Polycationic amphiphilic cyclodextrins for gene delivery: synthesis and effect of structural modifications on plasmid DNA complex stability, cytotoxicity, and gene expression

FULL PAPER

DOI: 10.1002/chem.200901149

Polycationic Amphiphilic Cyclodextrins for Gene Delivery: Synthesis and

Effect of Structural Modifications on Plasmid DNA Complex Stability,

AHCTUNGTRENGNCUN ytotoxicity, and Gene Expression

Alejandro Dꢀaz-Moscoso,^[a]Lçic Le Gourriꢁrec,^[b]Marta Gꢂmez-Garcꢀa,^[c]

Juan M. Benito,*^[a]Patricia Balbuena,^[c]Fernando Ortega-Caballero,^[c]

Nicolas Guilloteau,^[b]Christophe Di Giorgio,^[b]Pierre Vierling,*^[b]Jacques Defaye,*^[d]

Carmen Ortiz Mellet,*^[c]and Josꢁ M. Garcꢀa Fernꢃndez^[a]

Abstract: A molecular-diversity-orient-

ed approach for the preparation of

well-defined polycationic amphiphilic

cyclodextrins (paCDs) as gene-delivery

systems is reported. The synthetic strat-

egy takes advantage of the differential

reactivity of primary versus secondary

hydroxyl groups on the CD torus to re-

gioselectively decorate each rim with

cationic elements and lipophilic tails,

respectively. Both the charge density

and the hydrophobic–hydrophilic bal-

ance can be finely tuned in a highly

symmetrical architecture that is remi-

niscent of both cationic lipids and cat-

ionic polymers, the two most promi-

nent types of nonviral gene vectors.

The monodisperse nature of paCDs

and the modularity of the synthetic

scheme are particularly well suited for

structure–activity relationship studies.

Gel electrophoresis revealed that

paCDs self-assemble in the presence of

plasmid DNA (pDNA) to provide ho-

in vitro on BNL-CL2 and COS-7 cell

lines in the absence and presence of

serum and found to be intimately de-

pendent on architectural features.

Facial amphiphilicity and the presence

of a cluster of cationic and hydrogen-

bonding centers for cooperative and re-

versible complexation of the polyan-

ionic DNA chain is crucial to attain

high transgene expression levels with

very low toxicity profiles. Further en-

hancement of gene expression, eventu-

ally overcoming that of polyplexes

from commercial polyethyleneimine

(PEI) polymers (22 kDa), is achieved

by building up space-oriented dendritic

polycationic constructs.

mogeneous,

stable

nanoparticles

(CDplexes) of 70–150 nm that fully

protect pDNA from the environment.

The transfection efficiency of the re-

sulting CDplexes has been investigated

Keywords: amphiphiles · cyclodex-

trins · gene delivery · nanoparticles ·

self-assembly

[a] A. Dꢀaz-Moscoso, Dr. J. M. Benito, Prof. J. M. Garcꢀa Fernꢁndez

Instituto de Investigaciones Quꢀmicas

CSIC and Universidad de Sevilla

[c] Dr. M. Gꢃmez-Garcꢀa, Dr. P. Balbuena, Dr. F. Ortega-Caballero,

Prof. C. Ortiz Mellet

Departamento de Quꢀmica Orgꢁnica, Facultad de Quꢀmica

Universidad de Sevilla

Av. Amꢂrico Vespucio 49

Isla de la Cartuja, 41092 Sevilla (Spain)

Fax : (+34)954-460-565

Calle Prof. Garcꢀa Gonzꢁlez 1, 41012 Sevilla (Spain)

Fax : (+34)954-624-960

E-mail: juanmab@iiq.csic.es

E-mail: mellet@us.es

[b] Dr. L. Le Gourriꢂrec, N. Guilloteau, Dr. C. Di Giorgio, Dr. P. Vierling

LCMBA UMR 6001, CNRS

[d] Dr. J. Defaye

Dꢂpt. de Pharmacochimie Molꢂculaire

Institut de Chimie Molꢂculaire de Grenoble, CNRS

Universitꢂ de Grenoble, UMR 5063, FR 2607

Bꢄt. E. Andrꢂ Rassat, BP 53, 38041 Grenoble (France)

Fax : (+33)476-635-313

Universitꢂ de Nice Sophia Antipolis

28 Av. Valrose, 06100 Nice (France)

Fax : (+33)476-041-013

E-mail: vierling@unice.fr

E-mail: jacques.defaye@ujf-grenoble.fr

Supporting information for this article is available on the WWW

under http://dx.doi.org/10.1002/chem.200901149.

Chem. Eur. J. 2009, 15, 12871 – 12888

ꢅ 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

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Introduction

vance would be seriously impaired due to the complex legal

regulations that polydisperse entities have to meet.^[15]

The successful delivery of therapeutic genes to target cells

and their availability at the intracellular site of action are

crucial requirements for gene therapy. Naked plasmid DNA

(pDNA) has been shown to transfect cells both in vitro and

in vivo; however, the transfection efficiency is generally low

due to rapid degradation by serum nucleases and limited

membrane-permeation potential.^[1]In an effort to increase

transfection capabilities, much research has been carried out

on the development of effective carrier vectors, traditionally

divided into viral and nonviral systems, that compact, pro-

tect, and deliver genes.^[2]The U.S. Food and Drug Adminis-

tration (FDA) has not approved any viral-vector-based ther-

apeutics up to date due to immunogenicity, oncogenicity,

and potential virus recombination concerns.^[3]Nonviral

DNA condensing agents are currently the subject of increas-

ing attention because of their relative safety and simplicity

of use, though they are still far from achieving the gene ex-

pression efficiencies of viral systems.^[4]The design of artifi-

cial carriers that could prove as efficient as their viral coun-

terparts but that are safer to use, homogeneous, nonimmu-

nogenic, and more readily adapted to tailor-made elabora-

tion represents the ultimate challenge for the future devel-

opment of gene therapy.

Multifunctional molecular vectors have emerged as a new

generation of gene-delivery systems prone to chemical tai-

loring and systematic structural modification, thereby facili-

tating the elucidation of structure–activity relationships. A

general approach is based on the use of preorganized mac-

rocyclic scaffolds to achieve a precise alignment of function-

al elements, a strategy that has proven to be extremely

useful over the years in the design of artificial receptors/li-

gands that emulate the supramolecular events occurring in

living organisms. Thus, calixarene^[16]and cyclodextrin-cen-

tered^[17]starlike polycations have been synthesized and

shown to exhibit promising pDNA delivery abilities. The

tubular framework of CDs, which exhibit well-differentiated

faces, can be further amended to bidirectional functionaliza-

tion while keeping full homogeneity. The installation of seg-

regated polycationic and hydrophobic domains (Figure 1)

Most of the currently available nonviral gene vectors

belong to two main groups: cationic lipids and cationic poly-

mers.^[5]Both types of compounds can condense pDNA into

multimolecular complexes, named lipoplexes or polyplexes,

respectively, that show a range of sizes and physicochemical

properties in solution. Lipoplexes and polyplexes usually are

positively charged particles that efficiently enter the cell

after binding to negatively charged proteoglycans on the

outer face of the membrane, thereby resulting in improved

pharmacokinetics and pharmacodynamics and, eventually,

active intracellular delivery.^[6]Further functional elements

can be incorporated onto the vector or the preformed

pDNA–vector complex by means of covalent or supramolec-

ular interactions to help the system to overcome the cellular

barriers and the immune defense mechanisms, thus prevent-

ing undesired side effects or targeting specific tissues.^[7]The

cyclodextrin (CD)-containing polycationic polymers devel-

oped by Pun and Davis are paradigmatic examples in this

respect.^[8]The cyclomaltooligosaccharide framework imparts

biocompatibility, significantly alleviating the cytotoxicity of

cationic polymers^[9]both in vitro and in vivo,^[10]behaves as a

transfection enhancer, and offers unique possibilities associ-

ated with the intrinsic molecular inclusion capabilities of the

CD cavity,^[11,12]which has been translated into therapeutic

applications.^[13]Polyrotaxanes consisting of cationic CD de-

rivatives threaded onto poly(ethyleneglycol) (PEG) or

poly(ethyleneimine) (PEI) chains have also shown efficient

gene-delivery capabilities.^[14]Unfortunately, the essentially

polydisperse nature of such polymeric materials seriously

hampers drawing conclusions on structure–activity relation-

ships to provide feedback on new vector designs. Further-

more, even for the most efficient systems, their clinical rele-

Figure 1. Schematic representation of skirt- (left) and jellyfish-type

(right) polycationic amphiphilic CDs (paCDs). The circles and the rec-

tangular boxes represent positively charged groups and spacer segments,

respectively. The basket-shaped scaffold represents a cyclodextrin core

(see Scheme 1).

could be exploited, then, to generate facial amphiphilicity,^[18]

thereby endowing the system with self-assembling properties

and biomimetic^[19]cell-membrane-crossing aptitudes.^[20]We

have now implemented this concept in the preparation of a

library of monodisperse polycationic amphiphilic cyclodex-

trins (paCDs) that self-organize in the presence of pDNA to

form stable paCD–pDNA complexes (CDplexes). A modu-

lar synthetic approach has been settled that allows systemat-

ic modification of the charge density, spacer length and

nature, hydrophilic–hydrophobic balance, and overall archi-

tecture with high efficiency and relatively low synthetic cost.

The effects of structural variations on the molecular con-

struct in the CDplex stability and physicochemical proper-

ties, transfection efficacy, and cytotoxicity (BNL-CL2 and

COS-7 cell lines) have been assessed and compared to that

observed for commercial polycationic polymers (branched

PEI, 25 kDa;^[21]and linear JetPEI, 22 kDa).^[22]

Results and Discussion

Design criteria and synthesis: The construction of multi-

head/multitail paCDs critically depends on the availability

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Chem. Eur. J. 2009, 15, 12871 – 12888

Polycationic Amphiphilic Cyclodextrins

FULL PAPER

of efficient face-selective functionalization methods of the

CD scaffold. In principle, both possible relative orientations

of the cationic groups and the hydrophobic elements, that is,

the skirt or the jellyfish arrangements (Figure 1, left and

right, respectively),^[23]could be considered. Although a vari-

ety of synthetic strategies for regioselective manipulation of

the primary hydroxyl groups (narrower rim) are at hand,^[24]

elaboration of the secondary CD hydroxyl groups (wider

rim) has been scarcely explored.^[25]Even relatively simple

reactions, such as acylation, have been reported to lead to

mixtures of undersubstituted or other undesired products

when applied to this face,^[26]which severely hampers purifi-

cation of uniformly functionalized compounds that preserve

the original C_nsymmetry. Moreover, functionalization of the

secondary hydroxyl groups generally requires prior protec-

tion of the more accessible primary hydroxyl groups, thus

implying longer reaction sequences. We therefore focused

on a skirt-type architecture for the purpose of this work.

Since face-selective hydroxyl manipulation methodologies

are common to the three commercially available CDs (a, b,

and gCD), the most interesting representative from the

commercial point of view, namely, cyclomaltoheptaose

(bCD), was our chosen platform.

A semiconvergent, diversity-oriented strategy that allows

modification of each of the constituent parts of the paCD

construct in a modular manner has been disclosed. It in-

volves: 1) replacement of the seven primary hydroxyl

groups in bCD by tert-butoxycarbonyl (Boc)-protected cys-

teaminyl segments; 2) acylation of the fourteen secondary

hydroxyl groups; 3) hydrolysis of the carbamate groups and;

4) further modification of the cationic heads by thiourea-

forming reactions. In this way, two sublibraries were gener-

ated that consist of polyamino and polyaminothiourea bCD

derivatives, respectively (Scheme 1).

The first transformation was accomplished in two steps

from the commercially available cyclooligosaccharide by

heptabromination with the N-bromosuccinimide (NBS)/tri-

phenylphosphine (TPP) system (!1),^[27]followed by cesium

Scheme 1. CD-scaffolded library members with indication of their structure-correlated notation.

Chem. Eur. J. 2009, 15, 12871 – 12888

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12873

J. M. Benito, P. Vierling, J. Defaye, C. Ortiz Mellet et al.

carbonate promoted nucleophilic displacement of bromine

by N-Boc-cysteamine (!2). We next found that acylation of

the secondary hydroxyl groups could be achieved very effi-

ciently by using fatty acid anhydrides in N,N-dimethylforma-

mide (DMF) and N,N-dimethylaminopyridine (DMAP) as a

non-nucleophilic base catalyst, which is significantly differ-

ent from that reported when the corresponding acyl chlor-

ides are employed.^[20a]The tetradecahexanoate 3 and the tet-

radecamyristoate 4 were thus prepared. Subsequent acid-

promoted hydrolysis of the tert-butyl carbamate afforded

the corresponding heptacationic amphiphilic derivatives

HexCD-N and MyrCD-N in a four-step process involving a

single chromatographic purification on a multigram scale

(Scheme 2).

In view of the above considerations, the tetradecahexa-

noate derivative HexCD-N was chosen as the key precursor

to enlarge the collection of amphiphilic CD architectures.

The amine–isothiocyanate coupling reaction^[29]was selected

for this purpose for two main reasons. First, it generally pro-

ceeds in high yield and has already proven to be suitable for

the preparation of homogeneous CD-scaffolded hyper-

branched derivatives, thus avoiding overwhelming separa-

tions.^[28,30]Second, the resulting adducts will incorporate a

belt of hydrogen-bonding thiourea centers appropriately lo-

cated to participate in cooperative binding to the phosphate

groups in the plasmid chain. It is widely accepted that anion

recognition and binding in biological systems does not rely

on charge compensation exclusively, but rather depends on

the presence of cooperative networks of hydrogen bonding

and electrostatic interactions,^[31]and this is true also for

phosphates.^[32]Actually, artificial oligosaccharide mimics in-

corporating thiourea connectors have been shown to bind

phosphate anions even in aqueous media.^[33]

In principle, poly(N,N’-disubstituted thiourea) derivatives

can be synthesized by directly reacting the heptaamine with

appropriately functionalized isothiocyanates. In the case of

isothiocyanate partners susceptible of undergoing intramo-

lecular reactions^[34]or for the preparation of N,N’,N’-trisub-

stituted thiourea adducts, however, the corresponding hep-

taisothiocyanate 5, prepared by isothiocyanation of HexCD-

N with thiophosgene (Scheme 3), was instrumental. A set of

amine (6–11) and isothiocyanate building blocks (12–15)

was chosen to be able to investigate the effect that systemat-

ic variations in the distance between phosphate binding

motifs (amine and thiourea functionalities), their relative

disposition, the flexibility of the linkers, and the absence or

the multiple presence of any of them might have in CD–

DNA complex formation and delivery.

The individual Boc-protected sublibrary 2 members 16–24

were synthesized in parallel by coupling of 7–15 with the

complementary heptaamine or heptaisothiocyanate CD re-

agent. In all cases the reaction proceeded to completion, as

seen by TLC and MS of the reaction mixtures, in relatively

short times. No side products arising from the self-condensa-

tion of the isothiocyanates, a source of side products when

long reaction times or high temperatures are required,^[34]

were detected, and purification could be accomplished in all

cases by normal flash chromatography (64–99% isolated

yields). Trifluoroacetic acid (TFA)-catalyzed cleavage of the

carbamate protecting groups in 16–24 and freeze-drying of

the crude products from diluted HCl solutions afforded the

target amphiphilic polyaminothiourea–CDs (sublibrary 2),

as poly(hydrochloride) salts in virtually quantitative yield.

NMR spectroscopy, ESIMS (see the Supplementary Infor-

mation for a selection of spectra), and elemental microanal-

yses unequivocally demonstrated the structure and homoge-

Scheme 2. Synthesis of polyamino cyclodextrins (sublibrary 1). Reagents

and conditions: a) cysteamine hydrochloride, Et₃N, DMF, RT, 86%

(ref. [28]); b) N-Boc cysteamine, Cs₂CO₃, DMF, 708C, 66%; c) hexanoic

(n=1) or myristic (n=9) anhydride, DMAP, DMF, 45 min, 76 and 67%,

respectively; d) 1:1 TFA/CH₂Cl₂, RT, 2 h; then HCl, 99%.

The above compounds are the first representatives of the

cysteaminyl–paCD series. The presence of the cysteamine

segment in the structure is of importance since we already

know that the accessibility of the amino groups on the CD

core is much improved after insertion of the ethylene spacer

between the nitrogen atom and the CD nucleus.^[28]Such a

reactivity enhancement should facilitate the formation of in-

termolecular salt bridges with phosphate anions in the

pDNA skeleton. The length of the acyl chain was also ex-

pected to be a critical parameter. Actually, the compound

bearing fourteen hexanoyl functionalities at the secondary

face efficiently condensed the plasmid into nanometric

CDplexes suitable for gene delivery (see below), whereas

the myristoyl groups fully abolished the formation of self-as-

sembled paCD:pDNA complexes, probably due to irreversi-

ble aggregation in aqueous media.

1

neity of each library member. As an example, the H NMR

and ESIMS spectra of tetradecacationic CD HexCD-T-

AHCTUNGTREN[GNNU C₂N]₂, with the typical single-spin system for a fully sym-

metric molecule and the peaks for the multiply charged mo-

lecular ions, respectively, are shown in Figure 2.

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Polycationic Amphiphilic Cyclodextrins

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Scheme 3. Parallel synthesis of amphiphilic sublibrary 2 members. Reagents and conditions: a) CaCO₃, CH₂Cl₂/H₂O, RT, 2.5 h, 64%; b) Et₃N, CH₂Cl₂,

RT, 16 h, 64–99%; c) 1:1 TFA/CH₂Cl₂, RT, 2 h, then HCl, 99%.

and electrostatic interactions in DNA complexation. In ad-

dition, a nonamphiphilic control, CD-T-C₂N, was also in-

cluded in sublibrary 2 to estimate the effect of the presence

of the hexanoyl chains. Its synthesis involved acetylation of

the secondary hydroxyl groups in the N-(Boc)cysteaminyl–

bCD derivative 2 (!25), followed by TFA-promoted carba-

mate hydrolysis, the coupling of the resulting heptaamine 26

with 2-(tert-butoxycarbonylamino)ethylisothiocyanate (27;^[35]

!28), deacetylation (!29), and finally, N-deprotection

(Scheme 4).

pDNA complexation and CDplex characterization: Com-

pounds in sublibraries 1 and 2 constitute a structurally di-

verse series of monodisperse paCDs very well suited for

structure–gene transfection relationship studies. The CD-N

and CD-T-C₂N derivatives, which lack the fatty acyl chains

at the secondary face of the bCD core, were included in the

polyamino and polyaminothiourea series, respectively, as

nonamphiphilic control compounds. The capability of the

various CDs shown in Scheme 1 to form stable CDplexes

with pDNA (luciferase-encoding pTG11236) was deter-

mined at N/P ratios ranging from 5 to 30.^[36,37]To avoid self-

aggregation phenomena, the CD stock solutions were pre-

Figure 2. ¹H NMR spectra (500 MHz, 313 K, MeOD; top) and ESI mass

spectra (bottom) of HexCD-T[C₂N]_2.

pared in DMSO and further diluted with the pDNA solution

in 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid

a

AHCTUNGTRENNUNG

(HEPES) buffer (the final DMSO content never exceeded

1% v/v). These formulations were analyzed by agarose gel

electrophoresis, with staining by the ethidium bromide inter-

calating agent, for assessing DNA complex formation and

protection as well as DNA integrity. The paCD–pDNA

The amphiphilic hepta(hydroxyethylthiourea) derivative

HexCD-T-C₂O was prepared as a neutral control compound

to evaluate the relative contribution of hydrogen-bonding

Chem. Eur. J. 2009, 15, 12871 – 12888

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12875

J. M. Benito, P. Vierling, J. Defaye, C. Ortiz Mellet et al.

Figure 3. CD-mediated protection of pDNA from ethidium bromide in-

tercalation at different N/P ratios (5, 10, and 30) for representative CDs

Scheme 4. Synthesis of the nonamphiphilic control CD-T-C₂N. Reagents

and conditions: a) Ac₂O/pyridine, RT, 24 h, 81%; b) 1:1 TFA/CH₂Cl₂,

RT, 2 h, 99%; c) Et₃N, CH₂Cl₂, RT, 24 h, 70%; d) NaOMe in MeOH, RT,

16 h, 99%; e) HCl, 99%.

(HexCD-TCAHUTTGNRENG[UN C₂N]₂, CD-N, CD-T-C₂N, HexCD-T-C₂NACHUTGNTREN[NUNG C₂N]₂, and HexCD-

T-C₂N[C₂N]₂. Naked DNA is used for comparative purposes.

AHCTUNGTRENNUNG

bromide staining (lanes 8 and 9). Intact recovery of pDNA

upon SDS-induced CDplex dissociation demonstrated the

reversibility of the complexation in all cases (data not

shown).

The CD–pDNA nanoparticle hydrodynamic diameters

and z potentials were next determined for different N/P

ratios. Though all assayed polycationic CDs featured

pDNA-complexing capabilities, the DLS results confirmed

that the nonamphiphilic derivatives CD-N and CD-T-C₂N

do not result in stable nanoparticles, further supporting the

need of an amphiphilic architecture to promote the self-as-

sembling process (Figure 4). Moreover, the CD-N:pDNA

nanoparticles were further characterized by dynamic light

scattering (DLS) for average hydrodynamic size (an impor-

tant parameter to select potential candidates for in vivo

uses),^[38]mixed-mode measurement-phase analysis light scat-

tering (M3-PALS) for zeta (z)-potential measurements, and

transmission electron microscopy (TEM) for particle size

and morphology.

Whereas the paCDs prepared in this study tend to form

irregular aggregates in aqueous media, a templating effect

resulting in compact, ordered, and stable nanoparticles was

observed in the presence of the plasmid. The tetradecamyr-

istoate MyrCD-N and the neutral derivative HexCD-T-C₂O,

which were not dispersible in an aqueous environment, are

two notable exceptions. The presence of a polycationic clus-

ter and an appropriate hydrophilic/hydrophobic balance are,

therefore, crucial features for triggering effective interac-

tions with pDNA, thereby leading to compaction. Formation

of the corresponding CDplexes was only achieved for the

polycationic amphiphilic CDs, as shown by the agarose gel

electrophoresis shift assay (Figure 3). Indeed, gel electro-

phoresis of the polycationic HexCD-based CDplexes indi-

cated the absence of “free” mobile plasmid. Moreover, if

pDNA is efficiently compacted and protected in the

CDplexes, it becomes inaccessible to the ethidium bromide

intercalating agent used as staining reagent for N/P ratios

above 5, as demonstrated by the absence of fluorescent

staining in the corresponding lanes. On the contrary, the

gene in CDplexes formulated with the nonamphiphilic hep-

taamine derivative CD-N remained accessible to ethidium

bromide in the whole range of N/P values from 5 to 30

(Figure 3, lanes 4–6). The presence of a thiourea segment in

CD-T-C₂N results in a more efficient covering of the DNA

surface, probably due to the interplay of electrostatic and

hydrogen-bonding interactions. Thus, although the plasmid

was still accessible to the intercalating agent for N/P 5

(Figure 3, lane 7), higher N/P values prevented ethidium-

Figure 4. Size (hydrodynamic diameters, bars [nm]) and z potential (*,

mV) of CDplexes determined by dynamic light scattering and M3-PALS

analysis, respectively. Unfilled and filled bars correspond to nanoparticle

size at N/P 5 and 10, respectively, while dots (*) represent the z poten-

tial at N/P 10. The measured size and z potential at N/P 30 did not differ

significantly from those at N/P 10. Attempts to determine the size for

CDplexes obtained from the nonamphiphilic control compounds CD-N

and CD-T-C₂N were unsuccessful (nonreproducible and erratic measure-

ments).

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Polycationic Amphiphilic Cyclodextrins

FULL PAPER

and CD-T-C₂N:pDNA CDplexes exhibited z-potential

values close to neutrality even at a high N/P ratio, which is

in agreement with the low pDNA-compacting potency. In

sharp contrast, the paCD-based formulations displayed fur-

ther positive z potentials in the range 20–50 mV, which in-

creased upon increasing the N/P ratio and reached a plateau

for N/P 10 (the z potentials measured for N/P 30 are identi-

cal to those at N/P 10; data not shown). These results sug-

gest the prevalence of an ideal paCD–pDNA complex stoi-

chiometry that occurs at an N/P ratio of approximately 10,

above which the excess of paCD does not incorporate to the

CDplex nanostructure.

was more pronounced when CDplexes were exposed to salt

concentrations above 50 mm (8-fold and 3-fold for the

HexCD-T-C₂N:pDNA and HexCD-TACTHNUGTRNEUNG[C₂N]₂:pDNA com-

plexes, respectively, at 250 mm NaCl), probably due to nano-

particle aggregation (Figure 6). In any case, the pDNA re-

mained complexed and fully protected under these condi-

tions, as highlighted by agarose gel electrophoresis (data not

shown), which is significantly different from that reported

for nonamphiphilic systems.^[44]

Excepting the case of the HexCD-T-C₂N:pDNA complex

at N/P 5, all the CDplexes prepared from paCDs exhibited

rather small hydrodynamic diameters as compared with the

polymer:pDNA complexes (polyplexes) obtained using

branched poly(ethyleneimine) (bPEI, 25 kDa), one of the

most efficient commercial gene-delivery systems^[21](50–

80 nm vs. 150 nm). Additionally, quasi-monodisperse popu-

lations of cationic nanoparticles were observed in each indi-

vidual experiment. Such remarkable behavior (low particle

size and monodispersity) has only been reported for a

monoACHTUNGTRENNUNGmolecular condensation process that occurs upon

Figure 6. Salt-induced hydrodynamic diameter variation for HexCD-T-

C₂N:pDNA (*) and HexCD-TACHTNUTRGNE[UNG C₂N]₂:pDNA (&) CDplexes at N/P 7.

mixing DNA with dimerizable, polycationic detergents.^[39,40a]

Transmission electron microscopy confirmed the small

size and homogeneous distribution of CDplex formulations

(Figure 5).^[41]At high magnification, a snail-like ultrastruc-

pDNA delivery and transfection: The transfection efficiency

of the self-assembled paCD:pDNA complexes was evaluat-

ed using the luciferase-encoding reporter gene (pTG11236,

pCMV-SV40-luciferase-SV40pA) in BNL-CL2, COS-7, and

KB cells in HEPES buffer (20 mm, pH 7.4). CDplexes were

formulated at different N/P ratios. Branched PEI (bPEI,

25 kDa) or linear JetPEI (22 kDa) and naked pDNA were

used for comparative purposes (positive and negative con-

trols, respectively). Stepwise structure–activity relationship

(SAR) analyses were performed to assess the influence of

each structural element on transfection efficiency.

Figure 5. Transmission electron microscopy (TEM) images of HexCD-T-

Operating conditions were preliminarily optimized for

CDplexes formulated with HexCD-N (sublibrary 1) or

HexCD-T-C₂N (sublibrary 2). Maximum transfection effi-

ciencies and cell viabilities were detected for N/P ratios 10

and 5, respectively. Higher N/P ratios not only did not im-

prove performance, but also resulted in higher toxicity,

probably due to aggregation of the free paCD. The CDplex-

es based on the nonamphiphilic polycationic analogues CD-

N and CD-T-C₂N showed negligible gene-delivery properties

(Figure 7), which was in sharp contrast to reported results

for discrete nonamphiphilic CD polycations.^[17]Most proba-

bly, the much poorer gene-condensing and -protecting capa-

bilities featured by nonamphiphilic CDs together with their

poor membrane-fusiogenic properties accounts for this inef-

ficiency. Membrane-fusiogenic properties in lipoplexes are

known to play a decisive role in favoring cell uptake and en-

dosomal escape of DNA, by destabilizing anionic lipid-

based biological membranes.^[45]

ACHTUNGTRENNUNG[C₂N]₂:pDNA CDplexes.

ture was observed. These structures were probably made of

alternating lamellar arrangements of paCDs and electron-

dense densities corresponding to the pDNA molecule, a sce-

nario that is reminiscent of the onionlike structure encoun-

tered in multimolecular siRNA:liposome complexes.^[42]The

rather small size and homogeneity of CDplexes makes them

promising candidates for the development of systemic appli-

cations in vivo.^[5,40]Thus, their gene-delivery properties

would not suffer due to size-restricted diffusion and epithe-

lial permeation.^[43]

The effect of serum and of a saline medium on CDplex

size and stability was next investigated to further assess

their potential behavior under physiological conditions. A

size increase from approximately 70–80 nm to 130–200 nm

was observed when the HexCD-T-C₂N:pDNA or HexCD-T-

A

SAR analysis demonstrated the superior abilities of am-

phiphilic polyaminothiourea CD derivatives (sublibrary 2)

Chem. Eur. J. 2009, 15, 12871 – 12888

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12877

J. M. Benito, P. Vierling, J. Defaye, C. Ortiz Mellet et al.

Figure 7. a) In vitro transfection efficiency and b) cell viability in BNL-

CL2 cells of CDplexes formulated with HexCD-N, HexCD-T-C₂N, or the

nonamphiphilic CD-N and CD-T-C₂N controls in comparison with data

for naked DNA (=pTG11236) and bPEI (25 kDa)-based polyplexes at

N/P 5 (unfilled bars and ~) and 10 (filled bars and *).

Figure 8. a) In vitro transfection efficiency and b) cell viability in BNL-

CL2 cells of CDplexes formulated with heptaaminothioureido amphiphil-

ic CDs differing in spacer length and flexibility versus naked DNA

(=pTG11236) and bPEI (25 kDa)-based polyplexes at N/P 5 (unfilled

bars and ~) and 10 (filled bars and *).

relative to polyamino CD derivatives (sublibrary 1) as trans-

fection systems. Thus, the N/P 5 CDplexes made from

HexCD-T-C₂N were over 100-fold and 10-fold more effi-

cient than those obtained from HexCD-N at N/P 5 and 10,

respectively (Figure 7). The fact that such an improvement

in gene delivery and expression can be nanoengineered by

inserting a rationally designed recognition element operat-

ing at the atomic level is remarkable. The performance of

HexCD-T-C₂N at N/P 5 is actually similar to that shown by

branched PEI at N/P 10, but featuring a far less toxic profile

(cell viability ꢀ100%) than this commercial polycationic

polymer (60% cell viability; Figure 7).

To assess the effect of different spacer arms between the

thiourea moiety and the cationic centers, a further SAR

evaluation within heptaammonium/heptathiourea subli-

brary 2 members was conducted by comparing the transfec-

tion efficiency mediated by CDplexes formulated with

HexCD-T-C_nN (n=2, 4, 6), or HexCD-T-p and mXN. From

data in Figure 8, it can be inferred that luciferase expression

decreases when increasing the distance between the amino

and thioureido functions. At N/P 5, a 100-fold decrease in

transfection efficiency was observed for HexCD-T-C₆N rela-

tive to HexCD-T-C₂N. At N/P 10, there is no decrease when

going from HexCD-T-C₂N to HexCD-T-C₄N, but it was still

above one order of magnitude from HexCD-T-C₄N to

HexCD-T-C₆N. This observation highlights the benefits that

a suitable preorganization of phosphate-recognition ele-

ments in the individual branches exert over the transfection

efficiency of the corresponding supramolecular aggregates.

That becomes more evident in the case of the compounds

bearing aromatic spacers HexCD-T-pXN and HexCD-T-

mXN, in which a concerted operation of thiourea and amino

motifs in phosphate binding is probably impaired. Under

the same assay conditions, luciferase expression for the cor-

responding CDplexes is reduced over 100-fold relative to

HexCD-T-C₂N-based CDplexes. Moreover, toxicity turns to

be much higher at N/P 10 (Figure 8).

The above results led us to identify the shorter and flexi-

ble ethylene segment as the optimal tether between thiourea

and amine groups for gene delivery and expression mediat-

ed by CDplexes formulated with the paCDs. Keeping this

element fixed, we next explored the effect of increasing the

density of the amine and thiourea motifs. Deceptively, the

higher-valent linear arrangements HexCD-T-C₂N-C₂N^[46]

and HexCD-T-C₂-T-C₂N resulted in lower transfection capa-

bilities as compared with the heptaaminothiourea HexCD-

T-C₂N in BNL-CL2 cells; this was in contrast with previous

reports proposing a valency-dependent enhanced interaction

with oligonucleotides for linear arrangements of both oli-

goethyleneimines^[17a]and oligothioureas.^[47]On the contrary,

increasing the number of protonable amine groups in a den-

dritic display represented a significant improvement in

vector design. In the case of HexCD-T-C₂N

ACHTUGNTRNEN[UGN C₂N]₂, though

Figure 9 shows a performance similar to that of HexCD-T-

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Polycationic Amphiphilic Cyclodextrins

FULL PAPER

Figure 9. a) In vitro transfection efficiency and b) cell viability in BNL-

CL2 cells of CDplexes formulated with amphiphilic polyaminothiourea

CDs differing in the number and disposition of amine groups versus

naked DNA (=pTG11236) and bPEI (25 kDa)-based polyplexes at N/P 5

(unfilled bars and ~) and 10 (filled bars and *).

Figure 10. a) In vitro transfection efficiency and b) cell viability in COS-7

cells of HexCD-T-C₂N:DNA and HexCD-TACHTUNRTGNEUNG[C₂N]₂:DNA CDplexes

versus naked DNA (=pTG11236) and JetPEI-based polyplexes at N/P 5

(unfilled bars and ~) and 10 (filled bars and *).

C₂N, the fact that the structure of the former features 21

protonable groups (only 7 in HexCD-T-C₂N) implies that

only one third of the paCD is required to achieve the same

efficiency in molar terms. The better suited three-dimension-

transgene expression up to two orders of magnitude in

COS-7 cells (Figure 10). At N/P 10, the transfection efficien-

cy for HexCD-TACHTNUGTRNEG[UN C₂N]₂-based CDplexes was similar to that

observed for JetPEI-based polyplexes, but for a compound

that is now perfectly homogeneous.

al arrangement was that of HexCD-T

ing one of the hydrogen-bond donor centers at the thiourea

moiety, HexCD-T[C₂N]₂:pDNA complexes at N/P 5 or 10

ACHTUGNTRENN[NUG C₂N]₂. Despite lack-

Often, many artificial gene vectors that exhibit successful

gene delivery in vitro fail when trying to reproduce gene de-

livery under physiological conditions,^[48]which is usually as-

cribed to complex instability in the presence of serum com-

ponents. The serum saline stress (typically 150 mm) often

promotes aggregation of cationic complexes, thereby leading

to vascular blockage.^[49]Additionally, cationic complexes

readily bind with serum proteins, which hinders cellular

uptake, promotes aggregation, and eventually leads to

ACHTUNGTRENNUNG

featured a 10-fold increase in luciferase production relative

to the corresponding HexCD-T-C₂N:pDNA ones. In molar

terms, that means that half the amount of HexCD-TACTHNUTRGNE[UNG C₂N]₂

(14 protonable amine groups) is sufficient to achieve even a

higher transfection efficiency as compared with HexCD-T-

C₂N (7 protonable amine groups). HexCD-TACTHNUGTREN[UNG C₂N]₂:pDNA

complexes at N/P 5 are also one order of magnitude more

efficient than bPEI-based polyplexes at its best N/P ratio of

10, while preserving a far less toxic profile (Figure 9). The

transgene expression mediated by these CDplexes is over

three orders of magnitude higher than that of bPEI-based

N/P 5 polyplexes.

phagoACHTNUGRTNEUNG

cytosis.^[50]Shielding nanoparticles from salt and serum

components is a way to achieve successful artificial gene

vectors. In this context, the CDplex amphiphilic shell should

contribute to protect nanoparticle fate. In fact, the above-

mentioned nanoparticle stability assays have revealed the

Preliminary results indicated that paCD-based CDplexes

behave as broad-scope gene-delivery systems, with very sim-

ilar architecture-dependent efficiency profiles in different

cell lines, including COS-7 (Figure 10) and KB cells (data

not shown). Paralleling results with BNL-CL2, multiplica-

tion of the phosphate-binding motifs in a dendritic manner

almost inertness of HexCD-TACHTGUNETRN[NUG C₂N]₂-based CDplexes toward

saline stress and serum. To confirm this hypothesis, gene-de-

livery experiments in the absence and in the presence of

serum were conducted. Luciferase expression in BNL-CL2

cells showed a decrease of three orders of magnitude with

HexCD-T-C₂N in the presence of serum (Figure 11) relative

to the assay in serum-free medium. Interestingly, in the case

(e.g., from HexCD-T-C₂N to HexCD-TACHTUNRTGNEU[GN C₂N]₂) pushed

Chem. Eur. J. 2009, 15, 12871 – 12888

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J. M. Benito, P. Vierling, J. Defaye, C. Ortiz Mellet et al.

for BNL-CL2 and COS-7 cell lines by using paCDs that

present a dendritic arrangement of cationic elements as in

HexCD-TACTHNUGTRNE[UNG C₂N]₂. The versatility of the synthetic scheme

makes it potentially amenable to the selective installation of

additional functional elements (e.g., for cell targeting, nucle-

ar localization, monitoring cell uptake and fate) over the

CD core, thus offering further opportunities for gene target-

ing that will merit investigation.^[52]

It is worth stressing that the correlation of gene-vector

chemical design and final transfection efficiency is only an

overview of the whole process leading to transgene expres-

sion. Thus, comparable results on transfection efficiency en-

countered for dendritic paCD and PEI-based polyplexes in

this study do not necessarily imply identical internalization

mechanisms and intracellular trafficking routes. Investiga-

tion of the privileged pathways for cell uptake, escape of the

intracellular endosomal compartments, dissociation of plas-

mid and carrier, nuclear translocation of the nucleic acids,

and, finally, transcription of the transfected pDNA and pro-

tein expression is on the whole necessary to obtain the rele-

vant information for optimization of the system. Research

in that direction is currently in progress in our laborato-

ries.^[53]

Experimental Section

Figure 11. a) In vitro transfection efficiency and b) cell viability in BNL-

CL2 of HexCD-T-C₂N:pDNA and HexCD-T[C₂N]₂:pDNA CDplexes at

AHCTUNGTRENNUNG

General methods: Reagents and solvents were purchased from commer-

cial sources and used without further purification, with the following ex-

ceptions: dichloromethane was distilled under an Ar stream over CaH₂.

Optical rotations were measured at 208C in 1 cm or 1 dm tubes using a

Perkin–Elmer 141 MC polarimeter. IR spectra were recorded using an

FTIR spectrometer. ¹H (and ¹³C NMR) spectra were recorded at 500

(125.7) and 300 (75.5) MHz. 2D COSY, 1D TOCSY, and HMQC experi-

ments were used to assist with the NMR spectroscopy assignments. NMR

spectra and a guide to the notation used for the assignments can be

found in the Supporting Information. Thin-layer chromatography (TLC)

was carried out on aluminum sheets coated with Kieselgel 60 F₂₄₅(E.

Merck), with visualization by UV light and by charring with 10% H₂SO₄

or 0.1% ninhydrin in EtOH. Column chromatography was carried out on

silica gel 60 (E. Merck, 230–400 mesh). Electrospray mass spectra

(ESIMS) were obtained using a Bruker Esquire6000 instrument. Elemen-

tal analyses were performed at the Instituto de Investigaciones Quꢀmicas

(Sevilla, Spain).

N/P 10 in the absence (unfilled bars) and in the presence (filled bars) of

serum versus naked DNA and JetPEI-based polyplexes.

of HexCD-TACHTUNGTRENNUNG[C₂N]₂CDplexes, the efficiency drop is very

limited and remains close to that of JetPEI but with a lower

cytotoxic profile.

Conclusion

In summary, we have implemented a straightforward design

of polycationic CD-based facial amphiphiles as monodis-

perse molecular systems for efficient gene delivery and a di-

versity-oriented synthetic strategy suitable for SAR studies.

The overall architecture of these paCDs can be finely tuned

in terms of density of cationic groups, flexibility, and the

presence of additional hydrogen-bonding functionalities

while keeping a C₇-symmetric disposition. Control of the hy-

drophilic/hydrophobic balance between the CD primary and

secondary faces proved crucial for pDNA complexation and

nanoparticle (CDplex) formation. Most importantly, mono-

disperse populations of very small DNA nanoparticles

(<100 nm), which is a prerequisite when in vivo systemic

applications are sought, can be easily formulated from these

paCDs.^[51]Nanoparticle stability and transfection efficiency

can be rationally modulated by judicious tailoring of the

molecular topology. Remarkably, transfection efficiencies

that surpass those of the polycationic polymers bPEI and

JetPEI with lower cytotoxicity profiles have been achieved

Preparation of complexes composed of CD derivatives and plasmid

pTG11236: The plasmid pTG11236 (pCMV-SV40-luciferase-SV40pA),

used for the preparation of the DNA complexes and for transfection

assay, is a plasmid of 5739bp (base pairs). The quantities of compound

used were calculated according to the desired DNA concentration of

0.1 mgmL^ꢁ1(303 mm phosphate), the N/P ratio, the molar weight, and the

number of protonable nitrogen atoms in the selected CD derivative or

cationic polymer (bPEI, 25 kDa or JetPEI). Experiments were performed

for N/P 5, 10, 30, and 50. For the preparation of the DNA complexes

from CD derivatives and PEI, DNA was diluted in HEPES (20 mm,

pH 7.4) to a final concentration of 303 mm, then the desired amount of

CD derivative was added from 10 or 20 mm stock solution (DMSO) and

bPEI (25 kDa) was added from a 0.1m stock solution (H₂O). For JetPEI

polyplexes, DNA was diluted in a 150 mm NaCl solution to a final phos-

phate concentration of 303 mm, then the desired amount of JetPEI was

added from a 7.5 mm water solution. The preparation was vortexed for

2 h and used for characterization or transfection experiments.

Measurement of the size of the complexes by dynamic light scattering

(DLS) and of the zeta potential: The average sizes of the CDplexes were

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Polycationic Amphiphilic Cyclodextrins

FULL PAPER

measured using a Zetasizer nano (Malvern Instruments, Paris, France)

with the following specification: sampling time, automatic; number of

small drops of the CDplex samples (HEPES 20 mm, pH 7.4, DNA 303 mm

phosphate) prepared as described above. After 1–3 min of contact, grids

were negatively stained with a few drops of 1% aqueous solution of

uranyl acetate. The grids were then dried and observed using a Philips

CM12 electron microscope working under standard conditions. All these

experiments were reproduced twice on each formulation.

measurements,

3 per sample; medium viscosity, 1.054 cP; refractive

index, 1.33; scattering angle, 1738; l=633 nm; temperature, 258C. Data

were analyzed using the multimodal number distribution software includ-

ed in the instrument. Results are given as volume distribution of the

major population by the mean diameter with its standard deviation.

Zeta-potentials measurements on the CDplexes were made using the

same apparatus with “mixed-mode measurement” phase analysis light

scattering.

Synthesis: The starting materials, heptakis(6-bromo-6-deoxy)cyclomalto-

heptaose (1),^[27]heptakis[6-(2-aminoethylthio)-6-deoxy]cyclomaltohep-

taose (CD-N),^[28]4- and 3-(tert-butoxycarbonylaminomethyl)benzyl iso-

thiocyanate (12, 13),^[55]and 2-(tert-butoxycarbonylamino)ethyl isothiocya-

nate (27)^[35]were prepared as described previously.

“Mixed-mode measurement” phase analysis light scattering (M3-PALS):

M3 consists of both slow field reversal and fast field reversal measure-

ments, hence the name “mixed-mode measurement”; it improves accura-

cy and resolution. The following specifications were applied: sampling

time, automatic; number of measurements, 3 per sample; medium viscosi-

ty, 1.054 cP; medium dielectric constant, 80; temperature, 258C.

Compound 2: tert-Butyl N-(2-mercaptoethyl)carbamate (5.3 mmol,

1.4 equiv) was added to a suspension of heptakis(6-bromo-6-deoxy)cyclo-

maltoheptaose^[27](1, 0.84 g, 0.53 mmol) and Cs₂CO₃(1.71 g, 5.25 mmol)

in dry DMF (10 mL). The suspension was heated, under an Ar atmos-

phere, at 708C for 48 h. The reaction mixture was cooled to RT, poured

into ice water (30 mL), and stirred overnight. The resulting solid was fil-

tered and washed with a large volume of water, and then with a small

amount of cold Et₂O. The residue was purified by flash chromatography

(40:10:1!30:10:1 CH₂Cl₂/MeOH/water). Yield: 787 mg (66%); R_f=0.60

(40:10:1 CH₂Cl₂/MeOH/water); [a]_D=+79.7 (c=0.8 in MeOH);

¹H NMR (500 MHz, MeOD, 323 K): d=4.95 (d, J_1,2=3.5 Hz, 7H; H-1),

4.00 (m, 7H; H-5), 3.86 (t, J_2,3=J_3,4=9.0 Hz, 7H; H-3), 3.48 (dd, 7H; H-

Before each series of experiments, the performance of the instruments

was checked with either a 90 nm monodisperse latex beads (Coulter) for

DLS or with DTS 50 standard solution (Malvern) for zeta potentials.

Agarose gel electrophoresis: Each CD derivative/DNA sample (20 mL,

0.4 mg of plasmid) was submitted to electrophoresis for about 30 min

under 150 V through a 0.8% agarose gel in 1ꢆ tris(hydroxymethyl)ami-

nomethane (Tris)/acetate/ethylenediaminetetraacetic acid (EDTA)

(TAE) buffer and stained by spreading an EtBr (Sigma) solution in TAE

buffer (20 mL ethidium bromide of a 10 mgmL^ꢁ1solution in 200 mL

TAE). The DNA was then visualized after photographing using an UV

transilluminator. The plasmid integrity in each sample was confirmed by

electrophoresis after decomplexation with sodium dodecyl sulfate (SDS,

8%).

2), 3.26 (t, ³J

ACHTUNGRTENUNG(H,H)=7.5 Hz, 21H; H-4, CH₂N_Cyst), 3.20 (m, 7H; H-6a),

2.91 (m, 7H; H-6b), 2.77 (m, 14H; CH₂S_Cyst), 1.45 ppm (brs, 63H;

CMe₃); ¹³C NMR (125.7 MHz, MeOD, 323 K): d=156.1 (CO), 104.1 (C-

1), 86.5 (C-4), 80.3 (C_q), 74.6 (C-3), 74.4 (C-2), 73.3 (C-5), 41.8

(CH₂N_Cyst), 34.9 (CH₂S_Cyst), 34.5 (C-6), 29.1 ppm (CMe₃); ESIMS: m/z:

1147.4 [M+2Na]²⁺, 2271.8 [M+Na]⁺; elemental analysis calcd (%) for

C₉₁H₁₆₁N₇O₄₂S₇: C 48.58, H 7.21, N 4.36; found: C 48.44, H 6.80, N 4.27.

Statistical analysis: Statistical tests were performed with STATGRAPH-

ICS Plus 5.0 software.^[54]Analysis of variance (Anova) was run on the

logarithmic transformation of transfection levels (log10(fg luciferase per

mg protein)) and on the cell viability to fit normal distributions of the

data. Two factors, that is, the nature of the complexing agent (CD deriva-

tive and PEI) and the N/P ratio, were analyzed as the source of the varia-

tion of logarithmic transformation of the transfection levels and of cell

variability percentages using a multiple comparison procedure. Tukeyꢇs

honestly significant difference (HSD) method was used to discriminate

among the means of cell viability percentages and the logarithmic trans-

formation of luciferase expression levels.

Compound 3: DMAP (4.56 g, 37.3 mmol, 3 equiv) and hexanoic anhy-

dride (12 mL, 49.8 mmol, 4.0 equiv) were added to a solution of 2 (2.0 g,

0.89 mmol) in dry DMF (10 mL) under Ar. The mixture was heated at

708C for 4–5 h. Then, MeOH (10 mL) was added and the mixture was

further stirred at 708C for 3 h. The mixture was poured into ice water

(50 mL) and extracted with CH₂Cl₂(4ꢆ50 mL). The organic phase was

washed with diluted H₂SO₄(2ꢆ50 mL), water, and cold saturated aque-

ous NaHCO₃(4ꢆ50 mL), dried (Na₂SO₄), concentrated and purified by

flash chromatography (1:3 EtOAc/petroleum ether). Yield: 2.44 g (76%);

R_f=0.45 (1:2 EtOAc/petroleum ether); [a]_D=+84.1 (c=0.9 in CHCl₃);

¹H NMR (500 MHz, CDCl₃, 323 K): d=5.45 (m, 7H; NH), 5.25 (t, J_2,3

_3,4=9.5 Hz, 7H; H-3), 5.08 (d, J_1,2=3.5 Hz, 7H; H-1), 4.76 (dd, 7H; H-

2), 4.12 (ddd, J_4,5=9.5 Hz, J_5,6b=5.5 Hz, J_5,6a=2.5 Hz, 7H; H-5), 3.77 (t,

7H; H-4), 3.30, 3.29 (2t, ³J

(H,H)=6.5 Hz, 14H; CH₂N_Cyst), 3.10 (dd,

_6a,6b=14.0 Hz, 7H; H-6a), 3.02 (dd, 7H; H-6b), 2.74, 2.72 (2dt, ²J-

(H,H)=13.5 Hz, ³J

(H,H)=7.0 Hz, 14H; CH₂S_Cyst), 2.38–2.11 (m, 28H;

CH₂CO), 1.57 (m, 28H; CH₂CH₂CO), 1.42 (brs, 63H; CMe₃), 1.30 (m,

56H; CH₃CH₂, CH₃CH₂CH₂), 0.89, 0.87 ppm (2t, ³J

(H,H)=7.0 Hz, 42H;

=

In vitro transfection: Twenty-four hours before transfection, BNL-CL2 or

COS-7 cells were grown at a density of 2ꢆ10⁴cells per well in 96-well

plates in Dulbelcco modified Eagle culture medium (DMEM; Gibco-

BRL) containing 10% fetal calf serum (FCS; Sigma), 10 mgmL^ꢁ1genta-

mycin for BNL-CL2 cells, or 100 units per mg penicillin and 100 mgmL^ꢁ1

streptomycin for COS-7 cells, in a wet (378C) and 5% CO₂/95% air at-

mosphere. The above-described CD:pDNA (=pTG11236) complexes

and PEI:pDNA polyplexes were diluted to 100 mL in DMEM or in

DMEM supplemented with 10% FCS so as to have 0.5 mg of pDNA in

the well (15 mm phosphate). The culture medium was removed and re-

placed by these 100 mL of the complexes. After 4 and 24 h, DMEM (50

and 100 mL) supplemented with 30% and 10% FCS, respectively, were

added. After 48 h, the transfection was stopped, the culture medium was

discarded, and the cells were washed twice with PBS (100 mL) and lysed

with lysis buffer (50 mL; Promega, Charbonniꢈres, France). The lysates

were frozen at ꢁ328C before the analysis of luciferase activity. This mea-

surement was performed using a luminometer (GENIOS PRO, Tecan

France S.A.) in dynamic mode, for 10 s on the lysis mixture (20 mL) and

using the “luciferase” determination system (Promega) in 96-well plates.

The total protein concentration per well was determined by the BCA test

(Pierce, MontluÅon, France). Luciferase activity was calculated as femto-

grams (fg) of luciferase per mg of protein. The percentage of cell viability

was calculated as the ratio of the total protein amount per well of the

transfected cells relative to that measured for untreated cells ꢆ100. The

data were calculated from three or four repetitions in two fully independ-

ent experiments (formulation and transfection).

J

AHCTUNGTRENNUNG

J

N

ACHTUNGTRENNUNG

AHCTUNGTRENNUNG

CH₃); ¹³C NMR (125.7 MHz, CDCl₃, 323 K): d=173.2, 171.6 (CO ester),

155.9 (CO carbamate), 96.7 (C-1), 79.2 (CMe₃), 78.8 (C-4), 71.4 (C-5),

70.6 (C-3), 70.3 (C-2), 40.5 (CH₂N_Cyst), 34.0 (C-6, CH₂CO), 33.9

(CH₂S_Cyst), 33.8 (CH₂CO), 31.5, 31.3 (CH₃CH₂CH₂), 28.5 (CMe₃), 24.4,

24.3 (CH₂CH₂CO), 22.3, 22.2 (CH₃CH₂), 13.8 ppm (CH₃); ESIMS: m/z:

1833.9 [M+2Na]²⁺; elemental analysis calcd (%) for C₁₇₅H₃₀₁N₇O₅₆S₇: C

58.00, H 8.37, N 2.71; found: C 57.79, H 8.19, N 2.50.

Compound 4: A mixture of 2 (0.2 g, 89 mmol), DMAP (0.46 g, 3.73 mmol,

3 equiv), and myristic anhydride (2.18 g, 4.97 mmol, 4.0 equiv) was dis-

solved in dry DMF (15 mL) at 08C under an Ar atmosphere. The result-

ing suspension was stirred at RT for 48 h, and the solvent was evaporated

under diminished pressure. The resulting residue was refluxed with

CH₂Cl₂/MeOH (5:95, 100 mL) for 1 h, decanted, and the residue was pu-

rified by flash chromatography (1:2 EtOAc/petroleum ether). Yield:

0.32 g (67%); R_f=0.56 (1:2 EtOAc/petroleum ether); [a]_D=+ 49.2 (c=

1.0 in CH₂Cl₂); ¹H NMR (500 MHz, CDCl₃, 313 K): d=5.38 (brs, 7H;

NH), 5.26 (t, J_2,3=J_3,4=8.9 Hz, 7H; H-3), 5.08 (d, J_1,2=3.8 Hz, 7H; H-1),

4.74 (dd, 7H; H-2), 4.13 (m, 7H; H-5), 3.76 (t, J_4,5=8.7 Hz, 7H; H-4),

Transmission electron microscopy (TEM): Formvar-carbon-coated grids

previously made hydrophilic by glow discharge were placed on top of

3.30 (brd, ³J

ACTHGNUETRNN(UG H,H)=6.1 Hz, 14H; CH₂N_Cyst), 3.10 (d, J_6a,6b=12.5 Hz, 7H;

ACTHNGUTERNNUG

H-6a), 3.01 (brd, 7H; H-6b), 2.72, 2.70 (2dt, ²J(H,H)=13.5 Hz, ³J-

Chem. Eur. J. 2009, 15, 12871 – 12888

ꢅ 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

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12881

J. M. Benito, P. Vierling, J. Defaye, C. Ortiz Mellet et al.

A

Compound HexCD-T-O: A solution of 5 (112 mg, 35 mmol) in dry

CH₂Cl₂(2 mL) was added dropwise to a solution of ethanolamine (6,

0.37 mmol, 22.2 mL, 1.5 equiv) in dry CH₂Cl₂(1 mL). The mixture was

stirred at RT for 16 h and the reaction mixture was quenched with water

(10 mL) and extracted with CH₂Cl₂(3ꢆ10 mL). The combined organic

layers were dried (Na₂SO₄), concentrated, and purified by flash chroma-

tography (9:1!6:1 CH₂Cl₂/MeOH). Yield: 82 mg (64%); R_f=0.34 (6:1

CH₂Cl₂/MeOH); [a]_D=+80.9 (c=1.0 in MeOH); ¹H NMR (500 MHz,

ACHTUNGTRENNUNG

CDCl₃, 313 K): d=173.3, 171.6 (CO ester), 155.9 (CO carbamate), 96.6

(C-1), 79.2 (CMe₃), 78.7 (C-4), 71.3 (C-5), 70.5 (C-3, C-2), 40.4

(CH₂N_Cyst), 34.2 (CH₂CO), 33.9 (C-6, CH₂S_Cyst), 32.0 (CH₂CH₂CH₃),

29.8–29.3 (CH₂), 28.5 (CMe₃), 24.8, 24.7 (CH₂CH₂CO), 22.6 (CH₂CH₃),

14.0 ppm (CH₃); ESIMS: m/z: 2619.5 [M+2Na]²⁺; elemental analysis

calcd (%) for C₂₈₇H₅₂₅₁N₇O₅₆S₇: C 66.36, H 10.19, N 1.89; found: C 66.01,

H 9.62, N 1.65.

MeOD, 313 K): d=5.32 (t, J_2,3=J_3,4=8.6 Hz, 7H; H-3), 5.14 (d, J_1,2

3.6 Hz, 7H; H-1), 4.80 (dd, 7H; H-2), 4.18 (m, 7H; H-5), 3.90 (t, J_4,5

=

8.5 Hz, 7H; H-4), 3.74 (m, 14H; CH₂N_cyst), 3.68 (t, ³J

ACHTUNGTRENNUNG

Compound HexCD-N: Treatment of carbamate 3 (0.71 g, 0.2 mmol) with

1:1 TFA/CH₂Cl₂(20 mL) at RT for 2 h, followed by evaporation of the

solvents and freeze-drying from a diluted HCl solution, gave HexCD-N

in virtually quantitative yield (0.6 g). [a]_D=+72.6 (c=1.0 in MeOH);

¹H NMR (500 MHz, MeOD): d=5.37 (t, J_2,3=J_3,4=9.5 Hz, 7H; H-3),

J

ACHTUNGTRENNUNG

AHCTUNGTRENNUNG

A

ACHTUNGTRENNUNG

5.16 (d, J_1,2=3.5 Hz, 7H; H-1), 4.79 (dd, 7H; H-2), 4.07 (brt, J_5,6a=J_5,6b

=

(125.7 MHz, MeOD, 313 K): d=183.8 (CS), 174.7, 173.6 (CO), 98.2 (C-

1), 80.0 (C-4), 73.2 (C-5), 72.0 (C-3), 71.7 (C-2), 61.8 (CH₂OH), 47.7

(CH₂NH), 45.3 (CH₂N_cyst), 35.2 (CH₂CO), 35.0 (C-6), 34.1 (CH₂S_cyst),

32.6, 32.5 (CH₂CH₂CH₃), 25.6 (CH₂CH₂CO), 23.5 (CH₂CH₃), 14.5,

14.3 ppm (CH₃); ESIMS: m/z: 1844.6 [M+2Na]²⁺; elemental analysis

calcd (%) for C₁₆₁H₂₈₀N₁₄O₄₉S₁₄: C 53.05, H 7.74, N 5.38; found: C 52.67,

H 7.61, N 5.19.

6.5 Hz, 7H; H-5), 3.90 (t, J_4,5=9.0 Hz, 7H; H-4), 3.22 (d, J_6a,6b=13.0 Hz,

3

7H; H-6a), 3.21 (brt, J

N

6b), 3.02, 2.94 (2dt, ²J(H,H)=14.5 Hz, ³J

ACHTUNGTRENNUNG ACHTUNGTRENNUNG

2.47–2.21 (m, 28H; CH₂CO), 1.62 (m, 28H; CH₂CH₂CO), 1.33 (m, 56H;

3

CH₃CH₂, CH₃CH₂CH₂), 0.92, 0.91 ppm (2t, J

N

¹³C NMR (125.7 MHz, MeOD): d=174.4, 174.3 (CO), 98.2 (C-1), 80.0

(C-4), 74.0 (C-5), 71.8 (C-2), 71.3 (C-3), 40.2 (CH₂N_Cyst), 35.2, 35.0

(CH₂CO), 34.4 (C-6), 32.6, 32.5 (CH₃CH₂CH₂), 31.7 (CH₂S_Cyst), 25.6, 25.5

(CH₂CH₂CO), 23.6, 23.5 (CH₃CH₂), 14.4, 14.3 ppm (CH₃); ESIMS: m/z:

1462.1 [M+2H]²⁺, 974.8 [M+3H]³⁺; elemental analysis calcd (%) for

Compound 16: A solution of 5 (114 mg, 35 mmol) in dry CH₂Cl₂(2 mL)

was added dropwise to a solution of N-tert-butoxycarbonylethylendi-

AHCTUNGTERNGUNaN mine (7, 0.37 mmol, 59 mg, 1.5 equiv) in dry CH₂Cl₂(1 mL). The mix-

ture was stirred at RT for 20 h and the reaction mixture was quenched

with water (10 mL) and extracted with CH₂Cl₂(3ꢆ10 mL). The com-

bined organic layers were dried (Na₂SO₄), concentrated and purified by

flash chromatography (20:1 CH₂Cl₂/MeOH). Yield: 129 mg (85%); R_f=

0.27 (20:1 CH₂Cl₂/MeOH); [a]_D=+78.4 (c=1.05 in CH₂Cl₂); ¹H NMR

(500 MHz, CDCl₃, 313 K): d=7.19 (brs, 1H; NH), 7.00 (brs, 1H; NH),

C

2.98.

₁₄₀H₂₅₂Cl₇N₇O₄₂S₇: C 52.91, H 7.99, N 3.09: found: C 52.66, H 7.84, N

Compound MyrCD-N: Treatment of the heptacarbamate

4 (112 mg,

21.6 mmol) with 1:1 TFA/CH₂Cl₂(2 mL) at RT for 2 h, followed by evap-

oration of the solvent and freeze-drying from a diluted HCl solution,

gave MyrCD-N in virtually quantitative yield (102 mg). [a]_D=+ 43.3

(c=1.0 in CH₂Cl₂); ¹H NMR (500 MHz, 9:1 MeOD/CDCl₃, 323 K): d=

5.36 (t, J_2,3=J_3,4=9.0 Hz, 7H; H-3), 5.12 (d, J_1,2=3.5 Hz, 7H; H-1), 4.77

5.46 (brs, 1H; NH), 5.25 (t, J_2,3=J_3,4=8.9 Hz, 7H; H-3), 5.08 (d, J_1,2

3.7 Hz, 7H; H-1), 4.76 (dd, 7H; H-2), 4.15 (m, 7H; H-5), 3.75 (t, J_4,5

=

8.7 Hz, 7H; H-4), 3.72 (m, 14H; CH₂N_Cyst), 3.58 (m, 14H; CH₂NH), 3.29

(m, 14H; CH₂NHBoc), 3.20 (brd, J_6a,6b=13.4 Hz, 7H; H-6a), 3.04 (dd,

(dd, 7H; H-2), 4.12 (m, 7H; H-5), 3.85 (t, J_4,5=9.0 Hz, 7H; H-4), 3.20

2

ACTHNGUTRENNUG ACHTUNGTRENNUNG(H,H)=

_5,6b=4.8 Hz, 7H; H-6b), 2.93, 2.81 (2dt, ²J(H,H)=13.3 Hz, ³J

(m, 21H; H-6a, CH₂N_Cyst), 3.05 (m, 7H; H-6b), 2.99, 2.95 (2dt, J

N

J

14.0 Hz, ³J

1.59 (m, 28H; CH₂CH₂CO), 1.30 (m, 280H; CH₂), 0.88 ppm (t, JACHTNUGTRNE(NUG H,H)=

ACHTUNGTRENNUNG(H,H)=7.0 Hz, 14H; CH₂S_Cyst), 2.45–2.18 (m, 28H; CH₂CO),

6.6 Hz, 14H; CH₂S_Cyst), 2.38–2.11 (m, 28H; CH₂CO), 1.56 (m, 28H;

3

CH₂CH₂CO), 1.42 (s, 63H; CMe₃), 1.24 (m, 56H; CH₃CH₂,

7.0 Hz, 42H; CH₃); ¹³C NMR (125.7 MHz, 9:1 MeOD-CDCl₃): d=174.3,

173.5 (CO), 98.2 (C-1), 80.3 (C-4), 73.6 (C-5), 71.9 (C-2), 71.4 (C-3), 40.0

(CH₂N_Cyst), 35.0 (CH₂CO), 34.3 (C-6), 33.2 (CH₂CH₂CH₃), 31.7

(CH₂S_Cyst), 31.3–30.1 (CH₂), 26.0, 25.9 (CH₂CH₂CO), 23.9 (CH₂CH₃),

14.8 ppm (CH₃); ESIMS: m/z: 4491.6 [M+H]⁺; elemental analysis calcd

(%) for C₂₅₂H₄₇₆Cl₇N₇O₄₂S₇: C 63.73, H 10.10, N 2.06; found: C 63.55, H

9.84, N 1.95.

CH₃CH₂CH₂), 0.90, 0.87 ppm (2t, ³J(H,H)=7.3 Hz, ³J

ACTHNUTRGENNUG ACHUTTGNREN(NUGN H,H)=6.9 Hz,

42H; CH₃); ¹³C NMR (125.7 MHz, CDCl₃, 313 K): d=182.0 (CS), 173.4,

171.6 (CO ester), 156.9 (CO carbamate), 96.7 (C-1), 79.8 (CMe₃), 79.0

(C-4), 71.5 (C-5), 70.5 (C-3), 70.4 (C-2), 44.6 (CH₂NH), 43.8 (CH₂N_Cyst),

40.0 (CH₂NHBoc), 34.0 (CH₂CO), 33.8 (C-6), 32.9 (CH₂S_Cyst), 31.4, 31.2

(CH₃CH₂CH₂), 28.5 (CMe₃), 24.4, 24.3 (CH₂CH₂CO), 22.3 (CH₃CH₂),

13.8 ppm (CH₃); ESIMS: m/z: 2191.2 [M+2Na]²⁺; elemental analysis

calcd (%) for C₁₉₆H₃₄₃N₂₁O₅₆S₁₄: C 54.26, H 7.97, N 6.78; found: C 54.10,

H 7.84, N 6.67.

Compound 5: CaCO₃(528 mg, 5.28 mmol, 4 equiv) and CSCl₂(207 mL,

2.64 mmol, 2 equiv) were added to a solution of the heptaamine HexCD-

N

(600 mg, 189 mmol) in a mixture of acetone (2.4 mL) and water

Alternatively, 16 was prepared from HexCD-N as follows: A solution of

2-(tert-butoxycarbonylamino)ethylisothiocyanate^[35](27, 39 mg, 192 mmol,

1.1 equiv) in CH₂Cl₂(1.5 mL) was added to a solution of HexCD-N

(80 mg, 25 mmol) and Et₃N (26 mL, 192 mmol, 1.1 equiv) in CH₂Cl₂

(1.5 mL). The reaction mixture was stirred at RT for 16 h. The solvent

was evaporated under diminished pressure. The residue was purified by

flash chromatography (50:1!20:1 CH₂Cl₂/MeOH). Yield: 98 mg (90%).

(3.6 mL). The reaction mixture was stirred for 2.5 h and then concentrat-

ed. The residue was dissolved in CH₂Cl₂(6 mL) and washed with saturat-

ed aqueous NaHCO₃(6 mL)_.The organic phase was decanted, dried

(Na₂SO₄), and concentrated. The residue was subjected to flash chroma-

tography (1:4!1:3 EtOAc/petroleum ether). Yield: 390 mg (64%); R_f=

0.34 (1:3 EtOAc/petroleum ether); [a]_D=+118.8 (c=1.0 in CHCl₃);

¹H NMR (500 MHz, CDCl₃): d=5.26 (t, J_2,3=J_3,4=9.5 Hz, 7H; H-3), 5.05

Compound 17: A solution of 5 (110 mg, 35 mmol) in dry CH₂Cl₂(2 mL)

was added dropwise to a solution of N-tert-butoxycarbonylbutylendi-

(d, J_1,2=4.0 Hz, 7H; H-1), 4.77 (dd, 7H; H-2), 4.13 (ddd, J_4,5=8.5 Hz,

3

J

_5,6b=5.5 Hz, J_5,6a=2.5 Hz, 7H; H-5), 3.81 (t, 7H; H-4), 3.77 (t, J

G

AHCTUNGTERNGUNaN mine (8, 75 mL, 0.37 mmol, 1.5 equiv) in dry CH₂Cl₂(1 mL). The mix-

7.0 Hz, 14H; CH₂N_Cyst), 3.16 (dd, J_6a,6b=14.5 Hz, 7H; H-6a), 3.04 (dd,

7H; H-6b), 3.00, 2.90 (2dt, ²J(H,H)=13.5 Hz, ³J

(H,H)=6.5 Hz, 14H;

CH₂S_Cyst), 2.38–2.11 (m, 28H; CH₂CO), 1.55 (m, 28H; CH₂CH₂CO), 1.27

(m, 56H; CH₃CH₂, CH₃CH₂CH₂), 0.88, 0.86 ppm (2t, ³J

(H,H)=7.0 Hz,

ture was stirred at RT for 16 h and the reaction mixture was quenched

with water (10 mL) and extracted with CH₂Cl₂(3ꢆ10 mL). The com-

bined organic layers were dried (MgSO₄), filtered, concentrated and puri-

fied by flash chromatography (20:1!15:1 CH₂Cl₂/MeOH). Yield: 114 mg

(72%); R_f=0.37 (9:1 CH₂Cl₂/MeOH); [a]_D=+65.8 (c=1.0 in CH₂Cl₂);

¹H NMR (500 MHz, CDCl₃, 313 K): d=6.89 (brs, 14H; NH), 5.25 (t,

G

ACHTUNGTRENNUNG

AHCTUNGTRENNUNG

42H; CH₃); ¹³C NMR (125.7 MHz, CDCl₃): d=173.4, 171.7 (CO), 132.2

(NCS), 96.7 (C-1), 78.5 (C-4), 71.6 (C-5), 70.2 (C-2, C-3), 45.6 (CH₂N_Cyst),

34.1 (C-6, CH₂S_Cyst), 34.0, 33.8 (CH₂CO), 31.4, 31.3 (CH₃CH₂CH₂), 24.4,

24.3 (CH₂CH₂CO), 22.4, 22.3 (CH₃CH₂), 13.9 ppm (CH₃); ESIMS: m/z:

1628.0 [M+2H]²⁺, 722.6 [M+3H]³⁺; elemental analysis calcd (%) for

J

_2,3=J_3,4=8.9 Hz, 7H; H-3), 5.08 (d, J_1,2=3.7 Hz, 7H; H-1), 4.95 (brs,

7H; NH), 4.75 (dd, 7H; H-2), 4.14 (m, 7H; H-5), 3.75 (t, J_4,5=8.8 Hz,

7H; H-4), 3.76 (m, 14H; CH₂N_cyst), 3.46 (m, 14H; CH₂NH), 3.19 (brd,

C

₁₄₇H₂₃₁N₇O₄₂S₁₄: C 54.88, H 7.24, N 3.05; found: C 54.98, H 7.37, N 3.02.

J_6a,6b=12.2 Hz, 7H; H-6a), 3.10 (m, 14H; CH₂NHBoc), 3.03 (dd, J_5,6b=

12882

www.chemeurj.org

ꢅ 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Chem. Eur. J. 2009, 15, 12871 – 12888

Polycationic Amphiphilic Cyclodextrins

FULL PAPER

5.6 Hz, 7H; H-6b), 2.92, 2.80 (2dt, ²J

(H,H)=13.3 Hz, ³J

E

was added to a solution of 5 (66 mg, 20.7 mmol) and Et₃N (22 mL,

160 mmol, 1.1 equiv) in CH₂Cl₂(1.5 mL). The reaction mixture was stirred

at RT for 16 h. The solvent was evaporated under diminished pressure.

The residue was purified by flash chromatography (50:1!20:1 CH₂Cl₂/

MeOH). Yield: 90 mg (88%); R_f=0.48 (20:1 CH₂Cl₂/MeOH); [a]_D=

+63.2 (c=1.1 in CH₂Cl₂); ¹H NMR (500 MHz, CDCl₃, 313 K): d=7.18

14H; CH₂S_cyst), 2.38–2.30 (m, 14H; CH₂CO), 2.28–2.21 (m, 7H; CH₂CO),

2.18–2.11 (m, 7H; CH₂CO), 1.60–1.49 (m, 56H; CH₂CH₂CO,

CH₂CH₂NH, CH₂CH₂NHBoc), 1.41 (s, 63H; CMe₃), 1.31–1.24 (m, 56H;

CH₃CH₂, CH₃CH₂CH₂), 0.89, 0.87 ppm (2t, ³J(H,H)=7.4 Hz, ³J

ACTHNUTRGENNUG AHCUTNGTREN(NUGN H,H)=

6.7 Hz, 42H; CH₃); ¹³C NMR (125.7 MHz, CDCl₃, 313 K): d=182.0 (CS),

173.4, 171.6 (CO ester), 156.4 (CO carbamate), 96.6 (C-1), 79.3 (CMe₃),

78.9 (C-4), 71.5 (C-5), 70.4 (C-2, C-3), 44.2 (CH₂NH), 43.8 (CH₂N_cyst),

40.3 (CH₂NHBoc), 34.1 (CH₂CO), 33.8 (C-6), 33.1 (CH₂S_cyst), 31.4, 31.2

(CH₃CH₂CH₂), 28.5 (CMe₃), 27.6 (CH₂CH₂NHBoc), 26.4 (CH₂CH₂NH),

24.4, 24.3 (CH₂CH₂CO), 22.3 (CH₃CH₂), 13.8 ppm (CH₃); ESIMS: m/z:

1533.9 [M+3 Na]³⁺, 2288.9 [M+2Na]²⁺; elemental analysis calcd (%) for

C₂₁₀H₃₇₁N₂₁O₅₆S₁₄: C 55.61, H 8.25, N 6.49; found: C 55.36, H 8.30, N

6.26.

(t, ³J

7.05 (d, J

NHCS), 5.24 (t, J_2,3=J_3,4=9.0 Hz, 7H; H-3), 5.18 (brs, 7H; NHBoc), 5.09

(d, _1,2=3.8 Hz, 7H; H-1), 4.75 (dd, 7H; H-2), 4.45 (brs, 14H;

(H,H)=9.4 Hz, 7H; H-5_Ph), 7.10 (d, ³J

ACHUTGTNRENNUG CAHTUNGTRENNUNG

3

AHCTUNGTRENNUNG

J

PhCH₂NHCS), 4.14 (m, 21H; H-5, PhCH₂NHBoc), 3.75 (t, J_4,5=9.0 Hz,

7H; H-4), 3.70 (brs, 14H; CH₂CH₂S_cyst), 3.21 (brd, J_6a,6b=13.1 Hz, 7H;

H-6a), 3.04 (dd, J_5,6b=5.5 Hz, 7H; H-6b), 2.82 (m, 14H; CH₂S_cyst), 2.40–

2.10 (m, 28H; CH₂CO), 1.60 (m, 28H; CH₂CH₂CO), 1.40 (s, 63H;

CMe₃), 1.28 (m, 56H; CH₃CH₂, CH₃CH₂CH₂), 0.89–0.88 ppm (m, ³J-

Compound 18: A solution of 5 (110 mg, 35 mmol) in dry CH₂Cl₂(2 mL)

was added dropwise to a solution of N-tert-butoxycarbonylhexylendi-

ACHTUNGTRENNUNGamine (9, 82 mL, 0.37 mmol, 1.5 equiv) in dry CH₂Cl₂(1 mL). The mix-

ture was stirred at RT for 12 h and the reaction mixture was quenched

with water (10 mL) and extracted with CH₂Cl₂(3ꢆ10 mL). The com-

bined organic layers were dried (MgSO₄), filtered, concentrated, and pu-

rified by flash chromatography (20:1!15:1 CH₂Cl₂/MeOH). Yield:

107 mg (65%); R_f=0.38 (9:1 CH₂Cl₂/MeOH); [a]_D=+65.6 (c=1.0 in

CH₂Cl₂); ¹H NMR (500 MHz, CDCl₃, 313 K): d=6.89 (brs, 7H; NH),

AHCTUNGTRENNUNG

(H,H)=7.0 Hz, 42H; CH₃); ¹³C NMR (125.7 MHz, CDCl₃, 313 K): d=

182.5 (CS), 173.7, 171.9 (CO ester), 156.7 (CO carbamate), 139.8, 138.3,

129.1, 126.8, 126.4, 126.0 (Ph), 96.9 (C-1), 80.0 (CMe₃), 79.2 (C-4), 71.8

(C-5), 70.7 (C-3, C-2), 48.4 (PhCH₂NCS), 44.8 (PhCH₂NHBoc), 44.2

(CH₂CH₂S_cyst), 34.3, 34.1 (CH₂CO, C-6), 33.2 (CH₂S_cyst), 31.7, 31.5

(CH₃CH₂CH₂), 28.7 (CMe₃), 24.7, 24.6 (CH₂CH₂CO), 22.6 (CH₃CH₂),

14.1 ppm (CH₃); ESIMS: m/z: 2455.3 [M+K+H]²⁺

,

1649.9

[M+K+2Na]³⁺; elemental analysis calcd (%) for C₂₃₈H₃₇₁N₂₁O₅₆S₁₄: C

58.68, H 7.68, N 6.04; found: C 58.62, H 7.66, N 5.85.

6.82 (brs, 14H; NH), 5.26 (t, J_2,3=J_3,4=8.9 Hz, 7H; H-3), 5.09 (d, J_1,2

Compound 21: A solution of 14^[56](89 mg, 259 mmol, 1.2 equiv) in CH₂Cl₂

(2 mL) was added to a solution of HexCD-N (98 mg, 31 mmol) and Et₃N

(33 mL, 238 mmol, 1.1 equiv) in CH₂Cl₂(2 mL), . The reaction mixture

was stirred at RT for 16 h. The solvent was evaporated under diminished

pressure. The residue was purified by flash chromatography (50:1!20:1

CH₂Cl₂/MeOH). R_f=0.48 (20:1 CH₂Cl₂/MeOH); [a]_D=++62.8 (c=0.8 in

CH₂Cl₂); ¹H NMR (500 MHz, CDCl₃, 313 K): d=7.42 ꢁ6.90 (m, 14H;

NHCS), 5.27 (t, J_2,3=J_3,4=8.9 Hz, 7H; H-3), 5.08 (d, J_1,2=3.7 Hz, 7H; H-

1), 5.06 (brs, 7H; NHBoc), 4.75 (dd, 7H; H-2), 4.15 (m, 7H; H-5), 3.80

(t, J_4,5=8.9 Hz, 7H; H-4), 3.70 (brs, 14H; CH₂CH₂S_cyst), 3.62 (brs, 14H;

BocNCH₂CH₂NHCS), 3.39 (m, 14H; CSNHCH₂CH₂NBoc), 3.32 (m,

3.7 Hz, 7H; H-1), 4.76 (m, 7H; NH), 4.75 (dd, 7H; H-2), 4.15 (m, 7H;

H-5), 3.76 (t, J_4,5=8.8 Hz, 7H; H-4), 3.72 (m, 14H; CH₂N_cyst), 3.43 (m,

14H; CH₂NH), 3.18 (brd, J_6a,6b=12.1 Hz, 7H; H-6a), 3.06 (m, 21H;

CH₂NHBoc, H-6b), 2.92, 2.79 (2dt, ²J(H,H)=13.3 Hz, ³J

ACTHNUTRGENNUG ACHUTTGNREN(NUGN H,H)=6.7 Hz,

14H; CH₂S_cyst), 2.38–2.30 (m, 14H; CH₂CO), 2.28–2.21 (m, 7H; CH₂CO),

2.18–2.11 (m, 7H; CH₂CO), 1.56 (m, 42H; CH₂CH₂CO, CH₂CH₂NH),

1.45 (m, 14H; CH₂CH₂NHBoc), 1.41 (s, 63H; CMe₃), 1.31–1.24 (m, 84H;

ACTHNUTRGNEUNG

CH₃CH₂, CH₃CH₂CH₂, CH₂), 0.89, 0.87 ppm (2t, ³J(H,H)=7.3 Hz, ³J-

ACHTUNGTRENNUNG

(H,H)=6.8 Hz, 42H; CH₃); ¹³C NMR (125.7 MHz, CDCl₃, 313 K): d=

182.0 (CS), 173.5, 171.6 (CO ester), 156.2 (CO carbamate), 96.6 (C-1),

79.1 (CMe₃), 78.8 (C-4), 71.4 (C-5), 70.4 (C-2, C-3), 44.5 (CH₂NH), 43.8

(CH₂N_cyst), 40.5 (CH₂NHBoc), 34.1 (CH₂CO), 33.8 (C-6), 33.1 (CH₂S_cyst),

31.4, 31.3 (CH₃CH₂CH₂), 30.0 (CH₂CH₂NHBoc), 29.0 (CH₂CH₂NH), 28.5

(CMe₃), 26.5, 26.4 (CH₂), 24.4, 24.3 (CH₂CH₂CO), 22.3 (CH₃CH₂),

13.8 ppm (CH₃); ESIMS: m/z: 2388.4 [M+2Na]²⁺; elemental analysis

calcd (%) for C₂₂₄H₃₉₉N₂₁O₅₆S₁₄: C 56.86, H 8.50, N 6.22; found: C 56.61,

H 8.3, N 6.10.

14H; BocNHCH₂CH₂NBoc), 3.23 (brq, ³J

ACTHNUTRGNE(NUG H,H)=5.4 Hz, 14H;

CH₂NHBoc), 3.17 (brd, J_6a,6b=10.9 Hz, 7H; H-6a), 3.08 (brd, 7H; H-6b),

2.85 (m, 14H; CH₂S_cyst), 2.40–2.10 (m, 28H; CH₂CO), 1.60 (m, 28H;

CH₂CH₂CO), 1.45 (s, 63H; NHCOOCMe₃), 1.41 (s, 63H; NCOOCMe₃),

1.32 (m, 56H; CH₃CH₂, CH₃CH₂CH₂), 0.91, 0.89 ppm (2t, ³J

ACHTUNGTRENNUNG(H,H)=

7.6 Hz, 42H; CH₃); ¹³C NMR (125.7 MHz, CDCl₃, 313 K): d=179.8 (CS),

173.6, 171.8 (CO ester), 156.4 (CO carbamate), 96.8 (C-1), 80.8 (CMe₃),

78.8 (C-4), 71.6 (C-5), 70.7 (C-3, C-2), 58.1 (BocNCH₂CH₂NHBoc), 57.5

(BocNCH₂CH₂NHCS), 44.0 (CH₂CH₂S_cyst), 43.5 (NCH₂CH₂NHCS), 40.1

(CH₂NHBoc), 34.3, 34.0 (CH₂CO, C-6), 33.0 (CH₂S_cyst), 31.6, 31.5

(CH₃CH₂CH₂), 28.7 (CMe₃), 24.6 (CH₂CH₂CO), 22.5 (CH₃CH₂),

14.0 ppm (CH₃); ESIMS: m/z: 2691.9 [M+2Na]²⁺, 1808.3 [M+2Na+K]³⁺

; elemental analysis calcd (%) for C₂₄₅H₄₃₄N₂₈O₇₀S₁₄: C 55.09, H 8.19, N

7.34; found: C 54.79, H 8.09, N 7.23.

Compound 19: A solution of 4-(tert-butoxycarbonylaminomethyl)benzyl

isothiocyanate^[55](12, 44.3 mg, 160 mmol, 1.1 equiv) in CH₂Cl₂(1.5 mL)

was added to a solution of HexCD-N (66 mg, 20.7 mmol) and Et₃N

(22 mL, 160 mmol, 1.1 equiv) in CH₂Cl₂(1.5 mL). The reaction mixture

was stirred at RT for 16 h. The solvent was evaporated under diminished

pressure. The residue was purified by flash chromatography (50:1!20:1

CH₂Cl₂/MeOH). Yield: 85 mg (84%); R_f=0.48 (20:1 CH₂Cl₂/MeOH);

1

[a]_D=+66.9 (c=1.1 in CH₂Cl₂); H NMR (500 MHz, CDCl₃, 313 K): d=

Compound 22: A solution of 15^[56](61 mg, 158 mmol, 1.2 equiv) in CH₂Cl₂

(1.5 mL) was added to a solution of HexCD-N (60 mg, 18.8 mmol) and

Et₃N (37 mL, 265 mmol, 2.0 equiv) in CH₂Cl₂(1.5 mL), and the reaction

mixture was stirred at RT for 16 h. The solvent was evaporated under di-

minished pressure. The residue was purified by flash chromatography

(50:1!20:1 CH₂Cl₂/MeOH). Yield: 90 mg (85%); R_f=0.46 (20:1

CH₂Cl₂/MeOH); [a]_D=+57.4 (c=1.0 in CH₂Cl₂); ¹H NMR (300 MHz,

7.10 (m, 28H; Ph), 6.95, 6.83 (2brs, 14H; NHCS), 5.25 (t, J_2,3=J_3,4

=

9.1 Hz, 7H; H-3), 5.16 (brs, 7H; NHBoc), 5.09 (d, J_1,2=3.7 Hz, 7H; H-

1), 4.74 (dd, 7H; H-2), 4.48 (brs, 14H; PhCH₂NHCS), 4.15 (m, 21H; H-

5, CH₂NHBoc), 3.73 (t,

CH₂CH₂S_cyst), 3.20 (brd,

J

_4,5=8.7 Hz, 7H; H-4), 3.64 (brs, 14H;

_6a,6b=13.2 Hz, 7H; H-6a), 3.01 (dd, J_6b,5

(H,H)=13.4 Hz, ³J

(H,H)=6.6 Hz, 14H;

J

=

5.5 Hz, 7H; H-6b), 2.80 (2dt, ²J

N

ACHTUNGTRENNUNG

CH₂S_cyst), 2.40–2.10 (m, 28H; CH₂CO), 1.60 (m, 28H; CH₂CH₂CO), 1.40

(s, 63H; CMe₃), 1.28 (m, 56H; CH₃CH₂, CH₃CH₂CH₂), 0.89, 0.88 ppm

CDCl₃, 313 K): d=7.22, 7.16 (2brs, 14H; NHCS), 5.29 (t, J_2,3=J_3,4

=

9.2 Hz, 7H; H-3), 5.24 (brs, 14H; NHBoc), 5.08 (d, J_1,2=3.5 Hz, 7H; H-

1), 4.77 (dd, 7H; H-2), 4.17 (m, 7H; H-5), 3.83 (t, J_4,5=9.2 Hz, 7H; H-4),

3.77 (brq, 14H; CH₂CH₂S_cyst), 3.54 (brq, 14H; NCH₂CH₂NHCS), 3.15

ACHTUNGTRENNUNG

(2t, ³J(H,H)=7.0 Hz, 42H; CH₃); ¹³C NMR (75.5 MHz, CDCl₃, 313 K):

d=182.5 (CS), 173.9, 172.0 (CO ester), 156.7 (CO carbamate), 138.8,

128.3, 127.9, 127.5 (Ph), 97.0 (C-1), 80.0 (CMe₃), 79.4 (C-4), 72.0 (C-5),

70.8 (C-3, C-2), 48.4 (PhCH₂NCS), 44.8 (PhCH₂NHBoc), 44.2

(CH₂CH₂S_cyst), 34.5, 34.2 (CH₂CO, C-6), 33.4 (CH₂S_cyst), 31.8, 31.6

(CH₃CH₂CH₂), 28.8 (CMe₃), 24.8, 24.7 (CH₂CH₂CO), 22.7 (CH₃CH₂),

3

(m, 42H; H-6a, H-6b, CH₂NHBoc), 2.87 (m, 14H; CH₂S_cyst), 2.67 (brt, J-

ACHUTNGRENNUG ACHTUTGNREN(NUGN H,H)=5.2 Hz,

(H,H)=5.5 Hz, 14H; NCH₂CH₂NHCS), 2.58 (brt, ³J

28H; CH₂CH₂NHBoc), 2.43–2.11 (m, 28H; CH₂CO), 1.60 (m, 28H;

CH₂CH₂CO), 1.44 (s, 126H; CMe₃), 1.30 (m, 56H; CH₃CH₂,

14.2 ppm (CH₃); ESIMS: m/z: 2454.8 [M+K+H]²⁺

,

1643.5

CH₃CH₂CH₂), 0.91, 0.89 ppm (2t, ³J

ACTHNUTRGNE(UNG H,H)=7.8 Hz, 42H; CH₃);

[M+K+Na+H]³⁺; elemental analysis calcd (%) for C₂₃₈H₃₇₁N₂₁O₅₆S₁₄: C

¹³C NMR (75.5 MHz, CDCl₃, 313 K): d=182.8 (CS), 173.8, 172.0 (CO

ester), 156.9 (CO carbamate), 97.0 (C-1), 79.8 (CMe₃), 79.0 (C-4), 71.8

(C-5), 70.9 (C-3, C-2), 55.1 (CH₂CH₂NHBoc), 54.0 (NCH₂CH₂NHCS),

44.5 (CH₂CH₂S_cyst), 42.9 (NCH₂CH₂NHCS), 39.5 (CH₂NHBoc), 34.4, 34.2

58.68, H 7.68, N 6.04; found: C 58.76, H 7.60, N 5.88.

Compound 20: A solution of 3-(tert-butoxycarbonylaminomethyl)benzyl

isothiocyanate^[55](13, 44.3 mg, 160 mmol, 1.1 equiv) in CH₂Cl₂(1.5 mL)

Chem. Eur. J. 2009, 15, 12871 – 12888

ꢅ 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

www.chemeurj.org

12883

J. M. Benito, P. Vierling, J. Defaye, C. Ortiz Mellet et al.

(CH₂CO, C-6), 33.6 (CH₂S_cyst), 31.8, 31.7 (CH₃CH₂CH₂), 28.9 (CMe₃),

24.8, 24.7 (CH₂CH₂CO), 22.7 (CH₃CH₂), 14.2 ppm (CH₃); ESIMS: m/z:

2820.3 [M+2H]²⁺, 1880.7 [M+3H]³⁺; elemental analysis calcd (%) for

1442.0 [M+2Na]²⁺; elemental analysis calcd (%) for C₁₁₉H₁₈₉N₇O₅₆S₇: C

50.63, H 6.71, N 3.45; found: C 50.21, H 6.46, N 3.35.

Compound 26: Treatment of 25 (56 mg, 20 mmol) with a mixture of TFA/

CH₂Cl₂(1:1, 2 mL) at RT for 2 h, followed by evaporation of the solvents

at diminished pressure afforded in virtually quantitative yield 26 (58 mg),

which was characterized as its heptakis(trifluoroacetate). [a]_D=+51.7

C

₂₅₉H₄₆₉N₃₅O₇₀S₁₄: C 55.13, H 8.38, N 8.69; found: C 54.76, H 8.21, N

8.50.

Compound 23: A solution of 5 (50 mg, 15.5 mmol) in CH₂Cl₂(2.5 mL)

(c=1.0 in water); ¹H NMR (500 MHz, MeOD, 313 K): d=5.38 (t, J_2,3

=

was added to

ACHTUNGTRENNUNG

a solution of bis(2-(tert-butoxycarbonylamino)ethyl)-

amine^[30e](10, 36 mg, 120 mmol, 1.1 equiv) and Et₃N (17 mL, 122 mmol,

J

_3,4=8.0 Hz, 7H; H-3), 5.24 (d, J_1,2=4.0 Hz, 7H; H-1), 4.82 (dd, 7H; H-

2), 4.14 (m, 7H; H-5), 3.96 (t, 7H; H-4), 3.20 (m, 14H; H-6), 3.12 (t, ³J-

AHCTUNGERTN(GNUN H,H)=7.0 Hz, 14H; CH₂N), 3.06 (t, 14H; CH₂S_Cyst), 2.10, 2.08 ppm (2s,

1.1 equiv) in CH₂Cl₂(2.5 mL), and the reaction mixture was stirred at RT

for 16 h. The solvent was evaporated under diminished pressure. The res-

idue was purified by flash chromatography (50:1!20:1 CH₂Cl₂/MeOH).

Yield: 83 mg (99%); R_f=0.41 (20:1 CH₂Cl₂/MeOH); [a]_D=+57.0 (c=

42H; MeCO); ¹³C NMR (125.7 MHz, MeOD, 313 K): d=172.0, 171.9

(CO), 97.9 (C-1), 79.9 (C-4), 73.8 (C-3), 72.2 (C-2), 71.9 (C-5), 40.6

(CH₂N_Cyst), 34.8 (C-6), 31.8 (CH₂S_Cyst), 21.2, 20.9 ppm (MeCO); ESIMS:

m/z: 2136.4 [M+H]⁺; 1069.2 [M+2H]²⁺; elemental analysis calcd (%) for

C₉₈H₁₄₀F₂₁N₇O₅₆S₇·2H₂O: C 39.61, H 4.88, N 3.30, S 7.65; found: C 39.57,

H 4.85, N 3.09, S 7.49.

1.0 in CH₂Cl₂); ¹H NMR (500 MHz, MeOD, 313 K): d=5.30 (t, J_2,3

_3,4=8.6 Hz, 7H; H-3), 5.13 (d, J_1,2=3.4 Hz, 7H; H-1), 4.79 (dd, 7H; H-

=

J

2), 4.17 (m, 7H; H-5), 3.93 (t, J_4,5=8.6 Hz, 7H; H-4), 3.86 (m, 14H;

CH₂CH₂S_cyst), 3.74 (m, 28H; CH₂NCS), 3.20 (m, 42H; H-6a, H-6b,

CH₂NHBoc), 2.98 (m, 14H; CH₂S_cyst), 2.40–2.10 (m, 28H; CH₂CO), 1.60

(m, 28H; CH₂CH₂CO), 1.44 (s, 126H; CMe₃), 1.30 (m, 56H; CH₃CH₂,

Compound 28: A solution of 2-(tert-butoxycarbonylamino)ethylisothio-

cyanate (27, 34 mg, 0.17 mmol, 1.2 equiv) in CH₂Cl₂(1.5 mL) was added

CH₃CH₂CH₂), 0.91, 0.89 ppm (2t, ³J

ACTHNUTRGNE(UNG H,H)=7.8 Hz, 42H; CH₃);

to

a solution of 26 (58 mg, 20 mmol) and Et₃N (29 mL, 0.21 mmol,

¹³C NMR (125.7 MHz, MeOD, 313 K): d=181.6 (CS), 173.6, 172.5 (CO

ester), 157.6 (CO carbamate), 97.2 (C-1), 80.3 (CMe₃), 79.0 (C-4), 72.1

(C-5), 71.0 (C-3, C-2), 50.9 (CH₂NCS), 45.9 (CH₂CH₂S_cyst), 38.4

(CH₂NHBoc), 34.2, 34.0 (CH₂CO, C-6), 32.8 (CH₂S_cyst), 31.7, 31.5

(CH₃CH₂CH₂), 28.9 (CMe₃), 24.7 (CH₂CH₂CO), 22.6 (CH₃CH₂),

1.5 equiv) in CH₂Cl₂(1.5 mL). The reaction mixture was stirred at RT for

24 h. The solvent was evaporated under diminished pressure. The residue

was purified by flash chromatography (50:1!20:1 CH₂Cl₂/MeOH).

Yield: 50 mg (70%); R_f=0.37 (20:1 CH₂Cl₂/MeOH); [a]_D=+69.0 (c=

0.4 in CHCl₃); ¹H NMR (500 MHz, CDCl₃, 313 K): d=7.20, 7.01 (2brs,

13.5 ppm (CH₃); ESIMS: m/z: 2701.3 [M+Na+K]²⁺

,

1813.9

14H; NH thiourea), 5.44 (brs, 7H; NH carbamate), 5.24 (t, J_2,3=J_3,4

=

[M+Na+2 K]³⁺; elemental analysis calcd (%) for C₂₄₅H₄₃₄N₂₈O₇₀S₁₄: C

8.8 Hz, 7H; H-3), 5.10 (d, J_1,2=3.6 Hz, 7H; H-1), 4.78 (dd, 7H; H-2),

4.17 (m, 7H; H-5), 3.75 (t, J_4,5=8.5 Hz, 7H; H-4), 3.74, 3.58 (2brs, 28H;

55.09, H 8.19, N 7.34; found: C 54.83, H 8.08, N 7.26.

CH₂NHCS), 3.29 (brq, ³J

ACHTUNGTRENNUNG

J

Compound 24: Compound 11^[56](68 mg, 261 mmol) was added to a solu-

tion of 5 (100 mg, 31 mmol) and Et₃N (36 mL, 261 mmol) in CH₂Cl₂

(3 mL), and the solution was stirred at RT for 16 h. The solvent was

evaporated under diminished pressure and the residue was purified by

flash chromatography (20:1 CH₂Cl₂/MeOH). Yield: 115 mg (73%);

(2m, 14H; CH₂S_Cyst), 2.05, 2.00 (2s, 42H; MeCO), 1.42 ppm (s, 63H;

CMe₃); ¹³C NMR (125.7 MHz, CDCl₃, 313 K): d=182.6 (CS), 171.0, 169.6

(CO ester), 157.2 (CO carbamate), 97.2 (C-1), 80.2 (C-4), 79.7 (CMe₃),

72.0 (C-3), 71.3 (C-2), 70.9 (C-5), 45.4, 45.0 (CH₂NHCS), 40.5

(CH₂NHBoc), 34.1 (CH₂S_Cyst), 33.3 (C-6), 28.9 (CMe₃), 21.0 ppm

(MeCO); ESIMS: m/z: 1818.8 [M+2 K]²⁺, 1806.8 [M+Na+K]²⁺, 1798.8

[M+2Na]²⁺; elemental analysis calcd (%) for C₁₄₀H₂₃₁N₂₁O₅₆S₁₄: C 47.32,

H 6.55, N 8.28, S 12.63; found: C 47.41, H 6.62, N 8.15, S 12.48.

1

[a]_D=+63.4 (c=1.0 in CH₂Cl₂); R_f=0.74 (6:1 CH₂Cl₂/MeOH); H NMR

(500 MHz, CDCl₃, 313 K): d=7.52–7.13 (m, 28H; NHCS), 5.44 (brs,

14H; NHBoc), 5.30 (t, J_2,3=J_3,4=9.2 Hz, 7H; H-3), 5.12 (d, J_1,2=3.5 Hz,

7H; H-1), 4.82 (dd, 7H; H-2), 4.19 (m, 7H; H-5), 3.83 (t, J_4,5=9.2 Hz,

7H; H-4), 3.90–3.50 (m, 56H; CH₂NHCS), 3.30 (m, 21H; H-6a,

CH₂NHBoc), 3.10 (m, 7H; H-6b), 2.93 (m, 14H; CH₂S_cyst), 2.50–2.13 (m,

28H; CH₂CO), 1.75–1.55 (m, 28H; CH₂CH₂CO), 1.44 (s, 63H; CMe₃),

1.40–1.20 (m, 56H; CH₃CH₂CH₂, CH₃CH₂), 1.00–0.97 ppm (m, 42H;

CH₃); ¹³C NMR (75.5 MHz, CDCl₃, 313 K): d=182.8 (CS), 173.8, 172.0

(CO ester), 156.9 (CO carbamate), 97.0 (C-1), 79.8 (CMe₃), 79.0 (C-4),

71.8 (C-5), 70.9 (C-2, C-3), 55.1 (NCH₂CH₂NHBoc), 54.0

(NCH₂CH₂NHCS), 44.5 (SCH₂CH₂NHCS), 42.9 (NCH₂CH₂NHCS), 39.5

(CH₂NHBoc), 34.3 (CH₂CO, C-6), 33.6 (SCH₂CH₂NHCS), 31.7

(CH₃CH₂CH₂), 28.9 (CMe₃), 24.7 (CH₂CH₂CO), 22.7 (CH₃CH₂),

Compound 29: A solution of 28 (50 mg, 14 mmol) in MeOH (3 mL) was

treated with methanolic NaOMe (1m, 25 mL). The reaction mixture was

stirred at RT for 16 h, then neutralized with Amberlite IR-120 (H⁺) ion-

exchange resin, the solution was filtered, and the solvent was removed

under diminished pressure. Yield: 42 mg (99%); R_f=0.59 (10:2:1 MeCN/

water/NH₄OH); [a]_D=+34.9 (c=0.24 in MeOH); ¹H NMR (500 MHz,

MeOD, 323 K): d=4.99 (d, J_1,2=3.4 Hz, 7H; H-1), 4.05 (m, 7H; H-5),

3.81 (t, J_2,3=J_3,4=9.3 Hz, 7H; H-3), 3.73, 3.58 (2brs, 28H; CH₂NHCS),

3.50 (m, 14H; H-2, H-4), 3.26 (m, 21H; H-6a, CH₂NHBoc), 2.98 (m, 7H;

H-6b), 2.92 (m, 14H; CH₂S_Cyst), 1.46 ppm (brs, 63H; CMe₃); ¹³C NMR

(125.7 MHz, MeOD, 323 K): d=183.7 (CS), 158.6 (CO), 104.1 (C-1), 86.4

(C-4), 80.6 (C_q), 74.5 (C-3), 74.4 (C-2), 73.5 (C-5), 45.3, 45.2 (CH₂NHCS),

41.2 (CH₂NHBoc), 35.0 (CH₂S_Cyst), 33.8 (C-6), 29.1 ppm (CMe₃); ESIMS:

14.2 ppm (CH₃). ESIMS: m/z: 2546.2 [M+2Na]²⁺, 1705.9 [M+3 Na]³⁺

;

elemental analysis calcd (%) for C₂₁₇H₃₈₅N₃₅O₅₆S₂₁: C 51.57, H 7.68, N

9.70; found: C 51.39, H 7.46, N 9.46.

m/z: 1501.8 [M+H+K]²⁺

;

elemental analysis calcd (%) for

Compound 25: Acetic anhydride (3 mL) was added to a solution of 2

(0.56 g, 0.25 mmol) in pyridine (6 mL), and the reaction was stirred at RT

for 24 h. Then, the mixture was poured into ice water (30 mL), the prod-

uct was extracted with CH₂Cl₂(2ꢆ20 mL), the organic layer was washed

successively with 2n H₂SO₄(2ꢆ20 mL) and saturated NaHCO₃(2ꢆ

20 mL), dried with Na₂SO₄, and the solvent was removed under dimin-

ished pressure. The resulting residue was purified by flash chromatogra-

phy (40:1!20:1 CH₂Cl₂/MeOH). Yield: 0.58 g (81%); R_f=0.80 (20:1

CH₂Cl₂/MeOH); [a]_D=+18.9 (c=1.0 in CH₂Cl₂); ¹H NMR (500 MHz,

CDCl₃, 313 K): d=5.39 (brs, 7H; NH), 5.28 (t, J_2,3=J_3,4=9.4 Hz, 7H; H-

3), 5.15 (d, J_1,2=3.9 Hz, 7H; H-1), 4.84 (dd, 7H; H-2), 4.20 (m, 7H; H-

C₁₁₂H₂₀₃N₂₁O₄₂S₁₄: C 45.37, H 6.90, N 9.92; found: C 45.11, H 6.69, N

9.80.

Compound CD-T-C₂N: Compound 29 (42 mg, 17 mmol) was treated with

a mixture of TFA/CH₂Cl₂(1:1, 2 mL) at RT for 2 h. Then the solvents

were evaporated under diminished pressure and further coevaporated

with water. The resulting residue was freeze dried from diluted HCl to

afford CD-T-C₂N (32 mg) in virtually quantitative yield as its heptahydro-

1

chloride. [a]_D=+46.5 (c=0.5 in H₂O); H NMR (500 MHz, D₂O, 333 K):

d=5.42 (d, J_1,2=3.1 Hz, 7H; H-1), 4.25 (brt, J_4,5=9.1 Hz, 7H; H-5), 4.20

(t,

CH₂CH₂NH₂), 3.99 (brs, 14H; CH₂NHCS), 3.96 (dd, 7H; H-2), 3.87 (t,

7H; H-4), 3.55 (t, ³J

(H,H)=6.0 Hz, 14H; CH₂NH₂), 3.52 (brd, J_6a,6b

13.8 Hz, 7H; H-6a), 3.28 (dd, J_5,6b=7.8 Hz, 7H; H-6b), 3.21 ppm (t, ³J-

(H,H)=6.9 Hz, 14H; CH₂S_Cyst); ¹³C NMR (125.7 MHz, D₂O, 333 K): d=

J ACHTUNTGNUER(GN H,H)=6.0 Hz, 14H;

_2,3=J_3,4=9.1 Hz, 7H; H-3), 4.14 (t, ³J

5), 3.82 (t, J_4,5=9.4 Hz, 7H; H-4), 3.63 (brq, ³J

CH₂N), 3.16 (dd, J_6a,6b=11.6 Hz, J_5,6a=2.1 Hz, 7H; H-6a), 3.06 (dd, J_5,6b

ACHTUNGTREN(NUGN H,H)=6.5 Hz, 14H;

=

A

=

6.0 Hz, 7H; H-6b), 2.82 (m, 14H; CH₂S_Cyst), 2.06, 2.02 (2s, 42H; MeCO),

1.42 ppm (s, 63H; CMe₃); ¹³C NMR (75.5 MHz, CDCl₃, 313 K): d=171.1,

169.8 (CO ester), 156.4 (CO carbamate), 97.1 (C-1), 79.7 (C-4), 79.4

AHCTUNGTRENNUNG

182.8 (CS), 101.6 (C-1), 84.1 (C-4), 73.6 (C-3), 72.5 (C-2), 72.0 (C-5),

(CMe₃), 71.7 (C-3), 71.3 (C-2), 70.9 (C-5), 40.6 (CH₂N_Cyst), 34.1 (CH₂S_Cyst

,

44.3, 41.6 (CH₂NHCS), 39.8 (CH₂NH₂), 34.1 (C-6), 32.4 ppm (CH₂S_Cyst);

C-6), 28.9 (CMe₃), 21.2 ppm (MeCO); ESIMS: m/z: 2860.5 [M+Na]⁺,

ESIMS: m/z: 1132.8 [M+2H]²⁺

;

elemental analysis calcd (%) for

12884

www.chemeurj.org

ꢅ 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Chem. Eur. J. 2009, 15, 12871 – 12888

Polycationic Amphiphilic Cyclodextrins

FULL PAPER

C₇₇H₁₅₄Cl₇N₂₁O₂₈S₁₄: C 36.71, H 6.16, N 11.68, S 17.82; found: C 36.48, H

5.95, N 11.44, S 17.49.

Ar), 5.32 (t, J_2,3=J_3,4=8.8 Hz, 7H; H-3), 5.15 (d, J_1,2=3.6 Hz, 7H; H-1),

4.80 (dd, 7H; H-2), 4.67 (brs, 14H; PhCH₂NHCS), 4.17 (m, 21H; H-5),

4.06 (s, 14H; PhCH₂NH₂), 3.90 (t, J_4,5=8.8 Hz, 7H; H-4), 3.72 (brs, 14H;

Compound HexCD-T-C₂N: Compound 16 (104 mg, 24 mmol) was dis-

solved in a mixture of TFA/CH₂Cl₂(1:1, 2 mL) and stirred for 2 h. The

acid was eliminated by repeated coevaporation with water and the result-

ing residue was dissolved in diluted HCl and freeze-dried to give pure

HexCD-T-C₂N. Yield: 90 mg (96%); [a]_D=+80.7 (c=1.0 in DMSO);

¹H NMR (500 MHz, [D₆]DMSO, 333 K): d=8.08 (brs, 2H; NH₂), 7.92

(brs, 1H; NH), 7.72 (brs, 1H; NH), 5.21 ((t, J_2,3=J_3,4=9.0 Hz, 7H; H-3),

5.05 (d, J_1,2=2.9 Hz, 7H; H-1), 4.67 (dd, 7H; H-2), 4.12 (m, 7H; H-5),

3.85 (t, J_4,5=8.6 Hz, 7H; H-4), 3.68 (m, 14H; CH₂NH), 3.59 (m, 14H;

CH₂CH₂S_cyst), 3.27 (brd,

J_6a,6b=14.2 Hz, 7H; H-6a), 3.16 (dd, J_5,6b=

5.2 Hz, 7H; H-6b), 2.90 (m, 14H; CH₂S_cyst), 2.50–2.20 (m, 28H; CH₂CO),

1.60 (m, 28H; CH₂CH₂CO), 1.30 (m, 56H; CH₃CH₂, CH₃CH₂CH₂), 0.90,

ACTHNUTRGNEUNG

0.89 ppm (2t, ³J(H,H)=7.2 Hz, 42H; CH₃); ¹³C NMR (125.7 MHz,

MeOD, 313 K): d=184.0 (CS), 174.8, 173.5 (CO), 141.3, 130.1, 129.3,

129.0 (Ph), 98.2 (C-1), 80.0 (C-4), 73.2 (C-5), 71.9 (C-3), 71.7 (C-2), 49.4

(PhCH₂NCS), 45.3 (CH₂CH₂S_cyst), 44.1 (PhCH₂NH₂), 35.2, 35.1 (CH₂CO,

C-6), 34.1 (CH₂S_cyst), 32.6, 32.4 (CH₃CH₂CH₂), 25.6 (CH₂CH₂CO), 23.5

(CH₃CH₂), 14.2 ppm (CH₃); ESIMS: m/z: 2085.2 [M+2H]²⁺

[M+3H]³⁺1043.0 [M+4H]⁴⁺

₂₀₃H₃₂₉N₂₁O₄₂S₁₄Cl₁₄: C 55.09, H 7.33, N 6.65; found: C 54.89, H 7.396, N

,

1390.4

CH₂N_Cyst), 3.10 (d, 14H; H-6a, H-6b), 2.98 (t, ³J

CH₂NH₂), 2.79, 2.77 (2dt, ²J(H,H)=13.5 Hz, ³J

(H,H)=7.0 Hz, 14H;

CH₂S_Cyst), 2.35–2.11 (m, 28H; CH₂CO), 1.50 (m, 28H; CH₂CH₂CO), 1.24

(m, 56H; CH₃CH₂, CH₃CH₂CH₂), 0.84, 0.83 ppm (2t, ³J

(H,H)=7.3 Hz,

³J(H,H)=6.9 Hz, 42H; CH₃); ¹³C NMR (125.7 MHz, [D₆]DMSO, 333 K):

ACHTUNGTREN(NUGN H,H)=6.3 Hz, 14H;

,

; elemental analysis calcd (%) for

A

ACHTUNGTRENNUNG

C

6.508.

AHCTUNGTRENNUNG

ACHTUNGTRENNUNG

Compound HexCD-T-mpXN: Treatment of 20 (30 mg, 6.2 mmol) with

d=183.4 (CS), 172.9, 171.9 (CO), 96.7 (C-1), 78.5 (C-4), 71.5 (C-5), 70.7

(C-3, C-2), 44.2 (CH₂N_Cyst), 41.7 (CH₂NH), 39.0 (CH₂NH₂), 34.0 (C-6),

33.7 (CH₂CO), 32.9 (CH₂S_Cyst), 31.3, 31.1 (CH₃CH₂CH₂), 24.3, 24.2

(CH₂CH₂CO), 22.2 (CH₃CH₂), 14.0 ppm (CH₃); ESIMS: m/z: 1819.4

TFA/CH₂Cl₂(1:1, 0.6 mL) at RT for 2 h, followed by evaporation of the

solvents and freeze-drying from

HexCD-T-mXN. Yield: 27 mg (99%); [a]_D=+47.8 (c=1.0 in MeOH);

¹H NMR (500 MHz, MeOD, 323 K): d=7.40 (m, 28H; Ar), 5.36 (t, J_2,3

_3,4=8.7 Hz, 7H; H-3), 5.19 (d, J_1,2=3.0 Hz, 7H; H-1), 4.85 (dd, 7H; H-

a diluted HCl solution, gave pure

=

[M+2H]²⁺

, ; elemental analysis calcd (%) for

1213.3 [M+3H]³⁺

J

C₁₆₁H₂₉₄Cl₇N₂₁O₄₂S₁₄: C 49.67, H 7.61 N 7.56; found: C 49.27 H 7.30 N

7.32.

2), 4.77 (brs, 14H; PhCH₂NHCS), 4.18 (m, 21H; H-5, PhCH₂NH₂), 3.95

(t, J_4,5=8.7 Hz, 7H; H-4), 3.79 (brs, 14H; CH₂CH₂S_cyst), 3.31 (brd, 7H;

H-6a), 3.20 (brd, J_6b,6b=13.0 Hz, 7H; H-6b), 2.94 (m, 14H; CH₂S_cyst),

Compound HexCD-T-C₄N: Treatment of 17 (58 mg, 13 mmol) with TFA/

CH₂Cl₂(1:1, 1 mL) at RT for 2 h, followed by evaporation of the solvents

and freeze-drying from a diluted HCl solution, gave pure HexCD-T-C₄N.

Yield: 52.8 mg (99%); [a]_D=+46.9 (c=0.5 in DMSO); ¹H NMR

(500 MHz, [D₆]DMSO, 333 K): d=7.89 (brs, 14H; NH₂), 7.68 (brs, 7H;

2.50–2.20 (m, 28H; CH₂CO), 1.66 (m, 28H; CH₂CH₂CO), 1.35 (m, 56H;

3

CH₃CH₂, CH₃CH₂CH₂), 0.96–0.94 ppm (m, J

N

¹³C NMR (125.7 MHz, MeOD, 323 K): d=182.4 (CS), 173.4, 172.1 (CO),

140.0, 133.1, 129.1, 128.0, 127.7, 127.3 (Ph), 96.9 (C-1), 78.7 (C-4), 71.9

(C-5), 70.6 (C-3), 70.3 (C-2), 48.4 (PhCH₂NCS), 44.1 (CH₂CH₂S_cyst), 43.1

(PhCH₂NH₂), 33.8, 33.7 (CH₂CO, C-6), 32.8 (CH₂S_cyst), 31.2, 31.1

(CH₃CH₂CH₂), 24.2 (CH₂CH₂CO), 24.2 (CH₃CH₂), 12.9 ppm (CH₃);

NH), 7.54 (brs, 7H; NH), 5.21 (t, J_3,4=9.0 Hz, 7H; H-3), 5.04 (d, J_1,2

=

3.1 Hz, 7H; H-1), 4.67 (dd, J_2,3=9.9, Hz, 7H; H-2), 4.11 (m, 7H; H-5),

3.85 (t, J_4,5=8.5 Hz, 7H; H-4), 3.58 (m, 14H; CH₂N_cyst), 3.37 (m, 14H;

CH₂NH), 3.10 (m, 14H; H-6a, H-6b), 2.76 (m, 28H; CH₂NH₂, CH₂S),

2.34–2.29 (m, 14H; CH₂CO), 2.21–2.11 (m, 14H; CH₂CO), 1.58–1.48 (m,

56H; CH₂CH₂CO, CH₂CH₂NH, CH₂CH₂NH₂), 1.25–1.22 (m, 56H;

ESIMS: m/z: 2085.2 [M+2H]²⁺, 1390.4 [M+3H]³⁺, 1043.0 [M+4H]⁴⁺

883.6 [M+5H]⁵⁺; elemental analysis calcd (%) for C₂₀₃H₃₂₉N₂₁O₄₂S₁₄Cl₁₄

C 55.09, H 7.33, N 6.65; found: C 54.88, H 7.29, N 6.24.

,

:

CH₃CH₂, CH₃CH₂CH₂), 0.84, 0.83 ppm (2t, ³J(H,H)=7.0 Hz, ³J

ACTHNUTRGENNUG AHCUTNGTREN(NUGN H,H)=

Compound HexCD-T-C₂N-C₂N: Treatment of the carbamate 21 (45.5 mg,

8.55 mmol) with 1:2 TFA/CH₂Cl₂at RT for 2 h, followed by evaporation

of the solvent and freeze-drying from diluted HCl solution, gave pure

HexCD-T-C₂N-C₂N. Yield: 38 mg (99%); R_f=0.05 (6:3:1 MeCN/H₂O/

NH₄OH); [a]_D=+29.4 (c=1.0 in MeOH); ¹H NMR (500 MHz, 5:1

MeOD/D₂O, 313 K): d=5.30 (t, J_2,3=J_3,4=8.7 Hz, 7H; H-3), 5.14 (d,

6.8 Hz, 42H; CH₃); ¹³C NMR (125.7 MHz, [D₆]DMSO, 313 K): d=183.1

(CS), 172.9, 171.9 (CO), 96.7 (C-1), 78.5 (C-4), 71.6 (C-5), 70.7 (C-2, C-

3), 44.1 (CH₂NH_yst), 43.5 (CH₂NH), 39.2 (CH₂NH₂), 33.9, 33.7 (C-6,

CH₂CO), 33.1 (CH₂S_cyst), 31.3, 31.1 (CH₃CH₂CH₂), 26.3, 25.0

(CH₂CH₂NH, CH₂CH₂NH₂), 24.3, 24.2 (CH₂CH₂CO), 22.2 (CH₃CH₂),

14.0 ppm (CH₃); ESIMS: m/z: 1917.3 [M+2H]²⁺, 1278.5 [M+3H]³⁺; ele-

mental analysis calcd (%) for C₁₇₅H₃₂₂Cl₇N₂₁O₄₂S₁₄: C 51.40, H 7.94, N

7.19; found: C 51.31, H 7.75, N 6.96.

J

_1,2=3.54 Hz, 7H; H-1), 4.80 (dd, 7H; H-2), 4.15 (m, 7H; H-5), 3.97 (brs,

14H; HNCH₂CH₂NHCS), 3.89 (t, J_4,5=8.7 Hz, 7H; H-4), 3.75 (brs, 14H;

CH₂CH₂S_cyst), 3.50 (t, ³J

(H,H)=6.3 Hz, 14H; CH₂NH₂), 3.46 (t, 14H;

NH₂CH₂CH₂NH), 3.41 (t, ³J

(H,H)=6.0 Hz, 14H; CSNHCH₂CH₂NH),

AHCTUNGTRENNUNG

Compound HexCD-T-C₆N: Treatment of 18 (33.4 mg, 7 mmol) with TFA/

CH₂Cl₂(1:1, 1 mL) at RT for 2 h, followed by evaporation of the solvents

and freeze-drying from a diluted HCl solution, gave pure HexCD-T-C₆N.

Yield: 30 mg (99%); [a]_D=+56.9 (c=0.3 in DMSO); ¹H NMR

(500 MHz, [D₆]DMSO, 333 K): d=7.79 (brs, 14H; NH₂), 7.54 (brs, 7H;

3.25 (brd, 7H; H-6a), 3.14 (m, 7H; H-6b), 2.91 (m, 14H; CH₂S_cyst), 2.50–

2.15 (m, 28H; CH₂CO), 1.60 (m, 28H; CH₂CH₂CO), 1.30 (m, 56H;

3

CH₃CH₂CH₂, CH₃CH₂), 0.91, 0.88 ppm (2t, J

N

¹³C NMR (125.7 MHz, MeOD, 323 K): d=184.3 (CS), 173.6, 172.2 (CO),

96.8 (C-1), 78.8 (C-4), 71.9 (C-5), 70.6 (C-3), 70.3 (C-2), 57.5

(HNCH₂CH₂NHCS), 44.7 (CH₂NH₂), 44.1 (CH₂CH₂S_cyst), 40.2

(NHCH₂CH₂NHCS), 35.7 (NHCH₂CH₂NHBoc), 33.9 (C-6), 33.8

(CH₂CO), 32.6 (CH₂S_cyst), 31.2, 31.0 (CH₃CH₂CH₂), 24.2 (CH₂CH₂CO),

22.1, 22.0 (CH₃CH₂), 13.1, 12.9 ppm (CH₃); ESIMS: m/z: 1968.9

[M+2H]²⁺, 1313.4 [M+3H]³⁺, 985.6 [M+4H]⁴⁺, 788.7 [M+5H]⁵⁺; ele-

mental analysis calcd (%) for C₁₇₅H₃₃₆N₂₈O₄₂S₁₄Cl₁₄: C 47.23, H 7.61, N

8.81; found: C 47.13, H 7.66, N 8.29.

NH), 7.43 (brs, 7H; NH), 5.22 (t, J_3,4=9.0 Hz, 7H; H-3), 5.04 (d, J_1,2

=

3.0 Hz, 7H; H-1), 4.67 (dd, J_2,3=10.0 Hz, 7H; H-2), 4.11 (m, 7H; H-5),

3.85 (t, J_4,5=8.5 Hz, 7H; H-4), 3.58 (m, 14H; CH₂N_cyst), 3.34 (m, 14H;

CH₂NH), 3.08 (m, 7H; H-6a, H-6b), 2.75 (m, 28H; CH₂NH₂, CH₂S_cyst),

2.34–2.29 (m, 14H; CH₂CO), 2.21–2.11 (m, 14H; CH₂CO), 1.55–1.45 (m,

56H; CH₂CH₂CO, CH₂CH₂NH, CH₂CH₂NH₂), 1.29–1.22 (m, 84H;

CH₃CH₂, CH₃CH₂CH₂, CH₂), 0.84, 0.83 ppm (2t, ³J

(H,H)=6.9 Hz, ³J-

ACHTUNGTRENNUNG

d=182.9 (CS), 172.9, 171.9 (CO), 96.7 (C-1), 78.4 (C-4), 71.6 (C-5), 70.6

(C-2, C-3), 44.1 (CH₂NH, CH₂N_cyst), 39.4 (CH₂NH₂), 33.9, 3.7 (C-6,

CH₂CO), 33.2 (CH₂S_cyst), 31.3, 31.1 (CH₃CH₂CH₂), 29.1 (CH₂CH₂NH),

27.4 (CH₂CH₂NH₂), 26.4, 26.1 (CH₂), 24.3, 24.2 (CH₂CH₂CO), 22.2

Compound HexCD-T-C₂NACHTNUGTRNEG[UN C₂N]₂: Treatment of the carbamate 22 (30 mg,

5.3 mmol) with 1:1 TFA/CH₂Cl₂at RT for 2 h, followed by evaporation of

the solvent and freeze-drying from diluted HCl solution, gave pure

HexCD-T-C₂N

MeOH); ¹H NMR (500 MHz, 5:1 MeOD/D₂O, 333 K): d=5.27 (t, J_2,3

_3,4=8.6 Hz, 7H; H-3), 5.13 (d, J_1,2=3.7 Hz, 7H; H-1), 4.82 (dd, 7H; H-

2), 4.14 (m, 7H; H-5), 3.89 (t, J_4,5=8.6 Hz, 7H; H-4), 3.73 (m, 28H;

CH₂CH₂S_cyst(H,H)=6.2 Hz, 28H;

NCH₂CH₂NHCS), 3.25 (t, ³J

CH₂NH₂), 3.14 (m, 14H; H-6a, H-6b), 3.14 (t, 28H; CH₂CH₂NH₂), 2.97

(t, ³J

(H,H)=6.5 Hz, 28H; NCH₂CH₂NHCS), 2.90 (m, 14H; CH₂S_cyst),

2.50–2.15 (m, 28H; CH₂CO), 1.60 (m, 28H; CH₂CH₂CO), 1.30 (m, 56H;

ACHTNUGERTN[NGNU C₂N]₂. Yield: 25 mg (99%); [a]_D=+48.2 (c=0.67 in

(CH₃CH₂), 14.0 ppm (CH₃); ESIMS: m/z: 2015.3 [M+2H]²⁺

, 1343.8

=

[M+3H]³⁺; elemental analysis calcd (%) for C₁₈₉H₃₅₀Cl₇N₂₁O₄₂S₁₄: C

52.96, H 8.23, N 6.86; found: C 53.02, H 8.18, N 6.63.

J

Compound HexCD-T-pXN: Treatment of 19 (30 mg, 6.2 mmol) with

TFA/CH₂Cl₂(1:1, 0.6 mL) at RT for 2 h, followed by evaporation of the

,

ACHTUNGTRENNUNG

solvents and freeze-drying from

a

diluted HCl solution, gave pure

ACHTUNGTRENNUNG

HexCD-T-pXN. Yield: 27 mg (99%); [a]_D=+54.9 (c=1.0 in MeOH);

3

¹H NMR (500 MHz, MeOD, 313 K): d=7.35 (2d, J

N

CH₃CH₂, CH₃CH₂CH₂), 0.91, 0.89 ppm (2t, JACTHNGUTERN(NUG H,H)=7.8 Hz, 42H; CH₃);

Chem. Eur. J. 2009, 15, 12871 – 12888

ꢅ 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

www.chemeurj.org

12885

J. M. Benito, P. Vierling, J. Defaye, C. Ortiz Mellet et al.

¹³C NMR (125.7 MHz, 5:1 MeOD-D₂O, 333 K): d=183.5 (CS), 175.0,

173.4 (CO), 98.1 (C-1), 80.1 (C-4), 73.1 (C-5), 71.9 (C-3), 71.5 (C-2), 53.5

(NCH₂CH₂NHCS), 52.3 (CH₂CH₂NH₂), 45.4 (CH₂CH₂S_cyst), 42.1

(NCH₂CH₂NHCS), 38.1 (CH₂NH₂), 35.2 (C-6), 35.0 (CH₂CO), 33.9

(CH₂S_cyst), 32.3, 32.2 (CH₃CH₂CH₂), 25.4, 25.3 (CH₂CH₂CO), 23.2, 23.1

G. J. Nabel, M. Manthorpe, J. Norman, Hum. Gene Ther. 1995 , 6 ,

55 3 –5 64.

[2] M. A. Behlke, Mol. Ther. 2006 , 13 , 64 4 –6 70.

[3] a) E. Mastrobatista, M. A. E. M. van der Aa, W. E. Hennink, D. J.

Crommelin, Nat. Rev. Drug Discovery 2006, 5, 115–121; b) K.

Kodama, Y. Katayama, Y. Shoji, H. Nakashima, Curr. Med. Chem.

2006 , 13 , 215 5 –2 161; c) M. Gore, Gene Ther. 2003 , 10 , 4; d) B. Gans-

bacher, J. Gene Med. 2003, 5, 261–262.

(CH₃CH₂), 14.2 ppm (CH₃); ESIMS: m/z: 2119.6 [M+2H]²⁺

, 1413.8

[M+3H]³⁺, 1060.3 [M+4H]⁴⁺, 848.7 [M+5H]⁵⁺, 707.3 [M+6H]⁶⁺, 606.3

[M+7H]⁷⁺; elemental analysis calcd (%) for C₁₈₉H₃₇₁N₃₅O₄₂S₁₄Cl₁₄: C

47.78, H 7.87, N 10.32; found: C 47.45, H 7.34, N 10.35.

[4] E. Mastrobattista, S. A. Bravo, M. A. van der Aa, D. J. A. Cromme-

lin, Drug Discovery Today Technol. 2005 , 2 , 10 3 –1 09.

Compound HexCD-TACHTUNGTRENNUNG[C₂N]₂: Treatment of the carbamate 23 (30 mg,

5.6 mmol) with 1:1 TFA/CH₂Cl₂at RT for 2 h, followed by evaporation of

the solvent and freeze-drying from diluted HCl solution, gave pure

[5] For a recent review, see: M. A. Mintzer, E. E. Simanek, Chem. Rev.

2009 , 109 , 25 9 –3 02.

HexCD-TACHTUNGTRENNUNG[C₂N]₂. Yield: 25 mg (99%); [a]_D=+19.5 (c=0.85 in MeOH);

[6] a) D. Putnam, Nat. Mater. 2006 , 5 , 43 9 –4 51; b) M. E. Davis, Z.

Chen, D. S. Shin, Nat. Rev. Drug Discovery 2008 , 7 , 77 1 –7 82; c) S.

Jin, K. Ye, Biotechnol. Prog. 2007 , 23 , 3 2 –4 1; d) M. Thomas, A. M.

Klibanov, Appl. Microbiol. Biotechnol. 2003 , 62 , 2 7 –3 4.

[7] a) Y. Yang, F. E. Nunes, K. Berencsi, E. E. Furth, E. Gçnczçl, J. M.

Wilson, Proc. Natl. Acad. Sci. USA 1994 , 91 , 440 7 –4 411; b) D. A.

Muruve, Hum. Gene Ther. 2004 , 15 , 115 7 –1 166; c) R. Tomanin, M.

Scarpa, Curr. Gene Ther. 2004, 4, 357–372.

[8] S. H. Pun, M. E. Davis, Bioconjugate Chem. 2002 , 13 , 63 0 –6 39.

[9] a) Y. H. Choi, F. Liu, J.-S. Kim, Y. K. Choi, J. S. Park, S. W. Kim, J.

Controlled Release 1998 , 54 , 3 9 –4 8; b) D. Fischer, Y. Li, B. Ahle-

meyer, J. Krieglstein, T. Kissel, Biomaterials 2003 , 24 , 112 1 –1 131;

c) D. Fischer, T. Bieber, Y. Li, H.-P. Elsꢊsser, T. Kissel, Pharm. Res.

1999 , 16 , 127 3 –1 279.

¹H NMR (500 MHz, MeOD, 313 K): d=5.32 (t, J_2,3=J_3,4=8.9 Hz, 7H; H-

3), 5.13 (d, J_1,2=3.6 Hz, 7H; H-1), 4.77 (dd, 7H; H-2), 4.14 (m, 7H; H-

5), 4.02 (m, 56H; CH₂NCS), 3.91 (t, J_4,5=8.6 Hz, 7H; H-4), 3.84 (t, ³J-

ACHTUNGTRENNUNG ACHTUNTGREN(NUGN H,H)=6.9 Hz, 56H;

(H,H)=6.9 Hz, 14H; CH₂CH₂S_cyst), 3.25 (t, ³J

CH₂NH₂), 3.22 (brd, J_6a,6b=13.2 Hz, 7H; H-6a), 3.22 (dd, J_5,6b=5.3 Hz,

7H; H-6b), 2.94 (t, 14H; CH₂S_cyst), 2.50–2.15 (m, 28H; CH₂CO), 1.60 (m,

28H; CH₂CH₂CO), 1.30 (m, 56H; CH₃CH₂, CH₃CH₂CH₂), 0.93,

ACHTUNGTRENNUNG

0.92 ppm (2t, ³J(H,H)=8.2 Hz, 42H; CH₃); ¹³C NMR (125.7 MHz,

MeOD, 313 K): d=182.8 (CS), 173.4, 172.1 (CO), 96.8 (C-1), 78.5 (C-4),

71.9 (C-5), 70.5 (C-3), 70.4 (C-2), 47.1 (CH₂NCS), 46.1 (CH₂CH₂S_cyst),

36.9 (CH₂NH₂), 33.9 (C-6), 33.8, 33.7 (CH₂CO), 32.2 (CH₂S_cyst), 31.2, 31.1

(CH₃CH₂CH₂), 24.2 (CH₂CH₂CO), 22.1 (CH₃CH₂), 13.2, 13.1 ppm (CH₃);

ESIMS: m/z: 1969.4 [M+2H]²⁺

; elemental analysis calcd (%) for

C₁₇₅H₃₃₆N₂₈O₄₂S₁₄Cl₁₄: C 47.23, H 7.61, N 8.81; found: C 47.64, H 7.59, N

8.85.

[10] M. E. Davis, M. E. Brewster, Nat. Rev. Drug Discovery 2004, 3,

1023–1035.

Compound HexCD-T-C₂-T-C₂N: Treatment of the carbamate 24 (30 mg,

5.9 mmol) with 1:1 TFA/CH₂Cl₂(2 mL) at RT for 2 h, followed by evapo-

ration of the solvent and freeze-drying from diluted HCl solution, gave

pure HexCD-T-C₂-T-C₂N. Yield: 21 mg (99%); [a]_D=++53.7 (c=1.0 in

[11] For a complete survey on CD molecular inclusion properties, see:

a) Comprehensive Supramolecular Chemistry, Vol. 3 (Eds.: J. Szejtli,

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MeOH); ¹H NMR (500 MHz, MeOD, 313 K): d=5.35 (brt, J_2,3=J_3,4

=

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8.9 Hz, 7H; H-3), 5.19 (d, J_1,2=3.0 Hz, 7H; H-1), 4.85 (dd, 7H; H-2),

4.21 (m, 7H; H-5), 3.94 (m, 21H; H-4, CH₂CH₂NH₂), 3.79 (brs, 14H;

CH₂CH₂S_Cyst),

3.73

(brs,

28H;

CSNHCH₂CH₂NHCS,

CSNHCH₂CH₂NHCS), 3.28 (m, 28H; CH₂NH₂, H-6a), 2.98 (m, 14H;

CH₂S_Cyst), 2.50–2.20 (m, 28H; CH₂CO), 1.75–1.55 (m, 28H; CH₂CH₂CO),

1.50–1.30 (m, 56H; CH₃CH₂CH₂, CH₃CH₂), 1.01–0.90 ppm (m, 42H;

CH₃); ¹³C NMR (125.7 MHz, MeOD, 313 K): d=186.3, 184.7 (CS), 176.0,

174.7 (CO), 99.4 (C-1), 81.3 (C-4), 74.5 (C-5), 73.2 (C-3), 72.9 (C-2), 46.6

(SCNHCH₂CH₂NH₂),

45.8

(SCNHCH₂CH₂NHCS,

SCNHCH₂CH₂NHCS), 43.9 (CH₂NH₂), 42.3 (CH₂CH₂S_Cyst), 36.4

(CH₂CO, C-6), 35.5 (CH₂S_Cyst), 33.7 (CH₃CH₂CH₂), 26.9 (CH₂CH₂CO),

24.8 (CH₃CH₂), 15.6 ppm (CH₃); ESIMS: m/z: 2176.2 [M+2H]²⁺, 1451.0

[M+3H]³⁺

, ; elemental analysis calcd (%) for

1088.5 [M+4H]⁴⁺

C₁₈₂H₃₃₈N₃₅O₄₂S₂₁Cl₇: C 47.43, H 7.35, N 5.39; found: C 47.11, H 7.23, N

10.39.

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This work was supported by the Spanish Ministerio de Innovaciꢃn y

Ciencia (MICINN; contract numbers CTQ2006-15515-C02-01/BQU and

CTQ2007-61180/PPQ), the Junta de Andalucꢀa (P06-FQM-01601), the

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Received: April 30, 2009

Published online: October 15, 2009

12888

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ꢅ 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Chem. Eur. J. 2009, 15, 12871 – 12888

Article Doi

Polycationic amphiphilic cyclodextrins for gene delivery: synthesis and effect of structural modifications on plasmid DNA complex stability, cytotoxicity, and gene expression

DOI: 10.1002/chem.200901149

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