Comparison of hydrazone heterobifunctional cross-linking agents for reversible conjugation of thiol-containing chemistry

Article abstract of DOI:10.1021/bc100049c

Reversible covalent conjugation chemistries that allow site- and condition-specific coupling and uncoupling reactions are attractive components in nanotechnologies, bioconjugation methods, imaging, and drug delivery systems. Here, we compare three heterob

Full text of DOI:10.1021/bc100049c

Bioconjugate Chem. 2010, 21, 1779–1787
1779
Comparison of Hydrazone Heterobifunctional Cross-Linking Agents for
Reversible Conjugation of Thiol-Containing Chemistry
R. James Christie, Diana J. Anderson, and David W. Grainger*
Department of Chemistry, Colorado State University, Fort Collins, Colorado 80523-1872. Received January 26, 2010;
Revised Manuscript Received June 12, 2010
Reversible covalent conjugation chemistries that allow site- and condition-specific coupling and uncoupling reactions
are attractive components in nanotechnologies, bioconjugation methods, imaging, and drug delivery systems.
Here, we compare three heterobifunctional cross-linkers, containing both thiol- and amine-reactive chemistries,
to form pH-labile hydrazones with hydrazide derivatives of the known and often published water-soluble polymer,
poly[N-(2-hydroxypropyl methacrylamide)] (pHPMA), while subsequently coupling thiol-containing molecules
to the cross-linker via maleimide addition. Two novel cross-linkers were prepared from the popular heterobi-
functional cross-linking agent, succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC), modified
to contain either terminal aldehyde groups (i.e., 1-(N-3-propanal)-4-(N-maleimidomethyl) cyclohexane carboxamide,
PMCA) or methylketone groups (i.e., 1-(N-3-butanone)-4-(N-maleimidomethyl) cyclohexane carboxamide, BMCA).
A third cross-linking agent was the commercially available N-4-acetylphenyl maleimide (APM). PMCA and BMCA
exhibited excellent reactivity toward hydrazide-derivatized pHPMA with essentially complete hydrazone conjugation
to polymer reactive sites, while APM coupled only ∼60% of available reactive sites on the polymer despite a
3-fold molar excess relative to polymer hydrazide groups. All polymer hydrazone conjugates bearing these
bifunctional agents were then further reacted with thiol-modified tetramethylrhodamine dye, confirming cross-
linker maleimide reactivity after initial hydrazone polymer conjugation. Incubation of dye-labeled polymer
conjugates in phosphate buffered saline at 37 °C showed that hydrazone coupling resulting from APM exhibited
the greatest difference in stability between pH 7.4 and 5.0, with hydrolysis and dye release increased at pH 5.0
over a 24 h incubation period. Polymer conjugates bearing hydrazones formed from cross-linker BMCA exhibited
intermediate stability with hydrolysis much greater at pH 5.0 at early time points, but hydrolysis at pH 7.4 was
significant after 5 h. Hydrazones formed with the PMCA cross-linker showed no difference in release rates at pH
7.4 and 5.0.
That these chemistries reside in a single coupling agent that
responds reliably to desired stimuli (e.g., local pH, thermal or
optical gradients, enzymes, electrochemical or redox inputs) to
couple and uncouple connected cargo is a challenging goal.
INTRODUCTION
Recent developments in nanomaterials, biotechnology, and
advanced drug delivery and imaging systems have prompted
innovation of new molecular building blocks and methods to
attach components together to achieve advanced materials with
specific functionality. For polymer-based therapeutics, in par-
ticular, the benefits of conjugating drugs to water-soluble
polymers are well-known and include the following: increased
circulation half-life, reduced nonspecific activity, enhanced
targeting ability, and physiological solubility (1). While these
benefits provide strong incentives for use of polymers and new
particle carriers in drug delivery, methods to chemically couple
therapeutic and imaging agents to such carriers have been
frequently inflexible and performance limiting. Yet, control and
versatility of the chemistry used to couple molecules together
is crucial to their function and purpose. Most therapeutic agents
are best released from carriers at the desired physiological site
of drug activity to be most effective, while retaining stability
in circulation until the construct reaches its therapeutic target.
Many cleverly designed coupling agents and cross-linkers have
therefore been developed for stable covalent attachment under
one set of conditions (i.e., normal circulation) and reversible
uncoupling chemistries under other sets of conditions (2-4).
Strategies for site-specific nanoparticle- or polymer-based
agent delivery often exploit biochemical conditions intrinsic to
cell internalization and processing pathways to specifically
release drug into the cellular cytoplasm (5). Polymer and
nanoparticle constructs internalized into cells by endocytosis
are transported to lysosomessmembrane-encapsulated cyto-
plasmic organelles more acidic (pH ∼5.0) than normal physi-
ological conditions, and containing a host of specific enzymes
(6). Therapeutic agents with activity beyond the lysosomal
compartment (i.e., most drugs) must achieve lysosomal release
at sufficient dosing in order to exert their desired activity. For
these reasons, polymer- and nanoparticle-drug conjugation
chemistry is often designed to degrade once inside the cell via
cleavage by lysosome-specific enzymes, as well as by nonspe-
cific pH-dependent and organelle-specific mechanisms. The Gly-
Phe-Lys-Gly (GFLG) peptide sequence has been shown to
degrade specifically by the lysosome enzyme, cathepsin B, and
is a distinguishing component in the poly[N-(2-hydroxypropyl)
methacrylamide] (pHPMA) copolymer-doxorubicin (DOX)
anticancer drug PK1, currently in clinical trials (7-11). Other
degradable peptide spacers such as the dipeptides valine-
citrulline, asparagine-D-lysine, and methionine-D-lysine have
been used to produce antibody-drug conjugates where the drug
is released by proteolysis of the peptide linker (12, 13).
Nonspecific pH-driven hydrolytic degradation and agent de-
* Corresponding author. D. W. Grainger. Current address: Depart-
ment of Pharmaceutics and Pharmaceutical Chemistry, College of
Pharmacy, Health Sciences Campus, University of Utah, Salt Lake City,
UT84112USA;Email:david.grainger@utah.edu.Phone:+1801.581.3715;
Fax: +1 801 0.581.3674.
10.1021/bc100049c  2010 American Chemical Society
Published on Web 08/09/2010

1780 Bioconjugate Chem., Vol. 21, No. 10, 2010
Christie et al.
Figure 1. Design strategy for reversible coupling of thiols to pHPMA and cross-linker structures.
coupling have been demonstrated for a variety of coupling
chemistries including hydrazone, acetal, carbamate, and cis-
aconityl moieties (14-17).
MATERIALS AND METHODS
General Procedures and Materials. All solvents were ACS-
certified grade purchased from Fisher Scientific (Fair Lawn,
USA) and used without further purification unless otherwise
noted. Trinitrobenzene sulfonic acid (5% w/v solution in
methanol) (TNBSA), cystamine hydrochloride (98%), diiso-
propylethylamine (DIPEA), ethyl carbazate (97%), N-hydroxy-
succinamide (NHS) (98%), 4-(aminomethyl)cyclohexane car-
boxylic acid (97%), maleic anhydride (98+%), acetic anhydride,
dicyclohexanecarbodiimide (DCC) (99%), hydrazine monohy-
drate (98%), 3-amino-1-propanol (99+%), triethyl amine (99.5%),
oxalyl chloride (99+%), and glacial acetic acid were all
purchased from Aldrich (St. Louis, USA). N-4-(Acetylphenyl)
maleimide (APM) (98+%) was purchased from Lancaster
Synthesis (Pelham, USA). 4-Amino-2-butanol (98%) was
purchased from Acros Organics (Geel, Belgium). Reducing gel
with immobilized tris(2-carboxyethyl) phosphine hydrochloride
(TCEP) was purchased from Pierce (Rockford, USA). Spectra/
Por 1, 6-8 kDa molecular weight cutoff dialysis tubing was
obtained from Spectrum Laboratories, Inc. (Rancho Dominguez,
USA). Sephadex G-25 and LH-20 size exclusion chromatog-
raphy gel were purchased from Amersham Biosciences (Upp-
sala, Sweden). Dimethyl sulfoxide-d6 containing 0.05% w/v
tetramethylsilane was purchased from Cambridge Isotope
Laboratories, Inc. (Andover, MA). 5(6)-Tetramethylrhodamine-
succinimidyl ester (TAMRA-NHS) was obtained from AnaSpec
(San Jose, USA). Poly(HPMA) molecular weight standards were
a gift from Prof. P. Kopeckova, Univ. Utah, USA. Water was
Millipore-filtered ASTM Type I quality. All NMR spectra were
recorded on a Magnex Scientific Inova 400 MHz instrument in
DMSO-d6 containing 5% w/v tetramethylsilane as an internal
standard. Absorbance measurements were obtained on a Varian
Carey 500 Scan instrument.
More specifically, hydrazone bonds have been successfully
used to generate a variety of pH-sensitive conjugates of the
anticancer drug doxorubicin with various polymers, particles,
micelles, and antibodies by reaction with hydrazide-derivatized
carriers and cross-linkers with the 13-keto position of this
drug (15, 16, 18-24). Hydrazone bonds are formed by
condensation of hydrazides with aldehydes or ketones after
elimination of the carbonyl oxygen as water, resulting in a
metastable imine bond. This reaction can be performed under
aqueous conditions with careful control of pH and reaction
stoichiometry but is most often performed under organic
conditions with an acid catalyst such as acetic acid or trifluo-
roacetic acid to facilitate dehydration and formation of the
hydrazone bond (19, 25, 26). While certain types of hydrazone
linkages are known to be stable at pH 7.4 and degrade at low
pH (i.e., <5.0), chemistries adjacent to both hydrazide and ketone
functionality greatly affect pH and hydrolytic stability. Hence,
specific substituents have been used to achieve different bond
cleavage rates and pH sensitivities (15, 16). This chemistry can
also be tailored to present other functional groups useful for
other conjugation strategies.
This work evaluates three heterobifunctional cross-linkers
designed to couple thiol-containing molecules to hydrazone-
derivatized pHPMA drug carriers enjoying a long history of
use (9, 10, 27). Thiol-based drug conjugation to polymer-linked
hydrazones is not widely studied. Efficiency of cross-linker
coupling to reactive sites on the polymer backbone, and their
subsequent stability at pH 7.4 and 5.0 were investigated. While
both polymer and cross-linker can be designed to provide the
hydrazide functionality, we placed this functional group on the
polymer carrier as previously described (19, 20). Crosslinkers
that facilitate reversible coupling of thiol-containing molecules
to pHPMA contain ketone or aldehyde functional groups on
one terminus for reaction with pHPMA hydrazide, and a
maleimide functionality at the other terminus to react with
sulfhydryl groups on the target adduct via a 1,4-Michael addition
reaction (19, 20). This thiol coupling strategy is advantageous
for coupling unmodified proteins/peptides lacking aldehyde/
ketone functionality, or other thiol-containing molecules, to
known hydrazide-derivatized pHPMA water-soluble carriers, or
analogous chemistry. Two heterobifunctional cross-linkers inves-
tigated, 1-(N-3-propanal)-4-(N-maleimidomethyl) cyclohexane car-
boxamide (PMCA), and 1-(N-3-butanone)-4-(N-maleimidomethyl)
cyclohexane carboxamide (BMCA) were prepared from the
popular commercial cross-linker succinimidyl-4-(N-maleimi-
domethyl) cyclohexane-1-carboxylate (SMCC), while the third
was a commercially available benzene derivative, N-4-acetylphe-
nyl maleimide. Illustration of the reversible coupling/release
strategy and cross-linking chemistry structures involving
hydrazone-thiol coupling are shown in Figure 1.
4-(N-Maleimidomethyl)cyclohexane Carboxylic Acid (MCCA)
(1). 4-(Aminomethyl)cyclohexane carboxylic acid (40.71 g, 0.26
mol, 1 equiv) was dissolved in dimethyl acetamide (DMAc,
100 mL). A solution of maleic anhydride (25.20 g, 0.26 mol, 1
equiv) was prepared in DMAc (50 mL), then added to the
4-(aminomethyl)cyclohexane carboxylic acid solution dropwise
while stirring. The reaction vessel was purged with nitrogen,
capped, and stirred for 24 h at room temperature. After 24 h,
sodium acetate (5.00 g, 0.06 mol, 0.24 equiv) and acetic
anhydride (43 mL, 0.455 mol, 1.75 equiv) were added and the
reaction was continued for an additional 24 h. After 24 h, the
reaction was heated to 65 °C and stirred for 5 h, then
concentrated to ∼125 mL under vacuum and poured into 1 L
cold water while stirring. The light-brown precipitate was
collected by filtration and dried under vacuum. The aqueous
portion was extracted twice with methylene chloride (500 mL),
and product was isolated after evaporation of solvent. Both solid
portions were combined and dissolved in methylene chloride
(500 mL), then silica gel (5 g) was added to the solution and

Hydrazone and Thioether-Forming Cross-Linkers
Bioconjugate Chem., Vol. 21, No. 10, 2010 1781
the suspension was stirred for 30 min. The resulting colorless
solution was filtered and solid product was obtained by
evaporation of solvent under vacuum. Crude product was further
purified by crystallization from ethyl acetate to yield a white
added and the mixture was stirred for 10 min. The organic layer
was separated, and the aqueous portion was re-extracted with 2
× 10 mL methylene chloride, and then all organic portions were
combined. The organic portion was washed with 1 M HCl, dilute
NaHCO3, and H2O, and then dried with Na2SO4. Product was
isolated after removal of Na2SO4 and evaporation of solvent.
1
solid. Yield: 14.70 g, 24%. H NMR, δ ppm: 0.926 (dd, 2H),
1.22 (dd, 2H), 1.517 (m, 1H), 1.6165 (d, 2H), 1.869 (d, 2H),
2.109 (m, 1H), 3.2355 (d, 2H), 7.012 (s, 2H), 12.010 (s, 1H).
13C NMR, δ ppm: 27.932, 29.112, 36.013, 42.105, 42.861,
134.273, 171.549, 176.497. MS calcd [M + H]+ (C12H15NO4):
238.107. Found: (FAB) 238.108.
1
Yield: 0.350 g, 66%, white powder. H NMR, δ ppm: 0.878
(dd, 2H), 1.232 (dd, 2H), 1.507 (m, 1H), 1.636 (m, 2H), 1.976
(m, 1H), 2.502, (solvent overlap) 3.23 (d, 2H), 3.284 (q, 2H),
7.011 (s, 1H), 7.796 (t, 1H), 9.604 (t, 1H). 13C NMR, δ ppm:
28.378, 29.286, 32.393, 36.037, 42.957, 43.265, 43.566, 134.274,
171.185, 174.958, 202.195. MS calcd [M + H]+ (C15H20N2O4):
293.1501. Found (FAB) 293.1507.
1-(N-3-Propanol)-4-(N-maleimidomethyl)cyclohexane Car-
boxamide (2). Cross-linker precursor MCAA (3.00 g, 1.0 equiv,
13 mmol) was dissolved in methylene chloride (75 mL), purged
with nitrogen, capped, and cooled to -15 °C. A suspension of
NHS was prepared (1.45 g, 1.0 equiv, 13 mmol) in methylene
chloride (25 mL), then cooled to -15 °C. Next, a solution of
DCC was prepared (2.87 g, 1.1 equiv, 14 mmol) in methylene
chloride (25 mL) and cooled to -15 °C. The DCC solution
was added to the solution of MCAA, followed by addition of
the NHS suspension. The reaction flask was purged with
nitrogen, stirred at -15 °C for 24 h, and then filtered to remove
dicyclohexylurea (DCU). The filtrate was retained and 3-amino-
1-propanol (1.98 mL, 1.9 equiv, 25 mmol) was added dropwise.
The reaction was purged with nitrogen, capped, and stirred at
room temperature for 3 h. After 3 h, the reaction mixture was
filtered to remove liberated NHS, and the filtrate was concen-
trated to ∼2/3 original volume under vacuum, cooled to -20
°C, and then refiltered. The resulting filtrate volume was doubled
by addition of diethyl ether to form a precipitate, which was
isolated by filtration. The solid product was further purified by
silica gel chromatography (10 × 40 cm, 90/10 methylene
chloride/methanol mobile phase) and isolated by evaporation
of solvent under vacuum. Yield: 1.91 g, 51.6%, white solid. 1H
NMR, δ ppm: 0.886 (dd, 2H), 1.265 (dd, 2H), 1.508 (q, 3H),
1.618 (d, 2H), 1.682 (d, 2H), 1.998 (m, 1H), 2.5 (solvent
overlap), 3.047 (q, 2H), 3.234 (d, 2H), 3.372 (q, 2H), 4.399 (t,
1H), 7.013 (s, 2H), 7.652 (t, 1H). 13C NMR, δ ppm: 28.488,
29.349, 32.386, 35.423, 36.060, 42.980, 43.704, 58.307, 134.277,
171.188, 174.825. MS calcd [M + H]+ (C15H22N2O4): 295.1657.
Found: (FAB) 295.1654.
1-(N-3-Butanone)-4-(N-maleimidomethyl)cyclohexane Car-
boxamide, BMCA (5). BMCA was prepared using the same
Swern procedure described above to prepare PMCA, except
starting with 3 (2.00 g, 1.0 equiv, 6.5 mmol) as the alcohol to
1
be oxidized. Yield: 1.34 g, 68%, white powder. H NMR, δ
ppm: 0.875 (q, 2H), 1.243 (q, 2H), 1.511 (m, 1H), 1.511 (m,
1H), 1.638 (m, 4H), 1.975 (m, 1H), 2.070 (s, 3H), 2.545 (t,
2H), 3.180 (q, 2H), 3.228 (d, 2H), 7.011 (s, 2H), 7.677 (t, 1H).
13C NMR, δ ppm: 28.407, 29.305, 29.752, 33.626, 36.043,
42.620, 42.962, 43.548, 134.273, 171.179, 174.796, 207.179.
MS calcd [M + H]+ (C16H22N2O4): 307.1657. Found (FAB):
307.1657.
Thiol-Containing Tetramethylrhodamine (6). Thiolated
tetramethylrhodamine was prepared by reaction of TAMRA-
NHS with cystamine hydrochloride activated with diisopropyl-
ethylamine (DIPEA) to form the TAMRA disulfide dimer
(ssTAMRA) as previously described (29). Briefly, cystamine
hydrochloride (9.6 mg, 0.9 equiv amines, 0.043 mmol) was
activated in dimethylformamide (DMF, 2.5 mL) containing
DIPEA (30 µL, 1.8 equiv, 0.17 mmol). This solution was added
to TAMRA-NHS (50.0 mg, 1.0 equiv, 0.095 mmol) in DMF
(1.5 mL) and stirred at room temperature in the dark for 30
min followed by addition of DIPEA (15 µL, 0.086 mmol, 1.0
equiv) and reaction for an additional hour. Product was
precipitated by addition of 50 mL cold diethyl ether, washed
five times, and dried under vacuum overnight. Further purifica-
tion was achieved by repeat precipitation from methanol into
cold ether. Yield: 24.81 mg, 54%, dark red solid. MS calcd
[M+H]+ (C54H52N6O8S2): 977.3366. Found (FAB): 977.3348.
Poly[N-(2-hydroxypropyl)methacrylamide], pHPMA (7).
PHPMA was synthesized to contain 5 mol % reactive nitro-
phenol ester terminated, nondegradable glycylglycine side chains
using previously described procedures (30-32). N-(2-Hydroxy-
propyl)methacrylamide (HPMA) and methacryloyl glycylglycine
nitrophenol ester (MAGGONP) monomers were prepared in
house and confirmed with NMR and mass spectrometry. A
detailed procedure for monomer synthesis and subsequent
polymerization is provided as Supporting Information. The
number of active esters in the resulting copolymer was estimated
to be 2.55 × 10-4 mol/g polymer (3.8 mol %) by measuring
the absorbance of released p-nitrophenolate anion at 400 nm (ε
) 1.74 × 104 M-1 cm-1) following hydrolysis of ester bonds
in 0.05 M sodium hydroxide for 3 h at 37 °C. No increase in
absorbance after the 3 h incubation period was observed,
showing complete cleavage of p-nitrophenol esters. Copolymer
molecular weight was estimated to be 18 200 Da by size
exclusion chromatography using a HP1050A HPLC equipped
with a HP1047A refractive index detector and BioSil 125
column calibrated with pHPMA samples of known molecular
weights. Samples were eluted at 0.5 mL/min with 0.1 M sodium
phosphate, 0.05 M NaCl, pH 7.4.
1-(N-3-Butanol)-4-(N-maleimidomethyl)cyclohexane Car-
boxamide (3). This product was obtained by an analogous
procedure used to synthesize compound 2 (vida infra) with the
only difference being the use of 4-amino-2-butanol (1.65 mL,
1.1 equiv, 17.3 mmol) in methylene chloride (30 mL) as the
amidation reagent. Yield: 2.88 g, 60%, white powder. 1H NMR,
δ ppm: 0.887 (dd, 2H), 1.036 (d, 3H), 1.267 (dd, 2H), 1.521
(m, 1H), 1.620 (d, 2H), 1.686 (d, 2H), 1.998, (m, 1H), 3.065
(q, 2H), 3.240 (d, 2H), 3.585 (septet, 1H), 4.443 (d, 1H), 7.018
(s, 2H), 7.645 (t, 1H). 13C NMR, δ ppm: 23.509, 28.459, 29.334,
35.489, 36.046, 38.719, 42.966, 43.694, 63.628, 134.267,
171.168, 174.776. MS calcd [M + H]+ (C16H24N2O4): 309.1814.
Found (FAB) 309.1819.
1-(N-3-propanal)-4-(N-maleimidomethyl)cyclohexane Car-
boxamide, PMCA (4). PMCA was prepared using the Swern
procedure for oxidation of alcohols to aldehydes (28). The
alcohol 2 (500 mg, 1.0 equiv, 1.72 mmol) was dissolved in
freshly distilled methylene chloride (20 mL) and cooled to -80
°C. An activated dimethyl sulfoxide (DMSO) solution was
prepared by combining DMSO (295 µL, 2.4 equiv, 4.13 mmol)
and oxalyl chloride (950 µL of 2.0 M solution in methylene
chloride, 1.1 equiv, 1.9 mmol) in methylene chloride (1.5 mL)
at -80 °C. The alcohol solution was slowly added to the
activated DMSO solution over 5 min. The reaction was allowed
to proceed for 15 min at -80 °C, followed by addition of
triethylamine (1.2 mL, 5.0 equiv, 8.5 mmol). The reaction was
equilibrated to room temperature, and then water (15 mL) was
Hydrazide Derivatized pHPMA (8). pHPMA copolymer
was further modified to contain hydrazide-terminated side chains
by reaction of p-nitrophenol ester-terminated MAGGONP side

1782 Bioconjugate Chem., Vol. 21, No. 10, 2010
Christie et al.
Scheme 1. Thiol-Reactive Crosslinker Synthesis
chains with excess hydrazine monohydrate using methods
previously described (19, 20). Briefly, p-nitrophenol activated
pHPMA (2.0 g, 1.0 equiv, 0.51 mmol active ester) was dissolved
in methanol (25 mL). The pHPMA solution was added dropwise
to a solution of hydrazine monohydrate (267 µL, 10.8 equiv,
5.5 mmol) in methanol (25 mL). The reaction was purged with
nitrogen, capped, and stirred for 3 h at room temperature. The
reaction solution turned dark yellow indicating reaction of active
ester groups and release of p-nitrophenol. After complete
reaction, the reaction mixture was dripped into cold water (50
mL) and low molecular weight byproducts were removed by
dialysis against water (SpectraPore, 6-8 kDa molecular weight
cutoff, 2 days). Polymer product was isolated by lyophilization.
Yield: 0.120 g, 60%, white powder. Hydrazide content: 2.63 ×
10-4 mol/g, 100.5% conversion.
1.25 h, followed by removal of TCEP gel by centrifugation.
The gel material was washed with 6 mL buffer (pH 7.0, 2.5
mM EDTA), centrifuged, and the supernatant retained and
combined with the first fraction.
Analysis of pHPMA Hydrazide Content. Hydrazide content
was estimated using a modified trinitrobenzene sulfonic acid
(TNBSA) assay and methacryloyl glycylglycine hydrazide
(MAGGH) standards (see Supporting Information for details
regarding synthesis of MAGGH standards). For each pHPMA
hydrazide content analysis, stock solutions of 0.1% w/v TNBSA
and ∼2 × 10-4 M MAGGH were prepared in 0.1 M NaHCO3,
pH 8.5. The MAGGH solution was further diluted with 0.1 M
NaHCO3 to generate standards in the range ∼(0.3-2) × 10-4
M. Next, hydrazide derivatized pHPMA solutions in 0.1 M
NaHCO3 were prepared at concentrations of 0.2-0.4 mg/mL
for samples prior to cross-linker reactions and 1-2 mg/mL for
cross-linker reacted samples. TNBSA stock solution (0.5 mL)
was combined with MAGGH standard or pHPMA solutions (1.0
mL) and incubated for 1.5 h at 37 °C. After incubation, sample
absorbance was measured at 505 nm and hydrazide content was
estimated from the MAGGH standard curve.
Reduced TAMRA solution (3 mL,∼1.1 equiv,∼5.9 µmol)
was added separately to pHPMA cross-linker conjugates 10,
11, and 12 (20.0 mg in 2 mL sodium phosphate, 0.05 M NaCl,
2.5 mM EDTA, pH 7.0, ∼1 equiv, ∼5.3 µmol). Reaction
mixtures were degassed, purged with nitrogen, capped, and
stirred at room temperature in the dark for 3 h. The coupling
procedure was terminated by separation of unreacted TAMRA
thiol after passage through an SEC column (Sephadex, G-25, 2
× 20 cm, H2O mobile phase). The polymer conjugate fraction
was collected as the first red band eluting from the column and
lyophilized. Typical yields of TAMRA conjugated pHPMA:
15.3-19.5 mg, 76-96%. TAMRA conjugation efficiency to
maleimide groups was determined by absorbance measurements
at 554 nm using ε ) 6.4 × 104 M-1 cm-1. TAMRA content
(µmol/g polymer): 13 ) 105.2, 14 ) 47.8, 15 ) 25.9.
Determination of Decoupling Hydrolysis Rates for Cross-
Linkers on Conjugates 13-15. Release of free TAMRA dye
from pHPMA was monitored at pH 5.0 (0.1 M sodium acetate
buffer) and pH 7.4 (0.1 M sodium phosphate buffer). TAMRA-
conjugated pHPMA samples (1.5-2.5 mg) were dissolved in
the appropriate buffer (8.5 mL) and incubated at 37 °C in the
dark while stirring. At various time points, 750 µL of polymer
solution was withdrawn and free TAMRA was separated from
polymer using a Sephadex G-25 SEC column (1 × 13 cm, H2O
mobile phase). The two resulting bands were collected and water
was evaporated using a speed-vac. The samples were then
dissolved in 500 µL water and the percentage of free TAMRA
was determined by the ratio of absorbance at 554 nm that of
the two fractions. For pHPMA conjugate 15, TAMRA release
was also determined after acidification (pH 3.0) of a sample
incubated for 72 h at pH 7.4 (0.1 M phosphate buffer). All
release experiments comprised 4 individual samples and data
was analyzed using Microsoft Excel.
Reaction of Hydrazide-Derivatized pHPMA with Cross-
Linkers (10, 11, 12). Hydrazide-derivatized pHPMA (8, 0.20 g,
1.0 equiv, 52 µmol hydrazide) was dissolved in 50/50 methylene
chloride/methanol (2 mL), and 100 mg Na2SO4 was added.
Cross-linker solutions (PMCA, 4, 46.0 mg; BMCA, 5, 48.0 mg;
APM, 9, 34.0 mg, 3.1 equiv, 160 µmol) were prepared in 50/
50 methylene chloride/methanol (2 mL) and then added drop-
wise to the pHPMA solution at room temperature. After
complete addition, acetic acid (5 µL of 34% v/v solution in
methylene chloride, 0.6 equiv, 30 µmol) was added, and the
reaction was stirred under nitrogen at room temperature for 24 h.
The reaction was terminated by precipitation of pHPMA product
into ether. Product was further purified by size exclusion
chromatography (Sephadex LH-20, 1 × 15 cm, methanol mobile
phase). Typical yields: pHPMA-PMCA 10, 116 mg 59%;
pHPMA-BMCA 11, 122 mg, 61%; pHPMA-APM 12, 126 mg,
63%; all as white powders. Hydrazide content postconjugation
was determined using a TNBSA assay, as described above.
Cross-linker content was determined by optical absorbance
measurement at λmax and using the molar extinction coefficients
of corresponding ethyl carbazate cross-linker hydrazone deriva-
tives: 10, 297 nm, ε ) 5.5 × 102 M-1 cm-1; 11, 299 nm, ε )
5.7 × 102 M-1 cm-1; 12, 288 nm, ε ) 1.9 × 104 M-1 cm-1
(see Supporting Information). Polymer cross-linker-bearing
RESULTS AND DISCUSSION
Cross-Linker Design and Synthesis. Although several
heterobifunctional cross-linkers are commercially available that
form hydrazone bonds and also contain thiol-coupling reactivity,
most are synthesized to contain hydrazide groups that react with
synthetic ketone-containing drugs such as doxorubicin or
oxidized sugar moieties present on biomolecules. We know of
only one commercially available cross-linker that contains both
ketone and maleimide functionality (APM), since hydrazide
groups are not a common target for reaction with biomolecules
or synthetic drugs. However, synthetic polymers, particles,
surfaces, and so forth can easily be prepared with hydrazide
functionality for reversible conjugation of thiolated molecules
using the cross-linkers described in this work.
1
conjugates were analyzed by H NMR to confirm maleimide
presence on the polymer backbone.
Coupling Thiolated TAMRA to Maleimide Activated
pHPMA (13, 14, 15). Dimeric TAMRA disulfide 6 was reduced
using TCEP-immobilized gel immediately prior to reaction with
cross-linker-derivatized pHPMA. TAMRA disulfide dimer
(17.46 mg, 0.036 mmol, 1 equiv) was dissolved in 1 mL DMSO
and added dropwise into an immobilized TCEP gel suspension
(5 mL gel, ∼1.1 equiv, ∼0.04 mmol TCEP) in 12 mL phosphate
buffer (0.1 M sodium phosphate, 0.05 M NaCl, 2.5 mM EDTA,
pH 7.0). The reduction reaction was allowed to proceed for
Synthesis of the aldehyde or methyl ketone SMCC-based
heterobifunctional cross-linkers is shown in Scheme 1. The

Hydrazone and Thioether-Forming Cross-Linkers
Bioconjugate Chem., Vol. 21, No. 10, 2010 1783
Table 1. Summary of pHPMA-Cross-Linker Coupling Reactionsa
cross-linker contentb
remaining hydrazidec
polymer conjugate
cross-linker
(µmol/g)
conversionb (%)
(µmol/g)
conversionc (%)
13
14
15
PMCA
BMCA
APM
373.0 ( 21.2
248.9 ( 8.1
166.7 ( 14.5
141.8 ( 8.1
94.6 ( 3.1
69.8 ( 6.1
19.5 ( 0.1
18.6 ( 0.1
90.9 ( 0.1
92.6 ( 4.3
92.9 ( 5.6
62.0 ( 1.9
a n ) 4 for all measurements. b Determined by cross-linker optical absorbance. c Determined by TNBSA assay.
cyclohexyl SMCC core was chosen as it is known to produce
a stable maleimide functional group. Maleimide groups are
subject to degradation by hydrolysis and the increased hydro-
phobicity and lack of a conjugated aromatic system in the
cyclohexyl spacer may reduce the hydrolysis rate (2, 33). Early
attempts to synthesize these cross-linkers involved direct
modification of commercially available SMCC, but synthesis
of SMCC precursors in-house allowed inexpensive scale-up.
Synthesis of the corresponding alcohols of SMCC was per-
formed in a one-pot reaction after activation of carboxylic acid
with NHS using well-known carbodiimide chemistry (2). Direct
coupling of 4-amino-2-butanol and 3-amino-1-propanol to 4-(N-
maleimidomethyl)cyclohexane carboxylic acid (1) following
DCC activation without NHS led to a mixture of products,
presumably due to addition of alcohol groups to the highly
reactive DCC-activated carboxylic acid. The choice of meth-
ylene chloride as the solvent aided in product purification due
to the limited solubility of NHS byproduct, which precipitated
from solution and was easily removed by filtration after
concentration and cooling the reaction mixture. Residual NHS
was removed by aqueous extraction for cross-linker precursor
3, but this purification strategy for 2 resulted in reduced yields
due to higher water solubility. Chromatographic purification of
2 resulted in markedly increased yields.
Figure 2. 1H NMR spectra of pHPMA conjugates showing the pHPMA
amide peak (broad) and cross-linker maleimide peak (sharp). Spectra
recorded at 400 MHz, 25 °C in DMSO-d6. (a) pHPMA-APM conjugate
12, (b) pHPMA-BMCA conjugate 11, (c) pHPMA-PMCA conjugate
10, (d) pHPMA-hydrazide 8.
conjugates as determined directly by cross-linker absorbance
and indirectly by determination of remaining hydrazide groups
using TNBSA measurements. Optical absorption measurements
of the pHPMA-PMCA cross-linker product 10 showed the
highest coupling efficiency, indicative of the higher reactivity
of aldehyde functional groups. Although more than stoichio-
metric conversion was observed for the aldehyde cross-linker,
the excess coupling was small compared to the total polymer
units in the pHPMA carrier, indicating that polymer units lacking
the hydrazide functionality were generally unreactive toward
the aldehyde. The amount of hydrazide containing monomer
units in pHPMA was ∼4 mol %; thus, the ∼140% coupling of
the aldehyde cross-linker observed under these reaction condi-
tions corresponds to modification of only ∼2% of the polymer
side chains in an unintended fashion. One possible explanation
for the higher coupling rate observed for the aldehyde cross-
linker is the formation of acetals with the alcohol groups
contained in pHPMA side chains or mixed acetals formed by
reaction with pHPMA and MeOH, as the reaction conditions
used for hydrazone formation are similar to those used for acetal
formation (i.e., acid catalyst and dehydrating agent). The
methylketone group of 5 reacted with pHPMA hydrazide groups
in a stoichiometric fashion, with absorbance and remaining
hydrazide content in good agreement for conjugate 11. This
demonstrates that the less reactive methyl ketone functionality
resulted in more readily controlled conjugation. The resonance
stabilized APM cross-linker 9 was less reactive and did not
stoichiometrically react with hydrazide-containing pHPMA 8
to produce conjugate 12. Only 60-70% conjugation was
observed in the cross-linker-derivatized product 12, with cross-
linker absorbance and remaining hydrazide measurement in good
agreement. A similar result was observed in the analogous
reaction of APM with ethyl carbazate (see Supporting Informa-
tion). 1H NMR analysis of the cross-linker polymer conjugates
10-12 also revealed a convenient analytical signal to confirm
conjugation due to the sharp maleimide peak at 7.0-7.2 ppm,
shown in Figure 2.
Oxidation of alcohol groups in cross-linker precursors 2 and
3 was readily achieved using the mild oxidation conditions of
the Swern procedure to yield the corresponding aldehyde- and
methylketone-containing reactive cross-linkers. No side reactions
were observed with other functional groups in the molecule.
Other attempts to oxidize the alcohol functional groups using
pyridinium chlorochromate resulted in oxidation of alcohol
groups, but removal of chromium salt byproduct was difficult,
resulting in low yields (data not shown).
Cross-Linker Coupling to pHPMA. Synthesis of hydrazide-
derivatized pHPMA (8) was easily achieved by reaction of active
p-nitrophenol esters on pendant side chains with hydrazine
monohydrate as previously described (19, 20). This construct
has been previously utilized to couple doxorubicin via a
hydrazone bond to allow pH-dependent release (19, 20). The
number of reactive sites in pHPMA was controlled by monomer
feed ratios in the free radical-initiated polymerization reaction
to produce pHPMA, with reactive sites kept fairly low so as to
not alter the polymer solubility after cross-linker conjugation.
Formation of hydrazone bonds has been reported for doxorubicin
conjugates with pHPMA and poly(ethylene glycol) in polar
protic conditions with methanol used as the solvent and acetic
acid as the catalyst (19, 20). Since the hydrophobic cross-linkers
used in this study were only minimally soluble in methanol,
and the more hydrophilic pHPMA was only minimally soluble
in solvents ideal for cross-linker solvation, a mixed solvent
system was used. The best methanol-based solvent system for
optimal cross-linker/polymer solubility was found to be 50/50
methanol/methylene chloride. PHPMA-cross-linker reactions
showing conjugation efficiency are summarized in Table 1. With
the reactive sites on the polymer controlled by monomer feed
ratio, an excess of cross-linker was used to maximize conjuga-
tion to the polymer.
For conjugates 10 and 11, the maleimide peak appeared in
1
an unobscured region of the H NMR spectrum allowing easy
Reaction of cross-linkers PMCA (4) and BMCA (5) resulted
in complete conversion of hydrazide groups to hydrazone
detection. Additionally, formation of conjugate 10 was con-
firmed by the disappearance of the aldehyde peak present in

1784 Bioconjugate Chem., Vol. 21, No. 10, 2010
Christie et al.
Scheme 2. Synthesis of pHPMA Conjugates
Figure 3. Hydrolysis rates for pHPMA-cross-linker-TAMRA conju-
gates. Graphs a-c represent hydrolysis at pH 7.4 (dashed) and pH 5.0
(solid): (a) pHPMA-PMCA-TAMRA conjugate 13, (b) pHPMA-
BMCA-TAMRA conjugate 14, (c) pHPMA-APM-TAMRA conjugate
15. (d) Hydrolysis of pHPMA-APM-TAMRA conjugate 15 upon
acidification following 75 h incubation at pH 7.4. Each data point
represents the average of 4 samples ( the standard deviation.
may increase with longer reaction times until hydrazone
degradation and release from the polymer competes with thiol
conjugation.
unreacted cross-linker 4 at 9.6 ppm. Although the maleimide
signal overlapped with the polymer backbone signal for
conjugate 12 containing APM, it was still visible protruding
above the broad amide peak. Additionally, the ability of each
cross-linker to form hydrazone linkages was modeled with the
hydrazide-containing small molecule ethyl carbazate, with
confirmation of hydrazone formation more analytically straight-
forward than with a small percentage of reactive sites in a
polymer conjugate (see Supporting Information). For PMCA
and BMCA, rapid, stoichiometric conversion to hydrazone
product was obtained, with only a simple purification procedure
needed to isolate pure hydrazone product. However, reaction
of APM with ethyl carbazate resulted in formation of several
products, and purification required a separation procedure for
isolation, which resulted in a lower overall yield.
Attachment of Thiolated TAMRA to pHPMA and
Subsequent Release. Synthesis of the pHPMA cross-linker
TAMRA conjugates 13-15 is shown in Scheme 2. Thiol-
modified TAMRA (6) was used as a model cargo compound to
react with maleimide groups resulting from cross-linker con-
jugation to pHPMA. This model system was convenient, as
TAMRA is fairly water-soluble, readily detected by simple
absorbance measurements, and released dye is sufficiently
smaller in size to allow rapid separation from the polymer-bound
fraction using size exclusion chromatography. Reduction of the
TAMRA disulfide 6 was performed with TCEP-immobilized
gel, allowing easy separation of reducing agent. Free TCEP,
DTT, or combinations of both resulted in lower conjugation of
TAMRA to the polymer (data not shown). Although free TCEP
is generally believed to be unreactive toward maleimide
functional groups, similar observations regarding interference
with maleimide coupling have been reported (34, 35). Reaction
times of TAMRA with cross-linker-derivatized pHPMA were
kept short (3 h) in this study, as degradation rates of the
hydrazones were unknown. TAMRA conjugation was not
efficient under these conditions, with couplings relative to cross-
linker content of ∼20% observed for all pHPMA-cross-linker
conjugates. Maleimide coupling efficiency for all cross-linkers
Release of conjugated TAMRA from the polymer backbone
at physiologically relevant temperature and pH [pH 7.4 (circula-
tion) and pH 5.0 (lysosomes), 37 °C] was influenced by cross-
linker structure as shown in Figure 3a-c. The conditions used
to observe hydrazone degradation are not physiological, only a
simple approximation, as the addition of other physiologically
relevant components such as serum interfere with the ability to
separate released TAMRA dye from the macromolecular
component of the mixture by size exclusion chromatography:
TAMRA can associate with high molecular weight serum
components in more complex milieu via nonspecific interactions.
Ideally, for systemic administration of a drug delivery system,
the hydrolytic stability of the hydrazone bond between the carrier
and therapeutic agent should be stable at systemic pH (∼7.4)
for the same period of time that it takes for the carrier to reach
its site of activity. However, if the carrier is rapidly cleared
from circulation, and the therapeutic target is also the site of
rapid accumulation (i.e., kidney or liver tissue), the hydrazone
does not necessarily need to exhibit extended stability at
systemic pH. For carriers targeting cancer tissue by the EPR
effect, the hydrazone used for preparation of the conjugate
should exhibit minimal hydrolysis at pH 7.4, as accumulation
into cancer tissue by the EPR effect requires a carrier with
extended circulation (36). In this regard, the stability of the
hydrazone used to couple the therapeutic agent to the carrier
depends on the properties of the carrier used.
Polymer conjugates 13 and 14 containing aliphatic acyl
hydrazone bonds were relatively unstable at physiological pH,
with more than 30% of conjugated TAMRA released after 5 h.
Release for conjugate 13 prepared with the aldehyde-containing
PMCA cross-linker showed no difference between pH 7.4 and
5.0, with a maximum of 40-60% release observed after only a
few hours, with little increase after 24 h. Hydrolysis of conjugate
13 rapidly plateaued at a value less than that for complete
hydrolysis, which may be attributed to the reversible nature of
the hydrazone bond and the closed system used to study the

Hydrazone and Thioether-Forming Cross-Linkers
Bioconjugate Chem., Vol. 21, No. 10, 2010 1785
release profile which could result in the highly reactive aldehyde-
containing PMCA cross-linker reaching equilibrium between
the aldehyde and hydrazone forms at this concentration and
temperature. Alternatively, the additional PMCA attached to the
polymer (∼140% conjugation to pHPMA was observed) could
be interfering with the observation of hydrazone bond hydroly-
sis, assuming that the additional conjugation resulted in a
hydrolytically unstable bond (i.e., acetal). Polymer conjugate
14 formed with the BMCA cross-linker showed a marked
difference in hydrazone degradation at early time points, with
more than 75% of cargo released after only 2 h at pH 5.0 while
only 25% of TAMRA was released at pH 7.4 in the same time
frame. This difference in initial pH stability for BMCA
containing conjugate 14 compared to the hydrazone formed with
PMCA in conjugate 13 is likely due to increased electron
donation ability of a methyl group relative to a hydrogen, which
would facilitate protonation of the imine nitrogen but also
sterically hinder the imine carbon electrophile toward nucleo-
philic attack by water. This property may be useful for targeting
or delivery devices that can efficiently reach a target site of
lower pH without prolonged residence in the off-target environ-
ment, with rapid release of cargo at the site of lower pH.
The desired difference in stability of polymer conjugates at
pH 7.4 and 5.0 was most pronounced for the pHPMA-APM
containing conjugate 15. Polymer conjugate 15 containing the
resonance-stabilized hydrazone was most stable at pH 7.4 with
less than 30% of degradation occurring after 24 h, while
degradation at pH 5.0 steadily increased during the same time
period (Figure 3c). Increased degradation rate of conjugate 15
upon acidification after extended incubation at pH 7.4 is shown
in Figure 3d; hydrolysis increased immediately upon lowering
the pH. This result shows that polymer-bound payload is likely
to be released within target cells in vivo following extended
circulation, cellular internalization, and trafficking to acidic
subcellular organelles (lysosomes).
Hydrazones investigated in this work showed the following
trend for increased difference in stability at pH 7.4 and 5.0 based
on the functional groups attached to the imine carbon: phenyl,
CH3 > alkyl, CH3 > alkyl, H. A similar trend has also been
observed for other hydrazone systems recently reviewed else-
where (15). This trend is directly related to cross-linker reactivity
toward polymer hydrazide groups, with highly reactive carbonyl
groups forming hydrazone bonds with decreased pH-dependent
hydrolysis. The acyl hydrazide functionality utilized on the pHPMA
component of the hydrazone forming system was not varied, but
the acyl hydrazide was convenient to prepare by simple aminolysis
of active esters by hydrazine and also likely aided in the stability
of the resulting hydrazones at pH 7.4. A recent detailed study of
hydrazone stability showed that an acyl hydrazone had a first-order
rate constant for hydrolysis a few hundred-fold lower relative to
an alkyl hydrazone at pH 7.0 (37).
The pH-dependent release mechanism for hydrazone hy-
drolysis involves (1) protonation of the imine nitrogen, (2)
nucleophilic attack of a water molecule at the imine carbon,
(3) formation of a tetrahedral carbinolamine intermediate, and
(4) decomposition of the carbinolamine and cleavage of the
C-N bond (15, 17, 37, 38). The rate at which this hydrolysis
occurs is influenced by substituents adjacent to the hydrazone.
In general, cross-linker electron-donating groups can facilitate
pH-dependent hydrolysis by encouraging protonation of the
imine nitrogen, which activates the imine carbon toward
nucleophilic attack from water, as the nitrogen can then accept
a pair of electrons from the imine bond to form the tetrahedral
carbinolamine intermediate. Electron withdrawing groups de-
crease pH sensitivity by decreasing electron density of the imine
carbon making it more susceptible to nucleophilic attack by
water (15, 37, 39, 40). Aromatic groups in π-bond conjugation
with hydrazones have also been shown to increase their stability,
due to resonance stabilization, as was observed in our results
(41). The desired stability for controlled pH-dependent hydroly-
sis is dependent on a delicate balance of groups adjacent to the
carbonyl and hydrazide reaction partners used to form the
hydrazone.
Modification of the carbonyl portion of cross-linkers described
here may result in increased stability at pH 7.4 and hydrolysis
rate at pH 5.0, while retaining excellent hydrazide reactivity.
For example, glyoxylyl aldehydes prepared from N-terminal
serine-modified amino acids have been recently used to form
pH-sensitive hydrazones with acyl hydrazides (42). These
findings suggest that further optimization of the SMCC-based
cross-linker chemistries described here could be achieved by
incorporation of different chemistries on carbon atoms adjacent
to the reactive ketone functionality.
CONCLUSIONS
This work demonstrates the feasibility of forming thiol-
reactive pHPMA conjugates with varying coupling and decou-
pling stability controlled by different chemical substitution
patterns in analogous heterobifunctional cross-linker chemistries.
Currently, the APM cross-linker is likely the best choice for
long-circulation in vivo application due to the high stability at
pH 7.4, with desired increased degradation occurring in acidic
conditions analogous to the lysosome environment within cells.
However, although APM shows the desired release properties
for a long circulating conjugate, this cross-linker has the inherent
disadvantage of lower hydrazide reactivity. BMCA is attractive
for use in rapid release of cargo at a target site where extended
stability is not desired (i.e., conjugates that rapidly accumulate
at their site of activity or those that need to be rapidly cleared
after use, i.e., gadolinium imaging agents). Alternatively,
modifications of chemistry contained in the hydrazide part of
synthetic constructs could result in conjugates with more stable
hydrazone linkages formed with PMCA and BMCA.
The utility of these thiol-reactive cross-linkers may be further
extended to produce nondegradable conjugates. In this case,
addition of a hydrazone reducing agent such as sodium
cyanoborohydride would result in a stable amide bond. Thus,
the excellent hydrazide and maleimide reactivity of PMCA and
BMCA would be attractive. Additionally, hydrazide reactivity
is different from amine reactivity due to the lower pKa of
hydrazides. Nonreversible conjugation of the cross-linkers
described here to hydrazides in the presence of primary amines
could be achieved by control of reaction pH and the use of a
hydrazone reducing agent.
In summary, the thiol-reactive cross-linkers described in this
work could prove to be useful new tools for preparation of
bioconjugates with specific coupling/decoupling properties.
Hydrazide functionality is readily incorporated into synthetic
systems, while thiol functionality is readily contained in
biological components targeted for conjugation and subsequent
release. These cross-linkers could be used for conjugation and
subsequent triggered release as described here, for nonreversible
conjugation by simply changing the reaction conditions, or serve
as a starting point for design of more advanced cross-linkers
with specific performance features.
ACKNOWLEDGMENT
We thank Andrew Higginbotham and Dr. Mark Kerr for
helpful discussion regarding organic procedures. Partial support
from NIH grant EB000894 is gratefully acknowledged.
Supporting Information Available: Experimental details
regarding pHPMA synthesis, synthesis of methacryloyl gly-
cylglycine TNBSA assay standards, reaction of cross-linkers

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