A convenient approach for solution-phase synthesis of water-soluble galactoside libraries

Article abstract of DOI:10.1016/S0040-4039(01)01180-7

A convenient approach for the solution-phase synthesis of water-soluble galactoside libraries through tributylphosphine promoted reaction of azido compounds with carboxylic acids is described.

Full text of DOI:10.1016/S0040-4039(01)01180-7

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 42 (2001) 6073–6076
A convenient approach for solution-phase synthesis of
water-soluble galactoside libraries
Feng Hong and Erkang Fan*
Department of Biological Structure and Biomolecular Structure Center, University of Washington, Box 357742, Seattle,
WA 98195, USA
Received 11 February 2000; revised 22 June 2001; accepted 27 June 2001
Abstract—A convenient approach for the solution-phase synthesis of water-soluble galactoside libraries through tributylphosphine
promoted reaction of azido compounds with carboxylic acids is described. © 2001 Elsevier Science Ltd. All rights reserved.
In the past decade, combinatorial chemistry has
emerged as powerful tool for drug discovery
Among many amide bond formation reactions,7 we
found that trialkylphosphine promoted reaction of
azido compounds with carboxylic acids8 involves only
organic soluble or volatile reagents and by-products.
No intermediate purification step is needed. The water-
soluble products are generated only in the final depro-
tection step with facile purification.
a
research.1 Its ability to generate large numbers of struc-
turally diverse compounds (a library) within a limited
period of time greatly enhances the efficiency of lead-
compound exploration. While early combinatorial
chemistry has primarily focused on solid-phase synthe-
sis, parallel library synthesis performed in solution
phase has become increasingly popular. Facing every
solution phase library synthesis is the need of efficient
purification methods. Consequently, a handful of inno-
vative purification approaches have been unveiled.
These include resin capture,2 acid/base wash,3 C18-sil-
ica extraction,4 and most notably, the fluorous phase
synthesis.5 We herein report a convenient approach for
the solution-phase parallel synthesis of water-soluble
galactoside derivatives. Such an approach was designed
by carefully choosing conditions so that all reagents
and by-products are either volatile or soluble in organic
solvent. Hence, the water-soluble products could be
obtained conveniently after removal of all by-products
and excess reagents by partitioning in water/organic
solvent and evaporation.
The analysis of reaction process is demonstrated in
Scheme 2. The first amide bond formation step was
carried out by using equal amounts of a carboxylic acid
and tributylphosphine in CH2Cl2, both in slight excess
to the azide (1) to ensure complete consumption of the
azide. Along with the desired intermediate (8), the
reaction also formed by-products: galactosylphos-
phazene (9), tributylphosphine oxide, as well as the
unreacted carboxylic acid and tributylphosphine. After
the removal of CH2Cl2, the mixture was directly sub-
jected to deprotection using NaOMe/HOMe, followed
by neutralization with Dowex-H+ resin. Besides the
water-soluble product (i.e. library compound 5), methyl
acetate and 1-amino-1-deoxy-galactose (10) were gener-
ated in this step. During the Dowex-H+ resin treatment,
10 and Na+ were bound to the resin and filtered off.
Subsequently, methanol and the by-product methyl ace-
tate were evaporated. At this stage, all the remaining
contaminants (tributylphosphine oxide, tributylphos-
phine and the acid) are soluble in organic solvent and
were removed by partitioning in CH2Cl2/H2O. The
water-soluble product was obtained after lyophilization.
We demonstrate this approach by using an amide bond
formation reaction, which generates a library of galac-
toside derivatives (5, 6, 7 in Scheme 1). Libraries of this
type can be used to explore the inhibition of the
receptor-binding process of bacterial toxins produced
by enterotoxigenic E. coli and by V. cholerae.6
Initially, the complete library synthesis (using 1, 2, 3
with 24 acids: 72 members) was performed at room
temperature without pre-drying the starting materials.
A typical procedure is described with compound 1.9
Keywords: amides; azides; carboxylic acids; derivatives; glycosides.
* Corresponding author.
0040-4039/01/$ - see front matter © 2001 Elsevier Science Ltd. All rights reserved.
PII: S0040-4039(01)01180-7

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F. Hong, E. Fan / Tetrahedron Letters 42 (2001) 6073–6076
OH
OAc
O
HO
AcO
AcO
O
i, ii, iii
O
HO
X
X
+
RCO2H
N
H
R
N3
OH
OAc
1, 2, 3
5, 6, 7
4
i). n-Bu3P, CH2Cl2, rt; ii). NaOMe/HOMe, rt; iii). Dowex-H+, HOMe, rt
1, 5: X = none, β
2, 6: X = OCH2CH2, α
3, 7: X = OCH2CH2, β
OMe
(CH2)2
MeO
(CH2)2
(CH2)2
R =
(CH2)2
a
c
b
d
O
Ph
S
O
N
N
(CH2)2
MeO
(CH2)2 CF3O
(CH2)2
(CH2)2
N
H
O
g
e
f
h
MeO
MeO
MeO
N
(CH2)2
(CH2)3
(CH2)2
(CH2)3
j
i
k
l
O2N
(CH2)3
MeO
MeO
(CH2)3
MeO
(CH2)3
m
n
o
O
(CH2)3
HN
(CH2)3
HN
N
(CH2)3
(CH2)4
Ph
Ph
O
p
q
r
s
O
(CH2)4
(CH2)5
(CH2)4
N
(CH2)5
(CH2)6
S
S
u
v
O
w
x
t
Scheme 1.
were made to improve the overall yields by varying
reaction conditions, including temperatures, solvents,
and reagents such as using triethylphosphine as pro-
moter. However, no dramatic improvement in yield was
observed. We noticed that pre-drying of starting mate-
rials by co-evaporation with toluene helped to improve
the isolated yields moderately to ꢀ15–55% in a later
round of library synthesis using azides 1–3 and acids
4k, 4n and 4o. These yields are in good agreement with
the general yields obtained for reactions carried out at
room temperature.8 Inazu et al. performed a thorough
investigation of reaction conditions and mechanisms for
this type of amide bond transformation.8b It was sug-
gested that carrying out the reaction at very low tem-
Overall yields ranged from 8.2 to 44% with high purity
of the final products for screening.6b All reactions were
monitored by TLC. The purities of the final compounds
were determined by HPLC and TLC. Most final prod-
ucts had purities between 70 and 95% based on HPLC
monitored at 220 nm, while TLC indicated purities of
>95% (single spot with ammonium molybdate staining).
Unreacted carboxylic acids were the major contami-
nants as identified using HPLC analysis by co-injection
with starting acids. All products gave the correct mass
spectra as determined by MALDI or ESI mass spec-
troscopy.10 In the initial synthesis, the overall yields
were quite disappointing, especially for library com-
pounds starting with the less reactive azide 1. Efforts

F. Hong, E. Fan / Tetrahedron Letters 42 (2001) 6073–6076
6075
OAc
O
OH
AcO
AcO
HO
HO
H
N
H
N
ii, iii
O
AcOMe
+
R
R
OAc
OH
O
O
OAc
O
AcO
AcO
5
8
i
N3
OAc
OH
HO
HO
OAc
O
AcO
AcO
AcOMe
PBu3
ii, iii
1
O
NH2
+
N
PBu3
O
OH
OAc
10
9
Scheme 2. (i) RCO2H, n-Bu3P, CH2Cl2, rt; (ii) NaOMe/HOMe, rt; (iii) Dowex-H+, HOMe, rt.
perature (−78°C) can facilitate the formation of desired
amide products, while reaction at higher temperature
(such as room temperature) can lead to the non-pro-
ductive glycosylphosphazene derivatives (9 in Scheme
2). From a library synthesis point of view, we did not
attempt to run the library synthesis at −78°C in the
absence of specialized equipment. In our library synthe-
sis at room temperature, we did observe the formation
of significant amounts of galactosylphosphazene deriva-
tives. Despite the presence of large amounts of phos-
phazene by-products, our purification procedure can
efficiently remove these impurities. At a small expense
of the yield, the use of excess Dowex-H+ resin ensures
the breakdown of phosphazenes and the subsequent
trapping of generated amines.11 For efficient removal of
the remaining neutral organic by-products, we also
found that in addition to CH2Cl2, ethyl acetate can also
be used to perform extraction from water.
97, 347–509; For a comprehensive list of literatures
online, see: (c) Lebl, M.; Leblova, Z. Dynamic Database
of References in Molecular Diversity. Internet http://
www.5z.com.
2. (a) Kaldor, S. W.; Siegel, M. G.; Fritz, J. E.; Dressman,
B. A.; Hahn, P. J. Tetrahedron Lett. 1996, 37, 7193; (b)
Flynn, D. L.; Crich, J. Z.; Devraj, R. J.; Hockerman, S.
L.; Parlow, J. J.; South, M. S.; Woodard, S. J. Am.
Chem. Soc. 1997, 119, 4878; (c) Brown, S. D.; Armstrong,
R. W. J. Am. Chem. Soc. 1996, 118, 6331; (d) Booth, R.
J.; Hodges, J. C. J. Am. Chem. Soc. 1997, 119, 4882.
3. (a) Boger, D. L.; Tarby, C. M.; Myers, P. L.; Caporale,
L. H. J. Am. Chem. Soc. 1996, 118, 2109; (b) Cheng, S.;
Comer, D. D.; Williams, J. P.; Myers, P. L.; Boger, D. L.
J. Am. Chem. Soc. 1996, 118, 2567.
4. Nilsson, U. J.; Fournier, E. J.-L.; Hindsgaul, O. Bioorg.
Med. Chem. Lett. 1998, 6, 1563.
5. (a) Studer, A.; Hadida, S.; Ferritto, R.; Kim, S.-Y.; Jeger,
P.; Wipf, P.; Curran, D. P. Science 1997, 275, 823; (b)
Curran, D. P.; Hadida, S.; He, M. J. Org. Chem. 1997,
62, 6714; (c) Studer, A.; Curran, D. P. Tetrahedron 1997,
53, 6681; (d) Luo, Z.; Zhang, Q.; Oderaotoshi, Y.; Cur-
ran, D. P. Science 2001, 291, 1766.
6. (a) For a review on related AB5 toxins, see: Merritt, E.
A.; Hol, W. G. J. Curr. Opin. Struct. Biol. 1995, 5,
165–171; (b) For the screening of the chemical libraries
reported here, see: Minke, W.; Hong, F.; Verlinde, C.;
Hol, W.; Fan, E. J. Biol. Chem. 1999, 274, 33469.
7. Bodanszky, M.; Bodanszky, A. The Practice of Peptides
Synthesis, 2nd ed.; Springer-Verlag: Berlin, 1994; pp.
75–126.
In summary, we have described a convenient approach
for the solution-phase synthesis of water-soluble galac-
toside libraries. By carefully choosing a reaction,
involving either organic-soluble or volatile reagents and
by-products, we have efficiently obtained water-soluble
pure galactoside derivatives through simple organic sol-
vent extraction, evaporation and lyophilization despite
of the low yielding nature of the reaction. The conve-
nience and efficiency might make such an approach a
useful alternative to the solution-phase library synthesis
of various water-soluble organic compounds.
8. (a) Inazu, T.; Kobayashi, K. Synlett 1993, 869; (b)
Mizuno, M.; Muramoto, I.; Kobayashi, K.; Yaginuma,
H.; Inazu, T. Synthesis 1999, 162; (c) Deras, I. L.;
Takegawa, K.; Kondo, A.; Kato, I.; Lee, Y. C. Bioorg.
Med. Chem. Lett. 1998, 8, 1763.
9. Typical procedure: To each acid (0.074 mmol) in a 1
dram glass vial was added 1 (25 mg, 0.067 mmol) in 1.5
ml of anhydrous CH2Cl2. After shaking for 5 min, n-
Bu3P (0.074 mmol) in 0.5 ml of CH2Cl2 was introduced
via a syringe through the cap. The resulting mixture was
shaken overnight at room temperature. All vials were
then placed in a desiccator that was connected to vacuum
to remove the solvent. To each residue in the same vial
was added 1.5 ml of methanol, followed by NaOMe/
Acknowledgements
We are grateful for support provided by the School of
Medicine of the University of Washington and the
National Institutes of Health (Grant AI44954). We also
thank Professor Wim Hol for helpful discussions.
References
1. For recent reviews, see the dedicated issues of: (a) Curr.
Opin. Chem. Biol. 1997, 1, 3–135; (b) Chem. Rev. 1997,

6076
F. Hong, E. Fan / Tetrahedron Letters 42 (2001) 6073–6076
HOMe (6.7 mmol). After 1 h, reaction mixture was treated
0.1% aqueous TFA and reaching 60% CH3CN in 0.1%
aqueous TFA in 20 min. For the nine compounds in the
second round of library synthesis, the HPLC gradient was
from 0% CH3CN in 0.1% aqueous TFA to 50% CH3CN
in 0.1% aqueous TFA in 30 min. HPLC peaks were
monitored by UV detector at 220 nm and at another
wavelength where the starting acids have significant
absorbance.
with Dowex-H+ resin (400 mg). The resin was filtered off
and the filtrate was collected in a 20 ml vial. Methanol was
evaporated the same way as for CH2Cl2. The residue was
partitioned in 10 ml of H2O and 5 ml of CH2Cl2 with
stirring for 5 min. The CH2Cl2 layer was removed, and the
extraction was repeated once. Water solution was subjected
to lyophilization to give the final product.
10. After library synthesis, all compounds were checked by
mass spectroscopy using either MALDI or electrospray
ionization. All library samples gave the corresponding
(M+H)+ and/or (M+Na)+ peaks. The purity of each sample
was determined by reverse phase HPLC using a C18
column with gradients starting with 10% (v/v) CH3CN in
11. Excessive amounts of Dowex resin can lower the isolated
yields of final products as the resin also traps product
non-specifically. For example, treatment of 30 mg of a
phosphazene-free product in 5 ml MeOH with 1 ml of
dry-volume Dowex resin can cause ꢀ30% loss of weight
after filtration and evaporation of solvent.
.