stirred for 2 h and then concentrated.
Silica column chromatography (5–10% methanol in dic-
hloromethane) provided the 11H as a yellow oil (50 mg,
81%): 1H NMR (400 MHz, CDCl3) δ 1.46 (s, 9H),
1.76–1.84 (m, 1H), 1.92 (br, 2H), 2.07–2.14 (m, 3H), 2.27
(t, J = 2.4 Hz, 1H), 2.36 (t, J = 8.0 Hz, 2H), 3.18–3.24 (m,
2H), 3.35–3.36 (m, 3H), 3.82 (s, 2H), 3.85 (s, 2H), 4.40 (t,
J = 6.8 Hz, 2H), 5.52 (s, 2H), 6.71 (t, J = 5.6 Hz, 1H),
7.26–7.28 (m, 2H), 7.35–7.40 (m, 3H), 7.53 (s, 1H), 7.68
(s, 1H); 13C NMR (100 MHz, CDCl3) δ 28.0, 30.1, 30.2,
32.9, 36.2, 42.1, 47.6, 47.7, 47.9, 54.1, 54.4, 73.8, 78.3,
81.4, 123.1, 123.6, 128.0, 128.7, 129.1, 134.7, 144.5,
144.9, 173.1, 174.7. HRMS (ESI) m/z calcd. for
C28H39N9NaO3 [M + Na]+ 572.3068, found 572.3069.
N-(1-Benzyl-1,2,3-triazol-4-yl)methyl-N-[1-(8-fluoro-
3,6-dioxaoctyl)-1,2,3-triazol-4-yl]methyl-N-{1-[3-(N-O-t-
butylglutamin-N’-yl)propyl]-1,2,3-triazol-4-yl}methyl-
amine (11). To the 11H (100 mg, 0.18 mmol) in acetoni-
trile (2 mL) was added 12f (64 mg, 0.36 mmol),
diisopropylethylamine (0.01 mL, 0.05 mmol) and CuI
(2 mg, 0.009 mmol). After 3 h, reaction mixture was
181.0. HRMS (ESI+) m/z calcd. for C34H51FN12NaO5 [M
+
+ Na]+: 749.3982, found 749.3986.
β-1-N-Boc-TBS-tripod-2,3,4,6-tetra-O-acetyl-D-glucose
(14). A solution of protected azidoglucose 12d (550 mg,
1.47 mmol) and 13 (632 mg, 1.47 mmol) in a 4:1 mixture
of t-BuOH–water (30 mL) was treated with CuSO4Á5H2O
(184 mg, 0.74 mmol) and sodium ascorbate (NaASC,
174 mg, 0.88 mmol). The reaction mixture was stirred at
23 ꢀC for 18 h. The reaction mixture was diluted with
EtOAc (300 mL), washed with water (3 × 30 mL) and satu-
rated aqueous NaCl (30 mL), dried (Na2SO4) and concen-
trated under reduced pressure to provide the crude product.
Flash column chromatography (EtOAc:hexane = 1:1)
afforded bis-protected tripod-glucose 14 as a white solid
1
(1.02 g, 87%): H NMR (400 MHz, CDCl3) δ 7.90 (s, 1H),
5.79 (d, J = 9.2 Hz, 1H), 5.54 (d, J = 3.1 Hz, 1H), 5.44 (t,
J = 9.7 Hz, 1H), 5.24 (dd, J = 10.3 Hz, 2.9 Hz, 1H), 4.74
(m, 1H), 4.62 (s, 2H), 4.23–4.11 (m, 3H), 4.06 (d,
J = 5.2 Hz, 2H), 3.32–3.24 (m, 2H), 3.21–3.14 (m, 2H),
2.24 (s, 3H), 2.09–2.04 (m, 5H), 2.00 (s, 3H), 1.88 (s, 3H),
1.43 (s, 9H), 0.94 (s, 9H), 0.13 (s, 6H); 13C NMR
(100 MHz, CDCl3) δ 170.2, 170.0, 169.7, 168,9, 155.8,
143.8, 121.1, 99.6, 89.6, 86.2, 79.2, 73.9, 70.4, 68.2, 66.8,
61.2, 49.5, 41.5, 38.8, 37.0, 28.3, 26.0, 23.8, 20.61, 20.56,
20.4, 20.1, 16.4, −4.8. HRMS (ESI+): m/z calcd. for
C34H55N5NaO13SSi+
824.3179.
[M + Na]+:
824.3179,
found
β-1-N-Boc-tripode-2,3,4,6-tetra-O-acetyl-D-glucose (15).
A solution of 14 (900 mg, 1.12 mmol) in THF (20 mL) at
0 ꢀC under Ar was treated with a solution of TBAF in THF
ꢀ
(1.4 mL, 1.0 M). The reaction mixture was stirred at 0 C
for 1 h and quenched with saturated aqueous NH4Cl
(10 mL). Resulting mixture was treated with EtOAc
(100 mL) and stirred for 1 h. Phases were separated, and
the organic phase was washed with water (3 × 20 mL) and
saturated aqueous NaCl (30 mL), dried (Na2SO4) and con-
centrated under reduced pressure to provide the crude prod-
uct. Flash column chromatography (EtOAc:hexane = 4:1)
afforded 15 as a white solid (647 mg, 84%): 1H NMR
(400 MHz, CDCl3) δ 7.89 (s, 1H), 5.80 (d, J = 9.1 Hz,
1H), 5.51 (s, 1H), 5.41 (t, J = 9.7 Hz, 1H), 5.24 (dd,
J = 10.3 Hz, 1.8 Hz, 1H), 4.87 (m, 1H), 4.61 (s, 2H),
4.24–4.10 (m, 3H), 4.01 (s, 2H), 3.26–3.22 (m, 2H),
Bull. Korean Chem. Soc. 2021, Vol. 42, 333–341
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KOREAN CHEMICAL SOCIETY
3.21–3.13 (m, 2H), 2.40 (s, 1H), 2.21 (s, 3H), 2.05–2.01
(m, 5H), 1.97 (s, 3H), 1.84 (s, 3H), 1.40 (s, 9H); 13C NMR
(100 MHz, CDCl3) δ 170.3, 170.0, 169.8, 169.0, 155.9,
143.8, 121.9, 86.3, 79.4, 77.4, 74.4, 74.1, 70.5, 68.2, 66.8,
61.2, 49.9, 41.7, 38.8, 36.0, 28.3, 24.1, 20.7, 20.6, 20.5,
20.2. HRMS (ESI+): m/z calcd. for C28H41N5NaO13S+ [M
+ Na]+: 710.2314, found 710.2314.
and the residue was dried under reduced pressure to pro-
vide deprotected compound, which used in the next step
without purification. Crude deprotected compound from the
previous step was dissolved in 4:1 = 0.1 M aqueous
Na2CO3-DMF (4 mL) and treated with commercially avail-
able NOTA-isothiocyanate 19 (20 mg, 0.036 mmol). The
reaction mixture was stirred at 23 ꢀC for 40 h under Ar and
then concentrated under reduced pressure. The residue was
purified with HPLC (Alltima 5 μm C18, 250 × 10 mm, gra-
dient, 5–50% MeCN-0.1% aqueous TFA buffer, 20 min,
4 mL/min, tR 23 min). The collected fraction was lyophi-
lized to afford the TFA salt of 20 as a white solid (28 mg,
51%): 1H NMR (500 MHz, DMSO) δ 9.63 (br, 1H),
847–8.44 (m, 1H), 8.37 (s, 1H), 8.24 (s, 1H), 8.18 (s, 1H),
8.17–8.11 (m, 2H), 8.00 (d, J = 7.1 Hz, 1H), 7.94 (s, 1H),
7.83 (d, J = 8.1 Hz, 2H), 7.65 (br, 2H), 7.41–7.36 (m, 4H),
7.24–7.18 (m, 4H), 7.16–7.11 (m, 4H) 5.66 (s, 2H), 5.50
(d, J = 9.1 Hz, 1H), 4.66–4.61 (m, 2H), 4.48–4.37 (m, 6H),
4.19–4.14 (m, 2H), 4.06–4.00 (m, 3H), 3.97–3.91 (m, 2H),
3.82–3.68 (m, 5H), 3.57–3.47 (m, 5H), 3.28–3.15 (m,
10H), 2.92–2.88 (m, 3H), 2.83–2.68 (m, 6H), 2.63–2.60
(m, 1H), 2.38–2.34 (m, 1H), 1.93–1.87 (m, 2H), 1.75–1.69
(m, 1H), 1.60–1.56 (m, 1H), 1.47–1.34 (m, 6H), 1.11–1.02
(m, 2H); 13C NMR (125 MHz, DMSO) δ 180.5, 173.4,
172.2, 171.7, 171.5, 171.2, 170.6, 170.0, 169.6, 169.3,
165.8, 162.3, 158.6, 158.4, 156.7, 142.7, 142.6, 138.9,
137.7, 137.3, 134.4, 129.2, 129.1, 128.1, 127.7, 127.6,
126.3, 124.5, 122.9, 117.8, 115.5, 88.2, 78.5, 73.6, 69.5,
68.5, 60.5, 58.7, 54.6, 54.4, 54.3, 53.2, 52.5, 51.8, 51.5,
50.9, 49.8, 48.9, 44.6, 43.3, 42.9, 42.2, 41.0, 40.3, 37.5,
β-1-N-Boc-tripode-D-glucose (16). Glucose derivative 15
(600 mg, 0.87 mmol) in a vial was treated with a solution of
NH3 in methanol (23 mL, 7 N). The reaction mixture was
stirred at 23 ꢀC for 18 h in a sealed condition. Volatiles were
removed under a stream of N2, and the residue was subject to
flash column chromatography (2–10% methanol in dic-
hloromethane) to provide 16 as a white film (430 mg, 95%):
1H NMR (400 MHz, CD3OD) δ 8.25 (s, 1H), 5.55 (d,
J = 9.2 Hz, 1H), 4.63 (s, 2H), 4.11 (t, J = 9.4 Hz, 1H), 4.05
(s, 2H), 3.95 (s,1H), 3.84–3.64 (m, 5H), 3.21–3.13 (m, 4H),
3.21–3.13 (m, 2H), 2.87 (s, 1H), 1.95–1.91 (m, 2H), 1.42 (s,
9H); 13C NMR (100 MHz, CD3OD) δ 158.3, 144.4, 124.2,
90.0, 80.1, 79.8, 78.4, 76.0, 75.0, 71.4, 70.2, 62.3, 50.7,
42.5, 39.7, 37.0, 28.8, 25.0. HRMS (ESI+): m/z calcd. for
C20H33N5NaO9S+ [M + Na]+: 542.1891, found 542.1893.
β-1-N-Boc-tripod-D-glucose-protected RGDfK (18). A
solution of 16 (119 mg, 0.23 mmol) and commercially
available 17 (200 mg, 0.22 mmol) in a 3:1 mixture of
MeOH-water (30 mL) was treated with CuSO4Á5H2O
(28 mg, 0.11 mmol) and sodium ascorbate (27 mg,
0.13 mmol). The reaction mixture was stirred at 23 ꢀC for
2 h and concentrated under reduced pressure to provide
crude product. The crude product was purified with HPLC
(Alltima 5 μm C18, 250 × 10 mm, gradient, 25–100%
MeCN-0.1% aqueous TFA buffer, 30 min, 4 mL/min, tR
16–19 min, broad peak) to afford 18 as a white solid
35.8, 35.1, 33.5, 31.0, 30.8, 28.5, 25.2, 22.9, 22.8. HRMS
(ESI+): m/z calcd. for C70H99N21O21S2
[M + 2H]2+:
2+
816.8377, found 816.8375.
1
(205 mg, 71%): H NMR (400 MHz, CD3OD) δ 8.22 (s,
1H), 7.95 (s, 1H), 7.83 (d, J = 8.3 Hz, 2H), 7.36 (d,
J = 8.3 Hz, 2H), 7.21–7.14 (m, 5H), 5.63–5.58 (m, 3H),
4.76–4.70 (m, 1H), 4.62–4.56 (m, 1H), 4.49 (br, 4H),
4.21–4.16 (m, 3H), 4.01–3.97 (m, 2H), 3.88–3.85 (m, 1H),
3.80–3.72 (m, 3H), 3.37–3.28 (m, 2H), 3.17–3.08 (m, 7H),
2.98–2.91 (m, 5H), 2.77–2.72 (m, 1H), 2.54–2.47 (m, 8H),
2.04 (s, 3H), 1.87–1.81 (m, 3H), 1.72–1.68 (m, 1H),
1.61–1.49 (m, 4H), 1.45–1.39 (m, 24H), 1.12–1.09 (m,
2H); 13C NMR (125 MHz, CD3OD) δ174.8, 173.8, 173.5,
172.4, 172.1, 171.1, 169.4, 161.2, 160.9, 158.4, 144.9,
144.6, 140.2, 137.8, 135.7, 130.3, 129.6, 129.2, 129.0,
127.9, 125.8, 124.4, 118.6, 90.1, 87.8, 82.3, 80.1, 79.9,
75.2, 71.5, 70.4, 62.5, 56.5, 56.3, 54.4, 54.0, 51.4, 51.0,
44.8, 43.9, 42.7, 40.5, 39.8, 38.2, 37.5, 31.9, 31.1, 29.8,
29.2, 28.8, 28.7, 28.3, 25.1, 24.3, 19.7, 18.4, 12.6. HRMS
Acknowledgments. This work was supported by the Radia-
tion Technology R&D program through the National
Research Foundation of Korea funded by the Ministry of Sci-
ence, ICT & Future Planning (2016M2A2A7A03913537)
and by a Sogang University Research Grant.
Supporting Information. Additional supporting informa-
tion is available in the online version of this article.
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