Artificial model for cystathionine β-synthase: Efficient β-replacement reaction with thiols employing a novel pyridoxal model compound having an imidazole function

Article abstract of DOI:10.1016/S0040-4020(03)00666-5

As a second-generation pyridoxal model compound for cystathionine β-synthase, we designed a novel model compound having an ionophore function and an imidazole function, application of which to the β-replacement reaction with various thiols smoothly took place to give S-substituted cysteines. Peptides having a serine-O-carbonate residue at the N-terminal position were also converted to the corresponding peptides having an S-substituted cysteine residue under the catalytic conditions of the novel pyridoxal model compound.

Full text of DOI:10.1016/S0040-4020(03)00666-5

Tetrahedron 59 (2003) 4873–4879
Artificial model for cystathionine b-synthase: efficient
b-replacement reaction with thiols employing a novel pyridoxal
model compound having an imidazole function
*
Kazuyuki Miyashita, Hidenobu Murafuji, Hiroshi Iwaki, Eito Yoshioka and Takeshi Imanishi
Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871, Japan
Received 12 March 2003; accepted 24 April 2003
Abstract—As a second-generation pyridoxal model compound for cystathionine b-synthase, we designed a novel model compound having
an ionophore function and an imidazole function, application of which to the b-replacement reaction with various thiols smoothly took place
to give S-substituted cysteines. Peptides having a serine-O-carbonate residue at the N-terminal position were also converted to the
corresponding peptides having an S-substituted cysteine residue under the catalytic conditions of the novel pyridoxal model
compound. q 2003 Elsevier Science Ltd. All rights reserved.
1. Introduction
A b-replacement reaction of the serine hydroxyl group with
a nucleophile catalyzed by pyridoxal plays an important role
in a biological system.1 Although this reaction system is
expected to be of great use for the synthesis of various
unnatural amino acids by artificial mimicking, only a
limited number of examples mimicking tryptophane
synthase, in which the nucleophile is indole, have been
Figure 2. Pyridoxal 1 catalyzed b-replacement reaction of serine-O-
carbonate 3 with thiols.
reported.2 In a preceding paper, we have described catalytic
transformation of serine-O-carbonate 3 into S-substituted
cysteines 4 employing pyridoxal model compound 1 (Fig. 1),
an alkyl group, the phenomenum appears to be strange. This
result could indicate that, as aromatic thiols are less
which is the first example mimicking cystathionine
nucleophilic but are more acidic than alkane thiols, the
formation of thiolate from thiol and/or the protonation
b-synthase as shown in Figure 2.3 However, there still
remains a problem in that compound 1 is a good catalyst for
procedure to an enolate species might be important to finish
the reaction with aryl thiols, but is less effective for that with
the reaction. In order to overcome this problem, we
alkane thiols. In order to extend the reaction system to a
designed model compounds 2 which have an imidazole
function. Introduction of the imidazole function was
expected to introduce activation of the elimination of the
general method, it is necessary to solve this problem. For the
purpose, we designed a novel second-generation model
compound of pyridoxal derivative 1.
carbonate moiety of 5 (Step 1) as well as activation of the
1,4-addition of alkane thiols to 6 (Step 2) as shown in Fig. 3.
Taking into account the distance between the imidazole
moiety and the reaction site, two model compounds 2a and
2b having different methylene lengths were designed.
Figure 1. Structures of pyridoxal model compounds 1 and 2.
On the other hand, we previously described regio- and
stereoselective a-alkylation of peptides at the N-terminal
position, employing a pyridoxal derivative.4 This is a unique
and useful method to synthesize peptides including
unnatural amino acids without preparing the corresponding
unnatural amino acids,5 and would be of great use for
construction of an unnatural peptide library.6 Accordingly,
we utilized the novel model compounds 2 to b-replacement
As, in general, alkane thiols are more nucleophilic than
aromatic thiols because of the electron-donating property of
Keywords: amino acids and derivatives; coenzymes; pyridines; substitution.
*
Corresponding author. Tel.: þ81-6-6879-8200; fax: þ81-6-6879-8204;
e-mail: imanishi@phs.osaka-u.ac.jp
0040–4020/03/$ - see front matter q 2003 Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0040-4020(03)00666-5

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K. Miyashita et al. / Tetrahedron 59 (2003) 4873–4879
Figure 3. Expected effect of the imidazole moiety on the b-replacement reaction.
reaction of peptides having a serine-O-carbonate residue at
the N-terminal position as well. In this paper, we would like
to describe the synthesis of pyridoxal model compounds 2
and their ability for b-replacement reaction of serine-O-
carbonate derivatives including peptides with thiols.7
are summarized in Table 1. Novel model compounds 2a and
2b efficiently catalyzed the b-replacement reactions with
benzyl and ethyl mercaptans, both of which were ineffective
substrates in the reactions catalyzed by the basic compound
1 (entries 1 and 2 vs entries 4–7).3 Particularly in the
reaction with ethyl mercaptan, 2a and 2b were about two
times as effective as 1 (entry 2 vs entries 5 and 7). These
results clearly show the effectiveness of the imidazole
function. Although no significant difference in reactivity
between 2a and 2b was observed (entries 4 and 5 vs entries 6
and 7), the proximity effect of the imidazole function in the
reactions with 2a and 2b is also evident from the result that
the yield for the reaction with 1 in the presence of 5 mol% of
imidazole was almost the same as that without imidazole
(entry 1 vs entry 3). Namely, the existence of the imidazole
function close to the aldehyde moiety is suggested to play an
important role in the present reactions. Other thiols also
reacted smoothly under the catalytic conditions of 2b
(entries 8 and 9).
2. Results and discussion
Synthesis of the model compounds 2 was achieved as shown
in Scheme 1. Alcohol 88 was protected with a tert-
butyldiphenylsilyl (TBDPS) group to give 9, the acetonide
group of which was removed by acidic treatment, affording
diol 10. Oxidation of 10 with MnO2 afforded aldehyde 11,
which was protected as dimethyl acetal 12. An ethoxyethyl
goup was introduced to 12, affording 13. In order to couple
with an imidazole moiety, the silyl group of 13 was removed
under conventional fluoride treatment to yield 14. Esteri-
fication of 14 with N-tosylimidazolecarboxylic acids 15a
and 15b smoothly took place to afford 16a and 16b,
respectively. Removal of the N-protecting group, a tosyl
group, afforded 17a and 17b, the dimethyl acetal group of
which was then hydrolyzed under the usual conditions to
give the desired model compounds 2a and 2b.
Encouraged by the results described above, we planned to
apply the reaction to peptides having a serine-O-carbonate
residue at the N-terminal position. At first, we applied 2b to
the b-replacement reaction of serine amide 18a. The
reaction, as expected, required longer time due to lower
reactivity of an amide group than that of an ester group, but
proceeded to give S-benzylcysteine amide 19a in almost the
Employing 2a and 2b, we examined the b-replacement
reaction of serine-O-carbonate 3 with thiols, and the results
Scheme 1. (a) TBDPSCI, imidazole, DMF, rt, 96%; (b) 60% AcOH, reflux, 72%; (c) MnO2, pyridine, rt, 65%; (d) HC(OMe)3, p-TsOH, MeOH, reflux, 94%; (e)
NaH, EtO(CH2)2Br, DMF, rt, 73%; (f) n-Bu4NF, THF, rt, 91%; (g) 15a or b, DCC, DMAP, CH2Cl2, rt, 16a: 74%, 16b: 60%; (h) Ac2O, pyridine, rt, 17a: quant.,
17b: 89%; (i) 60% AcOH, reflux, 2a: 58%, 2b: 81%.

K. Miyashita et al. / Tetrahedron 59 (2003) 4873–4879
Table 1. b-Replacement reaction of 3 with thiols (RSH) catalyzed by pyridoxal 1 or 2
4875
Entry
Pyridoxal
R
Time (h)
Product 4
Yield (%) of 4
Based on 3
Based on 1 or 2
1
2
3a
4
5
6
7
8
9
1
1
1
2a
2a
2b
2b
2b
2b
Bn
Et
Bn
Bn
Et
Bn
Et
HO(CH2)3
19
22
19
19
22
19
22
72
25
c
b
c
c
b
c
b
d
ec
61
41
63
83
76
84
80
49
60
1220
820
1260
1660
1520
1680
1600
980
b
HS(CH2)3
1200
a
Reaction was carried out in the presence of 5 mol% of imidazole.
Thiol (0.6 equiv.) was employed.
Both thiol groups were alkylated.
b
c
Table 2. b-Replacement reaction of amide and peptides 18 with thiols catalyzed by pyridoxal 2b
Entry
18: AA
R
Product 19
Yield (%) of 19
Based on 18
Based on 2b
1
2
3
4
5
6
7
8
a: NHBn
b: Gly-OBn
Bn
Bn
Bn
Bn
Bn
Ph
Et
a
b
c
d
e
f
81
77
76
70
68
81
70
44
1620
1540
1520
1400
1360
1620
1400
880
c: L-Ala-OBn
d: L-Val-OBn
e: L-Ala-L-Ala-OBn
c: L-Ala-OBn
c: L-Ala-OBn
c: L-Ala-OBn
g
h
HO(CH2)3
same yield as that for the ester 3 (entry 1 in Table 2 vs entry
6 in Table 1). Peptides 18b–e having a serine-O-carbonate
residue at the N-terminal position were also transformed
into those having an S-substituted cysteine residue under the
same conditions. Steric bulkiness of the neighboring amino
acid residue did not affect the reaction so much (entries
2–4), and the products 19c and 19d were obtained as a
diastereomeric mixture in a ratio of ca. 1:1 (entries 3 and 4).
Figure 4. b-Replacement reaction of 3 catalyzed by pyridoxals 1, 2a or 2b.

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K. Miyashita et al. / Tetrahedron 59 (2003) 4873–4879
Tripeptide 18e also reacted to give 19e in almost the same
yield (entry 5). Concerning the kind of thiols, the reaction
with thiophenol smoothly took place as expected (entry 6),
while those with alkane thiols also proceeded to give the
desired products (entries 7 and 8).
separated, and the aqueous phase was extracted with ether.
Combined ethereal layers were washed with H2O and
saturated NaCl solution, dried over Na2SO4, and concen-
trated under reduced pressure. The residue was passed
through a silica gel short column with ethyl acetate/hexane
(1:3) as an eluant to give the title compound 9 (18.8 g, 96%)
as a colorless oil. IR nmax (KBr): 2935, 2858, 1599,
In order to clarify the role of the imidazole function, we
1
traced the reactions of 1, 2a and 2b by H NMR spectrum,
1
1419 cm21. H NMR (CDCl3) d: 1.04 (9H, s, C(CH3)3),
and the results are shown in Fig. 4. Comparing the results of
1 and 2, it is obvious that the imidazole function does not
activate the elimination of the carbonate moiety (Step 1 in
Fig. 3), but activates the addition of the thiol. As described
in our preceding paper,3 it is obvious that the acidity of thiol
is highly related with the reactivity. Although the detailed
function of the imidazole moiety is not clear, hydrogen
transfer from thiol to the a-position, that involves
deprotonation of the thiol hydrogen and/or protonation to
the enolate, appears to be activated by the imidazole
function as indicated by Step 2 in Fig. 3.
1.55 (6H, s, C(CH3)2), 2.41 (3H, s, 2-CH3), 4.58 (2H, s,
5-CH2), 4.88 (2H, s, 4-CH2), 7.36–7.45 (6H, m, aromatic
H), 7.64–7.68 (4H, m, aromatic H), 7.88 (1H, s, 6-H). 13C
NMR (CDCl3) dC: 18.4, 19.2, 24.7, 26.7, 58.7, 61.6, 99.6,
125.5, 127.8, 128.3, 128.9, 129.9, 132.8, 135.5, 138.4,
147.3. EI-MS m/z (%): 447 (Mþ, 3.1). High-resolution MS
Calcd for C27H33NO3Si (Mþ) 447.2230. Found: 447.2232.
3.1.2. 5-[(tert-Butyldiphenylsilyloxy)methyl]-3-hydroxy-
4-(hydroxymethyl)-2-methylpyridine (10). A solution of 9
(18.7 g, 41.7 mmol) in acetic acid– H2O (3:2, 100 ml) was
refluxed for 4 h. After concentration under reduced
pressure, the resultant residue was dried by azeotropic
evaporation with benzene, and recrystallized from methanol
to give 10 (12.2 g, 72%) as colorless crystals, mp 206–
2088C (MeOH). IR nmax (KBr): 2952, 2637, 1467,
In conclusion, we have designed and synthesized novel
pyridoxal derivatives 2 having an ionophore function and an
imidazole moiety, which were clearly shown to be effective
for catalytic b-replacement reaction of serine-O-carbonate
with various thiols to S-substituted cysteines. This reaction
was also applied to peptides having a serine-O-carbonate
residue at the N-terminal position, and consequently various
S-substituted cysteine residues were directly introduced
without preparing the respective S-substituted cysteines
alternatively, which would be of great use for construction
of a peptide library in the field of combinatorial chemistry.
Applications of the present reaction system to the
b-replacement reaction with other nucleophiles than the
sulfur nucleophile and to a chiral system would be of great
use for the synthesis of unnatural amino acids, which is
under progress in our laboratory.
1423 cm21
.
1H NMR (DMSO-d6) d: 1.00 (9H, s,
C(CH3)3), 2.35 (3H, s, 2-CH3), 4.70 (2H, s, 5-CH2), 4.79
(2H, s, 4-CH2), 5.70 (1H, br s, 40-OH), 7.40–7.57 (6H, m,
aromatic H), 7.62–7.65 (4H, m, aromatic H), 7.92 (1H, s, 6-
H), 9.24 (1H, br s, 3-OH). 13C NMR (DMSO-d6) dC: 18.8,
19.4, 26.6, 56.2, 61.4, 128.0, 130.0, 131.0, 131.7, 132.7,
135.0, 138.2, 146.3, 149.4. EI-MS m/z (%): 408 (MþHþ,
0.3). Anal. Calcd for C24H29NO3Si: C, 70.73; H, 7.17; N,
3.44. Found: C, 70.67; H, 7.13; N, 3.42.
3.1.3. 5-[(tert-Butyldiphenylsilyloxy)methyl]-3-hydroxy-
2-methylpyridine-4-carbaldehyde (11). To a stirred sol-
ution of 10 (10.0 g, 24.6 mmol) in pyridine (200 ml) was
added MnO2 (6.4 g, 73.6 mmol), and the whole was stirred
at room temperature for 55 h. The reaction mixture was
filtered through a cerite pad, and the cerite pad was washed
with pyridine. After concentration under reduced pressure,
the resultant residue was purified by silica gel column
chromatography (ethyl acetate/hexane¼1:3) to give 11
(6.52 g, 65%) as pale yellow crystals, mp 77–788C
(AcOEt/hexane). IR nmax (KBr): 3325, 2932, 2857,
3. Experimental
3.1. General
Melting points (mps) were taken on a Yanagimoto micro-
melting point apparatus and are uncorrected. Infrared
spectra were measured on a JASCO FT/IR-200 Fourier-
transfer infrared spectrometer. 1H NMR spectra were
measured on a JEOL EX-270 (270 MHz) spectrometer
and tetramethylsilane (TMS) was used as an internal
standard. 13C NMR spectra were measured on the same
instrument (67.8 MHz) with CDCl3 as an internal standard
(77.0 ppm). Low and High resolution mass spectra (EI-MS
and HR-MS) were obtained by use of a JEOL D-300 mass
spectrometer. For silica gel column chromatography,
E. Merck Kieselgel 60 (0.063–0.200 mm) was used.
1
1561 cm21. H NMR (CDCl3) d:1.03 (9H, s, C(CH3)3),
2.53 (3H, s, 2-CH3), 4.93 (2H, s, 5-CH2), 7.39–7.46 (6H, m,
aromatic H), 7.62–7.65 (4H, m, aromatic H), 7.81 (1H, s,
6-H), 10.54 (1H, s, CHO), 11.44 (1H, br s, 3-OH). 13C NMR
(CDCl3) dC: 18.8, 19.1, 26.7, 61.2, 120.2, 127.9, 130.1,
132.1, 132.5, 135.5, 138.6, 151.9, 153.8, 197.3. EI-MS m/z
(%): 405 (Mþ, 0.5), 348 (Mþ2tBu, 100). Anal. Calcd for
C24H27NO3Si: C, 69.18; H, 7.32; N, 3.10. Found: C, 69.15;
H, 7.25; N, 3.11.
3.1.4. 5-[(tert-Butyldiphenylsilyloxy)methyl]-3-hydroxy-
2-methylpyridine-4-carbaldehyde dimethyl acetal (12).
To a stirred solution of 11 (6.00 g, 14.8 mmol) in methanol
(100 ml) and trimethyl orthoformate (50 ml) was added
p-toluenesulfonic acid (ca. 100 mg), and the whole was
refluxed for 30 h. After cooling, the reaction mixture was
neutralized with saturated NaHCO3 solution, and concen-
trated under reduced pressure. The resultant residue was
3.1.1. 5-[(tert-Butyldiphenylsilyloxy)methyl]-2,2,8-tri-
methyl-4H-1,3-dioxino[4,5-c]pyridine (9). To a stirred
solution of alcohol 8 (9.13 g, 43.7 mmol) in DMF (50 ml)
was added imidazole (7.43 g, 109 mmol) and tert-butyl-
diphenylsilyl chloride (13.2 g, 48.0 mmol), and the whole
was stirred at room temperature for 15 h. After concen-
tration under reduced pressure, the resultant residue was
partitioned with ether and H2O. The ethereal layer was

K. Miyashita et al. / Tetrahedron 59 (2003) 4873–4879
4877
partitioned with ethyl acetate and H2O, and extracted with
ethyl acetate. The combined organic layers were washed
with H2O and saturated NaCl solution, dried over Na2SO4,
and concentrated under reduced pressure. The resultant
residue was purified by silica gel column chromatography
(ethyl acetate/hexane¼1:2) to give 12 (6.25 g, 94%) as
colorless crystals, mp 77.5–798C (ether/hexane). IR nmax
(KBr): 3071, 2931, 2858, 1661 cm21. 1H NMR (CDCl3) d:
1.06 (9H, s, C(CH3)3), 2.47 (3H, s, 2-CH3), 3.35 (6H, s,
CH(OCH3)2), 4.71 (2H, s, 5-CH2), 5.84 (1H, s,
CH(OCH3)2), 7.36–7.44 (6H, m, aromatic H), 7.66–7.69
(4H, m, aromatic H), 7.97 (1H, s, 6-H), 8.74 (1H, br s,
3-OH). 13C NMR (CDCl3) dC: 19.2, 26.8, 53.6, 61.5, 96.1,
102.6, 124.1, 127.8, 129.9, 131.1, 133.1, 135.6, 139.4,
148.5, 150.2. EI-MS m/z (%): 451 (Mþ, 1.0), 150 (Mþ2tBu,
100). Anal. Calcd for C26H33NO4Si: C, 71.11; H, 6.67; N,
3.46; O, 11.85; Si, 6.91. Found: C, 70.88; H, 6.60; N, 3.44.
3.63 (2H, q, J¼7 Hz, OCH2CH3), 3.76–3.79 (2H, m,
OCH2CH2OEt), 3.94–3.97 (2H, m, OCH2CH2OEt), 4.75
(2H, br d, J¼6 Hz, 5-CH2), 5.88 (1H, s, CH(OCH3)2), 8.27
(1H, s, 6-H). 13C NMR (CDCl3) dC: 15.2, 19.3, 56.3, 60.6,
66.7, 69.3, 74.0, 101.8, 133.3, 137.4, 146.5, 150.7, 152.7.
EI-MS m/z (%): 285 (Mþ, 20.5), 253 (Mþ2MeOH, 100).
Anal. Calcd for C14H23NO5: C, 58.79; H, 7.96; N, 4.90.
Found: C, 58.95; H, 8.07; N, 4.91.
3.1.7. 3-(2-Ethoxyethoxy)-2-methyl-5-{[N-(p-toluene-
sulfonyl)imidazol-4(5)-yl]acetoxy}methylpyridine-4-car-
baldehyde dimethyl acetal (16a). To a stirred solution of
14 (232 mg, 0.815 mmol) and (N-tosylimidazolyl)acetic
acid (274 mg, 0.979 mmol) in CH2Cl2 (8 ml) were added
DCC (202 mg, 0.981 mmol) and DMAP (ca. 10 mg) at room
temperature, and the whole was stirred at room temperature
for 40 h. After being diluted with CH2Cl2, the reaction
mixture was filtered, washed with saturated NaHCO3
solution, H2O and saturated NaCl solution, and dried over
Na2SO4. After concentration under reduced pressure, the
resultant residue was purified by silica gel column
chromatography (ethyl acetate/acetone¼5:1) to give 16a
(330 mg, 74%) as a colorless oil. IR nmax (KBr): 3842, 3631,
3.1.5. 5-[(tert-Butyldiphenylsilyloxy)methyl]-3-(2-
ethoxyethoxy)-2-methylpyridine-4-carbaldehyde
dimethyl acetal (13). To a stirred suspension of NaH (60%
in oil, 293 mg, 7.33 mmol), which had been washed with
hexane, in DMF (7 ml) was added dropwise a solution of 12
(3.00 g, 6.65 mmol) in DMF (13 ml) under ice-cooling, and
the whole was stirred at room temperature for 30 min.
2-Bromoethyl ethyl ether (0.9 ml, 7.98 mmol) was added
dropwise to the reaction mixture at room temperature, and
stirring was continued at room temperature for 20 h. The
reaction mixture was diluted with H2O, and extracted with
ether. The combined ethereal layers were washed with 1 M
NaOH solution, H2O and saturated NaCl solution, dried
over Na2SO4, and concentrated under reduced pressure. The
resultant residue was purified by silica gel column
chromatography (ethyl acetate/hexane¼1:3) to give 13
(2.55 g, 73%) as a colorless oil. IR nmax (KBr): 2931,
1
3119, 2932, 1739, 1594 cm21. H NMR (CDCl3) d: 1.28
(3H, t, J¼7 Hz, OCH2CH3), 2.43 (3H, s, Ar-CH3), 2.54 (3H,
s, 2-CH3), 3.41 (6H, s, CH(OCH3)2), 3.62 (2H, q, J¼7 Hz,
OCH2CH3), 3.67 (2H, s, OCOCH2), 3.75–3.79 (2H, m,
OCH2CH2OEt), 3.95–3.98 (2H, m, OCH2CH2OEt), 5.45
(2H, s, 5-CH2), 5.75 (1H, s, CH(OCH3)2), 7.31 (1H, d,
J¼1 Hz, imidazole H), 7.35, 7.81 (each 2H, AA0BB0,
J¼8 Hz, aromatic H), 7.94 (1H, d, J¼1 Hz, imidazole H),
8.30 (1H, s, 6-H). 13C NMR (CDCl3) dC: 15.2, 19.3, 21.6,
34.0, 55.9, 61.9, 66.8, 69.3, 74.0, 101.1, 115.2, 127.3, 128.4,
130.4, 134.8, 136.1, 137.4, 137.6, 145.5, 146.2, 150.7,
152.9, 169.6. FAB-MS m/z: 548 (MþHþ). High-resolution
MS Calcd for C26H34N3O8 (MþHþ): 548.2066. Found:
548.0264.
2858, 1590 cm21 1H NMR (CDCl3) d: 1.10 (9H, s,
.
C(CH3)3), 1.26 (3H, t, J¼7 Hz, OCH2CH3), 2.54 (3H, s,
2-CH3), 3.24 (6H, s, CH(OCH3)2), 3.60 (2H, q, J¼7 Hz,
OCH2CH3), 3.73–3.76 (2H, m, OCH2CH2OEt), 3.92–
3.95 (2H, m, OCH2CH2OEt), 5.07 (2H, s, 5-CH2), 5.64
(1H, s, CH(OCH3)2), 7.32–7.41 (6H, m, aromatic H),
7.69–7.73 (4H, m, aromatic H), 8.79 (1H, s, 6-H). 13C
NMR (CDCl3) dC: 15.2, 19.2, 19.3, 26.8, 55.3, 60.9, 66.8,
69.38, 73.9, 101.2, 127.6, 129.5, 133.6, 133.9, 135.5, 135.6,
144.1, 150.4, 151.0. FAB-MS m/z: 524 (MþHþ). High-
resolution MS Calcd for C30H42NO5Si (MþHþ) 524.2832.
Found: 524.2847.
3.1.8. 3-(2-Ethoxyethoxy)-2-methyl-5-{[imidazol-4(5)-
yl]acetoxy}methylpyridine-4-carbaldehyde dimethyl
acetal (17a). To a solution of 16a (50 mg, 0.091 mmol) in
acetic anhydride (0.4 ml) was added pyridine (8 ml) and the
whole was stirred at room temperature for 3 h. After
concentration, the residue was dissolved in methanol
(0.8 ml) and stirred at room temperature for 2 h. The
methanol was evaporated off under reduced pressure, and
H2O was added to the residue, which was adjusted pH 8
with NaHCO3 and extracted with CHCl3. The organic layers
were washed with H2O and saturated NaCl solution, dried
over Na2SO4, and concentrated under reduced pressure to
give 17a (35.9 mg, quant.) as a colorless oil. IR nmax (KBr):
3.1.6. 3-(2-Ethoxyethoxy)-5-(hydroxymethyl)-2-methyl-
pyridine-4-carbaldehyde dimethyl acetal (14). To a
stirred solution of 13 (2.41 g, 4.61 mmol) in THF (10 ml)
was added dropwise tetrabutylammonium fluoride (1 M in
THF, 11.5 ml, 11.5 mmol), and the whole was stirred at
room temperature for 5 h. After concentration, the resultant
residue was partitioned with ethyl acetate and H2O, and
extracted with ethyl acetate. The combined organic layers
were washed with H2O and saturated NaCl solution, dried
over Na2SO4, and concentrated under reduced pressure.
Purification by silica gel column chromatography (ethyl
acetate) afforded 14 (1.16 g, 91%) as colorless crystals, mp
66–678C (AcOEt/Hexane). IR nmax (KBr): 3241,
1
3128, 2935, 1739 cm21. H NMR (CDCl3) d: 1.28 (3H, t,
J¼7 Hz, OCH2CH3), 2.53 (3H, s, 2-CH3), 3.43 (6H, s,
CH(OCH3)2), 3.62 (2H, q, J¼7 Hz, OCH2CH3), 3.75 (2H, s,
OCOCH2), 3.77–3.79 (2H, m, OCH2CH2OEt), 3.95–3.98
(2H, m, OCH2CH2OEt), 5.46 (2H, s, 5-CH2), 5.76 (1H, s,
CH(OCH3)2), 6.94 (1H, s, imidazole H), 7.53 (1H, s,
imidazole H), 8.26 (1H, s, 6-H). 13C NMR (CDCl3) dC: 15.2,
19.2, 33.3, 55.9, 61.9, 66.8, 69.3, 74.1, 96.1, 101.2, 117.5,
128.8, 135.1, 137.6, 145.0, 150.8, 152.7, 170.8. EI-MS m/z
(%): 393 (Mþ, 35.2), 81 (imidazole–CHþ2 , 100). High-
resolution MS Calcd for C19H27N3O6 (Mþ): 393.1899.
Found: 393.1899.
2932 cm21
.
OCH2CH3), 2.54 (3H, s, 2-CH3), 3.52 (6H, s, CH(OCH3)2),
1H NMR (CDCl3) d: 1.29 (3H, t, J¼7 Hz,

4878
K. Miyashita et al. / Tetrahedron 59 (2003) 4873–4879
3.1.9. 3-(2-Ethoxyethoxy)-2-methyl-5-{[imidazol-4(5)-
yl]acetoxy}methylpyridine-4-carbaldehyde (2a).
(2b). Compound 2b was obtained as a colorless oil in 81%
yield by the same method as that for 2a. IR nmax (KBr):
A
1
solution of 17a (35.5 mg, 0.090 mmol) in acetic acid–
H2O (3:2, 5 ml) was refluxed for 3 h. After being neutralized
with saturated NaHCO3 solution, the reaction mixture was
extracted with ethyl acetate. The organic layers were
washed with saturated NaHCO3 solution, H2O and saturated
NaCl solution, dried over Na2SO4, and concentrated under
reduced pressure. The resultant residue was purified by
silica gel column chromatography (CHCl3/MeOH¼10:1) to
give 2a (18.2 mg, 58%) as a colorless oil. IR nmax (KBr):
3103, 2871, 1738, 1697, 1448 cm21. H NMR (CDCl3) d:
1.20 (3H, t, J¼7 Hz, OCH2CH3), 2.61 (3H, s, 2-CH3), 2.74,
2.95 (each 2H, t, J¼7 Hz, COCH2CH2imidazole), 3.54 (2H,
q, J¼7 Hz, OCH2CH3), 3.75–3.77 (2H, m, OCH2CH2OEt),
4.13–4.16 (2H, m, OCH2CH2OEt), 5.42 (2H, s, 5-CH2),
6.80 (1H, s, imidazole H), 7.55 (1H, s, imidazole H), 8.38
(1H, s, 6-H), 10.61 (1H s CHO). 13C NMR (CDCl3) dC:
15.0, 19.4, 21.9, 34.0, 61.6, 66.8, 69.2, 75.1, 77.2, 117.6,
128.2, 132.1, 134.7, 144.6, 155.1, 155.2, 172.9, 192.3. EI-
MS m/z: 361 (Mþ, 0.9), 123 (imidazole CH2CH2COþ, 100).
High-resolution MS Calcd for C18H23N3O5 (Mþ):
361.1637. Found: 361.1624.
1
3133, 2876, 1739, 1698 cm21. H NMR (CDCl3) d: 1.20
(3H, t, J¼7 Hz, OCH2CH3), 2.60 (3H, s, 2-CH3), 3.54 (2H,
q, J¼7 Hz, OCH2CH3), 3.75 (2H, s, OCOCH2), 3.69–3.76
(2H, m, OCH2CH2OEt), 4.13–4.16 (2H, m, OCH2CH2OEt),
5.46 (2H, s, 5-CH2), 6.95 (1H, s, imidazole H), 7.55 (1H, s,
imidazol H), 8.38 (1H, s, 6-H), 10.60 (1H, s, CHO). 13C
NMR (CDCl3) dC: 15.0, 19.3, 33.2, 62.0, 66.8, 69.2, 75.1,
96.1, 117.2, 128.3, 132.1, 135.1, 144.4, 155.0, 155.11,
170.6, 192.3. EI-MS m/z (%): 347 (Mþ, 0.2), 81
(imidazole–CHþ2 , 100). High-resolution MS Calcd for
C17H21N3O5 (Mþ): 347.1481. Found: 347.1492.
3.2. General procedure for b-replacement reaction
b-Replacement reactions with 2 were carried out according
to the same procedure described in a preceding paper, and
the results are summarized in Tables 1 and 2. Spectral
properties of the products 4a–e are also shown in a
preceding paper. Spectral properties of amide 19a and
peptides 19b–h are as follows. Peptides 19c–h were
obtained as a diastereomeric mixture (ca. 1:1).
3.1.10. 3-(2-Ethoxyethoxy)-2-methyl-5-{3-[N-(p-toluene-
sulfonyl)imidazol-4(5)-yl]propanoyloxy}methylpyri-
dine-4-carbaldehyde dimethyl acetal (16b). Compound
16b was obtained as a colorless oil in 60% yield by the same
method as that for 16a. IR nmax (KBr): 2931, 1737,
3.2.1. S-Benzylcysteine benzylamide (19a). A colorless
oil. IR nmax (KBr): 3374, 1668, 1587 cm21 1H NMR
.
(CDCl3) d: 1.55 (2H, m, NH2), 2.42 (1H, dd, J¼7, 14 Hz,
CH2S), 2.81 (1H, dd, J¼5, 14 Hz, CH2S), 3.56 (1H, dd,
J¼5, 7 Hz, a-H), 3.68 (2H, s, SCH2Ph), 4.22–4.41 (2H, m,
NHCH2Ph), 7.20–7.36 (10H, m, aromatic H), 7.98 (1H, m,
amide NH). 13C NMR (CDCl3) dC: 36.0, 38.6, 55.0, 66.3,
126.7, 127.2, 127.8, 128.5, 129.0, 129.7, 135.4, 137.9,
170.6. EI-MS m/z: 300 (Mþ, 38). High-resolution MS Calcd
for C17H20N2OS (Mþ): 300.1296. Found: 300.1301.
1379 cm21
.
1H NMR (CDCl3) d: 1.28 (3H, t, J¼7 Hz,
OCH2CH3), 2.43 (3H, s, Ar-CH3), 2.54 (3H, s, 2-CH3), 2.69,
2.87 (each 2H, t, J¼8 Hz COCH2CH2imidazole), 3.42 (6H, s,
CH(OCH3)2), 3.62 (2H, q, J¼7 Hz, OCH2CH3), 3.75–3.78
(2H, m, OCH2CH2OEt), 3.95–3.98 (2H, m, OCH2CH2OEt),
5.40 (2H, s, 5-CH2), 5.75 (1H, s, CH(OCH3)2), 7.02 (1H, d,
J¼1 Hz, imidazole H), 7.34, 7.80 (each 2H, AA0BB0, J¼8 Hz,
aromatic H), 7.91 (1H, d, J¼1 Hz, imidazole H), 8.28 (1H, s,
6-H). 13C NMR (CDCl3) dC: 15.2, 19.3, 21.7, 23.4, 33.1,
55.9, 61.4, 66.8, 69.4, 74.0, 101.2, 113.3, 127.3, 128.3,
128.8, 130.3, 136.2, 137.5, 143.7, 145.4, 146.0, 150.7,
152.8, 172.2. EI-MS m/z: 561 (Mþ, 4.4), 277 [Mþ2
COCH2CH2imidazole(Tos), 100]. High-resolution MS
Calcd for C27H35N3O8S (Mþ): 561.2144. Found: 561.2155.
3.2.2. (S-Benzylcysteinyl)glycine benzyl ester (19b). A
colorless oil. IR nmax (KBr): 3370, 1741, 1650, 1592 cm21
.
1H NMR (CDCl3) d: 1.48 (2H, br s, NH2), 2.35 (1H, dd,
J¼7, 14 Hz, a-CH2S), 2.83 (1H, dd, J¼5, 14 Hz, a-CH2S),
3.56 (1H, dd, J¼5, 7 Hz, N-terminal a-H), 3.68 (2H, s,
SCH2Ph), 4.13–4.37 (2H, AB in ABX, J¼5, 13 Hz,
C-terminal a-H), 5.10–5.16 (2H, AB type, J¼11 Hz,
CO2CH2Ph), 7.20–7.48 (10H, m, aromatic H), 8.01 (1H,
br s, amide NH). 13C NMR (CDCl3) dC: 39.1, 42.0, 55.0,
58.2, 65.3, 124.9, 125.9, 127.8, 128.0, 128.5, 129.3, 133.3,
138.8, 169.5, 172.1. EI-MS m/z (%): 372 (Mþ, 7.6), 91
(Bnþ, 100). High-resolution MS Calcd for C20H24N2O3S
(Mþ): 372.1508. Found: 372.1510.
3.1.11. 3-(2-Ethoxyethoxy)-2-methyl-5-{3-[imidazol-
4(5)-yl]propanoyloxy}methylpyridine-4-carbaldehyde
dimethyl acetal (17b). Compound 17b was obtained as a
colorless oil in 89% yield by the same method as that for
17a. IR nmax (KBr): 3091, 2932, 1736, 1447 cm21. 1H NMR
(CDCl3) d: 1.28 (3H, t, J¼7 Hz, OCH2CH3), 2.54 (3H, s,
2-CH3), 2.73, 2.96 (each 2H, t, J¼7 Hz, COCH2CH2-
imidazole), 3.42 (6H, s, CH(OCH3)2), 3.62 (2H, q, J¼7 Hz,
OCH2CH3), 3.75–3.79 (2H, m, OCH2CH2OEt), 3.94–3.98
(2H, m, OCH2CH2OEt), 5.43 (2H, s, 5-CH2), 5.76 (1H, s,
CH(OCH3)2), 6.79 (1H, s, imidazole H), 7.52 (1H, s,
imidazole H), 8.27 (1H, s, 6-H). 13C NMR (CDCl3) dC: 15.2,
19.3, 21.8, 34.1, 55.9, 61.5, 66.8, 69.3, 74.1, 77.2, 96.1,
101.2, 128.8, 134.7, 137.6, 145.4, 150.8, 152.8, 173.1. EI-
MS m/z: 407 (Mþ, 42.9), 123 (imidazole CH2CH2COþ,
100). High-resolution MS Calcd for C20H29N3O6 (Mþ):
407.2056. Found: 407.2056.
3.2.3. (S-Benzylcysteinyl)-L-alanine benzyl ester (19c, ca
1:1 diastereomeric mixture). A colorless oil. IR nmax
(KBr): 3376, 1737, 1652, 1593 cm21. 1H NMR (CDCl3) d:
1.33 (3/2H, d, J¼7 Hz, a-CH3), 1.36 (3/2H, d, J¼7 Hz,
a-CH3), 1.49 (2H, br s, NH2), 2.30 (1/2H, dd, J¼7, 14 Hz,
a-CH2S), 2.36–2.87 (2/2H, AB in ABX, a-CH2S), 2.93
(1/2H, dd, J¼5, 14 Hz, a-CH2S), 3.58 (1/2H, dd, J¼5, 7 Hz,
N-terminal a-H), 3.62 (1/2H, dd, J¼5, 7 Hz, N-terminal
a-H), 3.68 (2/2H, s, SCH2Ph), 3.70 (2/2H, s, SCH2Ph), 4.37
(1H, qn, J¼7 Hz, C-terminal a-H), 5.11–5.16 (2H, AB
type, CO2CH2Ph), 7.20–7.48 (10H, m, aromatic H), 7.92
(1H, br s, amide NH). 13C NMR (CDCl3) dC: 17.2, 38.4,
40.1, 53.0, 55.6, 66.9, 126.8, 127.1, 127.5, 128.0, 128.4,
3.1.12. 3-(2-Ethoxyethoxy)-2-methyl-5-{3-[imidazol-
4(5)-yl]propanoyloxy}methylpyridine-4-carbaldehyde

K. Miyashita et al. / Tetrahedron 59 (2003) 4873–4879
4879
129.0, 134.8, 138.4, 171.8, 172.9. EI-MS m/z (%): 386 (Mþ,
5.2), 91 (Bnþ, 100). High-resolution MS Calcd for
C21H26N2O3S (Mþ): 386.1664. Found: 386.1666.
m, aromatic H), 7.98 (1H, br s, amide NH). 13C NMR
(CDCl3) dC: 17.1, 39.1, 53.6, 56.1, 67.8, 124.3, 125.6, 128.0,
128.5, 128.9, 132.1, 138.8, 171.07, 172.2. EI-MS m/z (%):
324 (Mþ, 5.3), 91 (Bnþ, 100). High-resolution MS Calcd for
C16H34N2O3S (Mþ): 324.1508. Found: 324.1511.
3.2.4. (S-Benzylcysteinyl)-L-valine benzyl ester (19d, ca
1:1 diastereomeric mixture). A colorless oil. IR nmax
(KBr): 3366, 1747, 1669, 1508 cm21. 1H NMR (CDCl3) d:
0.73, 0.75 (each 3/2H, each d, J¼7 Hz, CH(CH3)2), 0.88,
0.91 (each 3/2H, each d, J¼7 Hz, CH(CH3)2), 1.50 (2H, br s,
NH2), 2.11–2.20 (1H, m, CH(CH3)2), 3.13 (1/2H, dd, J¼7,
11 Hz, a-CH2S), 3.15–3.53 (2/2H, AB in ABX, a-CH2S),
3.61 (1/2H, dd, J¼5, 11 Hz, a-CH2S), 3.59–3.63 (1H, m,
N-terminal a-H), 3.67 (2/2H, s, SCH2Ph), 3.70 (2/2H, s,
SCH2Ph), 4.48 (1/2H, dd, J¼5, 9 Hz, C-terminal a-H), 4.53
(1/2H, dd, J¼5, 9 Hz, C-terminal a-H), 5.11–5.14 (2H, AB
type, CO2CH2Ph), 7.30–7.45 (10H, m, aromatic H), 8.12
(1H, br s, amide NH). 13C NMR (CDCl3) dC: 17.2, 19.4,
31.2, 37.3, 38.4, 56.81, 59.6, 67.6, 126.0, 127.1, 128.0,
128.3, 128.6, 129.0, 135.1, 138.4, 171.7, 172.8. EI-MS m/z
(%): 414 (Mþ, 13), 91 (Bnþ, 100). High-resolution MS
Calcd for C23H30N2O3S (Mþ): 414.1977. Found: 414.1973.
3.2.8. S-(3-Hydroxypropyl)cysteinyl]-L-alanine benzyl
ester (19h, ca 1:1 diastereomeric mixture). A colorless
oil. IR nmax (KBr): 3368, 3033, 1737, 1661 cm21. 1H NMR
(CDCl3) d: 1.36 (3/2H, d, J¼7 Hz,** a-CH3), 1.38 (3/2H, d,
J¼7 Hz, a-CH3), 1.61 (2H, br, NH2), 1.76 (2H, qn, J¼7 Hz,
SCH2CH2CH2OH), 2.59 (2H, t, J¼7 Hz, SCH2CH2CH2-
OH), 2.90–2.95 (2/2H, m, a-CH2S), 2.98–3.06 (2/2H, m,
a-CH2S), 3.68 (1H, m, N-terminal a-H), 3.72 (2H, t,
J¼7 Hz, SCH2CH2CH2OH), 4.40 (1H, m, C-terminal a-H),
5.08 (2H, br s, CO2CH2Ph), 7.24–7.42 (5H, m, aromatic H),
8.09 (1H, br s, amide NH). 13C NMR (67.8 MHz, CDCl3)
dC: 17.0, 31.2, 36.8, 36.8, 38.8, 53.9, 54.3, 56.0, 67.3, 125.9,
126.8, 128.0, 134.1, 171.8, 172.7. EI-MS m/z (%): 355
(MþHþ, 1.0), 91 (Bnþ, 100). High-resolution MS Calcd for
C16H34N2O3S (MþHþ): 355.1615. Found: 355.1610.
3.2.5. (S-Benzylcysteinyl)-L-alanyl-L-alanine benzyl
ester (19e, ca 1:1 diastereomeric mixture). A colorless
oil. IR nmax (KBr): 3363, 1741, 1668, 1506 cm21. 1H NMR
(CDCl3) d: 1.21 (3H, d, J¼7 Hz, C-terminal a-CH3), 1.34
(3/2H, d, J¼7 Hz, a-CH3), 1.37 (3/2H, d, J¼7 Hz, a-CH3),
1.48 (2H, br s, NH2), 3.22–3.38 (1H, m, a-CH2S), 3.55–
3.70 (1H, m, a-CH2S), 3.70 (2/2H, s, SCH2Ph), 3.73 (2/2H,
s, SCH2Ph), 3.88 (1H, m, N-terminal a-H), 4.22 (1H, qn,
J¼7 Hz, a-H), 4.42 (1H, qn, J¼7 Hz, C-terminal a-H),
5.04–5.12 (2H, AB type, CO2CH2Ph), 6.96 (1H, br s, amide
NH), 7.28–7.45 (10H, m, aromatic H), 8.12 (1H, br s, amide
NH). 13C NMR (CDCl3) dC: 17.0, 17.8, 36.5, 38.4, 47.5,
52.6, 55.2, 66.9, 126.8, 127.1, 128.2, 129.0, 135.0, 138.4,
170.6, 171.8, 173.1. EI-MS m/z (%): 443 (Mþ, 3.4), 91
(Bnþ, 100). High-resolution MS Calcd for C23H30N3O4S
(Mþ): 443.1879. Found: 443.1884.
Acknowledgements
A part of this work was supported by SUNBOR Grant.
References
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1:1 diastereomeric mixture). A colorless oil. IR nmax
(KBr): 3373, 1737, 1665, 1590 cm21. 1H NMR (CDCl3) d:
1.39 (3/2H, d, J¼7 Hz, a-CH3), 1.42 (3/2H, d, J¼7 Hz,
a-CH3), 1.55 (2H, br s, NH2), 3.22 (1/2H, dd, J¼7, 14 Hz,
a-CH2S), 3.25–3.60 (2/2H, AB in ABX, a-CH2S), 3.62
(1/2H, dd, J¼5, 14 Hz, a-CH2S), 3.66 (1H, m, N-terminal
a-H), 4.42 (1H, qn, J¼7 Hz, C-terminal a-H), 5.04–5.12
(2H, AB type, CO2CH2Ph), 7.30–7.45 (10H, m, aromatic
H), 8.12 (1H, br s, amide NH). 13C NMR (CDCl3) dC: 17.0,
39.0, 56.1, 57.9, 66.9, 125.9, 126.8, 127.2, 128.0, 128.5,
129.0, 129.7, 135.5, 138.8, 171.8, 172.7. EI-MS m/z (%):
354 (Mþ, 19), 263 (32), 91 (Bnþ, 100). High-resolution MS
Calcd for C17H26N2O4S (Mþ): 354.1613. Found: 354.1610.
3. Miyashita, K.; Murafuji, H.; Iwaki, H.; Yoshioka, E.; Imanishi,
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3.2.7. (S-Ethylcysteinyl)-L-alanine benzyl ester (19g, ca
1:1 diastereomeric mixture). A colorless oil. IR nmax
(KBr): 3381, 1756, 1653, 1500 cm21. 1H NMR (CDCl3) d:
1.13 (3H, d, J¼7 Hz, SCH2CH3), 1.31 (3/2H, d, J¼7 Hz,
a-CH3), 1.32 (3/2H, d, J¼7 Hz, a-CH3), 1.55 (2H, br s,
NH2), 2.51 (2H, q, J¼7 Hz, SCH2CH3), 2.73–3.11 (2/2H,
AB in ABX, a-CH2S), 2.81–2.96 (2/2H, AB in ABX,
a-CH2S), 3.68 (1H, m, N-terminal a-H), 4.40 (1H, m,
C-terminal a-H), 5.21 (2H, s, CO2CH2Ph), 7.30–7.45 (5H,
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T. Chem. Commun. 2002, 1922.
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