Synthesis of two bi-functional ligands for the QUEST three-hybrid system

Article abstract of DOI:10.1016/S0040-4039(00)01202-8

The synthesis is described for two bi-functional ligands (1) and (2) that have been used as chemical inducers of dimerization for QUEST,¹ a three-hybrid system designed to detect novel enzyme activity in vivo. (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0040-4039(00)01202-8

Tetrahedron Letters 41 (2000) 7009±7012
Synthesis of two bi-functional ligands for the QUEST
three-hybrid system
Stephen J. Baker, Steven M. Firestine, David Smithrud, Frank Salinas
and Stephen J. Benkovic*
414 Wartik Laboratory, Pennsylvania State University, University Park, PA 16802, USA
Received 17 June 2000; accepted 7 July 2000
Abstract
The synthesis is described for two bi-functional ligands (1) and (2) that have been used as chemical
inducers of dimerization for QUEST,1 a three-hybrid system designed to detect novel enzyme activity in
vivo. # 2000 Elsevier Science Ltd. All rights reserved.
Recently, there has been much interest in protein±protein and protein±ligand interactions.
While a number of methods exist to study these types of interactions, methods related to the yeast
two- and three-hybrid systems have proven especially attractive. Protein±protein interactions can
be studied using the yeast two-hybrid system2 while protein±ligand interactions can be studied
using the yeast three-hybrid system.3 In the yeast two-hybrid system2 (Fig. 1A), protein±protein
interactions are detected based upon the dimerization of transcriptional proteins that produce a
detectable signal.
Figure 1.
* Corresponding author. Tel: +1 814 865 2882; fax: +1 814 865 2973; e-mail: sjb1@psu.edu
0040-4039/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0040-4039(00)01202-8

7010
In the three-hybrid system3 (Fig. 1B), the dimerization domain is replaced by a ligand-binding
domain that is incapable of forming a dimer. Activation of the transcriptional event only occurs
in the presence of a bi-functional ligand otherwise known as a chemical inducer of dimerization
(CID), which results in the formation of an arti®cial dimer, causing transcription and an
observable response. Recently, we published a derivative of the three-hybrid system, termed
QUEST,1 (QUerying for EnzymeS using the Three-hybrid system) which can detect novel
enzymatic activities in vivo. Our initial target was to detect the fungal enzyme scytalone
dehydratase in bacteria. To investigate this system, a mutant non-catalytic scytalone dehydratase
(SD) was chosen as the ligand binding domain of the transcriptional protein. Two CIDs, (1) and
(2) (Fig. 2), were designed for QUEST based upon two inhibitors of SD that have di€erent Ki
values. From an X-ray crystal structure4 of SD, the ligands needed to be separated by at least
18A in order to overcome steric requirements and e€ect dimerization of the DNA transcription
proteins. To satisfy this parameter, the terminal ligands were separated by a central linking region
that was 23A in length. Here, we wish to report the synthesis of CIDs (1) and (2).
Figure 2.
CID (1) was synthesized as described in Scheme 1. Benzoyl chloride (3) was treated with
4-aminomethylbenzoic acid (4) in dichloromethane with triethylamine to a€ord 4-benzoylamino-
methylbenzoic acid (5)5 in 46% yield.
Scheme 1.

7011
The carboxylic acid group of compound (5) was then converted into a mixed anhydride using
isobutylchloroformate in tetrahydrofuran with triethylamine, which was subsequently treated
with 1,8-diaminooctane to furnish 4-benzoylaminomethyl-N-(aminooctyl)benzamide (6)6 in 71%
yield after column chromatography puri®cation on silica gel. Two molar equivalents of
compound (6) was then treated with 1 molar equivalent of ethylene diamine tetraacetic dianhydride
(7) to complete the synthesis of ligand (1),7 which was of sucient purity to be used directly in the
biological studies.
CID (2) was synthesized as described in Scheme 2. 4-Hydroxyquinazoline (8) was heated to
re¯ux in phosphorus oxychloride with phosphorus pentachloride8 to a€ord 4-chloroquinazoline
(9) in 85% yield. This quinazoline (9) was subsequently treated with 4-aminomethylbenzoic acid
(4) in iso-propanol with triethylamine to give 4-(p-carboxybenzyl)aminoquinazoline (10)9 in 55%
yield. The same methodology to synthesize the central linking region described for CID (1) was
attempted but this was met with limited success. After conversion of the carboxylic acid of the
4-aminoquinazoline (10) to a mixed anhydride and subsequent treatment with 1,8-diaminooctane,
we were unable to purify the product formed. This was due to the highly polar nature and low
solubility of the product, which prevented us from separating it from the excess 1,8-diaminooctane
used in the reaction. We decided to modify the central linking region to circumvent this problem.
The carboxylic acid of the 4-aminoquinazoline (10) was converted to a methyl ester by using
methanol saturated with hydrogen chloride giving 4-(p-methylbenzoate)aminoquinazoline (11)10
in 92% yield. The methyl ester (11) was then heated to re¯ux in ethylene diamine to furnish
4-(p-N-(2-aminoethyl)benzamido)aminoquinazoline (12)11 in quantitative yield. In the ®nal step,
2 molar equivalents of ethylene diamine tetraacetic dianhydride was treated with 1 molar
equivalent of diaminoethane in dimethylsulfoxide giving the dianhydride adduct (13). This was
then treated with 2 molar equivalents of the aminoethylbenzamide (12) to complete the synthesis
of ligand (2),12 which was also of sucient purity to be used directly in the biological studies.
Scheme 2.

7012
Acknowledgements
We gratefully acknowledge Douglas Jordon and Gregory Basarab (DuPont) for information
and materials. S.M.F. was funded by a fellowship from the Damon Runyon±Walter Winchell
foundation and F.S. was funded by a fellowship from the National Institutes of Health.
References
1. Firestine, S. M.; Salinas, F.; Nixon, A. E.; Baker, S. J.; Benkovic, S. J. Nature Biotech. 2000, 18, 544±547.
2. Vidal, M.; Legrain, P. Nucleic Acids Res. 1999, 27, 919±929.
3. Spencer, D. M.; Wandless, T. J.; Schreiber, S. L.; Crabtree, G. R. Science 1993, 262, 1019±1024.
4. Lundqvist, T.; Rice, J.; Hodge, C. N.; Basarab, G. S.; Pierce, J.; Lindqvist, Y. Structure 1994, 2, 937±944.
5. Selected data for compound (5): 1H NMR (CD3OD, 360 MHz) ꢀ 7.98 (d, J=8.3 Hz, 2H), 7.85 (m,2H), 7.55±7.43
(m, 5H), 4.63 (s, 2H); m/z (EI) 255 [M]+; found: 256.0988, C15H13NO3+H requires: 256.0904.
1
6. Selected data for compound (6): H NMR (CD3OD, 200MHz) ꢀ 7.87±7.75 (m, 4H), 7.49±7.42 (m, 5H), 4.61 (s,
2H), 2.88 (t, J=7.4 Hz, 2H), 1.62 (br s, 4H), 1.38 (br s, 10H); m/z (FAB, Glycerol) 382 [M+H]+; found: 382.2469,
C23H31N3O2+H requires: 382.2495.
7. Selected data for compound (1): m/z (FAB, Glycerol) 1019 [M+H]+, 1041 [M+Na]+.
8. Endicott, M. M.; Wick, E.; Mercury, M. L.; Sherrill, M. L. J. Am. Chem. Soc. 1946, 68, 1299.
1
9. Selected data for compound (10): H NMR (CD3OD, 360 MHz) ꢀ 8.63 (s, 1H), 8.29 (d, J=8.7 Hz, 1H), 7.92 (t,
J=7.8 Hz, 1H), 7.85 (d, J=8.3 Hz, 2H), 7.71±7.65 (m, 2H), 7.39 (d, J=8.2 Hz, 2H), 4.96 (s, 2H); m/z (CI) 279
[M]+; found: 280.1074, C16H13N3O2+H requires: 280.1086.
10. Selected data for compound (11): 1H NMR (CD3OD, 200 MHz) ꢀ 8.79 (s, 1H), 8.44 (d, J=8.7 Hz, 1H), 8.11±7.98
(m, 3H), 7.86±7.79 (m, 2H), 7.55 (d, J=8.2 Hz, 2H), 5.11 (s, 2H), 3.87 (s, 3H); m/z (CI) 294 [M+H]+; found:
294.1234, C17H15N3O2+H requires: 294.1243.
11. Selected data for compound (12): 1H NMR (CD3OD, 200 MHz) ꢀ 8.41 (s, 1H), 8.18 (d, J=8.6 Hz, 1H), 7.83±7.68
(m, 4H), 7.53 (m, 1H), 7.47 (d, J=8.4 Hz, 2H), 4.90 (s, 2H), 3.46 (t, J=6.2 Hz, 2H), 2.84 (t, J=6.5 Hz, 2H); m/z
(FAB, 3-NBA) 322 [M+H]+, 344 [M+Na]+, 643 [2M+H]+; found: 322.1657, C18H19N5O+H requires: 322.1668.
12. Selected data for compound (2): m/z (FAB, 3-NBA) 1215 [M+H]+.