Chromophoric azo reagents for amino acid and peptide labelling

Article abstract of DOI:10.1002/ejoc.200300692

Four carboxylic azo dyes, with spectroscopic absorption peaks ranging from 400 to 500 nm, are presented as new markers for amino acid and peptide labelling at their N-terminus. Labelling can also be performed at side-chain residues as is exemplified with lysine and serine. Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2004.

Full text of DOI:10.1002/ejoc.200300692

FULL PAPER
Chromophoric Azo Reagents for Amino Acid and Peptide Labelling
[a]
[a]
[a]
˜
Susana M. B. Fraga, Maria S. T. Goncalves,* Joao C. V. P. Moura, and
¸
Kavitha Rani[a]
Keywords: Amino acids / Azo dyes / Non-fluorescence / Peptides
Four carboxylic azo dyes, with spectroscopic absorption
peaks ranging from 400 to 500 nm, are presented as new
markers for amino acid and peptide labelling at their N-ter-
minus. Labelling can also be performed at side-chain res-
idues as is exemplified with lysine and serine.
( Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim,
Germany, 2004)
Introduction
zole[17] and quinoline[18] azo dyes have been suggested for
the study of dye-protein interactions and sequence analysis
The use of dyes in chemistry, biology, and medicine is of genes by Resonance Raman spectroscopy.
growing continuously, with many new applications in the
Another application of non-fluorescent dyes in peptide
diagnosis and treatment of disease.[1Ϫ4] In the last two dec- chemistry is the synthesis of colored peptide libraries lab-
ades, the sensitive detection, identification, and quantifi- eled with monocarboxylic blue anthraquinone and red azo
cation of amino acids has been achieved through simple dyes.[19,20]
and fast analytical methods. Although fluorometric meth-
With this in mind, and following on from our previous
ods are potentially much more sensitive than colorimetric work with a non-fluorescent dye as a temporary marker in
methods, the use of non-fluorescent dyes for quantitative peptide chemistry,[21,22] we decided to develop and test
and qualitative analyses can offer some advantages. First, some new markers with spectroscopic absorption peaks
histochemical applications often require reliable, sensitive, ranging from 400 to 500 nm for amino acid and peptide
and stable detection of targets in complex samples that may labelling.
have significant background from either the sample’s natu-
ral auto fluorescence or fluorescence created during sample
preparation. This problem can be avoided by the use of
non-fluorescent dyes. Second, the need for ultra violet radi-
Results and Discussion
ation for excitation of fluorescent dyes implies more expens-
ive equipment than chromophoric methods. Examples of
applications of non-fluorescent dyes include the use of am-
ino acid N-derivatizing groups such as 4-[(N,N-dimethylam-
inophenyl)-4Ј-diazenyl]phenyl isothiocyanate for quantitat-
ive analysis,[5,6] 5-formyl-1H-pyrrole-2-carboxylic acid,
which can be colored on demand by treatment with hydro-
cinnamoyl chloride,[7,8] and 4-[(N,N-dimethylaminophenyl)-
4Ј-diazenyl]benzenesulfonyl chloride (DABS-Cl) for amino
acid analyses by HPLC,[9Ϫ11] and capillary[12] and polyacryl-
amide gel[13,14] electrophoresis.
A method for staining proteins prior to polyacrylamide
gel electrophoresis with Remazol Brilliant Blue R[15] and
Drimarene Brilliant Blue,[16] two reactive dyes containing
an ethyl sulfone and a difluorochloropyrimidyl group,
respectively, has been described. Non-fluorescent benzotria-
The new chromophores used (Scheme 1) were obtained
by diazotization of 3-aminophenylacetic acid, 2-amino-4-
thiazoleacetic acid, and 3- and 4-aminobenzoic acid and
coupling of the resulting diazonium salt to N,N-dimeth-
ylaniline (1, 2) or to β-naphthol (3, 4).[23] The carboxylic
azo dyes (1Ϫ4) were bonded to the α-amine group of vari-
ous amino acid esters by coupling them with the aid of
dicyclohexylcarbodiimide (DCC) assisted by hydroxybenzo-
triazole (HOBt) under standard conditions. After purifi-
cation by chromatography (dry or flash) on silica gel, fol-
lowed by recrystallization, the corresponding acetyl azo de-
rivatives (5, 6) were obtained; the benzoyl azo derivatives
(7, 8) were isolated by precipitation from the reaction mix-
ture with water and recrystallized from acetone. All labeled
amino acids (5Ϫ8) were obtained as solid materials in yields
ranging from 56 to 99% (Table 1) and were characterized
by elemental analysis, and by NMR (1H and 13C), IR, and
visible spectroscopy. The visible spectra of compounds 5
show λmax values of 409 (5d) and 446 nm (5b), with ε values
of 31644 and 17282, respectively. The thiazole ring, com-
[a]
Department of Chemistry, University of Minho,
Gualtar, 4710-057 Braga, Portugal
Fax: (internat.) ϩ351-253678983
E-mail: msameiro@quimica.uminho.pt
1750
 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
DOI: 10.1002/ejoc.200300692
Eur. J. Org. Chem. 2004, 1750Ϫ1760

Chromophoric Azo Reagents for Amino Acid and Peptide Labelling
FULL PAPER
Scheme 1. Synthesis of labeled amino acid derivatives (5Ϫ8)
pared to the benzene ring, produces a bathochromic shift, racemization of 50% when the benzoyl azo derivative was
as exemplified by compound 6d where λmax is 495 nm. used.[22] As a result this marker represents a potential im-
When β-naphthol was used as the coupling component in- provement with respect to benzoyl azo derivatives in step-
stead of N,N-dimethylaniline, the resulting amino acid es- wise syntheses.
ters (7, 8) showed absorption peaks at 475 and 480 nm,
respectively.
Table 2. Yields obtained in the synthesis of labeled dipeptides 12,
14, and 16
Table 1. Yields obtained in the synthesis of labeled amino acid
derivatives (5Ϫ10)
Compound
Yield (%)
Ddp-Ala-Phe-OtBu (12a)
Ddp-Val-Phe-OtBu (12b)
Ddp-Phe-Val-OtBu (12c)
Ddp-Phe-Gly-OMe (14a)
Ddp-Phe-Ile-OMe (14b)
Ddp-Phe-Val-OMe (14c)
Ddp-Phe-Ala-OMe (14d)
Z-Lys(ω-Ddp)-Phe-OEt (16c)
Z-Lys(ω-Ddp)-Ala-OMe (16d)
84
66
94
81
65
60
84
91
98
Product (compound no.)
Yield (%)
Ddp-Gly-OMe (5a)
Ddp-Ile-OMe (5b)
Ddp-Phe-OEt (5c)
Ddp-Ala-OMe (5d)
Ddt-Gly-OMe (6a)
Ddt-Ile-OMe (6b)
Ddt-Phe-OEt (6c)
Mnf-Gly-OMe (7a)
Mnf-Ile-OMe (7b)
Mnf-Phe-OEt (7c)
Pnf-Gly-OMe (8a)
Pnf-Ile-OMe (8b)
77
79
91
71
85
75
77
59
56
58
75
99
76
70
86
Pnf-Phe-OEt (8c)
Z-Lys(ω-Ddp)-OMe (9)
Boc-Ser(Ddp)-OMe (10)
Following the same method (DCC/HOBt), colored pep-
tides 12 were obtained by acylation at their N-terminus
with chromophore 1 (Scheme 2, Table 2). Furthermore, di-
peptides 14 were obtained in yields of between 60% and
84% by reacting labeled phenylalanine derivative 13 with
several amino acid esters (Scheme 3, Table 2). 1H NMR
spectroscopy suggested that the racemization was as much
as 5% (see Figures 1 and 2). Previous work has shown a
Scheme 3. Synthesis of labeled dipeptides 14
In addition to labelling amino acids or peptides at their
N-terminus, an alternative acylation at a ω-amine group
was also investigated. Thus, the methyl ester of N-benzyl-
oxycarbonyl lysine was treated with 1 (Scheme 4) under the
conditions reported above, and the product (9) saponified
quantitatively to the C-deprotected amino acid 15, which
was then coupled with alanine and phenylalanine esters to
yield labeled dipeptides 16 (Table 2).
Another approach for side-chain labelling was under-
taken by reacting tert-butyloxycarbonylserine methyl ester
with dye 1 to yield 86% of the corresponding ester deriva-
tive 10. Coloured compounds were characterized as above
Scheme 2. Labelling of dipeptides 11 with dye 1
Eur. J. Org. Chem. 2004, 1750Ϫ1760
www.eurjoc.org
 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
1751

S. M. B. Fraga, M. S. T. Gonc¸alves, J. C. V. P. Moura, K. Rani
FULL PAPER
With the aim of testing the possibility of recovering the
initial amino acid esters by removal of the chromophore,
two of the resulting labeled products 5 were then treated
with di-tert-butyl pyrocarbonate (Boc2O) in the presence of
N,N-dimethylamino pyridine (DMAP) to give compounds
17 in fair yields. Aminolysis with 2-(N,N-dimethylamino)
ethylamine (DEAEA) in dry acetonitrile at room tempera-
ture gave the expected Boc-amino acid esters 18c and 18d
(Scheme 5, Table 3) isolated as colorless materials in 50%
and 58% yields, respectively.
Figure 1. 1H NMR spectrum of Ddp-Phe-Val-OMe (14c) before
purification
Scheme 5. Selective cleavage of labeled amino acids
Table 3. Selective cleavage of labeled amino acids
Starting material
Deprotection method
Product
Yield (%)
17c
17d
5c
DEAEA
DEAEA
Zn/HCO2H
Zn/HCO2H
18c
18d
19c
19d
50
58
77
65
5d
Labeled amino acid esters 5c and 5d were also treated
with zinc powder and thus converted into the correspond-
ing colorless 3-aminophenylacetyl derivatives 19 (Scheme 5,
Table 3). Despite difficulties in isolating the required prod-
ucts, the latter method of converting the labeled compounds
into colorless materials proved to lead to better yields than
the former.
1
Figure 2. H NMR spectrum of Dpa-Phe-Val-OMe before purifi-
cation
Stability tests carried out with colored alanine ester 5d
under forcing conditions similar to those usually required
for cleavage of protecting groups during peptide synthesis,
showed a good stability of the label to acidolysis, aminolysis
and hydrogenation catalyzed by Pd/C; treatment with
strong base cleaved the ester function but not the label.
Conclusion
It was possible to obtain suitable colored amino acid es-
ters with maximum absorption peaks ranging from 400 to
500 nm by choosing the appropriate azo chromophore. Ace-
Scheme 4. Synthesis of labeled lysine and serine residues
and the visible spectra of all labeled peptides showed λmax tyl analogs such as those obtained from 3-aminophenyl-
falling at 409 nm (ε ϭ 18532; 14a) and 415 (ε ϭ 21587; acetic acid and N,N-dimethylaniline strongly suggest step-
14d). All products were stable on storage at room tempera- wise synthesis with lower epimerization rates when coupling
ture without further precautions.
is carried out under similar conditions as for benzoyl azo
1752
 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org Eur. J. Org. Chem. 2004, 1750Ϫ1760

Chromophoric Azo Reagents for Amino Acid and Peptide Labelling
FULL PAPER
for 10 minutes and a mixture of propionic-acetic acids 1:5 (80 mL)
was added. 2-Amino-4-thiazoleacetic acid (2 g, 12.6 mmol) was ad-
ded in portions and the mixture was left stirring for 20 minutes,
with external cooling (5Ϫ10 °C). N,N-Dimethylaniline (1.9 mL) in
acetic acid (53 mL) was added to the diazonium solution slowly,
keeping external cooling at 10 °C for 4 hours. A saturated solution
of ammonium acetate was added until the mixture was neutral to
Congo Red paper and it was allowed to stand at 4 °C for one hour.
The mixture was poured into water and the separation of an oil
was noted. The oil was dissolved in 6  HCl and the addition of 6
 NaOH gave a red solid (0.94 g, 26%). M.p. 181.3Ϫ183.3 °C. TLC
markers. Thus, our results show that these chromophores
can be used for peptide and protein labelling.
Experimental Section
General Remarks: All melting points are uncorrected; they 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 visualized under UV
light or by exposure to vapourized iodine. Dry and column chro-
matography were carried out on Merck Kieselgel (230Ϫ240 mesh).
Light petroleum refers to the fraction boiling within the range
40Ϫ60 °C. IR spectra were determined on a PerkinϪElmer FTIR-
1600 and UV/Vis spectra were determined on a Hitachi U-2000
spectrophotometer. 1H NMR spectra were recorded on a Varian
300 spectrometer in 5% CDCl3 or [D6]DMSO (DMSO) solution at
25 °C. All chemical shifts are given in ppm relative to SiMe4 (δ ϭ
0 ppm) as reference and J values are given in Hz. Assignments were
made by comparison of chemical shifts, peak multiplicities, and J
values. 13C NMR spectra were run on the same instrument but at
75.4 MHz using the solvent peak as internal reference. Spectro-
metric analyses were performed at the ‘‘Unidad de Espectrometria
de Masas’’ of the University of Vigo, Spain. Elemental analyses
were carried out on a Leco CHNS 932 instrument. Serine methyl
ester hydrochloride and N-benzyloxycarbonyl lysine were commer-
cial products. All the other amino acid ester hydrochlorides were
prepared from thionyl chloride by the usual procedure. N-tert-bu-
tyloxycarbonylserine methyl ester hydrochloride was prepared with
di-tert-butylpyrocarbonate by the usual procedure. Dipeptide tert-
butyl esters 11 were prepared by catalytic hydrogenation of the cor-
responding N-benzyloxycarbonyldipeptides, which were synthe-
sized by standard methods. Dyes 3 and 4 were prepared following
the same procedure described before.[23]
(chloroform/methanol, 4:2): Rf ϭ 0.69. UV/Vis (MeOH): λmax
ϭ
490 nm (ε ϭ 26855 dm3·molϪ1·cmϪ1). IR (film): ν˜ ϭ 2675, 2558,
2494, 1727, 1605, 1550, 1527, 1456, 1367, 1325, 1307, 1270, 1207,
1159, 988, 943, 860, 814, 767, 747, 674 cmϪ1. 1H NMR (300 MHz,
DMSO): δ ϭ 3.11 (s, 6 H, NMe2), 3.75 (s, 2 H, CH2), 6.87 (d, J ϭ
9.6 Hz, 2 H, 2 ϫ Ar-H ortho NMe2), 7.41 (s, Ͻ 1 H, C-H), 7.80
(d, J ϭ 9.6 Hz, 2 H, 2 ϫ Ar-H meta NMe2) ppm.
General Method for the Acylation with Dyes 1 to 4
The carboxylic dye (1Ϫ4) was reacted on a 1.12-mmolar scale with
an amino acid methyl (or ethyl) ester hydrochloride in DMF by a
standard DCC/HOBt coupling. After evaporation of the solvent,
dry (or column) chromatography on silica gel, and recrystallization
from ethyl acetate/hexane, the required acetyl derivative (5, 6) was
obtained. Compounds 7 and 8 were precipitated with water and
recrystallized from acetone.
N-{3-[(N,N-Dimethylaminophenyl)-4Ј-diazenyl]phenylacetyl}glycine
Methyl Ester (5a): The product of the reaction of 1 with glycine
methyl ester hydrochloride (144 mg, 1.12 mmol) was chromato-
graphed with ethyl acetate/hexane (1:1) as the eluent to give the
ester 5a (305 mg, 77%). M.p. 140.0Ϫ142.3 °C. TLC (chloroform/
methanol, 5.5:0.5): Rf ϭ 0.81. UV/Vis (MeOH): λmax ϭ 422 nm
(ε ϭ 26929 dm3·molϪ1·cmϪ1). IR (KBr, 1%): ν˜ ϭ 3268, 2934, 1766,
1651, 1605, 1556, 1524, 1401, 1372, 1270, 1245, 1200, 1150, 1128,
3-[(N,N-Dimethylaminophenyl)-4Ј-diazenyl]phenylacetic Acid (1):
HCl (6 , 4.6 mL) was added, with stirring, to a suspension of 3-
aminophenyl acetic acid (3.11 g, 20 mmol) in 1  HCl (82 mL), fol-
lowed by 14 mL of a cold aqueous solution of sodium nitrite
(2.69 g; 30 mmol), the mixture was stirred at low temperature (Ͻ 5
°C) for 20 minutes. The diazonium salt was added to a solution of
N,N-dimethylaniline (4.6 mL, 36 mmol) in a mixture of glacial
acetic acid and water (1.5:1; 23 mL) at room temperature and the
mixture kept stirring for 2 h. A solution of 2  sodium acetate was
then added to pH 4 and the red oil thus obtained dissolved in the
minimum amount of 6  HCl and precipitated with aqueous 6 
NaOH. The solid was filtered off and washed with cold water and
light petroleum. The dye was obtained as an orange solid (5.55 g,
98%). M.p. 269.0Ϫ271.4 °C. TLC (chloroform/methanol, 6:1): Rf ϭ
1
1037, 819, 692 cmϪ1. H NMR (300 MHz, CDCl3): δ ϭ 3.11 (s, 6
H, NMe2), 3.73 (s, 5 H, CH2, OMe), 4.02 (d, J ϭ 6.1 Hz, 2 H, CH2
Gly), 5.95 (br. s, 1 H, α-NH Gly), 6.77 (d, J ϭ 8.4 Hz, 2 H, 2 ϫ
Ar-H ortho NMe2), 7.33 (d, J ϭ 7.2 Hz, 1 H, 6-H or 4-H), 7.49 (t,
J ϭ 7.2 Hz, 1 H, 5-H), 7.76Ϫ7.81 (m, 2 H, 2-H, 4-H or 6-H),
7.89 (d, J ϭ 8.4 Hz, 2 H, 2 ϫ Ar-H meta NMe2) ppm. 13C NMR
(75.4 MHz, CDCl3): δ ϭ 40.2 (NMe2), 41.2 (CH2 Gly), 43.2 (CH2),
52.2 (OMe), 111.4 (C-2Ј, C-6Ј), 121.6 (C-4), 122.8 (C-2), 125.0 (C-
3Ј, C-5Ј), 129.5 (C-5), 130.1 (C-6), 135.2 (C-1), 143.4 (C-4Ј), 152.4
(C-3), 153.5 (C-1Ј), 170.1 (CONH), 170.5 (CO2Me) ppm.
C19H22N4O3 (354.40): calcd. C 64.39, H 6.26, N 15.81; found C
64.39, H 6.44, N 15.50.
N-{3-[(N,N-Dimethylaminophenyl)-4Ј-diazenyl]phenylacetyl}-
isoleucine Methyl Ester (5b): The product of the reaction of 1 with
isoleucine methyl ester hydrochloride (203 mg, 1.12 mmol) was
chromatographed with ethyl acetate/hexane (4:6) as the eluent to
give the ester 5b (362 mg, 79%). M.p. 71.6Ϫ72.9 °C. TLC (ethyl
acetate/hexane, 6:4): Rf ϭ 0.75. UV/Vis (MeOH): λmax ϭ 446 nm
(ε ϭ 17282 dm3·molϪ1·cmϪ1). IR (film): ν˜ ϭ 3315, 2962, 1738,
1650, 1601, 1518, 1444, 1408, 1365, 1309, 1246, 1127, 1152, 1128,
1045, 945, 823 cmϪ1. 1H NMR (300 MHz, CDCl3): δ ϭ 0.82Ϫ0.94
(m, 6 H, δ-CH3 Ile, γ-CH3 Ile), 0.98Ϫ1.40 (2 ϫ m, 2 H, γ-CH2 Ile),
1.80Ϫ1.90 (m, 1 H, β-CH Ile), 3.10 (s, 6 H, NMe2), 3.70 (s, 5 H,
CH2, OMe), 4.58Ϫ4.61 (m, 1 H, α-CH Ile), 5.90 (d, J ϭ 8.3 Hz, 1
H, α-NH Ile), 6.77 (d, J ϭ 9.6 Hz, 2 H, 2 ϫ Ar-H ortho NMe2),
7.32 (d, J ϭ 6.4 Hz, 1 H, 6-H or 4-H), 7.48 (t, J ϭ 7.5 Hz, 1 H, 5-
H), 7.77Ϫ7.80 (m, 2 H, 4-H or 6-H, 2-H) ppm. 13C NMR
0.56. UV/Vis (MeOH): λmax
dm3·molϪ1·cmϪ1). IR (KBr, 1%): ν˜ ϭ 3406, 2910, 1604, 1566, 1556,
1517, 1403, 1376, 1361, 1229, 1152, 1120, 1071 cmϪ1 1H NMR
ϭ
405 nm (ε
ϭ
19200
.
(300 MHz, DMSO): δ ϭ 3.04 (s, 6 H, NMe2), 3.42 (s, 2 H, CH2),
6.82 (d, J ϭ 9.0 Hz, 2 ϫ Ar-H ortho NMe2), 7.23 (d, J ϭ 7.5 Hz,
1 H, 6-H or 4-H), 7.33 (t, J ϭ 7.5 Hz, 1 H, 5-H), 7.52 (d, J ϭ
7.5 Hz, 1 H, 4-H or 6-H), 7.63 (s, 1 H, 2-H), 7.78 (d, J ϭ 9.0 Hz,
2 H, 2 ϫ Ar-H meta NMe2) ppm. 13C NMR (75.4 MHz, DMSO):
δ ϭ 39.9 (NMe2), 46.0 (CH2), 111.6 (C-2Ј, C-6Ј), 119.2 (C-4), 122.4
(C-2), 124.6 (C-3Ј, C-5Ј), 128.3 (C-5), 130.8 (C-6), 141.0 (C-1),
142.7 (C-4Ј), 152.1 (C-3), 152.4 (C-1Ј), 174.5 (CO2H) ppm.
3-[(N,N-Dimethylaminophenyl)-4Ј-diazenyl]thiazoleacetic Acid (2):
Sodium nitrite (1.04 g, 15 mmol) was added to concentrated H2SO4
(12 mL) with external cooling (5 °C), the suspension was stirred
Eur. J. Org. Chem. 2004, 1750Ϫ1760
www.eurjoc.org
 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
1753

S. M. B. Fraga, M. S. T. Gonc¸alves, J. C. V. P. Moura, K. Rani
FULL PAPER
(75.4 MHz, CDCl3): δ ϭ 11.4 (δ-CH3 Ile), 15.3 (γ-CH3 Ile), 25.0
(γ-CH2 Ile), 37.7 (β-C Ile), 40.2 (NMe2), 43.5 (CH2), 52.0 (OMe),
56.4 (α-C Ile), 111.4 (C-2Ј, C-6Ј), 121.6 (C-4), 122.6 (C-2), 125.0
δ ϭ 3.16 (s, 6 H, NMe2), 3.74 (s, 3 H, OMe), 3.84 (s, 2 H, CH2),
4.08 (d, J ϭ 5.4 Hz, 2 H, CH2 Gly), 6.75 (d, J ϭ 9.0 Hz, 2 H, 2 ϫ
Ar-H ortho NMe2), 7.10 (s, 1 H, CH), 7.22 (br. s, 1 H, α-NH Gly),
(C-3Ј, C-5Ј), 129.5 (C-5), 130.0 (C-6), 135.4 (C-1), 143.4 (C-4Ј), 7.95 (d, J ϭ 9.3 Hz, 2 H, 2 ϫ Ar-H meta NMe2) ppm. 13C NMR
152.4 (C-3), 153.5 (C-1Ј), 170.4 (CONH), 172.2 (CO2Me) ppm. (75.4 MHz, CDCl3): δ ϭ 39.2 (CH2), 40.2 (NMe2), 41.4 (CH2 Gly),
C23H30N4O3 (410.50): calcd. C 67.29, H 7.37, N, 13.65; found C
67.22, H 7.45, N 13.40.
52.3 (OMe), 111.5 (C-2Ј, C-6Ј), 116.5 (C-3Ј, C-5Ј), 126.9 (C-5),
142.4 (C-4Ј), 148.9 (C-4), 153.9 (C-1Ј), 169.6 (CONH), 170.0
(CO2Me), 178.7 (C-2) ppm. The assignments were supported by
the Dept 135 technique. HRMS: calcd. for C16H19N5O3S [Mϩ]
361.1209; found 361.1214.
N-{3-[(N,N-Dimethylaminophenyl)-4Ј-diazenyl]phenylacetyl}-
phenylalanine Ethyl Ester (5c): The product of the reaction of 1
with phenylalanine ethyl ester hydrochloride (257 mg, 1.12 mmol)
was chromatographed with chloroform/methanol (5.5:0.5) as the
eluent to give the ester 5c (467 mg, 91%). M.p. 111.8Ϫ113.1 °C.
N-{3-[(N,N-Dimethylaminophenyl)-4Ј-diazenyl]thiazoleacetyl}-
isoleucine Methyl Ester (6b): The product of the reaction of 2 with
TLC (diethyl ether/hexane, 9:1): Rf ϭ 0.48. UV/Vis (MeOH): isoleucine methyl ester hydrochloride (203 mg, 1.12 mmol) was
λmax ϭ 425 nm (ε ϭ 24797 dm3·molϪ1·cmϪ1). IR (KBr, 1%): ν˜ ϭ chromatographed with chloroform/methanol (5.8:0.2) as the eluent
3320, 2931, 1730, 1642, 1602, 1542, 1518, 1455, 1362, 1344, 1231,
to give the ester 6b (350 mg, 75%). M.p. 117.3Ϫ117.6 °C. TLC
1
1154, 1115, 1039 cmϪ1. H NMR (300 MHz, CDCl3): δ ϭ 1.80 (t, (chloroform/methanol, 5.8:0.2): Rf ϭ 0.68. UV/Vis (MeOH):
J ϭ 7.5 Hz, 3 H, OCH2CH3), 3.05 (tap, J ϭ 7.0 Hz, 2 H, β-CH2 λmax ϭ 493 nm (ε ϭ 22253 dm3·molϪ1·cmϪ1). IR (KBr, 1%): ν˜ ϭ
Phe), 3.10 (s, 6 H, NMe2), 3.63 (s, 2 H, CH2), 4.10 (q, J ϭ 7.5 Hz, 3250, 2726, 1741, 1735, 1698, 1654, 1601, 1540, 1523, 1458, 1371,
2 H, OCH2CH3), 4.80Ϫ4.90 (m, 1 H, α-CH Phe), 5.87 (d, J ϭ
1305, 1258, 1148, 1131, 821, 666 cmϪ1 1H NMR (300 MHz,
.
7.5 Hz, 1 H, α-NH Phe), 6.78 (d, J ϭ 9.3 Hz, 2 H, 2 ϫ Ar-H ortho CDCl3): δ ϭ 0.85Ϫ0.95 (m, 6 H, δ-CH3 Ile, γ-CH3 Ile), 1.10Ϫ1.50
NMe2), 6.80Ϫ6.93 (m, 2 H, 2 ϫ Ar-H Phe), 7.11Ϫ7.19 (m, 3 H, 3 (m, 2 H, γ-CH2 Ile), 1.90Ϫ2.00 (m, 1 H, β-CH Ile), 3.16 (s, 6 H,
ϫ Ar-H Phe), 7.22 (d, J ϭ 8.1 Hz, 1 H, 6-H or 4-H), 7.44 (t, J ϭ NMe2), 3.71 (s, 3 H, OMe), 3.82 (s, 2 H, CH2), 4.57Ϫ4.60 (2 ϫ d,
7.8 Hz, 1 H, 5-H), 7.70 (br. s, 1 H, 2-H), 7.78 (d, J ϭ 8.0 Hz, 1 H,
4-H or 6-H), 7.90 (d, J ϭ 9.0 Hz, 2 H, 2 ϫ Ar-H meta NMe2) ppm. ortho NMe2), 7.10 (br. s, Ͻ 2 H, CH, α-NH Ile), 7.94 (d, J ϭ
13C NMR (75.4 MHz, CDCl3): δ ϭ 14.0 (OCH2CH3), 37.6 (β-C 9.3 Hz, 2 H, 2 ϫ Ar-H meta NMe2) ppm. 13C NMR (75.4 MHz,
J ϭ 6.3 Hz, 1 H, α-CH Ile), 6.76 (d, J ϭ 9.0 Hz, 2 H, 2 ϫ Ar-H
Phe), 40.2 (NMe2), 43.5 (CH2), 53.0 (α-C Phe), 61.4 (OCH2CH3), CDCl3): δ ϭ 11.5 (δ-C Ile), 15.5 (γ-CH3 Ile), 25.1 (γ-CH2 Ile), 37.6
111.4 (C-2Ј, C-6Ј), 121.6 (C-4), 122.8 (C-2), 125.2 (C-3Ј, C-5Ј), (β-C Ile), 39.6 (CH2), 40.2 (NMe2), 52.0 (OMe), 56.7 (α-C Ile),
126.9 (C-4 Phe), 128.4 (C-3 Phe, C-5 Phe), 129.1 (C-2 Phe, C-6
Phe), 129.5 (C-5), 130.1 (C-6), 135.2 (C-1), 135.1 (C-1 Phe), 143.5
111.5 (C-2Ј, C-6Ј), 116.2 (C-3Ј, C-5Ј), 126.8 (C-5), 142.4 (C-4Ј),
149.4 (C-4), 153.8 (C-1Ј), 169.0 (CONH), 172.1 (CO2Me), 178.4
(C-4Ј), 152.5 (C-3), 153.5 (C-1Ј), 170.1 (CONH), 171.1 (CO2Et) (C-2) ppm. The assignments were supported by the Dept 135 tech-
ppm. C27H30N4O3 (458.54): calcd. C 70.72, H 6.59, N 12.22; found
C 70.46, H 6.74, N 12.19.
nique. HRMS: calcd. for C20H27N5O3S [Mϩ] 417.1835; found
417.1832.
N-{3-[(N,N-Dimethylaminophenyl)-4Ј-diazenyl]phenylacetyl}alanine
Methyl Ester (5d): The product of the reaction of 1 with alanine
methyl ester hydrochloride (156 mg, 1.12 mmol) was chromato-
graphed with chloroform/methanol (5.5:0.5) as the eluent to give
the ester 5d (293 mg, 71%). M.p. 138.6Ϫ140.0 °C. TLC (chloro-
N-{3-[(N,N-Dimethylaminophenyl)-4Ј-diazenyl]thiazoleacetyl}-
phenylalanine Ethyl Ester (6c): The product of the reaction of 2
with phenylalanine ethyl ester hydrochloride (257 mg, 1.12 mmol)
was chromatographed with ethyl acetate/hexane (7:3) as the eluent
to give the ester 6c (401 mg, 77%). M.p. 124.0Ϫ124.5 °C. TLC
form/methanol, 5.5:0.5): Rf ϭ 0.86. UV/Vis (MeOH): λmax
409 nm (ε ϭ 31644 dm3·molϪ1·cmϪ1). IR (KBr, 1%): ν˜ ϭ 3334,
2928, 2847, 1754, 1745, 1650, 1604, 1531, 1434, 1406, 1366, 1217, 1723, 1718, 1691, 1643, 1606, 1558, 1542, 1523, 1466, 1451, 1370,
1163, 1156, 1056, 825 cmϪ1 1H NMR (300 MHz, CDCl3): δ ϭ 1339, 1288, 1157, 1125, 1109, 1034, 826 cmϪ1. 1H NMR (300 MHz,
1.35 (d, J ϭ 8.2 Hz, 3 H, β-CH3 Ala), 3.11 (s, 6 H, NMe2), 3.68 (s, CDCl3): δ ϭ 1.22 (t, J ϭ 7.0 Hz, 3 H, OCH2CH3), 3.05 (tap, J ϭ
ϭ
(ethyl acetate/hexane, 7:3): Rf ϭ 0.71. UV/Vis (MeOH): λmax
ϭ
495 nm (ε ϭ 25927 dm3·molϪ1·cmϪ1). IR (KBr, 1%): ν˜ ϭ 2921,
.
2 H, CH2), 3.72 (s, 3 H, OMe), 4.52Ϫ4.70 (m, 1 H, α-CH Ala),
6.02 (d, J ϭ 6.3 Hz, 1 H, α-NH Ala), 6.77 (d, J ϭ 8.1 Hz, 2 H, 2
7.0 Hz, β-CH2 Phe), 3.17 (s, 6 H, NMe2), 3.48Ϫ3.82 (m, 2 H, CH2),
4.10, 4.18 (2 ϫ d J ϭ 7.0 Hz, 2 H, OCH2CH3), 4.80Ϫ4.88 (m, 1
ϫ Ar-H ortho NMe2), 7.32 (d, J ϭ 7.8 Hz, 1 H, 6-H or 4-H), 7.49 H, α-CH Phe), 6.78 (d, J ϭ 9.6 Hz, 2 H, 2 ϫ Ar-H ortho NMe2),
(t, J ϭ 8.1 Hz, 1 H, 5-H), 7.75Ϫ7.81 (m, 2 H, 4-H or 6-H, 2-H), 6.90Ϫ7.20 (m, 6 H, 5 ϫ Ar-H Phe, CH), 7.96 (d, J ϭ 9.0 Hz, 2 H,
7.89 (d, J ϭ 8.1 Hz, 2 H, 2 ϫ Ar-H meta NMe2) ppm. 13C NMR
2 ϫ Ar-H meta NMe2) ppm. 13C NMR (75.4 MHz, CDCl3): δ ϭ
(75.4 MHz, CDCl3): δ ϭ 18.2 (β-C Ala), 40.2 (NMe2), 43.4 (CH2), 14.0 (OCH2CH3), 37.8 (β-C Phe), 39.6 (CH2), 40.3 (NMe2), 53.5
48.1 (α-C Ala), 52.4 (OMe), 111.4 (C-2Ј, C-6Ј), 121.5 (C-4), 122.8 (α-C Phe), 61.3 (OCH2CH3), 111.6 (C-2Ј, C-6Ј), 116.2 (C-3Ј, C-5Ј),
(C-2), 125.0 (C-3Ј, C-5Ј), 129.5 (C-5), 130.1 (C-6), 135.3 (C-1), 126.9 (C-5, C-4 Phe), 128.4 (C-3 Phe, C-5 Phe), 129.3 (C-2 Phe, C-
143.4 (C-4Ј), 152.4 (C-3), 153.5 (C-1Ј), 170.2 (CONH), 173.2
6 Phe), 135.8 (C-1 Phe), 142.5 (C-4Ј), 149.3 (C-4), 153.8 (C-1Ј),
(CO2Me) ppm. C20H24N4O3 (368.42): calcd. C 65.20, H 6.57, N 168.7 (CONH), 171.2 (CO2Et), 178.5 (C-2) ppm. The assignments
15.21; found C 65.14, H 6.74, N 14.98.
were supported by the Dept 135 technique. C24H27N5O3S (465.56):
calcd. C 61.91, H 5.85, N 15.04, S 6.89; found C 61.80, H 6.01, N
14.90, S 6.94.
N-{3-[(N,N-Dimethylaminophenyl)-4Ј-diazenyl]thiazoleacetyl}-
glycine Methyl Ester (6a): The product of the reaction of 2 with
glycine methyl ester hydrochloride (144 mg, 1.12 mmol) was chrom- N-{3-[(2-Hydroxy-1-naphthyl)-1Ј-diazenyl]benzoyl}glycine Methyl
atographed with chloroform/methanol (6:0.1) as the eluent to give
the ester 6a (344 mg, 85%). M.p. 161.2Ϫ162.7 °C. TLC (chloro-
form/methanol, 6:0.1): Rf ϭ 0.83. UV/Vis (MeOH): λmax ϭ 495 nm
(ε ϭ 26806 dm3·molϪ1·cmϪ1). IR (KBr, 1%): ν˜ ϭ 3433, 3330, 2926,
Ester (7a): Reaction of 3 with glycine methyl ester hydrochloride
(141 mg, 1.12 mmol) gave the ester 7a (240 mg, 59%). M.p.
205.2Ϫ205.6 °C. TLC (ethanol): Rf ϭ 0.80. UV/Vis (MeOH):
λ
max ϭ 475 nm (ε ϭ 18080 dm3·molϪ1·cmϪ1). 1H NMR (300 MHz,
2845, 1759, 1725, 1654, 1622, 1607, 1551, 1518, 1436, 1372, 1306, DMSO): δ ϭ 3.67 (s, 3 H, OMe), 4.02Ϫ4.10 (m, 2 H, CH2 Gly),
1229, 1202, 1163, 979, 823 cmϪ1 1H NMR (300 MHz, CDCl3):
.
6.92 (d, J ϭ 9.3 Hz, 1 H, 3Ј-H), 7.48 (t, J ϭ 7.5 Hz, 1 H, 6Ј-H),
1754
 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
Eur. J. Org. Chem. 2004, 1750Ϫ1760

Chromophoric Azo Reagents for Amino Acid and Peptide Labelling
FULL PAPER
7.60Ϫ7.70 (m, 2 H, 7Ј-H, 5-H), 7.80 (d, J ϭ 6.9 Hz, 1 H, 5Ј-H), (141 mg, 1.12 mmol) gave the ester 8a (269 mg, 74%). M.p.
7.85 (d, J ϭ 6.6 Hz, 1 H, 4-H), 7.98 (d, J ϭ 9.3 Hz, 1 H, 4Ј-H), 180.0Ϫ181.7 °C. TLC (ethanol): Rf ϭ 0.89. UV/Vis (MeOH):
8.04 (d, J ϭ 7.0 Hz, 1 H, 6-H), 8.31 (tap, J ϭ 1.8 Hz, 1 H, 2-H),
8.58 (d, J ϭ 8.0 Hz, 1 H, 8Ј-H), 9.19 (tap, J ϭ 4.8 Hz, 1 H, α-NH DMSO): δ ϭ 3.66 (s, 3 H, OMe), 4.03 (d, J ϭ 6.5 Hz, 2 H, CH2
λ
max ϭ 480 nm (ε ϭ 27752 dm3·molϪ1·cmϪ1). 1H NMR (300 MHz,
Gly) ppm. The assignments were supported by spin decoupling-
Gly), 6.82 (d, J ϭ 9.6 Hz, 1 H, 3Ј-H), 7.47 (dt, J ϭ 7.0, 1.2 Hz, 1
H, 6Ј-H), 7.61 (t, J ϭ 8.0 Hz, 1 H, 7Ј-H), 7.75 (d, J ϭ 7.8 Hz, 1
double resonance. 13C NMR (75.4 MHz, DMSO): δ ϭ 41.7 (CH2
Gly), 52.2 (OCH3), 117.7 (β-naphthol), 121.9 (C-2), 122.1 (β-naph- H, 5Ј-H), 7.91 (t, J ϭ 8.4 Hz, 2 H, 3-H, 5-H), 7.96 (s, 1 H, 4Ј-H),
thol), 124.5 (C-4), 126.5 (β-naphthol), 126.8 (β-naphthol), 128.3 8.00 (d, J ϭ 9.6 Hz, 2 H, 2-H, 6-H), 8.51 (d, J ϭ 7.5 Hz, 1 H, 8Ј-
(C-5), 129.4 (C-6), 129.6 (β-naphthol), 129.9 (β-naphthol), 130.4
(β-naphthol), 133.0 (β-naphthol), 135.5 (C-1), 140.9 (β-naphthol),
145.4 (β-naphthol), 157.0 (C-3), 166.2 (CONH), 170.3 (CO2CH3),
170.7 (CO naphthol) ppm. HRMS: calcd. for C13H24N2O [Mϩ]
363.1219; found 363.1224.
H), 9.04 (tap, J ϭ 6.0 Hz, 1 H, α-NH Gly), 15.99 (1 H, exchangeable
s, NH) ppm. The assignments were supported by spin decoupling-
double resonance. 13C NMR (75.4 MHz, DMSO): δ ϭ 41.7 (CH2
Gly), 52.1 (OCH3), 118.0 (β-naphthol), 122.1 (β-naphthol), 125.6
(C-3, C-5), 127.0 (C-2, C-6), 128.4 (β-naphthol), 129.4 (β-naph-
thol), 129.8 (β-naphthol), 130.3 (β-naphthol), 131.7 (C-1), 133.1 (C-
4), 142.2 (β-naphthol), 146.3 (β-naphthol), 166.2 (CONH), 170.8
(CO2CH3), 174.8 (CO naphthol) ppm. HRMS: calcd. for
C20H17N3O4 [Mϩ] 363.1219; found 363.1206.
N-{3-[(2-Hydroxy-1-naphthyl)-1Ј-diazenyl]benzoyl}isoleucine
Methyl Ester (7b): Reaction of 3 with isoleucine methyl ester hydro-
chloride (203 mg, 1.12 mmol) gave the ester 7b (263 mg, 56%). M.p.
201.9Ϫ203.9 °C. TLC (ethanol): Rf ϭ 0.83. UV/Vis (MeOH):
λ
max ϭ 475 nm (ε ϭ 18741 dm3·molϪ1·cmϪ1). 1H NMR (300 MHz,
N-{4-[(2-Hydroxy-1-naphthyl)-1Ј-diazenyl]benzoyl}isoleucine
Methyl Ester (8b): Reaction of 4 with isoleucine methyl ester hydro-
chloride (203 mg, 1.12 mmol) gave the ester 8b (465 mg, 99%). M.p.
177.0Ϫ177.8 °C. TLC (ethanol): Rf ϭ 0.83. UV/Vis (MeOH):
DMSO): δ ϭ 0.92 (t, J ϭ 6.3 Hz, 3 H, δ-CH3 Ile), 0.98Ϫ1.40 (m,
5 H, γ-CH3 Ile, γ-CH2 Ile), 1.90Ϫ2.10 (m, 1 H, β-CH Ile), 3.67 (s,
3 H, OMe), 4.40 (t, J ϭ 7.8 Hz, 1 H, α-CH Ile), 6.93 (d, J ϭ 9.3 Hz,
1 H, 3Ј-H), 7.48 (t, J ϭ 7.2 Hz, 1 H, 6Ј-H), 7.60Ϫ7.70 (m, 2 H, 7Ј-
H, 5-H), 7.75Ϫ7.90 (m, 2 H, 5Ј-H, 4-H), 7.97 (d, J ϭ 9.9 Hz, 1 H,
4Ј-H), 8.06 (d, J ϭ 8.1 Hz, 1 H, 6-H), 8.30 (s, 1 H, 2-H), 8.56 (d,
J ϭ 8.1 Hz, 1 H, 8Ј-H), 8.83 (d, J ϭ 7.8 Hz, 1 H, α-NH Ile) ppm.
The assignments were supported by spin decoupling-double reson-
ance. 13C NMR (75.4 MHz, DMSO): δ ϭ 10.9 (δ-C Ile), 15.6 (γ-
CH3 Ile), 24.5 (γ-CH2 Ile), 35.7 (β-C Ile), 51.6 (OCH3), 57.4 (α-C
Ile), 118.0 (β-naphthol), 121.0 (C-2), 121.3 (β-naphthol), 124.0 (C-
4), 126.0 (β-naphthol), 126.7 (β-naphthol), 127.8 (C-5), 128.8 (C-
6), 129.1 (β-naphthol), 129.2 (β-naphthol), 129.7 (β-naphthol),
132.5 (β-naphthol), 135.1 (C-1), 140.4 (β-naphthol), 144.7 (β-naph-
thol), 156.5 (C-3), 165.9 (CONH), 169.8 (CO2CH3), 172.0 (CO
naphthol) ppm. HRMS: calcd. for C24H25N3O4 [Mϩ] 419.1845;
found 419.1858.
λ
max ϭ 480 nm (ε ϭ 25321 dm3·molϪ1·cmϪ1). 1H NMR (300 MHz,
DMSO): δ ϭ 0.90 (t, J ϭ 7.2 Hz, 3 H, δ-CH3 Ile), 0.98Ϫ1.40 (m,
5 H, γ-CH3 Ile, γ-CH2 Ile), 1.90Ϫ2.10 (m, 1 H, β-CH Ile), 3.66 (s,
3 H, OMe), 4.36 (t, J ϭ 7.5 Hz, 1 H, α-CH Ile), 6.83 (d, J ϭ 9.9 Hz,
1 H, 3Ј-H), 7.47 (d, J ϭ 7.20 Hz, 1 H, 6Ј-H), 7.61 (d t, J ϭ 8.4,
1.5 Hz, 1 H, 7Ј-H), 7.75 (d, J ϭ 7.0 Hz, 1 H, 5Ј-H), 7.88 (d, J ϭ
9.0 Hz, 2 H, 3-H, 5-H), 7.95 (d, J ϭ 9.3 Hz, 1 H, 4Ј-H), 8.04 (d,
J ϭ 8.1 Hz, 2 H, 2-H, 6-H), 8.49 (d, J ϭ 8.1 Hz, 1 H, 8Ј-H), 8.68
(d, J ϭ 7.8 Hz, 1 H, α-NH Ile), 15.99 (1 H, exchangeable s, NH)
ppm. The assignments were supported by spin decoupling-double
resonance. 13C NMR (75.4 MHz, DMSO): δ ϭ 11.2 (δ-C Ile), 15.9
(γ-CH3 Ile), 25.7 (γ-CH2 Ile), 36.1 (β-C Ile), 52.0 (OCH3), 57.8 (α-
C Ile), 117.8 (β-naphthol), 122.0 (β-naphthol), 125.5 (C-3, C-5),
126.9 (C-2, C-6), 128.4 (β-naphthol), 129.5 (β-naphthol), 129.8 (β-
naphthol), 130.3 (β-naphthol), 131.9 (C-1), 133.0 (C-4), 142.1 (β-
naphthol), 146.3 (β-naphthol), 166.4 (CONH), 172.9 (CO2CH3),
174.7 (CO naphthol) ppm. HRMS: calcd. for C24H25N3O4 [Mϩ]
419.1845; found 419.1856.
N-{3-[(2-hydroxy-1-naphthyl)-1Ј-diazenyl]benzoyl}phenylalanine
Ethyl Ester (7c): Reaction of 3 with phenylalanine ethyl ester hydro-
chloride (257 mg, 1.12 mmol) gave the ester 7c (301 mg, 58%). M.p.
205.0Ϫ206.0 °C. TLC (ethyl acetate/hexane, 1:1): Rf ϭ 0.82. UV/
Vis (MeOH): λmax ϭ 475 nm (ε ϭ 12490 dm3·molϪ1·cmϪ1). 1H
NMR (300 MHz, DMSO): δ ϭ 1.15 (t, J ϭ 5.2 Hz, 3 H,
OCH2CH3), 3.10Ϫ3.20 (m, 2 H, β-CH Phe), 4.10 (q, J ϭ 5.2 Hz,
2 H, OCH2CH3), 4.60Ϫ4.72 (m, 1 H, α-CH Phe), 6.92 (d, J ϭ
9.0 Hz, 1 H, 3Ј-H), 7.20 (d, J ϭ 7.5 Hz, 1 H, 1 ϫ Ar-H Phe), 7.28
(t, J ϭ 7.5 Hz, 2 H, 2 ϫ Ar-H Phe), 7.36 (d, J ϭ 7.0 Hz, 2 H, 2 ϫ
Ar-H Phe), 7.48 (t, J ϭ 8.1 Hz, 1 H, 6Ј-H), 7.59Ϫ7.68 (m, 2 H, 7Ј-
H, 5-H), 7.78 (t, J ϭ 8.50 Hz, 2 H, 4-H, 5Ј-H), 7.98 (d, J ϭ 9.6 Hz,
1 H, 4Ј-H), 8.02 (d, J ϭ 9.6 Hz, 1 H, 6-H), 8.23 (br. s,1 H, 2-H),
8.59 (d, J ϭ 8.1 Hz, 1 H, 8Ј-H), 9.09 (d, J ϭ 7.5 Hz, 1 H, α-NH
Phe) ppm. The assignments were supported by spin decoupling-
double resonance. 13C NMR (75.4 MHz, DMSO): δ ϭ 14.1
(OCH2CH3), 36.2 (β-C Phe), 54.5 (α-C Phe), 60.6 (OCH2CH3),
117.3 (β-naphthol), 121.5 (C-2), 124.0 (β-naphthol), 125.2 (C-4),
126.0 (C-4 Phe), 126.4 (β-naphthol), 127.6 (β-naphthol), 128.1 (C-
5), 128.4 (C-3 Phe, C-5 Phe), 129.0 (C-6), 129.8 (β-naphthol), 130.6
(β-naphthol), 131.8 (β-naphthol), 132.5 (β-naphthol), 135.0 (C-1),
135.1 (C-1 Phe), 137.5 (C-2 Phe, C-6 Phe), 140.4 (β-naphthol),
144.7 (β-naphthol), 156.6 (C-3), 165.6 (CONH), 169.7
(CO2CH2CH3), 171.4 (CO naphthol) ppm. HRMS: calcd. for
C28H25N3O4 [Mϩ] 467.1845; found 467.1863.
N-{4-[(2-Hydroxy-1-naphthyl)-1Ј-diazenyl]benzoyl}phenylalanine
Methyl Ester (8c): Reaction of 4 with phenylalanine ethyl ester hy-
drochloride (257 mg, 1.12 mmol) gave the ester 8c (399 mg, 76%).
M.p. 187.5Ϫ188.2 °C. TLC (ethanol): Rf ϭ 0.83. UV/Vis (MeOH):
λ
max ϭ 480 nm (ε ϭ 20620 dm3·molϪ1·cmϪ1). 1H NMR (300 MHz,
DMSO): δ ϭ 1.10 (t, J ϭ 6.5 Hz, 3 H, OCH2CH3), 3.00Ϫ3.20 (m,
2 H, β-CH2 Phe), 4.10 (q, J ϭ 6.5 Hz, 2 H, OCH2CH3), 4.60Ϫ4.70
(m, 1 H, α-CH Phe), 6.92 (d, J ϭ 9.3 Hz, 1 H, 3Ј-H), 7.15Ϫ7.30
(m, 5 H, 5 ϫ Ar-H Phe), 7.47 (t, J ϭ 7.2 Hz, 1 H, 6Ј-H), 7.61 (t,
J ϭ 7.8 Hz, 1 H, 7Ј-H), 7.76 (d, J ϭ 7.8 Hz, 1 H, 5Ј-H), 7.87 (d,
J ϭ 8.4 Hz, 2 H, 3-H, 5-H), 7.94 (d, J ϭ 9.0 Hz, 3 H, 2-H, 4-H,
4Ј-H), 8.50 (d, J ϭ 8.1 Hz, 1 H, 8Ј-H), 8.89 (d, J ϭ 7.5 Hz, 1 H,
α-NH Phe), 15.90 (s, 1 H, NH) ppm. The assignments were sup-
ported by spin decoupling-double resonance. 13C NMR
(75.4 MHz, DMSO): δ ϭ 14.4 (OCH2CH3), 36.7 (β-C Phe), 54.9
(α-C Phe), 61.0 (OCH2CH3), 117.9 (β-naphthol), 122.1 (β-naph-
thol), 125.5 (C-3, C-5), 126.9 (C-4 Phe), 126.9 (C-2, C-6), 128.4 (β-
naphthol), 128.6 (C-3 Phe, C-5 Phe), 129.5 (β-naphthol), 129.8 (β-
naphthol), 130.3 (β-naphthol), 131.7 (C-1, C-1 Phe), 133.1 (C-4),
138.0 (C-2 Phe, C-6 Phe), 142.1 (β-naphthol), 146.3 (β-naphthol),
166.1 (CONH), 172.1 (CO2CH2CH3), 174.8 (CO naphthol) ppm.
HRMS: calcd. for C28H25N3O4 [Mϩ] 467.1845; found 467.1856.
N-{4-[(2-Hydroxy-1-naphthyl)-1Ј-diazenyl]benzoyl}glycine Methyl
Ester (8a): Reaction of 4 with glycine methyl ester hydrochloride
Eur. J. Org. Chem. 2004, 1750Ϫ1760
www.eurjoc.org
 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
1755

S. M. B. Fraga, M. S. T. Gonc¸alves, J. C. V. P. Moura, K. Rani
FULL PAPER
N-Benzyloxycarbonyl-ω-{3-[(N,N-dimethylaminophenyl)-4Ј-
diazenyl]phenylacetyl}lysine Methyl Ester (9): The product of the
reaction of 1 with N-(benzyloxycarbonyl)lysine methyl ester hydro-
H, CH2), 4.40Ϫ4.50 (m, 1 H, α-CH Ala), 4.64Ϫ4.74 (m, 1 H, α-
CH Phe), 6.03 (d, J ϭ 7.2 Hz, 1 H, α-NH Ala), 6.43 (d, J ϭ 7.8 Hz,
1 H, α-NH Phe), 6.77 (d, J ϭ 9.0 Hz, 2 H, 2 ϫ Ar-H ortho NMe2),
chloride (231 mg, 0.82 mmol) according to the above general 7.10Ϫ7.16 (m, 2 H, 2 ϫ Ar-H Phe), 7.20Ϫ7.32 (m, 4 H, 3 ϫ Ar-
method for acylation with dyes 1Ϫ4 was chromatographed with
ethyl acetate/light petroleum (mixtures of increasing polarity) as
H Phe, 6-H or 4-H), 7.46 (t, J ϭ 7.8 Hz, 1 H, 5-H), 7.72 (s, 1 H,
2-H), 7.78 (d, J ϭ 8.1 Hz, 1 H, 4-H or 6-H), 7.89 (d, J ϭ 9.3 Hz,
the eluent to give the ester 9 (319 mg, 70%). M.p. 130.0Ϫ132.0 °C. 2 H, 2 ϫ Ar-H meta NMe2) ppm. The assignments were supported
TLC (chloroform/methanol, 5.5:0.5): Rf ϭ 0.65.UV/Vis (MeOH): by spin decoupling-double resonance technique. 13C NMR
λ
max ϭ 410 nm (ε ϭ 28430 dm3·molϪ1·cmϪ1). 1H NMR (300 MHz,
(75.4 MHz, CDCl3): δ ϭ 18.1 (β-C Ala), 27.8 (CMe3), 37.7 (β-C
CDCl3): δ ϭ 1.40Ϫ1.50 (m, 2 H, γ-CH2 Lys), 1.54Ϫ1.72 (m, 2 H, Phe), 40.1 (NMe2), 43.1 (CH2), 48.7 (α-C Ala), 53.7 (α-C Phe), 82.1
β-CH2 Lys), 1.75Ϫ1.97 (m, 2 H, δ-CH2 Lys), 3.11 (s, 6 H, NMe2), (CMe3), 111.3 (C-2Ј, C-6Ј), 121.3 (C-4), 122.7 (C-2), 124.9 (C-3Ј,
3.14Ϫ3.29 (m, 2 H, ε-CH2 Lys), 3.65 (s, 2 H, CH2), 3.72 (s, 3 H, C-5Ј), 126.8 (C-4 Phe), 128.2 (C-3 Phe, C-5 Phe), 129.3 (C-2 Phe,
OMe), 4.25Ϫ4.36 (m, 1 H, α-CH Lys), 5.10 (s, 2 H, CH2 Z), 5.36
(d, J ϭ 8.1 Hz, 1 H, α-NH Lys), 5.53 (br. s, 1 H, ω-NH Lys), 6.77
C-6 Phe, C-5), 130.0 (C-6), 135.3 (C-1), 136.0 (C-1 Phe), 143.4 (C-
4Ј), 152.3 (C-3), 153.4 (C-1Ј), 170.1 (CONH), 170.6 (CONH), 171.6
(d, J ϭ 9.0 Hz, 2 H, 2 ϫ Ar-H ortho NMe2), 7.24Ϫ7.36 (2 ϫ m, 6 (CO2CMe3) ppm. C32H39N5O4 (557.67): calcd. C 68.92, H 7.05, N,
H, 5 ϫ Ar-H Z, 6-H or 4-H), 7.46 (t, J ϭ 7.8 Hz, 1 H, 5-H), 7.71 12.56; found C 68.95, H 7.04, N 12.37.
(s, 1 H, 2-H), 7.77 (d, J ϭ 8.1 Hz, 1 H, 4-H or 6-H), 7.88 (d, J ϭ
N-{3-[(N,N-Dimethylaminophenyl)-4Ј-diazenyl]phenylacetyl}-
valylphenylalanine tert-Butyl Ester (12b): The product of the reac-
tion of dye 1 (255 mg, 0.90 mmol) with valylphenylalanine tert-bu-
tyl ester 11b (288 mg, 0.90 mmol) under the conditions described
above for acylation with compounds 1Ϫ4 was chromatographed
with diethyl ether/hexane (4:6) as the eluent to give the labeled
dipetide 12b (348 mg, 66%). M.p. 187.5Ϫ188.8 °C. TLC (diethyl
ether/hexane, 6:4): Rf ϭ 0.63. UV/Vis (MeOH): λmax ϭ 414 nm
9.0 Hz, 2 H, 2 ϫ Ar-H meta NMe2) ppm. The assignments were
supported by spin decoupling-double resonance technique. 13C
NMR (75.4 MHz, CDCl3): δ ϭ 22.3 (γ-C Lys), 28.9 (β-C Lys), 32.1
(δ-C Lys), 39.2 (ε-C Lys), 40.3 (NMe2), 43.8 (CH2), 52.4 (OMe),
53.6 (α-C Lys), 67.0 (CH2 Z), 111.5 (C-2Ј, C-6Ј), 121.6 (C-4), 122.8
(C-3Ј, C-5Ј, C-2), 125.1 (C-3 Z, C-5 Z), 128.1 (C-4 Z), 128.5 (C-2
Z, C-6 Z), 129.7 (C-5), 130.2 (C-6), 135.7 (C-1), 136.2 (C-1 Z),
143.5 (C-4Ј), 152.6 (C-3), 153.6 (C-1Ј), 155.9 (CO2 Z), 170.9
(CONH), 172.8 (CO2Me) ppm. C31H37N5O5 (559.65): calcd. C
66.53, H 6.66, N 12.52; found C 66.37, H 6.90, N 12.31.
1
(ε ϭ 29146 dm3·molϪ1·cmϪ1). H NMR (300 MHz, CDCl3): δ ϭ
0.76 (d, J ϭ 6.9 Hz, 3 H, γ-CH3 Val), 0.86 (d, J ϭ 6.9 Hz, 3 H, γ-
CH3 Val), 1.40 (s, 9 H, CMe3), 1.93Ϫ2.05 (m, 1 H, β-CH Val), 3.04
(d, J ϭ 6.9 Hz, 2 H, β-CH2 Phe), 3.10 (s, 6 H, NMe2), 3.68 (d, J ϭ
4.8 Hz, 2 H, CH2), 4.17Ϫ4.40 (m, 1 H, α-CH Val), 4.66Ϫ4.78 (m,
1 H, α-CH Phe), 5.97 (d, J ϭ 8.7 Hz, 1 H, α-NH Val), 6.18 (d, J ϭ
7.8 Hz, 1 H, α-NH Phe), 6.77 (d, J ϭ 9.0 Hz, 2 H, 2 ϫ Ar-H ortho
N-tert-Butyloxycarbonyl-O-{3-[(N,N-dimethylaminophenyl)-4Ј-
diazenyl]phenylacetyl}serine Methyl Ester (10): The product of the
reaction of 1 with N-tert-butyloxycarbonylserine methyl ester hy-
drochloride (289 mg, 1.32 mmol) according to the above general
method for acylation with dyes 1Ϫ4 was chromatographed with NMe2), 7.10Ϫ7.14 (m, 2 H, 2 ϫ Ar-H Phe), 7.20Ϫ7.32 (m, 3 H, 3
chloroform/methanol (5.5:0.5) as the eluent to give the ester 10
(464 mg, 86%). M.p. 95.6Ϫ96.6 °C. TLC (ethyl acetate/light petro-
ϫ Ar-H Phe), 7.48 (t, J ϭ 8.3 Hz, 1 H, 5-H), 7.74Ϫ7.80 (m, 2 H,
6-H, 4-H), 7.89 (d, J ϭ 9.3 Hz, 2 H, 2 ϫ Ar-H meta NMe2) ppm.
leum, 4:6): Rf ϭ 0.79. UV/Vis (MeOH): λmax ϭ 415 nm (ε ϭ 23643 The assignments were supported by spin decoupling-double reson-
dm3·molϪ1·cmϪ1). IR (KBr, 1%): ν˜ ϭ 3370, 2977, 2910, 2812, 1741,
1714, 1601, 1565, 1520, 1446, 1408, 1367, 1310, 1246, 1224, 1154,
1063, 1023, 946, 913, 824, 785, 732, 690 cmϪ1. 1H NMR (300 MHz,
ance technique. 13C NMR (75.4 MHz, CDCl3): δ ϭ 17.9 (γ-C Val),
19.1 (γ-C Val), 27.8 (CMe3), 30.8 (β-C Val), 38.0 (β-C Phe), 40.2
(NMe2), 43.5 (CH2), 53.5 (α-C Phe), 58.3 (α-C Val), 82.2 (OCMe3),
CDCl3): δ ϭ 1.43 (s, 9 H, CMe3), 3.10 (s, 6 H, NMe2), 3.68 (s, 3 111.4 (C-2Ј, C-6Ј), 121.7 (C-4), 122.6 (C-2), 125.0 (C-3Ј, C-5Ј),
H, OMe), 3.91 (s, 2 H, CH2), 4.35Ϫ4.50 (m, 2 H, β-CH2 Ser), 126.9 (C-4 Phe), 128.4 (C-3 Phe, C-5 Phe), 129.4 (C-2 Phe, C-6
4.51Ϫ4.60 (m, 1 H, α-CH Ser), 5.32 (d, J ϭ 8.1 Hz, 1 H, α-NH Phe), 129.5 (C-5), 130.0 (C-6), 135.5 (C-1), 135.9 (C-1 Phe), 143.5
Ser), 6.75 (d, J ϭ 9.0 Hz, 2 H, 2 ϫ Ar-H ortho NMe2), 7.29 (d,
(C-4Ј), 152.4 (C-3), 153.5 (C-1Ј), 170.1 (CONH), 170.4 (CONH),
J ϭ 7.5 Hz, 1 H, 4-H or 6-H), 7.44 (t, J ϭ 7.5 Hz, 1 H, 5-H), 170.7 (CO2CMe3) ppm. C34H43N5O4 (585.72): calcd. C 69.72, H
7.70Ϫ7.80 (m, 2 H, 2-H, 6-H or 4-H), 7. 90 (d, J ϭ 9.0 Hz, 2 H, 2
ϫ Ar-H meta NMe2) ppm. 13C NMR (75.4 MHz, CDCl3): δ ϭ
28.2 (CMe3), 40.2 (NMe2), 40.9 (CH2), 52.7 (OMe), 52.8 (α-C Ser),
64.7 (β-C Ser), 80.2 (CMe3), 111.4 (C-2Ј, C-6Ј), 121.7 (C-4), 122.3
(C-2), 124.9 (C-3Ј, C-5Ј), 129.1 (C-5), 130.0 (C-6), 134.3 (C-1),143.4
(C-4Ј), 152.4 (C-3), 153.2 (C-1Ј), 155.1 (CO2CMe3), 170.1 (CONH),
170.7 (CO2CH2), 171.3 (CO2Me) ppm. C25H32N4O6 (484.54):
calcd. C 61.97, H 6.66, N 11.56; found C 62.04, H 6.75, N 11.41.
7.40, N 11.96; found C 69.53, H 7.44, N 11.95.
N-{3-[(N,N-Dimethylaminophenyl)-4Ј-diazenyl]phenylacetyl}-
phenylalanylvaline tert-Butyl Ester (12c): The product of the reac-
tion of dye 1 (255 mg, 0.90 mmol) with phenylalanylvaline tert-bu-
tyl ester 11c[24] (288 mg, 0.90 mmol) under the conditions described
above for acylation with compounds 1Ϫ4 was chromatographed
with diethyl ether/hexane (mixtures of increasing polarity) as the
eluent to give the labeled dipetide 12c (500 mg, 94%). M.p.
124.7Ϫ126.0 °C. TLC (diethyl ether/hexane, 6:4): Rf ϭ 0.69. UV/
Vis (MeOH): λmax ϭ 411 nm (ε ϭ 25029 dm3·molϪ1·cmϪ1). 1H
NMR (300 MHz, CDCl3): δ ϭ 0.82 (dd, J ϭ 6.6, 1.5 Hz, 6 H, γ-
N-{3-[(N,N-Dimethylaminophenyl)-4Ј-diazenyl]phenylacetyl}-
alanylphenylalanine tert-Butyl Ester (12a): The product of the reac-
tion of dye 1 (342 mg, 1.12 mmol) with alanylphenylalanine tert-
butyl ester 11a (327 mg, 1.12 mmol) under the conditions described CH3 Val), 1.46 (s, 9 H, CMe3), 2.02Ϫ2.14 (m, 1 H, β-CH Val), 3.00
above for acylation with compounds 1Ϫ4 was chromatographed
with diethyl ether/hexane (4:6) as the eluent to give the labeled dipe-
(d, J ϭ 6.90 Hz, 2 H, β-CH2 Phe), 3.11 (s, 6 H, NMe2), 3.63 (s, 2
H, CH2), 4.30Ϫ4.34 (m, 1 H, α-CH Val), 4.64Ϫ4.72 (m, 1 H, α-
tide 12a (524 mg, 84%). M.p. 119.7Ϫ120.4 °C. TLC (diethyl ether/ CH Phe), 5.95 (d, J ϭ 7.5 Hz, 1 H, α-NH Phe), 6.39 (d, J ϭ 8.1 Hz,
hexane, 8:2): Rf ϭ 0.82. UV/Vis (MeOH): λmax ϭ 410 nm (ε ϭ 1 H, α-NH Val), 6.78 (d, J ϭ 9.3 Hz, 2 H, 2 ϫ Ar-H ortho NMe2),
28430 dm3·molϪ1·cmϪ1). 1H NMR (300 MHz, CDCl3): δ ϭ 1.28 7.02 (dd, J ϭ 7.8, 1.8 Hz, 2 H, 2 ϫ Ar-H Phe), 7.12Ϫ7.27 (m, 4
(d, J ϭ 6.9 Hz, 3 H, β-CH3 Ala), 1.41 (s, 9 H, CMe3), 3.00Ϫ3.09
H, 3 ϫ Ar-H Phe, 6-H or 4-H), 7.42 (t, J ϭ 7.8 Hz, 1 H, 5-H), 7.65
(m, 2 H, β-CH2 Phe), 3.10 (s, 6 H, NMe2), 3.63 (d, J ϭ 1.5 Hz, 2 (tap, 1.8 Hz, 1 H, 2-H), 7.75Ϫ7.82 (m, 1 H, 4-H or 6-H), 7.90 (d,
1756
 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
Eur. J. Org. Chem. 2004, 1750Ϫ1760

Chromophoric Azo Reagents for Amino Acid and Peptide Labelling
FULL PAPER
J ϭ 9.0 Hz, 2 H, 2 ϫ Ar-H meta NMe2) ppm. The assignments tion of acylamino acid 13 (87 mg, 0.20 mmol) with isoleucine
were supported by spin decoupling-double resonance technique.13C
methyl ester hydrochloride (36.7 mg, 0.20 mmol) under the con-
NMR (75.4 MHz, CDCl3): δ ϭ 17.6 (γ-C Val), 18.7 (γ-C Val), 28.0 ditions described above for acylation with dyes 1Ϫ4 was chromato-
(CMe3), 31.2 (β-C Val), 37.4 (β-C Phe), 40.2 (NMe2), 43.5 (CH2), graphed with chloroform/methanol (5.8:0.2) as the eluent to give
54.2 (α-C Phe), 57.5 (α-C Val), 81.9 (CMe3), 111.4 (C-2Ј, C-6Ј), dipeptide 14b (73 mg, 65%). M.p. 165.5Ϫ167.0 °C. TLC (chloro-
121.7 (C-4), 122.7 (C-2), 125.0 (C-3Ј, C-5Ј), 126.8 (C-4 Phe), 128.6 form/methanol, 5.8:0.2): Rf ϭ 0.65. UV/Vis (MeOH): λmax
(C-3 Phe, C-5 Phe), 129.1 (C-2 Phe, C-6 Phe), 129.6 (C-5), 130.1
(C-6), 135.0 (C-1), 136.0 (C-1 Phe), 143.5 (C-4Ј), 152.5 (C-3), 153.5
ϭ
410 nm (ε ϭ 24986 dm3·molϪ1·cmϪ1). 1H NMR (300 MHz,
CDCl3): δ ϭ 0.78 (d, J ϭ 6.6 Hz, 3 H, γ-CH3 Ile), 0.86 (t, J ϭ
(C-1Ј), 170.3 (CONH), 170.7 (CONH, CO2CMe3) ppm. 7.50 Hz, 3 H, δ-CH3 Ile), 0.92Ϫ1.37 (2 ϫ m, 2 H, γ-CH2 Ile),
C34H43N5O4 (585.72): calcd. C 69.72, H 7.40, N 11.96; found C
69.76, H 7.60, N 11.96.
1.70Ϫ1.87 (m, 1 H, β-CH Ile), 3.01 (d, J ϭ 6.9 Hz, 2 H, β-CH2
Phe), 3.11 (s, 6 H, NMe2), 3.63 (s, 2 H, CH2), 3.70 (s, 3 H, OMe),
4.42Ϫ4.50 (m, 1 H, α-CH Ile), 4.65Ϫ4.70 (m, 1 H, α-CH Phe), 6.01
(d, J ϭ 7.0 Hz, 1 H, α-NH Phe), 6.45 (d, J ϭ 7.3 Hz, 1 H, α-NH
Ile), 6.78 (d, J ϭ 9.3 Hz, 2 H, 2 ϫ Ar-H ortho NMe2), 7.04 (dd,
J ϭ 7.7, 1.5 Hz, 2 H, 2 ϫ Ar-H Phe), 7.10Ϫ7.20 (m, 4 H, 3 ϫ Ar-
H Phe, 6-H or 4-H), 7.41 (t, J ϭ 7.3 Hz, 1 H, 5-H), 7.62 (tap, J ϭ
2.1 Hz, 1 H, 2-H), 7.78 (d, J ϭ 7.3 Hz, 1 H, 4-H or 6-H), 7.90 (d,
J ϭ 9.0 Hz, 2 H, 2 ϫ Ar-H meta NMe2) ppm. The assignments
were supported by spin decoupling-double resonance technique.
13C NMR (75.4 MHz, CDCl3): δ ϭ 11.5 (γ-CH3 Ile), 15.3 (δ-C Ile),
25.0 (γ-CH2 Ile), 37.3 (β-C Phe), 37.6 (β-C Ile), 40.3 (NMe2), 43.5
(CH2), 52.1 (OMe), 54.3 (α-C Phe), 56.6 (α-C Ile), 111.4 (C-2Ј, C-
6Ј), 121.8 (C-4), 122.7 (C-2), 125.1 (C-3Ј, C-5Ј), 126.9 (C-4 Phe),
128.7 (C-3 Phe, C-5 Phe), 129.2 (C-2 Phe, C-6 Phe), 129.6 (C-5),
130.0 (C-6), 134.9 (C-1), 136.0 (C-1 Phe), 143.5 (C-4Ј), 152.5 (C-
3), 153.6 (C-1Ј), 170.4 (CONH), 170.8 (CONH), 171.7 (CO2Me)
ppm. C32H39N5O4 (557.67): calcd. C 68.92, H 7.05, N 12.56; found
C 68.79, H 6.83, N 12.49.
N-{3-[(N,N-Dimethylaminophenyl)-4Ј-diazenyl]phenylacetyl}-
phenylalanine (13): NaOH (1 ; 1.23 mL, 1.23 mmol) was added to
the fully protected amino acid 5c (375 mg; 0.82 mmol) in 1,4-diox-
ane (4.10 mL) at 0 °C (ice bath). The solution was stirred for 6
hours at 0 °C and acidified to pH 2Ϫ3 with 1  KHSO4. After
extraction with ethyl acetate and evaporation of the solvent the
required acylamino acid 13 was obtained as an orange solid
(353 mg; 100%). M.p. 117.9Ϫ119.0 °C. IR (film): ν˜ ϭ 3405, 2925,
2854, 1726, 1648, 1601, 1519, 1459, 1376, 1152, 944, 823, 723 cmϪ1
.
1H NMR (300 MHz, CDCl3): δ ϭ 2.80Ϫ3.00 (m, 2 H, β-CH2 Phe),
3.08 (s, 6 H, NMe2), 3.61 (s, 2 H, CH2), 4.80Ϫ4.91 (m, 1 H, α-CH
Phe), 6.17 (d, J ϭ 7.5 Hz, 1 H, α-NH Phe), 6.76 (d, J ϭ 9.0 Hz, 2
H, 2 ϫ Ar-H ortho NMe2), 6.90Ϫ7.00 (m, 2 H, 2 ϫ Ar-H Phe),
7.08Ϫ7.20 (m, 4 H, 3 ϫ Ar-H Phe, 4-H or 6-H), 7.39 (t, J ϭ 7.8 Hz,
1 H, 5-H), 7.65 (s, 1 H, 2-H), 7.76 (d, J ϭ 8.4 Hz, 1 H, 6-H or
4-H), 7.89 (d, J ϭ 9.0 Hz, 2 H, 2 ϫ Ar-H meta NMe2) ppm. 13C
NMR (75.4 MHz, CDCl3): δ ϭ 36.9 (β-C Phe), 40.3 (NMe2), 43.4
(CH2), 53.2 (α-C Phe), 111.5 (C-2Ј, C-6Ј), 121.7 (C-4), 122.8 (C-2),
125.1 (C-3Ј, C-5Ј), 127.1 (C-4 Phe), 128.7 (C-3 Phe, C-5 Phe), 129.2
(C-2 Phe, C-6 Phe), 129.7 (C-5), 130.2 (C-6), 134.8 (C-1), 135.3 (C-
1 Phe), 143.5 (C-4Ј), 152.6 (C-3), 153.5 (C-1Ј), 171.4 (CONH),
173.8 (CO2H) ppm.
N-{3-[(N,N-Dimethylaminophenyl)-4Ј-diazenyl]phenylacetyl}-
phenylalanylvaline Methyl Ester (14c): The product of the reaction
of acylamino acid 13 (200 mg, 0.47 mmol) with valine methyl ester
hydrochloride (78 mg, 0.47 mmol) under the conditions described
above for acylation with dyes 1Ϫ4 was chromatographed with di-
ethyl ether/hexane (6:4) as the eluent to give dipeptide 14c (152 mg,
60%). M.p. 164.0Ϫ165.9 °C. TLC (diethyl ether/hexane, 8:2): Rf ϭ
N-{3-[(N,N-Dimethylaminophenyl)-4Ј-diazenyl]phenylacetyl}-
phenylalanylglycine Methyl Ester (14a): The product of the reaction
of acylamino acid 13 (300 mg, 0.70 mmol) with glycine methyl ester
hydrochloride (88 mg, 0.70 mmol) under the conditions described
above for acylation with dyes 1Ϫ4 was chromatographed with
chloroform/methanol (5.8:0.2) as the eluent to give dipeptide 14a
(284 mg, 81%). M.p. 171.5Ϫ173.6 °C. TLC (chloroform/methanol,
5.8:0.2): Rf ϭ 0.51. UV (MeOH): λmax ϭ 409 nm (ε ϭ 18532
0.63. UV/Vis (MeOH): λmax
ϭ
411 nm (ε
ϭ
20256
1
dm3·molϪ1·cmϪ1). H NMR (300 MHz, CDCl3): δ ϭ 0.81 (t, J ϭ
7.5 Hz, 6 H, γ-CH3 Val), 2.0Ϫ2.19 (m, 1 H, β-CH Val), 3.01 (d,
J ϭ 7.2 Hz, 2 H, β-CH2 Phe), 3.11 (s, 6 H, NMe2), 3.63 (s, 2 H,
CH2), 3.70 (s, 3 H, OMe), 4.38Ϫ4.45 (m, 1 H, α-CH Val),
4.64Ϫ4.76 (m, 1 H, α-CH Phe), 6.07 (d, J ϭ 7.5 Hz, 1 H, α-NH
Phe), 6.51 (d, J ϭ 8.4 Hz, 1 H, α-NH Val), 6.78 (d, J ϭ 9.3 Hz, 2
H, 2 ϫ Ar-H ortho NMe2), 7.05 (d, J ϭ 6.9 Hz, 2 H, 2 ϫ Ar-H
Phe), 7.10Ϫ7.23 (m, 4 H, 3 ϫ Ar-H Phe, 6-H or 4-H), 7.42 (t, J ϭ
7.8 Hz, 1 H, 5-H), 7.65 (s, 1 H, 2-H), 7.78 (d, J ϭ 8.1 Hz, 1 H, 4-
H or 6-H), 7.89 (d, J ϭ 9.0 Hz, 2 H, 2 ϫ Ar-H meta NMe2) ppm.
The assignments were supported by spin decoupling-double reson-
ance technique. 13C NMR (75.4 MHz, CDCl3): δ ϭ 17.7 (γ-C Val),
18.8 (γ-C Val), 31.0 (β-C Val), 37.3 (β-C Phe), 40.3 (NMe2), 43.5
(CH2), 52.1 (OMe), 54.3 (α-C Phe), 57.3 (α-C Val), 111.4 (C-2Ј, C-
6Ј), 121.8 (C-4), 122.7 (C-2), 125.1 (C-3Ј, C-5Ј), 126.9 (C-4 Phe),
128.6 (C-3 Phe, C-5 Phe), 129.2 (C-6 Phe, C-2 Phe), 129.6 (C-5),
130.0 (C-6), 134.9 (C-1), 136.0 (C-1 Phe), 143.5 (C-4Ј), 152.5 (C-
3), 153.6 (C-1Ј), 170.6 (CONH), 170.9 (CONH), 171.7 (CO2Me)
ppm. C31H37N5O4 (543.65): calcd. C 68.48, H 6.86, N 12; found C
68.27, H 6.81, N 12.88.
1
dm3·molϪ1·cmϪ1). H NMR (300 MHz, CDCl3): δ ϭ 3.02 (t, J ϭ
6.9 Hz, 2 H, β-CH2 Phe), 3.11 (s, 6 H, NMe2), 3.63 (s, 2 H, CH2),
3.72 (s, 3 H, OMe), 3.96 (t, J ϭ 6.0 Hz, 2 H, CH2 Gly), 4.64Ϫ4.75
(m, 1 H, α-CH Phe), 6.02 (d, J ϭ 7.5 Hz, 1 H, α-NH Phe), 6.56
(tap, J ϭ 5.1 Hz, 1 H, α-NH Gly), 6.78 (d, J ϭ 9.0 Hz, 2 H, 2 ϫ
Ar-H ortho NMe2), 7.04 (dd, J ϭ 6.0, 1.2 Hz, 2 H, 6-H or 4-H, 1
ϫ Ar-H Phe), 7.12Ϫ7.23 (m, 4 H, 4 ϫ Ar-H Phe), 7.43 (t, J ϭ
8.1 Hz, 1 H, 5-H), 7.63 (tap, J ϭ 1.8 Hz, 1 H, 2-H), 7.78 (d, J ϭ
8.1 Hz, 1 H, 4-H or 6-H), 7.90 (d, J ϭ 9.0 Hz, 2 H, 2 ϫ Ar-H meta
NMe2) ppm. The assignments were supported by spin decoupling-
double resonance technique. 13C NMR (75.4 MHz, CDCl3): δ ϭ
37.4 (β-C Phe), 40.3 (NMe2), 41.2 (CH2 Gly), 43.4 (CH2), 52.4
(OMe), 54.2 (α-C Phe), 111.5 (C-2Ј, C-6Ј), 121.8 (C-4), 122.7 (C-
2), 125.1 (C-3Ј, C-5Ј), 127.0 (C-4 Phe), 128.7 (C-3 Phe, C-5 Phe),
129.1 (C-2 Phe, C-6 Phe), 129.7 (C-5), 130.1 (C-6), 134.9 (C-1),
136.0 (C-1 Phe), 143.2 (C-4Ј), 152.5 (C-3), 153.6 (C-1Ј), 169.7
(CONH), 170.9 (CONH), 171.1 (CO2Me) ppm. C28H31N5O4
(501.57): calcd. C 67.05, H 6.23, N 13.96; found C 66.92, H 6.31,
N 13.62.
N-{3-[(N,N-Dimethylaminophenyl)-4Ј-diazenyl]phenylacetyl}-
phenylalanylalanine Methyl Ester (14d): The product of the reaction
of acylamino acid 13 (254 mg, 0.59 mmol) with alanine methyl ester
hydrochloride (82.4 mg, 0.59 mmol) under the conditions described
above for acylation with dyes 1Ϫ4 was chromatographed with
chloroform/methanol (5.8:0.2) as the eluent to give dipeptide 14d
N-{3-[(N,N-Dimethylaminophenyl)-4Ј-diazenyl]phenylacetyl}-
phenylalanylisoleucine Methyl Ester (14b): The product of the reac-
Eur. J. Org. Chem. 2004, 1750Ϫ1760
www.eurjoc.org
 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
1757

S. M. B. Fraga, M. S. T. Gonc¸alves, J. C. V. P. Moura, K. Rani
J ϭ 6.6 Hz, 2 H, 2 ϫ Ar-H Phe), 7.20Ϫ7.40 (m, 8 H, 5 ϫ Ar-H Z,
FULL PAPER
(252 mg, 84%). M.p. 166.8Ϫ168.5 °C. TLC (diethyl ether/hexane,
6:4): Rf ϭ 0.77. UV/Vis (MeOH): λmax ϭ 415 nm (ε ϭ 21587 3 ϫ Ar-H Phe), 7.43 (t, J ϭ 7.8 Hz, 1 H, 5-H), 7.70 (m, 2 H, 4-H,
1
dm3·molϪ1·cmϪ1). H NMR (300 MHz, CDCl3): δ ϭ 1.32 (d, J ϭ 6-H), 7.82 (s, 1 H, 2-H), 7.88 (d, J ϭ 9.0 Hz, 2 H, 2 ϫ Ar-H meta
7.2 Hz, 3 H, β-CH3 Ala), 3.00 (d, J ϭ 7.70 Hz, 2 H, β-CH2 Phe), NMe2) ppm. The assignments were supported by spin decoupling-
3.10 (s, 6 H, NMe2), 3.62 (s, 2 H, CH2), 3.71 (s, 3 H, OMe), double resonance technique. 13C NMR (75.4 MHz, CDCl3): δ ϭ
4.40Ϫ4.50 (m, 1 H, α-CH Ala), 4.63Ϫ4.72 (m, 1 H, α-CH Phe), 14.0 (OCH2CH3), 22.1 (γ-C Lys), 28.7 (β-C Lys), 33.8 (δ-C Lys),
6.17 (d, J ϭ 7.5 Hz, 1 H, α-NH Phe), 6.60 (d, J ϭ 7.5 Hz, 1 H, α- 37.7 (β-C Phe), 38.8 (ε-C Lys), 40.2 (NMe2), 43.5 (CH2), 53.2 (α-
NH Ala), 6.76 (d, J ϭ 9.3 Hz, 2 H, 2 ϫ Ar-H ortho NMe2),
C Phe), 54.5 (α-C Lys), 61.5 (OCH2CH3), 66.9 (CH2 Z), 111.4 (C-
7.01Ϫ7.08 (m, 2 H, 2 ϫ Ar-H Phe), 7.12Ϫ7.22 (m, 4 H, 3 ϫ Ar-H 2Ј, C-6Ј), 121.3 (C-4), 122.8 (C-3Ј, C-5Ј), 125.0 (C-3 Z, C-5 Z),
Phe, 6-H or 4-H), 7.42 (t, J ϭ 8.1 Hz, 1 H, 5-H), 7.65 (tap, J ϭ 127.0 (C-2), 127.9 (C-4 Phe), 128.1 (C-3 Phe, C-5 Phe), 128.4 (C-4
1.5 Hz, 1 H, 2-H), 7.77 (d, J ϭ 8.1 Hz, 1 H, 4-H or 6-H), 7.89 (d, Z, C-2 Z, C-6 Z), 129.2 (C-2 Phe, C-6 Phe), 129.5 (C-5), 130.1 (C-
J ϭ 9.3 Hz, 2 H, 2 ϫ Ar-H meta NMe2) ppm. The assignments
were supported by spin decoupling-double resonance technique.
6), 135.7 (C-1), 135.8 (C-1 Z), 136.1 (C-1 Phe), 143.4 (C-4Ј), 152.4
(C-3), 153.5 (C-1Ј), 156.1 (NHCO2CH2Ph), 171.0 (CONH Lys),
13C NMR (75.4 MHz, CDCl3): δ ϭ 18.1 (β-C Ala), 37.7 (β-C Phe), 171.3 (CONH Phe, CO2Et) ppm. C41H48N6O6 (720.84): calcd. C
40.4 (NMe2), 43.5 (CH2), 48.1 (α-C Ala), 52.4 (OMe), 54.2 (α-C 68.31, H 6.71, N 11.66; found C 68.05, H 6.95, N 11.57.
Phe), 111.5 (C-2Ј, C-6Ј), 121.7 (C-4), 122.7 (C-2), 125.0 (C-3Ј, C-
N-Benzyloxycarbonyl-ω-{3-[(N,N-dimethylaminophenyl)-4Ј-
diazenyl]phenylacetyl}lysylalanine Methyl Ester (16d): The product
of the reaction of 15 (70 mg, 0.13 mmol) with alanine methyl ester
hydrochloride (17.4 mg, 0.13 mmol) under the conditions described
above for acylation with dyes 1Ϫ4 was chromatographed with
chloroform/methanol (5.8:0.2) as the eluent to give the fully pro-
tected dipeptide 16d (77 mg, 98%). M.p. 152.6Ϫ154.6 °C. TLC
(chloroform/methanol, 5.8:0.2): Rf ϭ 0.30. UV/Vis (MeOH):
5Ј), 126.9 (C-4 Phe), 128.6 (C-3 Phe, C-5 Phe), 129.2 (C-2 Phe, C-
6 Phe), 129.6 (C-5), 130.1 (C-6), 135.1 (C-1), 136.0 (C-1 Phe), 143.5
(C-4Ј), 152.5 (C-3), 153.5 (C-1Ј), 170.2 (CONH), 170.8 (CONH),
172.8 (CO2Me) ppm. C29H33N5O4 (515.59): calcd. C 67.55, H 6.45,
N 13.58; found C 67.65, H 6.67, N 13.49.
N-Benzyloxycarbonyl-ω-{3-[(N,N-dimethylaminophenyl)-4Ј-
diazenyl]phenylacetyl}lysine (15): NaOH (1 ; 0.5 mL, 0.5 mmol)
was added to the fully protected amino acid 9 (187 mg, 0.34 mmol)
λ
max ϭ 414 nm (ε ϭ 22069 dm3·molϪ1·cmϪ1). 1H NMR (300 MHz,
in 1,4-dioxane (1.7 mL) at 0 °C (ice bath). The solution was stirred CDCl3): δ ϭ 1.26Ϫ1.40 (m, 3 H, β-CH3 Ala), 1.41Ϫ1.54 (m, 2 H,
at 0 °C for 6 hours and acidified to pH 2Ϫ3 with 1  KHSO4.
After extraction with ethyl acetate and evaporation of the solvent,
γ-CH2 Lys), 1.56Ϫ1.78 (m, 2 H, β-CH2 Lys), 1.80Ϫ2.00 (m, 2 H,
δ-CH2 Lys), 3.10 (s, 6 H, NMe2), 3.13Ϫ3.31 (m, 2 H, ε-CH2 Lys),
3.63 (s, 2 H, CH2), 3.73 (s, 3 H, OMe), 4.05Ϫ4.20 (m, 1 H, α-CH
acylamino acid 15 was obtained as an orange solid (178 mg, 98%).
1
M.p. 74.5Ϫ76.2 °C. H NMR (300 MHz, CDCl3): δ ϭ 1.30Ϫ1.50 Lys), 4.48Ϫ4.60 (m, 1 H, α-CH Ala), 5.11 (s, 2 H, CH2 Z),
(m, 2 H, γ-CH2 Lys), 1.60Ϫ1.81 (m, 2 H, β-CH2 Lys), 1.82Ϫ1.95 5.44Ϫ5.60 (m, 1 H, α-NH Lys), 5.66Ϫ5.75 (m, 1 H, ω-NH Lys),
(m, 2 H, δ-CH2 Lys), 3.00 (s, 6 H, NMe2), 3.10Ϫ3.20 (m, 2 H, 6.60Ϫ6.73 (m, 1 H, α-NH Ala), 6.76 (d, J ϭ 9.3 Hz, 2 H, 2 ϫ Ar-
ε-CH2 Lys), 3.60 (s, 2 H, CH2), 4.27Ϫ4.40 (m, 1 H, α-CH Lys),
5.10 (s, 2 H, CH2 Z), 5.72 (d, J ϭ 8.1 Hz, 1 H, α-NH Lys), 5.92 7.45 (t, J ϭ 7.8 Hz, 1 H, 5-H), 7.70Ϫ7.80 (m, 2 H, 4-H or 6-H, 2-
(tap, J ϭ 5.9 Hz, 1 H, ω-NH Lys), 6.73 (d, J ϭ 9.3 Hz, 2 H, 2 ϫ H), 7.87 (d, J ϭ 9.3 Hz, 2 H, 2 ϫ Ar-H meta NMe2) ppm. The
Ar-H ortho NMe2), 7.20Ϫ7.40 (m, 6 H, 5 ϫ Ar-H Z, 6-H or 4-H), assignments were supported by spin decoupling-double resonance
7.42 (t, J ϭ 7.8 Hz, 1 H, 5-H), 7.68 (s, 1 H, 2-H), 7.73 (d, J ϭ
technique. 13C NMR (75.4 MHz, CDCl3): δ ϭ 17.8 (β-C Ala), 22.0
8.1 Hz, 1 H, 4-H or 6-H), 7.86 (d, J ϭ 9.3 Hz, 2 H, 2 ϫ Ar-H meta (γ-C Lys), 28.7 (β-C Lys), 33.8 (δ-C Lys), 38.8 (ε-C Lys), 40.2
H ortho NMe2), 7.20Ϫ7.40 (m, 6 H, 5 ϫ Ar-H Z, 6-H or 4-H),
NMe2) ppm. The assignments were supported by spin decoupling-
(NMe2), 43.5 (CH2), 48.0 (α-C Ala), 52.4 (OMe), 54.4 (α-C Lys),
66.9 (CH2 Z), 111.4 (C-2Ј, C-6Ј), 121.3 (C-4), 122.8 (C-3Ј, C-5Ј),
double resonance technique. 13C NMR (75.4 MHz, CDCl3): δ ϭ
21.8 (γ-C Lys), 28.5 (β-C Lys), 31.7 (δ-C Lys), 39.1 (ε-C Lys), 40.2 125.0 (C-3 Z, C-5 Z), 128.0 (C-2), 128.1 (C-4 Z), 128.4 (C-2 Z, C-
(NMe2), 43.3 (CH2), 53.5 (α-C Lys), 66.8 (CH2 Z), 111.4 (C-2Ј, 6 Z), 129.5 (C-5), 130.2 (C-6), 135.8 (C-1), 136.2 (C-1 Z), 143.4 (C-
C-6Ј), 121.2 (C-4), 122.9 (C-3Ј, C-5Ј), 125.2 (C-2), 127.9 (C-3 Z, C- 4Ј), 152.5 (C-3), 153.4 (C-1Ј), 156.2 (NHCO2CH2Ph), 171.0
5 Z), 128.0 (C-4 Z), 128.4 (C-2 Z, C-6 Z), 129.6 (C-5), 130.3 (C-6), (CONH Lys), 171.4 (CONH Ala), 173.2 (CO2Me) ppm.
135.5 (C-1), 136.2 (C-1 Z), 143.1 (C-4Ј), 152.6 (C-3), 153.3 (C-1Ј), C34H42N6O6 (630.72): calcd. C 64.74, H 6.71, N 13.33; found C
156.1 (NHCO2CH2Ph), 171.7 (CONH Lys), 174.5 (CO2Me) ppm.
64.79, H 6.70, N 13.23.
N-Benzyloxycarbonyl-ω-{3-[(N,N-dimethylaminophenyl)-4Ј-
diazenyl]phenylacetyl}lysylphenylalanine Ethyl Ester (16c): The
product of the reaction of 15 (100 mg, 0.18 mmol) with phenylala-
nine ethyl ester hydrochloride (41.1 mg, 0.18 mmol) under the con-
ditions described above for acylation with dyes 1Ϫ4 was chromato-
N-{3-[(N,N-Dimethylaminophenyl)-4Ј-diazenyl]phenylacetyl}-N-tert-
butyloxycarbonylphenylalanine Ethyl Ester (17c): DMAP (4.3 mg,
0.035 mmol) was added to a solution of 5c (161 mg, 0.35 mmol) in
dry acetonitrile (5.0 mL) followed by di-tert-butyl pyrocarbonate
(368 mg, 1.68 mmol) and the mixture stirred vigourously for two
graphed with chloroform/methanol (5.8:0.2) as the eluent to give days at room temperature, the reaction being monitored by TLC.
the fully protected dipeptide 16c (117 mg, 91%). M.p. 142.7Ϫ144.7
Evaporation of the solvents under reduced pressure followed by
dry chromatography on silica gel with diethyl ether/hexane (6:4) as
°C. TLC (chloroform/methanol, 5.8:0.2): Rf ϭ 0.44. UV/Vis
1
(MeOH): λmax ϭ 412 nm (ε ϭ 18704 dm3·molϪ1·cmϪ1). H NMR the eluent gave ester 17c (91 mg, 46%). TLC (diethyl ether/hexane,
(300 MHz, CDCl3): δ ϭ 1.20 (t, J ϭ 6.9 Hz, 3 H, OCH2CH3), 6:4): Rf ϭ 0.53. UV/Vis (MeOH): λmax ϭ 416 nm (ε ϭ 17646
1.21Ϫ1.50 (m, 2 H, γ-CH2 Lys), 1.52Ϫ1.70 (m, 2 H, β-CH2 Lys), dm3·molϪ1·cmϪ1). IR (film): ν˜ ϭ 3252, 3000, 1749, 1732, 1714,
1.71Ϫ2.00 (m, 2 H, δ-CH2 Lys), 3.00Ϫ3.30 (2 ϫ m, 10 H, NMe2, 1692, 1676, 1602, 1520, 1456, 1371, 1355 cmϪ1
.
1H NMR
(300 MHz, CDCl3): δ ϭ 1.22 (t, J ϭ 7.0 Hz, 3 H, OCH2CH3), 1.44
OCH2CH3), 4.28Ϫ4.30 (m, 1 H, α-CH Lys), 4.74Ϫ4.88 (m, 1 H, (s, 9 H, CMe3), 3.09 (s, 6 H, NMe2), 3.40Ϫ3.52 (m, 2 H, β-CH2
α-CH Phe), 5.08 (s, 2 H, CH2 Z), 5.60 (d, J ϭ 7.8 Hz, 1 H, α-NH Phe), 4.00Ϫ4.30 (m, 4 H, OCH2CH3, CH2), 5.49Ϫ5.58 (m, 1 H, α-
Lys), 5.80 (br. s, 1 H, ω-NH Lys), 6.64 (d, J ϭ 8.3 Hz, 1 H, α-NH CH Phe), 6.77 (d, J ϭ 9.3 Hz, 2 H, 2 ϫ Ar-H ortho NMe2), 7.07
Phe), 6.74 (d, J ϭ 9.0 Hz, 2 H, 2 ϫ Ar-H ortho NMe2), 7.10 (d, (d, J ϭ 8.1 Hz, 1 H, 6-H or 4-H), 7.10Ϫ7.30 (m, 5 H, 5 ϫ Ar-H
ε-CH2 Lys, β-CH2 Phe), 3.60 (s, 2 H, CH2), 4.00Ϫ4.20 (m, 2 H,
1758
 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
Eur. J. Org. Chem. 2004, 1750Ϫ1760

Chromophoric Azo Reagents for Amino Acid and Peptide Labelling
FULL PAPER
Phe), 7.38 (t, J ϭ 8.1 Hz, 1 H, 5-H), 7.58 (t, J ϭ 1.5 Hz, 1 H, 2-
4-H Phe, 6-H Phe) ppm. 13C NMR (75.4 MHz, CDCl3): δ ϭ 14.0
H), 7.73 (d, J ϭ 8.1 Hz, 1 H, 4-H or 6-H), 7.88 (d, J ϭ 9.3 Hz, 2 (OCH2CH3), 37.6 (β-C Phe), 43.6 (CH2), 52.9 (α-C Phe), 61.4
H, 2 ϫ Ar-H meta NMe2) ppm. 13C NMR (75.4 MHz, CDCl3): (OCH2CH3), 114.1 (C-4), 115.8 (C-2), 119.4 (C-6), 126.9 (C-4 Phe),
δ ϭ 14.1 (OCH2CH3), 27.8 (CMe3), 35.7 (β-C Phe), 40.3 (NMe2), 128.4 (C-3 Phe, C-5 Phe), 129.2 (C-5), 129.9 (C-2 Phe, C-6 Phe),
44.1 (CH2), 57.4 (α-C Phe), 61.3 (OCH2CH3), 84.1 (CMe3), 111.4 135.4 (C-1), 135.7 (C-1 Phe), 146.9 (C-3), 170.5 (CONH), 171.3
(C-2Ј, C-6Ј), 121.1 (C-4), 123.0 (C-2), 124.8 (C-3Ј, C-5Ј), 126.6 (C- (CO2CH2CH3) ppm. HRMS: calcd. for C19H22N2O3 [Mϩ]
4 Phe), 128.4 (C-3 Phe, C-5 Phe), 129.4 (C-2 Phe, C-6 Phe), 129.5
(C-5), 130.6 (C-6), 135.6 (C-1), 137.4 (C-1 Phe), 143.6 (C-4Ј), 151.9
(C-3), 152.3 (C-1Ј), 153.1 (CO2CMe3), 170.2 (CONH), 173.4
(CO2CH2CH3) ppm. HRMS: calcd. for C32H38N405 [Mϩ]
558.2842; found 558.2818.
326.1630; found 326.1630.
N-(3-Aminophenylacetyl)alanine Methyl Ester 19d by Chemical Re-
duction of 5d: Reduction of 5d (100 mg, 0.27 mmol) with zinc dust
(100 mg, 1.53 mmol) according to the procedure described above
for compound 19c gave the corresponding ester 19d (42 mg, 65%)
as an oil. TLC (ethyl acetate/hexane, 8:2): Rf ϭ 0.31. IR (film): ν˜ ϭ
3360, 3042, 2986, 2953, 1739, 1658, 1651, 1607, 1538, 1494, 1461,
N-{3-[(N,N-Dimethylaminophenyl)-4Ј-diazenyl]phenylacetyl}-N-tert-
butyloxycarbonylalanine Methyl Ester (17d): The product of the re-
action of 5d (100 mg, 0.27 mmol) with DMAP and di-tert-butyl
pyrocarbonate under the conditions reported above for compound
17c was chromatographed with diethyl ether/hexane (6:4) as the
eluent to give ester 17d (56 mg, 48%) as an orange residue. M.p.
92.0Ϫ94.0 °C. TLC (diethyl ether/hexane, 6:4): Rf ϭ 0.53. UV/Vis
1436, 1375, 1357, 1300, 1245, 1216, 1168, 1055, 773 cmϪ1 1H
.
NMR (300 MHz, CDCl3): δ ϭ 1.34 (d, J ϭ 7.2 Hz, 3 H, β-CH3
Ala), 3.71 (s, 5 H, CH2, OMe), 4.50Ϫ4.64 (m, 1 H, α-CH Ala),
6.07 (d, J ϭ 5.4 Hz, 1 H, α-NH Ala), 6.56Ϫ6.70 (m, 3 H, 2-H, 4-
H, 6-H), 7.14 (t, J ϭ 7.5 Hz, 1 H, 5-H) ppm. The assignments were
supported by spin decoupling-double resonance technique. 13C
NMR (75.4 MHz, DMSO): δ ϭ 18.2 (β-C Ala), 43.6 (CH2), 48.0
(α-C Ala), 52.4 (OMe), 114.1 (C-4), 115.9 (C-2), 119.4 (C-6), 130.0
(C-5), 135.5 (C-1), 147.0 (C-3), 170.6 (CONH), 173.3 (CO2Me)
ppm. HRMS: calcd. for C12H16N2O3 [Mϩ] 236.1161; found
236.1160.
1
(MeOH): λmax ϭ 417 nm (ε ϭ 23090 dm3·molϪ1·cmϪ1). H NMR
(300 MHz, CDCl3): δ ϭ 1.46 (s, 9 H, CMe3), 1.48 (s, 3 H, β-CH3
Ala), 3.09 (s, 6 H, NMe2), 3.67 (s, 3 H, OMe), 4.39 (d, J ϭ 1.8 Hz,
2 H, CH2), 5.33, 5.37 (2 ϫ d, J ϭ 6.6, 6.0 Hz, 1 H, α-CH Ala),
6.76 (d, J ϭ 9.3 Hz, 2 H, 2 ϫ Ar-H ortho NMe2), 7.29 (d, J ϭ
7.2 Hz, 1 H, 6-H or 4-H), 7.43 (t, J ϭ 8.1 Hz, 1 H, 5-H), 7.72Ϫ7.78
(m, 2 H, 4-H or 6-H, 2-H), 7.88 (d, J ϭ 9.3 Hz, 2 H, 2 ϫ Ar-H
meta NMe2) ppm. 13C NMR (75.4 MHz, CDCl3): δ ϭ 15.4 (β-C
Ala), 27.8 (CMe3), 40.3 (NMe2), 44.2 (CH2), 51.8 (α-C Ala), 52.1
(OMe), 84.2 (CMe3), 111.5 (C-2Ј, C-6Ј), 121.1 (C-4), 122.9 (C-2),
124.9 (C-3Ј, C-5Ј), 128.8 (C-5), 130.6 (C-6), 135.6 (C-1), 143.6 (C-
4Ј), 151.9 (C-3), 152.3 (C-1Ј), 153.2 (CO2CMe3), 171.5 (CONH),
173.2 (CO2Me) ppm. HRMS: calcd. for C25H32N405 [Mϩ]
468.2373; found 468.2378.
Stability Tests with Starting Material 5d
Acidolysis with Trifluoroacetic Acid: Trifluoroacetic acid (0.56 mL)
was added to the fully protected amino acid 5d (113 mg;
0.31 mmol) and the mixture stirred vigorously for 14 hours. Evap-
oration of the solvents under reduced pressure gave a red solid
(113 mg; 100%). 1H NMR spectroscopy confirmed the structure of
the compound.
N-tert-Butyloxycarbonylphenylalanine Ethyl Ester 18c by Amino-
lysis of 17c: The colored substrate 17c (62 mg, 0.11 mmol) was
treated with DEAEA (62 ϫ 10Ϫ3 mL, 0.44 mmol) for two days
according to the procedure of Grehn[25] et al. The product was
chromatographed with diethyl ether/hexane (2:8) as the eluent to
give ester 18c (16 mg, 50%) as an oil. TLC (chloroform/methanol,
5.8:0.2): Rf ϭ 0.90. 1H NMR spectroscopic data compared well
with those of a genuine sample.[22]
Acidolysis with Hydrochloric Acid: HCl (6 , 0.50 mL) was added
to the fully protected amino acid 5d (50 mg; 0.14 mmol) and the
mixture stirred vigorously for 25 minutes. Evaporation of the sol-
vents under reduced pressure gave a red solid (47.6 mg; 84%). H
NMR spectroscopy confirmed the structure of the compound.
1
Catalytic Hydrogenation: A solution of 5d (100 mg; 0.28 mmol) in
methanol (3 mL) and 1,4-cyclohexadiene (92
ϫ
10Ϫ3 mL;
0.98 mmol) was mixed with 10% palladium on charcoal catalyst
(56 mg) and refluxed for 12 hours with stirring. The catalyst was
filtered off and washed with methanol; the combined liquids were
then evaporated to dryness under reduced pressure. Recrystalliza-
tion from ethyl acetate and hexane afforded the compound as a red
solid (100 mg, 100%). Its 1H NMR was identical to that of the
starting material.
N-tert-Butyloxycarbonylalanine Methyl Ester 18d by Aminolysis of
17d: The product of the reaction of 17d (46 mg, 0.10 mmol) with
DEAEA (60 ϫ 10Ϫ3 mL, 0.43 mmol) according to the procedure
described above for compound 18c was chromatographed with di-
ethyl ether/hexane (2:8) as eluent to give ester 18d (11.8 mg, 58%).
TLC (ethyl acetate/hexane, 2:8): Rf ϭ 0.67. 1H NMR spectroscopic
data compared well with those of a genuine sample.[22]
N-(3-Aminophenylacetyl)phenylalanine Ethyl Ester 19c by Chemical
Reduction of 5c: Reduction of 5c (180 mg, 0.39 mmol) with zinc
dust (144 mg, 2.2 mmol) in methanol in the presence of formic acid
according to the procedure described by Gowda[26] et al. gave the
corresponding ester 19c (97.9 mg, 77%), as an oil. TLC (ethyl acet-
ate/hexane, 8:2): Rf ϭ 0.53. IR (film): ν˜ ϭ 3357, 3062, 3030, 2981,
2935, 1737, 1659, 1652, 1606, 1591, 1538, 1520, 1495, 1463, 1455,
Acknowledgments
We thank the Foundation for Science and Technology (Portugal)
for financial support to the Institute of Biotechnology and Fine
Chemistry (University of Minho).
[1]
G. W. Case, P. J. Bekowies, US Patent 5413915 (1995).
J. C. V. P. Moura, in: Non-Antibiotics (Eds.: A. N. Chakrabarty,
[2]
1444, 1375, 1350, 1298, 1278, 1214, 1200, 1116, 1029, 701 cmϪ1
.
J. Molnar), NISCOM, New Delhi, 1998, pp. 327Ϫ356.
1H NMR (300 MHz, CDCl3): δ ϭ 1.23 (t, J ϭ 7.2 Hz, 3 H,
OCH2CH3), 2.98Ϫ3.12 (m, 2 H, β-CH2 Phe), 3.46 (s, 2 H, CH2),
4.14 (q, J ϭ 7.2 Hz, 2 H, OCH2CH3), 4.80Ϫ4.90 (m, 1 H, α-CH
Phe), 5.93 (d, J ϭ 8.3 Hz,1 H, α-NH Phe), 6.51 (s,1 H, 2-H),
6.53Ϫ6.68 (m, 2 H, 4-H, 6-H), 6.9Ϫ7.0 (m, 2 H, 3-H Phe, 5-H
Phe), 7.11 (t, J ϭ 7.2 Hz, 1 H, 5-H), 7.18Ϫ7.25 (m, 3 H, 2-H Phe,
[3]
G. M. Johnson, R. J. Schaeper, US Patent 5464739 (1995).
[4]
B. Altenberg-Greulich, G. Vriend, J. Mol. Struct. 2001, 598,
1Ϫ8.
J. Y. Chang, E. H. Creaser, K. W. Bentley, Biochem. J. 1976,
153, 607Ϫ611.
[5]
[6]
J. Y. Chang, Anal. Biochem. 1988, 170, 542Ϫ556.
Eur. J. Org. Chem. 2004, 1750Ϫ1760
www.eurjoc.org
 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
1759

S. M. B. Fraga, M. S. T. Gonc¸alves, J. C. V. P. Moura, K. Rani
FULL PAPER
[7]
[19]
´ ´ ´ ´
F. Sebestyen, G. Szendrei, M. Mak, M. Doda, E. Illyes, G.
A. D. Abell, B. C. H. May, B. K. Nabbs, Peptides 2001,
´
´ ´
ˆ
785Ϫ786.
Szokan, K. Kindla, W. Rapp, P. Szegö, E. Campian, A. Furka,
J. Pept. Sci. 1998, 4, 294Ϫ299.
[8]
A. D. Abell, D. C. Martyn, B. C. H. May, B. K. Nabbs, Tetra-
[20]
´
´
ˆ
hedron Lett. 2002, 43, 3673Ϫ3675.
E. H. J. M. Jansen, R. H. Van Den Berg, R. Both-Miedema,
F. Sebestyen, K. Kindla, W. Rapp, E. Campian, A. Furka, in:
Peptides: Chemistry, Structure and Biology (Eds.: P. T. P. Kau-
maya, R. S. Hodges), Mayflower Scientific Ltd., 1996; pp.
319Ϫ320.
[9]
L. Doorn, J. Chromatogr. 1991, 553, 123Ϫ133.
V. Stocchi, G. Piccoli, M. Magnani, F. Palma, B. Biagiarelli, L.
[10]
[21]
[22]
[23]
[24]
[25]
[26]
Cucchiarini, Anal. Biochem. 1989, 178, 107Ϫ117.
M. S. T. Goncalves, H. L. S. Maia, Tetrahedron Lett. 2001,
¸
[11]
J. Y. Chang, R. Knecht, G. Braun, Biochem. J. 1982, 203,
42, 7775Ϫ7777.
803Ϫ806.
M. S. T. Goncalves, H. L. S. Maia, Org. Biomol. Chem. 2003,
¸
[12]
T. W. Garner, E. S. Yeung, Anal. Chem. 1990, 62, 2193Ϫ2198.
1, 1480Ϫ1485.
[13]
D. Parkinson, J. D. Redshaw, Anal. Biochem. 1984, 141,
J. I. N. R. Gomes, J. Griffiths, H. L. S. Maia, J. C. V. P. Moura,
A. M. F. O. Campos, Dyes and Pigments 1991, 17, 269Ϫ278.
G. R. Pettit, Synthetic Peptides, Litton Education Publishing
Inc., New York, 1970, 3, 150.
L. Grehn, K. Gunnarsson, U. Ragnarsson, Acta Chem. Scand.,
Ser. B 1987, B41, 18Ϫ23.
121Ϫ126.
[14]
M. Tzeng, Anal. Biochem. 1983, 128, 412Ϫ414.
I. P. Griffith, Anal. Biochem. 1972, 46, 402Ϫ412.
H. F. Bosshard, A. Datyner, Anal. Biochem. 1977, 82, 327Ϫ333.
L. Fruk, A. Grondin, W. E. Smith, D. Graham, Chem. Com-
mun. 2002, 2100Ϫ2101.
B. Pal, P. K. Bajpai, T. S. B. Baul, Spectrochimica Acta Part A
[15]
[16]
[17]
S. Gowda, K. Abiraj, D. C. Gowda, Tetrahedron Lett. 2002,
43, 1329Ϫ1331.
[18]
2000, 56, 2453Ϫ2458.
Receiced October 30, 2003
1760
 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
Eur. J. Org. Chem. 2004, 1750Ϫ1760