Discovery of short peptides exhibiting high potency against Cryptococcus neoformans

Article abstract of DOI:10.1021/ml500011v

Rapid increase in the emergence of resistance against existing antifungal drugs created a need to discover new structural classes of antifungal agents. In this study we describe the synthesis of a new structural class of short antifungal peptidomimetcis, their activity, and plausible mechanism of action. The results of the study show that peptides 11e and 11f are more potent than the control drug amphotericin B, with no cytotoxicity to human cancer cells and noncancerous mammalian kidney cells. The selectivity of peptides to fungus is depicted by transmission electron microscopy studies, and it revealed that 11e possibly disrupts the model membrane of the fungal pathogen.

Full text of DOI:10.1021/ml500011v

Letter
pubs.acs.org/acsmedchemlett
Discovery of Short Peptides Exhibiting High Potency against
Cryptococcus neoformans
Amit Mahindra,† Nitin Bagra,† Nishima Wangoo,‡ Shabana I. Khan,§ Melissa R. Jacob,§ and Rahul Jain*,†
†Department of Medicinal Chemistry, National Institute of Pharmaceutical Education and Research, Sector 67, S. A. S. Nagar, Punjab
160 062, India
‡Center for Nanoscience and Nanotechnology, Panjab University, Sector 14, Chandigarh 160 014, India
§National Center for Natural Products Research, School of Pharmacy, The University of Mississippi, University, Mississippi 38677,
United States
S
* Supporting Information
ABSTRACT: Rapid increase in the emergence of resistance against existing
antifungal drugs created a need to discover new structural classes of antifungal
agents. In this study we describe the synthesis of a new structural class of short
antifungal peptidomimetcis, their activity, and plausible mechanism of action. The
results of the study show that peptides 11e and 11f are more potent than the control
drug amphotericin B, with no cytotoxicity to human cancer cells and noncancerous
mammalian kidney cells. The selectivity of peptides to fungus is depicted by
transmission electron microscopy studies, and it revealed that 11e possibly disrupts
the model membrane of the fungal pathogen.
KEYWORDS: Antifungal peptides, microwave, TEM, C. neoformans, arylation
antimicrobial peptides are not completely understood, but in
uring the past 20 years, the incidence of invasive fungal
Dinfections such as candidiasis, cryptococcosis, and most cases their biological effects are believed to involve
membrane disruption of the target cells.15 CAMPs are generally
positively charged and therefore bind preferentially to
negatively charged bacterial membranes rather than to
mammalian cell membranes, which are neutral.16 Because of
their unique mode of action, the development of resistance
against them is considered unlikely, and thus, they can be
promising alternatives of traditional known drugs.
As part of our ongoing effort in search of novel peptide and
peptidomimetics for infectious diseases,17,18 we examined the
sequence and mode of action of a number of antifungal
peptides (AFPs) reported in literature.19−25 As shown in Table
1, despite having large sequence diversity, these peptides have a
number of amino acids in common and sometimes a repeat of a
specific sequence. A large number of cationic amino acids such
as arginine (R, Arg), lysine (K, Lys), and histidine (H, His) are
present in the sequences; while at the same time, hydrophobic
amino acids like tryptophan (W, Trp), phenylalanine (F, Phe),
leucine (L, Leu), and isoleucine (I, Ile) are also present
abundantly. It is already known that a charge to bulk ratio is
essential for peptides to show antifungal activity. In the cases of
indolicidin22 and tritrpticin,23 it is observed that repeated Arg
aspergillosis in humans has increased considerably.1 They are
major causes of mortality and morbidity, especially in patients
whose immune systems are compromised by AIDS, cancer, or
organ transplant.2 Defense against such infectious agents has
relied mainly on the use of three classes of chemotherapeutic
agents, including azoles (e.g., fluconazole),3 macrocyclic
polyenes (e.g., amphotericin B),4 and candins (e.g., micafun-
gin).5 Each treatment option has several drawbacks, such as
drug related toxicity, emergence of resistant strains, nonoptimal
pharmacokinetics, poor solubility, and serious drug−drug
interactions.6 Therefore, there is a pressing need to discover
new antifungal agents that do not share the same structural
scaffold and the same line of mechanism as existing drugs.7
The quest for new antifungals is even more critical because
recently developed antifungal drugs also are developing
resistance against the most prevalent pathogens like Crypto-
coccus neoformans and Candida albicians.8 In that regard,
cationic antimicrobial peptides (CAMPs) offer promising
opportunities for the development of new antifungal agents.9
During the past 5−10 years, many CAMPs have been patented
for a variety of biological activities, such as antitumor,10
antiinflammatory,11 antiviral,12 and antifungal.13 Although they
are diverse in terms of sequence and structure, they share
common features such as a net positive charge and the
hydrophobic groups on different sides.14 The mechanisms of
Received: October 4, 2013
Accepted: January 15, 2014
Published: January 15, 2014
© 2014 American Chemical Society
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Table 1. Sequence and Mode of Action of Natural AFPs
peptide
sequence
target organism/mode of action
defensin NP-1
dermaseptin b
histatin-5
indolicidin
tritrpticin
Sub5
VVCACRRALCLPAQRRAGFCRIRGRIHPLCCRR
DVLKKIGTVALHAGKAALGAVADTISQ
DSHAKRHHGYKRKFHEKHHSHRGY
ILPWKWPWWPWRR-NH2
VRRFPWWWPFLRR
C. neoformans/Lysis
C. neoformans/Lysis
C. albicans/Lysis
C. neoformans/Lysis
C. albicans/Lysis
A. nidulans/Lysis
C. albicans/Lysis
C. albicans/Lysis
C. albicans/Lysis
C. neoformans/Lysis
RRWKIVVIRWRR
W3
VRWRIRVAVIRA
Pep15
VRLRIRVWVIRA
Bac2A
RLARIVVIRVAR
R3
RLRRIVVIRVAR
units are present on the terminal end, whereas the central core
of these peptides contains several Trp residues. It was also
noted that tritrpticin exhibits weak antifungal activity as
compared to indolicidin, possibly because C-terminus of
indolicidin is amidated, whereas C-terminus of tritrpticin is
carboxylated.25
A curious examination of the sequences of defensin NP-1,19
Sub5,24 W3,24 Pep 15,24 Bac2A,24 and R324 clearly indicates the
presence of Arg residue next to hydrophobic amino acids,
confirming the importance of a minimum charge versus bulk
ratio in the bioactivity. In specific cases, presence of RIR, RIH
(defensin NP-1), KKI (dermaseptin b), KRH, KRK (histatin-
5), WRR (indolicidin), RWR (sub5 and W3), and RLR (pep15,
Bac2, and R3) motifs containing both hydrophobic and
hydrophilic amino acids are observed. Despite having promising
activities, these peptides have some disadvantages in terms of
high cost of synthesis, proteolytic instability, high cytotoxicity,
nonspecific activity, and short circulation lifetime. One method
to overcome these obstacles is to synthesize peptides of shorter
length, while keeping the presumed bioactive core intact. The
truncation of native peptide to smaller fragments is one of the
most attractive strategies to discover smaller peptides, which
carry the bioactivity of the original sequence.26 This strategy
provides prefabricated structural frameworks for synthetic
manipulations that could be successfully optimized for
discovering short AFPs.26 A number of studies based on the
truncation approach have been reported, including peptoids,27
cyclic peptides,28 and synthetic peptidomimetics.17,18,29
Our aim was to develop shorter AFPs with enhanced activity
and potency. In this regard, we designed tripeptides by keeping
the arginine residue at the end terminals, and the centrally
located L-histidine residue was substituted with an aryl moiety
at the C-2 position. The presence of the guanidinium side-
chains of arginine and imidazole ring of the histidine provide
the necessary hydrophilicity. The presence of an aryl moiety at
the C-2 position of L-histidine and NHBzl or OMe groups at
the C-terminus provides the required bulk and hydrophobicity
for the membrane insertion. Furthermore, the introduction of
an aryl moiety in the imidazole ring of histidine creates an
amino acid with dual hydrophobichydrophilic character. The
general scaffold of the proposed peptides is shown in Figure 1.
A wide variety of lipophilic aryl substituents on the C-2 position
of L-histidine were explored. To observe the effect of C-
terminus capping on the bioactivity, two series of peptides were
synthesized having NHBzl and OMe groups at the C-terminus.
For the synthesis of the target peptides, a series of 2-arylated-
L-histidines (3a−e) were synthesized using a procedure
reported earlier, as depicted in Scheme 1.30 The removal of
protecting groups of 3a−e was achieved by refluxing in 6 N
Figure 1. Representative scaffold of the designed tripeptides.
Scheme 1. Overview of the Synthesis of N-α-Boc-2-aryl-L-
a
histidines
a
Reaction conditions: (i) 1 (1 equiv), 2a−e (2 equiv), AgNO3 (0.2
equiv), NH4(S2O8)2 (2 equiv), CF3CO2H (1.5 equiv), CH2Cl2/H2O
(1:1, v/v), 10−18 h, rt; (ii) 6 N HCl, 16−24 h, reflux; (iii) (Boc)2O,
1,4-dioxane/H2O (1:1), 12 h, rt.
HCl for 16−24 h. Finally, the synthesis of N-α-Boc-2-aryl-L-
histidines (5a−e) was achieved from 2-aryl-L-histidine dihydro-
chloride (4a−e).
The desired peptides were synthesized by a recently
developed, highly efficient and rapid microwave (MW)-assisted
peptide synthesis protocol, as depicted in Scheme 2.31,32 The
highlight was the successful synthesis of the desired peptides by
using amino acids bearing participating side-chain groups,
thereby confirming high reactive functional group tolerance and
atom economy of the procedure. In a 10 mL MW vial,
equipped with a magnetic stir bar, amino acid (Arg-NHBzl/
OMe, dissolved in DMF) and DIEA were added. Boc-2-aryl-
His-OH (5a−e) was then added, followed by DIC and HONB.
The reaction mixture was subjected to MW irradiation with gas
cooling for 28 min at 40 W with magnetic stirring and a
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ACS Medicinal Chemistry Letters
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temperature limit of 50 °C to give Boc-protected peptides 7−8
in 45−60% isolated yield.
Scheme 2. Synthesis of Arg-His(2-aryl)-Arg-NHBzl (11a−f)
and Arg-His(2-aryl)-Arg-OMe (12a−f) under MW
a
Irradiation
Boc-His(2-aryl)-Arg-NHBzl/OMe (7−8) upon reaction with
3 N HCl (5 mL) at 25 °C for 15 min resulted in the removal of
Boc group. The salt of dipeptide was neutralized in situ with
DIEA and to the resulting NH2-His(2-aryl)-Arg-NHBzl/OMe
in DMF was added Boc-Arg-OH, followed by HATU and
HOAt. Mixture was subjected to MW irradiation for 28 min at
40 W with magnetic stirring, and a temperature limit of 50 °C
to produce protected peptides 9−10 in 40−45% isolated yield.
The removal of the Boc group using 3 N HCl afforded the
designed peptides 11−12. We have also synthesized Boc-L-His-
Arg-OMe, Boc-D-His-Arg-OMe, and Boc-D,L-His-Arg-OMe
under MW irradiation and confirmed the extent of racemization
using HPLC. As evident from the HPLC chromatograms,
purified peptides were free of racemization (see Supporting
Information).
The synthesized peptides were evaluated for in vitro activity
against fungal (C. albicians, C. glabrata, C. krusei, Aspergillus
fumigates, and C. neoformans) and bacterial (E. coli, S. aureus,
and MRSA) strains, and the results are summarized in Tables 2
and 3 for Cryptococcus, S. aureus, and MRSA. All the peptides
were found to be inactive against Candida, Aspergillus, and E.
coli (results not included). The minimum inhibitory concen-
tration (MIC) was measured using a protocol suggested by the
Clinical and Laboratory Standard Institute (previously known
a
Reaction conditions: (i) Boc-His(2-aryl)-OH, DIEA, DIC, HONB,
DMF, 50 °C, 28 min, MW; (ii) 3 N HCl in MeOH, 15 min, rt; (iii)
Boc-Arg-OH, DIEA, HATU, HOAt, DMF, 50 °C, 28 min, MW.
Table 2. In Vitro Antifungal Activities of Peptides
d
e
f
C. neoformans (μg/mL)
cytotoxicity
selectivity index
a
b
c
peptide
Ar
R1
R
IC50
MIC
MFC
CTX (μg/mL)
C. neoformans
9a
H
NHBzl
NHBzl
NHBzl
NHBzl
NHBzl
NHBzl
OMe
Boc
Boc
Boc
Boc
Boc
Boc
Boc
Boc
Boc
Boc
Boc
Boc
H
5.35
0.67
2.15
0.68
0.18
0.20
NA
10.00
1.25
2.50
2.50
0.63
0.31
NA
10.00
1.25
2.50
5.00
0.63
0.31
NA
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>1.8
9b
C6H5
>14.9
>4.6
9c
4-CH3−C6H4
4-OCH3−C6H4
4-C(CH3)3−C6H4
4-C6H5−C6H4
H
9d
>14.7
>55.6
>50.0
9e
9f
10a
10b
10c
10d
10e
10f
11a
11b
11c
11d
11e
11f
12a
12b
12c
12d
12e
12f
C6H5
OMe
10.4
9.71
4.06
0.71
0.8
NA
NA
4-CH3−C6H4
4-OCH3−C6H4
4-C(CH3)3-C6H4
4-C6H5−C6H4
H
OMe
NA
NA
>1.0
OMe
5.00
1.25
1.25
NA
5.00
1.25
1.25
NA
>2.5
OMe
>14.1
>12.5
OMe
NHBzl
NHBzl
NHBzl
NHBzl
NHBzl
NHBzl
OMe
NA
C6H5
H
0.73
0.35
0.43
0.07
0.10
19.0
NA
1.25
0.63
0.63
0.16
0.16
NA
1.25
10.00
0.63
0.16
0.16
NA
>13.7
>28.6
>23.3
>142.8
>100.0
4-CH3−C6H4
4-OCH3−C6H4
4-C(CH3)3−C6H4
4-C6H5−C6H4
H
H
H
H
H
H
C6H5
OMe
H
NA
NA
4-CH3−C6H4
4-OCH3−C6H4
4-C(CH3)3−C6H4
4-C6H5−C6H4
OMe
H
19.7
3.93
0.61
0.59
0.69
NA
NA
OMe
H
5.00
1.25
1.25
1.25
5.00
1.25
1.25
1.25
>2.5
OMe
H
>16.3
>16.9
OMe
H
amphotericin B
a
b
IC50 is the concentration (μg/mL) that affords 50% inhibition of growth. MIC (minimum inhibitory concentration) is the lowest test
c
concentration (μg/mL) that allows no detectable growth. MFC (minimum fungicidal concentration) is the lowest test concentration (μg/mL) that
kills 100% of the organism. Compounds that did not exhibit activity at the highest tested concentration of 20 μg/mL were considered not active
(NA). The in vitro cytotoxicity was determined against four human cancer cell lines (SK-MEL, KB, BT-549, and SK-OV-3) and two noncancerous
mammalian kidney cells (VERO and LCC-PK1) up to a highest tested concentration of 10 μg/mL. Selectivity index was calculated as CTX divided
d
e
f
by IC50 values for C. neoformans.
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determined logP values using ACD/LogP software.36 The
results demonstrated a strong correlation between the bio-
logical activity and overall lipophilicity of the tripeptides (Table
4). The correlation of activity and lipophilicity of Boc-protected
a
Table 3. In Vitro Antibacterial Activity of Peptides
S. aureus (μg/mL)
MRSA (μg/mL)
peptide
IC50
MIC
MBC
IC50
MIC
MBC
9e
12.51
6.88
20.00
20.00
NA
NA
NA
NA
0.50
11.50
10.77
NA
20.00
20.00
NA
NA
9f
20.00
NA
Table 4. Correlation of Lipophilicity and Activity of Peptides
a
11f
cipro
15.41
0.08
Using RP-HPLC
0.25
0.09
0.25
0.50
peptide HPLC analysis tR (min)
logP
C. neoformans IC50 (μg/mL)
a
MBC (minimum bactericidal concentration) is the lowest test
10a
10b
10c
10d
10e
10f
9a
10.75
11.89
14.76
13.59
20.91
18.64
16.41
17.63
19.41
18.93
24.41
24.04
−1.63
0.41
0.87
0.57
2.09
2.05
−0.90
1.14
1.60
1.30
2.83
2.78
NA
concentration (μg/mL) that kills 100% of the organism. NA, not
active.
10.44
9.71
4.06
0.71
0.8
as the National Committee for Clinical Laboratory Standards,
NCCLS).33 Amphotericin B, which is used clinically for C.
neoformans infections but is associated with high toxicity toward
mammalian cells, served as a positive control in these studies.34
To have a better understanding of the structure−activity
relationship it was decided to also evaluate Boc-protected
intermediate peptides 9a−f and 10a−f for their activity, in
addition to target peptides 11a−f and 12a−f (Table 2). The
results from screening of activity against strains fungal and
bacterial strains showed that the peptides were in general more
selective against the C. neoformans than S. aureus and MRSA.
The data shows that the substituents attached to the phenyl
ring of the centrally placed amino acid, L-histidine, greatly
influenced the overall hydrophobicity and activity of the
peptides.
5.35
0.67
2.15
0.68
0.18
0.20
9b
9c
9d
9e
9f
a
Retention times are given for elution of the respective peptide on a
reversed phase analytical HPLC system using a C-18 column. NA, not
active. Method: C-18 column (25 cm × 4.6 mm, 5 μM) run for 40 min
with a flow of 1 mL/min, using a gradient of 85−5%, where buffer A
was 0.1% CF3CO2H (TFA) in H2O and buffer B was 0.1% TFA in
CH3CN and detection at 220 nm.
In series 1, peptide 11e containing a bulky t-butyl group at
the para-position of the phenyl moiety placed at the C-2
position of the imidazole ring was found to be the most potent
against C. neoformans with an IC50 of 0.07 μg/mL as compared
to 0.69 μg/mL for amphotericin B. It also showed potent MIC
and MFC value of 0.16 μg/mL. Peptide 11f containing a
biphenyl group at the C-2 position of the imidazole ring also
showed similar activity. Most noteworthy are the potent MFCs
of 9e, 9f, and 11d−f, which are between 2 and 8 times more
potent than amphotericin B. Peptides 9e and 9f also exhibited
weak antibacterial activity against S. aureus and MRSA with IC50
values in the range of 6.88 and 12.51 μg/mL and MIC of 20.00
μg/mL (Table 3).
As discussed above, in series 2 peptides (12e and 12f) having
bulky groups at C-2 position of the imidazole ring like t-
butylphenyl or biphenyl exhibit most potent activity against C.
neoformans with IC50 values of 0.61 and 0.59 μg/mL,
respectively. Other peptides of this series 10b−10f, 12a, and
12c−d also showed promising IC50 values against C. neoformans
in the range of 0.71−20 μg/mL. The presence of NHBzl group
appeared to be more optimal for antifungal activity compared
to OMe group at the C-terminus.
All synthesized peptides were also evaluated for cytotoxicity
in a panel of mammalian cell lines to determine their safety
profile. The in vitro cytotoxicity was determined against four
human cancer cell lines (SK-MEL, KB, BT-549, and SK-OV-3)
and two noncancerous mammalian cells (VERO and LLC-
PK1) by neutral red uptake assay.35 The results demonstrated
that the synthesized peptides were nontoxic up to a
concentration of 10.00 μg/mL, which is indicative of a higher
selectivity index (>1- to >142-fold) of anticryptococcal activity.
The activity of these compounds does not seem to be due to a
general cytotoxic effect (Table 2).
peptides 10a−f and 9a−f is provided in Table 4. The data show
that the central residue, histidine, when substituted with the
bulky t-butyl on the para position of phenyl ring shows highest
potency.
The peptides 10e and 9e exhibited highest tR and logP of
20.91 min/2.09 and 24.41 min/2.83, respectively. The peptides
containing a biphenyl ring, 10f and 9f (tR and logP = 18.64
min/2.05 and 24.04 min/2.78, respectively) are less hydro-
phobic than 10e and 9e, showed the second highest antifungal
potency against the C. neoformans. The effect on the
hydrophobicity is also due to the presence of nonpolar side
groups. A closer examination of the results also revealed that
peptide 10e containing an ester group at the C-terminus was
less active compared to 9e containing a more hydrophobic
benzylamide group at the C-terminus. The more hydrophobic
benzylamide group containing peptides, in general, exhibit
higher bioactivity. To conclude, in all the cases, peptides with 4-
t-butylphenyl and biphenyl substituents at the C-2 position of
the histidine residue exert more hydrophobicity than 4-
methylphenyl, 4-methoxyphenyl, and phenyl substituted
counterparts and thereby exhibit enhanced antifungal activity.
The study depicting correlation of lipophilicity and activity of
peptides 11a−f and 12a−f is provided in the Supporting
Information.
For preliminary mechanistic studies, the most potent peptide
11e was imaged by transmission electron microscopy (TEM)
with a view to gain some understanding on its behavior in terms
of interactions with the synthetic mimics of membranes.37,38
The small unilamellar vesicles (SUVs) were prepared using the
reported method in the literature.39 The morphology of the
SUVs was monitored in the presence and absence of the
peptide 11e by depositing, on to a carbon coated copper grid,
of the treated and untreated SUVs and negatively staining the
sample with 2% (w/v) phosphotungstic acid solution.
In order to examine the impact of lipophilicity on the
biological activity of the synthesized peptides, we measured
their retention time (tR) by HPLC using a C-18 column and
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The results indicated that the untreated SUVs were
uniformly shaped, with intact morphology (Figure 2a,c),
AUTHOR INFORMATION
■
Corresponding Author
*(R.J.) E-mail: rahuljain@niper.ac.in. Fax: +91 (172) 2214692.
Tel: +91 (172) 2292024.
Author Contributions
All authors have given approval to the final version of the
manuscript.
Funding
A.M. thanks the Council of Scientific and Industrial Research
(CSIR), New Delhi, for the award of a Senior Research
Fellowship. Antimicrobial testing was supported by the NIH,
NIAID, Division of AIDS, Grant No. AI 27094 (antifungal) and
the USDA Agricultural Research Service Specific Cooperative
Agreement No. 58-6408-1-603 (antibacterial).
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
Ms. Marsh Wright and Mr. John Trott are acknowledged for
excellent technical support in biological testing at NCNPR.
ABBREVIATIONS
■
AA, amino acid; AMB B, amphotericin B; DIC, 1,3-
diisopropylcarbodiimide; DIEA, N,N-diisopropylethyl-amine;
DMF, N,N-dimethylformamide; EYPC, egg yolk L-α-phospha-
tidylcholine; EYPG, egg yolk L-α-phosphatidyl-DL-glycerol;
HATU, (1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo-
[4,5-b]pyridinium 3-oxid hexafluorophosphate); HOAt, 1-
hydroxy-7-azabenzotriazole; HONB, N-hydroxy-5-norbornene-
2,3-dicarboxylic acid imide; IC50, concentration (μg/mL) that
affords 50% inhibition of growth; MFC/MBC, minimum
fungicidal/bactericidal concentration; MIC, minimum inhib-
itory concentration; TEM, transmission electron microscopy
Figure 2. TEM images of untreated and treated SUVs with 11e. (a)
Untreated EYPC/EYPG. (b) EYPC/EYPG treated with 11e. (c)
Untreated EYPC/cholesterol. (d) EYPC/cholesterol treated with 11e.
whereas SUVs treated with 11e resulted in the destruction of
their cell membrane. In the case of EYPC/EYPG, the integrity
of SUVs was disrupted thoroughly when treated with peptide
(Figure 2b), whereas in the case of EYPC/cholesterol the
integrity of the cell wall is conserved in few cases (Figure 2d).
These results indicate that the differential disintegration of the
cell membrane of pathogenic fungi and cell membrane of host
could be the basis for the preferential activity of these peptides
toward Cryptococcus. It is presumed that tested peptide being
cationic in nature preferably interacts with the negatively
charged membrane as compared to zwitterionic membrane
thereby exhibiting selectivity to Cryptococcus.
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In conclusion, we have synthesized a series of novel small
peptides exhibiting high selectivity against the fungal pathogen
C. neoformans. The tripeptides were highly potent against C.
neoformans, with two of them, 11e and 11f, being approximately
8-fold more potent than the standard drug. Most interestingly,
peptides containing Boc group at the N-terminus also showed
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studies. The results from TEM studies revealed that the most
potent peptide 11e possibly kills the fungal pathogen by
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ASSOCIATED CONTENT
* Supporting Information
■
S
Detailed synthetic procedures, characterization data, HPLC
chromatograms, and details on the biological assays. This
material is available free of charge via the Internet at http://
pubs.acs.org.
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