Expanding the Versatility of Microbial Transglutaminase Using α-Effect Nucleophiles as Noncanonical Substrates

Article abstract of DOI:10.1002/anie.202001830

The substrate promiscuity of microbial transglutaminase (mTG) has been exploited in various applications in biotechnology, in particular for the attachment of alkyl amines to glutamine-containing peptides and proteins. Here, we expand the substrate repertoire to include hydrazines, hydrazides, and alkoxyamines, resulting in the formation of isopeptide bonds with varied susceptibilities to hydrolysis or exchange by mTG. Furthermore, we demonstrate that simple unsubstituted hydrazine and dihydrazides can be used to install reactive hydrazide handles onto the side chain of internal glutamine residues. The distinct hydrazide handles can be further coupled with carbonyls, including ortho-carbonylphenylboronic acids, to form site-specific and functional bioconjugates with tunable hydrolytic stability. The extension of the substrate scope of mTG beyond canonical amines thus substantially broadens the versatility of the enzyme, providing a new approach to facilitate novel applications.

Full text of DOI:10.1002/anie.202001830

A Journal of the Gesellschaft Deutscher Chemiker
Angewandte
Chemie
International Edition
www.angewandte.org
Accepted Article
Title: Expanding the Versatility of Microbial Transglutaminase Using α-
Effect Nucleophiles as Noncanonical Substrates
Authors: Tak Ian Chio, Breanna R. Demestichas, Brittany M. Brems,
Susan L. Bane, and L. Nathan Tumey
This manuscript has been accepted after peer review and appears as an
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of the final Version of Record (VoR). This work is currently citable by
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.202001830
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10.1002/anie.202001830
Angewandte Chemie International Edition
RESEARCH ARTICLE
Expanding the Versatility of Microbial Transglutaminase Using α-
Effect Nucleophiles as Noncanonical Substrates
Tak Ian Chio,[a] Breanna R. Demestichas,[a] Brittany M. Brems,[b] Susan L. Bane,*[a] and L. Nathan
Tumey*[b]
Abstract: The substrate promiscuity of microbial transglutaminase
(mTG) has been exploited in various applications in biotechnology,
in particular for the attachment of alkyl amines to glutamine-
containing peptides and proteins. Here we expand the substrate
repertoire to include hydrazines, hydrazides, and alkoxyamines,
resulting in the formation of isopeptide bonds with varied
susceptibilities to hydrolysis or exchange by mTG. Furthermore, we
demonstrate that simple unsubstituted hydrazine and dihydrazides
can be used to install reactive hydrazide handles onto the side chain
of internal glutamine residues. The distinct hydrazide handles can be
transamidated product. Given the mechanistic role of the amine
as a nucleophile and the broad substrate tolerance of the
enzyme, we envisaged that the scope of the acyl acceptor
substrate could be extended to include other nucleophiles, such
as α-effect amines.
further
coupled
with
carbonyls,
including
ortho-
carbonylphenylboronic acids, to form site-specific and functional
bioconjugates with tunable hydrolytic stability. The extension of the
substrate scope of mTG beyond canonical amines thus substantially
broadens the versatility of the enzyme, providing a new approach to
facilitate novel applications.
Scheme 1. Transamidation reaction catalyzed by microbial transglutaminase
(mTG) using an amine or an α-effect nucleophile – (a) hydrazine, (b) hydrazide,
or (c) alkoxyamine as the acyl acceptor substrate.
Introduction
A number of enzyme-based methods have been developed to
incorporate novel functionalities into biomolecules.[1] These
strategies make use of the ability of the enzymes to accept
diverse substrate analogues. One prominent member of this
enzymatic toolbox is transglutaminase (TG), of which the simpler
single-domain and calcium-independent variants of microbial
origin are commonly used.[2] Microbial transglutaminase (mTG)
catalyzes a transamidation reaction between certain surface-
exposed glutamine (Gln) and lysine (Lys) residues on protein
substrates, crosslinking the two via an isopeptide bond. In
addition to Lys, mTG is also known to recognize a wide range of
primary amine substrates, thus providing the basis for mTG’s
utility to equip proteins with assorted functional moieties.[3] The
enzyme, for instance, has been employed to generate protein-
Indeed, evidence of hydrazine and hydrazide acting as TG
substrates can be found in early studies in the 1970s that aimed
to elucidate the mechanism of hydrazine-/hydrazide-
functionalized agents as inhibitors of mammalian tissue TG.[5]
Detection of isotopic labeling by C14-isonicotinic acid hydrazide
(isoniazid)
was
observed
for
N-benzyloxycarbonyl-L-
glutaminylglycine (ZQG, a common model acyl donor substrate
for TG) and a variety of proteins using guinea pig liver TG.
Hydrazinonaphthalazine (hydralazine) reportedly led to the same
result. Meanwhile, hydroxylamine is used as a substrate in the
classical hydroxamate assay used to measure TG activity.[6]
Despite these early indications that TG can recognize α-effect
nucleophiles, contemporary use of mTG in bioconjugation
chemistry has been exclusively focused on alkyl amine
substrates. Herein, we explore the expansion of the applied
substrate palette to include α-effect nucleophiles (Scheme 1)
and we show that the versatility of mTG can be bolstered by
using these “rediscovered” substrates.
polymer
and
antibody-drug
conjugates
(ADCs)
for
pharmaceutical applications.[4]
The catalytic mechanism of mTG involves a nucleophilic
attack of the target acyl donor, Gln, by the enzyme’s active site
cysteine to form
a
thioester intermediate. Subsequent
nucleophilic attack of the thioester by an amine substrate
serving as the acyl acceptor results in the formation of the
Results and Discussion
Coupling of α-effect nucleophiles to peptides/proteins by
mTG
[a]
[b]
T. I. Chio, B. R. Demestichas, Prof. Susan L. Bane
Department of Chemistry
Binghamton University, State University of New York
25 Murray Hill Rd, Vestal, NY 13850 (USA)
E-mail: sbane@binghamton.edu
We first set out to verify mTG’s substrate tolerance to α-effect
nucleophiles, as evidence from previous studies were primarily
based on mammalian TG. The ZQG acyl donor substrate was
incubated with mTG and benzoic hydrazide, the latter being
isostructural to isonicotinic acid hydrazide and used as our initial
α-effect amine-functionalized substrate. Analysis by ESI-MS
found a product with an m/z corresponding to ZQG incorporated
with benzoic hydrazide (Figure 1a; Figure S1). The structure of
B. M. Brems, Prof. L. Nathan Tumey
Department of Pharmaceutical Sciences
Binghamton University, State University of New York
96 Corliss Ave, Johnson City, NY 13790 (USA)
Email: ntumey@binghamton.edu
Supporting information for this article is given via a link at the end of
the document.
1
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10.1002/anie.202001830
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RESEARCH ARTICLE
the isolated product was determined by NMR (Figure S2), which
indicated that the terminal amino group of the hydrazide (not the
amide-like nitrogen) was the site of conjugation to the acyl group
of Gln in ZQG.
To determine whether antibody conjugation with the
hydrazide substrate can be extended to locations beyond the C-
terminus of Tras LC-Q, we tested another trastuzumab mutant,
referred as Tras HC-Q, which has the LLQG tag engineered at
the internal residues 234-237 of the heavy chain’s CH2
domain.[4c] Specific incorporation of benzoic hydrazide to the
heavy chain of Tras HC-Q was observed (~85%), demonstrating
that the recognition motif installed on other sites of the antibody
are amenable to conjugation with α-effect amines by mTG
(Figure 1a; Figure S5). We also attempted to modify the native
Gln295 residue of a deglycosylated trastuzumab.[4b] However, no
incorporation of benzoic hydrazide was observed on the
antibody under our experimental conditions (Figure S6).
To further demonstrate the scope of the mTG-promoted
reaction with noncanonical substrates, we employed another
protein, apomyoglobin (aMb), which has been previously shown
to undergo mTG-mediated modification by amines at Gln91 (fully
modified) and Gln152 (partially modified).[7] Consistent with this
study, incubation of aMb with mTG and benzoic hydrazide
yielded two product peaks on ESI-MS (Figure 1a; Figure S7),
with the major and minor products harboring one and two
modifications, respectively. Taken together, our results thus far
showcased the ability of mTG to recognize an α-effect amine-
functionalized substrate, which can be coupled enzymatically to
reactive Gln-containing peptides and proteins.
Having demonstrated that benzoic hydrazide is an
effective mTG substrate in a model dipeptide, we next examined
whether it could serve as a substrate in a more typical site-
specific protein conjugation. Given the growing prevalence of
mTG in the assembly of site-specific ADC therapeutics,[4b, 4c]
a
trastuzumab antibody targeting the oncogenic human epidermal
growth factor receptor 2 (HER2) was used as our model protein.
In particular, we used a mutant, herein referred as Tras LC-Q,
that has an LLQG tag engineered at the C-terminus of the light
chain. Since the endogenous Gln residues in native
immunoglobulin G antibodies lack reactivity to mTG, enzymatic
conjugation is known to occur site-specifically at the engineered
Gln on the light chain.[4c] Indeed, treatment of Tras LC-Q with
benzoic hydrazide under fairly typical mTG coupling conditions
resulted in a single conjugation per light chain, but not the heavy
chain (Figure 1a; Figure S3). Meanwhile, the native
trastuzumab, which does not carry the engineered Gln, was inert
when subjected to the same conditions (Figure S4), supporting
the specificity of the enzymatic reaction.
Kinetics of mTG-catalyzed conjugation of benzoic
hydrazide to Tras LC-Q was then compared with its amine
counterpart, phenethylamine. Both substrates demonstrated
very similar rates of conjugation (Figure 1b; Figure S8),
suggesting that the enhanced nucleophilicity of the hydrazide did
not result in an increase in the rate of displacement of the
thioester acyl-enzyme intermediate.
Conjugation efficiency and optimization for coupling of
assorted α-effect nucleophile substrates
We next surveyed the structure-reactivity relationship for a
series of hydrazide derivatives, including alkyl hydrazides (1a-b),
semicarbazides (1d-e), thiosemicarbazides (1f) and sulfonyl
hydrazides (1g), as well as
a few hydrazines (1h-j) and
alkoxyamines (1k-l). The efficiency of conjugation of the tested
substrates was calculated based on the loading on the light
chain of Tras LC-Q as determined by ESI-MS after antibody
reduction (Figure S9). Encouragingly, many of the assorted α-
effect nucleophile substrates (1b-d, 1h, 1l) exhibited high
efficiency of conjugation (>80%). Some were less efficient
substrates under our initial conditions (Table 1; Condition 1).
Compounds with
a
nearby carboxylic acid exhibited
comparatively lower extent of conjugation than their parent
molecule (compare 1d vs. 1e and 1h vs. 1i). This is consistent
with previous observations that having a carboxylic acid group
proximal to the amine hampers conjugation,[8] suggesting that
the structure-reactivity relationship for amine substrates may be
transferrable to α-effect nucleophiles.
In order to increase the yield for the less efficient
substrates, we performed a systematic optimization of a number
of parameters, including pH, temperature, and the equivalents of
mTG and substrate (Figure S10). Unexpectedly, reduced
temperature (4°C) significantly improved the loading as
compared to 22°C and 37°C (Figure S10b). We speculate that
this may be due to improved enzyme stability. A negligible effect
was observed when increasing the enzyme concentration
(Figure S10c), similar to results from a previous study.[4d]
Meanwhile, increased substrate concentration contributed
positively to loading (Figure S10d). Using thiosemicarbazide as
the representative substrate for this optimization, the loading
was increased from ~16% to ~95% (Table 1). The optimized
conditions enabled complete or near-complete loading for many
Figure 1. Coupling of α-effect nucleophiles to peptides/proteins by mTG. (a)
mTG-catalyzed conjugation of benzoic hydrazide to ZQG, Tras LC-Q, Tras
HC-Q, or aMb. For aMb, two products with modification on one (+1) or two
(+2) sites were detected. PBD entries 1HZH and 1YMB were used as
representative illustrations of the trastuzumab mutants and aMb, respectively.
Each chain of the antibody is color-coded, with red and yellow corresponding
to the light chain (LC) and light blue and dark blue corresponding to the heavy
chain (HC). The pinpointed sites are the approximate locations of the reactive
Gln on the protein based on the PBD entries. C term. = C-terminus; a.a. =
amino acid. Similar protein conjugations with other α-effect amine substrates
(phenylhydrazine and o-benzylhydroxylamine) are shown in Figure S5, S7, &
S9. (b) Comparison of the kinetics of enzymatic conjugation of phenethylamine
(circle) vs. benzoic hydrazide (triangle) to Tras LC-Q.
2
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10.1002/anie.202001830
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RESEARCH ARTICLE
of the substrates that were less efficiently conjugated under the
parent conditions (Table 1; Condition 2). The exception in our list
is p-toluenesulfonyl hydrazide (1g), which exhibited only 10%
loading even under the optimized conditions, suggesting that
(aryl) sulfonyl hydrazides are inherently poor substrates for
mTG.
contribute to enhanced proteolytic stability, hence their
application in peptidomimetics.[10] The stability improvement
associated with these “α-effect” pseudopeptide linkages thus
prompted us to explore whether the isopeptide bond analogues
formed from the α-effect amine substrates (Table 1) may confer
a similar protective ability against proteolysis, specifically by
mTG itself. Although TG is known for its bond-forming reaction,
it is also able to catalyze the reverse reaction.[11] In the absence
of excess amine substrates, the enzyme can hydrolyze the
isopeptide linkage, thereby irreversibly converting the original
Gln residue to a glutamic acid. Alternatively, in the presence of
an excess of another amine substrate, the enzyme can promote
transamidation, or the exchange of one amine for another. We
therefore examined whether the isopeptide analogues generated
by mTG and α-effect amine substrates can prevent this reversal
or exchange reaction, thus offering an advantage over traditional
amine substrates.
Table 1. Percent mTG-mediated conjugation of assorted α-effect
nucleophiles to Tras LC-Q
Substrate
Structure
Condition
1[a]
Condition
2[b]
1a
1b
1c
1d
1e
1f
Propanoic hydrazide
Cyanoacetohydrazide
Benzoic hydrazide
Semicarbazide
57
94
100
86
67
16
0
84
ND
ND
100
95
O
NH2
N
H
(Hydrazinecarbonyl)
glycine
S
Thiosemicarbazide
95
NH2
NH2
H2N
N
H
O
S
O
1g
p-Toluenesulfonyl
hydrazide
10
N
H
1h
1i
Phenylhydrazine
99
0
ND
ND
H
N
4-hydrazinobenzoic
acid
NH2
HO
O
1j
1k
1l
Benzylhydrazine
77
62
98
85
100
ND
o-Ethylhydroxylamine
o-Benzylhydroxylamine
[a] Condition 1: 1 mg/mL Tras LC-Q, pH = ~7.4, 6.5 wt. eq. mTG, 5%
DMSO, 50 eq. substrate (per reactive Gln), 22°C. [b] Condition 2: 1 mg/mL
Tras LC-Q, pH ~7.4, 65 wt. eq. mTG, 0% DMSO, 450 eq. substrate (per
reactive Gln), 4°C. ND = not determined. Note: The weight equivalent (wt.
eq.) of mTG corresponds to the ratio of the wt. of the formulated mTG
(which contains ~1% mTG by wt.) to the wt. of the antibody used in the
sample.
Figure 2. Stability of the isopeptide bond analogues toward hydrolysis or
exchange by mTG. (a) Scheme illustrating the Tras LC-Q conjugates prepared
for stability evaluation and the resultant structures should hydrolysis or
exchange occur. 2a-2e were incubated in 0.1 M PBS, pH 7.4 at 37 ˚C for 5
days in the presence of (b) mTG or (c) mTG and dansylcadaverine to assess
their susceptibilities to hydrolysis or exchange, respectively. The bar graphs
illustrate the average and the standard deviation from two independent
experiments.
Stability of the isopeptide bond analogues toward mTG-
mediated hydrolysis and exchange
To compare the stability of the different isopeptide
derivatives, we prepared a series of conjugates of Tras LC-Q
For the typical mTG-catalyzed transamidation of Gln with an
alkyl amine, the resultant linkage is an amide bond (known as an
isopeptide bond). On the other hand, transamidation with α-
effect amines result in the formation of isopeptide analogues,
which are akin to classical peptide bond surrogates employed
for peptide backbone modifications (e.g. hydrazinopeptide,
aminoxypeptide, and azapeptide).[9] The unique chemical and
structural properties of these pseudopeptide linkages can
with
phenethylamine
(2a),
benzoic
hydrazide
(2b),
benzylhydrazine (2c), phenylhydrazine (2d), and o-
benzylhydroxylamine (2e) (Figure 2a). The conjugates were then
subjected to incubation with mTG in phosphate-buffered saline
(PBS), pH 7.4 at 37 ˚C and their integrity was monitored over
time (up to five days) by ESI-MS. Reversibility was detected
based on the appearance of a lower molecular weight peak
3
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RESEARCH ARTICLE
corresponding to the deamidated species. The extent of
hydrolysis of the conjugates was observed in the order, from
most to least: benzoic hydrazide > o-benzylhydroxylamine >
phenethylamine > benzylhydrazine > phenylhydrazine (Figure
2b; Figure S11). The same trend was observed when excess of
a different amine substrate, dansylcadaverine, was incubated
along with the conjugate and the enzyme, though instead of the
deamidated species, the transamidated product with
dansylcadaverine was observed (Figure 2c; Figure S11).
Notably, in both scenarios of enzymatically induced hydrolysis or
exchange, the two conjugates of Tras LC-Q with hydrazine
substrates, benzylhydrazine (2c) and phenylhydrazine (2d),
exhibited the least turnover, with the latter showing ≤ ~10%
deamidation after five days under these forcing conditions.
Curiously, under identical conditions but in the absence of mTG,
2d and, to a small extent, 2c, underwent hydrolysis over the five-
day incubation (Figure S12). The reason for this deviation is
unclear given the general regard of the hydrazide linkage to be
relatively stable.[12]
modification of the engineered Gln on the light chain was
observed using unsubstituted hydrazine as substrate (Figure 3a;
S18). For carbohydrazide, in addition to the modified light chain,
a species with a mass equivalent to a dimer of two light chains
crosslinked via both ends of a carbohydrazide was observed on
ESI-MS (Figure S16). As expected based on the previous
study,[14] formation of the functionalized monomer can be
maximized by increasing the carbohydrazide concentration
(Figure
S16-17).
Meanwhile,
derivatization
with
thiocarbohydrazide can be achieved using the optimized
conditions in Table 1 (Condition 2) (Figure S18).
Since the reactive Gln in the LLQG tag of Tras LC-Q is
positioned within the C-terminus, we sought to illustrate further
that the hydrazide functionalization could be implemented on an
internal Gln residue. To that end, we treated the aMb protein
with mTG along with unsubstituted hydrazine, carbohydrazide,
or thiocarbohydrazide. Indeed, as determined by ESI-MS, aMb
could be derivatized by all three substrates, with full conversion
to the respective hydrazide group on one predominant site
particularly when paired with hydrazine and carbohydrazide
substrates (Figure S19). As aforementioned, Gln91 of aMb has
previously been shown to be the major mTG-reactive site.[7]
Therefore, the enzymatic modification likely stemmed from
conjugation to Gln91, supporting the ability to achieve hydrazide
functionalization at internal amino acid residues. A method to
install a hydrazide group internally on the side chain would
complement existing strategies that typically introduce the
reactive group on the C-terminus of the protein backbone.[15]
Such a method could find use in protein semisynthesis, where
the hydrazide is well known for serving as a stable thioester
precursor for native chemical ligation.[16] Notably, internal
installation of this thioester synthon on the side chain could
enable synthetic access to unique branched and lariat protein
structures distinct from the canonical linear scaffold.
The stability evaluation based on Tras LC-Q conjugates
2a-2e suggests that the isopeptide subtype generated by
conjugation of hydrazine substrates are less susceptible to
reversibility or exchange by mTG than the classical isopeptide
linkage. As enzymatic reactivity of the acyl donor substrate may
differ depending on nearby residues and its local conformation,[7,
13]
we note that the trend may differ depending on the site and
the protein. Nevertheless, the overall differential stabilities of the
isopeptide bond derivatives toward hydrolysis or exchange by
mTG present a new design consideration when using the
enzyme as a tool for bioconjugate assembly.
Functionalization of a peptide/protein with a side chain
hydrazide group
In an early study examining unsubstituted hydrazine as a
diamine substrate, the authors observed two products after
incubating it along with ZQG and guinea pig liver TG.[14] One of
Application of the installed hydrazide handles in
bioconjugation
the products was
a dimer crosslinked via both ends of
hydrazine. Another product, of interest to us, was a monomer in
which one end of the hydrazine was conjugated, transforming
Gln into γ-glutamic acid-hydrazide. Formation of this monomer
species could be favored over the dimer by increasing the
concentration of hydrazine, which presumably competes with the
monomer product as substrate. Therefore, in the presence of TG
and sufficient excess of hydrazine, the Gln side chain could be
converted into a hydrazide functional group. This previous work
thus inspired us to explore the application of unsubstituted
hydrazine as a TG substrate to introduce a side chain hydrazide
moiety in peptides and proteins.
We first verified that hydrazine also acts as a substrate for
mTG, resulting in the conversion of ZQG to Z(Q-hydrazide)G as
was found with mammalian TG (Figure S13). Furthermore,
continuing with the rationale of using difunctional hydrazines to
convert the carboxamide group of Gln into a hydrazide, the
Aside from potential application in protein semisynthesis, the
mTG-derived hydrazides could be used as a bioorthogonal
handle for coupling with aldehydes or ketones.[17] For example,
hydrazones have been widely employed as pH-sensitive linkers
in triggered release systems. To demonstrate similar
applications of mTG-derived hydrazides, Tras LC-Q was
derivatized with an alkyl hydrazide (3a), carbohydrazide (3b), or
thiocarbohydrazide (3c) and allowed to react with 10 eq. of an
aldehyde fluorophore (coumarin aldehyde) at pH ~7.4 in the
presence of 4-amino-phenylalanine as
a reaction catalyst
(Figure 3a).[18] Reducing SDS-PAGE analysis showed that only
the modified light chain was fluorescently labeled, consistent
with the chemoselective nature of the reaction (Figure 3b, lanes
1-4). Tras LC-Q-hydrazide (3a) and -carbohydrazide (3b) had
similar coupling efficiencies (~70% based on ESI-MS; Figure
S21). On the other hand, Tras LC-Q-thiocarbohydrazide (3c)
simplest
dihydrazides,
namely
carbohydrazide
and
had a relatively low conjugation yield (~30%) under the
thiocarbohydrazide, were also explored as substrates (Figure
3a). In both cases, ZQG was converted to the respective Z(Q-
carbohydrazide)G and Z(Q-thiocarbohydrazide)G as confirmed
by ESI-MS (Figure S14-15).
experimental conditions. We thus focus our discussion on 3a
and 3b, though we note that the sulfur-containing
thiocarbohydrazide handle (3c) may be useful for other
applications.
Next, the ability to install the various hydrazide functional
groups on a protein was examined, again using Tras LC-Q. Full
Periodic reimaging of the gel stored in acidic destaining
solution (10% acetic acid, 50% methanol) showed that the
4
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Figure 3. Introduction of assorted reactive hydrazides on the Gln side chain of Tras LC-Q and their applications in bioconjugation. (a) Scheme illustrating the use
of unsubstituted hydrazine and dihydrazides as mTG substrates to install assorted hydrazide handles and their subsequent coupling to an aldehyde- or 2fPBA-
functionalized fluorophore. (b) Reducing SDS-PAGE and in-gel fluorescence of the carbonyl coupling reactions. *The numbers on the side correspond to the
number of days that the gel was kept in an acidic destaining solution (10% acetic acid, 50% methanol). All in-gel fluorescence images were captured using the
same exposure time. The image of the gel after Coomassie staining is shown in Figure S23. HC = heavy chain; LC = light chain. (c) Fluorescence microscopic
images of SK-BR-3 or MDA-MB-231 cells that were treated with the antibody-fluorophore conjugate of 3b and Janelia Fluor 669-2fPBA. The cells were
counterstained with Hoechst 33342 nuclear dye. A diode 405 nm laser and a 633 nm laser were used for excitation. Scale bar = 10 µm. The structures of all the
fluorophores used in this figure are shown in Figure S20.
fluorescence of the bands corresponding to the conjugates of 3a
and 3b decreased over time (Figure 3b, lanes 2 & 3), indicating
hydrolysis of the hydrazone conjugates in the acidic milieu.
Importantly, fluorescence of the acyl hydrazone conjugate (lane
2) diminished more quickly than the carbohydrazone (lane 3),
suggesting that the carbohydrazone linkage is more
hydrolytically stable. Our observation is in line with the
reportedly shorter half-life of acyl hydrazone compared to
catalyzed or performed under acidic conditions.[17] Recent
developments in a variation of the classical aldehyde/ketone
condensation involving placement of a boronic acid ortho to an
aromatic aldehyde or ketone offer opportunities to improve both
product stability and reaction kinetics.[21] Intramolecular Lewis
acid catalysis from the ortho-boronic acid increases the
condensation rate by several orders of magnitude compared to
simple carbonyls. In particular, the reaction between 2-
semicarbazone,[19] the latter sharing resemblance to
a
formylphenylboronic acid (2fPBA) and a hydrazide rapidly
carbohydrazone. The different hydrazide handles (alkyl
hydrazide vs. carbohydrazide) therefore rendered variation in
the rates of hydrolysis of the resultant hydrazone conjugates.
This can be exploited to fine-tune the cleavage rate, which is an
important design factor for ADCs containing such hydrolytic
linkages.[20]
Although the reversible hydrazone construct has been
leveraged to enable controlled payload delivery, it is not ideal for
other applications where the stability of the linkage is essential.
Moreover, hydrazone formation is known to be slow unless
results in a hydrazone intermediate that further cyclizes into a
boron-nitrogen heterocycle.[22] Stability of this 2,3,1-
benzodiazaborine (DAB) product can vary by changing the
nature of the hydrazide, with certain substituted hydrazides and
semicarbazides capable of forming stable DABs that are suited
to serve as robust coupling scaffolds.[23] To extend the utility of
the mTG-modified protein-hydrazides in areas that are limiting
for conventional hydrazone conjugates, bioconjugation using the
2fPBA-based subtype of carbonyl chemistry was also examined.
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Tras LC-Q-hydrazide (3a), -carbohydrazide (3b), and -
thiocarbohydrazide (3c) were allowed to react with 5 eq. of a
2fPBA-functionalized fluorophore, Texas Red-2fPBA (Tx Red-
2fPBA), at pH 7.4 (Figure 3a). Reducing SDS-PAGE analysis
showed that, once again, only the modified light chain was
fluorescently labeled (Figure 3b, lanes 5-8; Figure S22). In-gel
fluorescence of the DAB conjugate of Tras LC-Q-hydrazide (lane
6) decreased over time when the gel was kept in acidic
destaining solution, consistent with our previous finding that the
DAB product of 2fPBA and an alkyl hydrazide is acid-labile.[22]
On the other hand, the fluorophore-labeled conjugate of Tras
LC-Q-carbohydrazide (3b) showed a negligible difference over
time (up to two weeks), demonstrating remarkable stability
(Figure 3b, lane 7). Notably, the DAB conjugate of 3b persisted
even when the hydrazone conjugate with coumarin aldehyde
had disassembled. (compare lane 2-3 with lane 7). This
demonstrates that the carbohydrazide-2fPBA ligation results in a
DAB linkage with stability that exceeds the hydrolytic limit of
typical hydrazones.
dihydrazides, which are widely accessible and inexpensive, can
be used as substrates for mTG to install assorted hydrazide
functionalities in peptides and proteins. The introduced
hydrazide handle can subsequently participate in bioorthogonal
ligation with conventional aldehydes/ketones or (the more
recently developed) ortho-carbonylphenylboronic acids to form
dynamic or stable bioconjugates. Importantly, the distinguished
side chain position at which the hydrazide is presented enables
functionalization of an internal amino acid residue, which
complements existing methods that typically introduce the
hydrazide at the C-terminus or the complementary carbonyl
group on the protein. The diversification of the substrate scope
of mTG with α-effect amines therefore further unravels the
versatility of this important enzyme and invites the possibility of
continued expansion of the applied substrate palette with other
nucleophilic moieties.
Acknowledgements
To ensure that the bioconjugate generated via the
chemoenzymatic process retains the function of the
biomolecule, Tras LC-Q-carbohydrazide coupled to a 2fPBA-
functionalized fluorophore (Janelia Fluor 669-2fPBA) was
prepared (Figure S24) and tested for its functional activity in
antigen binding. SK-BR-3 (HER2+) and MDA-MB-231 (HER2−)
breast cancer cells were treated with the antibody-fluorophore
conjugate (AFC). Clear fluorescence localization on the cell
membrane of SK-BR-3 cells, but not MDA-MB-231 cells, was
observed by fluorescence microscopy (Figure 3c), illustrating the
unperturbed function of the AFC in binding its HER2 target on
the cell surface.
Taken together, these results demonstrate the application
of the mTG-derived hydrazides in bioorthogonal conjugation with
carbonyls. Importantly, they also highlighted the impact that the
choice of the hydrazide and the participating carbonyl can have
on the hydrolytic stability of the resultant linkage. Appreciating
this tunability, our method of hydrazide functionalization affords
a handful of hydrazide handles to be elected for installation.
Furthermore, as a complement to strategies that commonly
append a carbonyl group on the biomolecule,[24] the alternative
introduction of the hydrazide functional group on the protein
enables rational selection of the carbonyl partner to cater to
specific applications.
Funding was provided by the NIH Grant R15 CA227747, the
SUNY Research Foundation Accelerator Fund (S.L.B.), and the
Research Foundation of the State of New York (L.N.T.). The
engineered antibodies used in this work were a gift from Pfizer,
Inc. The authors wish to acknowledge the Dr. G. Clifford &
Florence B. Decker Foundation for the generous donation of
equipment that were used in these studies. The Regional NMR
Facility at Binghamton University is supported by the NSF (CHE-
0922815). SK-BR-3 and MDA-MB-231 cells were a gift from Prof.
Tracy Brooks and Prof. Ming An, respectively. The authors
would like to thank Dr. Han Gu for the syntheses of
(hydrazinecarbonyl)glycine and Texas Red- and Janelia Fluor
669-2fPBA; Saptarshi Ghosh for the synthesis of coumarin
aldehyde; and David Tuttle for gel imaging assistance.
Keywords: α-effect nucleophile • click chemistry • enzyme
catalysis • isopeptide • microbial transglutaminase
References
[1]
aY. Zhang, K.-Y. Park, K. F. Suazo, M. D. Distefano, Chemical
Society Reviews 2018, 47, 9106-9136; bM. W. Popp, J. M. Antos,
G. M. Grotenbreg, E. Spooner, H. L. Ploegh, Nat Chem Biol 2007,
3, 707-708; cA. M. Weeks, J. A. Wells, Chem Rev 2020, 120,
3127-3160; dG. K. Nguyen, Y. Qiu, Y. Cao, X. Hemu, C. F. Liu, J.
P. Tam, Nat Protoc 2016, 11, 1977-1988.
Conclusion
[2]
[3]
[4]
Y. Zhu, J. Tramper, Trends in Biotechnology 2008, 26, 559-565.
P. Strop, Bioconjugate Chemistry 2014, 25, 855-862.
aA. Fontana, B. Spolaore, A. Mero, F. M. Veronese, Advanced
Drug Delivery Reviews 2008, 60, 13-28; bS. Jeger, K.
Zimmermann, A. Blanc, J. Grünberg, M. Honer, P. Hunziker, H.
Struthers, R. Schibli, Angew. Chem., Int. Ed. 2010, 49, 9995-9997;
cP. Strop, S. H. Liu, M. Dorywalska, K. Delaria, R. G. Dushin, T. T.
Tran, W. H. Ho, S. Farias, M. G. Casas, Y. Abdiche, D. Zhou, R.
Chandrasekaran, C. Samain, C. Loo, A. Rossi, M. Rickert, S.
Krimm, T. Wong, S. M. Chin, J. Yu, J. Dilley, J. Chaparro-Riggers,
G. F. Filzen, C. J. O'Donnell, F. Wang, J. S. Myers, J. Pons, D. L.
Shelton, A. Rajpal, Chem. Biol. 2013, 20, 161-167; dY. Anami, W.
Xiong, X. Gui, M. Deng, C. C. Zhang, N. Zhang, Z. An, K.
Tsuchikama, Org. Biomol. Chem. 2017, 15, 5635-5642; eS. R.
Benjamin, C. P. Jackson, S. Fang, D. P. Carlson, Z. Guo, L. N.
Tumey, Molecular Pharmaceutics 2019, 16, 2795-2807; fA. Ebenig,
N. E. Juettner, L. Deweid, O. Avrutina, H.-L. Fuchsbauer, H.
Kolmar, Chembiochem 2019, 20, 2411-2419.
In summary, we have demonstrated that hydrazines, hydrazides,
and alkoxyamines can be used as acyl acceptor substrates for
mTG, thus significantly broadening the substrate repertoire of
this promiscuous enzyme. Transamidation with these
noncanonical substrates produces diverse analogues of the
isopeptide bond. Their disparate propensities to bond
cleavage/exchange by mTG prompt new considerations when
using the enzyme to assemble bioconjugates, particularly those
that may encounter mTG or its homologues in their functioning
milieu. In addition to providing diversity in the linkages generated
via direct conjugation, the expanded substrate repertoire also
offers a way to directly introduce a reactive functional group for
chemoenzymatic conjugation. Unsubstituted hydrazine and
[5]
L. Lorand, L. K. Campbell, B. J. Robertson, Biochemistry 1972, 11,
434-438.
6
This article is protected by copyright. All rights reserved.

10.1002/anie.202001830
Angewandte Chemie International Edition
RESEARCH ARTICLE
[6]
[7]
[8]
J. E. Folk, P. W. Cole, Journal of Biological Chemistry 1965, 240,
2951-2960.
B. Spolaore, S. Raboni, A. Ramos Molina, A. Satwekar, N.
Damiano, A. Fontana, Biochemistry 2012, 51, 8679-8689.
aT. Ohtsuka, A. Sawa, R. Kawabata, N. Nio, M. Motoki, Journal of
Agricultural and Food Chemistry 2000, 48, 6230-6233; bM. T.
Gundersen, J. W. Keillor, J. N. Pelletier, Applied Microbiology and
Biotechnology 2014, 98, 219-230.
[9]
I. Avan, C. D. Hall, A. R. Katritzky, Chemical Society Reviews 2014,
43, 3575-3594.
[10]
aA. Amour, A. Collet, C. Dubar, M. Reboud-Ravaux, International
Journal of Peptide and Protein Research 1994, 43, 297-304; bL.
Guy, J. Vidal, A. Collet, A. Amour, M. Reboud-Ravaux, Journal of
Medicinal Chemistry 1998, 41, 4833-4843; cR. Vanderesse, L.
Thevenet, M. Marraud, N. Boggetto, M. Reboud, C. Corbier,
Journal of Peptide Science 2003, 9, 282-299; dC. P. Dorn, M.
Zimmerman, S. S. Yang, E. C. Yurewicz, B. M. Ashe, R.
Frankshun, H. Jones, Journal of Medicinal Chemistry 1977, 20,
1464-1468.
[11]
aK. N. Parameswaran, X.-F. Cheng, E. C. Chen, P. T. Velasco, J.
H. Wilson, L. Lorand, Journal of Biological Chemistry 1997, 272,
10311-10317; bS.-W. Qiao, J. Piper, G. Haraldsen, I. Øynebråten,
B. Fleckenstein, Ø. Molberg, C. Khosla, L. M. Sollid, The Journal
of Immunology 2005, 174, 1657-1663; cJ. Mimuro, S. Kimura, N.
Aoki, The Journal of Clinical Investigation 1986, 77, 1006-1013.
G. T. Hermanson, in Bioconjugate Techniques (Third Edition) (Ed.:
G. T. Hermanson), Academic Press, Boston, 2013, pp. 549-587.
aY. Sugimura, K. Yokoyama, N. Nio, M. Maki, K. Hitomi, Archives
of Biochemistry and Biophysics 2008, 477, 379-383; bN. M.
Rachel, D. Quaglia, É. Lévesque, A. B. Charette, J. N. Pelletier,
Protein Science 2017, 26, 2268-2279.
[12]
[13]
[14]
[15]
J. Schrode, J. E. Folk, Journal of Biological Chemistry 1978, 253,
4837-4840.
aJ. Thom, D. Anderson, J. McGregor, G. Cotton, Bioconjugate
Chem. 2011, 22, 1017-1020; bY.-M. Li, M.-Y. Yang, Y.-C. Huang,
Y.-T. Li, P. R. Chen, L. Liu, ACS Chemical Biology 2012, 7, 1015-
1022; cA. L. Adams, B. Cowper, R. E. Morgan, B. Premdjee, S.
Caddick, D. Macmillan, Angewandte Chemie International Edition
2013, 52, 13062-13066; dY.-M. Li, Y.-T. Li, M. Pan, X.-Q. Kong,
Y.-C. Huang, Z.-Y. Hong, L. Liu, Angewandte Chemie International
Edition 2014, 53, 2198-2202; eC. Komiya, A. Shigenaga, J.
Tsukimoto, M. Ueda, T. Morisaki, T. Inokuma, K. Itoh, A. Otaka,
Chemical Communications 2019, 55, 7029-7032.
[16]
aG. M. Fang, Y. M. Li, F. Shen, Y. C. Huang, J. B. Li, Y. Lin, H. K.
Cui, L. Liu, Angew Chem Int Ed Engl 2011, 50, 7645-7649; bD. T.
Flood, J. C. J. Hintzen, M. J. Bird, P. A. Cistrone, J. S. Chen, P. E.
Dawson, Angewandte Chemie International Edition 2018, 57,
11634-11639.
[17]
[18]
[19]
[20]
D. K. Kölmel, E. T. Kool, Chemical Reviews 2017, 117, 10358-
10376.
A. R. Blanden, K. Mukherjee, O. Dilek, M. Loew, S. L. Bane,
Bioconjugate Chemistry 2011, 22, 1954-1961.
J. Kalia, R. T. Raines, Angewandte Chemie International Edition
2008, 47, 7523-7526.
aL. N. Tumey, H. Sean, Current Topics in Medicinal Chemistry
2017, 17, 3444-3462; bJ. D. Bargh, A. Isidro-Llobet, J. S. Parker,
D. R. Spring, Chemical Society Reviews 2019, 48, 4361-4374.
S. Cambray, J. Gao, Acc. Chem. Res. 2018, 51, 2198-2206.
H. Gu, T. I. Chio, Z. Lei, R. J. Staples, J. S. Hirschi, S. Bane, Org.
Biomol. Chem. 2017, 15, 7543-7548.
[21]
[22]
[23]
aT. I. Chio, H. Gu, K. Mukherjee, L. N. Tumey, S. L. Bane,
Bioconjug Chem 2019, 30, 1554-1564; bH. Gu, S. Ghosh, R. J.
Staples, S. L. Bane, Bioconjugate Chemistry 2019, 30, 2604-2613;
cA. Bandyopadhyay, S. Cambray, J. Gao, J Am Chem Soc 2017,
139, 871-878.
[24]
R. J. Spears, M. A. Fascione, Org. Biomol. Chem. 2016, 14, 7622-
7638.
7
This article is protected by copyright. All rights reserved.