Triphenylphosphinecarboxamide: An effective reagent for the reduction of azides and its application to nucleic acid detection

Article abstract of DOI:10.1021/ol402832w

A series of triphenylphosphinecarboxamide (TPPc) derivatives were designed and synthesized as alternative reagents to triphenylphosphine for the facile reduction of azides. The TPPc derivatives performed as efficient reducing agents for the synthesis of primary amines without the need for an additional hydrolysis procedure. The TPPc derivatives were also applied to nucleic acid sensing using a RhAz-oligonucleotide conjugate in a DNA-templated fluorogenic reaction.

Full text of DOI:10.1021/ol402832w

Letter
pubs.acs.org/OrgLett
Triphenylphosphinecarboxamide: An Effective Reagent for the
Reduction of Azides and Its Application to Nucleic Acid Detection
Hisao Saneyoshi,†,∥ Tatsuya Ochikubo,† Takushi Mashimo,†,⊥ Ken Hatano,⊥ Yoshihiro Ito,*,†,∥
and Hiroshi Abe*,†,‡,§,∥,⊥
†Nano Medical Engineering Laboratory, RIKEN, 2-1, Hirosawa, Wako, Saitama 351-0198, Japan
‡Faculty of Pharmaceutical Sciences, Hokkaido University, Kita-12, Nishi-6, Kita-ku, Sapporo 060-0812, Japan
§PRESTO, Japan Science and Technology Agency, 4-1-8, Honcho, Kawaguchi, Saitama 332-0012, Japan
∥Emergent Bioengineering Materials Research Team, RIKEN Center for Emergent Matter Science, 2-1, Hirosawa, Wako, Saitama
351-0198, Japan
⊥Division of Material Science, Graduate School of Science and Technology, Saitama University, 255 Shimo-Ohkubo, Sakura-ku,
Saitama 338-8570, Japan
S
* Supporting Information
ABSTRACT: A series of triphenylphosphinecarboxamide (TPPc) deriva-
tives were designed and synthesized as alternative reagents to triphenyl-
phosphine for the facile reduction of azides. The TPPc derivatives performed
as efficient reducing agents for the synthesis of primary amines without the
need for an additional hydrolysis procedure. The TPPc derivatives were also
applied to nucleic acid sensing using a RhAz-oligonucleotide conjugate in a
DNA-templated fluorogenic reaction.
he reduction of an azide group to the corresponding
amine represents an indispensable reaction in organic
is not possible to use post-treatment processes of this type in a
cellular context. One of the alternatives to using triphenyl-
phosphine is to use trialkyl phosphines such as triscarbox-
ymethylphosphine (TCEP), which has been widely used as a
reducing reagent in biochemistry.17 Unfortunately, alkyl
phosphines are unstable and can be readily oxidized in air as
well as aqueous solution.11
With this particular limitation in mind, our attention became
focused on the impact of introducing substituents at the ortho
position of the phenyl rings of triphenylphosphine (TPP) to
assist in the hydrolysis of the intermediate iminophosphorane
through neighboring group participation (NGP). One of the
most successful examples of NGP in this context was a
Staudinger-Bertozzi ligation, where an o-methylcarboxydiphen-
yl phosphine derivative was used to facilitate an intramolecular
migration to form a covalent amide bond.3−5 Interestingly, the
results of this particular study indicated that the addition of
water was also required to facilitate this reaction. This study
inspired us to want to develop a new triphenylphosphine-based
derivative for the reduction of azides. As shown in Figure 1, it
was envisaged that the use of a carboxamide instead of an ester
T
synthesis, and the Staudinger reaction, especially, continues to
be an important transformation in both modern organic
synthesis and bioorganic chemistry.1,2 There are several
attractive features to the Staudinger reaction, including the
fact that the reaction requires only very mild conditions that are
compatible with other hydrogenolytically sensitive functional
groups such as benzyl type protecting groups and double bond
moieties. In addition, the bioorthogonal properties of the
Staudinger reaction have been employed in chemical ligations
(i.e., Staudinger-Bertozzi ligation)3−5 and molecular sensing,
where azide masked fluorogenic compounds were used in
oligonucleotide templated reactions.6−15 The iminophosphor-
ane intermediate formed during the Staudinger reaction,
however, can be particularly stable toward hydrolysis with
H2O and can require relatively long reaction times to be
completely converted to the corresponding primary amines.
For convenience, acidic or basic conditions are generally used
to hydrolyze the iminophosphoranes.16 During the synthesis of
multifunctional target molecules or analytical systems such as
those used in DNA detection, for example, the requirement for
additional treatments can be time-consuming, and the treat-
ments themselves could have an adverse impact on some of the
other functional groups present in the system. Furthermore, it
Received: October 2, 2013
Published: December 3, 2013
© 2013 American Chemical Society
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Table 1. Model Staudinger Reaction using TPP and the
a
TPPc Derivatives
entry
R
solvent
time (h)
6 (%) 7 (%) 8 (%)
1
2
3
4
5
H
H
H
A
B
B
A
B
B
A
B
A
B
2
98
94
95
2
24
CONH2
CONH2
CONH2
2
65
91
93
51
58
38
45
28
2
b
6
10 min
7
CONHCH3
CONHCH3
CON(CH3)2
CON(CH3)2
2
2
2
2
21
5
8
9
Figure 1. Azide reduction via neighboring group participation.
10
a
Conditions: 5 (0.1 M), TPP or TPPc 2−4 (1.1 equiv); solvent: A
as the ortho substituent would prevent the migration of this
substituent because of its poor migratory aptitude of the
carboxamide group. Furthermore, the carboxamide moiety has
been shown to assist in NGP18 and could promote the rapid
hydrolysis of the iminophosphorane intermediate without the
requirement for any additional treatment processes. In this
study, a series of triphenylphosphine carboxamide (TPPc)
derivatives were designed and synthesized to investigate the
feasibility of our new mechanism. These TPPc derivatives were
subsequently used to reduce azides without the requirement for
an additional hydrolysis treatment in nucleic acid sensing.
For the synthesis of the TPPc derivatives, o-diphenyl
phosphino benzoic acid 1 was subjected to a series of coupling
reactions with ammonia, methyl amine, and dimethyl amine
using a mixed anhydride intermediate to give the desired TPPc
derivatives 2−4, as shown in Scheme 1.
{THF (5 mL) and quenched with H2O} or B {THF/H2O (5 mL),
9:1, v/v}. 1.5 equiv of 2 was used.
b
hand, TPPc 2 gave the reduced compound 6 (65%) as well as
the ligated product 8 (28%) under condition A. When aqueous
conditions were used, the reduced compound 6 was obtained in
excellent yield (91%) without any of the aza-ylide complex or
ligated product being detected. Furthermore, when a slight
excess of reducing reagent 2 (1.5 equiv) was used, the reaction
reached completion within 10 min to give compound 6 in 93%
yield (Table 1, entry 5). Pseudo-first-order condition was
applied and checked by 31P NMR. The result revealed that the
phosphine was rapidly converted to the phosphine oxide (5
min) with the aza-ylide complex not being detected
(Supplementary Figure S1). The difference between these
results was attributed to the amide group, which presumably
assisted in the nucleophilic attack of water on the aza-ylide
complex. To clarified the effect of the amide structure, we also
tested the secondary and tertiary amides 3 and 4. Pleasingly,
similar tendencies were observed for these amides in terms of
the formation of the aza-ylide complex (Table 1, entries 7−10).
A comparison of the reactivity of these TPPc derivatives
revealed that their reactivity was dependent on the number of
substituents on the nitrogen atom. Thus, as the number of
substituents on the nitrogen increased, there was a reduction in
the yield and reactivity of the TPPc derivative toward the azide
group.To assess the overall scope and applicability of this new
method, several substrates were tested, including a functional
alkyl azide, fluorescent molecule, aromatic azide, carbohydrate,
and nucleoside. Pleasingly, the results revealed that the
reduction conditions described above were tolerant of a variety
of different functional groups, including benzyl, silyl, imide,
dimethoxytrityl, double bond, acetal, and carbamate moieties
(Table 2). Furthermore, the current reduction system
eliminated the requirement for an additional treatment process
to decompose the aza-ylide complex typically formed when
TPP is used as the reducing agent. Taken together, these results
demonstrate that the TPPc derivative 2 could be useful for the
Scheme 1. Synthesis of Triphenylphosphine Carboxamide
Derivatives
At first, we compared the reduction ability of TPP with those
of the TPPc derivatives (2−4) under conditions A (anhydrous
THF and then H2O) and B (THF/H2O, 9:1, v/v) (Table 1).
TPP reacted with methyl azide benzoate 519 to give aza-ylide
complex 7 in quantitative yield under both sets of conditions.
Surprisingly, the aza-ylide complex 7 remained intact even in
the presence of water (Table 1, entry 2). Furthermore, when
the reaction time was extended to 24 h, the aza-ylide complex
was obtained in 95% yield (Table 1, entry 3). On the other
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Table 2. Azide Reduction of Different Azide-Containing
a
Compounds
Figure 2. DNA-templated Staudinger reaction by conventional RhAz-
TPP or new RhAz-TPPc. (A) Fluorescence spectrum of reaction
mixture after 30 min. (B) Time course of DNA-templated Staudinger
reaction. Conditions: 100 nM RhAz probe, 300 nM TPP or TPPc
probe, 100 nM DNA target in 20 mM Tris-HCl (pH 7.2) containing
100 mM MgCl2 and 10 μg/mL BSA, 37 °C, 30 min, ex 490 nm, em
525 nm. Yield was calculated from reduced RhAz probe by
dithiothreitol (DTT).
The reaction yield of the RhAz-TPPc pair was determined to
be almost quantitative after 30 min. The greatest advantage of
the RhAz-TPPc pair over the RhAz-TPP pair was its signal
amplification ability, as shown in Supplementary Figures S2 and
S3. Even when the target DNA was present as 0.1 equiv, the
same reaction yield was similar to that achieved under
stoichiometric conditions. In contrast, the conventional RhAz-
TPP pair provided a much lower level of signal amplification.
The proposed mechanism for this transformation is shown in
Supplementary Figure S4. The TPP-RhAz pair (conventional)
would generate an aza-ylide complex, which would disturb the
strand exchange process and result in reduced levels of signal
amplification. These results indicated that the RhAz-TPPc pair
provided an enhanced level of signal amplification based on fast
aza-ylide hydrolysis, making it much more suitable for use in
nucleic acid detection compared with the conventional TPP-
RhAz probe pair.
In summary, we have developed a series of new TPPc
derivatives that can be used to reduce azides to the
corresponding amines under very mild conditions in a single
step. In contrast to the conditions traditionally used for the
reduction of azides by TPP, where an additional hydrolysis
reaction is required to break down any intermediate aza-ylide
complex, our new strategy successfully avoids this additional
step and allows for direct access to primary amines in complex
functional molecules. Further applications, such as those
involving the reduction of the azide group in the protein, are
currently being conducted in our laboratory.
a
Conditions: 9a−9g (0.1 M), phosphine 2 (1.1 equiv), THF/H2O
(9:1, v/v), rt, 2 h.
reduction of azide groups during the synthesis of multifunc-
tional molecules. We then proceeded to investigate the use of
TPPc for the detection of nucleic acid species using a
rhodamine azide (RhAz)-oligonucleotide probe in a biological
buffer. Several other groups including our own have already
reported the formation of an aza-ylide complex during a DNA
templated reaction using a TPP-oligonucleotide probe and its
fluorescence peak, which appears around 550 nm during
fluorescence analysis instead of 525 nm, which was previously
reported for rhodamine110.8,15 If the TPPc derivative worked
as suggested in Scheme 1, then none of the aza-ylide complex
would be formed. In addition, the fluorescence intensity of the
aza-ylide complex would be lower than that of the fully reduced
species.8
The new RhAz-TPPc probe pair gave a fluorescence signal
that was very similar to that of dithiothreitol, indicating that
none of the aza-ylide complex had been formed (Figure 2). In
contrast, the RhAz-TPP pair gave a smaller fluorescence signal
because of the formation of the aza-ylide complex.
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Letter
ASSOCIATED CONTENT
* Supporting Information
■
S
Experimental procedures and full spectroscopic data for all new
compounds. This material is available free of charge via the
Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Authors
■
*E-mail: y-ito@riken.jp.
*E-mail: h-abe@pharm.hokudai.ac.jp.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
H.A. was financially supported by the Ministry of Education,
Culture, Sports, Science, and Technology (MEXT), Precursory
Research for Embryonic Science and Technology (PREST),
and the New Energy and Industrial Technology Development
Organization (NEDO). H.S. was financially supported by a
Grant-in-Aid for Young Scientists (B). We are grateful for the
support received from the Brain Science Institute (BSI)
Research Resource Center for mass spectrum analysis.
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