Agrochemical Discovery - American Chemical Society

Chapter 20

Biorational Design, Synthesis, and Inhibition of Photosystem II Inhibitors

Downloaded by UNIV OF PITTSBURGH on May 3, 2015 | http://pubs.acs.org Publication Date: December 28, 2000 | doi: 10.1021/bk-2001-0774.ch020

Huayin Liu, Yinlin Sha, Aiming Yu, Huifen Tan, and Huazheng Yang

1

State Key Laboratory of Elemento-Organic Chemistry, Institute of Elemento-Organic Chemistry, Nankai University, Tianjin 300071, China 1Corresponding author.

Molecular modeling of cyanoacrylates(cyanoacrylamides) with D1 protein of Pisum sativum have been presented. Studies show that the binding force includes mainly: H-bond interaction, Van der Waals and π-ring stacking interaction. It was found that SER 268 in D1 protein might be an important binding site. Thus some new cyanoacrylates (cyanoacryl amides) were designed and synthesized. For rapid optimization, combinatorial methods were introduced to synthesize 3-Nsubstituted (2-thio)hydrouracils library by the acidic cycli zation-cleavage of ureas and thioureas from Wang resin. Their Hill inhibition is discussed.

Photosynthesis is a special and important physiological and biochemical phenomenon for plants. Hence the selection of photosynthesis as the herbicidal target is desirable to get nontoxic pesticides. Recently, Deisenhofer has successfully elucidated the three dimensional structure of L protein of the photosynthetic reaction center of Rps. Viridis with X-ray diffraction(l), and this important result makes it possible to design new photosystem II (PS II) inhibitors based on the receptor structure. A large number of inhibitors are known to block electron transport in PS II by displacing plastoquinone from Q -binding niche of the D l protein in the B

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© 2001 American Chemical Society

In Agrochemical Discovery; Baker, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2000.

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reaction center(l,2). Based on the three dimensional structure of L protein of photosynthetic reaction center of Rps. Viridis, the 3D-structure of Pisum sativum 32Kdal(Dl) was constructed by homology modeling method, and the properties, such as steric, hydrophobic, electrostatic properties of active sites region of the D l protein were also characterized by the grid search method with probes of methyl group, H 0 , amino and proton. For molecular modeling , the software SYBYL(Version 6.2) from Tripos Associates Inc. was used(3,4). Cyanoacrylates are potent inhibitors of photosynthetic electron transport. A number of studies concerning the inhibition of photosynthetic electron flow in PS II with a series of cyanoacrylate inhibitors have shown that the potency of cyanoacrylates in blocking photosynthetic electron flow is extremely sensitive to minor structural variation(5,6). The structures and inhibition(pl value) of cyanoacrylates(cyanoacrylamides) are shown in Table 1. It was considered that these inhibitors would be useful probes for determining the nature of the receptor topography.


2

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Table 1 The structures and inhibition(pl value) of cyanoacrylates(6) R ^ H ^ C O X R 50

E f C N No. la Hi 1c

1

R P-C1C H -C1C H CH P-C1C H CH 6

P

6

4

6

4

4

2

2

ι

XR OCzHtOEt OC H OEt NHC^OEt 2

4

PI50

5.70 8.20

Thus, based on the constructed receptor model, cyanoacrylates(cyanoacrylamides) were selected for molecular modeling in order to find additional active sites in D l protein.

Binding of Cyanoacrylates(Cyanoacrylamides) with Dl Protein Plate 1 shows compound l b in the Q binding pocket of D l protein. It appears that the oxygen atom of the ester carbonyl group in cyanoacrylates binds to the hydroxyl of SER 264 residues in agreement with the literature(7,8), and the nitrogen atom in cyano group binds to LEU 271 residues. On the other hand, there are significant hydrogen bond and electrostatic interaction of the oxygen atom in an ethoxyethyl group with hydroxyl group of SER 268, resulting in higher Hill inhibition than those with the only alkyl ester. It is obvious that an oxygen atom in the alkyl group of ester is very important, and this can be B



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Plate 1 Simple Complex map of Compound lb interacted with Dl protein Pisum Sativum. Van der Waals and π-ring stacking interaction between PHE 211, PEE 255, PEE 265, PEE 274; Ε-bonding interaction between it a SER 264, SER 268, LEU271. (Reproduced with permission from reference 9. Copyright 1999.)

(This plate is printed in color in the color insert between pages 246 and 2



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Plate 2 Simple Complex map of Compound lç interacted with Dl protein Pisum Sativum. Van der Waals and π-ring stacking interaction between i PHE 211, PHE 255, PHE 265, PHE 274; Η-bonding interaction between it a SER 264, SER 268, LEU271. (Reproduced with permission from reference 9. Copyright 1999.)

(This plate is printed in color in the color insert between pages 246 and 2


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explained by the binding of an electronegative atom with SER 268 hydroxyl group. The phenyl moiety of cyanoacrylates is oriented into the pocket composing of PHE 211, PHE 255, PHE 265 and PHE 274, by mainly Van der Waals and πstacking interactions. The aromatic interaction is influenced by the existence of methylene groups separating a phenyl from the amino group. Compared to compound l a , compound l h has one methylene between the phenyl and the amino group, and the phenyl moiety goes into the pocket more deeply. So compound l b has a closer interaction with the D l protein, and henceforth has a higher p l value (pl =8.20) than compound l a (pl =5.70). So cyanoacrylates with benzylamine group could have improved interaction and binding with D l protein, and thus have high inhibition. It could be concluded that those cyanoacrylates with an electronegative atom in alkyl of ester group and benzyl amine group have good interaction with D l protein, and thus exhibit high inhibition. Based on above result, the complex model of acrylamides 1c with D l protein was constructed(Plate 2). Acrylamide 1c has the key structure as acrylates l h necessary for binding with D l protein, such as an electronegative atom in side amide, and benzyl amino group. So it could be expected that this acrylamide should have Hill reaction inhibition.


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Synthesis and Bioassay of Cyanoacrylates(amides) The synthetic route(9,10) to the designed compounds is shown as Figure 1.

NCCH COOH 2

HXR

^NCCH COXR 2

Me2S0

MeS,

,COXR

>==< CN MeS

Figure 1 The synthesis route to the designed cyanoacrylates(cyanoacrylami


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Table 2 The structures of cyanoacrylates(cyanoacrylamides), binding energies of interaction between inhibitors and D l protein and their Hill inhibition (pl value) 50

R*NH

.COXR


MeS No.

R

2a

CH

2b 2c 2d 2s 2f 2g 2h 2i

C6H5CH2

6

1

2

CN

XR OC2H5

5

C6H5CH2 C6H5CH2 C6H5CH2 C6H5CH2 C6H5CH2

OC H 0 CH CH 0 CH 0 CH CH 0 CH CH NH CH CH 0 CH NH CH CH CH 0 CH 2

5

2

2

2

2

2

2

C6H5CH2

21

C6H5CH2

C6H5CH2

3

2

2

6

4

Binding Energy(kJ/moL)

5.93 6.66 7.73 8.00 7.42 7.28 6.35

-370.10 -410.73 - 607.48 - 632.94 - 564.47 - 483.92

7.47

3

OCH CH OC H (CH ) -5, 4

7.20

OC H (OCH M OCH CH=CH

4.33 7.10 6.41

2

2i 2k

3

2

0CH CH C1 OCH CH OC H (CH )-2 2

C6H5CH2

3

2

2

2

C6H5CH2

3

2

Plso

2

6

6

4

3

3 2

3

2

2

OCH OCH Source: Reproduced with permission from reference 9. Copyright 1999. Compounds were assayed for Hill inhibition using suspensions of chloroplasts isolatedfromthe leaves of 20 day old plants of Pisum sativum. The experimental procedure was as the method described elsewhere(ll). The activity of a compound as a Hill inhibitor was expressed in terms of its p l value i. e. lgIC , where IC was the molar concentration required to decrease the rate of dye reduction under illumination of saturating intensity to 50% that obtained in the absence of the compound. All the Hill inhibition of compounds 2a -21 are recorded in Table 2. Compound 2a and 2b were also synthesized for comparison. Bioassay indicate that all the synthesized compounds have high Hill inhibition and compound 2a and 2h show lower inhibition (pl =5.93 and 6.66) as expected. Compound 2d has higher inhibition (pl =8.00) than compound 2£(pl =7.73), and the binding energy of compound M and 2Ê with D l protein are lower than that of compound 2a and 2b. These results show that the binding site of D l protein with alkyl of ester group in cyanoacrylate is large, and that the hydrophobic property might effect inhibition. On the other hand, compound 2e 2

50

50

50

50

50


50

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and 2f also show high Hill inhibition, and cyanoacrylamides might be a new kind of photosynthesis inhibitors. The inhibition of compound 2e and 2f are higher, and the binding energy of compound 2e and 2f with D l protein are lower than that of compound 2a and 2h. This indicates that the distance between carbonyl group and electronegative atom in alkyl chain might effect inhibition.


Combinatorial Synthesis of Hydrouracil Library As we know, various heterocyclic compounds containing nitrogen atoms, especially uracils, have biologically interesting properties in medicinal and pesticidal chemistry for example as fungicides and herbicides(12). Our studies(13) show that these uracils were lead compounds as PS II inhibitors. Meanwhile, combinatorial chemistry has emerged as an efficient tool to synthesize "compounds libraries" for the rapid identification and optimization of new lead compounds in drug and agrochemical discovery(14). Solid-phase organic synthesis provides a rapid means for preparation of compounds libraries, and has been successfully used for the construction of oligomeric compounds and small organic molecules(15), especially the heterocyclic compounds(16) libraries. So combinatorial synthesis was introduced to optimize lead compounds uracils. The solid-phase synthesis approach was depicted in Figure 2(17). The acryloyl chloride or acid was coupled to Wang resin 3 to afford the acryloyl ester 4. Resin bound ester 4 was reacted with primary amines to give secondary amines 5, which were converted to the ureas 6 by the treatment with isocyanates. Preparation of the 3-substituted hydrouracils 7 was achieved by acidic cyclization-cleavagefromthe resin.

Figure 2 The synthesis route to hydrouracils (Reproduced with permission from reference 18. Copyright 1998.)



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The reaction time course was monitored using an improved KBr pellet FTIR method and MAS *HNMR method(18). Microwave technique was introduced to improve the efficiency of solid phase organic reactions. The reaction "real time" under normal condition and under microwave irradiation are determined by analyzing the reaction time course obtained. The results indicate that the reaction process is greatly enhanced under microwave irradiation, which shows as an efficient approach for solid phase organic synthesis. In order to improve molecular diversity of compounds library, other kinds of reagents were used as building blocks in this synthesis. Isothiocyanates were reacted with Wang resin bound amines 5 to afford thioureas 8, which formed 2thiohydrouracils 9 after the cleavage from the resin (Figure 2). A small library containing 40 (2-thio)hydrouracil derivatives(Ri=H, R =H) was synthesized with 10 primary amines(Table 3) and 4 iso(thio)cyanates(Table 4). GC-MS analyses of the library indicated that all the desired compounds were contained in them. 2

The small libraries were submitted to test their photosynthesis inhibition. Using "mix and split" method(19), the active component (Figure 3, 1050=8.63 ppm) was foundfromthe library(IC =10.96ppm) with 40 compounds. 50

Table 3 Structure of 10 primary amines used in combinatorial libray


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Table 4 Structure of 4 iso(thio)cyanates used in combinatorial libray

Figure 3. The structure of active component 10 from the library

Conclusions Interaction modes of the cyanoacrylates(cyanoacrylamides) with QB binding pocket in D l protein of Pisum Sativum have been presented. The essence of these modes include mainly two binding regions: phenyl moiety(Van der Waals and π-ring stacking interaction), carbonyl group and its alkyl substituent(H-bond interaction). It was found that SER 268 in D l protein might be an important binding site. In order for compounds to exhibit strong inhibition, it is necessary to have an electronegative atom as a substituent in the alkyl ester which can undergo a Η-bond interaction with SER 268 of the D l protein. It was also found that cyanoacrylamides with the key sturcture feature of cyanoacrylates are photo synthesis inhibitors with potent activity. We also have developed a general method for the solid-phase synthesis of (2-thio)hydrouracil analogues, which have been shown to inhibit the Hill reaction. The solid-phase approach has been applied to the combinatorial synthesis of a small library containing 40 (2-thio)hydrouracil analogs. Using "mix and split" methodology, the active component was foundfromthe library.


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Acknowledgement This project is supported by the National Natural Science Foundation, P. R. China( 29702006, 29832050), the Research Fund for the Doctoral Program of Higher Education, P. R. China(98005519), the Key Fund of Technology of Ministry of Education, P. R. China and the Key Fund of Nature Science of Tianjin, P. R. of China.


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