Base-mediated one-pot synthesis of aliphatic diazirines for photoaffinity labeling

Article abstract of DOI:10.3390/molecules22081389

Aliphatic diazirines have been widely used as prominent photophores for photoaffinity labeling owing to their relatively small size which can reduce the steric effect on the natural interaction between ligands and proteins. Based on our continuous efforts

Full text of DOI:10.3390/molecules22081389

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molecules
Article
Base-Mediated One-Pot Synthesis of Aliphatic
Diazirines for Photoaffinity Labeling
Lei Wang 1, Zetryana Puteri Tachrim 2, Natsumi Kurokawa 2, Fumina Ohashi 2,
Yasuko Sakihama 2 ID , Yasuyuki Hashidoko 2 and Makoto Hashimoto 2,
*
1
Center for Drug Design, Academic Health Center, University of Minnesota, Minneapolis, MN 55455, USA;
lei870610@gmail.com
Division of Applied Bioscience, Graduate School of Agriculture, Hokkaido University, Kita 9, Nishi 9,
Kita-ku, Sapporo 060-8589, Japan; z317_style@live.com (Z.P.T.); natsumi.k0420@gmail.com (N.K.);
fumina28ohsei@gmail.com (F.O.); sakihama@abs.agr.hokudai.ac.jp (Y.S.);
yasu-h@abs.agr.hokudai.ac.jp (Y.H.)
2
*
Correspondence: hasimoto@abs.agr.hokudai.ac.jp; Tel./Fax: +81-11-706-3849
Received: 2 August 2017; Accepted: 21 August 2017; Published: 22 August 2017
Abstract: Aliphatic diazirines have been widely used as prominent photophores for photoaffinity
labeling owing to their relatively small size which can reduce the steric effect on the natural interaction
between ligands and proteins. Based on our continuous efforts to develop efficient methods for
the synthesis of aliphatic diazirines, we present here a comprehensive study about base-mediated
one-pot synthesis of aliphatic diazirines. It was found that potassium hydroxide (KOH) can also
promote the construction of aliphatic diazirine with good efficiency. Importantly, KOH is cheaper,
highly available, and easily handled and stored compared with the previously used base, potassium
tert-butoxide (t-BuOK). Gram-scale study showed that it owned great advantages in being used
for the large-scale production of aliphatic diazirines. This protocol is highly neat and the desired
products can be easily isolated and purified. As the first comprehensive study of the base-mediated
one-pot synthesis of aliphatic diazirines, this work provided good insight into the preparation and
utilization of diazirine-based photoaffinity labeling probes.
Keywords: photoaffinity labeling; one-pot synthesis; aliphatic diazirine; base-mediated reaction
1. Introduction
Photoaffinity labeling [1–5] has emerged as a powerful methodology in the field of biology
due to its availability for elucidating the interactions between bioactive molecules and proteins.
Various photophores—including phenyl azides, benzophenones, and diazirines—are commonly
employed in this field [6–11]. Under UV irradiation, photophores are converted into highly
reactive intermediates which can form covalent bonds between ligands and proteins. The stable
links in the components allow for further separation and detection. Among these photophores,
aliphatic diazirines [12
thus minimizing the steric effect of the natural interaction between ligands and proteins. For the
conventional synthesis procedure of aliphatic diazirines [17 18], the ketones precursor was first treated
–16] have attracted tremendous attention due to their relatively small size,
,
with ammonia for several hours, then it was reacted with hydroxylamine-O-sulfonic acid (NH2OSO3H)
to form diaziridines. After removal of the ammonia, diaziridines were further converted to diazirine in
the presence of I2-TEA or Ag2O (Scheme 1a). Despite the wide applications, this protocol suffers many
issues such as low yield and tedious procedures. To solve these issues, we have recently developed
a novel one-pot synthesis of aliphatic diazirines by using t-BuOK under air [19]. In spite of its good
efficiency, t-BuOK is a flammable base and it should be commonly handled and stored under inert
gas. Furthermore, its relatively high price also limits the large-scale production of diazirines and the
Molecules 2017, 22, 1389; doi:10.3390/molecules22081389
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corresponding labeling probes. Herein, for the first time, we carried out a comprehensive study to
screen the bases and other conditions, and it was found that KOH also could be used for one-pot
synthesis of aliphatic diazirines with great efficiency (Scheme 1b). Significantly, it is cheaper, easily
handled, and stored as well as being highly available in many labs. Gram-scale study confirmed its
usability for the large-scale production of aliphatic diazirines. Furthermore, a kinetic study to elaborate
the one-pot process was also performed on the basis of NMR analysis.
Scheme 1. Previous (a) and base-mediated one-pot (b) synthesis of aliphatic diazirines.
2. Results and Discussion
2.1. Reaction Optimization
We initiated our studies by using levulinic acid (1a) as a model substrate for reaction optimization,
which was treated with 1.1 equivalent of NH2OSO3H in liquid ammonia at room temperature for
12 h to generate the corresponding diaziridine in situ. Then, base (3.3 equiv.) was gradually added
into the mixture and the reaction was further stirred at room temperature under air for 2 h. Lithium
amide was first tested and it was able to afford aliphatic diazirine (2a) at a 36% yield (Table 1,
entry 1). Sodium amide was examined and a slightly higher yield of 2a was obtained (44%, entry 2).
Several metal hydrides were able to mediate the construction of diazirine (2a), albeit with moderate
yields (entries 3–5). Alkoxides were also utilized to promote the formation of products and the
results indicated that a potassium base is preferred to sodium one (entries 6–8). Furthermore, NaOH
and KOH [20–25] were employed and it was found that KOH was able to afford 2a in 67% yield
(entries 9 and 10). Although its efficiency was slightly lower compared to t-BuOK [19], KOH is easily
handled, stored, and highly available in many labs. Its relatively low price makes it feasible for
large-scale synthesis of aliphatic diazirines. Moreover, the by-products derived from OH anion can
easily be removed in this reaction. Thus, we optimized KOH for the subsequent study. The reaction
carried out at either low temperature (entry 11) or in methanolic ammonia (entry 12) caused a low
yield of product (2a), indicating that both temperature and concentration of ammonia are crucial for
the generation of diazirine in this protocol.
2.2. Scope Investigation
With the optimized condition in hand, the generality of the protocol was evaluated with different
ketone procedures. A wide range of substituted ketones—including acid, alcohol, ester, and hexatomic
rings—were tolerated under this condition to be converted into the corresponding diazirine products.
When monoacid substrates were tested, 3.3 equivalents of KOH were required to create the desired
product and the corresponding diazirines were obtained in moderate to good yield (Scheme 2, 2a–d).
A binary acid derivative (1e) was subjected to the optimal reaction with 4.3 equivalents of KOH, and the
desired product (2e) was isolated in 35% yield. Neutral ketones with aliphatic chain or aromatic ring are
good substrates (2f–h) and up to an 89% yield was given for compound (2g). 3-Azibutanol (2i), a widely
utilized photophore, was readily synthesized with a 75% yield. Compared to the previously synthetic
method for 2i, the KOH-mediated procedure showed a relatively good efficiency. Furthermore, t-butyl

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alcohol ester was also tolerated in the optimal condition, and the desired product was isolated in
61% yield (2j). The spectroscopic data were summarized in Supplementary Materials.
Table 1. Reaction optimization of one-pot synthesis for aliphatic diazirine 2a a.
Yield (%) b
Entry
Solvent
Base
Temp
1
2
3
4
5
6
7
8
9
NH3
NH3
NH3
NH3
NH3
NH3
NH3
NH3
NH3
NH3
NH3
LiNH2
NaNH2
NaH
MgH2
CaH2
EtONa
MeONa
MeOK
NaOH
KOH
RT
RT
RT
RT
RT
RT
RT
RT
RT
RT
36
44
47
20
17
45
45
63
28
67
11
10
11
◦
KOH
0 C
NH3/MeOH
12
KOH
RT
5
c
a
Unless otherwise mentioned, 1a (2 mmol) and NH2OSO3H (1.1 equiv.) was added to liquid ammonia (8 mL)
at
−
78 ◦C and the reaction mixture was stirred at room temperature for 12 h until base (3.3 equiv.) was added.
b
After the addition of base, the reaction was further stirred at room temperature under air for 2 h. Isolated yields
were presented. Ammonia gas was bubbled into MeOH (8 mL) at 0 ◦C for 30 min.
c
a
a
Scheme 2. Scope investigation of one-pot synthesis of aliphatic diazirine with KOH . Unless
otherwise mentioned, (2 mmol) and NH2OSO3H (1.1 equiv.) was added into liquid ammonia (8 mL)
at
78 ◦C and the reaction mixture was stirred at room temperature for 12 h until base was added.
1
−
After the addition of base, the reaction was further stirred at room temperature under air for 2 h.
b
4.3 equivalent of KOH was used.

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2.3. Kinetic Study of One-Pot Synthesis of Aliphatic Diaizirine
To investigate the one-pot process, we performed a kinetic study based on 1H-NMR analysis.
Aliphatic ketone (1f) (Scheme 3a) was initially treated with liquid ammonia at room temperature, while
no imine (1f0) was observed although the treatment was continued to 12 h. When 1f was subjected
to the reaction with NH2OSO3H in liquid ammonia at room temperature for 12 h, the corresponding
diaziridine (1f00) was detected (Scheme 3b). To be noted, a trace amount of diazirine (2f) was generated
at this stage which can be detected by 1H-NMR spectrum. Following with further treatment by KOH
for 2 h, the formation of diazirine (2f) was dramatically improved (Scheme 3c). Furthermore, no
other by-products were distinctly detected in the spectrum during the formation of diaziridine as well
as diazirine. These results indicated that diaziridine as the precursor can be directly converted to
diazirine in KOH-mediated one-pot reaction. This method is useful with the isolation and purification
of the final products being more convenient.
Scheme 3. One-pot synthesis of aliphatic diazirine 2f. The reaction stages were monitored using
1H-NMR with CDCl3.
(a) starting material 1f; (b) 1f was treated with NH2OSO3H in liquid ammonia
for 12 h; (c) The residue was further treated with KOH in liquid ammonia under air for 2 h.
2.4. Gram-Scale Synthesis
To explore the behavior of this protocol for large-scale synthesis of aliphatic diazirines, we
designed gram-scale reactions. Compound 1f (1.03 g) was subjected to the optimized conditions and
the desired product (2f) can be readily isolated at a 90% yield (Scheme 4a). It undoubtedly confirmed
the advantages and applicability of KOH-mediated synthetic strategy for the large-scale synthesis
of aliphatic diazirines. 3-Azibutanol (2i) is a widely used photophore that labels active molecules
in the field of photoaffinity labeling [26–30]. While many syntheses suffered relatively low yields.
By using the KOH-mediated one-pot strategy, we can synthesize 2i in a 75% yield (Scheme 2). It is
a great improvement compared with previous synthetic methods as well as the t-BuOK-mediated