Orth et al.
hydroxylamine derivatives, which are considered highly reactive
R-nucleophiles. Benzohydroxamate anion represents a unique
type of reactive R-nucleophile since it behaves as a one-cut self-
destructive molecular scissor, which loses its nucleophilic ability
after destroying the phosphate diester target. This type of
reactive nucleophilic reagent may be of utility in the design of
special scissors for gene therapy, and we are currently inves-
tigating the reactivity of other hydroxamate anions in similar
systems.
delivered the reagent solution into the ESI source at a flow rate of
10 µL/min. ESI and the QqQ (linear trap) mass spectrometer was
operated in the negative-ion mode. Main conditions: curtain gas
nitrogen flow ) 20 mL min ; ion spray voltage ) -4500 eV;
declustering potential ) -21 eV; entrance potential ) -10 eV;
collision cell exit potential ) -12 eV. Some of the main anionic
species detected by ESI-MS were subjected to ESI-MS/MS by using
collision-induced dissociation (CID) with nitrogen and collision
energies ranging from 5 to 45 eV.
-1
Other MS analyses were performed to detect nonanionic products
-
in the reaction medium. Reaction of BDNPP with BHO (1:1) was
followed in aqueous solution at pH 10 and 25 °C for 50 min, and
the products were extracted with diethyl ether, and after evaporation,
the EI-MS spectrum was obtained in a low-resolution instrument
by using the direct injection mode, with electron ionization (EI) at
70 eV. The samples were placed in a sample vial fixed onto the
probe. Probe temperatures were programmed as follows: 10 °C
Experimental Section
Materials. BDNPP as the pyridinium salt was prepared as
5
+
described, and the pyridinium ion was exchanged for Na on the
cation-exchange resin in the Na+ form. Benzohydroxamic acid
1
1
(
BHOH) was prepared following a described procedure, with
-1
modifications summarized in the Supporting Information, resulting
min up to 85 °C and held for 3 min, increasing from 85 to 300
25
-1
in the formation of white crystals: mp 125-126 °C (lit. 124-125
C). All other inorganic and organic reagents, such as 2,4-
at 20 °C min and held for 3 min.
NMR Spectroscopy. All 1H spectra were monitored on a
°
dinitrophenol and 1-chloro-2,4-dinitrobenzene, were of the highest
purity and were used as purchased.
spectrometer (400 MHz) at 25 °C, in D O, except for reactions
2
with 1-chloro-2,4-dinitrobenzene, where 10% CD CN was used.
3
1
Kinetics. Reactions followed spectrophotometrically were started
Some aromatic H signals were obscured by signals of the excess
BHO . The H chemical shifts are referred to internal sodium
-
3
-
1
by adding 10.0 µL of a stock solution of the substrate (4 × 10
M) in MeCN to 3.0 mL of the aqueous reaction mixture, with a
large excess of the nucleophile, assuring first-order kinetics for the
initial nucleophilic attacks upon the substrate. Solutions were self-
buffered from pH 8-10 with BHOH/BHO- and aqueous 0.1 M
NaOH and for pH 6-8, with 0.01 M phosphate buffer.
3-(trimethylsilyl) propionate (TSP), and pD was the observed pH
2
8
+0.4 in D O at 25 °C.
2
Potentiometric Titration. Potentiometric titrations were carried
out with a pH meter and a automatic buret, in a 150.0 mL
thermostatted cell, under N at 25 °C. A solution of 20.0 mL of
2
5.0 mM of BHO- was acidified with 1.0 mL of 0.1 M HCl and
Formation of 2,4-dinitrophenoxide ion (DNP) was monitored at
4
0
00 nm in the water jacketed thermostatted cell holder, at 25.0 (
titrated with small increments of CO -free 0.1008 M KOH with
2
2
9
.1 °C, of a diode-array spectrophotometer. Observed first-order
0.1 ionic strength (KCl). The FITEQL v.4.0 program was used
to calculate the constants.
rate constants (kobs) were calculated from linear plots of ln(A
∞
t
-A )
against time for at least 90% of the reaction by using an iterative
least-squares program; correlation coefficients were >0.999 for all
kinetic runs. Reactions of BDNPP with BHOH as a function of
pH were first-order with respect to BDNPP, and in all cases, more
than 1.5 mol of DNP was always formed in comparison to reactant.
Mass Spectrometry: In order to identify intermediates and
Acknowledgment. We are grateful to INCT-Cat a´ lise, PRON-
EX, FAPESC, FAPESP, CNPq, and CAPES for support of this
work.
Supporting Information Available: Preparation of benzo-
hydroxamic acid, UV absorption spectra for the reaction of
-
reaction products of BDNPP with BHO , direct infusion electro-
spray ionization mass spectrometry analyses were performed with
1
-chloro-2,4-dinitrobenzene (CDNB) with benzohydroxamate
1
,26,27
a hydrid triple quadrupole linear ion-trap mass spectrometer.
For typical electrospray ionization (ESI-MS) conditions, 1 mL of
-
anion (BHO ), low-resolution mass data for product identifica-
-
6
tion, NMR spectral data for product identification, and kinetic
1
× 10 M BDNPP, in aqueous medium at pH 10, was mixed
-
-1 -
data reaction of BDNPP with BHO . This material is available
with 100 µL min of 0.1 M aqueous BHO . A microsyringe pump
free of charge via the Internet at http://pubs.acs.org.
(
25) Reddy, A. S.; Kumar, M. S.; Reddy, G. R. Tetrahedron Lett. 2000, 41,
285–6288.
26) Abella, C. A. M.; Benassi, M.; Santos, L. S.; Eberlin, M. N.; Coelho,
F. J. Org. Chem. 2007, 72, 4048–4053.
27) Santos, L. S.; Pavam, C. H.; Almeida, W. P.; Coelho, F.; Eberlin, M. N.
Angew. Chem., Int. Ed. 2004, 43, 4330–4333.
JO9007354
6
(
(28) Fife, T. H.; Bruice, T. C. J. Phys. Chem. 1961, 65, 1079–1080.
(29) Heberlin, A. L.; Westall, J. C. FITEQL, version 4.0, report 99-01;
Department of Chemistry, Oregon State University: Corvalis, OR, 1999.
(
5
016 J. Org. Chem. Vol. 74, No. 14, 2009