From solution to in-cell study of the chemical reactivity of acid sensitive functional groups: A rational approach towards improved cleavable linkers for biospecific endosomal release

Article abstract of DOI:10.1039/c6ob00846a

pH-Sensitive linkers designed to undergo selective hydrolysis at acidic pH compared to physiological pH can be used for the selective release of therapeutics at their site of action. In this paper, the hydrolytic cleavage of a wide variety of molecular structures that have been reported for their use in pH-sensitive delivery systems was examined. A wide variety of hydrolytic stability profiles were found among the panel of tested chemical functionalities. Even within a structural family, a slight modification of the substitution pattern has an unsuspected outcome on the hydrolysis stability. This work led us to establish a first classification of these groups based on their reactivities at pH 5.5 and their relative hydrolysis at pH 5.5 vs. pH 7.4. From this classification, four representative chemical functions were selected and studied in-vitro. The results revealed that only the most reactive functions underwent significant lysosomal cleavage, according to flow cytometry measurements. These last results question the acid-based mechanism of action of known drug release systems and advocate for the importance of an in-depth structure-reactivity study, using a tailored methodology, for the rational design and development of bio-responsive linkers.

Full text of DOI:10.1039/c6ob00846a

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From solution to in-cell study of the chemical reactivity of acid
sensitive functional groups: a rational approach towards improved
cleavable linker for biospecific endosomal release
Sylvain A. Jacques,a† Geoffray Leriche,a† Michel Mosser,a Marc Nothisen,b
Christian D. Muller,c Jean-Serge Remy,b and Alain Wagnera*
a.
LFCS laboratory, CAMB UMR 7199 CNRS University of Strasbourg, LabEx Medalis,
icFRC, Faculty of Pharmacy, 74 route du Rhin, 67400 Illkirch, France. E-mail:
alwag@unistra.fr
b.
V-SAT laboratory, CAMB UMR 7199 CNRS University of Strasbourg, LabEx
Medalis, icFRC, Faculty of Pharmacy, 74 route du Rhin, 67400 Illkirch, France.
c.
Laboraroire d’Innovation Thérapeutique, UMR 7200, CNRS University of
Strasbourg, Faculty of Pharmacy, 74 route du Rhin, 67400 Illkirch, France.
† Both authors contributed equally to the work.

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Abstract
pH-sensitive linkers designed to undergo selective hydrolysis at acidic pH compared to
physiological pH can be used for selective release of therapeutics at their site of
action. In this paper, the hydrolytic cleavage of a wide variety of molecular structures
that have been reported for their use in pH-sensitive delivery systems was examined .
A wide variety of hydrolytic stability profiles was found among the panel of tested
chemical functionalities. Even within a structural family, a slight modification of the
substitution pattern has an unsuspected outcome on the hydrolysis stability. This
work lead us to establish a first classification of these groups based on their
reactivities at pH 5.5 and their relative hydrolysis at pH 5.5 vs. pH 7.4. From this
classification, four representative chemical functions belonging eand compare
lysosomal cleavage in living cells..and revealed that only the most reactive functions
underwent significant lysosomal cleavage, according to flow cytometry
measurements. These last results question the acid-based mechanism of action of
known drug release systems and advocate for the importance of in-depth structure-
reactivity study, using tailored methodology, for the rational design and development
of bio-responsive linkers.
Introduction
Specific release of bioactive compounds has become a major issue in modern drug
development. The efficacy and the selectivity of the bond breaking process is a
paramount component for the success of a prodrug1 and targeted therapeutics such
as Antibody Drug Conjugates (ADC).2 For a delivery system to be effective, it must
remain stable and active until reaching its site of action. As a strategy for selective
bond cleavage mechanisms, one can exploit singularities linked to pathologies or sub-
cellular compartments including enzymatic activity3 or high glutathione level.4
In the field of pH-sensitive drug delivery systems, several acid-sensitive linkers have
been developed to respond to a pH-shift. Typical examples are shown in Error! Reference
source not found.. For delivery systems, hydrazone,5 cis-aconityl6 and trityl bonds7 have
been the most widely investigated, but other linkers such as dialkyl and
diaryldialkoxysilane,8 orthoester,9 acetal,10
β
-propionate,11 phosphoramidate,12

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imine,13 vinyl ether,14 imidazole15 and polyketal16 have also been described and
reviewed.17
Fig. 1 Examples of acid sensitive bonds.
We have recently published a study on a homo-bifunctional spiro di-orthoester
(SpiDo) linker derivative which has shown a fast lysosomal hydrolysis and a high
stability in Human plasma.18 In the course of this work we have noticed that despite
many reports on acid-labile delivery systems, the choice of a suitable starting point
for the design of biospecific release system remains mostly intuitive.
Most of the acid-sensitive linkers have been described for different applications and
in different model systems. Each study mostly focused on the use of one type of acid-
sensitive linker, for one particular application, in one specific biological system. The
majority of the time, the pH-sensitive linkers are first characterized in solution with
their hydrolysis profiles at pH 5.5 and 7.4, and then directly used in specific biological
applications. The scarce and disparate data do not provide a precise and
comprehensive overview on the chemical reactivity, allowing the direct comparison of
chemical structures and guiding the rational design of acid-sensitive biospecific
linkers. For this reason, we report herein the pH sensitivity profiles of the main acid-

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labile structures under standardized conditions in solution, and further in cellular
environments using turn-on fluorescent probes.
Results and discussion
Determination of hydrolysis profiles
Following a literature review, we designed analogs representative of known acid-
sensitive chemical linkers. For the most widely reported linkers, several derivatives
have been also synthesized to further expand the Structure-Acidlability-Relationship.
Each of these derivatives includes a UV traceable moiety to allow HPLC-UV detection.
Hydrolytic profiles of all compounds have been monitored in aqueous buffer solution
at pH 5.5 (to simulate lysosomal pH) and pH 7.4 (to simulate serum conditions). A 10
mM solution of each molecule was prepared in DMSO and diluted in buffer to obtain
a 1 mM solution. For pH 7.4 NaH2PO4/Na2HPO4 phosphate buffers and for pH 5.5
KH2PO4/Na2HPO4 were prepared at 100 mM. Each solution was immediately analyzed
by analytical HPLC (Eluent A/B, A: NH3HCOOH solution (10 mM, pH 8.5)/B: ACN.
Gradient: 5% B to 95% B in 20 minutes and 10 minutes of re-equilibration). Reaction
was performed at 25 °C and crude was injected every 30 minutes for up to 8 hours.
The first measurement t1 was recorded after 30 seconds which is a result of the delay
of sample preparation. For each recorded time tx the amount of non-hydrolyzed
product was measured by the calculation of the area under the peak. Comparing with
the calculated area at t1 the percentage of hydrolyzed product was calculated
following the formula:
(At1 − Atx)
% hydrolysis = 100 ×
At1
For each pH the variation of % hydrolysis within the time of the experiment was
plotted. The half-live times t1/2 were estimated and the hydrolysis selectivity was
measured by the ratio:
ꢂ
ꢀꢁ ꢄꢅꢆꢇ.ꢈ
ꢃ
.
ꢂ
ꢀꢁ ꢄꢅꢆꢉ.ꢉ
ꢃ
When compounds were completely hydrolyzed at the first measurement t1, a half-life
time of less than 0.01 hour (0.5 minute) was attributed. In these particular cases we

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were not able to determine if the compounds were hydrolyzed within the 10 seconds
before the analysis (pH 5.5 or 7.4) or during the analysis itself (pH 8.5). However
considering the required plasma stability (pH = 7.4) such functional groups were not
considered as potent leads to efficient delivery systems.
Synthesis of the model compounds is straightforward (See SI for a full description of
their syntheses) but requires careful attention, requiring the use of mild acidic
conditions when handling pH-sensitive groups especially upon purification.
Carbazate, oxime, imine, hydrazone and acylhydrazone derived compounds
Compounds containing a carbon-nitrogen double bond (e.g. hydrazone) are the most widely
used structures for acid-labile pro-drugs and controlled delivery of doxorubicin, cisplatin and
other agents.5n-5v In a similar manner, the imine (Schiff base) itself has already been used to
achieve acid-triggered drug release.19 With compounds 1-8 in hand, we studied the lability of
the carbon-nitrogen double bond towards hydrolysis at pH 5.5 and 7.4 (Fig. 2). This
particular case gave binary results. On one hand, oxime 1, carbazate 4 and hydrazone
pyridine 7 were completely stable at both pH; on the other hand imines 2, 3 and
acylhydrazones 5 and 6 were completely hydrolyzed at the first measurement. It is known
that pyridine can be protonated in acidic media and thus can lead to a specific acidic
cleavage.20 However, in our conditions we found that replacing a benzene ring (2) to a
pyridine ring (7) did not improve the stability.

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Fig. 2 Hydrolysis kinetics of compounds
1 – 7 at pH 5.5 and 7.4. (S = Compound stable
during the time of the experiment (0 % hydrolysis)).
Substituted heterocycles derived from benzaldehyde
Kinetics stability studies of heterocycles 9 to 11 were done to further examine the effect of
the heteroatoms on the hydrolytic profiles of five membered rings in aqueous media (Fig. 3
and Fig. S1). At t1, imidazoline 9 and oxazolidine 10 showed complete hydrolysis at both pH,
while dioxolane 11 displayed distinct hydrolysis profiles. At pH 5.5, 50% of hydrolysis was
detected after two hours while less than 5% of hydrolysis was observed at pH 7.4.
Extrapolation of the data recorded at pH 7.4 gave a half-life of about 33 hours.

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Fig. 3 Influence of heteroatoms on the hydrolysis of five membered ring systems.
Hydrolysis kinetics of compounds 11 at pH 5.5 and 7.4.
9
–
The influence of ring size and steric effects was studied using dioxolane compounds 11 to 13
(Fig. 4 and Fig. S2). Compound 12 bearing one extra methyl compared to compound 11
showed a better stability at pH 5.5 with a half-life of 4.2 hours. However, it led to a 2-fold
decrease in half-life at pH 7.4. 1,3-dioxane type compound 13 revealed to be the most stable
of this series at both pH, with no trace of p-anisaldehyde being observed after 6 hours. This
result is consistent with previous work by Harper21 and others.22
Fig. 4 Influence of steric effects and size of the ring on dioxane and dioxolane type
ring systems. Hydrolysis kinetics of compounds 11
– 13 at pH 5.5 and 7.4. (S =
Compound stable during the time of the experiment (0 % hydrolysis)).
Following the work by Bundgaard and co-workers who reported the hydrolysis kinetics of
several oxazolidines, and assessed their suitability as prodrug forms for beta-amino alcohols

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and/or carbonyl-containing compounds,22 oxazolidines 15 - 21 were synthesized and their
pH sensitivity recorded. Derivatives 15 and 16 showed complete hydrolysis at t1 at both pH
(Fig. 5 and Fig. S3 and S4). Oxazolidine 17 was very reactive toward hydrolysis with a half-life
time of less than 20 min at pH 5.5. The addition of a second electron donating group did not
increase the rate of hydrolysis, and half-lives of 18 were very similar at both pH. As expected,
the substitution of benzaldehyde with an electron withdrawing group in 19 decreased the
rate of hydrolysis at both pH. A similar effect was also observed with the pyridine derivative
20. It is known in the literature that the stability of the oxazolidine can be tuned by the steric
hindrance on the heterocycles.23 However in this case, derivative 21 revealed to be very
unstable at both pH.
Fig. 5 Effects of the substitution on the benzaldehyde moiety on the hydrolysis rate of
oxazolidine derivatives. Hydrolysis kinetics of compounds 15 – 21 at pH 5.5 and 7.4.
We next turned our attention to the hydrolysis of the ketal moiety (Fig. 6 and Fig. S5 and S6).
They are well known acid-sensitive moieties that were originally found to be useful as
protecting groups for aldehydes.24 Derivative 22 had a half-life of 0.8 hours and 12.2 hours at
pH 5.5 and 7.4 respectively. The rate of hydrolysis was increased by adding an electron
donating methoxy group 23. Moreover, both compounds showed a good stability at pH 7.4.
Derivative 24 presented the highest ratio of hydrolysis between both pH. The N-

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ethoxybenzymidazole derivative 25 showed a slower hydrolysis kinetics at pH 5.5 and a
better stability at pH 7.4 compared to its parent diacetal compound 24. These last results are
in agreement with previous study by Yang and coworkers.15a
Fig. 6 Effects of the substitution on the hydrolysis rate of ketal derivatives. Hydrolysis
kinetics of compounds 22 – 25 at pH 5.5 and 7.4.
Siloxane, acetal, β-thiopropionate THP and carbamate derived compounds
Widely used in organic chemistry as protecting groups,24 silyl ethers have also been used as
hydrolysable monomers in biodegradable polymers,8 and it is known that the increase in the
size of the substituents on the silicon atom significantly reduces the linker hydrolysis rate
under acidic conditions. Derivative 26 hydrolyzed 4 times slower at physiological pH as
compared to pH 5.5. (Fig. 7 and Fig. S7). Two additional siloxane acetals derived from 26
(where the two phenyl rings in 26 were substituted by two methyl or two n-butyl groups)
were synthesized but could not be purified due to their high instability. Based on a similar
scaffold, acetonide 27 was found to be very unstable and was completely hydrolyzed at t1. In
2004, Hubbell and co-workers were able to show that 3-sulfanylpropionyl or 4-
sulfanylbutyryl esters could be used for oligodesoxynucleotide vectorisation.25 In our study,
β-thiopropionate derivative 28 showed moderate stability at both pH but suffered from poor
selectivity with a ratio of 1.5. Finally, THP-based and carbamate compounds 29 and 30 were
completely stable at both pH as expected.

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Fig. 7 Hydrolysis rates of siloxane, acetal,
β
-thiopropionate, tetrahydropyranyl ether
and carbamate derivatives. Hydrolysis kinetics of compounds 26
– 30 at pH 5.5 and
7.4. (S = Compound stable during the time of the experiment (0% hydrolysis)) R =
(CH2)2-O-(CH2)2-NHCO-C5H6.
Our results reveal that albeit these molecular structures were described to possess good
stability at pH 7.4 and increased lability at pH 5.5, important discrepancies in their relative
profiles could be observed. The hydrolysis rate can change dramatically, not only from one
family to the other, but also within a specific family depending on the substituent pattern of
the tested derivatives. However based on that study it seems possible to categorize the
different structural molecular motif according to their average hydrolysis pattern.
All tested structures have thus been ranked according to the following criteria (Fig. 8):
-
-
Non-hydrolysable when no hydrolysis was observed under our conditions at pH 5.5
Fast hydrolysis when the half-life time at pH 5.5 was below 1 hour.
ꢀ
ꢂ
-
-
-
Highly selective hydrolysis when the ratio ꢀ ꢊꢃ(ꢋꢌ ꢍ.ꢎ) was more than 15.
ꢂ
ꢊ (ꢋꢌ ꢏ.ꢏ)
ꢃ
ꢀ
ꢂ
Selective hydrolysis when the ratio ꢀ ꢊꢃ(ꢋꢌ ꢍ.ꢎ) was between 2 and 15.
ꢂ
ꢊ (ꢋꢌ ꢏ.ꢏ)
ꢃ
ꢀ
ꢂ
Non-selective hydrolysis when the ratio ꢀ ꢊꢃ(ꢋꢌ ꢍ.ꢎ) was below 2.
ꢂ
ꢊ (ꢋꢌ ꢏ.ꢏ)
ꢃ
We then decided to go further in this study and validate if the in-solution reactivity could be
translated to usable data for the design of endosomal release systems. For that purpose, we
decided to incorporate selected acid-labile structures into turn-on FRET-based probes that

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will enable us to measure in-cell endosomal release under standardized and comparable
conditions. More specifically, we chose four derivatives belonging to four different
categories:
-
-
-
An acylhydrazone derivative which hydrolyzes rapidly but not selectively according to
pH.
A dialkyl acetal derived from o-anisaldehyde with kinetic profiles similar to the spiro
di-orthoester already studied but showing an improved selectivity for low pH.
A tetrahydropyranyl ether derivative which showed no hydrolysis in our conditions.
Fig. 8 Classification of pH-sensitive structures depending on rate of hydrolysis at pH
5.5 and selectivity of hydrolysis measured by the ratio of hydrolysis at pH 7.4 vs. pH
5.5.

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Design and synthesis of FRET-type probes
On the basis of the previously reported TAMRA-Orthoester-(BHQ)-2 probe,18 we designed
four FRET-based probes including the acid-sensitive linker between a fluorophore TAMRA,
and a quencher (BHQ)-2 (TAMRA-Acid sensitive linker-(BHQ)-2). It is worth noting that
particular attention has to be taken when purifying the acid sensitive intermediates and final
probes. Synthesis of TAMRA-Acylhydrazone-(BHQ)-2, TAMRA-Orthoester-(BHQ)-2 and
TAMRA-Amide-(BHQ)-2 have been previously reported (Fig. 9).18
Synthesis of TAMRA-THP-(BHQ-2) probe P1
The synthesis started from (3,4-dihydro-2H-pyran-2-yl)methanol 31 which was converted to
carboxylic acid 33 in two steps (Scheme 1). Coupling of 33 with propargylamine gave the
corresponding amide 34 in good yield. Reaction of 34 with alcohol 35 in the presence of
catalytic amount of p-TsOH gave the THP derivative 36. A microwave assisted Click reaction
between alkyne 36 and an azide derivative of BHQ-2 gave 37 which was further
functionalized by Michael addition with TAMRA-SH to give probe TAMRA-THP-(BHQ-2) P1
after careful purification by preparative HPLC (NH3HCO2H/ACN, pH 8.5).
Synthesis of TAMRA-Acetal-(BHQ-2) probe P2
Following the procedure described by Zhang and Zhao,26
o-Anisaldehyde 38 was reacted
with chloroethanolamine at 90 °C to give the corresponding acetal 39 which was further
functionalized to the corresponding bis-azide 40 after treatment with sodium azide. Finally, a
double copper catalyzed cycloaddition of bis-azide 40 with (BHQ-2)-alkyne and TAMRA-
alkyne derivatives26 gave TAMRA-Acetal-(BHQ-2) probe P2 in 10% yield. The yield of the last
reaction was unfavorably low because of solubility issues which favored the formation of
TAMRA-acetal-TAMRA side product in 72% yield.

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Scheme 1 Synthesis of TAMRA-THP-(BHQ)-2 and TAMRA-Acetal-(BHQ-2) probes P1
and P2. Reagents and conditions: a) 32, NaH, THF, 0 °C, 3 h, 77%; b) LiOH, MeOH, H2O,
rt, 5 h, quant.; c) propargylamine, HBTU, DIEA, DMF, rt, 16 h, 71%; d) 35, p-TsOH,
t
CH2Cl2, 0 °C to rt, 2 h, 77%; e) Sodium ascorbate, CuSO4, BuOH, H2O, THF, 4Å
molecular sieves, microwave, 50 °C, 1 h, 74%; f) CH2Cl2, MeOH, Et3N, rt, 2 h, 62%; g)
chloroethanolamine, p-TsOH, benzene, Dean-Stark, 90 °C, 16 h, 40%; h) NaN3, DMF,
80 °C, 16 h, 26%; i) CuSO4, sodium ascorbate, tBuOH, THF, H2O, rt, 3 h, 10%.
Fig. 9 Previously reported acid sensitive FRET based probes18
Effect of pH on hydrolysis of FRET-based probes
Before engaging in-cell studies, we validated that the FRET probes had similar in-solution
hydrolytic profiles to their parent molecules. Kinetic fluorescence measurements were
conducted at pH 3 to 7 to evaluate the stability of the different probes for 15 hours in

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aqueous buffer solution (Fig. 10). For the THP-based probe, no significant hydrolysis was
observed within the range of tested pH. The acetal-based probe P2 was respectively
hydrolyzed at 34 % and 16 % at pH 3 and 4 after 15 hours and showed little hydrolysis at pH
5 and 6. In agreement with the results of the small analogs, the hydrolysis of the
acylhydrazone derivative did not display a strong pH-selectivity; only two different profiles
were observed from pH 3-7. Alternatively, the hydrolysis of the Spiro diorthoester based
probe P5 showed a strong pH-dependency. It was totally and instantly hydrolyzed below pH
3, whereas 1 and 4.5 hours were necessary to get complete hydrolysis at pH 4 and 5,
respectively. At pH 6 and 7, 65 % and 24 % of hydrolysis, respectively, were observed after
15 hours. As expected the general profile of hydrolysis rate is in agreement with data
obtained by HPLC for the corresponding analogs of THP (non hydrolysable), Acylhydrazone
(hydrolysable, non selective), Orthoester and Acetal (hydrolysable, selective).
However, the lability of the pH-sensitive linkers under acidic conditions appears to be much
less important after incorporation into the FRET-based probe. Interestingly, these four
probes showed slightly different profiles (from completely stable to quickly hydrolysable and
from selective to non-selective). The next goal was to reveal which one will be the most
suitable to achieve specific endosomal release.
Fig. 10 Hydrolysis kinetics of FRET-based probes P1
, P2, P4 and P5 at pH 3 (red lines),
4 (green lines), 5 (light blue lines), 6 (dark blue lines) and 7 (purple lines). All data

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were recorded in triplicate at room temperature on a microplate reader and with a
probe concentration of 0.5 µM in buffers (100 mM citric acid/phosphate buffer; pH 3
- 7).
Confocal microscopy and flow cytometry analysis
The biosensitivities of the different probes (P1 – P5) were further studied in tissue cultures
and cells were analyzed by both confocal microscopy to confirm lysosomal cleavage, and
flow cytometry to provide quantitative information about intracellular probe hydrolysis. First
BNL-CL2, a mouse liver cell line, was loaded with the different probes (1 µM, 90 min) and the
nuclei were stained with Hoechst 33258. The cells were imaged by confocal microscopy and
representative images are shown in Fig. 11 TAMRA-Amide-(BHQ-2) probe P3 was used as
non-hydrolysable control. Preliminary control experiments were performed using a small
TAMRA derivative, such as the released reporter generated after hydrolysis of the acid-
sensitive probes. This derivative was incubated in the presence of BNL CL.2 hepatocytes at 1
µM for 2 hours and imaging of the cells revealed an absence of fluorescence compare with
the acid-sensitive probe P5 (Fig. S8). Therefore, the intracellular fluorescence signal can be
directly correlated to the intracellular hydrolysis of the probes, even if extracellular
hydrolysis of the probe happens.
TAMRA-Orthoester-(BHQ-2) and TAMRA-Acylhydrazone-(BHQ-2) probes P5 and P4
displayed strong staining distributed predominantly in the peri-nuclear region of cytoplasm
that are consistent with lysosomal cleavage (Fig. 11).18 To get quantitative evaluation of the
probe hydrolysis, NIH/3T3 cells were incubated with probes P1 – P5 for 4 hours, suspended
in media and analyzed by flow cytometry (Fig. 12). This experiment showed that TAMRA-
Orthoester-(BHQ-2) probe P5 was the most efficient probe followed by TAMRA-
Acylhydrazone-(BHQ-2) probe P4. TAMRA-Acetal-(BHQ-2) probe P2 showed only very little
hydrolysis compared to the non-hydrolysable control probe. These results are in good
agreement with hydrolysis rates measured for probes P1 – P5 in solution (Fig. 10). Indeed
acylhydrazone and orthoester-based probes showed significant hydrolysis at pH 5 and 6
after 1.5 hours while the acetal-based probe showed little to no hydrolysis at pH 5 and 6
after the same period of time. TAMRA-Acetal-(BHQ-2) probe P2 did not shown any lability
with little to no fluorescence observed (Fig. 11). Interestingly, TAMRA-THP-(BHQ-2) probe
P1 show a fluorescence signal similar to the TAMRA-Acylhydrazone-(BHQ-2) probe P4.

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Fig. 11 In-vitro imaging of BNL CL.2 cells loaded with probes P1
– P5; Red channel:
activable probe (1 µM, 90 min), blue channel: Hoechst nuclei staining (5 µg/mL, 30
min); Scale bar: 25 µM.
Fig. 12 Flow cytometry analysis (2000 cells/assay) of NIH/3T3 cells loaded with probes
P1
– P5 (1 µM, 4h). One-way ANOVA using Dunnett’s post-test were performed to
compare all probes to the control amide and the orthoester to the acylhydrazone; *p
< 0.05, ***p < 0.001.

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Conclusion
In this study, the hydrolytic cleavage of a wide variety of molecular structures that were
reported for their use in pH-sensitive delivery systems was carefully examined. Standardized
data measured by HPLC enabled the establishment of a first classification of these groups
based on their hydrolysis profiles at pH 5.5 and their relative hydrolysis at pH 5.5 vs. pH 7.4.
From this classification four chemical functions belonging to three different categories were
selected and used as core motifs to design FRET-based probes. These probes were used to
quantify and compare the lysosomal cleavage of the different acid-sensitive structures
(orthoester, acylhydrazone, acetal and tetrahydropyranyl ether).
Surprisingly, a wide variety of hydrolytic stability profiles could be found among the chemical
functionality supposedly suitable for pH release systems. Even within a structural family, a
slight modification of the substitution pattern could have an unsuspected outcome on the
hydrolysis stability. Furthermore, we also showed that incorporating these small motifs in
larger molecular constructs slowed down their hydrolysis without modifying their relative
reactivity. These observations underline the need to carefully design a reporting probe to
validate the actual stability of the cleavable group in the environment of the delivery system.
Our tests also showed that only the most reactive functional groups underwent significant
lysosomal cleavage according to flow cytometry measurements. Interestingly, these are the
motifs that proved to be very difficult to synthesize and to store. They showed almost
complete hydrolysis in our HPLC assay after 30 seconds. These last results question the
validity of the claimed acid-based mechanism of action of some described drug release
systems. We believe that structure-reactivity study and the methodology assessing in-cell
hydrolysis of the main functional groups will provide a stimulating insight for the rational
design and development of improved acid-sensitive linkers.
Acknowledgments
This work was supported through a research contract funded by the French Ministry of
Research. The International Center for Frontier Research in Chemistry (icFRC) is
acknowledged for financial support to LFCS.

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Notes and references
1. J. Rautio, H. Kumpulainen, T. Heimbach, R. Oliyai, D. Oh, T. Järvinen, J. Savolainen,
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