Syntheses and reactivities of non-symmetrical "active ester" bi-dentate cross-linking reagents having a phthalimidoyl and acid chloride, 2-benzothiazole, or 1-benzotriazole group

Article abstract of DOI:10.1039/c0ob00671h

We have newly synthesized the non-symmetrical "phthalimidoyl active ester" bi-dentate cross-linking reagents having an acid chloride, 2-benzothiazole, or 1-benzotriazole group (i.e., 9, 15, and 16) on the basis of the reactivity study of the "active ester" model compounds, 11-14, toward the various nucleophiles and examined their reaction selectivity towards the same nucleophiles. Then, we applied for the modification of cholesterol at the more reactive site of the bi-dentate linkers to give 3β-cholesteryl 4-(phthalimidoyloxycarbonyl)butyrate (39), and the subsequent reaction of 39 with several amines, such as benzylamine, 4-chlorobenzylamine, 2-phenylethylamine, l-phenylalanine methyl ester, or diphenylalanine benzyl ester as a protein model of the cholesterol antigen.

Full text of DOI:10.1039/c0ob00671h

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PAPER
Syntheses and reactivities of non-symmetrical “active ester” bi-dentate
cross-linking reagents having a phthalimidoyl and acid chloride,
2-benzothiazole, or 1-benzotriazole group†
Md. Chanmiya Sheikh,*a Shunsuke Takagi,a Mebumi Sakai,a Tasuya Mori,a Naoto Hayashi,b Tetsuo Fujie,a
Shin Ono,a Toshiaki Yoshimura*a and Hiroyuki Morita*a
Received 4th September 2010, Accepted 2nd November 2010
DOI: 10.1039/c0ob00671h
We have newly synthesized the non-symmetrical “phthalimidoyl active ester” bi-dentate cross-linking
reagents having an acid chloride, 2-benzothiazole, or 1-benzotriazole group (i.e., 9, 15, and 16) on the
basis of the reactivity study of the “active ester” model compounds, 11–14, toward the various
nucleophiles and examined their reaction selectivity towards the same nucleophiles. Then, we applied
for the modification of cholesterol at the more reactive site of the bi-dentate linkers to give
3b-cholesteryl 4-(phthalimidoyloxycarbonyl)butyrate (39), and the subsequent reaction of 39 with
several amines, such as benzylamine, 4-chlorobenzylamine, 2-phenylethylamine, L-phenylalanine methyl
ester, or diphenylalanine benzyl ester as a protein model of the cholesterol antigen.
for the purpose of modification of versatile compounds it is
Introduction
very interesting and challenging to develop new type of non-
To date, various cross-linking reagents have been used not
symmetrical cross-linking reagents having two different reactivities
only in the field of bio-conjugation of proteins, but also in
toward the various nucleophiles, such as alcohol, amine, thiol,
other macromolecules, such as glycoprotein, nucleic acids, or
phenol, and so on.
even synthetic polymers etc. Particularly, covalent linking of
small molecules to proteins has been a target of numerous
synthetic endeavours since this process converts non-immunogenic
molecules into immunogenic materials. Among many other com-
mercially available cross-linking reagents, active ester linkers1 such
as disuccinimidyl glutarate (DSG), and m-maleimidobenzoyl-
As the first example of the non-symmetrical bi-dentate
cross-linking reagent, we have aimed to synthesize phthalim-
ido 4-chloroformylbutanoate (9) by the chlorination of ph-
thalimido 4-carboxybutanoate (10).5 After using several chlo-
rination methods and carefully repeated recrystallization, 9
was successfully obtained as a pure solid form. Furthermore,
in order to develop the more stable non-symmetrical bi-
dentate cross-liking reagents, we have studied the reactivities of
model compounds of so called “active ester” derivatives, i.e.,
N-(3-phenylpropionyloxy)benzotriazole (11), N-(3-phenylpro-
pionyloxy)phthalimide (12), 3-phenylpropionyloxy)benzothiazole
(13), and N-(3-phenylpropionyl)benzotriazole (14) towards
N-hydroxysuccinimide ester (MBS) have become very useful
in the area of bio-conjugation chemistry.2 The MBS linker is
particularly useful as a non-symmetrical cross-linking reagent.
However, apparently its limitation is that the maleimidoyl group is
useful fundamentally only for the Michael addition of SH group.
Previously, using MBS linker we have succeeded in synthesizing
various nucleophiles.5–8 From these results, we have de-
signed and synthesized the new class of non-symmetrical
bi-dentate cross-linking reagents, i.e., phthalimido 4-(2-
benzothiazolyloxycarbonyl)butanoate (15) and phthalimido 4-(1-
benzotriazolylcarbonyl)butanoate (16) which are stable, safe for
handling, and longer storage.5
the particular antigen bearing oxidized cholesterol moiety (1a–c)
after tremendous efforts to introduce SH group in the cholesterol
framework.3 Seeking more convenient and shorter synthetic route,
we have finally used the stepwise route. Though we could not
find good conditions to obtain 7a, resulting in the substantial
undesired formation of 8a (Scheme 1).4 Under these backgrounds,
Herein, we report the full details synthesis of new class of
non-symmetrical cross-linking reagents 9, 15, and 16 and their
reactivities towards several nucleophiles. We also describe the pre-
liminary examination of introducing the linkers 9, 15, and 16 into
b-OH of cholesterol and the further reaction with several amines
such as benzylamine, 4-chlorobenzylamine, 2-phenylethylamine,
L-phenylalanine methyl ester, or diphenylalanine benzyl ester as a
protein model for the cholesterol antigen.
aDepartment of Applied Chemistry, Faculty of Engineering, University
of Toyama, 3190 Gofuku, Toyama 930-8555, Japan. E-mail: chan-
sheikh@yahoo.com; Fax: +81-76-445-6850; Tel: +81-76-445-6850
bDepartment of Chemistry, Graduate School of Science and Engineering,
University of Toyama, 3190 Gofuku, Toyama 930-8555, Japan
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c0ob00671h
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Scheme 1 Preparation of antigens.
DMAP) in 87% yield, or (C) the reaction of 10 with dichloromethyl
methyl ether in the presence of Ph3P in 88% yield, or (D) the
reaction of 10 with trichloroacetic acid ethyl ester in the presence
of Ph3P in 90% yield (Scheme 2).
The target linker 9 was obtained as a pure solid form after
repeated recrystallization from AcOEt–hexane. Although, linker
9 is sensitive to moisture and decomposed gradually, however, it
can be stored under N2 for a long time.
Results and discussion
Synthesis of acid chloride linker with “active ester” moiety 9
In order to synthesize non-symmetrical cross-linking reagent, first
we have prepared acid chloride linker 9 having “active ester”
group. Linker 9 was prepared by the following four procedures:
(A) the one step reaction of N-hydroxyphthalimide (we selected N-
hydroxyphthalimide instead of N-hydroxysuccimide by the reason
of ease to monitor the reaction by TLC) with glutaryl dichloride
(17) in the presence of pyridine in 44% yield, where undesired bis-
(1,3-diphthalimidyl glutarate) (18) was obtained in 30% yield, or
(B) the reaction of thionyl chloride with 10 in 92% yield, where
10 was synthesized by the reaction of N-hydroxyphthalimide with
glutaric anhydride in the presence of 4-dimethylaminopyridine (4-
Reactivities of the model “active ester” compounds 11–14 towards
various nucleophiles
Previously,
reported
N-acyloxybenzotriazole9
and
N-
acylbenzotriazole10,11 are revealed to be efficient acylating
reagents. However, there has been no report about the reactivity
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Table 1 Reactivities of the model "active ester" compound towards
various nucleophiles
Entry Compound NuH (1.0 equiv)
Time
Product Yieldb (%)
90
1
11
20 min
2
3
4
5
12
13
14
11
20 h
2 h
5 h
19
20
82
89
90
86
15 min
6
7
8
9
12
13
14
11
38 h
25 min
3 h
83
94
81
91
15 min
10
11
12
13
12
13
14
11
40 h
2 h
14 h
5 min
82
86
92
90c
21
22
14
15
16
17
12
13
14
11
6 h
87c
96c
95c
89c
5 min
5 min
5 min
Scheme 2 Synthesis of 4-(Phthalimidoyloxycarbonyl)butanoyl chloride
(9).
18
19
20
21
12
13
14
11
8 h
23
71c
96c
89c
94
5 min
25 min
5 min
difference between the so-called “active ester” groups. For the
preparation of more stable non-symmetrical cross-linkers having
two different “active ester” groups, it is necessary to determine
the reactivities of the “active ester” groups towards nucleophiles.
Therefore, at first we examined the reactivity difference of
several so-called “activated carbonyl” groups using the model
compounds toward benzyl alcohol as a nucleophile in the presence
of 4-DMAP as a base.5 For careful inspection of the reactivity
further we studied the reactivity towards various nucleophiles, i.e.,
4-methylbenzyl alcohol, 4-chlorobenzyl alcohol, 2-phenylethanol,
2-phenylethylamine, 4-chlorobenzylamine, and benzyl mercaptan.
The results are summarized in Table 1. These results indicate
clearly that the reactivities of 11 towards all the above mentioned
nucleophiles are highest in view of both the yields of the desired
esters 19–24 in short reaction times although the yields were not
optimized (entries 1, 5, 9, 13, 17, and 21).12–16 Careful inspection
of the data reveals that the reactivity order towards nucleophiles
is 11 ꢀ 13 > 14 > 12 (cf. entries 1–24).
22
23
24
12
13
14
12 h
5 min
1 h
77
95
82
24
a Phth = N-Phthalimidoyl, Bt = 1-Benzotriazolyl, Btz = 2-Benzothiazolyl.
b Isolated yields (not optimized). c Reaction proceeded without base
out in the presence of 1.0 equiv. of 4-DMAP in CDCl3 to give
the benzyl 3-propionate5 under the pseudo-first-order reaction
condition. The yield was determined by 1H-NMR and the Pseudo-
first order rate constant was calculated graphically.
Then, a leaving ability of four compounds were determined in
the order of the compounds are shown in Fig. 1.
Considering the difference of the reactivity, we have se-
lected the combinations among benzotriazolyloxycarbonyl,
N-phthalimidoyloxycarbonyl, and 2-benzothiazolyloxycarbonyl
groups as the candidate of the “active ester” groups for the
synthesis of bi-dentate cross-linking reagents.
Further, their leaving ability by reacting with nucleophilic agents
was estimated by determining reaction rate constants by using 1H
NMR spectrum. The reaction of model compounds (11–14) with
10 equiv. of benzyl alcohol as a nucleophilic agent was carried
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Scheme 3 Synthesis of 4-(2-benzothiazolyloxycarbonyl)butyric-N-hydro-
xyphthalimide ester (15).
Fig. 1 Plots of kobs for the reaction of 11, 12, 13 or 14 (1 equiv) with
benzyl alcohol (10 equiv) in the presence of 4-DMAP (1 equiv) in CDCl3
at 20.5 ◦C.
Synthesis of the non-symmetrical “active ester” bi-dentate
cross-linking reagents 15 and 16
In our initial experiment, we attempted to synthesize the
cross-linkers having benzotriazolyloxy carbonyl as one of the
“active ester” group, such as phthalimido 4-(1-benzotriazoly-
loxycarbonyl)butanoate (25) (Fig. 2). When 9 was reacted with
N-hydroxybenzotriazole at room temperature in CH2Cl2 in the
presence of 4-DMAP. The reaction proceeded smoothly and the
Scheme 4 Synthesis of 4-(1-benzotriazoleoxa)butyric-N-hydroxyphtha-
limide ester (16).
X-Ray crystallographic analysis of phthalimido
4-(1-benzotriazolylcarbonyl)butanoate 16
A detailed structural analysis of 16 was performed by an X-ray
crystallographic analysis. Selected bond distances and bond angles
of 16 are collected in Table 2. An ORTEP drawing of 16 is depicted
in Fig. 3.
1
crude H NMR spectrum showed that the crude product of 25
contained an inseparable by-product. However, we were not able
to isolate as a pure form at this stage, probably due to the very
reactive nature of benzotriazolyloxycarbonyl group.
Reactions of linker 9, 15, or 16 with various nucleophiles in the
presence of 4-DMAP
We examined the reactivities of the linkers 9, 15, or 16 toward
the various common nucleophiles, such as alcohols, amines,
phenol, and thiol in the presence of 4-DMAP as a base in
CH2Cl2. The reaction times and yields are summarized in Table
3. From these results it is evident and important that the desired
Fig. 2 The cross-linking reagent having benzotriazolyloxy group (25).
As a result, we planned to synthesize the linkers having the
other combinations of PhthOCO-, BtzOCO-, and BtCO-groups
as activated carbonyl groups. After examination of the reaction
and separation conditions, we have successfully synthesized 15
by the reaction of 9 with 2-hydroxybenzothiazole in the presence
of 1,4-diazabicyclo[2,2,2]octane (DABCO) to generate 15 in 46%
yield which was reported previously.5 We now report the linker
15 is produced in 78% yield by direct treatment of 2-hydroxy-
benzothiazole with 10 in the presence of DCC (Scheme 3).
Next, the target reagent 16 was synthesized by the follow-
ing two procedure: (A) the one step reaction of 10 with 1-
(methanesulfonyl)benzotriazole (26) in the presence of pyridine,
where 26 was prepared by the reaction of benzotriazole with
methanesulfonyl chloride in the presence of pyridine, or (B) the
reaction of 10 with benzotriazole in the presence of DCC (Scheme
4). Expectedly, linkers 15 and 16 thus obtained were found to be
stable, safe for handling, and longer storage.
Table 2 Selected structural data for phthalimido 4-(1-benzotriazolyl-
carbonyl) butanoate (16)a
˚
˚
Bond angle(A)
Bond distance(A)
N(3)–N(2)
N(3)–C(5)
N(3)–C(7)
C(7)–O(1)
C(7)–C(8)
C(8)–C(9)
C(9)–C(10)
C(10)–C(11)
C(11)–O(2)
O(11)–O(3)
O(3)–N(4)
1.384(2)
1.381(2)
1.406(2)
1.196(2)
1.501(3)
1.511(3)
1.514(3)
1.491(
N(2)–N(3)–C(7)
N(2)–N(3)–C(5)
C(5)–N(3)–C(7)
N(3)–C(7)–O(1)
N(3)–C(7)–C(8)
O(1)–C(7)–C(8)
C(10)–C(11)–O(3)
C(10)–C(11)–O(2)
O(2)–C(11)–O(3)
120.8(1)
109.3(1)
129.8(2)
118.5(2)
115.8(2)
125.7(2)
109.1(2)
130.2(2)
120.8(2)
1.163)
1.376(2)
1.388(2)
a Number in parentheses are estimated standard deviation in the least
significant digits.
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Table 3 Reactions of linker 9, 15, or 16 with various nucleophiles in the
presence of 4-DMAP
Entry Linker NuH (1.0 equiv)
Time
Product Yielda (%)
73
1
9
30 min
2
3
4
15
16
9
1 h
4 h
1 h
27
79
73
65
5
6
7
15
16
9
10 h
38 h
40 min
28
29
71
62
73
Fig. 3 ORTEP drawing of 16.
mono-substituted esters 27–38, which was formed by the pref-
erential attacking of nucleophiles to more reactive site of the
compound, was obtained exclusively in high yield.
8
9
10
15
16
9
2 h
5 h
20 min
77
74
79
Concerning the reactivity, it is apparent the acid chloride linker
9 exhibited the highest reactivity towards all nucleophiles used in
view of the relatively higher yields in the shortest reaction time
(entries 1, 4, 7, 10, 13, 16, 19, 22, 25, 28, 31, and 34). As compared
with the results in Table 3 the reactivity order of the cross-linking
reagents thus prepared was revealed to be 9 ꢀ 15 > 16.
11
12
13
15
16
9
30 min 30
3 h
30 min
80
65
71
14
15
16
15
16
9
2 h
14 h
31
68
71
82
Reactions of linker 9, 15, or 16 with cholesterol and the successive
reaction of 39 with various amines in the presence of 4-DMAP
10 min
In order to test and apply in the actual biological molecule system,
we studied the reaction of the linker 9, 15, or 16 with cholesterol
in the presence of 4-DMAP or DABCO as a base. However,
we selected DABCO, because of the easy removal after the
reactions. The corresponding yield of the product 3b-cholesteryl
4-(phthalimidoyloxycarbonyl)butyrate (39)5 are summarized in
Table 4.
17
18
19
15
16
9
10 min 32
4.5 h
10 min
79
70
74
20
21
22
15
16
9
10 min 33
70 min
10 min
74
67
83c
As the result, relatively higher yield of 39 was obtained in the
cases of 9 and 15 (entries 1, 2, 4 and 5). The low yield in the
case of 16 (entries 3 and 6) will be explained by the relatively low
leaving ability of benzotriazole group (cf. Table 1). In comparison
with the results of Table 3 (for example, in the case of benzyl
alcohol as a nucleophile; entries 1–3). It is noticeable that in all
cases (Table 4, entries 1–6) both the yields and reactivities became
lower substantially, probably due to the nucleophilic attacking by
sterically hindered secondary hydroxyl group at 3-b position of
cholesterol.
23
24
25
15
16
9
10 min 34
15 min
10 min
78c
75c
79c
26
27
28
15
16
9
10 min 35
40 min
10 min
76c
73c
84c
Successively, we studied the reaction of 39 with benzylamine (a
model compound of protein) in the presence of DABCO as a model
reaction for the synthesis of an antigen, the corresponding product
was 40 in high yield.5 Furthermore, we studied the reaction of 39
with several amines, such as benzylamine, 4-chlorobenzylamine,
2-phenylethylamine in the absence of base and the corresponding
products 40–42 was relatively same compared with the presence
29
30
31
15
16
9
10 min 36
15 min
50 min
77c
75c
67b
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Table 3 (Contd.)
Table 5 Reactions of 39 with various amines in the presence of 4-DMAP
Entry Linker NuH (1.0 equiv)
Time Product Yielda (%)
Entry Amine (1.0 equiv)
1
Time (h)
10.5 h
Product Yielda (%)
32
33
34
15
16
9
1.5 h
5 h
1 h
37
61
61
64
40
82b
2
3
4
12.5 h
11 h
41
64b
35
36
15
16
2.5 h
6 h
38
59
56
42
43
65b
68
a OBtz = 2-Benzothiazolyl, Bt = 1-Benzotriazolyl. b Isolated yields (not
optimized). c Reaction proceeded without base.
10 h
Table 4 Reactions of linker 9, 15 and 16 with cholesterol in the presence
of DABCO or 4-DMAP
5
Overnight 44
59
a Isolated yields (not optimized). b Reaction proceeded without base.
Table 6 Reaction of 37 with various nucleophiles in the presence of 4-
DMAP
Entry
Linker
Base
Equiv (base)
Time
Yieldb (%)
1
2
3
4
5
6
9
7 h
62
79
56
68
80
58
15
16
9
4-DMAP
DABCO
1.0
1.3
19 h
24 h
7 h
20 h
25 h
15
16
Entry
1
NuH (1.0 equiv)
Time
32 h
Product
Yieldb (%)
75
a OBtz = 2-Benzothiazolyl, Bt = 1-Benzotriazolyl. b Isolated yields (not
optimized).
45
2
25 h
46
78
of base after long reaction time (Table 5, entries 1–3). Base
revealed to be rapid formation of product. Then, we examined the
reaction of 39 with L-phenylalanine methyl ester hydrochloride, or
diphenylalanine benzyl ester trifluoroacetic acid in the presence of
1.0 equiv of 4-DMAP as a base the corresponding products 43–44
in high yields (Table 5, entries 4–5).
3
4
28 h
47
48
78
10.5 h
85c
Reaction of 37 with various nucleophiles in the presence of
4-DMAP
5
Overnight
49
72
In order to apply for the modification of various compounds, it is
interesting to extend the preparative utility and generality of these
multicomponent reactions. We carried out the reaction of 37 with
various nucleophiles, i.e., benzyl alcohol, 4-methylbenzyl alcohol,
phenol, benzylamine, and benzyl mercaptan under the same
reaction conditions, and obtained the corresponding products,
45–49 in high yields and the results are shown in Table 6. All the
a Phth = N-Phthalimidoyl. b Isolated yields (not optimized). c Reaction
proceeded without base.
products were purified by flash column chromatography on silica
gel.
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Previously, we reported3 several antigens 7 having oxidized
cholesterol as haptens as shown in Scheme 1. We have demon-
strated that the target monoclonal antibodies were successfully
obtained by the in situ immunization with the antigens 7a–c
obtained by the reaction of 2a–c with a suitable protein. Therefore,
it is expected that many kinds of monoclonal antibodies can be
easily obtained by using the non-symmetrical cross-linkers 9, 15,
and 16 which were newly synthesized by the authors.
solution of dichloromethyl methyl ether (47.8 mL, 0.54 mmol) in
CH2Cl2 (1 mL) and C6H6 (1 mL) at reflux under N2, and stirred
for 5 h. Then, the solvent was removed under vacuum. The residue
was purified by repeated re-crystallization from AcOEt–hexane to
yield acid chloride 9 (46.8 mg, 88%) as a colorless solid; Method
(D): Compound 10 (115.0 mg, 0.42 mmol) was added to a stirred
solution of trichloroacetic acid ethyl ester (114.3 mL, 0.83 mmol)
in the presence of Ph3P (217.7 mg, 0.83 mmol) in ClCH2CH2Cl
(3 mL) at rt under N2, and the reaction mixture was stirred for
5 h. Then, the solvent was removed under vacuum. The residue
was purified by repeated re-crystallization from AcOEt–hexane
to yield acid chloride 9 (111.7 mg, 90%) as a colorless solid; mp
Conclusions
To conclude, we have synthesized the new class of bi-dentate
cross-linking reagents 9, 15 and 16, and studied their selective
reactivities towards various nucleophiles. Also, we introduced the
linkers into cholesterol through selective reaction with 3b-OH,
and successively with amines as protein model of the cholesterol
antigen. A simple route to conjugateable model derivatives 39
which is able to link to suitable protein covalently under mild
reaction conditions by using these cross-linking reagents described
here. In addition, it is interesting to modify sugar derivatives,
such as cellulose (include modified celluloses), starch (modified
starches), cyclodextrin etc., using these linkers. We are now
applying to introduce these linkers into filter papers and modified
starches to develop the useful functionalized papers and starches.
◦
88–90 C; IR (KBr)) 1805, 1791, 1741 cm-1; H NMR (CDCl3,
400 MHz) d 2.13–2.21 (m, 2H), 2.80 (t, J = 7.9 Hz, 2H), 3.13 (t,
J = 7.2 Hz, 2H), 7.79–7.83 (m, 2H), 7.88–7.92 (m, 2H); 13C NMR
(CDCl3, 100 MHz) d 19.9, 29.2, 45.2, 123.9, 124.0, 128.7, 134.7,
134.8, 161.7, 168.5, 173.1. Anal. Calcd for C13H10ClNO5: C, 52.81;
H, 3.41; N, 4.74. Found: C, 53.02; H, 3.56; N, 4.78.
1
Phthalimido 4-carboxybutanoate (10)
Colorless solid; mp 119–119.8 ◦C (from CH2Cl2–hexane); IR
(KBr) 3131, 1787, 1741, 1704 cm-1; 1H NMR (CDCl3, 400 MHz)
d 2.09–2.16 (m, 2H), 2.58 (t, J = 7.2 Hz, 2H), 2.80 (t, J = 7.2 Hz,
2H), 7.77–7.82 (m, 2H), 7.87–7.91 (m, 2H); 13C NMR (CDCl3,
100 MHz) d 19.5, 29.9, 32.3, 124.0, 124.0, 128.8, 134.7, 161.8,
168.9, 177.8; Anal. Calcd for C13H11NO6: C, 56.32; H, 4.00; N,
5.05. Found: C, 55.89; H, 4.02; N, 5.18.
Experimental section
General
All the melting points were uncorrected using a micro melting
point apparatus. 1H NMR (400 MHz) and 13C NMR (100
MHz) spectra were recorded in CDCl3 using TMS as an internal
standard. All the reactions were monitored with TLC and the
products were separated by column chromatography using Silica
Gel 60 and by preparative layer chromatography using Silica Gel
60 PF254 with UV or PMA and DNP detection. Mass spectra were
obtained on a JEOL-JMS-D300 mass spectrometer. The elemental
analyses were performed in the Micro Analytical Laboratory of
the Department of Material Systems Engineering and Life Science,
University of Toyama. All the reagents were the highest quality
and were further purified by distillation, or recrystallization.
Modified procedure for the preparation of phthalimido
4-(2-benzothiazolyloxycarbonyl) butanoate (15)
DCC (96.5 mg, 0.46 mmol) was added to a stirred solution of
10 (100.0 mg, 0.36 mmol) and 2-hydroxybenzothiazole (54.4 mg,
0.36 mmol) in CH2Cl2 (2 mL) and the reaction mixture was
◦
stirred for 3 h under N2 at 0 C. Then, the reaction mixture was
neutralized by dil. AcOH solution and extracted with CH2Cl2.
The organic layer was washed with water, dried over anhydrous
MgSO4, and concentrated under vacuum. The residue was purified
after repeated recrystalization from AcoEt-hexane to afford 15
(115.1 mg, 78%) as a colorless solid; mp 150–151 ◦C (from CH2Cl2–
hexane); IR (KBr) 1787, 1739, 1714 cm-1; 1H NMR (CDCl3, 400
MHz) d 2.23–2.30 (m, 2H), 2.86 (t, J = 7.4 Hz, 2H), 3.32 (t, J = 7.0
Hz, 2H), 7.25–7.28 (m, 1H), 7.32–7.39 (m, 2H), 7.78–7.81 (m, 2H),
7.87–7.90 (m, 2H), 8.33 (d, J = 8.4 Hz, 1H); 13C NMR (CDCl3, 100
MHz) d 19.3, 29.9, 37.5, 117.8, 121.8, 121.9, 123.9,125.5, 127.0,
128.8, 134.7, 161.8, 169.0, 172.9; Anal. Calcd for C20H14N2O6S: C,
58.53; H, 3.44; N, 6.83. Found: C, 57.94; H, 3.52; N, 6.81.
Preparation of phthalimido 4-chloroformylbutanoate (9)
Method (A): Glutaryl dichloride 17 (1457.4 mg, 8.62 mmol) was
added to a stirred solution of N-hydroxyphthalimide (469.8 mg,
2.87 mmol) and pyridine (749.0 mL, 2.87 mmol) in CH2Cl2 (7 mL)
at rt under N2, and the reaction mixture was stirred for 3 h. Then,
hexane was added to the reaction mixture and the precipitate
was removed by glass funnel. The solution was concentrated
by evaporation and purification by kugelrohr distillation and
finally repeated re-crystallization from AcOEt–hexane to yield
acid chloride 9 (373.8 mg, 44%) as a colorless solid; Method
(B): Thionyl chloride (316.1 mL, 4.33 mmol) was added to a
stirred solution of 10 (1.00 g, 3.60 mmol) in ClCH2CH2Cl (13 mL)
under N2 and the reaction mixture was refluxed with stirring for
3 h. Then, the solvent was removed under vacuum. The residue
was purified by repeated re-crystallization from AcOEt–hexane to
yield acid chloride 9 (979.0 mg, 92%) as a colorless solid; Method
(C): Compound 10 (50.0 mg, 0.18 mmol) was added to a stirred
Preparation of phthalimido 4-(1-benzotriazolylcarbonyl)butanoate
(16)
Method (A): To a stirred solution of 26 (156.2 mg, 0.79 mmol)
in CH2Cl2 (4 mL) was added to a solution of 10 (200.0 mg,
0.72 mmol) in CH2Cl2 (4 mL) in the presence of pyridine (75.5 mL,
0.93 mmol) at rt under N2, and stirred for 9 h. Then, the solvent
was removed under vacuum. The residue was purified by repeated
re-crystallization from CH2Cl2–hexane to yield 16 (145.6 mg, 55%)
as a colorless solid; Method (B): Compound 10 (1.00 g, 3.82 mmol)
was dissolved in CH2Cl2 (8 mL) and this solution was added
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to a solution of benzotriazole (456.4 mg, 3.82 mmol) and DCC
(948.1 mg, 4.59 mmol) in CH2Cl2 (17 mL) and stirred for 1 h under
N2 at 0 ◦C. The precipitate was filtered and washed with CH2Cl2.
Then, the removal of solvent afforded crude product, which were
purified by flash chromatography (AcOEt–hexane; 1 : 1) to yield 16
(1.215 g, 84%) as a colorless solid; mp 155.5–157 ◦C (from CH2Cl2–
hexane); IR (KBr) 1812, 1785, 1752 cm-1; 1H NMR (CDCl3, 400
MHz) d 2.37–2.44 (m, 2H), 2.95 (t, J = 7.2 Hz, 2H), 3.65 (t, J =
7.2 Hz, 2H), 7.50–7.54 (m, 1H), 7.65–7.69 (m, 1H), 7.78–7.82 (m,
3-Phenyl propionic acid 2 phenylethyl ester (21)
Colorless liquid; IR (neat) 1734 cm-1; 1H NMR (CDCl3, 400 MHz)
d 2.49–2.54 (m, 2H), 2.79–2.84 (m, 4H), 4.17–4.21 (m, 2H), 7.06–
7.21 (m, 10H); 13C NMR (CDCl3, 100 MHz) d 30.8, 35.0, 35.8,
64.8, 126.1, 126.4, 128.2, 128.4, 128.8, 137.7, 140.4, 172.7; HRMS
(EI) calcd for C17H18O2: 254.1307; found: m/z 254.1282.
N-Benzyl-3-phenylpropionamide (22)
2H), 7.87–7.90 (m, 2H), 8.12–8.15 (m, 1H), 8.29–8.31(m, 1H); 13
C
Colorless solid; mp 76.5–77.0 ◦C (from CH2Cl2–hexane); IR (KBr)
3292, 1639 cm-1; 1H NMR (CDCl3, 400 MHz) d 2.51 (t, J = 7.6 Hz,
2H), 2.99 (t, J = 6.8 Hz, 2H), 4.39 (d, J = 6.0 Hz, 2H), 5.62 (s, 1H),
7.13–7.31 (m, 10H); 13C NMR (CDCl3, 100 MHz) d 31.6, 38.5,
43.6, 126.2, 127.4, 127.7, 128.3, 128.5, 128.6, 138.1, 140.7, 171.8;
Anal. Calcd for C16H17NO: C, 80.30; H, 7.16; N, 5.85. Found: C,
80.34; H, 7.16; N, 5.90.
NMR (CDCl3, 100 MHz) d 19.1, 30.0, 34.1, 114.3, 120.2, 124.0,
126.2, 128.8, 130.5, 134.7, 146.1, 161.8, 168.8, 171.3; Anal. Calcd
for C19H14N4O5: C, 60.32; H, 3.73; N, 14.81. Found: C, 60.46; H,
3.73; N, 14.85. HRMS (EI) calcd for C19H14N4O5: 378.0964; found:
m/z 378.0949.
X-Ray crystal data; Empirical formula: C19H14N4O4; Formula
¯
weight 362.34; Crystal system = triclinic; Space group P1 (#2);
˚
˚
Lattice parameters: a = 8.781(2) A; b = 14.847(2) A, c = 7.735(2)
N-4-Chlorobenzyl-3-phenylpropionamide (23)
◦
◦
◦
3
˚
˚
A; a ◦= 90.96(2) , b = 114.70(2) , g = 74.50(1) ; V = 877.7(3) A ; T =
23.0 C; Z = 2; m (MoKa) = 0.99 cm-1; 5427 reflections measured,
2839 unique (Rint = 0.048); final R value 0.066. Crystallographic
data has been deposited with the Cambridge Crystallographic
Data Centre, CCDC No. 751600
Colorless solid; mp 114.5–115.5 ◦C (from CH2Cl2–hexane); IR
(KBr) 3282, 1636 cm-1; 1H NMR (CDCl3, 400 MHz) d 2.48 (t, J =
7.8 Hz, 2H), 2.93 (t, J = 7.8 Hz, 2H), 4.28 (s, 2H), 6.11 (s, 1H),
6.96–6.99 (m, 2H); 7.00–7.27 (m, 7H); 13C NMR (CDCl3, 100
MHz) d 31.5, 38.1, 42.5, 126.1, 128.2, 128.4, 128.5, 128.7, 132.9,
136.6, 140.5, 172.0; HRMS (EI) calcd for C17H16ClNO: 273.0920;
found: m/z 273.0917.
Bis(1,3-diphthalimidyl glutarate) (18)
Colorless solid; mp 216.5-217.5 ◦C (from CH2Cl2–hexane); IR
(KBr) 1816, 1787, 1741 cm-1; 1H NMR (CDCl3, 400 MHz) d 2.24–
2.31 (m, 2H), 2.89 (t, J = 7.4 Hz, 2H), 7.78-7.82 (m, 2H), 7.87-7.92
(m, 2H); 13C NMR (CDCl3,100 MHz) d 19.7, 29.7, 124.0, 128.9,
134.8, 161.8, 168.7; Anal. Calcd for C21H14N2O8: C, 59.72; H, 3.34;
N, 6.63. Found: C, 59.77; H, 3.58; N, 6.66.
3-Phenylthiopropionic S-benzyl ester (24)
Colorless liquid; IR (neat) 1686 cm-1; 1H NMR (CDCl3, 400 MHz)
d 2.85-2.89 (m, 2H), 2.99 (t, J = 7.6 Hz, 2H), 4.12 (s, 2H), 7.15–7.31
(m, 10H); 13C NMR (CDCl3, 100 MHz) d 31.3, 33.1, 45.2, 126.3,
127.2, 128.2, 128.5, 128.6, 128.7, 137.5, 139.9, 197.8; HRMS (EI)
calcd for C16H16OS: 256.0922; found: m/z 256.0920.
Reaction of model “active ester” compound 11–14 with several
nucleophiles (product 19 as an example)
Reaction of linker 9, 15, or 16 with various nucleophiles in the
present of 4-DMAP (product 27 as an example)
Typical procedure: To a solution of 4-DMAP (86.1 mg, 0.70 mmol)
was added to a stirred solution of 4-methylbenzyl alcohol (86.1 mg,
0.70 mmol) and 13 (200.0 mg, 0.70 mmol) in CH2Cl2 (4 mL)
under N2 at rt and stirred for 2 h. Then, the reaction mixture was
neutralized by dil. AcOH solution and extracted with CH2Cl2.
The organic layer was separated, successively washed with water
and brine, and dried over anhydrous MgSO4. Removal of solvent
in vacuum, to give oil crude product, which was purified by flash
chromatography yielded 3-phenylpropionic acid 4-methylbenzyl
ester 19 (159.7 mg, 89%) as a colorless liquid; IR (neat) 1735 cm-1;
1H NMR (CDCl3, 400 MHz) d 2.31 (s, 3H), 2.61–2.65 (m, 2H),
2.93 (t, J = 7.8 Hz, 2H), 5.04 (s, 2H), 7.11–7.26 (m, 9H); 13C
NMR (CDCl3, 100 MHz) d 21.0, 30.8, 35.7, 66.0, 126.1, 128.1,
128.2, 128.3, 129.0, 132.8, 137.8, 140.3, 172.5; HRMS (EI) calcd
for C17H18O2: 254.1307; found: m/z 254.1304.
Typical procedure: To a stirred solution of 4-DMAP (29.3 mg,
0.24 mmol) and benzyl alcohol (25.3 mL, 0.24 mmol) in CH2Cl2
(1 mL) dropwise added to a stirred solution of 15 (100.0 mg,
0.24 mmol) in CH2Cl2 (1 mL) at rt under N2, and stirred for
1 h. Then, the reaction mixture was neutralized by NaHCO3
solution and extracted with CH2Cl2, and dried over anhydrous
MgSO4, and concentrated under vacuum, to give crude product
which was purified by flash chromatography yielded, phthalimido
4-benzyloxycarbonylbutanoate 27 (69.6 mg, 79%) as a colourless
solid; mp 33.5–35 ◦C (from CH2Cl2–hexane); IR (KBr) 1814, 1787,
1743 cm-1; 1H NMR (CDCl3, 400 MHz) d 2.09–2.16 (m, 2H), 2.55
(t, J = 7.2 Hz, 2H), 2.76 (t, J = 7.2 Hz, 2H), 5.15 (s, 2H), 7.30–7.38
(m, 5H), 7.76–7.80 (m, 2H), 7.85–7.90 (m, 2H); 13C NMR (CDCl3,
100 MHz) d 19.8, 29.9, 32.6, 66.3, 123.9, 128.1, 128.2, 128.5, 128.8,
134.7, 135.7, 161.8, 168.9, 172.3; Anal. Calcd for C20H17NO6: C,
65.39; H, 4.66; N, 3.81. Found: C, 65.71; H, 4.75; N, 3.72.
3-Phenylpropionic acid 4-chlorobenzyl ester (20)
Colorless liquid; IR (neat) 1736 cm-1; 1H NMR (CDCl3, 400 MHz)
d 2.66 (t, J = 7.6 Hz, 2H), 2.94 (t, J = 7.8 Hz, 2H), 5.03 (s, 2H),
7.15–7.29 (m, 9H); 13C NMR (CDCl3, 100 MHz) d 30.8, 35.6,
65.2, 126.2, 128.1, 128.4, 128.5, 129.4, 133.9, 134.3, 140.1, 172.4;
HRMS (EI) calcd for C16H15ClO2: 274.0761; found: m/z 274.0741.
Phthalimido 4-(4-methylbenzyloxycarbonyl)butanoate (28)
Colorless liquid; IR (neat) 1814, 1788, 1745 cm-1; 1H NMR
(CDCl3, 400 MHz) d 1.54–1.61 (m, 3H), 2.08–2.13 (m, 2H),
2.50–2.54 (m, 2H), 2.69–2.75 (m, 2H), 5.88–5.91 (m, 1H),
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7.25–7.37 (m, 5H), 7.77–7.80 (m, 2H), 7.87–7.90 (m, 2H); 13C
NMR (CDCl3, 100 MHz) d 19.8, 22.2, 29.9, 32.9, 72.6, 123.9,
126.0, 126.1, 127.9, 128.5, 128.6, 128.8, 134.7, 141.5, 161.8, 169.0,
171.7; HRMS (EI) calcd for C21H19NO6: 381.1212; found: m/z
381.1198.
Phthalimido 4-(N-benzyloxycarbonyl)butanoate (34)
Colorless solid; mp 149.0–149.5 ◦C (from CH2Cl2–hexane); IR
(KBr) 3316, 1808, 1787, 1747 cm-1; 1H NMR (CDCl3, 400 MHz)
d 2.14–2.21 (m, 2H), 2.40 (t, J = 7.2 Hz, 2H), 2.74 (t, J = 6.8 Hz,
2H), 4.46 (d, J = 5.6 Hz, 2H), 6.27 (s, 1H), 7.21–7.32 (m, 5H),
7.78–7.82 (m, 2H), 7.85–7.89 (m, 2H); 13C NMR (CDCl3, 100
MHz) d 20.9, 29.9, 34.4, 43.6, 124.0, 127.4, 127.8, 128.6, 128.7,
134.8, 138.1, 162.0, 169.3, 171.5; Anal. Calcd for C20H18N2O5: C,
65.57; H, 4.95; N, 7.65. Found: C, 65.13; H, 4.88; N, 7.52.
Phthalimido 4-(1-methylbenzyloxycarbonyl)butanoate (29)
Colorless solid; mp 48.5–50.5 ◦C (from CH2Cl2–hexane); IR (KBr)
1817, 1787, 1738 cm-1; 1H NMR (CDCl3, 400 MHz) d 1.98–2.05
(m, 2H), 2.25 (s, 3H), 2.44 (t, J = 7.2 Hz, 2H), 2.66 (t, J = 7.2 Hz,
2H), 7.05–7.08 (m, 2H), 7.12–7.17 (m, 2H), 7.67–7.70 (m, 2H),
7.75–7.79 (m, 2H); 13C NMR (CDCl3, 100 MHz) d 19.7, 21.0, 29.9,
32.6, 66.2, 123.8, 128.3, 128.7, 129.0, 129.1, 132.7, 134.6, 138.0,
161.7, 168.9, 172.2; HRMS (EI) calcd for C21H19NO6: 381.1212;
found: m/z 381.1220.
Phthalimido 4-(N-4-cholorobenzyloxycarbonyl)butanoate (35)
Colorless solid; mp 144.5–147 ◦C (from CH2Cl2–hexane); IR
(KBr) 3293, 1807, 1783, 1743 cm-1; 1H NMR (CDCl3, 400 MHz) d
2.12–2.22 (m, 2H), 2.40 (t, J = 7.0 Hz, 2H), 2.74 (t, J = 6.6 Hz, 2H),
4.43 (d, J = 5.6 Hz, 2H), 6.33 (s, 1H), 7.21–7.27 (m, 4H), 7.80–7.83
(m, 2H), 7.86–7.89 (m, 2H); 13C NMR (CDCl3, 100 MHz) d 20.9,
29.8, 34.3, 42.9, 123.5, 124.0, 128.7, 129.2, 134.3, 134.9, 171.6,
147.9; HRMS (EI) calcd for C20H17ClN2O5: 400.0826; found: m/z
400.0868.
Phthalimido 4-(4-chlorobenzyloxycarbonyl)butanoate (30)
Colorless liquid; IR (neat) 1813, 1788, 1744 cm-1; 1H NMR
(CDCl3, 400 MHz) d 1.99–2.06 (m, 2H), 2.46 (t, J = 7.4 Hz, 2H),
2.67 (t, J = 8.0 Hz, 2H), 5.01 (s, 2H), 7.18–7.24 (m, 5H), 7.67–7.71
(m, 2H), 7.75–7.79 (m, 2H); 13C NMR (CDCl3, 100 MHz) d 19.7,
29.8, 32.4, 65.4, 123.8, 128.4, 128.6, 129.5, 134.0, 134.2, 134.7,
161.7, 168.8, 172.1; HRMS (EI) calcd for C20H16ClNO6: 401.0666;
found: m/z 401.0668.
Phthalimido 4-(N-phenylethyloxycarbonyl)butanoate (36)
Colorless solid; mp 100.5–102 ◦C (from CH2Cl2–hexane); IR
(KBr) 3312, 1816, 1791, 1750 cm-1; 1H NMR (CDCl3, 400 MHz)
d 1.99–2.06 (m, 2H), 2.22 (t, J = 7.0 Hz, 2H), 2.60 (t, J = 6.8 Hz,
2H), 2.75 (t, J = 7.0 Hz, 2H), 3.48 (q, J = 12.8 Hz, 2H), 6.00 (s,
1H), 7.03–7.19 (m, 5H), 7.70–7.77 (m, 2H), 7.78–7.81 (m, 2H);
13C NMR (CDCl3, 100 MHz) d 20.8, 29.8, 34.4, 35.4, 40.4, 123.9,
126.3, 128.4, 128.6, 128.7, 133.9, 134.7, 134.8, 138.6, 161.9, 169.2,
171.6; HRMS (EI) calcd for C21H20N2O5: 380.1372; found: m/z
380.1414.
Phthalimido 4-phenylethyloxycarbonylbutanoate (31)
Colorless liquid; IR (neat) 1814, 1788, 1745 cm-1; 1H NMR
(CDCl3, 400 MHz) d 2.03–2.12 (m, 2H), 2.45–2.49 (m, 2H), 2.68–
2.72 (m, 2H), 2.95 (t, J = 7.2 Hz, 2H), 4.30–4.34 (m, 2H), 7.21–7.31
(m, 5H), 7.77–7.80 (m, 2H), 7.87–7.89 (m, 2H); 13C NMR (CDCl3,
100 MHz) d 19.7, 29.9, 32.6, 35.0, 123.9, 126.5, 128.4, 128.7, 128.8,
134.7, 137.6, 161.8, 168.9, 172.3; HRMS (EI) calcd for C21H19NO6:
381.1212; found: m/z 381.1194.
Phthalimido 4-(N-phenylalaninocarbonyl methyl ester)butanoate
(37)
Colorless solid; mp 94.5–95.5 ◦C (from CH2Cl2–hexane); IR (KBr)
3293, 1785, 1745, 1639 cm-1; 1H NMR (CDCl3, 400 MHz) d 2.04–
2.12 (m, 2H), 2.33–2.37 (m, 2H), 2.59–2.75 (m, 2H), 3.06 (dd, J =
6.8 Hz, 14.0 Hz, 1H), 3.19 (dd, J = 5.6 Hz, 14.0 Hz, 1H), 3.73 (s,
3H), 4.93 (q, J = 13.6 Hz, 1H), 6.21 (d, J = 7.6 Hz, 1H), 7.13 (d, J =
7.2 Hz, 2H), 7.18–7.29 (m, 3H), 7.80–7.82 (m, 2H), 7.87–7.91 (m,
2H); 13C NMR (CDCl3, 100 MHz) d 20.7, 29.8, 34.2, 37.8, 52.3,
53.0, 124.0, 127.0, 128.5, 128.8, 129.1, 134.8, 135.8, 161.9, 169.2,
171.2, 172.0; Anal. Calcd for C23H22N2O7: C, 63.01; H, 5.06; N,
6.39. Found: C, 63.02; H, 5.10; N, 6.40.
Phthalimido 4-phenyloxycarbonylbutanoate (32)
Colorless solid; mp 105–106 ◦C (from CH2Cl2–hexane); IR (KBr)
1808, 1781, 1751 cm-1; 1H NMR (CDCl3, 400 MHz) d 2.19–2.26
(m, 2H), 2.78 (t, J = 7.2 Hz, 2H), 2.86 (t, J = 7.2 Hz, 2H), 7.08–
7.12 (m, 2H), 7.21–7.26 (m, 2H), 7.21–7.26 (m, 1H), 7.36–7.41 (m,
2H), 7.77–7.82 (m, 1H), 7.87–7.91 (m, 2H); 13C NMR (CDCl3, 100
MHz) d 19.7, 29.9, 32.6, 121.5, 124.0, 125.8, 128.8, 129.4, 134.8,
150.5, 161.8, 168.9, 171.0; Anal. Calcd for C19H15NO6: C, 64.59;
H, 4.28; N, 3.96. Found: C, 64.34; H, 4.33; N, 4.04. HRMS (EI)
calcd for C19H15NO6: 353.0899; found: m/z 353.0868.
Phthalimido 4-(N-diphenylalaninocarbonyl benzyl ester)butanoate
(38)
Colorless solid; mp 134–136 ◦C (from CH2Cl2–hexane); IR (KBr)
3289, 1813, 1788, 1747 cm-1; 1H NMR (CDCl3, 400 MHz) d 1.99–
2.05 (m, 2H), 2.29 (t, J = 8.0 Hz, 2H), 2.57–2.64 (m, 2H), 2.97–3.10
(m, 4H), 4.64–4.70 (m, 1H), 4.78–4.85 (m, 1H), 5.10 (s, 2H), 6.34
(d, J = 8.0 Hz, 1H), 6.40 (d, J = 8.0 Hz, 1H), 6.93–6.96 (m, 2H),
7.16–7.37 (m, 13H), 7.80–7.83 (m, 2H), 7.83–7.91 (m, 2H); 13C
NMR (CDCl3, 100 MHz) d 20.6, 29.6, 29.8, 34.2, 37.8, 53.3, 54.2,
67.2, 124.0, 127.0, 127.0, 128.5, 128.5, 128.6, 128.6, 128.8, 129.2,
129.2, 134.8, 135.0, 135.5, 136.4, 161.9, 169.2, 170.4, 170.7, 171.6;
HRMS (EI) calcd for C38H35N3O8: 661.2424; found: m/z 661.2433.
Phthalimido 4-(S-benzyloxycarbonyl)butanoate (33)
Colorless solid; mp 49–50 ◦C (from CH2Cl2–hexane); IR (KBr)
1810, 1745, 1683 cm-1; 1H NMR (CDCl3, 400 MHz) d 2.11–2.19
(m, 2H), 2.73–2.78 (m, 4H), 4.14 (s, 2H), 7.21–7.31 (m, 5H), 7.77–
7.81 (m, 2H), 7.86–7.90 (m, 2H); 13C NMR (CDCl3, 100 MHz)
d 29.9, 33.2, 41.9, 123.9, 127.3, 128.6, 128.7, 128.8, 134.7, 137.3,
161.8, 168.8, 197.5; Anal. Calcd for C20H17NO5S: C, 62.65; H,
4.47; N, 3.65. Found: C, 63.04; H, 4.54; N, 3.59.
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Preparation of 3b-cholesteryl
4-(phthalimidoyloxycarbonyl)butyrate (39)
43.1, 49.9, 56.1, 56.6, 74.3, 122.7, 128.8, 129.2, 133.4, 136.3, 139.4,
172.8; HRMS (EI) calcd for C39H58ClNO3: 623.4105; found: m/z
623.4081.
Typical procedure: To a stirred solution of 4-DMAP (59.8 mg,
0.48 mmol) and cholesterol (188.3 mg, 0.48 mmol) in CH2Cl2
(2 mL) was added dropwise to a stirred solution of 15 (200.0 mg,
0.48 mmol) in CH2Cl2 (2 mL) at rt under N2 and stirred for 19 h.
Then, the reaction mixture was neutralized by NaHCO3 solution
and extracted with CH2Cl2, and dried over anhydrous MgSO4,
and concentrated under vacuum, to give crude product which was
purified by flash chromatography yielded 39 (248.4 mg, 79%) as
a colorless solid; mp 83–85 ◦C (from CH2Cl2–hexane); IR (KBr)
1814, 1789, 1741 cm-1; 1H NMR (CDCl3, 400 MHz) d 0.67 (s, 3H),
0.86 (dd, J = 1.6 Hz, 6.4 Hz, 6H), 0.91 (d, J = 6.4 Hz, 3H), 0.94–
1.04 (m, 5H), 1.05–1.17 (m, 6H), 1.23–1.37 (m, 6H), 1.42–1.58 (m,
7H), 1.78–1.88 (m, 3H), 1.93–2.03 (m, 2H), 2.06–2.13 (m, 2H),
2.33 (d, J = 7.6 Hz, 2H), 2.47 (t, J = 7.2 Hz, 2H), 2.76 (t, J = 7.2
Hz, 2H), 4.60–4.68 (m, 1H), 5.38 (d, J = 4 Hz, 1H), 7.77–7.81 (m,
2H), 7.86–7.91 (m, 2H); 13C NMR (CDCl3, 100 MHz) d 11.8, 18.7,
19.3, 19.9, 21.0, 22.5, 22.8, 23.8, 24.3, 27.8, 27.9, 28.2, 30.1, 31.8,
31.9, 33.1, 35.8, 36.2, 36.6, 36.9, 38.1, 39.5, 39.7, 42.3, 49.9, 56.1,
56.7, 74.2, 122.7, 123.9, 128.8, 134.8, 139.6, 161.8, 169.1, 171.9;
Anal. Calcd for C40H55NO6: C, 74.38; H, 8.58; N, 2.17. Found: C,
74.79; H, 8.71; N, 2.12.
3b-Cholesteryl 4-(2-phenylethylaminocarbonyl)butyrate (42)
Colorless solid; mp 128–129.5 ◦C; (from CH2Cl2–hexane); IR
1
(KBr) 3292, 1729, 1637 cm-1; H NMR (CDCl3, 400 MHz) d
0.67 (s, 3H), 0.86 (dd, J = 1.6 Hz, 6.6 Hz, 6H), 0.91 (d, J = 6.4
Hz, 4H), 0.92–1.03 (m, 6H), 1.04–1.20 (m, 7H), 1.20–1.34 (m,
4H), 1.40–1.58 (m, 7H), 1.78–1.99 (m, 7H), 2.19 (t, J = 7.4 Hz,
2H), 2.30 (t, J = 7.4 Hz, 4H), 2.81 (t, J = 6.8 Hz, 2H), 4.55–4.65
(m, 1H), 5.36 (d, J = 3.6 Hz, 2H), 5.65 (s, 1H), 7.17–7.32 (m,
5H); 13C NMR (CDCl3, 100 MHz) d 11.8, 18.6, 19.2, 20.9, 21.0,
22.5, 22.7, 23.7, 24.2, 27.7, 28.1, 31.7, 31.8, 33.5, 35.4, 35.6, 36.1,
36.5, 36.9, 38.0, 39.4, 39.6, 40.5, 42.2, 49.9, 56.0, 56.6, 73.9, 122.6,
126.4, 128.5, 128.6, 138.7, 139.5, 172.1, 172.5; HRMS (EI) calcd
for C40H61NO3: 603.4651; found: m/z 603.4631.
3b-Cholesteryl 4-(L-phenylalaninocarbonyl methyl ester)butyrate
(43)
Colorless solid; mp 80–82 ◦C (from CH2Cl2–hexane); IR (KBr)
3431, 1789, 1733, 1716 cm-1; 1H NMR (CDCl3, 400 MHz) d 0.60
(s, 3H), 0.78 (dd, J = 1.6 Hz, 6.8 Hz, 6H), 0.84 (d, J = 6.4 Hz,
3H), 0.87–0.97 (m, 5H), 0.98–1.09 (m, 6H), 1.10–1.27 (m, 6H),
1.36–1.51 (m, 7H), 1.73–1.95 (m, 7H), 2.16 (t, J = 7.0 Hz, 2H),
2.19–2.24 (m, 4H), 2.98 (dd, J = 6.0 Hz, 14.2 Hz, 1H), 3.10 (dd, J =
6.0 Hz, 13.8 Hz, 1H), 3.65 (s, 3H), 4.49–4.57 (m, 1H), 4.79–4.84
(m, 1H), 5.30 (d, J = 4.4 Hz, 1H), 5.90 (d, J = 8.0 Hz, 2H), 7.01–
7.03 (m, 2H), 7.15–7.24 (m, 3H); 13C NMR (CDCl3, 100 MHz)
d 11.8, 18.6, 19.2, 20.7, 20.9, 22.5, 22.8, 24.2, 27.7, 27.9, 28.2,
31.8, 31.9, 33.5, 35.2, 36.1, 36.9, 37.8, 38.0, 39.4, 39.6, 42.2, 49.9,
52.3, 52.9, 56.0, 56.6, 73.9, 122.6, 123.4, 127.1, 128.5, 128.6, 128.8,
129.1, 134.0, 135.8, 139.5, 171.7, 172.0, 172.4; HRMS (EI) Calcd
for C42H63NO5: 661.4706; found: m/z 661.4717.
Reaction of 39 with several amines (product 40 as an example)
Typical procedure: To a stirred solution of benzylamine (16.5 mL,
0.15 mmol) in CH2Cl2 (1 mL) was added dropwise to a stirred
solution of 39 (100.0 mg, 0.15 mmol) in CH2Cl2 (1 mL) at rt
under N2 and stirred for 10.5 h. Then, the reaction mixture was
neutralized by dil. AcOH solution and extracted with CH2Cl2, and
dried over anhydrous MgSO4, and concentrated under vacuum, to
give solid crude product which was purified by flash chromatog-
raphy yielded, 3b-cholesteryl 4-(benzylaminocarbonyl)butyrate 40
(74.4 mg, 82%) as a colorless solid; mp 115–117 ◦C (from CH2Cl2–
hexane); IR (KBr) 3276, 1729, 1639 cm-1; 1H NMR (CDCl3, 400
MHz) d 0.67 (s, 3H), 0.86 (dd, J = 1.6 Hz, 7.2 Hz, 6H), 0.91 (d,
J = 6.4 Hz, 3H), 0.94–1.04 (m, 5H), 1.08–1.21 (m, 6H), 1.24–1.39
(m, 6H), 1.42–1.61 (m, 7H), 1.78–1.87 (m, 3H), 1.94–2.02 (m, 4H),
2.25–2.29 (m, 4H), 2.35 (t, J = 7.0 Hz, 2H), 4.43 (d, J = 5.6 Hz,
1H), 4.55–4.63 (m, 1H), 5.36 (d, J = 4.4 Hz, 2H), 5.86 (s, 1H),
7.26–7.37 (m, 5H); 13C NMR (CDCl3, 100 MHz) d 11.8, 18.6,
19.2, 20.9, 22.5, 22.8, 23.7, 24.2, 27.7, 27.9, 28.2, 31.8, 31.9, 33.6,
35.4, 35.7, 36.1, 36.5, 36.9, 38.1, 39.4, 39.6, 42.2, 43.6, 49.9, 56.0,
56.6, 74.0, 122.6, 127.5, 127.8, 128.7, 138.2, 139.5, 171.9, 172.5;
Anal. Calcd for C39H59NO3: C, 79.41; H, 10.08; N, 2.37. Found:
C, 78.93; H, 9.87; N, 2.35.
3b-Cholesteryl 4-(diphenylalaninocarbonyl benzyl ester)butyrate
(44)
Colorless solid; mp 145.5–147 ◦C (from CH2Cl2–hexane); IR
(KBr) 3295, 1728, 1638 cm-1; 1H NMR (CDCl3, 400 MHz) d 0.67
(s, 3H), 0.86 (dd, J = 2.0 Hz, 6.8 Hz, 6H), 0.91 (d, J = 6.4 Hz, 3H),
0.93–1.04 (m, 6H), 1.05–1.21 (m, 7H), 1.20–1.34 (m, 5H), 1.41–
1.58 (m, 7H), 1.80–1.87 (m, 5H), 1.90–2.02 (m, 2H), 2.16–2.22 (m,
4H), 2.30 (d, J = 8.0 Hz, 2H), 2.99 (d, J = 6.8 Hz, 3H), 3.08 (dd, J =
14.0 Hz, 6.0 Hz, 1H), 4.55–4.65 (m, 2H), 4.77 (q, J = 6.4 Hz, 1H),
5.09 (s, 2H), 5.36 (d, J = 4.4 Hz, 1H), 6.31 (s, 1H), 6.42 (s, 1H),
6.94 (t, J = 3.4 Hz, 2H), 7.13–7.27 (m, 10H), 7.34–7.36 (m, 2H);
13C NMR (CDCl3, 100 MHZ) d 11.8, 18.6, 19.2, 20.7, 21.0, 22.5,
23.8, 24.2, 27.7, 27.9, 28.2, 31.8, 31.9, 33.4, 35.0, 35.7, 36.1, 36.9,
37.7, 38.0, 38.1, 39.4, 39.6, 42.2, 49.9, 53.5, 54.4, 56.1, 56.6, 67.2,
74.0, 122.7, 127.0, 127.1, 128.5, 128.5, 128.6, 129.2, 129.3, 135.0,
135.4, 136.2, 139.5, 170.5, 170.6, 172.4; HRMS (FAB) Calcd for
C42H63NO5 (M+1): 885.5703; found (M+1): 885.6105.
3b-Cholesteryl 4-(4-cholorobenzylaminocarbonyl)butyrate (41)
Colorless solid; mp 122–124 ◦C (from CH2Cl2–hexane); IR (KBr)
1
3262, 1724, 1638 cm-1; H NMR (CDCl3, 400 MHz) d 0.67 (s,
3H), 0.86 (dd, J = 2.0 Hz, 6.4 Hz, 6H), 0.91 (d, J = 6.4 Hz, 3H),
0.94–1.0 (m, 6H), 1.05–1.16 (m, 7H), 1.20–1.35 (m, 5H), 1.41–1.58
(m, 7H), 1.80–1.90 (m, 3H), 1.95–2.02 (m, 4H), 2.27–2.37 (m, 6H),
4.41 (d, J = 5.6 Hz, 1H), 4.40–4.61 (m, 1H), 5.36 (d, J = 4.8 Hz,
1H), 6.38 (s, 1H), 7.20–7.31 (m, 4H); 13C NMR (CDCl3, 100 MHz)
d 11.8, 18.7, 19.2, 20.9, 21.0, 22.5, 22.8, 23.8, 24.2, 27.7, 27.9, 28.2,
31.8, 31.9, 33.4, 35.1, 35.7, 36.1, 36.5, 36.9, 38.0, 39.5, 39.7, 42.2,
Reaction of 37 with various nucleophiles (product 45 as an example)
Typical procedure: 4-DMAP (55.7 mg, 0.45 mmol) was added to
a stirred solution of 37 (200.0 mg, 0.45 mmol) and benzyl alcohol
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(47.1 mL, 0.45 mmol) in CH2Cl2 (2 mL) at rt under N2 and stirred
for 32 h. Then, the reaction mixture was neutralized by dil. AcOH
solution and extracted with CH2Cl2, and dried over anhydrous
MgSO4, and concentrated under vacuum, to give oil crude product
which was purified by flash column chromatography yielded,
O-benzyl 4-(L-phenylalaninocarbonyl methyl ester)butyrate (45)
(130.8 mg, 75%) as a colorless oil; IR (neat) 3301, 1814, 1787,
S-Benzyl 4-(L-phenylalaninocarbonyl methyl ester)butyrate (49)
Colorless syrup; IR (KBr) 3291, 1743, 1683, 1652 cm-1; 1H NMR
(CDCl3, 400 MHz) d 1.92–1.99 (m, 2H), 2.18–2.22 (m, 2H), 2.50–
2.62 (m, 2H), 3.05 (dd, J = 6.0 Hz, 13.8 Hz, 1H), 3.15 (dd, J = 5.6
Hz, 14.0 Hz, 1H), 3.72 (s, 3H), 4.11 (s, 2H), 4.85–4.90 (m, 1H),
5.96 (d, J = 8 Hz, 1H), 7.07–7.20 (m, 2H), 7.20–7.31 (m, 8H);
13C NMR (CDCl3, 100 MHz) d 21.2, 33.1, 34.8, 37.8, 42.3, 52.3,
52.9, 127.1, 127.2, 128.5, 128.6, 128.7, 129.1, 135.7, 137.4, 171.3,
172.0, 198.2; Anal. Calcd for C22H25NO4S: C, 66.14; H, 6.31; N,
3.51. Found: C, 66.35; H, 6.10; N, 3.61. HRMS (EI) Calcd for
C22H25NO4S: 399.1504; found: m/z 399.1519.
1
1743 cm-1; H NMR (CDCl3, 400 MHz) d 1.89–1.97 (m, 2H),
2.21 (t, J = 7.2 Hz, 2H), 2.34–2.40 (m, 2H), 3.05 (dd, J = 6.0 Hz,
14.0 Hz, 1H), 3.14 (dd, J = 5.6 Hz, 14.0 Hz, 1H), 3.72 (s, 3H),
4.85–4.90 (m, 1H), 5.10 (s, 2H), 5.92 (d, J = 7.6 Hz, 1H), 7.07–
7.09 (m, 2H), 7.20–7.38 (m, 8H); 13C NMR (CDCl3, 100 MHz) d
20.6, 33.1, 35.0, 37.8, 52.3, 52.9, 66.2, 127.1, 128.2, 128.2, 128.5,
128.6, 129.1, 135.7, 135.8, 171.5, 172.0, 172.8; HRMS (EI) Calcd
for C22H25NO5: 383.1733; found: m/z 383.1734.
Notes and references
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O-4-Methlbenzyl 4-(L-phenylalaninocarbonyl methyl
ester)butyrate (46)
Colorless solid; mp 53.5–55 ◦C (from CH2Cl2–hexane); IR (KBr)
3299, 1733, 1652 cm-1; 1H NMR (CDCl3, 400 MHz) d 1.88–1.96
(m, 2H), 2.21 (t, J = 7.0 Hz, 2H), 2.30–2.39 (m, 2H), 2.33 (s, 3H),
3.05 (dd, J = 6.0 Hz, 13.8 Hz, 1H), 3.14 (dd, J = 6.0 Hz, 13.8
Hz, 1H), 3.72 (s, 3H), 4.85–4.90 (m, 1H), 5.06 (s, 2H), 5.91 (d,
J = 7.2 Hz, 1H), 7.06–7.08 (m, 2H), 7.16 (d, J = 7.2 Hz, 2H),
7.23–7.29 (m, 5H); 13C NMR (CDCl3, 100 MHz) d 0.6, 21.1,
33.1, 35.1, 37.8, 52.3, 52.9, 66.1, 127.1, 128.3, 128.5, 129.1, 129.2,
132.8, 135.7, 138.1, 171.6, 172.0, 172.9; HRMS (EI) Calcd for
C23H27NO5: 397.1889; found: m/z 397.1886.
O-Phenyl 4-(L-phenylalaninocarbonyl methyl ester)butyrate (47)
Colorless solid; mp 43–45 ◦C (from CH2Cl2–hexane); IR (KBr)
3315, 1749, 1646 cm-1; 1H NMR (CDCl3, 400 MHz) d 2.00–2.07
(m, 2H), 2.30–2.33 (m, 2H), 2.55–2.59 (m, 2H), 3.07 (dd, J = 6.0
Hz, 13.8 Hz, 1H), 3.17 (dd, J = 5.6 Hz, 14.0 Hz, 1H), 3.73 (s,
3H), 4.88–4.93 (m, 1H), 5.97 (d, J = 7.6 Hz, 1H), 7.04–7.11 (m,
4H), 7.20–7.30 (m, 4H), 7.35–7.39 (m, 2H); 13C NMR (CDCl3,
100 MHz) d 20.5, 33.1, 34.9, 37.8, 52.3, 52.9, 121.4, 125.7, 127.1,
128.5, 129.1, 129.3, 135.7, 150.5, 171.5, 171.6, 172.0; HRMS (EI)
Calcd for C21H23NO5: 369.1576; found: m/z 369.1579.
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N-Benzyl 4-(L-phenylalaninocarbonyl methyl ester)butanamide
(48)
Colorless solid; mp 100–101 ◦C (from CH2Cl2–hexane); IR (KBr)
3289, 1747, 1643, 1546 cm-1; 1H NMR (CDCl3, 400 MHz) d 1.90–
1.98 (m, 2H), 2.14–2.25 (m, 4H), 2.92 (dd, J = 7.2 Hz, 14.0 Hz,
1H), 3.09 (dd, J = 5.6 Hz, 14.0 Hz, 1H), 3.72 (s, 3H), 4.33–4.45 (m,
2H), 4.83 (q, J = 6.9 Hz 1H), 5.92 (d, J = 7.6 Hz, 1H), 6.28 (s, 1H),
7.07 (d, J = 6.8 Hz, 2H), 7.20–7.35 (m, 8H); 13C NMR (CDCl3,
100 MHz) d 21.7, 34.8, 34.9, 37.6, 43.4, 52.4, 52.9, 127.1, 127.5,
127.8, 128.6, 128.7, 129.0, 135.8, 138.5, 172.2, 172.3, 172.4; Anal.
Calcd for C22H26N2O4: C, 69.09; H, 6.85; N, 7.32; found: C, 68.81;
H, 6.64; N, 7.23.
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