Noncanonical amino acids to improve the pH response of pHLIP insertion at tumor acidity

Article abstract of DOI:10.1002/anie.201409770

The pH low insertion peptide (pHLIP) offers the potential to deliver drugs selectively to the cytoplasm of cancer cells based on tumor acidosis. The WT pHLIP inserts into membranes with a pH₅₀ of 6.1, while most solid tumors have extracellular pH (pH_e) of 6.5-7.0. To close this gap, a SAR study was carried out to search for pHLIP variants with improved pH response. Replacing Asp25 with α-aminoadipic acid (Aad) adjusts the pH₅₀ to 6.74, matching average tumor acidity, and replacing Asp14 with γ-carboxyglutamic acid (Gla) increases the sharpness of pH response (transition over 0.5 instead of 1 pH unit). These effects are additive: the Asp14Gla/Asp25Aad double variant shows a pH₅₀ of 6.79, with sharper transition than Asp25Aad. Furthermore, the advantage of the double variant over WT pHLIP in terms of cargo delivery was demonstrated in turn-on fluorescence assays and anti-proliferation studies (using paclitaxel as cargo) in A549 lung cancer cells at pH 6.6.

Full text of DOI:10.1002/anie.201409770

Angewandte
Chemie
DOI: 10.1002/anie.201409770
Drug Delivery
Noncanonical Amino Acids to Improve the pH Response of pHLIP
Insertion at Tumor Acidity**
Joab O. Onyango, Michael S. Chung, Chee-Huat Eng, Lukas M. Klees, Rachel Langenbacher,
Lan Yao,* and Ming An*
Abstract: The pH low insertion peptide (pHLIP) offers the
potential to deliver drugs selectively to the cytoplasm of cancer
cells based on tumor acidosis. The WT pHLIP inserts into
membranes with a pH50 of 6.1, while most solid tumors have
extracellular pH (pHe) of 6.5–7.0. To close this gap, a SAR
study was carried out to search for pHLIP variants with
improved pH response. Replacing Asp25 with a-aminoadipic
acid (Aad) adjusts the pH50 to 6.74, matching average tumor
acidity, and replacing Asp14 with g-carboxyglutamic acid
(Gla) increases the sharpness of pH response (transition over
0.5 instead of 1 pH unit). These effects are additive: the
Asp14Gla/Asp25Aad double variant shows a pH50 of 6.79,
with sharper transition than Asp25Aad. Furthermore, the
advantage of the double variant over WT pHLIP in terms of
cargo delivery was demonstrated in turn-on fluorescence
assays and anti-proliferation studies (using paclitaxel as
cargo) in A549 lung cancer cells at pH 6.6.
cells within a tumor[2] and rapid development of resistance.[3]
An alternative approach is to target cancer cells based on
universal features of the tumor microenvironment (for
example, hypoxia),[4] which may have broad applications in
many different types of cancers. Tumor acidosis is such
a property that may be exploited.[5] Many solid tumors have
interstitial acidity (pHe of 6.5–7.0), compared to normal tissue
pHe of 7.2–7.5, whereas the intracellular pH of cancer and
healthy cells are similar (both pH 7.0–7.5).[5,6]
Tumor acidosis results from a) the altered metabolism of
cancer cells, that is, ATP production via glycolysis with
reduced rate of respiration, at first, in response to hypoxia
(the Pasteur effect), but eventually even under conditions of
plentiful oxygen (the Warburg effect); and b) a fast rate of
metabolism coupled with poor clearance of waste products.[7]
Low tumor pHe may confer growth advantages on cancer
cells, as healthy tissue and their extracellular matrix remodel
under acidic stress, clearing space for local tumor invasion and
eventual metastasis.[8] Cancer cells living in acidic and hypoxic
environments are especially resistant to radiation and chemo-
therapy, which in turn contribute to tumor recurrence.[4,7a,9]
Thus using pHLIP to target acidic cancer cells for destruction
can offer a complementary approach to established therapies.
At the physiological pH of 7.2–7.5, pHLIP is unstructured
in solution (State I in Figure 1) or peripherally bound to
O
ne approach to targeted cancer therapy relies on the use
of a drug carrier that can distinguish between normal and
cancer cells. The recent success of antibody–drug conjugates
validates this approach.[1] However, targeting specific tumor-
associated proteins can be hampered by heterogeneity among
[*] Dr. J. O. Onyango,[+] M. S. Chung,[+] C.-H. Eng, L. M. Klees,
R. Langenbacher, Prof. Dr. M. An
Department of Chemistry
State University of New York (SUNY), Binghamton University
P.O. Box 6000, Binghamton, NY 13902 (USA)
E-mail: aming@binghamton.edu
Prof. Dr. L. Yao
Department of Physics, Applied Physics and Astronomy
State University of New York (SUNY), Binghamton University
P.O. Box 6000, Binghamton, NY 13902 (USA)
E-mail: lanyao@binghamton.edu
Figure 1. Insertion of the pHLIP peptide into a membrane in response
to acidity.
[+] These authors contributed equally to this work.
[**] We thank Prof. Dr. Donald M. Engelman (Yale) for discussion and
support and Prof. Dr. Eriks Rozners and Prof. Dr. Susan Bane (both
of SUNY-Binghamton) for frequent use of their fluorimeter and
plate reader, respectively. We also thank Raemer J. Lapid (for
assistance in processing data), Rebecca A. Chandler, Meghan M.
Bell, Vladyslav Nazarenko, Ilana G. Bandler (for assistance in cell
assays), and Emma A. Gordon (for assistance in synthesis). This
work was supported by SUNY-Binghamton University (BU) (Start-
up funds to M.A. and L.Y., various financial supports to J.O.O.,
L.M.K., and M.S.C., HHMI-BU undergraduate summer fellowships
to C-H.E. and R.L.), NIH (R01-GM073857 for training of M.A. and
initial work), and NSF grant CHE-0922815 for the Regional NMR
Facility (600 MHz instrument) at SUNY-Binghamton.
membrane surfaces (State II); under slightly acidic condi-
tions, pHLIP inserts across a lipid bilayer, forming a trans-
membrane (TM) helix (State III).[10] The insertion is unidirec-
tional with C-terminus translocated across the membra-
ne,[10c,11] rapid (equilibrium reached in minutes),[12] and
mediated mainly by the protonations of Asp14 and Asp25
carboxyl sidechains in the TM region.[10b,13] Both d- and l-
pHLIP peptides can target acidic tumors in vivo.[13,14] Fur-
thermore, pHLIP does not cause membrane leakage,[11a,15]
and no toxic effects have been oberved in cell culture (up to
10 mm) or mice (5 mgkgꢀ1).[11a,13,16] Owing to its small size (36–
38 amino acids, ꢁ 4 KD), pHLIP can penetrate to the core of
tumor where hypoxia and acidosis are most prominent.[16]
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201409770.
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These properties favor pHLIP as a drug carrier worthy of
further development.
plasma-membrane in response to tumor pHe can benefit from
increased pH50 because most solid tumors exhibit an average
pHe of 6.8.[5,6]
One challenge in targeting tumor acidosis is the small pH
difference between healthy tissue and tumor. Thus, the drug
carrier should be a pH sensor with sharp transition. Previous
efforts in targeting tumor pHe have been focused on pH-
responsive polymers. Taking advantage of the facts that the
imidazole side-chain of histidine is protonated to the imida-
zonium cation with a pKa of 7.0 (neutral to positive at low
pHe) whereas certain sulfonamides have pKa of 6.8 for the
NH a-proton (negative to neutral at low pHe), Bae and co-
workers engineered a wide variety of polymer-based molec-
ular devices (for example, micelles) and behaviors (such as
unveiling of cell-penetrating peptides) to respond to the
slightly acidic environment of tumors for drug delivery.[17]
Another recently reported approach is based on the acid-
catalyzed, b-carboxyl neighboring-group assisted hydrolysis
of N-alkyl maleamic acids (negative to positive conversion in
response to low pHe).[18] However, the stability of this charge
reversal system at pH 7.4 is a concern.[18a,b,e] Compared to
these polymer-based systems, pHLIP is simpler and more
chemically defined, which can have significant practical
advantages in drug carrier development.
Our structure–activity relationship (SAR) study contains
9 novel pHLIP variants, the structures of which are shown in
Figure 2. Their insertion behavior into POPC membrane were
Figure 2. Side-chain structural variations at pHLIP position 14 and 25.
WT, D14E, and D25E are previously known.[10b,19]
characterized using established Trp fluorescence meth-
ods.[19–20,24,26] The basis of the assay is that pHLIP insertion
(State II to III in Figure 1) leads to an increase in Trp
fluorescence intensity and a blue-shift in emission lmax
,
When cargo, such as a small dye molecule, a cyclic
peptide, or a peptide nucleic acid (PNA), is attached to the C-
terminus of pHLIP, it can be carried across the membrane
during pHLIP insertion.[11,20] Thus, pHLIP not only can target
cancer cells based on low tumor pHe but also deliver the cargo
directly into the cytoplasm. Such insertion-mediated delivery
of phalloidin (and other toxins) inhibited the proliferation of
cancer cells in a pH-dependent fashion.[20a–c] Furthermore,
when many copies of pHLIP are attached to the surface of
13 nm gold[21] or 140 nm mesoporous silica nanoparticles,[22]
they seem to be able to work in concert to bring such large
cargo into cells. Recently, as the first example of in vivo
efficacy, pHLIP-mediated delivery of PNA (anti-miR)
silenced miR-155 onco-miR in a mouse lymphoma model.[23]
We believe pHLIP also presents an opportunity to
improve cancer chemotherapy. Many drugs, such as paclitaxel
(Taxol) or doxorubicin, have dose-limiting toxicity in off-
target sites (for example, bone marrow, heart). Our goal is to
use pHLIP to deliver such drugs selectively to cancer cells. In
the current study we aim to create pHLIP variants that insert
more effectively in response to tumor pHe through incorpo-
ration of noncanonical amino acids. The best of these variants
are further evaluated in cellular assays to demonstrate its
advantage over WT pHLIP.
The pHLIP peptide is derived from the TM helix C of
bacteriorhodopsin and has the following sequence: GGEQN-
PIYWARYADWLFTTPLLLLDLALLVDADEGT.[10b] For
the original “WT” pHLIP, the apparent pH50 of insertion
(that is, the pH at which 50% of pHLIP are in the inserted
State III) across 1-palmitoyl-2-oleoylphosphatidylcholine
(POPC) membrane is about 6.1 (Figure 3).[10b,19,24] When
pHLIP peptides interact with cells, insertion may take place at
plasmamembrane or endosomal membranes, and likely
both.[25] Given its pH50 even the WT pHLIP is able to
efficiently deliver cargo into the cytoplasm in response to
endosomal acidity. Yet drug delivery by insertion in the
reflecting the more hydrophobic environment the Trp side-
chains experience after insertion (especially W15). Further,
circular dichroism (CD) measurements were carried out to
confirm the pH-dependent conformational change: random
coil in States I and II but a-helical in State III.[10b,19] The
apparent pH50 values are calculated by fitting the transition
curve of “pH vs. lmax” (Figure 3 left column) to the
Henderson–Hasselbalch equation (albeit with pH50 in place
of pKa):
lmax ¼ lmax-III þ ðlmax-IIꢀlmax-IIIÞ=ð1 þ 10nðpH ꢀpHÞÞ
50
ð1Þ
where n is the Hill coefficient (which reflects the sharpness or
cooperativity of insertion into POPC membrane), and lmax-II
and lmax-III are the wavelengths of maximum emission in the
membrane-bound State II and the inserted State III, respec-
tively. For each novel variant, the Trp fluorescence assay is
repeated at least three times and the average pH50 and Hill
coefficient values are reported along with standard deviations
(s.d.) in Table 1.
The D25E and D14E variants have been described to
insert with pH50 of about 6.4–6.5.[19] To minimize aggregation,
we carried out experiments with lower ionic strength (11 mm
vs. 68 mm) and peptide concentration (2 mm vs. 7 mm) than
reported procedures. Under such conditions, D25E and D14E
showed pH50 of 6.27 ꢂ 0.03 and 6.14 ꢂ 0.05, respectively
(Table 1; see the Supporting Information, Table S1 for
sequence details and Figure S2 for Trp fluorescence and CD
data). The D25E variant is an important precedent for our
SAR study as it demonstrates that lengthening the D25 side-
chain can increase pH50.
To find out to what extent can side-chain extension at
position 25 be tolerated, the Cys side-chain of a D25C pHLIP
was lengthened by reaction with bromoacetic acid or 3-
bromopropionic acid to give variant D25C-2C or D25C-3C
(Figure 2). The D25C-2C has a pH50 of 6.05 ꢂ 0.04 and a Hill
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D25C-3C vs. 8–12 nm for all other variants), and the pH vs.
lmax transition is poorly defined (n = 0.9). Such data indicate
that D25C-3C is not a typical pHLIP, possibly because the
additional methylene group increases the tendency of aggre-
gation. In D25C-2C, the electron withdrawing ability of the
sulfur atom increases side-chain acidity, shifting the pH50
unfavorably down. However, D25C-2C gave hope that its
carbon isostere Asp25Aad would still behave as a pHLIP.
Indeed, Asp25Aad pHLIP has clear coil-to-helix transi-
tion from State II to III (Supporting Information, Figure S1).
More importantly, Asp25Aad has a pH50 of 6.74 ꢂ 0.14
(Figure 3), closely matching average tumor pHe in vivo.
Asp25Aad is also consistent with the trend observed from
WT to D25E: as the number of methylene groups in the side-
chain increased, the pH50 rose (WT 6.16 to D25E 6.27 to
Asp25Aad 6.74). Since the peptide backbone is electron-
withdrawing, as it became more distant, the innate acidity of
the side-chain carboxylic acid decreased, shifting pH50 up.
When D14 instead of D25 is replaced with Aad, the resulting
Asp14Aad variant has proper pH-dependent insertion (Sup-
porting Information, Figure S3), yet the pH50 is only 6.37 ꢂ
0.03. Therefore, factors other than the inherent side-chain
pKa, such as the precise location and the polarity of the
protonation environment in State II, also influence the pH50.
The side-chain at position 25 seems to dominate the setting of
pH50.
However, the Hill coefficient of Asp25Aad insertion into
POPC membrane is 1.73 ꢂ 0.17, lower than that of WT (2.48).
Insertion at neutral pH may hamper the selectivity of pHLIP-
mediated drug delivery. Thus, a sharp pH-response, that is,
insertion over a narrow pH range, is desirable. Engelman and
co-workers postulated that more carboxylic acid residues in
the TM region may increase the insertion cooperativity.[19]
Although their attempts at introducing additional carboxyl
group were not successful, we thought to revisit this hypoth-
esis. First, the Asp14 or Asp25 residue was replaced with
a pair of Glu residues, giving the variants D14EE or D25EE.
CD confirmed that D14EE and D25EE have pH-dependent
coil-to-helix transition characteristic of a pHLIP (Supporting
Information, Figure S3). But to our disappointment, D14EE
has a pH50 of 6.19 ꢂ 0.04 with a Hill coefficient of 1.41 ꢂ 0.02
while D25EE has a pH50 of 6.60 ꢂ 0.14 with a Hill coefficient
of 1.34 ꢂ 0.26. Both of these variants insert over a wider range
than WT pHLIP (Supporting Information, Figure S3).
Next, the D14 or D25 residue was substituted with the
noncanonical amino acid Gla, which has two carboxyl groups
appended to the g-carbon of the side-chain (Figure 2),
resulting in the variants Asp14Gla or Asp25Gla. Compared
with D14EE and D25EE variants, this modification allows for
more precise introduction of the additional carboxyl group at
locations critical for pHLIP insertion. CD showed that
Asp14Gla and Asp25Gla both have clearly defined pH-
dependent coil-to-helix transition (Supporting Information,
Figures S1 and S3). Asp25Gla turned out to be unremarkable,
with pH50 of 6.20 ꢂ 0.02 and a Hill coefficient of 1.90 ꢂ 0.19
(Supporting Information, Figure S3). On the other hand,
Asp14Gla has a pH50 of 6.24 ꢂ 0.05 but with a transition Hill
coefficient of 3.98 ꢂ 0.40 (Figure 3). Thus, the Asp14Gla
variant has the sharpest pH response recorded to date for
Figure 3. Trp fluorescence of Asp25Aad, Asp14Gla, and Asp14Gla/
Asp25Aad pHLIP variants, which show improved pH response at the
tumor pHe range of 6.5–7.0. Left column (top to bottom): insertion is
monitored through Trp fluorescence lmax blue-shift (different colors
denote experimental repeats); compared to the WT, the Asp25Aad
insertion shifted to a higher pH range closely matching tumor acidity
but suffers from poor cooperativity; the Asp14Gla variant has strikingly
sharp pH response; the Asp14Gla/Asp25Aad double variant maintains
a high pH50 of 6.8 with cooprativity restored to WT level. Right
column: For the pHLIP variants studied, Trp fluorescence spectra of
State I (black), II (blue), and III (red) show an increase in fluorescence
intensity and lmax blue-shift from State II to State III (that is, pHLIP
insertion into POPC liposome membrane).
coefficient of 2.44 ꢂ 0.10 (similar to WT), with pH-dependent
helix formation characteristic of a pHLIP (as confirmed by
CD; Supporting Information, Figure S2). However, the
D25C-3C variant lost the coil-to-helix transition: its CD
signal is already weakly helical at pH 8 and there is no
increase in helicity at pH 4 (Supporting Information, Fig-
ure S2). Compared to State I, its “State II” Trp fluorescence
shows significant increase in intensity and emission lmax blue-
shift (Supporting Information, Figure S2). Further lmax blue-
shift from “State II” to “III” is unusually narrow (5.5 nm for
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Table 1: Insertion pH50, Hill coefficient n, and Trp fluorescence lmax-II/III of pHLIP variants studied.
linkage on its C2’ OH (Fig-
ure 4b; Supporting Information,
Scheme S2). As attachment at this
site would interfere with paclitaxel
binding to tubulin,[27] a traceless
linker is constructed following
known methods[28] in which upon
cleavage of the disulfide bond in
cells, the resulting linker SH group
would cyclize onto the paclitaxel
C2’ ester carbonyl to release the
cargo exactly as paclitaxel (Fig-
ure 4b). This trace-less linker strat-
egy was tested in a chemical cleav-
age assay by incubating WT
pHLIP variants
pH50
n
lmax-II [nm]
lmax-III [nm]
WT
6.16
2.48
345.2
334.5
D25E
D14E
6.27ꢂ0.03
6.14ꢂ0.05
6.05ꢂ0.04
6.18
6.74ꢂ0.14
6.37ꢂ0.03
6.20ꢂ0.02
6.24ꢂ0.05
6.60ꢂ0.14
6.19ꢂ0.04
6.79ꢂ0.04
2.14ꢂ0.20
2.47ꢂ0.33
2.44ꢂ0.10
0.9
1.73ꢂ0.17
2.05ꢂ0.13
1.90ꢂ0.19
3.98ꢂ0.40
1.34ꢂ0.26
1.41ꢂ0.02
2.38ꢂ0.30
344.9ꢂ0.3
345.6ꢂ0.3
344.6ꢂ1.2
343.5
344.5ꢂ1.9
344.4ꢂ1.5
346.6ꢂ0.6
347.4ꢂ0.2
345.0ꢂ1.5
346.7ꢂ1.0
346.4ꢂ0.1
334.9ꢂ0.2
335.6ꢂ0.2
334.5ꢂ0.5
338.0
336.1ꢂ0.4
336.0ꢂ0.6
335.1ꢂ0.2
336.2ꢂ0.2
335.5ꢂ1.6
335.6ꢂ0.3
338.1ꢂ0.3
D25C-2C
D25C-3C
Asp25Aad
Asp14Aad
Asp25Gla
Asp14Gla
D25EE
D14EE
Asp14Gla/Asp25Aad
a pHLIP, with insertion occurring over half pH unit vs. one pH
unit for the WT.
To see whether higher insertion pH50 and sharper pH
response could be tuned separately in an additive fashion, the
Asp14Gla/Asp25Aad double variant was tested. The CD data
showed clear pH-dependent coil-to-helix transition character-
istic of a pHLIP (Supporting Information, Figure S1); and
indeed, this double variant maintains a high insertion pH50 of
6.79 ꢂ 0.04, similar to that of Asp25Aad, while the transition
Hill coefficient is restored to 2.38 ꢂ 0.30, close to the WT level
of sharpness in pH-response (Figure 3).
With this best variant in hand, we set out to demonstrate
its advantage over WT pHLIP in cultures of A549 human lung
cancer cells. WT or Asp14Gla/Asp25Aad pHLIP containing
C-terminal Lys and Cys residues were used to synthesize turn-
on fluorescence probes (with rhodamine TAMRA attached to
the Lys side-chain by an amide bond and quencher QSY9
attached to the adjacent Cys side-chain through a disulfide
bond) and pHLIP-paclitaxel conjugates (the drug paclitaxel is
conjugated to the Cys side-chain through a disulfide bond
traceless linker; Figure 4). Details of their syntheses are
described in the Supporting Information, Schemes S1 and S2.
In the self-quenched turn-on fluorescence probe, the
quencher QSY9 serves as a model cargo of intracellular drug
delivery. As the disulfide linker is selectively cleaved inside of
cells, presumably by glutathione (GSH), QSY9 release would
lead to dequenching and increase in TAMRA fluorescence
(Figure 4a), thus reporting the level of pHLIP insertion in
cellular membranes (because the C-terminus is translocated
across membrane during pHLIP insertion). In the WT vs.
Asp14Gla/Asp25Aad comparison, data shown in Figure 4a
establish that dequenching levels (and thus inferred pHLIP
insertion levels) are considerably higher for the double
variant at all acidic pHs but similar at neutral pH. In
particular, the pH 7.4 vs. 6.6 comparison reveals that the
amount of pH-dependent cargo release, which represents the
therapeutic window in pHLIP-mediated drug delivery in vivo,
is much greater for the double variant (9.2- vs. 5.2-fold) than
the WT (5.9- vs. 4.3-fold). Thus, the Asp14Gla/Asp25Aad
variant should be able to deliver drug in a more pH-
dependent fashion than WT.
Figure 4. Evaluation of WT pHLIP vs. Asp14Gla/Asp25Aad variant in
A549 cancer cells. a) Turn-on fluorescence assays: At all acidic pHs,
Asp14Gla/Asp25Aad pHLIP released more disulfide-linked QSY9
quencher than WT pHLIP. The TAMRA fluorescence levels plotted were
measured 72 h after treatment with 0.5 mm of self-quenched probe for
30 min (n=8, error bar is s.d.). b) Anti-proliferation assays: At pH 6.6,
Asp14Gla/Asp25Aad pHLIP-paclitaxel inhibited the growth of A549
cancer cells to a greater extent than either WT pHLIP-paclitaxel or the
free drug itself (30 min treatment with 0.5 mm of drug or pHLIP-drug,
n=8, error bar is s.d.).
pHLIP-paclitaxel (4 mm) with glutathione (11 mm) in DPBS
buffer (pH 7.6). Cargo release was monitored with HPLC,
which showed that within 2 h up to 90% of paclitaxel cargos
were released as the free drug (Supporting Information,
Figure S4). In the growth inhibition assay, A549 cells were
treated with WT or Asp14Gla/Asp25Aad pHLIP-paclitaxel
(0.5 mm, 30 min) at the simulated tumor pH of 6.6–6.7 (pH
measured before and after experiment) or the healthy tissue
pH of 7.4. The results of these experiments are shown in
Figure 4b. At pH 6.6, the double variant has decisive
advantage over WT pHLIP in delivering paclitaxel into the
cell, both in terms of absolute amount delivered (thus leading
to more growth inhibition) and pH dependence. Interestingly,
To test this hypothesis in antiproliferation assays, the drug
paclitaxel is conjugated to pHLIP by an ester–disulfide
4
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even WT pHLIP-paclitaxel can inhibit cell growth as well as
free drug but the pH-dependence is largely absent. On the
other hand, Asp14Gla/Asp25Aad pHLIP-paclitaxel can
inhibit cell growth to a greater extent than free drug (74%
vs. 37% growth inhibition) at pH 6.6 and its anti-proliferative
effects are very pH sensitive: 74% inhibition at pH 6.6 vs.
56% at pH 7.4.
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In summary, to find pHLIP variants with improved
insertion properties for drug delivery at tumor average pHe
of 6.8, we carried out a SAR study in which the D14 and D25
residues critical for insertion were modified. The variants
D25EE, Asp25Aad, and Asp14Gla/Asp25Aad insert with
pH50 of 6.60, 6.74, and 6.79, respectively. These three variants
insert into POPC membrane at higher pH than all previously
known pHLIP peptides. Thus, they may insert more effec-
tively at the tumor pHe range of 6.5–7.0, with the potential to
deliver more drugs into the cytoplasm of cancer cells via
plasmamembrane insertion. To reduce pHLIP-mediated drug
delivery at neutral pH (which may lead to off-target effects in
healthy tissues), insertion over a narrow pH range is desirable.
To this end, we found that the Asp14Gla variant can insert
into membrane with the sharpest pH-response observed so far
for a pHLIP peptide: over half a pH unit vs. one pH unit for
WT. This Asp14Gla variant may improve pHLIP-based
tumor imaging by reducing background signals. Thus,
adding carboxyl group to the 14th position side-chain can
sharpen the pH response while lengthening the side-chain at
position 25 can raise the pH50 of insertion. The Asp14Gla/
Asp25Aad double variant shows that these effects are
independent of each other and can be used additively to
tune for desired pHLIP insertion properties. Furthermore, the
successful introduction of additional carboxyl groups in the
TM region, as in D25EE and Asp14Gla, may also improve
pHLIP solubility and reduce aggregation, which are param-
eters critical for future applications. We have also shown that
the double variant Asp14Gla/Asp25Aad has considerble
advantage over WT pHLIP in cargo delivery at true tumor
pHe of 6.6, as demonstrated with turn-on fluorescence assays
and anti-proliferation studies in A549 cells using paclitaxel as
a model drug.
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D25C-2C and D25C-3C variants, capping of C-terminal Lys and Cys
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the Supporting Information.
Received: October 5, 2014
Revised: November 27, 2014
Published online: && &&, &&&&
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Keywords: biosensors · cancer · drug delivery · peptides ·
pH low insertion peptide
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These are not the final page numbers!

Angewandte
Chemie
Communications
Drug Delivery
The pH low insertion peptide (pHLIP)
allows drugs to be delivered selectively to
the cytoplasm of cancer cells based on
tumor extracellular acidity (pHe). Incor-
poration of noncanonical amino acids
into pHLIP can increase the pH at which
it inserts into membranes, thus better
matching tumor pHe, without compro-
mising the sharpness of transition.
J. O. Onyango, M. S. Chung, C.-H. Eng,
L. M. Klees, R. Langenbacher, L. Yao,*
M. An*
&&& — &&&
Noncanonical Amino Acids to Improve
the pH Response of pHLIP Insertion at
Tumor Acidity
Angew. Chem. Int. Ed. 2015, 54, 1 – 7
ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
7
These are not the final page numbers!