Role of linkers in tertiary amines that mediate or catalyze 1,3,5-triazine-based amide-forming reactions

Article abstract of DOI:10.1021/jo500376m

We studied 1,3,5-triazine-based amide-forming reactions that are mediated or catalyzed by various tert-amines. The representative tert-amine was trimethylamine, which has amido, 1,2,3-triazolyl, aryl, and alkyl linkers. It was found that electron-deficient aryl and heteroaryl linkers, particularly 1,2,3-triazolyl linkers, are superior. On the basis of our findings, we synthesized ligand catalysts, including a 1,2,3-triazolyl linker that connects a protein ligand to a trimethylamine moiety, and found that fluorescent-labeling of a targeting protein using the ligand catalysts proceeded in good yields.

Full text of DOI:10.1021/jo500376m

Note
pubs.acs.org/joc
Role of Linkers in Tertiary Amines That Mediate or Catalyze
1,3,5-Triazine-Based Amide-Forming Reactions
Masanori Kitamura, Fumitaka Kawasaki, Kouichi Ogawa, Shuichi Nakanishi, Hiroyuki Tanaka,
Kohei Yamada, and Munetaka Kunishima*
Faculty of Pharmaceutical Sciences, Institute of Medical, Pharmaceutical, and Health Sciences, Kanazawa University, Kakuma-machi,
Kanazawa 920-1192, Japan
S
* Supporting Information
ABSTRACT: We studied 1,3,5-triazine-based amide-forming reactions
that are mediated or catalyzed by various tert-amines. The representative
tert-amine was trimethylamine, which has amido, 1,2,3-triazolyl, aryl, and
alkyl linkers. It was found that electron-deficient aryl and heteroaryl linkers,
particularly 1,2,3-triazolyl linkers, are superior. On the basis of our findings,
we synthesized ligand catalysts, including a 1,2,3-triazolyl linker that con-
nects a protein ligand to a trimethylamine moiety, and found that fluorescent-
labeling of a targeting protein using the ligand catalysts proceeded in good
yields.
mides and esters are two of the most fundamental and
significant functional groups in chemistry, biochemistry,
To date, we have employed N,N-dimethylglycine ester as a
linker and a tert-amine moiety (Figure 1a) for the amide-
forming reaction for the following three reasons. (1)
Introduction of the N,N-dimethylaminomethyl group to func-
tional molecules via the ester bond is convenient. (2) The
electron-withdrawing β-carbonyl group decreases the pKa of the
conjugate acid of the N,N-dimethylaminomethyl group; thus, it
should be present in a reactive nonprotonated form in a protic
solvent. The resulting nonprotonated N,N-dimethylamino-
methyl group reacts smoothly with CDMT to form the
dehydrocondensing reagent 2 and shows good catalytic
activity.12 (3) We have reported that the structure of tert-
amines affects the reactivity toward CDMT and proposed the
gauche β-alkyl group effect; i.e., the existence of a β-alkyl group
in a gauche relationship against the electron pair of the amine
nitrogen decreases the reactivity of tert-amines.13 The N,N-
dimethylaminomethyl group does not have the gauche β-alkyl
group effect.
However, the ester bonds might be hydrolyzed during a
dehydrocondensing reaction in an aqueous solvent or storage
under atmospheric conditions. Thus, an alternative method for
tethering between a N,N-dimethylaminomethyl group and
various functional molecules is required.
Herein, we report on the synthesis of various tert-amines (1
in Figure 1b) that have amido, 1,2,3-triazolyl, aryl, and alkyl
linkers, and their reactivity for amide-forming reactions.
Trimethylamine was chosen as the representative tert-amine
moiety because there is no influence of the gauche β-alkyl group
effect, which changes the reactivity of the tert-amines. Thus,
the observed result will indicate the role of the linkers.
A
and materials science. Therefore, many dehydrocondensing
reagents for synthesizing amide and ester bonds from
carboxylic acids and amines or alcohols have been developed
over the past century.1−3 We have developed a dehydrocon-
densing reagent, 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methyl-
morpholinium chloride (DMT-MM), which selectively forms
an amide in alcoholic or aqueous media.4−7 As shown in Scheme 1a,
it has also been found that tertiary amines (tert-amines, 1)
catalyze similar dehydrocondensing reactions.8−11 Triazinylam-
monium salt (2), generated in situ from the tert-amines and
2-chloro-4,6-dimethoxy-1,3,5-triazine (CDMT), reacts with a
carboxylic acid (3) to form an activated acyloxytriazine (4),
which then undergoes attack by an amine (5) to give a cor-
responding amide (6). The tert-amine (1) is regenerated by a
proton capture agent (PCA).
On the basis of this catalytic condensing reaction, we have
exploited artificial enzymes by introducing a cyclodextrin or a
crown ether to tert-amines as substrate recognition sites.8,9 In
addition, tert-amines connected to ligands or drugs with specific
affinity for target proteins, i.e., a ligand catalyst, enable a
modular method for affinity labeling (MoAL method), as
shown in Scheme 1b.10,11 A triazinylammonium salt, formed
from a ligand catalyst (7) and 2-chloro-4,6-dimethoxy-1,3,5-
triazine (CDMT), reacts with a protein to give a complex (8).
Because of a linker of suitable length, the triazinylammonium
moiety in 8 can reach a carboxy group of aspartic acid or
glutamic acid at the protein surface to form an activated ester
(9). Reaction of 9 with a primary amino group of a labeling
module affords a labeled protein (10). Consequently, it is
necessary to use appropriate linkers between tert-amines and
ligand moieties for successful labeling of various targeting
proteins.
Received: February 24, 2014
Published: March 20, 2014
© 2014 American Chemical Society
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Note
Scheme 1. Reaction Mechanisms for (a) the Catalytic Amide-Forming Reaction and (b) Modular Method for Affinity Labeling
(MoAL)
To examine the reactivity of tert-amines (1) for the amide-
forming reaction, a carboxylic acid (3a) and amines (5a, 5b)
were used as model reactants under stoichiometric conditions
because these compounds have primary aliphatic substituents
and the phenyl group allows their detection by UV absorption.
The amide (6a) from 3a and 5a was obtained in 75% yield
using 1a and CDMT in methanol at rt (Table 1, entry 1).
When we examined tert-amine 1b containing a secondary
amide, which is more stable for hydrolysis than the ester, the
reaction gave rather disappointing results in terms of product
yields (entries 2 and 3, 16% yield for 6a and 15% yield for 6b).
The lower yields using 1b are possibly due to the migration of
the 1,3,5-triazinyl group to carbonyl oxygen in the intermediate
2b to form an imidate ester 1214 (Scheme 2). In fact, 1c, which
Scheme 2. Plausible Byproduct Formation from 2b
consists of a tert-amide moiety, and, therefore, does not
undergo such migration, improved the product yield (entry 4).
We then investigated tert-amines having a hydrolytically
stable and electron-deficient15 1,2,3-triazolyl group (1d−1f),
which are readily synthesized by 1,2,3-triazole forming reactions
between azides and alkynes, the so-called click chemistry.16−22
The electron-deficient 1,2,3-triazolyl group effectively decreases
the pKa of the conjugate acid of N,N-dimethylaminomethyl
moieties, as shown in Figure 1b. We have previously reported
that weakly basic tert-amines afford higher yields of the amide 6
Figure 1. (a) Conventional tert-amines. (b) Structure of tert-amines used
in this study. The pKa values of conjugate acids of tert-amines shown in
parentheses were determined by potentiometric pH titration experiments
or were obtained from the literature; see the Supporting Information.
Furthermore, on the basis of our findings, ligand catalysts were
synthesized and examined for fluorescent-labeling of a targeting
protein.
Table 1. Dehydrocondensing Reactions Mediated by tert-Amines 1
a
a
a
entry
5
6
1
yield (%)
entry
5
6
1
yield (%)
entry
5
6
1
yield (%)
1
2
3
4
5a
5a
5b
5a
6a
6a
6b
6a
1a
1b
1b
1c
75
16
15
60
5
6
7
8
5a
5a
5a
5a
6a
6a
6a
6a
1d
1e
1f
84
84
95
88
9
10
11
5a
5a
5a
6a
6a
6a
1h
1i
88
76
74
1j
1g
a
1
Yields based on H NMR analysis.
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Table 2. Catalytic Dehydrocondensing Reactions of Carboxylic Acid (3a) and Amine (5a) in MeOH
a
a
a
entry
1 (eq)
yield (%)
entry
1 (eq)
yield (%)
entry
1 (eq)
yield (%)
1
2
3
4
5
1a (0.2)
1c (0.2)
1d (0.2)
1e (0.2)
77
50
86
81
83
6
7
1f (0.2)
93
85
89
74
91
11
12
13
14
15
1h (0.05)
1i (0.2)
78
84
66
76
48
1f (0.05)
1g (0.2)
1g (0.05)
1h (0.2)
8
1i (0.05)
1j (0.2)
9
1e (0.05)
10
1j (0.05)
a
1
Yields based on H NMR analysis.
compared with strongly basic tert-amines in protic solvents
because weakly basic tert-amines are present in a reactive
nonprotonated form toward CDMT.12 It is considered that a
greater proportion of a nonprotonated N,N-dimethylamino-
methyl moiety by the electron-deficient 1,2,3-triazolyl group
results in a smooth formation of the dehydrocondensing re-
agent 2 in situ, and consequently, 1d−1f furnished the product
6a in good yields (entries 5−7 in Table 1). Similarly, aryl
linkers with electron-withdrawing groups expand the amount of
nonprotonated form and the yield gradually decreased with
increased pKa of the conjugate acids of 1g−1j, as shown in
entries 8−11 in Table 1.
Figure 2. Catalytic dehydrocondensing reactions of 3a and 5a in 50%
aqueous MeOH. The details of the results are shown in Table S3 in
the Supporting Information.
To perform the catalytic dehydrocondensing reaction using
tert-amines, it is necessary to add proton capture agents (PCAs)
in the reaction mixture to regenerate nonprotonated tert-amines
(Scheme 1a). Sodium hydrogen carbonate and N,N-diethylani-
line, which are weakly basic, were chosen as the PCAs in protic
solvents for minimal levels of byproduct formation (Table S2 in
the Supporting Information).12 The results of the catalytic
reactions using various tert-amines 1 in absolute methanol are
shown in Table 2. Although tert-amines that have ester, amido,
and alkyl linkers (1a, 1c, and 1j) produced 6a in moderate
yields (entries 1, 2, and 14), the catalysts with 1,2,3-triazolyl
(1d−1f) and aryl linkers (1g−1i) provided 6a in good yields
(entries 3, 4, 6, 8, 10, and 12). In contrast to the cases of 1g−1j
(entries 9, 11, 13, and 15), the reaction using 1e and 1f (entries
5 and 7) can be conducted with a lower catalyst amount (0.05
equiv) without any decrease in reactivity.
which have similar linker length and an ester group, were
also examined.23 Avidin was treated with the ligand catalysts,
CDMT, and Cascade Blue ethylenediamine (CBA), which is a
fluorescent primary amine, at rt for 24 h. The results are
summarized in Table 3. Compared with ligand catalysts 7c and
Table 3. Avidin Labeling Study Using Ligand Catalysts That
a
Have 1,2,3-Triazolyl or Ester Linkers
One of the characteristics of the triazine-based amide-
forming reaction is that the reaction can be conducted in
aqueous solutions that can dissolve polar substrates. In
50% aqueous methanol, it is considered that salts, such as
ammonium and carboxylate ions, in the reaction mixture are
more stabilized by solvation than in methanol. Therefore, it is
anticipated that influence of the pKa of the conjugated acids
of 1 would be increased in the catalytic amide-forming reaction.
Indeed except for 1a, a linear relationship between the yields
and pKa is observed, as shown in Figure 2. Similar to the
catalytic reaction in absolute methanol mentioned above, tert-
amines with 1,2,3-triazolyl linkers (1d−1f) produced the
product 6a in good yields in 50% aqueous methanol. The
lower yield of 1a could be partly explained by the hydrolysis of
the ester, resulting in the loss of catalyst.
With information regarding the 1,2,3-triazolyl linkers in
hand, we further examined the MoAL method (Scheme 1b) for
labeling of avidin, which has high affinity for biotin. We first
synthesized ligand catalysts in which tert-amines are connected
to a biotin moiety via the 1,2,3-triazolyl linker (synthetic schemes
for 7a, 7b are shown in Schemes S2, S3 in the Supporting
Information). To investigate whether the 1,2,3-triazolyl linker is
effective for the MoAL method, ligand catalysts 7c and 7d,11
a
The labeling yields (%) of avidin.
7d, 7a and 7b effectively labeled avidin, indicating that the
1,2,3-triazolyl linker is more efficient for the MoAL method
than the ester linker.
In summary, we have studied the triazine-based dehydro-
condensing reaction in protic solvents using trimethylamines
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Note
1
isomers. H NMR (270 MHz, CDCl3/TMS) δ 7.39−7.17 (m, 5H),
4.70 and 4.59 (s, 2H), 3.17 and 3.16 (s, 2H), 2.98 and 2.91 (s, 3H),
2.34 and 2.30 (s, 6H) ppm; 13C NMR (100 MHz, CDCl3) δ 169.70,
169.29, 136.80, 136.38, 128.26, 127.97, 127.46, 126.89, 126.72, 126.00,
61.74, 61.46, 52.29, 50.23, 45.13, 45.10, 33.80, 33.06 ppm; IR (CHCl3)
2943, 2862, 2823, 2773, 1647, 1496, 1454, 1404, 1263, 1172, 1120,
1038, 862, 737, 700 cm−1; HRMS (DART-TOF) m/z [M + H]+ Calcd
for C12H19N2O 207.1497; found 207.1497.
that have amido, 1,2,3-triazolyl, aryl, and alkyl linkers. It was
found that the 1,2,3-triazolyl and substituted aryl linkers were
superior for the amide-forming reactions. In the case of the
1,2,3-triazolyl linkers, various functionalities were readily
introduced to the N,N-dimethylaminomethyl group because
of the availability of click chemistry, and thus, the ligand
catalysts were conveniently prepared. In addition, the electron-
deficient 1,2,3-triazolyl group increases the reactivity of the
ligand catalysts in the MoAL method, which resulted in better
yields of the fluorescent-labeled avidin compared with the
yields afforded by the ester linker. We are currently
investigating the application of catalysts with 1,2,3-triazolyl
linkers for various dehydrocondensing reactions.
N-(1-Benzyl-1H-1,2,3-triazol-5-ylmethyl)-N,N-dimethylamine
(1d).27 To a stirred THF (10 mL) solution of benzyl azide (173 mg,
1.30 mmol) and N,N-dimethylpropargylamine (0.167 mL, 1.56 mmol)
was added pentamethylcyclopentadienylbis(triphenylphosphine)-
ruthenium(II) chloride (Cp*RuCl(PPh3)2, 20.6 mg, 0.0259 mmol),
and the mixture was refluxed for 4 h. The reaction mixture was
concentrated under reduced pressure. The residue was purified by
column chromatography on silica gel (CHCl3/MeOH = 95:5) to give
1d (231 mg, 82% yield) as a colorless oil.
N-(1-Benzyl-1H-1,2,3-triazol-4-ylmethyl)-N,N-dimethylamine
(1e).28 Benzylbromide (1.80 mL, 15.1 mmol) was added dropwise to a
DMSO (33 mL) solution of sodium azide (1.07 g, 16.5 mmol), and
the mixture was stirred for 1.5 h at rt. After addition of water (50 mL),
the mixture was extracted with diethyl ether. The combined organic
layer was washed with water and brine and then dried over MgSO4 and
filtrated. Concentration of the organic layer under reduced pressure
afforded benzyl azide (1.87 g) as a colorless oil. To a stirred solution of
benzyl azide (1.87 g), N,N-dimethylpropargylamine (2.26 mL, 21.0
mmol) in t-BuOH (10 mL) and water (5 mL) were added sodium
ascorbate (277 mg, 1.40 mmol) and CuSO4·5H2O (175 mg, 0.70
mmol) at rt. After stirring for 2 h, the reaction mixture was extracted
with CH2Cl2. The combined organic layer was washed with saturated
aqueous Na2CO3, water, and brine. The extract was dried over MgSO4,
filtrated, and concentrated under reduced pressure. The residue was
purified by column chromatography on silica gel (1% Et3N in CH2Cl2)
to give 1e (2.58 g, 79% yield for two steps) as a colorless solid. mp
41−42 °C.
EXPERIMENTAL SECTION
■
General Experimental Methods. 2-Chloro-4,6-dimethoxy-1,3,5-
triazine (CDMT),24 N,N-dimethyl-4-nitrobenzylamine (1g),25 ligand
catalysts (7c, 7d),11 and Cascade Blue ethylenediamine26 were
prepared by procedures reported in the literature. N,N-Dimethylben-
zylamine (1i), N,N-dimethylbutylamine (1j), dry dichloromethane
(CH2Cl2), dry acetonitrile (CH3CN), dry dimethylsulfoxide (DMSO),
and other chemicals and solvents were obtained from commercial
1
sources and used without further purification. H NMR spectra were
recorded on 270, 400, and 600 MHz spectrometers using CDCl3 or
CD3OD. Chemical shifts (δ) were determined relative to an internal
1
reference of tetramethylsilane for H NMR and solvent peaks for 13C
NMR. Analytical TLC was performed using a Merck silica gel 60 F254
TLC plate. Silica gel column chromatography was performed using
Kanto Silica Gel 60 Spherical (63−210 μm) and Merck Silica Gel 60
(<63 μm, 7729).
2-(Dimethylamino)acetic Acid Benzyl Ester (1a). To a stirred
solution of N,N-dimethylglycine hydrochloride (2.09 g, 15.0 mmol),
benzylalcohol (1.55 mL, 15.0 mmol), and N-methylmorpholine
(NMM, 1.65 mL, 15.0 mmol) in CH2Cl2 (44 mL) were added 4-
dimethylaminopyridine (DMAP, 0.92 g, 7.5 mmol) and N,N′-
dicyclohexylcarbodiimide (DCC, 4.64 g, 22.5 mmol) at 0 °C under
a N2 atmosphere. After warming to rt, the mixture was stirred
overnight. The reaction mixture was filtrated on Celite and
concentrated. The concentrate was dissolved in CH2Cl2, washed
with brine (2 times), dried over MgSO4, filtrated, and concentrated
under reduced pressure. The crude product was purified by silica gel
chromatography (hexane/EtOAc = 1:1) to give 1a (2.52 g, 87% yield)
as a colorless oil. 1H NMR (400 MHz, CDCl3/TMS) δ 7.37−7.33 (m,
5H), 5.18 (s, 2H), 3.23 (s, 2H), 2.36 (s, 6H) ppm; 13C NMR
(100 MHz, CDCl3) δ 170.30, 135.59, 128.40, 128.21, 128.14, 66.12,
60.21, 45.10 ppm; IR (neat) 2944, 2871, 2823, 2773, 1749, 1456,
1284, 1234, 1191, 1149, 1063, 858, 739, 698 cm−1; LRMS (ESI-
hexapole) m/z 194 [M + H]+, 216 [M + Na]+; HRMS (DART-TOF)
m/z [M + H]+ Calcd for C11H16NO2 194.1181; found 194.1171.
N-Benzyl-2-(dimethylamino)acetamide (1b). 1b was prepared in a
similar manner as that for 1a except for a dehydrocondensing reagent.
N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride
(EDC) was used as a dehydrocondensing reagent instead of DCC.
The crude product was purified by column chromatography on silica
gel (MeOH/CHCl3 = 1:5) to give 1b (1.32 g, 98% yield) as a pale
yellow oil. 1H NMR (270 MHz, CDCl3/TMS) δ 7.47 (brs, 1H), 7.38−
7.24 (m, 5H), 4.48 (d, J = 6.1 Hz, 2H), 3.01 (s, 2H), 2.28 (s, 6H)
ppm; 13C NMR (150 MHz, CDCl3) δ 170.55, 138.44, 128.67, 127.72,
127.39, 63.13, 46.04, 42.93 ppm; IR (CHCl3) 3363, 2950, 2852, 2833,
2787, 1668, 1603, 1537, 1508, 1456, 1419, 1269, 1149, 930, 733,
727 cm−1; LRMS (ESI-hexapole) m/z 193 [M + H]+, 215 [M + Na]+;
HRMS (DART-TOF) m/z [M + H]+ Calcd for C11H17N2O 193.1341;
found 193.1346.
N-(1-Octyl-1H-1,2,3-triazol-4-ylmethyl)-N,N-dimethylamine (1f).
1f was prepared in a similar manner as that for 1e. The crude prod-
uct was purified by column chromatography on silica gel (hexane/
ethyl acetate = 1:1 to 1% Et3N in ethyl acetate) to give 1f (1.94 g, 72%
1
yield for two steps) as a colorless oil. H NMR (270 MHz, CDCl3/
TMS) δ 7.47 (s, 1H), 4.33 (t, J = 7.3 Hz, 2H), 3.61 (s, 2H), 2.28 (s,
6H), 1.89 (tt, J = 7.3, 7.3 Hz, 2H), 1.31−1.26 (m, 10H), 0.87 (t, J =
6.7 Hz, 3H) ppm; 13C NMR (100 MHz, CDCl3) δ = 145.11, 121.98,
54.38, 50.17, 45.09, 31.57, 30.18, 28.92, 28.82, 26.34, 22.47 ppm; IR
(neat) 2927, 2856, 2817, 2767, 1458, 1377, 1331, 1263, 1219, 1174,
1144, 1097, 1045, 1016, 850, 814, 787 cm−1; LRMS (ESI-hexapole)
m/z 239 [M + H]+; HRMS (DART-TOF) m/z [M + H]+ Calcd for
C13H27N4 239.2236; found 239.2226.
N,N-Dimethyl-4-bromo-2-fluorobenzylamine (1h). To a stirred
solution of 4-bromo-2-fluorobenzylbromide (1.00 g, 3.73 mmol) and
potassium carbonate (3.10 g, 22.4 mmol) in CH3CN (19 mL) was
added dimethylamine (11.09 M solution in water, 1.01 mL, 11.2 mmol) at
0 °C. After stirring at rt for 10 min, the reaction mixture was concentrated
and water was added, and the resulting mixture was extracted with ethyl
acetate. The combined organic layer was washed with brine, dried over
MgSO4, and concentrated to obtain 0.795 g of 4-bromo-2-fluoro-N,N-
dimethylbenzylamine (1h) as a colorless oil in quantitative yield. A
portion of the product was filtered over silica gel using ethyl acetate as the
eluent, and it was purified with recycling preparative HPLC (CHCl3) for
potentiometric pH titrations. 1H NMR (400 MHz, CDCl3/TMS) δ
7.28−7.21 (m, 3H), 3.44 (s, 2H), 2.25 (s, 6H) ppm; 13C NMR (100
MHz, CDCl3) δ 161.12 (d, JC−F = 251.1 Hz), 132.58 (d, JC−F = 5.8 Hz),
127.22 (d, JC−F = 3.8 Hz), 124.71 (d, JC−F = 14.4 Hz), 120.97 (d, JC−F
=
9.6 Hz), 118.93 (d, JC−F = 26.0 Hz), 56.14, 45.20 ppm; IR (neat) 2976,
2945, 2860, 2819, 2771, 1605, 1577, 1483, 1456, 1404, 1367, 1267, 1219,
1174, 1149, 1109, 1068, 1025, 879, 852, 816 cm−1; Anal. Calcd for
C9H11BrFN: C, 46.58; H, 4.78; N, 6.03. Found: C, 46.48; H, 4.64; N,
6.00; LRMS (ESI-hexapole) m/z 232, 234 [M + H]+.
N-Benzyl-N-methyl-2-(dimethylamino)acetamide (1c). 1c was
prepared in a similar manner as that for 1b. The crude product was
purified by column chromatography on silica gel (CHCl3/MeOH =
7:3) to give 1c (3.00 g, 97% yield) as a pale yellow oil. Two sets of
NMR signals are attributable to the presence of amide cis and trans
Potentiometric pH Titrations. The preparation of the test solu-
tions and the method used for calibration of the electrode system
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Note
(Potentiometric Automatic Titrator 794 Basic Titrino, Metrohm) with
a Combined LL pH glass electrode (Metrohm) have been described in
ref 29. All of the test solutions (50 mL) were maintained under a
nitrogen atmosphere. The potentiometric pH titrations were per-
formed with I = 0.1 (NaNO3) at 25.0 °C (0.1 M aqueous NaOH was
used as the base). The deprotonation constants were determined using
and 8-azidooct-1-ylamine32 in a similar manner as that for 13. The
crude product was purified by column chromatography on silica gel
(CHCl3/MeOH = 12:1) to give 14 (23 mg, 30% yield). H NMR
1
(400 MHz, methanol-d4/TMS) δ 7.94 (brs, 1H) 4.49 (dd, J = 7.8, 4.5
Hz, 1H), 4.30 (dd, J = 7.8, 4.5 Hz, 1H), 3.29−3.13 (m, 7H), 2.92 (dd,
J = 12.8, 5.0 Hz, 1H), 2.70 (d, J = 12.8 Hz, 1H), 2.19 (t, J = 7.1 Hz,
2H), 2.17 (t, J = 7.6 Hz, 2H), 1.76−1.34 (m, 24H) ppm; 13C NMR
(100 MHz, methanol-d4) δ 176.02, 175.97, 166.13, 63.39, 61.63, 57.02,
52.45, 41.05, 40.34, 40.21, 37.01, 36.82, 30.39, 30.27, 26.76, 30.20,
30.14, 29.90, 29.80, 29.50, 27.90, 27.77, 27.57, 26.94 ppm; HRMS
(FAB) calcd for C24H44N7O3S [M + H]+ 510.3221; found 510.3222.
Ligand Catalyst (7b). This compound was synthesized from 14
and N,N-dimethylpropargylamine in a similar manner as that for 7a.
The crude product was purified by preparative thin layer column
chromatography (CHCl3/MeOH = 10:1 containing 0.5% Et3N) to
give 7b (18 mg, 73% yield). 1H NMR (400 MHz, methanol-d4/TMS)
δ 7.97 (s, 1H) 4.48 (dd, J = 7.8, 4.5 Hz, 1H), 4.41 (t, J = 7.1 Hz, 2H),
4.30 (dd, J = 7.8, 4.5 Hz, 1H), 3.81 (s, 2H), 3.21 (dd, J = 9.7, 5.0 Hz,
1H), 3.15 (t, J = 7.8 Hz, 2H), 3.14 (t, J = 7.8 Hz, 2H), 2.92 (dd, J =
12.8, 5.0 Hz, 1H), 2.70 (d, J = 12.8 Hz, 1H), 2.40 (s, 6H), 2.18 (t, J =
7.8 Hz, 2H), 2.17 (t, J = 7.8 Hz, 2H), 1.77−1.26 (m, 24H) ppm; 13C
NMR (100 MHz, methanol-d4) δ 175.97, 175.94, 166.10, 142.73,
125.92, 63.37, 61.61, 57.02, 53.82, 51.39, 44.36, 41.06, 40.30, 40.19,
36.99, 36.82, 31.24, 30.35, 30.16, 30.13, 29.97, 29.80, 29.50, 27.83,
27.55, 27.39, 26.94, 26.74 ppm; IR (KBr) 3430, 3310, 2927, 2854,
1698, 1633, 1558, 1471, 1458, 1261, 1149, 1054, 1037 cm−1; HRMS
(FAB) calcd for C29H53N8O3S [M + H]+ 593.3961; found 593.3962.
General Procedure for Affinity Labeling of Avidin. Avidin
(0.75 nmol) in a phosphate buffer (10 μL, 50 mM, pH 8.0) and ligand
catalyst (3.0 nmol) in a phosphate buffer (5 μL, 50 mM, pH 8.0) were
mixed in a microtube and left to stand at rt for 30 min. To this
solution, Cascade Blue ethylenediamine (60 nmol) in a phosphate
buffer (20 μL, 50 mM, pH 8.0), CDMT (60 nmol) in a mixture
(10 μL) of a phosphate buffer (50 mM, pH 8.0) and MeOH (5% v/v),
and a phosphate buffer (30 μL, 50 mM, pH 8.0) were added. The re-
sulting solution was shaken using a vortex mixer. The final concentration
of each component in the reaction mixture was as follows: avidin, 10 μM;
ligand catalyst, 40 μM; Cascade Blue ethylenediamine, 800 μM; CDMT,
800 μM. The reaction mixture was then left to stand at rt for 24 h. It was
then subjected to gel chromatography at rt (support: Sephadex G-50
medium; column size: 350 × 7 mm; elution: 50 mM phosphate buffer at
pH 8.0), and avidin-containing fractions were collected. The yields of
labeling of avidin were determined by UV absorption spectra of the
collected fractions.10,11
the “BEST” software program.30 The KW (equivalent to aH aOH ), KW
+
−
́
(equivalent to [H+][OH−]), and f H values used at 25 °C were
+
10−14.00, 10−13.79, and 0.825, respectively. The corresponding mixed
−
+
constants K2 (= [HO -bound species]aH /[H2O-bound species]),
+
+
+
were derived using [H ] = aH /f H
.
General Procedure for the Amide-Forming Reaction. To a
stirred solution of 3-phenylpropionic acid (38 mg, 0.25 mmol),
benzylamine (30 μL, 0.28 mmol), and tert-amine 1 (0.30 mmol) in
MeOH (1.0 mL) was added a MeOH solution of CDMT (48 mg,
0.28 mmol) at rt. After stirring at rt, the reaction mixture was
quenched with 1 M aqueous solution of KHSO4 (3 mL) and was then
concentrated under reduced pressure. The residue was extracted with
Et2O, and the organic phase was washed sequentially with water, 1 M
aqueous HCl, water, 1 M aqueous NaOH, water, saturated aqueous
NaHCO3, and brine. The extract was dried over MgSO4, filtrated, and
concentrated under reduced pressure. The yields of 6a and 6b7 were
determined using 1H NMR measurements of the crude product, which
contained imidazole or (E)-N,N-dimethylcinnamamide as an internal
standard.
In the case of entry 2 in Table 1, an unstable imidate ester 12, which
could be analyzed only by 1H NMR and mass spectroscopy, was
obtained (13.7 mg, 15% yield based on CDMT). 1H NMR (400 MHz,
CDCl3/TMS) δ 7.37−7.19 (m, 5H), 5.29 (s, 2H), 3.99 (s, 6H), 3.91
(s, 2H), 2.29 (s, 6H) ppm; LRMS (ESI-hexapole) m/z 332 [M + H]+.
N-(17-Azido-3,6,9,12,15-pentaoxaheptadecyl)biotinamide (13).
To a solution of 17-azido-3,6,9,12,15-pentaoxa-1-heptadecylamine31
(180 mg, 0.588 mmol) and (+)-biotin (144 mg, 0.588 mmol) in
MeOH (5 mL) was added DMT-MM4−7 (244 mg, 0.881 mmol) at rt.
After stirring overnight at rt, the solvent was removed under vacuum.
The crude mixture was purified by silica gel column chromatography
(CHCl3/MeOH = 9:1) to give the desired compound as colorless
1
crystals (179 mg, 57% yield). mp 96−97 °C. H NMR (400 MHz,
methanol-d4/TMS) δ 8.03 (s, 1H), 4.49 (dd, J = 7.8, 4.6 Hz, 1H), 4.30
(dd, J = 7.8, 4.6 Hz, 1H), 3.81−3.64 (m, 18H), 3.54 (t, J = 4.8 Hz,
2H), 3.37−3.30 (brs, 4H), 3.23−3.18 (m, 1H), 2.93 (dd, J = 12.6, 4.8
Hz, 1H), 2.70 (d, J = 12.8 Hz, 1H), 2.22 (t, J = 7.1 Hz, 2H), 1.76−1.61
(m, 4H), 1.48−1.40 (m, 2H) ppm; 13C NMR (100 MHz, methanol-
d4) δ 176.08, 166.06, 71.62, 71.55, 71.51, 71.23, 71.13, 70.56, 63.33,
61.59, 57.00, 51.76, 41.05, 40.34, 36.71, 29.77, 29.49, 26.84 ppm; IR
(CHCl3) 2931, 2102, 1702, 1648, 1452, 1107 cm−1; LRMS (ESI-
hexapole) 533 [M + H]+; HRMS (ESI-TOF) m/z [M + Na]+ Calcd
for C22H40N6NaO7S 555.2577; found 555.2554.
ASSOCIATED CONTENT
* Supporting Information
Potentiometric pH titration experiments for 1 (Figures S1 and
■
S
Ligand Catalyst (7a). To a solution of 13 (80 mg, 0.15 mmol),
N,N-dimethylpropargylamine (19 mg, 0.23 mmol), and sodium
ascorbate (3.0 mg, 0.015 mmol) in t-BuOH/H2O (2:1, 2 mL) was
added CuSO4·5H2O (1.9 mg, 0.0076 mmol) at rt. After stirring for
20 h at rt, the solvent was removed under vacuum. The crude mixture
was purified by silica gel column chromatography (CHCl3/MeOH =
85:15 containing 1% Et3N) to give 65 mg of the desired compound as
S2, Table S1, and Scheme S1); Schemes S2 and S3; Tables S2
1
and S3; and H and 13C NMR spectra for 1a−1h, 7a, 7b, 13,
and 14. This material is available free of charge via the Internet
at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*E-mail: kunisima@p.kanazawa-u.ac.jp.
■
1
colorless crystals (71% yield). mp 116−117 °C. H NMR (400 MHz,
CDCl3/TMS) δ 7.70 (s, 1H), 6.89 (brs, 1H), 6.29 (brs, 1H), 5.39 (brs,
1H), 4.55 (t, J = 5.3 Hz, 2H), 4.51 (dd, J = 7.1, 4.8 Hz, 1H), 4.32 (dd,
J = 7.1, 4.4 Hz, 1H), 3.88 (t, J = 5.0 Hz, 2H), 3.63 (t, J = 5.0 Hz, 18H),
3.56 (t, J = 5.0 Hz, 2H), 3.46−3.41 (m, 2H), 3.15 (dd, J = 11.9, 7.3 Hz,
1H), 2.91 (dd, J = 12.8, 5.0 Hz, 1H), 2.74 (d, J = 12.8 Hz, 1H), 2.29 (s,
6H), 2.23 (t, J = 7.3 Hz, 2H), 1.80−1.61 (m, 4H), 1.48−1.40 (m, 2H)
ppm; 13C NMR (100 MHz, CDCl3) δ 173.32, 163.93, 144.63, 123.79,
70.55, 70.53, 70.51, 70.46, 70.44, 70.38, 70.07, 69.96, 69.54, 61.75,
60.16, 55.59, 54.26, 50.21, 45.03, 40.55, 39.14, 35.92, 28.19, 28.10,
25.60 ppm; IR (CHCl3) 2931, 1701, 1658, 1458, 1103 cm−1; LRMS
(ESI-hexapole) 616 [M + H]+; HRMS (FAB) calcd for C27H50N7O7S
[M + H]+ 616.3492; found 616.3495.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
This work was supported by JSPS KAKENHI Grant (Nos.
23590004 and 25460013).
■
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