Carbohydrate Research 393 (2014) 9–14

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Carbohydrate Research

journal homepage: www.elsevier .com/locate /carres

Access to bifunctionalized biomolecular platforms using oximeligation q

http://dx.doi.org/10.1016/j.carres.2014.04.0200008-6215/� 2014 Elsevier Ltd. All rights reserved.

q Note: We previously reported the synthesis of original neoglycopeptide conju-gates and the evaluation of their immunological properties toward NK cells. Due tothe inability to reproduce the immunological data (Int. J. Mol. Sci. 2014, 15, 1271–1283), we have recently decided to fully retract this paper (J. Am. Chem. Soc. 2014,136, 1156–1156). The present manuscript describes this synthetic strategy whichwas fully validated, updated, and enhanced with additional experiments.⇑ Corresponding authors.

E-mail addresses: kren@biomed.cas.cz (V. Kren), olivier.renaudet@ujf-grenoble.fr(O. Renaudet).

Karel Krenek a, Radek Gazák a, Gour Chand Daskhan b, Julian Garcia b, Michele Fiore b, Pascal Dumy b,Miroslav Šulc a, Vladimír Kren a,⇑, Olivier Renaudet b,c,⇑a Institute of Microbiology, Academy of Sciences of the Czech Republic, Vídenská 1083, CZ-14220 Praha 4, Czech Republicb Département de Chimie Moléculaire, UMR CNRS 5250 & ICMG FR 2607, Université Joseph Fourier, BP53, 38041 Grenoble Cedex 9, Francec Institut Universitaire de France, 103 boulevard Saint-Michel, 75005 Paris, France

a r t i c l e i n f o

Article history:Received 28 March 2014Received in revised form 28 April 2014Accepted 29 April 2014Available online 9 May 2014

Keywords:Chemoselective ligationGlycoclusterCyclopeptideMultivalencyOxime

a b s t r a c t

This paper describes an efficient oxime ligation strategy to prepare multivalent conjugates wherein pep-tides alone or in combination with carbohydrate or oxime groups were coupled to a cyclopeptide scaffold.To demonstrate the versatility of this approach, two classes of conjugates have been prepared. In oneclass, we attached two or four peptide sequences to the cyclopeptide core together with free oximegroups, while the second class contains an additional substitution with four or two monosaccharides.The well-defined structure of these conjugates was confirmed by high-resolution mass spectrometry.

� 2014 Elsevier Ltd. All rights reserved.

1. Introduction

Carbohydrates and proteins are key structural motifs thatexist in all living systems where they play central roles in a widerange of biological events.1–3 Synthetic carbohydrates alone orcombined with peptides and proteins represent promising toolsto decipher these complex biological processes as well as todevelop potential therapeutic and diagnostic agents.4–15 How-ever, chemical construction of such structures is complicateddue to difficulties associated with the low compatibility ofpeptide/protein and carbohydrate chemistries, the fragility ofglycosidic linkage and the presence of multiple functionalities.Chemoselective and bioorthogonal strategies have been devel-oped to circumvent these difficulties and provide access to com-plex bioconjugates.16–20 Among these methods, we and othergroups have focused on oxime ligation that consists in using

two functionalities namely, aldehyde and aminooxy groups thatare highly reactive in aqueous buffers.21 This strategy has beenused successfully to synthesize numbers of original glycosylatedconstructs for applications going from cell targeting to surfacemodifications.22–25

As a part of a long term program, we have demonstrated theutility of cyclopeptide scaffolds, commonly known as Regioselec-tively Addressable Functionalized Templates (RAFT),26 for themultivalent presentation of carbohydrate or peptide ligands inwell-defined spatial orientation.27,28 The utilization of this scaf-fold is indeed interesting to us due to its conformational stabilityand the presence of two independent domains that can befunctionalized regioselectively. In particular, we demonstratedrecently the robustness of oxime ligation to prepare well-definedcyclopeptide-based glycodendrimers displaying clusters of carbo-hydrates on the upper domain of the scaffold.29–33 In this paper,we show that both addressable domains of the cyclopeptide canbe functionalized successively with peptides and carbohydratesusing an oxime-based ligation process. To demonstrate the versa-tility of our strategy we have prepared two classes of compounds(Fig. 1). In the first one, we have combined two or four peptidestogether with free oxime groups, that is compounds 1 and 3respectively. The second classes display an additional substitutionwith four or two GalNAc attached through an oxime ether link-age, that is compounds 2 and 4, respectively.

Figure 1. Structure of oxime–peptide (1 and 3) and carbohydrate–peptide (2 and 4)conjugates.

Scheme 1. Synthesis of oxime-peptide (1) an

10 K. Krenek et al. / Carbohydrate Research 393 (2014) 9–14

2. Results and discussion

We have previously showed that the synthesis of glycodendri-mers31 using successive oxime conjugations is efficient but shouldbe performed following a strict sequence to avoid side reactions. Inparticular, we have observed that the simultaneous presence ofboth aminooxy and oxime functionalities within a single moleculecan induce trans-oximation reactions.31 To avoid this problem, twodifferent cyclodecapeptide cores with orthogonal protectinggroups have been prepared (Scheme 1) as key intermediates forthe nonglycosylated and glycosylated peptide conjugates 1–4.

The first cyclopeptide 5 displays four lysines pre-functionalizedwith serines34 on the upper domain and two lysines protected withN-(1-(4,4-dimethyl-2,6-dioxocyclohexylidene)ethyl) (Dde)35 onthe lower domain. Dde protecting groups were first removed usinga solution of 3% of hydrazine in DMF. N-Hydroxysuccinimidyl esterof ethoxyethylidene aminooxy acetic acid linker (Eei-Aoa-OSu)36

was next coupled to the free lysines to afford compound 6. Finaldeprotection of the four serine and two aminooxy moieties wasperformed by acidolysis with a solution of trifluoroacetic acid/triisopropylsilane/water (TFA/TIS/H2O, 95:2.5:2.5) to provide thefully deprotected scaffold 7.

The molecular assembly was performed using successive oximecoupling and aldehyde formation. For this purpose, we have syn-thesized a pentapeptide that contains diverse functionalities (i.e.,carboxylic acids and alcohol). This model peptide was terminatedwith a serine which can be easily converted into glyoxylic aldehyde(–COCHO) by oxidative cleavage with sodium periodate.37,38 Theoxime coupling was performed with an excess of the peptide–COCHO and the aminooxy-containing scaffold 7 in a mixture ofCH3CN/H2O containing 0.1% of TFA. The conversion was completeand we obtained quantitatively a peptide conjugate displayingtwo peptide and four serine moieties. Additional treatment withsodium periodate afforded four COCHO functions that were usedto conjugate either hydroxylamine or aminooxy aGalNAc39 to thesecond addressable domain of the scaffold. The oxime coupling

d carbohydrate–peptide (2) conjugates.

Figure 2. HPLC profile (linear gradient: 5–90% CH3CN in 15 min, k = 250 nm) of the crude reaction mixture of (a) compound 1; (b) compound 2.

Scheme 2. Synthesis of oxime-peptide (3) and carbohydrate-peptide (4) conjugates.

K. Krenek et al. / Carbohydrate Research 393 (2014) 9–14 11

was performed under similar conditions and the desired com-pounds 1–2 were obtained quantitatively as confirmed by analyti-cal HPLC of the crude mixtures (Fig. 2).

We followed the same procedure to prepare the conjugates 3and 4 from the scaffold 8, which contains reverse functionalities,i.e. two pre-functionalized and four protected lysines (Scheme 2).No difference of reactivity was observed and both compounds 3and 4 were obtained as pure products after HPLC purification insimilar yields.

The fragmentation of oxime ether linkages during ionizationprocess of mass spectroscopy spectrometry analysis is well docu-mented in the literature.31,33,40 To avoid this problem and receivehigh resolution MS data we have chosen matrix-assisted laserdesorption/ionization ion source combined with Fourier transformion cyclotron resonance as detector, time-of-flight mass spectros-copy (MALDI-TOFFT-ICR-MS) for mass spectrometry experiments.The average monoisotopic molecular weight of the synthesizedconjugates 1–4 was measured by MALDI-TOF FT-ICR MS mass

Table 1MALDI-TOF FT-ICR MS analysis of conjugates 1–4

Compd Formula Calcd mass Found mass(m/z)

Error(ppm)

1 C118H188N32O46 2812.324768 [M+Na]+ 2812.31664 2.92 C150H240N36O66 3624.642258 [M+Na]+ 3624.64305 0.33 C174H278N42O70 4076.955767 [M+H]+ 4076.95899 0.84 C190H304N44O80 4483.114512 [M+H]+ 4483.19100 17.0

Figure 3. Molecular modeling of (a) compound 2; (b) compound 4.

12 K. Krenek et al. / Carbohydrate Research 393 (2014) 9–14

spectroscopy analysis using a-cyano-4-hydroxycinnamic acid(CCA) or 2,5-dihydrobenzoic acid (DHB) as MALDI matrices forcompounds 1–3 and 4, respectively (Table 1).

We next performed molecular modeling study with peptide-glycoconjugates 2 and 4 (Fig. 3). Energy minimizations were calcu-lated in vacuo using Insight II/Discover. We first observed thatminimized structures are conformationally stable and the cyclo-peptide remains rigid in both cases. In addition, no steric clasheswere observed between the peptides fragments and the carbohy-drates attached onto both addressable domains of the cyclopep-tide. This suggests that such scaffolds can be functionalized withdiverse recognition or structural elements in well-defined spatialorientation without interfering with their biological function.

3. Conclusion

In summary, we have described a straightforward strategy forthe synthesis of bifunctionalized cyclopeptide scaffolds bearing

peptides and carbohydrates on two separated addressabledomains. By employing successive and reproducible oximeligations, new classes of peptide-conjugates 1–3 and 2–4 were pre-pared using attachment of either peptide–aldehyde, hydroxyl-amine, or aminooxy aGalNAc onto RAFT scaffolds displayingaminooxy and masked aldehyde groups. The well-defined struc-ture of these conjugates was determined by high-resolution massspectrometry. Molecular modeling study also confirmed the spatialseparation of both addressable domains, thus providing an attrac-tive biomolecular platform for diverse biomedical applications.

4. Experimental

4.1. General methods

All chemical reagents were purchased from Aldrich (Saint-Quentin Fallavier, France) or Acros (Noisy-Le-Grand, France) andwere used without further purification. Protected amino acidsand Fmoc-Gly-Sasrin resin were obtained from AdvancedChemTech Europe (Brussels, Belgium), Bachem Biochimie SARL(Voisins-Les-Bretonneux, France) and France Biochem S.A.(Meudon, France). PyBOP was purchased from France Biochem.Reaction progress was monitored by reverse-phase HPLC onWaters equipment using C18 columns. Analytical HPLC (Nucleosil120 Å 3 lm C18 particles, 30 � 4.6 mm2) was performed at1.3 mL/min and preparative HPLC (Delta-Pak 300 Å 15 lm C18 par-ticles, 200 � 25 mm2) at 22 mL/min with UV monitoring (214 nmand 250 nm) using a linear A–B gradient (buffer A: 0.09% TFA inH2O; buffer B: 0.09% TFA in 90% CH3CN). Routine mass spectrawere recorded on a VG Platform II by electron spray ionization inthe positive mode. Accurate mass spectra were obtained for com-pounds 1–4 on a MALDI-APEX Qe-FT-ICR mass spectrometerequipped with a 9.4 T superconducting magnet and a ion CombiSource (Bruker-Daltonics, Bremen, Germany). Positive mass spec-tra were obtained by accumulating ions in the collision hexapoleand running the quadrupole mass filter in non-mass-selectiveRF-only mode so that ions of a broad m/z range (850–5000) wereallowed to enter the analyzer cell. All spectra were calibratedexternally using the monoisotopic [M+H]+ ions of Pepmix2 cali-brant (Bruker-Daltonics, Germany). A 5 mg/mL solution of CCA or10 mg/mL solution DHB in 50% CH3CN and 0.3% CH3CO2H was usedas matrix. A 1 lL of sample dissolved in CH3OH was mixed with a1.0 lL of the matrix solution. A 0.3 lL of mixture was loaded on thetarget and allowed to dry at ambient temperature.

4.2. Synthesis of compound 5

The linear precursor peptide (0.25 mmol; analytical RP-HPLC:Rt = 12.2 min (5–100% B in 15 min, 214 nm); ESI-MS: calcd forC118H203N20O31 2397.5 [M+H]+; found: m/z 2397.9) was cyclizedin CH2Cl2 (500 mL) with PyBOP (156 mg; 0.3 mmol) and DIPEA(82 lL; 0.5 mmol; pH 8). After stirring for 1 h at room temperature,the solution was evaporated and peptide 5 was recovered by pre-cipitation in Et2O. Analytical RP-HPLC: Rt = 14.9 min (5–100% B in15 min, 214 nm); ESI-MS: calcd for C118H201N20O30 2378.5[M+H]+; found: m/z 2378.1.

4.3. Synthesis of compound 6

Protected cyclodecapeptide 5 (0.25 mmol) was treated for 1 h atroom temperature with a solution of 3% hydrazine in DMF (50 mL).After evaporation and precipitation in Et2O, Eei-Aoa-OSu (129 mg;0.5 mmol) and DIPEA (83 lL; 0.5 mmol) were added and the solu-tion was stirred in DMF (25 mL). The coupling reaction was moni-tored by analytical HPLC and reached completion after 2 h. The

K. Krenek et al. / Carbohydrate Research 393 (2014) 9–14 13

solvent was then evaporated and the excess of the activated esterremoved by precipitation in Et2O. Yield: 52% (300 mg) from thecorresponding linear peptide sequence (3 steps); analytical RP-HPLC: Rt = 14.1 min (5–100% B in 15 min, 214 nm); ESI-MS: calcdfor C110H195N22O32 2336.4 [M+H]+; found: m/z 2336.2.

4.4. Synthesis of compound 7

Crude compound 6 (196 mg; 0.084 mmol) was treated with acocktail of TFA/TIS/H2O (50 mL; 95:2.5:2.5). After 2 h of stirringat room temperature, the solution was evaporated and fully depro-tected cyclopeptide 7 was recovered by precipitation in Et2O. Ana-lytical RP-HPLC: Rt = 5.7 min (5–60% B in 15 min, 214 nm); ESI-MS:calcd for C66H119N22O22 1571.9 [M+H]+; found: m/z 1571.9.

4.5. Synthesis of compound 8

Cyclodecapeptide 8 was synthesized for the linear peptide pre-cursor (0.25 mmol; analytical RP-HPLC: Rt = 10.5 min (5–100% B in15 min, 214 nm); ESI-MS: calcd for C114H185N18O27 2239.3 [M+H]+;found: m/z 2238.6) by following the procedure described for 5.Analytical RP-HPLC: Rt = 12.9 min (5–100% B in 15 min, 214 nm);ESI-MS: calcd for C114H183N18O26 2220.4 [M+H]+; found: m/z2220.1.

4.6. Synthesis of compound 9

Compound 9 was synthesized by following the proceduredescribed for 7. Analytical RP-HPLC: Rt = 5.7 min (5–60% B in15 min, 214 nm); ESI-MS: calcd for C64H115N22O22 1543.8[M+H]+; found: m/z 1543.6.

4.7. Synthesis of compound 1

Peptide 7 (14 mg; 0.0069 mmol) was dissolved in a mixture ofCH3CN/H2O/TFA (2 mL; 1:1:0.1) and peptide–CHO (27 mg;0.041 mmol) was added to the solution. The mixture was stirredat 37 �C overnight then acetone (1 mL) was added. The resultingpeptide conjugate (was used without further treatment. AnalyticalRP-HPLC: Rt = 12.6 min (5–40% B in 15 min, 214 nm); ESI-MS: calcdfor C122H205N32O46 2854.5 [M+H]+; found: m/z 2854.3. Sodium per-iodate (59 mg; 0.28 mmol) was then added to the crude solutionand the mixture purified by RP-HPLC after 1 h to isolate the corre-sponding oxidized conjugate in 95% yield (18 mg) from 7 (2 steps).Analytical RP-HPLC: Rt = 12.9 min (5–60% B in 15 min, 214 nm).This compound (9 mg; 0.0033 mmol) was finally stirred at 37 �Cwith hydroxylamine hydrochloride (1.8 mg; 0.026 mmol) in CH3-

CN/H2O/TFA (2 mL; 1:1:0.1). After RP-HPLC purification, compound1 was obtained with a 70% yield (6.5 mg). Analytical RP-HPLC:Rt = 7.6 min (5–100% B in 15 min, 214 nm); MALDI-FT-ICRTOFHRMS: calcd for C118H188N32O46 2812.324768 [M+Na]+; found:2812.31664.

4.8. Synthesis of compound 2

Compound 2 was obtained from the previous oxidized peptide(9 mg; 0.0033 mmol) and aminooxy aGalNAc (8 mg; 0.033 mmol)in CH3CN/H2O/TFA (2 mL; 1:1:0.1) following the proceduredescribed for 1. Yield: 80% (9.5 mg); analytical RP-HPLC: Rt = 7.1 min(5 to 100% B in 15 min, 214 nm); MALDI-FT-ICRTOF HRMS: calcd forC150H240N36O66 3624.642258 [M+Na]+; found: 3624.64305.

4.9. Synthesis of Compound 3

Compound 3 was prepared following the 3-step proceduredescribed for 1. Compound 9 (12 mg; 0.0068 mmol) was first

treated with peptide–CHO (28 mg; 0.041 mmol). The resultingconjugate was then oxidized with sodium periodate (29 mg;0.14 mmol) in water (7 mL) and purified by RP-HPLC after1 h. Yield: 91% (25 mg) from 9 (2 steps); analytical RP-HPLC:Rt = 8.3 min (5–100% B in 15 min, 214 nm). This peptide (11 mg;0.0027 mmol) was reacted with hydroxylamine hydrochloride(0.8 mg; 0.011 mmol) in CH3CN/H2O/TFA (2 mL; 1:1:0.1) to obtain3 after purification. Yield: 68% (7.5 mg); analytical RP-HPLC:Rt = 8.4 min (5–100% B in 15 min, 214 nm); MALDI-FT-ICRTOFHRMS: calcd for C174H278N42O70 4076.955767 [M+H]+; found:4076.95899.

4.10. Synthesis of compound 4

Compound 4 was obtained from the previous oxidized peptide(14 mg; 0.0034 mmol) and aminooxy aGalNAc (3 mg; 0.014 mmol)in CH3CN/H2O/TFA (2 mL; 1:1:0.1) following the proceduredescribed for 3. Yield: 66% (10 mg); analytical RP-HPLC:Rt = 8.1 min (5–100% B in 15 min, 214 nm); MALDI-FT-ICRTOFHRMS: calcd for C190H304N44O80 4483.114512 [M+H]+; found:4483.19100.

4.11. Molecular modeling

Structure calculations were performed in vacuo using InsightII/Discover (Version 2005, Accelrys, SanDiego, CA, USA) software, andthe energy of the system was calculated by the consistent CVFFforce field (version 2.3). To shorten the range of Coulomb interac-tion, a distance-dependent relative dielectric constant, er, was used(er = 4r). The resulting molecule was subjected to 2000 iterations ofsteepest descent minimization, followed by 3500 iterations of con-jugate gradient minimization and the convergence of minimizationwas followed until the RMS derivative was less than0.01 kcal mol�1.

Acknowledgements

This work was supported by the Université Joseph Fourier (UJF),the Centre National de la Recherche Scientifique (CNRS), the ‘Com-munauté d’agglomération Grenoble-Alpes Métropole’ (NanobioProgram), the Ligue contre le cancer (MF) and the ANR-12-JS07-0001-01 ‘VacSyn’ (GCD). O.R. acknowledges support from theLabex Arcane (ANR-11-LABX-003), and O.R. & V.K. acknowledgeEuropean Project ‘MultiGlycoNano’ ESF COST chemistry actionCM1102 (MSMT LD13042).

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