TECHNICAL NOTES

Anchorage of Synthetic Peptides onto Liposomes via Hydrazone andr-Oxo Hydrazone Bonds. Preliminary Functional Investigations

Line Bourel-Bonnet,*,† Eve-Isabelle Pecheur,‡,| Cyrille Grandjean,†,⊥ Annick Blanpain,†Thorsten Baust,§,∇ Oleg Melnyk,† Bernard Hoflack,‡,§,∇ and Helene Gras-Masse†

UMR 8525 CNRS, Institut Pasteur de Lille et Universite de Lille 2, France, FRE 2377 CNRS,Institut de Biologie de Lille, 1 rue du Pr. Calmette, 59021 Lille, France, and Biotec, Proteomics,TU Dresden, Max-Planck-Institute for Molecular Cell Biology and Genetics, Pfotenhauerstrasse 108,01307 Dresden, Germany. Received April 13, 2004; Revised Manuscript Received January 6, 2005

Synthetic peptidoliposomes have been designed and prepared according to a chemoselective ligation.Two aldehyde-functionalized lipidic anchors were synthesized and incorporated into the lipidic bilayersof unilamellar liposomes during their preparation. Complementary hydrazino acetyl peptides weresynthesized on the solid phase using N,N′,N′-tri(tert-butyloxycarbonyl)-hydrazino acetic acid andfurther coupled to the aldehyde groups displayed at the surface of the vesicles. Coupling yields weremeasured by amino acid hydrolysis following total acid hydrolysis. The ligation methodology provedsuperior to the simple insertion of lipopeptides, which was performed for comparison in terms of yields,implementation, and reproducibility. To check whether the grafted-peptides were accessible andfunctional, cytoplasmic sequences of LAMP protein (lysosomal associated membrane protein), whichis involved in intracellular membrane trafficking, have been selected. Using this model, wedemonstrated in vitro the specific interaction of the synthetic LAMP-peptidoliposomes with thecytoplasmic adaptor protein AP-3, a result that contributes to the understanding of protein sorting incells. Thus, these results clearly indicate the usefulness of such peptidoliposomes, easily prepared byhydrazone chemoselective ligation, as a tool for biological investigation.

INTRODUCTION

The modification of the surface properties of liposomesby peptides or proteins has been extensively studied (1-3). Considering peptides, most of the time, their stableanchorage into the phospholipid bilayers requires theirderivatization by a lipidic foot (4, 5) composed of at leasttwo alkyl chains (6, 7). However, it is a challenging taskdue to the generally low solubility of the resultingconjugates. To circumvent these difficulties, alternativestrategies have been developed where the purified pep-tide is ligated to a functionalized lipidic anchor alreadyinserted in the membrane (8). In these strategies, thechemical bond between anchor and peptide is oftenchosen among the amide (9) or thioether bonds (3, 5, 10-12), disulfide bridge (13), and, sometimes, imine bond(14). However, peptides are randomly oriented, usually

by more than one side chain if their sequence comprisesmore than one lysine and/or cysteine.

Alternatively, the hydrazone bond has been proposedby some authors to site-specifically immobilize peptidesor proteins onto vesicles or microspheres (15). Conjugatesare prepared by mild periodate oxidation of N-terminusSeryl or Threonyl peptides (16) or sugar of antibodies’Fc fragment (17) followed by the incubation of the productwith hydrazide-phosphatidylethanolamine (18) containedin the bilayers. Advantageously, this method does notrequire any activating agent, nor does it generate anyside-product. The reverse reaction consisting of thecoupling of amphiphilic aldehyde-like anchors with syn-thetic hydrazino acetyl peptides remains unprecedented(Scheme 1).

This strategy can now be envisaged because of therecent progress concerning the preparation of the lattercompounds. In particular, the use of the N,N′,N′-tri(tert-butyloxycarbonyl)-hydrazino acetic acid allows the facileintroduction of the hydrazino-group on peptides usingsolid-phase peptide synthesis (19, 20). Notably, thisapproach does not require extra steps to functionalize thepeptides after cleavage from the resin and purificationas compared to the synthesis of glyoxyl-peptides. More-over, sulfur-containing peptides are more easily acces-sible because no deleterious periodate oxidation is per-formed. At any case, this reverse strategy will probablycomplement and reinforce the attractiveness of thehydrazone chemoselective ligation.

* To whom correspondence should be addressed. Phone: 0033 (0)3 20 96 49 49. Fax: 00 33 (0)3 20 96 47 09. E-mail:lbourel@pharma.univ-lille2.fr.

† UMR 8525 CNRS.‡ FRE 2377 CNRS.§ Max-Planck-Institute for Molecular Cell Biology and Genet-

ics.| Present address: Structural NMR and Bioinformatics, UMR

5086 CNRS, IBCP, Lyon, France.⊥ Present address: Unite de Chimie Organique, URA CNRS

2128, Institut Pasteur, Paris, France.∇ Present address: Biotechnologisches Zentrum der TU-

Dresden, BioInnovationsZentrum, Dresden, Germany.

450 Bioconjugate Chem. 2005, 16, 450−457

10.1021/bc049908v CCC: $30.25 © 2005 American Chemical SocietyPublished on Web 02/25/2005

To test its feasability, we recently reported the solid-phase synthesis of an R-oxo aldehyde anchor 1a (Figure1), within 25% overall yield. As expected, 1a was suc-cessfully coupled to a model hydrazino acetyl peptidewithin 81% RP-HPLC conversion and 41% isolated yieldin an organic-aqueous solution (21). Moreover, we re-cently performed a comprehensive physicochemical studyof 1a reactivity to form hydrazone bonds on colloids justas the stability of the latter (22). Briefly, we demon-strated in that paper that hydrazone ligation offered highchemoselectivity and versatility. As we intended to usethe hydrazone ligation to target the controlled release oftherapeutic agents by vesicles, an accurate measure ofthe ligation bond stability was performed to ensure itwould stand long exposures to physiological conditions.The mechanism of the reaction in solution as well as indifferent self-organized systems (micelles, liposomes, andmultilamellar vesicles) was investigated. In solution,submicromolar stability was achieved as well as half-livesof several weeks (as an example, the hydrolysis rateconstants estimated at pH 7.4 and 25 °C gave for ourligation products half-lives of 23 to 800 days and shouldbe long enough at 37 °C to touch and attach their celltarget in vivo). The kinetics and stability were bothenhanced in colloidal media thanks to autoassociationeffects. Finally, the grafting of synthetic mannose recep-tor-ligands onto onion-like vectors to target humandendritic cells provided a first example of the efficiencyof this strategy (23).

To further demonstrate the usefulness and the versa-tility of our strategy, and particularly for probing biologi-cal processes, we describe herein the synthesis of a secondaldehyde anchor (1b, Scheme 2), the inclusion of 1a and1b into liposomes, and their reaction with hydrazino

acetyl peptides, via R-oxo hydrazone (A) or hydrazone (B)bonds, respectively, to obtain peptidoliposomes (Scheme1).

Peptido- or proteoliposomes have been used for variouspurposes in the cell biology of membranes, in particularto reconstitute in vitro several aspects of intracytoplasmicmembrane trafficking. In eukaryotes, vesicular intracel-lular transport is mediated by several coat components(COPs) and adaptor proteins (APs), the latter functioningwith clathrin (32-35). These cytosolic coat componentsbind to cytoplasmic domains of transmembrane proteinsto segregate them into transport vesicles. Previously, theinteraction of COPs had been reconstituted in vitro usingchemically defined proteoliposomes (36-38).

The AP-3 complex interacts in vivo with a tyrosine-based sorting motif in the cytoplasmic domain of trans-membrane LAMP (lysosomal-associated membrane pro-tein) (39). We show here that AP-3 can specificallyinteract in vitro with liposomes onto which a syntheticcytoplasmic domain of LAMP has been coupled.

To demonstrate this, hydrazone-based peptidolipo-somes were incubated with pig brain cytosol. Thesepeptidoliposomes bore either the wild-type sequence ofthe LAMP cytoplasmic domain (wt) or a mutated se-quence in which the key tyrosine residue of the sortingmotif had been replaced by an alanine, thereby producinga nonfunctional sorting signal. In this context, AP-3 wasspecifically recruited onto wt LAMP-bearing liposomes,demonstrating that synthetic peptidoliposomes are excel-lent versatile tools for the in vitro study of membranetrafficking. More generally, in a context of reconstitutedconditions in vitro, the necessity or the role of eachpartner, reagent, and/or condition can be studied.

EXPERIMENTAL PROCEDURES

Chemicals and Antibodies. All Fmoc protectedamino acids (L-Arg(Pmc),1 L-Asn(Trt), L-Asp(OtBu), L-Gln-(Trt), L-Glu(OtBu), L-His(Trt), L-Lys(Boc), L-Lys(Fmoc),L-Ser(tBu), L-Thr(tBu), L-Tyr(tBu)), coupling agents (HOBt,HBTU, BOP), and resins (Wang, Novasyn) were pur-chased from Novabiochem. Chemicals were from Sigma-Aldrich except cyanuric fluoride, which was from Lan-caster. Solvents were from Acros.

Lipids (phosphatidylcholine (PC), phosphatidyletha-nolamine (PE), phosphatidylserine (PS), and cholesterol(chol)) were from Avanti Polar Lipids. The antiproteasecocktail, GTP, and GTP-γ-S (the nonhydrolyzable GTPanalogue) were from Roche Biochemicals, and reagents

Scheme 1. r-Oxo Hydrazone (A) or Hydrazone Ligation Principles (B), and Corresponding Peptido Liposomes

Figure 1. Amphiphilic R-oxo aldehyde 1a (with X ) Tyr-NH-(CH2)3O[(CH2)2O]2-(CH2)3-NH-COCH2OCH2CO-Gly-NH-(CH2)3-).

Scheme 2. Solution-Phase Synthesis of Anchor 1ba

a Conditions and reagents: (i) Swern oxidation (35%); (ii) Dess-Martin oxidation (86%).

Technical Notes Bioconjugate Chem., Vol. 16, No. 2, 2005 451

for chemiluminescence detection were from Pierce. Theantibodies used in this study were as follows: affinity-purified rabbit polyclonal 2583 against the σ subunit ofAP-3, recognizing a doublet on Western blots (the σ3 Aand B isoforms); monoclonal antibody 100/3 against theγ-subunit of AP-1 (Sigma Immunochemicals); monoclonalantibody maD against the â subunit of COP I (SigmaImmunochemicals). The secondary antibodies used weregoat anti-mouse and goat anti-rabbit conjugated tohorseradish peroxidase (Sigma Immunochemicals).

RP-HPLC Eluents. A: TFA 0.05% in H2O. B: TFA0.05% in CH3CN/H2O 80/20 (v/v). C: TFA 0.05% inpropan-2-ol/H2O 40/60 (v/v). D: CH3CN/potassium phos-phate buffer 50 mM, pH 7.21 20/80 (v/v). E: CH3CN/potassium phosphate buffer 50 mM, pH 7.21 75/25 (v/v).

Buffers for Biological Experiments. F: 1.79 mL ofcitric acid 0.1 M + 3.21 mL of Na2PO4 0.2 M + 5 mL ofwater to reach 100 mM, pH 6.4. G: 25 mM Hepes-KOHpH 7.2 + 125 mM KOAc + 2.5 mM MgOAc + 1 mM DTT.

Synthesis of anchor 1a was performed as describedin ref 21.

Synthesis of Anchor 1b. Di-O-hexadecyl-rac-glycer-aldehyde was obtained using Dess-Martin’s oxidationreagent. CH2Cl2 was distilled over P2O5. Dess-Martinperiodinane, 15% weight solution in CH2Cl2 (287 mg, 0.10mmol), was added to a solution of di-O-hexadecyl-rac-glycerol (50 mg, 0.09 mmol) in CH2Cl2 (2 mL) under N2,with stirring. After 30 min, the resulting suspension wasdiluted with 9 mL of CH2Cl2/heptane 2/1 (v/v) and addedto 10 mL of 1 M NaOH. After 10 min of stirring, theorganic layer was washed with water, dried over Na2-SO4, and concentrated under reduced pressure. The whiteresidue was purified by chromatography on silica gel(eluent: heptane/AcOEt 95/5 (v/v)) to give 1b (43 mg,86%) as a white solid. 1H NMR (300 MHz, CDCl3) δppm: 9.24 (d, 1H, J ) 1.4 Hz), 3.81 (ddd, 1H, J ) 1.4,4.0 and 5.4 Hz), 3.71 (dd, 1H, J ) 4.0 and 10.5 Hz), 3.68(dd, 1H, J ) 5.4 and 10.5 Hz), 3.63 (dt, 1H, J ) 6.7 and9.2 Hz), 3.56 (dt, 1H, J ) 6.6 and 9.2 Hz), 3.48 (dt, 1H,J ) 6.5 and 9.3 Hz), 3.40 (dt, 1H, J ) 6.6 and 9.3 Hz),1.64 (q, CH2, J ) 6.6 Hz), 1.55 (q, CH2, J ) 6.9 Hz), 1.25(br s, 26CH2), 0.88 (t, 2 × 3H, J ) 6.4 Hz, 2CH3). 13CNMR (75 MHz, CDCl3) δ ppm: 203.3, 84.0, 72.1, 71.5,70.0, 32.1, 29.9, 29.8, 29.6, 29.4, 26.2, 22.9, 14.3. TOF-PDMS: m/z 539 [M + H]+.

Synthesis of the Dipalmitoyl Peptides 2a and 2b.A Fmoc/tBu peptide synthesis was performed on 0.25mmol of Wang resin in a 431A syntheziser (AppliedBiosystems, UK) with a double coupling 4 equiv in situactivation of amino acid in the presence of HBTU (4equiv), HOBt (4 equiv), DIEA (16 equiv), and Fmoc-protected amino acids to obtain the two expected Fmocpeptidyl resins. The last Fmoc was removed manuallyby a piperidine/NMP treatment 2/8 (v/v) within 2 and 20min, and the resin was washed with NMP. Fmoc-Lys-(Fmoc)-OH was introduced manually as above. Follow-

ing an ultimate deprotection, palmitic acid (8 equiv)reacted first under HBTU (8 equiv), HOBt (8 equiv)activation with DIEA (32 equiv) onto the deprotected di-amino peptidyl resin. To prepare the second acylation,palmitic acid (8 equiv) was activated by cyanuric fluoride(32 equiv) in the presence of pyridine (8 equiv) underargon for 2 h in CH2Cl2/tBuOMe 4/1 (v/v) (24). Followingan aqueous washing, CH2Cl2 was evaporated, and thefluoride was solubilized in DMF and reacted in parallelon the resins in the presence of DIEA (16 equiv) for 3 h.A total acid hydrolysis of the peptidyl resin conductedwith HCl/propionic acid 1/1 (v/v) at 130 °C for 3 h gavequantification results as expected. The dipalmitoyl pep-tidyl resins were cleaved by TFA in the presence of H2O/phenol/ethanedithiol/thioanisole: 0.5 mL/0.75 g/0.25 mL/0.5 mL in 10 mL of TFA for 2 h. Small amounts (37 mgout of 600 mg) of crude lipopeptides 2a and 2b werepurified by preparative RP-HPLC (C3 Zorbax column,300 Å, 1.8 µm, 15 × 500 mm, 4 mL min-1, λ ) 225 nm,50 °C, eluents A and B, B 0 to 100% in 30 min, 100% Bfor 10 min). 7 mg of 2a (19%) and 14 mg of 2b (38%) wereobtained. Identity was controlled by MALDI-TOF massspectrometer and purity by RP-HPLC (C3 Zorbax, 4.6 ×200 mm, 1 mL min-1, λ ) 215 nm, 50 °C, eluents A andB, B 0 to 100% in 30 min, 100% B for 10 min). Totalacidic hydrolyses in evacuated sealed tubes with 6 N HCl/phenol 10/1 (v/w) at 110 °C for 24 h followed by aminoacid analysis were in agreement with expected results.

The synthesis of hydrazino acetyl peptides 3 wasreported in refs 19, 20 for other applications. Briefly,Fmoc-tBu peptide syntheses were performed on 0.1mmol of Wang resin in a Pioneer syntheziser (AppliedBiosystems, UK) with a 10 equiv in situ activation ofamino acid in the presence of HBTU, HOBt, DIEA, andprotected amino acids to obtain the Fmoc peptidyl resins.Fmoc was removed automatically, and the deprotectedN-terminal amine was acylated by N,N′,N′-tri(tert-buty-loxycarbonyl)-hydrazino acetic acid using a HBTU/HOBtactivation. TFA/H2O/TIS 9.5/0.25/0.25 (v/v/v) allows thedeprotection and cleavage of peptides 3 then precipitatedin cold Et2O, washed and purified by preparative RP-HPLC (C18 Nucleosil column, 15 × 500 mm, 4 mL‚min-1,λ ) 225 nm, 50 °C, eluents A and C, C 0 to 100% in 30min, 100% C for 10 min). The peptides’ purities werecontrolled by RP-HPLC, and their identities were con-trolled on a MALDI-TOF spectrometer. 3a: >95%, m/zobserved 1446.6 ([M + H]+). 3b: >97%, m/z observed1353.82 ([M + H]+).

Chemoselective ligation between anchor 1a andLAMP wt peptide 3a in solution was performed asreported in ref 21. Briefly, 4.5 mg of anchor 1a (3.5 µmol)in CHCl3/MeOH 1/1 (v/v) (105 µL) was added to 6.6 mgof hydrazino acetyl peptide 3a (3.5 µmol) solubilized inpropan-2-ol/acetate buffer 10 mM pH 5:8/2 (v/v) (500 µL)at 37 °C. For a better stirring of the suspension, 1.3 mLof propan-2-ol/acetate buffer 1/1 (v/v) was added. RP-HPLC analyses were performed on a C3 Zorbax column(4.6 × 300 mm, 1 mL‚min-1, λ ) 215 nm, 70 °C, eluentsA and B, gradient: 0-100% B over 30 min, 100% B for5 min, 0% B for 10 min). After 3 h, the RP-HPLCconversion could be calculated as high as 81% on thebasis of peptide 3a disappearance. The crude compoundwas purified on a C3 Zorbax column, (15 × 500 mm, 4mL‚min-1, λ ) 225 nm, 50 °C, same eluents and gradi-ents) to obtain 41% isolated yield.

Chemoselective Ligation between Di-O-hexadec-yl-rac-glyceraldehyde 1b and LAMP wt Peptide 3ain Solution. 1b (1 mg, 1.8 µmol) and 3a (3.53 mg, 1.8µmol) were added to 330 µL of a mixture of CHCl3/2-

1Abbreviations: Boc, tertiobutyloxycarbonyl; BOP, benzo-triazole-1-yl-oxy-tris-(dimethylamino)-phosphonium hexafluoro-phosphate; DBU, 1,8-diazabicyclo [5,4,0]undec-7-ene; DIEA,diisopropylethylamine; DMAP, 4-N,N-(dimethylamino)pyridine;DMF, N,N-dimethylformamide; DMSO, dimethyl sulfoxide;DTT, dithiothreitol; ES-MS, electrospray mass spectrometry;Fmoc, 9-fluorenylmethyloxycarbonyle; HBTU, 2-(1H-benzotri-azole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate;HOBt, N-hydroxybenzotriazole; IPT, isopropylidene tartrate;Mtt, 4-methyltrityl; NMP, N-methyl pyrrolidone; OSu, N-hydroxysuccinimide ester; Pam, palmitoyl; PE, phosphatidylethanolamine; tBu, tertiobutyl; TFA, trifluoroacetic acid; TIS,triisopropyl silane; TNBS, 2,4,6-trinitrobenzenesulfonic acid.

452 Bioconjugate Chem., Vol. 16, No. 2, 2005 Technical Notes

methyl-2-propanol/citrate-phosphate buffer 10 mM, pH5.19, 8/10/15 (v/v/v). The suspension was stirred at roomtemperature for 4 h and at 37 °C overnight. Conversionwas estimated to 60% from analytical RP-HPLC. Theresulting mixture was then diluted with water andlyophilized. The pale yellow powder was purified by RP-HPLC on a C3 Zorbax column (15 × 500 mm, 4 mL‚min-1,λ ) 225 nm, 50 °C, eluents D and E, gradient: 0-100%over 40 min) to give the expected compound in about 15%isolated yield.

Functionalized Liposomes Preparation. Anchors1a or 1b were solubilized in CHCl3/MeOH 1/1 (v/v) at a10 mM final concentration. A mixture of PC/PE/PS/chol/anchor 1a or 1b 40/30/10/10/10 (by mole) in a CHCl3/MeOH 1/1 (v/v) solution was evaporated to dryness undera stream of nitrogen and resuspended in buffer F forfurther coupling to peptides such as 1 mL of suspensioncontains 5 µmol of total lipids and, among them, 0.5 µmolof anchor. After vigorous vortexing, the lipid suspensionwas submitted to 10 freeze-and-thaw cycles in liquidnitrogen and in a 37 °C water bath, respectively, andfiltered through polycarbonate 400 nm-membranes to sizethe liposomes homogeneously. In control liposomes,anchor was replaced by PC.

Next, 0.5 µmol‚mL-1 (1 equiv considering total anchor)of hydrazino acetyl peptide solution (3 mM in buffer F)was reacted to the suspension. The liposome/peptidemixture was left overnight at room temperature forcoupling to occur, and unbound peptide was removed bygel filtration over a Sephadex G-25 column with bufferG (NAP 5 columns, Amersham Pharmacia Biotech).

Amino Acid Analysis. 100 µL of liposome suspensionswas submitted to a 48 h total acid hydrolysis with 1 mLof HCl 6 N/phenol 10/1 (v/w) at 110 °C in the presence of10 µL of norleucine 0.5 mM in evacuated sealed tubes.The quantification of each immobilized peptide wasachieved by the determination of the subsequent aminoacid composition using a Beckman amino acid analyzermodel 7300 with ninhydrin detection.

Coat Protein Recruitment Assay. Pig brain cytosolwas prepared from fresh brains obtained at the localslaughterhouse. Brain slices were transferred to homog-enization buffer (25 mM Hepes-KOH pH 7.4, 250 mMsucrose, 1 mM EDTA supplemented with an antiproteasecocktail) on ice. Homogenization was done with a Waringblender in a 2-to-1 buffer-to-tissue (w/w) ratio. The crudehomogenate was centrifuged at 4 °C at 3000g for 10 min,then at 10 000g for 20 min, and finally at 100 000g for60 min. The final supernatant served as the cytosolicfraction and was flash-frozen in liquid nitrogen beforestorage at -80 °C. Before use, cytosol was rapidly thawedand desalted over Sephadex G-25 (PD-10) columns previ-ously equilibrated with buffer G. Any aggregated mate-rial was then removed by centrifugation at 250 000g for30 min at 4 °C.

Recruitment reactions were performed in siliconizedmicrofuge tubes in a total volume of 200 µL. Typical

recruitment reaction mixtures contained buffer G and acombination of gel-filtered cytosol (5 mg‚mL-1 final),liposomes (200 µg‚mL-1), GTP (1 mM) or GTP-γ-S (0.1mM), and/or ARF1 (ADP Ribosylation Factor-1, a myris-toylated G-protein that acts as membrane anchor for theassembly of protein coats, 3 µg‚mL-1 final). Tubes werekept on ice, and binding reactions were initiated bytransfer at 37 °C. After 20 min, reactions were stoppedon ice, and membranes were recovered by centrifugationat 4 °C for 15 min at 20 000g. Pellets were solubilized inSDS sample buffer, boiled for 5 min, and analyzed bySDS-PAGE followed by immunoblotting using the rel-evant antibodies. Visualization was performed by chemi-luminescence, and quantitation was obtained by scanningand spot analysis with the NIH software version 1.60/PPC.

RESULTS AND DISCUSSION

Ligation versus Incorporation of Lipopeptidesinto Preformed Vesicles. In a first strategy, we envis-aged the preparation of synthetic peptidoliposomes bymixing all liposomal components including the peptidesas lipopeptides. To this aim, we envisaged the synthesisof peptidic sequences modified at the N-terminus positionby two palmitoyl (Pam) groups using standard solid-phase protocols (25, 26). The RP-HPLC purification oflipopeptides is often difficult due to their generally lowsolubility and to the line broadening induced by the fattyacid chain(s). Thus, the major difficulty we encounteredduring the lipopeptides’ production was associated withthe preparative RP-HPLC steps. The overall yields weremodest and hardly reproducible. Thus, synthetic dipalmi-toyl-peptides corresponding to the intracytoplasmic LAMPsequences 2a, and its control 2b where the tyrosineresidue was replaced by alanine, were produced as Pam-L-Lys(Pam)-peptides under moderate to average yields(19% for 2a and 38% for 2b, Table 1).

Once obtained, 2a and 2b lipopeptides were insertedinto preformed liposomes. No more than 3% insertion wasreached, calculated by amino acid analysis following totalacid hydrolysis (Table 1). This phenomenon was previ-ously reported by Elliott and Prestwich (27). In their case,as much as 90% lipopeptide was aggregated in a micellarform rather than inserted into liposomes. A weak solubil-ity and a propensity to aggregate are responsible for thebad insertion of lipopeptides into liposomes. Thus, dueto synthetic difficulties, this strategy turned out to beunapplicable to systematic and iterative studies.

We then envisaged a second strategy based on thechemical ligation principle, where peptides are selectivelyattached onto liposomes. In a project aimed at anchoringvarious and sometimes complex polyfunctional moleculesto lipid membranes, we have examined the utility of thehydrazone and R-oxo hydrazone bonds. The hydrazonelink was chosen because it could be achieved in theabsence of activating agent and did not generate any toxic

Table 1. Peptide Sequences, Synthesis Yields, and Insertion (I) or Ligation (L) to Liposomes Yields (Measured by theSame Quantitative Method, by Amino Acid Analysis, Mean of Three Experiments)a

name sequencesynthesis

yieldinsertion or ligation to

liposomes yield

2a (Pam)2-LAMP wild type (wt) Pam-K(Pam)-GRKRSHAGYQTI-OH 19% <3% (I)2b (Pam)2-LAMP mutant (Y-A) Pam-K(Pam)-GRKRSHAGAQTI-OH 38% <3% (I)3a LAMP wild type (wt) H2N-GGRKRSHAGYQTI-OH 35% 44% (L)3b LAMP mutant (Y-A) H2N-GGRKRSHAGAQTI-OH 40% 58% (L)a Liposomes compositions were as follows: (I) PC/PE/PS/chol 50/30/10/10 (by mol.); (L) PC/PE/PS/chol/anchor 1b 40/30/10/10/10 (by

mol.). Pam ) palmitoyl.

Technical Notes Bioconjugate Chem., Vol. 16, No. 2, 2005 453

or remaining side-product. We immobilized synthetichydrazine-like peptides onto aldehyde or R-oxo aldehyde-liposomes, a strategy that was unprecedented. Thisstrategy not only required the design and synthesis ofamphiphilic aldehyde-like anchors to be manipulated aslipids when preparing the liposome suspension (Figure1), but also those of hydrazino acetyl peptides (Scheme1). Concerning the ligation process, a first and compre-hensive work had been performed on the thermodynamicand kinetic parameters of the reaction (22, see Introduc-tion). In this study, the R-oxo hydrazone bond formedbetween vesicles and hydrazino compounds in aqueousbuffer was found to be totally chemoselective and quan-titative. Its stability in physiological conditions was inthe submicromolar range, and nonspecific adsorptioncould be diminished to 10-15% on average. These resultsformed the basis of our present work.

Anchors’ Design and Synthesis. A first anchor 1aof type Pam-L-Lys(Pam)-spacer-CO-CHO was pre-pared as previously described (Figure 1 (21)). The dif-ficulty in preparing amphiphilic compounds such as 1aresides in the fine-tuning of the physicochemical proper-ties to allow their solubilization, purification, and char-acterization. In addition, depending on the biomolecularinteractions studied, the anchored peptide should be closeto or distant from the membrane surface. Thus, to allowthe spacer arm to be easily modulated, anchors of thetype 1a were elaborated on solid phase using standardFmoc/tBu protocols (28, 29). Compound 1a was isolatedwith a 25% overall yield.

A further anchor 1b presenting the basic elementsrequired for this application was synthesized in solution(Scheme 2). It contained a double hexadecyl arm to beinserted among liposome phospholipids and an aldehydereactive head. Di-O-hexadecyl-rac-glyceraldehyde 1b wasfirst obtained starting from di-O-hexadecyl-rac-glycerolfollowing Swern’s oxidation procedure (30). 1b wasobtained after purification together with unreacted di-O-hexadecyl-rac-glycerol (35%). The reaction did not cometo its completion, which was assigned to the poor solubil-ity of the lipophilic molecules in cold CH2Cl2. Indeed, ashigh as 86% isolated yield could be obtained when theoxidation was performed at room temperature usingDess-Martin’s reagent (31).

To summarize, two types of synthetic anchors are nowavailable. Those of the type 1a are versatile in such away that four analogues were synthesized (10-50%overall yield according to the spacer X in 100-500 mgscale). Anchor 1b is simple and cheap and was alsoobtained in a 100 mg scale. Some PE derivatives arecurrently under investigation. This should reinforce theinterest of using a hydrazone bond in the preparation ofpeptidoliposomes.

Hydrazino Acetyl Peptides’ Synthesis. In parallel,R-hydrazino acetyl peptides were automatically produced.Their sequences corresponded to a part of the cytoplasmicdomain of the lysosome-associated membrane proteinLAMP (wild-type LAMP, wt) or to a mutated sequencewhere the tyrosine sorting motif was replaced by analanine (Table 1, entries 3a,b). A conventional Fmoc/tert-butyl solid-phase peptide strategy (28, 29) and ourrecently described tri-protected compound N,N′,N′-tri-(tert-butyloxycarbonyl)-hydrazino acetic acid, (Boc)2N-N(Boc)-CH2-COOH, were employed for the unambigu-ous introduction of the hydrazino acetyl group upon theselectively deprotected R-amino group of the N-terminalamino acyl resins (19, 20). Each crude R-hydrazino acetylpeptide was easily purified by RP-HPLC, freeze-dried asa TFA salt, and could be thoroughly characterized by

amino acid analysis, RP-HPLC, and MALDI-TOF-MS(yields 3a, 35%, 3b, 40%, amino acid analyses in ac-cordance with expected results, RP-HPLC purity superiorto 95%). It is noteworthy that hydrazino acetyl peptidesare unstable in the presence of acetonitrile and shouldbe purified with RP-HPLC eluents containing 2-propanolas was exposed in ref 19.

Thus, the solid-phase synthesis of hydrazino acetylpeptides requires fewer steps than that of N-terminusglyoxylyl peptides, where a deleterious periodic oxidationand a second purification are necessary. Here, hydrazinoacetyl peptides were readily obtained after cleavage fromthe resin and RP-HPLC purification. Moreover, in ourhands, hydrazino acetyl peptide synthesis offered a totalflexibility, and as many as 10 sequences from threemodels were easily assembled and functionalized in goodyields.2 These advantages are good reasons for applyingthe hydrazone chemical ligation.

In-Solution Ligation. 1a and 1b were engaged in achemoselective hydrazone ligation in solution with hy-drazino acetyl peptide 3a to control both the accessibilityand the reactivity of the functional groups. The reactionof 1a or 1b with 3a led to the formation of the corre-sponding R-oxo hydrazone with 81% and 60% RP-HPLCconversion and 41% and 15% isolated yield following RP-HPLC purification, respectively. Although successful,these two reactions remained very difficult to handle andrequired heating and a mixture of organo-aqueous sol-vents.

Ligation onto Liposomes. Ligation of hydrazinoacetyl peptides proved to be more convenient and efficientonto vesicles, as anchors 1a and 1b’s aldehyde headseemed to be more “available” in liposomes than inorgano-aqueous solution. These results are consistentwith what we reported in our physicochemical study:when the ligation was performed in self-organized media,its speed and its stability were enhanced due to autoas-sociation of the reagents.

The liposomes were prepared by integrating 10% (mol/mol) of one of the anchors 1a or 1b. Assuming that thevesicles were unilamellar, 0.25 mM aldehyde functionscould be reacted on the outer membrane (see Experi-mental Procedures for further numerical data). Theproteoliposomes were obtained by simple stirring of thefunctionalized liposomes with the R-hydrazino acetylpeptides 3 (1 equiv toward total aldehydes, i.e., 2 equivtoward outer exposed aldehydes, Scheme 3). The incuba-tion mixture was left overnight for the reaction to becompleted. However, we routinely observed that 5 h weresufficient for a total conversion.

Unbound peptide was removed by gel filtration. Theaddition of the peptidic reagent and the workup imposeda 2.5-fold dilution of the liposome suspension. Thus, 0.1µmol of peptides was the maximum to be anchored permilliliter of recovered liposome suspension.

Quantification of the amount of peptide associated toliposomes was performed by amino acid analysis follow-ing total acid hydrolysis using norleucine as an internalstandard. As the medium was not totally homogeneous,the acidolysis was left over 48 h to be as complete aspossible and the analyses were performed three times(three samples) for more accuracy.

2Baust, T., Bourel-Bonnet, L., Gras-Masse, H., and Hoflack,B. (2004) Selective assembly of AP-1 and AP-3 coats ontosynthetic membranes: Requirement for ARF1, sorting signalsof specific cargo transmembrane proteins and phosphatidyli-nositol-phosphates. Unpublished results.

454 Bioconjugate Chem., Vol. 16, No. 2, 2005 Technical Notes

Liposomes containing anchor 1b (1b-liposomes) werefirst coupled to hydrazino peptide 3a, and a reproducible44% ( 3% coupling yield was measured (mean 44%,Table 1). When these vesicles were coupled to hydrazinopeptide 3b, the coupling yield was comprised between49% and 70% (mean 58%, Table 1). Apart from uncom-plete amino acid hydrolysis, these variations may be dueto the dilution of the suspensions occurring during thepurification by gel filtration.

Therefore, to further confirm these results, the peptideremaining in the supernatants after ligation onto 1b-liposomes was quantified by RP-HPLC. The amount ofrecovered peptide was measured (assuming that thepeptide amount that was not recovered was anchoredonto the liposome) and was compared to the amino acidanalyses of the suspensions. A good correlation could beestablished between the average amount of peptideremaining in the supernatant and that of the peptidepresent in the suspension (51% against 50%). Obtainedeither by RP-HPLC quantification or by amino acidanalysis, these results demonstrate that ligation isgreatly superior to insertion of lipopeptides for thepreparation of peptidoliposomes. These results are re-ported in Table 1 (lanes 2a and 2b as compared to lanes3a and 3b).

Biological Evaluation of the Vesicles. The scopeof our strategy stretches to ligand/receptor interactions.We chose to illustrate it in the field of cellular biology.Precisely, our peptidoliposomes were used to reconstitutein vitro a particular aspect of membrane trafficking. Inthis work, we demonstrated that AP-3 complex canspecifically interact in vitro with liposomes onto whichthe cytoplasmic domain of LAMP has been coupled.

Coat Recruitment Assay with the Peptidolipo-somes. 1b-Liposomes were coupled to 3a or 3b, twohydrazino acetyl dodecapeptides displaying either thetyrosine sorting motif of LAMP cytoplasmic tail or analanine, respectively (Scheme 3). After the free peptidewas eliminated by gel filtration, the suspensions wereincubated with pig brain cytosol. Vesicles were recoveredafter centrifugation at high speed. After washing, specificmonoclonal antibodies were used to detect and identifywhich cytosolic proteins were immobilized (in otherwords, “recruited”) onto the liposomes by western blotting(Figure 2).

When comparing lanes (a) and (b) (LP control with noanchor and LP-LAMP Y-A exposing the anchored mu-tated peptide, respectively) to lane (c) (LP-LAMP wtexposing the anchored wt LAMP peptide), it becomes

clear that AP-3 recruitment is higher when liposomesbear an intact LAMP cytosolic domain with a tyrosine-based sorting signal. Most notably, no AP-3 was recruitedonto liposomes bearing the mutated LAMP peptidelacking the tyrosine sorting motif, emphasizing thespecificity of our recruitment reaction. LP control repre-sents liposomes without anchor (1b was replaced by PC,see Experimental Procedures) exposed to peptide 3a andgel-filtered. For this type of suspension, the amino acidquantification was not reproducible, and peptide amountswavered between 10% and 40%, due to peptide adsorptionin a nonspecific manner. Because we observed that AP-3could not be efficiently recruited onto these liposomes,we concluded that covalent binding of the peptide wasnecessary for the peptide to interact with the AP-3 coat.These results are consistent with some previously re-ported work. As an example, Pecheur and Hoekstrashowed that liposomes’ fusion could only happen whena fusion-inducing peptide was covalently bound to thelipid bilayer and not when it was adsorbed to the surface(40). We suggest that, in our case, this covalent bindingallowed a correct exposition of the peptide to the cytosol,thus presenting the sorting motif in a proper way.Additional controls showed that, in the absence of pep-tide, even when the anchor 1b was present, AP could not

Scheme 3. Onto-Liposome Ligation of Anchor 1b to Peptides 3a or 3b

Figure 2. AP-3 membrane recruitment is LAMP-dependent.(a) LP control represents liposomes containing no anchorexposed to peptide 3a. (b) LP-LAMP Y-A and (c) LP-LAMP wtwere prepared with peptides 3b and 3a, respectively. Afterincubation with a pig brain cytosol, AP-1, AP-3, and COP boundto liposomes were detected by western blotting using specificantibodies.

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get recruited onto the liposomes, reinforcing the specific-ity of our recruitment reaction (see Supporting Informa-tion).

Selective AP-3 Recruitment onto LAMP-Peptido-liposomes Is a Function of LAMP Coupling Yield.1b-Liposomes containing a fixed amount of lipidic anchor(10 mol %) were incubated with increasing amounts ofLAMP wt peptide 3a, and 1 equiv means 1 equiv towardthe overall anchor (see experimental conditions for liga-tion). Figure 3 shows that AP-3 binding depends on theconcentration of LAMP wt peptide present on liposomes.AP-3 binding onto liposomes exposing the mutated LAMPpeptide was not observed, whatever the amount ofmutated peptide was. This confirmed the specificity ofour in vitro system.

CONCLUSION

We successfully prepared hydrazone-based peptidol-iposomes. We designed and synthesized two differentaldehyde-functionalized anchors able to covalently im-mobilize hydrazino acetyl peptides onto liposomes. Thisligation strategy was more efficient and versatile thanthe direct insertion of lipopeptides into liposomes. Severaltypes of peptidoliposomes could be prepared in parallel.As an example to illustrate the functionality of thevesicles, we have successfully reconstituted the selectiveinteraction of the AP-3 coat with cytoplasmic domainsof transmembrane proteins covalently immobilized onliposomes. This methodology can be applied to differentcoat components involved in different aspects of mem-brane trafficking and in a more general manner to anybiological problem into which transmembrane or mem-brane-associated proteins are implicated.

ACKNOWLEDGMENT

We thank Dr. Pascale Chenevier for fruitful scientificdiscussions, and Frederic Malingue, Eric Diesis, andHerve Drobecq (UMR 8525) for technical assistance. Wegratefully acknowledge financial support from the Min-istere de la recherche (L.B.-B. received a young scientist

“ACI jeune chercheur” financial support in 2001), Uni-versite de Lille 2, Centre National de la RechercheScientifique (CNRS), Institut Pasteur de Lille, Agencepour la Recherche contre le Cancer (ARC), and AgenceNationale de Recherche sur le SIDA (ANRS).

Supporting Information Available: Additional experi-mental data. This material is available free of charge viathe Internet at http://pubs.acs.org.

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