Azaphilone α-Bromoacetates (AzαBs): Fluorescent linchpin reagents for the inter- and intramolecular cross-linkage of primary amines to thiols

Article abstract of DOI:10.2174/157017809788681428

α-Bromoacetate esters of the azaphilone isochromene-6, 8-dione core exhibit a pH-dependent orthogonal reactivity allowing for the one-pot cross-linkage of primary amines to thiols in an inter- or intramolecular fashion to afford fluorescent products. This

Full text of DOI:10.2174/157017809788681428

Letters in Organic Chemistry, 2009, 6, 367-371
367
Azaphilone ꢀ-Bromoacetates (AzꢀBs): Fluorescent Linchpin Reagents for
the Inter- and Intramolecular Cross-Linkage of Primary Amines to Thiols
Jason J. Chruma*, Sung-Ju Moon and William E. Sanford, Jr.
Department of Chemistry, University of Virginia, Charlottesville, VA 22904-4319, USA
Received February 24, 2009: Revised May 04, 2009: Accepted May 04, 2009
Abstract: ꢀ-Bromoacetate esters of the azaphilone isochromene-6,8-dione core exhibit a pH-dependent orthogonal
reactivity allowing for the one-pot cross-linkage of primary amines to thiols in an inter- or intramolecular fashion to
afford fluorescent products. This represents a new potential strategy for bioconjugate chemistry.
Keywords: Azaphilones, bioconjugation, orthogonal reactivity, cross-linkage, fluorescent.
The facile and selective cross-linkage of two functionally
distinct molecular entities is a fundamental component of
bioconjugate chemistry, molecular imaging, affinity chroma-
tography, and bioanalytical techniques [1]. The selective
reactivity of thiols with maleimides and amines with N-
hydroxysuccinate esters are two of the most commonly
employed protocols for bimolecular cross-linkage. ꢀ-
Haloacetates and acetamides are also capable of trapping
either thiols or amines. Recently, the copper-catalyzed [3+2]
cycloaddition of azides with alkynes or nitriles ("click
chemistry") has been added to the arsenal of selective
conjugation techniques [2,3]. Combinations of such orthogo-
nally reactive groups in a single molecule afford useful
bifunctional cross-linkage reagents [4]. Such bifunctional
linchpins, however, typically offer no intrinsic value beyond
covalently joining two molecules of interest [5]. Our goal is
to develop novel multifunctional scaffolding reagents that
can serve as fluorescent tags as well as linchpins for
bimolecular cross-linkage. Herein, we report that azaphilone
ꢀ-bromoacetates (AzꢀBs) display pH-dependent orthogonal
reactivity between primary amines and thiols, allowing for
the inter- and intramolecular cross-linkage of these functio-
nalities. Moreover, preliminary investigations demonstrate
that the resultant AzꢀB cross-linked bimolecular conjugates
and macrocycles exhibit pronounced fluorescence activity,
with Stokes' shift values nearing 100 nm.
study of organelle movements [8-10]. This precedent led us
to consider the azaphilone framework as a potential scaffold
for multi-functional cross-linkage reagents. We were parti-
cularly encouraged by recent reports of a flexible and
enantioselective synthesis of 3-substituted 7-hydroxy-7-
methylisochromene-6,8-diones [11].
Our initial studies focused on the synthesis of azaphilone
ꢀ-bromoacetates (AzꢀBs) 2a and 2b (Scheme 1). Enantio-
selective construction of the azaphilone core was achieved in
two steps and good yield was obtained from the o-bromo-
benzaldehyde 1 and the requisite alkyne following published
procedures [11]. Esterification of the 7-hydroxyl moiety with
bromoacetic anhydride afforded the desired AzꢀBs 2 [12].
When AzꢀB 2a was dissolved in MeCN and treated with
benzylamine in the presence of dodecylthiol (to test selective
reactivity), the expected vinylogous ꢁ-pyridone product 4
was isolated in a surprisingly low yield (Scheme 2), even
though the starting AzꢀB was consumed within 5 minutes,
as determined by TLC analysis. 1H-NMR and LC/MS
analysis of the reaction mixture revealed that the majority of
material remained as the enamino ketone intermediate 3.
This intermediate was relatively unstable to work-up and
chromatography, decomposing to desired bicycle 4 and other
unidentified highly-polar products. We were encouraged that
the ꢀ-bromoacetate moiety survived the reaction conditions
unscathed, even in the presence of the reactive thiol
nucleophile. To identify conditions to accelerate the
conversion of 3 to 4, we screened a wide selection of Lewis
and Brønsted acids in various solvents [13]. The addition of
Sc(OTf)3 (3 equiv) in MeCN as solvent proved to be the
optimum reaction conditions; AzꢀB 2a was converted within
10 minutes to 4 in 86% isolated yield (Table 1, entry 1) [14].
Alternatively, THF or THF/H2O mixtures are also effective
solvents when substrate solubility dictates the necessity
(entries 4 & 6). As catalogued in Table 1, these optimized
procedures allowed for the coupling of a variety of
biologically-relevant monosubstituted amines to the AzꢀB
framework. Peptidal ꢀ-amines (entries 2, 3 & 5), lysine ꢂ-
amines (entry 6), and amino sugars (entry 4) all react with
the AzꢀB core to afford the desired vinylogous ꢁ-pyridones,
though significant differences in reaction rates were
observed. The substantial reaction rate difference between
The azaphilones are a large family of fungal metabolites
typified by
a highly oxygenated 7-hydroxy-7-methyl-
isochromene-6,8-dione core [6]. The azaphilones derive their
name from the reactivity of the 4H pyran ring toward
ammonia and primary amines, resulting in the formation of
vinylogous ꢁ-pyridones. The reactivity of the azaphilone core
is quite selective for primary amine nucleophiles [7].
Furthermore, the azaphilones and azaphilone-amine conjug-
ates are typically fluorescent, with the latter exhibiting more
intense fluorescence. The selective reactivity and fluorescent
behavior of the azaphilone epicocconone recently were
exploited to enable the real-time imaging of live cells and the
*Address correspondence to this author at the University of Virginia,
Department of Chemistry, P.O. BOX 400319, Charlottesville, VA 22904-
4319, USA; E-mail: jjc5p@virginia.edu
1570-1786/09 $55.00+.00
© 2009 Bentham Science Publishers Ltd.

368 Letters in Organic Chemistry, 2009, Vol. 6, No. 5
Chruma et al.
R
O
O
R
HO
Me
Br
1) ref. 11
Br
O
CHO
2)
O
Me
O
Br
O
OH
O
2
1
2a: R = C7H15 (26%)
2b: R = Ph (44%)
Scheme 1. Synthesis of AzꢀBs 2 (total isolated yields over three steps).
O
O
C7H15
BnNH2, C12H25SH,
Br
O
2a
MeCN, r.t.
Me
O
O
NHBn
3
O
O
C7H15
C
12H25SH
Br
N
+
Bn
Me
O
O
4
Scheme 2. ꢁ-Pyridone Formation.
Table 1. ꢁ-Pyridone Formation
O
O
R
O
R
R'-NH2,
O
Br
N
Br
O
R'
Sc(OTf)3, solvent,
Me
O
Me
O
O
O
20 °C
2
– H2O
Entry
AzꢀB
R'–NH2
Solvent
Time
Yieldc
1
2
3
2aa
2aa
2bb
Bn–NH2
CH3CN
CH3CN
CH3CN
10 min
2–3 h
3 h
86%
95%
93%
H2N-Gly-Phe-OMe
H2N-Gly-Phe-OMe
OH
O
2aa
THF/H2O
5 h
49%d
HO
HO
4
OH
NH2
5
6
2aa
2aa
H2N-Phe-OMe
Cbz-Lys(NH2)-Phe-OMe
CH3CN
THF
4 h
58%d
93%
24 h
aR = C7H15; bR = Ph; cIsolated yield after chromatographic purification; dPurified by preparative reverse-phase HPLC.
peptidal ꢀ-amines, e.g. entries 2 and 5, and lysine ꢂ-amines
(entry 6) imply an opportunity for the selective function-
alization of peptidal amine termini. Similar to the non-
catalyzed reaction, the ꢀ-bromoacetate moiety survived the
presence of Lewis acid, as long as only ꢀ1 equivalent of
amine nucleophile was employed. This selective reactivity
obviates the need for protection of hydroxyl groups (entry 4)
or thiols (vide infra) on the nucleophilic amine coupling
partner.
e.g. dodecylthiol. The water-soluble Lewis acid can be
removed readily from the reaction mixture by dilution with
EtOAc and extraction with brine. Concentration of the
resultant organic layer, which contains the ꢁ-pyridone and
thiol, followed by dissolution in CH3CN and addition of 1-2
equivalents of DBU effected the quantitative conversion of 4
to the thioether 5a within 20 minutes (Table 2, entry 1) [15].
As demonstrated in Table 2, this pH-dependent linchpin
coupling strategy can be employed to crosslink a variety of
amines with thiols in either an inter- or intramolecular
fashion [16,17]. The degree of cross-linkage, as determined
by yield of the isolated product, is most influenced by the
As noted, the Sc(OTf)3-mediated ꢁ-pyridone formation
can be accomplished in the presence of a nucleophilic thiol,

Azaphilone ꢀ-Bromoacetates: Fluorescent Linchpin Reagents
Letters in Organic Chemistry, 2009, Vol. 6, No. 5
369
Table 2. Two-Step Bifunctionalization
R'-NH2
O
R
+
1) Sc(OTf)3, MeCN;
O
N
2
+
R'
brine wash
Me
R"S
O
O
2) DBU, DMF
5
R"-SH
Entry
AzꢀB
1° Amine
Thiol
Product
Yieldc
1
2
2a
2b
Bn–NH2
Bn–NH2
C12H25–SH
C12H25–SH
5aa
5bb
86%
37%
SH
3
2a
2a
H2N-Phe-OMe
5ca
54%
Cbz-Cys-OMe
H
SH
H
N
S
O
4
H2N-Gly-Phe-OMe
5da
traced
N
H
H
H
HN
4
O
6
SH
H
H2N
N
5
6
2a
2a
5ea
66%
33%
NHCbz
O
7
O
SH
H
N
H2N
5fa
N
H
O
NHCbz
8
aR = C7H15 bR = Ph; cIsolated yield after two steps, see Table 1 for isolated yield for ꢁ-pyridone formation; dThe corresponding disulfide of thiol 6 in addition to the ꢀ-bromoacetate
;
ꢁ-pyridone were isolated in near quantitative yields.
propensity of the thiol partner to oxidatively dimerize to the
corresponding disulfide. For example, HPLC/MS analysis of
the reaction mixture in entry 3 suggested that all of the thiol,
Cbz-Cys-OMe, was either converted to 5c or the Cbz-
protected cystine methyl ester. Likewise, HPLC/MS analysis
revealed that the biotin derivative 6 dimerized under the
basic reaction conditions significantly faster than nucleo-
philic substitution of the ꢀ-bromoacetate moiety (entry 4);
the corresponding ꢁ-pyridone ꢀ-bromoacetate was recovered
in nearly quantitative yield. We were particularly gratified
that macrocyclization of the peptidal aminothiols 7 and 8
could be accomplished following our two-step protocol with
AzꢀB 2a (entries 5 & 6, respectively). Given this precedent,
it is conceivable that AzꢀBs could be used to cross-link and
conformationally constrain native peptides with spatially
proximate amine and thiol substituents; efforts toward this
end are on-going.
epicocconone is also provided [8]. Notably, all of the newly
described compounds studied exhibit significant Stokes'
shifts (56–86 nm), though not as pronounced as the epicoc-
conone butylamine adduct (210 nm). More importantly, the
absorption and emission properties of all three compounds
fall within readily accessible regions of the UV-visible
spectrum. Detailed investigations into the structural factors
governing the observed fluorescence of AzꢀBs 2 and cross-
linked products 5 will be described in due course.
In summary, we have demonstrated that the pH-
dependent orthogonal reactivity of the AzꢀBs 2 allows for
the relatively facile cross-linkage of biologically-relevant
monosubstituted amines and thiols. Moreover, preliminary
studies indicate that these novel cross-linkage reagents can
double as fluorescent labels for the newly constructed bioco-
njugate species. Future efforts focused on the incorporation
of fluorescent AzꢀB–peptide conjugates into lipid membr-
anes will be reported in a future communication.
As expected, the AzꢀB linchpin reagents 2 and, to a
greater extent, the corresponding ꢁ-pyridone thioethers 5
demonstrated pronounced fluorescence. Preliminary excita-
tion and emission data for selected compounds (2a, 5a & 5e)
in CH3CN are outlined in Table 3. For comparison, data for
the butyl amine adduct of the azaphilone natural product
ACKNOWLEDGEMENTS
Support for this research provided by the Thomas F. and
Kate Miller Jeffress Memorial Trust (J-808) and the UVA

370 Letters in Organic Chemistry, 2009, Vol. 6, No. 5
Chruma et al.
Table 3. Fluorescence Data
Compound
Excitation ꢄmax (nm)
Emission ꢄmax (nm)
O
C7H15
O
O
364
423
485
Me
Br
O
O
2aa
O
C7H15
Ph
O
N
399
Me
C12H15
S
O
O
5aa
O
O
C7H15
O
N
Me
O
S
353
429
O
H
N
O
N
H
O
5ea
O
R
OH
OH
O
OH
400
610
O
Me
O
HN
C4H9
epicocconone (butyl amine adduct)b
Me
R =
aDetermined from a ~0.01M solution in CH3CN. bIn water + BSA, as reported in reference [10].
[7]
For a recent example: Wei, W.-G.; Yao, Z.-J. J. Org. Chem., 2005,
70, 4585.
Bell, P. J.; Karuso, P. J. Am. Chem. Soc., 2003, 125, 9304.
Coghlan, D. R.; Macintosh, J. A.; Karuso, P. Org. Lett., 2005, 7,
2401.
Dept. of Chemistry. S. Payne and Prof. J. Demas (UVA)
provided valuable assistance and instrumentation for
obtaining and interpreting absorption and emission spectra.
[8]
[9]
[10]
[11]
Choi, H.-Y.; Veal, D. A.; Karuso, P. J. Fluoresc., 2006, 16, 475.
Zhu, J.; Grigoriadis, N. P.; Lee, J. P.; Porco, J. A., Jr. J. Am. Chem.
Soc., 2005, 127, 9342.
REFERENCES AND NOTES
[1]
For representative examples: (a) Ansell, S. M.; Tardi, P. G.;
Buchkowsky, S. S. Bioconjug. Chem., 1996, 7, 490; (b) Burgener,
M.; Sänger, M.; Candrian, U. Bioconjug. Chem., 2000, 11, 749; (c)
Glynou, K.; Ioannou, P. C.; Christopoulos, T. K. Bioconjug. Chem.,
2003, 14 1024; (d) Jin, L.; Horgan, A.; Levicky, R. Langmuir,
2003, 19, 6968.
[12]
General reaction conditions: To a stirred solution of the requisite
7-hydroxy-6H-isochromene-6,8(7H)-dione (0.35 mmol), 4-
dimethyl-aminopyridine (DMAP, 35 ꢀmol) and bromoacetic
anhydride (0.70 mmol) in CH2Cl2 (3 mL) at 0 °C was added
dropwise via syringe a solution of i-Pr2NEt (0.35 mmol) in CH2Cl2
(1 mL). The reaction mixture was stirred at 0 °C for 15 min, diluted
with EtOAc, washed sequentially with 1N KHSO4, conc. NaHCO3,
and brine, dried (Na2SO4), filtered and concentrated in vacuo. The
resultant product was immediately purified by flash
chromatography (1% MeOH in 3:2 hexanes:EtOAc) to afford the
[2]
[3]
[4]
Kolb, H. C.; Finn, M. G.; Sharpless, K. B. Angew. Chem. Int. Ed.
Engl., 2001, 40, 2004.
Wan, Q.; Chen, G.; Danishefsky, S. J. J. Org. Chem., 2006, 71,
8244.
For two seminal examples: (a) Melton, R. G.; Boyle, J. M. B.;
Rogers, G. T.; Burke, P.; Bagshawe, K. D.; Sherwood, R. F. J.
Immun. Methods, 1993, 158, 49; (b) Tournier, E. J. M.; Wallach, J.;
Blond, P. Anal. Chim. Acta, 1998, 361, 33.
desired AzꢀB as a dark yellow solid in the indicated isolated yield.
2a: 53%; 1H-NMR (CDCl3, 500 MHz) ꢁ 7.88 (s, 1H), 6.09 (s, 1H),
5.50 (s, 1H), 3.96 (ABq, JAB = 12.9 Hz, ꢂꢃ = 16.7 Hz, 2H), 2.39 (t,
J = 7.5 Hz, 2H), 1.60 (m, 2H), 1.58 (s, 3H), 1.27 (m, 9H), 0.87 (t, J
= 6.7 Hz, 3H); 13C-NMR (CDCl3, 125 MHz) ꢁ 192.5, 191.7, 166.2,
162.8, 143.1, 115.1, 108.6, 106.5, 85.7, 33.1, 31.6, 28.8, 26.5, 24.9,
22.6, 22.4, 14.1.
[5]
[6]
For a notable exception: Xia, B.; Kawar, Z. S.; Ju, T.; Alvarez, R.
A.; Sachdey, G. P.; Cummings, R. D. Nat. Methods, 2005, 2, 825.
For a recent review: Sturdikova, M.; Slugen, D.; Lesova, K.;
Rosenberg, M. Chem. Listy, 2000, 94, 105.

Azaphilone ꢀ-Bromoacetates: Fluorescent Linchpin Reagents
Letters in Organic Chemistry, 2009, Vol. 6, No. 5
371
2b: 73%; 1H-NMR (CDCl3, 500 MHz) ꢀ 8.04 (s, 1H), 7.72 (m, 2H),
7.50 (m, 3H), 6.76 (s, 1H), , 5.68 (s, 1H), 3.98 (ABq, JAB = 12.9
Hz, ꢁꢂ = 17.1 Hz, 2H), 1.61 (s, 3H); 13C-NMR (CDCl3, 125 MHz)
ꢀ 191.5, 168.2, 157.2, 153.8, 142.8, 131.6, 130.1, 129.1, 125.6,
115.2, 107.8, 107.2, 85.8, 60.4, 24.8, 22.5, 22.5, 21.0, 14.2.
Porco reported that 3 equiv of AcOH in THF was necessary for
high yielding ꢃ-pyridone formation in a related system; Zhu, J.;
Gemain, A. R.; Porco, J. A., Jr. Angew. Chem. Int. Ed. Engl., 2004,
43, 1239.
General reaction conditions: To a stirred solution of AzꢄB 2a or
2b (60 ꢀmol) and amine (60 ꢀmol) in the indicated solvent (0.5
mL) at 24 °C was added Sc(OTf)3 (180 ꢀmol, 3 equiv) in one
portion. The reaction mixture was stirred until the dark red color,
corresponding to the enamino ketone intermediate, dissipated into a
burnt orange. Alternatively, reaction progress was monitored by
HPLC/MS analysis of reaction aliquots. The resultant solution was
diluted with EtOAc, washed sequentially with 1N KHSO4 and
brine, dried (Na2SO4), filtered and concentrated in vacuo.
Purification by either flash chromatography (EtOAc) or preparative
intermediate was redissolved in 0.5 mL CH3CN and
diazabicyclo[5,4,0]undec-7-ene (DBU, 50 ꢀmol). After stirring at
24 °C for 20-30 min, the reaction mixture was diluted with EtOAc,
washed sequentially with 1N KHSO4 and brine, dried (Na2SO4),
filtered, and concentrated in vacuo. The crude products 5 were
purified by flash chromatography (EtOAc/hexanes).
[13]
[14]
1
5a: yellow oil; H-NMR (CDCl3, 300 MHz) ꢀ 7.75 (s, 1H), 7.53-
7.33 (m, 3H), 7.20–7.07 (m, 2H), 6.28 (s, 1H), 5.33 (s, 1H), 4.95 (s,
2H), 3.35 (d, J = 4.2 Hz, 2H), 2.70 (t, J = 7.2 Hz, 2H), 2.36 (t, J =
7,5 Hz, 2H), 1.62–1.50 (m, 4H), 1.55 (s, 3H), 1.24 (m, 28H), 0.87
(m, 6H); 13C-NMR (CDCl3, 75 MHz) ꢀ 194.8, 191.1, 169.5, 148.9,
148.8, 142.7, 134.5, 129.5, 128.8, 126.0, 116.5, 114.9, 99.5, 85.1,
56.5, 32.5, 32.1, 31.9, 31.6, 29.6, 29.5, 29.3, 29.2, 29.0, 28.8, 28.3,
23.1, 22.6, 22.5, 14.1, 14.0.
[17]
General intramolecular cross-linkage reaction conditions: To a
stirred solution of AzꢄB 2a (130 ꢀmol) in CH3CN (5 mL) at 24 °C
were added the requisite amino thiol 7 or 8 (130 ꢀmol) and
Sc(OTf)3 (260 ꢀmol). After 30 min, the mixture was diluted with
EtOAc (150 mL), washed with brine (2 x 100 mL), dried (MgSO4),
filtered and concentrated in vacuo. The resultant residue was
redissolved in CH3CN (100 mL) and treated with DBU (130 ꢀmol).
After stirring for 30 min, the solvent was removed in vacuo and the
residue was immediately purified by flash chromatography (1%
MeOH in 2:1 EtOAc/hexanes) to afford the macrocyclic product.
5e: orange solid; mp=158-161 °C; 1H-NMR (d6-acetone, 500 MHz)
ꢀ 8.30 (s, 1H), 7.91 (s, 1H), 7.81-7.72 (m, 1H), 7.44-7.11 (m, 9H),
6.71 (d, J = 8.1 Hz, 1H), 6.42 (s, 1H), 5.14 (s, 1H), 5.08 (dd, J =
35.6, 12.7 Hz, 1H); 4.87 (d, J = 16.2 Hz, 1H), 4.76 (d, J = 16.3 Hz,
1H), 4.68 (dd, J = 14.3, 7.3 Hz, 1H), 4.50-4.40 (m, 2H), 4.25 (dd, J
= 14.4, 8.6 Hz, 1H), 4.18 (dd, J = 14.4, 4.4 Hz, 1H), 3.40 (dd, J =
40.4, 14.9 Hz, 2H), 3.21 (dd, J = 13.8, 5.8 Hz, 1H), 2.81 (dd, J =
13.8, 9.1 Hz, 1H), 2.67 (dd, J = 15.7, 7.6 Hz, 2H), 1.73-1.61 (m,
2H), 1.51–1.21 (m, 11H), 0.88 (t, J = 6.9 Hz, 3H); 13C-NMR (d6-
acetone, 125 MHz) ꢀ 197.9, 191.6, 172.2, 172.1, 171.0, 168.0,
157.9, 152.2, 150.7, 145.1, 141.3, 141.1, 138.9, 130.2, 129.6,
129.5, 129.4, 129.2, 128.4, 127.6, 124.2, 121.7, 116.8, 116.5,
100.1, 87.5, 67.9, 67.9, 56.2, 55.9, 55.9, 45.0, 44.9, 44.2, 36.5,
34.5, 33.5, 33.4, 29.5, 24.4, 24.2, 15.3.
HPLC afforded spectroscopically pure ꢃ-pyridone product in the
yields indicated in Table 1.
4: 1H-NMR (CD3OD, 300 MHz) ꢀ 7.77 (s, 1H), 7.12 (d, J = 7.3 Hz,
3H), 7.51-7.35 (m, 2H), 6.30 (s, 1H), 5.34 (s, 1H), 4.97 (s, 2H),
4.00 (d, J = 4.0 Hz, 2H), 2.38 (t, J = 8.0 Hz, 2H), 1.58 (s, 1H), 1.57-
1.48 (m, 2H), 1.31-1.25 (m, 8H), 0.88 (t, J = 6.5 Hz, 3H); 13C-NMR
(CDCl3, 75 MHz) ꢀ 194.4, 190.3, 170.6, 166.2, 149.3, 142.8, 134.3,
129.6, 128.9, 126.1, 116.7, 115.1, 99.4, 85.8, 56.7, 31.7, 31.5, 29.0,
28.8, 28.3, 25.4, 23.2, 22.5, 14.0.
[15]
[16]
Thioetherification can also be initiated with 1 equivalent of
Cs2CO3, though 4 hours are required for complete conversion of 4
to 5a.
General intermolecular cross-linkage reaction conditions: To a
stirred solution of AzꢄB 2a (50 ꢀmol) in CH3CN (0.5 mL) at 24 °C
was added sequentially amine (50 ꢀmol), thiol (50 ꢀmol) and
Sc(OTf)3 (150 ꢀmol). Upon complete conversion to the
corresponding ꢃ-pyridone (HPLC/MS), the reaction mixture was
diluted with EtOAc. washed with brine to remove the Lewis acid,
dried (MgSO4) and concentrated in vacuo. The resulting