Chloride transport activities of trans- and cis-amide-linked bisureas

Article abstract of DOI:10.1039/c5cc02757h

Of the bisurea compounds linked through trans- and cis-benzanilide spacers, the cis-amide derivatives were found to be effective in chloride transport, using which a stimuli-responsive mobile carrier was devised. This journal is

Full text of DOI:10.1039/c5cc02757h

ChemComm
View Article Online
View Journal | View Issue
COMMUNICATION
Chloride transport activities of trans- and
cis-amide-linked bisureas†
Cite this: Chem. Commun., 2015,
51, 9197
Eun Bit Park and Kyu-Sung Jeong*
Received 2nd April 2015,
Accepted 29th April 2015
DOI: 10.1039/c5cc02757h
www.rsc.org/chemcomm
Of the bisurea compounds linked through trans- and cis-benzanilide anion transporters,10 we have herein prepared a series of bisurea
spacers, the cis-amide derivatives were found to be effective in compounds 1–7 containing trans- or cis-amides, and their transport
chloride transport, using which a stimuli-responsive mobile carrier abilities for chloride ions across a lipid membrane have been
was devised.
revealed. Furthermore, it is also demonstrated that the transport
activity can be modulated by chemical and enzymatic reactions,
Nature uses amide bonds to connect amino acids and synthesize leading to the conversion of cis-to-trans amide.
peptides and proteins. Most of the amide bonds predominantly
The syntheses of bisurea compounds 1–7 are outlined in
adopt trans conformations but the population of cis conformations Scheme 1. Coupling of 10 and 11 in the presence of 1-ethyl-3-(3-
tends to increase in the tertiary amides prepared from proline and dimethylaminopropyl)carbodiimide (EDCI) and 1-hydroxybenzo-
N-substituted amino acids such as sarcosine.1 The relative stabilities triazole (HOBt) afforded compound 12.11 After removal of the
of trans and cis conformers are reversed in some unnatural amides t-butyloxycarbonyl (Boc) protecting groups, the resulting amine
such as N-methyl acetanilide and benzanilide, showing a strong was directly coupled with p-cyanophenyl isocyanate to afford
preference for the cis conformation in the solid state and in bisurea 1. On the other hand, N-methylation of 12 yielded
solution.2 This feature has been nicely utilised for the construc- compound 13, which was converted to bisurea compounds 2–7
tion of amide-based foldamers and molecular switches.3
Transmembrane anion transport is essential to control the
via sequential deprotection and coupling reactions.
We first compared the chloride transport abilities of two urea
cellular ion concentration, osmotic pressure, volume and pH in compounds 1 and 2, which contain trans- and cis-amides, respec-
living organisms, and it is also associated with signal production, tively. Both compounds have identical p-cyanophenyl substituents,
cell-to-cell communication, neuronal proliferation, differentiation, which are known to greatly enhance the anion transport activities of
etc.4 It is well known that anion transport in the biological systems urea receptors across lipid membranes.8a,9 The chloride transport
is facilitated by proteins functioning as anion channels, trans- abilities of 1 and 2 were estimated using unilamellar 1-palmitoyl-2-
porters, and exchangers. A variety of synthetic molecules that can oleoyl-2-oleoylphosphatidyl-choline (POPC) vesicles which contain a
facilitate anion transport across lipid and cell membranes have phosphate buffer (10 mM, pH = 7.2), lucigenin (1 mM) and NaNO3
been described in the last few decades.5,6 These synthetic trans- (200 mM). These vesicles were suspended in a phosphate buffer (10
porters may have applications in biomedical research and potential mM, pH = 7.2) containing NaNO3 (200 mM) and NaCl (30 mM).
therapeutics for diseases related to defective anion transport.7
Chloride influx into vesicles upon addition of each compound in
In recent years, we have synthesized small synthetic molecules DMSO solution (2 mol% to lipid) was monitored via fluorescence
that can facilitate anion transport across lipid membranes via quenching of lucigenin. As shown in Fig. 1a, compound 1 with
antiport or symport.8 It was also demonstrated that the transmem- a trans-amide is nearly inactive while compound 2 with a cis-
brane transport of chloride ions could be controlled by light amide is highly efficient to facilitate chloride transport across a
stimulus using diazobenzene-based synthetic transporters.9 As part POPC membrane. The Hill plot afforded the EC50 value of 0.24 mol%
of our continuing efforts to develop stimuli-responsive synthetic (2 to lipid), which is the concentration needed to achieve 50%
completion of chloride transport at 300 s. A dramatic difference
in the chloride transport activities of 1 and 2 is attributed in part to
their binding affinities with the chloride ion. In compound 2
with a cis-amide, two urea functional groups can be involved in
Department of Chemistry, Yonsei University, Seoul, 120-749, Korea.
E-mail: ksjeong@yonsei.ac.kr; Fax: +82-2-364-7050; Tel: +82-2-2123-2643
† Electronic supplementary information (ESI) available: Synthetic procedures and
the binding of the chloride ion in a chelate manner, thus
simultaneously forming four hydrogen bonds. The association
characterization, experimental details for titrations and transports. See DOI:
10.1039/c5cc02757h
This journal is ©The Royal Society of Chemistry 2015
Chem. Commun., 2015, 51, 9197--9200 | 9197

View Article Online
Communication
ChemComm
Fig. 1 (a) Chloride influxes facilitated by 1 and 2 (2 mol% to lipid) (b) chloride
influxes facilitated by 2–7 (2 mol% to lipid). Vesicles which contain a phosphate
buffer (10 mM, pH = 7.2), lucigenin (1 mM) and NaNO3 (200 mM) were
suspended in a phosphate buffer (10 mM, pH = 7.2) containing NaNO3
(200 mM) and NaCl (30 mM).
Scheme 1 Reagents and conditions: (a) EDCI, HOBt, Et3N, CH2Cl2, r.t., 5 h,
72%; (b) TFA, then p-cyanophenyl isocyanate, anhydrous Et3N, CH2Cl2, r.t., 6 h,
56% (for two steps); (c) KOH, MeI, acetone, reflux, 1 h, 97%; (d) TFA, then each
isocyanate, anhydrous Et3N, CH2Cl2, r.t., 4–7 h, 49–76% (for two steps).
Next, we investigated the substituent effects on the chloride
transport activities using bisurea compounds 2–6 with different
substituents on the terminal phenyl rings (Fig. 1b). The transport
activities increase in the order of 6 (OCH3) B 5 (H) o 4 (Cl) o 3
(CO2CH3) { 2 (CN). This trend matches well with the increasing
order of association constants between 2–6 and tetrabutylammonium
chloride; that is 6 (250 MÀ1) o 5 (370 MÀ1) o 4 (860 MÀ1) o 3
(1230 MÀ1) o 2 (3950 MÀ1) in 10% (v/v) DMSO-d6/CDCl3
saturated with water (o0.1%) at 24 Æ 1 1C (Fig. S1–S5, ESI†).13
In addition, we also prepared thiourea compound 7 with the
cyano substituent, which bound the chloride ion more strongly
(9500 MÀ1) than the corresponding urea compound 2 (Fig. S6,
ESI†). Compound 7 was found to be most efficient in the chloride
transport and the EC50 value was 0.08 molar ratio of 7 to POPC.
These results suggest that the transport ability of synthetic
transporters of the same molecular framework can be enhanced
by increasing the binding affinity with a target ion. In order to
reveal the transport mechanism, we investigated the chloride
transport of compound 2 using POPC vesicles containing Na2SO4
(67 mM), instead of NaNO3 (200 mM). The transport activity of 2
was practically ineffective under these conditions (Fig. S10,ÀESI†),
indicating that the chloride transport occurs via ClÀ/NO3 anti-
port mechanism. Furthermore, we carried out transport experi-
ments with vesicles prepared from a 7 : 3 POPC/cholesterol
mixture. It has been known that cholesterol may reduce the
membrane fluidity due to its rigid structure, thus slowing down
the chloride transport.14 Both compounds 2 and 7 exhibited
constant between 2 and tetrabutylammonium chloride was
determined to be 3950 MÀ1 in 10% (v/v) DMSO-d6/CDCl3
saturated with water (o0.1%) at 24 Æ 1 1C (Fig. S1, ESI†). However,
such a chelate binding mode is not possible in compound 1 with a
trans-amide. Instead, compound 1 showed complex binding iso-
therms and therefore monourea compound 8 was prepared as a
reference compound, which showed the association constant of
170 MÀ1 under the identical conditions (Fig. S7, ESI†). Another
possible reason for different transport activities is because com-
pound 2 has a folded, C-shaped conformation with an internal
binding cavity wherein the chloride ion binds. This geometric
feature may reduce the contact area with the lipid surface, which
may facilitate the shuttling movement of 2 and its chloride complex
inside the lipid membrane.9,12
9198 | Chem. Commun., 2015, 51, 9197--9200
This journal is ©The Royal Society of Chemistry 2015

View Article Online
ChemComm
Communication
One was the addition of a good nucleophile, hydrazine for deacetyla-
tion. The transport ability of 9 was found to be significantly dropped
(Fig. 2) when treated for 10 min with hydrazine hydrate (10 equiv.,
3 : 1 (v/v) DMSO : a phosphate buffer (pH = 7.2), 21 1C). Under these
conditions, compound 9 was gradually converted to compound 1
1
with the trans conformation as demonstrated by H NMR spectro-
scopy (Fig. S12, ESI†). Another approach examined here was more
bio-relevant degradation via the enzymatic hydrolysis of the ester
residue. Upon incubation of 9 with porcine liver esterase (PLE) for
30 min in a phosphate buffer (pH = 7.2, 21 1C), the transport activity
of chloride ions was significantly suppressed as a result of the
conversion of 9 to 1 under the given conditions (Fig. 2).
In conclusion, we have shown that the chloride transport
activities of amide-linked bisurea compounds depend strongly
on the central amide conformation as well as the binding affinities
with the chloride ion. The trans-amide with an unfolded, extended
structure shows negligible transport ability while the N-methylated
cis-amide with a folded, curved shape functions as a very potent
mobile carrier across POPC membranes. Anion transports in living
organisms are controlled by biological stimuli such as concen-
tration gradient changes, ligand binding, and chemical reactions.16
In this context, it is also demonstrated that the transport activity
can be modulated by the cis-to-trans conversion by the chemical
and enzymatic degradation.
This study was supported by the National Research Foundation
of Korea (NRF) grant funded by the Korea government (MEST)
(NRF-2013R1A2A2A05005796). E.B.P. acknowledges the fellowship
of the BK 21-plus program from the Ministry of Education and
Human Resources Development.
Scheme 2 Plausible mechanism for the cis-to-trans isomerization of 9
upon deacetylation by chemical and enzymatic reactions.
noticeably decreased activities of chloride transport in the presence
of cholesterol (Fig. S11, ESI†), supporting the functioning of the
compounds as mobile carriers. Furthermore, the Hill coefficients of
2 and 7 were determined to be 1.09 and 1.06, respectively, indicative
of monomeric carriers (Table S1, ESI†).15
Utilizing different transport activities between trans- and cis-
amide-linked bisureas, we have designed and prepared com-
pound 9 as a stimulus-responsive anion transporter. To adopt
a cis-amide conformation, the N-methyl moiety is replaced by
p-acetyloxybenzyl group which can be degraded via deacetylation
to produce compound 1 with a trans-amide (Scheme 2). We first
examined the chloride transport ability of compound 9 using POPC
vesicles under the conditions described earlier. As anticipated,
compound 9 iÀtself is very efficient to transport chloride ions
via a ClÀ/NO3 antiport (Fig. 2). To show stimuli-responsive
chloride transport, we examined two different approaches.
Notes and references
1 (a) Y. Takeuchi and G. R. Marshall, J. Am. Chem. Soc., 1998, 120,
5363–5372; (b) G. Fischer, Chem. Soc. Rev., 2000, 29, 119–127;
(c) C. Dugave and L. Demange, Chem. Rev., 2003, 103, 2475–2532;
(d) M. Kuemin, Y. A. Nagel, S. Schweizer, F. W. Monnard, C. Ochsenfeld
and H. Wennemers, Angew. Chem., Int. Ed., 2010, 49, 6324–6327.
2 (a) A. Itai, Y. Toriumi, N. Tomioka, H. Kagechika, I. Azumaya and
K. Shudo, Tetrahedron Lett., 1989, 30, 6177–6180; (b) I. Azumaya,
H. Kagechika, K. Yamaguchi and K. Shudo, Tetrahedron, 1995, 51,
5277–5290.
3 (a) R. Yamasaki, A. Tanatani, I. Azumaya, S. Saito, K. Yamaguchi and
H. Kagechika, Org. Lett., 2003, 5, 1265–1267; (b) A. Tanatani, A. Yokoyama,
I. Azumaya, Y. Takakura, C. Mitsui, M. Shiro, M. Uchiyama, A. Maranaka,
N. Kobayashi and T. Yokozawa, J. Am. Chem. Soc., 2005, 127, 8553–8561;
(c) I. Okamoto, M. Nabeta, Y. Hayakawa, N. Morita, T. Takeya, H. Masu,
I. Azumaya and O. Tamura, J. Am. Chem. Soc., 2005, 129, 1892–1893;
(d) I. Okamoto, R. Yamasaki, M. Sawamura, T. Kato, N. Nagayama,
T. Takeya, O. Tamura, H. Masu, I. Azumaya, K. Yamaguchi,
H. Kagechika and A. Tanatani, Org. Lett., 2007, 9, 5545–5547;
(e) H. Kakura, I. Azumaya, H. Masu, M. Matsumura, K. Yamaguchi,
H. Kagechika and A. Tanatani, Tetrahedron, 2010, 66, 8254–8260;
( f ) N. Fujimoto, M. Matsumura, I. Azumaya, S. Nishiyama, H. Masu,
H. Kagechika and A. Tanatani, Chem. Commun., 2012, 48, 4809–4811.
4 (a) T. J. Jentsch, V. Stein, F. Weinreich and A. A. Zdebik, Physiol. Rev.,
2002, 82, 503–568; (b) L. Puljak and G. Kilic, Biochim. Biophys. Acta,
2006, 1762, 404–413; (c) A. P. Davis, D. N. Sheppard and B. D. Smith,
Chem. Soc. Rev., 2007, 36, 348.
5 (a) For reviews, see: P. R. Brotherhood and A. P. Davis, Chem. Soc.
Rev., 2010, 39, 3633–3647(b) J. T. Davis, O. Okunola and R. Quesada,
Chem. Soc. Rev., 2010, 39, 3843–3862; (c) P. A. Gale, Acc. Chem. Res.,
2011, 44, 216–226; (d) S. Matile, A. V. Jentzsch, J. Montenegro and
A. Fin, Chem. Soc. Rev., 2011, 40, 2453–2474; (e) F. De Riccardids,
I. Izzo, D. Montesarchio and P. Tecilla, Acc. Chem. Res., 2013, 46,
Fig. 2 Decrease in transport activity of 9 when treated with hydrazine in a
3 : 1 (v/v) DMSO : a phosphate buffer (pH = 7.2) at 21 1C for 10 min or PLE in
a phosphate buffer (pH = 7.2) at 21 1C for 30 min.
This journal is ©The Royal Society of Chemistry 2015
Chem. Commun., 2015, 51, 9197--9200 | 9199

View Article Online
Communication
ChemComm
2781–2790; ( f ) A. V. Jentzch, A. Henning, J. Mareda and S. Matile,
8 (a) Y. R. Choi, M. K. Chae, D. Kim, M. S. Lah and K.-S. Jeong, Chem.
Commun., 2012, 48, 10346–10348; (b) J. H. Lee, J. H. Lee, Y. R. Choi,
P. Kang, M.-G. Choi and K.-S. Jeong, J. Org. Chem., 2014, 79, 6403–6409.
9 Y. R. Choi, G. C. Kim, H.-G. Jeon, J. Park, W. Namkung and K.-S. Jeong,
Chem. Commun., 2014, 50, 15305–15308.
´
´
Acc. Chem. Res., 2013, 46, 2791–2800; (g) P. A. Gale, R. Perez-Tomas
and R. Quesada, Acc. Chem. Res., 2013, 46, 2801–2813; (h) G. W.
Gokel and S. Negin, Acc. Chem. Res., 2013, 46, 2824–2833; (i) H. Valkenier
and A. P. Davis, Acc. Chem. Res., 2013, 46, 2898–2909; ( j) D. S. Kim and
J. Sessler, Chem. Soc. Rev., 2015, 44, 532–546.
10 (a) P. V. Santacroce, J. T. Davis, M. E. Light, P. A. Gale, J. C. Iglesias-
´
6 (a) For some selected recent papers, see: S. J. Moore, C. J. E. Haynes,
Sanchez, P. Prados and R. Quesada, J. Am. Chem. Soc., 2007, 129,
´
J. Gonazalez, J. L. Sutton, S. J. Brroks, M. E. Light, J. Herniman,
1886–1887; (b) O. A. Okunola, J. L. Seganish, K. J. Salimian,
P. Y. Zavalij and J. T. Davis, Tetrahedron, 2007, 63, 10743–10750;
(c) P. V. Jog and M. S. Gin, Org. Lett., 2008, 10, 3693–3696;
(d) N. Busschaert, R. B. P. Elmes, D. D. Czech, X. Wu, I. L. Kirby,
E. M. Peck, K. D. Hendzel, S. K. Shaw, B. Chan, B. D. Smith,
K. A. Jolliffe and P. A. Gale, Chem. Sci., 2014, 5, 3617–3626.
´
´
G. J. Langley, V. Soto-Cerrato, R. Perez-Tomas, I. Marques, P. J. Costa,
´
V. Felix and P. A. Gale, Chem. Sci., 2013, 4, 103–117(b) I. Alfonso and
R. Quesada, Chem. Sci., 2013, 4, 3009–3019; (c) C.-R. Elie, A. Hebert,
M. Charbonneau, A. Haiun and A. R. Schmitzer, Org. Biomol. Chem., 2013,
11, 923–928; (d) C. Chhun, J. Richard-Daniel, J. Kempf and A. R.
´
Schmitzer, Org. Biomol. Chem., 2013, 11, 6023–6028; (e) A. V. Jentzsch 11 R. Hrdina, E. C. Mu¨ller, R. C. Wende, L. Wanka and P. R. Schreiner,
and S. Matile, J. Am. Chem. Soc., 2013, 135, 5302–5303; ( f ) P. B. Cranwell, Chem. Commun., 2012, 48, 2498–2500.
J. R. Hiscock, C. J. E. Haynes, M. E. Light, N. J. Wells and P. A. Gale, 12 L. W. Judd and A. P. Davis, Chem. Commun., 2010, 46, 2227–2229.
Chem. Commun., 2013, 49, 874–876; (g) N. Busschaert, S. J. Bradberry, 13 (a) A. V. Koulov, T. N. Lambert, R. Shukla, M. Jain, J. M. Boon, B. D. Smith,
M. Wenzel, C. J. E. Haynes, J. R. Hiscock, I. L. Kirby, L. E. Karagiannidis,
S. J. Moore, N. J. Wells, J. Herniman, G. J. Langley, P. N. Horton,
H. Li, D. N. Sheppard, J. B. Joos, J. P. Clare and A. P. Davis, Angew.
Chem., Int. Ed., 2003, 42, 4931–4933; (b) B. A. MacNally, A. V. Koulov, B. D.
Smith, J.-B. Joos and A. P. Davis, Chem. Commun., 2005, 1087–1089;
(c) N. Busschaert, I. L. Kirby, S. Young, S. J. Coles, P. N. Horton,
M. E. Light and P. A. Gale, Angew. Chem., Int. Ed., 2012, 51, 4426–4430;
(d) L. Adriaenssens, J. Am. Chem. Soc., 2013, 135, 8324–8330.
´
M. E. Light, I. Marques, P. J. Costa, V. Felix, J. G. Frey and P. A. Gale,
Chem. Sci., 2013, 4, 3036–3045; (h) C. Chhun, J. Richard-Daniel, J. Kempf
and A. R. Schmitzer, Org. Biomol. Chem., 2013, 11, 6023–6028; (i) C. J. E.
Haynes, N. Busschaert, I. L. Kirby, J. Herniman, M. E. Light, N. J. Wells,
´
I. Marques, V. Felix and P. A. Gale, Org. Biomol. Chem., 2014, 12, 62–72; 14 (a) W. F. D. Bennett, J. L. MaCallum and D. P. Tieleman, J. Am.
´
´
´
( j) E. Hernando, V. Soto-Cerrato, S. Cortes-Arroyo, R. Perez-Tomas and
R. Quesada, Org. Biomol. Chem., 2014, 12, 1771–1778; (k) J. Shang, W. Si,
W. Zhao, Y. Che, J.-L. Hou and H. Jiang, Org. Lett., 2014, 16, 4008–4011;
Chem. Soc., 2009, 131, 1972–1978; (b) N. Busschaert, M. Wenzel,
´
´
´
M. E. Light, P. Iglesias-Hernandez, R. Perez-Tomas and P. A. Gale,
J. Am. Chem. Soc., 2011, 133, 14136–14148.
(l) J. A. Cooper, S. T. G. Street and A. P. Davis, Angew. Chem., Int. Ed., 2014, 15 (a) S. Matile and N. Sakai, in Analytical Methods in Supramolecular
53, 5609–5613; (m) H. Valkenier, L. W. Judd, H. Li, S. Hussain, D. N.
Sheppard and A. P. Davis, J. Am. Chem. Soc., 2014, 136, 12507–12512;
(n) H. Valkenier, N. L. Mora, A. Kros and A. P. Davis, Angew. Chem., Int.
Ed., 2015, 54, 2137–2141.
7 (a) N. Busschaert and P. A. Gale, Angew. Chem., Int. Ed., 2013, 52,
1374–1382; (b) S.-K. Ko, S. K. Kim, A. Share, V. M. Lynch, J. Park,
W. Namkung, W. V. Rossom, N. Busschaert, P. A. Gale, J. L. Sessler
and I. Shin, Nat. Chem., 2014, 6, 885–892.
Chemistry, ed. C. A. Schalley, Wiley, Weinheim, 2007, pp. 391–418;
(b) A. V. Jentzsch, D. Emery, J. Mareda, S. K. Nayak, P. Metrangolo,
G. Resnati, N. Sakai and S. Matile, Nat. Commun., 2012, 3, 905–913.
16 (a) M. Chebib and G. A. R. Johnston, J. Med. Chem., 2000, 43,
1427–1447; (b) A. S. Verkman and L. J. V. Galietta, Nat. Rev. Drug
Discovery, 2009, 8, 153–171; (c) K. T. Kahle, T. Z. Deeb, M. Puskarjov,
L. Silayeva, B. Liang, K. Kaila and S. J. Moss, Trends Neurosci., 2013,
36, 726–737.
9200 | Chem. Commun., 2015, 51, 9197--9200
This journal is ©The Royal Society of Chemistry 2015