Improved synthesis of 4-amino-7-nitrobenz-2,1,3-oxadiazoles using NBD fluoride (NBD-F)

Article abstract of DOI:10.1016/j.tetlet.2011.02.111

The 7-nitrobenz-2,1,3-oxadiazole (NBD) unit is a highly useful fluorescent tag with wide application in biology. Installation of the NBD group typically proceeds via the S_NAr reaction between an amine and an NBD halide. Herein, we demonstrate that NBD-F 1 results in significantly higher yields than NBD-Cl 2, and that triethylamine in dimethylformamide at 23 °C overnight is a broadly applicable set of conditions for this reaction. In particular, the highly useful fluorescent carbohydrate 2-NBD-glucosamine (2-NBDG, 3) can now be prepared in 75% yield with NBD-F as compared to 12% with NBD-Cl.

Full text of DOI:10.1016/j.tetlet.2011.02.111

Tetrahedron Letters 52 (2011) 2533–2535
Contents lists available at ScienceDirect
Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
Improved synthesis of 4-amino-7-nitrobenz-2,1,3-oxadiazoles using
NBD fluoride (NBD-F)
⇑
Michael E. Jung , Timothy A. Dong , Xiaolu Cai
Department of Chemistry and Biochemistry, University of California, Los Angeles, CA 90095-1569, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
The 7-nitrobenz-2,1,3-oxadiazole (NBD) unit is a highly useful fluorescent tag with wide application in
biology. Installation of the NBD group typically proceeds via the SNAr reaction between an amine and
an NBD halide. Herein, we demonstrate that NBD-F 1 results in significantly higher yields than NBD-Cl
2, and that triethylamine in dimethylformamide at 23 °C overnight is a broadly applicable set of condi-
tions for this reaction. In particular, the highly useful fluorescent carbohydrate 2-NBD-glucosamine
(2-NBDG, 3) can now be prepared in 75% yield with NBD-F as compared to 12% with NBD-Cl.
Ó 2011 Elsevier Ltd. All rights reserved.
Received 2 February 2011
Revised 23 February 2011
Accepted 24 February 2011
Available online 2 March 2011
First introduced in 1968,1a the 7-nitrobenz-2,1,3-oxadiazole
(NBD) unit is a very useful fluorescent tag that has found wide
application in biology,1b for example, in the labeling of amino
acids,2 lipids,3 biotin,4 sugars,5 proteins,6 and other molecules.7 It
is typically installed via an SNAr reaction between an amine (either
primary or secondary) and the corresponding halide (either the
chloride 1 or fluoride 2) to produce the highly fluorescent product.
While both simple primary and secondary aliphatic amines gener-
ally react with NBD-Cl to afford the corresponding SNAr products in
good yield (50–100%),8 other amines have been known to result in
very low yields,9 possibly due to steric hindrance or to the presence
of competing hydroxyl functional groups (Fig. 1). In particular, 2-
NBD-glucosamine (2-NBDG, 3) is a small molecule that has been
used extensively to study the uptake of glucose in a variety of
organisms. Used in conjunction with flow cytometry, it has become
a very useful method for measuring glucose uptake in real time in
bacteria,10 yeast,11 and mammalian cells.5a During the course of a
study, we needed a large quantity of this compound. Surprisingly,
the best reported literature yield of 2-NBDG 3 was only 12%, and
involved the reaction of glucosamine hydrochloride and NBD-Cl
in an aqueous sodium bicarbonate solution.5a Similarly, a synthesis
of the NBD-fructosamine 4 was also reported in low yield (<5%),5b
as were several other amines of biological relevance.9 There have
been numerous studies in the literature (mainly involving the
pre-column derivatization of amino acids for HPLC) that have dem-
onstrated the increased reactivity of NBD-F compared to NBD-Cl.12
However, many of these studies were carried out on extremely
reactivities of NBD-F 2 and NBD-Cl 1. Thus, we sought to quantita-
tively demonstrate the utility of NBD-F by carrying out the synthe-
sis of very low-yielding NBD-labeled amines, specifically with the
aim of improving the synthesis of 2-NBDG 3.
NO2
OH
OH
O
N
N
NH
O
N
N
HO
OH
HO
O
X
1 X = Cl (NBD-Cl)
2 X = F (NBD-F)
NO2
3 2-NBDG
O
N
H
N
HO
N
O
NO2
OH
HO
4 NBD-fructosamine
Figure 1.
NO2
F
N
N
b
c
small scale (lmol) and in most cases, the reaction products were
not isolated nor the yields reported. To the best of our knowledge,
O
O
X
N
N
there has not yet been a systematic comparison of the relative
F
F
F
5 X = NH2
6 X = NO
7
2
a
⇑
Corresponding author.
E-mail address: jung@chem.ucla.edu (M.E. Jung).
Scheme 1. Reagents and conditions: (a) mCPBA, DCM, 0 °C to 23 °C, 2 h, 100%; (b)
NaN3, DMSO, 23 °C, 2 h; (c) NaNO3, H2SO4 (aq), 0 °C, 3 min, 24% over two steps.
NIH Chemistry–Biology Interface (CBI) trainee, 2007–10.
0040-4039/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2011.02.111

2534
M. E. Jung et al. / Tetrahedron Letters 52 (2011) 2533–2535
Table 1
Comparison of the yields of the synthesis of the NBD-amines using NBD-Cl 1 versus NBD-F 2
X
NH-R
R-NH2
N
N
N
8-17
O
O
base
N
solvent
NO2
NO2
1 X = Cl (NBD-Cl)
2 X = F (NBD-F)
3-4, 18-25
Entry
Amine
Conditions
Product
Isolated yield (%)
Cl
(R = NBD)
F
OH
OH
O
O
HO
HO
HO
HO
OH
1
aq NaHCO3 CH3CN 23 °C/18 h
125a
75
OH
NH2 HCl
NH
R
•
3
8
OAc
OAc
O
O
AcO
AcO
AcO
AcO
OAc
OAc
2
3
NEt3 CH3CN 23 °C/18 h
NEt3 DMF 23 °C/49 h
Trace
61
71
NH2
NH
18
9
R
OBn
OBn
O
O
BnO
BnO
BnO
BnO
OBn
OBn
3
NH2 • HCl
NH
R
19
10
HO
NH
OH
CO H)
2 • (
H
2
2
HO
N
O
HO
R
O
HO
4
5
aq NaHCO3 CH3CN 23 °C/14 h
55b
62
85
OH
OH
11
OH
4
Me
Me
Me
Me
O
O
O
O
O
O
NEt3 DMF 23 °C/2.5 days
46
NH2
HN
O
O
R
O
O
Me
Me
20
Me
Me
12
N
COOH
13
N
R
COOH
21
6
7
aq Na2B4O7 CH3CN 23 °C/21 h
2712b
84
95
H
Me
Me
Me
Me
OH
R
OH
NEt3 DMF 23 °C/2.5 h
189a
H2N
N
H
14
22
23
8
NEt3 DMF 23 °C/2.5 h
199a
82
R
OH
OH
N
H
H2N
HO
15
OH
NH2
OH
HO
H
N
9
EtOH 50 °C/2.5 h
R
229b
58
79
HO
HO
16
17
24
25
H
NH2
N
10
NaHCO3(s) MeOH 23 °C/18 h
529b
N
N
R

M. E. Jung et al. / Tetrahedron Letters 52 (2011) 2533–2535
2535
The synthesis of NBD-F 2 was first reported by Di Nunno et al. in
1970.13 Starting from 2,6-difluoroaniline 5, NBD-F is produced in
three steps in 24% overall yield (Scheme 1). Oxidation of the aniline
5 gives the nitrosoarene 6 in quantitative yield. SNAr reaction of 6
with sodium azide in DMSO and subsequent cyclization at ambient
temperature affords the benzoxadiazole 7. Final nitration com-
pletes the synthesis of 2. The analogous three step sequence from
2,6-dichloroaniline yields NBD-Cl in 20% yield.14 While 2,6-difluo-
roaniline is only four times more expensive than 2,6-dichloroani-
line, it is surprising that NBD-F 2 is 300 times more expensive
than NBD-Cl 1.15 Using Di Nunno’s procedure, we were able to con-
veniently produce one gram of NBD-F 2 in a single synthetic se-
quence. Due to its susceptibility to hydrolysis, NBD-F 2 was
stored in the cold under argon and allowed to warm to ambient
temperature just prior to use.
24 h worked well and led to comparable yields (entries 1–3, 5,
and 7–9, Table 2). However, for unknown reasons, this set of con-
ditions led to poor yields for the reaction of fructosamine 11 to give
4, of proline 13 to give 21, and of 2-pyridylmethylamine 17 to give
25 (entries 4, 6, and 10).
In conclusion, we have found that the reaction of primary and
secondary amines with NBD-F 2 yields significantly higher yields
of the NBD-labeled amines than reaction with NBD-Cl 1. These re-
sults confirm the qualitative results known for some time of the
higher reactivity of NBD-F compared to that of NBD-Cl and are also
in agreement with the large body of literature regarding the higher
reactivity of aryl fluorides compared to aryl chlorides in the SNAr
reaction.16 Using NBD-F 2, we were able to produce the highly use-
ful fluorophores 2-NBDG 3 and NBD-fructosamine 4 in good yields
(72% and 62%, respectively).
We repeated each literature synthesis, using reaction conditions
identical to those reported to prepare each NBD-labeled amine,
except that NBD-F 2 was used in place of NBD-Cl 1. As shown in
Table 1, for a given set of reaction conditions, NBD-F afforded sig-
nificantly higher yields of the desired NBD-labeled amines than
NBD-Cl. This was true for highly water-soluble amines, for exam-
ple, glucosamine 8 (entry 1), fructosamine 11 (entry 4), proline
13 (entry 6), and tris(hydroxymethyl)methylamine (Tris) 16 (entry
9), as well as for the highly organic-soluble amines, for example,
the protected glucosamines 9 and 10 (entries 2–3), protected fruc-
tosamine 12 (entry 5) and the aminoalcohols, valinol 14 and phe-
nylalininol 15 (entries 7–8). In particular, we found that 2-NBDG 3
and NBD-fructosamine 4 were produced in yields of 75% and 62%,
respectively,––each a significant improvement over the previously
reported literature yields. Since the NBD unit is used fairly often to
analyze amino acids, the fact the NDB–F gave greatly improved
yields of the NBD–proline 21 is likewise important. Finally it
should be pointed out that hydrolysis of the bis(acetonide) 20 gave
the NBD–fructosamine 4 in 77% yield.
Interestingly, while the reaction of NBD–Cl with unprotected
fructosamine 11 gave low yield (5%), the bis(acetonide) protected
fructosamine 12 gave the significantly higher yield of 46% (entry
4 and 5). Reaction of other protected sugars, for example, glucosa-
mine tetraacetate 9 and tetra-O-benzyl glucosamine hydrochloride
10, with NBD–Cl produced consistently low yields. As can be seen
in Table 1, many different reaction conditions for the SNAr reaction
of NBD-halides have been reported in the literature. We wanted to
find a single set of reaction conditions that could be applied to a
wide range of amine substrates. Encouraged by the high yields ob-
tained from the reaction conditions of triethylamine in DMF at
ambient temperature overnight9a (entries 7 and 8) and also by
the ability of DMF to dissolve both water-soluble and organic-
soluble amines, we applied those same reaction conditions to the
aforementioned substrates. The results are given in Table 2.
In most cases, the reactions of the amines (1.1 equiv) using
2–18 equiv of triethylamine in DMF (0.6 M in amine) at 23 °C for
Acknowledgments
The authors are grateful to the NIH–CBI Training Grant (USPHS
National Research Service Award GM08496) for generous support
of this work. High-Res Mass Spectrometers were purchased with
support from Grant Number S10RR025631 from the National Cen-
ter for Research Resources.
Supplementary data
Supplementary data (experimental procedures and spectral
data for new compounds) associated with this article can be found,
in the online version, at doi:10.1016/j.tetlet.2011.02.111.
References and notes
1. (a) Ghosh, P. B.; Whitehouse, R. D. Biochem. J. 1968, 108, 155–156; For a review,
see: (b) Lavis, L. D.; Raines, R. T. ACS Chem. Biol. 2008, 3, 142–155.
2. (a) Cooper, S. J.; Finney, G. L.; Brown, S. L.; Nelson, S. K.; Hesselberth, J.;
MacCoss, M. J.; Fields, S. Genome Res. 2010, 20, 1288–1296; (b) Aoyama, C.;
Santa, T.; Tsunoda, M.; Fukushima, T.; Kitada, C.; Imai, K. Biomed. Chromatogr.
2004, 18, 630–636.
3. (a) Novotny´, J.; Pospechová, K.; Hrabálek, A.; Cáp, R.; Vávrová, K. Bioorg. Med.
Chem. Lett. 2009, 19, 6975–6977; (b) Babià, T.; Ledesma, M. D.; Saffrich, R.; Kok,
J. W.; Dotti, C. G.; Egea, G. Traffic 2001, 2, 395–405.
ˇ
ˇ
´
4. Volny, M.; Elam, W. T.; Ratner, B. D.; Turecek, F. Anal. Chem. 2005, 77, 4846–
4853.
5. (a) Yamada, K.; Saito, M.; Matsuoka, H.; Inagaki, N. Nat. Protoc. 2007, 2, 753–
762; (b) Levi, J.; Cheng, Z.; Gheysens, O.; Patel, M.; Chan, C. T.; Wang, Y.;
Namavari, M.; Gambhir, S. S. Bioconjugate Chem. 2007, 18, 628–634.
6. (a) Doi, Y.; Hashimoto, T.; Yamaguchi, H.; Vertut-Doï, A. Eur. J. Biochem. 1991,
199, 277–283; (b) McCabe, R. T.; Wilson, B. R.; Rhodes, C. A. PCT WO 9423301.;
(c) Shi, Z.-D.; Karki, R. G.; Oishi, S.; Worthy, K. M.; Bindu, L. K.; Dharmawardana,
P. G.; Nicklaus, M. C.; Bottaro, D. P.; Fisher, R. J.; Burke, T. R. Bioorg. Med. Chem.
Lett. 2005, 15, 1385–1388.
7. (a) Tao, L.; Kaddis, C. S.; Ogorzalek Loo, R. R.; Grover, G. N.; Loo, J. A.; Maynard,
H. D. Chem. Commun. 2009, 2148; (b) Chong, H.-S.; Song, H. A.; Ma, X.; Lim, S.;
Sun, X.; Mhaske, S. B. Chem. Commun. 2009, 3011; (c) Wilson, J. J.; Fedoce Lopes,
J.; Lippard, S. J. Inorg. Chem. 2010, 49, 5303–5315; (d) You, L.; Gokel, G. W.
Chem. A Eur. J. 2008, 14, 5861–5870.
8. (a) Heberer, H.; Kersting, H.; Matschiner, H. J. Prakt. Chem. 1985, 327, 487–504;
(b) Uchiyama, S.; Santa, T.; Fukushima, T.; Homma, H.; Imai, K. J. Chem. Soc.,
Perkin Trans. 2 1998, 2165–2174.
Table 2
9. (a) Schmidinger, H.; Birner-Gruenberger, R.; Riesenhuber, G.; Saf, R.; Susani-
Etzerodt, H.; Hermetter, A. ChemBioChem 2005, 6, 1776–1781; (b) Bem, M.;
Badea, F.; Draghici, C.; Caproiu, M. T.; Vasilescu, M.; Voicescu, M.; Beteringhe,
A.; Caragheorgheopol, A.; Maganu, M.; Constantinescu, T.; Balaban, A. T.
ARKIVOC 2007, 87–104.
Reaction of amines with NBD-F in DMF using triethylamine as base
Entry
Amine
Product
Yield (%)
1
2
3
4
5
6
7
8
9
10
8
9
3
65
86
71
0
85
0
95
82
51
0
18
19
4
20
21
22
23
24
25
10. Natarajan, A.; Srienc, F. Metab. Eng. 1999, 1, 320–333.
10
11
12
13
14
15
16
17
11. Achilles, J.; Müller, S.; Bley, T.; Babel, W. Cytometry 2004, 61A, 88–98.
12. (a) Imai, K.; Watanabe, Y. Anal. Chim. Acta 1981, 130, 377–383; (b) Watanabe,
Y.; Imai, K. Anal. Biochem. 1981, 116, 471–472; (c) Watanabe, Y.; Imai, K. J.
Chromatogr., A 1982, 239, 723–732; (d) Toyo’oka, T.; Watanabe, Y.; Imai, K.
Anal. Chim. Acta 1983, 149, 305–312.
13. Di Nunno, L.; Florio, S.; Todesco, P. E. J. Chem. Soc. C 1970, 1433–1434. NBD-F is
also commercially available but relatively expensive, for example, Sigma: 5 mg,
$147.
14. He, J.; He, J. Zhongguo Yiyao Gongye Zazhi 2002, 33, 425–426.
15. Calculated using prices from Aldrich Chemical.
16. Vlasov, V. M. Russ. Chem. Rev. 2003, 72, 681.
Conditions: NBD-F (1.0 equiv), amine (1.1 equiv), NEt3 (2–18 equiv), DMF (0.6 M in
amine), 23 °C, overnight in dark.