Biotin-phenyldiazomethane conjugates as labeling reagents at phosphate in mono and polynucleotides

Article abstract of DOI:10.1016/j.bmc.2004.12.046

Molecules 2-5 that include in their structure a biotin moiety as detectable unit and differently substituted phenyl diazo functions as reactive group were prepared as reagents for labeling the phosphate group in mono and polynucleotides. These molecules w

Full text of DOI:10.1016/j.bmc.2004.12.046

Bioorganic & Medicinal Chemistry 13 (2005) 1453–1461
Biotin-phenyldiazomethane conjugates as labeling reagents
at phosphate in mono and polynucleotides
a
Cecile Bourget, Emmanuelle Trevisiol, Isabelle Bridon, Mitsuharu Kotera,
a
b
a,
*
´
´
Jean Lhommea and Ali Laayounb,*
a
´
´
LEDSS—UMR5616 associe au CNRS, BP53, Universite JosephFourier, 38041 Grenoble, France
b
´ ´
BioMerieux, sa—Diagnostic Moleculaire, Chemin de l’Orme, 69280 Marcy l’Etoile, France
Received 19 October 2004; revised 15 December 2004; accepted 20 December 2004
Abstract—Molecules 2–5 that include in their structure a biotin moiety as detectable unit and differently substituted phenyl diazo
functions as reactive group were prepared as reagents for labeling the phosphate group in mono and polynucleotides. These mole-
cules were shown to react selectively and quantitatively with the model nucleotide 30-UMP. They were used successfully in the
labeling step of DNA and RNA analysis using high-density DNA-chips (or microarrays) technology.
ꢀ 2004 Elsevier Ltd. All rights reserved.
1. Introduction
yldiazomethane, a reagent described for derivatizing
carboxylic acids, is a reagent of choice to alkylate selec-
tively the phosphate group in nucleotide monophos-
phates. Thus 30-UMP, 30-CMP, 30-AMP, 30-GMP, 30-
TMP, as well as the 20- or 50- analogs 20-UMP and 50-
UMP were selectively and quantitatively converted to
the corresponding pyrenyl phosphate esters by treat-
ment with pyrenyldiazomethane in buffered borate
aqueous solutions. No reaction at the nucleophilic sites
of the nucleic bases could be detected.20 These observa-
tions seemed of great interest to introduce labels or re-
porter molecules at phosphate in polynucleotides.
Molecule m-BioPMDAM 1 that includes in its structure
the biotin moiety as detectable unit and the diazo func-
tion as reactive group was thus designed and shown to
react selectively and efficiently with phosphate in nucleo-
tide monomers, oligonucleotides, DNA, and RNA.
The molecule fulfills the requirements for hybridiza-
tion-based analysis and especially for detection on
high-density DNA chips.21,22 In order to evaluate fur-
ther the possible effects of substitution on the properties
of the newly designed label m-BioPMDAM in terms of
reactivity, selectivity, stability, and water solubility. . .
notably for applications using DNA-chip technology
we prepared analogs 2–5. Molecules o-BioPMDAM 2
and p-BioPMDAM 3 are the ortho- and para-isomers
of m-BioPMDAM 1. m-BioDPDAM 4 was designed
to study the effect of phenyl substitution. We had previ-
ously seen that replacement of the methyl group by
hydrogen led to instability of the diazo function.20 The
Labeling of nucleotides, oligonucleotides, and nucleic
acids is essential in many applications, notably in nucleic
acid based technologies. Most described methods in-
volve labeling reagents of electrophilic nature, halides,
sulfonates, isocyanates, isothiocyanates, metal (plati-
num) derivatives. . . that react essentially at the nucleo-
philic sites of the nucleic bases.1–5 This generally leads
to modifications in the hybridization properties of the
labeled bases, which constitutes severe limitation for
many applications, for example, those using DNA-chips
(or microarrays) requiring a high specificity level. Label-
ing at the phosphate represents an attractive alternative
to avoid this problem. However selective reaction at
phosphate is poorly documented, due essentially to the
weak nucleophilicity of such a group.6–14 In the course
of a program aimed at devising new labeling reagents
for DNA-chip analysis,15–18 an application in which it
is of utmost importance to preserve the hybridization
properties of the labeled nucleic acid targets, we became
interested in diazo compounds by analogy with their use
for alkylation of carboxylate groups.19
We recently explored the use of diazo chemistry to alkyl-
ate the phosphate group. We showed notably that pyren-
Keywords: Nucleic acids; Alkylation; Biotin labeling; Diazomethane.
*
Corresponding authors. Tel.: +33 390 244 175; fax: +33 390 244
306; e-mail: kotera@bioorga.u-strasbg.fr
0968-0896/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2004.12.046

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C. Bourget et al. / Bioorg. Med. Chem. 13 (2005) 1453–1461
stabilizing effect of a strong electron-withdrawing group
was thus evaluated with the nitro-substituted analog
BioNPDAM 5. In the present paper we describe the syn-
thesis of molecules 2–5, we show that they react selec-
tively at phosphate with the model nucleotide
phosphate 30-UMP with significant differences in the
reaction kinetics. All give positive results using amplified
nucleic acid targets and DNA-chips analysis.
2. Results and discussion
2.1. Synthesis of the labeling molecules 2–5
o-BioPMDAM 2, p-BioPMDAM 3, and m-BioDPDAM
4 were prepared using the same synthetic route (Scheme
1) by coupling D-biotin with the respective commercial
aminoketones in DMF using isobutyl chloroformate
and N-methylmorpholine as condensing agent (yield:
50–70%). The resulting ketones were transformed into
the hydrazones, by warming with hydrazine monohy-
drate in ethanol, in 80–90% yields. Oxidation of the
hydrazones into the diazo compounds proved quite deli-
cate. The reaction was best achieved by stirring the
hydrazones with MnO2 at room temperature for a short
period (10–15 min). Poor results were observed when
using AgO,23 BaMnO4,24 or NiO2 as oxidizing agents
25
in different solvents. The diazo compounds, as pink
powders, were characterized notably by their 1H
and 13C NMRspectra. They exhibited a strong charac-
teristic band at 2038 cmꢀ1 in the IRspectra (see e.g.,
NMRand IRspectra of o-BioPMDAM in Fig. 1).
Figure 1. IR(a) and 1H NMR(b) 200 MHz, in DMSO- d6 spectra of o-
BioPMDAM 2.
1
isomers were characterized by their H NMRspectrum.
The nitro analog BioNPDAM 5 was obtained (Scheme
2) starting from 2,4-dinitrobenzaldehyde ethylene acetal
6. Controlled reduction with sodium sulfide in ethanol
gave a mixture of the two mono reduced derivatives
from which the major 2-nitro-4-aminoacetal 7 was iso-
lated and purified in 45% yield. The minor 2-amino-4-ni-
tro derivative 8 was obtained in 9% yield. The two
The 2-nitro-4-aminoethylene acetal 7 was coupled to D-
biotin and the acetal function was hydrolyzed with di-
lute sulfuric acid (90% yield). Hydrazonation followed
by MnO2 oxidation gave the diazo compound BioNP-
DAM 5 characterized as previously by the NMRand
21,22
IRdata.
Scheme 1. Synthesis of the labeling molecules 2–4.

C. Bourget et al. / Bioorg. Med. Chem. 13 (2005) 1453–1461
1455
the sugar protons. These data are quite comparable to
those reported for the adduct formed in the reaction
with the meta-isomer m-BioPMDAM 1.21,22
Capillary electrophoresis also allowed comparison of
the kinetics of the reactions with the different diazo mole-
cules. Half-lives for disappearance of the UMP nucleo-
tide in identical reaction conditions as indicated could
be evaluated from the evolution of the peaks as a func-
tion of time. Quite different velocities were measured for
the different labels (Table 1). Taking the already re-
ported m-BioPMDAM label 1 as reference, it appears
that replacing the methyl substituent by phenyl (in
DPDAM 4) or introducing the electroattracting nitro
group (in BioNPDAM 5) decreases the reaction kinetics.
The ortho substituted molecule o-BioPMDAM 2 exhib-
its unexpected high reactivity compared to the para-
isomer p-BioPMDAM (this acceleration could
hypothetically be due to rapid internal proton transfer
from the NH amido group to the neighboring diazo car-
bon atom C@N2 in the first step of the alkylation
reaction).26
Scheme 2. Synthesis of the labeling molecule BioNPDAM 5.
2.3. Hydrolytic stability of adducts
Simple diazo alkanes are unstable compounds while aryl
substitution generally increases the stability. It was thus
necessary to test the stability of the new diazo com-
pounds. Stability was evaluated in solution by monitor-
In the course of studies on DNA-chips aimed at evaluat-
ing the efficiency of molecules 2–5 to label RNA targets
(see infra), we noticed that the signals measured on the
chip exhibited intensity decrease with time. This phe-
nomenon was variable depending on the structure of
the labeling molecule. Decrease was particularly rapid
for the o- and p-BioPMDAM labels 2 and 3. This was
not observed with DNA targets, which suggests hydro-
lysis of the RNA adducts assisted by participation of
the 20-OH with loss of the biotin unit. We thus studied
hydrolysis in basic conditions of the UMP-adducts
formed with ortho-, meta-, para-BioPMDAM, and
meta-BioDPDAM. Capillary electrophoresis appeared
again as a method of choice to monitor the reaction
by measuring the kinetics for disappearance of the ad-
ducts. The half-lives for hydrolysis in 0.2 M aqueous
NaOH are given in Table 2. The hydrolytic stability of
the m-BioPMDAM adduct is clearly higher than the
ortho- and para-isomers. These observations could ac-
count for the instability of labeled RNA fragments for
their chip analysis.
1
ing the H NMRspectra of 2–5 as a function of time in
DMSO-d6 at room temperature from which the half-
lives for decomposition were determined. They average
to 3 days for molecules 1, 2, and 5. Molecule 3 is slightly
less stable with a half-life evaluated to 1 day.
2.2. Reactivity with the model nucleotide 30-UMP
We have reported that the marker m-BioPMDAM 1 re-
acts with nucleotides to form unique adduct correspond-
ing to alkylation at the phosphate.21,22 This was
observed with all nucleotides studied, independently of
the nature of the base and of the position of the phos-
phate group. The same observations had been done
for reaction of the pyrenyldiazomethane molecule.20
Using 30-UMP as representative nucleotide, capillary
electrophoresis had proved to be the method of choice
to monitor the reaction. We used the same methodology
to examine the reaction of the new labeling molecules 2–
5. Reactions were run in an homogenous solvents mix-
ture (DMSO–CH3CN–H2O) to dissolve both the hydro-
philic 30-UMP nucleotide and the lipophilic diazo
reagents, at pH 7.3 using borate buffer, at 60 ꢁC. The
reaction profiles were quite comparable for the four la-
bels 2–5. One very major (if not unique) peak appeared
in the electropherograms with concomitant disappear-
ance of the nucleotide peak (Fig. 2). The reaction prod-
ucts corresponding to the observed peaks were isolated
in the case of the o- and p-BioPMDAM reagents. The
1H NMRspectra ( Fig. 3) of corresponding adducts 12
and 13 exhibited all peaks corresponding to the indi-
cated structures with alkylation at phosphate, notably
a multiplet at 5.4 ppm corresponding to the CH proton
vicinal to the phosphate. The presence of two diastereo-
isomers in a 1/1 ratio was indicated by the splitting of
2.4. DNA and RNA labeling
DNA and RNA labeling by molecules 2–5 was exam-
ined using targets generated by enzymatic amplification,
PCRand post-PCRin vitro transcription, of 16S rDNA
hypervariable region of Mycobacterium tuberculosis
(Mtb, 202 nt).27 Following the procedure previously
used to test the labeling efficiency of m-BioPMDAM,
the amplified targets were reacted with the diazo re-
agents 2–5 and fragmented prior to their hybridization
to the Mycobacterium DNA-chip. Cleavage of labeled
DNA and RNA targets into smaller fragments is a pre-
requisite for uniformity and hybridization specificity on
the DNA-chip. RNA targets were cleaved with Mn2+
and imidazole. The DNA targets were fragmented by
acidic treatment that promoted random depurination

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C. Bourget et al. / Bioorg. Med. Chem. 13 (2005) 1453–1461
Figure 2. Adduct formation between 30-UMP (0.04 mM) and labels 2–5 (concentrations as indicated) in DMSO–CH3CN–H2O (1:3:1), H3BO3
(2 mM) at 60 ꢁC monitored by capillary electrophoresis. (a) Only traces of adduct are detected when 4 is used at 2 mM concentration.
and cleavage. After hybridization on DNA-chip, DNA
and RNA fragments were stained with streptavidin
bearing fluorescent R-phycoerythrin.21 The chip results
(Table 3) indicate that the two target types, the DNA
amplicons and the RNA transcripts were successfully
labeled. The signal intensities (rfu) were strong with
signal/background ratios high. With the exception of
diphenyl diazomethane 4, base-calling values, that

C. Bourget et al. / Bioorg. Med. Chem. 13 (2005) 1453–1461
1457
Table 3. Chip analysis of DNA amplicons labeled with molecules 1–5
Labeling molecule BC (%)a Median intensity Signal/
(rfu)
background
m-BioPMDAM 1
o-BioPMDAM 2
p-BioPMDAM 3
m-BioDPDAM 4
BioNPDAM 5
97
97
22,960
25,951
43,785
32,359
24,392
39.5
31.6
36.3
9.1
95
93
100
27.1
a In this model study, BC (%) P 95% correspond to a correct identi-
fication of DNA target.
value observed with diphenyl compound 4 is probably
due to the steric hindrance of such a structure affecting
the hybridization properties. It is also to be noted that
the signal intensities are in all cases too high to be solely
attributed to the labeling of the terminal phosphate
generated during the cleavage treatment. As already
reported for m-BioPMDAM these two observations,
the signal intensities and the base-calling values, are
strong indication that labeling also occurred at the inter-
nucleotidic phosphates.28
3. Conclusion
All these results show that diazo chemistry opens the
route to the design of new, versatile reagents for labeling
of nucleotides, oligonucleotides, and nucleic acids at
phosphate. Depending on the application, target of
interest (DNA or RNA), detection format (high com-
plexity or low complexity chip), . . .the reactivity of the
reagents can be modulated by introducing structural
modifications to the diazo moiety. The stability of la-
beled fragments can be controlled as well. The nature
of the label can be easily varied following the indicated
scheme, and such physical properties as water solubi-
lity. . . can be controlled by varying the nature and/or
the length of the linking chain between the diazo group
and the detectable moiety. The improvement of the sta-
bility in water of these labels is also a major goal. Work
is in progress in the design of new reagents that fulfill the
solubility and stability requirements.
Figure 3. 1H NMRspectra (in D O) of adducts 12 (a, 200 MHz) and
2
13 (b, 300 MHz, HDO peak suppressed by PRESAT), formed between
30-UMP and, respectively, o-BioPMDAM 2 and p-BioPMDAM 3.
Table 1. Kinetics for adduct formation between labeling molecules 1–5
and 30-UMP
Labeling molecule
t1/2
m-BioPMDAM 1
o-BioPMDAM 2
p-BioPMDAM 3
m-BioDPDAM 4
BioNPDAM 5
15 min
ꢁ5 min
5 min
ꢂ2 h
45 min
Half-lives for transformation of 30-UMP (0.04 mM) in the presence of
labels (2 mM), H3BO3 (2 mM) in DMSO-CH3CN-H2O (1:3:1) at
60 ꢁC.
4. Experimental
Table 2. Hydrolysis of adducts formed between 30-UMP and labels 1–
4.1. General remarks
4
All commercially available chemical reagents were used
without purification unless otherwise indicated. TLC:
Merck Kieselgel 60 F254, layer thickness 0.25 mm. Visu-
alization by UV light (254 nm) or by phosphomolybdic
acid solution. Preparative column chromatographies:
Macherey-Nagel Kieselgel, 230–400 mesh. Mp: Reichert
Thermovar (uncorrected). IR: Nicolet Impact 400. UV/
Vis: Varian Cary 400 scan. NMR: Bruker AC 200
Avance 300 and Varian U+500 spectrometers. NMR
spectra were referenced to the residual solvent peak,
chemical shifts d in ppm, apparent scalar coupling con-
stants J in Hz. MS: Esquire 3000+ (ES-MS) and Ther-
mofinningan Polaris Q (DCI and EI)—Elemental
analysis were performed by ÔService central de microana-
lyse du CNRSÕ.
Labeling molecule
t1/2
m-BioPMDAM 1
o-BioPMDAM 2
p-BioPMDAM 3
m-BioDPDAM 4
198 min
30 min
8 min
54 min
Half-lives for disappearance of adducts (1.6 mM) in aqueous NaOH
(200 mM) at 60 ꢁC.
measure the percent homology between the experimen-
tally derived sequence and the 202 nt sequence tiled on
the chip were superior or equal to 95% for all four mol-
ecules. This latter observation indicates that the hybrid-
ization properties of labeled targets have not been
altered by the presence of the label. The low BC (%)

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C. Bourget et al. / Bioorg. Med. Chem. 13 (2005) 1453–1461
4.2. 2-(N-Biotinoylamino)acetophenone (2a)
2.54 (d, J = 12.5 Hz, 1H), 2.24 (t, J = 7.2 Hz, 2H), 2.12
(s, 3H), 1.70–1.29 (m, 6H).
To a solution of D-biotin (1.0 g, 4.1 mmol) in dry DMF
(45 mL) cooled at 0 ꢁC under argon, were added succes-
sively N-methylmorpholine (590 lL, 5.33 mmol) and
isobutyl chloroformate (840 lL, 6.60 mmol). The solu-
tion was stirred for 30 min, and then 2-aminobenzophe-
none (824 mg, 6.10 mmol) was added. The solution was
stirred at room temperature for 3.5 h and then the sol-
vent was removed in vacuo. The residue was triturated
with cold water (50 mL). The resulting precipitate was
filtered, washed with water, and recrystallized from
MeOH to give 2a (1.1 g, 72%) as a white powder. Mp
105–110 ꢁC. IR(KBr): 3248, 2930, 2857, 1700, 1670,
1652, 1582, 1528, 1448, 1354, 1310, 1245, 1165, 962,
4.5. 4-(N-Biotinoylamino)acetophenone (3a)
The compound 3a was prepared by the same procedure
as described for 2a starting from D-biotin (1.0 g,
4.1 mmol)
and
4-aminoacetophenone
(824 mg,
6.10 mmol). Yield: 888 mg (60%). Mp 235–236 ꢁC. IR
(KBr): 1706, 1688, 1667, 1608, 1589, 1526, 1461, 1401,
1276, 1262, 1180, 833, 700, 680, 595 cmꢀ1. H NMR
1
(300 MHz, DMSO-d6): d = 10.18 (br s, 1H), 7.89 (d,
J = 8.7 Hz, 2H), 7.69 (d, J = 8.8 Hz, 2H), 6.40 (br s,
1H), 6.33 (br s, 1H), 4.27 (m, 1H), 4.11 (m, 1H), 3.11
(m, 1H), 2.80 (dd, J = 5.0, 12.3 Hz, 1H), 2.55 (d,
J = 12.3 Hz, 1H), 2.50 (s, 3H), 2.35 (t, J = 7.4 Hz, 2H),
1.67–1.28 (m, 6H). MS-DCI (NH3/isobutane):
m/z = 361.9 [M+H]+. C18H23N3O3S–1.5H2O (388.5):
calcd C 55.65, H 6.75, N 10.82; found C 55.37, H
6.35, N 11.26.
1
860, 759, 606 cmꢀ1. H NMR(200 MHz, DMSO- d6):
d = 11.24 (br s, 1H), 8.33 (d, J = 7.9 Hz, 1H), 7.97 (d,
2H, J = 6.9 Hz), 7.57 (t, J = 7.7 Hz, 1H), 7.18 (t,
J = 7.0 Hz, 1H), 6.44 (br s, 1H), 6.35 (br s, 1H), 4.30
(m, 1H), 4.14 (m, 1H), 3.12 (m, 1H), 2.80 (dd, J = 5.0,
12.5 Hz, 1H), 2.61 (s, 3H), 2.55 (d, J = 12.5 Hz, 1H),
2.37 (t, J = 7.2 Hz, 2H), 1.67–1.30 (m, 6H). MS-DCI
(NH3/isobutane): m/z = 361.9 [M+H]+. C18H23N3O3S–
0.5H2O (370.5): calcd C 58.36, H 6.53, N 11.34; found
C 58.29, H 6.54, N 11.25.
4.6. 4-(N-Biotinoylamino)acetophenone hydrazone (3b)
To a solution of the ketone 3a (870 mg, 2.4 mmol) in
absolute ethanol (8 mL), hydrazine monohydrate
(995 lL, 19.5 mmol) was added. The solution was re-
fluxed for 3 h. After cooling at room temperature,
resulting precipitate was filtered, washed, and dried in
vacuo to give 3b (820 mg, 90%). Mp 283–285 ꢁC. IR
(KBr): 3336, 3282, 3183, 2931, 2857, 1701, 1659, 1593,
1521, 1459, 1405, 1325, 1264, 1187, 1111, 828,
4.3. 2-(N-Biotinoylamino)acetophenone hydrazone (2b)
To a solution of the ketone 2a (500 mg, 1.38 mmol) in
absolute ethanol (8 mL), hydrazine monohydrate
(572 lL, 11.1 mmol) was added. The solution was re-
fluxed for 50 min and then solvents were evaporated.
The residue was suspended in water, filtered, washed
with water and with ether, and dried in vacuo to give
2b (416 mg, 80%). Mp 156–158 ꢁC. IR(KBr): 3412,
3241, 2931, 2909, 2857, 1706, 1677, 1590, 1532, 1444,
719 cmꢀ1 1H NMR(200 MHz, DMSO- d6): d = 9.86
.
(br s, 1H), 7.52 (s, 4H), 6.43 (br s, 1H), 6.35 (br s,
1H), 6.21 (br s, 2H), 4.29 (m, 1H), 4.12 (m, 1H), 3.12
(m, 1H), 2.81 (dd, J = 5.0, 12.5 Hz, 1H), 2.56 (d,
J = 12.5 Hz, 1H), 2.32 (t, J = 7.2 Hz, 2H), 1.97 (s, 3H),
1.69–1.30 (m, 6H). MS-DCI (NH3/isobutane):
m/z = 376.1 [M+H]+. C18H25N5O2S (375.5): calcd C
57.58, H 6.71, N 18.65; found C 57.68, H 6.74, N 18.59.
1
1303, 1270, 1169, 1114, 751, 643, 606 cmꢀ1. H NMR
(200 MHz, DMSO-d6): d = 11.97 (br s, 1H), 8.35 (d,
J = 7.9 Hz, 1H), 7.45 (d, J = 6.9 Hz, 1H), 7.19 (t,
J = 7.7 Hz, 1H), 7.04 (t, J = 7.2 Hz, 1H), 6.61 (s, 2H),
6.42 (br s, 1H), 6.35 (br s, 1H), 4.32 (m, 1H), 4.14 (m,
1H), 3.12 (m, 1H), 2.81 (dd, J = 5.0, 12.5 Hz, 1H),
2.56 (d, J = 12.5 Hz, 1H), 2.31 (t, J = 7.4 Hz, 2H), 2.09
(s, 3H), 1.72–1.30 (m, 6H). MS-DCI (NH3/isobutane):
m/z = 376.1 [M+H]+. C18H25N5O2S–0.5H2O (384.5):
calcd C 56.23, H 6.82, N 18.21; found C 56.12, H
6.77, N 18.09.
4.7. 4-(N-Biotinoylamino)phenylmethyldiazomethane (3)
The compound 3 was prepared by the same procedure as
described for 2 starting from the hydrazone 3b (200 mg,
0.53 mmol). Yield: 190 mg. IR(KBr): 3257, 2930, 2857,
2032, 1698, 1597, 1524, 1510, 1457, 1404, 1307, 1259,
1180, 1114, 1071, 828 cmꢀ1
.
1H NMR(200 MHz,
DMSO-d6): d = 9.82 (br s, 1H), 7.57 (d, J = 8.7 Hz,
2H), 6.83 (d, J = 8.7 Hz, 2H), 6.41 (br s, 1H), 6.34 (br
s, 1H), 4.28 (m, 1H), 4.12 (m, 1H), 3.11 (m, 1H), 2.80
(dd, J = 4.8, 12.7 Hz, 1H), 2.55 (d, J = 12.3 Hz, 1H),
2.25 (t, J = 7.0 Hz, 2H), 2.10 (s, 3H), 1.70–1.25 (m, 6H).
4.4. 2-(N-Biotinoylamino)phenylmethyldiazomethane (2)
To a solution of hydrazone 2b (200 mg, 0.53 mmol) in
DMF (10 mL), MnO2 (800 mg) was added and the sus-
pension was stirred for 15 min at room temperature. The
mixture was suction filtered through Celite and molecu-
4.8. 3-(N-Biotinoylamino)benzophenone (4a)
˚
lar sieves 3 A (powder, 0.5 cm thickness). After evapora-
The compound 4a was prepared by the same procedure
as described for 2a starting from D-biotin (1.0 g,
tion in vacuo (0.5 mmHg, T < 40 ꢁC), the oily residue
was triturated with ether to afford 2 (130 mg) as a pink
powder. IR(KBr): 3248, 2930, 2857, 2038, 1699, 1521,
4.1 mmol)
and
3-aminobenzophenone
(1.6 mg,
8.1 mmol). Yield: 1.3 g (75%). Mp 97–100 ꢁC. IR
(KBr): 3269, 3079, 2920, 2857, 1698, 1595, 1555, 1428,
1456, 1113, 754, 469 cmꢀ1
.
1H NMR(200 MHz,
1
DMSO-d6): d = 9.37 (br s, 1H), 7.26–7.00 (m, 4H),
6.43 (br s, 1H), 6.35 (br s, 1H), 4.30 (m, 1H), 4.15 (m,
1H), 3.12 (m, 1H), 2.82 (dd, J = 5.0, 12.5 Hz, 1H),
1325, 722, 642 cmꢀ1. H NMR(200 MHz, DMSO- d6):
d = 10.10 (br s, 1H), 8.00–7.39 (m, 9H), 6.43 (br s,
1H), 6.35 (br s, 1H), 4.27 (m, 1H), 4.13 (m, 1H), 3.12

C. Bourget et al. / Bioorg. Med. Chem. 13 (2005) 1453–1461
1459
(m, 1H), 2.84 (dd, J = 5.0, 12.3 Hz, 1H), 2.55 (d,
J = 12.3 Hz, 1H), 2.31 (t, J = 7.3 Hz, 2H), 1.70–1.30
(m, 6H). MS-DCI (NH3/isobutane): m/z = 424.1
[M+H]+.
After evaporation of solvents, the residue was purified
by flash chromatography (elution with cyclohexane/
ethyl acetate 60:40). From the faster eluting fractions,
the isomer 8 was obtained (0.5 g, 9%) and from the fol-
lowing fractions, the isomer 7 (2.42 g, 45%).
4.9. 3-(N-Biotinoylamino)benzophenone hydrazone (4b)
Isomer (7): 1H NMR(200 MHz, CDCl 3): 7.49 (d,
J = 8.6 Hz, 1H), 7.09 (d, J = 2.4 Hz, 1H), 6.80 (dd,
J = 2.4, 8.6 Hz, 1H), 6.27 (s, 1H), 4.05 (br s, 2H), 3.99
(m, 4H). MS-DCI (NH3/isobutane): m/z = 211.0
[M+H]+. C9H10N2O4 (210.2): calcd C 51.43, H 4.80, N
13.33; found C 50.96, H 4.46, N 13.72.
To a solution of the ketone 4a (400 mg, 0.94 mmol) in
absolute ethanol (8 mL), hydrazine monohydrate
(366 lL, 7.5 mmol) was added. The solution was re-
fluxed overnight. After evaporation, the residue was
purified by flash chromatography (elution with MeOH/
CH2Cl2 5–7%). Yield: 207 mg (50%). Mp 110–112 ꢁC.
IR(KBr): 3285, 3071, 2920, 2857, 1690, 1539, 1484,
1
Isomer (8): H NMR(200 MHz, CDCl ): 7.60–7.40 (m,
3H), 5.84 (s, 1H), 4.50 (m, 2H), 4.09–4.07 (m, 4H).
3
1420, 1333, 1253, 1063, 777, 698 cmꢀ1
.
1H NMR
(200 MHz, DMSO-d6): d = 9.99 and 9.80 (two br s,
1H), 7.70–6.75 (m, 9H), 6.42 (br s, 1H), 6.35 (br s,
1H), 6.26 and 6.22 (two br s, 1H), 4.28 (m, 1H), 4.13
(m, 1H), 3.12 (m, 1H), 2.78 (dd, J = 5.0, 12.0 Hz, 1H),
2.59 (d, J = 12.0 Hz, 1H), 2.30 and 2.24 (two t,
J = 7.3 Hz, 2H), 1.67–1.26 (m, 6H). MS-DCI (NH3/iso-
butane): m/z = 438.1 [M+H]+.
4.13. 4-Biotinoylamino-2-nitrobenzaldehyde
ethyleneacetal (9)
To a suspension of D-biotin (1.0 g, 4.1 mmol) in dry
DMF (20 mL) cooled at 0 ꢁC under argon, were added
successively N-methylmorpholine (600 lL, 6.40 mmol)
and isobutyl chloroformate (700 lL, 5.5 mmol). The
solution was stirred for 30 min, and the nitroamine
(1.0 g, 4.75 mmol) was added. The solution was stirred
at room temperature for 4 h and then the solvent was re-
moved in vacuo. The oily residue was purified by flash
chromatography (elution with MeOH/CH2Cl2 7–10%)
4.10. 3-(N-Biotinoylamino)diphenyldiazomethane (4)
To a solution of the hydrazone 4b (50 mg, 0.11 mmol) in
THF (2 mL), MnO2 (80 mg) was added and the suspen-
sion was stirred for 5 min. The mixture was filtered
1
˚
through Celite and molecular sieves 3 A (powder,
to afford 9 (1.1 g, 62%) H NMR(200 MHz, DMSO-
0.5 cm thickness). After evaporation in vacuo
(0.5 mmHg, T < 40 ꢁC), the residue was triturated with
ether to afford 4 (45 mg) as a pink powder. IR(KBr):
3253, 2928, 2039, 1698, 1611, 1547, 1500, 1436, 1261,
d6): d = 10.40 (br s, 1H), 8.31 (d, J = 2.0 Hz, 1H), 7.77
(dd, J = 2.0, 7.2 Hz, 1H), 7.68 (d, J = 7.2 Hz, 1H), 6.43
(br s, 1H), 6.36 (br s, 1H), 6.23 (s, 1H), 4.28 (m, 1H),
4.14 (m, 1H), 3.92 (m, 4H), 3.12 (m, 1H), 2.85 (dd,
J = 5.0, 12.0 Hz, 1H), 2.76 (d, J = 12.0 Hz, 1H), 2.34
(t, J = 7.0 Hz, 2H), 1.69–1.30 (m, 6H). MS-DCI (NH3/
isobutane): m/z: 437.0 [M+H]+. C19H24N4O6S–0.5H2O
(445.50): calcd C 51.23, H 5.66, N 12.58; found C
50.98, H 5.57, N 12.22.
753, 690 cmꢀ1
.
1H NMR(200 MHz, DMSO- d6):
d = 9.95 (br s, 1H), 7.60–7.20 (m, 8H), 6.92 (m, 1H),
6.42 (br s, 1H), 6.35 (br s, 1H), 4.28 (m, 1H), 4.14 (m,
1H), 3.12 (m, 1H), 2.83 (dd, J = 5.0, 12.0 Hz, 1H),
2.59 (d, J = 12.0 Hz, 1H), 2.27 (t, J = 8 Hz, 2H), 1.68–
1.25 (m, 6H).
4.14. 4-Biotinoylamino-2-nitrobenzaldehyde (10)
4.11. 2,4-Dinitrobenzaldehyde ethyleneacetal (6)
To a solution of the acetal 9 (768 mg, 1.76 mmol) in
THF (25 mL), H2SO4 aqueous solution (2 N, 4 mL)
was added. After stirring at room temperature for 4 h,
the reaction mixture was concentrated and the residue
was triturated with water. The resulting yellow powder
was filtered, washed, and dried in vacuo. Yield:
694 mg (76%). IR(KBr): 3369, 3259, 2928, 2849, 1697,
A
solution of 2,4-dinitrobenzaldehyde (5.0 g,
25.5 mmol), ethylene glycol (20 mL), and p-toluenesulf-
onic acid (150 mg) in toluene (250 mL) was refluxed with
Dean–Stark apparatus for 6 h. After cooling at room
temperature, the reaction mixture was diluted with
AcOEt (150 mL) and washed with water. Organic phase
was dried over MgSO4 and evaporated to give yellow oil
(6.4 g). This crude product was used without purifica-
tion. 1H NMR(200 MHz, CDCl 3): d = 8.70 (d,
J = 2.1 Hz, 1H), 8.44 (dd, J = 2.1, 8.6 Hz, 1H), 8.02 (d,
J = 8.6 Hz, 1H), 6.49 (s, 1H), 4.13–3.96 (m, 4H).
1
1587, 1531, 1405, 1319, 1248, 837, 751 cmꢀ1. H NMR
(200 MHz, DMSO-d6): d = 10.69 (br s, 1H), 10.09 (s,
1H), 8.43 (d, J = 2.0 Hz, 1H), 7.91 (s, 2H), 6.42 (br s,
1H), 6.35 (br s, 1H), 4.29 (m, 1H), 4.13 (m, 1H), 3.12
(m, 1H), 2.84 (dd, J = 5.0, 12.0 Hz, 1H) 2.78 (d,
J = 12.0 Hz, 1H), 2.39 (t, J = 7.0 Hz, 2H), 1.69–1.30
(m, 6H). MS-DCI (NH3/isobutane): m/z = 392.9
[M+H]+.
4.12. 4-Amino-2-nitrobenzaldehyde ethyleneacetal (7)
To a solution of dinitroacetal 6 (6.4 g, 25.5 mmol) in
EtOH–H2O (6:1, 350 mL), Na2S–9H2O (12.3 g,
51.1 mmol) was added. The solution was refluxed for
30 min and then concentrated to about 100 mL. The
aqueous residue was diluted with 50 mL of water and
then extracted three times with CH2Cl2. Combined ex-
tracts were washed with brine and dried over MgSO4.
4.15. 4-Biotinoylamino-2-nitrobenzaldehyde hydrazone
(11)
To a suspension of the aldehyde 10 (694 mg, 1.77 mmol)
in ethanol (12 mL), hydrazine monohydrate (686 lL,
14.1 mmol) was added. The resulting orange solution

1460
C. Bourget et al. / Bioorg. Med. Chem. 13 (2005) 1453–1461
was refluxed for 1 h. After cooling, the resulting precip-
itate was filtered, washed, and dried in vacuo to give 11
(700 mg, 97%). Mp 165–167 ꢁC. IR(KBr): 3442, 3388,
3251, 2936, 2852, 1694, 1584, 1529, 1478, 1338, 1250,
the resulting mixture was added CH2Cl2 (30 mL) and
H2O (3 mL). Aqueous phase was separated and further
washed twice with CH2Cl2 (30 mL) and then concen-
trated. The crude product was purified by reverse-phase
silica gel chromatography (LiChroprep RP 18 silica gel,
40–63 lm, eluant: 0–20% MeOH/H2O) to afford 9 mg
(65%) of solid 12. Two sets of signals for two diastereo-
1
1090 cmꢀ1. H NMR(300 MHz, DMSO- d6): d = 10.31
(br s, 1H), 8.31 (d, J = 1.9 Hz, 1H), 7.96 (s, 1H), 7.87
(d, J = 8.8 Hz, 1H), 7.68 (dd, J = 1.9, 8.8 Hz, 1H), 7.31
(br s, 2H), 6.42 (br s, 1H), 6.34 (br s, 1H), 4.29 (m,
1H), 4.13 (m, 1H), 3.12 (m, 1H), 2.84 (dd, J = 5.0,
12.2 Hz, 1H), 2.78 (d, J = 12.2 Hz, 1H), 2.33 (t,
J = 7.2 Hz, 2H), 1.69–1.30 (m, 6H). MS-DCI (NH3/iso-
butane): m/z = 407.0 [M+H]+. C17H22N6O4S–0.5H2O
(415.5): calcd C 49.15, H 5.58, N 20.23; found C
49.35, H 5.55, N 19.81.
1
isomers were observed in the H NMRspectrum. 1H
NMR(300 MHz,
D 2O): d = 7.80 and 7.77 (2d,
J = 7.9 Hz, 1H), 7.58 (m, 1H), 7.40–7.30 (m, 3H), 5.88
and 5.83 (2d, J = 7.9 Hz, 1H), 5.72 (d, J = 4.9 Hz,
0.5H), 5.68 (d, J = 3.7 Hz, 0.5H), 5.48 (m, 1H), 4.62
(m, 1H), 4.58 (m, 1H), 4.35–3.30 (m, 6H), 3.00 (dd,
J = 13.1 and 4.9 Hz, 1H), 2.78 (d, J = 12.8 Hz, 1H),
2.52 (t, J = 7.2 Hz, 2H), 1.50 and 1.48 (2d, J = 6.4 Hz,
3H), 1.35–1.75 (m, 6H).
4.16. 4-Biotinoylamino-2-nitrophenyldiazomethane (5)
To a solution of the hydrazone 11 (200 mg, 0.492 mmol)
in DMF (8 mL), MnO2 (341 mg) was added and the sus-
pension was stirred for 10 min at room temperature. The
mixture was filtered through Celite and molecular sieves
Adduct 13: The compound 13 was obtained by the same
procedure as described for 12 starting from 30-UMP
(8.8 mg) and p-BioPMDAM (3, 75 mg, 0.20 mmol).
Yield: 8.4 mg (61%). 1H NMR(300 MHz, D 2O):
d = 7.72 and 7.69 (2d, J = 7.8 Hz, 1H), 7.46 (s, 4H),
5.86 (d, J = 7.8 Hz, 1H), 5.70 (d, J = 4.5 Hz, 0.5H),
5.66 (d, J = 2.6 Hz, 0.5H), 5.46 (m, 1H), 4.62 (m, 1H),
4.58 (m, 1H), 4.30–3.52 (m, 5H), 3.33 (m, 1H), 3.00
(dd, J = 12.7 and 4.5 Hz, 1H), 2.78 (d, J = 12.7 Hz,
1H), 2.45 (t, J = 7.0 Hz, 2H), 1.58 and 1.53 (2d,
J = 5.4 Hz, 3H), 1.35–1.75 (m, 6H).
˚
3 A (powder, 0.5 cm thickness). After evaporation in
vacuo (0.5 mmHg, <40 ꢁC), the residue was triturated
with ether to afford 5 (180 mg) as a pink powder. IR
(KBr): 3281, 2922, 2848, 2367, 2073, 1697, 1583, 1525,
1
1460, 1330, 1297, 1158, 831, 759 cmꢀ1. H NMR(200
MHz, DMSO-d6): d = 10.21 (br s, 1H), 8.60 (d, J =
2.2 Hz, 1H), 7.77 (dd, J = 2.2, 8.8 Hz, H5); 7.22 (d,
1Haro, J = 8.8 Hz, H6), 6.60 (s, 1H), 6.41 (br s, 1H),
6.33 (br s, 1H), 4.29 (m, 1H), 4.13 (m, 1H), 3.12 (m,
1H), 2.84 (dd, J = 5.1, 12.5 Hz, 1H), 2.78 (d, J =
12.3 Hz, 1H), 2.29 (t, J = 7.3 Hz, 2H), 1.69–1.30 (m, 6H).
Acknowledgements
´
We thank Dr. A. Troesch, BioMeriex sa and Prof. Pas-
cal Dumy, Universite Joseph Fourier for their interest in
4.17. Adduct formation between 30-UMP and labels 2–5
´
this project.
Analytical: 30-UMP (0.04 mM) was incubated with la-
bels 2–5 (2 mM for 2, 3 and 5; 30 mM for 4) in a mixture
of DMSO–CH3CN–H2O (1:3:1, 2.5 mL total) contain-
ing 2 mM H3BO3 (pH 7.3) at 60 ꢁC. Aliquots (250 lL)
in appropriate time intervals were washed three times
with CH2Cl2 and the analyzed using capillary
electrophoresis.
Supplementary data
IRand 1H NMRspectra of 3, 4, and 5, electrophore-
grams monitoring hydrolysis of adducts formed between
30-UMP and labels 1–4. Supplementary data associated
with this article can be found, in the online version, at
doi:10.1016/j.bmc.2004.12.046.
Capillary electrophoresis (CE) procedure: CE experi-
ments were performed with a Beckman P/ACE 5000
capillary electrophoresis instrument (Beckman Coulter,
Fullerton, CA). An untreated fused silica capillary
(75 lm · 50 cm) was used. The applied voltage was
30 kV (normal polarity) and the capillary temperature
maintained at 23 ꢁC. The electrophoregrams were re-
corded at 254 nm. Borate buffer solution (0.1 M,
pH 8.3) was prepared from boric acid by adjusting pH
with NaOH solution and filtered through 0.2 lm filter.
Samples were injected by pressure (0.5 psi, 5 s). Before
each run, the capillary was regenerated by using succes-
sively NaOH solution (0.1 N, 2 min), pure water (2 min)
and borate buffer (2 min) by pressure (20 psi).
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