1,3-Dipolar Cycloadditions of Diazo Compounds in the Presence of Azides

Article abstract of DOI:10.1021/acs.orglett.6b00278

The diazo group has untapped utility in chemical biology. The tolerance of stabilized diazo groups to cellular metabolism is comparable to that of azido groups. However, chemoselectivity has been elusive, as both groups undergo 1,3-dipolar cycloadditions with strained alkynes. Removing strain and tuning dipolarophile electronics yields diazo group selective 1,3-dipolar cycloadditions that can be performed in the presence of an azido group. For example, diazoacetamide but not its azido congener react with dehydroalanine residues, as in the natural product nisin.

Full text of DOI:10.1021/acs.orglett.6b00278

Letter
pubs.acs.org/OrgLett
1
,3-Dipolar Cycloadditions of Diazo Compounds in the Presence of
Azides
Matthew R. Aronoff,†,§ Brian Gold,†,§ and Ronald T. Raines*,†,‡
†
‡
Department of Chemistry and Department of Biochemistry, University of WisconsinMadison, Madison, Wisconsin 53706,
United States
*
S Supporting Information
ABSTRACT: The diazo group has untapped utility in chemical biology. The tolerance of
stabilized diazo groups to cellular metabolism is comparable to that of azido groups.
However, chemoselectivity has been elusive, as both groups undergo 1,3-dipolar
cycloadditions with strained alkynes. Removing strain and tuning dipolarophile electronics
yields diazo group selective 1,3-dipolar cycloadditions that can be performed in the
presence of an azido group. For example, diazoacetamide but not its azido congener react
with dehydroalanine residues, as in the natural product nisin.
1
ecent developments in the Huisgen azide−alkyne 1,3-
(IED) cycloadditions, the dipole has relatively high electro-
12
2
R
dipolar cycloaddition have led to an apogee of interest in
philicity. Selectivity from electronic demand has been attained
13 14
the azido group. Now, azides dominate the landscape of
using a diversity of azides as well as nitrones. Here, we
report a strategy to differentiate a diazo group and analogous
azido group through chemoselective NED cycloadditions.
Diazo compounds undergo uncatalyzed cycloadditions with
3
,4
chemoselective reactions in chemical biology. Similar but
distinct reactivity makes the diazo group an attractive
alternative, especially when multicomponent selectivity is
desirable.
5b,6,8,15
numerous dipolarophiles other than strained alkynes.
The diazo group has been of tremendous utility to the field
of organic chemistry for well over a century. This functional
The decreased HOMO−LUMO gap imparted by strain enables
efficient reactions with azides. For example, electron-rich azides
react more rapidly than do electron-deficient azides in a
5
group is capable of varied reactivity, including alkylation,
carbene generation, nucleophilic addition, homologation, and
13,16
cycloaddition with DIBAC.
DIBAC also reacts faster than
5
d
6
ring expansions. A stabilized diazo group can also serve as a
reporter for chemical biology, undergoing cycloadditions with
the cyclooctynes now in common use in strain-promoted
do other cyclooctynes with the diazoacetamide functionality.
To search for preferential reactivity of the more electron-rich
diazo group over the azido group, we performed a competition
experiment. One equivalent each of both a diazoacetamide (1)
and its azide analogue (2) were combined with 1 equiv of
DIBAC and allowed to react until the octyne was consumed
completely (Scheme 1). Despite the ∼5-fold faster rate of the
diazo group, which was consistent with calculated values of
6
azide−alkyne cycloadditions (SPAACs) (Scheme 1).
With its compact size, facile preparation, and stability in an
aqueous environment, a pendant diazoacetamide group is well-
suited for biological investigations. This type of diazo group is
7
capable of faster cycloadditions with common cyclooctynes
8
⧧
ΔG (Figure 1), a significant percentage of the azido group
than is its azido analogue. Nevertheless, the reactivity of these
reacted as well. The modest rate difference did not produce
selectivity.
two groups is redundant, and the overlap encouraged us to
search for a cycloaddition that is selective for only one.
We sought a reaction that not only differentiated the
reactivity of diazo and azido groups but also was compatible
with physiological conditions. Known strategies exist for diazo-
specific reactions in aqueous conditions, such as metal−
From this precedent, we reevaluated the need for strain to
effect 1,3-dipolar cycloadditions with diazoacetamide 1.
Specifically, we hypothesized that we could replace strain
with electronic activation. In this modality, dipolarophiles
possessing a lower energy LUMO, generated from bond
polarization of adjacent electron-withdrawing groupsrather
than through strainwould complement the higher nucleo-
philicity of diazo compounds.
We first examined ethyl propiolate b and its alkene analogue,
ethyl acrylate a, under conditions identical to those in the
competition reaction with DIBAC. In this case, selective
reactivity for the diazoacetamide over the azide was observed,
9
10
carbenoid additions/insertions, esterifications, and carbonyl
1
1
additions, but each of these processes has drawbacks that
prevent broad application in a biological context. Frustrated by
the lack of selectivity generated with SPAAC-type reactions
between azido and diazo groups, we sought a simple
cycloaddition reaction that would favor the characteristics of
a diazo group.
In theory, orthogonal cycloadditions could be generated
through modulation of the electronics of the reacting partners.
In normal-electron-demand (NED) cycloadditions, the dipole
has relatively high nucleophilicity; in inverse-electron-demand
Received: January 27, 2016
©
XXXX American Chemical Society
A
DOI: 10.1021/acs.orglett.6b00278
Org. Lett. XXXX, XXX, XXX−XXX

Organic Letters
Letter
Scheme 1. (Left) 1,3-Dipolar Cycloaddition of a 1:1 Mixture of Diazoacetamide 1 and Azidoacetamide 2 with a Strained
Dipolarophile (R = CH CH NH ) Was Not Selective; (Right) 1,3-Dipolar Cycloaddition with Unstrained Dipolarophiles Was
2
2
2
a
Selective for the Diazo Compound
a
Conditions: 1:1 CH CN/H O; ≤ 72 h; ambient temperature.
3
2
Ethyl acrylate (a) reacted more quickly than did ethyl
−
3
−1 −1
propiolate (b) with diazoacetamide 1 (k = 1.6 × 10
M
s
and k = 5.8 × 10−
4
M
−1 −1
s , respectively). This result was
surprising, as previous reports had suggested similar reaction
18
rates for alkenes and alkynes, despite different FMO energies.
The difference that we observed is likely due to a smaller
distortion energy (ΔE ) for dipolarophile a, along with a
dist
19
slightly more favorable interaction energy (ΔE ) (Figure 1).
Although alkynes are easier to bend than alkenes, an alkyne
int
2
0
must be bent to a larger degree to reach a TS geometry. The
difference in dipolarophile distortion energy of 1.7 kcal/mol is
similar to the value of 2.1 kcal/mol reported previously for the
cycloadditions of diazomethane with ethylene and acetylene
calculated at the B3LYP/6-31G(d) level of theory. These
results are consistent with the smaller HOMO−LUMO gap of
alkenes relative to alkynes.
18
2
0
In light of the faster rates observed for the alkene, we
focused our attention on ethyl acrylate and related alkenyl
21
compounds. Upon examining the reaction kinetics of a
carbonyl series of electron-deficient alkenes, we verified that the
rate of the reaction was dependent upon the electron-
withdrawing capabilities of the substituent. Cycloadditions
with a variety of dipolarophiles (a−f, see Supporting
Information) produced rate constants comparable to those of
Figure 1. Computational analysis of cycloadditions with N-methyl-2-
diazoacetamide and N-methyl-2-azidoacetamide. Optimized geo-
metries, activation energies, distortion−interaction energies, and
changes in dipolarophile charge (italics) were calculated at the M06-
early SPAACs (that is, k ≈ 10−
2
M
−1 −1 4b,22
s ).
We next sought to challenge the selectivity in a simple
context. To do so, we synthesized a compact molecule
containing both a diazo group and an azido group. We reacted
diazo azide 3 with an excess of ethyl acrylate a. The results were
gratifyingthe diazo group reacted completely to form the
pyrazoline product 3a, while the azido group remained intact
and free for subsequent functionalization or derivation (Scheme
2
X/6-31+G(2d,p) level of theory. Energies (kcal/mol) and NBO
charges on dipolarophiles (italics) include solvation corrections
water) on gas-phase geometries using IEFPCM model (radii = UFF).
(
2
).
even in the presence of a 5-fold excess of dipolarophile
Scheme 1). These experimental results are consistent with a
(
Scheme 2. Competition for a Dipolarophile by a Diazo
Group and Azido Group in the Same Molecule
computational analysis, which suggests that the cycloaddition of
the diazo group proceeds by a transition state (TS) of lower
ΔG (Figure 1). Although the dipoles of both 1 and 2 favor
a
⧧
NED cycloadditions, the greater nucleophilicity of the diazo
group of 1 leads to chemoselectivity. Cycloaddition of the diazo
group appears to be less synchronous, as its TS exhibits greater
transfer of charge from the dipole to the dipolarophile as well as
a longer terminal N···C bond distance (Figure 1). Notably, the
relative reactivity of the diazo group is amplified in aqueous
6
,8
a
conditions (Figure S3), which favor the more polar TS of the
The reaction was selective for the diazo group. Conditions: CH CN/
2
3
1
7
diazo group.
H O 1:1; 24 h.
B
DOI: 10.1021/acs.orglett.6b00278
Org. Lett. XXXX, XXX, XXX−XXX

Organic Letters
Letter
⧧
Lastly, we considered applications of this chemoselective
process within the arena of chemical biology. Acrylate esters are
too reactive for most biological applications. The tempered
The Curtin−Hammett principle predicts a ΔG of 29.1 kcal/
mol for the lower energy C-5 conformation, but enforcement of
the C-7 conformation would provide a lower overall ΔG of
6
‡
reactivity of acrylamides, however, enables their metabolic
2
7.4 kcal/mol.
2
3
incorporation into biomolecules. For example, the 2-
acetamidoacrylamide group in dehydroalanine (Dha) is the
Inspired by the reactivity of diazoacetamide 1 with 2-
acetamido-N-methylacrylamide, we investigated its reactivity
with a natural product. Dehydroalanine is found in lantibiotics,
including the peptide antibiotic nisin. Nisin is well suited to
test the biological relevance of the cycloaddition with the diazo
group, as it contains two Dha residues and one dehydro-
drobutyrine (Dhb) residue (which is derived from threonine)
along with myriad common peptidic functional groups (Figure
S5, Supporting Information).
product of a post-translational elimination of H O from serine
2
24
or H S from cysteine. The alkenyl group of dehydroalanine is
2
25
in conjugation with both an amide carbonyl group and an
amide nitrogen. We hypothesized that this functionality might
be a good substrate for reaction with a diazo group.
We first tested the reactivity of diazoacetamide 1 with an O-
acyl derivative of Dha, methyl 2-acetamidoacrylate. The
reaction rate exceeded that of previously tested alkenes by an
order of magnitude, proceeding with k = 6.7 × 10−
2
M
−1 −1
s .
We began our work with nisin by assessing the ability of
diazoacetamide 1 to label a purified sample of the natural
product. After 16 h, most of the peptide had experienced a
single cycloaddition, and some had reacted twice (Figure 3).
Notably, the full mass of the diazo compound had been added
to the peptide, ruling out the possibility of reaction by
This enhanced rate was recapitulated in computational
investigations, as calculated free energies of activation with a
2-acetamido-N-methylacrylamide model system were similar to
those predicted for N-alkylacrylamides (Figure 2 and Table 1S).
1
0
esterification, as is possible with diazo groups.
Finally, we tested the utility of this cycloaddition for natural-
product discovery. To do so, we reacted a crude mixture of
denatured protein solids containing 2.5% w/w nisin with
diazoacetamide−biotin conjugate. We observed a change in
mass expected for the labeling of nisin by cycloaddition (Figure
S33, Supporting Information). No reaction was observed with
the analogous azidoacetamide−biotin conjugate.
In conclusion, we report on a simple means to differentiate
the reactivity of an azido group and a diazo group. Whereas
strain is necessary to obtain useful cycloaddition rates with
azides, electronic activation of alkenes allows for rapid reactivity
with diazoacetamides. This insight enables cycloadditions to
dehydroalanine, such as that in nisin. To our knowledge, this
example is the first of a cycloaddition to a natural amino acid
residue. The chemoselectivity of the diazo−Dha cycloaddition
could aid in applications of known natural products and the
discovery of new ones.
Figure 2. Computational analysis of the cycloaddition of 2-acetamido-
N-methylacrylamide with N-methyl-2-diazoacetamide. Optimized geo-
metries and free energies were calculated at the M06-2X/6-
31+G(2d,p) level of theory. Energies include solvation corrections
(
water) on gas-phase geometries using IEFPCM model (radii = UFF).
Figure 3. MALDI−TOF spectra of the products of the cycloaddition of nisin with N-benzyl-2-diazoacetamide (1) and N-benzyl-2-azidoacetamide
(2). Conditions: 10 mM sodium phosphate buffer (pH 7.3), 16 h, ambient temperature.
C
DOI: 10.1021/acs.orglett.6b00278
Org. Lett. XXXX, XXX, XXX−XXX

Organic Letters
Letter
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Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.or-
glett.6b00278.
(
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The authors declare no competing financial interest.
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This work was supported by Grant No. R01 GM044783 (NIH)
and made use of the National Magnetic Resonance Facility at
Madison, which is supported by Grant No. P41 GM103399
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Org. Lett. XXXX, XXX, XXX−XXX