学位申請論文

C
大環状テトラアミン亜鉛錯体を用いた
リ ン 酸 エ ス テ ル の￨加水分解
ーアルカリホスファターゼ、における亜鉛イオンとセリン残基の役割‑

広島大学大学院医学系研究科分子薬学系専攻活性構造化学

指導教授

木村栄一

玉

児

論文要旨
アルカリホスファターゼ (AP)は、リン酸モノエステルの加水分解やリン酸転移反応を触媒する亜鉛酵素であり、その活性中心は 2つの亜鉛イオンとそれらを同定するア
ミノ酸残基および亜鉛イオン近傍のセリン残基とから構成されている O 現在、この酵素の反応機構として、リン酸モノエステルのリン酸がセリン残基に転移したホスホセリン中間体の存在が確認されている O この中間体の形成には、亜鉛イオンとセリン残

I
‑
‑
O
R
)の関与が推察されているが明確な証拠は得
基とから生成したアルコキシド(ZnI
られていない 。また、他の多くの亜鉛酵素による加水分解反応における I厄鉛イオンに配位した水酸化物イオン (Znll‑OH‑)による l段階反応に比べ、中間体を経由する反応機構の有利な点も得られていない 。
私は、 AP のセリン残基や亜鉛イオンの役割および反応機構の解明を日的として、

1)ベンジルアルコールをペンダントに持つサイクレンを新規合成し、 2)その亜鉛錯休を単離した O 亜鉛錯体は、アルコールペンダントが亜鉛イオンに配位したアルコール型錯体およびそのアルコール型錯体のアルコールからプロトンが解離したアル
商定法により亜鉛錯体の亜鉛イオンに配位したコキシド型錯体を各々単離した 。pH i.
5
1 と決定され、アルコールは亜鉛イオンに
アルコールベンダントの酸解離定数は 7
配位することによって生理 pH付近でアルコキシドを形成できることが示唆された 。

3)さらに、アルコキシド型錯体の X 線結品構造解析より、この錯体においてアルコ
キシドが亜鉛イオンに配位していることがわかった 。 4)亜鉛錯体とピス (4‑ニトnll‑
ロフェニル)リン酸 (BNP)との反応を N加R によって追跡した結果、この反応は、 Z‑ORが BNP を求核攻撃することによってリン酸エステルがアルコールペンダントに転移したリン酸ジエステル中間体を形成したのち、その中間体のリン酸ジ、エステルペンダントが分子内 Znll‑OHーによって加水分解され、リン酸モノエステルペンダントを持つ最終生成物が生成することがわかった 。最終生成物においては、リン酸モノエス
テルベンダントの亜鉛イオンへの配位によって亜鉛錯体の加水分解活性が失われていた 。 このことから、リン酸モノエステルペンダントの加水分解には、 Znll‑cyclen などの求核体の分子内へ導入が必要性であることがわかった 。 5)中間体は単離、同定した 。 6) BNPと亜鉛錯体とから中間体が生成する反応を pH6‑10で検討した 。得られたシグモイド曲線から速度論的酸解離定数は 7.
4 となり、 pH滴 定 法 か ら 求 め た 亜
鉛錯体の酸解離定数 7.
3とほぼ一致した 。 この結果、この反応の活性種はアルコキシ
1
S‑
1 と決定
.
5X 1
0‑4 M‑
ド型錯体であると結論された 。 この反応の 2次反応速度定数は 61され、 ZnII‑methylcyclenによる BNPの加水分解反応速度定数 5.
2x 10.{j M‑
1
s‑
2
5倍
の1
にもなり、 ZnII‑‑ORはZnII‑OHーよりも強い求核体であることがわかった 。 7)中間体か
ら最終生成物へのリン酸ジエステルペンダント加水分解反応を pH6‑
10で検討した O
得られたシグモイド曲線から速度論的酸解離定数は 9.
0 となり、 pH 滴定法から求め.
1 とほぼ一致した 。 この結果、この反た中間体の亜鉛イオンの配位水の酸解離定数 9応の活性種は分子内 ZnII‑OH‑で、あると結論された 。この反応の l次反応速度定数は 3.
5X1
0‑S‑1 と決定した 。 8)同様の加水分解反応が分子間で起きた場合のモデルとして
5

I
I
Zn
‑
m
e
t
h
y
l
c
y
c
l
e
n と 4‑ニ ト ロ フ ェ ニ ル エ チ ル リ ン 酸 と の 反 応 (2次 反 応 速 度 定 数 :

7M‑
1
7.9x1
0
‑
s
‑
l
) を考えると 、速度定数の比較から、リン酸ジエステルと亜鉛錯体との分子間反応、が分子内で起きることによる有効モル濃度は 45Mとなった O この結果、基質を求核体の非常に近い位置に固定することによって反応性が上がることがわかった。
これら新規亜鉛錯体を用いた実験から、 APの反応機構において、 1)Z
n
l
l
‑‑
ORは生

理 pHの水溶液中で容易に生成すること、 2)ZnII‑‑ORは ZnII‑OHーよりも求核性が強く、リン酸転移反応が容易に起きること、 3)ホスホセリン中間体は 、亜鉛イオン近傍にあり、ZnII‑OHーによる加水分解の反応性が高いこと、

4)リン酸モノエステルと亜鉛錯体とは 1:1の複合体を形成するため、リン酸モノエステルの加水分解には 2分 子の亜鉛錯体が必要であることがわかった 。このモデル化学的反応の検討により生体機能の解明に大きく貢献できたと考える O

Contents

Abs tract
1. In trod uction
2. Results and Discution
2-1. Synthesis of ~enzyl Alcohol-Pendant Cyclen
2-2. Protonation and Zinc(II) Complexation Constants of the Benzyl AlcoholPendan t Cyclen
2-3. X-ray Crystal Structure of the Zn II Complex of the Deprotonated Benzyl
Alcohol-Pendant Cyclen
2-4. Net Reaction of the Benzyl Alcohol-Pendant Cyclen-ZnII Complex with
Bis(4-nitrophenyl) Phosphate
2-5. A Kinetic Study of the Initial Reaction
2-6. Isolation of the Phosphoryl Intennediate
2-7. Spontaneous Hydrolysis of the Pendant Phosphodiester in the Phosphoryl
Intennediate to a Phosphomonoester by the Intramolecular Zn ll-OH2-8. Hydrolysis of Ethyl (4-Nitrophenyl) Phosphate with an Intennolecular
Nucleophile Zn II-OH-

page
1
2

5
5
7
10
12
14
14
16

3. Summary and Conclusions

17

4. Experimental Section

18

References and Notes

23

Acknowledgement25

Phosphodiester Hydrolysis by a New Zinc(II) Macrocyclic Tetraamine Complex withan Alcohol Pendant:

Elucidation of the Roles of Ser-102 and Zinc(II) in Alkaline

Phosphatase
Abs tract:

A new benzyl alcohol-pendant cyclen (cyclen = 1,4,7, 10-tetraazacyclododecane) ligand,(S)-1-(2-hydroxy-2-phenylethyl)-1,4, 7, 10-tetraazacyclododecane (L) has been synthesized. The
complexation of L with Zn ll yielded 1: 1 five-coordinate complexes (isolated as its perchlorate salts
with the pendant alcohol either undissociated (ZnL) or dissociated (ZnH_I L) from acidic (pH 6.0) or
basic (pH 9.5) aqueous solution, respectively).

The pK a value for the pendant alcohol (ZnL~

ZnH_1 L + H+) was determined by potentiometric pH titration to be 7. 30 ± 0.02 at 35 °C with I = 0 . 10
(NaN03 ).

The X-ray crystal study of ZnH _1 L has shown two crystallographically distinct structureswith the alkoxide closely coordinated at the fifth coordination site, where an average distance of
Zn-O- is 1. 91 A.

The Znll-bound alkoxide anion in ZnH_IL is a more reactive nuc1eophile thanN-methylcyclen-Znll- OH- species (Znll- Me-cyclen).

In the kinetic study using ZnL in aqueous

solution (pH 6.0-10.3) at 35 °C with 1= 0.10 (NaN03 ), the rate-pH profile for a phosphoryl transfer
reaction from bis( 4-nitrophenyl)phosphate (BNP-) to ZnH_IL gave a sigmoidal curve with an
inflection point at pH 7.4, which corresponds to the pKa value for ZnL ~ ZnH_1 L + H+. Theof (6.5 ± 0.1) x 10- 1 M- 1 S-1 is 125 times greater than thesecond-order rate constant k
BNPcorresponding value of (5.2 ± 0.2) x 10- 6 M- 1 S-1 for BNP- hydrolysis catalyzed by Zn I1- Me-cyc1en.
The product of the phosphoryl transfer reaction from BNP- to ZnH _l L is the pendant alcoholphosphorylated ZnH_lL-NPP, which was isolated as its perchlorate salts ZnL-NPP by reacting
ZnH_l L with BNP- in DMF. In anhydrous DMF solution, the phosphoryl transfer (k BNP of 1. 1 ±O. 1 M- 1 S-1 at 35 °C) is 1700 times faster than that in aqueous solution. In the subsequent reactionof ZnL- NPP, the pendant phosphodiester undergoes an intramolecular nucleophilic attack by the
Znll-bound OH- of ZnH_1L-NPP to yield a phosphomonoester product ZnL-P.

From the sigmoidal

rate- pH relationship (pH 7.4- 10.5), the kinetic pKa value of 9.0 was estimated for ZnL-NPP~

ZnH_IL-NPP + H+, which is almost the same value (pKa = 9.10 ± 0.05) determined by potentiometricpH titration at 35 °e.

The first-order rate constant for the reaction ZnH_lL- NPP -...- ZnL-P is(3.5 ± O. 1) x 10- S- I at 35 °e with I = 0.10 (N aN03 ).
5

As a reference to this intramolecular

phosphodiester hydrolysis, ethyl (4-nitrophenyl) phosphate (NEP-) was hydrolyzed by Zn I1- Mecyclen. The second-order rate constant kNEP was (7.9 ± 0.3) x 10- 7 M- 1 S- 1 at 35 °e with I = O. 10(N aN03).

Thus, the intramolecular hydrolysis is 45 000 times faster than the intennolecular NEP-

hydrolYSis with 1 mM Zn I1-Me-cyclen. The present findings that demonstrate the potential of theproximate alcohol by Zn ll in the initial phosphoryl transfer and the potential of the Znll-bound water in
the intramolecular phosphate hydrolysis may well serve to elucidate the collaborative functions of
Ser-102 and Zn II ions in alkaline phosphatase.

11. Introduction

Alkaline phosphatase (AP) is a ZnII-containing enzyme that nonspecifically hydrolyzes
phosphate monoesters (ROP03 2 - ) at alkaline pH. 1

Intensive studies have been done on E. coli

On the basis of X-ray structure2 and NMR study 3 of native and metallosubstituted AP, it is now accepted that, at the AP active center consisting of two Zn II ions (ca 4 A
alkaline phosphatase.

separation), a substrate monophosphate is initially attacked by Ser-l02 in 1 to yield a phosphoserylenzyme intermediate 2, which subsequently is attacked by the adjacent ZnII-OH- to complete thehydrolysis 3~

4 and reproduce the free form of serine to reinitiate the catalytic cycle (see Scheme

1).4

Scheme 1
ASP327
OH2\. I
R
His331 - Znl~, /A site

./

'0)

/

\ - 0

HlS412·0" ~-

, ,"

ASPS1

II

. /

o·

/

ASP369- Zn - -B site\-Znll-OH2

o·

-ROH

0

\.

----- -z'\ 11--·0 -PIIIn)

HIS3700 '0·Ser102

S
er102

1

J

2
Phosphoseryl
Intermediate-HOP032.

-H+jl +H+ pKa -7.5

+ROP032'\-Znll-·OH,,/ /o~ ,,0

...

-

Zn ll

I

,P,O·

~---O

./

Ser102

4
3
Alkaline Phosphatase Active Center
In Scheme 1, the A-site Zn II serves to coordinate the phosphate substrate to make it vulnerable tothe attack of Ser-l 02 that is potentiated by the B site Zn II (1). After the phosphate is transferred fromthe substrate to Ser(l02) (2), the vacated coordination site of the A-site Zn II activates an H 2 0 as Zn IIOH-, which becomes an intramolecular nucleophile in the final dephosphorylation (3 ~ 4).

Thewild-type AP reaches maximal activity around pH 8, where the rate-limiting step is release of thetightly bound inorganic phosphate (the product) from the enzyme-product complex 4.
inorganic phosphate is a competitive inhibitor.
stable. 3d ,sAccordingly,

At pH < 5.5, the phosphoseryl intermediate (2) is2

There are some intrinsic questions concerning the AP mechanism, such as (i) what is the special
advantage in forming the phosphoseryl intermediate 2 for indirect hydrolysis and (ii) how does the
Ser-l02 associated with the Zn II ion become a nucleophile? Recently, the Ser-l02 in AP was
replaced using site-directed mutagenesis by Leu or Ala. 6

The mutant enzymes still catalyzed thephosphate hydrolysis, with similar rate-pH profiles, although the catalytic efficiency is 1/500 to
1/1000 of that of the wild-type enzyme, for which the direct hydrolysis of the substrate by ZnII-OH
was proposed.
more prevalent.

In other hydrolytic metalloenzymes, the direct hydrolysis by M-OH- species seems
7

There have been numerous studies of phosphatase model systems using simple metal
complexes,8,9 but most of these models have been built for the sole M-OH- systems as nucleophiles,

while few were concerned with the net reaction initiated by the metal-bound alcohol, followed by themetal-bound water, as was revealed by AP.
Recently, our laboratory discovered that ZnII-1,5,9-triazacyclododecane ([l2]aneN 3) complex
10
5a ,1l and ZnII-1,4,7,10-tetraazacyclododecane (cyclen) complex 6a l2 can activate an H 20 as the
ZnII-bound OH- species 5b (pKa = 7.3) and 6b (pKa = 7.9) and that both catalyze hydrolysis ofcarboxyesters, IDa< f3-lactams,12 phosphotriesters, and phosphodiesters. loc Our laboratory further
disclosed that the alcohol-pendant N -hydroxyethyl on [l2]aneN3 and cyclen formed 1: 1 Zn II
complexes 7 13 and 8, 14 which more efficiently catalyze 4-nitrophenyl acetate hydrolysis. These
were the novel tTIodels of metallocatalysts that indirectly hydrolyze the carboxyl ester via the ratelimiting "acyl intermediates" 9 and 1 0, respectively.

Using 8 and bis( 4-nitrophenyl) phosphate(BNP-), we also had checked the phosphatase activity in aqueous solution.

The phosphoryl transfer

reaction from BNP- to 8 could be followed as 4-nitrophenolate production,15 but the following
reaction was too complex to allow the elucidation of the total reaction mechanism.
X

Czl0

NH

L~-->

x

/1---t?"Z:N)
HN'------./NH

Sa: X = H20
b: X = HO-

6a: X = H20
b: X = HO-

7

8

9

10

3

In this study, I synthesized a new benzyl alcohol-pendant cyclen 11, bearing a chual carbon
adjacent to the phenyl group. I have discovered that 11 yields a 1: 1 Zn II complex whose
phosphoester bond cleavage activity has a very distinct reaction mechanism.

I herein describe anovel chemical model for ZnII-involving serine enzymes as part of our series of studies on the intrinsic
chemical prope11ies of Zn II in alkaline phosphatase.

114

2. Results and Discussion
2.1. Synthesis of (S)·I.(2.Hydroxy.2.phenylethyI).1,4, 7, 10-tetraazacyclododecane(Benzyl Alcohol-Pendant Cyclen, 11) (Scheme 2). The macrocyclic dioxotetraamine 12 16
and (S)-styrene oxide were heated to reflux in EtOH for 1 day to obtain (S)-1-(2-hydroxy-2phenylethyl)-5, 9-dioxo-1 ,4,7, 10-tetraazacyclododecane 13 in 46% yield.

Both amide groups werereduced with BH3-THF complex in THF to give (S)-1-(2-hydroxy-2-phenylethyl)-1,4,7,10tetraazacyclododecane (11), which was isolated as its tetrahydrochloride salt from 6 M HCl aqueous
solution in 620/0 yield.

Using rae-styrene oxide as the starting material, the corresponding racemic
For all the following studies, we used the enantiomeric product 11.

ligand was yielded in 24 %.

Scheme 2i) BHyTHF..

ii) 6M He}

1213

11·4HCl

2·2. Protonation and Zinc(II) Complexation Constants of the Benzyl AlcoholPendant Cyclen (11).

The protonation constants (KJ of 11 were determined by potentiomenic

pH titrations of 11· 4HCl (1 mM) using 0.10 M NaOH with I = 0.10 (NaCl04) at 25 °c.
pH titration curve is shown in Figure 1a.

A typical

The titration data were analyzed for equilibria 1-4.

The

mixed protonation constants K j-K 4 (C4r+ is the activity of H+) are defined as follows:
L+ H+

~

HL+H+

: K j = [HL]/[L]~+

(1)

H2L

: K 2 = [H 2L]/[HL]C4r+

(2)

H3 L

: K 3 = [H3L]/[H2L]~+

(3)

H4 L

: K 4 = [H4L]/[H3L]~+

(4)

HL

~

H2L + H+---

H3L + H+~

Table 1 summarizes the obtained protonation constants (log K n) in comparison with the reported
K n values of cyclen and N -(hydroxyethyl)cyclen (HE-cyclen) under the same conditions.

The K 1and K 2 values of 11 are extremely large with respect to K 3 and K 4 values, which are roughly the sameas those of cyclen and HE-cyclen.

5

11

9

pH
7

5

3

Figure 1. Typical titration curves for 11 at 25°C with I = 0.10
(NaCl04 ): (a) 1.0 mM of 11·4HCl; (b) a + 1.0 mM ZnS04 •Table 1. Comparison of the Protonation Constants of Cyclen Ligands and Zn ll Complexation
Constants (a)

11

(a) K

cyclenHE-cyclen

methylcyclen

log Kl10.92 ± 0.05 (b)11.04 (f)10.72 (f)log K 2

8.87 ± 0.03 (b)9.86 (f)9.28 (f)log K 3< 2 (b)< 2 (f)< 2 (f)log K4< 2 (b)< 2 (f)< 2 (f)log K(ZnL)

13.6 ± 0.1 (e)15.3 (f)13.8 (f)15.1 (i)pKa 25 °C

7.51 ± 0 .02 (d)7.86 (g)7.60 (h )7.68 (i)35 °C

7.30 ± 0.02 (e)7.64 (g)7.41 (h)7.50 ± 0.02 U)

K(ZnL) = [ZnLJ;TL][ZnIl].

pKa = -log([ZnH_ILJawttZnL]).

n= [HnL]ttHn--IL]aW'

1= 0.10 (NaCl0 4 ).

0.10 (NaCl0 4).

(d)

(e)

Determined with 1.0 mM of 14a and I = 0.10 (NaCl0 4).

with 1= 0 .10 (NaCl0 4 ).
(j)

At 25°C with

Determined with 1.0 mM of 14a and 4 equiv amount of HCl0 4 at 25°C with I =mM of 14a and 1= 0.10 (NaN0 3).
(NaCI0 4 ).

(b )

( h)

(f)

(e)

Determined with 1.0

From ref 14 at 25°C with I = 0 .10 (NaCl0 4 ).

From ref 14 with 1= 0 .10 (NaCl04 ).

(1)

Detem1ined with 1.0 mM of 15a and 1= 0.10 (NaN0 3 )6(g)

From ref 12

From ref 17 at 25°C with 1= 0.10

The potentiometric pH titration curve of 11· 4HCI in the presence of an equimolar amount ofZnil using O. 10M N aOH (Figure 1b) revealed two distinct equilibria: the first is the ZnL complex
formation at 4 < pH < 6 until a = 4, and the second is monodeprotonation from ZnL (4 < a < 5).

Up

to a = 4, the equilibration was extremely slow, so that we had to wait more than 2 h for each titration
point.

The titration data were treated for the 1:1 ZnL (14a) complex (eq 5) and its

monodeprotonated complex ZnH_1L (14b) (eq 6), where H_1L denotes alcoholic OH-deprotonatedligand (Scheme 3).

The obtained values log K (ZnL) at 25 °C and deprotonation constants pK a at 25

and 35 °C are listed in Table 1.

Any further deprotonation or precipitation of Zn(OH)2 was notobserved over pH 12, indicating the monodeprotonated species to be stable until pH ca. 12.

The

deprotonation constants pKa (eq 6) of 7.51 ± 0.02 and 7.30 ± 0.02 determined by the pH-metrictitration respectively at 25 °C with I = 0.10 (NaCI04 ) and 35 °C with 1= 0.10 (NaN03 ) are near thoseof6a 12 and Znll-N-methylcyclen 15a 17 (see Table 1). Fortunately, both the ZnL (14a) and ZnH_1L(14b) complexes were crystallized as their perchlorate salts from pH 6 and 9.5 aqueous solutions,
respectively.

The structure of the monodeprotonated complex 14 b was confirmed by the X-ray

crystal analysis (vide infra).

As the solution pH became higher, the chemical shifts of the benzyl

proton Ha for 14 moved upfield from 8 5.14 at pD 6.0 (14a) to 84.86 atpD > 9.5 (14b).

Fromthese facts and the following results, we assigned the deprotonated structure to be 14 b rather thanZnll-OH- species in aqueous alkaline solution.
~ ZnL

L+ Zn ll
ZnL~

ZnH_1L + H+

Scheme 3

11

Zn II

..

pH 6

/

HO~Ha
I---...

: K(ZnL) = [ZnL]j[L][ZnII](5): K a = [ZnH_1L]~+j[ZnL](6)

pKa 7.51

c;zn~:) ~::;iXHa

t7n~N)

H '---./ H

HN'---./NH

ZnL
14a

ZnH_IL14bx/1---... ,CH 3~-znlI-N(HN'---./NH/)"

ISa: X = H2 O
b: X = HO-

2·3. X-ray Crystal Structure of the Zn II Complex of the Deprotonated Benzyl
Alcohol-Pendant Cyclen (14b).

When the benzyl alcohol-pendant cyclen 11 (in the acid-freeform L, see the Experimental Section) in water was mixed with 1 equiv of Zn lI (CI04 )2 6H 2 0 at 60 °C,
14a· (Cl04)2 was obtained as colorless crystals.

Addition of 1 equiv of NaOMe to 14a· (CI04)2 in

MeOH gave 14b·Cl04 , which was recrystallized from aqueous alkaline solution (pH 9.5).

7

Figure 2 shows an ORTEP drawing of 14 b· CI0 4 with 30% probability thelmal ellipsoids.
Selected crystal data and collection parameters are displayed in Table 2. In this Zn ll complex, there
The Zn II ions Zn l and Zn 2 'respectively, lie above the four nitrogen atoms (Np N 4, N 7 , N10and N Z2 ' N 2S ' N Z8 ' N 31 ), and are
are two crystallographically distinct molecules (Figure 2a, b).

apically bound with the pendant alkoxide oxygens 0IS and 036' respectively.

The angles N I-Zn l-N 7

and N 22-Znz-N 28 and N 4-Zn l-N lo and N2S-Zn2-N31 are respectively bent at (138.5 and 139.2°)
and (135.0 and 134.5°) indicating distorted tetragonal-pyramidal structures.

The average Zn-CJ

bond distance of 1.91 A is much shorter than the Zn-N bonds (2.056-2. 173 A).

The Zn-o-distance

is shorter in the Znll-anion donor than in other ZnII-macrocyclic polyaminecomplexes. lob ,13,14,18 The earlier Zn-O bond distance with the neutral alcohol pendant in 8 was
1.994 A.14

The apical Zn-O bond is bent with the NI-Znl-O ls and N n -Zn 2-036 angles of 86.8 and

86.7°, respectively.

N7 , andOne may view 14b as having a distorted trigonal-bipyramidal structure with Np°15 as equatOlial donors and N4 and N IO as axial donors.

Although the pendant alkoxide donor binds firmly with Zn II in the solid state, this bonding
would be kinetically labile in DMF and H 20 solutions, so that this alkoxide anion can be a good
nucleophile for the phosphoryl transfer reaction with BNP-.

Table 2. Selected Crystallographic Data for 14 b· CI04.
empirical formula (fOlmula weight)

CI6H27N40sCIZn (456.25)

crystal color, habit

colorless, prismatic

crystal system

orthorhombic

space group

P212121 (no. 19)

lattice parameters

Q= 16.977(4), b= 18.135(4), c = 13.173(3) A

V = 4055(1) A3
2=8p(Cu Ka)

31.96 em- I

radiation

Cu Ka (A = 1.541 78 A), graphite monochromatedscan type(J}-28scan rate (in (j))

16.0 deg/min (five scans)scan width(1.42 + 0.30 tan et2emax

120.2°

no. of reflsn measd

3414 (total)

refinement

full-matrix least squares

no. of obs (I>3.00a(I))

2862

residuals: R; R w

0.050; 0.077

8

(a)

(b)

Figure 2. ORTEP drawing (30% probability ellipsoids) of the two crystallographically distinct
molecules (a and b) of 14 b· Cl04.

004 anions were omitted for clarity.

Bond distances: (a) Zn(l)-0(15) 1. 915(5), Zn(1)-N(l) 2.172(7), Zn(1)-N(4) 2.173(8), Zn(1)-N(7)2.067(7), Zn(l)-N(10) 2.143(8) A; (b) Zn(2)-N(22) 2.155(7), Zn(2)-N(25) 2.161(8), Zn(2)-N(28)2.056(7), Zn(2)-N(31) 2.169(7) A.

Bond angles: (a) N(1)-Zn(1)-N(4) 81.2(3), N(l)-Zn(1)-N(10) 81.4(4), N(4)-Zn(l)-N(7) 84.0(4), N(7)-Zn(1)-N(l0) 82.2(4), O(lS)-Zn(l)-N(l) 86.8(2r;(b) N(22)-Zn(2)-N(25) 82.8(4), N(22)-Zn(2)-N(31) 80.6(3), N(2S)-Zn(2)-N(28) 82.1(3), N(28)-Zn(2)-N(31) 82.8(3), 0(36)-Zn(2)-N(22) 86.7(2r.

92.4. Net Reaction of the Benzyl Alcohol-Pendant Cyclen-ZnlI Complex 14b with
Bis(4-nitrophenyl) Phosphate (BNP-).
simplified model of AP.

The ZnH IL complex 14b has been tested as aSince the phosphomonoester (e.g., 4-nitrophenyl phosphate (NPp 2 - )) was

hydrolyzed impractically slowly, we used a more reactive substrate, phosphodiester bis(4nitrophenyl) phosphate (BNP-).
The overall reaction of BNP- (25 mM) with 14b (20 mM) was followed by the IH NMR of the
benzyl protons in D2 0 at 35°C and pD = 10.3 (0.1 M CHES buffer) (see Figure 3).

The initial

product 16 (a new triplet at 8 5.56), which later was proven to be the phosphoryl intennediate,
increased as the starting 14 b (8 4.83) decreased.
subsequently.

The final product 17 (8 5.90, see blow) appeared

After 192 h, 14b and 16 diminished to 2.50/0 and 7.50/0, respectively, and the

majority (90%) of the initial Zn ll complex was converted to 17, where almost 2 equiv of 4nitrophenolate was released (191% based on initial concentration of 14b).

The same reaction was

followed by 31 P NMR under the same conditions, and the products 16 and 1 7 were identified at 8

-5.7 and 5.9, respectively.

No other side product was detected.

We assigned the net reaction

scheme as depicted in Scheme 4, with the aid of the following results.

100

%
80
60

40
20

~~~

---

.

.

~-~-------O~--~-----~====~==~

o

150

100

50

200

hr
Figure 3. Time course of the relative concentrations of Zn ll
complexes 14b (open square), 16 (solid circle), and 17 (solid
trigone) for the reaction of BNP- (25 mM) with 14 b (20 mM) in
D2 0 at 35°C, I = 0.10 (NaN03 ), and pD = 10.3 (0.1 M CHES
buffer).

The relative concentrations (%) are based on the initialconcentration of 14 b.

10

Scheme 4

16a

17

16b

11

16c

2-5. A Kinetic Study of the Initial Reaction 14 b~

16.

The initial phosphorylation

rate in aqueous solution at 35°C, I = 0.10 (NaN03 ), and pH 6.0-10.3 (20 mM Good's buffer) was
followed by the appearance of 4-nitrophenolate at 400 nm.

The second-order dependence of the rate

constant k'BNP on the total concentration of Zn II complex (= [14a] + [14 bJ) and [BNP ] fits the kinetic
eq 7, where v is the 4-nitrophenolate releasing rate.

The second-order rate constant, k' BNP is plotted

as a function of pH (see Figure 4).

The resulting sigmoidal curve indicates a kinetic processcontrolled by an acid-base equilibrium.

The inflection point at pH 7.4 is almost the same as the pKavalue of 14 for the pendant alcohol deprotonation (eq 6).
concluded to be the deprotonated complex 14 b.

Therefore, the reactive species is

The second-order rate constant k BNP (see eq 8) of

4(6.5 ± 0.1) x 10- M- 1 S- 1 was determined from the maximum k' BNP values.
\'II= k' BNP[total Zn complex] [BNP-](7)= k BNP [14 b][BNP-](8)

For a reference, the hydrolysis of the same substrate BNP- (to NPp 2 - ) with Znll- N -methylcyclen 15
has been detennined by the same method.

[BNP-] and [15 b].

The kinetics followed the second-order dependence on

The rate constant is (5.2 ± 0.2) x 10- 6 M- 1 S- 1 at 35°C, 1= 0.10 (N aN~), andpH 9.3 (20 mM CHES buffer), which demonstrates that the nucleophilic reaction catalyzed by 14b is
125 times Jaster than by 15 b.

It is understood that the Znll-alkoxide anion is a better nucleophilethan Zn"-hydroxide anion toward the phosphate substrate, just as toward 4-nitrophenyl acetate
substrate. 13 ,14

It should be noted, however, that the reaction with Znn-alkoxide 14 b is a phosphoryltransfer to form a phosphoryl intermediate 16 b (see Scheme 4), as the previously found acyl transfer
with 7 and 8. 13 , 14

On the other hand, the reaction with ISb is a hydrolysis that yields NPp2 - .

12

8
~

rFl

~

::;

6

~

=
~

4

X

~
~

~

2
0
6

7

10

9

8

pH
Figure 4. Rate- pH profile for the second-order rate constantsof the phosphoryl transfer from BNP- to 14 (see eq 7) at 35 °C
with I = 0.10 (NaN0 3 ) in aqueous solution.

Table 3. Comparison of the Phosphodiester Bond Cleavage Rate Constants, k BNP (M- 1 s 1), for
14 b, 8, 15 b, 5 b, and 6 b, and Aqueous OH- Ion in Aqueous Solution
catalyst

(a )

k BNP

catalyst

k BNP

14b

(6.5 ± 0.1) x 10-4 (a)5b8.5 x 10- 5 (d )815b5.0 X 10-4 (b)6bOH-

2.1 X 10- 5 (d)(5.2 ± 0.2) X 10- 6 (e)at

2.4 X 10- 5 (d )Detemlined with 2.0,1.0, and 0.5 mM of 14b and 10,5.0, and 2.5 mM of BNP- at 35 °C with I = 0.10

(NaN0 3 ).

(b)

From ref 15 at 35 °C with I = 0.10 (NaN0 3).

(c)

Determined with 16,8.0, and 4.0 mM 14b

and 10, 5.0, and 2.5 mM BNP- at 35 °C with I = 0.10 (NaN0 3 ).
(NaCI0 4) .

13

(d)

From ref 10c at 35°C with I = 0.20

2-6. Isolation of the Phosphoryl Intermediate 16a from the BNP- Reaction with 14b
in DMF.

The phosphoryl intermediate 16a was unequivocally determined by independent isolationof 16a·CI04 by the reaction of BNP- and 14b in dry OMF.
elemental analysis (C, H, N) and 1H, 13C, and 31 P NMR.
with J = 0.10 (NaN03 ) using 0.1 M aqueous NaOH
9.10± 0.05, which is assigned to 16a
15a

~

~

16b.

I9

The structure was identified by

The pH -metric titration of 16a at 35 °C

showed the monodeprotonation and it<; pKa of

ThepKa value is higher than 7.50 ± 0.02 for

ISb at the same conditions, which is possibly due to the proximate phosphate anion

binding to the Zn11 , see 16c.

The 3 1p NMR chemical shift of phosphoryl intermediate 16 in H 2 0changed from 8 -3.6 at pO 6.5 (16a) to 8 -6.5 at pO 11 (16b).
The reaction of BNP- with 14 b (the isolated monoperchlorate salt was used) in dry OMF at 35°C was kinetically studied by observing the appearance of 4-nitrophenolate at 430 nm.

The second-order rate constant kBNP (= 1.1 ± 0.1 MiS-I) with respect to [BNP ] and [14b] was obtained.

1l1e

comparison of the rate constants k BNP in OMF and aqueous solution points out that the Zno-boundalkoxide nucleophile acts 1700 times more efficiently in this aprotic solvent than in aqueous solution,
which is accounted for by less interfering solvations in OMF. 20 This observation suggests that inhydrophobic environments a phosphoryl transfer at the enzyme active center might occur quite
effectivel y.
2-7.

Spontaneous

Hydrolysis

of

the

Pendant

Phosphodiester

Phos phomonoes ter 17 by the Intramolecular ZnJI-OH-.

in

16b

to

a

The pendant phosphodiester in

16, the initial phosphorylation product resulting from the phosphotransfer reaction, was found toundergo spontaneous hydrolysis in alkaline aqueous solution to yield a phosphomonoester 17 (seeScheme 4).

We failed to isolate 17 as a solid.

spectral changes.

This reaction was followed by the 1H and 31 P NMR

The disappearance of the reactant 16 (5 mM) (8 5.56 (OCHC), 6.85 and 8.03

(02NArH) for IH; 8 - 5.7 for 3 1p) matched the appearance of the product 17 (8 5.09 (OCHC) for lH;°

85.9 for 3 1P) and 4-nitrophenolate (8 6.53 and 8.06) in O2 at 35 °C and pO = 10.3 (0. 1 M CHES
buffer).
The hydrolysis rate (v 2 ) of the phosphodiester pendant in 16 was followed by UV
spectroscopic measurement (at 400 nm) at pH 7.0- 10.5 (20 mM Good's buffer), 1 = 0.10 (NaN03 ),and 35 °C.

The first-order dependence on the total concentration of 16 (= [16a] + [16b] + [16c])

is consistent with the kinetic equation 9.
of pH in Figure 5.

The first-order rate constants k' POE are plotted as a function

The sigmoidal curve indicates characteristic of a kinetic process control1ed by anacid- base equilibrium and exhibits an inflection point at pH 9.0, which is almost the same as the pK avalue of 9.1 for the coordinate water of 16a.

Therefore, just as all the previous Zn IT- OH

species, 9d, loc the Zn D- OH in 16 b must be a good nucleophile to the intramolecular phosphate.

The

first-order rate constant kPDE of (3.5 ± 0.1) x lO s s I was obtained from the maximum k' POE (eq 10).

14

V2 = k' PDE[tOtal ZnL complex 16](9)= k pDE [16 b](10)

A prolonged (ca. 1 week) alkaline reaction at 35°C in 0.1 M aqueous NaOD solution did notchange the 31p NMR of 17 (8 5.9), indicating that 17 is inert and undergoes no more hydrolysis.
We assign the final product to the intramolecular phosphomonoester coordinating structure 17.
Earlier,

we foundthat the ZnII-cyc1en complex 6a tends to strongly

bind to dianionicphosphomonoesters, e. g., log K = 3.3 for 1:1 the NPp2--ZnII-cyc1en complex. 14°(5 mM)21 with EOTA (25 mM) in O2

Treatment of 17
at pO 10.3 (0.1 M CHES buffer) stripped Zn II to free theligand showing a singlet 31 P signal at 8 4.2.

4
~
I

r.r.J
lI)

3

<:>
~

X
~

2

Q

Q..

~

1

0
7

9

8

10

11

pH
Figure S.

Rate-pH profile for the first-order rate

constants of intramolecular phosphodiester hydrolysis of

16 (see eq 9) at 35°C with I = 0.10 (NaN03 ) in aqueous
solution.

15

2-8. Hydrolysis of Ethyl (4-Nitrophenyl) Phosphate (NEP-) with an Intermolecular
Nucleophile ZnII-OH- ISb (Scheme S).
Hydrolysis of 16b.

A Reference Reaction to the Intramolecular

In order to see the efficiency of the intramolecular attack of ZnIl- OH at thependant phosphodiester in 16 b~

17, we have measured the rate of an intermolecular reaction

between ethyl (4-nitrophenyl) phosphate (NEP-) and (N-methylcyclen)-ZnII- OH- (ISb) (Scheme 5)
under the same conditions.

The kinetics, followed by the appearance of 4-nitrophenolate at pH 9.3,

showed the second-order rate constant kr-..'fP of (7.9 ± 0.3) x 10 7 M 1 s 1.

One can calculate theeffective molality of 45 M (= kPDE/kNEP) for the intramolecular phosphate of 16 b.

In other words,

the hydrolysis by the intramolecular Znll-OH- (16 b) is 45 000 times faster than by the intermolecularZnll-OH- (l mM ISb).

Scheme 5
02 N

Oq

k

O·

NEP

0=)-0, - - - - . 02 N
0'
E t lSb

O O ' + O=~_O
o. "Et

NEPThe hydrolysis of the phosphoryl intermediate 16 by the intramolecular ZnII-OH- nucleophile issomewhat analogous to the Lindoy and Sargeson's Co III model system (Scheme 6). 9b

pH dependence of the hydrolysis rate (pH 6-9) implies the 18a~

The sigmoidal

18b equilibrium with pKa of

7.6, and the intramoleculal' Com-OH- nucleophile efficiently attacks the Co III -bound monophosphatewith the first-order rate constant of 7.8 x 10-4 S - 1 to fOlm the product 19.

Scheme 6_......:::::::-_ _ _
•

'\

18a

18b

16

/0" °

cis-(en) COllI
2

'"

0'

19

p.~ •

/"

°

3. Summary and Conclusions
The two-step mechanism of phosphate ester hydrolysis by ZnII-containing alkaline phosphatase(AP) (Scheme 1) is well mimicked by the newly designed complex 14: (i) a phosphoryl intermediate
16 is generated by attack of the hydroxy moiety of the alcohol pendant at the BNP (one of the ester
group is concomitantly hydrolyzed) and (ii) the phosphoryl intermediate 16 is hydrolyzed by the
intramolecular ZnII-OH-. The attack at the BNP- substrate and hydrolysis of the intermediate both
require Zn II . For the fIrst step, the hydroxyl group is activated by ZnII at physiological pH to 14 b

(pKa = 7.4), which is a 125 times more effective nucleophile to the phosphate substrate than the Zn IIactivated water of the reference ISb. For the second step, the intramolecular nucleophile isgenerated from Zn II-OH 16a with a pK a value of 9. This intramolecular hydrolysis is 45 000 times
2

faster than the intermolecular hydrolysis of NEP- with 1 mM ISb. In the AP enzyme (Scheme 1),
these two functions of Zn II are performed separately by two proximate Zn ll atoms; one is involved in
the activation of Ser-l 02 to yield phosphoryl intermediate 2, and the other is involved in the activationof H 20 3 to attack at the intermediate 2.

The intramolecular arrangement of these two Zn D ions in

AP is more advantageous than our single-ZnII system in order to provide this dual role, wherein the

pKa value of 9.0 (due to the close phosphate anion or the phosphate-binding) for 16ahigher than the reported pK a value of 7.4 for 2~~

16b is3 in the enzyme.

Scheme 4 summarizes the whole reaction mechanism of the P-O bond cleavage of thephosphodiester (BNP-) by 14.

The final phosphomonoester product 17, unfortunately, was foundto be very inert under normal conditions (all the attempts to hydrolyze it failed, including raising the
pH as high as 11).

Therefore, we could not use 14 as a catalyst.

However, the present results

may well serve the novel elucidation of the collaborative roles of Ser-l02 and Znll in alkaline
phosphatase.

174. Experimental Section

General Information.

All reagents and solvents used were of analytical grade.

The

Good's buffers (Dojindo) were commercially available and used without further purification: MES (2(N-morpholino)ethanesulfonic acid, pKa = 6.2), MOPS (3-(N-morpholino)propanesulfonic acid, pKa== 7.2), HEPES(N-(2-hydroxyethyl)piperazine-N'-2-ethanesulfonic acid, pKa = 7.6), EPPS (N-(2-hydroxyethyl)piperazine-N'-3-propanesulfonic acid, pKa = 8.0), TAPS (N-(tris(hydroxymethyl)methyl)amino-3-propanesulfonic acid, pK a = 8.4), CHES (2-(cyclohexylamino)ethanesulfonic acid,pKa = 9.5), CAPSO 3-(N -cyclohexylamino)-2-hydroxypropanesulfonic acid, pKa = 10.0), CAPS 3(N -cyclohexylamino)propanesulfonic acid, pK a = 10.4).

Sodium bis(4-nitrophenyl) phosphate wascrystallized from an aqueous solution of bis( 4-nitrophenyl) phosphoric acid (BNP) and equimolar
Lithium ethyl (4-nitrophenyl) phosphate was prepared by the reported method. 22

NaOH.

was distilled in vacuo over anhydrous MgS04 and stored in the dark.

DMF

All aqueous solutions wereprepared using deionized and distilled water.
Kinetic study was carried out using a Hitachi U -3500 spectrophotometer equipped with athermoelecuic cell temperature controller (±O. 5°C).
4200.

IR spectra were recorded on a Shimadzu FTIR-IH (400 rvn-Iz), 13C (100 MHz), and 31p (162 MHz) NMR spectra were recorded on a JEOL

a-400 specu·ometer.

3-(Trimethylsilyl)propionic-2,2,3,3-d4 acid sodium salt (Aldrich) in 0 2 0 andtetramethylsilane (Merck) in organic solvent were used as internal references for I H and 13C NMR

measurements.

°

A O2

NMR measurement.

solution of 80% phosphoric acid was used as an external reference for 31 P

Opitical rotations were recorded on a Union Giken Automatic Digital

Polarimeter PM-101 at 22.0 ± 0.5 °C.

Melting points were determined by using a Yanaco micro

melting apparatus without any conections.
Corder Mf-3.

Elemental analysis was performed on a Yanaco CHNThin layer (TLC) and silica gel column chromatographies were carried out on Merck

Alt. 5554 (silica gel) TLC plates and Wakogel C-300 (silica gel), respectively.

Synthesis of (S)-I-(2-Hydroxy-2-phenylethyl)-1,4, 7, lO-tetraazacyclododecane(11). 2,6-Dioxo-1,4,7,10-teraazacyclododecane (12) (2.00 g, 10 mmol)16 and (S)-styrene oxide(1. 32 g, 11 lTIlTIol) were heated to reflux in EtOH (100 mL) for 1 day.

evaporated to dryness.

The reaction mixture was

The residue was pmified by silica gel column chromatography (eluent

CH2C~!MeOH/280/0 aqueous NH3 = 40:3:0.1) followed by crystallization from CH 3CN to yield 1-(2-hydroxy-2-phenylethyl)-5,9-dioxo-1,4,7, 10-teu'aazacyclododecane (13) as colorless prisms (1.47 g,
4.6 mmol, 460/0 yield):

mp 192.0-193.0 °C; IR (KBr pellet) 3362, 3086,2975,2955,2822, 1655,

1539, 1455, 1435, 1348, 1316, 1291, 1269, 1227, 1200, 1169, 1071,920, 870,843,774,758,

706 em-I;

TLC (eluent CH 2 C1 2!MeOH/28% aqueous NH3 = 5:1:0.2) R f = 0.4;

IH NMR (CDCl 3)

81.83-2.06 (lH, br, amine NH), 2.51 (2H, dt, J = 13.2, 4.6 Hz, NCH), 2.64 (lH, dd, J = 13.6,
4.0 Hz, NCHCAr), 2.77 (2H, ddd, J = 13.2,9.0, 4.2 Hz, NCH), 2.84 (1H, dd, J = 13.6,9.4 Hz,
NCHCAr), 3.16-3.24 (1H, br, alcohol OH; 2H, m, CONCH), 3.29 (lH, d, J = 16.2 Hz, CHCON),18

3.34 (lH, d, J = 16.2 Hz, NCOCH), 3.44 (2H, m, CONCH), 4.79 (lH, dd, J = 9.4, 4.0 Hz,
CHAr), 7.30 (lH, tt, J = 6.4,2.0 Hz, ArH), 7.35 - 7.42 (4H, m, ArH), 7.43-7.48 (2H, br, amide
0NH); [a]D -55.9 (c 1.00, MeOH).
To a suspended solution of dioxo macrocycle 13 (1.92 g, 6.0 mmol) in dry THF (40 mL) was
added slowly a THF solution (65 mL) of 1 M BH3-THF complex at 0 °C.
at room temperature for 1 h and then heated at 60 0 C for 1 day.

The mixture was stirred

After decomposition of the excess

amount of the hydroborane complex with water at 0 °C, the solvent was evaporated.

The residue

was dissolved in 6 M aqueous HCI (70 mL) and then the solution was heated at 70 °C for 2 h.
mixture was washed with CH 2Cl2 (30 mL x 2) and evaporated to dryness.

The

The residue was passed

through an anion exchange column of Amberlite IRA-400 with water to obtain 11 as a colorless oi1.
Crystallization of the oil from 6 M aqueous HCI afforded colorless needles as its tetrahydrochloric
acid salts (11·4HCI) in 62% yield (1.63 g, 3.7 mmol):dec 210 °C; IR (KBr pellet) 3420, 2998,
2793,2448, 1576, 1495, 1439, 1066, 1028, 766, 704 cm- I; IH NMR (D20, pO 1.0) 8 2.82-3.21(18H, m, NCHJ, 4.89 (lH, t, J = 6.2, OCHC), 7.40-7.54 (5H, m, ArH); 13C NMR (02 pD 1.0)°,

84l.4, 41.5,44.5, 44.6, 45.4, 47.0, 52.3, 52.4,62.0, 73.8, 129.0, 131.5, 132.2, 144.8; [a]D--49.10 (c 1.00, H 20).

Anal. Calcd for C16H32N4OCl4·1 /2H20: C, 43.0; H, 7.44; N, 12.5.

Found:C, 43.1; H, 7.49; N, 12.4.
S ynthes is of (5 )-1- (2- Hydroxy-2-phenylethyl)-l, 4, 7, 10-tetraazacyclododecane
Zinc(II) Complex (14a' (CI0 4)2).

11·4HCl (438 mg, 1.0 mmol) was passed through an anion

exchange column of Amberlite IRA-400 with water to obtain the free ligand 11 as a colorless oil.
After the oil was dissolved in water (4 mL), Zn(CI04)2·6H20 (391 mg, 1.1 mmol) was added in thesolution.

The solution was stirred at 60 °C for 1 h.

After the solvent was evaporated, the residue

was recrystallized from water to obtain colorless prisms as diperchlorate salts 14a· (Cl04)2 in 91 %yield (507 mg, 0.91 mmol):IR (KBr pellet) 3420, 3293, 2930, 2886, 1480, 1458, 1379, 1365,
1358, 1298, 1283, 1267, 1096, 968, 928, 860, 777, 756, 742,708, 625 cm- I; 1H NMR (02 pO
6.0) 82.75-3.28 (18H, In, NCH), 5.14 (lH, dd, J = 10.1, 3.2 Hz, OCHC), 7.43-7.55 (5H, m,°,

ArH); 13C NMR (02°,pO 6.0) 845.97,46.00,46.2,48.4,48.5,53.7,55.0,61.9,72.4, 128.9,

131.4,131.8, 144.0; [a]D ---64.40 (c 1.00, H 2 0).
5.1; N, 10.1.

Found: C, 34.7; H, 5.2; N, 10.0.

Preparation(14b' CI0 4 ).

Anal. Calcd for CI6H28N409C~Zn: C, 34.5; H,of

Alkoxide

Pendant

Attached

Zinc(II)

Complexwith11

To a solution of 14a· (Cl04)2 (278 mg, 0.50 mmol) in MeOH (11 mL) was added

0.5 mL of 1 M methanolic NaOMe.

Colorless solid was precipitated by slow evaporation andrecrystallized from aqueous solution (pH 9.5) to obtain 14 b· Cl04 as colorless prisms in 960/0 yield(219 mg, 0.48 mmol):IR (KBr pellet) 3295, 2922, 2874, 1489, 1453, 1350, 1144, 1094, 982,
932,855,795,774,756,710,625 cm- I; IH NMR (D20, pO 9.5) 82.69-3.09 (17H, m, NCH 2),

19

3.22 (lH, ddd, I = 13.8, 11.1,4.3 Hz, NCH 2), 4.86 (lH, t, I = 6.6 Hz, OCHC). 7.34-7.49 (5H,°,m, ArH); J3c NMR (02pO 9.5) 845.2, 46.2,46.6,46.8,47.6,48.1, 52.6, 54.9, 63.5, 74.3,

129.2, 130.8, 131.6, 146.0; [a]o -65.8° (c 1.00, H 20).
42.1; H, 6.0; N, 12.3.
S ynthes isAnal. Calcd for CI6H27N40SC1Zn: C,

Found: C, 42A; H, 6.0; N, 12.2.

of(S) -1- (2- (4 - Nitro phenyl phos phory 1)-2- pheny lethy I) -1,4, 7, 10-

tetraazacyclododecane Zinc(II) Complex (16a' CI0 4 ).

A OMF solution (8 mL) of

14 b· CI04 (91. 3 mg, 0.20 mmol) and bis( 4-nitrophenyl)phosphate sodium salt (72.4 mg, 0.20 mmol)
was stilTed at 35°C for 1 day.

After the solvent was evaporated, the residue was dissolved in water(50 mL) and the pH was adjusted to 6.0 with 0.1 M aqueous HCI04 •washed with diethyl ether (15 mL x 3) and evaporated to dryness.

The aqueous solution was

The residue was recrystallized

from water to obtain 16a· CI04 as colorless prisms (98 mg, 0.15 mmol, 730/0 yield):dec 175°C; IR(KBr pellet) 3303,2934,2885, 1611, 1591, 1514, 1493, 1458, 1346, 1252, 1146, 1090,916, 864,°,

793,754,741,700,625,550 cm- I ; IH NMR (02pD 6.5) 82.93-3.30 (l7H, m, NCH 2), 3.37

(lH, dd, I = 15.3, 9.8 Hz, NCH 2), 5.47 (lH, m, OCH), 7.02 (2H, dtd, I = 9.3, 2.0, 0.9 Hz,

OArHN02), 7.33-7.46 (5H, m, CArH), 8.05 (2H, dtd, I = 9.3, 2.0,0.6 Hz, OArHN02); IJC NMR(020, pD 6.5) 8 45.6, 45.7, 46.9, 47.0, 47.8, 47.9, 53.3, 56.7, 65.1 (IpC = 5.9 Hz), 80.2 (IpC =6.6 Hz), 123.0 (IpC = 5.1 Hz), 128.4, 129.1, 131.6, 131.7,140.3, 146.5, 159.0 (I pC = 5.9 Hz);
31p NMR (02°,pO 6.5) 8 -3.59 (JHP = 9.2,2.7 Hz); [a]o -43.2° (c 1.00, MeOH).

for C22H33NSOjjClPZn: C, 39.1; H, 4.9; N, lOA.

Anal. Calcd

Found: C, 38.9; H, 4.9; N, 10.4.

Syntheses of Racemic Ligand and its ZnIl Complexes.

The racemic dioxocyc1en

delivative was prepared by the same method as that for 13 using rae-styrene oxide to give colorless
Plisms in 43% yield.

Mp, TLC, IR, and NMR are the same as those for 13.

U sing this dioxocyclen derivative, the racemic ligand (cyclen derivative) was prepared by thesame method as 11 to give colorless needles as its tetrahydrochloric acid salts in 60% yield.
and NMR are the same as those for 11·4HCI.

[a]D = 0° (c 1.00, H 30).

C16H32N/)C14·1/2H20: C, 43.0; H, 7A4; N, 12.5.

Found: C, 43.0; H, 7A1; N, 12.1.

Dec, IR,

Anal. Calcd for

The pendant alcohol undissociated ZnIJ complex with the racemic ligand was prepared by thesame method as that for 14a using the racemic ligand to give colorless prisms as its diperchlorate salts

in 93% yield.

IR and NMR are the same as those for 14a· (CI04)2'

Anal. Calcd forCI6H28N40gCI2Zn: C, 34.5; H, 5.1; N, 10.1.

[a 10 = 0° (c 1. 00, H 20).

Found: C, 34.8; H, 5.2; N, 10.1.

U sing this Zn complex, the alkoxide pendant attached Zn fl complex was prepared with theIIsame method as that for 14 b to give colorless prisms as perchlorate salts in 90% yield.

NMR are the same as those for 14b·CI04 .
C16H27N40sClZn: C, 42.1; H, 6.0; N, 12.3.

[aJ o = 0° (c 1.00, H 20).

Found: C, 42.0; H, 6.3; N, 12.3.

20

IR andAnal. Calcd forCrys tallog raphic Study.

A colorless prismatic crystal of 14 b· CI04 (0.40 x 0.40 x 0.20mm) was used for data collection.

The lattice parameters and intensity data were measured on a

Rigaku AFC7R diffractometer with graphite monochromated Cu Ka radiation and a 12-kW rotating
anode generator.

The structure was solved by a Patterson orientation/translation search andexpanded using Fourier techniques.

The non-hydrogen atoms were refined anisotropically.

Hydrogen atoms were included but not refined.

The fmal cycle of full-matrix least-squares

refinement was based on 2862 observed reflections (/ > 3.00a(/) to giveR = 0.050 and Rw = 0.077.

All calculations were performed using the teXsan crystal structure analysis package developed by
Molecular Stluctw'e Corp. (1985 and 1992).

Potentiometric pH Titration.

The preparation of the test solutions and the calibration

method of the electrode system were described earlier. 10,13

All test solutions (50 mL) were kept

under an argon (>99.9990/0 purity) atmosphere at 25.0 ± O. 1 °C with I = 0.10 (N aCI04 ) and 35 ± 0.1°C with / = 0.10 (NaN03 ).

The potentiometric pH titrations were carried out at [total ligand] = 1
mM in the presence or absence of equimolar ZnS0 , and [total Zn ll complex] = 1 mM, and at least
4

three independent titration were made.

The calculation methods for ligand protonation constants

II(K n)' Zn complexation constants (K (ZnL», and the deprotonation constant of the Znll complex (K a)were the same as described previously. 10,13

The protonation constants Kn are defined as[HnL]/[H n _ 1L]aH+, the 1: 1 metal complexation constant K(ZnL) as [ZnL]/[Znll][L], and the
deprotonation constant K a as [ZnH_1L]aH+/[ZnL]. The used values of K w' (= [H+] [OH-]) and fH+
were 10- 13 .79 and 0.825 at 25°C, and 10- 13 .48 and 0.823 at 35 °C, respectively.

Kinetics Procedure for the Phosphodiester Cleavage Reaction with Zinc(II)Complexes in Aqueous Solution. The phosphodiester cleavage reaction (i.e., 4-nitrophenolate
release reaction) rates of bis( 4-nitrophenyl) phosphate (BNP-) and ethyl (4-nitrophenyl) phosphate(NEP-) were measured by an initial slope method (following the increase in 400-nm absorption of
released 4-nitrophenolate) in aqueous solution at 35.0 ± 0.5 °C.

Buffer solutions containing 20 mM

Good's buffer (MES, pH 6.0; MOPS, pH 7.0; HEPES, pH 7.4; EPPS, pH 7.9; TAPS, pH 8.4;
CHES, pH 9.3; CAPSO, pH 10.0) were used, and the ionic strength was adjusted to 0.10 with

NaN 0 3 (ca. 90 mM).
employed:

For the initial rate determination, the following typical procedure was

BNP- (l0, 5.0, and 2.5 mM) and 14b (2.0, 1.0, and 0.50 mM) were mixed in thebuffered solution, the UV absorption increase recorded immediately and then followed generally until

ca 0.1 % formation of 4-nitrophenolate, where log £ values for 4-nitrophenolate were 3.23 (pH 6.0),
3.94 (pH 7.0),4.11 (pH 7.4), 4.20 (pH 7.9),4.24 (pH 8.4), 4.26 (pH 9.3), and 4.26 (pH 10.0) at

400 nm.

The observed first-order rate constant k obsd (S- l) was calculated from the decay slop (4-nitrophenolate release rate/[BNP-J).
rate constant k' B;-";P (M- 1

S- I)

The value of kobsi[total Zn ll complex] gave the second-orderfor BNP- hydrolysis.

21

The second-order rate constant kBNP wasdetennined from the maximum k' B~P values.
The second-order rate constants, kB~P and k t>."EP' for Zn II-N -methylcyclen 15 b were detenninedby the same method for that for 14b with BNP- (10,5.0, and 2.5 mM) and ISb (16, 8.0, and 4.0roM) and NEP- (20, 10, and 5.0 mM) and ISb (20, 10, and 5.0 mM), respectively.

Kinetics Procedure for the Phosphodiester Cleavage Reaction with 14b in DMF.
The phosphodiester cleavage rate of bis( 4-nitrophenyl) phosphate (BNP ) was measured by an initialslope method (following the increase in 430-nm absorption of released 4-nitrophenolate) in DMF at
35.0 ± 0.5 °C.

For the initial rate determination, the following procedure was employed.

BNP(1.0, 0.50, and 0.25 mM) and 14b (0.20, 0.10, and 0.050 mM) were mixed in DMF, the UV
absorption increase recorded immediately and then followed generally until ca. 1% formation of
4-nitrophenolate, where log £ of 4-nitrophenolate was 4.45 at 430 nm.

The second-order rate

constant k BNP in DMF was detennined by the similar method for k BNP in aqueous solution.

Kinetics Procedure for Intramolecular Hydrolysis with 16b.

The hydrolysis (i.e.,

4-nitrophenolate release reaction) rate of 16 b was measured by an initial slope method (following theincrease in 400-nm absorption of released 4-nitrophenolate) in aqueous solution at 35.

°± 0.5 °C.

Buffer solutions containing 20 mM Good's buffer (HEPES, pH 7.4; EPPS, pH 7.9; TAPS, pH 8.3
and 8.6; CHES, pH 9.1 and 9.5; CAPSO, pH 10.0; CAPS, pH 10.5) were used, and the ionicsU'ength was adjusted to 0.10 with NaN03 (ca. 90 mM).
following procedure was employed.

For the initial rate determination, the16b (1.0, 0.50, and 0.25 mM) were mixed in the buffered

solution, the UV absorption increase recorded immediately and then followed generally until ca. 1%
formation of 4-nitrophenolate, where log £ values for 4-nitrophenolate were 4.11 (pH 7.4), 4.24 (pH8.3),4.25 (pH 8.6),4.26 (pH 9.1),4.26 (pH 9.5),4.26 (pH 10.0), and 4.27 (pH 10.5).
order rate constant k' PDE (S- I) was calculated from the decay slop.
(S- I) was obtained from the maximum k' POE'

22

The first-

The ftrst-order rate constant kPDE

References and Notes(1) Coleman, J. E. Annu. Rev. Biophys. Biomol. Struct. 1992, 21, 441-483.

(2) Kim, E. E.; Wyckoff, H. W. 1. Mol. Bioi. 1991,218,449-464.

(3) (a) Gettins, P.; Coleman, J. E. 1. Bioi. Chem . 1984, 259, 4991-4997.

(b) Hull, W. E.;

Halford, S. E.; Gutfreund, H.; Sykes, B. D. Biochemisl1y 1976, 15, 1547-1561. (c) Bock, J.

L.; Cohn, M. 1. Bioi. Chern. 1978, 253, 4082-4085.

(d) Coleman, J. E.; Gettins, P. Zinc

Enzyme; Birkha user: Boston, MA, 1986; Chapter 6, p 77.

(4) For review; Fenton, D. E.; Okawa, H. 1. Chem. Soc., Dalton Trans. 1993, 1349-1357.
(5) Ried, T. W.; Wilson, L. B. Enzyme 1971, 4, 373-415.

(6) Butler-Ransohoff, J. E.; Rokita, S. E.; Kendall, D. A.; Banzon, J. A.; Carano, K. S.; Kaiser, E.
T.; Matlin, A. R. 1. Org. Chem. 1992, 57, 142-145.
(7) (a) Mueller, E. G.; Crowder, M. W.; Averill, B. A.; Knowles, J. R.I. Am. Chem. Soc. 1993,
115,2974-2975.

(b) Coleman, J. E. Zinc Enzyme; Birkha user: Boston, MA, 1986; Chapter 4,

p 49.
(8) For a review of bimetallic enzyme models: (a) Go bel, M. W. Angew. Chem., Int. Ed. Eng/.

1994,33,1141-1143.

(b) Karlin, K. D. Science 1993, 261, 701-708.

(9) (a) For rrrncomplexes: Hendry, P.; Sargeson, A. M. 1. Am. Chem. Soc. 1989,111,2521-2527.

(b) For Coll complexes: Jones, D. R.; Lindoy L. F.; Sargeson, A. M. 1. Am. Chem. Soc. 1983,
105, 7327-7336.

Hendry, P.; Sargeson, A. M. lnorg. Chem. 1990, 29, 92-97.

Banaszczyk, M.; Jubian, V.; Zou, X. 1. Am. Chem. Soc. 1989, 111, 186-190.

Chin. J.;

Kim, J. H.;

Chin, J. 1. Am. Chem. Soc. 1992,114,9792-9795.

Connolly, J. A.; Banaszczyk, M.; Hynes,

R. C.; Chin, J. lnorg. Chem. 1994, 33, 665-669.

(c) For Lnll complexes: Schneider, H.-J.;

Rammo, J.; Hettich, R. Angew. Chem., Int. Ed. Eng/. 1993,32, 1716-1719.
Govan, N. 1. Chem. Soc., Chem. Commun. 1990, 714-715.

Mon-ow, J. R.; Buttrey, L. A.;

Shelton, V. M.; Berback, K. A. f. Am. Chem. Soc. 1992,114, 1903-1905.
MOlTow, J. R. Inorg. Chern. 1994, 33, 5036-5041.

Chin, K. O. A.;

Tsubouchi, A.; Bruice, T. C. f. Am.

II

Chem. Soc. 1994,116, 11614-11615.

(d) For Zn complexes: Sigman, D. S.; Jorgensen, C.

T.1. Am. Chem. Soc. 1972, 94, 1724-1731.
Chem . Soc. 1986,108,2388-2394.

Hay, R. W.;

Gellman, S. H.; Petter, R.; Breslow, R. 1. Am.

Hikichi, S.; Tanaka, M.; Moro-oka, Y.; Kitajima, N. 1.

Chem. Soc., Chem. Commun. 1992, 814-815.
Soc., Chem. Commun. 1994, 135-136.

Ruf, M.; Weis, K.; Vahrenkamp, H. 1. Chem.

(e) For Felli complexes: Wilkinson, E. C.; Dong, Y.;

Que, L. Jr. 1. Am. Chem. Soc. 1994,116,8394-8395.
(10) (a) Kimura, E.; Shiota, T.; Koike, T.; Shiro, M.; Kodama, M. 1. Am. Chem. Soc. 1990,112,5805-5811.

(b) Kimura, E.; Koike, T.; Shionoya, M.; Shiro, M. Chem. Lett. 1992,787-790.

(c) Koike, T.; Kimura, E. 1. Am. Chem. Soc. 1991,113,8935-8941.

(d) Zhang, X.; van

Eldik, R.; Koike, T.; Kimura, E. l!lorg. Chem . 1993,32,5749-5755.

(e) Koike, T; Kimura,E.; Nakamura, 1.; Hashimoto, Y.; Shiro, M.1. Am. Chem. Soc. 1992, 114, 7338-7345.

23

(f) Kimura, E.; Shionoya, M.; Hoshino, A.; Ikeda, T. ; Yamada, Y.l. Am. Chem. Soc. 1992,

114,10134-10137.
(11 ) (a) Kimura, E.; Koike, T. Comments 1norg. Chem. 1991,11, 285-301.

(b) Kimura, E. In

Progress in Inorganic Chemisay; Karlin, K. D. , Ed.; John Wiley & Sons: New York, 1994; Vol.
41, 443-492.
(12) Koike, T.; Takamura, M.; Kimura, E. 1. Am . Chern. Soc. 1994, 116, 8443-8449.
(13) Kimura, E.; Nakamura, 1.; Koike, T.; Shionoya, M.; Kodama, Y.; Ikeda, T.; Shiro, M.1. Am.

Chem. Soc. 1994, 116,4764-4771.
(14) Koike, T.; Kajitani, S.; Nakamura, I.; Kimura, E.; Shiro, M. 1. Am. Chern. Soc. 1995, 117,
1210-1219.
(15) The second-order rate constant for the 4-nitrophenolate release reaction from BNP- with 8(determined by initial slop method) is (5.0 ± 0.1) x 10-4 M- 1 S - 1 in aqueous solution at 35 °C
with I = 0.10 (NaN0 3 ) .

Kimura, E.; Koike, T. Unpublished results.

(16) Kimura, E.; Kuramoto, Y.; Koike, T.; Fujioka, H; Kodama, M. 1. Org. Chern . 1990, 55, 4246 .
(17)Shionoya, M.; Ikeda, T.; Kimura, E.; Shiro, M. 1. Arn. Chern. Soc. 1994, 116,3848-3859.
The Zn II complexation constant log K(ZnL) for 15a is 15.1 ± 0.1 at 25 °C with I = 0.10
(NaCI04 ) .

Kimura, E.; Ikeda, T. Unpublished results.

(18) Kimura, E.; Koike, T.; Toriumi, K. Inorg. Chern. 1988,27, 3687-3688.
(19) Hence, the pK a was determined using the titration data before 0.5% hydrolysis of pendant
phosphate.
(20) (a) A second-order rate constant for hydrolysis of 4-nitrophenyl acetate with 14 b in OMF ((1.1± O. 1) X 102 M- 1 S- I at 35 °C) is also 350 times greater than the rate in aqueous solution (0.31 ±0.01) M- 1 S - 1 at 35 °C with I = 0.10 (NaN03 ».

Kimura, E.; Kodama, Y. Unpublished results.

(b) We have given a thought to running the hydrolysis with ZnII-OH- complex 15b in dry DMF
as a reference reaction.

However, we could not isolate 15b in any of the attempts using

various counteranions such as CI04 - , PF 6 - , Ct, etc.

Generation of 15 b in situ makes thereaction more complex and difficult to interpret.
(21) The solution of 17 was prepared by the reaction of 16a (5 mM) in 0 2 0 at 50°C and pO 10. 3(0 .1 M CHES buffer) for 1 day.
(22) (a) MOlTow, 1. R.; Trogler, W. C. Inorg. Chern . 1988, 27, 3387-3394.
Younas, M. 1. Chem. Soc. B 1970,1165-1172.

24(b) Kirby, A. J.;謝辞

本研究を行うにあたり、終始御指導、御鞭援を賜わりました広島大学
医学部総合薬学科教授

木村栄一先生に心より謹んで感謝の意を表わし

ます O

研究のみならず公私共に御指導、御鞭援を賜わりました広島大学医学部総合薬学科助教授
科学研究所教授

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ます O

X線結晶構造解析を行っていただきました、理学電機株式会社

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先生に深謝致します O

平 成 7年 12月

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