Flow and Microwave-Assisted Synthesis of N-(Triethylene glycol)glycine Oligomers and Their Remarkable Cellular Transporter Activities

Article abstract of DOI:10.1021/acs.bioconjchem.5b00307

Peptidomimetics, such as oligo-N-alkylglycines (peptoids), are attractive alternatives to traditional cationic cell-penetrating peptides (such as R₉) due to their robust proteolytic stability and reduced cellular toxicity. Here, monomeric N-alkylglycines, incorporating amino-functionalized hexyl or triethylene glycol (TEG) side chains, were synthesized via a three-step continuous-flow reaction sequence, giving the monomers N-Fmoc-(6-Boc-aminohexyl)glycine and N-Fmoc-((2-(2-Boc-aminoethoxy)ethoxy)ethyl)glycine in 49% and 41% overall yields, respectively. These were converted into oligomers (5, 7, and 9-mers) using an Fmoc-based solid-phase protocol and evaluated as cellular transporters. Hybrid oligomers, constructed of alternating units of the aminohexyl and amino-TEG monomers, were non-cytotoxic and exhibited remarkable cellular uptake activity compared to the analogous fully TEG or lysine-like compounds.

Full text of DOI:10.1021/acs.bioconjchem.5b00307

Article
pubs.acs.org/bc
Flow and Microwave-Assisted Synthesis of N‑(Triethylene
glycol)glycine Oligomers and Their Remarkable Cellular Transporter
Activities
ThingSoon Jong, Ana M. Perez-Lopez, Emma M. V. Johansson, Annamaria Lilienkampf,
́
́
and Mark Bradley*
School of Chemistry, EaStCHEM, University of Edinburgh, Joseph Black Building, King’s Buildings, West Mains Road, EH9 3FJ
Edinburgh, United Kingdom
S
* Supporting Information
ABSTRACT: Peptidomimetics, such as oligo-N-alkylglycines (pep-
toids), are attractive alternatives to traditional cationic cell-penetrating
peptides (such as R9) due to their robust proteolytic stability and
reduced cellular toxicity. Here, monomeric N-alkylglycines, incorporat-
ing amino-functionalized hexyl or triethylene glycol (TEG) side chains,
were synthesized via a three-step continuous-flow reaction sequence,
giving the monomers N-Fmoc-(6-Boc-aminohexyl)glycine and N-
Fmoc-((2-(2-Boc-aminoethoxy)ethoxy)ethyl)glycine in 49% and 41%
overall yields, respectively. These were converted into oligomers (5, 7,
and 9-mers) using an Fmoc-based solid-phase protocol and evaluated as
cellular transporters. Hybrid oligomers, constructed of alternating units
of the aminohexyl and amino-TEG monomers, were non-cytotoxic and
exhibited remarkable cellular uptake activity compared to the analogous
fully TEG or lysine-like compounds.
therefore restricting the formation of secondary structures
based on backbone hydrogen bonding.29,30 Cationic peptoids
have highly efficient cell delivery profiles and are nontoxic in
vivo,31−33 making them attractive candidates for cellular
delivery.
INTRODUCTION
■
Cellular delivery of chemical and biological cargos is of great
importance in biology and medicine;1−3 however, cellular
access is often limited by cargo solubility and physico−chemical
properties.4,5 To overcome these problems, a number of
cellular delivery techniques have been developed,6−9 including
Peptoids are typically synthesized via solid-phase synthesis
using N-substituted N-Fmoc-glycine monomers in an
Fmoc/tBu type strategy.34,35 However, this “monomer”
approach requires the multistep synthesis of N-Fmoc-glycine
monomers, which are conventionally performed in batch. Flow
chemistry36−38 enables the integration of individual synthetic
steps into a linear sequence to improve productivity and
synthetic efficiency.39−43 This is mainly due to improved
physical transport phenomena during chemical reactions as a
result of the intrinsic properties of flow reactors (10−1000 μm
inner dimensions).44−46 Recently, we reported the efficient
flow-mediated synthesis of Boc, Fmoc, and Ddiv monopro-
tected aliphatic diamines, highlighting enhanced reaction
selectivity in a tubular flow reactor.47
the use of protein-derived cell-penetrating peptides10−12
12,13
(CPPs) such as those based on Tat49−57
,
Penetratin,14
and Transportan.15 CPPs are capable of translocating across the
cell membrane due to their interaction with the plasma
membrane, with a host of mechanistic uptake possibilities
hypothesized: including endocytosis, direct translocation, and
“surface rafts”.16−18 Polylysines and polyarginines have been
shown to increase cellular uptake of a number of molecules
(e.g., methotrexate and cyclosporine A),19,20 with the 9-mer of
L-arginine and D-arginine reportedly being 20- and >100-fold
more efficient than HIV Tat49−57, respectively.21
Although the aforementioned CPPs work relatively well, they
can suffer from cytotoxicity (a common issue with poly-
guanidinium motifs) and cargos often remain trapped in
endosomes.22 In order to improve cellular delivery alongside
increased serum stability, peptidomimetics such as oligo-N-
alkylglycines (commonly referred to as peptoids) have been
investigated as alternatives to traditional CPPs.23−28 Peptoids
offer structural flexibility due to the absence of chirality and
hydrogen bond donors on their backbone, while the presence
of the tertiary amides decreases the polarity of the oligomer,
Here, in order to improve the efficiency of peptoid synthesis,
a scalable continuous flow procedure for the monomer N-
Fmoc-(6-Boc-aminohexyl)glycine 1 and its triethylene glycol
(TEG) analogue N-Fmoc-((2-(2-Boc-aminoethoxy)ethoxy)-
Received: June 1, 2015
Revised: June 30, 2015
© XXXX American Chemical Society
A
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Chart 1. “Hybrid” (H5, H7, and H9), TEG (T5, T7, and T9), and “Lysine-Like” (L5, L7, and L9) Peptoids, and the Structure of
the Monomers, N-Fmoc-(6-Boc-aminohexyl)glycine 1 and N-Fmoc-((2-(2-Boc-aminoethoxy)ethoxy)ethyl)glycine 2, Used in
a
Their Solid-Phase Synthesis
a
The peptoids were synthesized and isolated as a mixture of 5(6)-isomers of carboxyfluorescein.
ethyl)glycine 2 is reported. These two monomers were used to
synthesize a new class of “hybrid peptoids”, constructed from
alternating units of the aminohexyl and amino-TEG based
monomers 1 and 2, with a focus on optimizing cellular uptake
(Chart 1). It is known that the amphiphilicity of typically highly
cationic peptoids influences their cell permeability and
subcellular localization,48,49 and the current designs of new
cellular transporters involves achieving a delicate balance
between hydrophilic and hydrophobic structural compo-
nents.50−52 Here, the TEG moiety was introduced as a peptoid
side chain to improve solubility, hence potentially enhancing
their cellular uptake.53−55 These new “hybrid peptoids” (H5,
H7, and H9) showed significantly increased cellular uptake
compared to the traditional lysine-like peptoids (L5, L7, and
L9).
Continuous Flow Synthesis of Peptoid Monomers.
The four-step flow synthesis of the peptoid monomers 1 and 2
was adapted from the batch route and performed in a self-
assembled flow system, which incorporated PTFE tubular
reactors, packed scavengers, and catalyst (SI, Figure S1).
Monomer 1 was used as an optimization target and the
developed route was applied to the synthesis of the TEG
monomer 2. As reported previously, the mono-Boc carbama-
tion of 1,6-diaminohexane under flow conditions gave the
monoprotected product 3 in 61% yield,47 which was used as a
building block in a series of flow reactions. The next two steps,
monoalkylation and Fmoc carbamation, were linked into a
tandem sequence using in-line scavengers, followed by a
transfer hydrogenolysis using an immobilized Pd catalyst.
Flow Mediated Monoalkylation and Fmoc Carbama-
tion. Alkylation of 3 with 1 equiv of benzyl 2-bromoacetate was
explored by varying the concentration, solvent, and base (Table
1). The residence time and temperature were set at 3 min and
100 °C, respectively; as a preliminary screen showed that a
longer residence time (5 min) resulted in a lower conversion to
4, and a temperature range between 90 and 100 °C was well
tolerated (SI, Figure S2). The highest conversion to
monoalkylated 4 (85%) was observed using 40 mM of 3 and
3.0 equiv of DIPEA in MeCN (working pressure <2 bar).
Changing the solvent to DMF reduced both the conversion to
4 and the monoselectivity of the reaction (entries a vs b, Table
1), as did replacing DIPEA with piperidine (PIP), DBU, or
TEA (DBU and PIP gave dismal conversion) (entries c−e,
Table 1). A lower conversion (70% and 69%, respectively) was
detected at a higher reactant concentration (entry g, Table 1)
and when the starting materials were not preheated before
mixing (entry f, Table 1), emphasizing the importance of
reactant preconditioning.
RESULTS AND DISCUSSION
■
The monomer N-Fmoc-(6-Boc-aminohexyl)glycine 1, which is
used to synthesize the lysine-like peptoids, is typically
synthesized in batch from 1,6-diaminohexane in four steps.34
Mono-Boc protection of 1,6-diaminohexane with Boc2O gives
N-Boc-1,6-diaminohexane 3, which is followed by N-alkylation
with benzyl 2-bromoacetate to give benzyl 2-[(6-{[(tert-
butoxy)carbonyl]amino}hexyl)amino]-acetate 4. Fmoc carba-
mation of the secondary amine in 4 with Fmoc-OSu (to give 5)
and subsequent catalytic hydrogenolysis gives monomer 1. A
key challenge of this route is to achieve a high degree of
reaction selectivity for the first two steps, which can be hard to
achieve in conventional batch synthesis due to system
inhomogeneity. Similarly, the hydrogenolysis of the benzyl
protecting group in 5 can be adversely affected by a competing
side reaction resulting in cleavage of the Fmoc group.56,57
B
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Fmoc-Cl. Here, 15 mM of Fmoc-Cl (1.2 equiv) in MeCN was
used, the concentration adjusted based on the 85% conversion
into 4 during the first reaction step. With a residence time of 3
min for both reactions, 4.9 mmol of 3 gave a 69% isolated yield
of 5 over two steps (Scheme 1).
Table 1. Optimization of the Flow-Mediated Mono-
Alkylation Reaction of N-Boc-1,6-diaminohexane.
a
Flow Mediated Catalytic Transfer Hydrogenolysis.
The final step of the monomer synthesis was the catalytic
hydrogenolysis of the benzyl ester moiety to give the
corresponding carboxylic acid. Batch synthesis of 1 demon-
strated that the Fmoc protecting group in 5 is (partially) labile
toward Pd-catalyzed hydrogenolysis, resulting in a mixture of
products depending on the reaction conditions and activity of
the catalyst. The facile manipulation of contact time between
the active catalyst and the substrate in a flow system was
expected to promote reaction selectivity and thus reduce the
occurrence of the Fmoc deprotection. With the aim of
improving the scope of hydrogenolysis reactions in flow, the
transfer hydrogenolysis strategy was chosen over the more
commonly used H2. Flow transfer catalytic hydrogenolysis of 5
was achieved by passing the reactants through an immobilized
catalyst (3.0 × 0.4 cm, loading ∼150 mg/cartridge), with the
reaction screened for the optimal catalyst, substrate concen-
tration, and temperature, using 2.5 equiv of 1,4-cyclohexadiene
as the hydrogen donor. The use of ammonium formate as a
hydrogen donor was incompatible with the flow system
resulting in fluctuations of operating pressure. Four different
catalysts (10% Pd/C, 20% Pd(OH)2/C, 5% Pt/C, and 5% Ru/
Al2O3) and three substrate concentrations (15, 30, and 50 mM)
were tested (Table 2). 10% Pd/C as a catalyst required heating
to 40 °C to give good conversion to 1 with 15 mM being the
optimal reactant concentration (entry b, Table 2); however, at
the elevated temperature, conversion to the Fmoc deprotected
side product 7 was also observed (entries b−d, Table 2).
Furthermore, this catalyst proved unsuitable for larger-scale
reactions (3.4 mmol of 5) as saturation of the cartridge was
observed after 20 min (∼14% of total reaction progress) along
with unstable working pressures (fluctuating between 9 and 11
bar).
b
c
entry
solvent
base
conc 3
4
(%)
69
ratio 4:6
a
DMF
DIPEA
DIPEA
PIP
40 mM
40 mM
40 mM
40 mM
40 mM
40 mM
100 mM
1:0.16
1:0.10
−
b
c
MeCN
MeCN
MeCN
MeCN
MeCN
MeCN
85
6
d
e
DBU
10
69
69
70
−
TEA
1:0.06
1:0.08
1:0.11
d
f
DIPEA
DIPEA
g
a
20 μmol scale, 1 equiv of benzyl 2-bromoacetate, flow rate 0.67 mL/
min. Average conversion measured by HPLC with UV detection at
254 nm (n = 3, variation in conversions within 3%), with methyl
benzoate used as an internal standard. Integrated peak ratio of 4 to 6.
b
c
d
No preheating of reactants before mixing.
The single step Fmoc carbamation of 4 was achieved at room
temperature using 1.0 equiv of Fmoc-Cl (both reagents at 20
mM in MeCN) with a residence time of 3 min to give the
Fmoc-protected benzyl acetate 5 in 98% isolated yield. Fmoc-
Cl was chosen due to its better reactivity compared to Fmoc-
OSu, especially with sterically hindered secondary amines.58
Using the optimized parameters for the alkylation and Fmoc
carbamation reactions, the two steps were joined into a
cascading flow sequence. The unreacted alkylating reagent from
the first reaction stream was removed prior to the Fmoc
carbamation step by directly connecting the exiting stream from
the alkylation reaction of 3 (immediately after the back-
pressure regulator) to a scavenging column (6.0 × 0.5 cm),
packed with 0.6 g of Trisamine-Si (loading 1.58 mmol/g).
Based on HPLC analysis, Trisamine-Si was more effective in
removing excess benzyl 2-bromoacetate than Thiol-Si scav-
engers. After the scavenging column, the reaction stream was
cooled to room temperature prior to the mixing of 4 with
Pearlman’s catalyst (20% Pd(OH)2/C) produced the best
results with ≥70% conversion across the whole range of
Scheme 1. Continuous Flow Synthesis of N-Fmoc-(6-Boc-aminohexyl)glycine 1 and N-Fmoc-((2-(2-Boc-
a
aminoethoxy)ethoxy)ethyl)glycine 2
a
The continuous flow protocol was completed in three key steps with 49% and 41% overall yields for 1 and 2, respectively.
C
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the hydrogenolysis of monomer 1 was achieved in 70% isolated
yield (overall yield 49% over three steps) (Scheme 1). The
combined reaction time (∼10 h, excluding purifications)
required to synthesize 1 by this flow method was approximately
four times faster than that typically required for the
corresponding batch method.
Table 2. Catalyst Screening for the Flow-Mediated O-Bn
Hydrogenolysis
a
Based on the optimized continuous flow procedures for
monomer 1, the TEG-based monomer 2 was synthesized using
the same experimental setup using mono-Boc protected 2,2′-
(ethylenedioxy)bis(ethylamine)47 8 as the starting material.
The alkylation and Fmoc-carbamation sequence (4.9 mmol
scale) gave the protected intermediate 9 (62% isolated yield)
and the subsequent debenzylation produced monomer 2 in
68% yield (41% overall yield over three steps) (Scheme 1). The
synthetic yields for the TEG monomer 2 were comparable to
the yields obtained with the lysine-like monomer 1, thus
demonstrating the consistency of the continuous flow protocol.
Design and Synthesis of Hybrid Peptoids. In an effort
to correlate the cellular penetration efficiency of molecular
transporters with their structural characteristics, new cationic
“hybrid peptoids”, featuring an alternating sequence of
monomers 1 and 2, were synthesized and compared to
lysine-like peptoids and to peptoids constructed solely from
TEG-monomer 2. Three different oligomer lengths (5-, 7-, and
9-mer) of each peptoid type were synthesized to allow direct
comparison of their cellular uptake.
b
c
entry
catalyst
5 (mM) temp (°C)
1 (%) ratio 1:7
a
b
c
d
e
f
10% Pd/C
15
15
30
50
15
30
50
15
15
rt
23
nd
10% Pd/C
40
40
40
40
40
40
40
40
78
69
54
96
82
70
−
1:0.25
1:0.15
1:0.10
1:0.00
1:0.08
1:0.10
−
10% Pd/C
10% Pd/C
20% Pd(OH)2/C
20% Pd(OH)2/C
20% Pd(OH)2/C
5% Pt/C
g
h
i
5% Ru/Al2O3
−
−
a
7.5 μmol scale, 2.5 equiv 1,4-cyclohexadiene, flow rate 1 mL/min.
Average conversion measured by HPLC with UV detection at 254
nm (n = 3, variation in conversions within 3%), with aniline used as
an internal standard. Integrated peak ratio of 1 to 7.
b
c
substrate concentrations tested (15−50 mM). At 15 mM of 5,
no Fmoc cleavage was detected with 96% conversion to 1
(entry e, Table 2) along with a low and stable system pressure
(1 bar). For both Pd catalysts, reduced conversion was
observed as the concentration of substrate was increased
from 15 to 50 mM. 5% Pt/C and 5% Ru/Al2O3 were
unsuccessful in flow transfer hydrogenolysis despite their ability
to promote the debenzylation process in batch chemistry.59
Using 20% Pd(OH)2/C and 2.5 equiv of 1,4-cyclohexadiene,
The hybrid peptoids H5, H7, and H9 and TEG peptoids T5,
T7, and T9 were synthesized on a Rink-amide functionalized
aminomethyl polystyrene resin (1% DVB, 100−200 mesh,
loading 1.23 mmol/g) using the Fmoc/tBu-strategy with DIC/
Oxyma as coupling reagents (SI, Scheme S1). The synthesis of
Figure 1. Time-dependent cellular uptake of lysine-like, TEG, and hybrid peptoids in HeLa, HEK293T, and CHO cells (y-axis represents relative
fluorescence intensity (RFI) and x-axis incubation time in hours). The cells were incubated with 10 μM of each peptoid for 2, 4, and 24 h at 37 °C (n
= 3) and fluorescence intensity measured by flow cytometry (λEx/λEm 488/530 nm). Prior to the analysis, the samples were treated with Trypan Blue
to quench extracellular fluorescence.
D
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peptoids T5, T7, and T9, containing only the TEG-based
monomer 2, proved challenging with the coupling reactions
requiring microwave (μw) heating at 75 °C for 25 min.
Similarly, the subsequent Fmoc deprotection steps (20% PIP in
DMF) with T5, T7, and T9 required μw heating at 60 °C (2 ×
10 min). The same μw assisted deprotection procedure was
successful with the hybrid peptoids H5, H7, and H9 up to
heptamer sequences on the resin, after which the Fmoc
deprotection required treatment with DBU−PIP (2% DBU and
2% PIP in DMF for 60 °C μw for 10 min, followed by 4% DBU
and 4% PIP for 10 min). The lysine-like peptoids L5, L7, and
L9 were synthesized in similar fashion with deprotection at
room temperature using 20% PIP in DMF (2 × 10 min).34 6-
Aminohexanoic acid (εAhx) as a spacer and 5(6)-carboxy-
fluorescein were consecutively coupled to the N-terminus of all
the peptoids to allow evaluation of their cellular uptake. After
deprotection and cleavage from the resin (TFA/TIS/H2O), the
peptoids were purified by semipreparative HPLC and analyzed
by MALDI-TOF MS and HPLC (SI, Table S1). The hybrid
peptoids were isolated in low yields (5−12%) compared to the
other peptoid types, which may be attributed to the aggregation
of the peptoid chains in the sterically demanding, resin-bound
environment.
shows fluorescent images of HeLa incubated with H7 at 1 μM.
Similarly to HEK293T, H9 outperformed L9 and T9.
Interestingly, with CHO cells, the highest cellular uptake of
H9 was only observed after 24 h of incubation.
To better establish the effect of the hybrid structure, the
cellular uptake of H9, L9, and T9 were directly compared. After
2 h incubation at 1 and 10 μM, the cells were analyzed by flow
cytometry. Based on the fluorescence (λEx/λEm 488/530 nm)
intensity of the cells, H9 showed notably better uptake in all
three cell lines at both concentrations when compared to L9
and T9 (Figure 3), with L9 outperforming T9. Overall, the
Cellular Uptake of Hybrid Peptoids. The cellular uptake
of the fluorescein-tagged hybrid peptoids (H5, H7, H9) was
evaluated with human embryonic kidney (HEK293T), human
cervical carcinoma (HeLa), and Chinese hamster ovary (CHO)
cell lines, and compared with the lysine-like peptoids (L5, L7,
L9) and TEG peptoids (T5, T7, T9). The cells were incubated
with peptoids at 10 μM and analyzed at 2, 4, and 24 h by flow
cytometry (Figure 1).
With all peptoid types, the uptake was dependent on the
number of monomer units with longer oligomers yielding
greater cellular uptake as shown by the fluorescence intensity,
which increased over time in a nonlinear manner (with rapid
accumulation within the first 4 h), suggesting an active
transport mechanism.
Figure 3. (A) Flow cytometry analysis (λEx/λEm 488/530 nm) of
cellular uptake of nonamer peptoids L9, T9, and H9 in HEK293,
HeLa, and CHO cells after 2 h incubation at 1 and 10 μM (n = 6). (B)
Representative flow cytometry histograms of cells treated with 10 μM
of H9. Control populations are shown in dark gray, cells treated with
H9 in green (x-axis represents relative fluorescence intensity).
A clear difference in the level of uptake was observed with
different cells lines, with CHO cells demonstrating notably
higher uptake than HeLa and HEK293T with all peptoid types.
In HeLa cells, which showed the lowest uptake of the three cell
lines, the hybrid nonamer H9 exhibited the highest cellular
uptake, with 4-fold higher fluorescence intensity after 4 h
compared to H7 (Figure 1). After 24 h, a decrease in the
fluorescence was observed with the cells incubated with H9
suggesting saturation of the cells and efflux of H9 during
prolonged incubation, a phenomenon that was not observed
with H5 and H7, which exhibited lower initial uptake. Figure 2
hybrid nonamer H9 was the most efficient peptoid with ∼100%
of the cell populations labeled within 2 h at low μM
concentrations, whereas TEG-based peptoids exhibited the
lowest uptake. The hybrid peptoids H5, H7, and H9 were non-
cytotoxic, with cell viability ≥96% at 10 μM in PI assays (SI,
Figure S3).
CONCLUSIONS
■
To enable the efficient solid-phase synthesis of peptoids by the
“monomer” approach, a flow-based method for the gram-scale
synthesis of peptoid monomers, N-Fmoc-(6-Boc-aminohexyl)-
glycine and its ethylene glycol analogue N-Fmoc-((2-(2-Boc-
aminoethoxy)ethoxy)ethyl)glycine, was developed. N-Boc
monoprotected diamines, synthesized in flow, were selectively
N-alkylated with benzyl 2-bromoacetate, directly followed by
Fmoc carbamation of the secondary amine under flow
conditions. Finally, flow transfer hydrogenolysis, using 20%
Pd(OH)2/C as a catalyst and 1,4-cyclohexadiene as the
hydrogen donor, resulted in excellent yields of the debenzylated
products without notable Fmoc cleavage. This flow procedure
offers a continuous mode of processing, which is highly
attractive due to its scalability potential, and allows gram-scale
access to peptoid monomers. These monomers were used to
Figure 2. Confocal images of HeLa cells incubated with 1 μM of
hybrid peptoid H7 for 12 h. The cells were fixed, nuclei were stained
with Hoechst 33342, and the cells imaged (λEx 407 nm for Hoechst
33342 and 488 nm for fluorescein). (A) λEx 488 nm. (B) λEx 407 nm.
(C) Composition of fluorescence images at λEx 407 and 488 nm.
E
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* Supporting Information
Supporting figures and schemes, and experimental procedures
and analytical data for all compounds are included. The
Supporting Information is available free of charge on the ACS
Publications website at DOI: 10.1021/acs.bioconj-
chem.5b00307.
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AUTHOR INFORMATION
(2011) Mechanisms of cellular uptake of cell-penetrating peptides. J.
Biophys. 2011, 1−10.
■
Corresponding Author
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*E-mail: mark.bradley@ed.ac.uk.
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS
■
We thank the MTEM School of Chemistry Studentship for
funding TSJ, Caja Madrid and Ramon Areces Foundation for
funding AMPL, and University of Edinburgh (Innovation
Initiative Grant) for financial support.
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