From the Department of Pharmacology, University of Melbourne, Victoria 3010, Australia
Received for publication, March 28, 2003
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ABSTRACT |
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INTRODUCTION |
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BDNF, like the other neurotrophins, produces its effects on neurons through
two transmembrane receptors. Binding of a neurotrophin to a specific member of
the trk family of receptor tyrosine kinases (NGF binds to trkA, BDNF and
NT-4/5 bind to trkB, and NT-3 preferentially binds to trkC) results in the
step-wise homodimerization of the receptor, leading to receptor
autophosphorylation and the initiation of multiple signal transduction
pathways, including those leading to neuronal survival
(6). In contrast, the
glycoprotein p75 acts as a common low-affinity receptor
(KD 109
M) for all the neurotrophins
(7). Unlike the trk family, p75
signals apoptosis via a unique intracellular death domain, Chopper
(8), although its precise
biological function remains controversial. The high affinity binding sites for
BDNF and the other neurotrophins on neurons (KD
1011 M) probably consist of a
combination of the appropriate trk member and p75. The overall response to a
neurotrophin therefore depends on the balance of signaling through a trk
family member and p75, with the opportunity for modulation and cross-talk at
multiple levels of the signaling process.
The neurotrophins are homodimers consisting of two monomers, each of
120 residues. X-ray crystal structures of NGF
(9), NT-3
(10), NT-4/5, and a
BDNF/NT-4/5 heterodimer (11)
reveal a common fold for the neurotrophins. Each monomer consists of seven
-strands (contributing to three longitudinal antiparallel
-sheets)
connected by three solvent-exposed hairpin loops (loops 1, 2, and 4) and a
longer loop (loop 3) and contains three disulfide bridges between six fully
conserved cysteine residues arranged in a cystine-knot motif, characteristic
of this growth factor superfamily. However, in contrast to most of the other
members of the superfamily, the neurotrophin monomers are arranged in the
dimer in a parallel fashion, and are held together solely by non-covalent
(largely hydrophobic) interactions.
A number of studies implicate the solvent-exposed loops of BDNF and the other neurotrophins in mediating their biological effects. Site-directed mutagenesis analyses have revealed that the ability to bind and activate trkB can be conferred to NGF by replacing residues in loop 2 with the corresponding residues from BDNF (12). The crystal structure of NGF in complex with one of the Ig domains of trkA shows direct contact between residues in the receptor and loop 1 of NGF (13), although the authors of this article have since conceded that additional regions of the neurotrophins may well interact with other trk domains (14). In support of the functional role for loop 2, we have reported that conformationally constrained peptides (of which the monocyclic monomeric peptide 1 (Table I) was identified as the most effective) designed to mimic a single loop 2 of BDNF are inhibitors of BDNF-mediated survival of sensory neurons in culture, probably by acting as competitive trkB antagonists (15).
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Despite promising preclinical data, clinical trials with recombinant BDNF in patients with amyotrophic lateral sclerosis have proven unsuccessful (16). This failure is likely to have been caused, at least in part, by the unfavorable pharmacokinetics of BDNF; for example, the plasma half-life of recombinant BDNF in rats is less than 1 min (17). These and similar data from other neurotrophic factors have led to the view that low molecular weight drugs, with more appropriate pharmacokinetic properties than the parent proteins, might prove a more fruitful means of harnessing neurotrophic action for therapeutic use (18).
As a step toward the development of such neurotrophic drugs, we describe here a structure-based approach for the discovery of potent mimetics of BDNF. Given our previous data with monocyclic monomeric BDNF inhibitors (15), we reasoned that appropriately designed dimeric loop 2 mimetics should be able to bring about trkB homodimerization and thus mimic the actions of BDNF through this receptor. Using this approach, three classes of peptides were designed and synthesized: bicyclic dimeric peptides linked by a disulfide bridge internal to the sequence of monomeric monocyclic peptide 1; a bicyclic dimer linked by an amide bond external to the peptide 1 sequence; and a highly constrained tricyclic dimeric peptide containing both disulfide and amide dimerizing linkages. The latter compound exhibited extremely high potency, comparable with BDNF, in promoting the survival of chick sensory neurons in culture. The results suggest that this compound is worthy of further preclinical development and that our structure-based approach may be of general utility in developing potent mimetics of other growth factors and cytokines that dimerize their receptors.
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EXPERIMENTAL PROCEDURES |
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Peptide Synthesis
Assembly, Cleavage, and Cyclization of Monomeric
PrecursorsLinear peptide precursors were synthesized manually (0.1
mmol scale) from fluorenylmethoxycarbonyl-protected amino acids using standard
solid-phase synthesis methods, with the following points of note: peptides for
the synthesis of bicyclic disulfide-linked dimers 24 were
assembled on chlorotrityl resin, preloaded with Cys. Cys residues involved in
cyclization were trityl protected; the Cys residue needed for the dimerizing
linkage was acetamidomethyl (Acm)-protected. Peptides required for the
preparation of the bicyclic amide-linked dimer 5 and tricyclic dimer
6 were synthesized on Rink amide 4-methylbenzhydrylamine resin, which
upon cleavage yields the peptide amide. The C-terminal Glu and Lys residues
were incorporated as trifluoroacetic acid (TFA)-labile O-t-butyl- and
t-butyloxycarbonyl-protected derivatives. To improve reaction
efficiency, the resins were combined after coupling of Glu(O-t-butyl)
and Lys(t-butyloxycarbonyl) residues, and subsequent assembly,
cleavage, and purification steps were carried out on the mixture. Ser and the
additional Lys residue were incorporated as the TFA-stable benzyloxycarbonyl
(Z) and benzyl (Bzl) derivatives. For the tricyclic dimer 6, the Cys
residue required for the third cycle was incorporated as the Acm derivative.
The N termini of peptides 5 and 6 were acetylated before
cleavage. To reduce the likelihood of racemization, the N-terminal Cys residue
was coupled to all peptides as the preformed symmetrical anhydride
(19). All peptides were
cleaved from the resin with TFA/ethanedithiol/H2O (18:1:1). The
crude partially protected linear peptides were oxidized to the corresponding
partially protected monocyclic monomers by oxidizing the peptide (1 mg/ml) in
a solution of dimethyl sulfoxide (10%) in 0.1 M aqueous
NH4HCO3, pH 8
(20).
Synthesis of Bicyclic Disulfide-linked DimersBicyclic disulfide-linked dimers 24 were prepared using a modification of the method of Kamber et al. (21) by dissolving the appropriate Acm-protected monocyclic monomeric peptide (8 µmol) in aqueous acetic acid (40 µl, 50%) containing HCl (10 µl, 1 M), adding I2 (400 µl, 50 mM in 50% acetic acid) and stirring the mixture at room temperature under N2. Upon completion (typically 6 h), the reaction mixture was quenched with ascorbic acid (10 µl, 1 M), and the desired bicyclic disulfide-linked dimer was purified by HPLC.
Synthesis of Partially Protected Bicyclic Amide-linked
DimerThe Bzl/Z-protected bicyclic amide-linked dimer was prepared
by dissolving the two Bzl/Z-protected monocyclic monomers (6 µmol each) in
dimethylformamide (500 µl) before the addition of
O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium
hexafluorophosphate (6.6 µmol) and diisopropylethylamine (9.9 µmol) and
stirring. Upon completion (6 h), the reaction was diluted 20-fold with
10% acetonitrile, and the desired Bzl/Z-protected bicyclic amide-linked dimer
was purified by HPLC.
Synthesis of Partially Protected Tricyclic DimerThe Z-protected tricyclic dimer was prepared using a combination of the dimerization methods described for disulfide- and amide-linked dimers above. Firstly, the two Acm/Z-protected monocyclic monomers (6 µmol each) were coupled in the presence of O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate/diisopropylethylamine. The resultant Acm/Z-protected bicyclic dimer (2.8 µmol) was converted to the Z-protected tricyclic dimer by treatment with I2.
HF CleavageRemaining Bzl and Z protecting groups were removed from partially protected bicyclic amide-linked dimer and partially protected tricyclic dimer by treating the crude peptides with HF in the presence of m-cresol (10:1) using standard methods.
Purification and Characterization of PeptidesGeneral reaction progress was monitored and peptides purified as appropriate by HPLC using UV detection (220 nm) on analytical (150 x 2.1 mm) or semi-preparative (250 x 10 mm) C18 columns. All final peptides were eluted as single peaks after purification. Identity of all peptide intermediates and final products was confirmed by mass spectrometry, using a Micromass platform II triple quadrupole mass spectrometer with an electrospray source (carried out at the Victorian College of Pharmacy, Monash University, Australia). All peptides (intermediates and final products) gave molecular ions with a mass-to-charge ratio within 0.1% of calculated values.
Sensory Neuron CulturesPeptides were assayed in primary cultures of dorsal root ganglion sensory neurons prepared from 8-day-old embryonic chicks as described previously (15). Peptides (1 x 1011 to 104 M) were added to cells in triplicate wells 1 h after plating either alone (concentration-response studies) or with mouse recombinant BDNF (4 x 1011 M; competition studies). Positive control wells contained BDNF only; negative control wells contained neither BDNF nor peptide. Initial viable neuron numbers were determined by counting phase-bright cell bodies in 40 microscope fields (0.25 x 0.25 mm, at 200x magnification) in four randomly chosen wells. After 48 h of incubation, surviving neurons (phase-bright cells with neurites) were counted.
Data AnalysisCell counts for individual experiments were normalized by setting neuronal survival in the BDNF-only positive controls (typically 3540% of the initial number of viable neurons plated) to 100%, and survival in negative controls (typically 46%) to 0%. Neuronal survival was expressed as the mean ± S.E. from four or five different preparations. pEC50 and pIC50 values were estimated from logistical sigmoidal curves fit to concentration-response data. Neuronal survival in the presence of peptides was compared with negative controls (concentration-response studies) or positive controls (competition studies) by analysis of variance followed by Bonferroni's multiple comparisons test.
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RESULTS |
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Synthesis of Bicyclic Dimeric PeptidesThe bicyclic dimers 25 were prepared using appropriate orthogonal side-chain protection strategies, to allow the selective formation of cyclizing (i.e. disulfide) and dimerizing (i.e. disulfide or amide) constraints. The disulfide-linked dimers 24 were synthesized using TFA-labile trityl protection for Cys required for the formation intramolecular disulfide bonds and TFA-stable Acmprotection for Cys involved in the dimerizing disulfide bridges. The success of the iodine-mediated removal of Acm and subsequent oxidation to the dimer was found to be dependent on the nature of the iodine scavenger used; best results were obtained after quenching with ascorbic acid solution. Use of other scavengers (e.g. sodium thiosulfate) resulted in multiple reaction products.
The amide-linked dimer 5 was synthesized using a combination of tertiarybutyl-(i.e. TFA labile) and benzyl-(i.e. TFA stable) derived protection. After cyclization of the N- and C-terminally protected monomers, dimerization was effected with the amide bond forming reagent O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate. Other condensation agents and conditions gave rise to multiple and/or incorrect products. The final step in the synthesis of 5 was the removal of the remaining benzyl-derived protecting groups on Ser and Lys by treatment with HF. Mass spectral data relating to the bicyclic dimers 25 can be found in Table I.
Effects of Bicyclic Dimeric Peptides on Sensory Neuron Survival in VitroThe effects of the bicyclic dimers 25 on neuronal survival were assessed in primary cultures of embryonic chick dorsal root ganglion sensory neurons, an assay used routinely in the literature to characterize the survival effects of BDNF and the other neurotrophins. When added alone to primary cultures of embryonic chick sensory neurons, the bicyclic disulfide-linked dimeric peptides 2 and 4, and the bicyclic amide-linked dimeric peptide 5 produced significant and concentration-dependent increases in neuronal survival (Fig. 2A). Although peptides 2, 4, and 5 gave a similar maximal survival effect (around 30% of the maximal survival effect of BDNF), peptide 5 was clearly the most potent of the bicyclic dimers (pEC50 of 10.0 ± 0.15), being more than 100-fold more potent than either 2 or 4. For peptides 2, 4, and 5, the survival effect dropped after reaching a maximum, giving their concentration-response curves a distinct bell-shape. In contrast to the survival promoting effects of 2, 4, and 5, neither the bicyclic disulfide-linked dimer 3 (Fig. 2A) nor the monocyclic monomeric precursors to the bicyclic dimers (data not shown) significantly promoted neuronal survival compared with negative controls over the concentration range tested.
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To assess the possible partial agonist behavior of the bicyclic dimeric peptides, the bicyclic disulfide-linked dimeric peptide 4 was added to chick sensory neuron cultures in competition with BDNF. Similar to its loop 2-based monocyclic monomeric counterpart 1 (15), dimeric bicyclic peptide 4 caused significant and concentration-dependent inhibition of BDNF-mediated neuronal survival (Fig. 2B; maximal inhibition of 49% ± 7 at 1 x 107 M; pIC50 of 9.43 ± 0.25). Together, these data suggest that bicyclic disulfide-linked dimer 4 acts as a partial agonist on the majority of neurons that respond to BDNF, rather than being a full agonist on a subset (3035%) of BDNF responsive neurons.
Design of Tricyclic Dimeric PeptidesWe reasoned that the relatively low potency and partial agonist activity of the bicyclic dimers 25 may be caused, at least in part, by conformational flexibility about the dimerizing linkages, meaning that the two monomeric units within the peptides are likely to be capable of 360° rotation relative to one another (Fig. 3). The bicyclic dimers would probably behave as trkB agonists only when they existed in a conformation able to cause the in-plane dimerization of trkB (i.e. in a roughly parallel conformation). When the relative orientation of the two monomeric units is not favorable to receptor homodimerization (e.g. when the units are antiparallel), the bicyclic dimers would probably behave as antagonists, leading to a reduction in maximum, decrease in slope and rightward shift of the concentration-response curve, as previously shown for monocyclic monomeric peptides acting as competitive trkB antagonists (15).
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To explore this hypothesis and to attempt to obtain a BDNF mimetic with greater potency and efficacy, we designed tricyclic dimeric peptides (effectively, hybrids of the disulfide-linked and amide-linked dimers) in which two monocyclic, monomeric loop 2 units were linked by both an amide and a disulfide linkage (Fig. 3). Molecular dynamics simulations of these peptides showed that they possessed greatly reduced conformational freedom compared with their bicyclic counterparts, resulting in significantly greater similarity to the native loop 2 region of BDNF (data not shown). One of these tricyclic peptides, 6, was chosen for synthesis.
Synthesis of Tricyclic Dimeric PeptideThe challenging synthesis of the tricyclic dimeric peptide 6 was approached using a combination of the strategies used to synthesize the bicyclic disulfide- and amide-linked dimers, allowing complete control to be exercised over the formation of the multiple cyclizing and dimerizing linkages (Fig. 4). Using this approach, the two linear precursors to peptide 6 were assembled (Fig. 4a), cleaved from the resin and partially deprotected (Fig. 4b) and cyclized (Fig. 4c), and the cyclic monomers dimerized (Fig. 4d) as described for the bicyclic amide-linked dimer 5. The third cycle was then introduced by treating the bicyclic peptide with iodine (Fig. 4e), bringing about the step-wise removal of the Acmprotected Cys residues and the subsequent oxidation of the free thiols to the disulfide, as described for bicyclic disulfide-linked dimers 24. Treatment of the partially protected tricyclic dimer with HF (Fig. 4f) yielded the crude desired tricylic dimeric peptide 6, which was readily purifiable by HPLC.
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To maximize the efficiency of this synthetic approach, we carried out steps a to d (Fig. 4) on the mixture of the two peptide building blocks. To do this, the two portions of resin, one preloaded with fluorenylmethoxycarbonyl-Glu(O-t-butyl), the other with fluorenylmethoxycarbonyl-Lys(t-butyloxycarbonyl), were combined, and the subsequent peptide elongation reactions carried out on the resin mixture. The linear protected peptides were cleaved, cyclized, and dimerized as a mixture, halving the number both of the reactions and chromatographic purifications. The remaining steps (Fig. 4, e and f) proceeded as described above yielding a product identical to that obtained after synthesis of discrete compounds. Using this optimized, quasi-one-pot approach, it is possible to prepare purified tricyclic dimeric peptide 6 from scratch in under a week.
Effects of Tricyclic Dimer on Sensory Neuron Survival When
added alone to primary cultures of embryonic chick sensory neurons, the
tricyclic dimeric peptide 6 produced significant and
concentration-dependent increases in neuronal survival
(Fig. 2A; maximum
survival 35% at 1 x 108 M). The
tricyclic dimer 6 was particularly potent, with pEC50 of
10.96 ± 0.32 (corresponding to an EC50 of 11 pM),
making it 10-fold more potent than the bicyclic amide-linked dimer
5, and 1000-fold more potent than the bicyclic disulfide-linked dimer
4. However, like the bicyclic dimers, tricyclic dimer 6 also
displayed a bell-shaped concentration-response curve, although the curve width
was
2-fold greater than either 4 or 5.
In competition with BDNF, the tricyclic dimer 6 caused significant and concentration-dependent inhibition of BDNF-mediated neuronal survival (Fig. 2B; maximal inhibition 47% ± 6 at 1 x 106 M; pIC50 10.13 ± 0.08). These values were not statistically different from those of either the bicyclic dimeric peptide 4 or the monocyclic monomeric peptide 1 (analysis of variance, Bonferroni's multiple comparison test).
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DISCUSSION |
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There are now several examples in the literature of dimeric peptides able to mimic the effects of larger protein agonists, presumably by being able to bring about receptor dimerization, including bicyclic peptide agonists of the erythropoietin receptor (2224) and the thrombopoietin receptor (25), bicyclic peptide antagonists of interleukin-5 (26), and monocyclic dimeric agonists of the cell recognition molecule N-cadherin (27, 28). In the case of erythropoietin agonists, monocyclic peptides discovered through a phage display process could promote erythropoietin receptor homodimerization by virtue of the ability of the peptides to non-covalently self-associate into dimers (23). In this system, potency could be improved by including a single covalent dimerizing linkage between the peptides (24). It is of interest to note that the three-dimensional structure of a noncovalently dimerized erythropoietin peptide bound to the erythropoietin receptor bears some resemblance to the modeled structure of the tricyclic dimer 6 (data not shown), even though the receptors they activate have no apparent homology. This observation supports the hypothesis that the molecular design approach we describe in this article, using two appropriately incorporated dimerizing linkages to greatly restrict conformational freedom, could be of general utility in improving the potency of a range of dimeric peptide ligands.
Despite the ability of the bicyclic and tricyclic dimers to promote sensory neuron survival in vitro, they are partial agonists with respect to BDNF, in that they are able to support maximally about 35% of those neurons that would be kept alive by BDNF and inhibit the neuronal survival effect of BDNF when added in competition. One mechanism for this behavior is that the compounds are partial agonists of trkB, possibly because they are less efficient than BDNF in bringing about the dimerization of trkB or because of inappropriate conformational flexibility of the dimerizing linkages (although the retention of partial agonism by the highly constrained tricyclic compound would argue against this). Indeed, subtle conformational effects on receptor dimerization have been noted for a number of systems (29, 30) and three-dimensional structural data for the interaction of the neurotrophins with the trks indicate that a symmetrical complex is formed (13, 31). A consequence of reduced dimerization efficiency could be a reduced ability to cause the autophosphorylation of tyrosine residues in trkB. In the cytoplasmic domain of trkB, five tyrosine residues (Tyr484, Tyr670, Tyr674, Tyr675, and Tyr785) have been shown to undergo autophosphorylation upon exposure of cells expressing trkB to BDNF (32, 33). It is feasible that the dimeric peptides do not bring about optimal levels of autophosphorylation at all tyrosines and/or give a time course of autophosphorylation that differs from that of BDNF. These differences may cause a reduced activation of downstream signaling pathways and/or activation of specific pathways only, either of which could lead to partial agonism through trkB. Although bulk measurement of autophosphorylation of trkB (e.g. 34, 35) would help confirm the hypothesized mechanism of action of the bicyclic dimeric peptides through activation of trkB, it is unlikely to be sensitive enough to detect the possible subtle differences in autophosphorylation levels that might lead to partial trkB agonism. A more meaningful investigation might require studies with mutant trkB isoforms lacking individual phosphorylation sites (33), the use of site specific antibodies such as those developed by Segal et al. (36), or the analysis of the specific downstream signal components.
An alternative mechanism for the partial agonist behavior of the dimeric
peptides is that they act through trkB only, even as full trkB agonists, and
not p75. The bicyclic and tricyclic dimeric peptides all gave bell-shaped
concentration-response curves, thus resembling compounds that produce their
action through homodimerization of a single receptor, such as growth hormone
and typical growth factors
(37,
38). In contrast, BDNF and the
other neurotrophins show a more complex concentration response relationship
for neuronal survival (39),
indicating the involvement of multiple receptors (i.e. a trk member
plus p75), although effects known to be elicited through a trk member alone
(e.g. neurite outgrowth) do exhibit a simple bell-shaped
concentration-response relationship
(40). Pharmacodynamic modeling
studies we have performed suggest that the dimeric peptides, by acting solely
as trkB agonists, may be failing to recruit antiapoptotic action through p75,
giving rise to the reduced maximal survival
effect.2 To help
elucidate the mechanism of this partial agonistic behavior, a variety of
experimental approaches could be used. First, the binding characteristics of
the bicyclic dimeric peptides to trkB and p75 need to be examined. A
functional contribution (or lack thereof) of p75 toward the action of the
bicyclic dimeric peptides could be studied using known p75 antagonists, such
as blocking antibodies (e.g. REX)
(41) or examining the effects
of the peptides on such downstream targets of p75 as c-Jun
NH2-terminal kinase-p53-Bax, ceramide, and nuclear factor-B
(42). Similar experiments
using known trkB inhibitors, such as K252a
(43), could be used to confirm
the involvement of trkB.
The tricyclic dimeric peptide described here is the most potent member of a
growing class of small molecule neurotrophin mimetics, including a trkA
mimetic developed from a small cyclic peptide based on loop 4 of NGF
(44), and a series of dimeric
-turn mimetics that act as agonists of trkC
(45). To date, the
neurotrophins have failed to achieve clinical success in the treatment of
neurodegenerative diseases. It remains to be seen whether the currently
available neurotrophin mimetics will lead to drugs that effectively harness
neurotrophin actions for therapeutic use.
In conclusion, the structure-based drug design approach we describe has yielded potent low-molecular weight peptide mimetics of BDNF with picomolar potency comparable with that of the native protein. These compounds could serve as a springboard for the development of therapeutically useful BDNF mimetics. Furthermore, given the similarities between them and other growth factor/cytokine receptor dimerizing ligands, the approach may have general utility across a range of ligand receptor systems involving receptor homodimerization.
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FOOTNOTES |
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Recipient of a Melbourne Research Scholarship.
Directors and shareholders of Calibre Biotechnology Pty. Ltd.
¶ To whom correspondence should be addressed. Tel.: 61-3-8344-8604; Fax: 61-3-8344-0241; E-mail: rahughes{at}unimelb.edu.au.
1 The abbreviations used are: BDNF, brain derived neurotrophic factor; NGF,
nerve growth factor; neurotrophin 3, NT-3; NT-4/5, neurotrophin 4/5; r.m.s.,
root mean square; Acm, acetamidomethyl; TFA, trifluoroacetic acid; Z,
benzyloxycarbonyl; Bzl, benzyl.
2 P. D. O'Leary and R. A. Hughes, manuscript in preparation.
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REFERENCES |
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