Structure-based design and synthesis of triazole-based macrocyclic inhibitors of norovirus protease: Structural, biochemical, spectroscopic, and antiviral studies

Article abstract of DOI:10.1016/j.ejmech.2016.04.013

Outbreaks of acute gastroenteritis caused by noroviruses constitute a public health concern worldwide. To date, there are no approved drugs or vaccines for the management and prophylaxis of norovirus infections. A potentially effective strategy for the development of norovirus therapeutics entails the discovery of inhibitors of norovirus 3CL protease, an enzyme essential for noroviral replication. We describe herein the structure-based design of the first class of permeable, triazole-based macrocyclic inhibitors of norovirus 3C-like protease, as well as pertinent X-ray crystallographic, biochemical, spectroscopic, and antiviral studies.

Full text of DOI:10.1016/j.ejmech.2016.04.013

Accepted Manuscript
Structure-Based Design and Synthesis of Triazole-based Macrocyclic Inhibitors of
Norovirus Protease: Structural, Biochemical, Spectroscopic, and Antiviral Studies
Pathum M. Weerawarna, Yunjeong Kim, Anushka C. Galasiti Kankanamalage, Vishnu
C. Damalanka, Gerald H. Lushington, Kevin R. Alliston, Nurjahan Mehzabeen, Kevin
P. Battaile, Scott Lovell, Kyeong-Ok Chang, William C. Groutas.
PII:
S0223-5234(16)30295-1
10.1016/j.ejmech.2016.04.013
EJMECH 8528
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 21 January 2016
Revised Date: 4 April 2016
Accepted Date: 6 April 2016
Please cite this article as: P.M. Weerawarna, Y. Kim, A.C. Galasiti Kankanamalage, V.C. Damalanka,
G.H. Lushington, K.R. Alliston, N. Mehzabeen, K.P. Battaile, S. Lovell, K.-O. Chang, W.C. Groutas.,
Structure-Based Design and Synthesis of Triazole-based Macrocyclic Inhibitors of Norovirus Protease:
Structural, Biochemical, Spectroscopic, and Antiviral Studies, European Journal of Medicinal Chemistry
(2016), doi: 10.1016/j.ejmech.2016.04.013.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Structure-Based Design and Synthesis of Triazole-based
Macrocyclic Inhibitors of Norovirus Protease: Structural,
Biochemical, Spectroscopic, and Antiviral Studies.
Pathum M. Weerawarna,^ Yunjeong Kim,+ Anushka C. Galasiti Kankanamalage,^
^
#
Vishnu C. Damalanka, Gerald H. Lushington, Kevin R. Alliston,^ Nurjahan
@
$
@
Mehzabeen, Kevin P. Battaile, Scott Lovell, Kyeong-Ok Chang,*+ William C.
Groutas.*^
^Department of Chemistry, Wichita State University, Wichita, Kansas 67260, USA
+
Department of Diagnostic Medicine & Pathobiology, College of Veterinary Medicine,
Kansas State University, Manhattan, Kansas 66506, USA
#
LiS Consulting, Lawrence, KS 66046, USA
@
Protein Structure Laboratory, The University of Kansas, Lawrence, Kansas 66047,
USA
$
IMCA-CAT, Hauptman-Woodward Medical Research Institute, APS Argonne National
Laboratory, Argonne, IL 60439, USA
*
authors to whom correspondence should be addressed.
Department of Chemistry, Wichita State University, Wichita, KS 67260
Tel. (316) 978 7374; Fax: (316) 978 3431
e-mail: bill.groutas@wichita.edu
Department of Diagnostic Medicine & Pathobiology, Manhattan, KS 66506
Tel. (785) 532 3849; Fax: (785) 532 4039
e-mail: kchang@vet.ksu.edu
1

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Abstract
Outbreaks of acute gastroenteritis caused by noroviruses constitute a public health
concern worldwide. To date, there are no approved drugs or vaccines for the
management and prophylaxis of norovirus infections. A potentially effective strategy for
the development of norovirus therapeutics entails the discovery of inhibitors of norovirus
3
CL protease, an enzyme essential for noroviral replication. We describe herein the
structure-based design of the first class of permeable, triazole-based macrocyclic
inhibitors of norovirus 3C-like protease, as well as pertinent X-ray crystallographic,
biochemical, spectroscopic, and antiviral studies.
2

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1. Introduction
Noroviruses are the leading cause of foodborne illness in the U.S., accounting for >21
million infections per year and resulting in >70000 hospitalizations and nearly 800
deaths [1,3]. They are also the primary cause of sporadic and epidemic acute
gastroenteritis worldwide [4,5].The morbidity and mortality associated with norovirus
infection impacts disproportionately the elderly, immunocompromised individuals [6]
and, in developing countries, children [7]. Combating noroviruses presents a major
challenge because noroviruses are highly contagious, genetically diverse,
environmentally stable, display prolonged and profuse viral shedding, evolve rapidly,
and are capable of spreading in many different ways [8,9]. There are currently no
effective vaccines or norovirus-specific antivirals for norovirus infection. Consequently,
there is a pressing need for the discovery and development of small molecule
therapeutics for the treatment and prophylaxis of norovirus infection [10,13].
Norovirus is an RNA virus that belongs to the Norovirus genus of the Caliciviridae
family. It carries a positive sense single stranded RNA genome (7.7 Kb) that consists of
three open reading frames (ORF1-3). ORF1 encodes a 200 kDa precursor polyprotein
which is processed by a virus-encoded cysteine endopeptidase to generate at least six
mature non-structural proteins [14,16]. Polyprotein processing by norovirus 3C-like
protease (3CLpro) is vital for the replication of the virus, consequently, norovirus 3CLpro
has emerged as an attractive target for the discovery and development of antiviral
agents against norovirus infections [10-11,17].
3

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Norovirus 3CLpro is a chymotrypsin-like cysteine protease with an active site consisting
of the catalytic triad of Cys139, His30 and Glu54 and located in a groove that is
sandwiched between a β-sheet domain and a β-barrel domain [17,21].The β-barrel
domain accounts for most of the binding interactions involved between a peptide
substrate and 3CLpro. A hallmark of norovirus 3CLpro, as well as other related 3C or
3
CL viral proteases, is that its primary substrate specificity is dictated by the presence
of a P glutamine (and less frequently glutamic acid), at the S subsite [22].
1
1
Furthermore, previous studies involving the mapping of the substrate specificity of
CLpro have revealed that the protease prefers small hydrophobic amino acids such as
3
a leucine residue at P2 and that the plasticity of its S subsite allows the protease to
3
accommodate His, Gln or Glu [16,18-19].
In continuing our studies in this area [12-13,17,23], we describe herein the structure-
based design of cell-permeable macrocyclic transition state inhibitors (I) and (II) (Figure
1
) of norovirus 3CLpro [23,24], as well as relevant structural, biochemical,
spectroscopic, and cell-based studies.
2. Results and Discussion
2
.1 Inhibitor Design. Inspection of crystal structures of norovirus 3CLpro with peptidyl
pro
inhibitors reveals that peptidyl inhibitors of norovirus 3CL bind to the enzyme via a
network of backbone hydrogen bonds that mimic an antiparallel β-sheet [25-26]. Among
these hydrogen bonds, the backbone NH and carbonyl O of the Ala160 residue forms two
hydrogen bonds with the backbone carbonyl O and NH of the P residue of the inhibitor
3
(Figure 2A). In addition, a third hydrogen bond exists between the backbone O of the
Ala158 residue and the backbone NH of the P residue of the inhibitor. This hydrogen
1
4

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bonding arrangement resembles the hydrogen bonding pattern found in antiparallel β-
sheets and serves to correctly orient and position the inhibitor (or substrate) backbone to
the active site. The hydrogen bonding associated with an antiparallel β-sheet is more
feasible when an inhibitor (or substrate) binds to the active site of a protease in a β-strand
0
conformation, the extended “saw tooth” conformation of a peptide (ϕ = -120 ± 20 and ψ =
0
+
120 ± 20 ) where the backbone NHs are exposed to the environment [27]. As a
consequence, the aforementioned binding mode permits proteases to universally recognize
their ligands in the β-strand conformation [25-26].
Based on the forgoing, we recently reported the results of preliminary studies related to the
design of triazole-based macrocyclic inhibitors of 3C and 3C-like proteases of picornavirus,
norovirus and coronavirus [24]. Macrocyclization of linear peptidyl inhibitors was
accomplished by tethering the P and P residues of the linear inhibitor by a suitable linker,
1
3
thereby pre-organizing the macrocycle in a β-strand conformation prior to binding to the
enzyme active site (Figure 2B) [27-28]. This approach results in the formation of a pre-
organized and semi-rigid entity that displays the amino acid side chains in a well-defined
vector relationship for optimal binding and enhances the affinity of the inhibitor to the target
enzyme by minimizing the entropy loss upon binding. Additional potential advantages of
macrocyclic inhibitors include high stability to metabolizing enzymes, high selectivity and,
frequently, increased cellular permeability [29-32]. We describe herein the structure-based
design of macrocyclic inhibitors of norovirus 3CLpro where the side chain of the P1 Gln
primary substrate specificity residue is tethered to the P3 residue via a triazole linker to
yield macrocycles (I) (Figure 1) and explore the effect of ring size on conformation and its
impact on pharmacological activity using high field NMR and X-ray crystallographic studies.
5

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We also report the first examples of macrocyclic inhibitors of norovirus 3CLpro that are
cellurarly permeable.
2
.2 Chemistry. The synthesis of macrocyclic compounds 1-11 (Table 1) was
accomplished by first constructing key intermediates 15a-c, 18a-e, 19a-e, and 20a-b
using well-established methodologies (Scheme 1). The commercially available N-Boc
protected L-propargylglycine was reacted with L-leucine methy lester, L-
cyclohexylalanine methyl ester or L-phenylalanine methyl ester, in the presence of EDCI
and HOBt to provide the corresponding dipeptide esters. Deblocking of the resulting
esters was accomplished by treatment with 4M HCl in dioxane. The resulting amine
hydrochlorides were reacted with benzylchloroformate followed by LiOH to yield the
corresponding N-Cbz protected dipeptide acid intermediates 15a-c. Preparation of the
HCl salts of amino azides 18a-e was effected by sequential reaction of commercially
available N-Boc protected amino alcohols with methanesulfonyl chloride and sodium
azide followed by deprotection with 4M HCl in dioxane. Coupling of N-Boc protected
glutamic acid with the corresponding HCl salts of amino azides 18a-e in the presence of
EDCI and HOBt gave intermediates 19a-e, in good yields.
The macrocyclization step was effected in two different ways. Specifically, the synthesis
of macrocycles 2-3 entailed click chemistry-mediated macrocyclization using
intermediates 15a-c and 20a-b (Scheme 2), while compounds 1 and 4-11 were
obtained through macrolactamization starting with key intermediates 15a-c and 19a-e
(Scheme 3). In general, the macrolactamization methodology was found to be superior,
particularly in terms of ease of purification of the resultant cyclized products.
Intermediates 15a and 20a-b were coupled in the presence of EDCI and HOBt to yield
6

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the corresponding linear precursors 24a and 24b of the macrocyclic esters 25a and
2
5b. Intramolecular click reactions of the linear precursors 24a-b were carried out using
a CuI/DBU mixture in dichloromethane under a nitrogen atmosphere at room
temperature for 24 h to provide macrocyclic esters 25a-b in fairly good yields (45-50%).
Intermediates 15a-c and 19a-e were coupled using intermolecular click reaction in the
presence of CuSO and sodium ascorbate in THF/H O solution to obtain the
4
2
corresponding linear precursors 27a-i of macrocyclic esters 28a-i. Macrolactamization
of compounds 27a-i was carried out using EDCI and HOBt in DMF at room temperature
for 24 h to yield macrocyclic esters 28a-i in good yields (~60%). In both cyclization
methods, the dilution factor of the reaction mixture was critical in controlling the
intramolecular cyclization reaction over the intermolecular reaction. Reduction of
macrocyclic esters using LiBH followed by Dess-Martin periodinane oxidation provided
4
the corresponding macrocyclic aldehydes 1-11 in fairly good yields (~50%).
The orientation of the triazole ring and its effect on pharmacological activity and
conformation was probed by synthesizing macrocycle 12, via intermediates 21 and 23,
as illustrated in Scheme 4. The acyclic variant of macrocycle 3 was synthesized
according to Scheme 5 in order to determine the effect of macrocyclization on antiviral
activity. As dictated by the primary substrate specificity of norovirus 3CLpro, the P Gln
1
residue remained invariant in all synthesized inhibitors. Furthermore, norovirus 3CLpro
prefers a P Leu or Cha [12,23a-c], consequently, the convergent nature of the synthetic
2
plan allowed for variations in the P residue via the judicious selection of appropriate
2
natural or unnatural amino acid esters, thereby permitting the targeting of related viral
proteases in the Picornaviridae and Coronaviridae families.
7

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2
.3 Biochemical Studies. The inhibitory activity of compounds 1-12 against norovirus
CLpro in vitro and norovirus in a cell-based replicon system was evaluated as
3
previously described [12-13] and the results are summarized in Table 1.
2
.4 X-ray crystallography. In order to establish the precise mechanism of action of
inhibitors (I) and gain insight and understanding into the structural determinants that
impact potency and permeability that can be used to facilitate the structure-guided
optimization of pharmacological activity, high-resolution X-ray crystal structures of NV
3
CLPro with bound inhibitors 1, 3, and 10 were determined. Attempts to obtain crystal
structures of permeable inhibitors 7 and 8 were unsuccessful, precluding a structural
comparison between the permeable and non-permeable macrocyclic inhibitors that
might perhaps illuminate the structural determinants responsible for the observed
differences in potency and cellular permeability.
Examination of the active site revealed the presence of an antiparallel β-sheet-like
hydrogen bonding network associated with the backbone of all three inhibitors,
covalently bound to the catalytic Cys139 residue (Figure 3). The coordinates of the
Ramachandran plot (see Figure S8 in Supporting information) of six ϕ and ψ angle pairs
of the corresponding P and P residues, extracted from the X-ray crystal structures, lie
2
3
in the upper left quadrant of the plot, indicating that bound inhibitors 1, 3, and 10
assume an extended β-strand conformation. The pseudo ϕ angles of the P residue
1
o
o
covalently bound to the Cys139 residue range from -129.5 to -137.8 . The electron
density maps for the three bound inhibitors are shown in Figure 4A-C. As expected, the
backbones of all three inhibitors are structurally more rigid compared to the atoms
forming the macrocyclic linker, which is more flexible. This is evident from an analysis
8

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of the B-factors for the inhibitors in each structure, which are slightly lower in the
backbone region as shown in the right panels of Figure 4A-C. The electron density for
the “cap” phenyl ring was not observed for 1 and 3 due to disorder and could not be
modeled, while inhibitor 10 has somewhat weak electron density that permitted
modelling of the phenyl ring. The triazole ring of inhibitor 3 has reasonable electron
density, however, it is more disordered and its exact orientation is not clearly discernible
(Figure 4).
Perusal of Table 1 indicates that, with the exception of compounds 8 and 11, the rest of
the compounds displayed low micromolar IC values. The observed degree of inhibition
50
prompted us to delineate the structural determinants associated with binding in order to
gain insight and understanding into the relative potency of the inhibitors. Toward that
33
end, superposition with GESAMT of the high-resolution X-ray crystal structure of the
NV 3CLpro:10 complex (largest ring size) with the NV 3CLPro:1 complex (smallest ring
size) shows that the enzyme structures in both complexes are similar, with an RMSD
between Cα atoms of 0.76 Å (167 residues), except for the flexible loop spanning
Lys121 to Glu140 (Figure 5A). The binding of inhibitor 10 (larger ring size) to the active
site is accompanied by the movement of the flexible loop away from the inhibitor
resulting in the loss of a hydrogen bond between the P side chain (Gln C=O) of the
1
inhibitor with Thr134 (backbone NH) which forms the S pocket. His157, which is the
1
other constituent of the S pocket but is not a part of the flexible loop, maintains a
1
hydrogen bond interaction with the P Gln C=O (Figure 5B). It should be noted that the
1
interaction of NV 3CLpro with acyclic dipeptidyl and tripeptidyl inhibitors involves the
formation of two crucial hydrogen bonds between the carbonyl oxygen of the glutamine
9

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surrogate C=O in the inhibitor and the Thr134 and His157 residues [12]. These two
hydrogen bonding interactions, as well as the flexible backbone of the T123-G133 loop
[
34], appear to play a critical role in enzyme-substrate recognition and are conserved in
all norovirus proteases. Thus, in the NV 3CLpro:1 complex, the movement of the flexible
loop and the shorter linker length of the inhibitor which positions the carbonyl oxygen of
the P Gln side chain away from His157 and Thr134, results in the loss of the Thr134 P1
1
Gln C=O hydrogen bond (Figure 5C). Conversely, the binding of inhibitor 1 allows the
flexible loop to move closer to the inhibitor such that a contact can be formed (3.3 Å)
between Pro136 and the carbonyl oxygen of the warhead (Figure 5B).
The compact nature of the inhibitor positions the NH group of the P Gln side chain
1
within hydrogen bonding distance (3.2 Å) of the carbonyl oxygen of the Ala160 residue
(see Figure S1 in Supporting information for distance information related to the
hydrogen bonds). The subtle structural shifts associated with the P1 Gln side chain may
account for the lower inhibitory activity observed with this series of macrocyclic triazole
compounds, as compared to previously reported triazole-based macrocyclic calpain
inhibitors [35] specifically and macrocyclic inhibitors generally [29,31]. In the former
case, potency is solely dependent on the combined effect of hydrophobic interactions
and the network of hydrogen bonding interactions between the inhibitor backbone and
the enzyme active site which is facilitated by the β-strand conformation of the inhibitor.
The enhancement in the IC values associated with the replacement of the P isobutyl
50
2
side chain with a cyclohexyl group observed with linear dipeptidyl inhibitors [12] is
diminished in compound 10 (IC50 6.1 µM) and is partly due to the loss of the key
hydrogen bonding interaction with Thr134, as revealed by the X-ray crystal structure
1
0

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and the larger flexible ring (see discussion of NMR studies). On the other hand, as
mentioned above, the compact, rigid ring structure and the additional hydrogen bond
with Ala160, discernible from the X-ray crystal structure, may account for the 4-fold
decrease in the IC value (1.6 µM) of compound 1 as compared to compound 10. The
50
in vitro inhibitory activity of compounds 1 and 3 which form the same number of direct
hydrogen bonds with the enzyme (7 hydrogen bonds) was found to be 1.6 µM and 2.4
µM, respectively. Interestingly, compound 32, the acyclic variant of compound 3, was
devoid of any inhibitory activity. Finally, the 18-membered macrocycle 12 (Figure 1) in
which the triazole ring was constructed in a reverse fashion and presumably assumes a
different orientation than inhibitors 1-11, was effective against both 3CLpro and
norovirus in replicon cells having IC50 and EC50 values 15.5 µM and 8.5 µM,
respectively.
In summary, the interaction of macrocyclic inhibitors (I) with NV 3CLpro induces
conformational changes in the flexible loop and active site of the enzyme which are
dependent on macrocycle ring size and structure. These changes result in sub-optimal
angles and distances in forming the critical intermolecular hydrogen bonds between the
P1 Gln side chain and the active site His157 and Thr134 residues, consequently,
maximal binding potency and effective compensation for the energy penalty required to
desolvate the molecule, are not attained.
2
.5 NMR Studies. In order to gain further insight into the solution conformation and
binding of the synthesized macrocyclic inhibitors, as well as confirm the absence of
alternate structures, NMR studies were carried out using the precursor cyclic esters.
1
Assignment of the proton resonances was done using H, COSY, ROESY and HMBC
1
1

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spectral details. The ROESY spectra of the compounds show H to NH (i+1) type
iα
correlation, where the Gln and Leu NH show correlation to the Leu Hα and carbamate
Hα, respectively (see Figures S1-S5 in Supporting Information). This observation is
indicative of a prearranged structure within the macrocycle [28]. The temperature
dependence of amide NH chemical shifts experiments were performed using
compounds 28a (ring size: 17), 28e (ring size: 20) and 28g (ring size: 21), revealing
important information about the solution structures of the macrocycles. Solvent shielded
NH protons typically have ∆δ/T values less than 4 ppb/K while solvent exposed NH
protons have ∆δ/T values greater than 4 ppb/K [28]. According to the results, the Gln,
Leu, and propargylglycine NH protons have ∆δ/T values greater than 4 ppb/K in all
cases (Table 3). At larger ring sizes, the carbamate NH shows a higher temperature
dependence (-7.0 and 6.4 ppb/K for ring sizes 20 and 21, respectively), indicating
greater solvent exposure. However, for smaller ring size it shows comparatively low
temperature dependence (4.2 ppb/K at 17 ring size), indicating less solvent exposure.
This is further supported by the observed line broadening of the carbamate NH proton
at elevated temperatures for compounds 28e and 28g, and the lower line broadening
observed for 28a (Figure S6 in Supporting Information). Previous studies have shown
that amide signals exchanging with water impurity present in DMSO solvent show
considerable broadening of the line width at elevated temperatures due to the enhanced
exchange rate at higher temperatures [28]. At smaller ring size, all three NH protons
show less temperature dependence, indicating solvent shielding which is partly due to
the compact nature of the compound. The absence of very low temperature dependent
NH protons (~ 2 ppb/K) would appear to rule out the presence of alternative turn
1
2

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structures, such as γ-turns. Finally, the conformations of the peptide backbone of
3
macrocycles 28a and 28h were determined based on 1D J
coupling constants
NH-CHα
3
(
Table 4). The coupling constant J
correlates with the dihedral angle (ψ)
NH-CHα
according to the Karplus equation [28]. For unstructured random coil conformations the
coupling constant is around 7 Hz while unusually high coupling constants (> 9 Hz) are
observed for well-structured β-strands. According to the results shown in Table 4, the
expected β-strand conformation was observed only for the 17-membered ring.
3. Conclusion
The studies described herein demonstrate the feasibility of generating cell-permeable
triazole-based macrocyclic inhibitors of norovirus 3Cl protease. Valuable insights into
the mechanism of action, conformation, and pharmacological activity were gained using
an array of structural, spectroscopic, and antiviral techniques and methodologies. The
interaction of inhibitor (I) with NV 3CLpro results in subtle structural shifts in the active
site topography that impair hydrogen bonding interactions between the P1 Gln residue
in the inhibitor and key amino acid residues and contribute to lower pharmacological
activity. The results of ongoing studies are likely to illuminate further the interplay of ring
size, conformation, and cellular permeability, and will reported in due course.
1
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4. Experimental Section
4
.1 General. Reagents and dry solvents were purchased from various chemical
suppliers (Aldrich, Acros Organics, ChemImpex, TCI America, and Bachem) and were
used as obtained. Silica gel (230-450 mesh) used for flash chromatography was
purchased from Sorbent Technologies (Atlanta, GA). Thin layer chromatography was
performed using Analtech silica gel plates. Visualization was accomplished using UV
1
light and/or iodine. The H NMR were recorded in CDCl or DMSO-d using a Varian
3
6
XL-400 spectrometer and are reported relative to TMS (δ = 0.00 ppm). Chemical shifts
H
are reported in ppm and spin multiplicities are represented by the following signals: s
(singlet), d (doublet), dd (doublet of doublets), t (triplet), q (quartet) and m (multiplet).
Melting points were recorded on a Mel-Temp apparatus and are uncorrected. High
resolution mass spectrometry (HRMS) was performed at the University of Kansas Mass
Spectrometry lab using an LCT Premier mass spectrometer (Waters, Milford, MA)
equipped with a time of flight mass analyzer and an electrospray ion source. The purity
of the compounds was established using HPLC and was >95%.
4
.1.1. General Procedure A. Peptide coupling reaction. To a solution of N-protected
amino acid derivative (1.0 eq) in dry DMF was added EDCI (1.22 eq), HOBt (eq) and
the reaction mixture was stirred at room temperature for 40 minutes (Solution A). In the
0
meantime, O-protected amino acid / amine (1.0 eq) in dry DMF kept at 0 C was added
DIEA (4 eq) and stirred for 25 minutes (Solution B). After that Solution B was transferred
to Solution A and stirred overnight at room temperature. The solvent was removed on
the rotary evaporator and residue was taken up with ethyl acetate, washed sequentially
with saturated sodium bicarbonate, 5% HCl and brine. The organic layer was dried over
1
4

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anhydrous sodium sulfate, filtered and the solvent was removed on the rotary
evaporator. See individual experiments for details.
4
.1.2. General Procedure B. Hydrolysis of methyl esters. A solution of compound
methyl ester (1.0 eq) in dry THF kept in an ice bath was treated with a solution of 1M
0
lithium hydroxide (3.5 eq) and stirred for 4 hours at 0 C until the starting material
disappeared (as shown by TLC). The solvent was removed and the residue was diluted
with water. The pH of the aqueous solution was adjusted to ~2 with 5% HCl and
acidified solution was extracted with ethyl acetate and washed with brine. Organic
extract was dried using anhydrous sodium sulfate, filtered and solvent was removed on
the rotary evaporator. For details, see individual experiments.
4
.1.3. General Procedure C. Removal of N-Boc protection. A solution of N-Boc
protected compound (1.0 eq) in dry DCM was treated with 4M HCl in dioxane (10 eq)
and stirred at room temperature for 3 hours while monitoring the disappearance of the
starting material by TLC. Solvent was removed on the rotary evaporator, residue was
treated with ether and concentrated. See individual experiments for details.
4
.1.4. General Procedure D. Conversion of N-Boc protected alcohol to mesylate.
To a solution of N-Boc protected alcohol (60 mmol) and TEA (60 mmol) in dry DCM
0
(
100 mL) kept in an ice bath (0 C) was slowly added methane sulfonyl chloride (60
0
mmol)) and reaction mixture was stirred for 30 minutes at 0 C. The ice bath was
removed and reaction mixture was further allowed to stir at room temperature for 5
hours (Completion of the reaction was monitored by TLC). Then water (50 mL) was
added with stirring, the resulting solution was transferred to a reparatory funnel, organic
1
5

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layer was separated and aqueous layer was extracted with DCM (2 x 75 mL). The
combined organic layers were washed with brine (50 mL) dried over anhydrous sodium
sulfate and concentrated on rotary evaporator. See individual experiments for more
details.
4
.1.5. General Procedure E. Conversion of N-Boc protected mesylate to azide. To
a solution of compound N-Boc protected mesylate (60 mmol) in dry DMF (150 mL) was
added in small portions NaN (180 mmol) with stirring over 30 minutes. Resulting
3
0
solution was heated to 50 C for 1 hour and it was further allowed to react overnight at
room temperature. Then water (50 mL) was added with stirring, solvent was removed
and residue was extracted with ethyl acetate (2 x 175 mL). The combined organic layers
were washed sequentially with water (2 x 50 mL), brine (2 x 50 mL) and solvent was
dried over anhydrous sodium sulfate and evaporated on rotary evaporator. See
individual experiments for more details.
4
.1.6. General Procedure F. Intermolecular click reactions. To a stirred solution of
alkyne (5 mmol) in a mixture of water and THF (1:1, 25 mL) was added corresponding
azide (5 mmol) followed by addition of sodium ascorbate (0.5 mmol) and CuSO .5H O
4
2
(0.25 mmol). Resulting solution was further allowed to react 20 h at room temperature.
See individual experiments for more details.
4
.1.7. General Procedure G. Intramolecular click reaction. To a solution of
macrocycle precursor (0.512 mmol) in dry DCM (1.2 M) under N atmosphere, was
2
added DBU (1.53 mmol) while solution was stirring vigorously at room temperature.
After 15 minutes Cu(I)Br (0.512 mmol) was added and reaction mixture was stirred for
1
6

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1
2 hours at room temperature. After that it was quenched by adding 3M HCl (100 mL),
aqueous layer was separated and extracted with DCM (2 x 500 mL). The combined
organic layers were washed sequentially with water (300 mL), brine (300 mL), dried
over anhydrous sodium sulfate, filtered and concentrated on rotary evaporator. See
individual experiments for more details.
4
.1.8. General Procedure H. Macrolactamization reaction. To a solution of
macrocycle precursor (1 mmol) in dry DMF (2 M) was added DIEA (4 mmol) and stirred
for 30 minutes. After that, was added EDCI (1.25 mmol) followed by HOBt (1.25 mmol)
and resulting solution was stirred for 20 hours at room temperature. The solvent was
removed on the rotary evaporator and residue was taken up with DCM, washed
sequentially with saturated sodium bicarbonate, 5% HCl and brine. The organic layer
was dried over anhydrous sodium sulfate, filtered and the solvent was removed on the
rotary evaporator. See individual experiments for details.
4
.1.9. General Procedure I. Reduction of macrocycle esters. To a solution of
compound macrocycle ester (1 mmol) in dry THF (6 mL) was added a solution of 2M
LiBH (3.0 mmol) drop wise followed by drop wise addition of absolute ethanol (11 mL)
4
and was stirred at room temperature for 16 hours while monitoring the disappearance of
the starting material by TLC. After that solvent was removed on the rotary evaporator
and residue was partitioned between ethyl acetate and 1M KHSO . The aqueous phase
4
was extracted twice more chloroform and each organic extract was washed with brine
separately, dried over anhydrous sodium sulfate and evaporated to give corresponding
alcohol. See individual experiments for more details.
1
7

ACCEPTED MANUSCRIPT
4
.1.10. General Procedure J. Oxidation of macrocyclic alcohols. Corresponding
macrocycle alcohol (0.36 mmol) was suspended in dry DCM (40 mL) under N2
atmosphere and was added Dess-martin periodinane (1.08 mmol). Reaction mixture
was stirred for 4 hours at room temperature and quenched with saturated sodium
bicarbonate solution containing 10% Sodium thiosulfate (10 mL). Organic layer was
separated and aqueous layer was extracted with DCM (2 x 60 mL), combined organic
layers were washed with brine (40 mL), dried over anhydrous sodium sulfate and
solvent was evaporated on rotary evaporator. See individual experiments for more
details.
4
.1.11. Methyl ((S)-2-((tert-butoxycarbonyl)amino)pent-4-ynoyl)-L-leucinate 13a.
Boc-L-Propargylglycine ( 6.39 g; 30 mmol) in dry DMF ( 70 mL) was reacted with L-Leu-
OMe(HCl) (30 mmol) according to general procedure A and crude product was purified
using flash chromatography (silica gel; hexane/ethyl acetate 75:25) to give 13a as a
0
1
white solid (9.0 g ; 89% yield); M.p. 76-78 C; H NMR (400 MHz, DMSO-d ): δ ppm
6
0
2
.75 - 0.97 (m, 6 H), 1.38 (s, 9 H), 1.44 - 1.74 (m, 3 H), 2.43 (dd, J=8.70, 2.29 Hz, 1 H),
.51 - 2.57 (m, 1 H), 2.82 (br s, 1 H), 3.52 - 3.71 (m, 3 H), 4.12 (d, J=5.19 Hz, 1 H), 4.29
(
td, J=4.96, 2.90 Hz, 1 H), 6.97 (d, J=8.24 Hz, 1 H), 8.24 (d, J=7.63 Hz, 1 H); HRMS
ESI, m/z) calcd for C H N O [M+H]+ 341.2076. found: 341.2069.
(
17
29
2
5
Compounds 13b and 13c were prepared using a similar procedure as the one
described above.
4
.1.12.
Methyl
(S)-2-((S)-2-((tert-butoxycarbonyl)amino)pent-4-ynamido)-3-
0
1
cyclohexylpropanoate 13b. White solid (9.2 g ; 81% yield); M.p 85-87 C; H NMR
1
8

ACCEPTED MANUSCRIPT
(
400 MHz, DMSO-d ): δ ppm 0.71 - 0.99 (m, 2 H), 1.02 - 1.25 (m, 3 H), 1.29 - 1.44 (m, 9
6
H), 1.45 - 1.76 (m, 8 H), 2.43 (dd, J=8.79, 2.15 Hz, 1 H), 2.48 (dd, J=5.08, 2.73 Hz, 1
H), 2.83 (br. s., 1 H), 3.60 (s, 3 H), 4.11 (d, J=5.47 Hz, 1 H), 4.32 (d, J=5.08 Hz, 1 H),
6
.97 (d, J=8.20 Hz, 1 H), 8.24 (d, J=7.42 Hz, 1 H). HRMS (ESI, m/z) calcd for
C H N O [M+H]+ 381.2389. found: 381.2382.
20
33
2
5
4
1
.1.13. Methyl ((S)-2-((tert-butoxycarbonyl)amino)pent-4-ynoyl)-L-phenylalaninate
0
1
3c. White solid (8.3 g ; 74% yield); M.p 81-83 C; H NMR (400 MHz, DMSO-d ): δ
6
ppm 1.38 (s, 9 H), 2.37 (dd, J=8.85, 2.14 Hz, 1 H), 2.45 (dd, J=4.43, 2.59 Hz, 1 H), 2.82
br s, 1 H), 2.89 - 2.98 (m, 1 H), 2.99 - 3.12 (m, 1 H), 3.58 (s, 3 H), 4.06 - 4.21 (m, 1 H),
(
4
.37 - 4.57 (m, 1 H), 6.90 (d, J=8.85 Hz, 1 H), 7.17 - 7.24 (m, 3 H), 7.25 - 7.33 (m, 2 H),
.31 (d, J=7.63 Hz, 1 H). HRMS (ESI, m/z) calcd for C H N O [M+H]+ 375.1920.
8
20
27
2
5
found: 375.1909.
4
.1.14. Methyl ((S)-2-(((benzyloxy)carbonyl)amino)pent-4-ynoyl)-L-leucinate 14a.
Compound 13a (6.8 g; 20 mmol) in dry CH Cl (30 mL) was treated with 1M HCl in
2
2
dioxane to remove Boc protection according to general procedure C and solvent was
removed after the reaction, under high vacuum to dryness. To a solution of resulting
HCl salt of the amine in dry THF (70 mL) was added DIEA (12.9 g; 100 mmol) and
stirred for 30 minutes. After that, benzyl chloroformate (3.75 g; 22 mmol) was added
and the resulting solution was further reacted for 16 h at room temperature. The solvent
was removed on the rotary evaporator and the residue was taken up in ethyl acetate
(
150 mL) and washed sequentially with saturated NaHCO (50 mL), 5% HCl (2 x 50 mL)
3
and brine (50 mL). The organic layer was dried over anhydrous sodium sulfate, filtered
and solvent was removed on the rotary evaporator to give 14a as a white solid (6.7 g:
1
9

ACCEPTED MANUSCRIPT
0
1
9
1
0% yield); M.p 79-80 C. H NMR (400 MHz, DMSO-d ): δ ppm 0.79 - 0.93 (m, 6 H),
6
.40 - 1.72 (m, 3 H), 2.36 - 2.47 (m, 1 H), 2.52 - 2.60 (m, 1 H), 2.84 (s, 1 H), 3.55 - 3.66
(
m, 3 H), 4.15 - 4.32 (m, 2 H), 4.98 - 5.12 (m, 2 H), 7.24 - 7.42 (m, 5 H), 7.54 (d, J=8.54
Hz, 1 H), 8.36 (d, J=7.63 Hz, 1 H). HRMS (ESI, m/z) calcd for C H N O [M+H]+
20
27
2
5
3
75.1920. found: 375.1916.
Compounds 14b and 14c were prepared according to the same procedure as above.
4
.1.15.
Methyl
(S)-2-((S)-2-(((benzyloxy)carbonyl)amino)pent-4-ynamido)-3-
0
1
cyclohexylpropanoate 14b. White solid (7.6 g: 92% yield); M.p 86-88 C. H NMR (400
MHz, DMSO-d ): δ ppm 0.70 - 0.99 (m, 2 H), 1.02 - 1.24 (m, 3 H), 1.33 (br. s., 1 H), 1.43
6
-
1.56 (m, 2 H), 1.62 (d, J=12.21 Hz, 5 H), 2.37 - 2.48 (m, 1 H), 2.85 (s, 1 H), 3.60 (s, 3
H), 4.22 (td, J=8.85, 4.88 Hz, 1 H), 4.27 - 4.35 (m, 1 H), 5.04 (s, 2 H), 7.34 - 7.41 (m, 5
H), 7.54 (d, J=8.54 Hz, 1 H), 8.35 (d, J=7.63 Hz, 1 H). HRMS (ESI, m/z) calcd for
C H N O [M+H]+ 415.2233. found: 415.2228.
23
31
2
5
4
.1.16. Methyl ((S)-2-(((benzyloxy)carbonyl)amino)pent-4-ynoyl)-L-
phenylalaninate 14c.
0
1
White solid (6.9 g: 85% yield); M.p 106-108 C. H NMR (400 MHz, DMSO-d ): δ ppm
6
2
2
1
.31 - 2.44 (m, 1 H), 2.54 (dd, J=4.49, 2.54 Hz, 1 H), 2.84 (s, 1 H), 2.89 - 2.98 (m, 1 H),
.99 - 3.08 (m, 1 H), 3.52 - 3.63 (m, 3 H), 4.23 (td, J=8.98, 4.69 Hz, 1 H), 4.41 - 4.54 (m,
H), 4.99 - 5.10 (m, 2 H), 7.15 - 7.23 (m, 3 H), 7.24 - 7.29 (m, 2 H), 7.30 - 7.42 (m, 5
H), 7.50 (d, J=8.98 Hz, 1 H), 8.44 (d, J=7.42 Hz, 1 H). HRMS (ESI, m/z) calcd for
C H N O [M+H]+ 409.1763. found: 409.1754.
23
25
2
5
4
.1.17. ((S)-2-(((Benzyloxy)carbonyl)amino)pent-4-ynoyl)-L-leucine 15a. A solution
of compound 14a (5.6 g; 15 mmol) in in dry THF (60 mL) was hydrolyzed according to
2
0

ACCEPTED MANUSCRIPT
general procedure B. The crude product was purified by Flash chromatography (Silica
1
gel; Hexane/Ethyl acetate 50:50) to give 15a as a colorless oil (4.5 g; 84% yield); H
NMR (400 MHz, DMSO-d ): δ 0.80 - 0.95 (m, 6 H), 1.44 - 1.57 (m, 2 H), 1.59 - 1.70 (m,
6
1
H), 1.72 - 1.81 (m, 2 H), 2.35 - 2.48 (m, 1 H), 2.52 - 2.66 (m, 1 H), 2.79 - 2.86 (m, 1
H), 3.52 - 3.68 (m, 2 H), 4.11 - 4.32 (m, 2 H), 5.04 (s, 2 H), 7.25 - 7.43 (m, 6 H), 7.54 (d,
J=8.59 Hz, 1 H), 8.18 (d, J=8.20 Hz, 1 H), 12.58 (br. s., 1 H). HRMS (ESI, m/z) calcd for
C H N O [M+H]+ 361.1763. found: 361.1758.
19
25
2
5
Compounds 15b and 15c were prepared using the same procedure as described
above.
4
.1.18.
(S)-2-((S)-2-(((Benzyloxy)carbonyl)amino)pent-4-ynamido)-3-
1
cyclohexylpropanoic acid 15b. Colorless oil (5.2 g; 88% yield); H NMR (400 MHz,
DMSO-d ): δ 0.69 - 0.98 (m, 2 H), 1.05 - 1.21 (m, 3 H), 1.27 - 1.41 (m, 1 H), 1.53 (t,
6
J=7.17 Hz, 2 H), 1.56 - 1.73 (m, 5 H), 2.36 - 2.48 (m, 1 H), 2.52 - 2.62 (m, 1 H), 2.82 (s,
1
8
H), 4.16 - 4.32 (m, 2 H), 5.05 (s, 2 H), 7.27 - 7.43 (m, 5 H), 7.55 (d, J=8.85 Hz, 1 H),
.18 (d, J=7.93 Hz, 1 H), 12.50 (br. s., 1 H). HRMS (ESI, m/z) calcd for C H N O
5
22
29
2
[
M+H]+ 401.2076. found: 401.2068.
4
.1.19. ((S)-2-(((benzyloxy)carbonyl)amino)pent-4-ynoyl)-L-phenylalanine 15c.
1
Colorless oil (5.1 g; 86% yield); H NMR (400 MHz, DMSO-d ): δ 2.32 - 2.44 (m, 1 H),
6
2
.51 - 2.59 (m, 1 H), 2.82 (s, 1 H), 2.86 - 2.97 (m, 1 H), 3.00 - 3.11 (m, 1 H), 4.23 (td,
J=9.18, 4.30 Hz, 1 H), 4.42 (td, J=8.10, 5.27 Hz, 1 H), 5.01 - 5.07 (m, 2 H), 7.15 - 7.29
m, 5 H), 7.30 - 7.42 (m, 5 H), 7.52 (d, J=8.59 Hz, 1 H), 8.21 (d, J=7.81 Hz, 1 H), 12.69
(
2
1

ACCEPTED MANUSCRIPT
(
br. s., 1 H). HRMS (ESI, m/z) calcd for C H N O [M+H]+ 395.1607. found:
22
23
2
5
3
95.1599.
4
.1.20. Methyl N5-(3-azidoropyl)-N2-(tert-butyoxycarbonyl)-L-glutaminate 19a. A
solution of 2-(Boc-amino)-1-ethanol (9.6 g: 60 mmol) in dry DCM (100 mL) was
converted in to mesylate according to general procedure D to yield 16b as a colorless
oil (13.4 g; 94% yield).
Compound 16a (11.9 g; 50 mmol) was dissolve in dry DMF (120 mL) and converted in
to azide according to general procedure E to yield 17a as a light yellow oil (7.2 g; 78%
yield).
A solution of compound 17a (6.8 g; 37 mmol) in dry CH Cl (30 mL) was treated with 1M
2
2
HCl in dioxane to remove Boc protection according to general procedure C and solvent
was removed after the reaction under high vacuum to dryness resulting compound 18a.
Boc-Glu-OMe (9.67 g; 37 mmol) in dry DMF (100 mL) was reacted with compound 18a
(4.5 g: 37 mmol) according to general procedure A and the crude product was purified
by flash chromatography (silica gel; hexane/ethyl acetate 70:30) to give 19a as a
1
colorless oil (9.5 g ; 78% yield); H NMR (400 MHz, DMSO-d6): δ 1.27 - 1.47 (m, 9 H),
1
.68 - 1.83 (m, 1 H), 1.86 - 1.97 (m, 1 H), 2.16 (t, J=7.42 Hz, 2 H), 3.23 (q, J=5.60 Hz, 2
H), 3.30 - 3.40 (m, 2 H), 3.62 (s, 3 H), 3.96 (d, J=4.69 Hz, 1 H), 7.24 (d, J=7.42 Hz, 1
H), 8.06 (br. s., 1 H). HRMS (ESI, m/z) calcd for C H N O [M+H]+ 330.1777. found:
13
24
5
5
3
30.1769.
Compounds 19b-e were prepared using the same procedure as described above.
2
2

ACCEPTED MANUSCRIPT
5
2
4
.1.21. Methyl N -(3-azidopropyl)-N -(tert-butoxycarbonyl)-L-glutaminate 19b.
1
Colorless oil (8.2 g; 72% yield); H NMR (400 MHz, DMSO-d ): δ 1.27 - 1.44 (m, 9 H),
6
1
.63 (q, J=6.84 Hz, 2 H), 1.69 - 1.81 (m, 1 H), 1.85 - 1.98 (m, 1 H), 2.14 (t, J=7.42 Hz, 2
H), 3.01 - 3.18 (m, 2 H), 3.28 - 3.41 (m, 2 H), 3.62 (s, 3 H), 3.94 (dd, J=8.01, 3.32 Hz, 1
H), 7.23 (d, J=7.81 Hz, 1 H), 7.87 (d, J=4.69 Hz, 1 H). HRMS (ESI, m/z) calcd for
C H N O [M+H]+ 344.1934. found: 344.1929.
14
26
5
5
5
2
4
.1.22. Methyl N -(4-azidobutyl)-N -(tert-butoxycarbonyl)-L-glutaminate 19c.
1
Colorless oil (9.0 g: 85% yield); H NMR (400 MHz, DMSO-d ): δ 1.29 - 1.40 (m, 9 H),
6
1
3
.40 - 1.58 (m, 4 H), 1.66 - 1.80 (m, 1 H), 1.84 - 1.97 (m, 1 H), 2.13 (t, J=7.42 Hz, 2 H),
.04 (q, J=6.25 Hz, 2 H), 3.32 (t, J=6.84 Hz, 2 H), 3.62 (s, 3 H), 3.94 (dd, J=8.01, 3.71
Hz, 1 H), 7.23 (d, J=7.81 Hz, 1 H), 7.71 - 7.91 (m, 1 H). HRMS (ESI, m/z) calcd for
C H N O [M+H]+ 358.2090. found: 358.2083.
15
28
5
5
5
2
4
.1.23. Methyl N -(5-azidopentyl)-N -(tert-butoxycarbonyl)-L-glutaminate 19d.
1
Colorless oil (7.9 g: 69% yield); H NMR (400 MHz, DMSO-d ): δ 1.24 - 1.34 (m, 3 H),
6
1
.35 - 1.44 (m, 10 H), 1.52 (q, J=7.13 Hz, 2 H), 1.66 - 1.81 (m, 1 H), 1.84 - 1.97 (m, 1
H), 2.13 (t, J=7.42 Hz, 2 H), 3.02 (q, J=6.12 Hz, 2 H), 3.26 - 3.37 (m, 2 H), 3.62 (s, 3 H),
.94 (d, J=4.30 Hz, 1 H), 7.23 (d, J=7.81 Hz, 1 H), 7.78 (t, J=5.27 Hz, 1 H). HRMS (ESI,
m/z) calcd for C H N O [M+H]+ 372.2247. found: 372.2241.
3
16
30
5
5
5
2
4
.1.24. Methyl N -(6-azidohexyl)-N -(tert-butoxycarbonyl)-L-glutaminate 19e.
1
Yellow oil (6.5 g: 68% yield); H NMR (400 MHz, DMSO-d ): δ 1.24 - 1.30 (m, 3 H), 1.31
6
-
(
1.35 (m, 3 H), 1.36 - 1.41 (m, 9 H), 1.44 - 1.60 (m, 2 H), 1.73 (br. s., 1 H), 1.86 - 1.96
m, 1 H), 2.12 (t, J=7.48 Hz, 2 H), 3.01 (q, J=6.21 Hz, 2 H), 3.22 - 3.36 (m, 2 H), 3.62 (s,
2
3

ACCEPTED MANUSCRIPT
3
H), 3.94 (d, J=4.27 Hz, 1 H), 7.23 (d, J=7.63 Hz, 1 H), 7.76 (d, J=5.19 Hz, 1 H). HRMS
(
ESI, m/z) calcd for C H N O [M+H]+ 386.2403. found: 386.2401.
17 32 5 5
5
4
.1.25. Methyl N -(3-azidopropyl)-L-glutaminate hydrochloride 20a. A solution of
compound 19a (4.4 g: 15 mmol) in dry DCM (20 mL) was treated with 4M HCl in
dioxane according to general procedure C to obtain compound 20a as a yellow oil (4.0
1
g; 95% yield); H NMR (400 MHz, DMSO-d ): δ 1.63 (q, J=6.88 Hz, 2 H), 1.67 - 1.80 (m,
6
1
2
H), 1.85 - 1.98 (m, 1 H), 2.14 (t, J=7.42 Hz, 2 H), 3.01 - 3.18 (m, 2 H), 3.28 - 3.41 (m,
H), 3.62 (s, 3 H), 3.94 (dd, J=8.01, 3.32 Hz, 1 H), 7.23 (d, J=7.81 Hz, 1 H), 7.87 (d,
J=4.69 Hz, 1 H). HRMS (ESI, m/z) calcd for C H ClN O [M+H]+ 280.1176. found:
9
19
5
3
2
80.1172.
Compound 20b was prepared using the same procedure as described above.
5
4
9
1
.1.26. Methyl N -(4-azidobutyl)-L-glutaminate hydrochloride 20b. Yellow oil (4.8 g;
1
4% yield); H NMR (400 MHz, DMSO-d ): δ 1.39 - 1.57 (m, 4 H), 1.66 - 1.80 (m, 1 H),
6
.84 - 1.97 (m, 1 H), 2.13 (t, J=7.42 Hz, 2 H), 3.04 (q, J=6.25 Hz, 2 H), 3.33 (t, J=6.84
Hz, 2 H), 3.62 (s, 3 H), 3.94 (dd, J=8.01, 3.71 Hz, 1 H), 7.23 (d, J=7.81 Hz, 1 H), 7.71 -
7
2
.91 (m, 1 H). HRMS (ESI, m/z) calcd for C H ClN O [M+H]+ 294.1333. found:
10 21 5 3
94.1330.
34
4
.1.27. 5-azido-2-(((benzyloxy)carbonyl)amino)pentanoic acid 21. To a solution of
0
sodium azide (2.27 g; 35 mmol) suspended in anhydrous acetonitrile (35 mL) at 0 C
was added triflic anhydride (8.18 g; 29 mmol) dropwise over 10 minutes and the cooled
solution was stirred for 2 hours. The mixture was filtered through cotton wool to give a
crude triflic azide solution. To a vigorously stirred suspension of Z-L-ornithine (6.39 g;
2
4

ACCEPTED MANUSCRIPT
2
4 mmol) in acetonitrile (100 mL) and water (40 mL) mixture was added trimethylamine
0
(
7.38 g; 73 mmol) and CuSO (24 mmol). The resulting solution was cooled to C and
4
the triflic azide solution was added dropwise. After 30 minutes, cooling was removed
and the reaction mixture was stirred at room temperature overnight. The solvent was
removed on the rotary evaporator and the aqueous solution was washed with ethyl
acetate (2 x 100 mL). The aqueous phase was then acidified with 2M HCl followed by
concentrated HCl and extracted with ethyl acetate (3 x 100 mL). The organic layer was
dried over anhydrous sodium sulfate, filtered, and concentrated to yield a crude product.
After that chloroform was added and the solution was refrigerated and the precipitate
was filtered off. The solvent was removed on the rotary evaporator and the crude
product was purified using flash chromatography to obtain compound 21 as a light
1
yellow oil (2.14 g; 30% yield); H NMR (400 MHz, DMSO-d ): δ 1.45 - 1.71 (m, 3 H),
6
1
7
.72 - 1.87 (m, 1 H), 3.32 (t, J=6.41 Hz, 2 H), 3.96 - 4.02 (m, 1 H), 5.05 (s, 2 H), 7.23 -
.46 (m, 5 H), 7.62 (d, J=7.93 Hz, 1 H), 12.10 (b. s., 1H). HRMS (ESI, m/z) calcd for
C H N O [M+H]+ 293.1250. found: 293.1248.
13
17
4
4
2
5
4
.1.28. Methyl N -(tert-butoxycarbonyl)-N -(prop-2-yn-1-yl)-L-glutaminate 22. To a
solution of Boc-Glu-OMe (5.2 g; 20 mmol) in dry DMF (60 mL) was added EDCI (4.67 g;
4.4 mmol), HOBt (3.7 g; 24.4 mmol) and the reaction mixture was stirred at room
2
temperature for 30 minutes. Propargylamine (1.1 g; 20 mmol) was added to the mixture
and the reaction mixture was stirred overnight at room temperature. The solvent was
removed on the rotary evaporator and residue was taken up with DCM (400 mL) and
washed sequentially with 10% aqueous citric acid (150 mL) and brine (150 mL). The
organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated to
2
5

ACCEPTED MANUSCRIPT
yield a crude product which was purified by flash chromatography (silica gel;
1
hexane/ethyl acetate 50:50) to give 22 as a colorless oil (3.8 g ; 64% yield); H NMR
(
400 MHz, DMSO-d ): δ ppm 1.35 (s, 9 H), 1.96 - 2.10 (m, 2 H), 2.19 - 2.30 (m, 1 H),
6
2
2
.30 - 2.42 (m, 1 H), 3.12 (t, J=2.29 Hz, 1 H), 3.69 - 3.78 (m, 3 H), 3.85 (dd, J=5.34,
.44 Hz, 2 H), 3.96 - 4.08 (m, 1 H), 7.81 (d, J=6.81 Hz, 1H), 8.48 (t, J=5.19 Hz, 1 H).
HRMS (ESI, m/z) calcd for C H N O [M+H]+ 299.1607. found: 299.1602.
14
23
2
5
2
5
4
2
1
.1.29. Methyl N -((tert-butoxycarbonyl)leucyl)-N -(prop-2-yn-1-yl)-L-glutaminate
3. A solution of compound 22 (8.91 g; 30 mmol) in dry DCM (90 mL) was treated with
M HCl in dioxane according to general procedure C. The resulting compound was
coupled with Boc-Glu-OMe (7.74 g; 30 mmol) in dry DMF (70 mL) according to general
procedure A and the crude product was purified by flash chromatography (silica gel;
1
hexane/ethyl acetate 50:50) to give 19a as a yellow oil (8.0 g ; 65% yield); H NMR (400
MHz, CDCl ): δ 0.95 (t, J=6.56 Hz, 6 H), 1.38 - 1.56 (m, 10 H), 1.65 (dd, J=13.58, 5.19
3
Hz, 2 H), 1.85 - 2.03 (m, 1 H), 2.18 - 2.39 (m, 4 H), 3.74 (s, 3 H), 4.04 (td, J=5.04, 2.59
Hz, 2 H), 4.08 - 4.20 (m, 1 H), 4.54 (br. s., 1 H), 5.02 (d, J=7.63 Hz, 1 H), 6.80 - 6.92 (m,
1
H), 6.94 - 7.07 (m, 1 H). HRMS (ESI, m/z) calcd for C H N O [M+H]+ 412.2448.
20 34 3 6
found: 412.2445.
5
2
4
.1.30. Methyl N -(3-azidopropyl)-N -(((S)-2-(((benzyloxy)carbonyl)amino)pent-4-
ynoyl)-L-leucyl)-L-glutaminate 24a. A solution of compound 15a (11.7 g; 31 mmol) in
dry DMF (100 mL) was coupled with compound 20a according to general procedure A
and the crude product was purified using flash chromatography (silica gel;
DCM/methanol 95:5) to obtain compound 24a as a white solid (8.3 g ; 46% yield); M.p
0
1
1
10-111 C. H NMR (400 MHz, DMSO-d ): δ 0.86 (dd, J=15.43, 6.44 Hz, 6 H), 1.45 (t,
6
2
6

ACCEPTED MANUSCRIPT
J=6.84 Hz, 2 H), 1.63 (t, J=6.64 Hz, 3 H), 1.73 - 1.87 (m, 1 H), 1.89 - 2.02 (m, 2 H), 2.07
- 2.21 (m, 2 H), 2.35 - 2.48 (m, 2 H), 2.52 - 2.63 (m, 1 H), 2.82 (br. s., 1 H), 3.01 - 3.15
(m, 2 H), 3.60 (s, 3 H), 4.11 - 4.25 (m, 2 H), 4.31 (d, J=7.03 Hz, 1 H), 5.05 (s, 2 H), 7.23
- 7.41 (m, 5 H), 7.59 (d, J=7.81 Hz, 1 H), 7.82 (br. s., 1 H), 7.99 (d, J=7.78 Hz, 1 H),
8
.29 (d, J=7.42 Hz, 1 H). HRMS (ESI, m/z) calcd for C H N O [M+H]+ 586.2989.
28 40 7 7
found: 586.2986.
Compound 24b was prepared using the same procedure as described above.
5
2
4
.1.31. Methyl N -(4-azidobutyl)-N -(((S)-2-(((benzyloxy)carbonyl)amino)pent-4-
0
1
ynoyl)-L-leucyl)-L-glutaminate 24b. White solid (9.6 g; 58% yield); M.p 106-108 C. H
NMR (400 MHz, DMSO-d ): δ 0.75 - 0.94 (m, 6 H), 1.34 - 1.53 (m, 6 H), 1.54 - 1.69 (m,
6
2
2
4
H), 1.72 - 1.87 (m, 2 H), 1.94 (dd, J=13.19, 5.49 Hz, 2 H), 2.04 - 2.21 (m, 2 H), 2.77 -
.90 (m, 1 H), 3.04 (q, J=6.29 Hz, 2 H), 3.54 - 3.67 (m, 3 H), 4.10 - 4.25 (m, 2 H), 4.26 -
.39 (m, 2 H), 5.01 - 5.08 (m, 2 H), 7.28 - 7.44 (m, 5 H), 7.61 (d, J=8.42 Hz, 1 H), 7.81
(
t, J=5.40 Hz, 1 H), 8.01 (d, J=8.24 Hz, 1 H), 8.31 (d, J=7.14 Hz, 1 H). HRMS (ESI, m/z)
calcd for C H N O [M+H]+ 600.3146. found: 600.3144.
29
42
7
7
4
.1.32.
Methyl
(3S,6S,9S,Z)-3-(((benzyloxy)carbonyl)amino)-6-isobutyl-4,7,12-
1
trioxo-1 H-5,8,13-triaza-1(4,1)-triazolacyclohexadecaphane-9-carboxylate 25a. A
solution of compound 24a (0.3 g: 0.512 mmol) in dry DCM (420 mL) was cyclized
according to general procedure G and the crude product was purified using flash
chromatography (silica gel; DCM/methanol 95:5) to give compound 25a as a white solid
0
1
(
0.14 g; 45% yield); M.p 210-211 C. H NMR (DMSO-d ): δ 0.73 - 0.99 (m, 6 H), 1.45
6
(q, J=6.60 Hz, 3 H), 1.62 - 1.80 (m, 2 H), 1.83 - 1.94 (m, 1 H), 2.00 - 2.30 (m, 4 H), 2.60
2
7

ACCEPTED MANUSCRIPT
-
2.76 (m, 1 H), 3.04 (d, J=4.30 Hz, 2 H), 3.57 - 3.68 (m, 3 H), 4.08 - 4.20 (m, 1 H), 4.26
4.45 (m, 4 H), 5.04 (d, J=9.76 Hz, 2 H), 7.03 (d, J=7.03 Hz, 1 H), 7.24 - 7.42 (m, 5 H),
.53 (s, 2 H), 8.16 (d, J=7.42 Hz, 1 H), 8.53 (d, J=8.20 Hz, 1 H). HRMS (ESI, m/z) calcd
-
7
for C H N O [M+H]+ 586.2989. found: 586.2984.
28
40
7
7
Compound 25b was prepared using the same procedure as described above.
4
.1.33.
Methyl
(3S,6S,9S,Z)-3-(((benzyloxy)carbonyl)amino)-6-isobutyl-4,7,12-
trioxo-1 H-5,8,13-triaza-1(4,1)-triazolacycloheptadecaphane-9-carboxylate 25b.
White solid (0.15 g; 50% yield); M.p 213-215 C. H NMR (400 MHz, DMSO-d ): δ 0.87
1
0
1
6
(dd, J=12.69, 6.44 Hz, 6 H), 1.14 - 1.32 (m, 2 H), 1.35 - 1.56 (m, 3 H), 1.57 - 1.83 (m, 4
H), 2.03 - 2.32 (m, 3 H), 2.73 (t, J=6.25 Hz, 1 H), 2.98 - 3.14 (m, 2 H), 3.54 - 3.70 (m, 3
H), 4.11 - 4.32 (m, 3 H), 4.32 - 4.44 (m, 2 H), 4.92 - 5.16 (m, 2 H), 6.91 (d, J=7.81 Hz, 1
H), 7.26 - 7.41 (m, 5 H), 7.67 - 7.83 (m, 2 H), 8.22 (d, J=7.42 Hz, 1 H), 8.36 (d, J=7.81
Hz, 1 H). HRMS (ESI, m/z) calcd for C H N O [M+H]+ 600.3146. found: 600.3142.
29
42
7
7
4
.1.34.
((S)-2-(((benzyloxy)carbonyl)amino)-3-(1-(2-((S)-4-((tert-
butoxycarbonyl)amino)-5-methoxy-5-oxopentanamido)ethyl)-1H-1,2,3-triazol-4-
yl)propanoyl)-L-leucine 27a. Compound 15a (2.04 g; 5 mmol) was reacted with
compound 19a (1.92 g; 5 mmol) according to general procedure F and the crude
product was purified using flash chromatography (silica gel; DCM/methanol 95:5) to give
0
1
compound 27a as a white solid (3.17 g; 80% yield); M.p 48-49 C. H NMR (400 MHz,
DMSO-d ): δ 0.68 - 0.94 (m, 6 H), 1.25 - 1.41 (m, 9 H), 1.42 - 1.81 (m, 4 H), 1.84 - 2.01
6
(m, 1 H), 2.13 (t, J=7.23 Hz, 1 H), 2.76 - 3.12 (m, 2 H), 3.56 - 3.68 (m, 3 H), 3.94 (d,
J=5.08 Hz, 1 H), 4.15 - 4.26 (m, 1 H), 4.27 - 4.42 (m, 3 H), 4.89 - 5.07 (m, 2 H), 7.24 (d,
2
8