Synthesis of chlorophyll-amino acid conjugates as models for modification of proteins with chromo/fluorophores

Article abstract of DOI:10.1016/j.bmc.2013.12.059

A chlorophyll-a derivative bonded directly with epoxide at the peripheral position of the chlorin π-system was reacted with N-urethane and C-ester protected amino acids bearing an alcoholic or phenolic hydroxy group as well as a carboxy group at the residue to give chlorophyll-amino acid conjugates. The carboxy residues of N,C-protected aspartic and glutamic acids were esterified with the epoxide in high yields. The synthetic conjugates in dichloromethane had absorption bands throughout the visible region including intense red-side Qy and blue-side Soret bands. By their excitation at the visible bands, strong and efficient fluorescence emission was observed up to the near-infrared region. The chromo/fluorophores are promising for preparation of functional peptides and modification of proteins.

Full text of DOI:10.1016/j.bmc.2013.12.059

Bioorganic & Medicinal Chemistry 22 (2014) 1421–1428
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis of chlorophyll–amino acid conjugates as models
for modification of proteins with chromo/fluorophores
⇑
Hitoshi Tamiaki , Yasuaki Isoda, Takuya Tanaka, Shinnosuke Machida
Graduate School of Life Sciences, Ritsumeikan University, Kusatsu, Shiga 525-8577, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
A chlorophyll-a derivative bonded directly with epoxide at the peripheral position of the chlorin p-sys-
Received 28 November 2013
Revised 24 December 2013
Accepted 24 December 2013
Available online 3 January 2014
tem was reacted with N-urethane and C-ester protected amino acids bearing an alcoholic or phenolic
hydroxy group as well as a carboxy group at the residue to give chlorophyll–amino acid conjugates.
The carboxy residues of N,C-protected aspartic and glutamic acids were esterified with the epoxide in
high yields. The synthetic conjugates in dichloromethane had absorption bands throughout the visible
region including intense red-side Qy and blue-side Soret bands. By their excitation at the visible bands,
strong and efficient fluorescence emission was observed up to the near-infrared region. The chromo/fluo-
rophores are promising for preparation of functional peptides and modification of proteins.
Ó 2013 Elsevier Ltd. All rights reserved.
Keywords:
Fluorescence emission data
Labeling
Pigment
Pyropheophorbide
Visible absorption spectra
1. Introduction
Here, we report preparation of adducts of N,C-protected amino
acids to methyl 3-oxiranyl-pyropheophorbide-a (4, Fig. 1), one of
Interaction of porphyrinoids (typically, chlorophylls and hemes)
with peptides is important for the construction of various func-
tional proteins in photosynthesis, respiration, oxygenative metab-
olism, and so on. Cyclic tetrapyrroles as their cofactors bind
peptides through a covalent bond as well as a non-covalent bond
including coordination of any peptidyl residues to the central met-
als from the axial site. Therefore, a variety of conjugates of por-
phyrinoids with amino acids, oligopeptides and proteins were
prepared and investigated from the viewpoints of functional mate-
rials for photoinduced energy/electron transfer and luminescence:
artificial photosynthesis, photodynamic therapy, bioimaging, etc.1
Some of the biocompatible conjugates are utilized for cancer
therapy.
the chlorophyll-a derivatives, through an ether or ester linkage at
the residue and their optical properties including visible absorp-
tion and fluorescence emission data in a diluted solution.
We recently reported synthesis of epoxide–chlorin 4 and its
high reactivity with alcohols and carboxylic acids.5 No other highly
chemically reactive chlorophyll derivatives have been isolated, to
our best knowledge, except for activated 17-propionate ester of
pyropheophorbide-a with N-hydroxysuccinimide.6 In this report,
we demonstrate the efficient modification of hydroxy and carboxy
groups in the residues of all natural L-a-amino acids by fluorescent
chlorophyllous pigment 4.
2. Results and discussion
Most of the conjugates reported so far are amides of a carboxy
group at the peripheral position of porphyrinoids with an N-termi-
nal amino group of amino acids and peptides. Some of them are
conjugates with the residue of (un)natural amino acids and their
peptides.2 Chlorophylls and their derivatives are useful for photo-
synthesis and photodynamic therapy,3 but have been less available
for such bioconjugates. Attachment of chlorophyll derivatives to
amino acids and peptides at their residues is especially limited.4
2.1. Synthesis of chlorophyll–amino acid conjugates
Commercially available hydroxylated
L-amino acids, Fmoc-
AA(OH)-OH, protected with 9-fluorenylmethyloxycarbonyl
a
(Fmoc) group at the N-terminal in diethyl ether was treated with
ethereal diazomethane to give the corresponding serine (Ser), thre-
onine (Thr) and tyrosine (Tyr) methyl esters, Fmoc-AA(OH)-OMe
5a–c, possessing a hydroxy group at the residue (see the upper
drawing of Scheme 1). tert-Butoxycarbonylated L-amino acids,
Abbreviations: APCI, atmospheric pressure chemical ionization; CD, circular
dichroism; FCC, flash column chromatography; Fmoc, 9-fluorenylmethyloxycar-
bonyl; NP, normal phase; RP, reverse phase.
Fmoc-AA(OtBu)-OH, were similarly methylated (see the lower
drawing of Scheme 1) and the resulting methyl esters in dichloro-
methane were subjected to trifluoroacetic acid in the presence of
⇑
Corresponding author. Fax: +81 77 561 2659.
E-mail address: tamiaki@fc.ritsumei.ac.jp (H. Tamiaki).
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.12.059

1422
H. Tamiaki et al. / Bioorg. Med. Chem. 22 (2014) 1421–1428
no remote diastereomeric control at the 31-position was observed
from the three chiral positions, 17S and 18S of the chlorin part (sit-
uated seven and six covalent-bonds distant from) as well as S-ste-
reochemistry at the
apart).
a-position of the L-serine part (three-bonds
The isolated yields of 1a1 and 1a2 were 20% and 21%, respec-
tively, their combined total being 41% (see Table 1). The total yield
was comparable to the 43% isolated yield for 3a obtained from the
similar reaction of 4 with one serine analog 6a lacking a FmocNH
group and lower than the 60% yield of 3b from 4 with the other ser-
ine analog 6b lacking a COOMe group (see Scheme 3). The suppres-
sion of the yields in 1a and 3a would be ascribable to lower
reactivity of 5a and 6a through intramolecular six-membered
hydrogen bonding between the hydroxy and ester groups, while
the intramolecular seven-membered hydrogen bonding between
OH and urethane moiety and five-membered H-bond of carbamate
NH with serine-residue O might be less effective in 5a and 6b.
Extensive HPLC gave no 32-adducts in any of the three products.
The regioselectivity in exclusive formation of such 31-adducts 1a,
3a, and 3b is consistent with the previous results in the ring-open-
ing reaction of 4 with various alcohols.5
The reaction of 4 with Fmoc-Thr(OH)-OMe 5b regioselectively
afforded 31-adducts 1b1 (10% isolated yield) and 1b2 (10%). The to-
tal yield (20%) was half of that in 1a (41%). The reduction was due
to the steric hindrance around the secondary alcoholic hydroxy
group in Thr residue, compared with the primary OH in Ser. The
R-configuration at the b-position of Thr was two bonds apart from
the 31-position in 1b but did not affect the 31-stereoselectivity. It is
noteworthy that no racemization of the b-position occurred in the
present etheration.
In addition to alcohols, phenols were similarly reacted with 4 to
give the corresponding 31-adducts. Using Fmoc-Tyr(OH)-OMe 5c
and PhOH 6c instead of 5a/b and 6a/b led to the sole production
of 31-equimolar mixtures of 1c (59% isolated yield) and 3c (70%),
respectively. The comparable yields showed that the reactivity of
the phenolic OH in 5c was less disturbed with FmocNH and COOMe
in a molecule. Their 31-epimers could not be separated by either
HPLC or FCC under the present conditions.
Addition of Fmoc-Asp(OH)-OMe 5d (5 equiv) to 3-oxiranyl-
chlorin 4 was done without any acid catalyst. After stirring the
dichloromethane solution at room temperature for 1 h, epoxide 4
disappeared. The resulting reaction mixture was purified with
NP-FCC to give the corresponding adduct as the product. The prod-
uct was separated by RP-HPLC to give two fractions. Their 1H NMR
spectra indicated that the first-eluting fraction was 31-adduct 1d
(58% isolated yield) and the second was 32-adduct 2d (35%). Both
the isolated products were 31-epimeric mixtures (1:1) which could
Figure 1. Chemically reactive pyropheophorbides.
triethylsilane to afford N,C-protected aspartic acid (Asp) and glu-
tamic acid (Glu), Fmoc-AA(OH)-OMe 5d and e, bearing a free car-
boxy group.
Epoxide 4 in dichloromethane was reacted with 5 equiv of alco-
hol 5a in the presence of p-toluenesulfonic acid (0.1 equiv) at room
temperature in the dark under nitrogen to consume 4 completely
after stirring for 2 h and give the corresponding ring-opened ad-
duct (see Scheme 2), confirmed by its mass spectrometry: m/
z = 669.3 (MH+). The visible absorption spectra (see Fig. 2A) indi-
cated that the product possessed a 131-oxo-chlorin
p-system and
a 3-CH(OY)CH2(OZ) moiety:5,7 k = 664 and 410 nm in CH2Cl2. The
1H NMR spectra showed that the product was a 1:1 mixture of
two stereoisomers. Normal phase flash column chromatography
(NP-FCC) on silica gel could partially separate the two isomers
and reverse-phase high performance liquid chromatography (RP-
HPLC) on octadecylated silica gel achieved their full separation
(see the details in Section 4.3). The first-eluting fraction in RP-HPLC
(slowly eluting on NP-FCC) gave one set of proton resonance sig-
nals and contained a single stereoisomer. From 1H-1H COSY and
NOESY, the molecular structure was identified to be 31-adduct
1a. 1D and 2D 1H NMR spectra indicated that the second-eluting
fraction in RP-HPLC (fast eluting on NP-FCC) was the other 31-ad-
duct 1a. Hereafter, the former stereoisomer is called 1a1 and the
latter 31-epimer is referred to as 1a2.
1H NMR spectroscopy and HPLC showed that the above
separated samples were diastereomerically pure and thus no
racemization occurred at the chiral center in the
a-position of
L
-serine during the acid-catalyzed ring-opening reaction. Circular
dichroism (CD) spectra of 1a1 and 1a2 in dichloromethane were
slightly but apparently different (see Fig. 2B), confirming them to
be 31-epimers. The stereoisomers were fully separated by RP-HPLC,
but the stereochemistry could not be determined from the present
data. Since the product was an equimolar mixture of 1a1 and 1a2,
Scheme 1. Reagents and conditions: Synthesis of N,C-protected
L-amino acids 5: (i) CH2N2/Et2O; (ii) CF3COOH, Et3SiH/CH2Cl2.

H. Tamiaki et al. / Bioorg. Med. Chem. 22 (2014) 1421–1428
1423
Scheme 2. Reagents and conditions: Synthesis of N,C-protected
or no acid catalyst (for d and e).
L-amino acids possessing a chlorophyll derivative at the residue 1/2: (i) CH2Cl2, cat. p-CH3C6H4SO3H (for a–c)
Scheme 3. Reagents and conditions: Synthesis of chlorophyll derivatives 3a/b and
3c as analogs of 2a and 2c: (i) CH2Cl2, cat. p-CH3C6H4SO3H.
Figure 2. Visible absorption (A) and CD spectra (B) of 1a1 (solid line) and 1a2
(broken line) in dichloromethane. All the spectra were normalized by the intense
peaks around 410 nm.
Table 1
Isolated yield of chlorophyll–amino acid conjugates 1/2 and their analogs 3 from the
reaction of 4 with 5 and 6, respectively (see Schemes 2 and 3 as well as Section 4.3)
ROH
Yield/% (conjugated product)
5a (Fmoc-Ser-OMe)
5b (Fmoc-Thr-OMe)
5c (Fmoc-Tyr-OMe)
5d (Fmoc-Asp-OMe)
5e (Fmoc-Glu-OMe)
6a (HOCH2CH2COOMe)
6b (HOCH2CH2NHFmoc)
6c (HOC6H5)
41 = 20 (1a1) + 21 (1a2)
20 = 10 (1b1) + 10 (1b2)
59 (1c)a
Scheme 4. Acid-catalyzed acyl migration of 1d/e to 2d/e.
93 = 58 (1d)a + 35 (2d)a
79 = 33 (1e1) + 33 (1e2) + 13 (2e)a
43 (3a)a
not be successfully separated under the present HPLC conditions.
The carboxylate anion of 5d first attacked the 31-position of
protonated 4 at the epoxide oxygen-atom to give 31-adduct 1d
(see the upper drawing of Scheme 4), similarly as in the above
60 (3b)a
70 (3c)a
a
31R:31S = 1:1.

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H. Tamiaki et al. / Bioorg. Med. Chem. 22 (2014) 1421–1428
reactions of 4 with 5a–c to 1a–c. The resulting 31-adduct 1d was
transformed through acid-catalyzed acyl migration to regioisomer-
ic 32-adduct 2d (see the lower drawing of Scheme 4). The rear-
rangement readily occurred at room temperature. Since the
reaction completed within 1 h and the total isolated yield was
93%, the addition of 5d to 4 proceeded smoothly and cleanly.
Similar to the addition of 5d to 4, the reaction of Fmoc-Glu(OH)-
OMe 5e with 4 gave 1e1 (33% isolated yield), 1e2 (33%), and 2e
(13%) after purification with FCC followed by HPLC. The total iso-
lated yield was good (79%) and comparable to that in the case of
Asp.
emission quantum yields of 1/2 were 27 + 1% and their lifetimes
were 6.1 + 0.3 ns. Although the emission spectra were dependent
on the Qy absorption bands, all the synthetic conjugates inten-
sively emitted the light at a visible to near-infrared region to give
similar emission spectra.
3. Concluding remarks
Alcoholic and phenolic residues of N,C-protected amino acids
5a–c were added to methyl 3-devinyl-3-oxiranyl-pyropheophor-
bide-a (4) by the catalytic action of an acid to give epoxide-ring
opened 31-ethers 1a–c. Esterification of 4 with carboxy residues
of protected amino acids 5d/e without any acid catalyst afforded
31-esters 1d/e and their acyl-migrated 32-isomers 2d/e in very good
yields. The synthetic chlorophyll derivative–amino acid conjugates
1/2 absorbed ultraviolet to visible lights and emitted visible to
near-infrared lights efficiently. The conjugates could be used for
construction of oligopeptides with chemo/fluorophores by conven-
tional peptide synthesis. Epoxide 4 would be useful for modifying
and labeling proteins at the residues of aspartic and glutamic acids.
2.2. Optical properties of chlorophyll–amino acid conjugates
In a diluted dichloromethane solution (ca. 10 lM), synthetic
chlorophyll–amino acid conjugates 1/2 gave electronic absorption
bands from the visible to ultraviolet regions (see Figs. 2 and 3A
and S1–10A). The absorption bands are typical of monomeric spe-
cies.7 One of the two intense bands at the red side is called the Qy
band, while the other at the blue side is referred to as the Soret
band. In the region between these two bands, Qx bands were ob-
served. Both the main Qy and Qx bands had two vibrational bands
at their blue side. In 1d, for example, absorption maxima (kabs) of
Qy(0,0), Qy(0,1), and Qy(0,2) bands were situated at 665, 608,
and ꢀ560 nm, respectively, and Qx(0,0)/(0,1)/(0,2) maxima at
537/506/473 nm (see Fig. 3A and Table 2). The shapes of absorp-
tion spectra of all the conjugates are almost the same: typically,
kabs(Qy(0,0)) À kabs(Qy(0,1)) = 57 nm and relative Qy(0,0) peak
absorbances to the Soret maxima (Arel) = 0.525 + 0.015 (see
Table 2). Their peak positions are slightly shifted within 2 nm:
kabs(Qy(0,0)) = 663–665 nm and kabs(Soret) = 410–411 nm. The
absorption spectra are little dependent on the substituents Y and
Z in 3-CH(OY)CH2(OZ) of 1/2. It is noted that kabs(Qy)s of the first
fractions of 31-adducts 1a1, 1b1, and 1e1 are red-shifted slightly
but noticeably (1 nm) from those of the second 1a2, 1b2, and
1e2, respectively, and also that those of 31-adduct 1d are larger
by 2 nm than those of 32-adduct 2d.
4. Experimental
4.1. General
Visible absorption and CD spectra were measured with a Hitachi
U-3500 spectrophotometer and a Jasco J-720 W spectropolarime-
ter, respectively. Fluorescence emission spectra and quantum
yields were obtained by a Hamamatsu Photonics C9920-03G spec-
trometer. Fluorescence emission lifetimes were determined by a
Hamamatsu Photonics C7990S system. 1H NMR spectra in chloro-
form-d were recorded with
a JEOL ECA-600 spectrometer
(600 MHz); residual non-deuterated chloroform (d = 7.26 ppm)
was used as an internal reference. Time-of-flight mass data were
obtained using direct laser desorption/ionization by a Shimadzu
AXIMA-CFR plus spectrometer. High resolution mass spectra
(HRMS) were recorded on a Bruker micrOTOF II spectrometer;
atmospheric pressure chemical ionization (APCI) and positive mode
in an acetonitrile solution. HPLC was performed on a packed octa-
decylsilylated silica gel column (Cosmosil 5C18-AR-II, Nacalai Tes-
que), with a Shimadzu LC-10ADvp pump as well as SPD-M10Avp
photodiode-array. FCC was carried out on silica gel (Merck, Kiesel-
When each highly diluted dichloromethane solution (ca. 1 lM)
of 1/2 was irradiated with the light at the Soret maxima, strong
fluorescence emission was observed at the red side of the Qy re-
gion (see Figs. 3B and S1–10B). Fluorescence spectra were nearly
mirror images of the Qy bands. The full widths at half maximum
of the main emission bands at 668–670 nm were 370–400 cmÀ1
and each value was slightly larger by just 20 cmÀ1 than that of
the corresponding Qy(0,0) band (see Table 2). Their Stokes shifts
gel 60, 40–63 lm, 230–400 mesh). Thin layer chromatography
(TLC) was performed with silica gel (Merck, Kieselgel 60 F254).
Methyl 3-hydroxypropanoate8 and methyl 31,32-epoxy-meso-
pyropheophorbide-a (4)5 were prepared according to reported
(D) were also identical (5 nm) for all the conjugates. Fluorescence
Figure 3. Visible absorption (A) and fluorescence emission spectra (B, excitation at the Soret maxima) of 1d (solid line) and 2d (broken line) in dichloromethane. The
absorption spectra were normalized by the Soret peaks and each emission maximum around 670 nm was fitted into the corresponding Qy(0,0) peak intensity.

H. Tamiaki et al. / Bioorg. Med. Chem. 22 (2014) 1421–1428
1425
Table 2
Optical properties of 1/2 in air-saturated dichloromethane at room temperaturea
Compound
kabs (nm)
Arel
kem
D
Uem
sem
Soret
Qx
Qy
(nm)
(nm)
(%)
(ns)
(0,1)
(0,0)
(0,1)
(0,0)
1a1 (31-Ser⁄)
1a2 (31-Ser⁄)
1b1 (31-Thr⁄)
1b2 (31-Thr⁄)
1c (31-Tyr)
411
410
410
410
410
411
410
410
410
410
506
506
505
506
506
506
506
506
506
506
537
537
536
537
536
537
536
536
537
537
608
607
607
606
607
608
606
607
606
606
665 [360]
664 [350]
664 [350]
663 [350]
664 [350]
665 [360]
663 [360]
664 [380]
663 [360]
663 [360]
0.52
0.53
0.54
0.52
0.54
0.54
0.52
0.51
0.51
0.52
670 [380]
668 [370]
669 [370]
668 [370]
669 [370]
670 [380]
668 [380]
669 [400]
668 [380]
668 [380]
5
5
5
5
5
5
5
5
5
5
27
26
27
28
26
27
27
27
27
27
6.4
6.2
6.0
6.3
6.0
6.4
6.1
6.0
5.8
6.2
1d (31-Asp)
2d (32-Asp)
1e1 (31-Glu⁄)
1e2 (31-Glu⁄)
2e (32-Glu)
a
kabs, absorption maxima; Arel, relative Qy(0,0) peak absorbances to the Soret maxima; kem, emission maxima (excited at Soret maxima);
D
, Stokes shifts = kem À kabs(Qy
(0,0)); Uem, emission quantum yields (excited at Soret maxima);
sem, emission lifetimes (excited at 403 nm). The value in a square bracket indicates a full width at half
maximum (cmÀ1).
procedures. N-Fmoc-glycinol and N-Fmoc-
L
-amino acids (Fmoc-
0.1 equiv) was stirred at room temperature in the dark under nitro-
gen: in the case of carboxylic acids 5d/e, pTSA was not necessary as
the catalytic additive. After the disappearance of 4 determined by
checking TLC (stirring for 1–3 h), the reaction was quenched by
addition of distilled water. The separated organic phase was
washed with an aqueous saturated sodium bicarbonate solution
and water, dried over sodium sulfate, and filtered. All the solvent
was evaporated and the residue was purified with FCC (5–10%
Et2O–CH2Cl2 or 50% AcOEt–hexane) and recrystallization (CH2Cl2–
hexane) to give desired conjugates 1–3 as analytically pure black
solids. All the samples were purified by HPLC (1% THF-14% H2O–
CH3CN) just before their optical measurements.
AA-OH) were purchased from Watanabe Chemical Industries.
Commercially available acetonitrile, dichloromethane, diethyl
ether, ethyl acetate, hexane, and petroleum ether (Nacalai Tesque)
were used without alteration. Other reagents were purchased from
commercial suppliers.
Dichloromethane for optical spectroscopy was purchased from
Nacalai Tesque as a reagent prepared specially for spectroscopy
and used without further purification. Optical properties were
measured in air-saturated dichloromethane at room temperature.
4.2. Synthesis of N-Fmoc-L-amino acid methyl ester (5, Fmoc-
AA(OH)-OMe)
4.4. Spectral data
4.2.1. N-Fmoc-
group 5a–c
L
-amino acid methyl ester possessing a hydroxy
4.4.1. Methyl 32-hydroxy-31-[2-(methoxycarbonyl)ethoxy]-
mesopyropheophorbide-a (3a)
A
diethyl ether solution (100 mL) of Fmoc-AA(OH)-OH
43% isolated yield (31R/S = 1/1); UV–vis (CH2Cl2) kmax = 663 (rel-
ative intensity, 0.51), 606 (0.08), 537 (0.09), 506 (0.09), 474 (0.03),
410 (1.00), 399 (0.80, sh), 379 (0.58, sh), 318 nm (0.20); 1H NMR
(CDCl3) d = 9.67 (1H, s, 5-H), 9.56 (1H, s, 10-H), 8.58 (1H, s,
20-H), 5.984/982 (1H, dd, J = 9, 4 Hz, 3-CH), 5.273/266, 5.127/124
(each 1H, d, J = 19 Hz, 131-CH2), 4.60/59 (1H, dt, J = 2, 9 Hz,
31-CH), 4.50/49 (1H, dq, J = 2, 7 Hz, 18-H), 4.31 (1H, m, 17-H),
4.15–4.10 (1H, m, 31-CH), 4.11–4.06 (1H, m, 31-OCH), 4.02/01
(1H, dt, J = 10, 5 Hz, 31-OCH), 3.72 (2H, q, J = 8 Hz, 8-CH2), 3.68
(3H, s, 12-CH3), 3.624/622 (3H, s, 31-OC2CO2CH3), 3.604/600 (3H,
s, 172-CO2CH3), 3.430/428 (3H, s, 2-CH3), 3.27 (3H, s, 7-CH3),
3.09/07 (1H, br, 32-OH), 2.83/81 (1H, dt, J = 8, 5 Hz, 31-OCCH),
2.77–2.72 (1H, m, 31-OCCH), 2.72–2.65, 2.59–2.52, 2.34–2.21
(1H+1H+2H, m, 17-CH2CH2), 1.811/808 (3H, d, J = 7 Hz, 18-CH3), 1.71
(3H, t, J = 8 Hz, 81-CH3), 0.25, À1.84/85 (each 1H, s, NH Â 2); HRMS
(APCI) found: m/z = 669.3292, calcd for C38H45N4O7: MH+, 669.3283.
(0.8 mmol, AA = Ser, Thr, and Tyr) was treated with ethereal diazo-
methane (10 mL) at room temperature. After a check of TLC
showed that the starting carboxylic acid had disappeared (stirring
for 30 min), the reaction was quenched by addition of distilled
water. The separated organic phase was washed with water, dried
over sodium sulfate, and filtered. All the solvent was evaporated
and the residue was purified with FCC (10% Et2O–CH2Cl2) and
recrystallization (CH2Cl2–hexane) to give 5a–c (ca. 80–90%).
4.2.2. N-Fmoc-L-amino acid methyl ester possessing a carboxy
group 5d/e
Similar to the synthesis of Fmoc-AA(OH)-OMe, Fmoc-AA(OtBu)-
OH (AA = Asp and Glu) was transformed to Fmoc-AA(OtBu)-OMe
(ca. 90%) after purification of sole recrystallization (AcOEt–petro-
leum ether).
A dichloromethane solution (10 mL) of Fmoc-AA(OtBu)-OMe
(0.3 mmol) was treated with trifluoroacetic acid (1.5 mL) and tri-
ethylsilane (0.25 mL) at room temperature. After a check of TLC
showed that the starting tert-butyl ether had disappeared (stirring
for 2–3 h), the reaction was quenched by addition of distilled
water. The separated organic phase was washed with water, dried
over sodium sulfate, and filtered. All the solvent was evaporated
and the residue was purified with FCC (10% Et2O–CH2Cl2) and
recrystallization (CH2Cl2–hexane) to give 5d/e (ca. 50–70%).
4.4.2. Methyl 31-[2-(9-fluorenylmethoxycarbonyl)amino-
ethoxy]-32-hydroxy-mesopyropheophorbide-a (3b)
60% isolated yield (31R/S = 1/1); UV–vis (CH2Cl2) kmax = 663 (rel-
ative intensity, 0.51), 606 (0.08), 537 (0.09), 506 (0.10), 474 (0.04),
411 (1.00), 399 (0.80, sh), 379 (0.57, sh), 318 nm (0.20); 1H NMR
(CDCl3) d = 9.70 (1H, s, 5-H), 9.48/45 (1H, s, 10-H), 8.58 (1H, s,
20-H), 7.70–7.61, 7.39–7.29, 7.27–7.21, 7.19–7.15, 7.05–7.02,
6.97–6.89 (8H, m, 1-, 2-, 3-, 4-, 5-, 6-, 7-, 8-H of Fmoc), 5.99/97
(1H, dd, J = 6, 4 Hz, 3-CH), 5.25/24, 5.21/07 (each 1H, d, J =19 Hz,
131-CH2), 5.19/17 (1H, t, J = 6 Hz, Fmoc-NH), 4.77–4.71 (1H, m,
31-CH), 4.50/48 (1H, dq, J = 2, 7 Hz, 18-H), 4.30 (1H, m, 17-H),
4.21 (1H, m, 31-CH), 4.13, 4.03/01 (each 1H, dd, J = 11, 7 Hz, 9-
CH2 of Fmoc), 3.92–3.85, 3.84–3.79 (2H, m, 31-OCH2), 3.74/73
(1H, t, J = 7 Hz, 9-H of Fmoc), 3.65 (2H, q, J = 7 Hz, 8-CH2),
4.3. Synthesis of conjugates 1–3
A dichloromethane solution (15 mL) of epoxide 4 (28.2 mg,
50
lmol) and Fmoc-AA(OH)-OMe 5 or ROH 6 (250 lmol, 5 equiv)
with(out) p-toluenesulfonic acid monohydrate (pTSA, 1 mg,

1426
H. Tamiaki et al. / Bioorg. Med. Chem. 22 (2014) 1421–1428
3.65–3.58, 3.51–3.46 (2H, m, 31-OCCH2), 3.62, 3.60, 3.434/429,
3.284/281 (each 3H, s, 172-CO2CH3, 2-, 7-, 12-CH3), 2.88
(1H, br, 32-OH), 2.73–2.64, 2.61–2.51, 2.32–2.21 (1H+1H+2H,
m, 17-CH2CH2), 1.83/79 (3H, d, J = 7 Hz, 18-CH3), 1.66/64 (3H,
t, J = 7 Hz, 81-CH3), 0.17/13, À1.84/86 (each 1H, s, NH Â 2);
HRMS (APCI) found: m/z = 848.4033 calcd for C51H54N5O7: MH+,
848.4018.
(1H, t, J = 7 Hz, 9-H of Fmoc), 4.18 (1H, dd, J = 9, 8 Hz, 31-CH),
4.10, 4.06 (each 1H, dd, J = 4, 10 Hz, b-H of Ser), 3.70 (2H, q,
J = 7 Hz, 8-CH2), 3.68, 3.62, 3.40, 3.27, 3.17 (each 3H, s, 2-, 7-, 12-
CH3, 172-CO2CH3, Ser-OCH3), 3.15 (1H, br, 32-OH), 2.74–2.67,
2.61–2.55, 2.35–2.24 (1H+1H+2H, m, 17-CH2CH2), 1.81 (3H, d,
J = 7 Hz, 18-CH3), 1.68 (3H, t, J = 7 Hz, 81-CH3), 0.20, À1.86 (each
1H, s, NH Â 2); HRMS (APCI) found: m/z = 906.4063, calcd for
C
53H56N5O9: MH+, 906.4073.
4.4.3. Methyl 32-hydroxy-31-phenoxy-mesopyropheophorbide-
a (3c)
4.4.6. Methyl 31-[2-(9-fluorenylmethoxycarbonyl)amino-2-(me-
thoxycarbonyl)-1-methylethoxy]-32-hydroxy-mesopyropheo-
phorbide-a (Thr-adduct) as the first fraction of HPLC (1b1)
10% isolated yield (31-epimerically pure, slow-eluting fraction
on FCC with Rf = 0.14); UV–vis (CH2Cl2) kmax = 664 (relative inten-
sity, 0.54), 607 (0.07), 536 (0.09), 505 (0.10), 475 (0.04), 410
(1.00), 399 (0.80, sh), 379 (0.58, sh), 318 nm (0.20); 1H NMR
(CDCl3) d = 9.55 (1H, s, 10-H), 9.48 (1H, s, 5-H), 8.59 (1H, s, 20-
H), 7.79, 7.77 (each 1H, d, J = 7 Hz, 4-, 5-H of Fmoc), 7.62, 7.50 (each
1H, d, J = 7 Hz, 1-, 8-H of Fmoc), 7.42, 7.38 (each 1H, t, J = 7 Hz, 3-,
6-H of Fmoc), 7.32, 7.20 (each 1H, t, J = 7 Hz, 2-, 7-H of Fmoc), 6.00
(1H, m, 3-CH), 5.80 (1H, d, J = 9 Hz, Fmoc-NH), 5.28, 5.14 (each 1H,
d, J = 19 Hz, 131-CH2), 4.82 (1H, m, 31-CH), 4.52 (1H, dq, J = 1, 7 Hz,
18-H), 4.42, 4.40 (each 1H, dd, J = 11, 7 Hz, 9-CH2 of Fmoc), 4.35–
4.30 (1H, m, 17-H), 4.32 (1H, dq, J = 2, 6 Hz, b-H of Thr), 4.23 (1H,
70% isolated yield (31R/S = 1/1); UV–vis (CH2Cl2) kmax = 665 (rel-
ative intensity, 0.52), 608 (0.07), 537 (0.09), 506 (0.10), 475 (0.04),
411 (1.00), 399 (0.81, sh), 380 (0.58, sh), 318 nm (0.19); 1H NMR
(CDCl3) d = 9.71 (1H, s, 5-H), 9.554/551 (1H, s, 10-H), 8.56 (1H, s,
20-H), 7.171/169 (2H, d, J = 8 Hz, o-H of 31-OPh), 7.08/07 (2H, dd,
J = 8, 7 Hz, m-H of 31-OPh), 6.81/80 (1H, t, J = 4 Hz, 3-CH), 6.78/77
(1H, t, J = 7 Hz, p-H of 31-OPh), 5.254/250, 5.111/108 (each 1H, d,
J = 19 Hz, 131-CH2), 4.82/80 (1H, ddd, J = 9, 4, 3 Hz, 31-CH), 4.47/
46 (1H, dq, J = 2, 7 Hz, 18-H), 4.39/37 (1H, dd, J = 9, 4 Hz, 31-CH),
4.29 (1H, m, 17-H), 3.72 (2H, q, J = 8 Hz, 8-CH2), 3.68/67 (3H, s,
12-CH3), 3.591/590 (3H, s, 172-CO2CH3), 3.46 (3H, s, 2-CH3),
3.320/314 (3H, s, 7-CH3), 2.77/76 (1H, d, J = 3 Hz, 32-OH), 2.70–
2.63, 2.57–2.49, 2.31–2.19 (1H+1H+2H, m, 17-CH2CH2), 1.79/78
(3H, d, J = 7 Hz, 18-CH3), 1.712/711 (3H, t, J = 8 Hz, 81-CH3), 0.19,
À1.90 (each 1H, s, NH Â 2); HRMS (APCI) found: m/z = 659.3238,
calcd for C40H43N4O5: MH+, 659.3228.
dd, J = 9, 2 Hz,
a-H of Thr), 4.18 (1H, t, J = 7 Hz, 9-H of Fmoc),
4.17 (1H, m, 31-CH), 3.72 (2H, q, J = 8 Hz, 8-CH2), 3.69, 3.62, 3.49,
3.42, 3.30 (each 3H, s, 2-, 7-, 12-CH3, 172-CO2CH3, Thr-OCH3),
2.75–2.69 (1H, br, 32-OH), 2.75–2.69, 2.63–2.56, 2.35–2.28
(1H+1H+2H, m, 17-CH2CH2), 1.80 (3H, d, J = 7 Hz, 18-CH3), 1.69
(3H, t, J = 8 Hz, 81-CH3), 1.59 (3H, d, J = 6 Hz, b-CH3 of Thr), 0.20,
À1.85 (each 1H, s, NH Â 2); HRMS (APCI) found: m/z = 920.4261,
calcd for C54H58N5O9: MH+, 920.4229.
4.4.4. Methyl 31-[2-(9-fluorenylmethoxycarbonyl)amino-2-(me-
thoxycarbonyl)ethoxy]-32-hydroxy-mesopyropheophorbide-a
(Ser-adduct) as the first fraction of HPLC (1a1)
20% isolated yield (31-epimerically pure, slow-eluting fraction
on FCC with Rf = 0.07); UV–vis (CH2Cl2) kmax = 665 (relative inten-
sity, 0.52), 608 (0.08), 537 (0.09), 506 (0.10), 474 (0.04), 411
(1.00), 399 (0.80, sh), 379 (0.58, sh), 318 nm (0.20); 1H NMR
(CDCl3) d = 9.68 (1H, s, 5-H), 9.36 (1H, s, 10-H), 8.59 (1H, s, 20-
H), 7.56, 7.47, 7.00, 6.18 (each 1H, d, J = 7 Hz, 1-, 4-, 5-, 8-H of
Fmoc), 7.27, 7.10, 7.05, 6.57 (each 1H, t, J = 7 Hz, 2-, 3-, 6-, 7-H of
Fmoc), 5.92 (1H, dd, J = 9, 4 Hz, 3-CH), 5.67 (1H, d, J = 8 Hz, Fmoc-
NH), 5.23, 5.03 (each 1H, d, J = 19 Hz, 131-CH2), 4.78 (1H, dd,
4.4.7. Methyl 31-[2-(9-fluorenylmethoxycarbonyl)amino-2-(me-
thoxycarbonyl)-1-methylethoxy]-32-hydroxy-mesopyropheo-
phorbide-a (Thr-adduct) as the second fraction of HPLC (1b2)
10% isolated yield (31-epimerically pure, fast-eluting fraction on
FCC with Rf = 0.16); UV–vis (CH2Cl2) kmax = 663 (relative intensity,
0.52), 606 (0.07), 537 (0.09), 506 (0.09), 475 (0.04), 410 (1.00),
399 (0.81, sh), 379 (0.58, sh), 318 nm (0.20); 1H NMR (CDCl3)
d = 9.68 (1H, s, 5-H), 9.58 (1H, s, 10-H), 8.60 (1H, s, 20-H), 7.71
(2H, d, J = 7 Hz, 4-, 5-H of Fmoc), 7.56, 7.54 (each 1H, d, J = 7 Hz,
1-, 8-H of Fmoc), 7.30 (2H, t, J = 7 Hz, 3-, 6-H of Fmoc), 7.18, 7.15
(each 1H, t, J = 7 Hz, 2-, 7-H of Fmoc), 6.02 (1H, dd, J = 10, 4 Hz,
3-CH), 5.88 (1H, d, J = 9 Hz, Fmoc-NH), 5.28, 5.14 (each 1H, d,
J = 13, 9 Hz, 31-CH), 4.52 (1H, dt, J = 8, 3 Hz,
a-H of Ser), 4.47 (1H,
dq, J = 1, 7 Hz, 18-H), 4.29 (1H, m, 17-H), 4.27, 4.04 (each 1H, dd,
J = 10, 3 Hz, b-H of Ser), 4.18 (1H, ddd, J = 13, 9, 4 Hz, 31-CH), 3.96
(3H, s, Ser-OCH3), 3.62 (2H, q, J = 8 Hz, 8-CH2), 3.60 (3H, s,
12-CH3), 3.58 (3H, s, 172-CO2CH3), 3.54, 3.24 (each 1H, t, J = 8 Hz,
9-CH2 of Fmoc), 3.44 (3H, s, 2-CH3), 3.34 (1H, s, 7-CH3), 2.75 (1H,
d, J = 9 Hz, 32-OH), 2.71–2.62 (1H, m, 9-H of Fmoc), 2.71–2.62,
2.57–2.51, 2.29–2.21 (1H+1H+2H, m, 17-CH2CH2), 1.86 (3H, d,
J = 7 Hz, 18-CH3), 1.62 (3H, t, J = 8 Hz, 81-CH3), 0.05, À1.87 (each
1H, s, NH Â 2); HRMS (APCI) found: m/z = 906.4097, calcd for
J = 19 Hz, 131-CH2), 4.72 (1H, dd, J = 9, 2 Hz,
a-H of Thr), 4.63
(1H, dd, J = 13, 10 Hz, 31-CH), 4.51 (1H, dq, J = 2, 7 Hz, 18-H), 4.49
(1H, dq, J = 2, 6 Hz, b-H of Thr), 4.45, 4.39 (each 1H, dd, J = 11,
7 Hz, 9-CH2 of Fmoc), 4.32 (1H, ddd, J = 9, 3, 2 Hz, 17-H), 4.22
(1H, t, J = 7 Hz, 9-H of Fmoc), 4.05 (1H, m, 31-CH), 4.02, 3.70,
3.60, 3.45, 3.31 (each 3H, s, 2-, 7-, 12-CH3, 172-CO2CH3, Thr-
OCH3), 3.71 (2H, q, J = 8 Hz, 8-CH2), 3.13 (1H, br, 32-OH), 2.73–
2.67, 2.58–2.53, 2.34–2.22 (1H+1H+2H, m, 17-CH2CH2), 1.82 (3H,
d, J = 7 Hz, 18-CH3), 1.69 (3H, t, J = 8 Hz, 81-CH3), 1.06 (3H, d,
J = 6 Hz, b-CH3 of Thr), 0.21, À1.87 (each 1H, s, NH Â 2); HRMS
C
53H56N5O9: MH+, 906.4073.
4.4.5. Methyl 31-[2-(9-fluorenylmethoxycarbonyl)amino-2-(me-
thoxycarbonyl)ethoxy]-32-hydroxy-mesopyropheophorbide-a
(Ser-adduct) as the second fraction of HPLC (1a2)
21% isolated yield (31-epimerically pure, fast-eluting fraction on
FCC with Rf = 0.12); UV–vis (CH2Cl2) kmax = 664 (relative intensity,
0.53), 607 (0.07), 537 (0.09), 506 (0.09), 473 (0.03), 410 (1.00),
399 (0.81, sh), 379 (0.58, sh), 318 nm (0.19); 1H NMR (CDCl3)
d = 9.54 (2H, s, 5-, 10-H), 8.56 (1H, s, 20-H), 7.78 (2H, d, J = 7 Hz,
4-, 5-H of Fmoc), 7.76, 7.61 (each 1H, d, J = 7 Hz, 1-, 8-H of Fmoc),
7.42, 7.41 (each 1H, t, J = 7 Hz, 3-, 6-H of Fmoc), 7.34, 7.30 (each 1H,
t, J = 7 Hz, 2-, 7-H of Fmoc), 6.11 (1H, d, J = 8 Hz, Fmoc-NH), 5.97
(1H, dd, J = 9, 4 Hz, 3-CH), 5.28, 5.13 (each 1H, d, J =19 Hz, 131-
(APCI) found: m/z = 920.4262, calcd for
C
54H58N5O9: MH+,
920.4229.
4.4.8. Methyl 31-[4-{2-(9-fluorenylmethoxycarbonyl)amino-2-
(methoxycarbonyl)ethyl}phenoxy]-32-hydroxy-mesopyropheo-
phorbide-a (Tyr-adduct, 1c)
59% isolated yield (31R/S = 1/1); UV–vis (CH2Cl2) kmax = 664 (rel-
ative intensity, 0.54), 607 (0.07), 536 (0.09), 506 (0.09), 474 (0.04),
410 (1.00), 399 (0.81, sh), 380 (0.59, sh), 318 nm (0.20); 1H NMR
(CDCl3) d = 9.69 (1H, s, 5-H), 9.561/555 (1H, s, 10-H), 8.57/56
(1H, s, 20-H), 7.57–7.48, 7.41, 7.36–7.32, 7.28–7.10, 7.01–6.97
CH2), 4.62 (1H, dt, J = 8, 4 Hz, 31-CH), 4.62 (1H, dt, J = 8, 4 Hz,
a-H
of Ser), 4.51 (1H, dq, J = 2, 7 Hz, 18-H), 4.48, 4.44 (each 1H, dd,
J = 10, 7 Hz, 9-CH2 of Fmoc), 4.38 (1H, dt, J = 9, 2 Hz, 17-H), 4.25

H. Tamiaki et al. / Bioorg. Med. Chem. 22 (2014) 1421–1428
1427
(8H, m, 1-, 2-, 3-, 4-, 5-, 6-, 7-, 8-H of Fmoc), 7.074/071 (2H, d,
J = 9 Hz, 3-H of 31-OPh), 6.77/75 (2H, d, J = 9 Hz, 2-H of 31-OPh),
6.76 (1H, m, 3-CH), 5.26, 5.12/11 (each 1H, d, J = 19 Hz, 131-CH2),
5.06/4.99 (1H, d, J = 8 Hz, Fmoc-NH), 4.82–4.76 (1H, m, 31-CH),
d = 9.60 (1H, s, 5-H), 9.53 (1H, s, 10-H), 8.60 (1H, s, 20-H), 7.65,
7.39–7.28, 7.22–7.16 (8H, m, 1-, 2-, 3-, 4-, 5-, 6-, 7-, 8-H of Fmoc),
7.34 (1H, m, 3-CH), 5.41 (1H, d, J = 8 Hz, Fmoc-NH), 5.25, 5.10 (each
1H, d, J = 19 Hz, 131-CH2), 4.88–4.82 (1H, m, 31-CH), 4.59 (1H, m,
a-
4.51 (1H, dq, J = 2, 8 Hz, 18-H), 4.45 (1H, m,
a
-H of Tyr), 4.38/36
H of Glu), 4.47 (1H, dq, J = 2, 7 Hz, 18-H), 4.33 (1H, ddd, J = 13, 8,
4 Hz, 31-CH), 4.28 (1H, m, 17-H), 4.23, 4.20 (each 1H, dd, J = 11,
7 Hz, 9-CH2 of Fmoc), 3.86 (1H, t, J = 7 Hz, 9-H of Fmoc), 3.70 (2H,
q, J = 8 Hz, 8-CH2), 3.67, 3.66, 3.60, 3.49, 3.27 (each 3H, s, 2-, 7-,
12-CH3, 172-CO2CH3, Glu-OCH3), 2.80 (1H, br, 32-OH), 2.76–2.69
(1H, dt, J = 10, 3 Hz, 31-CH), 4.30 (1H, m, 17-H), 4.27, 4.18/13 (each
1H, dd, J = 10, 7 Hz, 9-CH2 of Fmoc), 3.99/89 (1H, t, J = 7 Hz, 9-H of
Fmoc), 3.72 (2H, q, J = 8 Hz, 8-CH2), 3.69/68, 3.590/586, 3.51/49,
3.45/44, 3.31/30 (each 3H, s, 2-, 7-, 12-CH3, 172-CO2CH3, Tyr-
OCH3), 2.83 (2H, m, b-H of Tyr), 2.71/69 (1H, d, J = 3 Hz, 32-OH),
2.69–2.64, 2.56–2.50, 2.31–2.21 (1H+1H+2H, m, 17-CH2CH2),
1.79/77 (3H, d, J = 8 Hz, 18-CH3), 1.71 (3H, t, J = 8 Hz, 81-CH3),
0.17, À1.90 (each 1H, s, NH Â 2); HRMS (APCI) found: m/
z = 982.4400, calcd for C59H60N5O9: MH+, 982.4386.
(2H, m, c-H of Glu), 2.71–2.64, 2.57–2.51, 2.28–2.21 (1H+1H+2H,
m, 17-CH2CH2), 2.35–2.29, 2.11–2.05 (each 1H, m, b-H of Glu),
1.78 (3H, d, J = 7 Hz, 18-CH3), 1.70 (3H, t, J = 8 Hz, 81-CH3), 0.11,
À1.94 (each 1H, s, NH Â 2); HRMS (APCI) found: m/z = 948.4185,
calcd for C55H58N5O10: MH+, 948.4178.
4.4.9. Methyl 31-[2-(9-fluorenylmethoxycarbonyl)amino-2-(me-
thoxycarbonyl)ethyl]carbonyloxy-32-hydroxy-mesopyropheo-
phorbide-a (Asp-adduct, 1d)
4.4.12. Methyl 31-[3-(9-fluorenylmethoxycarbonyl)amino-3-(me-
thoxycarbonyl)propyl]carbonyloxy-32-hydroxy-mesopyropheo-
phorbide-a (Glu-adduct) as the second fraction of HPLC (1e2)
33% isolated yield (31-epimerically pure, fast-eluting fraction on
FCC with Rf = 0.30); UV–vis (CH2Cl2) kmax = 663 (relative intensity,
0.51), 606 (0.07), 537 (0.09), 506 (0.10), 474 (0.03), 410 (1.00),
398 (0.80, sh), 379 (0.58, sh), 318 nm (0.19); 1H NMR (CDCl3)
d = 9.59 (1H, s, 5-H), 9.54 (1H, s, 10-H), 8.57 (1H, s, 20-H), 7.74
(2H, d, J = 8 Hz, 4-, 5-H of Fmoc), 7.54 (2H, d, J = 7 Hz, 1-, 8-H of
Fmoc), 7.38 (2H, t, J = 7 Hz, 2-, 7-H of Fmoc), 7.29 (2H, t, J = 7 Hz,
3-, 6-H of Fmoc), 7.43 (1H, dd, J = 9, 3 Hz, 3-CH), 5.67 (1H, d,
J = 8 Hz, Fmoc-NH), 5.26, 5.12 (each 1H, d, J = 19 Hz, 131-CH2),
58% isolated yield (31R/S = 1/1); UV–vis (CH2Cl2) kmax = 665 (rel-
ative intensity, 0.54), 608 (0.07), 537 (0.09), 506 (0.10), 473 (0.04),
411 (1.00), 399 (0.81, sh), 380 (0.59, sh), 318 nm (0.20); 1H NMR
(CDCl3) d = 9.60/57 (1H, s, 5-H), 9.50/37 (1H, s, 10-H), 8.60/59
(1H, s, 20-H), 7.65, 7.59, 7.42, 7.35–7.17, 7.11–7.07, 7.03, 6.97,
6.72, 6.55, 6.41 (8H, m, 1-, 2-, 3-, 4-, 5-, 6-, 7-, 8-H of Fmoc), 7.31
(1H, m, 3-CH), 5.78/66 (1H, d, J = 8 Hz, Fmoc-NH), 5.28/27, 5.12/
11 (each 1H, d, J = 18 Hz, 131-CH2), 4.94/73 (1H, dt, J = 8, 4 Hz,
a-
H of Asp), 4.85/78, 4.33–4.27 (each 1H, m, 31-CH2), 4.50/49 (1H,
dq, J = 2, 7 Hz, 18-H), 4.33–4.27 (1H, m, 17-H), 4.26/15, 3.94/70
(each 1H, dd, J = 11, 7 Hz, 9-CH2 of Fmoc), 3.82, 3.67/65, 3.59,
3.49/48, 3.31 (each 3H, s, 2-, 7-, 12-CH3, 172-CO2CH3, Asp-OCH3),
3.80/18 (1H, t, J = 7 Hz, 9-H of Fmoc), 3.70/48 (2H, q, J = 8 Hz, 8-
CH2), 3.42–38/25–21, 3.27–24/13–08 (each 1H, m, b-H of Asp),
2.95/53 (1H, br, 32-OH), 2.79–2.54, 2.42–2.22 (each 2H, m, 17-CH2-
CH2), 1.82/78 (3H, d, J = 7 Hz, 18-CH3), 1.70/67 (3H, t, J = 8 Hz, 81-
CH3), 0.07/À0.13, À1.96/À2.08 (each 1H, s, NH Â 2); HRMS (APCI)
found: m/z = 934.4002, calcd for C54H56N5O10: MH+, 934.4022.
4.84 (1H, ddd, J = 13, 9, 4 Hz, 31-CH), 4.62 (1H, m,
a-H of Glu),
4.48 (1H, dq, J = 2, 8 Hz, 18-H), 4.41, 4.32 (each 1H, dd, J = 11,
7 Hz, 9-CH2 of Fmoc), 4.29 (1H, dt, J = 8, 2 Hz, 17-H), 4.24 (1H,
ddd, J = 13, 9, 3 Hz, 31-CH), 4.11 (1H, t, J = 7 Hz, 9-H of Fmoc),
3.77, 3.69, 3.60, 3.46, 3.19 (each 3H, s, 2-, 7-, 12-CH3, 172-CO2CH3,
Glu-OCH3), 3.76 (1H, dd, J = 9, 4 Hz, 32-OH), 3.68 (2H, q, J = 8 Hz, 8-
CH2), 2.82–2.76, 2.59–2.52 (each 1H, m,
c-H of Glu), 2.72–2.66,
2.57–2.52, 2.31–2.23 (1H+1H+2H, m, 17-CH2CH2), 2.43–2.37 (2H,
m, b-H of Glu), 1.79 (3H, d, J = 8 Hz, 18-CH3), 1.69 (3H, t, J = 8 Hz,
81-CH3), 0.16, À1.93 (each 1H, s, NH Â 2); HRMS (APCI) found:
m/z = 948.4187, calcd for C55H58N5O10: MH+, 948.4178.
4.4.10. Methyl 32-[2-(9-fluorenylmethoxycarbonyl)amino-2-(me-
thoxycarbonyl)ethyl]carbonyloxy-31-hydroxy-mesopyropheo-
phorbide-a (Asp-adduct, 2d)
4.4.13. Methyl 32-[3-(9-fluorenylmethoxycarbonyl)amino-3-(me-
thoxycarbonyl)propyl]carbonyloxy-31-hydroxy-mesopyropheo-
phorbide-a (Glu-adduct, 2e)
35% isolated yield (31R/S = 1/1); UV–vis (CH2Cl2) kmax = 663 (rel-
ative intensity, 0.52), 606 (0.07), 536 (0.09), 506 (0.09), 473 (0.03),
410 (1.00), 398 (0.80, sh), 379 (0.58, sh), 318 nm (0.19); 1H NMR
(CDCl3) d = 9.69/65 (1H, s, 5-H), 9.48 (1H, s, 10-H), 8.56/55 (1H, s,
20-H), 7.64, 7.53, 7.32–7.22 (8H, m, 1-, 2-, 3-, 4-, 5-, 6-, 7-, 8-H of
Fmoc), 6.50/45 (1H, m, 3-CH), 5.94/93 (1H, d, J = 8 Hz, Fmoc-NH),
5.23/21, 5.08/07 (each 1H, d, J = 19 Hz, 131-CH2), 5.02/4.96 (1H,
13% isolated yield (31R/S = 1/1); UV–vis (CH2Cl2) kmax = 663 (rel-
ative intensity, 0.52), 606 (0.07), 537 (0.09), 506 (0.10), 475 (0.04),
410 (1.00), 398 (0.80, sh), 379 (0.58, sh), 318 nm (0.20); 1H NMR
(CDCl3) d = 9.73/67 (1H, s, 5-H), 9.50/49 (1H, s, 10-H), 8.549/546
(1H, s, 20-H), 7.66–7.54, 7.34–7.21, 7.03 (8H, m, 1-, 2-, 3-, 4-, 5-,
6-, 7-, 8-H of Fmoc), 6.58/50 (1H, dd, J = 9, 3 Hz, 3-CH), 5.64/60
(1H, d, J = 8 Hz, Fmoc-NH), 5.25/24, 5.10/09 (each 1H, d, J = 19 Hz,
131-CH2), 5.16/4.74 (1H, dd, J = 12, 9 Hz, 31-CH), 4.89/62 (1H, dd,
dd, J = 12, 9 Hz, 31-CH), 4.84 (1H, ddd, J = 8, 5, 4 Hz,
a-H of Asp),
4.82/80 (1H, m, 31-CH), 4.48/47 (1H, dq, J = 3, 7 Hz, 18-H), 4.44–
4.32 (2H, m, 9-CH2 of Fmoc), 4.26 (1H, m, 17-H), 4.13/09 (1H, t,
J = 7 Hz, 9-H of Fmoc), 3.81/80, 3.65, 3.62, 3.45/44, 3.25 (each 3H,
s, 2-, 7-, 12-CH3, 172-CO2CH3, Asp-OCH3), 3.69 (2H, q, J = 8 Hz, 8-
CH2), 3.65/44 (1H, br, 31-OH), 3.14/11 (1H, dd, J = 16, 5 Hz, b-H of
Asp), 3.08/06 (1H, dd, J = 16, 4 Hz, b-H of Asp), 2.71–2.63, 2.59–
2.53, 2.32–2.22 (1H+1H+2H, m, 17-CH2CH2), 1.80/79 (3H, d,
J = 7 Hz, 18-CH3), 1.693/691 (3H, t, J = 8 Hz, 81-CH3), 0.17, À1.89
(each 1H, s, NH Â 2); HRMS (APCI) found: m/z = 934.4030, calcd
for C54H56N5O10: MH+, 934.4022.
J = 12, 3 Hz, 31-CH), 4.68–4.62 (1H, m,
a-H of Glu), 4.54–4.44 (2H,
m, 9-CH2 of Fmoc), 4.48 (1H, m, 18-H), 4.28 (1H, m, 17-H), 4.25/
14 (1H, br, 31-OH), 4.18/17 (1H, t, J = 7 Hz, 9-H of Fmoc), 3.83/81,
3.653/651, 3.613/610, 3.45/44, 3.25/23 (each 3H, s, 2-, 7-, 12-CH3,
172-CO2CH3, Glu-OCH3), 3.69/68 (2H, q, J = 8 Hz, 8-CH2),
2.72–2.52, 2.35–2.24 (each 2H, m, 17-CH2CH2), 2.49–2.34 (2H, m,
c
-H of Glu), 2.07–1.93 (2H, m, b-H of Glu), 1.80/79 (3H, d,
J = 7 Hz, 18-CH3), 1.694/687 (3H, t, J = 8 Hz, 81-CH3), 0.30/24,
À1.82/85 (each 1H, s, NH Â 2); HRMS (APCI) found:
m/z = 948.4187, calcd for C55H58N5O10: MH+, 948.4178.
4.4.11. Methyl 31-[3-(9-fluorenylmethoxycarbonyl)amino-3-(me-
thoxycarbonyl)propyl]carbonyloxy-32-hydroxy-mesopyropheo-
phorbide-a (Glu-adduct) as the first fraction of HPLC (1e1)
33% isolated yield (31-epimerically pure, slow-eluting fraction
on FCC with Rf = 0.23); UV–vis (CH2Cl2) kmax = 664 (relative inten-
sity, 0.51), 607 (0.07), 536 (0.09), 506 (0.10), 473 (0.03), 410
(1.00), 399 (0.82, sh), 379 (0.59, sh), 319 nm (0.20); 1H NMR (CDCl3)
Acknowledgment
This work was partially supported by a Grant-in-Aid for Scien-
tific Research (A) (No. 22245030) from the Japan Society for the
Promotion of Science (JSPS).

1428
H. Tamiaki et al. / Bioorg. Med. Chem. 22 (2014) 1421–1428
Science; Ferreira, G. C., Kadish, K. M., Smith, K. M., Guilard, R., Eds.; World
Supplementary data
Scientific: Singapore, 2014; p 303. Vol. 27, Chapter 135.
2. Recent reports: (a) Tamiaki, H.; Kozawa, T.; Kitamoto, R.; Kunieda, M. J.
Porphyrins Phthalocyanines 2010, 14, 375; (b) Chaleix, V.; Sol, V.; Krausz, P.
Tetrahedron Lett. 2011, 52, 2977; (c) Melomedov, J.; von Leupoldt, A. W.; Meister,
M.; Laquai, F.; Heinze, K. Dalton Trans. 2013, 42, 9727.
Supplementary data (optical spectra of 1 and 2) associated with
this article can be found, in the online version, at http://dx.doi.org/
10.1016/j.bmc.2013.12.059.
3. Kashiyama, Y.; Tamiaki, H. Chem. Lett. 2014, 43, in press.
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