Solvatochromic Reagents for Multicomponent Reactions
COMMUNICATION
isocyanides in multicomponent reactions, our findings
should be applicable to many different molecular scaf-
folds.[16]
Gly) (Cha=cyclohexylalanine) was chosen to study if a sim-
ilar localization would be observed for
a macrocyclic
system. The peptide was constrained using an aziridine alde-
hyde and fluorescent isocyanide 3 to give 25 in 94% yield
(Scheme 2). The aziridine ring was reduced by a two-step
protocol involving ring-opening with thiobenzoic acid, fol-
lowed by desulfurization in the presence of Raney nickel.
This reaction “deleted” the aziridine ring by a formal ring-
opening with “HÀ”. After deprotection of the arginine side-
chains, an analysis of the cellular localization of 26 (Fig-
ure 1a) in HeLa cells, a cervical carcinoma cell line, was
conducted. The probe exhibited excellent mitochondrial lo-
calization, which was confirmed by co-localization of the
peptide with a mitochondrion-selective fluorescent probe,
MitoTracker CMXRos (Figure 1b). As a control, fluorescent
formamide 2 exhibited no cellular fluorescence.
Macrocyclization conditions were then applied to a series
of short peptides to generate the macrocycles depicted in
Scheme 2. Each linear peptide studied was constrained in
good yield and with excellent diastereoselectivity. Using this
methodology, peptide macrocycles equipped with a fluores-
cent tag as well as an accessible electrophile can be synthe-
sized in a single step from a wide pool of linear peptides.[17]
In cases where chromatographic purification was required,
the brightly colored product was easily identified by visual
inspection of collected fractions. In THF, the constrained di-
and tripeptides were found to display a sequence-dependent
lmax in the 495–500 nm range. The diastereomeric pair Pro-
l-TrpACHTUNGTRENNUNG(allyl) and Pro-d-TrpAHCTUNGTRENN(UGN allyl) were also constrained and
the fluorescence properties of the product macrocycles 16
and 17 were examined. Macrocycle 16 exhibited a signifi-
cantly greater fluorescence excitation at 212 nm than 17
when studied in THF, indicating a possibility that fluores-
cence is responsive to macrocyclic configuration. This obser-
vation, as well as the observed shift in lmax of the naphthali-
mide fluorophore upon macro-
To determine whether a cyclic MPP offered a significant
advantage over a linear one, the side-chain deprotection and
hydrolysis of the aziridine amide of macrocycle 25 was used
to give a linear counterpart (lin-26). Evaluation of localiza-
tion of lin-26 in HeLa cells revealed that the linear peptide
produced less mitochondrial fluorescence than cycle 26
within the same concentration range (Figure 1). In the con-
focal microscopy images for both 26 and lin-26, bright spots
are observed outside of the mitochondria, and in some cases
outside of the cell. These arise from aggregation of the
probes, as well as endosomal uptake. To better understand
the localization results, we quantified the cellular uptake of
2, 26, and lin-26 using flow cytometry. Importantly, whereas
many fluorophores are known to increase the membrane
permeability of probes, 2 showed minimal cellular uptake
(See the Supporting Information, Figure S1). Furthermore,
cyclic 26 consistently showed higher uptake than lin-26.
In conclusion, solvatochromic isocyanides are novel tools
for the preparation of macrocycles using aziridine aldehyde-
induced macrocyclization. We expect that these reagents
will find a wide range of applications in other isocyanide-
based multicomponent reactions[12] to generate libraries of
fluorescently-tagged products. Our method should also be
conducive to generating diverse libraries using recently de-
signed microfluidic platforms.[19] A wide range of physiologi-
cally relevant linear peptide sequences are known and many
of them avoid extended conformation upon binding to pro-
tein targets. These systems will provide excellent starting
points for the one-step synthesis of solvatochromic macrocy-
cles. The possibility of developing conformation-responsive
macrocycles and ionophores[20] is equally exciting. Aziridine-
containing fluorescent macrocycles may also find an applica-
tion as irreversible probes of enzyme acitivity. Our current
efforts are aimed at capitalizing on the increased amphipa-
thicity of our macrocycles, conjugation of various types of
molecular cargo using straightforward aziridine ring-opening
transformations, and attenuation of the undesired aggrega-
tion and endosomal uptake observed with our current
probes.
cyclization, aroused our curiosi-
ty about a possible reporter for
chemical transformations of the
macrocycle. Linear control lin-
16 (Scheme 3) displayed lmax of
507 nm (THF), outside of the
observed range for the studied
macrocycles. This trend was
found to be general for lin-15
and lin-20 as well (Table 2).
Scheme 3. Linear control pep-
tide lin-16.
Table 2. Fluorescence properties of macrocycles and derivatives.
Compound[a]
lexc [nm]
THF
lem [nm]
THF
lexc [nm]
lem [nm]
H2O:TFE[b]
H2O:TFE[b]
3
16
17
18
19
20
21
lin-15
lin-16
lin-20
474
457
467
473
469
472
463
455
468
458
516
500
501
497
497
498
495
508
507
516
508
–
–
506
506
505
505
–
532
–
–
518
519
515
515
–
–
–
–
–
[a] Solutions were prepared to a concentration of 0.5 mm [b] 1:1 (v/v).
To demonstrate the potential of fluorescent macrocyclic
peptide probes in cellular systems, we pursued organelle-se-
lective macrocycles. Our previous investigations into linear
MPPs had uncovered a series of sequences capable of trans-
porting cargo across the mitochondrial membrane. By bal-
ancing positively charged and hydrophobic residues, synthet-
ic hexa- and octapeptides exhibited localization in the mito-
chondria.[18] A representative peptide (Pro-(Cha-d-Arg)4-
Chem. Eur. J. 2011, 17, 12257 – 12261
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