Direct peptide bioconjugation/PEGylation at tyrosine with linear and branched polymeric diazonium salts

Article abstract of DOI:10.1021/ja211855q

Direct polymer conjugation at peptide tyrosine residues is described. In this study Tyr residues of both leucine enkephalin and salmon calcitonin (sCT) were targeted using appropriate diazonium salt-terminated linear monomethoxy poly(ethylene glycol)s (mPEGs) and poly(mPEG) methacrylate prepared by atom transfer radical polymerization. Judicious choice of the reaction conditions-pH, stoichiometry, and chemical structure of diazonium salt-led to a high degree of site-specificity in the conjugation reaction, even in the presence of competitive peptide amino acid targets such as histidine, lysines, and N-terminal amine. In vitro studies showed that conjugation of mPEG ₂₀₀₀ to sCT did not affect the peptide's ability to increase intracellular cAMP induced in T47D human breast cancer cells bearing sCT receptors. Preliminary in vivo investigation showed preserved ability to reduce [Ca²⁺] plasma levels by mPEG₂₀₀₀-sCT conjugate in rat animal models.

Full text of DOI:10.1021/ja211855q

Article
pubs.acs.org/JACS
Direct Peptide Bioconjugation/PEGylation at Tyrosine with Linear
and Branched Polymeric Diazonium Salts
Mathew W. Jones,† Giuseppe Mantovani,*,‡ Claudia A. Blindauer,† Sinead M. Ryan,§ Xuexuan Wang,§
David J. Brayden,§ and David M. Haddleton*,†
†Department of Chemistry, University of Warwick, Coventry CV4 7AL, U.K.
‡School of Pharmacy, University of Nottingham, Nottingham NG7 2RD, U.K.
§UCD School of Agriculture, Food Science and Veterinary Medicine and UCD Conway Institute, University College Dublin, Belfield,
Dublin 4, Ireland
S
* Supporting Information
ABSTRACT: Direct polymer conjugation at peptide tyrosine
residues is described. In this study Tyr residues of both leucine
enkephalin and salmon calcitonin (sCT) were targeted using
appropriate diazonium salt-terminated linear monomethoxy
poly(ethylene glycol)s (mPEGs) and poly(mPEG) methacry-
late prepared by atom transfer radical polymerization.
Judicious choice of the reaction conditionspH, stoichiom-
etry, and chemical structure of diazonium saltled to a high
degree of site-specificity in the conjugation reaction, even in
the presence of competitive peptide amino acid targets such as histidine, lysines, and N-terminal amine. In vitro studies showed
that conjugation of mPEG2000 to sCT did not affect the peptide’s ability to increase intracellular cAMP induced in T47D human
breast cancer cells bearing sCT receptors. Preliminary in vivo investigation showed preserved ability to reduce [Ca2+] plasma
levels by mPEG2000-sCT conjugate in rat animal models.
onjugation of poly(ethylene glycol), or PEGylation, of
biologically relevant peptide and protein drugs is a
the many components responsible for the overall therapeutic
efficacy of these biohybrid materials, the development of site-
specific conjugation strategies which are able to circumvent this
problem is now considered a priority in PEGylation and
conjugation science. To this end, in recent years considerable
research effort has been spent in identifying and developing
PEGylation strategies alternative and complementary to those
being used clinically. One breakthrough was the work by
Brocchini and co-workers, who first described the site-specific
PEGylation of disulfide bridges of a number of proteins and
peptides using novel α,β-unsaturated sulfone derivatives.7,8
The growing demand for the synthesis of well-defined
bioconjugates via site-specific coupling has increased interest in
tyrosine as a target for protein modification. During the past
decade, a number of reports have emerged whereby tyrosine
C
strategy that prolongs therapeutic half-life and reduces
immunogenicity.1−4 A number of PEG−protein therapeutics
are currently prescribed for the treatment of a range of
prevalent conditions, including hepatitis C, chemotherapy-
associated neutropenia, and leukemia, and many more are
currently in clinical and preclinical development.2 PEGylation
generally occurs by targeting nucleophilic amino acids, such as
the primary amine at N-termini in hystidine and lysine and
thiols at free cysteine residues with end-functional PEGs.5,6 In
order for PEGylated proteins and peptides to retain an
acceptable proportion of their original activity, conjugation
must not occur at, or in the immediate vicinity of, crucial points
in the polypeptide sequence. These would include the binding
sites of hormones and the catalytic sites of enzymes. It is
therefore key that polymer conjugation occurs preferentially, if
not exclusively, at specific amino acids known for not being part
of (poly)peptide active sites. PEGylated protein therapeutics
consisting of a mixture of positional isomers (PEGamers)i.e.,
PEGylated IFN alfa-2a and 2b, currently prescribed as first-line
treatment for hepatitis C and some forms of melanomahave
received in the past regulatory approval. This is due to largely
improved pharmacokinetic profiles and clinical efficacy
compared to the native protein therapeutics. However, upon
polymer conjugation, some of these therapeutics retain only a
fraction of the activity of the original protein (as low as 7% for
PEGylated IFN-alfa-2a). Although in vitro activity is only one of
́
residues have indeed been targeted. Gonzalez et al. reported the
selective mono-iodination of tyrosine residues using iodinating
reagent IPy2BF4,9 and this has been exploited for the
introduction of further functionality using Suzuki−Miyaura
coupling.10 Recently, Barbas and co-workers have reported an
elegant ene-type conjugation of cyclic azadicarboxylate small
molecules to Tyr residues of model proteins.11
Francis and co-workers have reported a number of efficient
strategies whereby tyrosine residues were targeted via a three-
Received: December 27, 2011
Published: April 12, 2012
© 2012 American Chemical Society
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component Mannich-type reaction, as well as alkylation of the
residue via the hydroxide group using Trost allylation
conditions12 and coupling with diazonium reagents. This has
rejuvenated the field of diazonium coupling, and several reports
have described the modification of the tyrosine residues on
bacteriophage MS2 viral capsids13 and tobacco mosaic virus
(TMV).14 Wang et al. have since reported a study into the
functionalization of TMV involving diazonium coupling at
tyrosine, and this has been adapted to modify the tyrosine
residues of M13 bacteriophage.15
Scheme 1. Synthesis of Poly(ethylene glycol)-Functional
Anilines as Precursors for Diazonium-Terminated
Conjugating PEGs
Of the three techniques developed by Francis, diazonium-
mediated targeting of tyrosine residues appeared to us to be the
most attractive for direct polymer conjugation of (poly)-
peptides. The three-component Mannich-type approach is
generally a slow reaction, which makes it less attractive for the
coupling of macromolecules, a process that in itself is slower
compared to coupling with small molecules. Moreover, this
generally requires a large excess of coupling reagents,16−18
which again is less than ideal for polymer conjugation of
proteins and peptides. Pd-catalyzed O-alkylation by Trost
allylation is an efficient process, although the need for a
potentially toxic transition metal catalyst (albeit at very low
concentration) may make this less attractive for the direct
preparation of protein and peptide derivatives of pharmaceut-
ical interest. Conversely, diazonium couplings are metal-free,
generally fast processes that have the potential for being applied
to polymer conjugation targeting of medically relevant proteins
and peptides. In addition, they have been employed to generate
pharmaceutically relevant therapeutics, including the sulfona-
midochrysoidine prodrug Prontosil.
Despite these promising recent advances, to date, no report
has described direct polymer conjugation, including PEGylation,
to tyrosine residues of (poly)peptides. In this current work we
have developed a novel and potentially general route to
polymer−peptide biohybrid materials via preferential targeting
of peptide tyrosine residues with appropriate diazonium salt-
terminated polymers with various macromolecular architec-
tures.
Boc-protected 4-aminobenzoic acid (1) was reacted with an
excess of hexa(ethylene glycol) for 2a, or a monomethoxy
poly(ethylene glycol) (mPEG, 1.9 kDa) for 2b, in the presence
of EDC and DMAP. Removal of the Boc protecting group with
trifluoroacetic acid in dichloromethane afforded the desired
aniline-terminated diazonium precursors 3a and 3b featuring
electron-withdrawing ester moieties para to the aromatic amino
group. For 3b, overall yields were high (79% from mPEG2000
starting material), with extremely high purity confirmed by
MALDI-TOF analysis (see Supporting Information).
The range of polymers available for use in diazonium
conjugation of peptides was further expanded by preparing a
grafted PEG-based polymer with different macromolecular
architecture, p(mPEG MA), bearing the same α-functionality.
Conjugating polymers with this structure have emerged in
recent years as key macromolecular intermediates for the
preparation of polymer−(poly)peptide conjugates20−24 and
others.25−28 In this study, a relatively low molecular weight
comb polymer was prepared, in order to facilitate the
purification and characterization of the peptide conjugates.
Polymerization29−31 of mPEG(475) MA in the presence of
Boc-protected p-aminobenzoic ester initiator 4 and Cu(I)Br/
pyridinylimine ligand afforded the polymer intermediate 5,
Mn(NMR) = 8.0 kDa and PDI = 1.24. Removal of the Boc
protecting group afforded the final aniline-terminated poly-
(mPEG(475) MA) polymer (6, Scheme 2), which was used
later as a diazonium-terminated polymer precursor.
Conjugation to Tyrosine-Containing Peptides. Cou-
pling of diazonium derivatives to (poly)peptides is a technique
that has been exploited for more than 100 years.32 The high
reactivity of these substrates makes them ideal for protein
labeling, although one of the major drawbacks in using this
approach lies in the very poor selectivity often observed in
RESULTS AND DISCUSSION
■
Design of Polymer Diazonium Coupling Agents.
Francis and co-workers highlighted the importance of utilizing
highly reactive diazonium salts in order to achieve efficient Tyr
targeting.14 The reactivity of diazonium coupling agents can be
increased by introducing electron-withdrawing groups (EWGs)
in appropriate positions of their aromatic rings. In this regard,
Francis et al. reported a decrease in the efficiency of diazonium
decoration of the exterior protein shell of the TMV, from >90
to ∼30%, by replacing the diazonium salt of p-amino-
nitrobenzene with the one obtained from p-aminobenzamide,
i.e., replacement of a NO2 group with a less electron-
withdrawing C(O)NH2.
a
Scheme 2
Diazonium reagents are generally prepared by reacting
aromatic amines with in situ-generated HNO2 or nitrous
esters.19 In this work a range of aniline-terminated polymers
were generated and converted to the required diazonium
derivatives in situ during the peptide conjugation experiments.
An ester moiety was chosen as the para group as, in addition to
its electron-withdrawing properties, it allowed for facile linking
of the synthetic polymers employed with the aniline diazonium
precursor moieties. In particular, two aniline-terminated
poly(ethylene glycol)s, 3a and 3b, with FW = 401.5 Da and
Mn = 2.0 kDa, respectively, were prepared (Scheme 1). Briefly,
a
Reagents and conditions: (a) Cu(I)Br/N-(ethyl)-2-pyridylmethani-
mine, mPEG475MA, toluene, 50 °C; [(4)]:[mPEG475MA]:[Cu]:
[ligand] = 1:10:1:3; (b) CF3COOH, dichloromethane, 25 °C.
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these conjugation reactions.33−35 Diazonium derivatives have
been used for nonspecific targeting of a number of proteins for
a range of applications spanning the introduction of haptens
onto protein conjugates36 to the support of proteins on PVA
polymer matrices.37
Tryptophan has been reported to react efficiently with
diazonium salts such as 3-diazonium-1,2,4-triazole (3-DT) at
very low pH (<3), with reactivity decreasing with an increase in
pH.38 All of the other amino acid targets, N-nucleophiles such
as lysine, (poly)peptide N-termini, and arginine, C-nucleophiles
tyrosine and histidine, and S-nucleophile free cysteine, present
acid/base moieties which affect their reactivity toward
diazonium coupling reagents.33 A general trend for the N-
nucleophiles (Lys, Arg, and N-terminal amine) is that with
lower pH, higher proportions of the nucleophilic nitrogen
centers will be in their protonated form, which is nonreactive
toward electrophilic reagents.
from 2.3 to 9.2, which correlate with burial within proteins.41
Due to the high reactivity of tyrosinate/tyrosine residues
toward diazonium reagents, the coupling has been reported to
occur at pH as low as 4.37 In the same study, the authors
reported that over the 4.0−9.0 interval of pH investigated, Tyr
analogues had higher reactivity than the corresponding His
derivatives.
In this present work we aimed at reducing the pH at which
the conjugation reaction was conducted to a point at which all
nitrogen-containing amino acids would be in their protonated
and nonreactive form, while utilizing reactive diazonium
coupling agents able to react with Tyr residues under these
conditions. Tracey and Shuker have shown that even at the
higher pH of 8.8, EWG-activated diazonium salts have the
tendency to react with Tyr and His, even in the presence of
unprotected Trp, Lys, and a terminal amino group.42
For the first conjugation experiment, a model peptide was
chosen, [D-ala2]-leucine enkephalin, an opioid pentapeptide
containing a single tyrosine residue (Scheme 3). In addition to
Histidine and tyrosine follow the same trend,37 albeit for
different reasons. Protonation of the His basic nitrogen
removes electronic density from its aromatic ring, which can
reduceor even eliminate, depending on the pHits
nucleophilic character. Tyrosine residues can, in principle,
react with nucleophiles in both their neutral Tyr and
deprotonated tyrosinate forms. The latter is more reactive
than the former, and an increase in the rate of diazo coupling
with an increase in pH has been observed. For naphthols it has
been reported that the reactivity of the naphtholate ion is
appoximately 1010 times higher than that of the undissociated
naphthol, implying that it always reacts with diazonium salts in
its TyrO− form unless at pH < 1−2, where the rate of the
coupling reaction makes it almost negligible. Selective
modification of protein Tyr residues so far has been restricted
either to proteins lacking exposed competitive conjugation sites
(i.e., Lys, Cys, and His)14 or to less reactive diazonium salts
bearing a ligand, uridine 2′(3′)-phosphate, able to direct them in
the immediate vicinity of a specific Tyr residue in an enzyme
binding site (Tyr73 of ribonuclease A).39
Herein we reasoned that preferential (or ideally exclusive)
selectivity in targeting tyrosine residues could have been
obtained by utilizing appropriate diazonium reagents, by
judiciously choosing the experimental conditions at which the
coupling reactions are performed. The pKa of protonizable
amino acids can vary dramatically, depending on where these
fragments are within a (poly)peptide sequence. Remarkable
differences from expected values are generally observed in non-
solvent-exposed amino acids, in which the presence of
hydrophobic/hydrophilic local environments in addition to
the presence of hydrogen bonds can alter remarkably their
acid/basic behavior.
Scheme 3. Conjugation of Diazo-Functional Model PEG 3a
to Tyr1 Residue of [D-ala2]-Leucine Enkephalin
the substituted phenol, Tyr1 presents an N-terminal amino
group that could represent a competitive attachment site for
polymer conjugation. Hexa(ethylene glycol) precursor 3a was
used as the conjugating agent, due to its intrinsically
monodisperse nature and small size, which facilitated the
study of the coupling reaction and the characterization of the
final adduct(s). The conjugation reaction was carried out at pH
4.5, where all the amino groups were expected to be mainly in
their protonated and nonreactive form. The process was
1
monitored by H NMR and was found to be extremely neat,
with rapid and quantitative synthesis of diazonium derivatives
from the parent anilines (see Supporting Information). The
required diazonium salts were generated in situ by sequential
addition of trifluoroacetic acid and NaNO2 at 0 °C. After 30
min sulfamic acid was added, so as to destroy excess nitrous
acid generated in the previous step. The pH was adjusted to 4.5
with 500 mM acetate buffer, and [D-ala2]-leucine enkephalin
was finally added. The reaction was performed at 4 °C for 24 h,
during which time the reaction solution gradually turned from
colorless to orange due to formation of the azobenzene linker.
After quenching of the excess diazonium coupling agent with p-
cresol and purification by size exclusion chromatography,
MALDI-TOF-MS analysis revealed the presence of a single
product with the expected m/z = 982 Da, [M+H]+, for the
monoadduct 3a-enkeph (see Supporting Information). It is
important to note that 3a-enkeph monoadduct was the only
product observed, even though a 10:1 excess of diazonium
conjugating agent was employed in the coupling reaction.
Following this successful modification of [D-ala2]-leucine
enkephalin, the versatility of the conjugation strategy was tested
with a more complex peptide, salmon calcitonin (sCT). sCT is
a 32-amino-acid hormone currently prescribed for the treat-
Very broadly speaking, protein N-nucleophiles all feature a
relatively high pKa in aqueous environments, where the
guanidine fragment of Arg has pKa ≈ 12, ε-amino groups of
Lys residues pKa ≈ 10, and N-terminal amino acid residues pKa
≈ 8.40 In their protonated form these fragments are not reactive
toward electrophilic agents (i.e., conjugating polymers).
Selective PEGylation of (poly)peptide N-terminal amino acids
can be achieved by reductive amination with α-aldehyde PEGs
at pH 5−6, where a higher proprortion of more basic protein
reactive sites, i.e., ε-amino groups of Lys residues, are all in their
nonreactive protonated form.
The pKa values of C-nucleophiles can be rather diverse, Tyr
typically having pKa ≈ 10 and His pKa ≈ 6, although for the
latter an extremely wide range of values have been observed,
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ment of bone-related disorders including osteoporosis, Paget’s
disease, and hypercalcemia. sCT’s hypocalcemic propertities
have been ascribed to a number of concurrent phenomena,
including its ability to inhibit the activity of osteoclasts in bones
and to promote Ca2+ renal excretion.43 Most importantly for
the scope of this study, sCT is a very well characterized peptide
which presents only one tyrosine residue (Tyr22) for polymer
conjugation, in addition to a number of other potential
conjugating sites, including Cys1 (N-terminal amine), Lys11
and Lys18, His17, and Arg24, which make sCT an ideal
substrate for testing the selectivity of the diazonium
conjugation process. Moreover, Tyr22 is included in a short
portion of the primary structure of sCTfrom Leu19 to
Tyr22that can be modified, or even completely removed,
with no loss of bioactivity.44 This makes Tyr22 an ideal
targeting site, as its modification is expected to have minimal
impact on the bioactivity of sCT.
ing to the loss of 17 mass units from the conjugates, commonly
observed in diazonium coupling with proteins and peptides.14
Although the exact nature of such an elimination product has
never been established, one explanation for this could be a
formal elimination of ammonia, possibly via reaction of the
diazonium derivative with either the N-terminal amine or a Lys
residue to generate a monoalkyl-triazene, followed by its β-
elimination, a common decomposition route for these
intermediates45,46 (although other degradation processes are
also possible).47
The influence of the pH on the selectivity of the coupling
process was then investigated by performing the conjugation
reaction at pH 7.0 (500 mM). Upon incubation of sCT, an
immediate color change was observed, with the solution
turning dark purple. RP-HPLC analysis indicated that sCT had
been immediately consumed, and MALDI-TOF-MS analysis of
the reaction solution showed a mixture of products, ranging
from a small peak corresponding to native sCT to the
polypeptide conjugated to as many as five molecules of
hexa(ethylene glycol) diazonium (3a) (Figure 2). The presence
Initial investigations were again conducted using the
hexa(ethylene glycol) precursor 3a under conditions analogous
to those previously optimized for [D-ala2]-leucine enkephalin.
Upon addition of sCT, no immediate color change was
observed, but over a period of time, the solution became
orange, due to the formation of the azo-linker (Scheme 4).
Scheme 4. Conjugation of mPEG Diazonium Precursors 3a
and 3b to Salmon Calcitonin (sCT)
Samples were taken periodically and analyzed by MALDI-TOF-
MS. Analysis of the reaction mixture after 8 h confirmed the
presence of unreacted sCT, along with a single peak
corresponding to the mass of 3a-sCT conjugate (Figure 1). It
should be noted that MALDI-TOF-MS spectrometry is
remarkably more sensitive to unmodified sCT than to 3a-
sCT, with the latter found to be the major product at the end of
the diazonium coupling reaction. Neither for the synthesis of
3a-enkeph nor for 3a-sCT was the peak observed correspond-
Figure 2. Hyperfine MALDI-TOF-MS spectrum of the diazonium
coupling reaction of sCT with hexa(ethylene glycol) diazonium (3a)
performed at pH 7.0, at [3a]0:[sCT]0 = 10:1.
of up to five molecules of conjugating molecules per sCT may
be ascribed to conjugation to Tyr22, His17, the N-terminal
amine, Lys11, and Lys18, while the pKa of Arg24 may be too
high to ensure efficient conjugation of this residue at pH 7.0.
The coupling reaction conducted at intermediate pH values,
namely 5.4 and 6.2, led to multiple conjugations similar to that
observed at pH 7.0, although the observed rate of conjugation
was significantly slower (see Supporting Information).
Conjugation with mPEG2000 (3b) at pH 4.5 was performed
at 2.5, 10, and 20 3b:sCT molar ratios in order to optimize the
rate and efficiency of the conjugation reaction. The reaction
was monitored by RP-HPLCequipped with a C18 column.
Conversion was found to be 21, 54, and 78%, respectively, after
56 h.
Pure 3b-sCT was isolated, with relatively low purification
yield (24%), after ion-exchange FPLC. MALDI-TOF-MS
analysis (Figure 3) showed a single distribution of peaks with
the observed mass in the range expected for 3b-sCT. No
conjugates with higher molecular mass were detected, neither
by RP-HPLC nor MALDI-TOF-MS, suggesting again that
under the conditions employed a 1:1 polymer:sCT was
obtained.
Finally, the same coupling reaction was performed utilizing
poly(mPEG(475) methacrylate) (6) as the diazonium
Figure 1. MALDI-TOF-MS spectrum of the diazonium-coupling
reaction of sCT with 3a at pH 4.5, at [sCT]0:[3a]0 = 1:20.
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Figure 3. (Top) MALDI-TOF-MS analysis of purified sCT-3b
(PEG2000). (Bottom) Detail of sodiated and protonated 3b-sCT. A
more detailed account is reported in the Supporting Information.
precursor. The coupling process proved to be as efficient as the
ones previously performed with the structurally simpler linear
polymers 3a and 3b, reaching 90% after 72 h at 4 °C. Again, the
conjugate was purified by ion-exchange FPLC, affording pure
conjugate. Biohybrid materials consisting of protein or peptide
conjugates with comb mPEG (meth)acrylates are extremely
difficult to characterize by MALDI-TOF mass spectrometry,48
the only known example reported to date being restricted to
conjugates with low Mn.49 The conjugate sCT-6 was
characterized by cleavage of the diazo linker followed by
analysis of the resulting modified peptide and its tryptic digests
(vide infra). All polymer conjugations were repeated several
times (3−5, depending on the conjugate), and the results were
found to be reproducible.
Characterization of sCT-Polymer Conjugates: Attach-
ment Site. Following observations that under specific
conditions sCT-polymer monoconjugates could be synthesized,
it was necessary to confirm the conjugation site. An
immediately evident feature of all the conjugation reactions is
the change of the solution from colorless to orange, indicative
of the formation of diazo adducts. In addition, the color seemed
to assume a more intense orange coloration with increasing pH.
UV−vis analysis of isolated 3b-sCT showed a maximum at λ =
471 nm, and upon gradually lowering the pH to 5.0, this peak
was observed to disappear, with the formation of a band at λ =
372 nm (Figure 4). For comparison purposes, the diazonium
salt of 3b was conjugated with water-soluble model histidine
(8) and tyrosine (9) mimics. UV−vis spectra of the isolated
conjugates 3b-8 prepared from a His mimic showed very little,
if any, dependence on the pH. Conversely, analysis of 3b-9
bearing a Tyr mimic showed a marked change with the pH,
with a band at approximately 500 nm with intensity that
increased with the pH, in line with the behavior observed for
the calcitonin conjugate 3b-sCT. This is in agreement with
literature data for other diaza-Tyr conjugates, in which the
appearance of a band at approximately 500 nm was ascribed to
Figure 4. UV−vis spectra of 3b-sCT and purified conjugates of 3b
with histidine (8) and tyrosine (9) mimics at various pH.
a chromophore formed by deprotonation of a diaza-phenolic
moiety.39 Further titration studies allowed us to estimate pKa ≈
11 for both the model diaza-Tyr conjugate 3b-9 and 3b-sCT,
again suggesting a strong involvement of Tyr22 in the
diazonium polymer conjugation reaction.
Further investigations involved the isotopic labeling of the
peptide conjugates using Na15NO2 for the preparation of
diazonium salts used for the coupling reactions. This allowed
for the investigation of the peptide’s amino acid attachment site
by NMR, and also provided a means to discriminate the
nitrogen atom introduced in the peptides from the nitrogens
already present, facilitating the characterization of the peptide
conjugates by mass spectrometry.
Conjugation reactions of 3a to [D-ala2]-leucine enkephalin,
and aniline-functional linear 3b and comb 6 polymers to sCT,
were repeated as described previously, this time using
Na15NO2. TOCSY NMR of 3a-[D-ala2]-leucine enkephalin
confirmed the conjugation of 3a to the model pentapeptide.
Although the presence of conformational isomers makes the
analysis of the conjugate rather complicated, spin systems
corresponding to a 1,3,4-trisubstituted benzene ring further
provide strong evidence of conjugation to Tyr1 (see Supporting
Information).
An additional potential advantage offered by peptide
conjugates bearing a diazo linker is the possibility for selective
cleavage in the presence of specific external reducing stimuli,
i.e., colon azoreductase enzymes. Several currently prescribed
therapeutics, i.e., Ipsalazide, Balsalazide, and Sulfasalazine, are
administered as prodrugs containing diazo linkers that can be
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cleaved by colon azoreductases, releasing the required active
drugs.50,51 Several drug delivery systems based on polymer-
containing diazo linkers have also been extensively inves-
tigated.51−53 Reversible PEGylation of peptides and colon
release are beyond the scope of the present work.
signals corresponding to unmodified sCT and traces of higher
mass species were also observed, which may be derived from
higher conjugated species. Qualitative analysis of MALDI-TOF-
MS spectra showed that smaller amounts of these minor
impurities were present when poly(mPEG(475) MA) (6) was
employed, suggesting that its branched structure may provide
better protection toward further polymer conjugation.
The polymers were cleaved from the conjugated peptides
using sodium dithionite. The reaction was optimized and
investigated using a model tyrosine conjugate prepared by
reacting the diazonium derivative obtained from mPEG aniline
3b and p-cresol. This was then used in conjugation reactions as
a means to quench the excess of diazonium polymers.
Conjugates with p-cresol were therefore produced at the end
of every conjugation run, and appeared to be ideal simple
substrates for the optimization of the reductive cleavage of
diaza linkages. Upon addition of 1 equiv of sodium dithionite
reducing agent, an immediate loss of the characteristic orange
color of the model conjugate was observed, indicating rapid and
quantitative cleavage of the azobenzene linker, yielding the
parent aniline functional polymers and 2-amino-4-methylphe-
nol, as confirmed by 1H NMR, UV−vis, and MALDI-TOF-MS
analysis (see Supporting Information).
In order to determine the modification site of the amino-
modified polypeptides, sCT-15NH2 obtained by reductive
cleavage of sCT-polymer conjugates was subjected to trypsin
digestion, which cleaved it at the C-terminus of the Lys and Arg
residues. Under these conditions unmodified sCT yields four
fragments, Cys1-Lys11, Leu12-Lys18, Leu19-Arg24, and
Thr25-Pro(NH2)32. This approach to determine the attach-
ment site in sCT-polymer conjugates was conducted by analogy
to that previously described by us49 and Lee et al.54 His17 and
Tyr22 residues reside on separate fragments, Leu12-Lys18 and
Leu19-Arg24, respectively, and therefore the selectivity of the
conjugation process could be determined by MALDI-TOF-MS
analysis of the digest fragments. For both 15N 3b-sCT and 6-
sCT, analysis of the region corresponding to the Leu19-Arg24
tyrosine-containing fragment revealed a distribution corre-
sponding to a 15NH2-labeled fragment (addition of 16 Da) as
well as the analogous residue from the digestion of the traces of
unconjugated sCT present as an impurity in the isolated 6-sCT
biohybrid material and some secondary distributions (Figure
6).
The experiment was then repeated with 15N-labeled 3b-sCT
and 6-sCT, which resulted in the release of sCT with an
additional 15NH2 group (Scheme 5 for 3b-sCT). Again, upon
Scheme 5. 15N Isotopic Labeling of the Azobenzene Linker
in the Diazonium Coupling of Aniline mPEG 3b to sCT,
Followed by Release of 15NH2-sCT by Reductive Cleavage
with Na2S2O4
addition of sodium dithionite to a solution of all sCT
conjugates at pH 7.2, an immediate loss of the characteristic
orange color of the linker was observed, and the reduction was
confirmed by UV−vis spectroscopy.
MALDI-TOF-MS analysis of the cleaved conjugates (Figure
5 for 6-sCT) confirmed the presence of the amino-modified
sCT, with the expected mass gain of 16.0 Da observed due to
introduction of a −15NH2 group to the polypeptide. Small
Figure 6. Trypsin digestion of 6-sCT conjugate: MALDI-TOF-MS
analysis of Leu12-Lys18 and Leu19-Arg24.
All of the other trypsin digest fragments were identical to
those of native sCT, which is consistent with little, if any,
conjugation of the diazonium polymer at any other residues but
Tyr22.
In Vitro and in Vivo Preliminary Studies on the
Bioactivity of sCT-Polymer Conjugates. In order to assess
the biological activity of the conjugates prepared in this study,
in vitro tests were carried out by monitoring the ability of the
3b-sCT conjugate to recognize and activate the calcitonin
receptor in T47D human breast cancer cells.
Figure 5. MALDI-TOF spectrum of sCT-15NH2 adduct obtained by
cleavage of the 6-sCT azo-linker using sodium dithionite.
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In vitro results revealed that 3b-sCT was able to significantly
elevate intracellular cAMP levels (Figure 7) with no loss in
calcitonin model peptides. Experimental conditions, especially
in terms of the pH used for the peptide conjugation reactions,
were optimized for the specific targeting of tyrosine residues.
UV−vis and NMR experiments, in conjunction with mass
spectrometry analysis of the fragments obtained from trypsin
digestion of reduced polymer−peptide conjugates, indicated
good selectivity for Tyr conjugation. Preliminary in vitro
experiments aimed at monitoring the increase of intracellular
cAMP induced in T47D human breast cancer cells bearing sCT
receptors by 3b-sCT revealed a virtually identical potency for
the diazo protein−peptide conjugate compared to native sCT.
This PEGylation approach is reversible, and parent aniline
polymers can be recovered from their peptide conjugates in the
presence of appropriate reducing agents, including sodium
dithionite and, potentially, azoreductase enzymes like those
found in the gastrointestinal tract of mammals.55−57 which may
open the way for the use of our approach for the development
of colon-specific drug delivery systems. In vivo preliminary
experiments revealed that our diazo sCT-PEG2000 conjugate
displayed a comparable ability in lowering the plasma [Ca2+] of
male Wistar rats.
The approach for polymer−peptide conjugation presented in
this work could complement well the set of polymer
conjugation techniquesi.e., N-terminal amine conjugation
with aldehyde-functional polymersthat already allow prefer-
ential (or sometimes exclusive) targeting of specific peptide
amino acids in the presence of other possible peptide
conjugation sites. Direct polymer conjugation to tyrosine
residues appears to be a rather versatile approach and could
potentially be applied to conjugates which include a plethora of
different polymeric materials other than PEG or PEG-based
polymers. In addition, this strategy could be expanded to a
number of other biologically relevant peptides (and possibly
proteins), as long as the conjugation conditions are optimized
to take into account the specific characteristics (i.e., exposition
and pKa of relevant amino acid residues) of the conjugated
peptide. This work showed that, for the specific case of sCT
Tyr22 conjugation with linear PEG2000, no loss of bioactivity
occurred in vitro, as measured by the increase of intracellular
cAMP induced in T47D human breast cancer cells bearing sCT
receptors. Preliminary experiments in vivo showed that the
same conjugates had an ability to lower plasma total [Ca2+] in
Wistar rats comparable to that of sCT, further confirming that
polymer conjugation did not interfere with the bioactivity of
sCT. Tyr22 is included in a portion of the sCT peptide
sequence that can be modified or even removed with no loss of
bioactivity. This in itself highlights the importance of having an
ample toolkit of conjugation techniques that allow site-specific
conjugation at sequence domains that are not involved in the
active sites of proteins and peptides. The present study aimed
at complementing the existing polymer conjugation techniques
with, to the best of our knowledge, the first example of site-
specific direct polymer conjugation at Tyr residues.
Figure 7. ELISA measurement of the increase of intracellular cAMP
induced in T47D human breast cancer cells bearing sCT receptors by
3b-sCT and native sCT.
bioactivity compared to native sCT. In these experiments two
negative controls were employed, PBS alone and polymer 3b in
PBS. Both negative controls showed no cAMP was produced. A
positive control, forskolin, was also included to ensure that the
cell model was working correctly.
Following the very promising in vitro behavior of this
conjugate, in vivo tests aimed at investigating the ability of 3b-
sCT to lower [Ca2+] plasma levels in mammals were conducted
using male Wistar rats. Blood samples were taken periodically
up to 240 min after injections into the tail vein, and the plasma
total calcium was analyzed. The 3b-sCT conjugate was shown
to have ability for lowering plasma calcium comparable to that
of unmodified sCT (Figure 8). Negative control data using i.v.
Figure 8. Plasma total calcium profiles of male Wistar rats following
intravenous injections of sCT (blue triangles) and 3b-sCT conjugate
(green circles).
injection of PBS were without effect on plasma calcium levels.22
Homeostatic events mean that plasma calcium levels do not
move from 2.5 mM in untreated conditions.
In summary, this work describes a novel general approach to
direct polymer conjugation to peptides, based on diazonium-
terminated materials with various macromolecular architec-
tures. A short oligoethylene glycol, a well-defined linear PEG,
and poly(mPEG(475) methacrylate) prepared by atom transfer
radical polymerization were synthesized and employed to target
Tyr residues of [D-ala2]-leucine enkephalinamide and salmon
ASSOCIATED CONTENT
■
S
* Supporting Information
Experimental section, including synthetic procedures, character-
ization of polymers and all intermediates, bioconjugation
experiments, and in vitro and in vivo test procedures. This
material is available free of charge via the Internet at http://
pubs.acs.org.
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(25) Lele, B. S.; Murata, H.; Matyjaszewski, K.; Russell, A. J.
Biomacromolecules 2005, 6, 3380.
(26) Gao, W.; Liu, W.; Mackay, J. A.; Zalutsky, M. R.; Toone, E. J.;
Chilkoti, A. Proc. Natl. Acad. Sci. U.S.A. 2009, 106, 15231.
(27) Magnusson, J. P.; Bersani, S.; Salmaso, S.; Alexander, C.;
Caliceti, P. Bioconjugate Chem. 2010, 21, 671.
(28) Yasayan, G.; Saeed, A. O.; Fernandez-Trillo, F.; Allen, S.; Davies,
M. C.; Jangher, A.; Paul, A.; Thurecht, K. J.; King, S. M.; Schweins, R.;
Griffiths, P. C.; Magnusson, J. P.; Alexander, C. Polym. Chem. 2011, 2,
1567.
AUTHOR INFORMATION
■
Corresponding Author
D.M.Haddleton@warwick.ac.uk
Notes
The authors declare the following competing financial
interest(s): D. M. Haddleton is a Director of Warwick Effect
Polymers Ltd.
(29) Kato, M.; Kamigaito, M.; Sawamoto, M.; Higashimura, T.
Macromolecules 1995, 28, 1721.
(30) Wang, J.-S.; Matyjaszewski, K. J. Am. Chem. Soc. 1995, 117,
5614.
(31) Ouchi, M.; Terashima, T.; Sawamoto, M. Chem. Rev. 2009, 109,
4963.
(32) Pauly, H. Z. Hoppe-Seyl. Z. 1904, 42, 508.
(33) Howard, A. N.; Wild, F. Biochem. J. 1957, 65, 651.
(34) Sams, C. F.; Matthews, K. S. Biochim. Biophys. Acta 1984, 787,
61.
ACKNOWLEDGMENTS
■
We thank EPSRC and Warwick Effect Polymers Ltd for
funding; equipment used was supported by the Innovative Uses
for Advanced Materials in the Modern World (AM2), with
support from Advantage West Midlands (AWM) and partially
funded by the European Regional Development Fund (ERDF).
Dr. Francisco Fernandez-Trillo is thanked for useful discussion.
D.M.H. is a Royal Society/Wolfson Fellow.
(35) Schalk, I.; Ehret-Sabatier, L.; Le Feuvre, S.; Bon, S.; Massoulie,
J.; Goeldner, M. Mol. Pharmacol. 1995, 48, 1063.
(36) Szurdoki, F.; Szekacs, A.; Le, H. M.; Hammock, B. D. J. Agric.
Food Chem. 2002, 50, 29.
(37) Curreli, N.; Oliva, S.; Rescigno, A.; Rinaldi, A. C.; Sollai, F.;
Sanjust, E. J. Appl. Polym. Sci. 1997, 66, 1433.
(38) DeTraglia, M. C.; Brand, J. S.; Tometsko, A. M. Anal. Biochem.
1979, 99, 464.
(39) Gorecki, M.; Wilchek, M. Biochim. Biophys. Acta 1978, 532, 81.
(40) Wong, S. S. Reactive groups of proteins and their modifying
agents. Chemistry of Protein Conjugation and Crosslinking; CRC:
Boston, MA, 1991; p 8.
(41) Edgcomb, S. P.; Murphy, K. P. Proteins 2002, 49, 1.
(42) Tracey, B. M.; Shuker, D. E. Chem. Res. Toxicol. 1997, 10, 1378.
(43) Chesnut, C. H.; Azria, M.; Silverman, S.; Engelhardt, M.; Olson,
M.; Mindeholm, L. Osteoporosis Int. 2008, 19, 479.
(44) Epand, R. M.; Epand, R. F.; Stafford, A. R.; Orlowski, R. C. J.
Med. Chem. 1988, 31, 1595.
(45) White, E. H.; Scherrer, H. Tetrahedron Lett. 1961, 2, 758.
(46) Southam, R. M.; Whiting, M. C. J. Chem. Soc., Perkin Trans. 2
1982, 597.
(47) Vaughan, K. Chem. Soc. Rev. 1978, 7, 377.
(48) Lele, B. S.; Murata, H.; Matyjaszewski, K.; Russell, A. J.
Biomacromolecules 2005, 6, 3380.
(49) Sayers, C. T.; Mantovani, G.; Ryan, S. M.; Randev, R. K.; Keiper,
O.; Leszczyszyn, O. I.; Blindauer, C.; Brayden, D. J.; Haddleton, D. M.
Soft Matter 2009, 5, 3038.
REFERENCES
■
(1) Duncan, R. Nat. Rev. Drug Discov. 2003, 2, 347.
(2) Fishburn, C. S. J. Pharm. Sci. 2008, 97, 4167.
(3) Jain, R. K.; Stylianopoulos, T. Nat. Rev. Clin. Oncol. 2010, 7, 653.
(4) Aghemo, A.; Rumi, M. G.; Colombo, M. Nat. Rev. Gastroenterol.
Hepatol. 2010, 7, 485.
(5) Veronese, F. M. Biomaterials 2001, 22, 405.
(6) Roberts, M. J.; Bentley, M. D.; Harris, J. M. Adv. Drug Delivery
Rev. 2002, 54, 459.
(7) Brocchini, S.; Balan, S.; Godwin, A.; Choi, J.-W.; Zloh, M.;
Shaunak, S. Nat. Protocols 2006, 1, 2241.
(8) Zloh, M.; Shaunak, S.; Balan, S.; Brocchini, S. Nat. Protocols 2007,
2, 1070.
(9) Espuna, G.; Arsequell, G.; Valencia, G.; Barluenga, J.; Perez, M.;
́
Gonzalez, J. M. Chem. Commun. 2000, 1307.
(10) Vilaro, M.; Arsequell, G.; Valencia, G.; Ballesteros, A.;
Barluenga, J. Org. Lett. 2008, 10, 3243.
(11) Ban, H.; Gavrilyuk, J.; Barbas, C. F. J. Am. Chem. Soc. 2010, 132,
1523.
(12) Tilley, S. D.; Francis, M. B. J. Am. Chem. Soc. 2006, 128, 1080.
(13) Hooker, J. M.; Kovacs, E. W.; Francis, M. B. J. Am. Chem. Soc.
2004, 126, 3718.
(14) Schlick, T. L.; Ding, Z.; Kovacs, E. W.; Francis, M. B. J. Am.
Chem. Soc. 2005, 127, 3718.
(15) Li, K.; Chen, Y.; Li, S. Q.; Huong, G. N.; Niu, Z. W.; You, S. J.;
Mello, C. M.; Lu, X. B.; Wang, Q. A. Bioconjugate Chem. 2010, 21,
1369.
(16) Joshi, N. S.; Whitaker, L. R.; Francis, M. B. J. Am. Chem. Soc.
2004, 126, 15942.
(17) McFarland, J. M.; Joshi, N. S.; Francis, M. B. J. Am. Chem. Soc.
2008, 130, 7639.
(50) Aurora, J.; Talwar, N.; Vinayak, P. Eur. Gastroenterol. Rev. 2006,
1.
(51) Canevari, M.; Castagliuolo, I.; Brun, P.; Cardin, M.; Schiavon,
M.; Pasut, G.; Veronese, F. M. Int. J. Pharmaceutics 2009, 368, 171.
(52) Kopecek, J. J. Controlled Release 1990, 11, 279.
(53) Kopecek, J.; Kopeckova, P.; Brondsted, H.; Rathi, R.; Rihova, B.;
Yeh, P. Y.; Ikesue, K. J. Controlled Release 1992, 19, 121.
(54) Lee, K. C.; Moon, S. C.; Park, M. O.; Lee, J. T.; Na, D. H.; Yoo,
S. D.; Lee, H. S.; DeLuca, P. P. Pharm. Res. 1999, 16, 813.
(55) Jain, A.; Gupta, Y.; Jain, S. K. Crit. Rev. Ther. Drug. 2006, 23,
349.
(18) Romanini, D. W.; Francis, M. B. Bioconjugate Chem. 2008, 19,
153.
(19) Chemistry of the Diazonium and Diazo Groups, Part 1; Patai, S.,
Ed.; Wiley-Blackwell: Chichester, 1978; ISBN 0471994928. Chemistry
of the Diazonium and Diazo Groups, Part 2; Patai, S., Ed.; Wiley-
Blackwell: Chichester, 1978; ISBN 0471994936.
(20) Lecolley, F.; Tao, L.; Mantovani, G.; Durkin, I.; Lautru, S.;
Haddleton, D. M. Chem. Commun. 2004, 2026.
(21) Mantovani, G.; Lecolley, F.; Tao, L.; Haddleton, D. M.; Clerx, J.;
Cornelissen, J. J. L. M.; Velonia, K. J. Am. Chem. Soc. 2005, 127, 2966.
(22) Ryan, S. M.; Wang, X.; Mantovani, G.; Sayers, C. T.; Haddleton,
D. M.; Brayden, D. J. J. Controlled Release 2009, 135, 51.
(23) Sayers, C. T.; Mantovani, G.; Ryan, S. M.; Randev, R. K.; Keiper,
O.; Leszczyszyn, O. I.; Blindauer, C.; Brayden, D. J.; Haddleton, D. M.
Soft Matter 2009, 5, 3038.
(56) Brown, J. P. Appl. Environ. Microbiol. 1981, 41, 1283.
(57) Rafii, F.; Franklin, W.; Cerniglia, C. E. Appl. Environ. Microbiol.
1990, 56, 2146.
(24) Tao, L.; Mantovani, G.; Lecolley, F.; Haddleton, D. M. J. Am.
Chem. Soc. 2004, 126, 13220.
7413
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