THE JOURNAL OF BIOLOGICAL CHEMISTRY Q 1989 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 264, No. 11, Issue of April 15, pp. 6260-6267,1989 Printed in U.S.A.

The Chemical Synthesis of Cecropin D and an Analog with Enhanced Antibacterial Activity*

(Received for publication, November 17, 1988)

Jiirgen Fink$$, R. B. Memifield$, Anita Bomanll, and Hans G. Bomanll From the $Rockefeller University, New York, New York 10021 and nDepartment of Microbwlogy, the University of Stockholm, S-10691 Stockholm, Sweden

Cecropin D was synthesized by solid-phase methods and shown to be homogeneous and of correct composi- tion and molecular weight. It was indistinguishable from natural cecropin D and constitutes a structure proof for this peptide. Several analogs of cecropin D were synthesized and used to draw conclusions about the structural features contributing to antibacterial activity. They included [Lys’]cecropin D, [Gins, Leu‘] cecropin D, and cecropin D-(9-37). It was concluded that a strongly basic NHS-terminal segment is a pre- requisite for antibacterial activity. A hybrid analog cecropin A-(1-11) D-(12-37) was designed and pre- dicted to have enhanced potency. It was found to be 5 to 55 times as active as cecropin D against six of the bacteria tested and was slightly more active than ce- cropin A. However, against Bacillus subtilis Bsl l the analog was 6 times more active than cecropin A.

Cecropins are a family of basic antibacterial peptides pro- duced by the humoral immune response of certain insects (1). Cecropins, attacins, and lysozyme are induced in the hemo- lymph of the pupae of the giant silk moth Hyulophoru cecropia following injection of live bacteria (2, 3). Like cecropins A and B (4), cecropin D is a major component in this family (5). Cecropin D shows homology to cecropins A and B, since they each are about the same size (cecropin A, 37 residues; cecropin B, 35 residues; and cecropin D, 36 residues), and they each contain a hydrophilic amino-terminal region and a hydropho- bic amidated carboxyl terminus. However, cecropin D is less basic (net charge: +3) than cecropins A and B (net charges: +7 and +8). Furthermore, cecropin D is less active against most of the bacteria tested.

To confirm the deduced sequence of natural cecropin D and to understand the reasons for the altered antibacterial activity of cecropin D compared with the A and B forms, we have synthesized the parent compound and four analogs with sig- nificant structural changes in the NHz-terminal region’: [Lys’lcecropin D, [Gln3,Leu4]cecropin D, cecropin D-(9-37), and cecropin A-(1-11) D-(12-37) (peptide AD). The latter is a hybrid of the A and D forms containing the sequence of

* This work was supported in part by Grant DK01260 from the United States Public Health Service (to R. B. M.), Grant BU2453 from the Swedish Natural Science Research Council (to H. G. B.), and by a fellowship from the Deutsche Forschungsgemeinschaft (to J. F.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ Present address: Hoechst-AG, Frankfurt-Main, West Germany. We have used the numbering system for cecropin A throughout.

Thus, cecropin D is lacking residue 1 and begins with Trp’. The last amino acid of the 36-residue cecropin D is therefore number 37.

cecropin A from residues 1 to 11 and the sequence of cecropin D from residues 12 to 37. The amino terminus was selected for study both for synthetic convenience and for its previously established role in the activity of cecropins (6,7). The segment of residues 1-11 in cecropins A and B can be considered an almost perfect amphipathic helix (€9, and it contains most of the lysine residues that account for the basicity of these peptides. The NHz-terminal sequence of cecropin D differs significantly, lacking most of the basic lysine residues. Syn- thetic cecropin D and its four analogs were tested against Gram-negative and Gram-positive bacteria, and their anti- bacterial activities were related to structural features of the peptides.

EXPERIMENTAL PROCEDURES

Materials-p-Methylbenzhydrylamine resin (0.45 meq of nitrogen/ g) was purchased from Peptides International. tert-Butyloxycarbonyl (Boc)’ amino acids were obtained from Peninsula Laboratories. Boc- [3H]Ala was prepared from a 0.25-mCi sample of the free amino acid (ICN Biochemicals) by appropriate dilution with nonradioactive amino acid and subsequent reaction with BocnO (9). Other reagents were trifluoroacetic acid (Halocarbon Products), N,N-diisopropyl- ethylamine (DIEA) (Aldrich) distilled over CaH2, pyridine (Aldrich) distilled over ninhydrin (Pierce Chemical Co.), dicyclohexylcarbodi- imide (DCC) (Fluka), 1-hydroxybenzotriazole (HOBt) (Aldrich), di- chloromethane (Fisher) distilled from anhydrous NazCO dimethyl- formamide (DMF) (Burdick and Jackson) stored over 4-1 molecular sieves, acetonitrile, HPLC-grade (Burdick and Jackson), p-cresol (Aldrich), p-thiocresol (Fluka), dimethyl sulfide (Fluka), and anhy- drous HF (Matheson).

General Methods-Hydrolysis of free peptides was done with 6 N HC1 in evacuated, sealed tubes at 110 “C for 24 h (10). Peptide-resins were hydrolyzed in 12 N HCl/propionic acid (1:l) a t 130 ‘C for 15 h (11). After filtration, hydrolysates were analyzed on a Beckman 6300 amino acid analyzer. HF reactions were carried out in a Diaflon HF apparatus (Toho Kasei, Osaka, Japan). Gel filtration was done on a 1.5 X 100-cm Sephadex G-25 column, eluted with 1 M HOAc at 45 ml/h.

Preparative reverse-phase liquid chromatogra hy was done on CIS silica (Vydac, 218 TP, 15-20-pm spheres, 300- K pores) packed in a 2.2 X 13-cm Michel-Miller column (Ace Glass), equilibrated with 5% acetonitrile in 0.05% trifluoroacetic acid in water. The samples were loaded, and the column was washed for 1 h. Elution was then performed with linear gradients (range: 12-60%) of acetonitrile in 0.045% trifluoroacetic acid in water, a t 1 ml/min. The effluent was monitored at 220 nm on a Kratos Spectroflow 757 absorbance detec- tor.

Analytical high pressure liquid chromatography of the peptides was performed at room temperature on a Vydac C18 reverse-phase column (0.4 X 25 cm) with a Shimadzu 6A high performance liquid chromatographic system. Solution A contained 900 ml of water, 100 ml of CH3CN, and 0.44 ml of trifluoroacetic acid; solution B contained

’ The abbreviations used are: Boc, butyloxycarbonyl; DIEA, N,N- diisopropylethylamine; DCC, dicyclohexylcarbodiimide; HOBt, l-hy- droxybenzotriazole; DMF, dimethylformamide; HPLC, high pressure liquid chromatography; Bzl, benzyl; ClZ, 2-chlorobenzyloxycarbonyl; cHex, cyclohexyl; Tos, tosyl; For, formyl; Ac, acetyl.

6260

Cecropin D and Analogs 6261 200 ml of water, 800 ml of CH3CN, and 0.37 ml of trifluoroacetic acid. Linear gradients in the 20-60% range for 25-30 min, at 1.5 ml/ min, were used. Detection was at 220 nm. Radioactivity was deter- mined on a Beckman LS 355 liquid scintillation system.

Synthetic Protocols-Synthesis was performed manually in a re- action vessel described previously (12). The following standard double coupling protocol was used, based on 2.5 g of starting resin (0.21 mmol/g): 1) CHzClz, 50 ml, 4 X 1 min; 2) 50% trifluoroacetic acid/ CHZClz, 50 ml, 2 X 1 min; 3) 50% trifluoroacetic acid/CHZClZ, 50 ml, 1 X 20 min; 4) CH2Cl2, 50 ml, 6 X 1 min; 5) 5% DIEA/CHzC12, 50 ml, 2 X 2 min; 6) CHzClZ, 50 ml, 6 X 1 min; 7) protected amino acid, 4 eq in 20 ml of CHZCl2, add to reaction vessel, rinse with 5 ml of CH2Clz, and shake at room temperature for 5 min; 4 eq of DCC in 3 ml of CH2ClZ, add to reaction vessel, rinse with 2 ml of CHZC12, and shake for 100 min at room temperature; 8) CHzClz, 50 ml, 4 X 1 min; 9) 5% DIEA/CHZClz, 50 ml, 1 X 2 min; 10) CHZCL, 50 ml, 4 X 1 min; 11) DMF, 50 ml, 2 X 2 min; 12) protected amino acid, 8 eq in 3 ml of CHzClz, 0 "C, add DCC, 4 eq in 1 ml of CHzC12,O "C, rinse with 1 ml of CHzC12, 0 "C, after 10 min at 0 "C, filter, add 25 ml of DMF, 0 "C, add to reaction vessel, rinse with 5 ml of DMF, 0 'C, shake for 1 h at room temperature; 13) DMF, 50 ml, 2 X 2 min; 14) CHZC12, 50 ml, 4 X 1 min; 15) 5% DIEA/CH&l2, 50 ml, 1 X 2 min; 16) CH2C12, 50 ml, 4 X 1 min; 17) 3- to 5-mg sample for ninhydrin. Some modified procedures used to couple particular amino acids are described under "Results."

Polyacrylamide Gel Electrophoresis-Electrophoretic characteriza- tion of antibacterial substances was according to Hultmark et al. (4) on 15% polyacrylamide gels in &alanine acetate buffer, pH 4, with an acrylamide/bisacrylamide ratio of 75:l. Run at 200 V for 3.5 h.

Assay ofAntibacterial Actiuity-The analogs were tested for activity by an inhibition zone assay. Thin agar plates (8.7 cm diameter) were prepared with 6 ml of rich medium containing 1-4 X 10' viable cells of a test organism. Wells of 3 mm diameter were punched in the plates, and 3 pl of serially diluted samples was placed in the wells. The diameters of the zones of inhibition around the wells were measured after overnight incubation at 30 'C . For each peptide the squares of the zone diameters were plotted against the logarithm of the amounts applied, and from the slopes and intercepts the lethal concentrations were calculated according to Ref. 5.

RESULTS

Synthesis of Cecropin D-The synthesis followed the con- ventional stepwise solid-phase procedures (13) with the acid- labile Boc group for temporary Nu-protection and the more acid-stable benzyl (Bzl), 2-chlorobenzyloxycarbonyl (ClZ), and cyclohexyl (cHex) groups for side chain protection. In addition tosyl (Tos) and formyl (For) protecting groups were used for Arg and Trp, respectively. Since cecropin D is a peptide amide, the resin of choice was a benzhydrylamine- type resin (14); p-methylbenzhydrylamine-copoly(styrene- 1%-&vinylbenzene) resin was used because of increased sus- ceptibility to the final HF cleavage relative to the unsubsti- tuted benzhydrylamine resin (15).

The structure of the protected peptide resin was Boc- Trp(For)-Asn-Pro-Phe-Lys(ClZ)-Glu(OcHex)-Leu-Glu(Oc- Hex)-Lys(ClZ)-Val-Gly-Gln-Arg(Tos)-Val-Arg(Tos)-Asp- (OcHex)-Ala-Val-Ile-Ser(Bzl)-Ala-Gly-Pro-Ala-Val-Ala- Thr(Bzl)-Val-Ala-Gln-Ala-Thr(Bzl)-Ala-Leu-Ala-Lys(ClZ)- NH-CH(p-CH3-C6H4)-C6H4-resin. The desired substitution for the first amino acid (Lys3') was 0.20-0.25 mmol/g. In order to get this value, 0.75 mmol of Boc-Lys(C1Z) was coupled to 2.5 g of resin (0.45 mmol of nitrogen/g) using a single DCC coupling for 2 h. Unreacted amino groups were then blocked by acetylation with a mixture of acetic anhydride/pyridine/ CHzClz (1:1:2, v/v). A quantitative ninhydrin reaction (16) on 5 mg of the resin showed that only 0.0012 mmol/g free amine remained (0.25% of the original amino groups). The exact degree of substitution was determined by deprotection and ninhydrin analysis and found to be 0.21 mmol/g. The next amino acid (Ala36) was coupled to Lys3' and an amino acid analysis showed the expected substitution of Ala36 (0.22 mmol/g) but a low value for Lys3" (ratio Lys/Ala: 0.65). The

subsequent amino acids were added with a double coupling protocol that varied depending on the particular residue. For all residues except Boc-Arg(Tos1, Boc-Asn, Boc-Gln, and the residue coupled after Gln, the first coupling was with DCC in CHzClz and the second was with the preformed symmetric anhydride in DMF/CH,Cl, (6:1, v/v) (17). In some cases a third coupling was required; this was done with the preformed HOBt ester in DMF for 2 to 4 h at room temperature. For Boc-Arg(Tos) the second coupling was with the HOBt ester to avoid lactam formation. For Boc-Asn and Boc-Gln, the HOBt ester was used for both couplings to minimize nitrile and amidine formation (18), the first coupling in CH*C12/ DMF (21, v/v), the second in DMF. For coupling Boc-Ala3' to Gln31 and Boc-Gly" to Gln13, the first coupling was with the symmetric anhydride preformed in CHzCl2, filtered, evap- orated, and redissolved in DMF to minimize pyrrolidone carboxylic acid formation (19). For B~c-[~HlAla the first coupling was with DCC (0.8 eq of B~c-[~HlAla) and the second with unlabeled Boc-Ala symmetric anhydride (4 eq). The specific activity of this residue after incorporation was 0.09 mCi/mmol.

The quantitative ninhydrin reaction was routinely used throughout the synthesis to determine the extent of coupling (Table I). At some difficult sequences, such as e.g. ThrZS-ValZ9 or Gln13 to Arg14, a third coupling was required, sometimes the remaining low levels of amino groups were capped with AczO. For the main part of the synthesis the average coupling according to these data was 99.9% after correction for a background of -0.5%.

The progress of the synthesis as well as the substitution of the polymer was checked by amino acid analysis of acid- hydrolyzed samples of peptide-resin at several stages of the synthesis (Table 11). The molar ratios of the component amino acids showed the expected values except for Lys, which was low and Ser and Thr, which are partly destroyed under these conditions. Since the ratios of Glu relative to the residues before and after Gln were close to 1.0, there was no evidence of significant chain termination by pyrrolidone formation. Nevertheless, as indicated by amino acid analysis and by the ninhydrin data, there was a decrease in growing chains at the end of the synthesis, the final substitution of the peptide (corrected for weight gain) being 0.18 mmol/g, i.e. about 80% of the initial substitution. This is not unusual for the synthesis of cecropins, where similar decreases of substitution were observed in previous syntheses of cecropin A-(1-33) (12), cecropin A and analogs (6, 7), and cecropin B (20).

Cleavage and Purification-A sample of the fully protected cecropin D-resin was treated with trifluoroacetic acid to re- move the N*-Boc group and dried (317 mg). Cleavage of the peptide from the resin support was by the low/high HF method (21). Low HF was done with 5 ml of HF/dimethyl sulfidelp-cresollp-thiocresol (25:65:7.5:2.5), at 0 "C for 2 h. High HF was done with 10 ml of HF/p-cresollp-thiocresol (95:3.75:1.25), at 0 "C for 1 h. After evaporation of HF, the product was first washed with anhydrous ether to remove the scavengers and then dissolved in 10% HOAc in water. After lyophilization, 145 mg of crude material was obtained. The cleavage yield was 85% on the basis of radioactivity. The HPLC analysis of the crude cleavage product showed 62% to be in one main peak.

The peptide (80 mg) was partially purified by gel filtration on a Sephadex G-25 column in 1 M HOAc to remove low molecular weight impurities. After lyophilization 42.1 mg of peptide was obtained (53% of the material loaded on column). On the basis of radioactivity the recovery was 80%.

Further purification was by preparative reverse-phase chro-

6262 Cecropin D and Analogs

TABLE I Ninhydrin monitoring of the synthesis of cecropin D

Synthetic Protected Ninhydrin monitoring after step" residue Substituted* Extent of

2nd coupling 3rd coupling Acetylation coupling'

1.05 1.29

1.51 1.83 2.41

1.74

0.97

~~

mmol/g resin pmollg 7% 1 Lys(C1Z) 0.22 2 Ala 1.80 1.21 99.9 3 Leu 0.80 4 [3H]Ala 5

1.47 1.24 Thr(Bz1)

99.8 0.20

6 1.21 99.8

Ala 1.68 7

1.10 Gln

99.9

8 0.21 1.09

Ala 99.9

1.05 9

99.9 Val 0.20 2.62

10 Thr(Bz1) 1.77 99.6

11 1.92 1.24 99.9

Ala 0.97 12 Val 1.12 99.9 13 Ala 1.03 14

99.9 Pro 0.91 100

15 G ~ Y 16 Ala 1.06 99.9 17 Ser(Bz1) 1 .a0 99.9 18 Ile 1.49 19

99.8 Val 0.21 0.95 100

20 Ala 1.10 21

99.9 Asp(0cHex) 1.09 99.9

22 Arg(Tos) 1.33 99.8 23 Val 1.80 24

99.7 Arg(Tos) 1.81 99.5

25 Gln 4.04 99.2 26 G ~ Y 1.20 27

99.8 Val 1.88 99.6

29 Glu(0cHex) 0.96 100 30 Leu 1.07 99.9 31 Glu(0cHex) 1.26 99.8 32 Lys(C1Z) 1.14 99.9 33 Phe 1.02 99.9 34 Pro 0.87 100 35 Asn 36 T d F o r ) 0.18 0.72 100

1.15

100

100

28 Lys(C1Z) 0.19 0.91 100

0.97

0.71 0.91

1.07

"The first amino acid attached to the resin was Boc Lys(ClZ), it is residue 37 in the peptide sequence according to the cecropin A numbering that was used. The 36th synthetic step was with Boc Trp(For), which is designated residue 2 in the cecropin D sequence.

* Corrected for weight gain during peptide growth. Corrected for a background ninhydrin value of 0.9 pmollg.

matography on Cls silica (Fig. 1). The purity of each fraction was studied by analytical reverse-phase HPLC. According to this, fractions 39-41 contained pure material (8.3 mg). Side fractions 33-37 and 42-44 were combined and run again under the same conditions to yield an additional 2.2 mg of pure peptide. The overall yield of purified cecropin D was 10.5 mg. An analytical HPLC (Fig. 2) indicated clean material. The amino acid composition of purified cecropin D is shown in Table 111. Additionally, the correct molecular weight was established by fission-fragment mass spectrometry (22) (see Table IV).

Analogs-Four cecropin D analogs, [Lys'lcecropin D, [Gln3,Leu4]cecropin D, cecropin A-(1-11) D-(12-37) (called peptide AD), and cecropin D-(9-37) were synthesized (Fig. 3). They were prepared from the same batch of resin by assembling the 9-37 core sequence and dividing the peptide- resin into five portions and continuing the synthesis for each individual analog. The peptides were deprotected and cleaved from the resin under conditions, and with yields, similar to those for cecropin D and were purified to homogeneity by preparative reverse-phase chromatography, The amino acid analyses are given in Table 111. Analytical HPLC chromato- grams of the crude and purified analogs are shown in Fig. 4.

It can be seen that the retention times occur in the expected order with the most hydrophilic peptides eluting earliest. Fission-fragment mass spectrometry showed the expected val- ues for the molecular weight of each compound (Table IV).

Gel Electrophoresis-The electrophoretic mobility of the cecropin analogs at pH 4 (Fig. 5) correlated well with calcu- lated net charge but was also a function of size and hydropho- bicity. Thus, cecropin A moved slightly ahead of peptide AD, which is more hydrophobic.

Antibacterial Activity-The lethal concentration of cecro- pins A and D and the four analogs of cecropin D against several bacterial strains are summarized in Table V. Toward Escherichia coli D21 only cecropin A and cecropin A-(1-11) D-(12-37) were fully active (0.5-0.6 p ~ ) , while cecropin D and the [Lys'] and [Gln3,Leu4] analogs were 2 to 7 times less active. A and AD were also very active against Pseudomonas aeruginosa, Bacillus megaterium, and Micrococcus luteus, whereas the other analogs were 10 to 70 times less active than AD. E. coli D22 was the most susceptible organism and the natural cecropins and all but the shortest analog, cecropin D- (9-37), were essentially equally active. Strain D22, which is a mutant of D21, has an easily permeable outer membrane. Another method to increase the permeability of the outer

Cecropin D and Analogs 6263

+ 3 .s!

h

5 Y

B B

.3 3 bl 5;

B e 0 ." 3 W :

3

0 e - 0 ." .- y

; 3 m t - - 3 ~ c - ~ m W P W

000~0000 000 ?+?9I?I? ?I?

d m d 4 O d r c 00

E 8

0, 0 I

8 I 0

U2 * 0 1

m 1 10

0

10 t-

0 I

0 m 0 ?

P- m ? 0

r: 2

t- t-

0 I

0 W

2

0 m ? 0

N N

0 v

2 v 0

m ? t-

0

W 4

@! 0

s 0 v

w 2

W N

0 v

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-

- 40 z

I I 0 2 4 6 8 10 12

I I I

Elution time (hr) FIG. 1. Preparative reverse-phase chromatogram of syn-

thetic cecropin D. Sample: 42 mg of HF-cleaved peptide after passage through Sephadex G-25 in 1 M HOAc and lyophilization. Column: Vydac CIS, 15-20 bm, 300-A pores, in a 2.2 X 13-cm column. Linear gradient of 20 to 60% CH&N in 0.05% aqueous trifluoroacetic acid, 800 ml each solution, 1 ml/min, monitored at 220 nm.

0 a 0 I

I

0 IO 20 Time (min)

FIG. 2. Analytical HPLC chromatogram of purified syn- thetic cecropin D. Sample: center cut of preparative column (Fig. 1). Column: Vydac CIS silica (218 TP 54,5-pm spheres, 300-A pores) 0.46 X 25 cm. Linear gradient 2&60% acetonitrile in 0.05% aqueous trifluoroacetic acid in 30 min, 1.5 ml/min. Shimadzu 6A instrument.

6264 Cecropin D and Analogs TABLE 111

Amino acid analysis of purified cecropin D and its a d g s

Amino acid D [Lys'ID

21 h 12 h 24 h 72 h [GlnS,Leu']D, 24 h A-(1-11) D-(12-37), 24 h D-(9-37), 24 h

ASD 0.97 0.96 0.97 0.98 1.01 1.07 1.04 Thr Ser Glu Pro GlY Ala Val Ile Leu P he LYS Arg Trpb

0.92 0.97 1.04 1.05 1.01 1.01 0.94 0.69" 1.03 1.11 1.00 1.02

0.86" 0.72" 0.99 1.02 1.02 1.03 1.00 0.90 0.99 1.07 0.96 0.98

0.93 0.87" 0.92 0.75" 1.03 1.02 1.02 1.04 1.04 1.06 0.99 1.05 0.82" 0.94 0.72" 0.94 1.03 1.00 1.03 1.07 0.99 0.97 1.04 0.91

0.97 0.91 0.98 1.00 1.12 1.03 0.96 0.67" 1.04 0.97 0.99 0.98

1.02 1.03 1.03 0.78" 1.08 1.03 0.98 0.77" 1.01 1.05 0.96 0.91

0.99 0.97 0.98 0.74" 0.99 1.08 0.91 0.71" 1.07

0.99 0.91

Omitted from average. Tryptophan was not determined.

TABLE IV Molecular weights of cecropin D analogs

Molecular weight Peptide A

Found Calculated

Cecropin D 3793.8 3793.4 +0.4 [Lys']Cecropin D 3922.0 3921.5 +0.5 [Gln3,Leu4]Cecropin D 3823.7 3823.4 +0.3 Cecropin D49-37) 2877.7 2877.3 +0.4 Cecropin A-(1-11) D-(12-37) 3951.3 3950.7 +0.6

Determined by ='Cf fission fragment time of flight mass spectrom- etry from the mean of the most abundant isotopes.

membrane is to add EDTA to the medium (23). As shown in Table VI, 0.5 mM EDTA increased the susceptibility of E. coli D21 to the peptides, and it became as sensitive as the E. coli D22 mutant. Thus, in the outer membrane there must be a barrier that hinders penetration of cecropins.

The most interesting finding was the discovery that Bacillus subtilis, which is very insensitive (40-200 pM) to cecropin A or D, was quite susceptible (6.3 p ~ ) to the hybrid cecropin A- (1-11) D-(12-37).

DISCUSSION

Synthetic Proof of Structure of Cecropin D-This chemical synthesis of cecropin D has confirmed the previously reported sequence of the peptide (5). An independent proof was also obtained by sequencing a cDNA clone for preprocecropin D (24). The purity of the synthetic peptide was established by HPLC and electrophoresis, and its composition was con- firmed by amino acid analysis, amino acid sequencing, and molecular weight determination by mass spectrometry. The identity between natural and synthetic material was demon- strated by electrophoretic and chromatographic criteria and by the similarity of their spectra of antibacterial activities. Both samples were very active against E. coli, moderately active against P. aeruginosa, B. megaterium, and M. luteus, and without significant activity against B. subtilis (see also Ref. 5). The importance of a proof of structure of cecropins A and B and sarcotoxin IA by chemical synthesis has been well demonstrated in the past (6, 12, 20, 25, 26), and we believe it is important to have similar evidence for the structure of cecropin D.

Explanation of Differences in Antibacterial Activity of the Cecropins-Besides the synthesis of cecropin D, another main aspect of this work was to determine which structural differ- ences are responsible for the altered antibacterial activity of

cecropin D compared with cecropins A and B. As can be seen in Fig. 3, the sequences of cecropins A, B,

and D are quite similar. The identity between A and B is 67%, between A and D 63%, and between B and D 42%. However, these figures overemphasize the differences between the three forms because many of the amino acid replacements are conservative. Thus, the actual similarity is higher, being about 81% between cecropins A and B, 77% between A and D, and 65% between B and D. Nonetheless the antibacterial activities are significantly different.

The distribution pattern of hydrophilic and hydrophobic residues is conserved in all cecropins. They have a hydrophilic NHz-terminal region containing many charged residues and a relatively hydrophobic region in the COOH-terminal half. However, cecropin D differs from the A and B forms in two important aspects: cecropin D lacks most of the lysine resi- dues at the amino terminus and is therefore less basic than the two other forms; and D has a stretch of 19 residues (19- 37) without any charge. Both of these differences make the D form more hydrophobic. Applying empirical rules (27-29) for the prediction of secondary structure, both cecropin A and B have been shown to have a strong potential to form an amphipathic a-helix (30) between residues 1 and 11 (8, 12). In the case of cecropin D a similar structure can be predicted. However, since the peptide contains two helix breakers (Am3 and Pro4), the NH2-terminal helical segment should be shorter (residues 5-11). Each of the natural cecropins shows a helical part at the COOH terminus, beginning at residue 25, and, in addition, cecropin D also has a central region with potential for a-helix formation. Thus, cecropin D has a potential for existing largely in a helical conformation.

Taking the above structural differences of the three cecro- pins into consideration, the different biological activity of cecropin D compared with the A and B forms could be mainly due to three reasons. First, it could be caused by the lack of basic residues, i.e. a strongly positive net charge might be obligatory for cecropin function. Second, it could be due to the disruption of the NHz-terminal amphipathic helix by Asn3 and Pro4, because it is known that introduction of helix breakers in the 1-11 region of cecropin A causes a significant loss of antibacterial activity (7). Third, differences in the sequences of the central and COOH-terminal regions might be responsible for the different activities.

In order to discover which of these features is significant, we synthesized cecropin D and the analogs shown in Fig. 4. Each has an altered NH2 terminus but has the same sequence

Cecropin D and Analogs 6265

Peptide Sequence

1 Cecropin A 5 10 15 20

Lys-Trp-Lys-Leu-Phe-Lys-Lys-Ile-Glu-Lys-Val-Gly-Gln-Asn-Ile-Arg-Asp-Gly-Ile-Ile-

2 Cecropin B

3 Cecropin D

Lys-Trp-Lys-Val-Phe-Lys-Lys-Ile-Glu-Lys-Met-Gly-Arg-Asn-Ile-Arg-Asn-Gly-Ile-Val-

Trp-Asn-Pro-Phe-Lys-Glu-Leu-Glu-Lys-Val-Gly-Gln-Arg-Val-Arg-Asp-Ala-Val-Ile-

4 [ ~ y s l l c e c r o p i n D

Lys-Trp-Lys-Leu-Phe-Lys-Lys-Ile-Glu-Lys-Val-Gly-Gln-Arg-Val-Arg-Asp-Ala-Val-Ile- 7 Cecropin A(1-11) D(12-37)

Gly-Lys-Val-Gly-Gln-Arg-Val-Arg-Asp-Ala-Val-Ile- 6 Cecropin D (9-37)

Trp-Gln-Leu-Phe-Lys-Glu-Leu-Glu-Lys-Val-Gly-Gln-Arg-Val-Arg-Asp-Ala-Val-Ile- 5 ~ e u ~ lcecropin D Lys-Trp-Asn-Pro-Phe-Lys-Glu-Leu-Glu-Lys-Val-Gly-Gln-Arg-Val-Arg-Asp-Ala-Val-Ile-

1 c o n ' t . 25 30 35 37

-Lys-Ala-Gly-Pro-Ala-Val-Ala-Val-Val-Gly-Gln-Ala-Thr-Gln-Ile-Ala-Lys-NH~

2 c o n ' t .

-Ser-Ala-Gly-Pro-Ala-Val-Ala-Thr-Val-Ala-Gln-Ala-Thr-Ala-Leu-Ala-Lys-NH2 3-7 con ' t . -Lys-Ala-Gly-Pro-Ala-Ile-Ala-Val-Leu-Gly-Gln-Ala-Lys-Ala-Leu-NH~

A

FIG. 3. The amino acid sequences of cecropins A, B, and D and the cecropin D analogs. In all cases, cecropin A numbering is used.

IC u) *) r;

- I IO 20

11 10 20

8 "! N

1 A IO 20

0 0 2

- ! IO 20

D

0 0

il 10 20 !L IO 20

FIG. 4. Analytical HPLC chromatograms of crude and pu- rified cecropin D analogs. Column: Vydac Cl8 silica 218 TP54, 0.46 X 25 cm. Elutions were with 30-min linear gradients of acetoni- trile in 0.05% aqueous trifluoroacetic acid. Elution times are expressed in minutes. In each panel the crude product after HF cleavage is on the kft, and the purified peptide is on the right. Panel A , cecropin D- (9-37), gradients, 32-59% and 17-52% CH3CN, panel B, [Lys'] cec- ropin D, gradients, 24-58% and 27.5-66% CH&N panel C, [Gln3,Leu4]cecropin D, gradients, 24-59% and 24-59% CH&N panel D, cecropin A-(1-11) D-(12-37), gradients 24-59% and 24-59% CHsCN.

6266 Cecropin D and Analogs

TABLE V Lethal concentrations of cecropins A, B, D, and analogs

Lethal concentration

E. coli D21 E. coli D22 P. aeruginosa OT97 B. mgateriurn Bmll M. luteus MLll B. subtilis Ball W

Peptide

Cecropin D, natural 4.6 0.62 >go 28 35 >95 Cecropin D, synthetic 2.6 0.51 66 31 12 100 [Lys'ICecropin D 1.1 0.39 37 16 7 120 [Gln3,Leu']Cecropin D 3.2 0.54 86 24 11 190 Cecropin A-(1-11) D-(12-37) 0.50 0.35 1.2 0.83 0.71 6.3 Cecropin A, synthetic 0.58 0.48 1.7 1.2 1.0 41 Cecropin D-(9-37) >390 >390 >390 >390 >390 >390

a From Ref. 5.

TABLE VI Effect of EDTA on the lethal concentrations of cecropin D and

analogs toward E. coli 021 Lethal concentration

Peptide E. coli D21" E. coli D22'

No With No EDTA' EDTAd EDTA'

PM Cecropin D, natural 2.6 0.35 0.62 Cecropin D, synthetic 3.5 0.29 0.51 [Lys'ICecropin D 1.4 0.28 0.39 [Gln3,Leu4]Cecropin D 4.8 0.40 0.54 Cecropin A-(1-11) D-(12-37) 0.48 0.41 0.35 Cecropin A, synthetic 0.48 0.41 0.48 Cecropin D49-37) >390 >390 >390

"Notice that the exact LC values for D21 in this table differ somewhat from those shown in Table V. This is an indication of the range to be expected in such inhibition zone assays.

'The data for E. coli D22 are taken from Table V for easy comparison.

e Grown on plates of LB medium. Grown on plates of LB medium plus 0.5 mM EDTA.

ecules, while hydrophilic agents remain unaffected. The ad- dition of EDTA made the wild type E. coli D21 behave like the mutant D22. Since only hydrophobic molecules are af- fected by this alteration, the activities of the strongly hydro- philic cecropin A and cecropin A-(1-11) D-(12-37) did not change upon addition of EDTA. On the contrary, the more hydrophobic peptides, cecropin D, [Lys']cecropin D, and [Gln3,Leu4]cecropin D, significantly gained antibacterial po- tency against D21 after addition of EDTA. Since these pep- tides, as well as cecropin B (5), are almost equally active against E. coli, the structural differences among the com- pounds do not significantly affect the binding of cecropins to this bacterium. However, the total loss of antibacterial activ- ity for cecropin D-(9-37), which lacks the amphipathic helix at the NH2 terminus, indicates that this segment is a prereq- uisite for the antibacterial activity of the cecropins.

Nearly the same activities are observed for cecropin D and [Gln3,Leu4]cecropin D against P. aeruginosa, B. megaterium, M. luteus, and B. subtilis. Both peptides have the same net charge (+3) and almost the same sequence, the only difference being the absence of the helix breaker [Asn3,Pro4]- in [Gln3,Leu4]cecropin D. These replacements do not alter the amphipathicity of this segment; however, they increase the helicity. According to Chou-Fasman calculations ( P a ) of seg- ment 2-11 in [Gln3,Leu4]cecropin D is 1.21 and, for compar- ison, ( P a ) of segment 1-11 in cecropin A is 1.17. That means, in the [Gln3,Leu4] analog, the NH2-terminal amphipathic helix should be about the same length as in cecropin A. The fact that both compounds, cecropin D and [Gln3,Leu4]cecro-

pin D, show the same (low) activities could be due to two reasons. First, the different helicities in the NHz-terminal region do not affect the antibacterial activity. Second, the low basicity of the NHZ terminus is the dominant factor limiting the activity. The first explanation is not very likely, because it is known that incorporation of helix breakers in the amino- terminal region of cecropin A causes a loss of the broad spectrum activity (7). Thus, one can assume a strongly basic amino terminus is playing an important role for antibacterial activity.

Further evidence for this explanation comes from compar- ison of the activities of cecropin D, [Lys'lcecropin D, [Gln3,Leu4]cecropin D, and cecropin [A-(1-11) D-(12-37)]. Cecropin D and its [G1u3,Leu4] analog (net charges: +3) show the lowest activities against P. aeruginosa, B. megaterium, and M. luteus, whereas [Lys'lcecropin D (net charge: +4) is slightly more active, and cecropin A-(1-11) D-(12-37) (net charge: +7) is highly active. The only difference between cecropin D and [Lys'lcecropin D is the lysine residue at position 1. Since the helicity of [Lys'lcecropin D should not be affected by the additional lysine residue (there is a disrup- tion of the NHz-terminal helix by the helix breakers Am3 and Pro4), the main difference will be an additional positive charge. Apparently, this is the reason for the increasing activity in the case of [Lys'lcecropin D. The same effect, although more pronounced, should be responsible for the high activity of cecropin A-(1-11) D-(12-37) because the differ- ences at the NHz terminus between the highly active cecropin A-D hybrid and the less active [Gln3,Leu4]cecropin D are differences of charge; the predicted helicities of both analogs are similar ((Pa) of cecropin [A-(1-11) D-(12-37)]: 1.17, (Pa.) of [Gln3,Leu4]cecropin D: 1.21).

The Design of a Cecropin Analog with Enhanced Activity- It has been possible to construct a cecropin analog that exhibits greater antibacterial activity than the natural cecro- pins. Thus, the synthetic hybrid, cecropin A-(1-11) D 4 2 - 37) was slightly more active than cecropin A against E. coli D21 and D22, P. aeruginosa, B. megaterium, and M. luteus, but was clearly several times more active than cecropin A against B. subtilis. Since the hybrid has the same NH2- terminal sequence 1-11 and the same net charge (+7) as cecropin A, the enhancement of activity must be due to differences in the central and COOH-terminal regions that are the same as in cecropin D. Cecropin D is believed to be more helical than cecropins A or B, and it is more hydrophobic toward the COOH terminus. Both features are thought to contribute to the higher activity of cecropin A-(1-11) D-(12- 37) compared with the A form. Conversely, the enhancement of the activity of the analog relative to cecropin D must be due to the differences in the NHz-terminal region where the hybrid is more basic. The large effects (5-55-fold increases)

Cecropin D show the very important role of the basic NHp-terminal segment. This is the first example we know in which a cecropin analog has been successfully designed, on the basis of previous structure-activity studies, to have enhanced anti- bacterial potency.

The most remarkable property of the AD hybrid is its dramatically increased ability to form voltage-dependent channels in artificial membranes (32). We believe this may be the mechanism by which these peptides exert their anti- bacterial effects and are studying this question.

Acknowledgments-We thank Drs. Frank Field and Brian Chait for the mass spectrometry that was carried out at the Rockefeller University Mass Spectrometric Biotechnology Research Resource. The spectropolarimeter was purchased by the Rockefeller University with funds from National Science Foundation Grant PCM 84-00268.

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