Accepted Manuscript

Access to bifunctionalized biomolecular platforms using oxime ligation

Karel Křenek, Radek Gaž ák, Gour Chand Daskhan, Julian Garcia, MicheleFiore, Pascal Dumy, Miroslav Šulc, Vladimír Křen, Olivier Renaudet

PII: S0008-6215(14)00181-5DOI: http://dx.doi.org/10.1016/j.carres.2014.04.020Reference: CAR 6737

To appear in: Carbohydrate Research

Received Date: 28 March 2014Revised Date: 28 April 2014Accepted Date: 29 April 2014

Please cite this article as: Křenek, K., Gaž ák, R., Daskhan, G.C., Garcia, J., Fiore, M., Dumy, P., Šulc, M., Křen,V., Renaudet, O., Access to bifunctionalized biomolecular platforms using oxime ligation, CarbohydrateResearch (2014), doi: http://dx.doi.org/10.1016/j.carres.2014.04.020

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Access to bifunctionalized biomolecular platforms using oxime ligation

Karel Křenek,a Radek Gažák,a Gour Chand Daskhan,b Julian Garcia,b Michele Fiore,b

Pascal Dumy,b Miroslav Šulc,a Vladimír Křen*,a Olivier Renaudet*,b,c

a Institute of Microbiology, Academy of Sciences of the Czech Republic, Vídeňská

1083, CZ-14220 Praha 4, Czech Republic,

b Département de Chimie Moléculaire, UMR CNRS 5250 & ICMG FR 2607,

Université Joseph Fourier, BP53, 38041 Grenoble Cedex 9, France,

c Institut Universitaire de France, 103 boulevard Saint-Michel, 75005 Paris, France c

*E-mail: kren@biomed.cas.cz, olivier.renaudet@ujf-grenoble.fr

Keywords

Chemoselective ligation; glycocluster; cyclopeptide; multivalency; oxime

Note

We previously reported the synthesis of original neoglycopeptide conjugates and the

evaluation of their immunological properties towards NK cells. Due to the 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 which was fully

validated, updated and enhanced with additional experiments.

Abstract

This paper describes an efficient oxime ligation strategy to prepare multivalent

conjugates wherein peptides alone or in combination with carbohydrate or oxime

group were coupled to a cyclopeptide scaffold. To demonstrate the versatility of this

approach, two classes of conjugates have been prepared. In one class, we attached

two or four peptide sequences to the cyclopeptide core together with free oxime

groups, 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.

Introduction

Carbohydrates and proteins are key structural motifs that exist in all living systems

where they play central roles in a wide range of biological events [1-3]. Synthetic

carbohydrates alone or combined with peptides and proteins represent promising

tools to decipher these complex biological processes as well as to develop potential

therapeutic and diagnostic agents [4-15]. However, chemical construction of such

structures is complicated due to difficulties associated with the low compatibility of

peptide/protein and carbohydrate chemistries, the fragility of glycosidic linkage and

the presence of multiple functionalities. Chemoselective and bioorthogonal strategies

have been developed to circumvent these difficulties and provide access to complex

bioconjugates [16-20]. Among these methods, we and other groups have focused on

oxime ligation that consists in using two functionalities namely, aldehyde and

aminooxy groups that are highly reactive in aqueous buffers [21]. This strategy has

been used successfully to synthesize numbers of original glycosylated constructs for

applications going from cell targeting to surface modifications [22-25].

As a part of a long term program, we have demonstrated the utility of cyclopeptide

scaffolds, commonly known as Regioselectively Addressable Functionalized

Templates (RAFT) [26], for the multivalent presentation of carbohydrate or peptide

ligands in well-defined spatial orientation [27-28]. The utilization of this scaffold is

indeed interesting to us due to its conformational stability and the presence of two

independent domains that can be functionalized regioselectively. In particular, we

demonstrated recently the robustness of oxime ligation to prepare well-defined

cyclopeptide-based glycodendrimers displaying clusters of carbohydrates on the

upper domain of the scaffold [29-33]. In this paper, we show that both addressable

domains of the cyclopeptide can be functionalized successively with peptides and

carbohydrates using an oxime-based ligation process. To demonstrate the versatility

of our strategy we have prepared two classes of compounds (Figure 1). In the first

one, we have combined two or four peptides together with free oxime groups, i.e.

compounds 1 and 3 respectively. The second classes display an additional

substitution with four or two GalNAc attached through an oxime ether linkage, i.e.

compounds 2 and 4, respectively.

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

conjugates.

Results and Discussion

We have previously showed that the synthesis of glycodendrimers [31] using

successive oxime conjugations is efficient but should be performed following a strict

sequence to avoid side reactions. In particular, we have observed that the

simultaneous presence of both aminooxy and oxime functionalities within a single

molecule can induce trans-oximation reactions [31]. To avoid this problem, two

different cyclodecapeptide cores with orthogonal protecting groups have been

prepared (Scheme 1) as key intermediates for the nonglycosylated and glycosylated

peptide conjugates 1-4.

Scheme 1. Synthesis of oxime-peptide (1) and carbohydrate-peptide (2) conjugates.

The first cyclopeptide 5 display four lysines pre-functionalized with serines [34] on the

upper domain and two lysines protected with N-(1-(4,4-dimethyl-2,6-

dioxocyclohexylidene)ethyl) (Dde) [35] on the lower domain. Dde protecting groups

were first removed using a solution of 3% of hydrazine in DMF. N-

Hydroxysuccinimidyl ester of ethoxyethylidene aminooxy acetic acid linker (Eei-Aoa-

OSu) [36] was next coupled to the free lysines to afford compound 6. Final

deprotection of the four serine and two aminooxy moieties was performed by

acidolysis with a solution of trifluoroacetic acid/triisopropylsilane/water (TFA/TIS/H2O,

95:2.5:2.5) to provide the fully deprotected scaffold 7.

The molecular assembly was performed using successive oxime coupling and

aldehyde formation. For this purpose, we have synthesized a pentapeptide that

contains diverse functionalities (i.e. carboxylic acids and alcohol). This model peptide

was terminated with a serine which can be easily converted into glyoxylic aldehyde (-

COCHO) by oxidative cleavage with sodium periodate [37-38]. The oxime coupling

was performed with an excess of the peptide-COCHO and the aminooxy-containing

scaffold 7 in a mixture of CH3CN/H2O containing 0.1% of TFA. The conversion was

complete and we obtained quantitatively a peptide conjugate displaying two peptide

and four serine moieties. An additional treatment with sodium periodate afforded four

COCHO functions that were used to conjugate either hydroxylamine or aminooxy

αGalNAc [39] to the second addressable domain of the scaffold. The oxime coupling

was performed under similar conditions and the desired compounds 1-2 were

obtained quantitatively as confirmed by analytical HPLC of the crude mixtures (Figure

2).

(a)

(b)

Figure 2. HPLC profile (linear gradient: 5 to 90% CH3CN in 15 min, λ = 250 nm) of

the crude reaction mixture of (a) compound 1; (b) compound 2.

We followed the same procedure to prepare the conjugates 3 and 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 3 and 4 were obtained as pure products after HPLC purification in similar

yields.

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

The fragmentation of oxime ether linkages during ionization process of mass

spectroscopy spectrometry analysis are well documented in the literature [31, 33, 40].

To avoid this problem and received high resolution MS data we have chosen matrix-

assisted laser desorption/ionization ion source combined with Fourier transform ion

cyclotron resonance as detector, time-of-flight mass spectroscopy (MALDI-TOFFT-

ICR-MS) for mass spectrometry experiments. The average monoisotopic molecular

weight of the synthesized conjugates 1-4 was measured by MALDI-TOF FT-ICR MS

mass spectroscopy analysis using α-cyano-4-hydroxycinnamic acid (CCA) or 2,5-

dihydrobenzoic acid (DHB) as MALDI matrices for compounds 1-3 and 4,

respectively (Table 1).

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

Cpnd Formula Calcd. mass Found mass (m/z)

Error (ppm)

1 C118H188N32O46 2812.324768 [M+Na]+ 2812.31664 2.9

2 C150H240N36O66 3624.642258 [M+Na]+ 3624.64305 0.3

3 C174H278N42O70 4076.955767 [M+H]+ 4076.95899 0.8

4 C190H304N44O80 4483.114512 [M+H]+ 4483.19100 17.0

We next performed molecular modelling study with peptide-glycoconjugates 2 and 4.

Energy minimizations were calculated in vacuo using Insight II/Discover. We first

observed that minimized structures are conformationally stable and the cyclopeptide

remains rigid in both cases. In addition, no steric clashes were observed between the

peptides fragments and the carbohydrates attached onto both addressable domains

of the cyclopeptide. This suggests that such scaffolds can be functionalized with

diverse recognition or structural elements in well-defined spatial orientation without

interfering with their biological function.

(a)

(b)

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

Conclusion

In summary, we have described a straightforward strategy for the synthesis of

bifunctionalized cyclopeptide scaffolds bearing peptide and carbohydrates on two

separated addressable domains. By employing successive and reproducible oxime

ligations, new classes of peptide-conjugates 1-3 and 2-4 were prepared using

attachment of either peptide-aldehyde, hydroxylamine or aminooxy αGalNAc onto

RAFT scaffolds displaying aminooxy and masked aldehyde groups. The well-defined

structure of these conjugates was determined by high-resolution mass spectrometry.

Molecular modelling study also confirmed the spatial separation of both addressable

domains, thus providing an attractive biomolecular platform for diverse biomedical

applications.

Experimental

General methods. All chemical reagents were purchased from Aldrich (Saint-

Quentin Fallavier, France) or Acros (Noisy-Le-Grand, France) and were used without

further purification. Protected amino acids and Fmoc-Gly-Sasrin resin were obtained

from Advanced ChemTech 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 on Waters equipment using C18 columns. Analytical HPLC

(Nucleosil 120 Å 3 µm C18 particles, 30 × 4.6 mm2) was performed at 1.3 mL/min and

preparative HPLC (Delta-Pak 300 Å 15 µm C18 particles, 200 × 25 mm2) at 22

mL/min with UV monitoring (214 nm and 250 nm) using a linear A–B gradient (buffer

A: 0.09% TFA in H2O; buffer B: 0.09% TFA in 90% CH3CN). Routine mass spectra

were recorded on a VG Platform II by electron spray ionization in the positive mode.

Accurate mass spectra were obtained for compounds 1-4 on a MALDI-APEX Qe-FT-

ICR mass spectrometer equipped with a 9.4 T superconducting magnet and a ion

Combi Source (Bruker-Daltonics, Bremen, Germany). Positive mass spectra were

obtained by accumulating ions in the collision hexapole and running the quadrupole

mass filter in non-mass-selective RF-only mode so that ions of a broad m/z range

(850–5000) were allowed to enter the analyzer cell. All spectra were calibrated

externally using the monoisotopic [M+H]+ ions of Pepmix2 calibrant (Bruker-

Daltonics, Germany). A 5 mg/mL solution of CCA or 10 mg/mL solution DHB in 50%

CH3CN and 0.3% CH3CO2H was used as matrix. A 1 µL of sample dissolved in

CH3OH was mixed with a 1.0 µL of the matrix solution. A 0.3 µL of mixture was

loaded on the target and allowed to dry at ambient temperature.

Synthesis of compound 5. The linear precursor peptide (0.25 mmol; analytical RP-

HPLC: Rt = 12.2 min (5 to 100% B in 15 min, 214 nm); ESI-MS: calcd. for

C118H203N20O31 2397.5 [M+H]+; found: m/z 2397.9) was cyclized in CH2Cl2 (500 mL)

with PyBOP (156 mg; 0.3 mmol) and DIPEA (82 µL; 0.5 mmol; pH 8). After stirring for

1 h at room temperature, the solution was evaporated and peptide 5 was recovered

by precipitation in Et2O. Analytical RP-HPLC: Rt = 14.9 min (5 to 100% B in 15 min,

214 nm); ESI-MS: calcd. for C118H201N20O30 2378.5 [M+H]+; found: m/z 2378.1.

Synthesis of compound 6. Protected cyclodecapeptide 5 (0.25 mmol) was treated

for 1 h at room 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 µL; 0.5 mmol) were added and the solution was stirred in DMF (25 mL). The

coupling reaction was monitored by analytical HPLC and reached completion after 2

h. The solvent was then evaporated and the excess of the activated ester removed

by precipitation in Et2O. Yield: 52% (300 mg) from the corresponding linear peptide

sequence (3 steps); analytical RP-HPLC: Rt = 14.1 min (5 to 100% B in 15 min, 214

nm); ESI-MS: calcd. for C110H195N22O32 2336.4 [M+H]+; found: m/z 2336.2.

Synthesis of compound 7. Crude compound 6 (196 mg; 0.084 mmol) was treated

with a cocktail of TFA/TIS/H2O (50 mL; 95:2.5:2.5). After 2 h of stirring at room

temperature, the solution was evaporated and fully deprotected cyclopeptide 7 was

recovered by precipitation in Et2O. Analytical RP-HPLC: Rt = 5.7 min (5 to 60% B in

15 min, 214 nm); ESI-MS: calcd. for C66H119N22O22 1571.9 [M+H]+; found: m/z

1571.9.

Synthesis of compound 8. Cyclodecapeptide 8 was synthesized for the linear

peptide precursor (0.25 mmol; analytical RP-HPLC: Rt = 10.5 min (5 to 100% B in 15

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 to

100% B in 15 min, 214 nm); ESI-MS: calcd. for C114H183N18O26 2220.4 [M+H]+; found:

m/z 2220.1.

Synthesis of compound 9. Compound 9 was synthesized by following the

procedure described for 7. Analytical RP-HPLC: Rt = 5.7 min (5 to 60% B in 15 min,

214 nm); ESI-MS: calcd. for C64H115N22O22 1543.8 [M+H]+; found: m/z 1543.6.

Synthesis of compound 1. Peptide 7 (14 mg; 0.0069 mmol) was dissolved in a

mixture of CH3CN/H2O/TFA (2 mL; 1:1:0.1) and peptide-CHO (27 mg; 0.041 mmol)

was added to the solution. The mixture was stirred at 37°C overnight then acetone (1

mL) was added. The resulting peptide conjugate (was used without further treatment.

Analytical RP-HPLC: Rt = 12.6 min (5 to 40% B in 15 min, 214 nm); ESI-MS: calcd for

C122H205N32O46 2854.5 [M+H]+; found: m/z 2854.3. Sodium periodate (59 mg; 0.28

mmol) was then added to the crude solution and the mixture purified by RP-HPLC

after 1 h to isolate the corresponding oxidized conjugate in 95% yield (18 mg) from 7

(2 steps). Analytical RP-HPLC: Rt = 12.9 min (5 to 60% B in 15 min, 214 nm). This

compound (9 mg; 0.0033 mmol) was finally stirred at 37°C with hydroxylamine

hydrochloride (1.8 mg; 0.026 mmol) in CH3CN/H2O/TFA (2 mL; 1:1:0.1). After RP-

HPLC purification, compound 1 was obtained with a 70% yield (6.5 mg). Analytical

RP-HPLC: Rt = 7.6 min (5 to 100% B in 15 min, 214 nm); MALDI-FT-ICRTOF HRMS:

calcd for C118H188N32O46 2812.324768 [M+Na]+; found: 2812.31664.

Synthesis of compound 2. Compound 2 was obtained from the previous oxidized

peptide (9 mg; 0.0033 mmol) and aminooxy αGalNAc (8 mg; 0.033 mmol) in

CH3CN/H2O/TFA (2 mL; 1:1:0.1) following the procedure described 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 for C150H240N36O66 3624.642258 [M+Na]+; found:

3624.64305.

Synthesis of Compound 3. Compound 3 was prepared following the 3-step

procedure described for 1. Compound 9 (12 mg; 0.0068 mmol) was first treated with

peptide-CHO (28 mg; 0.041 mmol). The resulting conjugate was then oxidized with

sodium periodate (29 mg; 0.14 mmol) in water (7 mL) and purified by RP-HPLC after

1 hour. Yield: 91% (25 mg) from 9 (2 steps); analytical RP-HPLC: Rt = 8.3 min (5 to

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 obtain 3 after purification. Yield: 68% (7.5 mg); analytical RP-HPLC: Rt =

8.4 min (5 to 100% B in 15 min, 214 nm); MALDI-FT-ICRTOF HRMS: calcd for

C174H278N42O70 4076.955767 [M+H]+; found: 4076.95899.

Synthesis of compound 4. Compound 4 was obtained from the previous oxidized

peptide (14 mg; 0.0034 mmol) and aminooxy αGalNAc (3 mg; 0.014 mmol) in

CH3CN/H2O/TFA (2 mL; 1:1:0.1) following the procedure described for 3. Yield: 66%

(10 mg); analytical RP-HPLC: Rt = 8.1 min (5 to 100% B in 15 min, 214 nm); MALDI-

FT-ICRTOF HRMS: calcd for C190H304N44O80 4483.114512 [M+H]+; found:

4483.19100.

Molecular modeling. Structure calculations were performed in vacuo using InsightII /

Discover (Version 2005, Accelrys, SanDiego,CA,USA) software, and the energy of

the system was calculated by the consistent CVFF force field (version 2.3). To

shorten the range of Coulomb interaction, a distance-dependent relative dielectric

constant, εr, was used (εr = 4r). The resulting molecule was subjected to 2000

iterations of steepest descent minimization, followed by 3500 iterations of conjugate

gradient minimization and the convergence of minimization was followed until the

RMS derivative was less than 0.01 kcal.mol-1.

Acknowledgements

This work was supported by the Université Joseph Fourier (UJF), the Centre National

de la Recherche Scientifique (CNRS), the “Communauté d’agglomération Grenoble-

Alpes Métropole” (Nanobio Program), the Ligue contre le cancer (MF) and the ANR-

12-JS07-0001-01 “VacSyn” (GCD). O.R. acknowledges support from the Labex

Arcane (ANR-11-LABX-003), and O.R.&V.K. acknowledge European project

“MultiGlycoNano” ESF COST chemistry action CM1102 (MSMT LD13042).

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