Multifunctional bioconjugation by Morita-Baylis-Hillman reaction in aqueous medium

Article abstract of DOI:10.1039/c2cc17116c

An efficient approach for modular assembly of multifunctional bioconjugates from oligosaccharides, peptides and proteins with fluorescent probes/affinity tags based on Morita-Baylis-Hillman (MBH) reaction in aqueous medium has been developed.

Full text of DOI:10.1039/c2cc17116c

View Article Online / Journal Homepage / Table of Contents for this issue
Dynamic Article Links
ChemComm
Cite this: Chem. Commun., 2012, 48, 3527–3529
www.rsc.org/chemcomm
COMMUNICATION
Multifunctional bioconjugation by Morita–Baylis–Hillman reaction in
aqueous mediumw
Gai-Li Li, Karen Ka-Yan Kung, Lan Zou, Hiu-Chi Chong, Yun-Chung Leung, Ka-Hing Wong
and Man-Kin Wong*
Received 16th November 2011, Accepted 10th February 2012
DOI: 10.1039/c2cc17116c
An efficient approach for modular assembly of multifunctional
bioconjugates from oligosaccharides, peptides and proteins with
fluorescent probes/affinity tags based on Morita–Baylis–Hillman
(MBH) reaction in aqueous medium has been developed.
Selective modification of biomolecules is an important strategy
for modulating the function of biomolecules and studying
their roles in complex biological systems.1 However, selective
Scheme 1 MBH-based multifunctional bioconjugation.
biomolecule modification is difficult to achieve as the bio-
Through the MBH reaction, the aldehyde moiety of oligo-
conjugation reactions need to be chemoselective and efficient in
saccharides could be selectively modified by vinyl ketones via the
aqueous medium within a narrow pH and temperature range.
formation of a b-hydroxyl-a-methylene-carbonyl moiety. Here
Thus, the development of new chemical methods for selective
we first report the use of the MBH reaction for multifunctional
bioconjugation has attracted great attention.2 In particular,
modification of oligosaccharides and demonstrate that the
oligosaccharides exhibit important biological functions including
b-hydroxyl-a-methylene-carbonyl moiety could be further modified
cell signalling, host–pathogen interaction, and cancer cell
by thiol-based biophysical probes and cysteine-containing peptides
metastasis3 and are thus attractive targets for bioconjugation.4
and proteins. The present modular approach opens up a new
Aldehydes have long been used as bioorthogonal handles
direction for combinatorial assembly of multifunctional bio-
for bioconjugation of oligosaccharides.5 Through periodate or
conjugates with high structural diversity (Scheme 1).
galactose oxidase-mediated alcohol oxidation, aldehyde moieties
could be easily introduced into oligosaccharides,1 and then
The MBH reaction of unprotected D-raffinose aldehyde 1
(10 mM) and vinyl ketone 2 (3 equiv.) in the presence of
converted into oximes and hydrazones via CQN double bond
formation. However, the drawbacks including slow reaction
1,4-diazabicyclo[2.2.2]octane (DABCO) (1 equiv.) in PBS buffer
(50 mM, pH 7.4) at 40 1C for 3 h was conducted (Scheme 2).
Vinyl ketone-modified D-raffinose 4a with the hydroxyl groups
kinetics, acidic (pH 5–6) reaction conditions, cross-reactivity
with metabolites2b and hydrolysis of CQN double bonds6
restrict their use in biological applications.
remaining intact was obtained in 88% aldehyde conversion11 by
LC-MS analysis of the crude reaction mixture. In addition, our
studies indicated that good to excellent conversion (65–97%)
in the MBH coupling reaction of 1 and 2 could be achieved at
pH 5.1–9.3 (Table S1 in ESIw). Apart from DABCO, a number
of amines commonly used in catalyzing MBH reaction are also
found to be effective.
The assembly of multifunctional biomolecules through multiple
ligations provides an easy access to novel bioconjugates with high
structural complexity. These multifunctional bioconjugates may
contain alkyne moieties for further modification via click reaction,
fluorescent probes for molecular imaging, and/or biotin tags for
streptavidin-based immobilization.7 Along with our ongoing
direction toward bioconjugation research,8 we envisioned that
amine-catalyzed Morita–Baylis–Hillman (MBH) reaction9 could
be developed as an efficient approach for multifunctional
modification of aldehyde-based oligosaccharides owing to
its high selectivity and mild aqueous reaction conditions.10
We studied the time course of the MBH reaction of
D-raffinose aldehyde 1 and vinyl ketone 2 in the presence of
State Key Laboratory of Chirosciences and Department of Applied
Biology and Chemical Technology, The Hong Kong Polytechnic
University, Hung Hom, Kowloon, Hong Kong, China.
E-mail: bcmkwong@inet.polyu.edu.hk; Fax: +852 2364 9932
w Electronic supplementary information (ESI) available: Experimental
details and compounds characterization. See DOI: 10.1039/c2cc17116c
Scheme 2 Modification of D-raffinose aldehyde 1 with vinyl ketone 2.
Chem. Commun., 2012, 48, 3527–3529 3527
c
This journal is The Royal Society of Chemistry 2012

View Article Online
Fig. 1 Time course of the MBH reaction of D-raffinose aldehyde 1 with 2.
Scheme 5 Bifunctional modification of alkyne-bearing D-raffinose 4a
with thiol-based dansyl 8a and coumarin 8b followed by click reaction
with biotin-azide 10. Reaction conditions: (i) PBS buffer (pH 8.0),
40 1C, 2 h; (ii) CuSO4, sodium L-ascorbate, 25 1C, 12 h.
Cross reactivity with ketone-containing metabolites such as
pyruvate and oxaloacetate is an inherent problem in the
oxime-/hydrazone-based bioconjugation reactions especially
in cell-based applications.7 We are pleased to find that no
MBH adduct was detected in the reaction of methyl vinyl
ketone 6 with methyl pyruvate and oxaloacetic acid, respec-
Scheme 3 Modification of D-raffinose aldehyde 1 with vinyl ketone 3.
DABCO (1 equiv.) in NaHCO3 solution (pH 9.3) at 40 1C
(Fig. 1). When 3 equivalent of 2 was used, 73% aldehyde
conversion was observed in 15 min. Up to 80% conversion was
obtained in 1 h, and the conversion reached the maximum (98%)
in 4 h. Using 5 equivalent of 2, a slight increase in aldehyde
conversion (82%) could be achieved in 15 min. Using 1 equivalent
of 2, good overall conversion (69%) was obtained in 4 h.
Next, we proceeded to study the MBH-based oligosaccharide
bioconjugation using fluorescent vinyl ketone 3 (Scheme 3).
Notably, using 1 equivalent of 3, selective modification of
D-raffinose aldehyde 1 with DABCO (1 equiv.) in NaHCO3
solution (pH 9.3) gave fluorescent D-raffinose 4b with 92%
conversion at 40 1C in 6 h.
To provide support for the formation of the b-hydroxyl-a-
methylene-carbonyl moiety via the MBH reaction, milligram
scale reactions with monosaccharides 5a–5b were performed.
Reaction of protected galactose aldehyde 5a and methyl vinyl
ketone 6 (3 equiv.) with DABCO (1 equiv.) in 1,4-dioxane/water
(1 : 1) at 25 1C for 12 h gave the MBH adduct 7a in 70%
isolated yield with a diastereomeric ratio (dr) of 86 : 14 by
1H NMR analysis of the crude reaction mixture (Scheme 4).12
Aldehyde-bearing sialic acid 5b could be converted to the
corresponding MBH adduct 7b in 73% isolated yield with a
dr of 88 : 12, and the absolute configuration of the major
diastereomer of 7b was determined by X-ray crystallographic
analysis (Fig. S13, ESIw). The present MBH-based bio-
conjugation reaction features selective aldehyde modification
via stable C–C bond formation that addresses the intrinsic
problem of low hydrolytic stability of the CQN double bond
in oxime-/hydrazone-based bioconjugation reactions.6
1
tively, as confirmed by H NMR and ESI-MS analysis.
One unique advantage of the MBH-based bioconjugation
reaction is that the b-hydroxyl-a-methylene-carbonyl moiety
incorporated into oligosaccharides allows further ligation with
thiol-based biophysical probes via conjugate addition. As
illustrated in Scheme 5, alkyne-bearing D-raffinose 4a was
treated with thiol-based dansyl 8a (1 equiv.) and coumarin
8b (1 equiv.) at 40 1C for 2 h to give dansyl-D-raffinose 9a and
coumarin-D-raffinose 9b with 99% and 98% conversions,
respectively. Through click reaction with biotin azide 10, 9a
and 9b could be converted into the corresponding bifunctional
biotin-dansyl-D-raffinose 11a and biotin-coumarin-D-raffinose
11b with 86% and 70% conversions. It is envisioned that the
excellent versatility and compatibility of the MBH-based
bioconjugation reaction sequence significantly expands the
scope of multifunctional bioconjugate assembly.
The successful modification of the b-hydroxyl-a-methylene-
carbonyl moiety of the MBH adducts with thiol-based bio-
physical probes encouraged us to explore the possibility of a
more challenging ligation with cysteine-containing peptides.
Gratefully, the coupling reaction of an unprotected cysteine-
containing peptide STSSSCNLSK 12 (0.1 mM) with b-hydroxyl-
a-methylene-carbonyl-bearing MBH adducts 7a and 7b (5 equiv.)
in PBS buffer (50 mM, pH 8.0) at 40 1C for 2 h gave the corres-
ponding products 13a and 13b with 98% and 99% conversions,
respectively (Scheme 6). As confirmed by LC-MS/MS analysis,
7a and 7b were selectively ligated to the cysteine sulfhydryl group
of 12 without modification of the N-terminal a-amino group or
the side chains of serine and lysine.
Note that the ligation of the single, surface exposed cysteine
residue of bovine serum albumin (BSA) to fluorescent dansyl-
linked MBH adduct 14 (50 equiv.) was confirmed by
SDS-PAGE and ESI-MS analysis (Scheme 7). It was found that
14-modified BSA gave a fluorescent signal under UV excitation
in SDS-PAGE protein analysis (Fig. 2). No modification was
observed with lysozyme, which contains no free cysteine residue.
Scheme 4 MBH reaction of monosaccharide aldehydes 5a–5b with 6.
3528 Chem. Commun., 2012, 48, 3527–3529
c
This journal is The Royal Society of Chemistry 2012

View Article Online
2008, 37, 1423–1431; (c) R. A. Scheck, M. T. Dedeo,
A. T. Iavarone and M. B. Francis, J. Am. Chem. Soc., 2008, 130,
11762–11770; (d) Y. Zeng, T. N. C. Ramya, A. Dirksen,
P. E. Dawson and J. C. Paulson, Nat. Methods, 2009, 6,
207–209; (e) L. A. Canalle, D. W. P. M. Lowik and J. C. M. van
¨
Hest, Chem. Soc. Rev., 2010, 39, 329–353; (f) J. Alam, T. H. Keller
and T. P. Loh, J. Am. Chem. Soc., 2010, 132, 9546–9548;
(g) J. C. Jewett, E. M. Sletten and C. R. Bertozzi, J. Am. Chem.
Soc., 2010, 132, 3688–3690; (h) J. Rayo, N. Amara, P. Krief and
M. M. Meijler, J. Am. Chem. Soc., 2011, 133, 7469–7475;
(i) Q. Zhang, J. Sun, Y. Zhu, F. Zhang and B. Yu, Angew. Chem.,
Int. Ed., 2011, 50, 4933–4936; (j) E. M. Sletten and C. R. Bertozzi,
Acc. Chem. Res., 2011, 44, 666–676; (k) J. M. Chalker, G. J. L.
Bernardes and B. G. Davis, Acc. Chem. Res., 2011, 44, 730–741;
(l) L. S. Witus and M. B. Francis, Acc. Chem. Res., 2011, 44,
774–783.
Scheme 6 Selective modification of cysteine-containing peptide
STSSSCNLSK 12 with b-hydroxyl-a-methylene-carbonyl-bearing MBH
adducts 7a–7b.
3 (a) S. J. Danishefsky and J. R. Allen, Angew. Chem., Int. Ed., 2000,
39, 836–863; (b) D. P. Galonic and D. Y. Gin, Nature, 2007, 446,
´
1000–1007; (c) S. I. van Kasteren, H. B. Kramer, H. H. Jensen,
S. J. Campbell, J. Kirkpatrick, N. J. Oldham, D. C. Anthony and
B. G. Davis, Nature, 2007, 446, 1105–1109.
4 (a) R. J. Payne and C. H. Wong, Chem. Commun., 2010, 46, 21–43;
(b) C. Y. Wu and C. H. Wong, Chem. Commun., 2011, 47,
6201–6207.
5 (a) A. Dirksen, T. M. Hackeng and P. E. Dawson, Angew. Chem.,
Int. Ed., 2006, 45, 7581–7584; (b) I. S. Carrico, B. L. Carlson and
C. R. Bertozzi, Nat. Chem. Biol., 2007, 3, 321–322.
6 (a) J. Kalia and R. T. Raines, Angew. Chem., Int. Ed., 2008, 47,
7523–7526; (b) J. Kalia and R. T. Raines, Curr. Org. Chem., 2010,
14, 138–147.
Scheme 7 Selective modification of free cysteine-containing protein
BSA with fluorescent dansyl-linked MBH adduct 14.
7 (a) P. Kele, G. Mezo, D. Achatz and O. S. Wolfbeis, Angew.
¨
Chem., Int. Ed., 2009, 48, 344–347; (b) J. Morales-Sanfrutos,
F. J. Lopez-Jaramillo, F. Hernandez-Mateo and F. Santoyo-
Gonzalez, J. Org. Chem., 2010, 75, 4039–4047; (c) C. S. Tsai,
P. Y. Liu, H. Y. Yen, T. L. Hsu and C. H. Wong, Chem. Commun.,
2010, 46, 5575–5577; (d) M. E. B. Smith, F. F. Schumacher,
C. P. Ryan, L. M. Tedaldi, D. Papaioannou, G. Waksman,
S. Caddick and J. R. Baker, J. Am. Chem. Soc., 2010, 132,
1960–1965; (e) B. C. Sanders, F. Friscourt, P. A. Ledin,
N. E. Mbua, S. Arumugam, J. Guo, T. J. Boltje, V. V. Popik
and G.-J. Boons, J. Am. Chem. Soc., 2011, 133, 949–957;
(f) M. Galibert, O. Renaudet, P. Dumy and D. Boturyn, Angew.
Chem., Int. Ed., 2011, 50, 1901–1904; (g) C. P. Ryan, M. E. B.
Smith, F. F. Schumacher, D. Grohmann, D. Papaioannou,
G. Waksman, F. Werner, J. R. Baker and S. Caddick, Chem.
Commun., 2011, 47, 5452–5454; (h) M. L. Conte, S. Staderini,
A. Marra, M. Sanchez-Navarro, B. G. Davis and A. Dondoni,
Chem. Commun., 2011, 47, 11086–11088.
Fig. 2 SDS-PAGE of BSA, 14-modified BSA and lysozyme: fluores-
cence visualization (left) and Coomassie staining (right).
The peaks at 66 547 Da (BSA) and 67 140 Da (14-modified BSA)
revealed incorporation of one molecule of 14 per BSA.
In conclusion, we have developed an efficient MBH-based
aldehyde bioconjugation reaction for multifunctional modifica-
tion of oligosaccharides, peptides and proteins with fluorescent
probes/biotin tags. The excellent compatibility of the MBH-based
bioconjugation reaction with thiol-based bioconjugation and
click reaction allows modular assembly of multifunctional bio-
conjugates that would have widespread applications.
8 (a) W.-K. Chan, C.-M. Ho, M.-K. Wong and C.-M. Che, J. Am.
Chem. Soc., 2006, 128, 14796–14797; (b) H.-Y. Shiu, T.-C. Chan,
C.-M. Ho, Y. Liu, M.-K. Wong and C.-M. Che, Chem.–Eur. J.,
2009, 15, 3839–3850; (c) K. K.-Y. Kung, G.-L. Li, L. Zou, H.-C.
Chong, Y.-C. Leung, K.-H. Wong, V. K.-Y. Lo, C.-M. Che and
M.-K. Wong, Org. Biomol. Chem., 2012, 10, 925–930.
We thank the financial support from Hong Kong Research
Grants Council (PolyU 7052/07P) and The Hong Kong
Polytechnic University (PolyU Departmental General Research
Funds, Competitive Research Grants for Newly Recruited
Junior Academic Staff and SEG PolyU01).
9 (a) D. Basavaiah, K. V. Rao and R. J. Reddy, Chem. Soc. Rev.,
2007, 36, 1581–1588; (b) D. Basavaiah, B. S. Reddy and
S. S. Badsara, Chem. Rev., 2010, 110, 5447–5674.
10 (a) C. Yu, B. Liu and L. Hu, J. Org. Chem., 2001, 66, 5413–5418;
(b) J. Cai, Z. Zhou, G. Zhao and C. Tang, Org. Lett., 2002, 4,
4723–4725; (c) S. Luo, P. G. Wang and J. P. Cheng, J. Org. Chem.,
2004, 69, 555–558.
11 For the determination of aldehyde conversion by LC-MS analysis,
Notes and references
see ESIw.
1 G. T. Hermanson, Bioconjugate Techniques, Academic Press,
Amsterdam, 2nd edn, 2008.
2 (a) C. P. R. Hackenberger and D. Schwarzer, Angew. Chem.,
Int. Ed., 2008, 47, 10030–10074; (b) I. S. Carrico, Chem. Soc. Rev.,
12 The result is consistent with previous work by Krishna et al. using
protected sugar-derived aldehydes as chiral electrophiles in
MBH reaction, see: A. Manjuvani, V. Kannan and P. R. Krishna,
Tetrahedron: Asymmetry, 2005, 16, 2691–2703.
c
This journal is The Royal Society of Chemistry 2012
Chem. Commun., 2012, 48, 3527–3529 3529