Synthesis of selectively 15N- or 13C-labelled malachite green

Article abstract of DOI:10.1002/jlcr.554

Two complimentary syntheses of selectively ¹⁵N- or ¹³C-labelled malachite green hydrochloride were developed in order to provide labelled ligands for structural studies of RNA aptamer/ligand complexes. The ¹⁵N- and ¹³C-labelled versions of the dye have been used in NMR studies to probe the changes in the electronic charge distribution and dynamics of the dye upon binding to RNA. Copyright

Full text of DOI:10.1002/jlcr.554

JOURNAL OF LABELLED COMPOUNDS AND RADIOPHARMACEUTICALS
J Label Compd Radiopharm 2002; 45: 241–248.
DOI: 10.1002/jlcr.554
Research Article
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Synthesis of selectively N- or C-labelled
malachite green
Steven C. DeFina and Thorsten Dieckmann*
Department of Chemistry, University of California at Davis, One Shields Avenue,
Davis, CA 95616, USA
Summary
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Two complimentary syntheses of selectively N- or C-labelled malachite
green hydrochloride were developed in order to provide labelled ligands for
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structural studies of RNA aptamer/ligand complexes. The N- and C-
labelled versions of the dye have been used in NMR studies to probe the
changes in the electronic charge distribution and dynamics of the dye upon
binding to RNA. Copyright # 2002 John Wiley & Sons, Ltd.
1
Key Words: N-; C; aptamer; malachite green; NMR spectroscopy
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Introduction
Intramolecular dynamics of both ligand and target are of crucial
importance for recognition and stability in complexes between nucleic
1–4
acids and small molecule ligands. NMR spectroscopy is particularly
suitable to study structural changes and internal motions on the ns–ms
time scale in these systems. In comparison to the large number of studies
that look at the nucleic acid dynamics, there are no studies of the ligand
dynamics to date. This is, in part, due to the ease with which the nucleic
*Correspondence to: T. Dieckmann, Department of Chemistry, University of California,
One Shields Avenue, Davis, CA 95616, U.S.A. E-mail: dieckman@chem.ucdavis.edu.
Contract/grant sponsor: NSF; Contract/grant number: MCB 0110689
Contract/grant sponsor: NIH; Contract/grant number: RR11973
Copyright # 2002 John Wiley & Sons, Ltd.
Received 3 October 2001
Revised 21 November 2001
Accepted 21 November 2001

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S. C. DEFINA AND T. DIECKMANN
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acid can be labelled with NMR active isotopes, whereas the synthesis of
labelled ligand is often difficult orext re mely costly.
The malachite green binding aptamer is an RNA sequence that binds
the organic dye malachite green and related dyes with high affinity. The
molecule was identified by in vitro selection with the goal to provide a
6
laseractivated cleavage site module. Its three-dimensional structure in
complex with tetramethylrosamine was determined by X-ray crystal-
7
lography. However, it was not possible to crystallize the complex with
malachite green. NMR studies of the system were initiated in order to
determine the structure of the malachite green – RNA complex and to
study the dynamics of the dye in the complex.
Based on the lack of commercially available labelled malachite green
and a limited selection of labelled synthetic precursors, the syntheses of
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both N- and C-labelled malachite green were undertaken and are
described below. Synthetic precursors had to be labelled in the
appropriate place, they needed to be reasonably priced, and had to be
materials in which the necessary transformations leading to the dye
could be afforded in good yield and high purity. Molecules with labels
located in the N-methyl groups were chosen because of their central
location in the complex and the possibility of using them to characterize
conformational exchange processes. The synthetic schemes were initially
tested using non-labelled precursors. Emphasis was put on maximizing
yields rather than purity because small amounts of impurities are easily
identified and do not interfere with the NMR experiments. A
preliminary NMR investigation using the synthetic malachite green
free and in complex with the RNA aptamer is described below.
Discussion
The synthesis of both the nitrogen and carbon labelled dyes (Schemes 1
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and 2) were carried out using N-labelled aniline or C-labelled
formalin purchased from Isotec Inc. (Miamisburg, Ohio). All non-
labelled reagents were purchased from Sigma-Aldrich (Milwaukee,
Wisconsin). It was possible to shorten the synthesis scheme of the
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C-labelled dye by one step compared to the N-labelled dye because
unlabelled 4-Bromo-aniline was commercially available at low cost. In
addition to the two labelled versions presented here, there are a number
of labelled-reagent variations that can be purchased from Isotec Inc. to
produce additional labelled versions of malachite green. For example, a
Copyright # 2002 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2002; 45: 241–248

SYNTHESIS OF LABELLED MALACHITE GREEN
243
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Scheme 1. Synthesis of N-labelled malachite green
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Scheme 2. Synthesis of C-labelled malachite green
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fully C-labelled version could be synthesized by making the necessary
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transformations using fully C-labelled aniline, formalin and benzoic
acid. Additional variations could incorporate labels selectively into
rings A and B, or C (Figure 1).
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The use of specifically N- or C-labelled malachite green in
multidimensional NMR spectroscopy provides the possibility to probe
the electronic structure and internal dynamics of the dye by looking at
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the N- and C-chemical shift changes and line widths in the free and
bound states. Preliminary NMR studies were performed on the free dye
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and the RNA/ligand complex (Figure 2) using N- and C-labelled
13
malachite green. The C-labelled dye allows the unambiguous assign-
ment of the methyl groups in the free and the RNA bound dye. The
13
observed changes of the C-chemical shifts forthe methyl g ro ups in the
Copyright # 2002 John Wiley & Sons, Ltd.
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S. C. DEFINA AND T. DIECKMANN
Figure 1. Malachite green with carbon and nitrogen atoms numbered as well as
rings labelled to allow discussion of specific labelling
free and bound dye are consistent with a stabilization and thus greater
contribution of the malachite green resonance structure that carries the
charge on one of the nitrogen atoms. This observation is confirmed by
an analysis of the proton line widths for the methyl groups on rings A
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and B, which indicate a faster rate of rotation for ring B. More detailed
NMR studies and simulations of the electron density are currently in
progress.
Experimental
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9,10
Preparation of N-labelled N,N-dimethylaniline (4) (Scheme 1):
A solution containing 3 (0.233 g, 2.5 mmol), NaBH CN (0.943 g,
3
1
5.0 mmol) and THF (15 ml) was slowly added (over30 min) to a
solution of formalin (0.450 g of a 37% solution, 15.0 mmol) and H SO
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4
(2.02 ml, 3 M) on ice. The reaction was allowed to continue for 20 h
before being basified with 5 M NaOH and subsequently extracted with
Et O (3 Â 20 ml). The extract was dried over K CO , filtered and solvent
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2
3
removed by vacuum. The solid/oil was purified by silica gel
chromatography (MeOH/EtOAC/Hexane 1:1.5:7.5) separating 4 from
monomethylated aniline and 3, providing a mass of 0.142 g, a 47%
yield. The product was used without further purification.
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Preparation of
N-labelled 4-Bromo-N,N-dimethylaniline (5):
Selective bromination of the para position in N,N-dimethylaniline was
achieved by placing 4 (0.142 g, 1.17 mmol) in glacial AcOH (2.5 ml) at
Copyright # 2002 John Wiley & Sons, Ltd.
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SYNTHESIS OF LABELLED MALACHITE GREEN
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Figure 2. NMR studies of an RNA aptamer with specifically labelled malachite
green. (A) Secondary structure of the malachite green binding aptamer. (B)
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00 MHz 2D H– N- long-range HSQC correlation spectrum of 1.2 mM N-
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labelled MG dye in D O, 10 mM potassium phosphate buffer, 10 mM KCl, pH 5.8
2
at 258C. The crosspeak marked a denotes the signal originating from the two-
bond coupling between the nitrogens and their respective methyl hydrogens, while
c marks the signal originating from the three-bond coupling between the nitrogens
and hydrogens 3, 4 and 9, 10 (Figure 1). b marks the residual HDO resonance. (C)
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00 MHz 2D H– C HMQC correlation spectrum in D O of C-labelled MG
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dye, 0.8 mM RNA, ratio of RNA :dye 1 :1.2, 10 mM potassium phosphate buffer,
0 mM KCl, pH 5.8 at 258C. Signals associated with the four methyl groups are
labelled (A21, A22, B24, B25). X and * denote signals originating from residual
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starting material of the synthesis (4-Br-N,N-dimethylaniline) and monomethy-
1
lated dye, a by-product of the synthesis, respectively. The N- and C-chemical
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shifts are referenced relative to protons in TMS via the ratios of c to c or c ,
C
H
N
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4
respectively. The nitrogen chemical shift for the signal in (B) is À353.5 ppm if
referenced to nitromethane
Copyright # 2002 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2002; 45: 241–248

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S. C. DEFINA AND T. DIECKMANN
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58C, and slowly adding (over 1 h) the brominating agent 4,4-Dibromo-
-methylpyrazol-5-one (0.299 g, 1.17 mmol). After3 h the solution
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took on a purple hue with a small amount of white precipitate. At 24 h
the color had darkened and amount of precipitate greatly increased. The
mixture was filtered and the clear purple supernatant diluted with 30 ml
H O, neutralized with solid NaOH, extracted with Et O (2 Â 15 ml) and
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CH Cl (2 Â 15 ml), dried over K CO , filtered, and solvent removed.
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The composition was determined by H NMR. The mixture was
purified by silica gel chromatography (MeOH/EtOAC/Hexane
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:1.5:7.5) providing white crystals of mass 0.162 g, a 69% yield.
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Preparation of N-labelled malachite green hydrochloride (1): Fine
magnesium ribbon (0.03 g), 5 (0.162 g, 0.810 mmol), and dry THF (3 ml)
were refluxed for 1h in a dry flask. The solution was allowed to cool to
room temperature before addition of methyl benzoate (0.028 g,
0
.025 ml). The solution was refluxed for 10 min then cooled to room
temperature and 0.1 ml of 1 M HCl was added, whereupon the solution
turned a dark green. The volume of the solution was reduced under
vacuum and then purified by column chromatography (Silica gel, 230–
(
00 mesh, 60 A, n-propanol/EtOH/AcOH/H O 5/0.33/1/2). A 34%
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yield was determined by measuring absorbance of malachite green in
À1
H O at 425 nm using a molarextinction coefficient of 30413 cm
2
À1 13
M .
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Synthesis of C-labelled 4-bromo-N,N-dimethylaniline (7) (Scheme 2):
A solution containing 6 (0.143 g, 0.832 mmol), NaBH CN (0.310 g,
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.99 mmol) and THF (5 ml) was slowly added to a solution containing
C-labelled formalin (0.495 g of a 20% solution, 3.33 mmol) and
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H SO (0.337 ml, 6 M) stirring over an ice bath. The reaction was
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allowed to continue for20 h befo re being basified with 5 M NaOH and
subsequently extracted with Et O (3 Â 15 ml). The extract was dried
2
overK CO , filtered and solvent removed. This solid/oil was then
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purified by silica gel chromatography (MeOH/EtOAC/Hexane
1
6
(
:1.5:7.5) giving white crystals with mass 0.115 g, a percent yield of
9% with respect to 6 and of 17% with respect to the labelled precursor
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C-CH O). The product was used without further purification.
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Synthesis of C-labelled Malachite Green (2): Fine magnesium
ribbon (0.02 g), 7 (0.115 g, 0.577 mmol), and dry THF (2 ml) were
refluxed for 1 h in a dry flask. The solution was cooled to room
temperature before addition of methyl benzoate (0.020 g, 0.0179 ml).
The solution was refluxed for 10 min then cooled to room temperature
and 0.2 ml of 1 M HCl was added, whereupon the solution turned a
Copyright # 2002 John Wiley & Sons, Ltd.
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SYNTHESIS OF LABELLED MALACHITE GREEN
247
dark green. The volume of the solution was reduced under vacuum and
then purified by column chromatography (silica gel, 230–400 mesh,
(
0A, n-propanol/EtOH/AcOH/H O 5/0.33/1/2). A 58% yield was
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determined by measuring absorbance of malachite green in H O at
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25 nm using a molarextinction coefficient of 30413 cm À1 MÀ1.
Conclusion
The two synthesis schemes described here provide an easy and
inexpensive access to specific NMR probes for the study of ligand
structure and dynamics in malachite green RNA complexes. For the
purpose of NMR spectroscopy a maximum of material was more
important than purity in this case. The bound dye can easily be
identified by its NOEs to the RNA and the two majorcontaminants
(residual starting material, 4-Br-N,N-dimethylaniline and monomethy-
lated dye) do not interact with the aptamer. If necessary the dye can be
further purified by column chromatography, albeit at a substantial
reduction in yield.
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The NMR results outlined above demonstrate that the C-labelled
methyl groups on rings A and B are in unique electronic environments
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within the aptamer as becomes apparent by their different H and C-
chemical shifts in the bound state (Figure 2). The use of the specifically
labelled dye will allow a first detailed analysis of the ligand dynamics
and electronic structure in an RNA aptamer – ligand complex.
Acknowledgements
This work was supported by NSF grant MCB0110689 to T.D. The
Bruker 600 MHz spectrometer was paid for in part by funds from NIH
grant RR11973. The authors thank Ben Shen and Frank Osterloh for
shared equipment, and Janet Trang for technical assistance.
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