6430 Chem. Commun., 2013, 49, 6430--6432 This journal is c The Royal Society of Chemistry 2013

Cite this: Chem. Commun.,2013,49, 6430

N-Triethylene glycol (N-TEG) as a surrogate for theN-methyl group: application to Sansalvamide Apeptide analogs†‡

Ana I. Fernandez-Llamazares,ab Jesus Garcıa,a Vanessa Soto-Cerrato,c

Ricardo Perez-Tomas,c Jan Spengler*ab and Fernando Albericio*abde

Here we studied the N-triethylene glycol (N-TEG) group as a surrogate for

the N-Me group in Sansalvamide A peptide. The five N-TEG and N-Me

analogs of this cyclic pentapeptide were synthesized, and their biological

activity, lipophilicity and conformational features were compared.

The replacement of natural amino acids with N-methyl amino acidsin biologically active peptides has resulted in analogs with improvedpharmacological properties.1,2 Peptides containing N-methyl aminoacids show increased metabolic stability and higher hydrophobicity,which can enhance their bioavailability, thus amplifying theirtherapeutic potential.2 Furthermore, the incorporation of N-methylamino acids into bioactive peptides can have a substantial impacton their conformation and, as a result, increased biological activityand higher receptor selectivity may be achieved.3

Surprisingly, little attention has been devoted to the comparisonof the synthesis and properties of N-methylated peptides with otherN-alkylated peptides.4 We hypothesized that modification of a peptidewith an N-triethylene glycol (N-TEG) group would be comparable tomodification with the N-Me group in terms of structural andbiological effects, while providing some of the features associatedwith oligoethylene glycol (OEG). To test this notion, we sought toincorporate N-TEG amino acids at the different positions of Sansal-vamide A peptide (1), a cyclic pentapeptide that exhibits anti-tumoractivity against a variety of cancer cell lines (Fig. 1).5,6 This cyclic

peptide was considered a reasonable model, since its five N-Meanalogs (1b–5b) have been synthesized, and some of them arecytotoxic against certain cancer cell lines.6 Replacement of an N-Megroup with an N-TEG group does not alter the amide proton patternof the cyclic backbone with respect to the N-Me analogs; however, dueto the amphiphilic nature of OEG, an alteration of certain physico-chemical properties, such as hydrophilicity, was expected.

Here we report on the synthesis of all five N-TEG analogs (1a–5a) ofSansalvamide A peptide (1) (Fig. 1). The five N-Me analogs (1b–5b) werealso synthesized, and both sets of compounds were compared withrespect to biological activity, lipophilicity and conformational features.

Our synthetic strategy to prepare the N-TEG and N-Me analogs ofSansalvamide A peptide (1a–5a and 1b–5b) involved the use ofFmoc-protected N-TEG or N-Me amino acids as solid-phase building

Fig. 1 (A) Structure of Sansalvamide A peptide (1). (B) Structure of the N-TEGand N-Me analogs (1a–5a and 1b–5b).

a Institute for Research in Biomedicine (IRB), Barcelona Science Park (PCB), Baldiri

Reixac 10, 08028 Barcelona, Spain. E-mail: albericio@irbbarcelona.org,

jan.spengler@irbbarcelona.orgb CIBER-BBN, Networking Centre on Bioengineering, Biomaterials and

Nanomedicine, PCB, Baldiri Reixac 10, 08028 Barcelona, Spainc Department of Pathology and Experimental Therapeutics, University of Barcelona,

Pavello Central, LR 5101 C/Feixa Llarga s/n, E-08907 L’Hospitalet, Spaind Department of Organic Chemistry, University of Barcelona, Martı i Franques 1-11,

08028 Barcelona, Spaine School of Chemistry & Physics, University of KwaZulua-Natal, 4001 Durban, South

Africa

† To Professor Klaus Burger, a mentor and a friend, on occasion of his 75thanniversary.‡ Electronic supplementary information (ESI) available: Experimental details,characterization data, and copies of the HPLC, 1H-NMR and 13C-NMR spectraof selected compounds. See DOI: 10.1039/c3cc41788c

Received 9th March 2013,Accepted 24th May 2013

DOI: 10.1039/c3cc41788c

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This journal is c The Royal Society of Chemistry 2013 Chem. Commun., 2013, 49, 6430--6432 6431

blocks. All Fmoc-N-Me analogs of the proteinogenic amino acids arecommercially available. In contrast, we had to prepare the requiredFmoc-N-TEG amino acids. The Fmoc-N-TEG analogs of Val, Leu andPhe (6–8) were obtained from their corresponding amino acid tert-butyl esters, which were subjected to reductive alkylation with 3,6,9-trioxadecanaldehyde. In all cases, the reaction crude consisted ofunreacted starting material, N-monoalkylated product and N,N-dialkylated product. The last two were found to be inseparable;however, after Fmoc-protection of the amino group, the desiredproducts (6–8) could be isolated using flash chromatography.Finally, acidic cleavage of the tert-butyl ester yielded the requiredFmoc-N-TEG derivatives (9–11) in 40–69% overall yield (Scheme 1).

For the synthesis of the N-TEG and N-Me analogs of SansalvamideA peptide (1a–5a and 1b–5b), their corresponding linear precursors(p1a–p5a and p1b–p5b) were prepared by stepwise solid-phasepeptide synthesis (SPPS) on the 2-chlorotrityl resin and then cyclizedin solution (Scheme 2). The N-substituted residue was placed inthe middle of the pentapeptide sequence, which minimizes sterichindrance during cyclization and is expected to facilitate this processdue to the turn-inducing properties of N-alkyl amino acids.7

In the SPPS of the pentapeptides (p1a–p5a and p1b–p5b), Fmoc-N-TEG amino acids (9–11) and Fmoc-N-Me amino acids werecoupled to the peptidyl-resin using DIPCDI–OxymaPure activation.As expected, couplings onto the N-TEG residues were a challengingstep. These couplings are hampered by the triethylene glycol chain,which is sterically more demanding than a methyl group. Even forthe couplings onto N-Me residues, special conditions are required toovercome low coupling yields caused by steric hindrance.3 Wechecked several protocols that are reported to be efficient for difficultcoupling steps, like activation of the subsequent amino acid withHATU or as a symmetrical anhydride using DIPCDI. The best resultswere obtained using bis(trichloromethyl)carbonate (BTC) as anactivating reagent.8 In the case of BTC-mediated couplings ontothe N-Me residues, no unreacted peptide was detected in cleavedsamples after a single treatment with the activated amino acid (aschecked using HPLC-MS). For couplings onto the N-TEG residues,complete conversion was achieved after two treatments.

After cleavage from the solid support, the crude pentapeptides(p1a–p5a and p1b–p5b) were efficiently cyclized, and the desired cyclic

peptides (1a–5a and 1b–5b) were easily purified using semi-preparativeRP-HPLC. In all cases, sufficient amounts of peptide were obtained inyields that are commonly achieved for cyclic pentapeptides. Except forcompound 3a, the overall yields of the syntheses of the N-TEGcyclopeptides were not dramatically lower than those of the N-Mecyclopeptides. Thus, N-TEG peptides are accessible by the samesynthetic repertoire as that already established for N-Me peptides.

In order to study how replacement of the N-Me group by theN-TEG group affects the anti-cancer activities, all compounds(1, 1a–5a and 1b–5b) were tested for their cytotoxicity against GLC-4and MDA-MB-231 cancer cells. Treatment with 50 mM of somecompounds for 72 h decreased the viability of GLC-4 (1, 1a, 2a, 4a,2b, 4b, 5b) and MDA-MB-231 (1a, 4a, 3b) cells up to 50–60% (Fig. 2).The cytotoxic activity of the N-TEG analogs (1a–5a) was found to bewithin the same range as that of the N-Me analogs (1b–5b).

The incorporation of N-TEG or N-Me was expected to affect thelipophilicity of the original peptide (1), which is highly hydrophobic.One of the most common parameters to estimate lipophilicity is theoctanol/water partition coefficient (log P). However, in our attempts todetermine the logP of our compounds (1, 1a–5a and 1b–5b) by theshake-flask method, no compound was detected in the aqueous phase,thus impeding calculation of this coefficient. The relative hydrophobi-city of 1, 1a–5a and 1b–5b was evaluated by comparison of their RP-HPLC retention times.9 The increase in the time at which the N-TEG

Scheme 1 Synthesis of the Fmoc-N-TEG amino acids (9–11). Reagents andconditions: (a) CH3O–(CH2CH2O)2–CH2CHO, NaBH3CN; (b) Fmoc-Cl, DIEA; (c)TFA–DCM 1 : 1. Scheme 2 Synthesis of the N-TEG and N-Me analogs (1a–5a and 1b–5b).

Reagents and conditions: (a) Fmoc-aa1-OH, DIEA; (b) MeOH; (c) piperidine–DMF 1 : 4; (d) Fmoc-aa2-OH, DIPCDI, OxymaPure; (e) Fmoc-(N-TEG)aa3-OH,DIPCDI, OxymaPure; (f) Fmoc-(N-Me)aa3-OH, DIPCDI, OxymaPure; (g) Fmoc-aa4-OH, BTC, 2,4,6-trimethylpyridine, THF; (h) Fmoc-aa5-OH, DIPCDI, OxymaPure; (i)2% TFA in DCM; (j) EDC�HCl, 4-DMAP.

Fig. 2 Cell viability of GLC-4 and MDA-MB-231 cancer cells after 50 mMtreatment of 1, 1a–5a and 1b–5b for 72 h.

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6432 Chem. Commun., 2013, 49, 6430--6432 This journal is c The Royal Society of Chemistry 2013

and the N-Me analogs (1a–5a and 1b–5b) were eluted indicated that theN-alkylated analogs were slightly more lipophilic than the originalpeptide (1) (Table 1). Their enhanced lipophilicity can be attributedto the fact that N-alkylation of an amide bond decreases the number ofpotential intermolecular hydrogen bonds that can be formed with thepolar solvent (H2O). Surprisingly, all the N-TEG analogs (1a–5a) wereslightly more hydrophobic than their corresponding N-Me counterparts(1b–5b), presumably due to the N-TEG chain shielding the backboneamide groups from interaction with H2O. Since the decreased hydro-gen-bonding potential and the enhanced lipophilicity upon N-methyla-tion are considered to improve membrane permeability,10 we proposethat the incorporation of N-TEG may provide a novel modification togain bioavailability in therapeutic peptides.

The effect of the N-TEG group on the conformational state ofSansalvamide A peptide (1) was found to be the same as that of theN-Me group. An overlay of the 1H-NMR spectra (in CDCl3) of 1,1a–5a and 1b–5b in the HN- and Ha-regions clearly showed thatN-alkylation induced changes in the backbone conformation of theparent peptide (Fig. 3). Depending on the position of N-alkylation,different signal patterns were observed; however, the N-TEG and theN-Me analogs (1a–5a and 1b–5b) bearing the N-substituent at thesame position were found to have similar Ha-resonances. Theresemblance of their Ca-chemical shifts gives further evidence ofsimilar conformational preferences (see ESI‡).

For peptides 5a and 5b, a more detailed study was performed incomparison with the parent peptide (1) (see ESI‡). For the threepeptides, no evidence of conformational equilibria was found using1H-NMR in the temperature range between 5 and 45 1C. For eachpeptide, the HN-, Ha- and Ca-signals were unequivocally assigned, theinterproton NOEs were analyzed and the temperature coefficients ofthe amide protons were determined. These experimental parameters,which are sensitive to conformational changes, were almost identical

for the N-TEG and N-Me analogs 5a and 5b, whilst considerablydiffering from those of the unmodified Sansalvamide A peptide (1).

In conclusion, this study shows that peptides in which thebackbone N-Me group is replaced by a short oligoethylene glycolchain are accessible by the same synthetic repertoire as that alreadyestablished for N-Me peptides. Comparison of the NMR data ofN-Me and N-TEG peptides gives evidence of similar conformationalpreferences for those peptides with the same N-alkylation pattern,and the incorporation of an N-TEG chain or an N-Me group into apeptide provides a higher lipophilicity. Considering the high abun-dance of N-Me amino acids in biologically active peptides, wecontend that modification at this position is a feasible alternativeto introduce structural diversity or alter pharmacologically impor-tant parameters when modification at any other position of thepeptide is not wished or possible.

This work was partially supported by CICYT (CTQ2012-30930),the Generalitat de Catalunya (2009SGR 1024), the Institute forResearch in Biomedicine and the Barcelona Science Park. Ana I.Fernandez-Llamazares thanks Ministerio de Educacion y Ciencia fora FPU fellowship. We thank the Cancer Cell Biology Research groupfor their support for the cytotoxicity assays and we thank BarcelonaScience Park (Mass Spectrometry Core Facility, Nuclear MagneticResonance Unit) for the facilities.

Notes and references1 C. Gilon, M. A. Dechantsreiter, F. Burkhart, A. Friedler and

H. Kessler, in Houben-Weyl, Methods of Organic Chemistry, Synthesisof Peptides and Peptidomimetics, ed. M. Goodman, A. Felix,L. Moroder and C. Toniolo, Georg Thieme Verlarg, Stuttgart andNew York, 2002, vol. E22c, pp. 215–271 and references cited therein.

2 J. Chatterjee, F. Rechenmacher and H. Kessler, Angew. Chem., Int.Ed., 2013, 52, 254.

3 J. Chatterjee, C. Gilon, A. Hoffman and H. Kessler, Acc. Chem. Res.,2008, 10, 1331.

4 F. Hubler, T. Ru, L. Patiny, T. Muamba, J.-F. Guichou, M. Mutter andR. Wenger, Tetrahedron Lett., 2000, 41, 7193.

5 W. Gu, S. Liu and R. B. Silverman, Org. Lett., 2002, 4, 4171; C. L. Carroll,J. V. C. Johnston, A. Kekec, J. D. Brown, E. Parry, J. Cajica, I. Medina,K. M. Cook, R. Corral, P.-S. Pan and S. R. McAlpine, Org. Lett., 2005,7, 3481; T. J. Styers, A. Kekec, R. Rodriguez, J. D. Brown, J. Cajica,P.-S. Pan, E. Parry, C. L. Carroll, I. Medina, R. Corral, S. Lapera,K. Otrubova, C.-M. Pan, K. L. McGuireb and S. R. McAlpine, Bioorg.Med. Chem., 2006, 14, 5625; P.-S. Pan, K. L. McGuireb and S. R. McAlpine,Bioorg. Med. Chem. Lett., 2007, 17, 5072.

6 S. Liu, W. Gu, D. Lo, X.-Z. Ding, M. Ujiki, T. E. Adrian, G. A. Soff andR. B. Silverman, J. Med. Chem., 2005, 48, 3630.

7 O. Demmer, I. Dijkgraaf, M. Schottelius, H.-J. Wester and H. Kessler, Org.Lett., 2008, 10, 2015; C. J. White and A. K. Yudin, Nat. Chem., 2011, 3, 509.

8 E. Falb, T. Yechezkel, Y. Salitra and C. Gilon, J. Pept. Res., 1999,53, 507; B. Thern, J. Rudolph and G. Jung, Angew. Chem., Int. Ed.,2002, 41, 2307; B. Thern, J. Rudolph and G. Jung, Tetrahedron Lett.,2002, 43, 5013; M. M. Sleebs, D. Scanlon, J. Karas, R. Maharani andA. B. Hugues, J. Org. Chem., 2011, 76, 6686; J. Spiegel, C. Mas-Moruno, H. Kessler and W. D. Lubell, J. Org. Chem., 2012, 77, 5271.

9 J. M. R. Parker, D. Guo and R. S. Hodges, Biochemistry, 1986,25, 5425; T. J. Sereda, C. T. Mant, F. D. Sonnichsen andR. S. Hodges, J. Chromatogr., A, 1994, 676, 139.

10 T. R. White, C. M. Renzelman, A. C. Rand, T. Rezai, C. M. McEwen,V. M. Gelev, R. A. Turner, R. G. Linington, S. S. F. Leung,A. S. Kalgutkar, J. N. Bauman, Y. Z. Zhang, S. Liras, D. A. Price,A. M. Mathiowetz, M. P. Jacobson and R. S. Lokey, Nat. Chem. Biol.,2011, 7, 810; J. G. Beck, J. Chatterjee, B. Laufer, M. Udaya Kiran,A. O. Frank, S. Neubauer, O. Ovadia, S. Greenberg, C. Gilon,A. Hoffman and H. Kessler, J. Am. Chem. Soc., 2012, 134, 12125.

Table 1 RP-HPLC retention times of 1, 1a–5a, 1b–5ba

Entry tr (min)

1 1(SA-peptide): 5.972 1a: 7.29 1b: 7.123 2a: 7.22 2b: 7.074 3a: 6.98 3b: 6.825 4a: 7.34 4b: 6.976 5a: 7.05 5b: 6.86

a Linear gradient from 10% to 90% ACN over 8 min, C18 column.

Fig. 3 1H-NMR spectra (in CDCl3) of 1, 1a–5a, 1b–5b in the 5.5–3.5 ppm region.

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