Diazo compounds for the bioreversible esterification of proteins

Article abstract of DOI:10.1039/c4sc01768d

A diazo compound is shown to convert carboxylic acids to esters efficiently in an aqueous environment. The basicity of the diazo compound is critical: low basicity does not lead to a reaction but high basicity leads to hydrolysis. This reactivity extends to carboxylic acid groups in a protein. The ensuing esters are hydrolyzed by human cellular esterases to regenerate protein carboxyl groups. This new mode of chemical modification could enable the key advantages of prodrugs to be translated from small-molecules to proteins. This journal is

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Diazo compounds for the bioreversible esterification of
proteins†
Cite this: DOI: 10.1039/x0xx00000x
Nicholas A. McGrath,‡§a Kristen A. Andersen,‡b Amy K. F. Davisc
Jo E. Lomax||d and Ronald T. Raines*ac
Received 00th January 2014,
Accepted 00th January 2014
A diazo compound is shown to convert carboxylic acids to esters efficiently in an aqueous
environment. The basicity of the diazo compound is critical: low basicity does not lead to a reaction
but high basicity leads to hydrolysis. This reactivity extends to carboxylic acid groups in a protein. The
ensuing esters are hydrolyzed by human cellular esterases to regenerate protein carboxyl groups.
This new mode of chemical modification could enable the key advantages of prodrugs to be
translated from small-molecules to proteins.
DOI: 10.1039/x0xx00000x
www.rsc.org/
diazomethane.14 Here, we report on the reactivity of still more
stabilized diazo compounds with relevant carboxylic acids.
Introduction
Esters are nearly absent from human cells, aside from acylglycerols
and amino-acyl tRNAs. One reason is their lability in the presence of
intracellular esterases.1 This reactivity is the basis for the clinical
utility of the many prodrugs that are unmasked by enzyme-catalyzed
ester hydrolysis.2-5
Inspired by prodrugs, we envisioned esterification as a means to
modify proteins covalently but reversibly with desirable pendants,
such as pharmacokinetic enhancing, cell-targeting, or cell-
penetration moieties. The acetylation of serine and threonine
residues is a natural post-translational modification.6,7 There is not,
however, an efficient means to generate esters from protein carboxyl
or hydroxyl groups by chemical synthesis. Carbodimides and other
reagents can be used to activate protein carboxyl groups, but solvent
water and protein amino, hydroxyl, and sulfhydryl groups compete
effectively with exogenous alcohols for the ensuing activated acyl
groups.8-10 Accordingly, we sought a different strategy—one that
enlists O-alkylation rather than acyl transfer.
Results and discussion
We began by screening the reactivity of diazo compounds 1 and 2
with small-molecule carboxylic acids of varying acidity and bearing
a variety of reactive functional groups. These diazo compounds were
accessed from their corresponding azide by a deimidogenation
reaction that uses a phosphinoester to convert an azide into a diazo-
compound.25,26 In acetonitrile, both diazo compounds were
unreactive toward some nucleophiles common in biomolecules:
amino, hydroxyl, and sulfhydryl groups. Both, however, did react
with carboxylic acids to give comparable yields of ester product
(Scheme 1). In contrast to diazo compounds 1 and 2, diethyl 2-
diazomalonate was unreactive with a–h. Tellingly, β-alanine (h)
proved unreactive with all three diazo compounds under any tested
condition. This acid alone exists as a zwitterion, and its lack of
Scheme 1 Chemoselective esterification in acetonitrile
Diazo compounds are in widespread use in synthetic organic
chemistry.11-13 The simplest—diazomethane—readily converts
carboxylic acids into methyl esters with molecular nitrogen as the
only byproduct. Diazomethane suffers, however, from non-specific
reactivity. For example, diazomethane is known to react with water
and with the side chains of lysine and tyrosine residues.14 Other non-
stabilized diazo compounds or stabilized metal carbenoids are
capable of esterifying carboxylic acids,15-20 but their high reactivity
likewise limits utility with biomolecules.
We were aware of precedent for the use of stabilized diazo
compounds in
a
biochemical context. In the 1960s,
N-(diazoacetyl)glycinamide,23
and
2-diazoacetamide,21,22
diphenyldiazomethane24 were used to identify the most reactive
carboxylic acid groups in proteins. These efforts required adding a
vast molar excess (>103-fold) of the diazo compound and tedious
monitoring of reaction pH, all to achieve modest labelling. We
suspected that the problem was non-specific reactivity, as with
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Fig.
1
Putative mechanism for the O-alkylation of carboxylic acids by diazo
compounds.27-28
reactivity is consistent with esterification occurring via a diazonium-
carboxylate salt (Fig. 1).27,28 Moreover, we noted that a carboxylic
acid (a–g) is acidic enough to promote the reaction, but a phenolic
hydroxyl (d), sulfhydryl (f), or ammonium group (h) is not.
lacks chemoselectivity in an aqueous environment (Scheme 2). Only
the conjugate base of fluorene (pKa 22.6)34 is matched to the task,
enabling diazo compound 2 access to the mechanism of Fig. 1 with
high chemoselectivity.
Next, we investigated the analogous reactions in an aqueous
We used thioacetic acid to probe the mechanism of esterification.
Its acidic proton resides primarily on sulfur,35 but an intermediate
diazonium-thiocarboxylate salt could lead to either a thioester or
thionoester product. In anhydrous acetonitrile, complete selectivity
for thioester formation was obtained with diazo compound 2
(Scheme 3). This selectivity rules out a cyclic transition state
reminiscent of an ene reaction,36,37 which would provide the
thionoester as the product. On the other hand, diazo compound 1
produced a mixture of thio- and thionoester products. In an aqueous
environment, these same reactants led exclusively to a thio- rather
than a thionoester, along with the hydrolysis product from diazo
compound 1.38 The lack of thionoester formation can be attributed to
the greater nucleophlicity of sulfur on the diazonium salt and the
greater ability of oxygen to form a hydrogen bond with solvent
water,39 which would decrease its nucleophilicity still further.
Finally, we assessed the ability of diazo compounds 1 and 2 to
esterify the carboxyl groups present in a model protein, bovine
pancreatic ribonuclease (RNase A; Fig. 2A). The catalytic activity of
this well-characterized enzyme provides an extremely sensitive
measure of native structure and function.40 RNase A was incubated
for 4 h at 37 °C in a 1:1 mixture of 10 mM MES–NaOH buffer, pH
5.5, and acetonitrile containing a diazo compound (10 equiv). Under
these conditions, diazo compound 1 proved incapable of labelling
the protein. Only in the presence of 200 equiv was any esterification
observed with this reagent, consistent with its tendency towards
hydrolysis in an aqueous environment (Scheme 2). In contrast, diazo
environment (Scheme 2). Whereas diazo compound
1 was
competent for esterification in a 3:1 mixture of acetonitrile and
10 mM MES–NaOH buffer, pH 5.5, the major product was alcohol 5
formed when water attacks the diazonium ion. On the other hand,
diazo compound 2 gave primarily the desired ester 4 in all cases.
Interestingly, the ester:alcohol product ratios with 1 were variable,
whereas those with 2 were approximately two.29 These data are
consistent with the nascent diazonium-carboxylate salt being
maintained in a solvent cage by Coulombic forces (Fig. 1), as
described by others.28,30 Carrying out the reaction in a 1:1 mixture of
acetonitrile and 10 mM MES–NaOH buffer resulted in greatly
deleterious partitioning with 1 but not 2. Diethyl 2-diazomalonate
was found to be unreactive in these aqueous conditions, as in neat
acetonitrile.
What is the basis for this differential reactivity? The pKa values of
relevant acids have been measured in dimethyl sulfoxide.31 These
pKa values correlate with the observed reactivity via the mechanism
of Fig. 1. Specifically, the conjugate base of diethylmalonate (pKa
16.4)32 is weak, and diethyl 2-diazomalonate cannot abstract a
proton from a carboxylic acid. On the other hand, the conjugate base
of diethylacetamide (pKa 35)33 is strong, and diazo compound 1
Scheme 2 Chemoselective esterification in an aqueous environment
Fig. 2 (A) Three-dimensional structure of RNase A (PDB entry 6rsa) showing its
eleven carboxyl groups. The four residues labeled primarily by diazo compound 2 are
indicated explicitly. (B) Scheme for the bioreversible esterification of RNase A with
diazo compound 2.
Scheme 3 Thio- versus thionoester formation
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compound 2 esterified, on average, three of the eleven carboxylates This work was supported by grant R01 GM044783 (NIH). N.A.M.
(Fig. S1†). Using trypsin digestion coupled with mass spectrometry, was supported by postdoctoral fellowship F32 GM096712 (NIH).
we were able to identify the esterified residues. Diazo compound 2 K.A.A. was supported by a PhRMA predoctoral fellowship and by
(10 equiv) labelled Asp14, Glu49, Glu111, and Asp121, almost Molecular and Cellular Pharmacology Training Grant T32
exclusively. Diazo compound 1 (200 equiv) labelled a similar subset GM008688 (NIH). A.K.F.D. was supported by
a Hilldale
of residues (Glu9, Asp14, Glu49, Glu111, and the C terminus). The Undergraduate/Faculty Research Fellowship. J.E.L. was supported
basis for reaction with these particular residues is not clear, but by an NSF Graduate Research Fellowship. This work made use of
likely integrates the reactivity (i.e., high pKa) and accessibility of the National Magnetic Resonance Facility at Madison, which is
their carboxyl groups.
supported by grant P41 GM103399 (NIH), and the Biophysics
Most importantly, we probed the bioreversibility of our protein Instrumentation Facility, which was established with grants BIR-
esterification reaction (Fig. 2B). To enhance the relevance of this 9512577 (NSF) and S10 RR013790 (NIH).
experiment, we sought to employ esterases endogenous to human
Notes and references
cells rather than commercial enzymes. Accordingly, we used
recombinant DNA technology to add an 8-residue FLAG tag to the
N-terminus of RNase A, and we esterified the ensuing FLAG–
aDepartment of Chemistry, University of Wisconsin–Madison, 1101
University Avenue, Madison, WI 53706 USA. Email: rtraines@wisc.edu
RNase A with diazo compound 2 as described above. We found
bMolecular
& Cellular Pharmacology Graduate Training Program,
labelling to be comparable to that of wild-type RNase A (Fig. S12†),
and we observed that the enzyme lost half of its catalytic activity
upon esterification (Fig. S13†).41 Then, we incubated the esterified
protein with a HeLa cell extract. We recovered the enzyme from this
complex mixture with an anti-FLAG antibody immobilized on
magnetic beads. We could not detect any esters in the extract-treated
enzyme with mass spectrometry (Fig. S12†),42 and we found that the
enzyme had recovered all of its catalytic activity (Fig. S13†). These
data indicate that the protein esterification reaction was fully
bioreversible.
University of Wisconsin–Madison, 1300 University Avenue, Madison, WI
53706 USA
cDepartment of Biochemistry, University of Wisconsin–Madison, 433
Babcock Drive, Madison, WI 53706 USA
dGraduate Program in Cellular and Molecular Biology, University of
Wisconsin–Madison, 1525 Linden Drive, Madison, WI 53706 USA.
† Electronic Supplementary Information (ESI) available: Experimental
procedures, analytical data, and spectral data for novel compounds. See
DOI: 10.1039/b000000x/
‡ These authors contributed equally to this work.
Finally, we sought to demonstrate the generality of our
esterification method by employing a second protein substrate with a
different tag for its purification. Specifically, we applied diazo
compound 2 (10 equiv) and the conditions described above to the
mCherry variant of red fluorescent protein (RFP) from Discosoma sp.
We found that His6–RFP became esterified with 1–3 fluorenyl
groups. The protein did not lose its fluorescence during the
esterification reaction. As with RNase A, the esterification of RFP
was bioreversible. Incubation with a HeLa cell extract followed by
recovery with a Ni-affinity resin, yielded unmodified His6–RFP (Fig.
S13†). Success with both FLAG-labeled RNase A and His6-labeled
RFP indicates that our esterification method has a broad scope.
§ Present address for Prof. N. A. McGrath: Department of Chemistry,
University of Wisconsin–La Crosse, 1725 State Street, La Crosse, WI
54601 USA.
|| Present address for Dr. Jo E. Lomax: Science Exchange, 555 Bryant
Street, #939, Palo Alto, CA 94301 USA.
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protein can be hydrolysed by esterases that are endogenous to human
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42 We detected no evidence (e.g., ∆m/z = –18) for the intramolecular
ε on a nascent ester, consistent with carboxyl groups
attack of a lysine N
near lysine and other cationic residues having pKa values that are too
low for participation in the mechanism in Fig. 1.
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