Synthetic

Control of Cytokinin Signaling

2017.5.23 Letitia Sarah

[Introduction]

Cytokinins are a class of plant hormones involved in diverse plant processes, including cell division
(cytokinesis) and senescence delay. Up to date, there are almost 200 naturally occurring and
synthetic cytokinins known. Synthetic cytokinins are beneficial, since they enable selective
functionalization of the hormones in specific organs at a specific time once an appropriate promoter
is introduced. This spatio-temporal regulation allows utilization of cytokinin for obtaining desired
crop traits.
In this project, we will mainly focus on the synthesis of cytokinins substituted with different
functional groups at position 1, 2, 3 and 4 (see figure 1A). These positions are assumed to be
interacting with Val248, Tyr250, Asp262 and Tyr318 of the cytokinin-receptor complex. By modifying
the different positions on the cytokinin, we strive to get selective binding to the genetically modified
receptors over the natural receptors (see figure 1B). This project is in collaboration with the Torii
group.

Tyr318
4
OH
HN
N 3 Asp262
N
H
H N N
H
1 2
Val248

Tyr250

Figure 1. A. Cytokinin at its receptor’s binding pocket, B. Overview of research concept
[Previous work]

New approach towards the synthesis of trans-Zeatin

DMSO (4.0 eq)
Boc 2O (1.0 eq)
oxalyl chloride (2.0 eq)
Et 3N (1.50 eq) O O
H 2N Et 3N (5.0 eq)
OH 4.0 eq

O
OH O N O N
CH2Cl2, 0 °C to rt H H
CH2Cl2, -78 °C to rt
1 2 3
O O 88%
(EtO) 2P (3.0 eq)
OEt

DBU (1.2 eq) O

H 1. DIBAL (2.8 eq), CH2Cl2
LiCl (2.5 eq) O N OH
OEt HCl •H 2N
CH 3CN O 2. 2 M HCl, 2 h
rt, 5 h 4 5

OH
Cl 5 (1.2 eq) HN
N K 2CO 3 (2.5 eq) N
N N

N nBuOH N N
N H
H
110 °C, 5 h
6 7
Scheme 1. New approach towards the synthesis of trans-Zeatin

Reference:

Corder, A. L., Subedi, B. P., Zhang, S., Dark, A. M., Foss, F. W., & Pierce, B. S. (2013). Peroxide-shunt substrate-specificity for
the Salmonella typhimurium O 2-dependent tRNA modifying monooxygenase (MiaE). Biochemistry, 52(36), 6182–
6196

[Current work]

Total synthesis of trans-Zeatin

Trans-Zeatin was managed to be synthesized.
The synthetic route started with synthesis of alcohol 2 through Boc2O and amino alcohol reaction
and followed by Swern oxidation. After performing Swern oxidation and HWE olefination, 4 was
purified using column chromatography. Later, the ester was reduced using DIBAL to its
corresponding alcohol and then acid deprotection of N-Boc group was done. The final step was SN
arylation of 5 and 6 to obtain trans-Zeatin (7). Trans-Zeatin was purified by PTLC and freeze-drying
for DMSO-water removal.

DMSO (4.0 eq)
Boc2O (1.0 eq)
oxalyl chloride (2.0 eq)
Et3N (1.50 eq) O O
H 2N Et3N (5.0 eq)
OH 4.0 eq

O
OH O N O N
CH2Cl2, 0 °C to rt H H
CH2Cl2, -78 °C to rt
1 2 3
O O 88% (LS-28)
(EtO)2P (3.0 eq)
OEt

DBU (1.2 eq) O

H 1. DIBAL (2.8 eq), CH2Cl2
LiCl (2.5 eq) O N OH
OEt HCl •H2N
CH3CN O 2. 2 M HCl, 2 h
rt, 5 h 5
4
19% (over 2 steps, LS-34) 56% (LS-35)

OH
Cl 5 (1.2 eq) HN
N K2CO3 (2.5 eq) N
N N

N nBuOH
N N
N H
H
110 °C, 5 h
6 7
69% (LS-36)

The sample of trans-Zeatin (tZ) was handed to Prof. Uchida for further testing on transgenic
Arabidopsis carrying GFP reporter, which is controlled by TCS synthetic promoter (TCS::GFP). The
figure below showed expression of TCS::GFP in shoot meristem upon introduction of tZ which
confirmed the activity of synthesized tZ.

Figure 2 Dose responses of TCS::GFP by 0.1 μM and 1 μM trans-Zeatin

Syntheses of cytokinin derivatives

Scheme 3 Synthesis route toward cytokinin derivatives

Reagents and conditions: (a) 2, 6-dichloropurine, K2CO3, nBuOH, 110 °C, 4 h; (b) 6-Chloro-2-
fluoropurine, K2CO3, nBuOH, 110 °C, 4 h; (i) phenylboronic acid, palladium(II) acetate,
tetrabutylammonium bromide (iii), (v) pyrrolidine (neat), 80 °C, time to be determined; (iv), (vii)
piperidine (neat), 80°C, time to be determined; (viii) aniline (neat), 80 °C, time to be determined






Determination of 6-chloro-2-fluoropurine selectivity

(1.2 eq) Cl
Cl NH N
N N
N N or N
N
N N N
F N N K2CO3 (2.5 eq) H N
H F N
nBuOH
H
110 °C, 5 h

Determining the selectivity of 6-Chloro-2-fluoropurine is important for obtaining the correct
structure of trans-Zeatin derivatives with substitution at C2. After being analyzed with LC-MS, it is
confirmed that C6 is more susceptible to substitution than C2. Thus, direct substitution of the
functional group to 6-chloro-2-fluoropurine should be avoided.

Synthesis of 8a

OH (1.2 eq)
ClH.H2N
5
Cl OH
(2.5 eq) HN
N K2CO3
N N
nBuOH N
Cl N N
H Cl N N
110 °C, 4 h H
a 8a

under purification

SN arylation to make 8a was initially performed at 80 °C, but the reaction did not proceed. The
temperature was raised to 110 °C.
The targeted product 8a was purified using PTLC, but trace of silica was always found. Filtration over
celite or membrane filter did not seem to work. Purification using reverse phase Isolera will be tried.

Synthesis of 8b

OH (1.2 eq)
ClH.H2N
5 OH
Cl HN
K2CO3 (2.5 eq)
N N N
N
nBuOH
F N N F N N
H 110 °C, 4 h H
b 8b

Product was not detected by LC-MS.


[Future work]

· Purification of 8a
· Continue derivatization of trans-zeatin