O2-binding properties of double-sided porphinatoiron(II)s with polar substituents and their human serum albumin hybrids

Article abstract of DOI:10.1246/bcsj.74.1695

Double-sided porphinatoiron(II)s with polar substituents [R; hydroxy (FeDP(OH)), methoxy (FeDP(OMe)), and acetoxy (FeDP(OAc))] on the 2,2-dimethylpropanoyloxy-fence groups have been synthesized. FeDP(OMe) and FeDP(OAc) formed five-N-coordinated high-spin Fe²⁺ complexes with an intramolecularly bound axial imidazole in toluene (or CH₂Cl₂) under an N₂ atmosphere. Upon the addition of O₂, they produced stable O₂ adducts at 25 °C; their half-lives in water-saturated toluene (50-77 h) are 2-3 fold longer compared to that of the single-face encumbered porphinatoiron(II) (FeP). Their O₂-binding parameters are almost identical to that of FeDP(H), which has nonpolar substituents on the fences. In contrast, FeDP(OH) showed a significantly low O₂-binding affinity and was immediately oxidized to the Fe³⁺ state after contact with bubbling O₂ gas. The incorporation of these FeDPs into the human serum albumin (HSA) provided artificial hemoproteins, which can reversibly bind and release O₂ under physiological conditions (in aqueous media, pH 7.3, 37 °C) like hemoglobin and myoglobin. The half-life of the dioxygenated HSA-FeDP(H) reached 5 h (37 °C). This corresponded to a 2.5-fold increase compared to that of HSA-FeP. The time dependences of the absorption changes accompanying the O_2- and CO-rebindings to the HSA-FeDPs after laser flash photolysis were composed of two phases. These observations indicate that the recombination of O₂ and CO to the central Fe²⁺ ion is affected by the microenvironments around the FeDPs in the HSA structure, e.g. a steric hindrance of the amino acid residue and a difference in polarity. Furthermore, FeDP(H) incorporated into HSA showed a high stability against H₂O₂.

Full text of DOI:10.1246/bcsj.74.1695

c. Jpn.
© 2001 The Chemical Society of Japan
Bull. Chem. Soc. Jpn., 74, 1695–1702 (2001) 1695
e Chemical
ciety of Ja-
n
e Chemical
ciety of Ja-
n
O2-Binding Properties of Double-Sided Porphinatoiron(Ⅱ)s with Polar
Substituents and Their Human Serum Albumin Hybrids
O2-Binding of Double-Sided Porphinatoiron(Ⅱ)s
T. Komatsu et al.
Teruyuki Komatsu, Tomoyuki Okada, Miho Moritake, and Eishun Tsuchida*,#
01
95
02
SJA8
09-2673
056
Department of Polymer Chemistry, Advanced Research Institute for Science and Engineering, Waseda University,
Tokyo 169-8555
(Received February 15, 2001)
Double-sided porphinatoiron(Ⅱ)s with polar substituents [R; hydroxy (FeDP(OH)), methoxy (FeDP(OMe)), and
acetoxy (FeDP(OAc))] on the 2,2-dimethylpropanoyloxy-fence groups have been synthesized. FeDP(OMe) and
FeDP(OAc) formed five-N-coordinated high-spin Fe2+ complexes with an intramolecularly bound axial imidazole in tol-
uene (or CH2Cl2) under an N2 atmosphere. Upon the addition of O2, they produced stable O2 adducts at 25 °C; their half-
lives in water-saturated toluene (50–77 h) are 2–3 fold longer compared to that of the single-face encumbered porphina-
toiron(Ⅱ) (FeP). Their O2-binding parameters are almost identical to that of FeDP(H), which has nonpolar substituents
on the fences. In contrast, FeDP(OH) showed a significantly low O2-binding affinity and was immediately oxidized to
the Fe3+ state after contact with bubbling O2 gas. The incorporation of these FeDPs into the human serum albumin
(HSA) provided artificial hemoproteins, which can reversibly bind and release O2 under physiological conditions (in
aqueous media, pH 7.3, 37 °C) like hemoglobin and myoglobin. The half-life of the dioxygenated HSA–FeDP(H)
reached 5 h (37 °C). This corresponded to a 2.5-fold increase compared to that of HSA–FeP. The time dependences of
the absorption changes accompanying the O2- and CO-rebindings to the HSA–FeDPs after laser flash photolysis were
composed of two phases. These observations indicate that the recombination of O2 and CO to the central Fe2+ ion is af-
fected by the microenvironments around the FeDPs in the HSA structure, e.g. a steric hindrance of the amino acid resi-
due and a difference in polarity. Furthermore, FeDP(H) incorporated into HSA showed a high stability against H2O2.
01
01
A simple, but serious, problem often found in synthetic he-
moprotein models is the short lifetimes of their biological ac-
tivities under physiological conditions, namely in water (pH
7.3) at 37 °C. In order to alleviate this fault, both-faces encum-
bered porphinatoirons have been synthesized to inhibit unfa-
vorable side-reactions by a steric hindrance on both sides of
the porphine ring.1–5 We have also found that 5,10,15-tris[2,6-
bis(2,2-dimethylpropanoyloxy)phenyl]-20-{2-(2,2-dimethyl-
propanoyloxy)-6-[5-(1-imidazolyl)pentanoyloxy]phenyl}por-
phinatoiron(Ⅱ) [double-sided porphinatoiron(Ⅱ) (FeDP(H))]
formed a stable O2-adduct with a lifetime on the order of one
day in not only toluene solution, but also aqueous media, by
embedding into the bilayer membrane of phospholipid vesicles
(Chart 1).6
On the other hand, it has been shown that human serum al-
bumin (HSA) incorporating 5,10,15,20-tetrakis[α,α,α,α-o-
(2,2-dimethylpropanamido)phenyl]-2-[8-(2-methyl-1-imida-
zolyl)octanoyloxymethyl]porphinatoiron(Ⅱ) (FeP) (HSA–FeP)
can reversibly bind and release O2 under physiological condi-
tions, like Hb and Mb.7 Since the serum albumin is the abun-
dant plasma protein, the biological advantage is significant
compared to the phospholipid vesicles as a vehicle for porphi-
natoirons. The obtained HSA–FeP solution has good compati-
bility with human whole blood and can quantitatively transport
O2 in vivo.7b,c,8 At present, HSA–FeP has become one of the
most promising materials as a red cell substitute.
On the basis of these two findings, we expect that the com-
bination of the double-sided porphinatoirons (FeDPs) and
HSA can provide a novel O2-carrying hemoprotein, which can
form a more stable O2-adduct complex. Recently, new double-
sided porphinatoiron(Ⅱ)s with different polar substituents on
the 2,2-dimethylpropanoyloxy-fence groups have been synthe-
sized to increase the compatibility of porphyrin to HSA, and
control the O2-binding equilibrium. A polar substituent around
the O2-binding site of the synthetic heme generally increases
its O2-binding affinity, which is due to a decrease in the O2-dis-
sociation rate constant (polarity effect).9–11 This kind of work
was mostly done in the 1980’s, but has recently gained atten-
tion again for analogues to the site-directed mutagenesis of the
distal residues in O2-binding hemoproteins.12,13 This paper de-
scribes the O2-binding equilibria and kinetics of the newly syn-
thesized double-sided porphinatoiron(Ⅱ)s with different polar
substituents in organic solvents. The O2-binding properties of
the HSA hybrids incorporating these FeDPs in aqueous media
are also evaluated in detail. Furthermore, we have found that
FeDP(H) incorporated into HSA shows high stability against
H2O2.
Experimental
Materials and Apparatus. Infrared spectra were recorded
with a JASCO FT/IR-410 spectrometer. 1H NMR spectra were
measured using a JEOL Lambda 500 spectrometer. Chemical
shifts were expressed in parts per million downfield from Me4Si as
an internal standard. FAB-MS spectra were obtained from a
# CREST investigator, JST.

1696 Bull. Chem. Soc. Jpn., 74, No. 9 (2001)
O2-Binding of Double-Sided Porphinatoiron(ꢀ)s
= 0.78 (CHCl3). IR (NaCl) 1111 (COC (ether)), 1736 (CwO (es-
1
ter)) cm−1. H NMR (CDCl3) δ 1.1 (6H, s, –C(CH3)2–), 3.2 (3H, s,
CH3OCH2–), 3.3 (2H, s, CH3OCH2–), 3.6 (3H, s, –CO(wO)CH3).
Five% ethanolic KOH (34.0 mL) was added to a THF solution
(5 mL) of 2 (3.0 g, 20.5 mol) and stirred for 12 h at 45 °C. After
the solvent was evaporated, the residue was dissolved again to
CHCl3 and carefully neutralized by hydrochloric acid. The organ-
ic layer was washed with water and dried in vacuo to give 3-meth-
oxy-2,2- dimethylpropanoic acid (3) as transparent liquid (1.06 g,
62%). IR (NaCl) 1122 (COC (ether)), 1704 (CwO), 3100 (OH)
cm−1
.
1H NMR (CDCl3) δ 1.2 (6H, s, –C(CH3)2–), 3.3 (3H, s,
CH3OCH2–), 3.4 (2H, s, CH3OCH2–).
5-[6-Hydroxy-2-(3-methoxy-2,2-dimethylpropanoyloxy)-
phenyl]-10,15,20-tris[2,6-bis(3-methoxy-2,2-dimethylpropano-
yloxy)phenyl]porphine (4). Thionyl chloride (0.72 mL, 9.82
mmol) was added to 3 (259 mg, 1.96 mmol) under an argon atmo-
sphere and stirred for 1 h at room temperature. Excess of thionyl
chloride was removed in vacuo (5.3 kPa) and dissolved in dry
THF (30 mL). This solution was then added dropwise to a THF
solutionof5,10,15,20-tetrakis(2,6-dihydroxyphenyl)porphine (200
mg, 0.269 mmol) and 4-(dimethylamino)pyridine (230 mg, 1.88
mmol) at room temperature, and refluxed for 12 h. The obtained
mixture was brought to dryness on a rotary evaporator and extract-
ed with CHCl3. The organic layer was then washed with water
and the evaporated residue was chromatographed on silica-gel
flash-column using CHCl3/CH3OH (15/1 v/v) as the eluent. The
second band eluted was collected and reduced to a small volume
on a rotary evaporator. The residue was dried at room temperature
for several hours in vacuo to give compound 4 as purple crystals
(42.4 mg, 10%). Rf = 0.44 (CHCl3/CH3OH, 15/1 v/v). IR (NaCl)
1098 (COC (ether)), 1756 (CwO(ester)), 3454 (OH) cm−1. UV-vis
(CHCl3) λmax (10−3 ε (M−1 cm−1)) 417 (410), 510 (25), 541 (4.0),
586(7.8),638 nm(0.93). 1HNMR(CDCl3)δ −3.0 (2H, d, innerH),
−0.7–−0.2 (42H, m, –C(CH3)2–), 0.9–2.4 (21H, m, CH3O–), 2.5–
2.7 (14H, m, CH3OCH2–), 6.9–7.9 (12H, m, Phenyl), 8.8 (8H, d,
pyrrole-β). FAB-MS: m/z 1541.2 [M+].
Chart 1.
JEOL JMS-SX102A spectrometer. UV-vis absorption spectra
were recorded on a JASCO V-570 spectrophotometer. Thin-layer
chromatography (TLC) was carried out on 0.2 mm precoated
plates of silica-gel 60 F254 (Merck). Purification was performed
by a silica-gel 60 (Merck) column or flash-column chromatogra-
phy. 5,10,15,20-Tetrakis(2,6-dihydroxyphenyl)porphine, and 5-
(1-imidazolyl)pentanoic acid were prepared according to previ-
ously reported procedures.6 All solvents were purified by distilla-
tion before use. Other chemicals were of commercial high-purity
grades and not further purified. The water used was deionized us-
ing an ADVANTEC GS-200 system. An HSA was purchased
from Bayer Co., Ltd. (Albumin Cutter, 5 wt%). Isoelectric points
were measured by a Pharmacia Phastsystem using isoelectric fo-
cusing (IEF) in pH 3–9 Phast Gel IEF 3–9.7b The temperature
during the electrophoresis was maintained at 15 °C. The markers
used were from an Isoelectric Focusing Calibration Kit.
5-{6-[5-(1-Imidazolyl)pentanoyloxy]-2-(3-methoxy-2,2-di-
methylpropanoyloxy)phenyl}-10,15,20-tris[2,6-bis(3-methoxy-
2,2-dimethylpropanoyloxy)phenyl]porphine (5). After oxalyl
chloride (1.0 mL, 11.5 mmol) was added to a dry CH3CN (20 mL)
solution of 5-(1-imidazolyl)pentanoic acid hydrochloride5 (87.8
mg, 0.429 mmol) under an argon atmosphere, the mixture was
stirred for 1 h at 65 °C. The excesses of oxalyl chloride and
CH3CN were removed in vacuo to yield a pale-yellow solid. A
dry CH3CN (10 mL) solution of 4 (42.4 mg, 0.0286 mmol) and 4-
(dimethylamino)pyridine (78.6 mg, 0.643 mmol) was added drop-
wise to this acid chloride at room temperature under an argon at-
mosphere and darkness. The solution was then refluxed for a fur-
ther 12 h. After the solvent was evaporated, CHCl3 extracted the
residue, which was dried over anhydrous Na2SO4. The obtained
mixture was chromatographed on a silica-gel flash-column using
CHCl3/CH3OH (8/1 v/v) as the eluent. The major band was col-
lected and dried at room temperature for several hours in vacuo to
give compound 5 as purple crystals (35.4 mg, 74%). Rf = 0.50
(CHCl3/CH3OH, 8/1 v/v). IR (NaCl) 1102 (COC (ether)), 1760
(CwO (ester)) cm−1. UV-vis (CHCl3) λmax (10−3 ε (M−1 cm−1))
3-Methoxy-2,2-dimethylpropanoic Acid (3).
Sodium hy-
dride (60%) (6.0 g, 149.8 mmol) was dispersed to a dry THF solu-
tion (500 mL) of methyl 3-hydroxy-2,2-dimethylpropanoate (1)
(19.8 g, 149.8 mmol), and stirred for 2 h at 0 °C and 2 h at room
temperature. Iodomethane (14 mL, 224.7 mmol) was then added
dropwise to the mixture. After stirring for 12 h, the solvent was
brought to dryness on a rotary evaporator and CHCl3 extracted the
residue. The organic layer was washed with water several times
and dried over anhydrous Na2SO4. The vacuum distillation of this
mixture under 1.6 kPa at 43 °C yielded methyl 3-methoxy-2,2-
dimethylpropanoate (2) as a transparent liquid (6.83 g, 31%). Rf
1
416 (420), 510 (26), 541 (4.1), 586 (8.0), 640 nm (0.96). H NMR
(CDCl3)δ −3.0(2H, s, innerH), −0.6–−0.2 (42H, m, –C(CH3)2–),
0.8–2.2 (20H, m, ImCH2(CH2)3–, CH3OCH2–), 3.6–3.7 (2H, m,
ImCH2–), 6.6, 6.9, 7.2 (3H, 3s, Im), 7.4–7.9 (12H, m, phenyl), 8.8
(8H, m, pyrrole-β). FAB-MS: m/z 1691.7 [M+].
Fe3+DP(OMe) Cl−. Fe(CO)5 (0.082 mL, 630 µmol) and I2

T. Komatsu et al.
Bull. Chem. Soc. Jpn., 74, No. 9 (2001) 1697
(5.3 mg, 21 µmol) were added to a dry toluene solution (4.0 mL)
of 5 (17.7 mg, 10.5 µmol) under an argon atmosphere. After the
mixture was heated at 110 °C for 3 h, an aqueous NaCl solution
was added at room temperature. CHCl3 extracted the dispersion,
which was washed with water. After drying over anhydrous
Na2SO4, the organic layer was evaporated to dryness and the resi-
due was chromatographed on a silica-gel flash-column using
CHCl3/CH3OH (10/1 v/v) as eluent. The major band was collect-
ed and dried at room temperature for several hours in vacuo to
give Fe3+DP(OMe) Cl− as purple crystals (14.6 mg, 78%). Rf =
0.44 (CHCl3/CH3OH, 10/1 v/v). IR (NaCl) 1098 (COC (ether)),
1760(CwO(ester))cm−1. UV-vis(CHCl3)λmax (10−4 ε (M−1 cm−1))
341 (3.9), 416 (12), 508 (1.2), 575 nm (0.81). FAB-MS: m/z
1745.2 [M+−Cl]. Found: C, 64.78; H, 6.55; N, 4.23%. Calcd for
C94H108N6O23FeCl•C6H6: C, 64.60; H, 6.28; N, 4.52%
ImCH2–), 2.4–2.8 (14H, m, BzlOCH2–), 3.4 (4H, m,
Im(CH2)3CH2–), 3.9–4.3 (14H, m, PhCH2–), 6.6, 6.9 (2H, 2s, Im),
7.1–7.8 (47H, m, Phenyl, Im), 8.8 (8H, m, pyrrole-β). FAB-MS:
m/z 2224.2 [M+].
5-{2-(3-Hydroxy-2,2-dimethylpropanoyloxy)-6-[5-(1-imida-
zolyl)pentanoyloxy]phenyl}-10,15,20-tris[2,6-bis(3-hydroxy-
2,2-dimethylpropanoyloxy)phenyl]porphine (10). Boron trif-
luoride diethyl ether complex (332 µL, 2.63 mmol) and ethanethi-
ol (1.36 mL, 18.4 mmol) were added to the CH2Cl2 solution of 9
(16.2 mg, 7.28 µmol). After stirring for 2.5 h at room tempera-
ture, water was added to stop the reaction and CHCl3 extracted the
mixture. The organic layer was washed with water and dried over
anhydrous Na2SO4. The residue was chromatographed on a silica-
gel flash-column using CHCl3/CH3OH (6/1 v/v) as the eluent.
The major band was collected and dried at room temperature for
several hours in vacuo to give compound 10 as purple crystals
(13.6 mg, 44%). Rf = 0.40 (CHCl3/CH3OH, 6/1 v/v). IR (NaCl)
1756 (CwO (ester)), 3435 (OH (alcohol)) cm−1. UV-vis (CHCl3)
λmax (10−3 ε (M−1 cm−1)) 417 (390), 512 (23), 546 (3.8), 582
3-Benzyloxy-2,2-dimethylpropanoic Acid (7).
This com-
pound was prepared according to a similar manner of 3, except for
using benzyl bromide. From the crude material, methyl 3-benzyl-
oxy-2,2-dimethylpropanoate (6) was purified by removing the un-
reacted benzyl bromide and 1 under 1.7 kPa at 75 °C (5.65 g,
68%). After saponification, 3-benzyloxy-2,2-dimethylpropanoic
acid (7) was given as white crystals (921 mg, 38%). Rf = 0.54
(CHCl3/MeOH, 30/1 v/v). IR (NaCl) 1100 (COC (ether)), 1704
1
(8.6), 634 nm (0.92). H NMR (CDCl3) δ −3.3 (2H, s, innerH),
−0.1–0.4 (42H, m, –C(CH3)2–), 0.8–0.9 (4H, m, ImCH2(CH2)2–),
1.0–1.5 (16H, m, HOCH2–, Im(CH2)3CH2–), 3.6(2H, m, ImCH2–),
6.6, 6.8, 7.2 (3H, 3s, Im), 7.4–7.9 (12H, m, phenyl), 8.8 (8H, m,
pyrrole-β). FAB-MS: m/z 1593.2 [M+].
.
(CwO), 2900 (OH) cm−1 1HNMR (CDCl3) δ 1.2 (6H, s,
–C(CH3)2–), 3.5 (2H, s, BzlOCH2–), 4.6 (2H, s, PhCH2–), 7.3–7.4
(5H, m, phenyl).
Fe3+DP(OH)Cl−. Iron insertion to the compound 10 was
performed by the same procedure as described above.
Fe3+DP(OH) Cl− was obtained as a purple crystalline (12.9 mg,
95%). Rf = 0.32 (CHCl3/CH3OH, 6/1 v/v). IR (NaCl) 1753 (CwO
(ester)), 3372 (OH) cm−1. UV-vis (CHCl3): λmax (10−4 ε (M−1
cm−1)) 342 (2.5), 418 (9.0), 509 (0.74), 586 nm (0.42). FAB-MS:
m/z 1647.4 [M+−Cl]. Found: C, 61.69; H, 5.31; N, 4.32%. Calcd
for C87H94N6O23FeCl•H2O: C, 61.98; H, 5.64; N, 4.05%
5-[2-(3-Benzyloxy-2,2-dimethylpropanoyloxy)-6-hydroxy-
phenyl]-10,15,20-tris-[2,6-bis(3-benzyloxy-2,2-dimethylpro-
panoyloxy)phenyl]phenylporphine (8). Oxalyl chloride (1.23
mL, 14.1 mmol) was added to 7 (589 mg, 2.83 mmol) under an ar-
gon atmosphere and stirred for 1.5 h at room temperature. Excess
of oxalyl chloride was removed in vacuo, and the residue was dis-
solved in dry THF (30 mL). The obtained solution was slowly
added dropwise to a THF solution (100 mL) of 5,10,15,20-tet-
rakis(2,6-dihydroxyphenyl)porphine (300 mg, 0.404 mmol) and
4-(dimethylamino)pyridine (346 mg, 2.83 mmol) at room temper-
ature, and the mixture was refluxed for 3 h. After the evaporation
of THF, CHCl3 extracted the mixture. The organic layer was
washed with water and dried over anhydrous Na2SO4. The solvent
was then removed and the residue was chromatographed on a sili-
ca-gel flash-column using CHCl3/CH3OH (40/1 v/v) as the eluent.
The second band eluted was collected and dried at room tempera-
ture for several hours in vacuo to give compound 8 as purple crys-
tals (104 mg, 13%). Rf = 0.34 (CHCl3/CH3OH, 40/1 v/v). IR
(NaCl) 1100 (COC (ether)), 1759 (CwO (ester)), 3450 (OH (alco-
hol)) cm−1. UV-vis (CHCl3) λmax (10−3 ε (M−1 cm−1)) 416 (400),
5-{2-(3-Acetoxy-2,2-dimethylpropanoyloxy)-6-[5-(1-imida-
zolyl)pentanoyloxy]phenyl}-10,15,20-tris[2,6-bis(3-acetoxy-
2,2-dimethylpropanoyloxy)phenyl]porphine (11).
Acetyl
chrolide (0.186 mL, 2.64 mmol) was added to a dry THF solution
(8 mL) of 10 (20 mg, 0.0126 mmol) and pyridine (0.268 mL, 2.64
mmol). The mixture was stirred for 2 h at room temperature and
brought to dryness on a rotary evaporator. The residue was ex-
tracted with CHCl3 and the organic layer was washed with water.
After drying over anhydrous Na2SO4, the organic layer was evapo-
rated and chromatographed on a silica-gel flash-column using
CHCl3/CH3OH (6/1 v/v) as the eluent. The major band was col-
lected and dried at room temperature for several hours in vacuo to
give compound 11 as purple crystals (13.3 mg, 65%). Rf = 0.57
(CHCl3/CH3OH, 6/1 v/v). IR (NaCl) 1738, 1759 (CwO (ester))
cm−1. UV-vis (CHCl3) λmax (10−3 ε (M−1 cm−1)) = 416 (430),
510 (27), 541 (4.9), 586 (8.6), 638 nm (1.1). 1H NMR (CDCl3) δ
−3.1 (2H, s, innerH), −0.9–−0.4 (42H, m, –C(CH3)2–), 0.9 (4H,
m, ImCH2(CH2)2–), 1.3–1.8 (23H, m, CH3C(wO)O–, Im(CH2)3
CH2–), 3.0–3.4 (14H, m, AcOCH2–), 3.7 (2H, m, ImCH2–) 6.7,
6.9, 7.2 (3H, 3s, Im), 7.3–7.9 (12H, m, phenyl), 8.8 (8H, m, pyr-
role-β). FAB-MS: m/z 1887.0 [M+−H].
1
510 (27), 541 (4.0), 585 (8.4), 638 nm (0.93). H NMR (CDCl3) δ
−3.0 (2H, d, innerH), −0.9–−0.5 (42H, m, –C(CH3)2–), 2.1–2.8
(14H, m, BzlOCH2–), 3.8–4.3 (14H, m, PhCH2–), 6.8–7.8 (47H,
m, phenyl), 8.8 (8H, d, pyrrole-β) . FAB-MS m/z 2073.5 [M+
−H].
5-{2-(3-Benzyloxy-2,2-dimethylpropanoyloxy)-6-[5-(1-imi-
dazolyl)pentanoyloxy]phenyl}-10,15,20-tris[2,6-bis(3-benzyl-
oxy-2,2-dimethylpropanoyloxy)phenyl]porphine (9). The in-
troduction of the (1-imidazolyl)alkyl arm to 8 was carried out ac-
cording to the same procedure for 5, as described above. The
compound 9 was afforded as purple crystals (64.8 mg, 60%). Rf =
0.27 (CHCl3/CH3OH, 20/1 v/v). IR (NaCl) 1097 (COC (ether)),
Fe3+DP(OAc) Cl−. Iron insertion to compound 10 was car-
ried out using the same procedure as that for Fe3+DP(OMe)Cl−, as
described above. Fe3+DP(OAc) Cl− was obtained as purple crys-
tal (12.9 mg, 94%). Rf = 0.59 (CHCl3/CH3OH = 6/1 (v/v)). IR
(NaCl) 1737, 1759 (CwO (ester)) cm−1
. UV-vis (CHCl3) λmax
1760 (CwO (ester)) cm−1
.
UV-vis (CHCl3) λmax (10−3 ε (M−1
(10−4 ε (M−1 cm−1)) = 341 (3.4), 416 (11), 507 (1.1), 578 nm
(0.71). FAB-MS: m/z 1941.3 [M+−Cl]. Found: C, 62.22; H, 5.66;
N, 4.38%. Calcd for C101H108N6O30FeCl•C6H6: C, 62.53; H, 5.59;
N, 4.09%
1
cm−1)) 416 (430), 509 (27), 539 (4.2), 584 (8.2), 639 nm (1.0). H
NMR (CDCl3) δ −3.1 (2H, s, innerH), −1.1–−0.5 (42H, m,
–C(CH3)2–), 0.8–0.9 (4H, m, ImCH2(CH2)2–), 1.2 (2H, m,

1698 Bull. Chem. Soc. Jpn., 74, No. 9 (2001)
O2-Binding of Double-Sided Porphinatoiron(ꢀ)s
Reduction of Fe3+ Complex to Fe2+ Complex in Organic
Solvent. Reduction to the porphinatoiron(Ⅱ) complex was car-
ried out using toluene (or CH2Cl2) − aq Na2S2O4 in a heteroge-
neous two-phase system under aerobic conditions, as previously
reported.6,14 After separation of the two phases, the organic layer
containing the reduced compound was transferred into a cuvette
under an Ar atmosphre. A Karl Fisher's reagent (Kyoto Electronic
Ind.) measured the water content in the obtained organic solution.
The concentration of the water in a toluene solution was 0.048
wt% and 0.22 wt% in CH2Cl2, which shows the good agreement
with the literature values.
Preparation of HSA–FeDP Hybrid.
The HSA–FeDP hy-
brid was prepared by the following procedure. Aqueous ascorbic
acid (17 mM, 37 µL) was added to a DMSO solution of an
Fe(ꢀ)DP derivative (133 µM, 5 mL) under carbon monoxide
(CO). Partial reduction of the central Fe3+ ion occurred in this
stage. Then, the obtained solution was photoirradiated with a 250-
W ultra high-pressure Hg arc-lamp (Ushio UCH-250).15 After
complete reduction, the UV-vis absorption spectrum showed the
formation of a six-coordinated carbonyl complex. This CO com-
plex in DMSO was injected into the phosphate buffer solution (33
mM, pH 7.3) of HSA (17 µM, pH 7.3, 20 mL) under a CO atmo-
sphere, and the mixture was dialyzed with a cellulose membrane
against a phosphate buffer (pH 7.3) for 2 h and 15 h at 4 °C. At
the last, the total volume was adjusted to 33 mL, giving an HSA–
FeDP(CO) solution (FeDP/HSA = 8 (mol/mol), [Fe] = 20 µM).
Binding Numbers of FeDP into HSA. Based on the absor-
bance intensity of the UV-vis absorption spectra of the HSA–
FeDP(CO) hybrid with different FeDP/HSA mixing ratios, the
binding numbers of FeDP in the albumin host were assayed.7b,c
Fig. 1. Visible absorption spectral changes of FeDP(OMe)
in toluene solution at 25 °C.
O2- and CO-Coordination Equilibria and Kinetics.
The
O2- and CO-bindings to FeDP derivative are expressed by
L
kon
FeDP + L
FeDP(L)
(1)
L
koff
L
(K = konL/koff
)
The O2-binding affinity (the pressure at half O2-binding for FeDP,
L
P1/2 = 1/KL) of FeDPs in organic solvents or their HSA hybrids
in aqueous media was determined by spectral changes at various
partial pressures of O2 as in previous literature.6,14,16,17 The FeDPs
concentrations of 20 µM were normally used for UV-vis absorp-
tion spectroscopy. The spectra were recorded within the range of
350–700 nm. The O2- and CO-association and -dissociation rate
constants (kon, koff) were measured by a competitive rebinding
technique using a Unisoku TSP-600 laser-flash photolysis appara-
tus.6,14,16,17 Because of the absorption decays accompanied O2-
and CO-rebinding to FeDPs in organic solvents obeyed a single
exponential, we applied a first-order kinetics to calculate the rates.
On the other hand, HSA–FeDP hybrid in aqueous solution showed
triphases. We employed triple-exponential kinetics to analyze the
absorption decays.7d
Fig. 2. Visible absorption spectral changes of FeDP(OMe)
during the O2-titration in toluene solution at 25 °C.
the Fe2+ complex by reduction in a heterogeneous two-phase
system (toluene or CH2Cl2 with aq Na2S2O4) under an N2 at-
mosphere. For instance, the UV-vis absorption spectrum of the
orange solution of FeDP(OMe) showed the formation of a
five-N-coordinated complex (λmax: 437, 544, 560 nm, Fig. 1),
which was constant in the range from 5 µM–1 mM at 10–70
°C. The paramagnetic S = 2 state of FeDP(OMe) was deter-
mined by the β-pyrrolic proton signals at 50.2, 50.5, 56.0, 57.7
ppm downfield to TMS (25°C). Nopeaksbetween−5and−15
ppm demonstrated that a square-planar Fe2+ porphinate (S =
1) did not exist.18 We can conclude that FeDP(OMe) is a five-
coordinated high-spin Fe2+ complex with an intramolecularly
bound imidazole under an N2 atmosphere. These results are
consistent with the data of FeDP(H), which has no p olar sub-
stituents on the fences.6 FeDP(OAc) also showed the same re-
sults (λmax: 437, 544, 560 nm, and β-pyrrolic proton signals at
Results and Discussion
O2-Coordination Properties of FeDPs in Organic Sol-
vents. Double-sided porphinatoiron(Ⅱ)s with polar substitu-
ents [R; hydroxy (FeDP(OH)), methoxy (FeDP(OMe)), and
acetoxy (FeDP(OAc))] on the 2,2-dimethylpropanoyloxy-
fence groups have been synthesized according to the modified
procedure of FeDP(H) using the corresponding acid chlorides
(Scheme 1). The obtained Fe3+ compounds were converted to

T. Komatsu et al.
Bull. Chem. Soc. Jpn., 74, No. 9 (2001) 1699
Scheme 1.
51.1, 51.2, 56.2, 58.1 ppm). In contrast, the UV-vis absorption
maxima of FeDP(OH) appeared at different positions (426,
534, 559 nm), which may suggest partial coordination of the
hydroxy group to the Fe2+ center.
Upon exposure of these FeDPs solutions to O2 or CO, the
UV-vis absorptions immediately changed to those of the O2 or
CO adduct complex, respectively. The dioxygenations were
kinetically stable and reversible at 25 °C, depending on the O2-
partial pressure (Fig. 2); however, the oxidation to the Fe3+
porphinate slowly took place by a proton-driven process in wa-
ter-saturated toluene, and the final products were actually all
Fe3+OH complexes with λmax at 417 and 576 nm. The seven
fences on the ring plane obviously prevent µ-oxo porphine
dimer formation. The half-lives (τ1/2) of the O2-addducts were
determined to be 50–77 h. (25 °C, under 1 atm O2) for
FeDP(H), FeDP(OMe), and FeDP(OAc) (Table 1), which are
all 2–3-fold longer compared to that of FeP (24 h) under the
same conditions.14
On the other hand, the dioxygenated FeDP(OH) has very
short lifetime (τ1/2: 0.2 h). It has been reported that the intro-
duction of a flexible protic group around the O2-binding site
accelerates irreversible oxidation.10,12 The same situation
probably holds true for FeDP(OH).
O
The O2-binding affinities (P1/2 2), and association and disso-
Table 1. O2-Binding Parameters of Double-Sided Porphinatoiron(Ⅱ)s in Organic Solvents at 25 °C
O
O
O
CO
O
Solvent
P1/2
kPa
2
10−7 kon
2
10−3 koff
s−1
2
10−6 kon
τ1/2
h
2
M−1 s−1
2.4a)
0.0026
1.1
M−1 s−1
1.8a)
0.2
FeDP(H)
FeDP(OH)
FeDP(OMe)
FeDP(OAc)
FeP
toluene
CH2Cl2
toluene
toluene
toluene
1.7a)
33.5
2.0
2.4a)
77
0.2
50
77
24
0.055
2.1
1.3
1.7
2.7
0.99
1.7
5.1b)
16b)
46b)
2.9b)
a) From Ref. 6. b) From Ref. 14.

1700 Bull. Chem. Soc. Jpn., 74, No. 9 (2001)
O2-Binding of Double-Sided Porphinatoiron(ꢀ)s
O
O
ciation rate constants (kon 2, koff 2) were almost identical for
FeDP(H), FeDP(OMe), and FeDP(OAc), indicating that the
nonprotic substituents on the 2,2-dimethylpropanoyloxy-fence
groups did not cause any change in their O2-binding equilibria
and kinetics (Table 1). In contrast, FeDP(OH) showed an ex-
due to partial neutralization of the surface charge. The FeDPs
without any ionic residue interacts nonspecifically with a hy-
drophobic cavity of HSA. We concluded that the hydrophobic
interaction is the major molecular force of the FeDP binding to
HSA, and its incorporation does not induce any changes in the
surface charge distribution of the host albumin.
O
tremely low binding affinity for O2 (P1/2 2: 33.5 kPa), as op-
posed to the prediction that the H-bond would increase the O2-
binding affinity.1,12,13 This is consistent with Kyuno’s results.10
In our case, the hydroxy groups form interfence H-bondings
with the nearest carbonyls in the deoxy state; therefore, the ex-
tra binding energy of the O2 molecule by the H-bond formation
was cancelled out by cleavage of the former one. Our laser-
flash experiments for FeDP(OH) revealed that the low O2-
binding affinity is kinetically ascribed to the small association
rate constant by the steric hindrance, rather than by the de-
O2-Coordination Properties of HSA-FeDPs.
The UV-
vis absorption spectrum of the aqueous HSA including carbon-
yl FeDP(OMe) showed the formation of the typical CO-coor-
dinated low-spin tetraphenylporphinatoiron(Ⅱ) derivative
(λmax: 427, 546 nm) (Fig. 3). Light irradiation with a 500 W
incandescent lamp of this solution under flowing O2 led to CO
dissociation, producing the dioxygenated species (λmax: 426,
549 nm). Upon exposure of the dioxygenated HSA–
FeDP(OMe) to N2, the UV-vis absorption spectrum changed
to that of a five-N-coordinated Fe2+ complex with an intramo-
lecularly coordinated axial imidazole (λmax: 438, 540, 560 nm).
This dioxygenation was reversible and kinetically stable under
physiological conditions (pH 7.3, 37 °C). The same O2-adduct
formation was also observed for the HSA hybrids with
FeDP(H) and FeDP(OAc).
However, the autooxidation reaction of the oxy state slowly
occurred, and the absorption band of 549 nm almost disap-
peared after one day at 37 °C, leading to the formation of the
inactive Fe3+ porphinate. The half-life of the dioxygenated
species (τ1/2) of HSA–FeDP(H) was 5 h at 37 °C (pH 7.3, un-
der 1 atm O2), which is 2.5-fold longer compared to the τ1/2 of
HSA–FeP.7c
O
creased koff 2 due to the H-bonding. The space-filling model
showed that the four hydroxy end groups form H-bondings
with the nearest neighboring esters,19 so that each substituent
could be noncovalently linked to construct a narrow pocket
around the O2-binding site. Indeed, the stretching frequency of
the ester fences (νCwO) of FeDP(OH) are shifted to the low fre-
quency region relative to that of FeDP(H)(1760 → 1753 cm−1),
suggesting the H-bond formation. Moreover, the coordination
of the hydroxy group to the central iron may also retard the O2
association. These results showed that covalently attaching the
nonprotic polar (methoxy or acetoxy) substituents on the 2,2-
dimethylpropanoyloxy groups of the double-sided porphine
does not change its O2-binding parameter and the half-life of
the dioxygenated complex in water-saturated toluene. On the
other hand, the hydroxy groups significantly promote the irre-
versible oxidation and decrease the O2-binding affinity, which
is kinetically ascribed to the 1/103-fold lower association rate
constant compared to FeDP(H).
The association and dissociation rate constants (kon, koff) of
O2 and CO were again explored by laser flash photolysis. The
absorption decays that accompanied these gaseous recombina-
tions were composed of three-phases of the first-order kinetics
(Fig. 4), therefore the curves were fit by a triple-exponential
equation, which is similar to the previously reported HSA–
FeP.7d The minor, less than 10%, component, which has the
fastest rate constant, was independent of the O2 and CO con-
FeDPs Incorporations into HSA. Serum albumin is the
major transport protein, which binds a great variety of metabo-
lites and organic compounds in our blood stream.20,21 Despite
the tremendous research on ligand bindings, there are only a
few reports on the crystal structure of human serum albumin
(HSA). The 585 amino acids consist of a unique heart-shaped
structure, that is made of three repeating domains ꢁ to ꢀ, and
each one is constructed of two sub-domains.22,23 The majority
of the ligands is bound at one or both sites within special hy-
drophobic cavities of the subdomains ⅡA and ꢀA. A maxi-
mum of eight FeP molecules were incorporated into certain
domains of human serum albumin with binding constants from
106–104 M−1.7b,c FeDPs were also expected to bind to HSA in
a similar fashion, and the binding sites would also be identical.
From quantitative analyses of the absorption intensity for the
Soret band of aqueous HSA–FeDP(OMe), the maximum
binding numbers of FeDP(OMe) to an HSA were determined
to be eight. The other FeDPs [FeDP(H), FeDP(OAc)] also
showed the same results. The binding numbers are always
eight and independent of the polar substituents. The red-col-
ored solution of HSA–FeDPs could be stored without any ag-
gregation and precipitation for more than six months at 4 °C.
The isoelectric points (pI) of the HSA–FeDPs [FeDPs/HSA =
8 (mol/mol)] were all 4.8 for FeDP(H), FeDP(OMe) and
FeDP(OAc) which was exactly the same as that of HSA. Fatty
acid binding, for example, induced a reduction of the pI value
Fig. 3.
Visible absorption spectral changes of HSA–
FeDP(OMe) inphosphatebuffersolution (pH 7.3) at25°C.

T. Komatsu et al.
Bull. Chem. Soc. Jpn., 74, No. 9 (2001) 1701
O
(Table 2). The kon 2(fast) values are 4–7-fold greater than
O
CO
kon 2(slow), and konCO(fast) are 6–8-fold increase than kon
(slow). The concentration ratio of the fast and slow reactions
was approximately 3 for all the HSA–FeDPs [FeDP(H),
FeDP(OMe), and FeDP(OAc)]. Therefore, we concluded that
the O2 association to FeDPs incorporated into the certain do-
mains of serum albumin is mostly influenced by the molecular
microenvironments around each O2-coordination site, e.g. the
steric hindrance of the amino acid residue and the difference in
polarity. This completely fits the previous observation for
HSA–FeP.7d
O
The P1/2 2 of the HSA–FeDPs were determined on the basis
of the UV-vis spectral changes during O2 titration (Table 2).
According to the results of kinetics experiments, the P1/2 val-
ues were divided into two components using the previously re-
ported equation.7d As expected, the isolated FeDP in HSA did
not show a cooperative O2-binding profile like that seen in
O
Hbs; the Hill coefficient was 1.0. The P1/2 2 value of HSA–
FeDP(H), for example, showed a relatively low value (3.1
O
kPa) compared to those of HSA–FeP, the red cells (P1/2 2: 1.2
kPa), and FeDP(H) itself in the bilayer membrane of phospho-
lipid vesicles (1.3 kPa).6 It is noteworthy that the O2- and CO-
binding parameters are very similar for FeDP(H),
FeDP(OMe), and FeDP(OAc), indicating that the polar sub-
stituents on the fence groups did not cause any change in their
equilibria and kinetics, even in albumin. Although the O2-
binding affinity of HSA–FeDPs are slightly low, the O2-trans-
porting efficacy in vivo between the lungs (PO2: 14.7 kPa) and
muscle tissue (PO2: 5.3 kPa) is estimated to be ca. 20%, which
is nearly the same value as that of the red cells.
Fig. 4. Absorption decay of O2 and CO rebinding to HSA–
FeDP(H) in phosphate buffer solution after the laser flash
photolysis at 25 °C. The kinetics was composed of total
three phases and the relaxation curve can be well fitted by
triple-exponentials. The component of b.e. is related to the
base elimination. (a) The relaxation observed within ca.
1000 µs corresponds to the O2-association to the five-coor-
dinated FeDP(H), and the much slower decay is reorgani-
zation from the O2 complex to carbonyl complex. [O2]:
81.7 kPa and [CO]: 9.2 kPa. (b) The relaxation decay ob-
served within ca. 10 ms corresponds to the CO-association
to five-coordinated FeDP(H). [CO]: 101 kPa.
Degradation of FeDPs in HSA by H2O2. H2O2 is a reac-
tive oxygen species involved in the propagation of cellular in-
jury during various pathophysiological conditions. It is known
that the formation of H2O2 in red blood cells is associated with
the autooxidation of oxy Hb.24 Because most of the H2O2 is
eliminated by catalase and glutathione peroxidase, the concen-
tration of H2O2 in normal plasma is 4–5 µM (1 M = 1 mol
dm−3), but increases under inflammatory conditions.25,26 In the
presence of a small amount of H2O2 of 0.2 mM, the dioxygen-
ated HSA–FeDP(H) rapidly oxidized within 30 min at 37 °C.
Interestingly, following the oxidation of the Fe2+ ion, a very
slow bleaching of the porphinate absorption was observed
(Fig. 5). The solution became almost colorless after 5 days;
the half-life (τ1/2) of this degradation was ca. 17 h. The more
remarkable observation is that HSA–FeP bleached much faster
than HSA–FeDP(H) with a τ1/2 of 0.5 h under the same condi-
centrations. It is presumably correlated with a base elimina-
tion reaction.22 Based on an evaluation of the other two phas-
es, the association rate constants for the fast and slow rebind-
ings (kon(fast) and kon(slow)) of O2 and CO were calculated
Table 2. O2- and CO-Binding Parameters of HSA Incorporating Double-Sided Porphinatoiron(Ⅱ)s (FeDP/HSA = 8 (mol/
mol)) in Phosphate Buffer Solution (pH 7.3, 25 °C)
O
O
O
O
P1/2 2/kPa
10−7 kon 2/M−1 s−1
10−2 koff 2/s−1
10−6 konCO/M−1 s−1
τ1/2 2/h
fast slow
fast
slow
fast
slow
fast
slow
(at 37 °C)
FeDP(H)
FeDP(OMe)
FeDP(OAc)
FePa)
3.7
3.1
3.1
1.7
3.7
3.1
3.1
1.9
1.1
1.1
0.89
3.4
0.15
0.20
0.23
0.95
5.0
4.1
3.4
7.5
0.69
0.76
0.88
2.0
1.4
1.7
2.0
4.9
0.22
0.27
0.24
0.67
5
2
2
2
a) From Ref. 7d.

1702 Bull. Chem. Soc. Jpn., 74, No. 9 (2001)
O2-Binding of Double-Sided Porphinatoiron(ꢀ)s
121, 460 (1999).
a) J. T. Groves, R. Neumann, J. Am. Chem. Soc., 111, 2900
5
(1989). b) J. Lahiri, G. D. Fate, S. B. Unsashe, and J. T. Groves, J.
Am. Chem. Soc., 118, 2347 (1996).
6
E. Tsuchida, T. Komatsu, K. Arai, and H. Nishide, J. Chem.
Soc., Dalton Trans., 1993, 2465.
a) T. Komatsu, K. Ando, N. Kawai, H. Nishide, and E.
7
Tsuchida, Chem. Lett., 1995, 813. b) T. Komatsu, K. Hamamatsu,
J. Wu, and E. Tsuchida, Bioconjugate Chem., 10, 82 (1999). c) E.
Tsuchida, T. Komatsu, Y. Matsukawa, K. Hamamatsu, and J. Wu,
Bioconjugate Chem., 10, 797 (1999). d) T. Komatsu, Y.
Matsukawa, and E. Tsuchida, Bioconjugate Chem., 11, 772
(2000).
8
E. Tsuchida, T. Komatsu, K. Hamamatsu, Y. Matsukawa,
A. Tajima, A. Yoshizu, Y. Izumi, and K. Kobayashi, Bioconjugate
Chem., 11, 46 (2000).
Fig. 5. Absorption decays of the Soret bands of HSA–FeP
and HSA–FeDP(H) ([Fe]: 20 µM) in phosphate buffer so-
lution (pH 7.3, 37 °C, under Air) in the presence of H2O2
of 0.2 mM.
9
C. K. Chang, B. Ward, R. Young, and M. P. Kondylis, J.
Macromol. Sci.-Chem., A25, 1307 (1985).
10 a) H. Imai, A. Nakatsubo, S. Nakagawa, Y. Uemori, and E.
Kyuno, Synth. React. Inorg. Met.-Org. Chem., 15, 265 (1985). b)
H. Imai, S. Sekizawa, and E. Kyuno, Inorg. Chim. Acta., 125, 151
(1986).
11 T. Komatsu, S. Kumamoto, H. Nishide, and E. Tsuchida,
Bull. Chem. Soc. Jpn., 66, 1640 (1993).
12 G. E. Wuenschell, C. Tetreau, D. Lavalette, and C. A. Reed,
J. Am. Chem. Soc., 114, 3346 (1992).
13 C. K. Chang, Y. Ling, and G. Avilés, J. Am. Chem. Soc.,
117, 4191 (1995).
14 E. Tsuchida, T. Komatsu, S. Kumamoto, K. Ando, and H.
Nishide, J. Chem. Soc., Perkin Trans. 2, 1995, 747.
15 A. Nakagawa, T. Komatsu, and E. Tsuchida, Bioconjugate
Chem., 12, (2001) in press.
16 J. P. Collman, J. I. Brauman, B. L. Iverson, J. L. Sessler, R.
M. Morris, and Q. H. Gibson, J. Am. Chem. Soc., 105, 3052
(1983).
17 T. G. Traylor, S. Tsuchiya, D. Campbell, M. Mitchell, D.
Stynes, and N. Koga, J. Am. Chem. Soc., 107, 604 (1985).
18 J. P. Collman, J. I. Brauman, K. M. Doxsee, T. R. Halbert,
E. Bunnenberg, R. E. Linder, G. N. LaMar, J. D. Guadio, G. Lang,
and K. Spartalian, J. Am. Chem. Soc., 102, 4182 (1980).
19 The optimum structures of the double-sided porphina-
toirons were predicted by the ESFF forcefield simulation using an
Insight Ⅱ system (Molecular Simulations Inc.) on a Silicon Graph-
ics O2 computer. The structure was generated by alternative mini-
mizations and annealing dynamic calculations from 1000 to 100
K.
tions. This difference clearly showed that the double-sided
porphinatoiron has a high stability against H2O2. A detailed
analysis of the products in these reactions is now underway.
In conclusion, human serum albumin incorporating double-
sided porphinatoiron(Ⅱ) derivatives as O2-binding sites pro-
vides a synthetic O2-carrying hemoprotein with a relatively
long lifetime for the dioxygenated complexes under physiolog-
ical conditions. The FeDPs binding to HSA is a hydrophobic
interaction, which did not lead to a change in the surface
charge of the HSA molecule. All eight FeDP complexes, in
which the imidazolyl group is intramolecularly coordinated to
the central Fe2+, form reversible O2 adducts in the albumin.
The half-life of HSA–FeDP(H) became 5 h at 37 °C, which is
2.5-fold longer compared to that of HSA–FeP. Furthermore,
FeDP(H) incorporated into the HSA structure showed a high
stability against H2O2. The HSA incorporated double-sided
porphinatoiron series may be useful for the synthetic analogue
of the oxidation enzyme.
This work was partially supported by the Core Research for
Evolutional Science and Technology, JST, Health Science Re-
search Grants (Artificial Blood Project) of the Ministry of
Health and Welfare, Japan, and a Grant-in-Aid for Scientific
Research (No. 11650935) form the Ministry of Education, Sci-
ence, Sports and Culture.
20 U. Kragh-Hansen, Pharmacol. Rev., 33, 17 (1981), and ref-
erences therein.
21 T. Peters Jr., Adv. Protein Chem., 37, 161 (1985). T. Peters
Jr., “All about Albumin, Biochemistry, Genetics, and Medical Ap-
plications,” Academic Press, San Diego (1997).
22 S. Curry, H. Mandelkow, P. Brick, and N. Franks, Nat.
Struct. Biol., 5, 827 (1998).
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