Form Matters: Stable Helical Foldamers Preferentially Target Human Monocytes and Granulocytes

Article abstract of DOI:10.1002/cmdc.201600597

Some hybrid foldamers of various length, all containing the (4R,5S)-4-carboxy-5-methyloxazolidin-2-one (d-Oxd) moiety alternating with an l-amino acid (l-Val, l-Lys, or l-Ala), were prepared in order to study their preferred conformations and to evaluate their biological activity. Surprisingly, only the longer oligomers containing l-Ala fold into well-established helices, whereas all the other oligomers give partially unfolded turn structures. Nevertheless, they all show good biocompatibility, with no detrimental effects up to 64 μm. After equipping some selected foldamers with the fluorescent tag rhodamine B, a quantitative analysis was performed by dose– and time–response fluorescence-activated cell sorting (FACS) assays with human HeLa cells and primary blood lymphocytes, granulocytes, and monocytes. Among the cell types analyzed, the oligomers associated with monocytes and granulocytes with greatest efficacy, still visible after 24 h incubation. This effect is even more pronounced for foldamers that are able to form stable helices.

Full text of DOI:10.1002/cmdc.201600597

Accepted Article
Title: Form Matters: Stable Helix Foldamers Preferentially Target
Human Monocytes and Granulocytes
Authors: Claudia Tomasini, Nicola Zanna, Lorenzo Milli, Benedetta Del
Secco, Andrea Cornia, Regina Tavano, Emanuele Papini, and
Giulia Malachin
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the VoR from the journal website shown below when it is published
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To be cited as: ChemMedChem 10.1002/cmdc.201600597
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Form Matters: Stable Helical Foldamers Preferentially Target
Human Monocytes and Granulocytes
Benedetta Del Secco,[a] Giulia Malachin,[b] Lorenzo Milli,[a] Nicola Zanna,[a] Emanuele Papini,[b] Andrea
Cornia,*[c] Regina Tavano,*[b] Claudia Tomasini* [a]
Abstract: Some hybrid foldamers of different length all containing
the D-Oxd moiety alternated with a L-amino acid (L-Val, L-Lys or L-
Ala) have been prepared to study their preferred conformations and
to evaluate their biological activity. Surprisingly, only the longer
oligomers containing L-Ala fold into well-established helices, while all
the other oligomers give partially unfolded turn structures.
Nevertheless, they all show a good biocompatibility, with no
detrimental effects up to 64 µM concentration. After equipping some
selected foldamers with the fluorescent tag Rhodamine B, a
quantitative analysis was obtained by dose and time response
analysis by FACS of human HeLa cells, and primary blood
lymphocytes, granulocytes and monocytes. Among the analyzed cell
types, these oligomers associated with monocytes and granulocytes
with best efficacy, still visible after 24 h incubation. This effect is
even more pronounced for foldamers able to form stable helices.
peptide that is covalently coupled to a fluorophore. Its
measurement by fluorescence (fluorimetry) enables the peptide
indirect quantification and confocal microscopy allows its
localization inside living cells.
Rhodamine dyes are well known synthetic molecules used for
fabrics dyeing and belong to the class of xanthene dyes.[2]
These fluorescent markers are used as fluorescent probes in
biological studies, in structural microscopic studies,
photosensitizers and laser dyes. They may be successfully
introduced in synthetic structures,[3] called “foldamers”.[4,5] This
neologism refers mainly to medium-sized molecules (about 500–
5000 Dalton) that fold into definite secondary structures (i.e.,
helices, turns and sheets), thus being able to mimic
[6,7]
biomacromolecules despite their smaller size. Applications
have ranged from cellular penetration
[8]
and membrane
disruption to discrete molecular recognition.[9,10] Hybrid
foldamers, containing the (4R,5S)-4-carboxy-5-methyl-
oxazolidin-2-one (D-Oxd) unit alternated with an α- or β-amino
acid, have been extensively studied by our research group,[11] as
the D-Oxd moieties impart rigidity to the peptide chain,[12–14]
while the L-amino acids promote the folding.[15–17]
In the past, the Gellman group suggested that the formation of a
globally amphiphilic helix is not required for host-defense
peptide mimicry,[18] as also amphiphilic conformations of random
Introduction
The understanding of sophisticated interplays between structure
and function of molecules within complex systems is a great
challenge in life sciences. Selective labeling of biomolecules
with bio-physical probes in principle allows for investigation and
manipulation of proteins, enzymes or biochemical processes in
vitro as well as in vivo.[1] Various biological and biophysical
methods may be used to study the internalization mechanism, to
localize and to quantify the peptides internalization together with
their cargoes inside cells. Yet, any method has its pitfalls, so a
combination of different approaches is often needed to have
access to the large pictures.
copolymers without any helical structure were proven to exhibit
[19,20]
potent antibiotic activity. Other studies
demonstrated that
only the Aib-based foldamers forming a precise helical-spherical
domain can target human monocytes, macrophages and
dendritic cells more effectively than non-immune cells. In this
work we describe some hybrid foldamers of different length all
containing the D-Oxd moiety alternated with a L-amino acid (L-
Val, L-Lys or L-Ala), to check any possible relationship between
their preferred conformations and their behavior towards
leukocytes.
The foldamers were functionalized with Rhodamine B (RB)
(Figure 1) in order to measure their interactions with cells by
fluorescence techniques.[21] For an efficient synthesis of all the
conjugates, we developed a cheap and straightforward protocol
to introduce RB in a polypeptide chain, based on a microwave-
assisted reaction.
The fluorescence-based protocol requires the preparation of a
[a]
[b]
[c]
B. Del Secco, Dr. L. Milli, Dr. N. Zanna, Prof. C. Tomasini
Dipartimento di Chimica Ciamician
Alma Mater Studiorum Università di Bologna
Via Selmi 2, 40126 Bologna (Italy)
E-mail: claudia.tomasini@unibo.it
Dr. G. Malachin, Prof. E. Papini, Dr. R. Tavano
Dipartimento di Scienze Biomediche
Università di Padova
Viale G. Colombo 3, 35121 Padova (Italy)
E-mail: regina.tavano@unipd.it
Prof. A. Cornia
Dipartimento di Scienze Chimiche e Geologiche
Università di Modena e Reggio Emilia & INSTM
Via G. Campi 103, 41125 Modena (Italy)
E-mail: acornia@unimore.it
Supporting information for this article is given via a link at the end of
the document.
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Figure 1. General structure of the foldamers described in this work.
Results and Discussion
D-Oxd-OBn was obtained in high yield starting from D-Thr in
multigram scale,[17] and coupled with N-protected L-Val or L-Lys
in the presence of HBTU [O-(benzotriazol-1-yl)-N,N,N′,N′-
tetramethyluronium hexafluorophosphate] and DIEA (di-
isopropyl ethyl amine) to prepare 1 and 2 that were used to
access complex foldamers (for all the synthetic details, see SI)
(Scheme 1). These amino acids have been chosen as they have
very different polarity, being L-Val a lipophilic amino acid, while
L-Lys is a basic and hydrophilic amino acid. Additionally, they
have been extensively used in the past for the preparation of
bioactive peptides.[18,22,23]
Scheme 2. Reagents and Conditions: (i) H2, Pd/C (10%), MeOH, r.t., 16 h; (ii)
TFA (18 equiv.), dry CH2Cl2, r.t., 4 h; (iii) HBTU (1.1 equiv.), DIEA (2 equiv.),
dry CH3CN, 1 h, r.t.; (iv) Pd/C (10%), 11 M HCl, dry MeOH, 4 h, r.t; then 2,2,2-
trifluoroethanol, dry CH2Cl2, 3 h, r.t.
Using the same deprotection technique, we synthesized the
foldamers HCl.H-(L-Ala-D-Oxd)n-OH (n = 2, 4, 6) 9, 10 and 11,
starting from the already reported foldamers Boc-(L-Ala-D-
Oxd)n-OBn.[15]
The biocompatibility of fully deprotected oligomers of both series
was checked. HeLa cells were seeded and incubated for 24h
with compounds 4, 6, 8, 9, 10 and 11 at different concentrations,
then cells were washed, MTS solution was added and the
absorbances were read by an ELISA reader after 30 minute
incubation. All tested oligomers have demonstrated to be non-
toxic up to 64 µM concentration and after a 24 hours incubation
to HeLa cells, irrespective of length and charge (Figure 2). This
outcome suggests a good biocompatibility of our foldamers
formulations.
Scheme 1. Reagents and Conditions: (i) HBTU (1.1 equiv.), DIEA (2 equiv.),
dry CH3CN, 1 h, r.t.
Compound 2 was prepared using Boc-L-Lys(2Cl-Z)-OH, as the
2Cl-Z group is stable towards hydrogenolysis under mild
conditions (Scheme 1). Complex foldamers 3, 5 and 7 and the
corresponding fully deprotected oligomers 4, 6 and 8 have been
prepared in solution alternating the two moieties (Scheme 2).
For the biological essays, trifluoracetate counterions should be
avoided, so the deprotection of compounds 4, 6 and 8 with
trifluoroacetic acid was replaced with a protocol envisaging
hydrochloric acid,[24] that afforded fully deprotected oligomers as
hydrochloride salts in high yield.
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Figure 2. MTS assay performed on the fully deprotected foldamers HCl.H-(L-
Val-D-Oxd-L-Lys-D-Oxd)n-OH 4, 6 and 8 and HCl.H-(L-Ala-D-Oxd)n-OH 9, 10
and 11. Percentages of alive cells were obtained with respect to non-treated
cells. Results are representative of one experiment run in triplicate.
Synthesis and Biological Evaluation of the Rhodamine B
Containing Oligomers
To gain a deeper insight into the influence of the foldamer nature
on cellular uptake, foldamers 4, 8, 9 and 11 have been coupled
with a fluorescent moiety. They differ for chain length (4 and 9
are short, while 8 and 11 are long oligomers) and for the amino
acid choice, as 4 and 8 contain L-Val and L-Lys, while 9 and 11
contain only L-Ala. To avoid the use of expensive fluorescein
isothiocyanate,[25] we developed a new method to couple our
foldamers with the cheap RB.
By modification of a well-known procedure,[26,27] we added RB
base 12 to piperazine-trimethyl aluminium in toluene (Scheme 3).
The reaction mixture was stirred under microwave irradiation at
50 W for 1 h, then acidified, filtered and purified by crystallization.
The resulting derivative piperazine-Rhodamine B (P-RB) 13 was
obtained in excellent yield and was then coupled with Boc-L-Ala-
OH in the presence of HATU [1-[Bis(dimethylamino)methylene]-
1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate]
and DIEA in high yield to afford pure Boc-L-Ala-P-RB 14 after
flash chromatography. The labelled derivatives 16, 18, 20 and
22 have been obtained by standard coupling reaction in solution
with satisfactory overall yields (Scheme 4).
Scheme 4. Reagents and Conditions: (i) H2, Pd/C (10%), AcOEt, r.t., 2 h ; (ii)
TFA (18 equiv.), dry CH2Cl2, r.t., 4 h; (iii) HATU (1.1 equiv.), DIEA (3 equiv.),
dry CH3CN, 1 h, r.t.; (iv) H2, Pd/C (10%), 12M HCl (4 equiv.), AcOEt, r.t., 2 h.
Initially we performed a qualitative analysis of the ability of our
foldamers to target human monocytes purified from buffy coats
using confocal microscopy. Data indicated that all the analyzed
Rhodamine-labelled analogues are readily captured by
monocytes, and that the best result is obtained with 22 (Figures
3 and S1). The cytosolic distribution of the fluorescence signal of
labelled analogues is punctuated and clearly reminiscent of an
endocytic mechanism of cell accumulation in monocytes. Similar
experiments with granulocytes did not give interpretable
intracellular distribution information, because of the small
dimension and high background of these cells, while the signal
from lymphocytes and HeLa cells was not strong enough.
Scheme 3. Reagents and Conditions: (i) AlMe3 (2 equiv.), piperazine (4
equiv.), toluene, MW 50W, 1h; (ii) Boc-L-Ala-OH (1 equiv.), HATU (1.1 equiv.),
DIEA (2 equiv.), dry acetonitrile, 1 h, r.t.
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10.1002/cmdc.201600597
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cell selectivity, the kinetics of cell association was slower than
for 22.
Moreover, comparing the capacity of oligomers with different
length to accumulate in different cell types provides interesting
results, as the oligomers length strongly modulates the capture
by any cellular model. While short oligomers weakly interact with
cells, 22 shows a clearly improved cell-association capacity
already after 3 h incubation.
Finally, among the analysed cells types, monocytes and
granulocytes captured all the oligomers with best efficacy (6-7
times more effectively than HeLa and lymphocytes). Such a
sustained cell capture is still visible after 24 h incubation.
To explain the good results obtained with 22, we performed the
conformational analysis of our foldamer series, including 4, 8, 9
and 11. As described in the next section, we indeed found that a
distinctive feature of the long L-Ala-D-Oxd oligomer 11 is its
tendency to fold into stable helices.
Conformational Analysis
Figure 3. Confocal analysis of human monocytes after purification from buffy
coats of healthy donors, seeding and incubation for 18h (20 µM) with
foldamers 16, 18, 20 and 22. The cells were washed with PBS and directly
analyzed by confocal microscopy.
To check the presence of intramolecular N-H•••O=C hydrogen
bonds in the fully protected oligomers 3, 5 and 7, IR and 1H
NMR spectra were recorded in the structure supporting solvents
CH2Cl2 and CDCl3, respectively. From IR analysis, all studied
foldamers feature free N-H amide groups in CH2Cl2 solution.[28]
Therefore, to overcome these problems, a study was carried out
by a dose and time response analysis by more sensitive and
quantitative Fluorescence Activated Cell Sorting (FACS)
However, when moving from
3
to 7, the amount of
intramolecular N-H•••O=C hydrogen bonds increases as shown
by a broad band centered at about 3250 cm-1 (for a more
detailed discussion, see SI and Figure S3).
approach.
Purified human lymphocytes,
granulocytes,
monocytes and HeLa cells were incubated for 3 or 24 hours at
37°C with foldamers 16, 18, 20 and 22, and with H-L-Ala-P-RB
for comparison (Table 1). The cells were then washed with PBS,
recovered and directly analyzed by cytofluorimetry.
A confirmation of these results was obtained by investigating the
DMSO-d6 dependence of NH proton chemical shifts in NMR
spectra.[29] Compounds 3 and 5 give large  values of about 1
ppm, suggesting that their NH hydrogens are not involved in
intramolecular H-bonding. By contrast, the five NH groups of
longer foldamer 7 are less sensitive to DMSO-d6 addition and
display moderately to strongly reduced  values (0.77 to 0.33
ppm) (Table S1 and Figure S4).
In contrast, the conformational analysis of the fully protected
foldamers Boc-(L-Ala-D-Oxd)n-OBn has been previously
reported.[15] We unambiguously proved that the oligomers having
general formula Boc-(L-Ala-D-Oxd)n-OR (n = 1-6, R = Bn or H)
fold into ordered structures when n ≥ 5. From CD spectra we
could demonstrate that the secondary structure adopted is a -
bend ribbon spiral, which is a subtype of the 310-helix.
Then, we also relied on ECD spectroscopy to directly investigate
the conformation adopted by our new foldamers series in water
solution. Since the Rhodamine dye is a strong fluorophore and
inhibits the ECD analysis, we registered the ECD spectra of the
unlabelled foldamers 4, 8, 9 and 11 (Figure 4). In the labelled
compounds the Rhodamine moiety is connected to one end of
the chain and should not affect the preferred foldamer
conformation.
In the case of the L-Ala series, 22 shows the best cell
association especially after a 3 h incubation, while 20 capture
was not distinguishable from the background, estimated by the
cell association of H-L-Ala-P-RB, at any time. Moreover
inflammatory monocytes and granulocytes are targeted by 22
more effectively than HeLa cells and lymphocytes even after 24
h incubation. Such effect was strongly inhibited by decreasing
the incubation temperature to 0°C, a condition blocking active
endocytosis of foldamers and only permitting their binding to
cells (Figure S2). Collectively, data indicate that 22 is taken up
by cells better than shorter 20 analogue and can preferentially
target blood myeloid cells (monocytes and granulocytes) than
lymphoid and epithelial cells. Such selectivity is peculiar and is
reminiscent of observed in the same conditions, with the peptidic
part of Trichogin, a well-known foldamer with a stable α-helix
structure.[20]
Since cells have a negative superficial charge, the multiple
cationic sites in foldamers 16 and 18 should in principle favor
cellular association. As could be foreseen, the cell association of
the short 16 oligomer is increased compared to 20, although
with a slow kinetics, being the improvement better visible after
24 h than after 3 h. In contrast, the introduction of three
additional positive charges in foldamer 18 decreases its cell
capture efficacy compared to 22, and this effect is marked after
short incubation time. Although 16 and 18 displayed the same
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Table 1. Association of foldamers 16, 18, 20, 22 and of H-L-Ala-P-RB to different human cells after 3 hours incubation (left) and after 24 hours incubation (right).
Results are representative of one experiment run in duplicate. MFI: mean fluorescence intensity.
Foldamer
After 3 hours
After 24 hours
8000
6000
4000
2000
0
8000
6000
4000
2000
0
Lymphocytes
Lymphocytes
Granulocytes
Monocytes
HeLa
Granulocytes
Monocytes
HeLa
16
0
5
10
Concentration (M)
15
20
0
5
10
15
20
Concentration (M)
8000
6000
4000
2000
0
8000
6000
4000
2000
0
Lymphocytes
Lymphocytes
Granulocytes
Monocytes
HeLa
Granulocytes
Monocytes
HeLa
18
0
5
10
15
20
0
5
10
15
20
Concentration (M)
Concentration (M)
8000
6000
4000
2000
0
8000
6000
4000
2000
0
Lymphocytes
Granulocytes
Monocytes
HeLa
Lymphocytes
Granulocytes
Monocytes
HeLa
20
0
5
10
Concentration (M)
15
20
0
5
10
15
20
Concentration (M)
Lymphocytes
8000
6000
4000
2000
0
8000
6000
4000
2000
0
Lymphocytes
Granulocytes
Monocytes
HeLa
Granulocytes
Monocytes
HeLa
22
0
5
10
Concentration (M)
15
20
0
5
10
15
20
Concentration (M)
8000
6000
4000
2000
0
8000
6000
4000
2000
0
Lymphocytes
Granulocytes
Monocytes
HeLa
Lymphocytes
Granulocytes
Monocytes
HeLa
H-L-Ala-P-RB
0
10
20
0
10
Concentration (M)
20
Concentration (M)
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mixtures is a general phenomenon,[40] but racemic crystallization
has been extensively studied for proteins, short peptides[41] and
DNA.[42] Recently, this technique and X-ray crystallography have
been successfully applied to the determination of the unknown
structure of snow flea antifreeze protein (sfAFP),[43] and of the
crystal structures of seven tripeptides in enantiomeric and
racemic forms.[44]
After several attempts, X-ray quality crystals of rac-Boc-L-Ala-D-
Oxd-OBn, Boc-(D-Ala-L-Oxd)2-OBn and Boc-L-Val-D-Oxd-L-
Lys(2Cl-Z)-D-Oxd-OBn (3) were grown by slow evaporation of
ethyl acetate or chloroform solutions. Crystals of Boc-(D-Ala-L-
Oxd)2-OBn were obtained from the racemate and spontaneous
resolution thus occurred during crystallization (see Experimental
Section for details on absolute structure determination).
Unfortunately, we were not able to grow suitable crystals of the
longer foldamers. The molecular structures of Boc-L-Ala-D-Oxd-
OBn, Boc-(L-Ala-D-Oxd)2-OBn and 3 are depicted in Figures 5
and 6 (for an easier comparison, we show enantiomers with the
same absolute configuration at corresponding chiral centers).
Additional plots of molecular and crystal structures are provided
as Figures S6-S11. All crystal data and refinement parameters
are given in Table S2 while the geometry of hydrogen-bond
interactions and conformational data are gathered in Tables S3-
S5 and S6-S8, respectively.
40000
30000
20000
10000
0
4
8
9
11
-10000
-20000
-30000
-40000
200
220
240
260
280
300
Wavelength (nm)
Figure 4. Normalized per-residue ECD spectra of compounds 4, 8, 9 and 11 in
H2O solution (3 mM, room temperature).
The per-residue ECD spectra of foldamers 4, 8 and 9 are very
similar as they all display a strong negative signal at 182-185 nm
and a positive band at 202-204 nm. Foldamers 4 and 8 also
exhibit a broad and weak negative band centered at 240 nm,
while 9 shows a more intense broad negative band centered at
230 nm. The modest variations across the three spectra suggest
that these oligomers do not assume a helical conformation but
rather fold in a turn conformation.[30] Unlike the α-helix and β-
sheet, there is no unique ECD signature for turns because of the
range of conformers included in this structural category.[31]
Various spectral patterns were predicted, but the predominant
one had a weak negative n →π∗ band at 220–230 nm and two
strong π →π∗ bands, a positive one at 200–210 nm and a
negative one at 180–190 nm.[32]
In contrast, the spectrum of 11 displays a strong positive signal
at 193 nm and two negative bands centered at 208 and 228 nm
respectively, with R = []228/[]208 = 0.75, like the parent Boc-(L-
Ala-D-Oxd)6-OH compound.[15] This is the typical ECD signature
of 310 helices, in agreement with the work of Toniolo and
coworkers
experimental and theoretical studies point to the following main
characteristics of the 310-helix: (a) a negative ECD band centred
near 207 nm; (b) a negative shoulder in the vicinity of 222 nm;
(c) a R = [θ]222/[θ]207 ratio smaller than reported for the α-helical
peptides, where R is usually close to 1; (d) a positive maximum
al 198 nm.
Foldamers 8 and 11 have the same number of residues and
should adopt the same preferred conformation. The unexpected
turn structure in 8 may be attributed to the presence of sterically
demanding residues and of residues which can form side-chain
to main-chain intra-strand H-bonds.
To probe the conformation of these materials in the solid state,
we tried to crystallize foldamers of the two series in both
protected and deprotected forms. Since it is known that
crystallization is more difficult for enantiomerically pure chiral
molecules than for the corresponding racemates,[36–39] we also
synthesized enantiomers and prepared racemates. It is still not
clear whether more facile crystallization of racemic protein
In the solid state, the three compounds do not form
intramolecular N-H•••O=C hydrogen bonds. Amide nitrogen
atoms are involved only in intermolecular hydrogen bonds or in
no H interaction at all, like for the amide nitrogen N1 in Boc-(D-
Ala-L-Oxd)2-OBn. The emerging picture thus agrees well with
solution ECD data (see above and Ref. [15]). Secondary
structure relies just on C-H···O interactions: in the three studied
foldamers, the -carbon atom of each amino acid residue has an
H
contact with the endocyclic carbonyl oxygen of the
neighboring Oxd moiety (see captions to Figures 5 and 6 for
details). Such a nonclassical hydrogen bond is commonplace in
the solid state structure of Oxd-based foldamers[11,15,45,47] and
persists in solution, as judged by the chemical shift of -protons
in NMR spectra.[14,15,17,47]
Turning now to intermolecular interactions in the solid state, rac-
Boc-L-Ala-D-Oxd-OBn entails centrosymmetric supramolecular
dimers held together by a pair of N-H•••O=C bonds between the
two antiparallel peptide segments. In Boc-(D-Ala-L-Oxd)2-OBn
and 3, a classical H-bond is formed between the amide nitrogen
N3 and carbonyl oxygen O2 of the same peptide segment, but
belonging to a neighboring, antiparallel molecule in the crystal. A
similar chain-like supramolecular arrangement based on a single
classical H bond was previously found in Boc-(L-Phe-D-Oxd)n-
OBn, the alignment of peptide units being parallel and
antiparallel for n = 1 and 2, respectively.[48,49]
Figures 5 and 6 and inspection of backbone torsion angles
(Tables S6-S8) indeed show that the three compounds adopt
similar, partially extended conformations, in agreement with
ECD studies. Conformational parameters at L-Ala residues ( = -
87.4  -70.3°,  = 147.4  173.4°) correspond approximately to
those characteristic of PPII structure,[50] while the conformation
at the L-Val and L-Lys residues in 3 ( = -134.3  -132.2°,  =
150.6  172.0°) is more reminiscent of peptide  strands.[51]
and with theoretical calculations.[35] Both
[33,34]
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(°): H1···O9 = 2.28, C1-H1···O9 = 115.0, H5···O11 = 2.32, C5-H5···O11 =
113.5.
Conclusions
Some selected foldamers containing the D-Oxd moiety
alternated with a L-amino acid and coupled with Rhodamine B
(RB) have been prepared to check their behavior towards
purified human lymphocytes, granulocytes, monocytes and
HeLa cells. All these molecules show a good biocompatibility,
with no detrimental effects up to 64 µM concentration.
The conformational analysis performed in solution and in the
solid state suggests that only the longer oligomer of the L-Ala
series (22) folds into a well-established 310 helix, while the other
oligomers give partially unfolded turn structures. A quantitative
study was obtained by dose and time response analysis by
FACS of human HeLa cells, and primary blood lymphocytes,
granulocytes and monocytes incubated with foldamers using H-
L-Ala-P-RB as a comparison. Among the analyzed cell types,
monocytes and granulocytes associate with these oligomers
with best efficacy (6-7 times more effectively than HeLa and
lymphocytes). Such a sustained cell captured is still visible after
24 h incubation.
The unfolded foldamers 16, 18 and 20 display a slower kinetics
of cell association than 22. This outcome could be ascribed to
the tendency of 22 to fold into a stable helix, although the
presence of several positive charges in 16 and 18 should in
principle favor cellular uptake.
Figure 5. Molecular structure of (a) Boc-L-Ala-D-Oxd-OBn molecule in rac-
Boc-L-Ala-D-Oxd-OBn and of (b) Boc-(L-Ala-D-Oxd)2-OBn, obtained by
inversion of the experimental model. Atoms are drawn as spheres with
arbitrary radius. Labels are provided for all N and O atoms, for backbone
carbons as well as for C and H atoms relevant to H bonding. Color code: C =
dark grey, N = blue, O = red, H = light grey. Intramolecular nonclassical H
bonds are represented as yellow dotted lines. Selected interatomic distances
(Å) and angles (°): in (a), H1···O5 = 2.290(13), C1-H1···O5 = 121.2(10); in (b),
H1···O9 = 2.27, C1-H1···O9 = 119.1, H5···O11 = 2.38, C5-H5···O11 = 112.8.
Given the different chemical structure of the herein
characterized molecules, our new evidence further supports the
hypothesis that foldamers with helix-like frameworks can favor
the association to inflammatory cells, independent of their
charge and chemical nature. These oligomers may thus be
regarded as possible targeting agents of small drugs for several
reasons: they are intrinsically non-toxic to cells at relatively high
doses; moreover, if long enough, they show a peculiar cell
targeting preference, being rapidly and more effectively
internalized in human blood myeloid cells, like monocytes and
granulocytes. Although the biochemical basis for such cell
selectivity, especially displayed by long (> 2 units) and neutral
oligomers, is still poorly understood, the present formulations or
improved analogues may be used to target specific cell types.
For example, anti-inflammatory drugs may be coupled to
appropriate oligomers and directed more selectively to
monocytes/granulocytes. Similar foldamers may be refined and
serve as alternative targeting agent also for nanoparticles in
nanomedicine.
Experimental Details
Synthesis. Solvents were dried by distillation before use. All
reactions were carried out in oven-dried glassware. The melting
points of the compounds are uncorrected. High quality infrared
spectra (64 scans) were obtained with an ATR-FT-IR Bruker
Alpha System spectrometer. All spectra were measured in
Figure 6. Molecular structure of 3 with atoms drawn as spheres with arbitrary
radius. Labelling criteria, H-bond representation and color code are the same
as in Figure 5, plus Cl = green. Selected interatomic distances (Å) and angles
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10.1002/cmdc.201600597
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solutions at 3 mM concentration in dry CH2Cl2 using a 1 mm
NaCl cell, or at 1% w/w in dry KBr. All compounds were dried in
vacuo and all sample preparations were performed in a nitrogen
atmosphere. NMR spectra were recorded with a Varian Inova
400 spectrometer at 400 MHz (1H NMR) and at 100 MHz (13C
NMR). The measurements were carried out in D2O or in CDCl3.
The proton signals were assigned by gCOSY spectra. Chemical
shifts are reported in δ values relative to the solvent (D2O or
by anomalous dispersion effects using Mo-K radiation. In the
same conditions, the absolute structure of Boc-(D-Ala-L-Oxd)2-
OBn could not be established with certainty, but the Flack
parameter (x) suggests that the measured crystal is likely to
entail D-Ala and L-Oxd residues, as written (x = -1.4(5) vs. 2.4(5)
for the inverted model). CCDC 1493119-1493121 contain the
supplementary crystallographic data for this paper. These data
can be obtained free of charge from the Cambridge
CDCl3)
peak.
The
(4R,5S)-4-benzoxycarboxy-5-methyl-
Crystallographic
Data
Centre
via
oxazolidin-2-one (D-Oxd-OBn) was obtained by following the
previously reported procedure.[17]
www.ccdc.cam.ac.uk/data_request/cif.
Cell isolation and culture. HeLa cells were maintained in
DMEM medium (Gibco) supplemented with 10% FCS
(Euroclone) and antibiotics (penicillin and streptomycin,
Invitrogen) at 37°C in a humidified atmosphere containing 5%
(v/v) CO2; cells were split every 2-3 days.
ECD spectra. ECD spectra were recorded at room temperature
on a spectropolarimeter. Quartz cuvettes of 0.2 and 1 mm path
length were employed. Foldamers were dissolved in water,
yielding clear solutions at approximately 3 mM concentration.
The spectra were recorded between 180 and 300 nm for the far-
UV and near-UV region, with 0.1 nm resolution. 32 scans were
accumulated with a scanning speed of 100 nm/min and a time
constant of 1 s and the solvent baseline was subtracted from the
averaged spectra. Final spectra are presented in molar ellipticity.
Human monocytes, granulocytes and lymphocytes were purified
from buffy coats of healthy donors, kindly provided by the Centro
Immunotrasfusionale, Hospital of Padova. Briefly, for monocytes
purification, buffy coats were subjected to two sequential
centrifugations on Ficoll and Percoll gradient (GE Healthcare);
residual lymphocytes were removed by incubation in 2% FCS
RPMI at 37 °C and subsequently removed by washing. Unless
otherwise specified, cells were kept at 37 °C in a humidified
atmosphere containing 5% (v/v) CO2 in RPMI-1640
supplemented with 10% FCS. For granulocytes purification, the
pellet of cells obtained after the centrifugation on Ficoll gradient
was subjected to dextran erythrocyte precipitation; residual
erythrocytes were removed by hypotonic lysis in 155 mM NH4Cl,
10 mM KHCO3, and 100 mM Na2EDTA at pH 7.4 and cells were
cultured in RPMI medium, supplemented with 10% FCS. For
lymphocytes preparation, buffy coats were incubated with 50
μl/ml of Rosette Sep® Human T Cell Enrichment Cocktail
(StemCell Technologies). Blood was then centrifuged over a
Ficoll gradient and cells were cultured in RPMI medium,
supplemented with 10% FCS.
X-ray crystallography. Single-crystal X-ray diffraction studies
were carried out at 115(2) K on compounds rac-Boc-L-Ala-D-
Oxd-OBn (from chloroform), Boc-(D-Ala-L-Oxd)2-OBn (from
chloroform) and 3 (from ethyl acetate) with a four-circle Bruker
X8-APEX diffractometer equipped with a Mo-K generator ( =
0.71073 Å), an area detector and a Kryo-Flex cryostat, and
controlled by Bruker-Nonius X8APEX software. The structures
2
were solved and refined on Fo by standard methods using the
[52]
[53]
WINGX v2013.3 package
in conjunction with SIR92
and
SHELXL-2014/7 [54] programs. Compound rac-Boc-L-Ala-D-Oxd-
OBn crystallizes in monoclinic space group P21/n (Z = 4)
whereas crystals of Boc-(D-Ala-L-Oxd)2-OBn and 3 belong to
monoclinic space group P21 (Z = 2). Disorder effects were
limited to the 2Cl-Z substituent in 3, which was refined over two
positions with 0.919(2) and 0.081(2) occupancies. The minority
component was restrained to have the same geometry as the
majority component within 0.01 and 0.02 Å for 1,2- and 1,3-
distances, respectively. Furthermore, its NC(O)O and 2-Cl-Ph
moieties were restrained to be flat and all minority atoms were
refined with a common isotropic displacement (ID) parameter.
All remaining nonhydrogen atoms in the structures were refined
anisotropically. Hydrogen atoms were treated with ID
parameters but only in rac-Boc-L-Ala-D-Oxd-OBn they were
subject to unrestrained refinement. Amide hydrogens in Boc-(D-
Ala-L-Oxd)2-OBn and 3 were refined independently with N-H
distances restrained to 0.880(15) Å and a unique ID parameter
in 3. Aromatic, tertiary and methylene hydrogens in Boc-(D-Ala-
L-Oxd)2-OBn and 3 were added in idealized positions and
MTS cytotoxicity assay. HeLa cells (1 x 106 cells/mL) were
plated into a 96-well culture plate the day before the experiment.
Cells were then incubated for 18 h with different oligomers at
different concentrations (up to 50 µM) in DMEM additioned with
10% FCS. Cellular mitochondrial activity (indicator of cellular
viability) was evaluated by MTS assay (Promega) according to
the instruction manual.
Oligomer association to cells. Intracellular distribution of
labelled-oligomers was assessed by confocal microscopy.
Human purified monocytes (2x105) were seeded on cover
glasses and after 24h they were incubated for 18h at 37°C with
oligomers, washed with PBS and directly analyzed by confocal
allowed to ride on the parent carbon atom, with U(H) = 1.2Ueq(C). microscopy (Leica SP2). Images were processed using ImageJ
Methyl hydrogens were also treated as riding contributors but
with torsion angle refinement (AFIX 137), and a common ID
parameter per CH3 group in Boc-(D-Ala-L-Oxd)2-OBn or U(H) =
1.5Ueq(C) in 3.
Thanks to the chlorine atom in the partially-disordered 2Cl-Z
group, the absolute configuration expected for 3 was confirmed
software. Alternatively, monocytes, granulocytes, lymphocytes
or HeLa cells (2x105 cells/well) were incubated with labelled-
oligomers for
3 or 24 hours at 37°C. Cell MFI (mean
fluorescence intensity) values were obtained by cytofluorimetry
(FACSCanto, BD) and analyzed by FACSDiva Software (BD).
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10.1002/cmdc.201600597
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FULL PAPER
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Keywords: bioconjugates • conformational analysis • foldamers
• leukocytes • peptides
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Entry for the Table of Contents
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Text for Table of Contents
Benedetta Del Secco, Giulia Malachin,
Lorenzo Milli, Nicola Zanna, Emanuele
Papini, Andrea Cornia,* Regina Tavano,*
Claudia Tomasini*
Page No. – Page No.
Title
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