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Circular Dichroism Studies ofCarbopeptoid-Cyclodextrins

ALISON A. EDWARDS,1 GEORGE W.J. FLEET,2 BEN A. MAYES,2

STUART J. HUNTER,2 and GEORGE E. TRANTER1*1Biological Chemistry, Division of Biomedical Sciences, Imperial College, London, United Kingdom

2Chemistry Research Laboratory, Department of Chemistry, University of Oxford,Mansfield Road, Oxford, United Kingdom

ABSTRACT A series of sugar amino acids, based on open chain sugars, have beenoligomerised and cyclised. The resulting cyclic carbopeptoids have been examined fordesirable properties such as host–guest chemistry (as in cyclodextrins) or self-assembling properties (e.g., peptide nanotubes). Initial studies of these systems, bycircular dichroism and X-ray crystallography, have given valuable insight into theirstability and properties. One of the four cyclic species studied was found to interact withion/molecular probes. Chirality 17:S114–S119, 2005. A 2005 Wiley-Liss, Inc.

KEY WORDS: circular dichroism; sugar amino acids; binding; CPCD

Sugar amino acids (SAAs) and carbopeptoids (linearoligomers of SAAs) have been extensively employed aspeptidomimetics, as scaffolds, and as foldamers1 (oligo-mers that adopt compact conformations in relativelyshort sequences).2 – 5 There has been recent interest inthe cyclisation of carbopeptoids to form a novel class ofbiomaterials known as carbopeptoid-cyclodextrins(CPCDs).6 – 10 These novel systems have structural featurescommon to both macrocyclic carbohydrates and cyclicpeptides (Fig. 1).

SAAs have been incorporated into cyclic peptides togenerate biologically active analogues, e.g., somatostat-in11,12 and integrin inhibitors (RGD peptides).13 – 15 Cyclicpeptides have potential applications in nanotube technol-ogy16 – 20 and are commonly utilised as antibiotics21,22 andfor other chemotherapeutic purposes23,24; such roles canbe likewise envisaged for SAA-modified structures.

SAAs have also been prepared as cyclic carbopeptoids(CPCDs), as homo-oligomers using pyranose- or furanose-based SAAs7 – 9 and as hetero-oligomers—often containingproteinogenic amino acids.25 – 27 The biological potential ofthese systems may be vast; Kessler et al. have alreadyidentified a cyclic hexamer (of pyranose-based SAAs)which can form inclusion complexes with benzoic acidakin to the host–guest chemistry of cyclodextrins.7

Cyclodextrins, cyclic sugars of (1!4)-linked a-D-glucopy-ranose units, have widespread use in drugs, foods, andcosmetics.28,29

CPCDs6,10,30 have been examined as part of anextensive project to investigate the structural featuresgoverning properties of both linear and cyclic carbopep-toids. A wide range of spectroscopic techniques (includingcircular dichroism) and crystallography have been utilisedin this study. Many cyclic carbopeptoids have beenexamined for conformational flexibility and tested for

binding with a range of probes. Herein, we report theresults from initial studies of a set of dimeric cyclic carbo-peptoids containing both structural and chiral differ-entiation (Fig. 2).

MATERIALS AND METHODSEquipment

Circular dichroism (CD) spectra were recorded on aJASCO J600 CD spectrometer fitted with a custom builtthermostated cell holder. The sample cell was a quartzSuprasil cylindrical cell with a path length of 1.0 cm.Sample spectra were measured at 293 K (unless statedotherwise) in acetonitrile, methanol, 2,2,2-trifluoroethanol(TFE), or water and at an amide concentration of 100 mM.Amide concentration refers to the concentration of amidelinkages in solution, for example, a 50 mM solution ofcyclic dimer (two amide linkages per molecule) has anamide bond concentration of 100 mM. The following ac-quisition parameters were used: scan speed, 10 nm/min;time constant, 4 sec; spectral bandwidth, 1 nm; datainterval, 0.1 nm; scan range, 260–185 nm. A baselinespectrum was recorded in the same cell at proximal timeand subtracted from the sample spectra. The resultant

Contract grant sponsors: EPSRC; BBSRC; Smith & Nephew; Contractgrant number: GR/S44105/01 (EPSRC)This article includes Supplementary Material available via the Internet athttp://www.interscience.wiley.com/jpages/0899-0042/suppmat*Correspondence to: George E. Tranter, Biological Chemistry, Division ofBiomedical Sciences, Imperial College, London, SW7 2AZ, U.K. E-mail:[email protected] for publication 1 September 2004; Accepted 15 November 2004DOI: 10.1002/chir.20117Published online in Wiley InterScience (www.interscience.wiley.com).

A 2005 Wiley-Liss, Inc.

CHIRALITY 17:S114–S119 (2005)

spectra were normalised for path length and mean amideconcentration to give molar CD spectra.

Chemicals

Solvents were supplied by Romil at Super Purity grade.The probes utilised for binding studies were of suit-able grade (normally analytical) supplied by Aldrich, BDH,and Fluka. The syntheses of compounds 1–4 have beenpreviously reported.10,30 The stereochemistry of thesugars (when present) and the extent of hydroxyl pro-tection are detailed in Figure 2.

Temperature Studies

Temperature studies were conducted over 5 to 85jCfor the dimer species in water at an amide concentra-tion of 100 mM. The experiments were conducted asfollows. A spectrum was recorded of the dimer in aque-ous solution at an ambient temperature (20jC), andthen the sample cooled to 5jC. After allowing the sam-ple to equilibrate, a spectrum was recorded at 5jC.The solution was then heated to 15jC and allowed toequilibrate before another spectrum was recorded. This

procedure was repeated at 25, 35, 45, 55, 65, 75, and 85jC.After the spectrum at 85jC had been recorded, the sam-ple was allowed to cool to 20jC and a final spectrumwas recorded.

RESULTS AND DISCUSSION

A range of cyclic oligomers (dimeric to decameric)have been prepared from galactose in addition tohetero-oligomers of hexanoates and sugars.6,10,30 Herewe report the study of cyclic dimers by CD and crys-tallography. The amide bonds enable CD to be used tostudy the flexibility, stability, and binding features ofthese systems in an analogous fashion to the tech-nique’s application to protein studies.31 However, duecare should be taken during the interpretation of CDspectra of novel systems.32 Crystal structures can giveinformation such as the diameter of the ‘‘empty’’ core ofthe cyclic species and crystal packing that may beuseful for the exploration of molecular interactions.

The crystal structures of two of the dimers discussedhere have been reported: the unprotected galactose dimer2 and galactose–hexanoate dimer 3 (Fig. 3).10,30 Thedistances across the centre of the cyclic dimers (between

Fig. 1. Structural comparison of cyclic carbopeptoids with cyclic peptides and cyclodextrins.

S115CIRCULAR DICHROISM STUDIES OF CPCDS

atoms in the molecule with VDW radii subtracted)indicate that no substantial holes were present to allowconventional host–guest chemistry. No packing analo-gous to ‘‘peptide nanotubes’’ was observed in the crys-talline state.

CD spectra were recorded of each dimer in water,methanol, acetonitrile, and 2,2,2-trifluoroethanol (TFE) toassess solubility and the presence of any solvent effects.No dramatic changes appeared between the spectrawith solvent changes, and unsurprisingly the most sig-nificant change in most cases was found in the waterspectrum (Fig. 4).

On comparison of a spectrum of each dimer in thesame solvent, substantial conformational changes wereobserved (Fig. 5). This could be attributed to the alter-ation of the amide environments as a direct result ofmanipulation of protecting groups and/or degree offunctionalisation of the monomers (see Fig. 2). There-fore, CD is ideal for the study of these systems becausechanges in an amide environment can be directly cor-related to structural manipulations.

In order to examine the stability of such systems, tem-perature studies were conducted in water (see Materialsand Methods). In all cases, it was found that no dramaticconformational change occurred on heating of the dimerspecies, which was not unexpected considering the small(14-member) ring size. The small gradual change inspectra observed with heating was found to be reversibleon cooling to ambient temperature (Fig. 6). This wouldsuggest that the dimers are either very flexible or veryrigid, the latter being most likely.

The binding potential of the cyclic dimers wasexplored by testing several types of probe. The use ofthe word ‘‘probe’’ herein refers to the use of a range ofmetal ions (e.g., Na+, Cu2+, Mg2+), small molecules (e.g.,alanine), hydrophobic molecules (e.g., tyrosine), andhydrophilic molecules (e.g., sucrose) used to investigatethe binding potential of the cyclic dimer species.Aqueous solutions of the cyclic species were preparedwith a 10-fold excess of the probe. Reference spectra ofthe cyclic species and probe were recorded indepen-dently (with the same experimental parameters) and

Fig. 2. Dimeric cyclic carbopeptoids.

Fig. 3. Crystal structures of dimer 2 (A) and dimer 3 (B).

S116 EDWARDS ET AL.

used to generate a theoretical spectrum that would beobserved if no interaction had taken place between thecyclic species and probe. This ‘‘calculated’’ spectrumwas then compared against the observed spectrum forthe solution of the cyclic species with a 10-fold excessof probe to determine whether any interaction had

occurred (Fig. 7). Of the four dimers studied, onlydimer 2 exhibited differences between the observedand calculated spectra (for 6 of the 8 probes studied).Work is ongoing to establish the stoichiometry andnature of this interaction. The absence of a reasonable‘‘hole’’ in the crystal structure of dimer 2 and initial

Fig. 4. Spectra of dimer 1 in four different solvents.

Fig. 5. CD spectra of dimers 1–4 in water.


titration studies suggest that the interaction is not theresult of conventional host–guest chemistry.

CONCLUSION

CD and crystallography are powerful diagnostic toolsfor the study of CPCDs. One dimeric cyclic carbopeptoid

has already been observed to interact with probes.Further studies are required to establish the observed in-teraction and stoichiometry. Analysis of 1H NMR spec-tra before and after the addition of probe moleculesmay give further indication as to the type of interaction.7

These results have given a basis for the investigation ofrelated systems and future synthesis.

Fig. 6. Temperature experiment of dimer 2 in water.

Fig. 7. Sample spectra that show an interaction.

S118 EDWARDS ET AL.

ACKNOWLEDGMENTS

Thanks to the EPSRC (A.A.E. and S.J.H.), the BBSRC(B.A.M.), and Smith & Nephew (B.A.M.) for funding.

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