Synthesis and activity of thioether-containing analogues of the complement inhibitor compstatin

Article abstract of DOI:10.1021/cb2000378

Disulfide bonds are essential for the structural stability and biological activity of many bioactive peptides. However, these bonds are labile to reducing agents, which can limit the therapeutic utility of such peptides. Substitution of a disulfide bond w

Full text of DOI:10.1021/cb2000378

ARTICLES
pubs.acs.org/acschemicalbiology
Synthesis and Activity of Thioether-Containing Analogues
of the Complement Inhibitor Compstatin
||
||
Patrick J. Knerr,†, Apostolia Tzekou,§, Daniel Ricklin,§ Hongchang Qu,§ Hui Chen,§
Wilfred A. van der Donk,†,‡,* and John D. Lambris§,*
†Department of Chemistry and ‡Howard Hughes Medical Institute, University of Illinois at UrbanaꢀChampaign,
600 S. Mathews Avenue, Urbana, Illinois 61801, United States
§Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104, United States
S Supporting Information
b
ABSTRACT: Disulfide bonds are essential for the structural
stability and biological activity of many bioactive peptides. How-
ever, these bonds are labile to reducing agents, which can limit the
therapeutic utility of such peptides. Substitution of a disulfide
bond with a reduction-resistant cystathionine bridge is an attrac-
tive means of improving stability while imposing minimal struc-
tural perturbation to the peptide. We have applied this approach
to the therapeutic complement inhibitor compstatin, a disulfide-
containing peptide currently in clinical trials for age-related
macular degeneration, in an effort to maintain its potent activity
while improving its biological stability. Thioether-containing compstatin analogues were produced via solid-phase peptide synthesis
utilizing orthogonally protected cystathionine amino acid building blocks and solid-supported peptide cyclization. Overall, the
affinity of these analogues for their biological target and potent inhibition of complement activation were largely maintained when
compared to those of the parent disulfide-containing peptides. Thus, the improved stability to reduction conferred by the thioether
bond makes this new class of compstatin peptides a promising alternative for therapeutic applications. Additionally, the versatility of
this synthesis allows for exploration of disulfide-to-thioether substitution in a variety of other therapeutic peptides.
isulfide bonds play a critical role in the structural stability of
Cth is the methylene homologue of lanthionine, found in the
lantibiotic family of natural products.23,24 As such, the consider-
able body of work on the chemical synthesis of lanthionine-
containing cyclic peptides, including lactosin S,25 bis(desmethyl)
lacticin 3147 A2,26 and fragments of nisin27,28 and subtilin,29,30 is
directly applicable to the synthesis of Cth analogues of disulfide-
bridged peptides. Additionally, the structural constraint asso-
ciated with cyclic lanthionines has been utilized to improve the
activity and/or stability of bioactive peptides, including enke-
phalin,18 angiotensin,31 luteinizing hormone-releasing hormone,32
and somatostatin.33 Thus, incorporation of cyclic thioether-con-
taining amino acids is a valuable approach to the development and
optimization of therapeutic peptides, and improved methods of
constructing such compounds have application to a wide variety of
biological problems.
Compstatin (I[CVVQDWGHHRC]T-NH2) is a 13-residue,
disulfide-containing peptide inhibitor of complement activation
that was originally discovered via screening of a phage-display
library.34 Compstatin inhibits the proteolytic activation of com-
plement component C3 to the anaphylatoxin C3a and the
opsonin C3b by C3 convertase complexes (Figure 1a) via direct
D
peptides and proteins.1 Intricate conformations enforced by
cyclic disulfide networks are associated with the potent biological
activities of a variety of peptide natural products such as the
conotoxins,2 cyclotides,3 and defensins.4 In the case of many
bioactive peptides, the structural constraint introduced by disul-
fide cyclization has been demonstrated to improve resistance to
proteolysis and reduce the entropic cost of binding to their
targets.5,6 However, the lability of the disulfide bond to intra- and
extracellular reducing agents7ꢀ9 can reduce the biological activity
of disulfide-containing peptides and limit their usefulness as
therapeutics. To address this concern, substitution of the disul-
fide bond in bioactive peptides with a variety of different moieties
has been explored, including the use of diselenide,10,11
lactam,12,13 carba,14ꢀ16 and thioether bridges.17,18 A disulfide-
to-thioether substitution utilizing a cystathionine (Cth) bridge is
particularly attractive, since it involves the substitution of only a
single atom (-S- to -CH2-) and results in a carbonꢀsulfur bond
stable to reduction with minimal structural perturbation. Syn-
thetic replacement of disulfides with thioethers has produced
analogues of vasopressin,19 oxytocin,20 anticardiolipid antibody
binders,21 and VCAM/VLA-4 antagonists.22 However, as evi-
denced by lack of recent literature describing Cth-containing
peptides, a higher-yielding and more versatile synthetic strategy
is needed to allow greater access to these cyclic structures.
Received: February 3, 2011
Accepted: April 26, 2011
Published: April 26, 2011
r
2011 American Chemical Society
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Scheme 1. Synthesis of Fmoc-Cystathionine Isomer 11a
a Reagents and conditions: (a) 70% HClO4, tBuOAc; (b) Fmoc-OSu, N-
methylmorpholine, THF, 62% (two steps); (c) PBu3, H2O, THF, 86%;
(d) Alloc-Cl, Na2CO3, H2O, CH3CN; (e) allyl bromide, NaHCO3,
DMF, 77% (two steps); (f) PPh3, CBr4, CH2Cl2, 80%; (g) Bu4NBr,
NaHCO3, H2O, EtOAc, 84%; (h) CF3CO2H, PhSiH3, CH2Cl2, 95%.
Scheme 2. Synthesis of Fmoc-Cystathionine Isomer 17a
Figure 1. Complement-targeted inhibitory mechanism and peptide
sequences of compstatin analogues. (a) The complement cascade is
initiated through at least three different mechanisms, which all converge
in the proteolytic activation of C3 to C3b; C3b participates in a self-
amplified loop that enhances the effect of complement activation.
Inhibition by compstatin at the level of C3/C3b effectively stops the
activation of all initiation pathways, the amplification loop, and all
downstream effects of complement activation. C1ꢀC5: complement
components 1ꢀ5; FB: factor B; FD: factor D; MAC: membrane attack
complex; MBL: mannose-binding lectin; MASP: MBL-associated serine
protease; P: properdin. (b) The sequences of compstatin analogues 1ꢀ5
used in this study. The two cystathionine isomers are depicted as Ala-
Hcy or Hcy-Ala, depending on the position of the sulfur atom. Hcy:
homocysteine; Trp(Me): 1-methyltryptophan.
a Reagents and conditions: (a) Fmoc-OSu, Na2CO3, H2O, 1,4-dioxane;
(b) CCl3C(NH)OtBu, CH2Cl2, THF, 52% (two steps); (c) PPh3, CBr4,
CH2Cl2, 94%; (d) Aloc-Cl, Na2CO3, H2O, CH3CN; (e) allyl bromide,
NaHCO3, DMF, 70% (two steps); (f) CF3CO2H, Pr3SiH, CH2Cl2,
85%; (g) Bu4NBr, NaHCO3, H2O, EtOAc, 93%; (h) CF3CO2H,
PhSiH3, CH2Cl2, 98%.
i
binding to C3 as well as C3b.35ꢀ37 The central role of C3 and its
proteolytic fragments in all complement initiation and amplifica-
tion pathways makes compstatin an attractive potential thera-
peutic for the treatment of pathologies involving inappropriate
activation of the complement system,35,38 including a variety of
autoimmune, inflammatory, and neurodegenerative diseases as
well as sepsis, hemodialysis-associated thrombosis and transplan-
tation medicine. In particular, compstatin has shown promising
activity in treating early forms of age-related macular degenera-
tion and is currently in clinical trials for the treatment of this
disease that constitutes a major cause of blindness in the
elderly.39
Early work has shown that the activity of compstatin is
dependent on disulfide formation between cysteine residues at
positions 2 and 12, which enforces the cyclic conformation
critical for binding C3.36 Either reductionꢀalkylation of the
disulfide bond or replacement of the two cysteine residues with
alanine results in complete loss of C3 binding and complement
inhibition.34 Furthermore, cyclization protects compstatin from
proteolytic degradation in human blood.40 Extensive structureꢀ
activity relationship studies have resulted in derivatives with up to
1000-fold improvement in potency when compared to the original
compstatin sequence.35,41ꢀ43 Yet the increasing use of compstatin
in both systemic and local (e.g., intravitreal) clinical applications
also puts new demands on pharmacokinetic properties such as
metabolic stability. Here, we report the synthesis of a series of
compstatin analogues 2, 3, and 5 (Figure 1b) containing a
reduction-resistant Cth bridge in place of disulfide, which maintain
potent complement inhibitory activity and strong affinity for C3b.
’ RESULTS AND DISCUSSION
Cth Building Block Synthesis. We desired a versatile and
high-yielding synthetic strategy to produce a family of Cth-
containing compstatin analogues, which could also be applied
to the synthesis of other Cth-containing peptides. In this regard,
we adopted a 9H-fluorenylmethoxycarbonyl (Fmoc)-based so-
lid-phase peptide synthesis (SPPS) that was modified from the
approach used by Vederas and co-workers for the total synthesis
of lantibiotics.25,26 This strategy involved the synthesis of two
enantiomerically pure, orthogonally protected LL-Cth building
blocks, representing the two possible sulfur-to-methylene sub-
stitutions from the parent disulfide, followed by solid-supported
peptide assembly and cyclization. This building block approach
allows for complete positional freedom for the placement of the
Cth bridge within the cyclic peptide. Construction of Cth building
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Scheme 3. Synthesis of Cystathionine-Containing Compsta-
tin Analogues 2, 3, and 5a
Figure 2. Kinetic ranking of compstatin analogues. (a) Analogues 1-5 at
1 μM concentration were injected over captured C3b at a surface density
of 3000 RU. The signals are overlaid to show relative differences in their
association and dissociation phases. Each plot is representative of three
data sets. RU: resonance units. (b) Schematic representation of the
SPR assay.
a Reagents and conditions: (a) SPPS; (b) Pd(PPh3)4, PhSiH3, DMF; (c)
i
piperidine, DMF; (d) PyBOP, HOAt, Pr2EtN, DMF; (e) CF3CO2H,
i
H2O, Pr3SiH, HS(CH2)2SH. An asterisk indicates standard Fmoc-
orthogonal side chain protection: Gln(Trt), Asp(OtBu), Trp(Boc),
His(Trt), Arg(Pbf), Thr(tBu).
results from building block 17, yields a more potent complement
inhibitor. Based on those results, we used 17 in the synthesis of
analogue 5, which includes three additional mutations (Trp4/
Trp(Me), Gly8/Sar, and Thr13/N-MeIle) found in the most
active compstatin sequence reported to date, Compstatin 20
(control peptide 4, Figure 1b).41
block 11 (Scheme 1) proceeded in high yield via a convergent
synthesis. Phase-transfer condensation44 of Fmoc- and tert-butyl
ester-protected L-cysteine 7, derived from L-cystine, with allyloxy-
carbonyl (Aloc)- and allyl ester (OAll)-protected γ-bromo-L-am-
inobutyrate 9, derived from L-homoserine, yielded the protected
Cth building block 10. The tert-butyl ester of 10 was subsequently
removed to reveal the free carboxyl group in 11 necessary for SPPS.
Synthesis of the other Cth isomer 17 (Scheme 2) was similarly
performed via phase-transfer condensation of γ-bromoaminobuty-
rate derivative 13 with cysteine derivative 15.
The stereochemical purity and absolute confirmation of the
synthesized LL-Cth building blocks was assessed via chiral gas
chromatography/mass spectrometry (GC/MS) using a Varian
CP-Chirasil-L-Val column (Supporting Information). Com-
pound 17 was globally deprotected and derivatized as the
pentafluoropropionamide methyl ester.25,45 Comparison of this
compound with derivatized standards of LL- and DL-cystathionine
confirmed the expected LL-configuration of the synthetic Cth
building block with no detectable epimerization of the enantio-
merically pure starting materials during synthesis.
Peptide Synthesis. Three Cth-containing compstatin analo-
gues (2, 3, and 5) were prepared. Peptides 2 and 3 represent the
two possible Cth analogues of the second-generation compstatin
derivative 4W9A (control peptide 1, Figure 1b),36 with the Cth-
based thioether bridge replacing the original Cys2ꢀCys12
disulfide bond. Our initial assessment of binding data with
peptides 1ꢀ3 (see Kinetic Analyses below) indicated that the
location of the sulfur atom closer to the N-terminus, as in 3 that
Construction of compstatin analogues 2, 3, and 5 (Scheme 3)
utilized Fmoc-based SPPS on Rink amide AM resin and 2-(6-
chloro-1H-benzotriazole-1-yl)-1,1,3,3-tetramethylaminium hexa-
fluorophosphate (HCTU) as the coupling reagent. As reported
for the synthesis of the lanthionine-containing peptide bis-
(desmethyl)lacticin 3147 A2,26 intermolecular coupling between
the distal ends of two resin-bound lanthionine residues becomes
problematic during cyclization when using resin loadings greater
than 0.3 mmol/g. Therefore, in order to promote the desired
intramolecular cyclization, resin loading was reduced to
0.2 mmol/g by coupling a substoichiometric amount of the
Cth building block, followed by acetylation of the remaining free
resin sites with acetic anhydride (Methods). After coupling of the
remaining residues inside the incipient cystathionine ring, the
allyl protecting groups of the Cth residue were removed with
Pd(PPh3)4 and PhSiH3, and the N-terminal Fmoc group was
removed with piperidine. Cyclization of this deprotected resin-
bound fragment 18 was promoted with (benzotriazole-1-yloxy)-
tripyrrolidinophosphonium hexafluorophosphate (PyBOP),
1-hydroxy-7-azabenzotriazole (HOAt), and diisopropylethyl-
amine. Coupling of the N-terminal isoleucine, Fmoc removal,
amine acetylation, and trifluoroacetic acid-promoted global
deprotection/resin cleavage yielded the crude compstatin analo-
gues, which were purified by reversed-phase high-performance
liquid chromatography (RP-HPLC). The pure peptides were
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Figure 3. Single-cycle kinetics analysis of the thioether compstatin analogues and corresponding disulfide bond controls. Sets of five increasing
concentrations were consecutively injected over a C3b surface (3000 RU density) in a single cycle. In the case of peptides 1ꢀ3, two sets of
concentrations are superimposed for a better description of the full binding range and to achieve a well-defined affinity profile. The processed signals
were fitted to a 1:1 binding model (orange simulation curves) and kinetic rate constants ka and kd were extracted (Table 1). The compstatin analogue
used is shown in the top left corner of each plot. Plots are representative of at least six data sets.
obtained with average overall yields of 2.4ꢀ5.0%, or 88ꢀ90% per
Table 1. Kinetic Rate Constants, Binding Affinities, Inhibi-
step over 29 steps. Peptide 5 had the lowest overall yield, presumably
due to difficulty in coupling the Cth building block immediately after
a sterically demanding N-methylisoleucine residue.
tory Potential, and Relative Inhibitory Activities (rIC50) of
Compstatin Analogues
ka
kd
KD
(nM)a
IC50
(nM)b
Chiral GC/MS was again utilized in order to confirm that the
LL-stereochemistry of the Cth building blocks was maintained
during compstatin analogue synthesis (Supporting Information).
Peptide 5 was hydrolyzed in refluxing 6 M HCl, and the resulting
amino acids were derivatized as the pentafluoropropionamide methyl
esters and compared to the derivatized LL- and DL-cystathionine
standards as described above. The vast majority of the derivatized
cystathionine hydrolyzed from 5 contained the appropriate
LL-configuration, confirming that the Cth building blocks are con-
figurationally stable to the basic conditions employed during SPPS.
Kinetic Analyses. The resulting Cth-containing compstatin
analogues were then assayed to determine their affinity for C3b
and their effectiveness in inhibiting complement activation. Kinetic
characterization of the interaction of each compstatin analogue with
C3b was performed using surface plasmon resonance (SPR).
Biotinylated C3b was site-specifically captured on a streptavidin-
coated sensor chip to prevent surface heterogeneity.38,42 A kinetic
ranking of the compstatin analogues at 1 μM (Figure 2) indicated
that the association and dissociation profiles of peptide 3 were the
most comparable to those of its corresponding control peptide 1.
Peptide 2, on the other hand, displayed a significantly lower binding
activity compared to 1. In the case of Compstatin 20, analogue 5
showed a similar association but faster dissociation rate than the
corresponding control peptide 4.
peptide (105 Mꢀ1 sꢀ1
)
(10ꢀ2 sꢀ1
)
rIC50
a
a
1
2
3
5.6 ( 1.4
5.5 ( 0.7
4.6 ( 0.5
8.1 ( 1.1 150 ( 19 1900 ( 1200
46.8 ( 5.7 847 ( 59 >20000
11 ( 1.9 236 ( 20 3100 ( 1800
1
>10
1.6
4
5
18.6 ( 4.1
16.4 ( 3.8
0.4 ( 0.1
1.5 ( 0.4
2.4 ( 0.6
9 ( 1.8
130 ( 40
270 ( 80
1
2.1
a Results show average and standard deviations of at least six data sets.
b Results show average and standard deviations of at least five data sets.
subsequent injection or the use of regeneration solutions that
remove residual peptide yet may harm the activity of C3b. We
therefore opted for a single-cycle kinetics approach,46 which
allows for kinetic characterization of interactions with slow
dissociation rates in a shorter period of time than does the
traditional multicycle approach.40 This approach was used for all
peptides, so as to enable us to make direct comparisons of the
acquired results. Importantly, all compounds showed a binding
response that could be fitted to a 1:1 binding model. The single-
cycle kinetics analysis showed fast association and dissociation
rates for compounds 1, 2, and 3, and fast association but slower
dissociation for compounds 4 and 5 (Figure 3, Table 1). The
compounds mainly differ in their dissociation rates, whereas the
association rate remained rather constant within the correspond-
ing series of analogues. The resulting binding affinity is therefore
mainly defined by the changes in kd. In particular, peptide 2
displayed a 6-fold faster dissociation and a 6-fold reduced affinity
To further characterize and quantitate the relationship be-
tween the structure and activity of these compounds, we per-
formed a full kinetic analysis. The slow dissociation rate constant
of the current lead compstatin sequence 441 would require a long
assay time to allow the signal to reach baseline level before the
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their corresponding disulfide-containing control peptides
(Figure 4, Table 1). The ability of these compounds to inhibit
antigen/antibody-induced complement activation was assessed
by measuring the resulting reduction in C3b deposition on an
ELISA plate when compared to a control well with no peptide.
The percentage inhibition was then plotted against the peptide
concentrations and fitted to a logistic doseꢀresponse function.
These assays yielded results consistent with the data obtained in
the kinetic assays. Again, peptide 2 was identified as the least
active analogue, thereby confirming the unfavorable effect of
sulfur-to-methylene substitution on the C-terminal side of the
original disulfide. In both series, the thioether-containing peptides
3 and 5 showed a decreased inhibitory activity by a factor of only 2
when compared to their parent disulfide-linked peptides 1 and 4,
respectively (Table 1). In agreement with previous studies, these
results show a good correlation between the binding affinity for
C3b and the ability to inhibit complement activation. Importantly,
the combined analyses also confirm thioether-restricted compsta-
tin analogues as potent complement inhibitors.
Effect of Oxidation and Reduction on Peptide Activity.
The resistance of the cyclic structure of the Cth-containing
peptide 5 to reductive treatment and, conversely, the reductive
lability of the corresponding disulfide-linked analogue 4 was
confirmed by incubating the peptides with 500-fold molar excess
of the reducing agent tris(2-carboxyethyl)-phosphine hydro-
chloride (TCEP-HCl) and subsequent analysis by mass spectro-
metry (Supporting Information). In order to confirm that recent
compstatin analogues with largely increased potency and affinity
still depend on an intact ring structure, we subjected peptide 4
after reduction and alkylation to binding and complement
inhibition analysis as described above. This analysis is necessary
in the context that analogues containing sarcosine instead of
glycine at position 8 (including peptides 4 and 5) have been
implied to form a bound-like conformation in solution41 that may
have rendered maintenance of the disulfide less important.
However, consistent with previous analogues,40 peptide 4 was
rendered essentially inactive by the reduction process
(Supporting Information), thereby confirming the importance
both of the cyclic structure and of reductive stability. Whereas
cyclic, N-acetylated compstatin analogues have previously been
shown to exert high plasma stability under normal conditions,40
there are a variety of complement-related diseases that trigger
changes in the reductive status of blood. For example, compstatin
has shown promise as an organ-protecting agent in models of
bacterial sepsis,47 but this pathology also involves a large increase
in the plasma levels of thioredoxin that may endanger the
disulfide structure.48 Similarly, complement inhibition by comp-
statin has large potential in hemolytic conditions such as parox-
Figure 4. Inhibitory activity of compstatin analogues. (a) Representa-
tive assay result for inhibition of antigen/antibody complex-initiated
complement activation by compstatin analogues. The percentage in-
hibition is plotted against the peptide concentration and compared to
that for parent compstatin analogues 1 and 4. Plots are representative of
at least five assays. BSA: bovine serum albumin; OVA: ovalbumin; a-hC3
HRP: anti-human C3 antibody conjugated to horseradish peroxidase.
(b) Schematic representation of the assay.
when compared to control peptide 1. Peptide 3, on the other
hand, had a slightly slower association rate (1.2-fold) and slightly
faster dissociation rate (1.4-fold), yielding a ∼1.6-fold lower affinity
for C3b when compared to peptide 1. Finally, compound 5
displayed a comparable association rate but a ∼3-fold faster
dissociation rate than the corresponding control peptide 4 and thus
a ∼3.8-fold lower affinity for C3b. Importantly, the kinetic para-
meters for the control peptides 1 and 4 were very similar to those
published previously.40,41 The largely stable association rate con-
stants within each compound series suggest that the cystathionine
modification had little effect on complex formation with C3b and
appears to maintain the ability of compstatin peptides to change
between the distinct solution and bound conformers.36,41 However,
the slightly changed geometry between a disulfide and a thioether
bridge may have influenced the exact positioning of key residues
such as the tryptophan derivatives at position 4 and 7, thereby
affecting the complex stability as measured by kd. This effect appears
more pronounced in 5, as the mutations made in control peptide 4
better tailored the peptide to the binding site. Importantly, our
kinetic data clearly show that thioether-bridged analogues of
compstatin show high binding affinities in the range of the
compound currently in clinical trials (1Me-Trp;4 KD = 11 nM).42
Complement Inhibition Assays. The biological activity of
the Cth-containing compounds was also compared to that of
38
ysmal nocturnal hemoglobinuria or hemodialysis-induced
side effects,49 during which the large reservoir of reduced
glutathione (GSH) in erythrocytes (1ꢀ3 mM) is released and
increases the plasma pool of GSH.50,51 In these compstatin-
relevant therapeutic applications, protection of the peptide against
reduction may therefore become even more important.
Although thioethers are inert to reductive cleavage, their
lability to metabolic oxidation is well documented, especially in
the case of methionine in peptide and protein drugs.52,53 In
addition, thioether oxidation has been shown to severely decrease
the activity of the lantibiotic nisin,54 although the lantibiotic
actagardine contains an enzymatically oxidized methyllanthionine
bridge important for full activity.55 In our hands, the Cth-containing
building blocks and peptides appear stable to spontaneous oxidation
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during synthesis, purification, biological testing, and storage. How-
ever, to probe the effect of thioether oxidation on peptide binding
and inhibitory activities, we exposed peptides 4 and 5 to conditions
that allow selective oxidation of Cth to the corresponding sulfoxide
by hydrogen peroxide (Supporting Information). Oxidation of
peptide 5 led to a mass shift of þ16 Da, whereas peptide 4 was
largely unaffected by the treatment. As expected, the oxidative
treatment of peptide 4 did not significantly affect its inhibitory
potency (ΔIC50 < factor 2). Although Cth-oxidation of peptide 5
did induce some loss in both binding affinity and inhibition activity
(4ꢀ6-fold), the oxidized peptide was still a highly potent inhibitor
with a KD below 100 nM (Supporting Information).
Conclusion. We describe here the chemical synthesis of analo-
gues of the complement inhibitor compstatin with a reduction-
stable thioether bond in place of the reduction-labile disulfide bond.
This modification was installed via a versatile and high-yielding
solid-phase synthesis utilizing orthogonally protected cystathionine
building blocks. Furthermore, we have characterized the structure
ꢀactivity relationships of these cystathionine-containing analogues
and the disulfide-bonded parent peptides. Our results indicate that
the position of the sulfur atom within the thioether bridge has
consequences for activity, as δ-Cth-containing analogues largely
maintained the binding and inhibitory properties of the disulfide
parent, but γ-Cth-containing analogues did not. In comparing
potential metabolic alterations of these compounds, reduction of
the disulfide bond had a much more deleterious effect on potency
than potential oxidation of the cystathionine bridge, thereby under-
scoring the benefit of the substitution in this context. More gen-
erally, the impact of peptide-based drugs continues to grow, and
many of these compounds contain disulfide bonds.56,57 The versa-
tile nature of the synthesis described here allows for the production
of a wide variety of cyclic Cth-containing peptides in order to probe
the effects of disulfide substitution on the activity and stability of
other therapeutic peptides.
PhSiH3 (10 equiv) in 1:1 DMF/CH2Cl2 was added to the resin and reacted
for 2 h, protected from light. The reaction vessel was drained, and the resin
was washed with 0.5% diethyldithiocarbamate in DMF, DMF, and CH2Cl2.
The Fmoc group was removed with 20% piperidine in DMF (2 ꢁ 10 min).
Peptide cyclization was performed by treating the resin-bound linear peptide
with PyBOP (5 equiv), HOAt (5 equiv), and diisopropylethylamine
(10 equiv) in DMF for 2 ꢁ 1.5 h. The N-terminal Fmoc-Ile-OH was then
coupled, deprotected, and acetylated as described above.
The resin was washed with DMF and CH2Cl2 and then dried.
Concurrent side-chain deprotection and cleavage from resin were
achieved by stirring the resin-bound peptide in 91.5:5:2.5:1 trifluoroa-
cetic acid/water/triisopropylsilane/ethanedithiol for 2 h under N2. The
cleaved resin was removed by filtration, and the filtrate was concentrated
by a stream of N2. Crude peptide was precipitated with the addition of
cold Et2O, isolated, and lyophilized from 1:1 water/acetonitrile. The dry
crude peptide was purified by RP-HPLC employing the gradients de-
scribed below (solvent A is 0.1% trifluoroacetic acid in water; solvent B is
80% acetonitrile in water with 0.086% trifluoroacetic acid). Product-
containing fractions were pooled, lyophilized, and checked for identity
by MALDI-TOF MS.
Peptide 2. This compound was purified on a Waters Delta-Pak C18
preparative column with the following gradient: 2% solvent B for 1 min,
2ꢀ20% B over 2 min, 20ꢀ26% B over 3 min, 26ꢀ41% B over 30 min.
After lyophilization, 8 mg of pure peptide was obtained from a 0.1-
mmol-scale synthesis (5.0% total yield, 90% per step over 29 steps). tR =
31.9ꢀ32.8 min. LRMS (MALDI-TOF) calcd for C72H103N22O18S
1595.7, found 1595.7.
Peptide 3. This compound was purified in the same fashion as 2.
After lyophilization, 6 mg of pure peptide was obtained from a 0.1-mmol-
scale synthesis (3.8% total yield, 89% per step over 29 steps). tR
=
31.8ꢀ32.8 min. LRMS (MALDI-TOF) calcd for C72H103N22O18S
1595.7, found 1596.0.
Peptide 5. This compound was purified on a Phenomenex Luna
C18 preparatory column with the following gradient: 5% B for 1 min,
5ꢀ24% B over 2 min, 24ꢀ30% B over 3 min, 30ꢀ45% B over 30 min.
After lyophilization, 12 mg of pure peptide was obtained from a 0.3-
mmol-scale synthesis (2.4% total yield, 88% per step over 29 steps). tR =
26.7ꢀ27.8 min. LRMS (MALDI-TOF) calcd for C77H113N22O17S
1649.8, found 1649.9.
’ METHODS
Synthesis of Peptides 2, 3, and 5 via SPPS. Fmoc-protected
Rink amide AM resin (0.6 mmol/g loading) was swelled in dimethyl-
formamide (DMF) for 20 min and Fmoc-deprotected with 20%
piperidine in DMF (2 ꢁ 10 min). The first amino acid (Fmoc-Thr-
OH for 2 and 3, Fmoc-N-Me-Ile-OH for 5; 4 equiv to resin) was preac-
tivated in the presence of HCTU (4 equiv) and diisopropylethylamine
(8 equiv) in DMF for 5 min and then coupled to the resin for 1 h via
agitation with N2. The Fmoc group was then removed. In order to
prevent interstrand dimerization upon attempted cyclization, the resin
loading was manually reduced to 0.15 mmol/g by reacting a preactivated
solution of 11 or 17 (0.25 equiv to resin), HCTU (0.25 equiv), and
diisopropylethylamine (2 equiv) in DMF with the resin-bound peptide
for 3 h. The remaining free resin sites were acetylated with a solution of
1:1:3 acetic anhydride/diisopropylethylamine/DMF for 30 min, yield-
ing a negative Kaiser test.
After resin-loading modification, the resin-bound peptide was Fmoc-
deprotected with 20% piperidine in DMF (2 ꢁ 10 min), the reaction
vessel was drained, and the resin was washed with DMF and CH2Cl2.
Appropriately side chain-protected Fmoc-amino acids (4 equiv with
respect to the reduced-loading resin) were preactivated in the presence
of HCTU (4 equiv) and diisopropylethylamine (8 equiv) in DMF for 5
min and then coupled to the deprotected resin for 45ꢀ60 min. The
reaction vessel was drained, and the resin was washed with DMF and
CH2Cl2. A negative Kaiser test confirmed completed couplings.
Following the coupling of Fmoc-Val-OH to the resin-bound peptide,
a solution of Pd(PPh3)4 (1 equiv to the reduced-loading resin) and
Synthesis of Control Peptides 1 and 4. The synthesis and
characterization of these peptides has been described previously.41,58
Kinetic Analyses. C3 was purified from human plasma as described
before.43 C3b was generated by limited trypsinization and labeled with
biotin-maleimide using established protocols.59 The interaction of the
compstatin analogues with C3b was characterized using a Biacore 3000
instrument (GE Healthcare, Corp., Piscataway, NJ) at 25 °C. The
running buffer was PBS, pH 7.4 (10 mM sodium phosphate, 150 mM
NaCl) with 0.005% Tween-20. Biotinylated C3b was captured site-
specifically on a streptavidin chip at ∼3000, 4000, and 5000 RU density;
an untreated flow cell was used as a reference surface. An initial kinetic
ranking was performed by injecting the various peptides at a constant
concentration of 1 μM for 2 min. For detailed kinetic analysis, sets of five
increasing concentrations of a particular compound were consecutively
injected over the chip surface in a single cycle without waiting for full
dissociation between injections (kinetic titration).46 A 3-fold dilution
series (one set at 0.46ꢀ37 nM for analogues 4 and 5, and two sets at
0.46ꢀ37 and 111ꢀ9000 nM for all other analogues) was injected at a
flow rate of 30 μL/min; each injection was done for 2 min, allowing the
peptide to dissociate for 5 min each time before the next injection was
started. After the end of the last injection, a 10-min dissociation was used for
compounds 1, 2and 3, while a 15-min dissociation was used for compounds
4 and 5. Data processing and analysis was performed using Scrubber
(BioLogic Software, Campbell, Australia) and BIAevaluation (GE Health-
care). A series of blank injections was subtracted from the binding signals
758
dx.doi.org/10.1021/cb2000378 |ACS Chem. Biol. 2011, 6, 753–760

ACS Chemical Biology
ARTICLES
(double referencing) and the processed signals were fitted to a single cycle
1:1 binding model (kindly provided by GE Healthcare) to extract the
kinetic rate constants (ka, kd) and calculate the binding affinity (KD =kd/ka).
Complement Inhibition Assays. To assess the complement
inhibitory ability of the compstatin analogues, an enzyme-linked im-
munosorbent assay (ELISA) was performed as previously described60
with small modifications. In brief, an ovalbumin-based antigen/antibody
complex was used as an initiator of the classical pathway of complement
activation. Serial dilutions of the compstatin analogues were prepared,
and normal human plasma was added to a final dilution of 1:80 in
veronal-buffered saline containing MgCl2 and CaCl2. Incubation for
15 min followed to allow complement activation and amplification.
The deposition of C3b on the plate was measured with a polyclonal
antibody against C3. The percentage of inhibition was plotted against
the compstatin concentration and fitted to a logistic doseꢀresponse
function using Origin (OriginLab Corp., Northampton, MA).
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essential function of protein-disulfide isomerase is to unscramble non-
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P. T., Daly, N. L., Hu, S.-H., Armishaw, C. J., Wang, C.-I. A., Lewis, R. J.,
Martin, J. L., Noakes, P. G., Craik, D. J., Adams, D. J., and Alewood, P. F.
(2010) Solving the R-conotoxin folding problem: Efficient selenium-
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(1991) Design and synthesis of a specific endothelin 1 antagonist:
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’ ASSOCIATED CONTENT
(14) Elaridi, J., Patel, J., Jackson, W. R., and Robinson, A. J. (2006)
Controlled synthesis of (S,S)-2,7-diaminosuberic acid: A method for
regioselective construction of dicarba analogues of multicystine-contain-
ing peptides. J. Org. Chem. 71, 7538–7545.
(15) Derksen, D. J., Stymiest, J. L., and Vederas, J. C. (2006)
Antimicrobial leucocin analogues with a disulfide bridge replaced by a
carbocycle or by noncovalent interactions of allyl glycine residues. J. Am.
Chem. Soc. 128, 14252–14253.
S
Supporting Information. General materials and methods,
b
synthetic procedures and characterization for synthetic compounds
6-17, effects of oxidation/reduction on the activity of peptides 4
and 5, and GC/MS procedures and chromatograms. This material
is available free of charge via the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION
(16) Stymiest, J. L., Mitchell, B. F., Wong, S., and Vederas, J. C.
(2005) Synthesis of oxytocin analogues with replacement of sulfur by
carbon gives potent antagonists with increased stability. J. Org. Chem.
70, 7799–7809.
(17) Bondebjerg, J., Grunnet, M., Jespersen, T., and Meldal, M.
(2003) Solid-phase synthesis and biological activity of a thioether
analogue of conotoxin G1. ChemBioChem 4, 186–194.
Corresponding Author
*E-mail: vddonk@illinois.edu, lambris@upenn.edu.
Author Contributions
These authors contributed equally to this work.
(18) Rew, Y., Malkmus, S., Svensson, C., Yaksh, T. L., Chung, N. N.,
Schiller, P. W., Cassel, J. A., DeHaven, R. N., and Goodman, M. (2002)
Synthesis and biological activities of cyclic lanthionine enkephalin
analogues: Delta-opioid receptor selective ligands. J. Med. Chem.
45, 3746–3754.
(19) Jost, K., Prochazka, Z., Cort, J. H., Barth, T., Skopkova, J.,
Prusik, Z., and Sorm, F. (1974) Amino acids and peptides. CXXII.
Synthesis and some biological activities of analogs of deaminovasopres-
sin with the disulfide bridge altered to a thioether bridge. Collect. Czech.
Chem. Commun. 39, 2835–2856.
’ ACKNOWLEDGMENT
We thank Dr. Deborah McClellan for editorial assistance. This
work was supported by the Robert C. and Carolyn J. Springborn
Endowment (P.J.K.), the Howard Hughes Medical Institute
(W.A.V.). and National Institutes of Health grants T32
GM070421 (P.J.K.), GM062134 (J.D.L.), and AI068730 (J.D.L.).
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