Chemo-enzymatic Routes to Lipopeptides and Their Colloidal

May 23, 2014 - Department of Chemistry & Biomolecular Science, Clarkson University, Potsdam, New York 13699, United States. Langmuir , 2014, 30 (23), ...
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Chemo-enzymatic Routes to Lipopeptides and Their Colloidal Properties Geng Li,† Jun Wu,‡ Xu Qin,† Jianhui Zhu,† Kodandaraman Viswanathan,† He Dong,§ P. Somasundaran,‡ and Richard A. Gross*,† †

Department of Chemistry and Biology, Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute (RPI), 4005B BioTechnology Building, 110 Eighth Street, Troy, New York 12180, United States ‡ Department of Earth and Environmental Engineering, Henry Krumb School of Mines, NSF I/UCRC for Particulate and Surfactant Systems, Columbia University, New York, New York 10027, United States § Department of Chemistry & Biomolecular Science, Clarkson University, Potsdam, New York 13699, United States S Supporting Information *

ABSTRACT: A unique chemo-enzymatic route to lipopeptides was demonstrated herein that, relative to alternative methods such as solid-phase peptide synthesis (SPPS) and microbial synthesis, is simple, efficient, and scalable. Homoand co-oligopeptides were synthesized from amino acid ethyl esters via protease catalysis in an aqueous media, followed by chemical coupling to fatty acids to generate a library of lipopeptides. Synthesized lipopeptides were built from hydrophobic moieties with chain lengths ranging from 8 to 18 and peptides consisting of oligo(L-Glu) or oligo(L-Glu-co-L-Leu) with an average of seven to eight repeating units. The chemical structures of the lipopeptides were characterized and confirmed by NMR and matrix-assisted laser desorption/ionization (MALDI). The colloidal and interfacial properties of these lipopeptides were characterized and compared in terms of the hydrophobic chain length, oligopeptide composition, and solution pH. The results showed correlation between the interfacial activity of the lipopeptides and the hydrophobicity of the fatty acid and oligopeptide headgroup, the effects of which have been semiquantitatively described in the manuscript. Results from these studies provide insights into design principles that can be further expanded in future work to access lipopeptides from protease-catalysis with improved control over sequence and exploring a wider range of peptide and lipid compositions to further tune lipopeptide biochemical and physical properties.

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fermentation from recombinant organisms.10 These methods are important and versatile providing peptides with a precise sequence and chain length. To expand the repertoire of existing microbial surfactant structures, research has been conducted to prepare completely synthetic lipopeptides by solid-phase peptide synthesis with selective conjugation of a lipid moiety.11−14 For example, S-[2,3-bis(palmitoyloxy)propyl]cysteine (Pam2Cys), a lipid moiety, was conjugated to peptides prepared by SPPS by a convergent click chemistry approach.13 In another example, SPPS and lipid conjugation was used to prepare caspofungin mimics with high and selective antifungal activities against Candida strains.14 However, review of these and other related publications on solid and solution phase peptide synthesis shows these methods require tedious step-bystep reactions involving toxic solvents and reagents that lead to product costs that are only compatible with high value

INTRODUCTION

Biosurfactants are naturally produced surface-active agents that offer several advantages over chemically synthesized surfactants, such as low toxicity, inherently high biodegradability, and ecological acceptability. Among naturally occurring surfactants, lipopeptides are particularly interesting due to their high surface activities and potential for use as therapeutics. Lipopeptides are a class of oligopeptides that are covalently linked with lipid chains. The oligopeptides can be either linear or cyclic. A large variety of lipopeptides with surface activity and/or antibiotic activity have been isolated and reported1 including bacillomycin,2 iturin,3 mycosubtilin,4 plipastatin,5 surfactant BL866 and hallobacillin.7 They are usually produced extracellularly or are found within cell membranes of yeast, bacteria, or filamentous fungi.8 Despite their excellent potential to provide new therapeutics, significant challenges remain to achieve controlled microbial synthesis of lipopeptides in high purity, quantity, and with batch-to-batch reproducibility of molecular composition. Currently, chemical routes to synthesize oligopeptides relies primarily on solid phase peptide synthesis (SPPS)9 and © 2014 American Chemical Society

Received: February 3, 2014 Revised: April 20, 2014 Published: May 23, 2014 6889

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Scheme 1. Chemo-enzymatic Routes to Lipopeptides

Table 1. Lipopeptides Synthesized for Study in This Work product #

lipid

oligopeptidea

feed ratios (L-Glu/L-Leu) (mol/mol)

yield (%)b

1 2 3 4 5 6 7 8 9

lauric acid (C12) lauric acid (C12) lauric acid (C12) octanoic acid (C8) decanoic acid (C10) lauric acid (C12) myristic acid (C14) palmitic acid (C16) stearic acid (C18)

oligo(L-Glu-co-12 mol % L-Leu)7.5 oligo(L-Glu-co-21 mol % L-Leu)7.1 oligo(L-Glu-co-33 mol % L-Leu)7.4 oligo(L-Glu)7.9 oligo(L-Glu)7.9 oligo(L-Glu)7.9 oligo(L-Glu)7.9 oligo(L-Glu)7.9 oligo(L-Glu)7.9

90/10 80/20 70/30 n/a n/a n/a n/a n/a n/a

65 68 66 68 65 68 66 65 56

lipid-conjugated N-termini (%)c 96 95 96 96 97 95 96 95 91

± ± ± ± ± ± ± ± ±

2 2 1 2 1 2 2 2 1

a

Oligopeptides are written as oligo(L-Glu-co-X mol % L-Leu)y, where X is the mol content of Leu units and y is the DPavg. Both of these values were determined by 1H NMR. bYield (wt %) of lipopeptide was calculated after precipitation in cold water and washing the precipitate with ethanol (see Experimental Section). cPercent of terminal amine groups conjugated by the corresponding lipid moiety was determined by the ninhydrin quantitative method (see Experimental Section).

alternating peptides.25 This was accomplished by first preparing alanine-glycine ethyl ester (Ala-Gly-OEt) by standard chemical coupling. Subsequently, Ala-Gly-OEt was converted by papaincatalysis in 30s to (Ala-Gly)x (80%-yield, x = 9.4 ± 0.3). This methodology was extended to a wider range of alternating oligopeptides including (lysine-leucine)x.26 This paper describes the preparation of a unique family of lipopeptides via a chemo-enzymatic route and studies of their surface and interfacial properties. Homo and co-oligopeptides were synthesized by protease-catalyzed oligopeptide synthesis followed by conjugating a lipid chain to the oligopeptide Nterminus (Scheme 1). A small library of lipopeptides was prepared by simply changing the feeding ratio of the amino acid monomers and the length of the lipid chains used for postchemical modification. The surface and interfacial properties of these model compounds were investigated and correlated with structures of both the headgroup and the lipid tail. The successful ability to tune lipopeptide surface and interfacial properties herein validates further utilization of the synthetic approach in the fabrication of other functional lipopeptide based materials. Furthermore, results on interfacial properties obtained here provide the basis for further evaluation of the nanostructure assembly of these molecules.

applications such as for human therapeutics. Furthermore, use of recombinant organisms to produce peptides generally gives poor process efficiency that limits their use as above.15 New methods are needed that can extend the use of lipopeptides to a broad range of industrial and specialty chemical applications. This will require readily scalable peptide synthetic methods that can provide multipound peptide quantities at costs orders of magnitude below that of methods such as SPPS and microbial synthesis. To that end, our laboratory is exploring protease-catalyzed routes to oligopeptides from their corresponding amino acid ethyl ester monomers. Proteases have been successfully used to prepare a range of homo-16−21 and co-oligopeptides22−24 in aqueous media. Advantages of this approach relative to those above are (i) lower cost and high yields, (ii) reduced use of organic solvents, (iii) possible catalyst reuse, (iv) minimal racemization, (v) regio-selectivity that circumvents the need for amino acid side chain protection, and (vi) mild nonhazardous operating conditions. A potential disadvantage of protease-catalyzed oligopeptide synthesis is that oligopeptides are obtained as mixtures with respect to composition, sequence distribution and chain length. Recently, our group showed the potential to use protease-catalyzed oligopeptide synthesis to prepare strictly 6890

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RESULTS AND DISCUSSION

Synthesis and Chemical Structure Characterization. The molecular composition of synthesized lipopeptides is shown in Table 1. The oligopeptide headgroup contains either a homo- or co-oligopeptide, and the hydrophobic tail is composed of a fatty acid with varying chain lengths. The synthesis was achieved by papain-catalyzed homo- or cooligomerization of amino acid ethyl ester monomer(s) followed by covalent attachment of the fatty acid (Scheme 1). The 1H NMR spectra for all batches of homo-oligopeptides and cooligopeptides were identical to those displayed elsewhere.16,22 Peak assignments for these products were also based on those described previously by us elsewhere.16,22 To calculate the average chain length (DPavg) for oligo(γ-Et-L-Glu), comparison was made of the relative integration intensities of methine protons (NH−C[R]H−[CO]), associated with the α-carbon of γ-Et-L-Glu repeat units (4.3 ppm), and protons of N-terminal γ-Et-L-Glu units at 3.4 ppm. Similarly, for oligo(γ-Et-L-Glu-co-LLeu), direct comparison of the additive signal integrations due to methine resonances of L-Leu and γ-Et-L-Glu repeat units, both at 4.3 ppm, and the N-terminal methine resonances at 3.6 ppm, gave DPavg values of co-oligopeptides. The relative content of γ-Et-Glu and Leu repeat units along co-oligopeptides was determined from relative intensities of signals corresponding to the methyl protons ([CH3]2−CH−CH2) on the δcarbon of Leu residues (0.9 ppm), and the methylene protons (EtO[CO]−CH2−CH2) attached to the γ-carbon of γ-Et-LGlu repeat units. Experimentally determined compositions and DPavg values for the oligopeptide segment of lipopeptides are listed in Table 1. As shown in Scheme 1, lipopeptides were prepared by conjugating the aforementioned papain-catalyzed oligopeptides with N-hydroxysuccinimide activated fatty acids in the presence of DCC. Relative to spectra shown in previous studies of oligo(γ-Et-L-Glu) and oligo(γ-Et-L-Glu-co-L-Leu),16,22 conjugation of a fatty acid to the amino group at the peptide Nterminus forming amide bond led to changes in corresponding NMR spectra that are consistent with the formation of the resulting product (See 1H- and 13C NMR in Figures S1 and 2S recorded for the lipopeptide N-lauryl-oligo(γ-Et-L-Glu)7.9 (Product #6, Table 1)). For example, the 1H NMR spectrum of Product 6 in Figure S1 shows resonances at 2.11 ppm that correspond to the lipid α-methylene protons (CH2−CH2− CH2−[OC]−NH−oligopeptide). The 13C NMR spectrum (Figure S2b) of Product 6 has signals at 35.05, and 31.28, which correspond to carbons (−CH2−CH2−CH2−[OC]−NH−) and (−CH2−CH2−CH2−[OC]−NH−), respectively. In contrast, the α-methylene 13C NMR signal for carbon a of lauric acid appears at 34.10 ppm (−CH2−CH2−CH2−[O C]−OH) in Figure S2a. This downfield shift by 1.05 ppm upon amidation of lauric acid is constituent with lipopeptide product formation. The matrix assisted laser desorption/ionization-time-of-flight (MALDI-TOF) spectrum provided additional confirmation of product structures. For example, the MALDI-TOF chromatogram of N-lauryl-oligo(γ-Et-L-Glu)7.9 (Product #6), displayed in Figure 1, shows four series of ion peaks separated by mass 157 that equals the mass of a γ-Et-L-Glu repeat unit. The series of four isotopically resolved peaks correspond to [oligo(γ-Et-LGlu)x-C12+Na]+, [oligo(γ-Et-L-Glu)x-C12+K]+ and corresponding mass ions minus an ethyl moiety (associations with Na+ are marked by asterisks, associations with K+ appear as the

Figure 1. MALDI-TOF spectrum of N-lauryl-oligo(γ-Et-L-Glu)7.9 (Product #6).

fourth major peak in the cluster without any markers). That is, a fraction of synthesized oligopeptide chains lack one ethyl ester moiety, presumably hydrolyzed by protease catalysis during oligopeptide synthesis. Indeed, formation of populations of oligopeptides consisting of monocarboxyl oligomers and oligomers lacking free acid groups was similarly observed by us for papain-catalyzed γ-Et-L-Glu homo-oligomerizations.16 Figure S3 (Supporting Information) displays the MALDI-TOF spectrum of N-lauryl-oligo(γ-Et-L-Glu-co-21 mol %L-Leu)7.1 (Product #2, Table 1). The presence of lauryl units in molecular ions is consistent with the proposed product structure. Indeed, the absence of peaks above the signal-tonoise threshold corresponding to free peptides is further evidence that %-yields for conversion of peptides to lipopeptides are high. Moreover, the conversion of free peptides to lipopetides was determined by the ninhydrin method (see Experimental Section). Results of this analysis show that the percent of peptide N-termini that were converted to amides ranged between 95 and 97% with the exception of Product 8, which is 91%. Previously, our group studied the sequence distribution of oligo(γ-Et-L-Glu-co-L-Leu) by the combined methods of LCMS (to analyze the compositional distribution of triads generated by co-oligopeptide hydrolysis), comonomer relative reactivity ratios and MALDI-TOF.22 Indeed, study of Figure S3 shows that compositions of peptide moieties are highly variable and appear random, consistent with our previous work.22 Also, comonomer feed compositions are in excellent agreement with oligo(γ-Et-L-Glu-co-L-Leu) compositions (Table 1). This is also consistent with our previous results for this co-oligomerization.22 Chemo-physical Characterization of Colloidal Properties. Effects of the Chain Length on the Surface Activity. The equilibrium surface tension profiles for a series of N-acyloligo(L-Glu) lipopeptides where the N-acyl group consists of fatty acids with chain lengths varying from C8 to C18 (Products #4 to #8, Table 1) were determined by the Wilhelmy plate technique. Results of these measurements are shown in Figure 2. The CMC measurements were performed in DI water at pH values adjusted to 7.5, where glutamic acids units of oligopeptides are deprotonated to maintain solubility. Onset of the plateau range for surface tension/concentration curves identifies critical micelle concentration (CMC) values. Within the range of concentrations studied, micelle formation was not observed for C8/oligo(L-Glu). This is due to the relative short hydrophobic moiety along with the large polar headgroup. It is likely that the CMC of C8/oligo(L-Glu) is higher than its 6891

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sodium salts (6.9 mM, C14; 23 mM, C12; 95.5 mM, C10; 351 mM, C8).29 The CMC of a homologous series of surfactants is normally expressed as a function of the carbon chain length (straightchain) in the following form: log(CMC) = A − B·nc

(1)

Where A and B are constants specific to the homologous series and nc is the number of carbons on the hydrophobic chain of the surfactant. The plot of CMC (mM) versus N-acyl chain length in Figure 3 shows a linear relationship exists and gave values of A and B of 3.66 and 0.19, respectively. B is an indication of the free energy necessary to transfer a −CH2− group from vacuum into a micelle. The B value obtained from this family of lipopeptides is smaller than those of typical ionic surfactants,30 reflecting that head groups of the lipopeptides have a very strong affinity to water, which diminishes the repulsion between the carbon chain and water molecules. Influence of Oligopeptide Structure on the Surface Activity of N-Acyl-Oligopeptides. Surface active properties of lipopeptides were manipulated by keeping the N-acyl chain length constant at C12 while varying the hydrophilic oligopeptide headgroup polarity. To manipulate the polarity of the oligopeptide hydrophilic group, the average content of the hydrophobic amino acid L-Leu was varied within the oligo(L-Glu) segment. CMC values for N-lauryl-oligo(L-Glu-coL-Leu) co-oligomers with 12, 21, and 33 mol % L-Leu units (products 1, 2 and 3, Table 1) are summarized in Table S2 (see SI section). The results obtained for surface activity as a function of oligo(L-Glu-co-L-Leu) composition are plotted in Figure 4.

Figure 2. Surface activity profiles of N-acyl-oligo(L-Glu), where N-acyl groups consist of fatty acids with chain lengths from C8 to C18.

intrinsic aqueous solubility such that, as the concentration is increased, C8/oligo(L-Glu) precipitates, preventing determination of its CMC value. For C10, the plateau range onset is clearly seen. Increasing the fatty acid chain length resulted in a corresponding increase in lateral attraction between lipopeptide molecules, resulting in decreased CMC values. C12/oligo(LGlu) shows the highest surface activity, which reduces water/air interfacial tension down to ca. 31 mN/m when the concentration is above its CMC. The limited solubility at 25 °C of C18/oligo(L-Glu) preclude exploration of the full plateau range. The CMC and equilibrium surface tension results are summarized in Figure 3 and Table S1 (see SI section).

Figure 3. Relationship between chain length and CMC of N-acyloligo(L-Glu).

Figure 4. Surface activity profiles of lipopeptides with different oligopeptide compositions.

Interestingly, when the hydrophobic chain length increases above C12, the minimum surface tension values increase while the CMC decreases. This suggests looser packing of long lipopetides at the interface. Indeed, similar observations by others27,28 when studying amphiphilic molecules with large polar head groups was explained by the bending over of hydrophobic moieties resulting in loose packing of surfactant molecules at air/water interfaces. CMC values of N-acyloligo(L-Glu) are similar to those for fatty acid carboxylate

By increasing the average content of in C12/oligopeptides from 0 to 12, 21, and 33 mol %, CMC values decrease from 22.3 mM to 7.2 mM, 6.2 mM and 2.3 mM. Thus, the presence of leucine units in the polar headgroup decreases the headgroup hydrophilicity, resulting in significant increases in the corresponding lipopeptide surface activity. An empirical linear relationship (eq 2) was obtained by plotting CMC versus the mol %-L-Leu (Figure S4). 6892

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enhances solubility of the amphiphiles, and micelles form when the concentration reaches CMC. Increase of the solution pH to 9 results in a corresponding higher extent of peptide carboxyl group ionization. Consequently, the effective volume of head groups becomes larger as does electrostatic repulsion between lipopeptide molecules. Based on packing parameter theory,32

(2)

Micellar Size, Distribution, and Its Evolution. Figure 5 displays results obtained for the aggregate size distribution of

P = v /a0lc

(3)

where v is the volume occupied by the tail group, a0 is the section area of headgroup, and lc is the length of the hydrocarbon tail: when the effective volume increases, a0 increases, which leads to higher micelle curvature and a decrease in the number of molecules in a micelle. This result provides a working window for applications such as personal care formulations. Micellar Size of Different Lipopeptides by Dynamic Light Scattering Studies. The micelle size was further studied by dynamic light scattering (DLS), where dimensional information on micelles can be obtained. Micellar size distribution profiles of different lipopeptides are displayed in Figure S5, and the mean micelle sizes are listed in Table 2. For C8/oligo(L-Glu), Table 2. Mean Micellar Size of Different Lipopeptides by Dynamic Light Scattering Figure 5. Aggregate size distribution of N-lauryl-oligo(L-Glu) at 0.55M, 25 °C and pH 6.5, determined based on sedimentation velocity using AUC.

N-lauryl-oligo(L-Glu) at 0.55 M (25 °C, pH 6.5) using analytical ultracentrifugation (AUC). The sedimentation coefficient determined here is an indicator of aggregate size distribution. Results show that the lipopeptide molecules aggregate as small micelles. Calculations based on the sedimentation coefficient data31 reveal that the aggregation number is around 20−30 (detailed calculation method is provided in the Supporting Information file). The micellar size of N-lauryl-oligo(L-Glu) was also evaluated as a function of solution pH (Figure 6). Indeed, solution pH significantly effects micellization of N-lauryl-oligo(L-Glu). This is attributed to the ionization of hydrophilic head groups under different pH conditions. Aggregates are not observed at pH 3 due to their low solubility under acidic conditions. When the pH is increased to 6.5, ionization of peptide head groups

lipopeptide

D1 (nm)

D2 (nm)

C8/oligo(L-Glu) C12/oligo(L-Glu) C18/oligo(L-Glu) C12/oligo(L-Glu-co-21 mol %- L-Leu)

0 1.8 ± 0.6 4.9 ± 1.4 4.8 ± 1.2

0 17.8 ± 9.4 201.3 ± 59.7 131.9 ± 49.4

no peak was observed in the measurement range, indicating that no micelles are formed in the bulk solution. This observation agrees with results from surface tension reduction and CMC analysis (Figure 2, above). For lipopeptides C12/ oligo(L-Glu), C18/oligo(L-Glu), and C12/oligo(L-Glu-co-21 mol %-L-Leu), two peaks were observed in the tested range. The first peak, observed between 5 and 15 nm, is believed to be due to spherical micelles, while the second peak, between 75 and 400 nm, indicates formation of complex structures, e.g., multilayer vesicles. The assignment of the first peak between 5 and 15 nm to the formation of spherical micelles is due to strong repulsion between peptide headgroups; however, other morphologies cannot be excluded at this point. The driving force for aggregation into complex structures observed between 75 and 400 nm is most likely hydrogen bonding among peptide head-groups leading to decreased ΔH values. In future work we plan to define these structures by small-angle X-ray scattering and cryo-transmission electron microscopy studies. The coexistence of complex structure along with spherical micelles was not observed by AUC measurements, probably due to rapid sedimentation of large aggregates at the beginning of centrifugation experiments.

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EXPERIMENTAL SECTION

Materials. L-glutamic acid diethyl ester hydrochloride (L-(Et)2-Glu· HCl), L-leucine ethyl ester hydrochloride (L-Et-Leu·HCl) were purchased from Tokyo Kasei Co. Ltd. in the highest available purity and were used as received. Crude papain (cysteine protease; EC # 3.4.22.2; source-Carica papaya; 30 000 USP units/mg of solid; molecular weight 21K) was purchased from CalBioChem. Co. Ltd. Water-insoluble materials in the as-received papain were removed by dissolving 300 mg/mL crude papain powder in deionized water, centrifugation at 5000 rpm for 30 min, collecting the clear supernatant

Figure 6. Aggregation number of N-lauryl-oligo(L-Glu) as a function of pH determined at 0.55M, 25 °C, by AUC. 6893

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and discarding the insoluble precipitate. The clear supernatant was lyophilized overnight to obtain fully water-soluble papain as a beige powder that was used for all studies herein. N-hydroxysuccinimide (NHS), dimethyl sulfoxide (DMSO), dicyclohexylcabodiimide (DCC), 4-dimethylaminopyridine (DMAP, 2.3 mmol) and triethylamine were purchased from VWR International, Inc. and were used as received. Ninihydrin and hydrindantin were purchased from SigmaAldrich, Inc. and used as received. Octanoic acid, decanoic acid, lauric acid, myristic acid, palmitic acid, and stearic acid were purchased from Sigma-Aldrich, Inc. and used as is without further purification. Methods. General Procedure for Protease-Catalyzed Oligo(γ-EtL-Glu) Synthesis. The method for oligo(γ-Et-L-Glu) synthesis was performed as previously described in the literature.16 In summary, Lglutamic acid diethyl ester hydrochloride (600 mg, 2.5 mmol), papain (16 units/mL), and 5 mL phosphate buffer (pH 8.0, concentration 0.9 M) were transferred to a 15 mL Erlenmeyer flask. The flask was gently stirred in a water bath at 40 °C for 4 h. Then, the reaction mixture was cooled to room temperature, and deionized water (20 mL) was added. Insoluble product was separated by centrifugation (6000 rpm), washed once with dilute HCl (2% v/v) solution and then twice with deionized water. The resulting product was lyophilized giving a beige powder. General Procedure for Protease-Catalyzed Co-oligomerization of L-(Et)2-Glu·HCl and L-Et-Leu·HCl. The method for synthesis of oligo(LGlu-co-L-Leu) in various compositions was performed as previously described in the literature.22 In summary, (e.g., the co-oligopeptide moiety of Product #1, Table 1) a mixture of L-(Et)2-Glu·HCl (2.25 mmol), L-Et-Leu·HCl (0.25 mmol), papain (16 units/mL), and 5 mL of phosphate buffer solution (pH 8.0, concentration 0.9 M) were transferred to a 50 mL round-bottom flask. The mixture was gently stirred in a water bath at 40 °C for 4 h. Then, the reaction mixture was cooled to room temperature by addition of 20 mL deionized water. The precipitate was collected by centrifugation (6000 rpm), washed once with dilute HCl (2% v/v) solution and then twice with deionized water. The resulting material (product) was lyophilized giving a beige powder. Conjugation Oligopeptide with Fatty Acid. The oligopeptide Nterminal amine was conjugated to the fatty acid carboxylic acid moiety by first activating the carboxyl groups with NHS. The method followed is based on a literature procedure33 and summarized below. To activate the carboxyl groups of fatty acids (e.g., lauric acid), 0.36 g of lauric acid (1.8 mmol) and 0.2 g of NHS (1.8 mmol) was dissolved in 20 mL of DMSO in a dried 150 mL round-bottom flask. Subsequently, 0.47 g of DCC (2.3 mmol) and 0.28 g of DMAP (2.3 mmol) were transferred to the DMSO solution and the reaction was conducted for 24 h at room temperature. Then, solid dicyclohexylurea formed was removed by filtration. To synthesize lipopeptides, 1.8 mmol of activated fatty acid (e.g., lauric acid) and 2.0 g (1.5 mmol) of oligo(γEt-L-glutamate), or 1.5 mmol of an oligo(γ-Et-L-Glu-co-L-Leu) composition, is dissolved in 25 mL of DMSO. Triethylamine (0.6 mL, 4.4 mmol) was added, and the reaction was magnetically stirred at room temperature for 3 days. The product mixture was precipitated in an excess of cold water and centrifuged for about 30 min. The precipitate was then washed with 50 mL of cold methanol. Lipopeptide De-esterification. This method follows previously published methods for de-esterification of oligo(γ-Et-L-glutamate)16 and oligo(γ-Et-L-Glu-co-L-Leu)22 with the exception that the volume of the NaOH solution was decreased. In summary, dried lipopeptide powder (100 mg) was suspended in 1 N NaOH solution (5 mL) at 60 °C for 36 h; the resulting solution was neutralized with 6.0 M hydrochloride solution. Ninhydrin Method for Quantitative Determination of Oligopeptide. To prepare the ninhydrin solution, ninhydrin (2 g) and hydrindantin (0.3 g) were dissolved in 75 mL DMSO under a stream of nitrogen gas. Subsequently, 25 mL lithium acetate buffer (pH 5.2) was added, and the mixture was bubbled with nitrogen for at least 2 min, sealed, and stored in a refrigerator (4 °C). The method for measuring the free N-terminal amine groups follows that described in the literature.34 Briefly, 1 mL of a 1 mg/mL solution of oligopeptide in DMSO and 1 mL of the ninhydrin solution were transferred into a screw-capped test tube and heated in boiling water bath for 10 min.

After heating, tubes were immediately cooled in an ice-bath. Then, 5 mL of 50% ethanol was added into each tube, which was mixed thoroughly with a vortex mixer for 15 s. The absorbance (570 nm) of the reaction mixture was measured with a spectrophotometer (Spectra Max Plus Model 384). Three independent experiments each with duplicate samples were conducted, and the reported values were the mean of the three experiments. Linear regression was performed with commercial statistical software on a personal computer. Instrumental Methods. Nuclear Magnetic Resonance (NMR) Spectroscopy. Proton (1H) NMR spectra and carbon (13C) NMR were recorded on a Bruker DPX 300 spectrometer at 300 and 75.47 MHz, respectively. NMR experiments were performed in DMSO-d6 at 10 mg/mL (1H) and 100 mg/mL (13C). Data was collected by software BioSpin and analyzed by data processing software: MestRe-C. Chemical shifts were referenced to tetramethylsilane (TMS) at 0.00 ppm. (MALDI-TOF). MALDI-TOF spectra were obtained on an OmniFlex MALDI-TOF mass spectrometer (Bruker Daltonics, Inc.). The instrument was operated in a positive ion linear mode with an accelerating potential of +20kV. The TOF mass analyzer had pulsed ion extraction. The linear flight path was 120 cm. OmniFLEX TOF control software was used for hardware control and calibration. Spectra were acquired by averaging at least 200 laser shots. The pulsed ion extraction delay time was set at 200 ns. The spectrometer was externally calibrated using angiotensin II as a standard (1046.54 amu). To generate the matrix solution, a saturated solution of α-cyano-4hydroxycinnamic acid (CCA) was prepared in trifluoroacetic acid/ acetonitrile (TA, 1 to 10 v/v). Oligopeptide samples, dissolved in DMSO (5 μL), were diluted with TA solution to 1−5 pmol/μL and mixed with 5 μL saturated matrix solution. Then, 1 μL of this mixture was applied onto the clean target. The sample target was dried in a stream of cold air from a dryer. The abundance intensities of peaks vs m/z were collected via X-massOminFLEX 6.0.0 software and then were exported to an MS Excel spreadsheet for further calculations. Surface Tension−Wilhelmy Plate Method. The surface tension of the lipopeptide was measured at 25 ± 1 °C using the Wilhelmy vertical plate technique with a sandblasted platinum plate as the sensor. The pull exerted on the sensor was determined using a Cahn microbalance. The entire assembly was kept in a draft-free plastic cage at 25 ± 1 °C. For each measurement, the sensor was in contact with the solution for 30 min to allow equilibration. Dynamic Light Scattering. Dynamic light scattering was performed using a Brookhaven research-grade system with BI-900AT correlator and BI-200SM goniometer with adjustable angles of detection from 15 to 155°. A water-cooled Lexel argon laser light source was used at a wavelength of 488 Å. The samples were maintained at 25 °C. To minimize the dust effect encountered frequently in light scattering measurements, sample solutions were filtered through a 0.2 μm Nalgene membrane prior to use. The filtration process was found to produce no detectable effect on the surfactant concentration because of the relatively large membrane pore size. All measurements were made in dynamic mode (i.e., the instrument measures the diffusion coefficient of the micelles and back calculates the effective diameter assuming a spherical shape). Analytical Ultracentrifuge. Sedimentation velocity tests were performed using a Beckman Coulter Optima XL-I analytical ultracentrifuge equipped with both absorbance and interference optical detectors. The rotor speed was set at 40,000 rpm and the temperature was maintained at 25.0 °C. Tests were run after the vacuum reached below 5 μm Hg (∼0.65 Pa). SEDFIT 92 software developed by Schuck was used to analyze the sedimentation velocity data.

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CONCLUSIONS AND PERSPECTIVES A chemo-enzymatic method was developed that, relative to alternative methods such as SPPS, is simple, efficient, and scalable. The oligopeptide segment of lipopeptides was prepared by protease-catalyzed oligomerization of amino acid ethyl ester monomers in aqueous media under mild conditions. 6894

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This route enables the synthesis of a wide range of homo- and co-oligopeptide structures. Unlike natural lipopeptides or biomimetic synthetic lipopeptides prepared by methods such as SPPS, the lipopeptides studied herein are mixtures in chain length, co-oligopeptide composition as well as sequence distribution. This lack of uniformity in the polar headgroup including oligo(L-Glu-co-L-Leu) where chains having different compositions as well as randomly arranged sequences might intuitively be deemed not useful for the preparation of selfassembled systems. Hence, study of oligopeptide mixtures is important as it provides insight into how peptide uniformity, or a lack thereof, affects the physical properties and biological activities of these molecules. If peptide mixtures prepared by protease-catalysis can be components of lipopeptides that provide valuable properties, this would unlock numerous opportunities to develop such molecules for more cost-sensitive applications. The colloidal and interfacial properties of lipopeptides synthesized herein were characterized. These studies showed how lipopeptide colloidal properties can be regulated by changing the chain length of the lipid tail attached to oligo(LGlu) with DPavg around 7 to 8, or by increasing the hydrophobicity of oligo(L-Glu) segments by randomly interdispersing leucine units. One might have expected that random sequences of oligo(L-Glu-co-L-Leu) would not interact effectively in the polar oligopeptide component of selfassembled lipopeptide micelles. However, by systematic increases in the hydrophobic content within the oligopeptide segments of C12/oligo(L-Glu-co-L-Leu), regular decreases in the CMC from 22.3 mM to 2.3 mM was found. Furthermore, an empirical linear relationship was observed for CMC’s of this lipopeptide series. This paper represents the first study on what is a broad family of lipopeptides that can be prepared by a similar methodology as described herein. Indeed, there is broad canvas of design space for both the lipid and oligopeptide components. For example, by using perfectly alternating oligopeptide sequences that can now be accessed via protease-catalyzed oligopeptide synthesis, properties can be built into lipopeptides such as environmentally responsive gelation, antimicrobial properties and much more. Furthermore, recent advances in our laboratory provide methods by which oligopeptides prepared by protease-catalysis can be synthesized that have perfectly alternating sequences that enable other property attributes.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS G.L, X.Q, J.Z, W.L., J.L., V.K.R, and R.G. gratefully acknowledge financial support from NSF-1243313 entitled: “PIRE: Materials for Renewable Energy Nature’s Way”. J.W. and P.S. thank the NSF-I/UCRC for Particulate and Surfactant Systems (NSF grant# IIP-0749461) at Columbia University for financial support.

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ASSOCIATED CONTENT

S Supporting Information *

Tabulation of CMC and lowest surface tension reduction values for N-acyl-oligo(L-Glu) lipopeptides differing in fatty acid chain length, tabulation of CMC values of N-lauryl-oligo(L-Glu-co-LLeu) having different oligopeptide compositions, 1H NMR spectrum of N-lauryl-oligo(γ-Et-L-Glu)7.9, 13C NMR spectra of 13 C NMR spectrum of lauric acid and N-lauryl-oligo(γ-Et-LGlu)7.9, MALDI-TOF spectrum of N-lauryl-oligo(γ-Et-L-Glu-co21 mol %L-Leu), empirical linear relationship between CMC versus mol %-L-Leu, micellar size distribution of different lipopeptides by dynamic light scattering, and calculations from sedimentation coefficient data. This material is available free of charge via the Internet at http://pubs.acs.org. 6895

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