The effect of urea moiety in amino acid binding by β-cyclodextrin derivatives: A 1000-fold increase in efficacy comparing to native β-cyclodextrin

Article abstract of DOI:10.1016/j.carbpol.2017.02.018

Water soluble amphiphilic anion receptors based on urea-substituted β-cyclodextrin were synthesized via a copper(I) mediated azide-alkyne coupling reaction. The synthetic route was designed to minimize the number of operations of cyclodextrins. Stable pro

Full text of DOI:10.1016/j.carbpol.2017.02.018

Accepted Manuscript
Title: The effect of urea moiety in amino acid binding by
␤-cyclodextrin derivatives: A 1000-fold increase in efficacy
comparing to native ␤-cyclodextrin
Authors: Pawel Stepniak, Bruno Lainer, Kazimierz Chmurski,
Janusz Jurczak
PII:
S0144-8617(17)30138-8
DOI:
Reference:
http://dx.doi.org/doi:10.1016/j.carbpol.2017.02.018
CARP 11997
To appear in:
Received date:
Revised date:
Accepted date:
25-11-2016
2-2-2017
3-2-2017
Please cite this article as: Stepniak, Pawel., Lainer, Bruno., Chmurski, Kazimierz.,
& Jurczak, Janusz., The effect of urea moiety in amino acid binding by ␤-
cyclodextrin derivatives: A 1000-fold increase in efficacy comparing to native ␤-
cyclodextrin.Carbohydrate Polymers http://dx.doi.org/10.1016/j.carbpol.2017.02.018
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<AT>The effect of urea moiety in amino acid binding by β-cyclodextrin derivatives: a 1000-
fold increase in efficacy comparing to native β-cyclodextrin
<AU>Pawel Stepniaka, Bruno Lainera,b, Kazimierz Chmurskib, Janusz Jurczaka*
##Email##jurczak_group@icho.edu.pl##/Email##
<AU>
<AFF>aInstitute of Organic Chemistry, Polish Academy of Sciences, Kasprzaka 44/52, 01-
224 Warsaw, Poland
<AFF>bFaculty of Chemistry, University of Warsaw, Pasteura 1, 02-093 Warsaw, Poland
<PA>Tel.: +48 22 343 2330; fax: +48 22 632 66 81.
<ABS-HEAD>Highlights► Amphiphilic Urea-cyclodextrin derivatives bind amino acids ►
Amino acid binding is 1000-fold stronger in comparison to unsubstituted cyclodextrins ►
Side chains attached to the cyclodextrins cause formation of nano-sized aggregates
<ABS-HEAD>Abstract
<ABS-P>Water soluble amphiphilic anion receptors based on urea-substituted β-cyclodextrin
were synthesized via a copper(I) mediated azide-alkyne coupling reaction. The synthetic route
was designed to minimize the number of operations of cyclodextrins. Stable products were
obtained in 90% yield. They were successfully tested as amino acid receptors, showing
excellent affinity constants (103-104 M-1) in a highly competitive environment (pH 8
phosphate-buffered water solution). Isothermal titration calorimetry indicated that complex
formation strongly depends on the hydrophobic nature of the guest and that the urea moiety of
the receptor is necessary to efficiently bind amino acids.
<KWD>Keywords: cyclodextrin; azide-alkin coupling; host-guest chemistry; calorimetry;
amino acids; complexes
<H1>1. Introduction
Molecular recognition of biological molecules is a broadly studied and important area of
scientific interest. In particular, investigating selective interactions of amino acids or peptides
is very important in order to understand and mimic the mechanisms of biological processes
(Schneider, 2009). The utilization of hydrogen bond donors in the field of molecular
recognition is a commonly developed area of scientific interest (Amendola, Fabbrizzi, &
Mosca, 2010; Busschaert, Caltagirone, Van Rossom, & Gale, 2015; Choi & Hamilton, 2003).
Such donors provide short-contact, relatively strong and spatially oriented interactions with
negatively charged or polarized guests, allowing for selective and structurally-related binding.
1

However, this approach is often limited by the competing environment, in which the role of
the solvent cannot be neglected (Castronuovo, Elia, Pierro, & Velleca, 1999). As such, the
recognition employing hydrogen bond donating groups is executed mainly in mixed solvents,
where water’s hydrogen bond network is shared with other polar cosolvents (e.g. acetone,
acetonitrile, DMSO) (Caltagirone et al., 2008; Cametti & Rissanen, 2009; Dutta, Bose, &
Ghosh, 2013).
In this study we sought to provide a water soluble compartment in which hydrogen bond
donors are isolated from the solvent, so their interactions with hydrophobic L-amino acids (as
model amphiphilic carboxylates) should be enhanced. For this purpose we chose
β-cyclodextrin (β-CD) as a well-defined, water-soluble, amphiphilic vessel, that could be
selectively modified. β-CD has a concave structure, presenting its polar hydroxy groups to the
outside, while the cavity remains hydrophobic. Although several types of similar cavities have
been previously tested (Dziemidowicz, Witt, & Rachoń, 2008; Mutihac, Lee, Kim, & Vicens,
2011; Richard et al., 2008; Stone, Franz, & Lebrilla, 2002) only cucurbiturils gave
satisfactory (>>1000 M-1) affinities with amino acids (Logsdon, Schardon, Ramalingam,
Kwee, & Urbach, 2011; Rajgariah & Urbach, 2008). On the other hand, underivatized CDs
bind amino acids rather poorly, with Ka’s below 100 M-1 (Cooper & MacNicol, 1978;
Matsuyama, El-Gizawy, & Perrin, 1987; Rekharsky, Schwarz, Tewari, & Goldberg, 1994).
Therefore, we decided to incorporate additional hydrogen bond donors in the proximity of the
cavity in order to form an isolated amphiphilic environment for the recognition of amino
acids. We anticipated that a urea group, having two points of interaction, would serve as a
binding site efficient enough that the modification of just one of the 21 hydroxy groups of β-
CD would be necessary to provide well defined receptors.
We hypothesized that a decorating β-CD with a side-chain having both hydrophilic (urea) and
lipophilic (phenyl and isobutyl) moieties will give an appropriate environment for efficient
binding of amino acids, providing a receptor far more effective than underivatized β-CD.
Having such a tool in hand the recognition or separation of amino acids would not have to be
based on repetitive processes (as in chromatography or electrophoresis) but a single
interaction would be efficient enough for molecular discrimination of particular pairs of
substances.
<H1>2. Experimental
<H2>2.1. General
All solvents were used as received, unless stated otherwise. Purification of products was
performed using chromatography on silica gel (Merck Kieselgel 60, 230-400 mesh) with
mixtures of hexane/ethyl acetate, methanol/dichloromethane, acetonitrile/water/ammonia or
using gel filtration on styrene resin (Diaion® HP-20) with water/methanol gradient. Thin-
layer chromatography (TLC) was performed on silica gel plates (Merck Kieselgel 60 F254).
Visualization of the developed chromatogram was accomplished using UV light or ninhydrin
and cerium molybdate stains. Reported NMR spectra were recorded in CDCl3 or (CD3)2SO
using a Varian Unity Plus 200 MHz and Agilent 300 MHz and 500 MHz spectrometers.
Chemical shifts of 1H NMR and 13C NMR are reported as δ values relative to TMS (δ = 0.00)
and CDCl3 (δ = 77.0) or (CD3)2SO (δ = 39.5), respectively. The following abbreviations are
used to indicate the multiplicity: s - singlet; d - doublet; t - triplet; q - quartet; m – multiplet;
dm - doublet of multiplets. Mass spectra were measured on a Shimadzu LCMS-IT-TOF using
ESI technique.
Distilled water (>18 MΩ/cm grade) was supplied by Mili-Q water system. Phosphate buffers
were prepared by mixing specified amounts of NaH2PO4·2H2O and Na2HPO4·H2O and
subsequent titration with NaOH, to desired pH, using Elmetron CP-401 pH-meter.
2

Dialysis was performed using Spectrum Labs Biotech Grade Cellulose Ester Dialysis
Membrane, MWCO: 100-500 Da, preserved with 0.05% sodium azide.
<H2>2.2. Synthesis
Mono-2-propargyl-β-CD (1)
To a solution of dry β-CD (10 g, 10 mmol) in DMSO (100 mL) LiH (1 equiv.) was added and
stirred overnight, until the solution became clear. Next, propargyl bromide (1 equiv.) and LiI
(a few mg) were added and the mixture was stirred at 55°C for 5 h, avoiding light exposure.
The solvent was evaporated under reduced pressure, resulting slurry was dissolved in water
(20 mL) and precipitated with acetone (500 mL). Solid residue was filtered, air-dried and then
the resulting powder was dissolved in water and passed through styrene resin (Diaion® HP-
20) using gradient of water/methanol (0 to 10%) as solvent. Prior to use, the resin was
conditioned in methanol for 1h and rinsed with water. Fractions containing pure product were
evaporated under reduced pressure giving 4.1g of 1 (3.5 mmol, 35%). COSY, TOCSY, HSQC
and HMBC NMR spectra of 1 are appended in the ESI.
1HNMR (500MHz, DMSO) δ 5.80 (br s, 11H), 4.99 (d, J = 3.7 Hz, 1H), 4.82 (br s, 6H), 4.47
(dd, J = 15.8 Hz, J = 2.4 Hz, 1H), 4.46 (br s, 9H), 4.39 (dd, J = 15.8 Hz, J = 2.4 Hz, 1H), 3.79
(t, J = 9.2 Hz, 1H), 3.65–3.55 (m, 27H), 3.52 (t, J = 2.4 Hz, 1H), 3.43–3.40 (m, 2H), 3.37–
3.27 (m).
13C NMR (75 MHz, DMSO): δ 102.0–101.0, 100.1, 82.2–81.5, 79.9, 79.1, 77.8, 73.2–71.7,
72.6, 60.0–59.7, 58.7.
HRMS ESI+ m/z: 1195.3756 (MNa+, C45H72N35O3Na+ requires 1195.3752).
(S)-O-Tosyl-2-(Boc-amino)-4-methylpentanol
N-Boc-L-Leucinol (2.5 g, 11.5 mmol) was dissolved in dichloromethane (100 mL) and cooled
to 0°C. Triethylamine (1.1 equiv.) and p-tosyl chloride (1.05 equiv.) were added and the
resulting mixture was stirred overnight (allowing the mixture to warm up to rt). It was next
washed with saturated sodium bicarbonate (100 mL) and brine (20 mL). Organic layer was
then dried with MgSO4 and evaporated under reduced pressure. The product was purified on
silica gel using a 10:90 ethyl acetate:hexane mixture resulting with 2.91g of 2 (7.8 mmol,
68%).
Careful purification of the aminoalcohol 2 before the tosylation process is crucial, as mixed
anhydride (formed prior to the reduction step) acts as an inhibitor.
1H NMR (200 MHz, CDCl3) δ 7.78 (d, J = 8.4 Hz, 2H), 7.35 (d, J = 8.5 Hz, 2H), 4.70 – 4.34
(m, 1H), 3.97 (dd, J = 14.4, 11.0 Hz, 2H), 2.43 (d, J = 9.6 Hz, 3H), 1.64 (s, 2H), 1.50 – 1.35
(m, 9H), 1.14 (dd, J = 8.6, 5.9 Hz, 1H), 1.00 – 0.70 (m, 6H).
(S)-2-(Boc-amino)-1-azido-4-methylpentane (3)
2 (2.35 g, 5.9 mmol) was dissolved in DMF (20 mL) and NaN3 (3 equiv.) was added. The
addition of more than 3 equivs of NaN3 caused the elimination of the tosylated aminoalcohol
due to the basicity of azide ions in DMF. The resulting mixture was stirred at 60°C for 24 h. It
was then poured into water (300 mL) and extracted with diethyl ether. The combined organic
layers were then evaporated under reduced pressure. The oily residue was dried under vacuum
until it solidified resulting with 2.26 g (4.7 mmol, 80%) of 3.
1H NMR (300 MHz, CDCl3) δ 4.56 – 4.39 (m, 1H), 3.80 (d, J = 4.0 Hz, 2H), 3.54 – 3.10 (m,
2H), 1.71 – 1.51 (m, 1H), 1.49 – 1.40 (m, 9H), 0.92 (dd, J = 6.6, 2.2 Hz, 6H).
13C NMR (75 MHz, CDCl3) δ 155.40, 79.75, 70.81, 55.35, 51.03, 48.69, 44.53, 41.48, 28.49,
25.25, 24.88, 23.08, 22.26.
3

Synthesis of urea 4
(S)-2-(Boc-amino)-1-azido-4-methylpentane (600 mg, 2.5 mmol) was dissolved in 1:1
mixture of TFA and dichloromethane (5 mL) and the resulting mixture was stirred for 1 h,
after which all volatiles were evaporated. In order to neutralize residual TFA and deprotonate
the amine, the solid residue was treated with triethylamine (1 ml), which excess was
subsequently evaporated. Dichloromethane (20 mL) was added and the solution was treated
with the respective isocyanate (1 equiv., 2.5 mmol). After 24 h of stirring at rt the mixture was
washed with saturated sodium bicarbonate (50 mL) and brine (50 mL). The organic layer was
dried with MgSO4 and the product was purified on silica gel using a 3:97 mixture of
methanol:dichloromethane, yielding, after evaporation and vacuum drying, urea 4.
(S)-1-(1-Azido-4-methylpentan-2-yl)-3-phenylurea (4a)
Yield: 590 mg (2.25 mmol, 90%)
1H NMR (200 MHz, CDCl3) δ 7.49 – 7.19 (m, 5H), 7.06 (d, J = 6.9 Hz, 2H), 4.15 – 3.93 (m,
1H), 3.42 (ddd, J = 37.1, 12.1, 4.0 Hz, 2H), 2.14 (s, 2H), 1.64 (dt, J = 13.0, 7.5 Hz, 1H), 0.93
(d, J = 6.7 Hz, 6H).
13C NMR (50 MHz, CDCl3) δ 187.87, 138.38, 129.51, 124.06, 121.33, 55.67, 48.48, 41.51,
25.03, 22.35.
HRMS ESI+ m/z: 262.1609 (MH+, C13H19N5OH+ requires 262.1662).
(S)-1-(1-Azido-4-methylpentan-2-yl)-3-(4-nitrophenyl)urea (4b)
Yield: 705 mg (2.3 mmol, 92%)
1H NMR (200 MHz, CDCl3) δ 8.14 (d, J = 9.1 Hz, 1H), 7.92 (s, J = 9.6 Hz, 1H), 7.54 (d, J =
9.1 Hz, 1H), 5.59 (d, J = 8.4 Hz, 1H), 4.23 – 3.96 (m, 1H), 3.45 (ddd, J = 34.1, 12.3, 4.3 Hz,
1H), 1.67 (tt, J = 13.3, 6.5 Hz, 1H), 1.41 (dd, J = 9.5, 5.9 Hz, 2H), 0.93 (dd, J = 6.5, 2.0 Hz,
6H).
13C NMR (50 MHz, CDCl3) δ 154.47, 145.90, 142.02, 125.51, 117.97, 55.56, 48.28, 41.43,
29.83, 24.98, 23.01, 22.23.
HRMS ESI+ m/z: 307.1548 (MH+, C13H18N6O3H+ requires 307.1513).
(S)-1-(1-Azido-4-methylpentan-2-yl)-3-(3,5-di(trifluoromethyl)-phenyl)urea (4c)
Yield: 894 mg (2.25 mmol, 90%)
1H NMR (200 MHz, CDCl3) δ 7.76 (s, 2H), 7.54 (s, 1H), 7.45 (s, 1H), 5.30 (d, J = 8.6 Hz,
1H), 4.06 (s, 1H), 3.55 (dd, J = 12.4, 3.7 Hz, 1H), 3.37 (dd, J = 12.2, 4.6 Hz, 1H), 1.82 (bs,
1H), 1.75 – 1.53 (m, 1H), 1.53 – 1.31 (m, 2H), 0.93 (dd, J = 6.4, 1.8 Hz, 6H).
13C NMR (50 MHz, CDCl3) δ 154.77, 140.24, 132.73, 120.46, 118.81, 116.33, 55.60, 48.35,
41.44, 25.01, 22.90.
Synthesis of 1-(3,5-Bis(trifluoromethyl)phenyl)-3-(2-bromoethyl)urea (6)
2-Bromoethylamine hydrobromide (1 g, 4.88 mmol), 3,5-bis(trifluoromethyl)phenyl
isocyanate (0.93 mL, 5.37 mmol) and triethylamine (0.748 mL, 5.37 mmol) were dissolved in
dichloromethane (20 mL) at 0°C and the resulting mixture was stirred for 4h, after which all
volatiles were evaporated. The product was purified on silica gel using a 30:70 mixture of
ethyl acetate:hexanes, yielding, after evaporation and vacuum drying, 0.9 g (2.34 mmol,
48%).
1H NMR (400 MHz, DMSO) δ 9.42 (s, 1H), 8.08 (s, 2H), 7.56 (s, 1H), 6.73 (t, J = 5.2 Hz,
1H), 3.61 – 3.45 (m, 4H).
4

13C NMR (101 MHz, DMSO) δ 155.11, 142.83, 131.26, 130.94, 117.81, 114.17, 41.76, 33.47.
Synthesis of 1-(3,5-Bis(trifluoromethyl)phenyl)-3-(2-azidoethyl)urea (7)
1-(3,5-Bis(trifluoromethyl)phenyl)-3-(2-bromoethyl)urea (900 mg, 2.34 mmol) was dissolved
in DMF (40 mL) at rt and sodium azide (385 mg, 5.93 mmol), and a few mg of KI were
added. The resulting mixture was stirred for 24 h at 80°C. It was then poured into water (300
mL) and extracted with diethyl ether. The combined organic layers were then evaporated
under reduced pressure and purified on silica gel using a 30:70 mixture of ethyl
acetate:hexanes, affording 450 mg (1.3 mmol, 55%) of azide.
1H NMR (400 MHz, DMSO) δ 9.34 (s, 1H), 8.09 (s, 2H), 7.54 (s, 1H), 6.67 (t, J = 5.6 Hz,
1H), 3.42 (t, J = 5.8 Hz, 2H), 3.32 (q, J = 5.4 Hz, 2H).
13C NMR 13C NMR (101 MHz, DMSO) δ 154.79, 142.41, 130.75, 130.43, 117.34, 113.58,
50.38, 38.99.
Synthesis of receptors 8a-d
Mono-2-propargyl-β-CD 1 (1 g, 0.85 mmol) and the selected azide (1.1 equiv.) were
dissolved in a 4:1 mixture of DMSO and water (30 mL). Tris[(1-benzyl-1H-1,2,3-triazol-4-
yl)methyl]amine (TBTA, ca. 20 mg) was added. The mixture was deaerated by passing argon
through the solution. CuSO4 (0.1 equiv., aqueous solution) and sodium ascorbate (0.2 equiv.,
aqueous solution) were added dropwise into the stirred reaction mixture, which was further
stirred for 24 h at 50°C. After the removal of the catalyst (via the addition of ammonia to the
reaction mixture and its passage through a thin pad of silica gel) the solvents were evaporated
under reduced pressure and the crude product was dissolved in water (5 mL) and precipitated
with acetone (500 mL). The precipitate was filtered and air dried.
(S)-2-O-((1-(2-(3-Phenylureido)-4-methylpentyl)-1H-1,2,3-triazol-4-yl)methyl)-β-CD (8a)
1H NMR (500 MHz, DMSO) δ 8.40 (s, 1H), 8.07 (s, 1H), 7.36 (d, J = 7.6 Hz, 2H), 7.25 – 7.15
(m, 2H), 6.89 (t, J = 7.3 Hz, 2H), 6.08 (s, 3H), 5.90 (d, J = 6.5 Hz, 3H), 5.80 – 5.59 (m, 7H),
4.94 – 4.74 (m, 7H), 4.66 – 4.54 (m, 2H), 4.53 – 4.36 (m, 7H), 4.09 (dd, J = 16.5, 4.2 Hz, 2H),
3.74 – 3.48 (m, 21H), 3.32 (s, 22H), 1.73 – 1.59 (m, 2H), 1.32 – 1.11 (m, 1H), 0.86 (dd, J =
16.2, 6.6 Hz, 6H).
13C NMR (126 MHz, DMSO) δ 128.64, 124.93, 121.14, 117.70, 101.93, 101.53, 100.27,
82.07, 81.59, 81.52, 81.21, 79.43, 73.25, 73.04, 72.95, 72.67, 72.48, 72.38, 72.22, 72.02,
71.75, 71.69, 64.42, 60.02, 59.91, 53.52, 47.41, 42.88, 36.67, 29.02, 28.70, 24.12, 23.18,
21.64.
HRMS ESI+ m/z: 1434.5519 (MH+, C58H91N5O36H+ requires 1434.5517); 1456.5321 (MNa+,
C58H91N5O36Na+ requires 1456.5336).
(S)-2-O-((1-(2-(3-(4-Nitrophenyl)-ureido)-4-methylpentyl)-1H-1,2,3-triazol-4-yl)methyl)-β-
CD (8b)
1H NMR (500 MHz, DMSO) δ 9.25 (s, 1H), 8.11 (t, J = 13.8 Hz, 3H), 7.53 (dd, J = 53.5, 7.4
Hz, 2H), 6.48 (s, 1H), 6.09 – 5.47 (m, 11H), 4.99 – 4.66 (m, 7H), 4.66 – 4.30 (m, 7H), 4.09
(d, J = 43.9 Hz, 1H), 3.81 (t, J = 9.3 Hz, 1H), 3.75 – 3.46 (m, 22H), 3.46 – 3.09 (m, 22H),
1.64 (s, 2H), 1.27 (dd, J = 53.3, 40.5 Hz, 3H), 0.87 (dd, J = 17.3, 5.6 Hz, 6H).
13C NMR (126 MHz, DMSO) δ 128.76, 127.76, 125.14, 124.97, 116.98, 101.96, 101.54,
100.32, 84.40, 82.12, 81.63, 81.57, 81.46, 81.17, 79.46, 73.30, 73.07, 72.97, 72.71, 72.52,
5

72.40, 72.26, 72.04, 71.79, 71.70, 64.43, 60.06, 59.91, 59.79, 53.35, 47.72, 24.16, 23.18,
21.62.
HRMS ESI+ m/z: 1479.5350 (MH+, C58H90N6O38H+ requires 1479.5367); 1501.5163 (MNa+,
C58H90N6O38Na+ requires 1501.5187).
(S)-2-O-((1-(2-(3-(3,5-Di(trifluoromethyl))-phenyl)-ureido)-4-methylpentyl)-1H-1,2,3-
triazol-4-yl)methyl)-β-CD (8c)
1H NMR (500 MHz, DMSO) δ 9.21 (s, 1H), 8.23 – 7.80 (m, 3H), 7.52 (s, 1H), 6.49 (s, 2H),
6.19 – 5.37 (m, 11H), 5.01 – 4.67 (m, 7H), 4.66 – 4.31 (m, 7H), 4.06 (s, 1H), 3.81 (t, J = 9.4
Hz, 1H), 3.75 – 3.46 (m, 23H), 3.46 – 3.09 (m, 25H), 1.57 (d, J = 68.8 Hz, 2H), 1.43 – 1.09
(m, 3H), 0.86 (dd, J = 19.4, 6.4 Hz, 6H).
13C NMR (126 MHz, DMSO) δ 154.36, 143.41, 142.37, 130.73, 130.48, 125.02, 124.45,
122.28, 117.37, 101.96, 101.56, 100.26, 82.09, 81.58, 81.22, 79.35, 73.23, 73.06, 72.95,
72.68, 72.51, 72.40, 72.24, 72.04, 71.70, 64.32, 59.93, 53.28, 47.81, 24.14, 23.15, 21.64, 0.14.
HRMS ESI+ m/z: 1570.5255 (MH+, C60H89N5O36F6H+ requires 1570.5264).
2-O-((1-(2-(3-(3,5-Di(trifluoromethyl))-phenylureido)-ethyl)-1H-1,2,3-triazol-4-
yl)methyl)-β-CD (8d)
1H NMR (500 MHz, DMSO) δ 9.53 (s, 1H), 8.15 (d, J = 7.7 Hz, 1H), 8.08 (s, 2H), 7.55 (s,
1H), 6.72 (t, J = 5.6 Hz, 1H), 5.99 – 5.83 (m, 2H), 5.83 – 5.60 (m, 11H), 4.94 – 4.76 (m, 6H),
4.60 – 4.33 (m, 14H), 3.82 (t, J = 9.5 Hz, 1H), 3.76 – 3.49 (m, 20H), 3.48 – 3.15 (m, 22H).
13C NMR (126 MHz, DMSO) δ 155.32, 144.50, 144.12, 142.84, 131.43, 131.17, 130.91,
130.65, 127.04, 124.87, 124.82, 122.70, 120.53, 117.73, 114.08, 109.99, 102.37, 102.03,
100.60, 82.43, 82.01, 81.92, 81.69, 79.87, 73.61, 73.49, 73.38, 73.14, 72.92, 72.81, 72.65,
72.46, 72.22, 72.12, 64.88, 60.35, 49.70.
HRMS ESI+ m/z: 1514.4606 (MH+, C56H82N5O36F6H+ requires 1514.4644).
<H2>2.3. Isothermal Titration Calorimetry
Prior to the binding measurements, compounds 8 were purified via dialysis. 500 mg of
compound 8 was dissolved in 10 mL of water and placed in a dialysis tube. The sealed tube
was placed in 3 L of water, which was stirred during the purification process. Water was
replaced after 2, 12 and 24 hours. Finally the content of the bag was lyophilized.
Isothermal titration calorimetry (ITC) was performed using TA Instruments NanoITC
calorimeter. The solutions of all hosts and guests were prepared in 50 mM phosphate buffer
(pH = 8). In a typical experiment the calorimeter cell (0.949 mL) was filled with 2 mM
solution of host 8. The cell solution was stirred at 300 rpm. 10-16 mM amino acid solution
(250 µl) was titrated into the calorimeter cell in 25 injections, with 300 s intervals between
injections, at 25oC. Measured raw heats were corrected with the heat of dilution of amino acid
titrated into phosphate buffer. Heats were fitted to n-independent-sites binding model (H + nG
= HGn), which allowed to determine the Ka (affinity constant), ΔH (effective molar enthalpy
of the reaction) and n (stoichiometry of guest:host interaction). Mean parameters and their
uncertainties were calculated as the average of at least 3 independent experiments.
Model:
ITC measurements were conducted by the titration of amino acids into solutions of receptors.
The concentrations were restricted by the heat response upon titration and solubility of the
receptors. We have applied a model where one of the interacting species has n identical and
independent binding sites (n-independent-sites binding model), which allowed to compare the
strength of the interaction between the receptor and particular guests (amino acids).
6

Interaction parameter 휈̅ can be defined as a number of moles of the guest (G) bound to
one mole of the host (H), or in terms of concentration:
n
 i1
n
i
i  G
G
 
 
i
b
 
i
H
 
  G
 
T
i0
i
Transformation of this relation allows to link 휈̅ with the number of active sites of the host (n),
the microscopic affinity constant K (defined for elementary reaction G + H = GH) and the
unbounded guest concentration:
G
nK L
 
 
B
 

H
 
1 K L
 
T
Therefore, after transformations the injection heat exchange can be infered by estimating the
derivative:





Happ
1 G  nK H
   
1
dQ
1
Q
T
T




1

1 K G  nK H 2  4nK2 G
H
T
V d G
VC  G
 
2
 
C
T
T
       






T
T
T
Values of
K
,
Happ and
n
were calculated using nonlinear regression program
NanoAnalyzer supplied by TA Instruments. Errors in estimation were calculated using Monte
Carlo analysis and included in the analysis of measurement uncertainties.
<H2>2.4. Dynamic Light Scattering
Dynamic light scattering (DLS) measurements were conducted with a Zetasizer nanoseries
Nano-ZS-ZEN3600 instrument equipped with a laser of 633 nm using a ZEN-112 cuvette at
25 °C. For each measurement the number of scans was 10, the run duration was 35 s, the
equilibration time was 140 s, and the time delay was 4 s. Before measurements, the samples
were incubated for 2 h, then filtered through a 0.45 μm PTFE filter, sonicated for 1 min, and
finally filtered again.
<H1>3. Results and Discussion
<H2>3.1. Synthesis
Our aim in this study was to synthesize a class of compounds that can be easily obtained from
a simple and readily available β-CD derivative. We intended to incorporate moieties
containing both polar and hydrophobic fragments that would efficiently interact with model
amino acid guests. We previously reported that C-6 modified compounds are more likely to
undergo intramolecular self-association, which may hamper guest binding inside the cavity
(Chmurski, Stepniak, & Jurczak, 2016). In consequence, we decided to derivatize one of the
secondary OH groups of β-CD. Previous studies have reported that secondary-site
derivatization towards the synthesis of carbamates, esters, and ethers, results in moderate
yields (Krois, Brecker, Werner, & Brinker, 2004; Martina et al., 2010; Suzuki & Nozoe,
2002). Derivatives containing aromatic substituents have been obtained via amidation or
esterification reactions (Fukudome, Oiwane, Mori, Yuan, & Fujita, 2004; Hanessian, Benalil,
& Viet, 1995; Hao, Tong, Zhang, & Gao, 1995; McAlpine & Garcia-Garibay, 1996;
Mortellaro, Hartmann, & Nocera, 1996). Again, yields below 25% have been reported,
suggesting that it is not the character of the substituent residue, but the reaction mechanism
7

that is responsible for the low efficiency of monosubstitution processes. Some β-CDs
substituted with charged species (such as anionic sulfonates, EDTA derivatives, protonated
amines (Laferriere, Benalil, & Hanessian, 1995; Michels, Huskens, & Reinhoudt, 2002; Wenz
& Höfler, 1999; Yan, Atsumi, Yuan, & Fujita, 2002)) have also been synthesized. Usually, in
these systems, the β-CD moiety is responsible for interaction with the hydrophobic part of the
guest, while the side-chain bearing the polar group attracts ionized parts of the interacting
molecule. However, some residues attached to the upper rim of β-CD can also form self-
inclusion complexes. All the derivatizations described above are characterized by low yields
due to the many resulting side-products and the necessity of low efficiency purification.
Usually, the synthesis is limited by the presence of reactive groups in side-arms introduced to
the β-CD structure. An alternative, indirect way of derivatization is a perbenzylation-
debenzylation protocol (Wang et al., 2014). In view of these facts, a Sharpless click chemistry
approach (copper(I)-mediated azide-alkyne coupling – CuAAC) suggests itself as a versatile
and efficient method for β-CD derivatization (Aime et al., 2009; Faugeras et al., 2012;
Martos-Maldonado, Quesada-Soriano, Casas-Solvas, García-Fuentes, & Vargas-Berenguel,
2012; Rostovtsev, Green, Fokin, & Sharpless, 2002). It would allow us to minimize the
number of reaction steps performed on cyclodextrins as the more demanding synthetic
substrate, eliminating low yield procedures.
Due to the relatively higher acidity of the 2-OH groups (as compared to the others) we
deprotonated β-CD with lithium hydride; further treatment with propargyl bromide, according
to the known procedure (Casas-Solvas et al., 2009), gave mono-2-propargyl-β-CD (1)
(Scheme 1), which was then purified by gel-filtration on styrene resin. The procedure allowed
to obtain monosubstituted derivative (DS = 1). The propargyl substituent was positioned
solely at C-2 position of glucose subunit (for structural elucidation see: Appendix A.)
Next we prepared azide compounds of type 4 (Scheme 2a), which were to be partners for 1 in
the CuAAC method. Due to the anion binding properties of the urea group, we decided to
apply it as a pH-independent donor of hydrogen bonds, thus making it possible to synthesize a
class of receptors providing a metal-free binding of anions.
In order to introduce two additional attachment points into the target receptor, we modified
hydrophobic amino acid so it contained a urea moiety and a nonpolar fragment. The
replacement of the carboxyl group by the azide gave the intermediate for the conjugation
reaction. For this purpose we used N-Boc-L-leucine which was treated with iso-butyl
chloroformate, the resulting mixed anhydride being reduced with NaBH4 to give
aminoalcohol 2. Next, aminoalcohol 2 was tosylated and subsequently treated with sodium
azide, giving compound 3. Careful purification of aminoalcohol 2 before the tosylation
process is crucial, as mixed anhydride (formed prior to the reduction step) acts as an inhibitor.
The addition of more than 3 equivs of NaN3 caused elimination of tosylated aminoalcohol due
to the basicity of azide ions in DMF. The cleavage of the Boc group was carried out in a 1:1
TFA:CH2Cl2 mixture. The resulting crude aminium trifluoroacetate was reacted with aromatic
isocyanate under basic conditions, to yield urea-azide compound 4.
The conjugation reaction of 1 with 4 was carried out under conditions we have described
previously (Scheme 3) (Chmurski, Stepniak, & Jurczak, 2015). Using a 1:5 (v/v)
water:DMSO mixture allowed us to apply equimolar amounts of 1 and 4. Addition of 5 mol%
of copper catalyst provided reaction completion after 24 h. After removal of the catalyst (via
the addition of ammonia to the reaction mixture and its passage through a thin pad of silica
gel) and addition of acetone, the final product precipitated.
We synthesized three compounds of type 8, all of their side-arms containing a bulky
substituent (sec-butyl) and a urea grouping of varying acidity – due to the presence of phenyl
(8a), p-NO2-phenyl (8b), or 3,5-di-CF3-phenyl (8c) moieties.
8

We also prepared an analog 8d, bearing an achiral arm, in order to assess the role of the
isobutyl group in complexation process and the influence of the side-chain chiral center on the
chiral recognition of amino acids (Scheme 2b). Bromoethylamine (5) was reacted with a
selected isocyanate and the resulting bromourea 6 was transformed into azidourea 7, then
subsequently subjected to CuAAC reaction with 1.
<H2>3.2. Binding measurments
Next, we investigated the complexing properties of compounds 8a-d towards representative
hydrophobic L-amino acids. As we stressed above, β-CD derivatives would be the most
promising since a cavity of this size allows the formation of inclusion complexes with a vast
majority of substances possessing aromatic side-chains or hydrophobic moieties (Rekharsky
& Inoue, 1998). However, β-CD:L-phenylalanine complex has low stability in water (Ka is
ca. 18 M-1 in distilled water and 107 M-1 in a buffered solution (pH 11.3)). Several attempts
have been made to modify the β-CD structure in order to enhance these affinities. For
example, Tabushi, Kuroda & Mizutani (1986) regioselectively incorporated two ionic
residues that provided ammonium and carboxylate binding sites. Although this β-CD
derivative exhibits moderate chiral recognition of L-tryptophan, the affinity constant still does
not exceed 100 M-1. Because of both the high polarity and good solubility of amino acids in
water, multiple intermolecular interactions have to be formed by the receptor in order to
compete with the hydrogen bonding network and hydration shell stability (Whitesides &
Krishnamurthy, 2005). Usually this is achieved by the synthesis of receptors based on CD,
calixarene, and cucurbituril moieties or the addition of a metallic center able to introduce
metal-carboxylate or metal-amine interactions. Coordination complexes with affinity
constants up to 7.4×107 M-1 for L-phenylalanine:Cu2+:amino-β-CD-derivative ternary
complexes were reported as the most effective organometallic systems (Brown, Coates,
Easton, & Lincoln, 1994; Hoof, Russell, Mcnamara, & Darcy, 2000). Another way of making
complexes more stable is via the derivatization of the amino acid to increase its
hydrophobicity. However, this approach cannot be considered when the receptor is meant to
function in an environment that limits chemical modifications of substrates.
Receptors 8 were designed to operate in buffered aqueous solutions, so we decided to use
isothermal titration calorimetry (ITC) as the tool useful for simultaneous assessment of the
thermodynamic parameters, affinity constants and stoichiometry of the interacting species
(Czekalla et al., 1998; Schmidtchen, 2002). We were unable to conduct NMR titrations due to
the very small changes in shifts upon addition of the guest into the host solution. UV-VIS
spectrometry could also not be used as a general method for all the pairs of guests and hosts.
The overlapping of absorption bands of guests and hosts in some experiments caused
problems with fitting to the appropriate model. ITC, on the other hand, provided very reliable
data for the whole assay. Within the receptor solubility range, the detected heats were high
enough and the heat data showed a sigmoidal curvature (Figure 1).
Titration experiments carried out at pH 8 (50 mM phosphate-buffered solution) showed that
binding constants vary for amino acids with different hydrophobic side-chains (Figure 2). For
all amino acids, binding to receptors 8a-c was much stronger than binding to underivatized β-
CD. L-Alanine formed such complexes with moderate strength (2 300 to 5 000 M-1), while the
more hydrophobic L-phenylalanine exhibited much greater affinity to all receptors (up to
22 400 M-1). The synthesized CDs seemed to interact more strongly with an even larger
hydrophobic amino acid – L-tryptophan. Interestingly, more acidic urea moieties (in 8b and
8c) contributed to the formation of stronger complexes, indicating that hydrogen bonding of
this group plays a crucial role in the presented system. Reference compound 8d showed no
interaction with any of the amino acids investigated (Ka < 10 M-1), showing that the bulky
substituent is also a necessary part of the complexing system.
9

The ITC method also provided information about the thermodynamics and stoichiometry of
the complexation reaction (Table 1).
The enthalpies of all the reactions studied were slightly positive. The complex formation is
entropy-driven and this thermodynamic parameter mostly determines the equilibrium, since
the enthalpy values are similar for each amino acid (around 4.4 kJ/mol). In most cases in the
literature, the formation of CD inclusion complexes exhibits both negative enthalpy and
entropy. In the present study, however, the signs of both parameters were positive, as should
be typical of a classic hydrophobic effect. Therefore, we suspected a complexation
mechanism different from the inclusion process. The much stronger complexation of amino
acids by receptors 8a-c, in comparison to underivatized β-CD and compound 8d, showed that
the side-chain plays an important role and suggested that both its elements (the hydrogen
bond donor and the bulky hydrophobic substituent) are responsible for the complexation.
<H2>3.3. Structural studies
Amphiphilic derivatives of CDs are known to form aggregates which can influence their
properties and mode of interactions (Chmurski, Stępniak & Jurczak 2016, Tran et al., 2014).
In the case of similar CD derivatives we have also observed formation of intra- and
intermolecular self-inclusion complexes, thus affecting the stoichiometry of the guest binding.
Therefore, we carried out DLS measurements to assess the degree of aggregation of
compounds 8a-d in a 50 mM phosphate buffer (pH 8) as an imitation of cell physiological
conditions. This allowed us to identify polydispersed solutions of particles with average
diameter of 190, 142, and 122 nm for 8a, 8b, and 8c, respectively. The aggregation process
occurred also in diluted solutions (0.5 mM). Interestingly, underivatized β-CD and compound
8d did not form any aggregates. This provided us with evidence that both urea moiety and the
hydrophobic side-chain are important for interactions between obtained molecules and that
both affect self-association of these compounds. Provided that diameter of β-CD is about 1.5
nm, several dozens of molecules have to be involved in formation of the observed aggregates.
To elucidate the mode of self-interaction between molecules of compounds 8 we have
conducted ROESY experiments of 8c in D2O (Figure 3). We have found crosspeaks between
aromatic protons and CD cavity protons (7.47 with 3.97, 3.82 and 7.80 with 3.91, 3.75 ppm,
respectively). Moreover, we identified interactions between the isobutyl group and C-3 and C-
5 protons of CD (3.72-3.92 ppm). This clearly indicates that both substituents of the side-
chain participate in inclusion complex formation. We did not observe crosspeaks
corresponding to interaction between the triazole ring and CD protons that would suggest
intramolecular inclusion process. This is understandable considering the results of DLS
measurements. Most probably compounds 8a-c form aggregates via intermolecular binding of
amphiphilic side-chains by CD cavities. Presented data do not determine what higher ordered
structures are formed during the self-association process. Similar results were obtained for
alkaline aqueous solution of compound 8c. This shows that the hydrophobic side-chain is
crucial in the self-association process.
This hypothesis is supported by the stoichiometry of the reaction measured with ITC. It can
be inferred that approximately two molecules of a receptor of type 8 interact with one
molecule of an amino acid, although the n parameter varies between 0.35 and 0.92.
For such a stoichiometry we suppose that receptors of type 8 form shallow intermolecular
inclusion complexes, leading to aggregates owing to the presence of the hydrophobic
fragment of the side-chain. The thermodynamic data suggest that a binding pocket is created
in such a dimeric species, which behaves like a lipophilic environment isolated from the bulk
solvent (Figure 4). In such a construct, urea moieties provide undisturbed hydrogen bonding
allowing efficient interactions with the anionic guests, which may be the reason for such high
affinities in comparison to underivatized β-CD.
10

The ROESY experiments performed after addition of amino acid to the receptor solution
showed no signs of interaction between the protons of the CD cavity and either the receptor’s
side-chain or the amino acid. This seems to confirm the non-inclusion nature of the complex
formed.
<H1>4. Summary
In conclusion, we have synthesized amphiphilic monoderivatives of β-CD which are equipped
with a bifunctional side-chain responsible for the formation of active aggregates and tight
guest binding, due to presence of isopropyl and urea moieties, respectively. The concept of
the synthesis was to limit the number of reactions performed on CD, which lead to high
efficiency of the process. Receptors obtained in such a manner appeared to form aggregates.
The amphiphilic binding site is formed in the at least dimeric construct allowing the urea
moieties to act effectively, isolated from the bulk solvent. Typical entropy-driven
complexation is observed for the interaction with L-amino acids, proving that a classic
hydrophobic effect is responsible, rather than inclusion complex formation.
We can confirm that the designed β-CD decorated with a amphiphilic side-chain provide an
environment for the efficient binding of amino acids, however, the mode of interaction was
different than expected. Nevertheless, this approach allows hydrophobic L-amino acids to be
bound with thousand-fold greater strength than for the previously reported native β-CD.
<ACK>Acknowledgements
This research was financed by the European Union within the European Regional
Development Fund, Project POIG.01.01.02.-14-102/09. Authors thank Olga Staszewska-
Krajewska for the NMR measurements.
Appendix A.
Supplementary data Supplementary data associated with this article can be found, in the
online version.
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</BIBL>
<Figure>Figure 1. Representative thermogram of ITC experiment L-Phe was titrated to 8c.
<Figure>Figure 2. Affinity constants of receptors 8a-c and L-amino acid complexes
(estimation uncertainties are marked).
<Figure>Figure 3. ROESY spectrum of 8c in D2O.
<Figure>Figure 4. Illustration of the proposed binding mechanism.
<Figure>Scheme 1. Preparation of mono-2-propargyl-β-CD (1)
<Figure>Scheme 2. Synthesis of receptor side-chains.
<Figure>Scheme 3. Synthesis of receptors 8
<Table>Table 1. Thermodynamic data for L-amino acid and receptor 8a-c complexesa
ΔH
ΔS
K (103 M-1)b nc
(kJ/mol) (kJ/mol)
8a
Ala 2.3±0.4
Phe 7.0±1.6
Trp 9.8±5.6
8b
0.509±0.039 2.99±0.32 74.4
0.380±0.050 4.18±0.79 87.6
0.349±0.092 4.02±2.51 89.8
Ala 2.1±0.2
Phe 20.2±1.9
Trp 34.4±2.0
8c
0.773±0.030 4.89±0.27 79.8
0.481±0.006 4.9±0.09 98.8
0.393±0.003 5.42±0.07 105.0
Ala 5.0±0.9
Phe 22.4±1.4
Trp 52.4±3.0
0.922±0.026 3.72±0.28 83.3
0.520±0.002 5.51±0.07 101.7
0.457±0.002 4.46±0.05 105.3
a Aa (250 μL, 10-16 mM) was titrated to
8 (949 μL, 2
mM), stirred at 300 rpm. b Results fitted to n-
independent-sites binding model (where K is a
microscopic affinity constant). c Stoichiometry of the
interaction.
TDENDOFDOCTD
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