Deracemization of α-methylbenzylamine using an enzyme obtained by in vitro evolution

Article abstract of DOI:10.1002/1521-3773(20020902)41:17<3177::AID-ANIE3177>3.0.CO;2-P

Both the catalytic activity and enantioselectivity of the amine oxidase used in the deracemization, of D,L-α-methylbenzylamine (see scheme) have been enhanced by in vitro evolution methods to give α-methylbenzyamine in 77 % yield and 93 % ee.

Full text of DOI:10.1002/1521-3773(20020902)41:17<3177::AID-ANIE3177>3.0.CO;2-P

COMMUNICATIONS
[1] a) P. J. Stang, B. Olenyuk, Acc. Chem. Res. 1997, 30, 502 518;
b) Comprehensive Supramolecular Chemistry, Vols. 1 11 (Eds.: J.-M.
Lehn, J. L. Atwood, J. E. D. Davis, D. D. MacNicol, F. Vˆgtle)π
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istry: Concepts and Perspectives, VCH, Weinheim, 1995.
[8] W. L. Jˆrgensen, D. L. Severance, J. Am. Chem. Soc. 1990, 112, 4768
4774.
MeðSÞ
3þ
*
[9] Although the right-handed model structure of( P)-[Ag3L2
]
is
desirable for comparison with a left-handed X-ray crystal structure of
MeðSÞ
*
(M)-[Ag3L2
]
3þ, the right-handed structure was not available due
[2] a) O. D. Fox, M. G. B. Drew, P. D. Beer, Angew. Chem. 2000, 112, 139
144; Angew. Chem. Int. Ed. 2000, 39, 135 140; b) T. Kusukawa, M.
Fujita, Angew. Chem. 1998, 110, 3327 3329; Angew. Chem. Int. Ed.
1998, 37, 3142 3144; c) B. Olenyuk, J. A. Whiteford, A. Fechtenkot-
ter, P. J. Stang, Nature 1999, 398, 796 799; d) A. Ikeda, M. Yoshimura,
H. Udzu, C. Fukuhara, S. Shinkai, J. Am. Chem. Soc. 1999, 121, 4296
4297; e) P. Jacopozzi, E. Dalcanale, Angew. Chem. 1997, 109, 665 667;
Angew. Chem. Int. Ed. Engl. 1997, 36, 613 615; f) R. W. Saalfrank, H.
Glaser, B. Demleitner, F. Hampel, M. M. Chowdhry, V. Sch¸nemann,
A. X. Trautwein, G. B. M. Vaughan, R. Yeh, A. W. Davis, K. N.
Raymond, Chem. Eur. J. 2002, 8, 493 497.
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Cotton, L. M. Daniels, C. Lin, C. A. Murillo, Inorg. Chem. Commun.
2001, 4, 130 133; c) G. A. van Albada, I. Mutikainen, U. Turpeinen, J.
Reedijk, Eur. J. Inorg. Chem. 1998, 547 549; d) G. Lowe, S. A. Ross,
M. Probert, A. Cowley, Chem. Commun. 2001, 1288 1289; e) F. J.
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1072 1079.
[4] Chiral capsules based on hydrogen bonds: a) J. M. Rivera, T. MartÌn, J.
Rebek, Jr.,Science 1998, 279, 1021 1023; b) L. J. Prins, J. Huskens, F.
de Jong, P. Timmerman, D. N. Reinhoudt, Nature 1999, 398, 498 502;
chiral capsules based on metal ligand interactions: c) S. Hiraoka, M.
Fujita, J. Am. Chem. Soc. 1999, 121, 10239 10240; d) A. J. Terpin, M.
Ziegler, D. W. Johnson, K. N. Raymond, Angew. Chem. 2001, 113,
161 164; Angew. Chem. Int. Ed. 2001, 40, 157 160; circular helicates
based on metal ligand interactions: e) C. Provent, S. Hewage, G.
Brand, G. Bernardinelli, L. J. Charbonniõre, A. F. Williams, Angew.
Chem. 1997, 109, 1346 1348; Angew. Chem. Int. Ed. Engl. 1997, 36,
1287 1289; f) C. Provent, E. Rivara-Minten, S. Hewage, G. Brunner,
A. F. Williams, Chem. Eur. J. 1999, 5, 3487 3494; g) O. Mamula, A.
von Zelewsky, G. Bernardinelli, Angew. Chem. 1998, 110, 302 305;
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Constable, D. Fenske, C. E. Housecroft, T. Kulke, Chem. Commun.
1999, 195 196.
to the perturbed rotation along the C3 helical axis in (M)-
MeðSÞ
MeðRÞ
3þ
3þ
*
*
[Ag3L2
]
.
Instead,
a
model structure of( M)-[Ag3L2
]
MeðSÞ
*
energetically equivalent to (P)-[Ag3L2
]
3þ was obtained by simple
chirality inversion in the ligands from the solid structure of (M)-
MeðSÞ
*
[Ag3L2
]
3þ and compared with the solid structure; MacroModel 7.0
with modified MM2 force field. F. Mohamadi, N. G. J. Richards, W. C.
Guida, R. Liskamp, M. Lipton, C. Caufield, G. Chang, T. Hendrickson,
W. C. Still, J. Comput. Chem. 1990, 11, 440.
[10] Crystal
structure
of(
M)-[Ag3L MeðSÞ(CH3CN)2(NO3)3]/(P)-
*
2
[Ag3L MeðRÞ(CH3CN)2(NO3)3]: C40H48Ag3N11O15, Mw ¼ 1246.50, color-
*
2
less crystal 0.45 î 0.38 î 0.20 mm3, monoclinic C2/c, a ¼ 24.408(3), b ¼
14.0353(17), c ¼ 14.1561(17) ä; a ¼ 90, b ¼ 96.760(2), g ¼ 908; V¼
4815.7(10) ä3, Z ¼ 4, 1calcd ¼ 1.719 MgmÀ3(including solvent), m(MoKa
,
l ¼ 0.71073 ä) ¼ 1.285 mmÀ1, 2qmax ¼ 56.568; 14328 measured reflec-
tions, ofwhich 5623 were unique. The structure was solved by direct
methods and refined by full-matrix least squares calculations with
SHELX-97. The final R1 ¼ 0.0468, wR2 ¼ 0.0963 (I > 2s(I)); R1 ¼
0.1016, wR2 ¼ 0.1154 (all data); measurements: Siemens SMART
CCD equipped with a graphite crystal incident-beam monochromator
Lp. CCDC-182211 contains the supplementary crystallographic data
for this paper. These data can be obtained free of charge via
www.ccdc.cam.ac.uk/conts/retrieving.html (or from the Cambridge
Crystallographic Data Centre, 12, Union Road, Cambridge CB21EZ,
UK; fax: (þ 44)1223-336-033; or deposit@ccdc.cam.ac.uk).
[11] m/z 1290.0
of[Ag
3L*Ph(S)L*Me(S)(NO3)2]þ
and
1308.0
of
[Ag3L*Ph(S)L*Me(S)(NO3)2(H2O)]þ.
Deracemization of a-Methylbenzylamine Using
an Enzyme Obtained by In Vitro Evolution**
[5] a) R. Kramer, J.-M. Lehn, A. Marquis-Rigault, Proc. Natl. Acad. Sci.
USA 1993, 90, 5394 5398; b) B. Hasenknopf, J.-M. Lehn, G. Baum, D.
Fenske, Proc. Natl. Acad. Sci. USA 1996, 93, 1397 1400; c) D.
Caulder, K. N. Raymond, Angew. Chem. 1997, 109, 1508 1510;
Angew. Chem. Int. Ed. Engl. 1997, 36, 1440 1442; d) M. Albrecht,
M. Schneider, H. Rˆttele, Angew. Chem. 1999, 111, 512 515; Angew.
Chem. Int. Ed. 1999, 38, 557 559; e) M. A. Masood, E. J. Enemark,
T. D. P. Stack, Angew. Chem. 1998, 110, 973 977; Angew. Chem. Int.
Ed. 1998, 37, 928 932; f) T. W. Kim, M. S. Lah, J.-I. Hong, Chem.
Commun. 2001, 743 744.
Marina Alexeeva, Alexis Enright,
Michael J. Dawson, Mahmoud Mahmoudian, and
Nicholas J. Turner*
Enantiomerically pure chiral amines are valuable synthetic
intermediates, particularly for the preparation of pharma-
ceutical compounds. Traditionally, chiral amines have been
obtained by resolution-based procedures, for example, by
[6] Ligands were prepared according to the previous report:
H.-J. Kim, Y.-H. Kim, J.-I. Hong, Tetrahedron Lett. 2001, 42, 5049
5052.
[1,2]
kinetic resolution ofa racemate using an enzyme
or
crystallization ofa diastereomer using a chiral acid to form a
salt.[3] Increasingly, there is a desire to develop asymmetric
approaches, or their equivalents, which can in principal
deliver the product in 100% yield and 100% ee. For example,
transaminases have been utilized for the conversion of
[7] [Ag3L MeðSÞ](NO3)3 in the solid state has to be described as being
*
2
composed ofa cationic of rm, [Ag 3L2 MeðSÞ(NO3)2(CH3CN)4]þ, and an
*
anionic form, [Ag3L2 MeðSÞ(NO3)4(CH3CN)2]À. Crystal structure of
*
MeðSÞ
(CH3CN)4(NO3)2]}{(M)-[Ag3L MeðSÞ(CH3CN)2(-
*
*
{(M)-[Ag3L2
2
(NO3)4]}: C42H51Ag3N12O15, Mw ¼ 1287.56, colorless crystal, 0.45 î
0.38 î 0.20 mm3, monoclinic C2, a ¼ 23.7463(13), b ¼ 14.1186(8), c ¼
15.3235(9) ä; a ¼ 90, b ¼ 97.503(1), g ¼ 908; V¼ 5093.4(5) ä3, Z ¼ 4,
[*] Prof. N. J. Turner, M. Alexeeva, A. Enright
Department ofChemistry
1calcd ¼ 1.679 MgmÀ3(including solvent), m(MoKa
,
l ¼ 0.71073 ä) ¼
1.219 mmÀ1, 2qmax ¼ 56.568; 15905 measured reflections, of which
11383 were unique. The structure was solved by direct methods and
refined by full-matrix least squares calculations with SHELX-97. The
final R1 ¼ 0.0245, wR2 ¼ 0.0557 (I > 2s(I)); R1 ¼ 0.0278, wR2 ¼ 0.0572
(all data); measurements: Siemens SMART CCD equipped with a
graphite crystal incident-beam monochromator Lp. CCDC-182210
contains the supplementary crystallographic data for this paper. These
data can be obtained free of charge via www.ccdc.cam.ac.uk/conts/
retrieving.html (or from the Cambridge Crystallographic Data Centre,
12, Union Road, Cambridge CB21EZ, UK; fax: (þ 44)1223-336-033;
or deposit@ccdc.cam.ac.uk).
Centre for Protein Technology, The University of Edinburgh
King©s Buildings, West Mains Road, Edinburgh EH9 3JJ (UK)
Fax : (þ 44)131-650-4717
E-mail: n.j.turner@ed.ac.uk
Dr. M. J. Dawson, Dr. M. Mahmoudian
GlaxoSmithKline R&D, Medicines Research Centre
Gunnels Wood Road, Stevenage, Hertfordshire, SG1 2NY, (UK)
[**] We are grateful to the BBSRC and GlaxoSmithKline for funding a
postdoctoral fellowship (M.A.) and CASE award (A.E.). We also
thank the Wellcome Trust for financial support.
Angew. Chem. Int. Ed. 2002, 41, No. 17
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3177

COMMUNICATIONS
ketones into chiral amines[4] and chiral ruthenium catalysts
have been used for the asymmetric reduction of imines.[5]
Alternative strategies are based upon the dynamic kinetic
R2 ¼ Me) as a model system for study in view of its importance
as a chiral amine.[6] Our initial studies revealed that the
A. niger MAO-N enzyme possessed very low, but measurable,
activity towards l-a-methylbenzylamine and even slower
oxidation ofthe d enantiomer (see below). We reasoned that
such activity represented a viable starting point for improve-
ment ofboth the catalytic activity and enantioselectivity using
in vitro evolution methods.[17]
resolution ofamines, ofr example, by using
Burkholderia
plantarii lipase[6] or Candida antarctica lipase[7] with appro-
priate in situ racemization protocols.
Recently we described a method for the deracemization of
a range of a-amino acids that gave high yields and optical
purities[8,9] (Scheme 1, R2 ¼ CO2H). This deracemization
method involves the stereoinversion ofone enantiomer to
the other by repeated cycles ofenzyme-catalyzed oxidation to
The MAO-N gene from A. niger was obtained from
Schilling and Lerch[15] and subcloned into the E. coli PET16b
vector. Sequencing of the gene revealed four differences in
the amino acid sequence compared to the published sequence
(A300V, L304V, K348M, and R450G). Two of these differ-
ences (K348M and R450G) had previously been noted by
Sablin et al.[16] However, the activity ofthe enzyme (see
below) was found to be comparable to published data and
hence we used this plasmid as the starting point for the in vitro
evolution experiments. The initial priority was the develop-
ment of an effective high-throughput screen for amine oxidase
activity and, in this respect, we focussed on a method that
would allow approximately 2000 3000 colonies per plate to be
visualized. Amine oxidases are typical ofall members ofthe
oxidase family in that they evolve hydrogen peroxide as a by-
product. Many ofthe reported assays for oxidase activity are
based upon capture ofthe peroxide by a peroxidase in the
presence ofa substrate that yields a highly colored product
upon oxidation. Use of3,3 ’-diaminobenzidine (DAB) as the
peroxidase substrate gave rise to a dark pink, insoluble
product that gave both very high definition and contrast of the
active colonies (Figure 1).
Scheme 1. Process for the deracemization of chiral amines by employing a
cyclic sequence ofenantioselective oxidation using an amine oxidase
coupled with a nonselective reducing agent.
the imine, followed by nonselective reduction back to the
amino acid.[10] We demonstrated that both l- and d-a-amino
acids can be prepared in this way by employing enantiose-
lective d-[8] and l-amino acid oxidases,[9] respectively. A key
factor that determines the efficiency of the deracemization
process is the choice ofreducing agent. In the early reports [11]
sodium borohydride was employed, although the instability of
this reagent at pH 7 precludes its use on a practical scale.
However, this problem can be overcome by switching to
alternative, stable reductants such as sodium cyanoborohy-
dride,[8] ammonium formate with Pd/C,[9] and also amine bor-
ane complexes.[9] Herein we report a significant new extension
ofthe deracemization strategy by applying the method for the
first time to the deracemization of chiral amines.[12]
The key issue at the outset was the identification of
enantioselective amine oxidases that would be suitable for the
oxidation-reduction cycle. Amine oxidases have been classi-
fied into two groups, namely, Type I (Cu/TOPA-dependent)
and Type II (flavin-dependent).[13] In the catalytic cycle ofthe
Type I enzyme the intermediate imine remains covalently
bound to the protein and hence these enzymes were deemed
unsuitable for our use. The Type II enzymes generate ™free∫
imines, and although they are well known from mammalian
sources,[14] their microbial counterparts are poorly document-
ed; indeed at the outset ofthis work there were no reports of
enantioselective transformations. Schilling and Lerch[15] re-
ported the cloning and expression ofa Type II monoamine
oxidase from Aspergillus niger (MAO-N), and Sablin et al.[16]
subsequently purified the enzyme to homogeneity and carried
out substrate-specificity and kinetic studies. The enzyme was
reported to have high activity towards simple aliphatic amines
(for example, amylamine, butylamine) but was also active,
albeit at a lower rate, towards benzylamine. We decided to
select a-methylbenzylamine (AMBA, Scheme 1, R1 ¼ Ph,
Figure 1. Colorometric plate based screening assay for amine oxidase
activity by capture ofthe hydrogen peroxide produced using 3,3 ’-
diaminobenzidine with peroxidase: a) colonies with d-a-methylbenzyl-
amine and b) identical clones with the l enantiomer.
The library ofvariants was generated by carrying out
multiple cycles ofmutagenesis using the E. coli XL1-Red
mutator strain[18] followed by transformation of the plasmid
library into E. coli BL21. The library was plated out directly
onto nitrocellulose filters on agar plates (ca. 3000 colonies per
plate) and, following partial lysis of the cells, each plate was
treated with a cocktail containing both the assay mixture (l-
or d-AMBA, DAB) and also 2% agarose. The plates were
incubated at 378C after which they were inspected for positive
clones. By using this colorometric assay we were able to
identify clones that possessed higher activity towards l-
AMBA than the d enantiomer (Figure 1).
3178
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The result ofscreening a subset (ca. 150000 clones) ofthe
library led to the identification of 35 clones with improved
activity towards l-AMBA compared to the wild-type enzyme.
Each ofthese clones was grown on a small scale and assayed
against both l- and d-AMBA as cell-free extracts, which
resulted in the best 27 clones being selected for further study.
Each ofthese 27 clones was assayed against amylamine, l-
AMBA, and d-AMBA (Figure 2).
(ca. 17:1). Thus, the outcome ofthe in vitro evolution experi-
ments had been to simultaneously improve both the enantio-
selectivity and catalytic activity ofthe enzyme. For compar-
ison, the activity towards the best substrate for the wild-type
enzyme, namely amylamine, and also benzylamine, is pre-
sented. Sequencing ofthe mutant revealed a single change in
the amino acid sequence (Asn336Ser).
The substantial improvement in activity and selectivity of
the mutant was confirmed by chiral HPLC (Chiralcel
CrownPak CR þ ) in which complete oxidation ofthe l e-
nantiomer was apparent after 24 h, whereas there was no
detectable conversion ofthe d enantiomer. The final objec-
tive became the application ofthe mutant enzyme to the
deracemization of dl-AMBA. A range ofreducing agents was
screened (sodium borohydride, catalytic transfer hydrogena-
tion, amine borane complexes) using 1 mm dl-AMBA as the
substrate in the presence ofthe mutant MAO-N. This screen
identified the ammonia borane complex as the optimal
reagent which gave a 77% yield of d-AMBA with an ee value
of93%. Greater optical purities ofthe product (up to
99% ee) could be achieved although at the expense ofyield.
We also carried out the stereoinversion of l- to d-AMBA
(18% yield, 99% ee) and showed that under identical
conditions there was no conversion of d- into l-AMBA.
In conclusion we have successfully achieved the ™directed
evolution∫ ofan enzyme to meet the specific requirements of
a novel biotransformation. The decision to select the A. niger
MAO-N gene for in vitro evolution was based upon the
observation that the wild-type MAO-N enzyme showed
inherent selectivity for l- over d-AMBA (ca. 17:1), although
the overall rate was very low. An interesting aspect ofthe
present work is the identification of a mutant with enhanced
enantioselectivity by using a single enantiomer substrate in
the screen (l-AMBA). There is currently much effort directed
towards the development oftruly enantioselective screens in
which racemates are used, thereby mimicking the real-life
situation found in a kinetic resolution in which the two
enantiomers compete for the enzyme.[19] It may be that ifone
is able to screen large enough libraries ofvariant genes
(ca. 106), by using inherently simpler screens, then enzymes
with improved enantioselectivity can be identified in the
manner described here.
Figure 2. Activity ofthe best 27 clones identiiefd rofm screening
ca. 150000 colonies. Blue line: l-AMBA as substrate, pink: d-AMBA,
orange: ratio ofactivity towards l- versus d-AMBA, black: amylamine.
The left-hand y axis corresponds to absorption data (recorded at 510 nm)
for l-AMBA, d-AMBA, and the ratio of l/d-AMBA. The absorbance was
recorded after incubation for 96 h. The right-hand y axis corresponds to
data for amylamine. Initial rates were measured.
Examination ofthe polyacrylamide gels showed that
several ofthese 27 clones appeared to be ™expression
mutants∫, as evidenced by increased levels ofprotein
expression. However two (identical) clones were clearly
superior in terms oftheir selectivity and activity towards l-
versus d-AMBA and appeared not to have increased levels of
expression of MAO-N. This clone was therefore selected for
more detailed examination.
Both the wild-type and mutant MAO-N were purified by a
modification of the literature procedure to give protein of
approximately 10% purity. Each enzyme was then assayed
against l-AMBA, d-AMBA, benzylamine, and amylamine as
the substrate (Table 1).
The data revealed that the activity ofthe mutant towards l-
AMBA (kcat ¼ 8.0 minÀ1) was 47-fold higher than the wild-
type enzyme (kcat ¼ 0.17 minÀ1). Moreover, the selectivity of
the mutant for the l enantiomer versus d-AMBA (ca. 100:1)
had also increased (5.8-fold) relative to the wild-type enzyme
Received: April 4, 2002 [Z19043]
[1] A. Schmid, J. S. Dordick, B. Hauer, A. Kiener, M. Wubbolts, B.
Witholt, Nature 2001, 409, 258; Enzyme Catalysis in Organic Synthesis,
2nd ed. (Eds.: K.-H. Drauz, H. Waldmann), Wiley-VCH, Weinheim,
2002.
[2] S. Takayama, S. T. Lee, S. C. Hung, C.-H. Wong, Chem. Commun.
1999, 127; D. T. Guranda, L. M. van Langen, F. van Rantwijk, R. A.
Sheldon, V. K. Svedas, Tetrahedron: Asymmetry 2001, 12, 1645; L. M.
Langen, N. H. P. Oosthoek, D. T. Guranda, F. van Rantwijk, V. K.
Svedas, R. A. Sheldon, Tetrahedron: Asymmetry 2000, 11, 4593; L. E.
Iglesias, V. M. Sanchez, F. Rebolledo, V. Gotor, Tetrahedron: Asym-
metry 1997, 8, 2675.
Table 1. Km and kcat values for wild-type and best mutant monoamine
oxidase enzymes using l-AMBA, d-AMBA, benzylamine, and amylamine
as substrates.
Substrate
Wild-type
Mutant
kcat [minÀ1
]
Km[a] [mm]
kcat [minÀ1
]
Km [mm]
[3] J. Balint, G. Egri, M. Czugler, J. Schindler, V. Kiss, Z. Juvancz, E.
Fogassy, Tetrahedron: Asymmetry 2001, 12, 1511.
[4] J. S. Shin, B. G. Kim, A. Liese, C. Wandrey, Biotechnol. Bioeng. 2001,
73, 179; G. Matcham, M. Bhatia, W. Lang, C. Lewis, R. Nelson, A.
Wang, W. Wu, Chimia 1999, 53, 584; J. S. Shin, B. G. Kim, Biotechnol.
Bioeng. 1999, 65, 206.
l-AMBA
d-AMBA
benzylamine
amylamine
ND
ND
ND
ND
0.17
0.01
371
0.4
8.0
ND[a]
ND[a]
0.4
0.08
196
116
1000
[a] ND ¼ not determined.
Angew. Chem. Int. Ed. 2002, 41, No. 17
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0044-8249/02/4117-3179 $ 20.00+.50/0
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COMMUNICATIONS
[5] N. Uematsu, A. Fujii, S. Hashiguchi, T. Ikariya, R. Noyori, J. Am.
Chem. Soc. 1996, 118, 4916; J. Okuda, S. Verch, R. Sturmer, T. P.
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[7] M. T. Reetz, K. Schimossek, Chimia 1996, 50, 668; Y. K. Choi, M. J.
Kim, Y. Ahn, M. J. Kim, Org. Lett. 2001, 3, 4099.
[8] T. Beard, N. J. Turner, Chem. Commun. 2002, 246.
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D. J. Ager, N. J. Turner, Tetrahedron Lett. 2002, 43, 707.
[10] For recent reviews on the kinetics and stereochemistry ofderacem-
ization reactions, see U. T. Strauss, U. Felfer, K. Faber, Tetrahedron:
Asymmetry 1999, 10, 107; K. Faber, Chem. Eur. J. 2001, 7, 5005.
[11] E. W. Hafner, D. Wellner, Proc. Natl. Acad. Sci. USA 1971, 68, 987;
J. W. Huh, K. Yokoigawa, N. Esaki, K. Soda, J. Ferment. Bioeng. 1992,
74, 189; J. W. Huh, K. Yokoigawa, N. Esaki, K. Soda, Biosci.
Biotechnol. Biochem. 1992, 56, 2081.
[12] For an example ofthe deracemization ofchiral alcohols, see K. Soda,
T. Oikawa, K. Yokoigawa, J. Mol. Catal. B 2001, 11, 149.
[13] P. L. Dostert, M. S. Benedetti, K. F. Tipton, Med. Res. Rev. 1989, 9, 45.
[14] R. B. Silverman, J. M. Cesarone, X. Liu, J. Am. Chem. Soc. 1993, 115,
4955.
been investigated mainly by biophysical and biochemical
approaches, including X-ray crystallography and NMR spec-
troscopic studies ofcarbohydrate protein complexes, muta-
[2]
genesis, and synthesis ofnonnatural sugar analogues.
Despite the established approaches to investigate carbohy-
drate protein interactions, high-throughput methods have not
been developed.
In the last decade, biological microchips (biochips) have
been fabricated for a variety of applications. For instance,
DNA chips (gene chips) are extensively used for tracking the
activity ofmany genes at once and ofr studying changes in
gene expression in diseased states.[3] Additionally, protein
chips have been exploited for the high-throughput study of
molecular interactions and biochemical activities,[4,5] and
profiling protein expression in normal and diseased states.[6]
Ziauddin and Sabatini have developed cell microarrays for
the functional analysis of gene products in parallel.[7] Anal-
ogously, carbohydrate-based arrays may provide a new tool
for the high-throughput study of carbohydrate protein inter-
actions.[8]
[15] B. Schilling, K. Lerch, Biochim. Biophys. Acta. 1995, 1243, 529; B.
Schilling, K. Lerch, Mol. Gen. Genet. 1995, 247, 430. We are grateful to
Professor Schilling for the gift of the plasmid.
In many cases, carbohydrate-binding proteins recognize
terminal and/or penultimate saccharides. Furthermore, they
exhibit a strong affinity for multivalent carbohydrates with
suitable spacing and orientation (cluster effect) but bind to
monovalent oligosaccharides weakly.[9] Thus, we reasoned
that carbohydrate chips that contain immobilized mono- and
disaccharides with suitable distances between the carbohy-
drate probes on the glass surface could provide a useful tool
for elucidating recognition events between carbohydrates and
proteins at a molecular level.
An efficient immobilization technique of carbohydrates on
the surface is essential for successful fabrication of carbohy-
drate chips. Recently, we prepared glycopeptides/glycopro-
teins by chemoselective ligation ofmaleimide-linked sugars to
cysteine-possessing peptides/proteins.[10,11] Herein we report
how this method was applied to immobilize carbohydrates on
glass microscope slides (7.5 cm î 2.5 cm). As shown in
Scheme 1, maleimide-linked sugars are attached through
stable thioether linkages to the slide coated with thiol groups.
[16] S. O. Sablin, V. Yankovskaya, S. Bernard, C. N. Cronin, T. P. Singer,
Eur. J. Biochem. 1998, 253, 270.
[17] Directed Molecular Evolution of Proteins (Eds.: S. Brakmann, K.
Johnsson), Wiley-VCH, Weinheim, 2002; D. Zha, S. Wilensek, M.
Hermes, K.-E. Jaeger, M. T. Reetz, Chem. Commun. 2001, 2664; E. T.
Farinas, T. Bulter, F. H. Arnold, Curr. Opin. Biotechnol. 2001, 12, 545;
J. D. Sutherland, Curr. Opin. Chem. Biol. 2000, 4, 263.
[18] A. Greener, M. Callahan, B. Jerpseth, Mol. Biotechnol. 1997, 7, 189;
U. T. Bornscheuer, J. Altenbuchner, H. H. Meyer, Bioorg. Med.
Chem. 1999, 7, 2169.
[19] M. T. Reetz, Angew. Chem. 2001, 113, 292; Angew. Chem. Int. Ed.
2001, 40, 284; J.-L. Reymond, Chimia 2001, 55, 1049; M. Baumann, R.
St¸rmer, U. T. Bornscheuer, Angew. Chem. 2001, 113, 4329; Angew.
Chem. Int. Ed. 2001, 40, 4201.
Fabrication of Carbohydrate Chips for Studying
Protein Carbohydrate Interactions**
Sungjin Park and Injae Shin*
O
Carbohydrates play a central role in living organisms as
recognition markers to enable cell adhesion, fertilization,
differentiation, development, and tumor-cell metastasis
through carbohydrate protein interactions.[1] Interestingly, it
is through these biomolecular interactions that bacteria and
viruses adhere to the host cells and confer pathogenic
properties.[1] Therefore, it is important to determine the
molecular basis for specific protein carbohydrate recognition
events. Details ofcarbohydrate protein interactions have
O
O
O
N
O
tether
SH
SH
tether
N
tether
N
glass slide
coated by thiol
groups
O
O
O
O
S
S
Scheme 1. Immobilization ofmaleimide-linked carbohydrates on thiol-
derivatized slides.
[*] Prof. Dr. I. Shin, S. Park
Department ofChemistry, Yonsei University
Seoul 120-749 (Korea)
Carbohydrates were appended to linkers Ln ofvarious
lengths (L1, L2, L3, and L4) by coupling glycosylamines
obtained from one-pot amination of carbohydrates to bifunc-
tional cross-linkers such as 1 (Schemes 2 and 3).[10] Further-
more, we also prepared L2-NH2 and L4-NH2, which lack the
carbohydrate moiety, to examine the nonspecific binding of
lectins to linkers (Scheme 2).
Fax : (þ 82)2-364-7050
E-mail: injae@yonsei.ac.kr
[**] This work was supported by a grant from the Center for Integrated
Molecular Systems (KOSEF). We thank Dr. Srikanth Dakoji (Uni-
versity of California, San Francisco) for his critical reading of the
manuscript.
3180
¹ 2002 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
0044-8249/02/4117-3180 $ 20.00+.50/0
Angew. Chem. Int. Ed. 2002, 41, No. 17