Optimized Diazo Scaffold for Protein Esterification

Article abstract of DOI:10.1021/acs.orglett.5b00840

The O-alkylation of carboxylic acids with diazo compounds provides a means to esterify carboxylic acids in aqueous solution. A Hammett analysis of the reactivity of diazo compounds derived from phenylglycinamide revealed that the (p-methylphenyl)-glycinamide scaffold has an especially high reaction rate and ester/alcohol product ratio and esterifies protein carboxyl groups more efficiently than any known reagent. (Chemical Equation Presented).

Full text of DOI:10.1021/acs.orglett.5b00840

Letter
pubs.acs.org/OrgLett
Optimized Diazo Scaffold for Protein Esterification
Kalie A. Mix† and Ronald T. Raines*,†,‡
†Department of Biochemistry and ‡Department of Chemistry, University of Wisconsin−Madison, Madison, Wisconsin 53706, United
States
S
* Supporting Information
ABSTRACT: The O-alkylation of carboxylic acids with diazo
compounds provides a means to esterify carboxylic acids in aqueous
solution. A Hammett analysis of the reactivity of diazo compounds
derived from phenylglycinamide revealed that the (p-methylphenyl)-
glycinamide scaffold has an especially high reaction rate and ester/
alcohol product ratio and esterifies protein carboxyl groups more
efficiently than any known reagent.
to overcome hydrolytic decomposition. Moreover, the reaction
road reactivity has made diazo compounds one of the
Bmost versatile functional groups in synthetic organic was not chemoselective, as amino, sulfhydryl, and phenolic side
chemistry.1 Recently, this broad utility has been expanded into
the field of chemical biology. For example, the diazo group has
chains suffered alkylation. Such modifications are potentially
deleterious to protein function and not bioreversible.8
Previous work in our laboratory suggested that the obstacles
in reactivity can be overcome by tuning the reactivity of a diazo
group. In particular, we found that the basicity of 9-
diazofluorene endows this diazo compound with the ability to
label a protein in an aqueous environment.9 The fluorenyl
scaffold is, however, unduly large and not readily amenable to
synthetic modification, and its reaction rate and chemo-
selectivity are not necessarily maximal.
Accordingly, we sought a scaffold that is optimal for the
esterification of carboxyl groups in an aqueous environment.
Toward that end, we have examined derivatives of phenyl-
glycinamide (Figure 1A). This scaffold delocalizes the electron
density on Cα into an amidic carbonyl group as well as a phenyl
group that enables a Hammett analysis12 of the esterification
reaction. Moreover, the amide linkage enables facile installation
of useful moieties.
Diazo compounds 1−6 were accessed from derivatives of
phenylacetic acid (Figure 1B). Briefly, an azide was installed at
the benzylic position of the acid either through displacement of
a bromide or by diazo transfer to an existing amine. The
ensuing α-azido acids were then coupled to benzylamine and
converted to the diazo compound by deimidogenation using a
phosphinoester.10
been shown to undergo 1,3-dipolar cycloadditions with strained
alkynes in a tunable manner. The rates can greatly exceed those
of the analogous azide,2 and the reactions are chemoselective in
the presence of mammalian cells.3 In addition, diazo
compounds have been used to label proteins via C−H, N−H,
and S−H insertion reactions.4
Diazo compounds have another well-known mode of
reactivity: esterification of carboxylic acids. We realized that
this reactivity could provide unique opportunities in chemical
biology. For example, unlike the alkylation of other functional
groups, O-alkylation of a carboxyl group is bioreversible
because mammalian cells contain nonspecific esterases.5 The
esterification of carboxyl groups in proteins and other
biomolecules is, however, difficult to effect, as solvent water
competes effectively with alcohols for eletrophilic acyl groups.
In contrast, esterification reactions mediated by diazo groups
rely on the carboxyl group serving as a nucleophile (Scheme
1).6
The use of diazo compounds to label proteins was attempted
60 years ago.7 These initial results were not compelling. A large
molar excess (up to 103-fold) of diazo compound was required
Scheme 1
In initial experiments, we probed the effect of electron
distribution on the reactivity of diazo groups by measuring the
rate of esterification in acetonitrile. To do so, we reacted diazo
compounds 1−6 with BocGlyOH and measured the second-
1
order rate constants with H NMR spectroscopy (Figure S1,
Supporting Information). The effect of electron distribution on
the reaction rate was dramatic: rate constants spanned over 2
orders of magnitude and increased with the electron-donating
Received: March 22, 2015
Published: May 4, 2015
© 2015 American Chemical Society
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Letter
Figure 3. Effect of σp value on the chemoselectivity of diazo
compounds 1−6 in aqueous solution.
Figure 1. (A) Scaffold for testing the reactivity and selectivity of diazo
compounds. (B) Synthetic route to diazo compounds 1−6. Steps: (a)
NBS, AIBN; (b) NaN3, THF/H2O; (c) NHS, DCC, THF; (d)
PhCH2NH2, DCM; (e) N-succinimidyl 3-(diphenylphosphino)-
propionate, then NaHCO3 or DBU;10 (f) imidazole-1-sulfonyl azide
hydrochloride, DBU, CuSO4, MeOH.11
Figure 4. Chemoselectivity of esterification reactions in aqueous
solution.
character of the phenyl substituents (Figure 2A). Hammett
analysis of these rate constants gave a slope of ρ = −2.7 (Figure
2B). This value, which is comparable to those for typical SN1
reactions, indicates that the esterification reaction is highly
sensitive to substituents and that substantial positive charge
accumulates during its course,14 as expected from a mechanism
involving an intermediate diazonium ion (Scheme 1).6
Next, we sought to find the one compound that
demonstrates the greatest selectivity for esterification over
hydrolysis in an aqueous environment. Toward that end, we
reacted diazo compounds 1−6 with equimolar BocGlyOH in a
1:1 mixture of acetonitrile and 2-(N-morpholino)-
ethanesulfonic acid (MES)−HCl buffer at pH 5.5, and we
Figure 2. (A) Second-order rate constants for the esterification of
BocGlyOH by diazo compounds 1−6 in CD3CN. (B) Hammett plot
of the data in panel A. Values of σp are from ref 13. ρ = −2.7.
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model protein, ribonuclease A,16 with 10 equiv of each diazo
compound. The reactions were allowed to proceed for 4 h at 37
°C in 1:1 acetonitrile/10 mM MES−HCl buffer at pH 5.5. We
then determined the extent of esterification with MALDI−TOF
mass spectrometry. We found that diazo compound 2 was
approximately 2-fold more efficient than 9-diazofluorene in
effecting esterification (Figure 5).
We conclude that diazo compound 2 can be used to esterify
proteins in an aqueous environment more efficiently than any
other known reagent. Moreover, its modular design enables
facile modification with useful moieties. We are now using this
diazo compound to attach cell-type targeting, cell-penetration,
and pharmacokinetic enhancing modules to proteins of interest.
ASSOCIATED CONTENT
* Supporting Information
■
S
Experimental procedures for syntheses and kinetic analyses,
additional kinetic data, and compound characterization data.
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.or-
glett.5b00840.
AUTHOR INFORMATION
Corresponding Author
■
*E-mail: rtraines@wisc.edu.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by Grant No. R01 GM044783
(NIH). K.A.M. was supported by Molecular Biosciences
Training Grant No. T32 GM007215 (NIH). This work made
use of the National Magnetic Resonance Facility at Madison,
which is supported by Grant No. P41 GM103399 (NIH), and
the Biophysics Instrumentation Facility, which was established
with Grant Nos. BIR-9512577 (NSF) and S10 RR013790
(NIH). We thank Dr. N. A. McGrath (University of
WisconsinLa Crosse) for contributive discussions and critical
reading of the manuscript and Dr. B. VanVeller (Iowa State
University) for suggesting the phenylglycine scaffold.
Figure 5. MALDI−TOF mass spectrometry data for esterification of
RNase A with (A) 9-diazofluorene and (B) diazo compound 2.
determined the ratio of ester-to-alcohol product with 1H NMR
spectroscopy.
Surprisingly, the ester/alcohol ratio reached a maximum of
1.4:1 and remained unchanged despite increasing electron
withdrawal by the substituents (Figure 3). This result is
consistent with a sharp cutoff for the formation of a
carboxylate·diazonium intimate ion-pair intermediate that is
maintained in a solvent cage by a Coulombic interaction
(Scheme 1).6,15
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