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Tetrahedron Letters 55 (2014) 2917–2920
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Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
Mechanistic studies on the diazo transfer reaction
Arun Kumar Pandiakumar a, Siddhartha P. Sarma b,⇑, Ashoka G. Samuelson a,⇑
a
b
Department of Inorganic and Physical Chemistry, Indian Institute of Science, Bangalore 560012, India
Molecular Biophysics Unit, Indian Institute of Science, Bangalore 560012, India
a r t i c l e
i n f o
Article history:
Received 18 December 2013
Revised 11 March 2014
Accepted 12 March 2014
Available online 19 March 2014
a b s t r a c t
The mechanism of the diazo transfer reaction which converts amines to azides has been studied with
labeled amino acids and labeled imidazole-1-sulfonyl azides. Retention of amine nitrogen in the amine,
and transfer of the two terminal nitrogen atoms of the imidazole-1-sulfonyl azide to the product, were
unambiguously established.
Ó 2014 Elsevier Ltd. All rights reserved.
Keywords:
Imidazole-1-sulfonyl azideHCl
Labeled L-valine azide
Diazo (–N2) transfer
SNi mechanism
Labeled imidazole-1-sulfonyl azide
Azides are an important class of compounds performing key
roles in a variety of chemical transformations which include the
celebrated copper catalyzed ‘click’ reaction.1,2 An azide plays the
role of a protecting group for amines as it can be readily reduced
to regenerate the amine3, or converted to an amide4 or a carbamate5,6 using the Staudinger reaction.7 Often synthesis of an azide
by displacement of a nucleofuge results in elimination products,
and even if the azide is formed, the stereochemistry is unpredictable. To alleviate the loss in stereochemistry, better synthetic
methods have been developed for introducing an azide group.8,9
The most important of these is the ‘diazo’ transfer reaction, where
a diazo group (N2) is transferred to an amine to form an
azide.10,11 In this reaction, the stereochemistry of the starting
material is retained and there are no side products. Recent
examples12,13 where this has been used include the modification
of amino groups present in amino glycosides which are antibiotics
known to bind RNA.13
Among the various diazo transfer reagents developed14–18 the
most popular diazo transfer reagent is the hydrochloride salt of
imidazole-1-sulfonyl azide 1.18 It has several advantages over
other azidating reagents as it is inexpensive, easy to synthesize,
and not hazardous in the solid state. In spite of its importance,
the diazo transfer reaction has not been mechanistically elaborated. The first mechanistic proposal was made in 1967 by Anselme and co-worker.14 According to this mechanism, the amine
⇑ Corresponding authors. Tel.: +91 9844220898; fax: +91 8023601552 (A.G.S.).
E-mail address: [email protected] (A.G. Samuelson).
http://dx.doi.org/10.1016/j.tetlet.2014.03.057
0040-4039/Ó 2014 Elsevier Ltd. All rights reserved.
anion reacts with a tosyl azide giving a triazene anion intermediate
which then decomposes to give the azide. However, no experiments were conducted to confirm the structure of this intermediate that would identify the source of each N atom in the final
product.
The presence of an azide could be a useful spectroscopic probe
in proteins considering the fact that the azide has a clear IR signature in a region, that is, devoid of other bands characteristic of proteins.19,20 However, this possibility has not been explored
extensively. A clear understanding of the diazo transfer mechanism
presented in this Letter, would help one prepare specifically labeled proteins/reactants which might be useful for IR spectroscopic tagging of amines.
We first confirmed the diazo transfer as opposed to the SNi path
which would also result in retention of configuration. To the best of
our knowledge, it has not been shown that azide formation is not
following the SNi pathway. In order to conclusively show the identity of nitrogen atoms transferred during the azide formation, we
have carried out the diazo transfer reaction using 13C and 15N labeled amino acids such as valine 6 and isoleucine 8. The reaction
was carried out in the presence of imidazole-1-sulfonyl azideHCl
1 and copper(II) sulfate as shown in Scheme 1. The reaction was
optimized with unlabeled valine 2 and isoleucine 4, before carrying
out the reaction with labeled amino acids. Formation of azido
acids, 3 and 5, was confirmed by infrared spectroscopy (IR), multinuclear NMR spectroscopy, and mass spectrometry. The chemical
structures of labeled, unlabeled amino acids, and the corresponding azides are given in Table 1.
2918
A. K. Pandiakumar et al. / Tetrahedron Letters 55 (2014) 2917–2920
Scheme 1. Diazo transfer reaction of amino acids (labeled and unlabeled) mediated
by imidazole-1-sulfonyl azideHCl.
Table 1
Diazo transfer reaction of labeled, unlabeled L-valine and L-isoleucinea
Entry
Amino acid
Azido acid
Yield (%)
1
79
2
71
3
83
4
66
coupling constants of 34.3 Hz and 58.4 Hz, respectively. Further,
coupling to the 15N nucleus (J = 3.3 Hz) confirms that the 15N was
retained in its position. This splitting pattern is similar to the pattern observed for the labeled valine 6 confirming retention of 15N
after transformation to the azide 7 (Fig. 1).
The proton NMR spectrum of 9 showed a similar splitting pattern for Hb0 as observed for Ha0 in 7 at 3.64 ppm and is completely
consistent with a diazo ‘–N@N–’ transfer. This excludes the SNi
pathway, shown in Scheme 2, which will result in an azide without
a labeled nitrogen atom through an azide transfer with retention of
configuration.
To show where the diazo transfer reagent is being attacked, it is
necessary to use a labeled azidating reagent. The labeled imidazole-1-sulfonyl azide was prepared from a 15N labeled sodium azide
10 (NaN15
2 N) which was labeled at the terminal position
(Scheme 3). Interestingly, the small scale synthesis of the azidating
agent of imidazole-1-sulfonyl azide yielded better results than the
corresponding synthesis of the imidazole-1-sulfonyl azide-hydrochloride salt 1. The reverse is true in a large scale synthesis.
The diazo transfer reaction was carried out using the labeled
imidazole-1-sulfonyl azide 11 as an azidating reagent. The labeling
of 11 was confirmed by various spectroscopic techniques. The
a
Reaction condition: amino acid (0.03 mmol), K2CO3 (0.08 mmol), imidazole-1sulfonyl azideHCl 1 (0.036 mmol), CuSO45H2O (1 mol %) in methanol at room
temperature.
The IR spectrum of valine azide 3 and isoleucine azide 5 showed
a clear signature for an azide group at 2108 cm1 and at
2106 cm1, respectively. In the proton NMR spectrum of 3, Ha appeared as a doublet at 3.78 ppm with a coupling constant of 5.6 Hz.
The proton decoupled 13C NMR spectrum of 3 showed a singlet at
68.3 ppm corresponding to the carbon bearing the azide group as
reported in the literature.21–23 Similar chemical shift values and
coupling constants were observed for isoleucine azide 5 as
well.21,22
In the case of labeled azido acids, the presence of an azide was
confirmed by IR spectroscopy, where a band at 2106 cm1 a characteristic stretching frequency for the azide confirmed the structure of 7. Further, formation of an azide was confirmed by
multinuclear NMR spectroscopy and mass spectrometry. Unlike
unlabeled azides (3, 5), 1H NMR spectra of labeled azides (7, 9)
showed additional fine structure due to the one-bond coupled
13
C nucleus and two-bond coupled 15N nucleus. For instance, a
doublet at 3.63 ppm for the Ha0 proton in 7 was further split into
a multiplet due to coupling with the 15N nucleus and the neighboring 13C nucleus. A large coupling constant of 144 Hz supports the
coupling of the Ha0 proton present on the 13C atom directly connected to the 15N atom.
The proton decoupled 13C NMR spectrum of 7 in CDCl3 showed
a doublet of a doublet of a doublet (ddd) at 67.8 ppm corresponding to the carbon bearing the azide group. The carbon connected to
the azide moiety was split into a doublet of a doublet (dd) due to
coupling with the neighboring 13CH and the 13COOH groups with
Figure 1. 13C NMR spectra of carbon bearing amine group 6 (top) and azide group 7
(bottom) of labeled valine.
Scheme 2. SNi mechanism for the formation of valine and isoleucine azide that is
excluded by formation of 7 and 9.
Scheme 3. Synthesis of
15
N labeled isotopomers of imidazole-1-sulfonyl azide.
A. K. Pandiakumar et al. / Tetrahedron Letters 55 (2014) 2917–2920
proton decoupled 15N NMR spectrum showed two peaks at
247.93 ppm and at 137.07 ppm, corresponding to 15N nucleus directly attached to the –SO2 group and 15N nucleus at the terminal
position, respectively, in nearly 1:1 ratio. The IR spectrum showed
an intense broad band at 2119 cm1 confirming the presence of an
azide group. Deconvolution of the band revealed two bands at
2119 cm1 and at 2142 cm1 corresponding to the two labeled isotopomers of imidazole-1-sulfonyl azide 11.
A mechanistic proposal for the attack of the azide on the terminal nitrogen has been made by Wong and coworkers and is shown
in Scheme 4.24 This 15N labeled imidazole-1-sulfonyl azide 11 was
employed as an azidating reagent to probe the position of attack by
the amine group. The diazo transfer reaction was carried out with
unlabeled valine 2 and labeled imidazole-1-sulfonyl azide 11 in the
presence of a metal catalyst, CuSO45H2O under identical conditions. The IR spectrum of the azido acid synthesized using the labeled reagent exhibited two bands at 2108 cm1 and at
2067 cm1 showing the presence of two azide isotopomers
(Fig. 2). The band at higher stretching frequency corresponds to
the valine azide 3 which lacks a labeled 15N atom and the band
at lower stretching frequency corresponds to the 15N labeled azide
12.
To understand the position of attack, we analyzed the splitting
pattern of the hydrogen connected to the carbon bearing the azide
group in the proton NMR spectrum. It appeared as a multiplet at
3.77–3.79 ppm. The multiplet is a combination of a doublet at
3.78 ppm with a coupling constant of 5.6 Hz and another doublet
of a doublet at 3.77 ppm with a coupling constant of 4.4 Hz. A reasonable explanation is that the doublet corresponds to the
H
Tf
H
L2X2 Cu + RNH 2
- XH
X = Cl
L = Neutral ligand
L2 XCu N
R
TfN2 N
-L
N
L
Cu
X
N
N N
2919
unlabeled valine azide 3 and the doublet of a doublet corresponds
to the labeled valine azide 12 as shown in Scheme 5. This doublet
of a doublet (3.77 ppm) is due to the 3JN-H coupling of 15N and 1H
attached to the carbon bearing the azide group. Two valine azide
isotopomers 3 and 12 were present in a 1:1 ratio (confirmed by
integration of the doublet and the doublet of a doublet). The
appearance of a doublet of a doublet in the 1H NMR spectrum
due to coupling with a 15N atom was further verified by recording
15
N decoupled 1H NMR spectrum which showed the collapse of
doublet of a doublet to a doublet (Fig. 3).
Two peaks, of nearly equal intensity, with a mass difference of
one unit detected in the mass spectrometric studies, confirmed
the presence of unlabeled 3 and valine azide 12 with a 15N label.
The position of the label was ascertained from the proton decoupled 15N NMR spectrum which showed a singlet at 245.62 ppm
corresponding to the labeled valine azide 12. The proton coupled
15
N NMR showed a doublet at 245.53 ppm with a coupling constant of 4.4 Hz suggesting a 3JN-H coupling of 15N and 1H. To validate the assignment of the middle position for the labeled
nitrogen atom, 2D heteronuclear multiple bond correlation spectroscopy was carried out. The 3JN-H coupling between 15N and 1H
in the labeled azide 12 resulted in a correlation peak between
15
N at 245.62 ppm and the doublet of a doublet in the 1H NMR
spectrum at 3.77 ppm. These results conclusively showed that
the diazo transfer happens due to attack of the NH2 group on the
terminal nitrogen of the labeled imidazole-1-sulfonyl azide 11.
The diazo transfer reaction was also carried out with labeled valine 6 as a substrate to further confirm the generality of the amine
carrying out an attack on the terminal position of the azidating
agent. The product was analyzed as described above for 3 and
12. Analysis showed the formation of two valine azide isotopomers
13 and 7 (double 15N labeled and single 15N labeled valine azide) in
a 1:1 ratio. This shows that in the presence of a metal catalyst,
R
+ RNH 2 - TfNH 2
L2 XCu
+L
Tf
NH + R-N=N=N
Scheme 4. Plausible mechanism (after Wong and co-workers24) of copper catalyzed diazo transfer reaction illustrated with the terminally labeled isotopomer of
TfN3.
Scheme 5. Diazo transfer reaction of L-valine with labeled azidating reagent.
Figure 2. IR spectrum of the product obtained from reaction of
imidazole azide with valine.
15
N labeled
Figure 3. 15N coupled (bottom) and decoupled (top) 1H NMR spectrum of unlabeled
3 and middle 15N labeled 12 valine azide.
2920
A. K. Pandiakumar et al. / Tetrahedron Letters 55 (2014) 2917–2920
In summary, the present study provides strong evidence that
the diazo transfer reaction involves transfer of the two terminal
nitrogen atoms of the azidating agent to the amine to form the
azide as suggested by Anselme.14 This was confirmed by using labeled amino acids such as L-valine 6 and L-isoleucine 8. This study
precluded the possibility of an azide (–N3) transfer through SNi
mechanism. Labeled imidazole-1-sulfonyl azide 11 conclusively
proved that the attack of the amine group occurred on the terminal
nitrogen as suggested by Wong and co-workers.24 It was confirmed
that the absence or presence of a metal catalyst, did not alter the
site of amine attack.
Acknowledgement
Scheme 6. Plausible mechanism involving the attack of the amine on the middle
nitrogen of imidazole-1-sulfonyl azide in the absence of a metal ion.
A.G.S. thanks DST, New Delhi for the award of a research grant
and A.K. gratefully acknowledges a senior research fellowship from
CSIR. We thank DST, New Delhi, for providing us funds through its
FIST program for purchase of a 400 MHz NMR spectrometer.
Supplementary data
Supplementary data (all experimental procedures and characterization data for compounds are provided in the supplementary
data) associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.tetlet.2014.03.057. These data
include MOL files and InChiKeys of the most important compounds
described in this article.
References and notes
Scheme 7. A mechanistic scheme to explain attack of the amine on the terminal
nitrogen of imidazole-1-sulfonyl azide.
diazo transfer occurs due to the attack of the amine group on the
terminal nitrogen of the labeled imidazole-1-sulfonyl azide 11.
Thus all the data confirm the mechanism suggested by Wong
and co-workers for the formation of the azide shown in
Scheme 4.24
As reported in the literature the diazo transfer reaction can be
used to modify enzymes, antibiotics, and proteins to study their
functions or to modify their activity. But in these cases, the Cu(II)
catalyst is avoided lest it have any adverse effect on the structure
or biological function of the protein.25,26 To probe if these metalfree reactions proceed by a different mechanism such as the one
shown in Scheme 6, the reaction was carried out using unlabeled
valine 2 as a substrate and labeled imidazole-1-sulfonyl azide 11
as an azidating reagent without employing a metal catalyst. This
reaction also yielded two valine azide isotopomers 3 and 12 (unlabeled and middle 15N labeled valine azide) in the ratio 1:1 which
suggests that even in the absence of metal catalyst the reaction
proceeds through a mechanism where the amine group prefers
to attack the terminal nitrogen of the labeled imidazole-1-sulfonyl
azide 11. A plausible mechanism is given in Scheme 7.
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