Site-selective labeling of a lysine residue in human serum albumin

Article abstract of DOI:10.1002/anie.201405924

Conjugation to human serum albumin (HSA) has emerged as a powerful approach for extending the in vivo halflife of many small molecule and peptide/protein drugs. Current HSA conjugation strategies, however, can often yield heterogeneous mixtures with inadequate pharmacokinetics, low efficacies, and variable safety profiles. Here, we designed and synthesized analogues of TAK-242, a small molecule inhibitor of Toll-like receptor 4, that primarily reacted with a single lysine residue of HSA. These TAK-242-based cyclohexene compounds demonstrated robust reactivity, and Lys64 was identified as the primary conjugation site. A bivalent HSA conjugate was also prepared in a site-specific manner. Additionally, HSA-cyclohexene conjugates maintained higher levels of stability both in human plasma and in mice than the corresponding maleimide conjugates. This new conjugation strategy promises to broadly enhance the performance of HSA conjugates for numerous applications.

Full text of DOI:10.1002/anie.201405924

Angewandte
Chemie
DOI: 10.1002/anie.201405924
Bioconjugation
Site-Selective Labeling of a Lysine Residue in Human Serum
Albumin**
Shigehiro Asano, James T. Patterson, Thomas Gaj, and Carlos F. Barbas III†*
Abstract: Conjugation to human serum albumin (HSA) has
emerged as a powerful approach for extending the in vivo half-
life of many small molecule and peptide/protein drugs. Current
HSA conjugation strategies, however, can often yield hetero-
geneous mixtures with inadequate pharmacokinetics, low
efficacies, and variable safety profiles. Here, we designed and
synthesized analogues of TAK-242, a small molecule inhibitor
of Toll-like receptor 4, that primarily reacted with a single
lysine residue of HSA. These TAK-242-based cyclohexene
compounds demonstrated robust reactivity, and Lys64 was
identified as the primary conjugation site. A bivalent HSA
conjugate was also prepared in a site-specific manner. Addi-
tionally, HSA-cyclohexene conjugates maintained higher levels
of stability both in human plasma and in mice than the
corresponding maleimide conjugates. This new conjugation
strategy promises to broadly enhance the performance of HSA
conjugates for numerous applications.
varied in vivo efficacies, pharmacokinetics, and toxicities.[5] To
overcome this obstacle, we previously reported a serum-
stable alternative to maleimide-based protein conjugation
that allows site-specific labeling of cysteine residues.[6] Addi-
tionally, several compounds, such as b-lactam-based anti-
biotics, have been reported to react with lysine residues
present on the surface of HSA.[7] To date, however, none of
these compounds have displayed site specificity for lysine.
Recently, TAK-242 (1, Figure 1), a potent Toll-like
receptor 4 (TLR4) inhibitor,[8] was shown to form a covalent
lysine adduct with both rat and human albumin in plasma
Figure 1. Structures of TAK-242 (1) and PEG-modified TAK-242 deriva-
P
rotein conjugates to therapeutic moieties have been
tives used for HSA conjugation.
reported and evaluated in clinical trials.[1] Compared to the
parental small molecule, protein–drug conjugates offer sev-
eral advantages, including half-life extension, localization to
a target tissue, avoidance of drug–drug interactions, and
reduction of toxicity. Human serum albumin (HSA) has
proven to be a valuable protein for the conjugation of small
molecules, peptides, and proteins. For example, the half-lives
of the small molecules DOXO-EMCH (INNO-206)[2] and
AWO54[3] have been dramatically extended by conjugation to
HSA, facilitating their progression into clinical trials for the
treatment of cancer and rheumatoid arthritis, respectively.
Drug conjugation to target proteins is commonly achieved
through maleimide–cysteine chemistry or labeling of lysine
with N-hydroxysuccinimide ester. However, these methods
have several disadvantages, including the formation of
heterogeneous mixtures of protein conjugates[4] that have
after intravenous dosing.[9] TAK-242 inhibits protein–protein
interactions between TLR4 and its adapter proteins by
associating with a single cysteine residue within an intra-
cellular domain of TLR4.[10] A covalent binding mechanism
based on Michael addition with cysteine or lysine, and
subsequent elimination of the sulfonamide moiety through
allylic rearrangement has been proposed.[9] Based on this
potential mechanism, we hypothesized that linkers derived
from TAK-242 could react with lysine on HSA to enable the
formation of stable conjugates. Thus, we prepared polyethy-
lene glycol (PEG)-modified TAK-242 derivatives using
fluorobenzene sulfonamide as a leaving group to 1) maintain
labeling activity for HSA and 2) reduce TLR4 inhibitory
activity (Figure 1).
We evaluated the HSA reactivity of the TAK-242
analogues 5a–c and compared labeling to that of maleimide
compound 10 (Schemes 1 and 2). Recombinant HSA was
incubated with one or two equivalents of 5a–c or 10 at 378C
for two hours, then a click reaction was performed with the
azide–rhodamine compound 12a to give a fluorescent con-
jugate. Products were resolved by SDS-PAGE, and the
reaction rates were calculated by measuring the fluorescence
intensities of HSA-conjugate bands (Figures 2A and S1).
Additionally, we performed conjugation reactions between
HSA and compounds 5a–c or 10 (50 mm) in the presence of
human plasma (Figures 2B and S2). The PEG-modified
TAK-242 analogues reacted with recombinant HSA at rates
similar to the maleimide compound. Compound 5b, which
contains a 2,4-difluorophenyl sulfonamide, had the highest
reactivity. Following conjugation of 5b with HSA, the by-
[*] Dr. S. Asano,[+] Dr. J. T. Patterson,[+] Dr. T. Gaj,
Prof. Dr. C. F. Barbas III
The Skaggs Institute for Chemical Biology, Department of
Chemistry, and Department of Molecular and Cell Biology
The Scripps Research Institute
10550 North Torrey Pines Road, La Jolla, CA 92037 (USA)
E-mail: carlos@scripps.edu
[+] These authors contributed equally to this work.
[†] Deceased on June 24th, 2014
[**] This study was supported by The Skaggs Institute for Chemical
Biology. We thank Linh Truc Hoang for performing LC-MS/MS
analysis and Diane M. Kubitz for animal work. We also thank
Yoshihiro Ishihara for comments on the manuscript.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201405924.
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Scheme 1. Synthesis of PEG-linked TAK-242 analogues. Reagents and
conditions: a) Ba(OH)2 octahydrate, H2O, CH3CN, 608C, 1 h, 70% 4a,
83% 4b, 64% 4c; b) 6, DEAD, PPh3, THF, 08C!RT, overnight, 32%
5a, 44% 5b, 42% 5c. DEAD=diethyl azodicarboxylate.
Scheme 2. Synthesis of comparative compounds for HSA labeling.
Reagents and conditions: a) 6, DEAD, PPh3, THF, 08C!RT, overnight,
28%; b) triethylamine, EMCS, DMF, RT, overnight, 46%. DMF=N,N-
dimethylformamide; EMCS=N-(e-maleimidocaproyloxy)succinimide
ester.
product 2,4-difluoroaniline was detected in solution by HPLC
(Figure S3), suggesting that the mechanism of the reaction for
compounds 5a–c with HSA is similar to that of the reaction
with TAK-242 (Scheme S5).
Figure 2. Conjugation reactions of HSA with cyclohexene compounds
(5a–c) and with the maleimide compound (10). A) Normalized
intensities of conjugation reaction with recombinant HSA. B) Normal-
ized intensities of conjugation reaction with HSA in human plasma.
C) SDS-PAGE of conjugation reaction in HSA-depleted human plasma
(red arrow: HSA conjugate; yellow arrow: other protein conjugates).
In order to assess specificity for HSA, we incubated our
TAK-242 derivatives in human plasma depleted of HSA
(Figures 2C and S4). No proteins in the depleted serum
reacted with the TAK-242 compounds 5a–c (Figure 2C, red
arrow); however, maleimide (10) did react with several
proteins (Figure 2C, yellow arrows). Moreover, inclusion of
saturating amounts of myristic acid had no impact on
recombinant HSA labeling, thereby supporting the use of
cyclohexene compounds for in vivo bioconjugation (Fig-
ure S5). Evaluation of TAK-242 analogue stability indicated
that 5b was sufficiently stable for labeling applications
(Figure S6). Furthermore, structure–activity relationship
analyses showed that TLR4 inhibition requires the alkyl
group of the TAK-242 ester moiety and the hydrophilic
chain.[8] As expected, none of the cyclohexene compounds
inhibited TLR4 activity (Figure S7).
indicating that the sulfonamide group of 5b is an important
trigger for conjugation. Compound 13, which was modified to
contain rhodamine, was incubated with recombinant HSA in
various PBS solutions to evaluate dependence on the
pH value (Scheme 3). Conjugation was positively correlated
with the basicity of the solution, as the reaction rate at pH 9
was approximately two times higher than at pH 7.4 (Fig-
ure S9), likely as a result of an increase in amino acid
nucleophilicity.
To measure the equivalence and time dependence of the
labeling reaction, we conjugated HSA with the fluorescent
TAK-242 derivative 13. We calculated the dye/protein molar
ratios from the absorbance values of both the protein and dye,
which demonstrated that use of ten equivalents of compound
13 led to the highest rates of labeling, giving a dye/protein
molar ratio of higher than 1:1 after five hours (Figure S10).
The simple cyclohexene derivative compound 8 was
incubated with recombinant HSA to confirm the impact of
the sulfonamide group on conjugation (Scheme 2). No label-
ing was observed with this compound (Figure S8), thus
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Scheme 3. Synthesis of functionalized compounds for HSA labeling. Reagents and conditions: a) NEt3, 3-azidopropan-1-amine, CH3CN, RT,
overnight, 51% 12a, 31% 12b; b) 5b, CuSO4, THPTA, sodium ascorbate, tBuOH, H2O, RT, 3 h, 34% 13; 84% 15; c) N-(2-(2-(2-(2-
aminoethoxy)ethoxy)ethoxy)ethoxy)biotinamide, DMF, RT, 3 h, 46%. THPTA=tris(3-hydroxypropyltriazolylmethyl)amine.
This result suggests that the TAK-242 analogue may react
selectively with one amino acid residue of HSA, but that large
excesses of the electrophile can lead to labeling at secondary
positions. Our data also indicates that determining the
appropriate number of compound equivalents and reaction
kinetics is critical for sufficiently driving the reaction while
maintaining selectivity. Nonetheless, the cyclohexene linker is
highly site-selective, particularly with regard to the finding
that non-enzymatic glycation of lysine in HSA results in
modification at 21 sites.[11]
Next, we blocked the lysine residues of HSA with NHS-
biotin or the cysteine residues with iodoacetamide (IAA), and
then incubated each mixture with 5b to further establish that
TAK-242 reacts specifically with lysine and not cysteine.
After blocking with IAA, no change in labeling was observed
with 5b; however, the rate of conjugation with maleimide
compound 10 was greatly reduced (Figure S11). By contrast,
reaction of 5b with HSA was significantly reduced after
blocking with NHS–biotin (Figure S12). Furthermore, mono-
meric amino acids Lys, Cys, Ser, Thr, His, and Trp did not
react with 5b (data not shown), which suggests that the
tertiary structure of HSA is an important factor for conjuga-
tion.
cation at Lys64 in the sample reacted with 5b (Figure S14).
These findings were confirmed through the presence of b and
y fragment ions in the MS/MS spectrum. Given the stoichi-
ometry of HSA labeling and LC-MS/MS analysis, this data
indicates that Lys64 is the primary site of labeling.
We recently reported a phenyloxadiazole (ODA) com-
pound that efficiently labeled Cys34 of HSA and demon-
strated high stability in human plasma.[6] To test whether
conjugation at both Lys64 and Cys34 is feasible, recombinant
HSA was labeled with rhodamine-linked cyclohexene com-
pound 13 or fluorescein-linked ODA compound, and then
sequentially labeled at the second site with the respective
compound. The increase in fluorescence for the HSA
conjugates treated with both compounds relative to labeling
with 13 or ODA alone confirmed that each site was
conjugated (Figure 3A). To further validate that HSA was
modified at both sites, the fluorescence emission spectra were
collected for HSA conjugated with 13, ODA, or 13 + ODA
(Figure 3B). As anticipated, rhodamine fluorescence was
detected for HSA conjugated with 13 but not with ODA, and
fluorescein signal was observed for the ODA conjugate but
not with 13. Fluorescence for the rhodamine and fluorescein
labels was detected with 13 + ODA, demonstrating that HSA
can be simultaneously conjugated in a site-specific manner.
Finally, we determined the stability of HSA conjugates.
As a preliminary in vitro evaluation, we prepared HSA–
cyclohexene and HSA–maleimide fluorescent conjugates
based on compounds 13 and our previously described
fluorescein-linked maleimide linker,[6] respectively, and incu-
bated the conjugates in human plasma at 378C for one month
(Figure S15). The stability of the HSA–cyclohexene conju-
To determine the labeling site, we subjected the HSA–5b
conjugate to proteolysis and analyzed the digested fragments
by nano LC-MS/MS. One equivalent of 5b was used for the
preparation of the HSA conjugate to limit secondary labeling
(Figure S10). Forty-four unique peptides were identified, and
68% sequence coverage was obtained (Figure S13). The
peptide
containing
amino
acids
61
to
82
[NCDKSLHTLFGDKLCTVATLRE] had a 339 Da modifi-
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the application of cyclohexene compounds for site-selective
lysine conjugation of HSA appears to be a viable means for
improving the therapeutic half-life of drug moieties.
In conclusion, we designed and synthesized TAK-242
derivatives to overcome the limitations of maleimide con-
jugation in preparing HSA conjugates. TAK-242 analogues
possessed high reactivity and specificity for HSA, with Lys64
being identified as the primary labeling site. Additionally, the
HSA conjugates of cyclohexene compounds 13 and 15 showed
excellent stability in human plasma and in mice, respectively.
This conjugation framework should find great utility for the
generation of HSA conjugates with improved serum stability.
Received: June 4, 2014
Published online: && &&, &&&&
Keywords: chemoselectivity · drug conjugates ·
.
human serum albumin · lysine labeling ·
site-specific conjugation
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Figure 3. Site-specific labeling of recombinant HSA at Lys64 and
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Figure 4. Stability of HSA conjugate in mice. Top: representative
Western blot for cyclohexene (15) and maleimide (17) biotinylated
HSA conjugates. Bottom: averaged remaining biotin conjugate values.
gate (t1/2 > 28 d) proved to be much greater than that of the
corresponding HSA–maleimide conjugate (t1/2 = 7.2 ꢀ 0.4 d).
We next evaluated the in vivo stability of HSA conjugates
prepared with biotinylated compounds 15 and 17 (Scheme 3
and Figure 4). Biotinylated compounds were administered to
BALB/c mice at a single subcutaneous dose of 20 mgkgꢁ1 (n =
3). Blood was collected at 4, 8, 24, 48, 72, 96, and 168 hours,
and samples were analyzed by Western blot. Assuming the
linker did not influence clearance, the biotinylated cyclo-
hexene–HSA conjugate (t1/2 = 39.9 ꢀ 4.4 h) was more stable
than the maleimide–HSA conjugate (t1/2 = 25.0 ꢀ 2.8 h). Thus,
4
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Bioconjugation
S. Asano, J. T. Patterson, T. Gaj,
C. F. Barbas III†*
&&&& — &&&&
Site-Selective Labeling of a Lysine
Residue in Human Serum Albumin
Analogues of TAK-242, a small molecule
inhibitor of Toll-like receptor 4, were syn-
thesized and conjugated to human serum
albumin (HSA). These TAK-242-based
cyclohexene compounds demonstrated
high reactivity, and Lys64 was identified
as the primary conjugation site. A biva-
lent HSA conjugate was also prepared in
a site-specific manner.
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