Ditopic arginine-aspartate binders recognize RGD loops

FULL PAPER

DOI: 10.1002/ejoc.201201052

Ditopic Arginine-Aspartate Binders Recognize RGD Loops

Thomas Gersthagen,^[a]Johannes Hofmann,^[a]Frank-Gerrit Klärner,^[a]Carsten Schmuck,*^[a]

and Thomas Schrader*^[a]

Keywords: Supramolecular chemistry / Molecular tweezers / Host-guest systems / Receptors / Molecular recognition /

Peptides

A synthetic strategy was developed for the construction of

unsymmetrical arginine tweezers with an additional carb-

oxylate binding site. The ditopic receptor molecules 2 and 3

use two arms to bind arginine–glycine–aspartate (RGD) pept-

ides. However, contrary to expectations from molecular me-

chanics simulations, the tweezer cavity remains empty, and

arginine is bound through a simple guanidinium/phosphate

ion-pair formation. On the other hand, carboxylate recogni-

tion by the guanidiniopyrrole moiety operates very ef-

ficiently, leading to complexation of ionic amino acids in both

linear and cyclic RGD peptides with concentrations in the

20–30 μM). Truncation of the whole guanidiniopyrrole arm

leads to the simple unsymmetrical monophosphate tweezer

1, which lacks the aspartate binder, but restores the arginine

inclusion. Tweezer 1 displays a remarkable affinity and

selectivity towards cyclic RGD peptides, presumably by a

combination of arginine inclusion and additional cooperative

hydrogen bonds between its phenolic OH group and the

peptide backbone (NMR evidence, K_d= 15–30 μM). Compari-

son with symmetrical diphosphate tweezers 1a and 2–4

shows that in unsymmetrical tweezers the spacer group must

guarantee an open cavity.

low micromolar range in a buffered aqueous solution (K_d

=

velopment of artificial RGD receptor molecules, which can

cap the critical loop region in specific RGD proteins and

thus prevent their approach to the cell surface.

Introduction

In the past decades, a plethora of artificial receptor mole-

cules for amino acids have been developed. Crown ethers

adorned with a “recognition unit”,^[1]zinc porphyrins,^[2]

glycoluril clips (aromatic amino acids),^[3]polycyclic pyr-

idines (arginine),^[4]macrocyclic Tröger’s base derivatives (ly-

sine/arginine),^[5]zinc and copper chelate complexes (hist-

idine),^[6]boronic acids (homocysteine),^[7]and so forth all

represent powerful mono- or ditopic host molecules with

functionalities complementary to the side chains of pro-

teinogenic amino acids. Some of these also operate in water,

but very few cases have been reported for these receptor

molecules to be able to interfere with biological processes.

Arginine–glycine–aspartate (RGD) loops in proteins

docking onto the cell surface are particularly attractive tar-

gets.^[8]Because this small peptide sequence constitutes the

key recognition element for numerous cell-protein and cell-

cell communication events, it is a central research objective

of paramount biological and pharmaceutical relevance. In-

terference with major signaling pathways by the construc-

tion of RGD mimetics is promising as a new therapeutic

method against a large variety of related diseases.^[9]The op-

posite alternative is much less well explored, that is, the de-

One of the most efficient aspartate binders, which was

introduced by the Schmuck group,^[10]was further optimized

and developed for numerous bioorganic applications. It fea-

tures a guanidiniocarbonylpyrrole moiety that contains an

acylguanidinium group, which is conformationally locked,

for strong ionic hydrogen bonds to carboxylates and pres-

ents three contiguous hydrogen-bond donors in a crescent

shape towards its guest.^[11]Combinatorial techniques have

been employed to build this recognition element into a

number of elaborated receptor molecules, which selectively

bind key peptide fragments and protein domains.^[12]

Several years ago, the Schmuck and Schrader groups

joined forces and embarked on a program to develop artifi-

cial RGD receptor molecules that are tailored to interfere

with pathologic RGD recognition processes. We combined

a moderate bis(phosphonate) arginine binder with the

powerful guanidiniocarbonylpyrrole aspartate host. Ditopic

recognition of the linear RGD peptides was achieved in an

aqueous buffer solution, but the self-complementary nature

of the two binding sites led to extensive self-association,

and the tight solvent shell around the exposed bis(phos-

phonate) moiety lowered the absolute affinities (K_d

Ͼ

[a] Universität Duisburg-Essen, Fakultät für Chemie,

Universitätsstr. 7, 45117 Essen, Germany

200 μm).^[13]

Homepage: http://ww w .uni-due.de/akschmuck

In recent years, the Schrader and Klärner groups devel-

oped aromatic molecular tweezers for arginine and lysine,

which bind their guests by a unique threading mechanism

http://ww w .uni-due.de/chemie/ak_schrader/index.shtml

Supporting information for this article is available on the

WWW under http://dx.doi.org/10.1002/ejoc.201201052.

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Ditopic Arginine-Aspartate Binders Recognize RGD Loops

inside of the electron-rich cavity.^[14]This interaction is rein-

For the modeling experiments, we used molecular me-

forced by the pendant phosphate or phosphonate arms, chanics calculations of the ditopic tweezer molecules with

which lock the ammonium or guanidinium cation by ion- manual docking of both binding sites close to their guest

pair formation. We discovered inter alia that enzymes with moieties in the cyclopeptide and Monte-Carlo (MC) simu-

critical lysine residues in the vicinity of their active site or lations followed by molecular dynamics (MD) simulations

cofactor binding site could be effectively shut down with of the resulting complex structures (MacroModel 9.0, Am-

small amounts of these receptor molecules.

ber* force-field) and water (GBA solvation model for water

To use our molecular arginine tweezers for RGD recogni- approximation, 5000 steps for MC, 5 ns for MD at 300 K,

tion, it would be mandatory to develop a synthetic route no restraints).

towards an unsymmetrical prototype with the additional as-

Two spacer units (see Figure 1) led to favorable complex

partate binding site and to avoid by design its unwanted structures with cRGDfV. These complexes are characterized

self-association. A straightforward solution connects the ar- by a large energy stabilization, a minimum internal strain,

ginine tweezer to the guanidiniocarbonylpyrrole through a and a perfect retention of the RGD conformation. One unit

critical spacer of optimal length, polarity, and flexibility/ connects the tweezer OH group with the guanidiniocarbon-

rigidity. Ideally, this linker region would also render the ylpyrrole carboxyl through an ethyleneglycolamine, the

whole host molecule to be selective for a specific RGD loop other unit connects through an ethylaniline bridge. The for-

conformation and, hence, for a particular RGD protein.^[15]mer can be regarded as fairly flexible and able to adjust to

Herein, we report on the design, synthesis, and recogni- the peptide conformation by an induced fit. The latter is

tion properties of a new generation of RGD host molecules, fairly rigid and favors van der Waal contacts to the cyclo-

which contain two binding sites for RGD loops in peptides peptide. Both connections are ether structures, which are

and proteins.

Results and Discussion

Close inspection of the tweezer and guanidiniocarbonyl-

pyrrole structure leads to the phenolic hydroxy group on

the tweezer and a carboxylic acid or amine moiety on the

pyrrole unit as anchor points for covalent attachments.

Thus, a straightforward strategy protects one hydroquinone

OH group on the tweezer and uses the other to attach – by

ester or ether formation – a polar flexible or rigid spacer

that allows subsequent coupling with a suitable guanidini-

ocarbonylpyrrole moiety. After release of the second hy-

droxy group, phosphorylation, purification, and complete

removal of all of the protecting groups would furnish the

final receptor molecule in its zwitterionic form. Because

binding experiments with large proteins are difficult to ana-

lyze and do not provide access to structural information, it

would be advantageous to use a well-understood specific

model that contains the RGD sequence in a defined confor-

mation.

Several years ago, the Kessler group constructed various

cyclic RGD penta- and hexapeptides and demonstrated that

these were locked into stable and precisely defined confor-

mations that made them selective towards certain inte-

grins.^[16]Among others, the cyclic pentapeptides

cRGDf[NMe]V and cRGDfV were created as vitronectin

mimetics with an extended RGD loop.^[17]Transannular in-

tramolecular peptidic hydrogen bonds fix the cyclopeptide

into the desired conformation, which has been confirmed

by NMR experiments. This cyclopeptide adopts an all-trans

conformation and contains a βIIЈ turn as well as a γ turn.

We used this structure for docking experiments with ditopic

tweezers, and by using systematically varied spacers, we cal-

culated the resulting putative complexes. The best two

structures with respect to energy stabilization and retention

of the RGD loop conformation were selected and synthe-

sized.

Figure 1. Lewis structures of rationally designed unsymmetrical

phosphate tweezer molecules 2 (flexible) and 3 (rigid), as well as

reference compounds 1, 1a, and 4.

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hydrolytically stable under physiological conditions. The fi- ginine inclusion produced the two new arrangements de-

nal calculated complex structures feature perfect inclusion picted in Figure 4. In both, the guanidinium cation is still

of the arginine side chain in the tweezer cavity as well as involved as an ion pair with the tweezers phosphate anion,

efficient complexation of the aspartate residue by multiple but this time outside of the cavity. The remaining part of

hydrogen bonds with the guanidiniocarbonylpyrrole. In ad- the cyclopeptide nestles close to the tweezer arm, which car-

dition, the flexible ethyleneglycolamine forms some hydro- ries the carboxylate binding site, nearly similar to the ini-

gen bonds to the peptidic backbone, whereas the rigid ethyl- tially predicted structure. To switch between these two bind-

aniline unit undergoes dispersive interactions (see Figure 2a ing modes, the cavity only must be rotated away from the

and b).

peptide, which is easily affected by the simultaneous rota-

tion around both of the C–O bonds (see Figure 4a and b).

Figure 2. [(a) and (b)]. Calculated complexes between cRGDfV and

the two designed guanidiniocarbonylpyrrole phosphate tweezers 2

and 3. Both display the postulated threading mechanism for the

arginine side chain as well as aspartate recognition by multiple hy-

drogen bonds to the guanidiniocarbonylpyrrole (MacroModel 9.0,

Amber*, water/GBA solvation: Monte-Carlo simulation,

5000 steps, followed by 5 ns molecular dynamics simulation at

300 K). Hydrogen atoms have been omitted for clarity. Both com-

plexes are completely tension-free (no inner strain occurs), whereas

the RGD sequence is locked into an extended conformation.

Figure 4. The two putative complexes formed by receptor mole-

cules 2 (a) and 3 (b) with cRGDfV were calculated according to

the experimentally verified alternative mechanism involving the di-

rect ion-pair formation of the arginine guanidinium cation with the

tweezers phosphate moiety (MacroModel 9.0, Amber*, water/GBA

solvation: Monte-Carlo simulation, 5000 steps, followed by 5 ns

molecular dynamics simulation at 300 K). Hydrogen atoms have

been omitted for clarity. Note that in both final complexes the

RGD sequence is still locked into an extended conformation.

Superimposition of the RGD cyclopeptide prior to and

after docking to the ditopic tweezer reveals that the ex-

tended arrangement of both RGD arms is almost ideally

preserved in the complex (see Figure 3). In sharp contrast,

other cyclopeptides become severely distorted by this dock-

ing procedure and furnish less favorable complex structures

with higher energies.^[18]Thus, the in-silico-design was con-

sistent with the prediction that receptor molecules 2 and 3

would be able to recognize certain RGD cyclopeptides.

As a valuable reference compound, the unsymmetrical

monophosphate tweezer 1 was prepared first. We started

from the monoacetylated hydroquinone 6, which was ac-

cessible in quantitative yield by treating diacetoxy tweezer

5 with just 1 equiv. of aqueous sodium hydroxide (see

Scheme 1).^[19]The intermediate dimethyl phosphate 7 could

be obtained by either a classic two-step procedure with

phosphoryl chloride and methanol or by direct phosphoryl-

ation with dimethoxyphosphoryl chloride [O=P(OMe)₂Cl].

Sodium hydroxide selectively cleaved the acetate, which was

followed by the trimethylsilyl bromide-mediated (TMSBr-

mediated) removal of the two methyl ester groups in 8 to

furnish, after a final neutralization, the disodium salt of the

desired monophosphate tweezer 1.

The synthesis of our RGD receptor molecule also started

from monoacetylated hydroquinone 6 (see Scheme 2). The

S_N2 displacement by the appropriate spacer halide or

spacer tosylate furnished the unsymmetrical protected

tweezer derivatives 9a and 9b, which contained a terminal

tert-butoxycarbonyl-protected (Boc-protected) amine to at-

tach the aspartate binder.^[20]After quantitative acidic re-

moval of the Boc group [by treatment with trifluoroacetic

Figure 3. Superimposed conformations of free c-RGDfV and the

peptide complexed with 2 (pink, left) or 3 (pink, right; MC and

subsequent MD simulations with MacroModel 9.0, Amber* force-

field, water/GBA solvation, 5000 steps for MC, 5 ns for MD at

300 K, no restraints).

Unfortunately, experimental evidence from NMR studies acid (TFA) to give 10a and 10b], the peptide coupling with

of the complexes later ruled out that the arginine side chain Z-protected guanidiniocarbonylpyrrole carboxylic acid was

of the guest was inserted into the cavity of tweezers 2 and achieved in high yield through activation by O-(6-chloro-

3. In addition, the expected cooperativity between arginine benzotriazol-1-yl)-N,N,NЈ,NЈ-tetramethyluronium

hexa-

and aspartate binding events could not be observed Ϫ and fluorophosphate (HCTU)/N-methylmorpholine (NMM) in

dissociation constants did not reach the desired nanomolar warm dimethylformamide (DMF).^[21]The fully protected

range. Using Monte-Carlo simulations, a subsequent con- intermediates 11a and 11b were subsequently deacetylated

formational search for low-energy structures without the ar- under mild conditions (by treatment with NaOH in dioxane

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Ditopic Arginine-Aspartate Binders Recognize RGD Loops

Scheme 1. Synthesis of the monophosphate tweezer 1.

to give 12a and 12b) in nearly quantitative yield followed 50% yield over both steps).^[22]In two successive deprotec-

by phosphorylation with POCl₃/NEt₃. Both remaining tion steps, TMSBr/H₂O quantitatively liberated the free

chloride atoms were replaced by methanol to provide the phosphoric acids 14a and 14b,^[23]which was followed by

dimethyl phosphate derivatives 13a and 13b, which could be hydrogenolysis of the urethane moiety to afford free acyl-

separated by chromatography over silica gel (approximately guanidines 15a and 15b. In the last step, neutralization of

Scheme 2. Synthesis of unsymmetrical phosphate tweezers 2 and 3 with tethered guanidiniopyrrole moieties in their free zwitterionic form

(Cbz = benzyloxycarbonyl).

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15a and 15b with sodium hydroxide generated 2 and 3, observed from the usual linear decrease in emission inten-

respectively, as sodium salts with overall yields starting sity at 330 nm (excitation at 284 nm, 15–80 °C). We con-

from 6 of 36% (for 2) and 16% (for 3).

cluded that the steric demand of the attached guanidini-

In a similar protocol, by reversing the order of the reac- ocarbonylpyrrole moiety effectively prevented any self-asso-

tion steps, monomethyl phosphate 4 was obtained from 9a ciation of our ditopic monophosphate tweezer molecules.

through a simultaneous demethylation and unexpected Boc This remarkable behavior even tolerated flexible spacers

cleavage with 2 equiv. of dry lithium bromide in hot aceto- and paved the way towards a specific interaction with pept-

nitrile (55% yield, 17% overall yield from 6, see Scheme 3). ide guests.

Solubility tests immediately demonstrated the advantage

It is significant that the emission intensity of all of the

of the ethyleneglycol spacer in 2, which rendered the zwit- ditopic tweezers 2–4 in buffered aqueous solution was

terion highly water soluble (1 mm), but with a significantly roughly one order of magnitude lower than that of the hy-

smaller value in BisTris buffer at pH = 6.0 (0.05 mm).^[24]droquinone monophosphate tweezer 1 and two orders of

Because the acylguanidinium ion has a low pK (pK ≈ 6.5), magnitude lower than that of the diphosphate tweezer^[10]

the BisTris buffer is required for the binding experiments. (in DMSO, emission intensities of 1 and 1a were three times

Reference compound 1 with only one phosphate arm is also higher than those of 2–4). Increasing the methanol and, es-

very soluble in water (Ͼ1 mm) and in aqueous buffer (at pH pecially, the DMSO content in the solvent mixture rescued

= 7.4, 1 mm). On the contrary, 3 and 4 (with the 4-ethylanil- some (but by far not all) of the original fluorescence inten-

ine spacer or only one negative charge) are only soluble in sity and also led to considerable bathochromic shifts

an aqueous buffer containing 30% dimethyl sulfoxide (Ͼ10 nm). Clearly, the solvent environment influences the

(DMSO) or methanol (0.4 mm). This is a severe limitation concave arrangement of the fluorophoric aromatic groups

for biological applications, and, therefore, most of our rep- in the host. Most likely, a considerable energy transfer takes

resentative investigations were carried out with 2.

place from the tweezer to the pyrrole chromophore, from

In principle, the tweezer skeleton undergoes self-dimer- which it returns radiationless back to the ground state,

ization in a polar solution by interlocking two aromatic leaving only a small fraction for fluorescence emission.

cavities.^[25]However, for the diphosphate prototype as well

The complexes of ditopic hosts 2–4 with peptides are less

as for monophosphate tweezer 1, this behavior can only be soluble in water than the free hosts. Therefore, most of the

detected at elevated concentrations (Ͼ1 mm) and leads to titrations had to be carried out in a buffered aqueous solu-

small dimerization constants (for 1, 80 m^–1). By contrast, tion (pH = 6) that contained 50% DMSO as a cosolvent. In

the dimerization of the sterically more demanding ditopic pure BisTris buffer, only free arginine and the RGD peptide

tweezers 2–4 remains undetectable.

remained soluble throughout the entire binding experiment

Self-association phenomena proved to be much more with 2 and furnished dissociation constants below 100 μm

critical in the earlier zwitterionic systems for RGD recogni- (see Table 1). Surprisingly, upon transition to unbuffered

tion.^[13]Theoretically, flexible guanidinium phosphates water at an elevated pH (pH = 9.5), almost all of the RGD

could form intra- as well as intermolecular ion pairs, which affinity was lost. This behavior was also reproduced with a

would have to be cleaved before RGD recognition could linear RGD-containing hexapeptide. At this pH, the acylgu-

take place. Experimentally, we exploited NMR and fluores- anidinium ion of the host was deprotonated and showed

cence techniques to determine the self-association propen- poor binding to a carboxylate.

sity of our new receptor molecules. Tweezers 2 and 3 were

For a better comparison of 2–4 with each other, RGD

both examined by varying the concentration and tempera- penta- and hexapeptides as well as mutated versions were

ture, but no chemically induced ¹H NMR shift changes oc- titrated in buffered solvent mixtures with 50% DMSO. Ef-

curred [concentrations from 0.02 to 0.63 mm, temperatures ficient binding was observed for all of the peptides with

between 298 and 353 K (25–78 °C)]. Additionally, fluores- an intact RGD moiety. Molecule 2 formed the strongest

cence spectra were recorded at a lower concentration complexes (K_d= 20–40 μm), which was closely followed by

(0.03 mm) and temperature variation, but no deviation was 3 (K_d= 60 μm, see Table 2). However, complexes with 4 had

Scheme 3. Synthesis of monomethyl phosphate tweezer 4 by a reverse order protocol starting from 9a.

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Ditopic Arginine-Aspartate Binders Recognize RGD Loops

Table 1. Dissociation constants (K_d) determined by fluorescence ti- weaker binding (K_d= 100–250 μm, see Table 3).^[26]If the

tration experiments in different environments for complexes be-

tween 2 and RGD peptides.

aspartate in H-SRGDS-OH was replaced by alanine, the

affinity towards 2 remained unaltered, most likely reflecting

the ability of the receptor molecule to complex to the C-

terminus instead of the aspartate side chain (see related dis-

cussion for Table 4). In sharp contrast, the replacement of

arginine by alanine significantly weakened the attractive in-

teractions, demonstrating the influence of the tweezer moi-

ety.

Table 3. Dissociation constants (K_d) determined by fluorescence ti-

tration experiments in 10 mm BisTris buffer + 50% DMSO at pH

= 6.0 for complexes between 4 and RGD peptides.

RGD peptide

Solvent

K_d[μm]

H-Arg-OH

H-RGD-OH

BisTris buffer (10 mm, pH = 6.0) 70 (+/–08%)

BisTris buffer (10 mm, pH = 6.0) 65 (+/–30%)

unbuffered water (pH = 9.5)

BisTris buffer (10 mm, pH = 6.0) 80 (+/–26%)

unbuffered water (pH = 9.5) 560 (+/–20%)

buffer + 50% DMSO (pH = 6.0) 20 (+/–66%)

DMSO 20 (+/–31%)

buffer + 50% DMSO (pH = 6.0) 30 (+/–25%)

DMSO 15 (+/–32%)

buffer + 50% DMSO (pH = 6.0) 35 (+/–24%)

DMSO 10 (+/–18%)

buffer + 50% DMSO (pH = 6.0) 120 (+/–19%)

DMSO 26 (+/–11%)

buffer + 50% DMSO (pH = 6.0) 22 (+/–46%)

1100 (+/–30%)

H-GRGDTP-OH

H-SRGDS-OH

H-SRGAS-OH

H-SAGDS-OH

cGRGDSPA

RGD peptide

K_d[μm]

H-GRGDTP-OH

H-SRGDS-OH

H-SRGAS-OH

H-SAGDS-OH

140 (+/–28%)

250 (+/–35%)

710 (+/–18%)

Ͼ1000

Table 2. Dissociation constants (K_d) determined by fluorescence ti-

tration experiments in different environments for complexes be-

tween 3 and RGD peptides.

Table 4. On the left, affinities of model compound 1 (monophos-

phate tweezers) for protected and free arginine and lysine deriva-

tives displayed as dissociation constants (K_d). Fluorescence ti-

trations were carried out in phosphate buffer (10 mm) at pH = 7.4.

On the right, affinities of model compound 1 (monophosphate

tweezer) for linear and cyclic RGD peptides displayed as K_dvalues.

Fluorescence titrations were carried out in BisTris buffer (10 mm)

at pH = 6.0.

Amino acid

K_d[μm]

RGD peptide

K_d[μm]

H-Arg-OH^[a]

H-Arg-OH

H-Arg-OMe

Ac-Arg-OH

300 (+/–10%)

130 (+/–14%)

15 (+/–05%)

90 (+/–06%)

H-RGD-OH

H-GRGDTP-OH

H-SRGDS-OH

H-SRGAS-OH

H-SAGDS-OH

cRGDfV

130 (+/–18%)

120 (+/–23%)

240 (+/–15%)

150 (+/–10%)

Ͼ1000

20 (+/–13%)

15 (+/–15%)

30 (+/–60%)

30 (+/–32%)

Ac-Arg-OMe 110 (+/–18%)

H-Lys-OH

H-Lys-OMe

Ac-Lys-OH

150 (+/–09%)

15 (+/–21%)

50 (+/–06%)

cRGDfV^[b]

cRGDFPA^[b]

cGRGDfL^[b]

Ac-Lys-OMe 260 (+/–23%)

[a] BisTris buffer (10 mm, pH = 6.0). [b] Phosphate buffer (10 mm,

pH = 7.4). Because the phosphate group (OPO₃2Na⁺) of the

2–

phosphate tweezers reacts in slightly basic and in pure water (pH

≈ 8), these compounds are partially protonated in the buffered

aqueous solutions.

RGD peptide

Solvent

K_d[μm]

H-GRGDTP-OH

GRGDSPA

3-aminomethyl-benzoyl-cARGD DMSO

DMSO

30 (+/–40%)

60 (+/–28%)

150 (+/–15%)

buffer/DMSO^[a]

Unfortunately, cRGDfV and all of the other RGD cyclo-

peptides with aromatic moieties turned out to be suffi-

[a] 10 mm BisTris buffer (pH = 6.0) + 50% DMSO.

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ciently fluorescent under the conditions of tweezer exci-

Obviously, similar effects are operating in our case, and

tation (excitation at 280 nm, emission at 336 nm), which in- these may allow for a fine-tuning of the linker moieties with

terfered with the observed modest fluorescence change respect to the open or closed cavities. Stability considera-

upon complex formation of the new tweezers. Thus, only tions guided us to construct ether, and not ester bridges, on

one nonaromatic RGD cyclopeptide that could be subjected the central hydroquinone. In alternative synthetic routes,

to fluorescence titrations was available. It did not show a the latter turned out to be hydrolytically labile both in alka-

higher affinity towards 2 than its linear counterparts. How- line as well as in acidic solution.

ever, it should be noted that the ditopic receptor molecules

Monophosphate tweezer 1 structurally represents an in-

2 and 3 had been optimized for cRGDfV recognition. Al- termediate stage between the symmetrical diphosphate

though the RGD affinities of 2 already surpassed those of tweezer 1a and the above-discussed ditopic tweezers and,

our best earlier bisphosphonate-based receptor molecule by thus, allows comparison with both. To better understand

more than one order of magnitude,^[13]their absolute value the factors contributing to the unexpected binding mode of

was disappointing and did not agree with the expected re- 2–4, we decided to characterize its recognition profile rela-

cognition mode. Even noncooperative binding of the argi- tive to that of both tweezer types. To this end, 1 was titrated

nine side chain to the tweezer and the aspartate to guanid- with various arginine and lysine derivatives (comparison

iniocarbonylpyrrole unit should have resulted in the sum of with 1a, Table 4, left)^[10,29]and in a second series with vari-

the ΔG values and furnished dissociation constants in the ous RGD peptides (comparison to 2–4, Table 4, right).

high nanomolar range.

For the first series, neutral aqueous buffer was employed

For more structural information, NMR spectroscopic (10 mm phosphate buffer, pH = 7.4). Surprisingly, 1 seems

data was sought for the 1:1 complexes between 2 and the to bind comparable arginine and lysine derivatives equally

various RGD cyclopeptides. Although considerable changes strong, depending mostly on the number of cationic groups.

within the spectra were found in the vicinity of the aspartate Free basic amino acids as well as their N/C-protected deriv-

side chain (up to 0.32 ppm), the shift of the ¹H NMR signal atives, both carrying a net positive charge (at the side

that was induced by the presence of tweezers 2 and assigned chain), display affinities around 100–200 μm (K_d). For

to the arginine side chain (δ_CH2) never surpassed 0.11 ppm, amino acid esters with a free N-terminus, additional Cou-

ruling out its inclusion inside of the tweezer cavity. Similar lomb attractions become possible and increase the affinities

observations were made with complexes between 3 or 4 and by one order of magnitude (K_d≈ 15 μm). The K_dvalue de-

the RGD cyclopeptides. The observations indicate that the termined for the complex of Ac-Lys-OMe with monophos-

guest aspartate carboxylate is still surrounded by the host phate tweezer 1 (260 μm), however, is significantly larger

guanidiniopyrrole unit. Thus, multiple corresponding ionic than that of the symmetrical diphosphate tweezer (17 μm).

hydrogen bonds strongly contribute to the stability of the A similar, but smaller difference, was observed for the corre-

complex. On the other hand, the arginine side chain is not sponding complexes of the parent lysine, H-Lys-OH, (K_d=

threaded through the aromatic cavity of the tweezers, but 150 μm vs 40 μm).^[10]Evidently, the second phosphate group

most likely bound by a direct ion-pair formation between in the diphosphate tweezer strengthens the binding to the

the guanidinium cation and the tweezers phosphate anion. amino acids. The increased electrostatic host-guest interac-

At this point, the originally assumed complex structures tion is a consequence of the larger negative net charge at

had to be abandoned and were replaced by the ones de- the two phosphate groups and the necessity of the free

picted in Figure 4, which are in agreement with all of the amino acid guest to enter the cavity of monophosphate

experimental observations (vide supra).

tweezer 1 in the correct orientation for optimal intermo-

In complexes with RGD-loop mimicking zwitterionic cy- lecular host-guest ion-pair formation. This is entropically

clopeptides, the ditopic hosts 2–4 most likely avoid the in- less favored than the two possible guest orientations in the

tended rotaxane-like binding mode from the arginine side cavity of the diphosphate tweezer. The finding that the argi-

chain, because the tweezer cavity is sterically blocked.^[27]nine derivatives are almost as strongly bound by the mono-

¹H NMR experiments with simple C/N-protected arginine phosphate tweezer 1 as the corresponding lysine derivatives

confirmed, that even in this case, no inclusion takes place. (see Table 4, left) is remarkable, because the related

1

This is contrary to C/N-protected lysine, in which the H tweezer substituted with two methanephosphonate groups

–

NMR signals of the γ-, δ- and ε-methylene protons undergo [OP(Me)O₂Li⁺] in the central benzene spacer unit binds

large upfield shifts in the presence of ditopic tweezers 2 and lysine derivatives with a high degree of selectivity.^[9]

3 (Δδ Ͼ 2 ppm for ε-CH₂). This behavior, which was pre-

Mixtures of 1 and all of the amino acid derivatives (1:1

viously observed in organic solution, is reminiscent to that mixtures) showed strong upfield shifts of the δ- and ε-meth-

of the tweezers substituted either with alkyl (OCH₂–R) or ylene protons. Without the steric hindrance of the second

acyl groups [OC(=O)–R] at the central benzene spacer unit. arm in 2–4, the basic amino acid derivatives are all included

In this environment, which admittedly differs significantly inside the cavity of 1.

from aqueous solution, even a methoxy substituent points

The transition to RGD peptides was remarkable. Mono-

down toward the clip cavity and blocks the entrance of the phosphate tweezer 1 recognized the arginine residue in the

guest into the cavity, whereas an acyl carbonyl group stands free RGD peptide (H-RGD-OH) with the same efficiency

upright and keeps the cavity open so that inclusion com- as that integrated within a larger penta- or hexapeptide

plexes are formed.^[28]

framework (K_d= 100–200 μm, see Table 4, right). These val-

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Ditopic Arginine-Aspartate Binders Recognize RGD Loops

Figure 5. Calculated energy minimum for the 1:1 complex between 1 and cRGDfV. Note the reinforcing additional hydrogen bonds

(dotted lines; MacroModel 9.0, Amber*, H₂O, Monte-Carlo simulation, no restraints, 5000 steps).

ues are also comparable to those found for free arginine

Currently, we are synthesizing a variety of linkers for un-

and lysine (see Table 4, left). If the arginine in such a pept- symmetrical monophosphate tweezers to identify the opti-

ide was replaced by alanine, all of the affinity to 1 was lost mal spacer moiety, which combines chemical stability and

(H-SRGDS-OH vs. H-SAGDS-OH).^[30]

an open cavity. These will be used for another generation

Finally, 1 was titrated with various RGD cyclopeptides, of RGD receptor molecules, in which the double recogni-

which were developed as mimetics for important RGD pro- tion of the arginine and asparate side chain will hopefully

teins and constitute the best available test objects for their lead to nanomolar binding with high conformational pref-

RGD loops. Intriguingly, elimination of the N- and C-ter- erence for specific RGD loops, a prerequisite for pharma-

minus by cyclization of a linear RGD peptide shifts the af- ceutical relevance. In the future, we will test this new gener-

finities towards 1 into the low micromolar K_drange, with ation of ditopic RGD receptor molecules in antibody-based

1

cRGDfV taking the lead (15 μm). The H NMR spectrum cell assays aimed at optimizing their specific interference

of the 1:1 complex shows drastic upfield shifts only for pro- with RGD protein–integrin interactions.

tons assigned to the arginine side chain (Δδ Ͼ 2 ppm for γ-

CH₂), which indicates its specific inclusion in the cavity of

Experimental Section

1. A Monte-Carlo simulation indicates that after arginine

inclusion, the free (relatively acidic) phenolic OH group is

able to form two additional polar hydrogen bonds to amide

functions of the cyclopeptide without causing any steric

strain (see Figure 5). We tentatively explain the superior af-

General: All chemicals were used as supplied without further purifi-

cation. All reactions were carried out under argon. Solvents were

dried where necessary (according to the standard procedures).

Chemicals were purchased from Sigma Aldrich (München/Ger-

finity of 1 for the RGD cyclopeptides by these additional many), Bachem (Bubendorf/Switzerland), Fluka (Taufkirchen/Ger-

many), Acros Organics (Geel/Belgium), abcr GmbH & Co. KG

(Karlsruhe/Germany) and Novabiochem (Schwalbach/Germany).

hydrogen bonds, with strengths that obviously vary by their

amino acid composition (see Table 4, right).

Synthesis of the Flexible Guanidiniocarbonylpyrrole Phosphate

Tweezer 2

Conclusions

N-Boc-Aminoethoxyethyl Acetoxy Tweezer 9a: Monoacetoxy

tweezer

6 (200 mg, 0.33 mmol), N-Boc-ethoxyethyl tosylate

Monotopic tweezer 1 recognized RGD cyclopeptides

with the same efficiency as the designed ditopic receptor

molecules 2–4. The total loss of the aspartate binding site

can be fully compensated by the restoration of arginine in-

clusion inside the cavity of the tweezers. Moreover, a net-

work of hydrogen bonds renders this simple monophos-

phate tweezer selective for RGD cyclopeptides over their

linear counterparts.

(137 mg, 0.38 mmol), K₂CO₃(55.0 mg, 0.40 mmol), KI (catalytic

amount), and 18-crown-6 (catalytic amount) were dissolved in ab-

solute acetone (20 mL), and the resulting mixture was heated at

60 °C for 4 d. The solid precipitate was removed by filtration, and

the filtrate was concentrated under reduced pressure. The resulting

residue was purified by flash chromatography (cyclohexane/ethyl

acetate, 3:1) to afford product 9a (236 mg, 296 μmol, 90%) as a

white solid, m.p. Ͼ95 °C (decomposition).

Disappointingly, although its initial features were com-

plementary towards RGD loops in molecular simulations,

the elaborate design of the ditopic unsymmetrical molecular

tweezers did not produce the desired binding mode, because

none of the arginine side chains were included in the

tweezer cavity. It remains to be seen if steric reasons or per-

haps the suboptimal design of the spacer (with respect to

length, flexibility, solvation or polarity) can account for this

(blocking of the cavity entrance).

¹H NMR (500 MHz, CDCl₃): δ = 1.48 (s, 9 H, 34-H, 35-H, 36-H),

2.33 (dt, 2 H, 24-Ha, 25-Ha), 2.35 (s, 3 H, 28-H), 2.40–2.44 (m, 6

H, 23-Hi, 23-Ha, 26-Hi, 26-Ha, 24-Hi, 25-Hi), 3.33 (t, 2 H, 32-H),

3

3.51 (t, 2 H, 31-H), 3.606 (t, J_30-H,29-H= 5.07 Hz, 2 H, 30-H),

3.94–3.97 (m, 4 H, 29-H, 16-H, 22-H), 4.07 (t, 4 H, 5-H, 7-H, 9-

H, 11-H), 4.28 (d, 2 H, 18-H, 20-H), 6.735–6.773 (m, 4 H, 2-H, 3-

H, 13-H, 14-H), 7.06–7.08 (m, 4 H, 4-H, 12-H, 1-H, 15-H), 7.11 (s,

2 H, 17-H, 21-H), 7.15 (s, 2 H, 6-H, 10-H) ppm. ¹³C NMR

(125.7 MHz, CDCl₃): δ = 21.0 (C-28), 28.7 (C-34, C-35, C-36), 40.6

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H, 14-H), 7.07–7.09 (m, 2 H, 4-H, 12-H), 7.09–7.12 (m, 4 H, 1-H,

15-H, 17-H, 21-H), 7.15 (s, 2 H, 6-H, 10-H), 7.78 (br. s, 3 H,

NH) ppm. ¹³C NMR (125.7 MHz, [D₆]DMSO): δ = 20.5 (C-28),

38.6 (C-32), 47.4, 47.9 (C-18, C-20, C-7, C-9), 50.3 (C-16, C-22, C-

5, C-11), 66.9, 67.9, 68.9 (C-23, C-24, C-25, C-26), 69.6 (C-31), 72.1

(C-30), 72.9 (C-29), 116.3 (C-17, C-21), 116.5 (C-6, C-10), 121.5 (d,

C-4, C-12, C-1, C-15), 124.5 (C-3, C-13, C-2, C14), 134.2 (CF₃)

138.9, 141.1, 145.4 (C-5a, C-10a, C-16a, C-21a, C-7a, C-8a, C-18a,

C-19a), 146.3, 146.6, 147.2, 147.4 (C-6a, C-9a, C-17a, C-20a, C-8,

C-19), 150.2 (d, C-4a, C-11a, C-15a, C-22a), 157.9 (F₃COO), 168.9

(C-27) ppm. ¹⁹F NMR (282.4 MHz, [D₆]DMSO): δ = –73.81 ppm.

HRMS (ESI+, MeOH): calcd. for [M + H]⁺696.3119; found

696.3171.

Guanidiniocarbonylpyrrole Acetoxy Tweezer 11a: Compound 10a

(70.0 mg, 86.4 μmol), 5-(N-Cbz-guanidiniocarbonyl)-1H-pyrrole-2-

carboxylate (33.1 mg, 100 μmol), and HCTU (42.4 mg, 100 μmol)

were dissolved in absolute DMF (10 mL) under argon. After stir-

ring for 10 min, NMM (34.10 μL, 1.00 mmol) was added, and the

resulting mixture was heated at 40 °C for 48 h. The solvent was

evaporated, and the remaining yellow oil was purified by flash

chromatography (cyclohexane/ethyl acetate, 1:5) to afford product

11a (68.9 mg, 68.3 μmol, 79%) as a white solid, m.p. Ͼ200 °C (de-

composition).

(C-32), 48.6, 48.8 (C-18, C-20, C-7, C9), 51.4 (C-16, C-22, C-5, C-

11), 69.2 (C-23, C-24, C-25, C-26), 70.2 (C-31), 70.3 (C-30), 70.4

(C-29), 79.4 (C-33), 116.1 (C-17, C-21), 116.9 (C-6, C-10), 121.5

(C-4, C-12), 121.7 (C-1, C-15), 124.7 (C-3, C-13), 124.9 (C-2, C-

14), 136.6, 140.6, 141.1, 145.95 (C-5a, C-10a, C-16a, C-21a, C-7a,

C-8a, C-18a, C-19a), 146.8, 147.0, 147.7, 147.8 (C-6a, C-9a, C-17a,

C-20a, C-8, C-19), 150.4, 150.5 (C-4a, C-11a, C-15a, C-22a), 156.1

(NH-CO), 169.2 (C-27) ppm. HRMS (ESI+, MeOH): calcd. for [M

+ Na]⁺818.3550; found 818.3452.

Aminoethoxyethyl Acetoxy Tweezer 10a: Compound 9a (150 mg,

0.19 mmol) was dissolved in dichloromethane (DCM)/TFA (9:1,

10 mL), and the resulting mixture was stirred for 2 h at 0 °C. The

solvent was evaporated, and the residue was coevaporated with

benzene (3ϫ). After drying in vacuo, product 10a was obtained as

a white solid (quant.), m.p. Ͼ140 °C (decomposition).

¹H NMR (500 MHz, [D₆]DMSO): δ = 2.17–2.19 (m, 2 H, 24-Ha,

25-Ha), 2.22–2.29 (m, 6 H, 24-Hi, 25-Hi, 23-Ha, 23-Hi, 26-Ha, 26-

3

Hi), 2.35 (s, 3 H, 28-H), 3.46 (t, J_32-H,31-H= 5.07 Hz, 2 H, 32-H),

3

3.57 (t, J_31-H,32-H= 5.07 Hz, 2 H, 31-H), 3.61 (t, J_30-H,29-H

=

5.07 Hz, 2 H, 30-H), 3.97 (d, 2 H, 16-H, 22-H), 4.06 (d, 4 H, 5-H,

7-H, 9-H, 11-H), 4.11 (t, ³J_29-H,30-H= 5.07 Hz, 2 H, 29-H), 4.25 (d,

2 H, 18-H, 20-H), 5.13 (s, 2 H, 37-H), 6.69–6.77 (m, 4 H, 2-H, 3-

H, 13-H, 14-H), 6.88 (s, 1 H, 35-H), 7.019 (d, 2 H, 4-H, 12-H),

7.06–7.09 (m, 7 H, 1-H, 15-H, 17-H, 21-H, 6-H, 10-H, 34-H), 7.31–

7.39 (m, 5 H, 39-H, 40-H, 41-H, 42-H, 43-H), 8.58 (s, 1 H, NHCO),

8.80 (br. s, 1 H, NH), 9.34 (br. s, 1 H, NH), 11.15 (br. s, 1 H, NH),

12.33 (br. s, 1 H, NH) ppm. ¹³C NMR (125.7 MHz, [D₆]DMSO):

δ = 20.5 (C-28), 38.9 (C-32), 47.5, 47.8 (C-18, C-20, C-7, C-9), 50.3

(d, C-16, C-22, C-5, C-11), 67.9 (C-23, C-24, C-25, C-26), 68.9 (C-

31), 69.4 (d, C-30, C-37), 72.6 (C-29), 111.9 (C-35), 116.2 (C-17,

C-21, C-34), 116.5 (C-6, C-10), 121.5 (d, C-4, C-12, C-1, C-15),

124.4 (C-3, C-13, C-2, C-14), 127.6, 128.4 (C-39, C-40, C-41, C-42,

C-43), 134.3 (C-33, C-36), 135.9 (C-38), 139.5, 141.0, 145.2 (C-5a,

C-10a, C-16a, C-21a, C-7a, C-8a, C-18a, C-19a), 146.2, 146.6,

147.2, 147.3 (C-6a, C-9a, C-17a, C-20a, C-8, C-19), 150.1 (C-4a,

C-11a, C-15a, C-22a), 164.2 (NHCO), 168.8 (NHCO), 169.0 (C-

27) ppm. HRMS (ESI+, MeOH): calcd. for [M + H]⁺1008.3967;

found 1008.4035; calcd. for [M + Na]⁺1030.3786; found 1030.3842.

3

¹H NMR (500 MHz, CD₃OD): δ = –0.82 (t, J_32-H,31-H= 5.07 Hz,

3

2 H, 32-H), 1.55 (t, J_31-H,32-H= 5.07 Hz, 31-H), 2.35–2.40 (m, 4

H, 24-Ha, 25-Ha, 24-Hi, 25-Hi), 2.43–2.44 (m, 7 H, 28-H, 23-Ha,

23-Hi, 26-Ha, 26-Hi), 3.39 (t, J_30-H,29-H= 5.07 Hz, 2 H, 30-H),

4.09 (d, 2 H, 16-H, 22-H), 4.15 (d, 4 H, 1-H, 5-H, 7-H, 9-H, 11-

H), 4.31 (t, J_29-H,30-H= 5.07 Hz, 2 H, 29-H), 4.37 (d, 2 H, 18-H,

20-H), 6.88–6.96 (m, 4 H, 2-H, 3-H, 13-H, 14-H), 7.12 (s, 2 H, 4-

H, 12-H), 7.14 (d, 2 H, 1-H, 15-H), 7.18 (d, 2 H, 17-H, 21-H),

7.316 (s, 2 H, 6-H, 10-H) ppm. H NMR (500 MHz, [D₆]DMSO):

δ = 2.21–2.34 (m, 8 H, 24-Ha, 25-Ha, 24-Hi, 25-Hi, 23-Ha, 23-Hi,

3

1

3

26-Ha, 26-Hi), 2.36 (s, 3 H, 28-H), 2.99 (t, J_32-H,31-H= 5.07 Hz, 2

H, 32-H), 3.63 (t, J_31-H,32-H= 5.07 Hz, 2 H, 31-H), 3.69 (t,

3

³J_30-H,29-H= 5.07 Hz, 2 H, 30-H), 4.01 (d, 2 H, 16-H, 22-H), 4.11 Guanidiniocarbonylpyrrole Hydroxy Tweezer 12a: To a strongly

(t, 4 H, 5-H, 7-H, 9-H, 11-H), 4.17 (t, J_29-H,30-H= 5.07 Hz, 2 H, stirred solution of 11a (50.0 mg, 49.6 μmol) in dioxane (10 mL) was

3

29-H), 4.29 (d, 2 H, 18-H, 20-H), 6.75–6.79 (m, 4 H, 2-H, 3-H, 13- added NaOH (1 m solution, 450 μL) dropwise, and the resulting

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Ditopic Arginine-Aspartate Binders Recognize RGD Loops

mixture was stirred for an additional 16 h at room temp. The reac-

tion mixture was poured into a mixture of NH₄Cl solution/half-

concentrated HCl solution (1:1, 40 mL) and then extracted with

dichloromethane (3ϫ). The combined organic layers were washed

with water (3ϫ) and dried with Na₂SO₄. Concentration under re-

duced pressure afforded product 12a (45.0 mg, 46.6 μmol, 94%) as

a slightly yellow solid, m.p. 178 °C.

3

(s, 2 H, 16-H, 22-H), 4.08 (s, 2 H, 5-H, 11-H), 4.12 (t, J_29-H,30-H

= 5.0 Hz, 2 H, 29-H), 4.22 (s, 2 H, 9-H, 7-H), 4.25 (s, 2 H, 18-H,

20-H), 5.13 (s, 2 H, 37-H), 6.70–6.77 (m, 4 H, 2-H, 3-H, 13-H, 14-

H), 6.89 (s, 1 H, 35-H), 7.01–7.07 (m, 6 H, 4-H, 12-H, 1-H, 15-H,

17-H, 21-H), 7.14 (6-H, 10-H), 7.31–7.39 (m, 6 H, 34-H, 39-H, 40-

H, 41-H, 42-H, 43-H), 8.58 (s, 1 H, NHCO), 9.11 (br. s, 1 H, NH),

11.10 (br. s, 1 H, NH), 12.41 (br. s, 1 H, NH) ppm. ¹³C NMR

(125.7 MHz, CD₃OD): δ = 38.9 (C-32), 47.6, 47.8 (C-18, C-20, C-

7, C-9), 50.2 (d, C-16, C-22, C-5, C-11), 54.8 (d, C-27, C-28), 59.8

(C-37), 67.7, 68.6 (C-23, C-24, C-25, C-26), 69.3 (d, C-30, C-31),

72.6 (C-29), 111.7 (C-35), 116.2, 116.8 (C-17, C-21, C-6, C-10, C-

34), 121.1, 121.5 (C-4, C-12, C-1, C-15), 124.4 (d, C-3, C-13, C-2,

C-14), 127.6, 128.4 (C-39, C-40, C-41, C-42, C-43), 132.9 (C-33, C-

36), 140.2, 140.7 (C-5a, C-10a, C-16a, C-21a, C-7a, C-8a, C-18a,

C-19a), 144.8 (C-38), 146.3, 146.4, 147.2 (d, C-6a, C-9a, C-17a, C-

20a, C-8, C-19), 150.2 (d, C-4a, C-11a, C-15a, C-22a), 155.2

(NHCO), 159.6 (NHCO) ppm. ³¹P NMR (202 MHz, CD₃OD): δ =

–3.82 ppm. HRMS (ESI+, MeOH): calcd. for [M + H]⁺1074.3838;

found 1074.3957; calcd. for [M + Na]⁺1096.3657; found 1096.3779.

¹H NMR (500 MHz, [D₆]DMSO): δ = 2.15 (d, 2 H, 24-Ha, 25-

Ha), 2.22 (d, 2 H, 24-Hi, 25-Hi), 2.27 (s, 4 H, 23-Ha, 23-Hi, 26-

Ha, 26-Hi), 3.46–3.49 (m, 6 H, 30-H, 29-H, 28-H), 3.97–3.99 (m,

4 H, 16-H, 22-H, 27-H), 4.05 (s, 2 H, 5-H, 11-H), 4.14 (d, 2 H, 7-

H, 9-H), 4.19 (d, 2 H, 18-H, 20-H), 5.13 (s, 2 H, 35-H), 6.74–6.77

(m, 4 H, 2-H, 3-H, 13-H, 14-H), 6.89 (s, 1 H, 33-H), 6.99–7.07 (m,

9 H, 32-H, 4-H, 12-H, 1-H, 15-H, 17-H, 21-H, 6-H, 10-H), 7.32–

7.38 (m, 5 H, 37-H, 38-H, 39-H, 40-H, 41-H), 8.59 (s, 1 H, NHCO),

8.84 (br. s, 1 H, NH), 9.31 (br. s, 1 H, NH), 11.17 (br. s, 1 H, NH),

12.38 (br. s, 1 H, NH) ppm. ¹³C NMR (125.7 MHz, [D₆]DMSO):

δ = 34.4 (C-30), 46.7, 47.5 (C-18, C-20, C-7, C-9), 50.4 (C-16, C-

22, C-5, C-11), 66.4 (C-35) 67.7 (C-23, C-24, C-25, C-26), 69.1 (C-

29), 69.4 (C-28), 72.6 (C-27), 111.9 (C-33), 115.8 (C-17, C-21, C-

32), 116.4 (C-6, C-10), 121.2, 121.5 (C-4, C-12, C-1, C-15), 124.5

(C-3, C-13, C-2, C-14), 127.6, 128.0, 128.5 (C-37, C-38, C-39, C-

40, C-41), 136.5 (C-31, C-34), 139.2 (C-36), 139.5, 140.75, 141.0

(C-5a, C-10a, C-16a, C-21a, C-7a, C-8a, C-18a, C-19a), 146.7,

146.9, 147.1, 147.2 (C-6a, C-9a, C-17a, C-20a, C-8, C-19), 150.1,

150.4 (C-4a, C-11a, C-15a, C-22a), 164.3 (NHCO), 170.2

(NHCO) ppm. HRMS (ESI+, MeOH): calcd. for [M + H]⁺

966.3861; found 966.3948; calcd. for [M + Na]⁺988.3681; found

988.3752.

Guanidiniopyyrolo Phosphoric Acid Tweezer 14a: Under argon, 13a

(20.0 mg, 0.02 mmol) was dissolved in dichloromethane (10 mL) at

room temp., and bromotrimethylsilane (24.4 μL, 0.18 mmol) was

added through a syringe. The solution was stirred for 6 h at room

temp., and the solvent was removed under reduced pressure. After

drying in vacuo for several hours, the residue was dissolved in a

mixture of DCM/H₂O (1:1), and the two phases were strongly

stirred for 16 h. Evaporation of the solvent afforded product 14a

(quantitative) as a white solid, m.p. Ͼ200 °C (decomposition).

Phosphorylated Guanidiniocarbonylpyrrole Tweezer 13a: Under ar-

gon, 12a (50.0 mg, 51.8 μmol) was dissolved in absolute tetra-

hydrofuran (THF, 20 mL), and the resulting solution was cooled in

an ice bath to 0 °C. To this solution was added phosphorus oxych-

loride (66.0 μL, 0.72 mmol) through a syringe. After stirring for

an additional 10 min, triethylamine (97.0 μL, 0.72 mmol) was also

added through a syringe. The solution was stirred at 0 °C for 2 h.

After adding methanol (10 mL) and triethylamine (291 μL,

2.16 mmol), the solution was stirred at room temp. overnight. The

solution was concentrated under reduced pressure, and ethyl acet-

ate was added. The resulting precipitate was removed by filtration

and washed with ethyl acetate. The solvent of the combined organic

layers was removed under reduced pressure, and the resulting solid

was purified with flash chromatography (cyclohexane/ethyl acetate,

1:4) to afford product 13a (30.0 mg, 28.0 μmol, 54%) as a white

solid, m.p. 150 °C.

¹H NMR (500 MHz, [D₆]DMSO): δ = 2.18 (d, ³J_{24-Ha/24-Hi,25-Ha/25-Hi,}

= 7.3 Hz, 2 H, 24-Ha, 25-Ha), 2.26–2.30 (m, 6 H, 24-Hi, 25-Hi,

3

¹H NMR (500 MHz, CD₃OD): δ = 2.09 (d, J_{24-Ha/24-Hi,25-Ha/25-Hi,}23-H, 26-H), 3.45 (t, J_30-H,29-H= 5.0 Hz, 2 H, 30-H), 3.52–3.57

3

= 7.3 Hz, 2 H, 24-Ha, 25-Ha), 2.26–2.30 (m, 6 H, 24-Hi, 25-Hi,

(m, 4 H, 28-H, 29-H), 3.97 (t, J_27-H,28–H= 5.0 Hz, 2 H, 27-H),

3

23-H, 26-H), 3.47 (t, J_32-H,31-H= 5.0 Hz, 2 H, 32-H), 3.49 (d, 4.04 (s, 2 H, 16-H, 22-H), 4.08 (s, 2 H, 5-H, 11-H), 4.20 (s, 2 H, 7-

3

³J_{27-H/P,28-H/P}= 11.2 Hz, 6 H, 27-H, 28-H), 3.57 (t, J_31-H,32-H

=

H, 9-H), 4.29 (s, 2 H, 18-H, 20-H), 5.14 (s, 2 H, 35-H), 6.70–6.79

(m, 4 H, 2-H, 3-H, 13-H, 14-H), 6.89 (s, 1 H, 33-H), 7.00–7.10 (m,

3

5.0 Hz, 2 H, 31-H), 3.62 (t, J_30-H,29-H= 5.0 Hz, 2 H, 30-H), 4.04

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9 H, 4-H, 12-H, 1-H, 15-H, 17-H, 21-H, 6-H, 10-H, 32-H), 7.31–

NaOH (1.27 mg, 0.02 mmol) dissolved in H₂O (2 mL). The solu-

7.39 (m, 5 H, 37-H, 38-H, 39-H, 40-H, 41-H), 8.58 (s, 1 H, NHCO), tion was stirred for 6 h at room temp. The solvent was evaporated

8.87 (br. s, 1 H, NH), 9.45 (br. s, 1 H, NH), 12.00 (br. s, 1 H, to afford product 2 (quantitative yield) as a slightly yellow solid,

NH) ppm. ¹³C NMR (125.7 MHz, [D₆]DMSO): δ = 38.9 (C-30), m.p. Ͼ240 °C (decomposition).

47.6, 47.9 (C-18, C-20, C-7, C-9), 50.3 (d, C-16, C-22, C-5, C-11),

66.3 (C-35), 67.9, 68.9 (C-23, C-24, C-25, C-26), 69.1, 69.3 (C-28,

C-29), 72.7 (C-27), 112.0 (C-33), 115.8, 117.2 (C-17, C-21, C-6, C-

10, C-32), 121.2, 121.6 (C-4, C-12, C-1, C-15), 124.4 (d, C-3, C-13,

C-2, C-14), 127.6, 127.9, 128.4, (C-37, C-38, C-39, C-40, C-41),

135.3 (C-31, C-34), 140.0, 141.1 (C-5a, C-10a, C-16a, C-21a, C-7a,

C-8a, C-18a, C-19a), 144.2 (C-36), 146.7, 146.8, 147.1 (d, C-6a, C-

9a, C-17a, C-20a, C-8, C-19), 150.2 (d, C-4a, C-11a, C-15a, C-

22a), 159.6 (NHCO) ppm. ³¹P NMR (202 MHz, [D₆]DMSO): δ =

–4.86 ppm. HRMS (ESI+, MeOH): calcd. for [M + H]⁺1046.3525;

found 1046.3777; calcd. for [M – H]^–1044.3439; found 1044.3379.

Phosphorylated Free Guanidiniocarbonylpyrrole Tweezer 15a: Com-

pound 14a (30.0 mg, 287 μmol) was dissolved in MeOH/DCM (1:2,

15 mL), and a catalytic amount of Pd/C (10%) was added. The

suspension was stirred under H₂for 24 h at room temp. The cata-

lyst was removed by filtration, and the filtrate was concentrated

under reduced pressure. The resulting residue was dried in vacuo

to afford the product (quantitative) as a white solid, m.p. Ͼ235 °C

(decomposition).

¹H NMR (500 MHz, [D₆]DMSO): δ = 2.04 (d, 2 H, 24-Ha, 25-

Ha), 2.28 (s, 4 H, 23-H, 26-H), 2.33 (d, 2 H, 24-Hi, 25-Hi), 2.88

(br. s, 2 H, 30-H), 3.12 (br. s, 2 H, 28-H), 3.65 (br. s, 2 H, 29-H),

4.03 (ds, 4 H, 16-H, 22-H, 5-H, 11-H), 4.11 (br. s, 2 H, 7-H, 9-H),

4.18 (s, 2 H, 27-H), 4.53 (br. s, 2 H, 18-H, 20-H), 6.45 (d, 1 H, 33-

H), 6.53 (br. s, 1 H, 32-H), 6.73–6.78 (m, 4 H, 2-H, 3-H, 13-H, 14-

H), 6.99–7.10 (m, 8 H, 4-H, 12-H, 1-H, 15-H, 17-H, 21-H, 6-H,

10-H), 8.60 (br. s, 1 H, NH), 12.18 (br. s, 1 H, NH) ppm. ¹H NMR

3

(300 MHz, CD₃OD): δ = 2.20 (d, J_{24-Ha/24-Hi,25-Ha/25-Hi}= 7.3 Hz,

2

H, 24-Ha, 25-Ha), 2.28 (s, 4 H, 23-H, 26-H), 2.48 (d,

³J_{24-Hi/24-Ha,25-Hi/25-Hi}= 7.3 Hz, 2 H, 24-Hi, 25-Hi), 3.62–3.74 (m, 6

H, 28-H, 29-H, 30-H), 3.85 (m, 2 H, 27-H), 3.97 (ds, 4 H, 16-H,

22-H, 5-H, 11-H), 4.16 (s, 2 H, 7-H, 9-H), 4.73 (s, 2 H, 18-H, 20-

H), 6.74–6.79 (m, 5 H, 2-H, 3-H, 13-H, 14-H, 33-H), 6.86 (d,

³J_32-H,33-H= 4.0 Hz, 1 H, 32-H) 6.98–7.03 (m, 6 H, 4-H, 12-H, 1-

H, 15-H, 17-H, 21-H), 7.15 (s, 2 H, 6-H, 10-H) ppm. ¹H NMR

3

(300 MHz, D₂O): δ = 2.06 (d, J_{24-Ha/24-Hi,25-Ha/25-Hi}= 7.3 Hz,

¹H NMR (500 MHz, [D₆]DMSO): δ = 2.05 (d, ³J_{24-Ha/24-Hi,25-Ha/25-Hi,}

2

H, 24-Ha, 25-Ha), 2.11 (s, 4 H, 23-H, 26-H), 2.24 (d,

³J_{24-Hi/24-Ha,25-Hi/25-Hi}= 7.3 Hz, 2 H, 24-Hi, 25-Hi), 3.34 (s, 4 H, 28-

H, 30-H), 3.41 (s, 2 H, 29-H), 3.62 (s, 2 H, 27-H), 3.80 (s, 2 H, 16-

H, 22-H), 3.91 (s, 2 H, 5-H, 11-H), 4.00 (s, 2 H, 7-H, 9-H), 4.35 (s,

2 H, 18-H, 20-H), 6.24 (br. s, 4 H, 2-H, 3-H, 13-H, 14-H), 6.62 (s,

2 H, 17-H, 21-H), 6.74–6.82 (s, 6 H, 4-H, 12-H, 1-H, 15-H, 33-H,

32-H), 7.08 (s, 2 H, 6-H, 10-H) ppm. ¹³C NMR (125.7 MHz,

[D₆]DMSO): δ = 38.7 (C-30), 47.8 (C-18, C-20, C-7, C-9), 50.3 (C-

16, C-22, C-5, C-11), 66.0, 67.2 (C-23, C-24, C-25, C-26), 68.1 (C-

28, C-29), 69.5 (C-27), 111.6 (C-33), 116.1 (C-17, C-21, C-6, C-10,

C-32), 121.1 (d, C-4, C-12, C-1, C-15), 124.2 (d, C-3, C-13, C-2,

C-14), 134.5 (C-31, C-34), 139.1, 141.2 (C-5a, C-10a, C-16a, C-21a,

C-7a, C-8a, C-18a, C-19a), 146.6 (d), 147.0, 147.6 (C-6a, C-9a, C-

17a, C-20a, C-8, C-19), 150.4 (d, C-4a, C-11a, C-15a, C-22a) ppm.

³¹P NMR (202 MHz, [D₆]DMSO): δ = –4.79 ppm. ³¹P NMR

(121 MHz, CD₃OD): δ = –3.39 ppm. ³¹P NMR (121 MHz, D₂O):

3

= 7.3 Hz, 2 H, 24-Ha, 25-Ha), 2.16 (d, J_{24-Hi/24-Ha,25-Hi/25-Ha,}

=

7.3 Hz, 2 H, 24-Hi, 25-Hi), 2.27 (t, 4 H, 23-H, 26-H), 3.17 (t, 2 H,

30-H), 3.47 (t, 2 H, 28-H), 3.64 (t, 2 H, 29-H), 4.02 (s, 2 H, 16-H,

22-H), 4.05 (s, 2 H, 5-H, 11-H), 4.09 (s, 4 H, 27-H, 7-H, 9-H), 4.35

(s, 2 H, 18-H, 20-H), 6.71–6.79 (m, 5 H, 33-H, 2-H, 3-H, 13-H, 14-

H), 6.99–7.10 (m, 9 H, 4-H, 12-H, 1-H, 15-H, 17-H, 21-H, 6-H,

10-H, 32-H), 8.12 (s, 2 H, NHCO), 9.41 (br. s, 1 H, NH), 9.45 (br.

s, 1 H, NH), 12.88 (br. s, 1 H, NH) ppm. ¹³C NMR (125.7 MHz,

[D₆]DMSO): δ = 38.8 (C-30), 47.8, 48.5 (C-18, C-20, C-7, C-9),

50.3 (C-16, C-22, C-5, C-11), 67.5, 68.1 (C-23, C-24, C-25, C-26),

68.7 (C-28, C-29), 71.8 (C-27), 111.5 (C-33), 116.0, 116.3 (C-17, C-

21, C-6, C-10, C-32), 121.4 (d, C-4, C-12, C-1, C-15), 124.5 (d, C-

3, C-13, C-2, C14), 136.5 (C-31, C-34), 139.2, 141.1 (C-5a, C-10a,

C-16a, C-21a, C-7a, C-8a, C-18a, C-19a), 146.9 (d), 147.2 (d, C-

6a, C-9a, C-17a, C-20a, C-8, C-19), 150.2 (d, C-4a, C-11a, C-15a,

C-22a), 159.2 (NHCO) ppm. ³¹P NMR (202 MHz, [D₆]DMSO): δ

= –4.52 ppm. HRMS (ESI+, MeOH): calcd. for [M + H]⁺

912.3157; found 912.3596; calcd. for [M + Na]⁺934.2976; found

934.3399

δ = 1.05 ppm. IR [attenuated total reflectance (ATR)]: ν = 3332

˜

(NHCO), 2967, 2932, 2862 (-CH, -CH₂, -CH₃), 1633 (NH), 1588

(C=N), 1453, 1435 (aromatic vibration), 1356, 1303 (O=P–O, P–

O–Ar), 1279 (C–O) cm^–1. HRMS (ESI+, MeOH): calcd. for [M –

Na + H] 933, found 933; calcd. for [M] 955, found 955.

Guanidiniocarbonylpyrrole Phosphate Tweezer with Aminoethylene-

glycol Spacer 2, Na Salt: Compound 15a (10.0 mg, 0.01 mmol) was

dissolved in dioxane (10 mL), and to this solution was added

Self-Association Experiments: Dimerization propensities and puta-

tive intra- or intermolecular self-association tendencies of all un-

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Eur. J. Org. Chem. 2013, 1080–1092

Ditopic Arginine-Aspartate Binders Recognize RGD Loops

Figure 6. Concentration-dependent ¹H NMR spectra of monophosphate tweezer 1 during a 30-fold dilution of a 0.63 mm solution

(aromatic region).

symmetrical phosphate tweezer derivatives were probed with vari-

ous NMR and fluorescence spectroscopic methods. Inter alia,

chemical shift changes of characteristic sensor proton signals were

monitored against varied concentrations. Therefore, dilution of an

aqueous monophosphate tweezer solution produced typical down-

field shifts of the self-included terminal aromatic protons 2, 3, 13,

and 14 inside of the cavity of the partner molecule in the entangled

dimer (Δδ = 0.6 ppm, from 0.63 mm to 0.02 mm, see Figure 6).

¹H NMR Spectroscopic Characterization of the Host-Guest Com-

plex

Tweezer 1 Versus cRGDfV: A 1:1 mixture of tweezer 1 (0.38 mm)

and cRGDfV (0.38 mm) was dissolved in phosphate buffer (10 mm

solution, 0.6 mL) at pH = 7.4, and the ¹H NMR spectroscopic data

were recorded. This spectrum was compared to the ¹H NMR spec-

tra of the pure guest (0.38 mm) and the pure host. In the arginine

side chain, all of the signals from the β-, γ-, and δ-CH₂protons

were markedly shifted upfield. Because of simultaneous line broad-

ening, an unambiguous assignment was impossible, but the γ-CH₂

proton signal shifted at least Ͼ2 ppm. The signals from the tweezer

and all of the other CH protons of the cRGDfV peptide remained

unchanged.

Nonlinear regression analysis of the resulting dimerization iso-

therm furnished a 1:1 dimerization constant of K_dim= 80 m^–1. Be-

cause of the efficient π-stacking interactions of the naphthalene

bridges, this value is considerably lower than that for a related

naphthalene tweezer diphosphonate, which amounts to K_dim

=

2ϫ10⁶m^–1. Thus, it can be concluded that the free energy of dimer-

ization for 1 is negligible compared to that released by lysine and

arginine inclusion.

Tweezer 2/3 Versus Different RGD-Peptides: Mixtures of tweezers

2 or 3 and the RGD peptide guest (1:1 or 2:1) were dissolved in

Fluorescence Titrations

1

the appropriate deuterated solvent (0.6 mL), and a H NMR spec-

1

General Procedure: To keep the receptor concentration constant

over the entire titration, the peptide guest was dissolved in a tweez-

ers parent solution (400 μL). This solution (300 μL) was then ti-

trated in 11 steps to the tweezers parent solution (700 μL). After

each titration step, a new fluorescence spectrum was recorded.

Evaluation of the resulting binding isotherms by a standard nonlin-

ear regression algorithm furnished 1:1 binding constants as indi-

cated below.

troscopic data were recorded. This spectrum was compared to H

NMR spectra of pure guest and pure host. In the arginine side

chain, chemical shift changes remained smaller than 0.11 ppm,

which indicate that no inclusion into the host cavity took place. In

the aspartate side chain, chemical shift changes of up to 0.32 ppm

were observed, which most likely indicate an ion pairing with the

guanidinium ion of the tweezer. Various other peptide CH signals

were also slightly shifted upon the tweezer addition, which indicate

Eur. J. Org. Chem. 2013, 1080–1092

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1091

C. Schmuck, T. Schrader et al.

FULL PAPER

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Table 5. Tweezer 2 versus cRGDfV (0.46 mm/0.46 mm) in MeOD +

10% D₂O at pH = 6.0.

Guest protons

α-Gly

γ-Arg

β-Asp

Δδ [ppm]

0.09

0.11

0.32

Table 6. Tweezer 3 versus cRGDfV (0.43 mm/0.43 mm) in MeOD +

10% D₂O at pH = 6.0.

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Guest protons

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β-Asp

Δδ [ppm]

0.07

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0.21

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[22] The esterification detour was necessary to obtain pure prod-

ucts, and attempts to purify the free acid by preparative HPLC

were unsuccessful.

further complex formation without inclusion. All of the observed

chemical shifts are summarized in Tables 5 and 6.

Molecular Modeling: For the modeling experiments, we used mo-

lecular mechanics calculations of the ditopic tweezer molecules

with manual docking of both binding sites close to their guest moi-

eties in the cyclopeptide (cyclo-RGDfV) and Monte–Carlo simula-

tions followed by molecular dynamics simulations of the resulting

complex structures (MacroModel 9.0, Amber*) and water (GBA

solvation model for water approximation, 5000 steps for MC, 5 ns

for MD at 300 K, no restraints). All of the resulting complex struc-

tures are shown in the manuscript.

Supporting Information (see footnote on the first page of this arti-

cle): Full description of the synthetic routes to ditopic tweezers 3

and 4 as well as example fluorescence titrations.

[23] T. Morita, Y. Okamoto, H. Sakurai, Bull. Chem. Soc. Jpn.

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[24] 2,2-Bis(hydroxymethyl)-2,2Ј,2ЈЈ-nitrilotriethanol, pH range

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Acknowledgments

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[26] Generally, an increase in the DMSO content stabilized the

complexes between 8a and the RGD peptides: 50% DMSO,

0.4 K_d(water); 100% DMSO, 0.1 K_d(water). This behavior is in

line with the reinforced ionic hydrogen bonds when water is

removed.

[27] At this point, we cannot exclude that the reluctance for inser-

tion also stems from the nonoptimal design of the second bind-

ing arm. For clarification, a systematic linker study seems ap-

propriate.

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The authors thank the Deutsche Forschungsgemeinschaft (DFG)

for continued financial support of this project.

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[30] All of these titrations were carried out at pH = 6 to ensure

comparable conditions to subsequent ditopic receptor studies.

Unexpectedly, the replacement of aspartate by alanine in H-

SRGDS-OH does not lower the peptide’s affinity towards 1.

However, one should consider that the receptor molecule can

alternatively form a complex with the C-terminal carboxylate

instead of the aspartate side chain (see related discussion for

Table 2).

Received: August 3, 2012

Published Online: January 7, 2013

1092

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Eur. J. Org. Chem. 2013, 1080–1092

Article Doi

Ditopic arginine-aspartate binders recognize RGD loops

DOI: 10.1002/ejoc.201201052

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