Progress toward the assembly of the bicyclic theonellamide skeleton

Article abstract of DOI:10.1016/j.tet.2020.131127

An orthogonally protected τ-histidinoalanine residue was synthesized via regioselective alkylation of optically pure Boc-L-His-OTCE (TCE = trichloroethyl) with a sulfamidate electrophile derived from Fmoc-D-Ser-OBn. The goal was the synthesis of a non-natural theonellamide, invoking readily accessible variants of the other 10 amino acids. Peptide fragments corresponding to the east and west rings of “theonellamide X″ were synthesized in solution. Each ring was formed independently, providing insights into protecting group limitations, side reactions, and the optimal order of events to approach the formation of the bicyclic system. Ultimately, an undecapeptide was prepared, with the eastern ring formed. The twelfth amino acid, an L-α-aminoadipic acid building block, was prepared and preliminary investigations into its attachment to the undecapeptide are reported.

Full text of DOI:10.1016/j.tet.2020.131127

Journal Pre-proof
Progress toward the assembly of the bicyclic Theonellamide skeleton
Jyoti P. Mukherjee, Joyeeta Roy, Chyree S. Batton, Saroj Yadav, Douglas Wong,
Carol M. Taylor
PII:
S0040-4020(20)30251-9
https://doi.org/10.1016/j.tet.2020.131127
TET 131127
DOI:
Reference:
To appear in: Tetrahedron
Received Date: 9 February 2020
Revised Date: 8 March 2020
Accepted Date: 10 March 2020
Please cite this article as: Mukherjee JP, Roy J, Batton CS, Yadav S, Wong D, Taylor CM, Progress
toward the assembly of the bicyclic Theonellamide skeleton, Tetrahedron (2020), doi: https://
doi.org/10.1016/j.tet.2020.131127.
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Progress Toward the Assembly of the Bicyclic Theonellamide Skeleton
Jyoti P. Mukherjee, Joyeeta Roy, Chyree S. Batton, Saroj Yadav, Douglas Wong
and Carol M. Taylor*
Department of Chemistry, Louisiana State University, Baton Rouge, LA 70803
Abstract. An orthogonally protected τ-histidinoalanine residue was synthesized via
regioselective alkylation of optically pure Boc-
a sulfamidate electrophile derived from Fmoc-
L
-His-OTCE (TCE = trichloroethyl) with
D
-Ser-OBn. The goal was the synthesis of
a non-natural theonellamide, invoking readily accessible variants of the other 10 amino
acids. Peptide fragments corresponding to the east and west rings of “theonellamide X”
were synthesized in solution. Each ring was formed independently, providing insights
into protecting group limitations, side reactions, and the optimal order of events to
approach the formation of the bicyclic system. Ultimately, an undecapeptide was
prepared, with the eastern ring formed. The twelfth amino acid, an L-α-aminoadipic acid
building block, was prepared and preliminary investigations into its attachment to the
undecapeptide are reported.
1
.
Introduction
1
2
3
4
5
Theonellamides (TNMs) A-E, F (1), G and I, along with theonegramide and
6
theopalauamide, are bicyclic dodecapeptides that were isolated from sponges of the
Theonella genus and shown to inhibit the growth of prototypical fungi and cancer cell
lines. In the case of theopalauamide, it was established unequivocally that the peptide
7
,8
was produced by symbiotic filamentous bacteria.
The peptides are characterized by a

2
bridging τ-histidinoalanine residue and a high degree of post-translationally modified
2
residues. Since the disclosure of the structure of theonellamide F (1, Figure 1) in 1989,
many studies have been reported seeking to uncover the biological activity of these
architecturally novel dodecapeptides.
Figure 1
Theonellamides were the first low molecular weight compounds reported to
induce the formation of extraordinarily large vacuoles, i.e., fluid-filled compartments in
the cytoplasm. Watabe and coworkers made this observation in 3Y1 rat embryonic
fibroblasts, where TNMs did not kill the cells, giving the first indication of potential as
9
,10
good molecular probes to investigate intracellular membrane structures.
Vacuole
formation is a well-known marker of microbial activity, that often precedes cell lysis.
Through affinity chromatography, Wada et al. established that theonellamides A and F
bind to two enzymes from rabbit liver: 17β-hydroxysteroid dehydrogenase IV and
1
1
glutamate dehydrogenase. This 2000 discovery of affinity for a steroid dehydrogenase
was prophetic.

3
About 10 years ago, breakthrough chemical genetics studies revealed
theopalauamide to be a nanomolar antifungal agent that disrupts the cell membrane via
1
2,13
binding to ergosterol and up-regulates β-
D
-glucan synthesis.
In 2013, Espiritu et al.
1
4
confirmed that only 3β-hydroxy-functionalized steroids bind to TNM. They used solid-
state deuterium NMR, and surface plasmon resonance experiments, to demonstrate that
the steroid enhances the affinity of the TNM for lipid bilayers. The same year,
Nishimura et al. produced fluorescent conjugates of TNMs that served as probes for
1
5
31
sterols in liposomes. In 2016, Espiritu et al. used P NMR and dynamic light scattering
to demonstrate sterol concentration-dependent disruption of phosphatidylcholine
1
6
membranes by TNM A. Cornelio et al. recently reported NMR titration experiments to
determine the binding constant of TNM A and 25-hydroxycholesterol in aqueous DMSO.
They concluded that the steroid binds in the hydrophobic cavity of the TNM, reducing
1
7
the polarity of the latter, and thereby facilitating interactions with lipid membranes.
The vast majority of these highly collaborative studies have been conducted using TNM
A, from natural sources.
The chemical synthesis of theonellamides has the potential to produce useful
amounts of material and to probe the role of the various residues via structure-activity
studies. In the early 1990s, Tohdo, Hamada and Shioiri reported progress toward the
synthesis of theonellamide F (1), in the form of conference proceedings and
1
8-24
communications.
They formed each of the two rings independently but no plan was
enunciated for the formation of the bicycle. Cyclizations were performed between the p-
bromophenylalanine and β-alanine residues in the western ring and between the
phenylalanine and (3S,4S,5E,7E)-3-amino-4-hydroxy-6-methyl-8-bromophenyl)-5,7-

4
octadienoic acid (Aboa) residues in the eastern ring using diphenylphosphoryl azide at
high dilution in yields of 21 and 24% respectively.
We have recently reported the synthesis of some of the modified residues,
2
5
26
namely erythro-hydroxyasparagine,
τ-histidinoalanine,
and (2S,4R)-2-amino-4-
2
7
hydroxy-adipic acid.
We describe herein our strategy for the assembly of the bis-
macrocycle and its application to “theonellamide X” (2), a non-natural congener. With
the exception of τ-histidinoalanine, compound 2 invokes amino acids that are
commercially available, viz. the simplest representation of residues that occur in the
natural products, or surrogates thereof. Noteably, the Aboa/Apoa residue was replaced
by β-phenylalanine. This retains the ring size of the eastern ring, the configuration at C3
of the residue, and π-electron density in the truncated side chain.
Construction of the bicyclic structure requires a carefully choreographed
amalgamation of the fragments and building blocks (Scheme 1). Since the major element
of conformational restraint in the molecule is the τ-HAL residue, we set out to construct
this cornerstone and build on the two rings sequentially. We chose cyclization sites that
would involve non-epimerizable activation sites, viz. those of β-aminoacids. Moreover,
the linear precursors, with the τ-HAL residue embedded between peptide “arms” ought to
be able to achieve conformations amenable to cyclization. Our protecting group strategy
2
8
embraced Hirschmann’s concept of “coordinated orthogonal deprotection,” viz.,
Alloc/allyl ester in the western ring and Fmoc/β-cyanoethyl (βCE) ester in the eastern
ring. In each ring, the terminal protecting groups ought to be removed chemoselectively
and simultaneously. “Permanent” protection of the side chain functional groups with
traditional acid-labile groups, suffices for the current model system.

5
Scheme 1. Retrosynthetic analysis of theonellamide X (2) (solid/bold line
indicates site of cyclization; wavy line is a regular disconnection).
2
.
Results and Discussion
Pivotal to the successful synthesis of a theonellamide is an efficient synthesis of
2
9
the τ-histidinoalanine (τ-HAL) building block. We sought maximum flexibility vis-à-
vis protecting groups. The vast majority of precedent for protection of bis-amino acids
3
0
comes from the lantibiotic literature. There is much fanfare these days about protecting
group free synthesis. Wuts cautions, “There are, however, many classes of molecules
where our chemical technology is still not completely adequate to completely avoid the
use of protecting groups.” He identities peptides as one of the three classes of
3
1
compounds.
We are not aware of any system for bis-amino acids that conforms to
Barany and Merrifield’s rigorous definition of an orthogonal system as “a set of

6
completely independent classes of protecting groups. In a system of this kind, each class
3
2
of groups can be removed in any order and in the presence of all other classes.”
Following considerable thought and experimentation, we arrived at building block 3. For
3
3
the α-amino groups, the Fmoc and Boc groups were selected. The Fmoc group is
removed via β-elimination and the Boc group via acidolysis. Benzyl and trichloroethyl
3
4
(
TCE) esters were employed for the α-carboxylates. While both can be removed by
reduction, the former involves a hydrogenolytic process and the latter a two-electron
mechanism. The TCE ester was approached with well-founded caution (vide infra), but a
methyl ester – perhaps the more obvious choice – could not be selectively removed, even
3
5
with tin(II) hydroxide.
hydrolysis of such an ester was accompanied by “slight epimerization.”
Aware of the propensity for histidine to racemize on activation of the
Moreover, Shioiri and co-workers had reported that alkaline
2
1
3
6
carboxylate, we sought to protect the τ-nitrogen of the imidazole, initially with Boc
Scheme 2). Compound 12 was thus prepared in short order, but we were unable to
remove the N -Boc group to give 13, without accompanying side reactions of the TCE
ester. Treatment with sodium methoxide led to transesterification. Exposure to a
(
τ
3
7
3
8
weaker, non-nucleophilic base (Cs CO , imidazole, MeCN), led to cleavage of the TCE
2
3
ester and formation an insoluble material that was reminiscient of that described by
3
9
Sheehan et al. While TCE ester 13 could be prepared in good chemical yield, direct
from 9, it was indeed found to be a ~60:40 mixture of enantiomers by chiral HPLC; τ-
trityl protected 14 was key to that analysis. Ultimately, 13 was prepared from Boc-
4
0
41
His(N
τ-Ts)-OH (11) via carbodiimide-mediated esterification, followed by cleavage
4
2
of the tosyl amide with pyridinium hydrochloride, according to Beyerman et al.

7
Unfortunately, in our hands, unwanted formylation was observed with DMF as solvent.
Dimethylsulfoxide afforded the requisite solubility without this side reaction.
Scheme 2. Synthesis of orthogonally protected τ-histidinoalanine 3.
Sulfamidate 22 was prepared from Fmoc-D-Ser-OBn (17) by analogy to literature
4
3
procedures. Thus, treatment of 17 with thionyl chloride led to the formation of
sulfimidite 18, requiring base. Variation in the nature of the base and the temperature of
the reaction afforded little optimization; disappearance of starting material was not
complete before competing side reactions led to a complex mixture. Careful
chromatography of 18 and oxidation to sulfamidate 22 was satisfactory; but when crude
mixtures of 18 were subjected to oxidation, it was difficult to obtain useful amounts of
pure 22. Ultimately, it was discovered that disulfite ester 19 was a major side product of
the first step, isolable in ~25% yield. This side reaction was previously recognized by
4
4
Shiokawa et al. and could be minimized by the gradual addition of a solution of 17 to a

8
dilute solution of thionyl chloride and triethylamine. Chlorosulfite ester 20 and
dehydroalanine 21 were also observed in the NMR and mass spectra of product mixtures.
Alkylation of Boc-His-OTCE (13) with high purity sulfamidate 22, followed by
4
3a
hydrolysis of the sulfamic acid, afforded τ-3. As observed previously, none of the π-
regioisomer was observed. Despite considerable investigation and attempted
optimization of the coupling reaction, low yields prevailed. A contributing factor is the
instability of the sulfamidate. The reaction of 22 with 13 struggles to complete with the
degradation of 22 to an intractable mixture, probably involving a dehydroalanine
intermediate. The low affinity of an imidazole for a sulfamidate electrophile is
corroborated by De Luca and coworkers who reported that the thiol of the cysteine
residue in a pentapeptide (Ac-Cys-Gly-His-Val-Ala-NH ) reacts with the sulfamidate
2
4
5
derived from Boc-Ser-OMe to form a lanthionine, with the unprotected histidine residue
providing no competition. Preliminary experiments indicated that each protecting group
4
6
of τ-HAL 3 could be removed chemoselectively.
We next describe the assembly of the western hemisphere.
L-α-Amino-adipic
2
7
acid building block 4 was prepared by analogy to our previous report. Tetrapeptide
Fmoc-Asn(Trt)-Asn(Trt)-Phe-β-Ala-OAll (5) was prepared according to standard
solution phase protocols. Asparagine building block 24 was coupled with phenylalanine
benzyl ester (25) to give dipeptide 26. Hydrogenolysis of 26 afforded the corresponding
acid that was coupled with the hydrochloride salt of the allyl ester of β-alanine (27), to
afford tripeptide 28. The Fmoc-group was removed from the N-terminus of 28 and a
second asparagine appended to give compound 5. The N-terminus was deprotected
immediately prior to coupling with τ-HAL, to give amine 30.

9
Scheme 3. Assembly of the Western fragments of theonellamide X (2).
In parallel, the benzyl ester of τ-HAL 3 was removed hydrogenolytically.
Tetrahydrofuran was the solvent of choice for this reaction with complex reaction
mixtures arising when ethyl acetate or alcohols were used as solvent. It is likely that
reductive dehalogenation of the TCE group was the problem here. The resulting amine
47
3
0 and acid 31 were coupled using HATU and collidine to give 32. Treatment of
hexapeptide 32 with TFA led to removal of the Boc-group of the τ-HAL residue and
cleavage of the trityl amides. Condensation of freshly prepared acid 4 with the amine
derived from 32 afforded linear heptapeptide 33. We were aware of four previous
examples of simultaneous removal of two allyl-based protecting groups as a prelude to
48
macrocyclization: Yokokawa and Shiori’s synthesis of antillatoxin, Luesch and
4
9
coworkers’ synthesis of lyngbyastatin, the synthesis of somamide A by Yokokawa and
5
0
51
Shioiri and the assembly of an α-helical mimetic by Alewood and coworkers.
A
range of allyl acceptors were trialed and the reaction was found to be highly dependent
on the source and quality of Pd(PPh ) , with only traces of the macrocycle 34 isolated,
3
4

1
0
following cyclization. Over time, we came to realize that the TCE ester was not
surviving the Pd-catalyzed deallylation reaction conditions. This protecting group gives
rise to distinctive NMR signals, e.g., for linear heptapeptide 33 in DMSO-d : δ 4.84 and
6
2
1
4
.91 ppm ( J = 12.2 Hz) for the diastereotopic CH
2
protons in the H spectrum and δ 73.8
13
(
CH ) and 95.5 (CCl ) ppm in the C spectrum. The molecular ion cluster of TCE-
2
3
containing species is also characteristic. These “signatures” were typically missing from
the NMR and mass spectra of crude product mixtures. A small scale trial, replacing the
TCE ester with a methyl ester, led to successful deallylation and cyclization, confirming
the incompatibility of the TCE protecting group with the conditions required to remove
the Alloc and allyl ester protecting groups.

1
1
Scheme 4. Assembly of the Western ring of theonellamide X.
Independent investigation of the assembly of the Eastern hemisphere is depicted
in Schemes 5 and 6. Our original retrosynthetic analysis (Scheme 1) involved a β-
5
2
cyanoethyl (βCE) ester at the C-terminus, viz. fragment 6. Early reports had shown that
5
3
the βCE group could be removed by piperidine. Tetrapeptide 6 was synthesized, in
early studies, but upon coupling to the τ-HAL residue, gave a mixture of two peptides
that were inseparable and did not perform as expected in subsequent attempts to deprotect
and cyclize. Indeed, while this ester protecting group was advocated for use in peptide
5
2
synthesis as early as 1989, there have been no reports of its application in complex
molecule synthesis. Since our philosophy was for this ester to be removed under the
same conditions as an Fmoc group, two other esters of Nα-Boc-β-phenylalanine (35)
5
4
were prepared, viz. methyl ester 36 and fluorenylmethyl ester 37 (Scheme 5). The Boc
group was removed from each to give the corresponding trifluoroacetate salts, 38 and 39.

1
2
Scheme 5. Assembly of the Eastern fragment of theonellamide X (2).
Commercially available allo-threonine derivative 40 was activated as its NHS ester and
coupled with side-chain protected serine 41 to give dipeptide acid 42. Elongation in the
C-terminal direction with phenylalanine esters 43 (benzyl) or 44 (allyl) gave the
corresponding tripeptides 45 and 46. Hydrogenolysis of 45 and coupling with methyl
ester 38 gave tetrapeptide 47. Tetrapeptide 48, with the fluorenylmethyl ester at the C-
terminus, could be prepared by analogy. However, the Fmoc group could not be
5
5
removed selectively from the N-terminus. Thus, tripeptide 46 was employed and the β-
phenylalanine residue appended at a later stage.
Selective removal of the Fmoc group from τ-HAL 3 (Scheme 6, center) gave a
primary amine that was acylated with a commercially available serine derivative 50
(Scheme 6, clockwise from 3). Reductive cleavage of the trichloroethyl ester from

1
3
compound 51 gave a free acid that was coupled with tetrapeptide amine 49. Removal of
the Fmoc group from compound 52 was accompanied by cyclization to form a
5
6
diketopiperazine, with concomitant loss of the benzyl ester, as suggested by mass
spectrometry. Thus, to continue independent studies of the eastern ring, we converted
benzyl ester 52 to methyl amide 53. The methyl ester and the Fmoc carbamate could be
removed from 53 under alkaline hydrolysis. Cyclization was induced by HATU to give
the eastern macrocycle 54; this reaction was not optimized.
Scheme 6. Assembly of the Eastern ring of theonellamide X.
While the formation of 54 demonstrated the feasibility of cyclization between the
β-Phe and Ser residues in the eastern ring, compound 54 does not lend itself to further
5
7
elaboration. The necessary order of events, for assembly of the bicycle, was emerging.
Rather than introducing a methyl amide, we ought to introduce a relevant peptide, prior to
Fmoc removal. With the long-term view, favoring the fluorenylmethyl ester for the β-
Phe residue, pentapeptide 55 was produced, in good yield from τ-HAL 3. Addition of the

1
4
Ser residue gave rise to hexapeptide 56. Deallylation and incorporation of the β-Phe
residue led to heptapeptide 57.
Hydrogenolysis of the benzyl ester of the τ-HAL residue in compound 57 gave
the corresponding acid. While histidine epimerization was difficult to quantify, the
appearance of a second singlet (typically slightly downfield of that attributable the Boc
group in the desired epimer), in peptides derived from 3, alerted us to the likelihood of
epimerization at Cα of the histidine of τ-HAL. It is not widely appreciated that histidine
residues can epimerize during palladium-catalyzed reactions. Méry and Calas proposed
0
that Pd behaves as a Lewis acid, activating the His C=O, thereby increasing the acidity
5
8
of Hα, which is in close proximity to the Nπ that can serve as an internal base. The
inclusion of small volumes of acetic acid suppressed this side reaction. These concerns
about epimerization led to the realization that the basic Nπ could serve as an internal base
for the purposes of peptide couplings, averting the need to add an external base. Thus, in
the left-hand side of Scheme 6, and through Scheme 7, amide bond formation was
achieved without the addition of collidine or diisopropylethylamine.
The acid derived from 57 was coupled with tetrapeptide amine 30 (Scheme 3), to
give the branched undecapeptide 58 (Scheme 7). Gratifyingly, the Fmoc and Fm ester
protecting groups were removed, within 2 hours, under standard conditions in 84% yield.
Cyclization, with HATU was proceeded in an impressive 80% yield. This compares
favorably with the 24% yield reported by Shioiri and coworkers for cyclization of the east
2
1
ring between the Phe and Aboa residues.

1
5
Scheme 7. Construction of the bicycle and deprotection.
Removal of the Boc group was attempted under a range of conditions, following
literature precedents that gave us hope that this could be done chemoselectively. Given
the solubility issues experienced with compounds 53 and 54, we particularly sought
5
9
conditions that would leave the trityl amides intact. Jiang et al. had shown that
6
0
bromocatechol borane could removal a Boc group, leaving trityl amides intact.
Sakaitani and Ohfune first reported the combination of TBSOTf/lutidine to remove Boc
6
1
groups via a silyl carbamate. Rich and coworkers achieved this in the presence of a
6
2
trityl amide. Bastiaans et al. used the protocol in their cyclotheonamide B synthesis,
6
3
leaving a tert-butyl (aryl) ether unharmed.
The most promising conditions we
64
identified were those described by Tan and Ma in their salinamide A synthesis, viz.
neat formic acid. However, the major product of this reaction arose from loss of one tert-
butyl ether, as well as the Boc carbamate. Coupling with α-AAA building block 4 gave a
mixture that included decapeptides 60 and 61, as confirmed by mass spectrometry.
What remains, to complete the bicyclic structure of the theonellamides, is removal
of the two allyl-based protecting groups, cyclization of the western ring and global

1
6
deprotection of the side chain functional groups. It would be highly advantageous to
optimize the Boc-deprotection to produce a single compound prior to advancing these
goals.
3
.
Conclusions
We have described the synthesis of an orthogonally protected τ-HAL building
block that has served well in the assembly of the 12 amino acids of the theonellamide
skeleton. The trichloroethyl (TCE) ester has provided some complications in that it
suffers from nucleophilic attack and reductive cleavage. As Davies has said, “In peptide
6
5
synthetic circles dioxopiperazines are usually unwelcome;” that was certainly the case
during efforts to elaborate 52 to the eastern macrocycle. Fortunately, conversion of
benzyl ester 52 to undecapeptide 58, laid the foundation for cleavage of the Fmoc and Fm
ester protecting groups, followed by formation of the east ring. Preliminary studies are
described to append the last amino acid, α-amino-adipic acid. While this is the end of the
road for Taylor and coworkers, we hope that the lessons learned in our laboratory over
the past decade or so will inspire others to pursue the chemical synthesis of the
theonellamides, and study the structure-activity relationships of these beautiful and
valuable molecules.
4
.
Experimental Section
4
.1
General
Reactions were performed under a nitrogen atmosphere unless otherwise noted.
Reagents were obtained from commercial sources and used directly; exceptions are

1
7
noted. Diethylamine, diisopropylethylamine, 2,4,6-collidine and triethylamine were
dried and distilled from CaH and stored over KOH pellets. Compounds were visualized
2
by UV fluorescence or by staining with ninhydrin. Flash chromatography was performed
using silica gel from a range of sources, over the years, including Agela Technologies
(40-60µ). HPLC was performed on a Waters 600 system, interfaced with Empower™
software, and equipped with a Waters 2487 UV detector, monitoring absorption at 218
and 254 nm. Semi-preparative RP work was conducted on a 10 mm diameter Altima®
-1
C18 10µ column with a flow rate of 3 mL min ; eluants are detailed in each procedure.
NMR spectra were recorded on Bruker AV-400 or AV-500 liquid spectrometers. Proton
NMR data is reported in ppm downfield from TMS as an internal standard or calibrated
on solvent peaks. High resolution mass spectra were recorded using electrospray
ionization on an Agilent 6230 ESI TOF instrument. Rotations were measured on a Jasco
P2000 polarimeter.
4
.2
Synthesis of Trichloroethyl Esters
,2,2-Trichloroethanol (1.5 equiv.) was added dropwise to a solution of Boc-
His(X)-OH (1.0 equiv.) in CH Cl (0.1 mM) under N at 0 °C. EDC hydrochloride (1.2
2
2
2
2
equiv.) was added in portions, the mixture warmed to rt and stirred for 16 h.
Boc-His(Boc)-OTCE (12). On a 0.23 mmol scale, starting from Boc-His(Boc)-
OH (8). Dichloromethane (20 mL) and water (15 mL) were added. The aqueous layer
was further extracted with CH Cl (2 x 15 mL). The combined organic layers were
2
2
washed with sat’d aq. NaHCO (15 mL), sat’d aq. NH Cl (15 mL) and brine (15 mL),
3
4
dried over MgSO , filtered and concentrated. The yellow residue was subjected to flash
4

1
8
chromatography, eluting with 0.5-2.0% MeOH in CH
2
2
Cl to give Boc-His(Boc)-OTCE
2
5
(
12) as a viscous oil (105 mg, 93%). R 0.68 (95:5 CH Cl -MeOH). [α] +1.06 (c 0.8,
f
2
2
D
1
3 3
CHCl ). H NMR (CDCl , 400 MHz) δ 1.45 (s, 9H), 1.61 (s, 9H), 3.09 (dd, J = 14.8 4.7
Hz, 1H), 3.18 (dd, J = 14.8, 5.5Hz, 1H), 4.67 (d, J = 12.0 Hz, 1H), 4.67-4.74 (m, 1H),
1
3
4
.83 (d, J = 12.0 Hz, 1H), 6.03 (d, J = 8.2 Hz, 1H), 7.19 (s, 1H), 8.02 (s, 1H); C NMR
(
CDCl , 100 MHz) δ 27.9, 28.3, 29.6, 53.2, 74.6, 80.0, 85.8, 94.6, 114.9, 137.0, 138.1,
3
+
1
4
46.7, 155.5, 170.5. HRMS (ESI+) calcd for C18
86.0975.
H27Cl
3
N
3
O
6
(M+H) 486.0965, obsd
Boc-His(Trt)-OTCE (14). On a 0.20 mmol scale, starting from Boc-His(Trt)-
OH (10). Dichloromethane (20 mL) and water (15 mL) were added. The aqueous layer
was further extracted with CH Cl (2 x 15 mL). The combined organic layers were
2
2
washed with sat’d aq. NaHCO (15 mL), sat’d aq. NH Cl (15 mL) and brine (15 mL),
3
4
dried over MgSO
4
, filtered and concentrated. The yellow residue was subjected to flash
Cl to give Boc-His(Trt)-OTCE
14) as a colorless solid (110 mg, 87%). R 0.55 (95:5 CH Cl -MeOH). [α]
chromatography, eluting with 0.5-1.5% MeOH in CH
2
2
2
5
(
-10.3 (c
D
f
2
2
1
1
3
4
3 3
.0, CHCl ). H NMR (CDCl , 400 MHz) δ 1.43 (s, 9H), 3.07 (dd, J = 14.5, 4.1 Hz, 1H),
.17 (dd, J = 14.5, 4.8 Hz, 1H), 4.55 (d, J = 11.8 Hz, 1H), 4.60 (d, J = 11.8 Hz, 1H),
.63-4.71 (m, 1H), 6.34 (d, J = 7.8 Hz, 1H), 6.58 (s, 1H), 7.05-7.14 (m, 6H), 7.28-7.36
1
3
(
m, 9H), 7.43 (s, 1H); C NMR (CDCl , 100 MHz) δ 28.3, 29.6, 74.5, 75.3, 79.6, 94.5,
3
1
19.7, 128.1 (2C), 129.7, 136.1, 138.8, 142.2, 155.6, 170.4. HRMS (ESI+) calcd for
+
C
32
H
33Cl
3
N
3
O
4
(M+H) 628.1537, obsd 628.1541.
Boc-His(Ts)-OTCE (15). On a 3.66 mmol scale, starting from Boc-His(Ts)-
OTCE (11). Dichloromethane (40 mL) and water (30 mL) was added and the aqueous

1
9
2 2
layer further extracted with CH Cl (2 x 20 mL). The combined organic layers were
washed with sat’d aq. NaHCO (30 mL), sat’d aq. NH Cl (30 mL) and brine (30 mL),
3
4
dried over MgSO , filtered and concentrated. The yellow residue was subjected to flash
4
chromatography, eluting with 0.5-2.0% MeOH in CH Cl to give Boc-His(Ts)-OTCE
2
2
2
5
(
15) as a colorless solid (1.10 g, 83%). R 0.65 (95:5 CH Cl -MeOH). [α] +5.3° (c 0.6,
f 2 2 D
1
CHCl ). H NMR (CDCl , 400 MHz) δ 1.42 (s, 9H), 2.44 (s, 3H), 3.05 (dd, J = 15.0, 4.6
3
3
Hz, 1H), 3.14 (dd, J = 15.0, 4.6 Hz, 1H), 4.55 (d, J = 11.9 Hz, 1H), 4.68-4.73 (m, 1H),
4
7
5
.69 (d, J = 11.9 Hz, 1H), 5.91 (d, J = 8.3 Hz, 1H), 7.08 (s, 1H), 7.36 (d, J = 8.3 Hz, 2H),
1
3
.79 (d, J = 8.3 Hz, 2H), 7.92 (s, 1H); C NMR (CDCl
3
, 100 MHz) δ 21.7, 28.3, 29.7,
2.9, 74.5, 80.1, 94.4, 114.9, 127.4, 130.5, 134,7, 136.5, 139.8, 146.5, 155.4, 170.2.
35
HRMS (ESI+) calcd for C H Cl N O S (M+H) 540.0524; obsd 542.0540.
20
25
3
3
6
Boc-His-OTCE (13). Pyridinium hydrochloride (3.0 g, 25.9 mmol, 7.0 equiv)
was added to a stirred solution of 15 (2.0 g, 3.70 mmol, 1.0 equiv.) in DMSO (3 mL) at rt
2 2
under N . The mixture was stirred for another 2.5 h, poured onto H O (30 mL) and
extracted with EtOAc (3 x 60 mL). The combined organic layers were washed
sequentially with sat′d aq. NaHCO (30 mL), H O (30 mL), brine (30 mL), then dried
3
2
over MgSO , filtered, and concentrated to obtain a yellow residue that was purified by
4
flash chromatography, eluting with a gradient of 1-5% MeOH in CH Cl , to give Boc-
2
2
2
5
f 2 2 D
His-TCE (13) as colorless foam (1.2 g, 84%). R 0.35 (95:5 CH Cl -MeOH). [α] -
1
1
4
3.33 (c 1.0, CHCl ). H NMR (CDCl , 400 MHz) δ 1.38 (s, 9H), 3.02-3.18 (m, 2H),
3
3
.49-4.64 (m, 2H), 4.72 (d, J = 11.8 Hz, 1H), 6.07 (d, J = 7.5 Hz, 1H), 6.83 (s, 1H), 7.54
1
3
(
3
s, 1H); C NMR (CDCl , 100 MHz) δ 28.2, 29.2, 53.6, 74.4, 80.0, 94.4, 115.6, 133.9,

2
0
+
1
3
35.3, 155.6, 170.6. HRMS (ESI+) calcd for C13
86.0443.
3 3 4
H19Cl N O (M+H) 386.0441, obsd
4
.3
Synthesis of
Fmoc-D-Ser-OBn (17). Cesium carbonate (1.39 g, 4.27 mmol, 0.50 equiv.) was
added to a solution of Fmoc- -Ser-OH (16) (2.8 g, 8.54 mmol, 1.00 equiv.) in dry MeOH
35 mL). The mixture was stirred at rt for 2 h, concentrated, and the residue dissolved in
τ-Histidinoalanine Building Block 3
D
(
dry DMF (19 mL) under N . Benzyl bromide (1.2 mL, 875 mg, 10.29 mmol, 1.20 equiv.)
2
was added dropwise and the mixture stirred overnight. The mixture was partitioned
between EtOAc (70 mL) and H O (70 mL). The aqueous layer was further extracted with
2
EtOAc (70 mL), and the combined organic extracts washed with sat’d aq. NaHCO (3 x
3
2
0 mL) to remove Fmoc-
dried over MgSO , filtered and concentrated. The pale yellow oil was purified by flash
chromatography on silica gel, eluting with 2:1 hexanes-EtOAc, to give Fmoc- -Ser-OBn
17) as a clear oil that gave a colorless solid, following refrigeration overnight (2.90 g,
D-Ser-OH. The organic layer was washed with brine (40 mL),
4
D
(
2
5
1
8
2
4
7
2%). R 0.38 (1:1 EtOAc-hex). [α] -3.4 (c 1.0, MeOH). H NMR (400 MHz,CDCl ) δ
f D 3
.22 (br s, 1H), 3.91 (d, J = 9.8 Hz, 1H), 4.01 (d, J = 9.8 Hz, 1H), 4.20 (t, J = 6.5, 1H),
.36-4.48 (m, 3H), 5.21 (s, 2H), 5.77 (d, J = 7.5 Hz, 1H), 7.28-7.33 (m, 7H), 7.39 (t, J =
1
3
.4 Hz, 2H), 7.58 (d, J = 7.1 Hz, 2H), 7.75 (d, J = 7.5 Hz, 2H); C NMR (100 MHz,
) δ 47.1, 56.1, 63.3, 67.2, 67.5, 120.0, 125.1, 127.0, 127.1, 127.7, 128.2, 128.5,
28.6, 135.0, 141.3 (2C), 143.6, 143.8, 156.2, 170.3. HRMS (ESI) calcd for C25H24NO
5
CDCl
3
1
+
(
M+H) 418.1654, obsd 418.1670.

2
1
Sulfamidate 22. A solution of triethylamine (1.42 mL, 1.02 g, 10.1 mmol, 3.0
equiv) in CH Cl (28 mL) was stirred at -40 °C (MeCN/dry ice bath) under N in a two-
2
2
2
necked flask, fitted with a dropping funnel (vertical) and rubber septum (side). Distilled
thionyl chloride (500 µL, 800 mg, 6.7 mmol, 2.0 equiv) was added dropwise over 5 min
via the septum and stirring continued for 5 min. A solution of Fmoc-
D-Ser-OBn (17)
(
1.42 g, 3.4 mmol, 1.0 equiv) in CH Cl (55 mL) was added dropwise from the dropping
2
2
funnel over a period of 2 h. Following completion of the addition, the mixture was
maintained at -40 °C and stirred for another 2 h, then gradually warmed to rt over 2 h (ice
bath left in place, but no more dry ice added). Pyridine (1.00 mL, 1.06 g, 13.4 mmol, 4.0
equiv) was added dropwise and stirring continued for another 14 h. Water (70 mL) was
added and the mixture was extracted with CH Cl (55 mL). The organic layer was
2
2
2 4
washed with 5% citric acid (28 mL), H O (70 mL) and brine (30 mL), dried over MgSO ,
filtered and concentrated. The crude diastereomeric sulfimidites (18) were obtained as an
orange foam and used as such for the next step. R 0.47 (minor), 0.40 (major) (2:1
f
hexanes-EtOAc).
The residue was dissolved in MeCN (14 mL) and cooled to 0 °C. Sodium
periodate (790 mg, 3.6 mmol, 1.1 equiv) was added followed by ruthenium (III) chloride
(
70 mg, 0.34 mmol, 0.1 equiv). Water (14 mL) was added dropwise to the solution and
the mixture was left to stir vigorously for 1 h at 0 °C. The mixture was warmed to rt,
stirred an additional 1 h, and then water (28 mL) and Et O (55 mL) were added. The
two layers separated and the aqueous layer extracted further with Et O (50 mL). The
combined organic layers were washed with sat’d aq. NaHCO (80 mL), H O (80 mL) and
2
2
3
2
brine (80 mL), dried over MgSO , filtered and concentrated. The yellow residue was
4

2
2
purified by flash chromatography on silica gel, eluting with 5:1 hexanes-EtOAc, followed
by 3:1 hexanes-EtOAc, to give sulfamidate 22 as a colorless solid (980 mg, 61 % over
2
5
1
two steps). R 0.37 (2:1 hexanes-EtOAc). [α]
+11.1 (c 0.6, CHCl3). H NMR (400
D
f
MHz, CDCl ) δ 4.22-4.32 (m, 1H), 4.44 (app. t, J = 8.8 Hz, 1H), 4.56 (appt. t, J = 8.8 Hz,
3
1
2
2
1
H), 4.72 (d, J = 8.7 Hz, 1H), 4.78-4.93 (m, 1H), 4.83 (d, J = 8.7 Hz, 1H), 5.18-5.27 (m,
H), 7.30 –7.37 (m, 7H), 7.41 (t, J = 7.4 Hz, 2H), 7.67 (br s, 2H), 7.76 (d, J = 7.4 Hz,
1
3
3
H); C NMR (100 MHz, CDCl ) δ 46.4, 57.9, 68.0, 68.6, 70.6, 120.0, 125.2, 125.3,
27.3, 128.0, 128.2, 128.7, 128.8, 134.2, 141.2 (2C), 142.7, 142.9, 149.4, 166.4. HRMS
-
(ESI) calcd for C25
H20NO
7
S (M-H) 478.0966, obsd 478.0969.
τ-HAL derivative 3. Boc-
L-His-OTCE (13) (945mg, 2.46 mmol, 1.2 equiv.) was
added to a solution of sulfamidate 22 (980 mg, 2.05 mmol, 1.0 equiv.) in 1,2-DME (46
mL). This solution was heated at 80 °C under N and stirred for 5 h. A solution of
2
potassium dihydrogen phosphate (1 M, 30 mL) was added to the mixture which was
stirred at rt for 5 min. The mixture was partitioned between EtOAc (60 mL) and H O (60
2
mL) to dissolve the precipitate. The two layers were separated, and the aqueous layer was
extracted further with EtOAc (60 mL). The combined organic layers were dried over
MgSO , filtered and concentrated. The clear oil was subjected to flash chromatography
4
on silica gel, eluting with 2:1 hexanes-EtOAc, to give τ-HAL 3 as a colorless oil (590
2
5
1
mg, 36%). R 0.34 (95:5 CH Cl -MeOH). [α]
-3.9 (c 1.0, CHCl ). H NMR (CDCl ,
3 3
f
2
2
D
4
4
8
5
00 MHz) δ 1.42 (s, 9H), 3.03 (dd, J = 14.8, 4.5 Hz, 1H), 3.11 (dd, J = 14.8, 4.7 Hz, 1H),
.20 (t, J = 6.4 Hz, 1H), 4.32 (br s, 2H), 4.40 (appt. t, J = 8.6 Hz, 1H), 4.52 (appt. t, J =
.6 Hz, 1H), 4.60–4.64 (m, 2H), 4.68 (d, J = 12.1 Hz, 1H), 4.82 (d, J = 12.1 Hz, 1H),
.17 (d, J = 11.9 Hz, 1H), 5.28 (d, J = 11.8 Hz, 1H), 5.60 (br s, 1H) 6.18 (d, J = 7.8 Hz,

2
3
1
H), 6.36 (s, 1H), 7.27-7.42 (m, 9H), 7.58 (d, J = 7.3 Hz, 2H), 7.79 (d, J = 7.2 Hz, 2H);
1
3
C NMR (CDCl , 100 MHz) δ 28.3, 29.6, 47.0, 47.7, 53.5, 54.7, 67.2, 68.2, 74.5, 79.8,
3
9
1
4.7, 117.1, 120.0, 124.9, 125.0, 127.1, 127.2, 127.8, 128.9, 129.0, 129.1, 134.2, 137.6,
41.3, 143.4, 143.5, 155.5, 155.6, 168.7, 170.6. HRMS (ESI) calcd for C H Cl N O
8
3
8
40
3
4
+
(
M+H) 785.1906, obsd 785.1923.
4
.4
Synthesis of
L
-
α
-Aminoadipic Acid Building Block 4
-α-Aminoadipic acid (23) (250 mg, 1.55 mmol, 1.0
Alloc- -α-AAA-OH.
L
L
equiv.) was added to a solution of sodium hydroxide (223 mg, 5.60 mmol, 3.6 equiv.) in
water (12.5 mL) at 0 °C. Allyl chloroformate (247 μL, 281 mg, 2.35 mmol, 1.5 equiv.)
was added dropwise over 30 min and stirring continued for 4 h in an ice bath. The
mixture was washed with diethyl ether (20 mL). The aqueous layer was acidified to pH 2
by the addition of conc. HCl and extracted with EtOAc (4 x 20 mL). The combined
extracts were dried over MgSO4, filtered and concentrated to give 2-
(
allyloxycarbonylamino)-adipic acid (350 mg, 92%). R 0.29 (6:4:1 CHCl -MeOH-H O).
f 3 2
Alloc-L-α-AAA-OPMB (4). Triethylamine (139 μL, 106 mg, 1.05 mmol, 1.1
equiv) and p-methoxybenzyl chloride (128 μL, 162 mg, 0.95 mmol, 1.0 equiv.) were
added to a solution of Alloc-α-AAA-OH (255 mg, 0.95 mmol, 1.0 equiv.) in DMF (6
mL) at 38 °C and stirring continued at 38 °C overnight. Ethyl acetate (5 mL) was added
and the solution was filtered to remove the precipitate of triethylammonium
hydrochloride. The filtrate was diluted with ethyl acetate (20 mL), washed with water (10
mL), brine (10 mL), dried over MgSO , filtered and concentrated. The pale yellow oil
4
was purified by flash chromatography on silica gel, eluting with 98:2 (CH Cl -MeOH) to
2
2

2
4
afford the desired ester 4 as colorless oil (235 mg, 62%). R
f
0.32 (95:5 CH
2 2
Cl -MeOH).
2
5
1
[
(
α] -4.09 (c 1.0, CHCl ), H NMR (CDCl , 400 MHz) δ 1.65-1.74 (m, 3H), 1.80-1.96
D
3
3
m, 1H), 2.28-2.42 (m, 2H), 3.82 (s, 3H), 4.33-4.45 (m, 1H), 4.57 (d, J = 5.4 Hz, 2H),
5
.13 (q, J = 11.8 Hz, 2H), 5.22 (d, J = 10.4 Hz, 1H), 5.31 (d, J = 17.2 Hz, 1H), 5.47 (d, J
1
3
=
8.0 Hz, 1H), 5.86-5.94 (m, 1H), 6.89 (d, J = 8.6 Hz, 2H), 7.29 (d, J = 8.6 Hz, 2H); C
NMR (CDCl , 100 MHz) δ 20.3, 31.8, 53.6, 55.3, 65.9, 67.2, 114.0, 117.9, 127.3, 130.2,
3
+
1
3
32.6, 155.9, 159.8, 172.2. HRMS (ESI+) calcd for C18
66.1537.
H24NO
7
(M+H) 366.1547, obsd
4
.5
Synthesis of Tetrapeptide 5
Fmoc-Asn(Trt)-Phe-OBn (26). Phenylalanine benzyl ester hydrochloride (25)
(
(
(
200 mg, 0.68 mmol, 1.00 equiv.) was added to a solution of Fmoc-Asn(Trt)-OH (24)
160 mg, 0.68 mmol, 1.00 equiv.) in CH Cl (2 mL) at 0 °C under N . Triethylamine
2
2
2
189 µL, 137 mg, 1.36 mmol, 2.00 equiv.), EDC (138 mg, 0.7 mmol, 1.05 equiv.) and
HOBt (136 mg, 1.0 mmol, 1.50 equiv.) were added sequentially. After 20 min, the ice
bath was removed, the mixture warmed to rt and stirred for another 12 h. The mixture
was concentrated and applied to a flash column in a minimum volume of CH Cl , eluting
2
2
with 98:2 CH Cl : MeOH to give dipeptide 26 (295 mg; 51%). R 0.50 (95:5 CH Cl -
2
2
f
2
2
2
5
1
MeOH). [α]
-1.2 (c 0.8, CHCl ). H NMR (CDCl , 400 MHz) δ 1.53-1.74 (m, 1H),
3 3
D
2
.63 (dd, J = 15.0, 4.6 Hz, 1H), 2.94-3.03 (m, 2H), 3.07 (d, J = 15.0 Hz, 1H), 4.19 (app t,
J = 7.1 Hz, 1H), 4.28-4.37 (m, 2H), 4.50-4.60 (m, 1H), 4.78 (q, J = 6.6 Hz, 1H), 5.07 (d,
J = 12.0 Hz, 1H), 5.15 (d, J = 12.0 Hz, 1H), 6.42 (br s, 1H), 6.92 (br s, 1H), 6.99 (br s,
1
H), 7.12-7.18 (m, 3H), 7.22-7.30 (m, 23H), 7.42 (t, J = 7.1 Hz, 2H), 7.57 (d, J = 7.1 Hz,

2
5
1
3
2
6
1
3
H), 7.79 (d, J = 7.4 Hz, 2H); C NMR (CDCl , 125 MHz) δ 37.7, 38.1, 47.1, 51.3, 54.0,
7.2, 67.4, 70.9, 120.1, 125.3, 127.0, 127.2, 127.8, 128.1, 128.5, 128.6 (2C), 128.7,
29.3, 135.2, 135.7, 141.3, 143.8, 143.9, 144.4, 156.4, 170.6, 170.8, 170.9. HRMS
+
(
ESI+) calcd for C H N O (M+H) 834.3538, obsd 834.3526.
5
4
48
3
6
HCl.β-Ala-OAll (27). β-Alanine (502 mg, 5.6 mmol, 1.0 equiv.), allyl alcohol
(3.4 mL, 2.9 g, 27.0 mmol, 4.8 equiv.) and chlorotrimethylsilane (1.2 mL, 1.03 g, 9.4
mmol, 1.6 equiv.) were heated at 100 °C for 4 h. The mixture was cooled to rt, poured
onto diethyl ether (115 mL) and stirred at 0 °C for 24 h. The solid was collected by
f 2 2
filtration to give the hydrochloride salt 27 (800 mg, 86%). R 0.38 (9:1 CH Cl -MeOH).
1
H NMR (CDCl , 400 MHz) δ 2.96 (t, J = 6.7 Hz, 2H), 3.38 (t, J = 6.7 Hz, 2H), 4.63 (d, J
3
=
5.8 Hz, 2H), 5.24 (dd, J= 10.4, 1.1 Hz, 1H), 5.33 (dd, J = 17.2, 1.4 Hz, 1H), 5.86-5.96
13
(
m, 1H), 8.20 (br s, 2H); C NMR (CDCl , 100 MHz) δ 31.3, 35.7, 65.9, 118.8, 131.7,
3
+
1
70.9. HRMS (ESI+) calcd for C H NO (M+H) 130.0868, obsd 130.0863.
6
11
2
Fmoc-Asn(Trt)-Phe-β-Ala-OAll (28). 10% Pd-C (18 mg, 0.34 mmol, 2.0
equiv.) was added to a solution of dipeptide 26 (140 mg, 0.17 mmol, 1.0 equiv) in dry
2
MeOH (2 mL). The vessel was evacuated and then opened up to an atmosphere of H and
®
the mixture stirred at rt for 4 h. The mixture was filtered through a plug of Celite and the
filtrate concentrated to give the carboxylic acid as a colorless oil (100 mg, 0.14 mmol)
that was used directly in the next step without further purification. R 0.25 (9:1 CH Cl -
f
2
2
MeOH).
Fmoc-Asn(Trt)-Phe-OH (100 mg, 0.14 mmol, 1.05 equiv.) was dissolved in
(2 mL) and cooled to 0 °C under N . β-Alanine allyl ester hydrochloride (27) (22
mg, 0.13 mmol, 1.00 equiv.) was added, as a solid, followed by 2,4,6-collidine (35 µL, 32
CH
2
Cl
2
2

2
6
mg, 0.26 mmol, 2.00 equiv.) and HATU (52 mg, 0.14 mmol, 1.05 equiv.). After 20 min,
the mixture was warmed to rt and stirred for 12 h. The mixture was concentrated and
applied to a flash column in a minimum volume of CH Cl . Elution with a gradient of 2-
2
2
5
% MeOH in CH Cl yielded tripeptide 28 as a colorless foam (95 mg, 66% over 2
2 2
2
D
5
1
steps). R 0.49 (9:1 CH Cl -MeOH). [α] -5.2 (c 0.5, CHCl ). H NMR (CDCl , 400
f
2
2
3
3
MHz) δ 2.25-2.38 (m, 2H), 2.75-2.81 (m, 1H), 2.94-2.98 (m, 2H), 3.05-3.08 (m, 1H),
.26-3.31 (m, 2H), 4.18 (t, J = 7.0 Hz, 1H), 4.28-4.39 (m, 2H), 4.42-4.46 (m, 1H), 4.51
d, J = 5.6 Hz, 2H), 4.55-4.57 (m, 1H), 5.24 (d, J = 10.4 Hz, 1H), 5.30 (d, J = 16.2 Hz,
3
(
1
7
H), 5.83-5.93 (m, 1H), 6.22 (d, J = 6.7 Hz, 1H), 6.55 (t, J = 5.5 Hz, 1H), 6.85 (d, J =
.0 Hz, 1H), 7.00 (br s, 1H), 7.13-7.35 (m, 25H), 7.42 (d, J = 7.4 Hz, 2H), 7.58 (d, J = 7.2
1
3
Hz, 2H), 7.78 (d, J = 6.7 Hz, 2H); C NMR (CDCl , 100 MHz) δ 34.8, 37.5, 38.1, 47.0,
3
5
1
1.8, 54.6, 65.2, 67.4, 70.8, 118.4, 120.0, 125.1, 127.0, 127.1, 127.2, 127.8, 128.0, 128.7,
29.3, 132.0, 136.5, 141.3, 143.7 (2C), 144.2, 156.3, 170.0, 170.2, 170.6, 171.5. HRMS
+
(
ESI+) calcd for C H N O (M+H) 855.3752, obsd 855.3759.
5
3
51
4
7
Fmoc-Asn(Trt)-Asn(Trt)-Phe-β-Ala-OAll (5). Diethylamine (2 mL) was added
to a solution of tripeptide 28 (160 mg, 0.18 mmol, 1.00 equiv.) in acetonitrile (2 mL). The
mixture was stirred for 1 h at rt, after which time, TLC indicated that the starting material
had been consumed. The solution was concentrated, and then concentrated twice more
from CH CN (2 x 6 mL) to remove the excess Et NH. The residue was applied to a flash
3
2
column in a minimum volume of CH Cl , eluting with 9:1 CH Cl -MeOH to give the
2
2
2
2
f 2 2
amine, H-Asn(Trt)-Phe-β-Ala-OAll (118 mg, 0.18 mmol). R 0.42 (9:1 CH Cl -MeOH).
HATU (74 mg, 0.19 mmol, 1.05 equiv.) and 2,4,6-collidine (54 µL, 50 mg, 0.40
mmol, 2.2 equiv.) were added to a solution of the intermediate amine (118 mg, 0.18

2
7
mmol, 1.00 equiv.) and Fmoc-Asn(Trt)-OH (29) (116 mg, 0.19 mmol, 1.05 equiv.) in
CH Cl (4 mL) at 0 °C under N . After 20 min, the mixture was warmed to rt and stirred
2
2
2
1
4 h. The mixture was concentrated and applied to a flash column in a minimum volume
of CH Cl . Elution with 98:2 CH Cl -MeOH gave tetrapeptide 5 as a colorless foam (148
2
2
2
2
2
5
1
mg, 68% over two steps). R 0.52 (9:1 CH Cl -MeOH). [α]
-18.8 (c 0.5, CHCl ). H
f
2
2
D
3
NMR (DMSO-d , 400 MHz) δ 2.20 (t, J = 7.3 Hz, 1H), 2.40 (d, J = 11.7 Hz, 1H), 2.57-
6
2
.77 (m, 5H), 2.95-2.97 (m, 1H), 3.03 (d, J = 10.9 Hz, 1H), 3.08-3.10 (m, 1H), 4.21 (t, J
6.6 Hz, 1H), 4.25-4.38 (m, 3H), 4.42-4.45 (m, 1H), 4.48 (m, 1H), 4.53 (d, J = 5.0 Hz,
=
2
7
H), 5.22 (d, J = 10.4 Hz, 1H), 5.30 (d, J = 17.1 Hz, 1H), 5.89-5.94 (m, 1H), 7.06 (t, J =
.1 Hz, 1H), 7.11-7.27 (m, 39H), 7.25-7.29 (m, 1H), 7.39-7.46 (m, 1H), 7.66 (d, J = 8.2
Hz, 1H), 7.71-7.77 (m, 3H), 7.91 (d, J = 7.4 Hz, 2H), 8.10 (d, J = 8.0 Hz, 1H), 8.26 (d, J
1
3
=
7.4 Hz, 1H), 8.57 (s, 1H), 8.82 (s, 1H); C NMR (DMSO-d , 100 MHz) δ 33.7, 35.1,
6
3
1
1
7.1, 38.5, 38.7, 47.1, 50.4, 52.5, 54.7, 64.9, 66.4, 69.9, 118.3, 120.6, 121.4, 125.7 (2C),
26.5, 126.8, 126.9, 127.6, 127.9, 128.1, 128.5, 129.0, 129.4, 133.5, 138.6, 141.2, 144.2,
44.3, 145.1, 145.2, 156.2, 169.3, 170.1, 170.9, 171.0 (2C), 171.7. HRMS (ESI+) calcd
+
for C76
H
71
N
6
O
9
(M+H) 1211.5277, obsd 1211.5271.
4
.6
Construction of the Western Macrocycle
Hexapeptide 32. 10% Pd-C (90 mg, 30% w/w) was added to a solution of τ-
histidinoalanine 3 (300 mg, 0.4 mmol, 1.0 equiv.) in anhydrous THF (8 mL). The vessel
was evacuated and then opened to an atmosphere of H and the suspension stirred at rt for
2
3
h. The reaction mixture was filtered through a plug of Celite® and the filtrate was
concentrated. The residue was purified by flash chromatography eluting with 20% EtOH

2
8
in CH
2
Cl
2
to give the free acid 31 as a colorless oil (225 mg, 84%). R
f
0.20 (9:1 CH
2 2
Cl -
2
5
1
MeOH). [α]
-2.98 (c 1.0, CHCl ). H NMR (DMSO-d , 400 MHz) δ 1.35 (s, 9H),
D
3
6
2
.82-2.90 (m, 2H), 4.11-4.24 (m, 4H), 4.29 (t, J = 10.9 Hz, 2H), 4.35-4.40 (m, 1H), 4.82
d, J = 12.3 Hz, 1H), 4.91 (d, J = 12.3 Hz, 1H), 6.87 (s, 1H), 7.28 (d, J = 7.8 Hz, 1H),
.33 (t, J = 7.4 Hz, 2H), 7.41 (t, J = 7.4 Hz, 2H), 7.45 (s, 1H), 7.65 (t, J = 12.0 Hz, 2H),
(
7
7
5
1
6
13
.89 (d, J = 7.5 Hz, 3H); C NMR (DMSO-d , 100 MHz) 28.6, 29.8, 47.1, 47.3, 54.1,
6
5.4, 66.1, 74.0, 79.0, 95.5, 118.0, 120.6, 125.6, 125.7, 127.6, 128.1, 136.9, 138.1, 141.2,
+
44.3, 144.4, 155.7, 156.2, 171.3, 171.6; HRMS (ESI+) calcd for C31
95.1437, obsd 695.1442.
3 4 8
H34Cl N O (M+H)
Diethylamine (3 mL) was added to a solution of tetrapeptide Fmoc-Asn(Trt)-
Asn(Trt)-Phe-β-Ala-OAll (5) (480 mg, 0.38 mmol, 1.00 equiv.) in dry acetonitrile (3
mL). The mixture was stirred at rt for 2 h, concentrated, and concentrated twice more
from CH CN (2 x 6 mL) to remove the excess Et NH. The residue was then applied to a
3
2
flash column in a minimum volume of CH Cl , eluting with 9:1 CH Cl -MeOH to give
2
2
2
2
f 2 2
compound 30 (330 mg). R 0.30 (9:1 CH Cl -MeOH).
A solution of acid 31 (225 mg, 0.32 mmol, 1.00 equiv.) in CH
2
2
Cl (6 mL),
collidine (92 µL, 85 mg, 0.70 mmol, 2.20 equiv.) and HATU (138 mg, 0.36 mmol, 1.10
equiv.) were added sequentially to a solution of amine H-Asn(Trt)-Asn(Trt)-Phe-β-Ala-
OAll (30) (330 mg, 0.32 mmol, 1.00 equiv.) in CH Cl (8 mL) at 0 °C under N . After 20
2
2
2
min, the mixture was warmed to rt and stirred overnight. The mixture was concentrated
and the residue applied to a flash column in a minimum volume of CH Cl . Subsequent
2
2
2 2
elution with a gradient of 5-10% of MeOH in CH Cl gave hexapeptide 32 as a light
brown solid (282 mg, 53% over two steps from τ-HAL 3). R 0.35 (9:1 CH Cl -MeOH).
f
2
2