Stereoselective syntheses of fluorescent non-natural aromatic amino acids based on asymmetric michael additions

Article abstract of DOI:10.3390/12051170

Four fluorescent non-natural aromatic amino acids have been synthesized based on a key stereoselective Michael addition reaction. S-1-Phenylethylamine was employed as both the source of amine and the stereoselectivity controller. The overall yields were m

Full text of DOI:10.3390/12051170

Molecules 2007, 12, 1170-1182
molecules
ISSN 1420-3049
© 2007 by MDPI
www.mdpi.org/molecules
Full Paper
Stereoselective Syntheses of Fluorescent Non-Natural Aromatic
Amino Acids Based on Asymmetric Michael Additions
Heru Chen *, Xianbin Zhong and Jin Wei
Department of Chemistry, School of Sciences, Shantou University, Shantou, Guangdong 515063,
P. R. China
* Author to whom correspondence should be addressed; e-mail: hrchen@stu.edu.cn;
Tel. (+86) 754 2902772; Fax (+86) 754 2902767
Received: 10 May 2007; in revised form: 24 May 2007 / Accepted: 25 May 2007 / Published: 26 May
2007
Abstract: Four fluorescent non-natural aromatic amino acids have been synthesized based
on a key stereoselective Michael addition reaction. S-1-Phenylethylamine was employed as
both the source of amine and the stereoselectivity controller. The overall yields were
moderate (30-50%). Fluorescent properties of some of the fluorophores were also
investigated. It was found that compounds with a dimethylamino group bonded to the
aromatic ring display intramolecular charge transfer fluorescence.
Keywords: Aladan, fluorescent non-natural amino acids, asymmetric Michael addition,
stereoselective synthesis.
Introduction
Due to the development of the nonsense (stop codon) suppression technique, it has been possible to
site-specifically incorporate any non-natural amino acid into almost any protein [1]. One of the
significant features of this progress is that it offers a tool of almost limitless potential, promising
chemical biologists systematic protein structure/function studies of great precision [2]. The
development of fluorescent non-natural amino acids relates to this purpose. This new type of amino
acid could play both the roles of structural moiety and intrinsic fluorescent probe when incorporated

Molecules 2007, 12
1171
into target bioactive molecules. Consequently, much attention has been focused on the synthesis of
these novel amino acids. As an example, β-[6’-(N,N-dimethyl)amino-2’-naphthoyl]alanine [aladan
(Ald)] was designed and first synthesized by Cohen’s group and was further incorporated site
selectively at buried and exposed sites in the B1 domain of protein G [3]. We are aware of several
additional applications of aladan [4-5], including the one recently reported by our group [6].
Cohen’s group’s first synthesis of aladan [3] was based on a core reaction sequence involving
stereoselective condensation of 2-(α-iodoacetyl)-6-dimethylaminonaphthalene with N-(diphenyl-
methylene)glycine tert-butyl ester using O-(9)-allyl-N-(9-anthracenylmethyl) cinchonidinium bromide
as both phase transfer catalyst and stereocontrol device. However, several drawbacks were noted when
we repeated that synthesis. First, 2-(α-iodoacetyl)-6-dimethylaminonaphthalene was sensitive to light,
moisture and temperature, which is certainly inconvenient, especially in the workup step. The second
was that in order to guarantee the stereoselectivity, more than 20 mol% of the phase transfer catalyst
had to be used and a long reaction time (>24 hrs) at a very low temperature (-78 °C) avoiding light was
required. Moreover, usage of 1,2-dithioethanol as auxiliary reagent to deprotect the diphenylmethylene
group can generate environmental problems. Finally, the overall yield was not high (only 30%).
Consequently, we began to conceive a new scheme to prepare the title compounds (Scheme 1).
Moreover, this scheme has also been applied to the synthesis of several other non-natural amino acids.
Results and Discussion
Synthesis of non-natural fluorescent amino acids
As displayed in Scheme 1, steps (a) and (c) followed the same procedure as described in our
previous work [6]. In step (c), E-6'-(N,N-dimethylamino)-2'-naphthoylacryl acid was prepared via an
aldol reaction using a solution of tetramethylamine hydroxide in methanol as base and then
immediately dehydrated. The yield in this step was moderate (52%).
Scheme 1. New synthesis of aladan.
O
O
b
a
82%
50%
H3CO
N
H3CO
O
CH3
Ph
COOH
O
NH
d
c
52%
COOH
62%
N
N
2a
1a
3a
O
NH2
COOH
e
82%
N
(a) acetyl chloride, nitrobenzene, 0°C → r. t., 24 h; (b) 5 eq Li/HN(CH3)2, dry benzene/HMPA, r.t., 24 h;
(c) OHCCOOH, 20 wt% (CH3)4N+OH- in methanol, 0 °C → r. t.; followed by refluxing in p-
TosOH/benzene; (d) S-1-phenylethylamine, ethanol, 50-60 °C, 16 h; (e) 6N HBr, reflux, 12 h.

Molecules 2007, 12
1172
Step (d) is the key reaction of our new scheme. S-1-Phenylethylamine is used both as the amine
source of aladan and the stereocontroller. The reaction was carried out at a temperature of 50 to 60 °C
for 16 hours. The title compound, (αS,1S)-α-[N-(1-methyl)benzyl]-β-[(6’-dimethylamino)-2’-
naphthoyl]alanine (2a), began to precipitate from hot ethanol one hour later; as pointed out by Yamada
et al. [7], this dynamic preference was an important factor to resolve the (αS,1S) isomer and made it
the major product. The workup, in which only filtration and washing with cold ethanol are used, was
quite simple. Analytical results from RP-HPLC and HPLC on a chiral column (Chirex 3005,
Phenomenex) shown that purity and de value of the title compound was quite satisfactory (Table 2).
The yield was 62.0%. Removal of N-(1-methyl)benzyl was carried out in satisfactory yield by
refluxing the α-amino protected compound in 6N HBr. The absolute configuration of compound 3a
was confirmed as S by comparing the results of RP-HPLC and chiral HPLC analysis with those of the
standard sample prepared by Cohen’s method [3]. Scheme 1 has also been successfully applied to
synthesize several other non-natural amino acids (3b ~ 3d, Figure 1). The yields were moderate.
Figure 1.
O
NH2
COOH
O
NH2
COOH
O
NH2
COOH
N
HO
HO
3c
3d
3b
Efforts have also been made to prepare E-acryl acids 1a~1d by direct Friedel-Crafts reactions using
maleic anhydride as the starting material under anhydrous AlCl3 catalysis. Unfortunately, only
compound 1c could be successfully prepared by this method using 1,1,2,2-tetrachloroethane as solvent.
The yield was 60.5%.
Table 1 displays the results for synthesizing compounds 2a~2d via asymmetric Michael reaction. It
was shown that the yields for compounds 2b and 2c were higher than those for compounds 2a and 2d.
The reasons might be that with a dimethylamino group replacing the methoxyl group in the
corresponding site of E-acryl acid substrate, the nucleophilicity for Michael addition reaction was
lowered down due to the stronger combination of dimethylamino group to aluminum chloride.
The S-configuration of compound 2c was explained by Yamada et al. [7]. Therefore, the S
configurations of 2b and 2d were confirmed by a reasoned comparison of 2b with 2a, and 2d with 2c,
based on combined results of reaction mechanism, RP-HPLC and chiral HPLC analysis.
The four free non-natural fluorescent amino acids were easily turned into the corresponding
N-Fmoc protected derivatives by the interaction of 9-fluorenylmethyl chloroformate at room
temperature. The products were designated as 4a to 4d, respectively, according to the above order. The
yields were high, which lay between 82 to 93%. The N-acetyl amides are prepared followed a solid
phase synthesis assay. An amide type resins, TentaGel S RAM resins were used. The resulting
derivatives were therefore named 5a to 5d (Table 2).

Molecules 2007, 12
1173
Table 1. Data of yield, purity, and de values for (αS, 1S)-α-[N-(1-methyl)benzyl]alanine
derivatives 2a ~ 2d prepared via asymmetric Michael reactions *.
Yield
Entry
2a
Structure
Purity (%) de value (%)
(%)
CH3
Ph
O
NH
62
98
95
COOH
N
CH3
O
NH
Ph
76
86
99
99
96
97
2b
2c
COOH
H3CO
H3CO
N
CH3
O
NH
COOH
CH3
O
NH
COOH
63
97
95
2d
*calculation of yield was based on the amounts of precipitate; calculation of the purity and de
values were based on results from both RP-HPLC and chiral HPLC, respectively.
Fluorescence studies
Investigation of the steady-state fluorescence for the synthetic compounds had been carried out. As
shown in Table 2 and Figure 2, when the methoxyl group in 6-methoxyl-2-acetylnaphthalene was
replaced with a dimethylamino group, the maximum UV absorption and maximum fluorescent
emission wavelength red-shifted 50 and 78 nm, respectively. This means that dimethylamino group is a
better electron-donor group than a methoxyl group; it can affect the energy distribution of the
molecular orbital to a larger degree. On the other hand, the quantum yield of acedan (6-dimethylamino-
2-acetylnaphthalene) was almost three times lower that that of 6-methoxyl-2-acetylnaphthalene. This
phenomenon was ascribed to the fact that the dimethylamino moiety can increase the rate of
nonradiative decay (knr).
Quite interestingly, it was found that all three compounds, p-dimethylaminoacetopheneone, Ac-
Ald-NH2 and Ac-Alb-NH2 (β-[4-(N, N-dimethyl)aminobenzoyl]alanine, Alb), displayed intramolecular
charge transfer dual fluorescence in Tris-HCl buffer solution. Compared to a similar fluorophore,

Molecules 2007, 12
1174
p-dimethylaminobenzoylamide, which displayed strong charge transfer (CT) fluorescence at 480 nm
and a weak locally excited (LE) fluorescence at 350 nm (an experiment in which acetonitrile was used
as solvent) [8-10]; whilst in the case of p-dimethylaminoacetopheneone, it displayed a strong CT
fluorescence at 414 nm and a weak LE fluorescence at 554 nm.
When acedan was converted into Ac-Ald-NH2, the UV maximum absorption wavelength was
almost the same (from 361 nm to 364 nm), but the fluorescent maximum emission wavelength red-
shifted 15 nm. In the meantime, the quantum yield decreased by about two-fold and a new LE
fluorescence at 440 nm appeared. This revealed that the introduction of a glycyl moiety into acedan
increased the possibility of nonradiative decay. A same phenomenon was found when a glycyl moiety
was introduced into p-dimethylaminoacetophenone.
Table 2. Fluorescence spectroscopic parameters for some compounds in
Tris-HCl (20 mmol/L, pH 6.6, 20 °C) (λexc = 350 nm).
Abs
Em
Compd. structure
φ
λ
max (nm)
λ
max (nm)
O
311
361
444
522
0.392
MeO
O
0.143
0.112
0.069
N
O
301
364
414(s), 554(w)
440(w), 539(s)
N
O
NHAc
CONH2
N
5a
O
NHAc
CONH2
316
281
456
403
0.106
0.076
HO
5b
NHAc
O
CONH2
HO
5c
5d
O
NHAc
CONH2
305
432(s), 572(w)
0.053
N

Molecules 2007, 12
1175
Figure 2. Fluorescence spectra of four N-acetyl amides.
10000
5a
9000
8000
7000
6000
5000
4000
3000
2000
1000
0
5b
5c
5d
250 300 350 400 450 500 550 600 650 700 750
Wavelength (nm)
Determinations were carried out in Tris-HCl (20 mmol/L, pH
6.6, 20 °C) (λexc = 350 nm)
Conclusions
In summary, an efficient and simple alternative for the preparation of aladan and three other non-
natural fluorescent amino acids via asymmetric Michael addition using S-1-phenylethylamine was
reported. Prominent among the advantages of this new method are operational simplicity, inexpensive
reagents, shorter reaction time, higher overall yield, high stereoselectivity and easily workup. It was
disclosed that acedan, p-dimethylaminoacetophenone, Ac-Ald-NH2, Ac-Alb-NH2 display intra-
molecular charge transfer fluorescence.
Experimental
General
All chemicals were purchased from Aldrich Chemical Co. Nitrobenzene was freshly distilled from
4Å molecular sieve; Benzene, HMPA, and 1,1,2,-tetrachloroethane were dried over 4Å molecular
sieve; THF was freshly distilled from sodium granules. Other reagents were used as supplied. Melting
1
points were determined on an Electrothermal melting point apparatus. Both H-NMR and 13C-NMR
were acquired on Varian VXG-400 S equipment. Mass spectra was recorded on either VG-MM-1212
or MS 902/ZAB instruments. Optical rotations were measured on a Perkin Elmer Polarimeter 343
(PerkinElmer INC., USA). HPLC was carried on a Varian LC Star system (Varian Associates Inc.,
USA); The column used for reversed-phase analysis was purchase from Vydac (Hesperia, CA 92345,
USA), size: 0.46×25 cm packed with 5 µm C18 modified silica gel; the chiral column was available
from Phenomenex (Torrance, CA, USA), model: Chirex (R)-NGLY and DNB; size: 0.46×25 cm.
Method used for RP-HPLC analysis: isocratic using pH 2.5 aqueous phosphate buffer/acetonitrile (8/2,

Molecules 2007, 12
1176
V/V); flow rate: 0.5 mL/min; wavelength: 280 nm; Method used for chiral HPLC: isocratic using 0.03
mol/L NH4Ac in methanol; flow rate: 0.5 ml/min; wavelength: 280 nm.
Fluorescence determinations
The determinations were carried out on a Gemini EM instrument (Molecular Devices Corporation,
Sunnyvale, USA). The excitation wavelength was 350 nm, the cutoff was 420 nm, and the step was 2
nm. The spectral resolution was 2 nm for excitation and for emission. Solutions of sample compounds
and reference amino acid in Tris/HCl buffer (pH 6.6) at a concentration of 2×10-5 mol/L were used.
Fluorescence quantum yields (φ) were determined with N-acetyl-L-tryptophanamide (NATA) (φNATA =
0.14) as reference. The quantum yield was calculated based on the following equation:

2
nS
ES A
R 
ER AS


=
φS φR

nR


where the subscripts S and R refer to the sample and reference compound (NATA), respectively. E is
the integrated area under the corrected emission spectrum. A is the absorbance of the solution at the
excitation wavelength (A < 0.05) and (nS/nR)2 is the correction for the refractive index.
Synthesis of 6-methoxy-2-acetylnaphthalene
After following a procedure described in literature [6] the title compound was separated and
purified by flash liquid chromatography (ethyl acetate/n-hexane, 1:4, v/v). The fraction corresponding
to a Rf value of 0.42 was collected and further purified by crystallization (n-hexane) to give 12.6 g
1
(50%) of the title compound as colorless plates. Mp 106-108°C (lit. [11] 104-105°C); H-NMR
(CDCl3, 400 MHz) δ 8.39 (d, 1H, J=1.5 Hz), 8.0 (dd, 1H, J=8.6, 1.8 Hz), 7.85 (d, 1H, J=8.8 Hz), 7.76
13
(d, 1H, J=8.6 Hz), 7.2 (dd, 1H, J=8.9, 2.5 Hz), 7.15 (d, 1H, J=2.4 Hz), 3.95 (s, 3H), 2.7 (s, 6H); C-
NMR (100.6 MHz, CDCl3) δ 198.1, 160.0, 137.5, 132.8, 131.3, 130.3, 128.0, 127.3, 124.8, 119.9,
105.9, 55.6, 26.8.
Synthesis of 6-N,N-dimethyamino-2-acetylnaphthalene (acedan)
A literature procedure was followed [6]. The title compound was separated and purified by flash liquid
chromatography (ethyl acetate/n-hexane, 3:7, v/v). The fraction with a Rf value of 0.49 was then further
purified by crystallization (n-hexane/chloroform) to give 4.4 g (82%) of the title compound as needle-
like crystals; mp 154-156°C; 1H-NMR (400 MHz, CDCl3) δ 8.3 (s, 1H), 7.92 (dd, 1H, J=8.7, 1.8 Hz),
7.79 (d, 1H, J=9.2 Hz), 7.63 (d, 1H, J=8.6 Hz), 7.17 (d, 1H, J=6.2 Hz), 6.9 (s, 1H), 3.1 (s, 6H), 2.66 (s,
3H); 13C-NMR (106 MHz, CDCl3) δ 197.9, 150.0, 137.8, 131.2, 131.0, 130.6, 126.5, 125.2, 124.8,
116.5,105.8, 40.8, 26.7.
Synthesis of 4’-methoxyacetophenone
Anhydrous powdered AlCl3 (20 g, 0.15 mol) and acetyl chloride (10.7 mL, 0.15 mol) were
suspended in dried 1,1,2,2-tetrachloroethane (100 mL). The suspension was cooled down to 0 °C in an

Molecules 2007, 12
1177
ice bath, then anisole (10.8 mL, 0.1 mol) was added via syringe. The mixture was stirred at 0 °C for
another 4 hours and then warmed up to room temperature and stirred overnight. The reaction was
terminated by pouring the mixture into a solution of 4 M hydrochloric acid (100 mL) mixed with
cracked ice. The suspension was then extracted with ethyl acetate (3×50 mL). All the extracts were
combined and washed with water (3×20 mL) and brine (2×20 mL), respectively. The organic layer was
then dried over anhydrous MgSO4. After filtration and removal of ethyl acetate, the residue was
purified by flash liquid chromatography (ethyl acetate/n-hexane, 3:7, v/v). The fraction with an Rf value
of 0.51 was collected to give 13.6 g (91%) of the target compound as oil, which gradually solidified.
1
Mp 37-39 °C (lit. [12] 36-38 °C); H-NMR (400 MHz, CDCl3) δ 7.91 (dd, 2H, J = 1.5, 9.0 Hz), 6.90
(dd, 2H, J = 1.5, 9.0 Hz), 3.84 (s, 3H), 2.53 (s, 3H); 13C-NMR (106 MHz, CDCl3) δ 197.0, 163.7,
130.8, 130.5, 113.9, 44.8, 25.6.
Synthesis of 4’-(N, N-dimethyl)aminoacetophenone
The same procedure described for the synthesis of acedan was used employing 4’-methoxy-
acetophenone (3.42 g, 22.8 mmol). The crude residue was separated and purified by flash liquid
chromatography (ethyl acetate/n-hexane, 3:7, v/v). The fraction with Rf 0.45 was collected to give
1
1.213 g (33%) of the title compound as a light yellow solid. Mp 103-105 °C; H-NMR (400 MHz,
CDCl3) δ 7.89 (d, 2H, J = 8.6 Hz), 6.68 (d, 2H, J = 8.8 Hz), 3.06 (s, 6H), 2.51 (s, 3H); 13C-NMR (106
MHz, CDCl3) δ 196.6, 153.4, 130.7, 111.2, 40.5, 26.2.
Typical experimental procedure for the synthesis of E-acryl acids derivatives via an aldol reaction
Aryl 2-acetyl acetone (10 mmol) and glyoxylic acid monohydrate (0.92 g, 10 mmol) were stirred in
dried methanol and 20 wt% tetramethylammonium hydroxide in methanol (5.8 mL, 11 mmol) was
added via syringe. The temperature gradually rose to room temperature and the mixture was stirred for
24 h. The reaction mixture was then poured into 2 M aqueous H3PO4 solution (20 mL) mixed with
cracked ice. The mixture was concentrated to a volume of about 20 mL under reduced pressure at 39
°C. The residues were then extracted with chloroform (3×30 mL). All the extracts were combined and
washed with water (3×10 mL) and brine (2×10 mL). The organic layer was then dried over anhydrous
MgSO4. Removal of chloroform was followed by reduced pressure evaporation at room temperature.
To the residue p-toluenesulfonic acid (0.2 g, 1 mmol) and benzene (50 mL) were added, the flask was
equipped with a Dean-Stark trap and the solution was refluxed for 4 h, then the benzene was removed
by rotatory evaporation under reduced pressure at r.t. The residues was dissolved in chloroform (50
mL) and washed with water (3×10 mL) and brine (2×10 mL). The chloroform layer was then dried
over anhydrous MgSO4. After removal of chloroform, the residues were purified by liquid
chromatography (EtOAc/n-hexane/HOAc, 60/38/2, v/v/v).
6’-(N,N-Dimethylamino)-2’-naphthoylacryl acid (1a). Rf = 0.63; deep red solid; yield: 52%; Mp. 144-
146 °C; 1H-NMR (400 MHz, DMSO-d6) δ 8.60 (d, 1H, J = 1.5 Hz), 8.08 (d, 1H, J = 15.6 Hz), 7.97 (d,
1H, J = 9.2 Hz), 7.88 (m, 1H), 7.71 (m, 1H), 7.28 (m, 1H), 6.95 (m, 1H), 6.69 (d, 1H, J = 15.6 Hz),
3.06 (s, 6H); 13C-NMR (106 MHz, DMSO-d6) δ 188.2, 167.3, 151.3, 138.4, 137.1, 132.5, 131.9, 130.0,

Molecules 2007, 12
1178
127.2, 126.6, 125.0, 124.8, 117.1, 106.9, 40.7; FAB-MS m/z: calcd. for C16H14NO3 for [M-H]+ 268.11;
found 268.1; daughter peak: 224, 209, 196, 181.
6’-Methoxy-2’-naphthoylacryl acid (1b). Rf = 0.64; yellow solid; yield: 70%; Mp. 135-136 °C; 1H-
NMR (400 MHz, DMSO-d6) δ 8.75 (d, 1H, J = 1.5 Hz), 7.88-8.11 (m, 4H), 7.38-7.42 (m, 1H), 7.22-
13
7.28 (m, 1H), 6.72 (d, 1H, J = 15.6 Hz), 3.9 (s, 3H); C-NMR (106 MHz, DMSO-d6) δ 198.3, 189.2,
176.4, 167.1, 160.6, 160.1, 136.9, 133.2, 132.4, 128.3, 128.1, 125.0, 120.4, 106.9, 56.2; FAB-MS m/z:
calcd. for C15H13O4 ([M+H]+) 257.07, found 257.1; daughter peak: 211.07, 183.07, 159.07, 144.05.
1
p-Methoxybenzoylacrylic acid (1c). Rf = 0.5; white solid; yield: 66%; Mp. 126-128 °C; H-NMR (400
13
MHz, CDCl3) δ 8.03 (m, 3H), 7.01 (m, 2H), 6.86 (d, 1H, J = 15.6 Hz), 3.90 (s, 3H); C-NMR (106
MHz, CDCl3) δ 187.6, 171.0, 164.6, 138.8, 131.7, 131.0, 129.7, 114.5, 55.9; FAB-MS m/z: calcd. for
C11H10O4 ([M+H]+) 207.19, found 207.17.
1
4’-(N, N-Dimethyl)benzoylacryl acid (1d). Rf = 0.43; golden solid; yield: 63%; Mp. 146-148 °C; H-
NMR (400 MHz, DMSO-d6) δ 7.89 (m, 2H), 7.68 (m, 1H), 6.75 (m, 2H), 6.60 (d, 1H, J = 15.4 Hz),
3.03 (s, 6H); 13C-NMR (106 MHz, DMSO-d6) δ 186.1, 167.4, 154.5, 137.3, 131.7, 131.6, 130.8,
124.4,111.7, 40.5; FAB-MS m/z: calcd. for C12H13NO3 ([M+H]+) 219.24, found 219.21; daughter peak:
174.1, 146.1.
Typical experimental procedure for the synthesis of compounds 2a~2d by asymmetric Michael
addition.
To a vigorously stirred solution of trans-acrylic acid 1a~1d (2.0 mmol) in ethanol (60 mL) at 50 to
60 °C, S-1-phenylethylamine (0.28 mL, 2.2 mmol) was added dropwise via syringe. After the addition
was complete, the mixture was stirred at the same temperature for 24 h. Precipitation was first
observed ca. 1 h into the reaction and increased as time went on. The mixture was then cooled down to
room temperature, and the precipitates were collected by filtration and washed with cold ethanol. The
precipitates were quite pure, judged from both RP-HPLC and chiral HPLC analytical results. Further
purification by re-crystallization from acetonitrile/aqueous phosphate buffer (pH 2.5) was also
performed.
(αS,1S)-α-[N-(1-Methyl)benzyl]-β
-[(6’-dimethylamino)-2’-naphthoyl]alanine (2a). 1H-NMR (400
MHz, DMSO-d6/TFA) δ 8.43 (d, 1H, J=1.5 Hz,), 7.90 (d, J = 9.3 Hz, 1H), 7.80 (dd, 1H, J=8.8, 1.8 Hz)
7.68 (d, 1H, J=8.8 Hz), 7.40-7.54 (m, 5H), 7.28 (dd, 1H, J=9.2, 2.5 Hz), 6.97 (d, 1H, J=2.4 Hz), 4.58
(m, 1H), 3.93 (m, 1H), 3.72 (m, 2H), 3.06 (s, 6H), 1.61 (d, 3H, J=6.8 Hz); 13C-NMR (100.6 MHz,
DMSO-d6/TFA) δ 194.6, 170.5, 150.8, 138.2, 137.0, 131.5, 131.3, 129.9, 129.7, 128.6, 126.9, 125.2,
124.4, 117.3, 114.4, 105.8, 58.7, 53.5, 40.5, 20.6; FAB-MS m/z: calcd. for C24H27N2O3 ([M+H]+)
20
D
391.19, found 391.21; daughter peaks: 287, 214, 198, 172, 105;
[α
]
= +94.7 ° (c = 0.21,
methanol/0.1 mol/L H2SO4, 3/1, v/v).

Molecules 2007, 12
S,1S)- -[N-(1-Methyl)benzyl]-
1179
(α
α
β
-[(6’-methoxy)-2’-naphthoyl]alanine (2b). 1H-NMR (400 MHz,
DMSO-d6/TFA) δ 8.57 (s, 1H), 8.01 (d, 1H, J=9.2 Hz), 7.90 (m, 2H), 7.52 (d, 1H, J=8.0 Hz), 7.42 (m,
5H), 7.24 (m, 1H), 4.58 (m, 1H), 3.89 (s, 1H), 3.88 (s, 3H), 3.68 (m, 2H), 1.62 (d, 3H, J=6.8 Hz); 13C-
NMR (100.6 MHz, DMSO-d6/TFA) δ 195.2, 170.4, 160.4.9, 137.9, 136.9, 131.9, 131.1, 129.9, 129.7,
128.6, 128.0, 127.8, 124.6, 120.4, 120.1, 117.2, 114.3, 111.4, 106.6, 58.7, 55.9, 53.4, 20.5; FAB-MS
m/z: calcd. for C23H24NO4 ([M+H]+) 378.16, found 378.2; daughter peaks: 274, 201, 185, 159, 105;
20
D
[α
]
= +85.5 ° (c = 0.47, methanol/0.1 mol/L H2SO4, 3/1, v/v).
1
(αS,1S)-α-[N-(1-Methyl)benzyl]-β-[(4’-methoxy)benzoyl]alanine (2c). H-NMR (400 MHz, DMSO-
d6/TFA) δ 7.92 (dd, 2H, J=1.6, 8.8 Hz), 7.40-7.51 (m, 5H), 7.03 (dd, 2H, J=1.6, 8.8 Hz), 4.59 (m, 1H),
3.89 (m, 1H), 3.82 (s, 3H), 3.61-3.46 (m, 2H), 1.60 (d, 3H, J = 6.6 Hz); 13C-NMR (100.6 MHz,
DMSO-d6/TFA) δ 194.0, 170.4, 164.4, 136.9, 131.2, 129.9, 129.7, 128.8, 128.6, 120.1, 117.2, 114.6,
114.3, 111.4, 58.7, 56.1, 53.4, 20.5; FAB-MS m/z: calcd. for C19H22NO4 ([M+H]+) 328.15; found
20
D
328.2;
[α
]
= +63.9 ° (c = 0.49, methanol/0.1 mol/L H2SO4, 3/1, v/v)
(αS,1S)-
α-[N-(1-Methyl)benzyl]-β
-[(4’-dimethylamino)benzoyl]alanine (2d). 1H-NMR (400 MHz,
DMSO-d6/TFA) δ 7.75 (d, 2H, J=9.0 Hz), 7.38-7.50 (m, 5H), 6.68 (d, 2H, J=9.0 Hz), 4.59 (m, 1H),
3.82 (s, 1H), 3.41 (m, 2H), 2.98 (s, 6H), 1.59 (d, 3H, J=6.9 Hz); 13C-NMR (100.6 MHz, DMSO-
d6/TFA) δ 192.7, 170.5, 154.3, 136.9, 130.8, 129.9, 129.7, 128.5, 123.2, 120.0, 117.1, 114.3, 111.4,
58.7, 53.6, 40.4, 20.5; m/z (FAB-MS): calcd. for C20H25N2O3 ([M+H]+) 341.18; found 341.15;
daughter peak: 237, 220, 164, 148, 122, 105, 88, 74;
H2SO4, 3/1, V/V)
[α]
20 = +113.2° (c=0.28, methanol/0.1 mol/L
D
Typical deprotection procedure
To α-[N-(1-S-methyl)benzyl]alanine derivatives 2a~2d (2 mmol) in a 100 mL flask, 6 N aqueous
HBr solution (30 mL) was added. The flask was wrapped with aluminum foil. Under stirring, the
mixture was kept refluxing for 12 h. After cooling down, the mixture was concentrated to sticky oil
under reduced pressure at 45 °C and cold water (50 mL) was added. The precipitates were collected by
filtration and washed carefully with cold water, acetone and ether and then further purified by
crystallization using hot water.
(αS)-β
-[6’-(N,N-Dimethyl)amino-2’-naphthoyl]alanine (3a). Yield: 0.61 g (82%); 1H-NMR (400 MHz,
D2O+DCl) δ 8.68 (s, 2H), 8.19 (d, 1H, J=8.1 Hz), 7.96 (s, 2H), 7.78 (d, 1H, J=6.8 Hz), 4.35 (m, 1H),
3.84 (m, 2H), 3.16 (s, 6H); FAB-MS m/z: calcd. for C16H19N2O3 ([M+H]+) 287.33; found 287.1.
[α
]
20 = -42.3° (c=0.23, 1 mol/L HCl).
D
(α
S)-β
-(6’-Hydroxy-2’-naphthoyl)alanine (3b). Yield: 0.59 g (87%); 1H-NMR (400 MHz, D2O+DCl) δ
8.68 (s, 1H), 8.13 (d, 1H, J= 9.2 Hz), 8.02 (m, 2H), 7.63 (d, 1H, J=8.0 Hz,), 7.35 (m, 1H), 4.35 (m,
1H), 3.73 (m, 2H); FAB-MS m/z: calcd. for C14H14NO4 ([M+H]+) 260.08; found 260.10.
[α]
20 = -35.3°
D
(c=0.45, 1 mol/L HCl).

Molecules 2007, 12
1180
1
(
α
S)-
8.02 (dd, 2H, J=1.6, 8.8 Hz), 7.13 (dd, 2H, J=1.6, 8.8 Hz), 4.36 (m, 1H), 3.71 (m, 2H); FAB-MS m/z:
calcd. for C10H11NO4 ([M+H]+) 210.71; found 210.65. 20 = -17.6° (c=0.5, 1 mol/L HCl).
β-[(4’-Hydroxy)benzoyl]alanine (3c). Yield: 0.52 g (91%); H-NMR (400 MHz, D2O+DCl) δ
[ ]
α
D
(αS)-β
-[(4’-Dimethylamino)benzoyl]alanine (3d). Yield: 0.53 g (83%); 1H-NMR (400 MHz,
D2O+DCl) δ 7.86 (d, 2H, J=9.0 Hz), 6.89 (d, 2H, J=9.0 Hz), 4.36 (m, 1H), 3.52 (m, 2H), 3.11 (s, 6H);
FAB-MS m/z: calcd. for C10H12NO4 ([M+H]+) 237.12; found 237.05.
[α]
20 = -25.3° (c=0.52, 1 mol/L
D
HCl).
Typical experimental Fmoc-proctection procedure
To the above free amino acid HBr salt (2.0 mmol) dissolved in DMF (20 mL), aqueous Na2CO3
solution (10 mL, 0.53 g, 5.0 mmol) was added. The mixture was cooled down to 0 °C with an ice-
water bath. 9-Fluorenylmethyl chloroformate (0.62 g, 2.4 mmol) was added in small portions under
stirring in the ice-water bath for 4 hr. The solution was then warmed up to room temperature and
stirred for another 8 hr. After the end of reaction, the mixture was poured into solution of crushed ice
and water (100 mL) and concentrated hydrochloric acid was added slowly until the pH value was
around 1∼2. The mixture was extracted with chloroform (150 mL). The organics was washed with
water (2×30 mL) and brine, respectively, and dried over anhydrous MgSO4. After removal of solvent
by rotary evaporation, the residues were separated and purified by flash liquid chromatography with
ethyl acetate in chloroform (3:7, v/v).
α-N-Fmoc-(αS)-β-[6’-(N,N-Dimethyl)amino-2’-naphthoyl]alanine (4a). Rf = 0.62. Yield: 0.84 g (83%);
1H-NMR (400 MHz, CDCl3) δ 8.28 (s, 1H), 7.84 (br, 1H), 7.68 (m, 3H), 7.55 (m, 3H), 7.52-7.12 (m,
5H), 6.84 (s, 1H), 6.10 (s, 1H), 4.84 (br, 1H), 4.35-4.16 (m, 3H), 3.88 (m, 1H), 3.62 (m, 1H), 3.09 (s,
6H); 13C-NMR (106 MHz, CDCl3) δ 163.3, 156.5, 150.6, 144.2, 144.0, 141.4, 138.2, 131.2, 130.9,
129.6, 127.8, 127.3, 126.5, 125.5, 125.1, 124.5, 120.0, 116.4, 105.4, 67.4, 55.2, 52.3, 47.3, 36.9; FAB-
MS: calcd. C31H28N2O5 508.55, found 508.2.
1
α
-N-Fmoc-(αS)-
β
-(6’-Hydroxy-2’-naphthoyl)alanine (4b). Rf = 0.57; Yield: 0.86 g (89%); H-NMR
(400 MHz, CDCl3) δ 8.28 (s, 1H), 8.02 (s, 1H), 7.91-7.72 (m, 4H), 7.55 (m, 2H), 7.42-7.23 (m, 6H),
13
7.09 (m, 1H), 7.06 (s, 1H), 4.94 (br, 1H), 4.65-4.46 (m, 3H), 3.28 (m, 1H), 3.12 (m, 1H); C-NMR
(106 MHz, CDCl3) δ 165.6, 158.2, 156.5, 150.6, 145.1, 142.3, 138.2, 131.8, 130.6, 128.9, 127.0, 126.8,
126.5, 125.5, 118.4, 109.4, 66.8, 56.2, 50.9, 46.1; FAB-MS: calcd. C29H23NO6 481.5, found 482.3.
1
α
-N-Fmoc-(αS)-
β
-[(4’-Hydroxy)benzoyl]alanine (4c). Rf = 0.55; Yield: 0.79 g (92%); H-NMR (400
MHz, CDCl3) δ 7.91-7.8 (m, 4H), 7.53 (s, 2H), 7.41-7.23 (m, 5H), 6.86 (m, 2H), 5.04 (br, 1H), 4.71-
4.36 (m, 3H), 3.08 (m, 1H), 2.93 (m, 1H); 13C-NMR (106 MHz, CDCl3) δ 175.6, 168.2, 166.5, 158.6,
144.2, 142.7, 130.8, 128.9, 128.3, 126.8, 116.4, 66.8, 55.2, 51.4, 44.9; FAB-MS: calcd. C25H21NO6
431.44, found 432.5.

Molecules 2007, 12
-N-Fmoc-( S)-
-[(4’-Dimethylamino)benzoyl]alanine (4d). Rf = 0.60; Yield: 0.81 g (88%); 1H-NMR
1181
α
α
β
(400 MHz, CDCl3) δ 7.88-7.81 (m, 4H), 7.57 (m, 2H), 7.45-7.12 (m, 5H), 6.94 (s, 2H), 4.96 (br, 1H),
13
4.42-4.08 (m, 3H), 3.18 (m, 1H), 2.89 (m, 1H), 3.02 (s, 6H); C-NMR (106 MHz, CDCl3) δ 173.3,
168.5, 162.6, 154.2, 142.8, 141.4, 129.7, 128.6, 126.5, 126.2, 114.45, 68.2, 55.6, 50.7, 46.3, 38.9;
FAB-MS: calcd. C27H26N2O5 458.51, found 458.6.
Typical experimental procedure for the synthesis of N-acetylated aminoamides
N-acetylated and carboxamidated Ald, Alb and other two amino acids were synthesized on a
TentaGel S RAM resin (Sigma-Aldrich) respectively. After removal of the Fmoc group on the resins,
the N-Fmoc-protected amino acids were attached to the resin respectively by using PyBOP/HOBt as
coupling reagents. Fmoc deprotection, acetylation and cleavage from the resin by TFA/anisole
treatment yielded the title compounds. Crude residues were purified by preparative reversed-phase
HPLC using a linear gradient of 20-65% MeOH in 0.1% TFA over 25 min, respectively.
N-Ac-(αS)-β
-[6’-(N, N-Dimethyl)amino-2’-naphthoyl]alanyl amide (5a). 1H-NMR (400 MHz, DMSO-
d6) δ 8.43 (s, 1H), 8.06 (s, 1H), 7.9 (d, 9.2, 1H), 7.8 (d, 7.1, 1H), 7.66 (d, 8.8, 1H), 7.23 (s, 2H), 7.06
13
(d, 7.9, 1H), 6.94 (s, 1H), 4.70 (m, 1H), 3.25 (m, 1H), 2.93 (m, 1H), 3.05 (s, 6H), 1.79 (s, 3H); C-
NMR (100.6 MHz, DMSO-d6) δ 196.6, 174.0, 169.9, 150.9, 137.9, 131.4, 130.3, 129.5, 126.6, 125.2,
124.5, 117.1, 105.4, 49.7, 40.8, 39.5, 23.2; FAB-MS m/z: calcd. for C18H22N3O3 ([M+H]+) 328.16;
found 328.13.
1
N-Ac-(αS)-
β
-(6’-Hydroxy-2’-naphthoyl)alanyl amide (5b). H-NMR (400 MHz, DMSO-d6) δ 9.85 (s,
1H), 8.53 (s, 1H), 8.24 (s, 1H), 8.09 (d, 8.5, 1H), 7.92 (d, 6.9, 1H), 7.83 (d, 8.5, 1H), 7.26 (s, 2H), 7.09
13
(s, 1H), 7.06 (s, 1H), 5.11 (m, 1H), 3.25 (m, 1H), 2.96 (m, 1H), 1.86 (s, 3H); C-NMR (100.6 MHz,
DMSO-d6) δ 196.6, 174.0, 169.9, 159.9, 134.5, 131.6, 131.4, 130.7, 127.7, 126.2, 125.5, 118.6, 108.4,
50.7, 45.8, 23.2; FAB-MS m/z: calcd. for C16H17N2O4 ([M+H]+) 301.31; found 301.33.
N-Ac-(αS)-β
-[(4’-Hydroxy)benzoyl]alanyl amide (5c). 1H-NMR (400 MHz, DMSO-d6) δ 9.58 (s, 1H),
8.42 (s, 1H), 7.91 (d, 9.0, 2H), 7.24 (s, 2H), 6.78 (d, 9.0, 2H), 4.86 (m, 1H), 3.62 (m, 2H), 1.96 (s, 3H);
13C- NMR (100.6 MHz, DMSO-d6) δ 192.8, 174.2, 170.1, 162.6, 130.4, 129.5, 117.3, 51.5, 45.6, 22.8;
FAB-MS m/z: calcd. for C12H14N2O4 ([M+H]+) 251.25; found 251.23.
1
N-Ac-(αS)-
β
-[(4’-dimethylamino)benzoyl]alanyl amide (5d). H-NMR (400 MHz, DMSO-d6) δ 8.33
(s, 1H), 7.86 (d, 9.0, 2H), 7.21 (s, 2H), 6.89 (d, 9.0, 2H), 4.36 (m, 1H), 3.52 (m, 2H), 3.11 (s, 6H), 1.86
(s, 3H); 13C-NMR (100.6 MHz, DMSO-d6) δ 192.8, 174.2, 170.1, 154.3, 129.9, 125.2, 114.3, 51.2,
44.6, 39.7, 22.8; FAB-MS m/z: calcd. for C14H20N3O3 ([M+H]+) 278.14; found 278.11.
Acknowledgements
The authors would like to thank the financial support from the Accented Project for Natural
Scientific Research of Universities in Guangdong Province (No. 132112) and National Scientific

Molecules 2007, 12
1182
Foundation of China (No. 30672560). The author would like to express heartfelt thanks to Prof. Dr.
Peter W. Schiller for his kindness and very useful advice in this work. Thanks are also due to Prof. Dr.
Bogumil Zelent for his help in part of fluorescence determinations.
References and Notes
1. (a) Cornish, V. W.; Schultz, P. G. A New Tool for Studymg Protein Structure and Function. Curr.
Opin. Struct. Biol. 1994, 4, 601-607; (b) Cornish, V. W.; Mendel, D.; Schultz, P. G. Probing
Protein Structure and Function with an Expanded Genetic Code. Angew. Chem. Int. Ed. Engl.
1995, 34, 621-633; (c) Gillmore, M. A.; Steward L. E.; Chamberlin, A. R. Incorporation of
Noncoded Amino Acids by In Vitro Protein Biosynthesis. Topics Curr. Chem. 1999, 202, 77-99.
2. Dougherty, D. A. Unnatural Amino Acids as Probes of Protein Structure and Function. Curr. Opin.
Chem. Biol. 2000, 4, 645-652.
3. Cohen, B. E.; McAnaney, T. B.; Park, E. S.; Jan, Y. N.; Boxer, S. G.; Jan, L. Y. Probing Protein
Electrostatics with a Synthetic Fluorescent Amino Acid. Science 2002, 296, 1700-1703.
4. Vazquez, M. E.; Nitz, M.; Stehn, J.; Yaffe, M. B.; Imperiali, B. Fluorescent Caged Phosphoserine
Peptides as Probes to Investigate Phosphorylation-Dependent Protein Association. J. Am. Chem.
Soc. 2003, 125, 10150-1015.
5. Chen, H.; Chung, N. N.; Lemieux, C.; Zelent, B.; Vanderkooi, J. M.; Gryczynski, I.; Wilkes, B.
C.; Schiller, P. W. [Aladan3]TIPP: A Fluorescent δ Opioid Antagonist with High δ Receptor
Binding Affinity and δ Selectivity. Biopolymers (Pept. Sci.) 2005, 80, 325-331.
6. Chen, H.; Guo, X.; Zhong, X. Syntheses of Aladan and [Ald6] loloatin C and Study on their
Fluorescent Properties, Chin. J. Chem. 2006, 24, 1411-1417.
7. Yamada, M.; Nagashima, N.; hasegawa, J.; Takahashi, S. A highly efficient asymmetric synthesis
of methoxyhomophenylalanine using michael addition of phenethylamine. Tetrahedron Lett. 1998,
39(49), 9019-9022.
8. Wu, F. Y.; Zhang, X.; Jiang, Y. B. Recognition of Anion by Dual Fluorescent Sensor Based on the
Intramolecular Charge Transfer in p-Dimethylaminobenzoylhydrazine. Chem. J. Chin. Univ. 2003,
24, 1990-1992.
9. Wu, F. Y.; Jiang, Y. B. p-Dimethylaminobenzamide as an ICT Dual Fluorescent Neutral Receptor
for Anions under Proton Coupled Electron Transfer Sensing Mechanism Chem. Phys. Lett. 2002,
355, 438-444.
10. Braun D.; Rettig W.; Delmond S.; Létard, J. –F.; Lapouyade, R. Amide Derivative of DMABN :
A New Class of Dual Fluorescent Compound. J. Phys Chem. A 1997, 101, 6836-6841.
11. Haworth, R. D.; Sheldrich, G. The constituents of guaiacum resin. Part II. Synthesis of dl-
Guaiaretic Acid Dimethyl ether. J. Chem. Soc. 1934, 1423-1429.
12. Aldrich Chemical Index: 4’-methoxyacetophenone, m. p. 36-38 °C; b. p. 152-154 °C/26 mmHg
Sample Availability: Contact the authors.
© 2007 by MDPI (http://www.mdpi.org). Reproduction is permitted for noncommercial purposes.