Synthesis based on affinity separation (SAS): Separation of products having barbituric acid tag from untagged compounds by using hydrogen bond interaction

Article abstract of DOI:10.1055/s-2001-13392

A new method is described for affinity purification of synthetic compounds based on molecular recognition between bis(2,6-diaminopyridine)amide of isophthalic acid and a barbituric acid derivative. The desired compounds possessing the barbituric acid deri

Full text of DOI:10.1055/s-2001-13392

590
LETTER
Synthesis Based on Affinity Separation (SAS): Separation of Products Having
Barbituric Acid Tag from Untagged Compounds by Using Hydrogen Bond
Interaction
San-Qi Zhang, Koichi Fukase,* Minoru Izumi, Yoshiyuki Fukase, Shoichi Kusumoto
Department of Chemistry, Graduate School of Science, Osaka University, Machikaneyama 1-1, Toyonaka, Osaka 560-0043, Japan
E-mail: koichi@chem.sci.osaka-u.ac.jp
Received 6 March 2001
Polymer-supported reagents and scavenger resins have
Abstract: A new method is described for affinity purification of
made solution synthesis efficient but this strategy is not
synthetic compounds based on molecular recognition between
bis(2,6-diaminopyridine)amide of isophthalic acid and a barbituric
acid derivative. The desired compounds possessing the barbituric
always effective for multistep synthesis because of the
limited availability of the reagents.2
acid derivative as a tag were readily isolated from the reaction mix-
ture by the following procedure. After each reaction cycle, the reac-
brid method of solid-phase and solution-phase synthesis.3
tion mixture was applied to the polystyrene column possessing
Recently, “tag” methodology has been developed as a hy-
In this methodology, a compound having a tag group is
bis(2,6-diaminopyridine)amide of isophthalic acid as an artificial
easily separated from untagged molecules by using the af-
finity of the tag with a respective specific functionality.
Several tags such as fluorous affinity tags,3a-c hydrophobic
tags,3d-f a 2-pyridylsilyl tag for acidic extraction,3g and an
anthracene tag for Diels-Alder resin capture/release3h
have been reported. The “tag” methodology has advantag-
es over polymer-supported synthesis in the following
points. Namely, monitoring of reaction is easy by simple
and routine methods such as TLC and HPLC. Additional
purification of tagged intermediates is possible by other
methods after any reaction steps.
receptor. The compound possessing the barbituric acid tag was se-
lectively adsorbed on the column, whereas other impurities without
the tag such as excess reagents and byproducts were washed off.
Subsequent desorption with CH2Cl2-MeOH (1:1) afforded the de-
sired compound with high purity. This new strategy was applied to
the synthesis of a heterocycle, peptides, and oligosaccharides.
Key words: affinity purification, molecular recognition, molecular
tag, oligosaccharide synthesis, high throughput synthesis
Polymer supported synthesis has greatly simplified work-
up and purification procedure of organic reactions to es-
tablish combinatorial and parallel synthesis.1 In solid-
phase synthesis, the synthetic product at each step on solid
support is immediately separated from a reaction mixture
by filtration. Although utility of solid-phase synthesis has
been confirmed, there still exist several disadvantages: i)
some of conventional reaction conditions in solution are
not compatible with reactions on solid-supports, ii) the re-
action rates on solid-supports are generally lower than
those of the corresponding reactions in solutions. In liq-
uid-phase synthesis using soluble polymer support, the re-
activity of compounds on the support is similar to that in
solution but additional step, i.e., precipitation of the poly-
mer-supported products, is required for their isolation.1b
We previously reported a new “tag” strategy, termed
“synthesis based on affinity separation (SAS)”, in which
the desired tagged compound was separated from the re-
action mixture by solid-phase extraction using specific
molecular recognition (Figure 1).4 In that work, we em-
ployed the interaction between a crown ether (32-crown-
10) and ammonium ion for SAS.
In the present study, we investigated a new SAS method
by using interaction via multiple hydrogen bonds between
a guest residue and its artificial receptor. From many arti-
ficial receptors reported, we employed Hamilton’s recep-
tor, i.e., bis(2,6-diaminopyridine)amide of isophthalic
acid, which forms tight complex with barbituric acid via
b)
B
AB
B
a)
B
A
AB
C
B
Tag
Receptor
c)
d)
B
AB
C
Figure 1 Synthesis based on affinity separation. A: substrate, B: reagent, AB: product, C: byproduct. a) reaction in solution, b) adsorption of
tagged product, c) elution of excess reagent and untagged byproduct, d) desorption of tagged product.
Synlett 2001, No. 5, 590–596 ISSN 0936-5214 © Thieme Stuttgart · New York

LETTER
Synthesis Based on Affinity Separation (SAS)
591
six hydrogen bonds.5 A barbituric acid moiety was used as
a tag and the receptor moiety was bound to polystyrene
support (Figure 2).
OH
a, b
Cl
HO
AllylO
CO2Et
CO2Et
6
7
c
d
O
AllylO
NH
O
N
O
N
8
O
O
O
NH
NH
O
O
NH
NH
HN
HN
NH
NH
H
N
O
OH
O
O
e, f
NH
AllylO
O
HO
O
O
O
10: HOBA
NH
9
10: HOBA
1
O
Scheme 2 Reagents and conditions: a) xylylene glycol (2.0 equiv.),
NaH (2.2 equiv.), allylBr (1.0 equiv.), THF, 60°C, 7 h, 63%; b)
[Me2NCl]+Cl- (1.3 equiv), MeCN, reflux, 30 min, 99%; c) diethyl
ethylmalonate (1.06 equiv.), KOBut (1.06 equiv.), toluene, reflux,
6.5h, 98%; d) urea (10 equiv.), NaH (4 equiv.), DMSO, rt, 14 h, 81%;
Figure 2 Host-guest interaction of a polymer-supported receptor
with the barbituric acid tag.
-
e) [Ir(COD)(PMePh2)]+PF6 (0.045 equiv.), H2, THF, rt, 100 min, f)
Polymer-supported artificial receptor (1) was synthesized
from 5-hydroxyisophthalic acid (2) as illustrated in
Scheme 1.6 A highly cross-linked macroporous polysty-
rene resin ArgoPoreTM NH2 (1.16 mmol/g) was used as a
support since it gave better results than the usual 1%
cross-linked gel-type polystyrene resin in the previous
study. The barbituric acid tag 10 (HOBA) was synthe-
sized as shown in Scheme 2.7
I2/H2O, rt, 30 min, 87%.
pylcarbodiimide (DIC) and a catalytic amount of 4-
(dimethylamino)pyridine (DMAP) in CH2Cl2. The reac-
tion mixture was directly applied to the resin column 1.
The tagged 12 was retained in the column, whereas all un-
tagged compounds such as excess 11, DIC, DMAP, and
N,N’-diisopropylurea were washed out from the column
simply by elution with CH2Cl2. Elution of 12 with
CH2Cl2-methanol (1:1) and concentration gave purified
12. Fmoc group in 12 was then removed with 5% piperi-
dine in DMF. Peptide condensation was carried out by the
DIC method in CH2Cl2 to give dipeptide 13, which was
purified by the same affinity separation. After tripeptide
14 was formed in a similar manner, the removal of the
Fmoc group, N-acetylation, and purification by the affini-
ty separation afforded 15. The tag moiety of 15 was then
removed by transesterification to give 16, which was sep-
arated from the barbituric acid tag 10 by the affinity sepa-
ration: the desired compound 16 passed through the
The tagged compounds were effectively retained in the
resin column 1 by using nonpolar eluents such as CH2Cl2,
CHCl3, and toluene, which favors the formation of a com-
plex through hydrogen bonds. Since polar solvents disfa-
vor the complex formation, the tagged compounds were
readily desorbed from the column by using CH2Cl2
methanol as an eluent. The separation scale was 0.1
mmol~0.2 mmol for peptides and heterocycle and 0.05
mmol~0.11 mmol for oligosaccharides with 7 g of the res-
in 1.
This methodology was first applied to the synthesis of
small peptides (Scheme 3).8 The tag moiety 10 was cou-
pled to the starting Fmoc amino acid 11 by using diisopro-
O
CO2Et
CO2Et
CO2H
CO2H
N
H
N
N
NHCOPr
NHCOPr
a, b
c, d
e, f
O
O
HO
H
N
O2N
O2N
4
2
3
O
O
O
O
N
H
N
N
NHCOPr
NHCOPr
N
H
N
N
NHCOPr
g
O
O
H
N
H
N
H
N
H
N
NH
HO
NHCOPr
O
O
O
O
O
1
5
Scheme 1 Reagents and conditions: a) AcCl, EtOH, reflux, 4 h, 98%; b) p-nitrobenzyl bromide (1.12 equiv.), K2CO3, acetone, reflux, 2 h,
97%; c) n-BuLi (1.95 equiv.), 2,6-diaminopyridine (2.0 equiv.), THF, -78 °C to -60 °C, 8 h, 91%; d) PrCOCl (1.4 equiv.), DIEA, THF, rt, 4 h,
92%; e) NiCl2•6H2O, NaBH4, MeOH, THF, rt, 1.5 h; f) glutaric anhydride (2.0 equiv.), THF, rt, 5 h, 60% over two steps; g) ArgoPoreTM-NH2
(1.16 mmol/g), DIC, DMAP, DMF, rt, 3 d.
Synlett 2001, No. 5, 590–596 ISSN 0936-5214 © Thieme Stuttgart · New York

592
S.-Q. Zhang et al.
LETTER
column whereas 10 was adsorbed to the column. The
overall yield of the tripeptide 16 was 89% (from HOBA
(10)) with 81% purity (judged by HPLC analysis at 220
nm). Tripeptide Ac-Leu-Trp-Trp-OMe (22) (91% yield
with 86% purity) was also synthesized by the same SAS
strategy.
O
O
NH
NH
O
a, b
O
F
+
OH
HO
O2N
23
10: HOBA
c, b
O
O
F
N
OBA
OBA
a, b
O2N
c, d, b
O2N
Fmoc-Phe-OH + HOBA
Fmoc-Phe-OBA
25
24
12
11
10
O
d, b
c, e, b
c, d, b
N
Fmoc-Leu-Trp-Phe-OBA
Fmoc-Trp-Phe-OBA
OMe
14
13
O2N
f, b
91%; purity: 93%
26
Ac-Leu-Trp-Phe-OMe
Ac-Leu-Trp-Phe-OBA
15
16 Total yield : 89%; purity: 81%
Scheme 4 Reagents and conditions: a) DIC, DMAP, CH2Cl2;
b) affinity separation; c) piperidine (1.5 equiv.), NMP, rt, 2h;
d) 0.2 M MeONa in MeOH, rt, 10 min.
a, b
c, d, b
Fmoc-Trp-OH + 10
Fmoc-Trp-OBA
18
17
c, e, b
c, d, b
f, b
Fmoc-Leu-Trp-Trp-OBA
Fmoc-Trp-Trp-OBA
arylpiperidine 25 was thus effected without additional
steps for the removal of NMP. Transesterification and iso-
lation by affinity separation gave 26 in overall 91%
yield.10
20
19
Ac-Leu-Trp-Trp-OMe
Ac-Leu-Trp-Trp-OBA
Total yield : 91%; purity: 86%
21
22
Scheme 3 Reagents and conditions: a) Fmoc-amino acids (1.25
equiv.), DIC (2 equiv.), DMAP, CH2Cl2; b) affinity separation; c) 5%
piperidine in DMF, rt, 8 min; d) Fmoc-amino acids (1.25 equiv.), DIC
(2 equiv.), CH2Cl2; e) Ac2O, TEA, CH2Cl2; f) 0.2 M NaOMe in
MeOH, rt, 10 min.
We then investigated oligosaccharide synthesis by the
present SAS strategy (Scheme 5 and 6). The keys for the
success were the following two points: i) the tag moiety
was stable under glycosylation conditions and ii) oli-
gosaccharides possessing many protective groups were
effectively adsorbed on the resin.
This new synthetic protocol was then applied to synthesis
of N-arylpiperidine (Scheme 4). Since 1-methyl-2-pyr-
rolidone (NMP), which reduces the interaction of the arti-
ficial receptor with the tag, was used as a solvent for the
aromatic nucleophilic substitution of 24 with piperidine,9
the reaction mixture was first diluted with CH2Cl2 and
then subjected to the affinity separation. Purification of
The first example is synthesis of -(1-6)oligoglucosamine
(Scheme 5). The tag 10 was attached to the starting N-
trichloroethoxycarbonyl (Troc) glucosamine 27 through a
glutaric acid spacer. Removal of the benzylidene group of
29 gave a glycosyl acceptor 30. -Selective glycosylation
of 30 with glycosyl trichloroacetimidate 31 (1.5 equiv. to
30) was effected by using TMSOTf as a catalyst by virtue
O
O
O
O
O
O
O
O
O
O
O
O
O
a
b, c
Ph
Ph
Ph
+
OH
OBA
OH
O
O
O
O
O
O
O
O
76%
99%
NHTroc
NHTroc
NHTroc
27
28
29
O
NH
O CCl3
O
Ph
e)
OAc
O
HO
O
O
O
O
31
O
O
d, c
d, c
NHTroc
O
OAc
O
O
Ph
OBA
OBA
O
O
c
O
HO
O
HO
NHTroc
O
97%
NHTroc
NHTroc
32
30
HO
O
O
O
O
O
O
O
O
O
OAc
O
e)
, c
31
O
OAc
O
OAc
O
Ph
OBA
O
OBA
O
HO
HO
NHTroc
O
O
HO
NHTroc
HO
NHTroc
O
56% from 30
NHTroc
NHTroc
33
34
Scheme 5 Reagents and conditions: a) pyridine, DMAP, CH2Cl2, 3.5 h; b) HOBA (10), DIC, DMAP, CH2Cl2, 1.5 h; c) affinity separation;
d) 5% TFA, 1% H2O in CH2Cl2, 0 °C; e) TMSOTf (0.1 equiv.), MS4A, CH2Cl2, -20 °C, 10 min.
Synlett 2001, No. 5, 590–596 ISSN 0936-5214 © Thieme Stuttgart · New York

LETTER
Synthesis Based on Affinity Separation (SAS)
593
HO
HO
HO
O
O
O
O
O
OBn
O
OBn
b)
HO
OBn
a
OBA
BnO
BnO
BnO
OMe
OMe
OMe
O
O
O
O
c
NO2
TrocO
quant
NH2
N
H
OBA
94%
36
35
HO
TrocO
d)
O
O
OBn
O
OBn
OBn SPh
O
O
O
O
OBn
BnO
BnO
BnO
e
37
OBn
OBn
OBn
38
O
OBn
BnO
BnO
OMe
O
O
OMe
O
O
c
O
O
39
N
H
OBA
N
OBA
TrocO
H
TrocO
OBn
O
O
O
OBn SPh
BnO
BnO
d)
OBn
OBn
O
O
37
BnO
OBn
OBn
OBn
BnO
OMe
O
O
c
40
O
28.7% from 36
N
OBA
H
Scheme 6 Reagents and conditions. a) NiCl2•6H2O, NaBH4, MeOH; b) DIC, CH2Cl2; c) affinity separation; d) PhIO, TMSOTf, Et2O/dioxa-
ne; e) Zn-Cu couple, AcOH.
of the neighboring participation of the N-Troc group to af- coupling with the tag moiety (Scheme 6).16 Glycosylation
ford the disaccharide 32.11 Removal of the benzylidene of the tagged acceptor 36 with excess phenyl 6-O-Troc-
from 32 followed by glycosylation with 31 (5 equiv.) gave thioglycoside 37 (1.5 equiv.) using PhIO and TMSOTf
trisaccharide 34. After each reaction step, the product was gave disaccharide 38 ( : = 10:1).17 The 6-O-Troc group
rapidly separated from the reaction mixture by the affinity of 38 was removed and the resulting 6-O-free disaccha-
separation in this synthesis, too. Since there are two and ride 39 was then glycosylated with 37 to give the trisac-
three hydroxy groups in acceptors 30 and 33, respectively, charide 40 (28.7% from 36).18 After each reaction step,
some undesired byproducts were formed by their glyco- the product was rapidly separated from the reaction mix-
sylation at more hindered 4-positions. These byproducts ture by the affinity separation. Since the acylaminobenzyl
were not separated by affinity separation like solid-phase linker was partly cleaved at the glycosylation step, the fi-
synthesis, since they had the tag moiety. The desired nal product 40 was purified by silica-gel column chroma-
trisaccharide 34 was, however, readily purified by addi- tography after the affinity separation.
tional simple silica-gel chromatography. The purified 34
In summary, we have developed a new synthetic strategy
based on affinity separation. The present method can be
applied to the synthesis of relatively large molecules such
was thus obtained in 56% yield from 30 after silica-gel
column chromatography.12 In the case of solid-phase syn-
thesis, purification is only possible after products are
as protected oligosaccharides owing to the strong affinity
cleaved from solid-supports. By using the present SAS
of the barbituric acid tag with its artificial receptor, where-
method, products can be purified by affinity separation as
as previous tag strategies have been applied to synthesis
well as other purification methods. Purified synthetic in-
of small molecules.19 The strategy greatly facilitates the
termediates can be subjected to next reaction. This is im-
purification procedures in solution-phase synthesis and is
expected to be particularly useful for multiple parallel
portant feature of the present methodology.
As shown in Scheme 6, oligosaccharide synthesis was synthesis and combinatorial library preparation where ap-
carried out under the conditions for -selective glycosyl- plication of solid-phase synthesis is difficult.
ation by virtue of the solvent effect of ether using
2-O-benzylated thioglycosyl donors. Since the solubilities
of glycosyl acceptors having the barbituric acid tag in
Acknowledgement
The present work was financially supported in part by "Research for
the Future" Program No. 97L00502 from the Japan Society for the
Promotion of Science, by Special Coordination Funds of the Sci-
ence and Technology Agency of the Japanese Government, and by
Grants-in-Aid for Scientific Research No. 09044086 and No.
12640571 from the Ministry of Education, Science and Culture, Ja-
pan. We thank Dr. John A. Porco, Jr. (Boston University), Dr. W.
Clark Still (Columbia University), and Dr. H. Peter Nestler (Aventis
Pharma Deutschland GmbH) for helpful discussions.
ether were low, a mixture of ether-dioxane was used as the
reaction solvent.13 We previously found combinations of
iodosobenzene (PhIO) and various acids effectively pro-
mote glycosylation using thioglycosyl donors.14 Among
those found to be satisfactory, the combination of PhIO
and TMSOTf was employed in the present study.15
The tagged acceptor 36 was prepared from 4-nitrobenzyl-
glucoside 35 via reduction of the nitro group followed by
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594
S.-Q. Zhang et al.
LETTER
m, Ar-H), 5.47 (2H, s,-OCH2-), 2.37 (4H, t, J = 7.3 Hz, 2
COCH2-), 1.59 (4H, m, 2 CH2), 0.90 (6H, t, J = 7.3 Hz,
CH3).
N,N’-Bis[2-(6-butyrylaminopyridyl)]-5-[4-(4-carboxyl-
-
References and Notes
(1) a) Thompson, L. A.; Ellman, J. A. Chem. Rev. 1996, 96, 555.
b) Gravert, D. J.; Janda, K. D.; Chem. Rev. 1997, 97, 489. c)
Dolle, R. E.; Nelson, Jr. K. H. J. Comb. Chem. 1999, 1, 235.
d) Dolle, R. E. J. Comb. Chem. 2000, 2, 383.
2
butyryl)phenylmethyloxy]isophthaldiamide (5). To a solution
of NiCl2•6H2O (0.3 g, 1.26 mmol) in MeOH (80 ml) was
added of NaBH4 (0.1 g, 2.64 mmol) (the color of solution
changed from green to black) and a solution of 4 (2.0 g, 3.13
mmol) in THF (40 ml) was added. NaBH4 (4.0 g, 106 mmol)
was then added to the mixture by portions for 1.5 h. After that
1M NaOH (20 ml) and EtOAc (80 ml) were added, the organic
layer was washed with brine (60 ml 3), dried over Na2SO4,
and concentrated in vacuo. To the solution of the residue in
THF (30 ml) was added glutaric anhydride (1.0 g, 8.77 mmol)
and the mixture was stirred at room temperature for 5 h. The
desired 5 precipitated was collected by filtration and washed
with THF to give 1.10 g of 5. The filtrate was concentrated and
the residue was purified by silica-gel column chromatography
(CHCl3/MeOH = 20:1) to give another 0.25 g of the product.
Yield 1.35 g (59.7%). ESI-MS m/z 722.3 (negative) [(M-H)-].
1H NMR (600 MHz, DMSO), = 10.49 (2H, s, 2 CONH),
10.11 (2H, s, 2 CONH), 10.00 (1H, s, CONH), 8.13(1H, s,
Ph), 7.86-7.72 (5H, Ar-H),7.62 (2H, d, J = 7.7 Hz, PhNHCO),
7.42 (2H, d, J = 7.7 Hz, PhNHCO), 5.21(2H, s, -OCH2-), 2.37
(6H, m, 3 x CH2CO), 2.26 (2H, t, CH2CO), 1.75 (4H, m,
(2) a) Kaldor, S. W.; Siegel, M. G. Curr. Opin. Chem. Biol. 1997,
1, 101. b) Booth, J.; Hodges, J. C. Acc. Chem. Res. 1999, 32,
18. c) Thompson, L.A. Curr. Opin. Chem. Biol. 2000, 4, 324.
(3) a) Curran, D. P. Angew. Chem., Int. Ed. 1998, 37, 1174. b)
Curran, D. P.; Luo, Z. J. Am. Chem. Soc. 1999, 121, 9069.
c) Horváth, I. T. Acc. Chem. Res. 1998, 31, 641. d) Ramage,
R.; Swenson, H. R.; Shaw, K. T. Tetrahedron Lett. 1998, 39,
8715. e) Hay, A. M.; Hobbs-Dewitt, S.; MacDonald, A. A.;
Ramage, R. Tetrahedron Lett. 1998, 39, 8721. f) Nilsson, U.
J.; Fournier, E. J.-L.; Hindsgaul, O. Bioorg. Med. Chem. 1998,
6, 1563. g) Yoshida, J.; Itami, K.; Mitsudo, K.; Suga, S.
Tetrahedron Lett. 1999, 40, 3403. h) Wang, X.; Parlow, J. J.;
Porco Jr., J. A. Org. Lett. 2000, 2, 3509.
(4) a) Zhang, S.-Q.; Fukase, K.; Kusumoto, S. Tetrahedron Lett.
1999, 40, 7479. b) Zhang, S.-Q.; Fukase, K.; Kusumoto, S.
Peptide Science 1999 (Ed: N. Fujii), the Japanese Peptide
Society 2000, pp.151-154.
(5) a) Chang, S. K.; Hamilton, A. D. J. Am. Chem. Soc. 1988, 110,
1318. b) Moteshare, K.; Myles, D. C. J. Am. Chem. Soc. 1998,
120, 7328.
2
2
CH2), 1.61 (4H, m, 2 CH2), 0.90 (6H, t, J = 6.9 Hz,
CH3).
(6) The experimental procedure for the preparation of the
polymer supported receptor 1.
Preparation of the polymer supported receptor 1. To a
suspension ArgoPore-NH2-TM (1.16 mmol/g) (1.00 g, 1.16
mmol of NH2 group), 5 (1.25 g, 1.73 mmol), and DMAP (5.0
mg, 0.04 mmol) in DMF (10 ml) was added DIC (545 l, 3.48
mmol) at room temperature. After 3 d of agitation, the resin
was washed with DMF (20 ml 3), CH2Cl2-MeOH 1:1 (20
ml 3), and CH2Cl2 (20 ml 3) and dried under reduced
pressure to give the receptor-bound resin (1.42 g, 50.2%).
(7) The experimental procedure for the preparation of the tag 10.
4-Allyloxymethylbenzyl Alcohol. To a solution of p-xylylene
glycol (6) (11.1 g, 80.3 mmol) in anhydrous THF (200 ml) was
added NaH (60% oil suspension, 3.52 g, 88.0 mmol). The
mixture was stirred at room temperature for 1 h. Allyl bromide
(3.46 ml, 40.0 mmol) was added to the solution and the
mixture was stirred at 60 °C for 7 h. After addition of EtOAc
and aqueous saturated citric acid solution, the organic layer
was washed with water and brine, dried over Na2SO4, and
concentrated in vacuo. The residue was purified by silica-gel
column chromatography (CHCl3/acetone = 1:0 to 9:1) to give
5.64 g (63.4%) of the desired product. ESI-MS (positive) m/z
178.44 (M+); 196.44 [(M+H2O)+].
Diethyl 5-(4-Nitrophenylmethyloxy)isophthalate (3). The
mixture of diethyl 5-hydroxyisophthalate (7.68 g, 32.2 mmol),
p-nitrobenzyl bromide (7.80 g, 36.0 mmol), and K2CO3 (8.0 g,
58 mmol) in acetone (100 ml) was refluxed for 2 h. After the
mixture was cooled to room temperature, insoluble materials
were removed by filtration and the filtrate was concentrated in
vacuo. The crystalline residue was recrystallized from ethyl
acetate and hexane to give 3 (11.6 g, 96.5%). ESI-MS
(positive) m/z 396.1 [(M+Na)+].
N,N’-Bis[2-(6-aminopyridyl)]-5-(4-nitrophenyl-
methyloxy)isophthaldiamide. To a solution of 2,6-
diaminopyridine (8.00 g, 73.3 mmol) in anhydrous THF (150
ml) was added 25.0 ml of n-butyl lithium in hexane (2.52
mol•l-1) at 78 °C under N2. After the mixture was stirred for
20 min, a solution of 3 (6.00 g, 16.1 mmol) in anhydrous THF
(55 ml) was added dropwise. The mixture was stirred at
78 °C to 60 °C for 6 h and water (30 ml) was added to
quench the reaction. After addition of EtOAc (200 ml), the
organic layer was washed with water and brine, dried over
Na2SO4. Parts of the desired product were crystallized from
extracts. Filtration afforded 6.40 g of the product. The filtrate
was concentrated in vacuo and the residue was purified by
silica-gel column chromatography (EtOAc/hexane = 1:1) to
give another 0.90 g of the product. Yield: 7.30 g (90.9%). ESI-
MS (positive) m/z 500.2 [(M+H)+].
4-Allyloxymethylbenzyl Chloride (7). To a solution of
chloromethylenedimethylammonium chloride (1.30 g, 10.2
mmol) in anhydrous CH3CN (10 ml) was added dropwise a
solution of 4-allyloxyphenylmethanol (1.30 g, 7.29 mmol) in
anhydrous CH3CN (10 ml) under N2 and the mixture was
refluxed for 40 min. After the mixture was cooled to room
temperature, ice-cold water (10 ml) was added. The mixture
was extracted with EtOAc and the organic layer was washed
with water, dried over K2CO3, and concentrated in vacuo to
give 7 (1.14 g, 98.6%).
Diethyl 2-(4-Allyloxymethylbenzyl)-2-ethylmalonate (8). To
a solution of diethyl ethylmalonate (2.30 g, 12.2 mmol) in
anhydrous toluene (40 ml) was added potassium t-butoxide
(1.41 g, 12.6 mmol) under N2. After the mixture was refluxed
for 30 min, the solution of 7 (1.40 g, 7.12 mmol) in toluene (20
ml) was added. The mixture was refluxed for 6.5 h and then
cooled to room temperature. After addition of water and
EtOAc, the organic layer was washed with 0.5 M HCl,
saturated NaHCO3 solution, and brine, dried over Na2SO4, and
N,N’-Bis[2-(6-butyrylaminopyridyl)]-5-(4-
nitrophenylmethyloxy)isophthaldiamide (4). To a mixture of
N,N’-bis[2-(6-aminopyridyl)]-5-(4-nitrophenyl-methloxy)-
isophthaldiamide (3.32 g, 3.35 mmol) and N,N-
diisopropylethylamine (2.5 ml, 14.4 mmol) in THF (80 ml)
was added butanoyl chloride (2.0 ml, 19.3 mmol). The
mixture was stirred at room temperature for 4 h under N2.
After addition of water (50 ml) and EtOAc (200 ml), the
organic layer was washed with 0.5 M NaOH (50 ml 1) and
brine (100 ml 2), dried over Na2SO4, and concentrated. The
residue was purified by silica-gel column chromatography
(CHCl3/acetone = 10:1) to give 4 (3.8 g, 91.8%). ESI-MS
(positive) m/z 662.3 [(M+Na)]+. 1H NMR (600 MHz, DMSO),
= 10.50 (2H, s, 2 CONH), 10.09 (2H, s, 2 CONH), 8.30
(2H, d, J = 8.5 Hz, PhNO2), 8.16 (1H, s, Ph), 7.83 7.75 (10H,
Synlett 2001, No. 5, 590–596 ISSN 0936-5214 © Thieme Stuttgart · New York

LETTER
concentrated in vacuo. The residue was purified by silica-gel
Synthesis Based on Affinity Separation (SAS)
595
was added piperidine (20 l, 0.20 mmol). The mixture was
stirred at room temperature for 2 h, diluted with CH2Cl2 (8
ml), and then applied to the affinity separation. The desired 5-
ethyl-5-[4-(4-piperidino-3-nitrobenzoyl)oxymethyl-
benzyl]barbituric acid (25) was eluted at both CH2Cl2 and
CH2Cl2-MeOH (1:1) fractions. The CH2Cl2 fraction
column chromatography (hexane/EtOAc = 20:1 to 10:1) to
afford 8 (3.90 g, 97.7%). ESI-MS (positive) m/z 349.35
[(M+H)+, 18%]; 366.35 [(M+H2O)+, 40%]; 371.30 [(M+Na)+,
100%). 1H NMR (500 MHz, CDCl3), = 7.22 (2H, d, J = 7.7
Hz, Ar-H), 7.07 (2H, d, J = 8.0 Hz, Ar-H), 5.94 (1H, m,
CH2=CH-CH2-), 5.29 (1H, m, CH2=CH-CH2-), 5.19 (1H, m,
CH2=CH-CH2-), 4.47 (2H, s, -OCH2PhCH2-), 4.18 (4H, m, 2
-OCH2CH3), 4.01 (2H, m, CH2=CH-CH2-), 3.23 (2H, s, -
OCH2PhCH2-), 1.82 (2H, q, J = 7.6 Hz, -CH2CH3), 1.24 (6H,
t, J = 7.1 Hz, 2 -OCH2CH3), 0.92 (3H, t, J = 7.6 Hz, -
CH2CH3).
5-Ethyl-5-(4-allyloxymethylbenzyl)barbituric Acid (9). To a
solution of urea (7.00 g, 117 mmol) in DMSO (25 ml) was
added NaH (60% oil suspension, 2.0 g, 50 mmol) under N2.
After the mixture was stirred at room temperature for 30 min,
8 (4.10 g, 11.8 mmol) was added. The mixture was stirred at
room temperature for 14 h and then poured into 20 g of ice.
The mixture was acidified with 2M HCl and then extracted
with EtOAc. The organic layer was washed with water, dried
over Na2SO4, and concentrated in vacuo. The residue was
purified by silica-gel column chromatography (hexane/
EtOAc = 3:1 to 2:1) to give 9 (3.00 g, 80.6%). ESI-MS
(positive) m/z 339.15 [(M+Na)+]. 1H NMR (600 MHz,
CDCl3), = 7.86 (2H, s, NH), 7.22 (2H, d, J = 7.7 Hz, Ar-H),
7.08 (2H, d, J = 8 Hz, Ar-H), 5.93 (1H, m, CH2=CH-CH2-),
5.29 (1H, m, CH2=CH-CH2-), 5.20 (1H, m, CH2=CH-CH2-),
4.46 (2H, s, -OCH2PhCH2-), 4.00 (2H, m, CH2=CH-CH2-),
3.27(2H, s, -OCH2PhCH2-), 2.20 (2H, q, J = 7.6 Hz, -
CH2CH3), 0.92 (3H, t, J = 7.6 Hz, -CH2CH3).
containing 25 was concentrated and then applied to the
affinity separation again. The CH2Cl2-MeOH (1:1) fractions
were combined and concentrated. Compound 25 thus obtained
was dissolved in 0.2 M MeONa in MeOH (2.0 ml) and the
mixture was stirred at room temperature for 30 min. After
addition of AcOH (3 drops), the mixture was concentrated in
vacuo. The residue was dissolved in CHCl3 and then applied
to the affinity separation. The desired 26 was eluted with
CHCl3, whereas HOBA (10) was eluted with CH2Cl2/MeOH
(1:1). Evaporation of the solvent afforded 26 (24.0 mg,
90.9%). ESI-MS (positive) m/z 265.2 [(M+H)+]. 1H NMR
(600 MHz, CDCl3), = 8.42 (1H, s, Ar-H), 8.02 (1H, d,
J = 8.8 Hz, Ar-H), 7.05 (1H, d, J = 8.8 Hz, Ar-H), 3.90 (3H, s,
CH3), 3.15 (4H, t, NCH2), 1.72 (4H, m, CH2), 1.51 (2H, m,
CH2).
(11) Liu, W.-C.; Oikawa, M.; Fukase, K.; Suda, Y.; Kusumoto, S.
Bull. Chem. Soc., Jpn. 1999, 72, 1377.
(12) The experimental procedure for the preparation of
trisaccharide 34. The mixture of the glycosyl acceptor 30
(69.2 mg, 0.09 mmol), the glycosyl donor 31 (85 mg, 0.135
mmol), and MS 4A in CH2Cl2 (4 ml) was stirred at room
temperature for 1 h under N2 atmosphere and then cooled to
20 °C. TMSOTf (4.0 l, 22 mol) was added and the mixture
was stirred at the same temperature for 10 min. The mixture
was directly applied to the affinity separation to give
disaccharide 32. Disaccharide 32 was dissolved in CH2Cl2 (4
ml) containing 5% TFA and 1% water. The mixture was
stirred at 0 °C for 30 min, diluted with EtOAc (15 ml), washed
with saturated NaHCO3 solution and brine, dried over Na2SO4
and concentrated. The residue was applied to the affinity
separation to afford disaccharide acceptor 33. The mixture of
33 (59.2 mg, 0.0517 mmol), 31 (161 mg, 0.256 mmol), and
MS 4A in CH2Cl2 (5 ml) was stirred at room temperature for
1 h under N2, and then cooled to 20 C. TMSOTf (4.0 l, 22
mol) was added and the mixture was stirred at the same
temperature for 10 min. The mixture was directly applied to
the affinity separation to give 80.1 mg of the crude product,
which was then purified by silica-gel column chromatography
to afford the pure trisaccharide 34 (47.0 mg, 56.0%). ESI-MS
m/z 1630.0 [(M+Na)+]. 1H NMR (500 MHz, CDCl3), = 8.45
(2H, NH: barbituric acid), 7.45 (2H, m, Ph-H), 7.34 (3H, m,
Ph-H), 7.20 (2H, d, J = 7.6 Hz, Ph-H), 7.09 (2H, d, J = 8.1 Hz,
Ph-H), 6.28 (1H, br s, 2-NH: GlcNC), 5.92 (1H, m, CH2=CH-
CH2-), 5.51 (1H, s, PhCH=), 5.43 (1H, d, J = 9.9 Hz, 2-NH:
GlcNA), 5.37 (1H, br s, 2-NH: GlcNB), 5.33 (1H, m, CH2=CH-
CH2-), 5.24 (1H, m, CH2=CH-CH2-), 5.18 (1H, t, J = 9.7 Hz,
H-3: GlcNA), 5.13 (1H, t, J = 9.9 Hz, H-3: GlcNC), 5.05 (1H,
H-3: GlcNB), 5.02 (1H, d, J = 14 Hz, -OCH2PhCH2-), 4.97
(1H, d, J = 14 Hz, -OCH2PhCH2-). 4.90 (d, J = 3.7 Hz, 1H, H-
1: GlcNA), 4.79-4.65 (6H, CCl3CH2- 3), 4.65 (1H, d, J = 8.3
Hz, H-1: GlcNC), 4.63 (1H, d, J = 8.6 Hz, H-1: GlcNB), 4.37
(1H, dd, J = 5.6 Hz, 10.6 Hz, H-6: GlcNC), 4.22 (1H, m,
CH2=CH-CH2-), 4.21 (1H, H-6: GlcNB), 4.17 (1H, H-6:
GlcNA), 4.02 (1H, m, CH2=CH-CH2-), 3.96 (1H, H-5: GlcNA),
3.95 (1H, H-2: GlcNA), 3.84 (1H, H-6’: GlcNA), 3.82 (1H, H-
6’: GlcNC), 3.80 (1H, H-2: GlcNC), 3.79 (1H, H-6’: GlcNB),
3.71 (1H, H-4: GlcNC), 3.70 (1H, H-4: GlcNA), 3.62 (1H, H-
2: GlcNB), 3,53 (1H, H-5: GlcNB), 3.52 (1H, H-5: GlcNc), 3.51
(1H, H-4: GlcNB), 3.26 (2H, m, -OCH2PhCH2-), 2.35-2.22
(4H, m, -COCH2CH2CH2CO-), 2.18 (2H, q, J = 6.2 Hz,
CH3CH2-), 2.10 (3H, s, CH3CO-), 1.90 (2H, m, -
5-Ethyl-5-(4-hydroxymethylbenzyl)barbituric Acid (HOBA)
(10). To a degassed solution of 9 (2. 89 g, 9,13 mmol) in
anhydrous THF (100 ml) was added
[Ir(COD)(PMePPh2)]+PF6- (350 mg, 0.045 equiv) and the
solution was stirred under H2 at room temperature for 5 min
(the color of the solution changed from red to yellow) and then
for 100 min under N2. Water (45 ml) and iodine (4.4 g) was
added to the mixture successively. After the mixture was
stirred for 30 min, aqueous 10% Na2S2O3 solution (10 ml) was
added to quench the reaction. The mixture was extracted with
EtOAc and the organic layer was washed with brine, dried
over Na2SO4, and concentrated in vacuo. The residue was
purified by silica-gel column chromatography (CHCl3/
MeOH = 20:1) to give 10 (2.20 g, 87.3%). ESI-MS (positive)
m/z 575.3 [(2M+Na)+]; 315.1 [(M+K)+]; 299.2 [(M+Na)+]. 1H
NMR (600MHz, DMSO), = 7.18 (2H, d, J = 8 Hz, Ar-H),
6.94 (2H, d, J = 8 Hz, Ar-H), 5.15 (t, 1H, OH), 4.24 (2H, m,
-OCH2PhCH2-), 3.07 (2H, s, -OCH2PhCH2-), 1.97 (2H, q,
J = 7.6 Hz, -CH2CH3), 0.76 (3H, t, J = 7.6 Hz, -CH2CH3).
(8) General procedure for affinity separation. After completion of
the reaction, the reaction mixture was directly applied to the
resin 1 column (7.0 g: 1.5 cm 7 cm, CH2Cl2) unless
otherwise noted. After untagged compounds were washed off
with CH2Cl2 (70 ml), the tagged compound was eluted with
MeOH-CH2Cl2 (1:1) (30 ml). Evaporation of the solvents
afforded the desired product having the tag.
(9) Dankwardt, S. M.; Newman, S. R.; Krstenansky, J. L.
Tetrahedron Lett. 1995, 36, 4923.
(10) The experimental procedure for the preparation of methyl 4-
piperidino-3-nitrobenzoate (26). To a solution of HOBA (10)
(27.6 mg, 0.100 mmol), 4-fluruo-3-nitrobenzoic acid (22.0
mg, 0.119 mmol), and DMAP (1mg, 8 mol) in anhydrous
CH2Cl2 (3 ml) was added DIC (30 l, 0.19 mmol). The
mixture was stirred at room temperature for 2 h and then
applied to the affinity separation to give 5-ethyl-5-[4-(4-
fluruo-3-nitrobenzoyl)oxymethylbenzyl]barbituric acid (24).
To a solution of 24 in N-methylpyrrolidone (NMP) (0.8 ml)
Synlett 2001, No. 5, 590–596 ISSN 0936-5214 © Thieme Stuttgart · New York

596
S.-Q. Zhang et al.
LETTER
was washed with aqueous saturated NaHCO3 solution and
COCH2CH2CH2CO-), (3H, t, J = 6.2 Hz, CH3CH2-) (the
saccharide residues are designated as GlcNA, GlcNB, and
GlcNC (glucosamine residue) from reducing end).
brine, dried over Na2SO4, and concentrated. The residue was
then subjected to the affinity separation to give 39. The
mixture of 39, phenyl thioglucoside 37 (105.4 mg, 0.147
mmol), PhIO (48 mg, 0.22 mmol), and MS 4A in diethyl ether
(4.0 ml) and dioxane (2.0 ml) was stirred at r.t. for 1 h under
N2 and then cooled to 10 C. TMSOTf (20 l, 0.110 mmol)
was added and the reaction mixture was stirred for 1 h. The
reaction was quenched by addition of aqueous saturated
NaHCO3 solution and ethyl acetate. The organic layer was
washed with brine, dried over Na2SO4, and concentrated. The
residue was dissolved in CH2Cl2 and then subjected to the
affinity separation to give 119.2 mg of the crude product. The
product was purified by silica-gel column chromatography to
afford compound 40 (62.0 mg, 28.7%). ESI-MS m/z 1912.6
[(M+Na)+]; 1H NMR (600 MHz, CDCl3), (1-6) (1-6) :
= 8.12 (2H, NH: barbituric acid), 4.98 (1H, d, J = 3.3 Hz, H-
1: GlcC), 4.90 (1H, H-1: GlcB), 4.54 (1H, H-1: GlcA), 4.30 (2H,
H-6: GlcC), 3.96 (1H, dd, J = 9.3Hz,9.3 Hz, H-3: GlcC), 3.95
(1H, H-3: GlcA), 3.94 (1H, H-3: GlcB), 3.81 (m, 1H, H-5:
GlcC), 3.77 (1H, m, H-6, GlcA), 3.76 (1H, m, H-6: GlcB), 3.76
(1H, m, H-5: GlcB), 3.72 (1H, H-5: GlcA), 3.70 (1H, H-4:
GlcB), 3.66 (1H, H-4, GlcA), 3.48 (1H, H-2: GlcC), 3.48 (1H,
H-4: GlcC), 3.38 (1H, dd, J = 3.0 Hz, 9.3 Hz, H-2: GlcA), 3.37
(1H, dd, J = 3.0 Hz, 9.3 Hz, H-2: GlcB), 3.32 (3H, s, -OCH3),
3.24 (2H, s, -OCH2PhCH2-), 2.48-2.30 (4H,
(13) -Promoting solvent effect of dioxane was reported. a)
Demchenko, A. V.; Rousson, E.; Boons, G.-J. Tetrahedron
Lett. 1999, 40, 6523. b) Rademann, J.; Schmidt, R. R. J. Org.
Chem. 1997, 62, 3650.
(14) a) Fukase, K.; Kinoshita, I.; Kanoh, T.; Nakai, Y.; Hasuoka,
A.; Kusumoto, S. Tetrahedron 1996, 52, 3897. b) Fukase, K.;
Nakai, Y.; Kanoh, T.; Kusumoto, S. Synlett 1998, 84.
(15) -Selectivity is expected to be increased by using
combinations of PhIO with SnCl2-AgClO4, SnCl4-AgClO4,
BiCl3-AgClO4, and SbCl3-AgClO4, though these reagents
require careful handling.14
(16) An acylaminobenzyl linker, which can be cleaved by DDQ
oxidation, was used for solid-phase oligosaccharide synthesis.
Fukase, K.; Nakai, Y.; Egusa, K.; Porco Jr., J. A.; Kusumoto
S. Synlett 1999, 1074.
(17) We previously reported -orienting effect of the 6-O-Troc
group.14 Fukase, K.; Yoshimura, T.; Kotani, S.; Kusumoto S.
Bull. Chem. Soc. Jpn. 1994, 67, 473.
(18) The experimental procedure for the preparation of
trisaccharide 40. The mixture of the glycosyl acceptor 36
(97.4 mg, 0.114 mmol), phenyl thioglucoside 37 (122.8 mg,
0.171 mmol), PhIO (48 mg, 0.22 mmol), and MS 4A in diethyl
ether (5.0 ml) and dioxane (4.0 ml) was stirred for 1 h at r.t.
under N2 then cooled to 10 °C. TMSOTf (20 l, 0.110
mmol) was added and the reaction mixture was stirred for 1 h.
The reaction was quenched by addition of aqueous saturated
NaHCO3 solution and ethyl acetate. The organic layer was
washed with brine, dried over Na2SO4, and concentrated. The
residue was dissolved in CH2Cl2 and then subjected to the
affinity separation. Since the product 38 contained small
amount of the starting material 36, the mixture of 38 and 36
was subjected repeatedly to glycosylation and then affinity
separation to give 38 ( : = 10:1). ESI-MS m/z 1480.4
[(M+Na)+]; 1H NMR (600 MHz, CDCl3), -anomer: = 8.07
(2H, NH: barbituric acid), 4.95 (1H, d, J = 3.6 Hz, H-1: GlcB),
4.53 (1H, d, J = 3.6 Hz, H-1: GlcA), 4.32 (2H, d, J = 3.3 Hz,
H-6: GlcB), 3.972 (1H, dd, J = 9.3 Hz, 9.3 Hz, H-3: GlcA),
3.966 (1H, dd, J = 9.1 Hz, 9.1 Hz, H-3: GlcB), 3.86 (m, 1H, H-
5: GlcB), 3.79 (1H, m, H-6, GlcA), 3.77 (1H, m, H-5: GlcA),
3.71 (1H, H-6’: GlcA), 3.64 (1H, dd, J = 9.1 Hz, 9.3 Hz, H-4:
GlcA), 3.50 (1H, H-2: GlcB), 3.50 (1H, H-4: GlcB), 3.40 (1H,
dd, J = 3.6 Hz, 9.6 Hz, H-2: GlcA), 3.34 (3H, s, -OCH3),
3.257 (2H, s, -OCH2PhCH2-), 2.48-2.30 (4H, m,
-COCH2CH2CH2CO-), 2.19 (2H, CH3CH2-), 2.01 (2H, m,
-COCH2CH2CH2CO-), 0.91 (3H, t, J = 9.1 Hz, CH3CH2-).
(19) The trisaccharide 41 did not exhibit sufficiently strong affinity
to the receptor and leaked from the resin column when CH2Cl2
was used as an eluent. Large hydrophobic moiety in 41
decreases the relative affinity of the barbituric acid tag with
the receptor. When less polar toluene, in which the affinity is
expected to increase, was used as an eluent, 41 was effectively
retained in the column 1, which was then washed successively
with toluene and CH2Cl2. Compound 41 was not eluted during
the washing. Elution with CH2Cl2-methanol (1:1) afforded 41.
BnO
O
O
O
OBn
O
OBA
BnO
BnO
O
OBn
O
O
O
NHTroc
OBn
O
BnO
-COCH2CH2CH2CO-), 2.19 (2H, CH3CH2-), 2.0 (2H, m,
-COCH2CH2CH2CO-), 0.92 (3H, CH3CH2-). -anomer:
= 4.60 (1H, H-1: GlcA), 4.35 (1H, d, J = 8.4 Hz, H-1: GlcB),
3.44 (1H, H-2: GlcA), 3.31 (3H, S, -OCH3). To a solution of 38
in acetic acid (1 ml) was added Zn-Cu (260 mg) and the
mixture was stirred at r.t. for 1 h. Ethyl acetate was added and
the insoluble materials were removed by filtration. The filtrate
41
OBn
Article Identifier:
1437-2096,E;2001,0,05,0590,0596,ftx,en;Y06601ST.pdf
Synlett 2001, No. 5, 590–596 ISSN 0936-5214 © Thieme Stuttgart · New York