Carboxylic fused furans for amino acid fluorescent labelling

Article abstract of DOI:10.1016/j.tet.2006.07.003

Four carboxylic fused furans are presented as new fluorescent labels for the amino and hydroxyl functions of organic molecules. Various representative l-amino acids were chosen as models, labelled at their N-terminus and also at their side-chain. Fluoresc

Full text of DOI:10.1016/j.tet.2006.07.003

Tetrahedron 62 (2006) 9258–9267
Carboxylic fused furans for amino acid fluorescent labelling
*
Ana M. Piloto, Andrea S. C. Fonseca, Susana P. G. Costa and M. Sameiro T. Gonc¸alves
´
Centro de Quımica, Universidade do Minho, Campus de Gualtar, P-4710-057 Braga, Portugal
Received 12 May 2006; revised 29 June 2006; accepted 1 July 2006
Available online 4 August 2006
Abstract—Four carboxylic fused furans are presented as new fluorescent labels for the amino and hydroxyl functions of organic molecules.
Various representative L-amino acids were chosen as models, labelled at their N-terminus and also at their side-chain. Fluorescent derivatives
were obtained in high yields, and their absorption and emission properties were studied.
Ó 2006 Elsevier Ltd. All rights reserved.
1. Introduction
benzofurans have been the subject of more extensive studies
for the development of efficient routes for their synthe-
sis.17,18 Compared to benzofurans, synthetic methods for
the preparation of naphthofurans have been less re-
ported.19,20 The most applied synthetic protocol reported is
the intramolecular aldol-type condensation via dehydrative
cyclisation of properly substituted aromatic o-alkoxycar-
bonyl compounds16 or a-aryloxycarbonyl compounds.21
In order to enhance the sensitivity of analytical methodolo-
gies, the interest in fluorescent derivatisation has been
steadily growing in recent years, as fluorescence is far
more sensitive than common UV techniques. The develop-
ment of novel fluorescent derivatisation reagents and their
subsequent evaluation with model compounds has broad
application in biology and biochemistry, to investigate the
structure and dynamics of living systems.1,2
Following our interest in developing new chomophores and
to extend our preliminary results,22–24 we decided to inves-
tigate the application of fused furans in derivatisation reac-
tions with biomolecules. We now present a study involving
two systems, namely benzofuran and naphthofuran, bearing
different substituents and representative amino acids of
different character as models.
Amino acids are the building blocks in biological systems
like proteins, enzymes and many other molecules with bio-
logical activity. The understanding of their physiological
role is of utmost importance, given that a number of dis-
orders are directly associated with the presence or lack of
particular amino acids, and there is a practical interest in
their detection in areas such as clinical chemistry.3–5
The main purpose of our work is to present a label for amino
acids that have no (or low) intrinsic fluorescence or that are
not easily detectable by ultraviolet absorption, thus exclud-
ing tryptophan and tyrosine. Regarding phenylalanine, the
least UV-absorbing and fluorescent of the three aromatic
amino acids, there is a practical interest in its tagging in
order to enhance its properties.
As most amino acids are poor UV-absorbing, fluorescence
derivatisation is often employed for their determination. Al-
though, a large number of fluorescent tagging reagents have
been developed through the years, which cover the UV/vis
spectrum and are specifically reactive towards different
functional groups,6–10 there is only scarce information on
the application of benzofurans and naphthofurans in this
field.11,12
Evaluation of the fluorescence properties was carried out for
all labelled amino acids in ethanol and the influence of
solvent polarity in emission was measured for one of the
derivatives.
Arene ring-fused furans are found in various naturally occur-
ring compounds and a number of their natural and synthetic
derivatives are associated with diverse biological and
pharmacological activities.13–16 Regarding arenofurans,
2. Results and discussion
Keywords: Oxygen heterocycles; Fluorescent label; Amino acids; Furans.
* Corresponding author. Tel.: +351 253 604386; fax: +351 253 678983;
e-mail: msameiro@quimica.uminho.pt
Fluorophores 1 and 2a–c were obtained by an alkaline ring
contraction of the corresponding oxobenzopyran 3 and
0040–4020/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2006.07.003

A. M. Piloto et al. / Tetrahedron 62 (2006) 9258–9267
9259
oxobenzo[f]benzopyran 4a–c, respectively. The precursors
1-chloromethyl-6-methoxy-3-oxo-3H-benzopyran (3) and
1-chloromethyl-3-oxo-3H-benzo[f]benzopyrans (4a–c) were
prepared through a Pechmann reaction of 3-methoxyphenol
or 2-naphthol and its derivatives with ethyl 4-chloroaceto-
acetate by a known procedure,25 in good yields (71–92%).
Heating to 80 ꢀC compounds 3 or 4a–c in aqueous 2 M
sodium hydroxide solution yielded the 2-(5-methoxybenzo-
furan-1-yl)ethanoic acid, Bfm-OH (1) (92%) or 2-(naph-
tho[2,1-b]furan-1-yl)ethanoic acids (2a–c) (94–98%)
(Scheme 1, Table 1).
(C-1 and C-2) at d 113.83–115.94 ppm and d 142.31–
143.64 ppm, respectively. Signals between 171.97 and
172.62 ppm were assigned to the carboxyl group.
The carboxylic heterocyclic labels 1 and 2a were linked to
the a-amine group of L-phenylalanine and L-valine methyl
esters (5a and 5b), by coupling with the aid of N,N0-dicyclo-
hexylcarbodiimide (DCC) assisted by 1-hydroxybenzotri-
azole (HOBt) under standard conditions. After purification
by chromatography on silica gel, the corresponding acetyl
derivatives 6a,b and 7a,b were obtained (Scheme 2, Table 2,
entries 1–4).
CH2Cl
CH2CO2H
a)
a)
b)
Flu-Aaa-OCH3
H-Aaa-OCH3
Flu-OH
1,2
+
O
6,
7
5
H3CO
O
O
H3CO
H3CO
OH
1
3
Aaa = a Phe, b Val, c Ala, d Gly, e Asp(OMe), f Glu(OMe)
R
R
CH2Cl
a)
CH2CO2H
Ac-Lys(Nfm)-OMe
9
Flu-OH + Ac-Lys-OMe
2a
a)
b)
8
R
OH
O
O
O
a)
Boc-Ser(Flu)-OMe
Flu-OH + Boc-Ser-OMe
4
2
1,2a
Flu =
H3CO
11,12
10
a R = OCH3; b R = OH; c R = H
Scheme 1. Synthesis of fluorescent heterocycles 1–4. Reagents and
conditions: (a) ClCH2COCH2CO2Et, aq H2SO4 70%, rt; (b) aq 2 M
NaOH, 80 ꢀC.
-
R
CH2CO
2a R = OCH3, Nfm
2b R = OH, Nfh
2c R = H, Nfu
1, Bfm
-
CH CO
2
O
In the IR spectra of precursors 3 and 4a–c, a strong band
between 1739 and 1689 cmꢁ1 confirmed the presence of
the carbonyl group of the heterocycle. The 1H NMR spectra
showed the characteristic signal of proton 2 (H-2) of the
pyran ring at d 6.48 ppm (3) and 6.62–6.76 ppm (4a–c), in
addition to the expected signals of the fused rings, and the
chloromethylene group at d between 4.98 and 5.30 ppm. In
the 13C NMR spectra of these compounds, relevant signals
for the heterocycle were visible from d 150.87 to
152.27 ppm (C-1), 112.00 to 117.37 ppm (C-2) and 159.38
to 160.02 ppm (C-3). The protons corresponding to the
chloromethylene group appeared between d 41.34 and
46.17 ppm.
O
Scheme 2. Synthesis of labelled amino acid derivatives 6, 7, 9, 11 and 12.
Reagents and conditions: (a) DCC, HOBt, DMF, rt.
By comparison of fluorescence data obtained for these
derivatives, which will be discussed later, it was concluded
that the naphthofuran derivatives are more interesting than
their benzofuran counterparts for labelling applications.
The extension of its structure (incorporating to the molecule
one more aromatic ring) resulted in an expected increase in
the quantum yield of fluorescence (FF). Two other naphtho-
furan derivatives, 2b,c, were used in the reaction with model
valine methyl ester, under the same reaction conditions. The
resulting fluorogenic derivatives 7c,d (Scheme 2, Table 2,
entries 5 and 6) differ from 7a in the substituent attached
at position 8 of the naphthofuran moiety (OH and H, respec-
tively). Their fluorescence properties were inferior to those
For the target compounds 1 and 2a–c, strong stretching
vibration bands for the carboxyl group were present at
3550–3103 cmꢁ1 and 1703–1715 cmꢁ1. In the 1H NMR
spectra, the singlet corresponding to proton H-2 of the furan
ring at d 7.75–8.01 ppm, as well as a broad signal for the
carboxyl OH at high d (12.40 to 12.60 ppm) were visible.
13C NMR data confirmed the heterocyclic ring contraction
by showing the signals corresponding to carbons 1 and 2
Table 2. Synthesis of labelled amino acid derivatives 6, 7, 9, 11 and 12
Entry Label Amino Product
acid
Yield Mp (ꢀC)
(%)
1
2
3
4
5
6
7
8
1
1
5a
5b
5a
5b
5b
5b
5c
5d
5e
5f
6a
6b
7a
7b
7c
7d
7e
7f
7g
7h
9
11
12
Bfm-Phe-OMe
Bfm-Val-OMe
Nfm-Phe-OMe
Nfm-Val-OMe
Nfh-Val-OMe
Nfu-Val-OMe
Nfm-Ala-OMe
Nfm-Gly-OMe
Nfm-Asp(OMe)-OMe 98
Nfm-Glu(OMe)-OMe 98
Ac-Lys(Nfm)-OMe
Boc-Ser(Bfm)-OMe 65
Boc-Ser(Nfm)-OMe 54
87
71
72
89
57
83
90
95
128.9–130.6
Oil
Table 1. Synthesis of fluorescent heterocycles 1–4
2a
2a
2b
2c
2a
2a
2a
2a
2a
1
146.3–148.4
159.0–161.0
145.3–145.9
141.5–143.0
190.1–192.0
171.9–174.0
136.8–137.7
166.3–169.0
185.7–186.9
Oil
Compound
Yield (%)
Mp (ꢀC)
1
Bfm-OH
Nfm-OH
Nfh-OH
Nfu-OH
Opm-Cl
Obm-Cl
Obh-Cl
Obb-Cl
92
98
96
94
79
83
92
71
124.6–126.0
176.8–178.9
167.8–169.0
171.4–173.0
199.5–200.8
179.2–180.7
250.0–250.7
179.5–182.7
2a
2b
2c
3
4a
4b
4c
9
10
11
12
13
8
10
10
71
2a
Oil

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A. M. Piloto et al. / Tetrahedron 62 (2006) 9258–9267
of the 8-methoxy derivative and as a result, reaction of
compound 2a with the a-amine group of a set of representa-
tive L-amino acid methyl esters, namely alanine, glycine
and aspartic and glutamic acids (protected at their carboxyl
side-chain as methyl esters) was carried out in the same
conditions (Scheme 2, Table 2, entries 7–10).
at the wavelengths of maximum absorption found for each
one of the compounds to be tested.
The longest wavelength absorption maximum of all com-
pounds was located between 285–301 nm. Compound 2a
absorbs and emits at longer wavelength, when compared
to 1, the bathochromic shifts being 13 and 34 nm, respec-
tively. The fluorescence quantum yield for 2a (0.20) was
10 times higher than for 1 (0.02). When these two labels
were linked to phenylalanine and valine methyl esters, the
same trend was observed, the FF for compounds 6a–b being
about 0.07 and for compounds 7a–b higher than 0.3 (Table 3,
entries 5–8). Having in mind that our purpose was to obtain
a heterocycle for labelling applications, which required a
significant FF, we decided to use the naphthofuran moiety
in the following studies.
In addition to labelling amino acids at their N-terminus, the
alternative acylation at a lysine u-amine group was also in-
vestigated. Thus, the methyl ester of N-acetyl-lysine (8) was
reacted with 2a under the conditions reported above, to give
the expected fluorescent derivative 9 (Scheme 2, Table 2,
entry 11). Another approach for side-chain labelling was un-
dertaken by reacting N-tert-butyloxycarbonylserine methyl
ester 10 with compounds 1 and 2a to give the corresponding
fluorescent ester derivatives 11 and 12 (Scheme 2, Table 2,
entries 12 and 13).
In order to access the influence of substituents at this hetero-
cycle, absorption spectra in ethanol of compounds 2b,c and
7c,d showed that the insertion of more electron donating
substituents at position 8 shifted bathochromically the
absorption maximum by 5–8 nm (compare entries 2–4 and
8–10). In the emission spectra, a bathochromic shift is
observed when comparing compounds 7b–d (21–25 nm).
All labelled amino acids (6, 7, 9, 11 and 12) were obtained as
solid materials (except 6b, 11 and 12, which were oils) in
yields ranging from 54 to 98% (Table 2) and were character-
ised by elemental analyses or high-resolution mass spec-
trometry, NMR (1H and 13C), IR and UV/vis spectroscopies.
The IR spectra of labelled compounds showed bands due to
stretching vibrations of the carbonyl group from 1658 to
1628 cmꢁ1 (amide linkage) and from 1761 to 1707 cmꢁ1
(ester group). 1H NMR spectra showed signals of the amino
acid residues, such as a singlet for the methyl ester (d 3.45–
3.94 ppm), a multiplet for the a-CH (d 4.35–4.90 ppm, ex-
cept for 7f, which was a doublet at 3.97 ppm) and a doublet
for a-NH (d 5.18–6.69 ppm), in addition to the protons of the
heterocyclic moiety. In 13C NMR, signals of amide type
carbonyl were found from d 169.34 to 171.01 ppm and of
the ester type occurred from d 170.04 to 172.86 ppm.
The FF was also affected by the substituent, as naphtho-
furans 2a,b had higher FF than the unsubstituted 2c. We
expected that the substitution with an electron-donor would
result in an increase in fluorescence intensity and FF, as it
was observed for the OMe substituent. However, in the
case of OH substituent we observed that the values were
similar to that of the unsubstituted derivative probably due
to the presence of H-bonds to the solvent (Table 3, entries
3, 4 and 9, 10).
Taking these results into consideration, we studied the prop-
erties of the derivatives resulting from the reaction of 2a with
other representative amino acids. The resulting labelled ami-
no acids (7a,b,e–h, 9 and 12) exhibit moderate to good fluo-
rescence quantum yields (0.13<F<0.44) and the Stokes’
shifts are about 50 nm. There appears to be some influence
from the amino acid residue on FF, since labelled acidic
(7g and 7h) and polar uncharged (12) residues have lower
Electronic absorption and emission spectra of 10ꢁ6 M solu-
tions of compounds 1, 2, 6, 7, 9, 11 and 12 in degassed
absolute ethanol were measured, absorption and emission
maxima, and FF are also reported (Table 3). The quantum
yields were calculated using 9,10-diphenylanthracene as
standard (FF¼0.95 in ethanol).26 For the relative quantum
yields’ determination, 9,10-diphenylanthracene was excited
Table 3. UV and fluorescence data for compounds 1, 2, 6, 7, 9, 11 and 12
Entry
Compound
UV lmax (nm)
Fluorescence
Stokes’ shift (nm)
lem (nm)
FF
1
2
3
4
5
6
7
8
1
Bfm-OH
Nfm-OH
Nfh-OH
Nfu-OH
285
298
301
293
288
288
298
298
300
292
298
297
298
298
297
287
298
315
349
349
340
315
315
349
346
350
325
349
343
346
347
347
314
349
0.020ꢂ0.003
30
51
48
47
27
27
52
49
50
33
49
46
48
49
50
27
51
2a
2b
2c
6a
6b
7a
7b
7c
7d
7e
7f
7g
7h
9
11
12
0.20ꢂ0.01
0.062ꢂ0.003
0.076ꢂ0.008
0.064ꢂ0.007
0.070ꢂ0.006
0.32ꢂ0.02
0.37ꢂ0.03
0.10ꢂ0.01
0.13ꢂ0.01
0.24ꢂ0.02
0.24ꢂ0.01
0.14ꢂ0.01
0.14ꢂ0.02
0.44ꢂ0.03
0.064ꢂ0.007
0.13ꢂ0.01
Bfm-Phe-OMe
Bfm-Val-OMe
Nfm-Phe-OMe
Nfm-Val-OMe
Nfh-Val-OMe
Nfu-Val-OMe
Nfm-Ala-OMe
Nfm-Gly-OMe
Nfm-Asp(OMe)-OMe
Nfm-Glu(OMe)-OMe
Ac-Lys(Nfm)-OMe
Boc-Ser(Bfm)-OMe
Boc-Ser(Nfm)-OMe
9
10
11
12
13
14
15
16
17

A. M. Piloto et al. / Tetrahedron 62 (2006) 9258–9267
9261
1.2
1.0
0.8
0.6
0.4
0.2
0.0
values seem to be associated with aprotic solvents, either
apolar or polar, such as 1,4-dioxane, DMF or diethyl ether,
the highest being 0.57 (1,4-dioxane). In protic solvents
like ethanol and methanol, quantum yields were slightly
lower (0.32 and 0.33, respectively), which may be related
with the high ability for H-bond donating/electron density
acceptance power displayed by these solvents, which could
affect the conjugation on the heterocyclic moiety. For the
chlorinated solvent, the FF value was analogous to that of
the protic solvents.
7b
7c
7d
6b
250
300
350
Wavelength (nm)
400
450
With the aim of testing the possibility of using these fluoro-
phores as labels in peptide chemistry, stability tests, such as
hydrogenation catalysed by Pd/C, acidolysis (6 M HCl, tri-
fluoroacetic acid, TFA), aminolysis with 2-(N,N-diethyl-
amino)ethylamine (DEAEA)27 and reduction with metals
(Mg/MeOH),28 were carried out. Fluorescent valine methyl
ester, Nfm-Val-OMe (7b), used as model, was treated under
similar conditions to those usually required for cleavage of
protecting groups during peptide synthesis.29 In these condi-
tions, the compound showed good stability, being recovered
in yields from 95 to 100% (Pd/C, HCl, TFA and Mg) and
85% (DEAEA), as it was confirmed by 1H NMR. Treatment
with base (aqueous 1 M NaOH) was also performed, result-
ing in quantitative cleavage of the ester function, without
affecting the label. All labelled compounds were also stable
to prolonged storage at room temperature.
Figure 1. Normalised fluorescence spectra of valine labelled with different
fluorophores 6b and 7b–d.
FF than the free label 2a, probably due to the possibility of
occurrence of H-bonds between the label and the acidic or
polar side chains. For all other derivatives the linkage is
done through an amide bond and there are no extra effects
from the side chains. The higher electron donating character
of N and its resonance effect through the amide bond result
in higher FF when compared to the free label.
These results showed that 2-(8-methoxynaphtho[2,1-b]-
furan-1-yl)ethanoic acid, Nfm-OH (2a) was a suitable fluo-
rophore for the considered amino acids.
In Figure 1, the fluorescence spectra of valine labelled with
different fluorophores (6b, 7b, 7c and 7d) are shown.
3. Conclusions
In order to further elucidate the solvent interaction, absorp-
tion and emission spectra of model compound 7a, Nfm-
Phe-OMe, were measured in 10 other solvents of different
polarity and proton donor ability, such as n-hexane, diethyl
ether, toluene, 1,4-dioxane, ethyl acetate, methanol, acetoni-
trile, dichloromethane (DCM), N,N-dimethylformamide
(DMF) and dimethyl sulfoxide (DMSO). The wavelengths
of maximum absorption and emission and FF for this
compound are listed in Table 4.
A series of carboxylic fused furans were synthesised in
excellent yields by a simple procedure from the correspond-
ing oxobenzopyrans. These heterocycles were used in the
derivatisation of representative L-amino acids of different
character, such as nonpolar aliphatic (valine and alanine)
or aromatic (phenylalanine), basic (lysine), polar uncharged
(glycine and serine) and acidic (aspartic and glutamic acids)
amino acids. From the study of the absorption and emission
properties, carried out for all labelled residues, we con-
cluded that the 2-(8-methoxynaphtho[2,1-b]furan-1-yl)-
ethanoic acid (2a) was the most interesting fluorophore.
From the results, it can be seen that the polarity of the solvent
does not influence significantly the position of the band of
absorption and emission; the wavelengths of maximum ab-
sorption and emission range from 296 to 299 nm and from
343 to 351 nm, respectively. However, a more significant in-
fluence of the character of the solvent on FF was observed.
Concerning the FF obtained in the various solvents, higher
Considering the high yields of the synthesis and derivatisa-
tion reactions as well as the fluorescence properties and
also the stability to different deprotection conditions, usually
used in peptide synthesis, naphthofurans seem to be promis-
ing for fluorescent labelling purposes.
Table 4. UV and fluorescence data for compound 7a in different solvents
4. Experimental
4.1. General
Entry Solvent
UV lmax
(nm)
Fluorescence
Stokes’
shift (nm)
lmax (nm) FF
1
2
3
4
5
6
7
8
n-Hexane 296
Diethyl ether 297
343
345
346
346
348
346
347
348
347
350
351
0.29ꢂ0.02 47
All melting points were uncorrected and were measured on
a Gallenkamp melting point apparatus. TLC analyses were
carried out on 0.25 mm thick precoated silica plates (Merck
Fertigplatten Kieselgel 60F254) and spots were visualised
under UV light. Chromatography on silica gel was carried
out on Merck Kieselgel (230–240 mesh). IR spectra were de-
termined on a Perkin–Elmer FTIR-1600 using KBr discs or
Nujol. UV/vis spectra were run on a Hitachi U-2000 spectro-
photometer. 1H NMR spectra were recorded on a Varian 300
0.45ꢂ0.03 48
Ethanol
Toluene
298
298
0.32ꢂ0.02 48
0.50ꢂ0.03 48
0.57ꢂ0.04 51
0.36ꢂ0.03 49
0.33ꢂ0.03 50
0.40ꢂ0.03 51
0.30ꢂ0.04 48
0.55ꢂ0.03 51
0.36ꢂ0.04 52
1,4-Dioxane 297
Ethyl acetate 297
Methanol
Acetonitrile
DCM
DMF
DMSO
297
297
299
299
299
9
10
11

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A. M. Piloto et al. / Tetrahedron 62 (2006) 9258–9267
spectrometer in CDCl3 or DMSO-d6 at 300 MHz at 25 ꢀC.
All chemical shifts are given in parts per million using dH
Me4Si¼0 ppm as reference and J values are given in hertz.
13C NMR spectra were run in the same instrument at
75.4 MHz using the solvent peak as internal reference. As-
signments were made by comparison of chemical shifts,
peak multiplicities and J values, and were supported by spin
decoupling-double resonance and bidimensional hetero-
nuclear correlation HMBC and HMQC techniques. Mass
spectrometric analyses were performed at the C.A.C.T.I.—
Unidad de Espectrometria de Masas of the University of
Vigo, Spain, on a Hewlett–Packard 5989 A spectrometer
for low-resolution spectra and a VG Autospec M spectro-
meter for high-resolution mass spectra. Elemental analyses
were carried out on a Leco CHNS 932 instrument. Fluores-
cence spectra were collected using a Spex Fluorolog 1680
Spectrometer.
4.1.3. 2-(8-Hydroxynaphtho[2,1-b]furan-1-yl)ethanoic
acid, Nfh-OH (2b). Starting with Obh-Cl (4b) (0.205 g,
7.86ꢃ10ꢁ4 mol) and following the same procedure de-
scribed above for Bfm-OH (1), compound 2b was obtained
as a brown solid (0.183 g, 96%). Mp¼167.8–169.0 ꢀC.
TLC (chloroform/methanol, 5:2): Rf 0.36. 1H NMR
(DMSO-d6, 300 MHz): d¼3.97 (s, 2H, CH2), 7.05 (dd,
J¼8.7 and 2.4 Hz, 1H, H-7), 7.44 (d, J¼2.4 Hz, 1H, H-9),
7.49 (d, J¼8.7 Hz, 1H, H-4), 7.67 (d, J¼9.0 Hz, 1H, H-5),
7.85 (d, J¼9.0 Hz, 1H, H-6), 7.91 (s, 1H, H-2), 9.84 (s,
1H, OH), 12.57 (br s, 1H, CO2H). 13C NMR (DMSO-d6,
75.4 MHz): dC¼30.75 (CH2), 105.71 (C-9), 109.25 (C-4),
115.62 (C-1), 116.07 (C-7), 119.91 (C-3b), 124.49 (C-5a),
125.60 (C-5), 129.64 (C-5b), 130.44 (C-6), 142.79 (C-2),
153.11 (C-3a), 156.04 (C-8), 172.13 (CO2H). IR (KBr 1%,
cmꢁ1): n¼3365, 3107, 2926, 2860, 1715, 1636, 1533,
1471, 1419, 1388, 1360, 1334, 1286, 1259, 1239, 1193,
1173, 1107, 1033, 883, 860, 833. UV/vis (ethanol, nm):
lmax (3)¼301 (7970 Mꢁ1 cmꢁ1). HRMS (EI) calcd for
C14H10O4 [M+]: 242.0579; found: 242.0586.
4.1.1. 2-(5-Methoxybenzofuran-1-yl)ethanoic acid, Bfm-
OH (1).
A suspension of compound 3 (0.500 g,
2.23ꢃ10ꢁ3 mol) in aqueous 2 M sodium hydroxide solution
(10 mL) was stirred for 16 h at 80 ꢀC. After cooling, the re-
action mixture was acidified with aqueous 6 M hydrochloric
acid solution until pH¼5–6. The mixture was extracted with
ethyl acetate (3ꢃ20 mL), the organic extracts were com-
bined, dried with magnesium sulfate and evaporated under
vacuum in a rotary evaporator to yield compound 1 as a
off-white solid (0.422 g, 92%). Mp¼124.6–126.0 ꢀC. TLC
(chloroform/methanol, 95:5): Rf 0.30. 1H NMR (DMSO-d6,
300 MHz): d¼3.63 (s, 2H, CH2), 3.78 (s, 3H, OCH3), 6.87
(dd, J¼2.4 and 8.7 Hz, 1H, H-6), 7.15 (d, J¼2.4 Hz, 1H,
H-4), 7.44 (d, J¼8.7 Hz, 1H, H-7), 7.75 (s, 1H, H-2),
12.15 (br s, 1H, OH). 13C NMR (DMSO-d6, 75.4 MHz):
dC¼29.07 (CH2), 55.59 (OCH3), 95.94 (C-4), 111.54 (C-6),
113.83 (C-1), 120.30 (C-7), 120.98 (C-7a), 142.31 (C-2),
155.58 (C-3a), 157.72 (C-5), 171.97 (CO2H). IR (KBr 1%,
cmꢁ1): n¼3424, 3051, 3017, 2908, 2836, 1708, 1627,
1595, 1490, 1441, 1401, 1292, 1233, 1143, 1120,
1075, 1023, 935, 805. UV/vis (ethanol, nm): lmax (3)¼285
(1828 Mꢁ1 cmꢁ1). HRMS (EI): calcd for C11H10O4 [M+]:
206.0579; found: 206.0578.
4.1.4. 2-(Naphtho[2,1-b]furan-1-yl)ethanoic acid, Nfu-
OH (2c). Starting with Obh-Cl (4c) (0.207 g, 8.46ꢃ
10ꢁ4 mol) and following the same procedure described
above for Bfm-OH (1), compound 2c was obtained as
a brownish solid, which was recrystallised from ethyl ace-
tate/n-hexane. Compound 2c was obtained as a beige solid
(0.180 g, 94%). Mp¼171.4–173.0 ꢀC. TLC (chloroform/
methanol, 5.8:0.2): Rf 0.28. 1H NMR (DMSO-d6,
300 MHz): d¼4.04 (s, 2H, CH2), 7.51 (dt, J¼8.0 and
1.2 Hz, 1H, H-8), 7.60 (dt, J¼8.0 and 1.2 Hz, 1H, H-7),
7.77 (d, J¼9.0 Hz, 1H, H-4), 7.84 (d, J¼9.0 Hz, 1H, H-5),
8.01 (s, 1H, H-2), 8.04 (br d, J¼8.1 Hz, 1H, H-9), 8.18 (br
d, J¼8.1 Hz, 1H, H-6), 12.60 (br s, 1H, OH). 13C NMR
(DMSO-d6, 75.4 MHz): dC¼31.08 (CH2), 112.74 (C-4),
115.94 (C-1), 121.18 (C-3b), 123.14 (C-6), 124.44 (C-8),
125.67 (C-5), 126.49 (C-7), 127.96 (C-5a), 129.01 (C-9),
130.36 (C-5b), 143.64 (C-2), 152.69 (C-3a), 172.50
(CO2H). IR (KBr 1%, cmꢁ1): n¼3550–3000, 2900, 1707,
1623, 1583, 1524, 1413, 1387, 1323, 1286, 1232, 1197,
1177, 1158, 1120, 1110, 1024, 992, 949, 939, 857, 830.
UV/vis (ethanol, nm): lmax (3)¼293 (7442 Mꢁ1 cmꢁ1).
HRMS (EI) calcd for C14H10O3 [M+]: 226.0630; found:
226.0634.
4.1.2. 2-(8-Methoxynaphtho[2,1-b]furan-1-yl)ethanoic
acid, Nfm-OH (2a). Starting with Obm-Cl (4a) (0.232 g,
8.44ꢃ10ꢁ4 mol) and following the same procedure described
above for Bfm-OH (1), followed by recrystallisation from
ethyl acetate/n-hexane, compound2awas obtained as a brown
solid (0.212 g, 98%). Mp¼176.8–178.9 ꢀC. TLC (chloro-
form/methanol, 9.8:0.2): Rf 0.51. 1H NMR (DMSO-d6,
300 MHz): d¼3.90 (s, 3H, OCH3), 4.04 (s, 2H, CH2), 7.14
(dd, J¼9.0 and 2.5 Hz, 1H, H-7), 7.56 (s, 1H, H-9), 7.58 (d,
J¼9.0 Hz, 1H, H-4), 7.75 (d, J¼9.0 Hz, 1H, H-5), 7.93 (d,
J¼9.0 Hz, 1H, H-6), 7.96 (s, 1H, H-2), 12.60 (br s, 1H,
OH). 13C NMR (DMSO-d6, 75.4 MHz): dC¼31.03 (CH2),
55.06 (OCH3), 102.99 (C-9), 110.09 (C-4), 115.74 (C-1),
115.87 (C-7), 120.52 (C-3b), 125.21 (C-5a), 125.45 (C-5),
129.13 (C-5b), 130.41 (C-6), 143.20 (C-2), 153.22 (C-3a),
157.78 (C-8), 172.62 (CO2H). IR (KBr 1%, cmꢁ1): n¼3442,
3103, 3016, 2966, 2922, 1703, 1627, 1602, 1523, 1468,
1409, 1383, 1358, 1281, 1259, 1232, 1201, 1179, 1135,
1120, 1107, 1040, 1023, 947, 876, 838, 833. UV/vis (ethanol,
nm): lmax (3)¼298 (8436 Mꢁ1 cmꢁ1). Anal. Calcd for
C15H12O4 (256.25): C 70.30, H 4.72; found: C 70.48, H 4.81.
4.1.5. 1-Chloromethyl-6-methoxy-3-oxo-3H-benzopyran,
Opm-Cl (3). To a solution of 3-methoxyphenol (1.1 mL,
1.0ꢃ10ꢁ2 mol) in 70% aqueous sulfuric acid (10 mL), ethyl
4-chloroacetoacetate (2.02 mL, 1.5ꢃ10ꢁ2 mol) was added
and stirred at room temperature for 78 h. The reaction mix-
ture was poured into ice water and stirred for 2 h to give
a fine violet precipitate. The solid was collected by filtration,
washed with cold water and dried in a vacuum oven. Purifi-
cation by dry chromatography on silica gel using chloro-
form/n-hexane, 2:1 as eluent gave compound 3 as a white
solid (1.77 g, 79%). Mp¼199.5–200.8 ꢀC. TLC (chloro-
1
form): Rf 0.65. H NMR (DMSO-d6, 300 MHz): d¼3.85
(s, 3H, OCH3), 4.98 (s, 2H, CH2), 6.48 (s, 1H, H-2), 6.98–
7.03 (m, 2H, H-5 and H-7), 7.75 (d, J¼8.4 Hz, 1H, H-8).
13C NMR (DMSO-d6, 75.4 MHz): dC¼41.34 (CH2), 56.00
(OCH3), 101.06 (C-5), 110.44 (C-8a), 112.00 (C-2),
112.35 (C-7), 126.38 (C-8), 150.87 (C-1), 155.26 (C-4a),
160.02 (C-3), 162.61 (C-6). IR (KBr 1%, cmꢁ1): n¼3070,

A. M. Piloto et al. / Tetrahedron 62 (2006) 9258–9267
9263
3023, 2853, 1725, 1623, 1611, 1558. UV/vis (ethanol, nm):
lmax (3)¼325 (14125 Mꢁ1 cmꢁ1). HRMS (EI) calcd for
C11H9O335Cl [M+]: 224.0240; found: 224.0251; calcd for
C11H9O337Cl [M+]: 226.0194; found: 226.0202.
and 1.2 Hz, 1H, 7-H), 8.04 (d, J¼9.0 Hz, 1H, 6-H), 8.49
(br d, J¼9.0 Hz, 1H, 10-H). 13C NMR (CDCl3,
75.4 MHz): dC¼45.79 (CH2), 112.50 (C-4b), 117.37 (C-2),
117.78 (C-5), 124.90 (C-10), 125.80 (C-8), 128.51 (C-9),
128.75 (C-6b), 129.88 (C-7), 131.29 (C-6a), 134.33 (C-6),
151.22 (C-1), 155.10 (C-4a), 159.97 (C-3). IR (KBr 1%,
cmꢁ1): n¼3550, 3070, 1716, 1693, 1588, 1550, 1520,
1457, 1433, 1416, 1345, 1323, 1285, 1254, 1212, 1166,
1144, 1124, 1013, 1002, 918, 877, 862, 828. UV/vis (etha-
nol, nm): lmax (3)¼(11,449 Mꢁ1 cmꢁ1). HRMS (EI): calcd
for C14H9O325Cl [M+]: 244.0291; found: 244.0290; calcd
for C14H9O327Cl [M+]: 246.0262; found: 246.0267.
4.1.6. 1-Chloromethyl-9-methoxy-3-oxo-3H-benzo[f]-
benzopyran, Obm-Cl (4a). Starting with 7-methoxy-2-
naphthol (0.348 g, 2.0ꢃ10ꢁ3 mol) and following the same
procedure as described above for the preparation of com-
pound 3, using ethyl acetate/n-hexane 3:7, as the chromato-
graphy eluent, followed by recrystallisation from ethyl
acetate/n-hexane, compound 4a was obtained as an off-
white solid (0.456 g, 83%). Mp¼179.2–180.7 ꢀC. TLC
1
(ethyl acetate/n-hexane, 3:4): Rf 0.58. H NMR (CDCl3,
4.2. General method for the synthesis of fluorescent-la-
belled L-amino acids 6, 7, 9, 11 and 12
300 MHz): d¼4.02 (s, 3H, OCH3), 4.98 (s, 2H, CH2), 6.62
(s, 1H, H-2), 7.24 (dd, J¼9.0 and 2.4 Hz, 1H, H-8), 7.31
(d, J¼9.0 Hz, 1H, H-5), 7.79–7.90 (m, 2H, H-10 and H-7),
7.92 (d, J¼9.0 Hz, 1H, H-6). 13C NMR (CDCl3,
75.4 MHz): dC¼45.60 (CH2), 55.55 (OCH3), 105.71
(C-10), 111.80 (C-4b), 115.11 (C-5), 117.02 (C-8), 117.24
(C-2), 126.29 (C-6a), 130.23 (C-6b), 131.15 (C-7), 133.93
(C-6), 150.96 (C-1), 155.76 (C-4a), 159.69 (C-9), 159.99
(C-3). IR (KBr 1%, cmꢁ1): n¼3068, 1739, 1626, 1585,
1548, 1521, 1445, 1430, 1346, 1286, 1242, 1218, 1168,
1148, 1054, 1021, 912, 899, 863, 837, 734. UV/vis (ethanol,
nm): lmax (3)¼354 (12826 Mꢁ1 cmꢁ1). Anal. Calcd for
C15H11O3Cl (274.70): C 65.58, H 4.04; found C 65.50, H
4.21.
Carboxylic compounds (1, 2) were reacted with an amino
acid methyl ester (2 equiv) in DMF by a standard DCC/
HOBt coupling.29 After evaporation of the solvent and chro-
matography on silica gel, the required acetyl derivatives (6,
7, 9, 11 and 12) were obtained.
4.2.1. N-[(5-Methoxybenzofuran-1-yl)ethanoyl]phenyl-
alanine methyl ester, Bfm-Phe-OMe (6a). The product of
the reaction of Bfm-OH (1) (0.105 g; 5.1ꢃ10ꢁ4 mol) with
phenylalanine methyl ester hydrochloride (5a) was chroma-
tographed using chloroform as the eluent to give compound
6a as a yellow solid (0.162 g, 87%). Mp¼128.9–130.6 ꢀC.
1
TLC (chloroform): Rf 0.28. H NMR (CDCl3, 300 MHz):
4.1.7. 1-Chloromethyl-9-hydroxy-3-oxo-3H-benzo[f]ben-
zopyran, Obh-Cl (4b). Starting with 2,7-dihydroxynaph-
thalene (0.320 g, 2.0ꢃ10ꢁ3 mol) and following the same
procedure described above for compound 3, using ethyl ace-
tate/n-hexane 3:7, as the chromatography eluent, compound
4b was obtained as beige solid (0.482 g, 92%). Mp¼250.0–
d¼2.97–3.05 (m, 2H, b-CH2 Phe), 3.60 (s, 2H, CH2), 3.70
(s, 3H, OCH3 Phe), 3.88 (s, 3H, OCH3), 4.80–4.90 (m, 1H,
a-H Phe), 6.00 (d, J¼7.5 Hz, 1H, a-NH Phe), 6.80–6.84
(m, 2ꢃAr-H, 2H, Phe), 6.88 (dd, J¼2.4 and 8.4 Hz, 1H,
H-6), 7.04 (d, J¼2.1 Hz, 1H, H-4), 7.10–7.18 (m, 3ꢃAr-H,
3H, Phe), 7.33 (d, J¼8.7 Hz, 1H, H-7), 7.43 (s, 1H, H-2).
13C NMR (CDCl3, 75.4 MHz): dC¼31.60 (CH2), 37.50 (b-
CH2 Phe), 52.32 (OCH3 Phe), 52.87 (a-CH Phe), 55.71
(OCH3), 96.07 (C-4), 112.05 (C-6), 113.36 (C-3a), 119.75
(C-7), 120.45 (C-7a), 127.04 (C-4 Phe), 128.46 (C-3 and
C-5 Phe), 128.93 (C-2 and C-6 Phe), 135.32 (C-1 Phe),
142.18 (C-2), 156.43 (C-1), 158.41 (C-5), 169.34 (CONH),
171.68 (CO2CH3). IR (KBr 1%, cmꢁ1): n¼3289, 3064,
3030, 3008, 2956, 2930, 1750, 1659, 1629, 1602, 1587,
1544, 1494, 1455. UV/vis (ethanol, nm): lmax (3)¼288
(4717 Mꢁ1 cmꢁ1). HRMS (EI): calcd for C21H21NO5
[M+]: 367.1420; found: 367.1402.
1
250.7 ꢀC. TLC (chloroform/methanol 5.8:0.2): Rf 0.35. H
NMR (DMSO-d6, 300 MHz): d¼5.30 (s, 2H, CH2), 6.76
(s, 1H, H-2), 7.16 (dd, J¼8.7 and 2.1 Hz, 1H, H-8), 7.32
(d, J¼8.7 Hz, 1H, H-5), 7.81 (d, J¼2.1 Hz, 1H, H-10),
7.92 (d, J¼9.0 Hz, 1H, H-7), 8.09 (d, J¼9.0 Hz, 1H, H-6),
10.21 (s, 1H, OH). 13C NMR (DMSO-d6, 75.4 MHz):
dC¼46.17 (CH2), 108.66 (C-10), 110.67 (C-4b), 113.90
(C-5), 115.77 (C-2), 117.25 (C-8), 125.22 (C-6a), 130.31
(C-6b), 131.33 (C-7), 134.35 (C-6), 152.27 (C-1), 155.35
(C-4a), 157.80 (C-9), 159.38 (C-3). IR (KBr 1%, cmꢁ1):
n¼3287, 2930, 1689, 1624, 1597, 1542, 1467, 1438, 1406,
1363, 1306, 1256, 1234, 1218, 1195, 1138, 1044, 1001,
968, 850, 840, 773, 731, 702. UV/vis (ethanol, nm): lmax
(3)¼361 (12190 Mꢁ1 cmꢁ1). HRMS (EI) calcd for
C14H9O335Cl [M+]: 260.0240; found: 260.0246; calcd for
C14H9O337Cl [M+]: 262.0211; found: 262.0206.
4.2.2. N-[(5-Methoxybenzofuran-1-yl)ethanoyl]valine
methyl ester, Bfm-Val-OMe (6b). The product of the reac-
tion of Bfm-OH (1) (0.103 g, 5.0ꢃ10ꢁ4 mol) with valine
methyl ester hydrochloride (5b) was chromatographed using
chloroform as the eluent to give compound 6b as a yellow oil
(0.113 g, 71%). TLC (chloroform): Rf 0.27. 1H NMR
(CDCl3, 300 MHz): d¼0.73 (d, J¼6.9 Hz, 3H, g-CH3 Val),
0.84 (d, J¼6.9 Hz, 3H, g-CH3 Val), 2.00–2.08 (m, 1H, b-
CH Val), 3.66 (s, 3H, OCH3 Val), 3.77 (s, 2H, CH2), 3.86
(s, 3H, OCH3), 4.52–4.59 (m, 1H, a-CH Val), 6.11 (d,
J¼8.1 Hz, 1H, a-NH Val), 6.91 (dd, J¼2.1 and 8.7 Hz,
1H, H-6), 7.03 (d, J¼2.4 Hz, 1H, H-4), 7.43 (d, J¼8.7 Hz,
1H, H-7), 7.56 (s, 1H, H-2). 13C NMR (CDCl3,
75.4 MHz): dC¼17.62 (g-CH3 Val), 18.88 (g-CH3 Val),
31.09 (b-CH Val), 31.79 (CH2), 52.31 (OCH3 Val), 55.70
(OCH3), 57.10 (a-C Val), 96.12 (C-4), 112.06 (C-6),
4.1.8. 1-Chloromethyl-3-oxo-3H-benzo[f]benzopyran,
Obb-Cl (4c). Starting with 2-naphthol (1.0 g,
6.98ꢃ10ꢁ3 mol) and following the same procedure de-
scribed above for compound 3, using ethyl acetate/n-hexane
3:7, as the chromatography eluent, followed by recrystallisa-
tion from ethyl acetate/n-hexane gave compound 4c as a yel-
low solid (1.22 g, 71%). Mp¼179.5–182.7 ꢀC. TLC (ethyl
acetate/n-hexane, 2:8): Rf 0.36. 1H NMR (CDCl3,
300 MHz): d¼5.09 (s, 2H, CH2), 6.76 (s, 1H, 2-H), 7.52
(d, J¼8.7 Hz, 1H, 5-H), 7.61 (dt, J¼7.6 and 1.0 Hz, 1H, 8-
H), 7.72 (dt, J¼7.6 and 1.5 Hz, 1H, H-9), 7.96 (dd, J¼8.1

9264
A. M. Piloto et al. / Tetrahedron 62 (2006) 9258–9267
113.59 (C-3a), 119.76 (C-7), 120.47 (C-7a), 142.22 (C-2),
156.51 (C-1), 158.45 (C-5), 169.64 (CONH), 172.21
(CO2CH3). IR (KBr 1%, cmꢁ1): n¼3290, 1685, 1641. UV/
vis (ethanol, nm): lmax (3)¼288 (4888 Mꢁ1 cmꢁ1). HRMS
(EI) calcd for C17H21NO5 [M+]: 319.1420; found: 319.1426.
4.2.5. N-[(8-Hydroxynaphtho[2,1-b]furan-1-yl)etha-
noyl]valine methyl ester, Nfh-Val-OMe (7c). The product
of the reaction of Nfh-OH (2b) (0.090 g, 3.71ꢃ10ꢁ4 mol)
with valine methyl ester hydrochloride (5b) was chromato-
graphed using ethyl acetate/n-hexane 3:7, as the eluent to
give compound (7c) as a brown solid (0.075 g, 57%).
Mp¼145.3–145.9 ꢀC. TLC (ethyl acetate/n-hexane, 1:1):
Rf 0.65. 1H NMR (CDCl3, 300 MHz): d¼0.62 (d,
J¼6.9 Hz, 3H, g-CH3 Val), 0.74 (d, J¼6.9 Hz, 3H, g-CH3
Val), 2.00–2.10 (m, 1H, b-CH Val), 3.57 (s, 3H, OCH3
Val), 4.04 (s, 2H, CH2), 4.52–4.62 (m, 1H, a-CH Val),
6.45 (d, J¼8.7 Hz, 1H, a-NH Val), 7.13 (dd, J¼8.7 and
2.4 Hz, 1H, H-7), 7.47 (d, J¼8.7 Hz, 1H, H-4), 7.54 (d,
J¼2.4 Hz, 1H, H-9), 7.66 (d, 1H, J¼9.0 Hz, H-5), 7.69 (s,
1H, H-2), 7.82 (d, J¼8.7 Hz, 1H, H-6), 8.16 (br s, 1H,
OH). 13C NMR (CDCl3, 75.4 MHz): dC¼17.43 (g-CH3
Val), 18.71 (g-CH3 Val), 30.73 (b-CH Val), 33.56 (CH2),
52.27 (OCH3 Val), 57.57 (a-CH Val), 105.78 (C-9), 109.72
(C-4), 114.68 (C-1), 116.20 (C-7), 119.36 (C-3b), 125.29
(C-5a), 126.32 (C-5), 129.21 (C-5b), 130.79 (C-6), 142.74
(C-2), 154.39 (C-3a), 155.67 (C-8), 171.01 (CONH),
172.53 (CO2CH3). IR (KBr 1%, cmꢁ1): n¼3427, 3313,
2961, 2929, 1723, 1658, 1629, 1537, 1468, 1441, 1410,
1387, 1289, 1261, 1219, 1195, 1155, 1136, 1120, 1102,
1032, 994, 847, 834. UV/vis (ethanol, nm): lmax (3)¼300
(17650 Mꢁ1 cmꢁ1). HRMS (EI) calcd for C20H21NO5
[M+]: 355.1420; found: 355.1416.
4.2.3. N-[(8-Methoxynaphtho[2,1-b]furan-1-yl)etha-
noyl]phenylalanine methyl ester, Nfm-Phe-OMe (7a).
The product of the reaction of Nfm-OH (2a) (0.095 g,
3.71ꢃ10ꢁ4 mol) with phenylalanine methyl ester hydro-
chloride (5a) was chromatographed using ethyl acetate/n-
hexane 4:6, as the eluent to give compound 7a as a white
solid (0.111 g, 72%). Mp¼146.3–148.4 ꢀC. TLC (ethyl ace-
1
tate/n-hexane, 4:6): Rf 0.38. H NMR (CDCl3, 300 MHz):
d¼2.80–2.96 (m, 2H, b-CH2 Phe), 3.57 (s, 3H, OCH3
Phe), 3.93 (s, 3H, OCH3), 3.96 (s, 2H, CH2), 4.80–4.90
(m, 1H, a-CH Phe), 6.05 (d, J¼8.1 Hz, 1H, a-NH Phe),
6.47 (br d, J¼7.5 Hz, 2H, H-2 and H-6 Phe), 6.79 (br t,
J¼7.5 Hz, 2H, H-3 and H-5 Phe), 6.96 (br t, J¼7.5 Hz,
1H, H-4 Phe), 7.14 (dd, J¼9.0 and 2.1 Hz, 1H, H-7), 7.48
(d, J¼2.1 Hz, 1H, H-9), 7.52 (d, J¼9.0 Hz, 1H, H-4), 7.62
(s, 1H, H-2), 7.73 (d, J¼9.0 Hz, 1H, H-5), 7.85 (d,
J¼9.0 Hz, 1H, H-6). 13C NMR (CDCl3, 75.4 MHz):
dC¼33.97 (CH2), 37.57 (b-CH2 Phe), 52.15 (OCH3 Phe),
52.87 (a-CH Phe), 55.56 (OCH3), 102.32 (C-9), 109.99
(C-4), 115.04 (C-1), 116.77 (C-7), 120.00 (C-3b), 125.63
(C-5a), 126.26 (C-5), 126.85 (C-4 Phe), 128.13 (C-3 and
C-5 Phe), 128.52 (C-2 and C-6 Phe), 129.41 (C-5b),
130.37 (C-6), 134.90 (C-1 Phe), 142.66 (C-2), 154.44 (C-
3a), 158.65 (C-8), 169.75 (CONH), 171.38 (CO2CH3). IR
(KBr 1%, cmꢁ1): n¼3284, 2958, 2927, 2852, 1750, 1662,
1625, 1543, 1524, 1468, 1431, 1362, 1262, 1224, 1199,
1174, 1099, 1017, 823, 792. UV/vis (ethanol, nm): lmax
(3)¼298 (8261 Mꢁ1 cmꢁ1). HRMS (EI) calcd for
C25H23NO5 [M+]: 417.1576; found: 417.1588.
4.2.6. N-[(Naphtho[2,1-b]furan-1-yl)ethanoyl]valine
methyl ester, Nfu-Val-OMe (7d). The product of the reac-
tion of Nfu-OH (2c) (0.090 g, 3.71ꢃ10ꢁ4 mol) with valine
methyl ester hydrochloride (5b) was chromatographed using
chloroform/n-hexane 9:1, as the eluent to give compound
(7d) as a yellow solid (0.104 g, 83%). Mp¼141.5–
1
143.0 ꢀC. TLC (ethyl acetate/n-hexane, 1:1): Rf 0.85. H
NMR (CDCl3, 300 MHz): d¼0.56 (d, J¼6.9 Hz, 3H, g-
CH3 Val), 0.66 (d, J¼6.9 Hz, 3H, g-CH3 Val), 1.89–2.01
(m, 1H, b-CH Val), 3.51 (s, 3H, OCH3 Val), 4.03 (s, 2H,
CH2), 4.5–4.60 (m, 1H, a-CH Val), 6.24 (d, J¼9.0 Hz, 1H,
a-NH Val), 7.45 (dt, J¼7.2 and 1.0 Hz, 1H, H-8), 7.57 (dt,
J¼7.2 and 1.2 Hz, 1H, H-7), 7.65 (d, J¼9.0 Hz, 1H, H-4),
7.75 (d, J¼9.0 Hz, 1H, H-5), 7.76 (s, 1H, H-2), 7.95 (d,
J¼8.7 Hz, 1H, H-9), 8.16 (d, J¼8.4 Hz, 1H, H-6). 13C
NMR (CDCl3, 75.4 MHz): dC¼17.4 (g-CH3 Val), 18.6 (g-
CH3 Val), 31.0 (b-CH Val), 33.8 (CH2), 51.9 (OCH3), 57.2
(a-CH Val), 112.6 (C-4), 115.3 (C-1), 120.5 (C-3b), 123.0
(C-6), 124.5 (C-8), 126.3 (C-5), 126.7 (C-7), 128.04 (C-
5a), 129.0 (C-9), 130.7 (C-5b), 143.1 (C-2), 153.8 (C-3a),
169.8 (CONH), 171.9 (CO2CH3). IR (KBr 1%, cmꢁ1):
n¼3290, 3279, 2960, 2929, 2852, 1809, 1742, 1658, 1651,
1584, 1538, 1464, 1437, 1388, 1372, 1312, 1266, 1205,
1151, 1107, 1022, 990, 931, 857, 804. UV/vis (ethanol,
nm): lmax (3)¼292 (1758 Mꢁ1 cmꢁ1). HRMS (EI) calcd
for C20H21NO4 [M+]: 339.1471; found: 339.1471.
4.2.4. N-[(8-Methoxynaphtho[2,1-b]furan-1-yl)etha-
noyl]valine methyl ester, Nfm-Val-OMe (7b). The product
of the reaction of Nfm-OH (2a) (0.095 g, 3.71ꢃ10ꢁ4 mol)
with valine methyl ester hydrochloride (5b) was chromato-
graphed using chloroform/n-hexane 9:1, as the eluent to
give compound 7b as a brown solid (0.122 g, 89%).
Mp¼159.0–161.0 ꢀC. TLC (ethyl acetate/n-hexane, 4:6):
Rf 0.48. 1H NMR (CDCl3, 300 MHz): d¼0.54 (d,
J¼6.9 Hz, 3H, g-CH3 Val), 0.63 (3H, d, J¼6.9 Hz, g-CH3
Val), 1.85–2.00 (m, 1H, b-CH Val), 3.48 (s, 3H, OCH3
Val), 4.02 (s, 3H, OCH3), 4.03 (s, 2H, CH2), 4.46–4.56 (m,
1H, a-CH Val), 6.11 (d, J¼8.4 Hz, 1H, a-NH Val), 7.13
(dd, J¼9.0 and 2.4 Hz, 1H, H-7), 7.50 (s, 1H, H-9), 7.52
(d, J¼9.0 Hz, 1H, H-4), 7.70 (d, J¼8.7 Hz, 1H, H-5), 7.74
(s, 1H, H-2), 7.84 (d, J¼9.0 Hz, 1H, H-6). 13C NMR
(CDCl3, 75.4 MHz): dC¼17.47 (g-CH3 Val), 18.55 (g-CH3
Val), 30.91 (b-CH Val), 34.05 (CH2), 51.84 (OCH3 Val),
55.49 (OCH3), 57.27 (a-CH Val), 102.43 (C-9), 110.00
(C-4), 115.23 (C-1), 116.55 (C-7), 119.87 (C-3b), 125.54
(C-5a), 126.24 (C-5), 129.30 (C-5b), 130.34 (C-6), 142.65
(C-2), 155.45 (C-3a), 158.49 (C-8), 170.00 (CONH),
171.77 (CO2CH3). IR (KBr 1%, cmꢁ1): n¼3442, 3295,
3116, 3078, 2998, 2964, 1739, 1658, 1630, 1603, 1545,
1525, 1474, 1432, 1410, 1383, 1358, 1290, 1232, 1202,
1183, 1120, 1038, 1027, 832. UV/vis (ethanol, nm): lmax
(3)¼298 (10,196 Mꢁ1 cmꢁ1). HRMS (EI) calcd for
C21H23NO5 [M+]: 369.1576; found: 369.1560.
4.2.7. N-[(8-Methoxynaphtho[2,1-b]furan-1-yl)ethanoyl]-
alanine methyl ester, Nfm-Ala-OMe (7e). The product
of the reaction of Nfm-OH (2a) (0.104, 4.06ꢃ10ꢁ4 mol)
with alanine methyl ester hydrochloride (5c) (0.054 g,
3.90ꢃ10ꢁ4 mol) was chromatographed using ethyl acetate/
n-hexane 3:7, as the eluent to give compound 7e as a white
solid (0.125 g, 90%). Mp¼190.1–192.0 ꢀC. TLC (ethyl ace-
1
tate/n-hexane, 4:6): Rf 0.32. H NMR (CDCl3, 300 MHz):

A. M. Piloto et al. / Tetrahedron 62 (2006) 9258–9267
9265
d¼1.20 (d, J¼7.2 Hz, 3H, b-CH3 Ala), 3.50 (s, 3H, OCH3
Ala), 3.95 (s, 3H, OCH3), 3.99 (s, 2H, CH2), 4.50–4.65
(m, 1H, a-CH Ala), 6.22 (d, J¼7.5 Hz, 1H, a-NH Ala),
7.14 (dd, J¼9.0 and 2.7 Hz, 1H, H-7), 7.52 (d, J¼9.0 Hz,
2H, H-9 and H-4), 7.66 (d, J¼9.0 Hz, 1H, H-5), 7.72 (s,
1H, H-2), 7.84 (d, J¼9.0 Hz, 1H, H-6). 13C NMR (CDCl3,
75.4 MHz): dC¼18.00 (b-CH3 Ala), 33.90 (CH2), 48.08
(a-CH Ala), 52.16 (OCH3 Ala), 55.49 (OCH3), 102.42 (C-
9), 110.02 (C-4), 115.09 (C-1), 116.55 (C-7), 119.97 (C-
3b), 125.55 (C-5a), 126.19 (C-5), 129.33 (C-5b), 130.32
(C-6), 142.66 (C-2), 154.41 (C-3a), 158.48 (C-8), 169.71
(CONH), 172.74 (CO2CH3). IR (KBr 1%, cmꢁ1): n¼3435,
3299, 2961, 2926, 1741, 1654, 1630, 1601, 1543, 1475,
1454, 1407, 1384, 1359, 1261, 1232, 1201, 1185, 1154,
1021, 829, 802. UV/vis (ethanol, nm): lmax (3)¼298
(6456 Mꢁ1 cmꢁ1). HRMS (EI) calcd for C19H19NO5 [M+]:
341.1263; found: 341.1265.
(C-6), 142.75 (C-2), 154.43 (C-3a), 158.45 (C-8), 170.00
(CONH), 170.58 (CO2CH3 Asp, main chain), 170.70
(CO2CH3 Asp, side-chain). IR (KBr 1%, cmꢁ1): n¼3333,
2954, 2924, 2854, 1739, 1648, 1627, 1598, 1519, 1463,
1436, 1400, 1378, 1304, 1228, 1200, 1178, 1120, 1107,
1060, 1040, 1024. UV/vis (ethanol, nm): lmax (3)¼298
(7856 Mꢁ1 cmꢁ1). HRMS (EI) calcd for C21H21NO7 [M+]:
399.1318; found: 399.1319.
4.2.10. N-[(8-Methoxynaphtho[2,1-b]furan-1-yl)etha-
noyl]glutamic acid dimethyl ester, Nfm-Glu(OMe)-
OMe (7h). The product of the reaction of Nfm-OH (2a)
(0.10 g, 3.90ꢃ10ꢁ4 mol) with glutamic acid dimethyl ester
hydrochloride (5f) was chromatographed using ethyl ace-
tate/n-hexane 2:8, as the eluent to give compound 7h as
a white solid (0.160 g, 98%). Mp¼166.3–169.0 ꢀC. TLC
1
(ethyl acetate/n-hexane, 7:3): Rf 0.48. H NMR (CDCl3,
300 MHz): d¼1.60–1.80 (m, 2H, b-CH2 Glu), 1.82–2.00
(m, 2H, g-CH2 Glu), 3.49 (s, 3H, OCH3 Glu), 3.50 (s, 3H,
OCH3 Glu), 3.94 (s, 3H, OCH3), 3.99 (s, 2H, CH2), 4.52–
4.64 (m, 1H, a-CH Glu), 6.42 (d, J¼7.8 Hz, 1H, a-NH
Glu), 7.12 (dd, J¼8.9 and 2.4 Hz, 1H, H-7), 7.44–7.55 (m,
2H, H-9 and H-4), 7.65–7.75 (m, 2H, H-5 and H-2), 7.82
(d, J¼9.0 Hz, 1H, H-6). 13C NMR (CDCl3, 75.4 MHz):
dC¼26.63 (b-CH2 Glu), 29.34 (g-CH2 Glu), 33.93 (CH2),
51.58 (OCH3 Glu, main chain), 51.72 (a-CH Glu), 52.23
(OCH3 Glu, side-chain), 55.46 (OCH3), 102.28 (C-9),
110.02 (C-4), 115.00 (C-1), 116.52 (C-7), 119.94 (C-3b),
125.53 (C-5a), 126.19 (C-5), 129.28 (C-5b), 130.35 (C-6),
142.74 (C-2), 154.43 (C-3a), 158.55 (C-8), 170.31
(CONH), 171.61 (CO2CH3 Glu, main chain), 172.86
(CO2CH3 Glu, side-chain). IR (Nujol, cmꢁ1): n¼3311,
2954, 2925, 2854, 1761, 1715, 1651, 1628, 1531, 1463,
1377, 1271, 1230, 1177, 1134, 1120, 1035, 1018, 829.
UV/vis (ethanol, nm): lmax (3)¼298 (6716 Mꢁ1 cmꢁ1).
HRMS (EI) calcd for C22H23NO7 [M+]: 413.1475; found:
413.1476.
4.2.8. N-[(8-Methoxynaphtho[2,1-b]furan-1-yl)etha-
noyl]glycine methyl ester, Nfm-Gly-OMe (7f). The
product of the reaction of Nfm-OH (2a) (0.177 g, 6.91ꢃ
10ꢁ4 mol) with glycine methyl ester hydrochloride (5d)
was chromatographed using ethyl acetate/n-hexane 3:7, as
the eluent to give compound 7f as a white solid (0.215 g,
95%). Mp¼171.9–174.0 ꢀC. TLC (ethyl acetate/n-hexane,
1
7:3): Rf 0.46. H NMR (CDCl3, 300 MHz): d¼3.57 (s, 3H,
OCH3 Gly), 3.96 (s, 3H, OCH3), 3.97 (d, J¼4.5 Hz, 2H,
CH2 Gly), 4.04 (s, 2H, CH2), 6.18 (br s, 1H, a-NH Gly),
7.15 (dd, J¼9.0 and 2.4 Hz, 1H, H-7), 7.50–7.55 (m, 2H,
H-4 and H-9), 7.70 (d, J¼8.7 Hz, 1H, H-5), 7.74 (s, 1H,
H-2), 7.85 (d, J¼9.0 Hz, 1H, H-6). 13C NMR (CDCl3,
75.4 MHz): dC¼33.74 (CH2), 41.27 (CH2 Gly), 52.12
(OCH3 Gly), 55.51 (OCH3), 102.37 (C-9), 110.01 (C-4),
115.01 (C-1), 116.61 (C-7), 119.96 (C-3b), 125.53 (C-5a),
126.23 (C-5), 129.33 (C-5b), 130.34 (C-6), 142.75 (C-2),
154.40 (C-3a), 158.55 (C-8), 169.73 (CONH), 170.45
(CO2CH3). IR (Nujol, cmꢁ1): n¼3280, 2954, 2924, 2854,
1741, 1652, 1628, 1556, 1522, 1463, 1435, 1413, 1377,
1272, 1259, 1247, 1228, 1197, 1176, 826. UV/vis (ethanol,
nm): lmax (3)¼297 (7965 Mꢁ1 cmꢁ1). HRMS (EI) calcd
for C18H17NO5 [M+]: 327.1107; found: 327.1100.
4.2.11. N-Acetyl-u-[(8-methoxynaphtho[2,1-b]furan-1-
yl)ethanoyl]lysine methyl ester, Ac-Lys(Nfm)-OMe (9).
The product of the reaction of Nfm-OH (2a) (0.10 g,
3.90ꢃ10ꢁ4 mol) with N-acetyl-lysine methyl ester hydro-
chloride (8) was chromatographed using ethyl acetate/
n-hexane 3:7, as the eluent to give compound 9 as a white
solid (0.122 g, 71%). Mp¼185.7–186.9 ꢀC. TLC (chloro-
form/methanol, 5.8:0.2): Rf 0.46. 1H NMR (CDCl3,
300 MHz): d¼0.95–1.15 (m, 2H, g-CH2 Lys), 1.20–1.50
(m, 4H, b-CH2 Lys and d-CH2 Lys), 1.97 (s, 3H, CH3 Ac),
3.00–3.60 (m, 2H, 3-CH2 Lys), 3.69 (s, 3H, OCH3 Lys),
3.96 (s, 5H, OCH3 and CH2), 4.35–4.45 (m, 1H, a-CH
Lys), 5.82 (t, J¼6.0 Hz, 1H, NH Lys, side-chain), 5.95 (d,
J¼7.8 Hz, 1H, a-NH Lys), 7.15 (dd, J¼9.0 and 2.4 Hz,
1H, H-7), 7.49 (d, J¼2.4 Hz, 1H, H-9), 7.52 (d, J¼9.0 Hz,
1H, H-4), 7.70 (s, 1H, H-2), 7.72 (d, J¼9.0 Hz, 1H, H-5),
7.85 (d, J¼9.0 Hz, 1H, H-6). 13C NMR (CDCl3,
75.4 MHz): dC¼22.23 (g-CH2 Lys), 23.00 (CH3 Ac),
28.84 (b-CH2 Lys), 31.65 (d-CH2 Lys), 33.97 (CH2), 39.07
(3-CH2 Lys), 51.73 (a-CH Lys), 52.28 (OCH3 Lys), 55.57
(OCH3), 102.50 (C-9), 110.05 (C-4), 115.36 (C-1), 116.55
(C-7), 119.98 (C-3b), 125.52 (C-5a), 126.25 (C-5), 129.32
(C-5b), 130.40 (C-6), 142.72 (C-2), 154.41 (C-3a), 158.58
(C-8), 169.90 (CONH Ac), 170.32 (CONH), 172.84
(CO2CH3). IR (KBr 1%, cmꢁ1): n¼3289, 3077, 2951,
4.2.9. N-[(8-Methoxynaphtho[2,1-b]furan-1-yl)ethanoyl]-
aspartic acid dimethyl ester, Nfm-Asp(OMe)-OMe (7g).
The product of the reaction of Nfm-OH (2a) (0.10 g,
3.90ꢃ10ꢁ4) with aspartic acid dimethyl ester hydrochloride
(5e) (0.193 g, 9.75ꢃ10ꢁ4 mol) was chromatographed using
ethyl acetate/n-hexane 4:6, as the eluent to give compound
7g as a white solid (0.153 g; 98%). Mp¼136.8–137.7 ꢀC.
TLC (ethyl acetate/n-hexane, 6:4): Rf 0.46. 1H NMR
(CDCl3, 300 MHz): d¼2.60–2.70 (m, 1H, b-CH Asp),
2.80–2.90 (m, 1H, b-CH Asp), 3.15 (s, 3H, OCH3 Asp,
side-chain), 3.49 (s, 3H, OCH3 Asp, main chain), 3.95 (s,
3H, OCH3), 4.00 (s, 2H, CH2), 4.78–4.87 (m, 1H, a-CH
Asp), 6.69 (d, J¼7.5 Hz, 1H, a-NH Asp), 7.13 (dd,
J¼2.40 and 8.9 Hz, 1H, H-7), 7.45 (d, J¼2.4 Hz, 1H, H-
9), 7.50 (d, J¼9.0 Hz, 1H, H-4), 7.66 (d, J¼8.7 Hz, 1H, H-
5), 7.72 (s, 1H, H-2), 7.82 (d, J¼9.0 Hz, 1H, H-6). 13C
NMR (CDCl3, 75.4 MHz): dC¼33.86 (CH2), 35.60 (b-CH2
Asp), 48.45 (a-CH Asp), 51.49 (OCH3 Asp, side-chain),
52.50 (OCH3 Asp, main chain), 55.48 (OCH3), 102.34 (C-
9), 110.12 (C-4), 114.91 (C-1), 116.47 (C-7), 119.90 (C-
3b), 125.51 (C-5a), 126.06 (C-5), 129.30 (C-5b), 130.28

9266
A. M. Piloto et al. / Tetrahedron 62 (2006) 9258–9267
2931, 2859, 1748, 1651, 1633, 1601, 1550, 1525, 1474,
1464, 1454, 1434, 1415, 1375, 1359, 1290, 1258, 1231,
1200, 1180, 1146, 830. UV/vis (ethanol, nm): lmax
(3)¼297 (5333 Mꢁ1 cmꢁ1). HRMS (EI) calcd for
C24H28N2O6 [M+]: 440.1947; found: 440.1948.
catalyst (0.014 g) and refluxed for 7 h with stirring. The
catalyst was filtered off and washed with methanol; the
combined liquids were then evaporated under reduced pres-
sure affording the compound as a white solid (3.8ꢃ10ꢁ2 g,
95%). 1H NMR was well compared with the starting
material.
4.2.12. N-tert-Butyloxycarbonyl-O-[(5-methoxybenzo-
furan-1-yl)ethanoyl]serine methyl ester, Boc-Ser(Bfm)-
OMe (11). The product of the reaction of Bfm-OH (1)
(0.087 g, 4.22ꢃ10ꢁ4 mol) with N-tert-butyloxycarbonyl-
serine methyl ester (10) was chromatographed using chloro-
form as the eluent to give compound 11 as a brown oil
(0.111 g, 65%). TLC (chloroform): Rf 0.23. 1H NMR
(CDCl3, 300 MHz): d¼1.43 (s, 9H, C(CH3)3), 3.63–3.67
(m, 5H, CH2 and OCH3), 3.70 (s, 3H, OCH3 Ser), 3.83 (s,
3H, OCH3), 4.35–4.53 (m, 2H, b-CH2 Ser), 4.54–4.57 (m,
1H, a-CH Ser), 5.25 (d, J¼7.5 Hz, 1H, a-NH Ser), 6.89
(dd, J¼2.4 and 8.4 Hz, 1H, H-6), 6.99 (d, J¼2.4 Hz, 1H,
H-4), 7.39 (d, J¼8.4 Hz, 1H, H-7), 7.50 (s, 1H, H-2). 13C
NMR (CDCl3, 75.4 MHz): dC¼28.16 (C(CH3)3), 29.47
(CH2), 64.68 (b-CH2 Ser), 52.59 (OCH3 Ser), 52.74 (a-CH
Ser), 55.60 (OCH3), 80.25 (C(CH3)3), 95.94 (C-4), 111.83
(C-6), 112.45 (C-3a), 119.62 (C-7), 120.71 (C-7a), 141.87
(C-2), 155.02 (CO2C(CH3)3), 156.13 (C-3a), 158.15 (C-5),
170.04 (CO2CH3), 171.10 (CH2CO2). IR (KBr 1%, cmꢁ1):
n¼3293, 3044, 3030, 3000, 2946, 1752, 1733, 1660, 1629,
1612, 1597, 1554, 1498, 1450. UV/vis (ethanol, nm): lmax
(3)¼287 (5427 Mꢁ1 cmꢁ1).
4.3.2. Acidolysis with hydrochloric acid. To the fully pro-
tected amino acid Nfm-Phe-OMe (7a) (2.01ꢃ10ꢁ2 g,
4.36ꢃ10ꢁ5 mol) was added 6 M HCl (0.20 mL) under rapid
stirring over 1 h. Evaporation under reduced pressure gave
1
a white solid (2.01ꢃ10ꢁ2 g, 100%). H NMR confirmed
the structure of the compound.
4.3.3. Acidolysis with trifluoroacetic acid. To the fully pro-
tected amino acid Nfm-Phe-OMe (7a) (2.2ꢃ10ꢁ2 g,
5.27ꢃ10ꢁ5 mol) was added 0.74 mL of trifluoroacetic acid
under rapid stirring over 5 h. Evaporation under reduced
1
pressure gave a white solid (2.2ꢃ10ꢁ2 g, 100%). H NMR
confirmed the structure of the compound.
4.3.4. Aminolysis. A solution of Nfm-Phe-OMe (7a)
(2.0ꢃ10ꢁ2 g, 4.79ꢃ10ꢁ5 mol) in dry acetonitrile was treated
with DEAEA (4.1ꢃ10ꢁ2 mL, 2.87ꢃ10ꢁ4 mol) for 27 h ac-
cording to the procedure of Grehn et al.27 The product was
purified by flash chromatography, using chloroform/n-hex-
ane 6:1, as the eluent, to give the compound as an oily solid
1
(1.7ꢃ10ꢁ2 g, 85%). H NMR was well compared with the
starting material.
4.2.13. N-tert-Butyloxycarbonyl-O-[(8-methoxynaph-
tho[2,1-b]furan-1-yl)ethanoyl]serine methyl ester, Boc-
Ser(Nfm)-OMe (12). The product of the reaction of Nfm-
OH (2a) (0.095 g, 3.71ꢃ10ꢁ4 mol) with N-tert-butyloxycar-
bonylserine methyl ester (10) methyl ester hydrochloride
was chromatographed using ethyl acetate/n-hexane 2:8, as
the eluent to give compound 12 as an off-white oil
(0.092 g, 54%). TLC (ethyl acetate/n-hexane, 4:6): Rf 0.47.
1H NMR (CDCl3, 300 MHz): d¼1.42 (s, 9H, C(CH3)3),
3.45 (s, 3H, OCH3 Ser), 3.99 (s, 3H, OCH3), 4.07 (s, 2H,
CH2), 4.40–4.48 (m, 2H, b-CH2 Ser), 4.50–4.60 (m, 1H,
a-CH Ser), 5.18 (d, J¼7.5 Hz, 1H, a-NH Ser), 7.16 (dd,
J¼8.9 and 2.7 Hz, 1H, H-7), 7.51 (d, J¼9.0 Hz, 1H, H-4),
7.58 (d, J¼2.7 Hz, 1H, H-9), 7.67 (d, J¼9.0 Hz, 1H, H-5),
7.69 (s, 1H, H-2), 7.86 (d, J¼9.0 Hz, 1H, H-6). 13C NMR
(CDCl3, 75.4 MHz): dC¼28.20 (C(CH3)3), 31.73 (CH2),
52.42 (OCH3 Ser), 52.78 (a-CH Ser), 55.42 (OCH3), 65.06
(b-CH2 Ser), 80.31 (C(CH3)3), 103.08 (C-9), 110.22 (C-4),
114.23 (C-1), 115.65 (C-7), 120.15 (C-3b), 125.70 (C-5a),
125.77 (C-5), 129.36 (C-5b), 130.51 (C-6), 142.45 (C-2),
153.99 (C-3a), 155.07 (CO2C(CH3)3), 158.27 (C-8),
169.73 (CO2CH3), 170.43 (CH2CO2). IR (KBr 1%, cmꢁ1):
n¼3376, 2977, 2839, 1745, 1720, 1707, 1629, 1598, 1519,
1505, 1474, 1436, 1367, 1232, 1161, 1121, 1060, 1022,
950, 876, 830. UV/vis (ethanol, nm): lmax (3)¼298
(12705 Mꢁ1 cmꢁ1). HRMS (EI) calcd for C24H27NO8
[M+]: 457.1737; found: 457.1736.
4.3.5. Reduction with Mg/MeOH. To a solution of Nfm-
Phe-OMe (7a) (2.0ꢃ10ꢁ2 g, 4.33ꢃ10ꢁ5 mol) in dry metha-
nol (2 mL) magnesium powder (1.0ꢃ10ꢁ2 g, 4.11ꢃ
10ꢁ5 mol) was added and the resulting mixture was soni-
cated for 2.30 h, at room temperature. More magnesium
was added (3.2ꢃ10ꢁ2 g, 1.32ꢃ10ꢁ5 mol), in small portions
(1.0ꢃ10ꢁ2 g each) and the resulting mixture was sonicated
for another 7 h. The reaction mixture was quenched by the
addition of saturated aqueous NH4Cl (4 mL) and extracted
with ethyl acetate. The organic layer was dried with
MgSO4, concentrated to dryness to give an off-white oil
1
(1.9ꢃ10ꢁ2 g, 95%). H NMR was well compared with the
starting material.
4.3.6. Alkaline hydrolysis. To the fully protected amino
acid Nfm-Phe-OMe (7a) (2.0ꢃ10ꢁ2 g, 4.79ꢃ10ꢁ5 mol) in
1,4-dioxane (2 mL), 1 M NaOH (7.2ꢃ10ꢁ2 mL, 7.19ꢃ
10ꢁ5 mol) was added at low temperature. The solution was
stirred at 0 ꢀC for 5 h and acidified to pH 3 with 1 M
KHSO4. After extraction with ethyl acetate and evaporation
of the solvent, Nfm-Phe-OH (13) was obtained as an orange
solid (0.019 g, 100%). Mp¼191.0–193.0 ꢀC. 1H NMR
(CDCl3, 300 MHz): d¼2.80–2.90 (m, 2H, b-CH2 Phe),
3.87 (s, 3H, OCH3), 3.95 (s, 2H, CH2), 4.80–4.90 (m, 1H,
a-CH Phe), 6.12 (d, J¼7.8 Hz, 1H, a-NH Phe), 6.48 (d,
J¼6.9 Hz, 2H, H-2 and H-6 Phe), 6.73 (t, J¼7.5 Hz, 2H,
H-3 and H-5 Phe), 6.91 (t, J¼7.5 Hz, 1H, H-4 Phe), 7.13
(dd, J¼9.3 and 2.4 Hz, 1H, H-7), 7.41 (d, J¼2.4 Hz, 1H,
H-9), 7.52 (d, J¼9.0 Hz, 1H, H-4), 7.58 (s, 1H, H-2), 7.72
(d, J¼8.7 Hz, 1H, H-5), 7.85 (d, J¼9.0 Hz, 1H, H-6). 13C
NMR (CDCl3, 75.4 MHz): dC¼33.72 (CH2), 36.92 (b-CH2
Phe), 52.91 (a-CH Phe), 55.51 (OCH3), 102.20 (C-9),
109.99 (C-4), 114.63 (C-1), 116.80 (C-7), 119.84 (C-3b),
4.3. Stability tests with Nfm-Phe-OMe (7a)
4.3.1. Catalytic hydrogenation. A suspension of Nfm-
Phe-OMe (7a) (4.0ꢃ10ꢁ2 g, 9.58ꢃ10ꢁ5 mol) in methanol
(1.0 mL) and 1,4-cyclohexadiene (9.6ꢃ10ꢁ2 mL, 2.58ꢃ
10ꢁ4 mol) was mixed with 10% palladium on charcoal

A. M. Piloto et al. / Tetrahedron 62 (2006) 9258–9267
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~
We thank the Fundac¸ao para a Ciencia e Tecnologia (Portu-
gal) for financial support to the Centro de Quımica (Univer-
sidade do Minho).
^
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