An efficient synthesis of a class of heterobifunctional photo-reactive crosslinkers, labels, and probes

Article abstract of DOI:10.1016/S0040-4039(01)00131-9

Aryl esters are selectively reduced with DIBAL-H in the presence of an azide functional group to provide access to 4-azido-2-alkoxybenzaldehydes in five steps with overall yields ranging from 72 to 78%. This methodology improves traditional approaches to this class of compounds, which suffer from poor overall yields (4-11%). Our approach can be used to synthesize a variety of new aryl azide cross-linkers, labels, and probes.

Full text of DOI:10.1016/S0040-4039(01)00131-9

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 42 (2001) 2249–2251
An efficient synthesis of a class of heterobifunctional
photo-reactive crosslinkers, labels, and probes
Kyle W. Gano, Harold G. Monbouquetteb and David C. Mylesa,*
aDepartment of Chemistry and Biochemistry, University of California, Los Angeles, CA 90095-1569, USA
bDepartment of Chemical Engineering, University of California, Los Angeles, CA 90095-1592, USA
Received 6 October 2000; revised 17 January 2001; accepted 22 January 2001
Abstract—Aryl esters are selectively reduced with DIBAL-H in the presence of an azide functional group to provide access to
4-azido-2-alkoxybenzaldehydes in five steps with overall yields ranging from 72 to 78%. This methodology improves traditional
approaches to this class of compounds, which suffer from poor overall yields (4–11%). Our approach can be used to synthesize
a variety of new aryl azide cross-linkers, labels, and probes. © 2001 Elsevier Science Ltd. All rights reserved.
Heterobifunctional photoaffinity cross-linkers contain-
ing aryl azides, have simplified the study of protein–
protein interactions in multi-subunit proteins1 and have
found use as photoaffinity probes/labels due to their
ability to be made radioactive with high specific acitivi-
ties.2 In contrast to homobifunctional reagents, heterob-
ifunctional reagents allow the stepwise activation of
crosslinking groups. Thus, a single subunit or small
molecule can be activated with one functional group of
a photo-reactive heterobifunctional reagent (Fig. 1). This
protein or ligand can then be coupled to an adjacent
protein or protein active site3 via the selective activation
of the remaining functional group of the bifunctional
reagent.
Despite these useful applications, extensive employment
of the 4-azido-2-alkoxybenzaldehydes has been ham-
pered by the extremely laborious and inefficient synthesis
of these reagents. In this letter, we describe a rapid and
efficient synthesis of a series of derivatized azidobenz-
aldehydes for use as labeling and cross-linking reagents.
In our studies of new photolithographic methods, we
envisioned using 4-azido-2-alkoxybenzaldehydes as
agents for the manufacture of self-assembled monolayers
(SAM). Ultimately, by taking advantage of the photo-
reactive properties of the azide functional group, the
formed SAMs will undergo well-defined photoinduced
reactions. Photopatterning a SAM in the presence of a
Figure 1.
* Corresponding author. Associate Director Organic and Medicinal Chemistry, Chiron Corporation, 4560 Horton Street, Mail Stop 4.5,
Emeryville, CA 94608-2916, USA. E-mail: david myles@cc.chiron.com
–
0040-4039/01/$ - see front matter © 2001 Elsevier Science Ltd. All rights reserved.
PII: S0040-4039(01)00131-9

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K. W. Gano et al. / Tetrahedron Letters 42 (2001) 2249–2251
chosen trapping agent, opens the possibility of tailoring
surfaces with a variety of molecular functionalities.
HNO3 and H2SO4. Upon the introduction of NaN3, the
aryl azide 2 was formed in excellent yield without the
need for the traditional recrystallization. The acid 2 was
then converted to the methyl ester 3 without purifica-
tion with BF3·Et2O and methanol.6 Acid-catalyzed
methanolysis yielded the desired ester in only modest
yields, due to the formation of the unreacted acid 2 as
well as the decarboxylated azide.7 Alkylation of the free
hydroxy group with methyl iodide (MeI) in refluxing
acetone gave the methyl aryl ether in quantitative
yield.8 Treatment of the crude azide with DIBAL-H
resulted in the selective reduction of the ester to yield
the benzyl alcohol 4. Oxidation of this free alcohol
using Swern conditions9 gave the desired 4-azido-2-
methoxybenzaldehyde 5 in high yield after purification
Accordingly, we required multi-gram quantities of 4-
azido-2-alkoxybenzaldehydes and found the previously
described synthesis1e–g,2d to be plagued by the forma-
tion of undesired by-products and difficult purifica-
tions.1e–g,2d,4 Due to the poor yields achieved using
these methods, 4–11% for four steps, we conceived an
improved route to these compounds that would provide
both a facile set of reactions and a high overall yield for
the sequence of steps.
We found that the use of the methyl ester 3 (Scheme 1)
rather than the mixed anhydride, could avoid the for-
mation of the observed by-products of both a benzyl
amine and alcohol. The later products are the result of
over-reduction of the azide and acid functional groups
within 2 using literature methods.1e–g,2d,4 In this case, a
milder reducing agent could be used en route to the
desired aldehyde. Also, the diminished water solubility
and ease of reduction compared to the acid derivative
made the ester a desirable intermediate. To support
work to this end, we had to be certain that the ester
could be both synthesized in high yield and reduced
selectively in the presence of the azide. These tasks were
successfully completed.
10
by flash chromatography. In addition, MnO2 and
Dess–Martin11 reagents gave the aldehyde in quantita-
tive yields. The overall process of converting the start-
ing material to 4-azido-2-methoxybenzaldehyde 5,
includes five steps and one purification, and proceeds in
72–78% overall yield.12
Table 1 summarizes the results of the reactions of other
alkylating agents with 3 using K2CO3 in acetone. Since
the reaction conditions were optimized, inspection of
the table reveals both the alkyl iodide (entries 1–2) and
mesylates (entries 3–5) afford the desired compound in
high yields. The ideal reaction conditions for the alkyla-
tion include ten equivalents of the alkylating agent and
five equivalents of the base, K2CO3. Alkylations
employing the use of triethylamine as the base in DMF
do not go to completion.
We chose 4-azido-2-methoxybenzaldehyde 5 as our
model compound for the synthesis of 4-azido-2-alkoxy-
benzaldehydes (Scheme 1). Synthesis of this compound
would test if our approach would circumvent the afore-
mentioned problems.
As expected, alkyl bromides could serve adequately in
the desired alkylation, but required a longer reaction
time (70 hours) and gave lower yields of the desired
First, using a modified procedure of Smith and
Brown,5,6 4-aminosalicylic acid 1 was diazotized with
Scheme 1. (a) HNO2, NaN3, 91%; (b) BF3·Et2O, MeOH, reflux, 90%; (c) MeI, K2CO3, acetone, reflux; (d) DIBAL-H, CH2Cl2,
−78°C, 95% (two steps); (e) (COCl)2, DMSO, Et3N, CH2Cl2, −78°C, 92%.
Table 1. Alkylating agents used for the derivatization of aryl azide 3 in Scheme 1
Entry
Substrate
Equivalents
Base/equivalents
Time (h)
Yield (%)
1
2
CH2ꢀCH(CH2)7CH2I
CH2ꢀCH(CH2)7CH2I
CH2ꢀCH(CH2)7CH2OMs
OHC(CH2)8CH2OMs
TBSOCH2(CH2)8CH2OMsb
10
15
10
10
10
K2CO3/5
K2CO3/5
K2CO3/5
K2CO3/5
K2CO3/5
7.5
7.5
17
45
24
98
100
99
80
98
3
4a
5
a The aldol product of acetone, the solvent, reacting with the free aldehyde was observed.

K. W. Gano et al. / Tetrahedron Letters 42 (2001) 2249–2251
2251
product (25–80% with 3–10 equivalents of
CH2CH(CH2)7CH2Br) than the reactions with the cor-
responding alkyl iodide or mesylate (entries 1–5). Fur-
ther manipulation of the products isolated from these
reactions, by the strategies described within, allow the
possibility of using the azido-aldehyde derivatives as
surface-bound reagents. Currently, we our modifying
substrates of the type synthesized in Table 1 for their
use in SAMs. In general, by simply changing the nature
of the alkylating agent, one can gain access to numer-
ous derivatives of 4-azido-2-alkoxybenzaldehydes. In
summary, our facile, high yielding synthesis of 4-azido-
2-methoxybenzaldehyde 5 provides a potentially useful
approach to highly derivatized 4-azido-2-alkoxybenz-
aldehydes, and therefore, cross-linkers, labels, and
probes.
Conventional synthesis of 4-azido-2-alkoxybenzaldehydes.
(a) HNO2, NaN3, H2O, 70%; (b) CDI (carbon diimida-
zole), dioxane; (c) LiAlH4, Et2O, −20°C, 11–25% (two
steps); (d) R-I/R-Br, K2CO3/KOH, acetone/EtOH, 55–
65%.
5. 4-Aminosalicylic acid 1 (5.0 g, 33 mmol) was dissolved
in a solution of 25 mL H2SO4 and 130 mL deionized
water in a 2 L roundbottomed flask. The resulting mix-
ture was cooled to 0°C whereupon the amine was dia-
zotized with a solution of NaNO2 (2.8 g, 40 mmol)
in 25 mL deionized water. After stirring for 1 hour
at 0°C, a solution of NaN3 (3.6 g, 56 mmol) in 20 mL
deionized water was added dropwise to the chilled re-
action. The suspension was kept in the ice-bath and
stirred for 1 hour after the final addition, and was
then allowed to stand overnight at room tempera-
ture. The reaction mixture was washed with ethyl ace-
tate, then satd NaCl. After drying the organics, evapo-
ration yielded 5.3 g (91%) of the azide 2 as an orange
solid.
We believe the one extra step in the synthesis of the
mentioned class of azidobenzaldehydes is offset by the
ease of the reactions performed and the high overall
yield. In addition, to our knowledge, the reaction
described here involving the selective reduction of an
aryl ester in the presence of an aryl azide is the first
example using DIBAL-H. This approach may prove
useful in other systems.
6. Kadaba, P. K. Synth. Commun. 1974, 2, 167.
7. Doub, L.; Schaefer, J. J. J. Am. Chem. Soc. 1949, 71,
3564.
8. Our work has shown both diazomethane and
trimethylsilyldiazomethane can serve to transform acid 2
to the desired aryl ether 5. This method is not applicable
for the synthesis of 4-azido-2-alkoxybenzaldehydes in
general, however, because it does not allow the forma-
tion of different aryl ethers. Our strategy allows the
possibility to derivatize ester 3 with any number of
groups.
Acknowledgements
Financial support from the National Science Founda-
tion (NSF CTS-9816494) and (NSF CHE-9501728)
(D.C.M.), the Alfred P. Sloan Foundation (D.C.M.),
and the Rohm and Haas Company (D.C.M.) is grate-
fully acknowledged.
9. Huang, S.-L.; Mancuso, A. J.; Swern, D. J. Org. Chem.
1978, 43, 2480.
10. Fatiadi, A. J. Synthesis 1976, 2, 65.
11. Dess, D. B.; Martin, J. C. J. Am. Chem. Soc. 1991, 113,
7277.
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intermediates. Ester 3: 1H NMR (400 MHz, CDCl3) l
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J=2.3 Hz), 7.81 (d, 1H, J=8.6 Hz), 10.93 (s, 1H); 13C
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