1,3,5-Trisubstituted benzenes as fluorescent photoaffinity probes for human carbonic anhydrase II capture

Article abstract of DOI:10.1039/c3cc38251f

The synthesis of small molecule based 1,3,5-trisubstituted benzenes for photo-mediated capture of human carbonic anhydrase II with visualisation by fluorescence is described. The Royal Society of Chemistry 2013.

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1,3,5-Trisubstituted Benzenes as Fluorescent Photoaffinity Probes for Human
Carbonic Anhydrase II Capture
Partha Sarathi Addy,a Baisakhee Saha,b N. D. Pradeep Singh,a Amit K. Das,b*, Jacob T. Bush,c
Clarisse Lejeune,c Christopher J. Schofield c* and Amit Basaka*+
5
Received (in XXX, XXX) Xth XXXXXXXXX 20XX, Accepted Xth XXXXXXXXX 20XX
OI: 10.1039/b000000x
The synthesis of small molecule based 1,3,5-trisubstituted
benzenes for photo-mediated capture of human carbonic
10 anhydrase II with visualisation by fluorescence is described.
50
The ‘capture’ of proteins by small molecules via irreversible
crossꢀlinking mediated by photoꢀirradiation is of interest in the
field of proteomics (for reviews see ref. 1). The technique has the
15 potential for profiling proteinꢀbinding by small molecules, an
objective of importance both for basic cell biology and in
pharmaceutical science. Capture compounds, or photoaffinity
probes, are typically endowed with three functions comprising (i)
a selectivity function, such as an enzyme inhibitor, (ii) a photoꢀ
20 cross linking group (capture function) and (iii) a sorting group to
enable separation of the captured protein from biological
mixtures, such as biotin or an alkyne for subsequent modification.
The captured protein(s) can be isolated using streptavadin beads
and identified by mass spectrometry or western blotting (for
25 examples see ref. 2). We are interested in developing methods for
protein capture and visualisation without necessitating an
isolation or chemical conjugation step (Figure 1).3
55
60
65
70 Figure 1: Working principle of fluorescence based visualisation of
capture.
Various ‘tripodal’ molecules have been employed for protein
capture by small molecules, for example amino acids, peptides
30 and trisubstituted aromatics including 1,2,4ꢀtrisubstituted benzene
derivatives.4 However, there are few examples of the use of 1,3,5ꢀ
trisubstituted benzenes for this purpose.5 Here we report that 5ꢀ
amino dimethylisophthalate can be readily modified to produce
compounds (1, 2) suitable for fluorescence based monitoring of
35 protein capture (Figure 2) as exemplified by work on human
carbonic anhydrases (HCA), increases in the level of some
isoforms of which are indicative of disease especially those
related to hypoxia (CA IX).
It was envisioned that the three groups could be attached to 5ꢀ
amino dimethyl isophthalate acting as a tripodal template. A polar
linker (diamino ethylene) was introduced between the
75 visualization group and the template in order to distance the
former from the other two groups and to increase the
hydrophilicity (Figure 2).
O
O
HN
Y
Y
HN
N3
H
80
H
O
N
O
N3
N
H
H
O
N
O
O
HN
N
O
O
SO2NH2
SO2NH2
O
O
O
N
H
X
We employed two sulphonamide derivatives that are known
40 inhibitors for human carbonic anhydrase II (HCA II) as the
selectivity function.6 An aryl azide was chosen for photoꢀ
mediated crossꢀlinking due to its ability to react irreversibly via
X
X = Selectivity function
Y = Cross-linking function
Z = Visualization function
Z
85
Z
2
1
8
nitrene formation.7, A propargylated pyrene derivative was
Figure 2: Design of capture compounds
selected as a fluorescent visualisation function.
45
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60 Figure 3: Result of gel electrophoresis analysis of capture of HCA II by 1
and 2 at different protein concentrations, as visualised by UV (left) and
Coomassie blue (right). Lanes 1ꢀ5 represent incubation with 1 (100 ꢀM),
lanes 6ꢀ10 represent incubation with 2 (100 ꢀM). The final concentration
of protein in lanes 1ꢀ5 was 4, 6, 8, 20 and 40 ꢀM, respectively. HEPES
The synthesis started with BOPꢀ mediated amide coupling with
4ꢀazidobenzoic acid to link the crossꢀlinking function to amino
dimethyl isophthalate 4. The product, 5, was hydrolysed (NaOH)
to give acid 6. Subsequent coupling of 6 with monoꢀBoc
5
protected ethylenediamine afforded amide 7. Deprotection 65 buffer (pH 7.2) was used.
(CF3CO2H) followed by coupling of the resultant amine with acid
The results validate compounds 1 and 2 as fluorescent capture
3 gave amide 8 the ester of which was hydrolysed (NaOH). The
resultant free acid was coupled with 4ꢀaminomethylꢀ
benzenesulfonamide to furnish the capture compound, 1.
compounds for HCA II. Capture is visible by in gel analysis
under a UVꢀ transilluminator down to HCA II concentrations of 4
70 ꢀM, with the concentration of the capture compounds at 50 ꢀM.
We then investigated the selectivity of the probes. The process
of incubation and photolysis was carried with a mixture of
proteins i.e. HCA II, bovine serum albumin (BSA) and
Lysozyme. Under UVꢀtransillumination, only the fluorescence
75 band corresponding to HCA II was visible, whereas Coomassie
blue staining of the same gel showed bands corresponding to the
three proteins (Figure 4).
9
10 Reaction of 8 with chloro sulphonamide 10 using potassium
carbonate in DMF produced the other capture compound 2
(Scheme 1).9 The fluorescent tag 3 was prepared from 1ꢀ
bromopyrene via a 3ꢀstep protocol (see ESI).
O
O
NH2
HN
HN
c
b
a
15
20
25
30
N3
N3
COOMe
68%
74%
56%
MeOOC
COOMe
HOOC
HN
COOMe
MeOOC
6
4
5
O
O
O
4
3
2
1
4
3 2 1
HN
HN
N3
H
N3
H
N3
H
N
O
80
116 kDa
66.2 kDa
45 kDa
N
O
H
N
O
d
e
N
O
O
OMe
COOMe
BSA
O
OH
O
65%
O
8
9
HN
52%
O
7
O
N
O
f
35 kDa
25 kDa
HCA II
H
74%
O
85
O
HN
HN
18.4 kDa
14 kDa
g
N3
H
Lysozyme
N3
H
H
N
O
H
O
N
O
N
O
O
SO2NH2
N
O
O
O
HN
O
N
2
1
H
O
90 Figure 4: Results of gel electrophoresis analysis of capture of HCA II in a
mixture by 1 and 2 visualised using UV (left) and Coomassie blue (right).
Lane 1: mixture of BSA, HCA II and Lysozyme (all at 21 ꢀM), lane 2:
protein mixture (21 ꢀM) + 1 (50 ꢀM) with irradiation, lane 3: protein
mixture (21 ꢀM) + 2 (50 ꢀM) with irradiation, lane 4: molecular weight
95 marker. HEPES buffer (pH 7.2) was used.
COOH
O
O
SO2NH2
10
Cl
N
H
H2NO2S
3
Scheme 1: Synthesis of capture compounds 1 and 2. Reagents and
conditions: a) pꢀAzidobenzoic acid, BOPꢀreagent, diisopropylethylamine
(DIPEA), CH2Cl2 reflux, 24 h; b) NaOH, MeOH, 50 °C, 3 h; c)
35 MonoBOC protected ethylenediamine, BOPꢀreagent, DIPEA, CH2Cl2, 40
°C, 48 h; d) (i) CF3CO2H, CH2Cl2, 0 °C ꢀ rt, 20 min; (ii) Acid 3, BOPꢀ
reagent, DIPEA, CH2Cl2:DMF (5:1), rt, 20 h; e) NaOH, MeOH, 50 °C, 6
h; f) 4ꢀAminomethylꢀbenzenesulfonamide, BOPꢀreagent, DIPEA,
CH2Cl2:DMF (5:1), 40 °C, 48 h; g) K2CO3, 2ꢀChloroꢀNꢀ(4ꢀ
40 sulfamoylphenyl)acetamide 10, dry DMF, rt, 20 h.
The experiment was repeated with cell lysates of E. coli where
the selective capturing of HCA II was clearly apparent by PAGE
(Figure 5). Thus, both 1 and 2 showed high selectivity towards
HCA II demonstrating the suitability of our 1,3,5ꢀtrisubstituted
100 benzene template for capture and subsequent fluorescenceꢀbased
visualisation.
1
2
3
1
2
3
We then investigated the inhibition of HCA II by 1 and 2. Both
were found to be reversible (competitive) inhibitors with
compound 2 showing stronger inhibition (IC50: 1 16.7 ꢀM, 2 =
1.2 ꢀM, for kinetic plots see ESI). The issue of suitability of the
45 template for the proposed capture compounds and the efficacy of
the fluorescence based technique for visualising protein capture
was then addressed. Thus, 1 and 2 were incubated with HCA II at
various concentrations (15 min), irradiated (UV λ ≥ 300 nm, 7
min) and then directly subjected to polyacrylamide gel
50 electrophoresis (PAGE).10 Exposure of the gel to UVꢀ
transillumination showed clear fluorescence bands at the
expected region, which was further confirmed by Coomassie blue
staining (Figure 3).
105
Figure 5: Gel electrophoresis of capture of HCA II from a cell lysate by 1
110 and 2 visualised using UV (left) and Coomassie blue (right). Lanes 1: 0.5
mg cell + 1 (10 ꢁl, 1 mM) + 90 ꢁL buffer (HEPES); 2: 0.5 mg cell + 2
(10 ꢁl, 1mM) + 90 ꢁL buffer; 3: 0.5 mg cell + 100 ꢁL buffer.
The capture of HCA II by both compounds 1 and 2 was
115 validated by MALDI mass spectrometric analyses. Thus, the
incubated photoꢀreacted mixture was directly analysed using a
MALDIꢀTOF mass spectrometer which revealed a new peak at
m/z 29919.24 (1) or 29964.05 (2) corresponding to [M+ (HCA II)
+ (1 or 2) –N2 + H+)] (see ESI). The mass spectrum of the
120 incubated photo reacted mixture containing three proteins showed
the peak corresponding to capture of HCA II only (Figure 5 for
capture by 2; see ESI for 1).11
10 9 8 7 6 5 4 3 2 1
10
9
8
7 6
5 4 3 2 1
55
2
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65 Joseph, M. Humphrey and B. F. Cravatt, Proc. Natl. Acad. Sc. USA, 2004,
101, 10000. (b) E. W. S. Chan, S. Chattopadhaya, R. C. Panicker, X.
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Tetrahedron Lett., 2005, 46, 4053. For interaction between actin and the
70 antitumor agent Aplyronine see (d) T. Kuroda, K. Suenaga, A. Sakakura,
T. Handa, K. Okamoto and H. Kigoshi, Bioconjugate Chem., 2006, 17,
524. For a review on photoaffinity labeling see (e) L. Dubinsky, B. P.
Krom and M. M. Meijler, Bioorg. Med. Chem., 2012, 20, 554.
[4] (a) X. Li, JꢀH. Cao, L. Ying. P. Rondard, Y. Zhang, P. Yi, JꢀF. Liu and
75 FꢀJ Nan, J. Med. Chem., 2008, 51, 3057 and references therein.
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Endo and M. Suzuki, Bioorg. Med. Chem. Lett., 2002, 12, 3263. (b) T.
Hosoya, T. Hiramatsu, T. Ikemoto, H. Aoyama, T. Ohmae, M. Endo and
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80 Inoue, T. Hiramatsu, H. Aoyama, T. Ikemoto and M. Suzuki, Bioorg.
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Mioc, T. Numagaa, Y. Sawaguchia, T. Yoshidaa, M. Wakamoria, E.
Moria, T. Numataa, M. Ishiid, H. Takemotoe, A. Ojidae, K. Watanabeb,
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85 Hamachie, and Y. Moria, Proc. Natl. Acad. Sc. USA, 2009, 106, 5400.
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[7] K. L. Buchmueller, B. T. Hill, M. S. Platz and K. M. Weeks, J. Am.
90 Chem. Soc., 2003, 125, 10850.
In conclusion, we have described two readily accessible small
molecule probes, 1 and 2, for selective capture of HCA II via
photoꢀirradiation allowing fluorescent visualization. The ability
of 5ꢀamino dimethyl isophthalate to act as a tripodal template for
capture molecule design was demonstrated. We believe that
1,3,5ꢀtrisubstituted benzene based photoꢀreactive probes coupled
with fluorescence based visualisation offers a simple and
effective method for protein capture, which should be of utility in
evaluating drug toxicity by studying their offꢀtarget interactions,
5
10 inhibitor design and early disease diagnosis.
N
(I)
(II)
15
M
N
M=Lysozyme
N=HCAII
O=HCAII + 2 –N2 + H+
d
e
20
[8] C. J. Shields, D. E. Falvey, G. B. Schuster, O. Buchardt, and P. E.
Nielsen, J. Org. Chem., 1988, 53, 3501.
O
[9] Spectroscopic data: For 1: δH (d6ꢀDMSO): 10.52 (1H, s), 9.21 (1H, t,
J = 5.5 Hz), 8.66 (1H, bs), 8.42 (1H, d, J = 9.0 Hz), 8.39ꢀ8.27 (4H, m),
95 8.26ꢀ8.07 (5H, m), 8.02 (2H, d, J = 8.5 Hz), 7.77 (2H, d, J = 8.5 Hz), 7.49
(2H, d, J = 8.5 Hz), 7.22 (2H, d, J = 8.5 Hz), 4.72 (2H, s), 4.53 (2H, d, J =
5.5 Hz), 4.12 (3H, s), 3.16 (2H, s), 3.14 (3H, s). δC (CDCl3): 169.6, 166.8,
166.7, 166.2, 144.2, 143.6, 143.2, 139.9, 135.9, 135.6, 131.9, 131.6,
131.3, 131.2, 131.0, 131.4, 129.5, 129.0, 128.3, 127.8, 127.4, 126.7,
100 126.6, 126.3, 125.5, 125.2, 124.2, 123.9, 122.9, 122.7, 121.7, 119.7,
116.8, 91.9, 85.5, 69.4, 59.5, 43.1, 38.5, 31.9; νmax ( KBr, cmꢀ1): 3752,
3413, 2923, 2857, 2364, 2122, 1651, 1599, 1547, 1452, 1333, 1284, 1155,
1099, 1028; λmax (DMSO): 365 nm (ε 30,985 Mꢀ1cmꢀ1), 347 nm (ε 24.630
Mꢀ1cmꢀ1), 285 nm (ε 35,384 Mꢀ1cmꢀ1); HRMS: Calcd for
105 C45H36N8O7S+Na+ 855.2325, found 855.2346. For 2: δH (d6ꢀDMSO):
10.60 (2H, s), 8.83 (1H, s), 8.65 (1H, s), 8.57 (1H, s), 8.45 (1H, d, J = 8.5
Hz), 8.34 (1H, d, J = 7.0 Hz), 8.29 (2H, d, J = 9.5 Hz), 8.23ꢀ8.04 (8H, m),
7.76 (4H, bs), 7.25 (4H, bs), 4.99 (2H, s), 4.72 (2H, s), 4.14 (2H, s), 3.32
(4H, s); δC (CDCl3): 169.4, 166.2, 165.4, 146.5,
110 143.9, 141.7, 139.1, 136.4, 136.1, 135.6, 134.9, 133.9, 133.6, 132.3,
131.9, 131.6, 131.4, 130.8, 130.1, 129.7, 129.2, 128.8, 128.5, 127.6,
127.2, 126.4, 126.3, 125.2, 125.0, 124.2, 123.7, 119.4, 119.3, 116.5, 91.7,
85.3, 69.2, 59.3, 56.2, 38.3, 34.7; νmax ( KBr, cmꢀ1): 3855, 3751, 3398,
2924, 2853, 2357, 2127, 1745, 1696, 1606, 1542, 1399, 1308, 1243, 1155,
115 1102, 1021; λmax (DMSO): 365 nm (ε 27,369 Mꢀ1cmꢀ1), 347 nm (ε 21,200
Mꢀ1cmꢀ1), 285 nm (ε 34,615 Mꢀ1cmꢀ1); HRMS: Calcd for
C46H36N8O9S+Na+ 899.2224, found 899.2259.
25
Figure 5: MALDI spectra: (I) Mixture of HCA II, BSA and Lysozyme +
2, incubated and photoꢀreacted; (II) expanded spectrum.
30 We thank the Leverhulme Trust, UK for support to CJS and AB.
PSA is grateful to CSIR, Government of India (GoI), for a
research fellowship. BS is grateful to DST, GoI for her Young
Scientist Research Fellowship. CL and JTB were funded by the
Wellcome Trust and the EPSRC respectively.
35 aDepartment of Chemistry, bDepartment of Biotechnology, Indian Institute
of Technology Kharagpur 721302 India
cDepartment of Chemistry, Chemistry Research Laboratory, University of
Oxford, 12 Mansfield Road, Oxford, UK OX1 3TA
E-mail: absk@chem.iitkgp.ernet.in
40 Fax: +91 3222 282552; Tel: +91 3222 283300
†Electronic Supplementary Information (ESI) available: Experimental
procedure, compound characterization, copies of NMR, Inhibition
Kinetics, ESI and MALDIꢀTOF MS.
[10] Capture experiment protocol: For capture experiments, the HCA II
concentration was kept at 0.5 ꢁM and total volume was made up to 100
120 ꢁL with buffer (20 mM HEPES; pH 7.2). HCA II and capture compounds
(10ꢀ100 ꢁM) were mixed by vortexing followed by centrifugation. For
cell lysate preparation, 5 mL of induced, lag phase BL21(DE3)pLysS
cells carrying plasmid pACA/HCA II were resuspended in 0.5 mL of
buffer (50 mM Tris; pH 8.0 , 50 mM NaCl, 10 mM EDTA, 1 mM
125 dithiothreitol, 1 mM phenyl methanesulfonyl fluoride, 0.2 mM ZnSO4),
sonicated and centrifuged at 10,000 rpm for 10 minutes. The cell lysate
thus obtained was mixed with the 50 ꢁM capture compounds. To probe
the selectivity of the capture compounds towards HCA II, BSA and
lysozyme were used as controls. BSA and lysozyme were mixed at
130 equimolar concentrations with HCA II. Exactly same method was
followed as used with HCA II. The compounds were incubated with
proteins for 15 min, then photoꢀirradiated with medium pressure mercury
lamp at ≥ 300 nm for 7 minutes followed by SDSꢀPAGE and mass
spectrometric analysis.
45 Notes and references
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Florea and H. S. Overkleeft, Acc. Chem. Res., 2011, 44, 718 and
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Chem. Soc. Rev., 2011, 40, 246.
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Marappan and P. Yip, Assay Drug Dev. Technol., 2007, 5, 381. (b) J. J.
Fischer, C. Dalhoff, A. K. Schrey, O. Y. Graebner Neé Baessler, S.
55 Michaelis , K. Andrich, M. Glinski , F. Kroll, M. Sefkow, M. Dreger and
H. Koester, J. Proteomics, 2011, 75, 160. (c) C. Dalhoff, M. Hüben, T.
Lenz, P. Poot, E. Nordhoff, H. Köster and E. Weinhold, ChemBioChem.,
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2011, 75, 100. (e) D. Rotili, M. Altun, A. Kawamura, A. Wolf, R.
60 Fischer, I. K. Leung, M. M. Mackeen, Y. M. Tian, P. J. Ratcliffe, A. Mai,
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[3] Fluorescence based visualization on a gel of protein capture has been
reported: for metalloproteases see (a) A. Saghatelian, N. Jessani, A.
135 [11] The mass spectra didn’t show any peak for BSA probably due to its
high MW (66.5 kDa).
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