Preparation and use of a general solid-phase intermediate to biomimetic scaffolds and peptide condensations

Article abstract of DOI:10.3390/molecules23071762

The Distributed Drug Discovery (D3) program develops simple, powerful, and reproducible procedures to enable the distributed synthesis of large numbers of potential drugs for neglected diseases. The synthetic protocols are solid-phase based and inspired by published work. One promising article reported that many biomimetic molecules based on diverse scaffolds with three or more sites of variable substitution can be synthesized in one or two steps from a common key aldehyde intermediate. This intermediate was prepared by the ozonolysis of a precursor functionalized at two variable sites, restricting their presence in the subsequently formed scaffolds to ozone compatible functional groups. To broaden the scope of the groups available at one of these variable sites, we developed a synthetic route to an alternative, orthogonally protected key intermediate that allows the incorporation of ozone sensitive groups after the ozonolysis step. The utility of this orthogonally protected intermediate is demonstrated in the synthesis of several representative biomimetic scaffolds containing ozonolytically labile functional groups. It is compatible with traditional Fmoc peptide chemistry, permitting it to incorporate peptide fragments for use in fragment condensations with peptides containing cysteine at the N-terminus. Overall yields for its synthesis and utilization (as many as 13 steps) indicate good conversions at each step.

Full text of DOI:10.3390/molecules23071762

molecules
Article
Preparation and Use of a General Solid-Phase
Intermediate to Biomimetic Scaffolds and
Peptide Condensations
J. Geno Samaritoni 1,*, Jacek G. Martynow 2, Martin J. O’Donnell 1 and William L. Scott 1
1
Department of Chemistry and Chemical Biology, Indiana University Purdue University Indianapolis, 402 N.
Blackford St., Indianapolis, IN 46202, USA; modonnel@iupui.edu (M.J.O.); wscott@iupui.edu (W.L.S.)
Melinta Therapeutics, Inc., 300 George Street, S 301, New Haven, CT 06511, USA; jmartynow@melinta.com
2
*
Correspondence: jsamarit@iupui.edu; Tel.: +1-317-274-6872
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 23 June 2018; Accepted: 14 July 2018; Published: 18 July 2018
Abstract: The Distributed Drug Discovery (D3) program develops simple, powerful, and reproducible
procedures to enable the distributed synthesis of large numbers of potential drugs for neglected
diseases. The synthetic protocols are solid-phase based and inspired by published work.
One promising article reported that many biomimetic molecules based on diverse scaffolds with
three or more sites of variable substitution can be synthesized in one or two steps from a common
key aldehyde intermediate. This intermediate was prepared by the ozonolysis of a precursor
functionalized at two variable sites, restricting their presence in the subsequently formed scaffolds
to ozone compatible functional groups. To broaden the scope of the groups available at one
of these variable sites, we developed a synthetic route to an alternative, orthogonally protected
key intermediate that allows the incorporation of ozone sensitive groups after the ozonolysis
step. The utility of this orthogonally protected intermediate is demonstrated in the synthesis of
several representative biomimetic scaffolds containing ozonolytically labile functional groups. It is
compatible with traditional Fmoc peptide chemistry, permitting it to incorporate peptide fragments
for use in fragment condensations with peptides containing cysteine at the N-terminus. Overall yields
for its synthesis and utilization (as many as 13 steps) indicate good conversions at each step.
Keywords: distributed drug discovery; peptide fragment condensation; biomimetic scaffolds;
bicyclic thiazolidine lactams; cyclitive cleavage; homoserine lactones; diastereomers; acetal;
orthogonal protection; nuclear Overhauser enhancement
1. Introduction
Our Distributed Drug Discovery (D3) program seeks simple and powerful synthetic methodologies
to make large numbers of biomimetic molecules from diverse scaffolds. Solid-phase synthetic protocols
are ideal, because they enable multi-step syntheses to be carried out efficiently, on a small or large scale,
with quick, simple work-ups and a minimal loss of material. They are the most powerful when a single
protocol allows access through more than one variable step to multiple molecules with potential for
biological activity. The first dramatic example was Merrifield’s solid-phase synthesis of peptides [1,2],
where each variable position in the growing peptide chain provided an opportunity to substitute any of
20 amino acids, and the ultimate peptide could be many amino acids long. We developed a variation
on solid-phase peptide synthesis termed “unnatural peptide synthesis” [3–5], in which at a particular
step of a small peptide synthesis, an N-terminal glycine could be converted on-resin into an unnatural
amino acid that then became part of the growing sequence. Later, we extended this work with the
solid-phase conversion of natural amino acids (including glycine) into the key Merrifield resin intermediate
Molecules 2018, 23, 1762; doi:10.3390/molecules23071762
www.mdpi.com/journal/molecules

Molecules 2018, 23, 1762
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7 [6], which was subsequently transformed into multiple peptidomimetic and biomimetic scaffolds 1–5
(Scheme 1). The final release of product in all of these cases was by cyclitive cleavage. This permits the
use of acidic or basic conditions at any intermediate step [6].
Scheme 1. Synthesis and utilization of key intermediate 7 to multiple biomimetic scaffolds.
To make these scaffolds, our previously reported [6] synthesis of 7 (Scheme 2) required an
ozonolysis of precursor 10 that already contained the acylated residue R2. A major limitation of this
route is its incompatibility with any R2 groups that are ozonolytically labile, such as electron-rich
(hetero)aromatics, alkenes, and a number of amino acid side chains. In this report, we describe the
synthesis and utilization of the modified key intermediate 13 (Scheme 3), which is compatible with
both the introduction of ozone sensitive R2 (for example, the side chains on some amino acids such
as tyrosine or tryptophan) and classic solid-phase peptide synthesis. In addition to giving broader
access to the biomimetic scaffolds shown in Scheme 1, it also permits peptide fragments to be coupled
with N-terminal cysteine peptides, resulting in thiazolidine lactam scaffolds 3, in which either or
both R2 and R3 are amino acids or peptide derived. The latter case represents an example of peptide
fragment condensation.
Scheme 2. Previous synthesis of 7 (route is incompatible with unstable R2 during ozonolysis).

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Scheme 3. Preparation of alternative key intermediate 13 compatible with many R2.
The strategy used to overcome the limitation represented by Scheme 2 was to perform the
ozonolysis on the N-protected fluorenylmethyloxycarbonyl (Fmoc) derivative 11 (Scheme 3) prior to
introduction of the ozone-sensitive acyl group R2CO. The newly formed aldehyde 12 could then be
orthogonally protected as the key acetal intermediate 13
groups present in 13 can be selectively unmasked and functionalized, providing access to
ozonolytically labile compounds, and by a process compatible with Fmoc-based solid-phase peptide
synthesis (SPPS), a route to diverse unnatural peptides and peptidomimetics (Scheme 1), as well
[7
]. Now, the latent amino and aldehyde
7
with
1–5
as peptide fragment condensations. The potential for this methodology is illustrated in this report for
representative scaffolds 1 and 3.
Scaffold
1
compounds, N-acyl homoserine lactones (AHLs), have received considerable attention
compounds, such as N-acyl homoserine lactones,
over the last 10–15 years. Certain natural Scaffold
1
are known to be used by Gram-negative bacteria as signaling molecules to initiate quorum sensing [8,9].
In early efforts designed to mimic endogenous signaling lactones, non-native AHLs have been
identified as inhibitors of quorum sensing [
of biofilm formation in recent years [11–13].
Scaffold compounds, -bicyclic thiazolidine lactams, have been of particular interest and
9,10], and this has stimulated a growing search for inhibitors
3
γ
can be traced back to the years of the Second World War when the various structure–activity
relationships associated with the penicillins were being defined [14]. Subsequently, it was recognized
that fused bicyclic structures such as
Often, they adopt a -turn conformation [20
associated with various biological activities [17
3
can mimic conformationally-restricted peptides [15
22]: a secondary, reverse-turn structure that has been
19 23 24]. The replacement of dipeptide residues
–19].
β
–
–
, ,
with a conformationally-restricting bicyclic thiazolidine lactam core has found application in its
incorporation into the antimicrobial gramicidin S [25] and the hypertensive angiotensin II [26].
2. Results and Discussion
2.1. Preparation of Fmoc Acetal Intermediate Resins 13
Resin 9c (R1 = Bn) [
Scheme 3. Although amine 9c represents a potentially problematic or difficult coupling [27
virtue of its quaternary -carbon, there was no evidence in the subsequently released products of
6
] was converted to its Fmoc derivative 11c using Fmoc-Cl according to
30] by
–
α
incomplete acylation with the reactive Fmoc-Cl. The Fmoc functional group did not present any
complications during ozonolysis of 11c followed by reductive work-up [31]. The resultant aldehyde

Molecules 2018, 23, 1762
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functional group in 12c was then converted to its dimethylacetal 13c using methanol, trimethylsilyl
chloride, and trimethylorthoformate [7,32]. Similarly, 9a,b were converted to 13a,b.
2.2. Diverse Applications of Fmoc Acetal Intermediate Resins 13
2.2.1. Demonstrating Compatibility with Ozonolytically Labile R2
As a demonstration of the usefulness of resins 13a–c, examples of scaffolds 1 and 3 bearing
ozone-labile substructures are presented.
Scaffold 1
The approach to the preparation of homoserine lactone scaffolds
derivatives 19a (Scheme 4). Treatment of 13a with 20% piperidine followed by acylation of the
deprotected amine resin with 2-furoyl chloride gave the acylated acetal resins 16a . The latent
aldehyde was regenerated by a brief exposure (35 min) to aqueous trifluoroacetic acid, providing resins
17a . Reduction to the alcohol resins 18a was accomplished with sodium triacetoxyborohydride in
acetic acid, and this was followed by cyclitive cleavage at elevated temperatures in the presence of
diisopropylethylamine (DIEA, Hünig’s base) to give the desired lactones 19a bearing the ozone-labile
furan ring [33 34] in 14–25% overall yield (80–86% average yield per step over 11 steps from starting
substituted Merrifield resins).
1 is illustrated with the furanyl
–
c
–c
–
c
–
c
–c
–
c
,
Scheme 4. Introduction of the ozone-labile furan ring.
Scaffold 3
The synthesis of a scaffold
3
example featuring an ozone-sensitive group is illustrated in
Scheme 5. Fmoc acetal resin 13c was deprotected and acylated with the electron-rich, ozone-sensitive,
activated ester of the trimethoxybenzoic acid generated with 1-hydroxybenzotriazole (HOBt) and
diisopropylcarbodiimide (DIC) to give acetal resin 20c. Cyclitive cleavage (step b) at room temperature
failed, resulting in the recovery of starting material, but did proceed at elevated temperature to
give a 27% overall purified yield of 21c as a 2:1 mixture of diastereomers. The required thiazolidine
intermediate was formed through in situ acetic acid activation of the dimethylacetal and reaction with
cysteine ethyl ester.

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2.2.2. Demonstrating Compatibility with Peptide Chemistry
As a demonstration of the usefulness of resins 13a
–
c
as advanced intermediates in solid-phase
peptide synthesis and fragment condensation, examples of scaffolds
residues are presented.
1 and 3 bearing amino acid
Scheme 5. Introduction of an electron-rich aromatic ring.
Scaffold 1
Using standard Fmoc-based peptide synthetic procedures, advanced resins 13a
the opportunity to install amino acid or peptide residues into scaffold structures. Scheme 6 depicts
the successful incorporation of an N-capped amino acid residue at the N-terminus of the homoserine
lactone scaffold After deprotection of 13c, the amine was acylated using the anhydride of
–c also provide
1
1
.
Fmoc-Ala-OH. Subsequent deprotection and acylation with 4-chlorobenzoyl chloride gave the
N-capped, N-terminal alanine acetal resin 23c. Acetal hydrolysis, cyanoborohydride reduction,
and cyclitive cleavage then gave, in nearly equal quantities, the stereoisomers of 24c (54:46 ratio),
which were separated by reverse-phase chromatography to give the 3R and 3S diastereomers.
Scheme 6. Introduction of amino acid residue into Scaffold 1.

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Scaffold 3
Again, using standard Fmoc-based peptide synthetic procedures, amino acid or peptide residues
can be incorporated into advanced resins 13a at (1) the N-terminus of the bicyclic framework of
and at (2) the C-terminus of the bicyclic framework of during the bicyclization using N-terminal
cysteine peptides. This second opportunity was first fulfilled with the incorporation of a glycine
residue at the C-terminus of scaffold (Scheme 7). Following deprotection of 13c, the amine resin
–c
3,
3
3
was acylated with p-toluoyl chloride. Cyclitive cleavage using commercially available Cys-Gly-OH
at elevated temperature then afforded a 3:1 mixture of diastereomers, which when triturated with
dichloromethane and recrystallized from ethanol resulted in the isolation of a single diastereomer,
as evidenced by proton NMR spectroscopy. The structure of this stereoisomer, which was solved by
x-ray crystallography (Table 1), was found to be the 2R,5S,7S isomer
configuration pattern as the previously reported 27c (Figure 1) [ ]. Compound
first example of a fragment condensation [35 38] in which the C-terminal fragment is Cys-Gly, and the
β
-
26c; this bears the identical
β
-
6
β-26c represents our
–
N-terminal fragment is an N-acylated modified, resin-bound phenylalanine.
Scheme 7. Introduction of glycine residue into Scaffold 3.
Figure 1. Previously established stereochemistry of β-27c.

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Table 1. Crystal data and structure refinement for β-26c (Reciprocal Net report 08134).
Empirical formula
C25H28N3O5.50
490.56
S
Formula weight
Crystal color, shape, size
Temperature
colorless block, 0.25 × 0.22 × 0.17 mm3
150(2) K
Wavelength
0.71073 Å
Crystal system, space group
Monoclinic, P2(1)
◦
α = 90
a = 9.222 Å
b = 27.417 Å
c = 9.552 Å
◦
β = 91.73
Unit cell dimensions
◦
γ = 90
2414.0 Å3
4
Volume
Z
Density (calculated)
Absorption coefficient
F(000)
1.350 Mg/m3
−1
0.178 mm
1036
Data collection
Diffractometer
Theta range for data collection
Index ranges
APEX II Kappa Duo, Bruker
◦
1.49 to 28.39
−12 ≤ h ≤ 11, −36 ≤ k ≤ 27, −12 ≤ l ≤ 12
Reflections collected
Independent reflections
Observed Reflections
22656
10036 [R(int) = 0.0428]
8608
◦
Completeness to theta = 28.39
99.5%
Solution and Refinement
Absorption correction
Max. and min. transmission
Solution
Semi-empirical from equivalents
0.9704 and 0.9568
Direct methods
Full-matrix least-squares on F2
Refinement method
Weighting scheme
2
2
w = [σ2Fo + AP2 + BP]−1, with P = (Fo + 2 Fc2)/3, A =, B =
Data/restraints/parameters
Goodness of fit on F2
Final R indices [I > 2sigma(I)]
R indices (all data)
10036/259/638
1.044
R1 = 0.0404, wR2 = 0.0889
R1 = 0.0522, wR2 = 0.0946
0.04 (5)
Absolute structure parameter
Largest diff. peak and hole
2
−3
0.319 and −0.320 e.Å
Goodness-of-fit = [
Σ
[w(Fo
−
Fc2)2]/Nobservns
−
Nparams)]1/2, all data. R1 =
Σ
(|Fo|
−
|Fc|)/Σ |Fo|.
2
wR2 = [Σ[w(Fo − Fc2)2]/Σ [w(Fo2)2]]1/2
.
As demonstrated with the two examples in Schemes 5 and 7, cyclitive cleavage of dimethylacetal
resins can be exe◦cuted without initial conversion to the aldehyde resins. However, elevated
temperatures (90 C) were required. We later sought milder cyclitive cleavage conditions in an
effort to improve upon the diastereoselectivity. Consequently, all of the subsequent examples reported
below illustrate the cyclitive cleavage to Scaffold 3 compounds occurring from the aldehyde resins.
Compound 30b (Scheme 8) serves as an example of a scaffold
3 structure bearing alanine
residues at both the R2 and R3 positions, and represents another fragment coupling illustration
(dipeptide + dipeptide). The required Cys-Ala-OMe 32 was prepared using the mixed anhydride
method (Scheme 9). Originally 31, which is the precursor to 32, was prepared by a solid-phase
synthetic route in which cleavage from Wang resin was carried out in methanol/triethylamine at
◦
55–60 C for 42 h. However, concern for epimerization resulting from prolonged exposure to mild base
at elevated temperature was substantiated as 31, which when prepared by this method was found to be
a 95/5 mixture of diastereomers. When 31 was prepared by the mixed anhydride method (Scheme 9),
it was stereochemically pure.
2.2.3. Variation of R1, R2, and YR3 of Scaffold 3 Structures
Variations of the three substituents R1, R2, and YR3 of scaffold
and include R1 = H, Me, and Bn; R2 = aryl and aroyl-Ala; YR3 = NH-Gly-OH, NH-Leu-OMe,
3 are defined in Table 2

Molecules 2018, 23, 1762
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NH-Ala-OMe, and OEt. The synthetic routes used to prepare these compounds are described in
Schemes 10–12. Bicyclic thiazolidine lactams with ozone-incompatible groups are represented in
Scheme 10 (amino acid residues present) and Scheme 11 (acyl groups R2CO present), whereas
3
3,
with ozone-compatible R2CO, was prepared using Scheme 12. As Scheme 11 indicates, the bicyclic
thiazolidine lactam targets can be accessed in a single step (step d) from acetal resin 15 (vide supra).
If milder conditions are required, the two-step process (steps b and c) allows cyclitive cleavage to
proceed at slightly lower temperatures. Table 2 compounds are listed below each target structure in
the three schemes.
Scheme 8. Fragment condensation of an Fmoc acetal resin with Cys-Ala-OMe.
Scheme 9. Preparation of stereochemically pure Cys-Ala-OMe.
Scheme 10. Synthesis of Scaffold 3 compounds with ozone-incompatible groups and amino acid residues.

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Scheme 11. Synthesis of Scaffold 3 = amino acid residues.
compounds with ozone-incompatible groups and R2
Scheme 12. Synthesis of Scaffold 3 compounds with ozone-compatible R2.

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Table 2. Bicyclic Lactams 3.
%Overall
Yield (Steps)
29 (10)
Entry Cmpd
R1
R2
YR3
Cleavage Method
dr (β/α) a
∆δ (ppm)
R1
1
β-27c
α-27c
α-33a
β-33a
β-33b
α-33b
β-33c
α-33c
β-26c
β-34a
α-34a
α-35a
β-35a
β-35b
α-35b
β-35c
α-35c
β-36a
α-36a
β-36b
α-36b
α-36c
β-36c
α-30b
β-30b
β-30c
α-30c
α-37b
β-37b
benzyl
benzyl
H
H
Me
4-MePh
4-MePh
4-ClPh
4-ClPh
4-ClPh
4-ClPh
4-ClPh
4-ClPh
4-MePh
4-FPh
4-FPh
4-ClPh
4-ClPh
4-ClPh
4-ClPh
4-ClPh
4-ClPh
4-ClPh-Ala
4-ClPh-Ala
4-ClPh-Ala
4-ClPh-Ala
4-ClPh-Ala
4-ClPh-Ala
4-NO2Ph-Ala NH-Ala-OMe
4-NO2Ph-Ala NH-Ala-OMe
4-NO2Ph-Ala NH-Ala-OMe
4-NO2Ph-Ala NH-Ala-OMe
OEt
OEt
OEt
OEt
OEt
OEt
OEt
OEt
A
A
C
C
B
E
F
F
80:20
0.69
0.24
0.37
1.24
0.36
0.59
0.71
0.23
0.26 b
1.21
0.09
0.09
1.21
0.26
0.54
0.58
0.06
1.07
0.08
0.16
0.52
0.00
0.45
0.52
0.17
0.47
0.00
0.51
0.16
β
α
α
β
β
α
β
α
β
β
α
α
β
β
α
β
α
β
α
β
α
α
β
α
β
β
α
α
β
2
3
4
5
6
7
8
9
49:51
87:13
87:13
8 (11)
8 (11)
5 (11)
4 (11)
13 (11)
1 (11)
7 (10)
17 (5) c
15 (5)
6 (11)
4 (11)
9 (11)
9 (11)
8 (11)
3 (11)
4 (13) d
3 (13)
4 (13)
4 (13)
2 (13)
1 (13)
9 (13)
7 (13)
4 (13)
2 (13)
3 (11)
4 (11)
Me
benzyl
benzyl
benzyl
H
H
H
H
Me
Me
benzyl
benzyl
H
H
Me
NHCH2CO2H
NH-Leu-OMe
NH-Leu-OMe
NH-Ala-OMe
NH-Ala-OMe
NH-Ala-OMe
NH-Ala-OMe
NH-Ala-OMe
NH-Ala-OMe
NH-Ala-OMe
NH-Ala-OMe
NH-Ala-OMe
NH-Ala-OMe
NH-Ala-OMe
NH-Ala-OMe
A
C
C
D
D
D
D
D
D
D
D
D
D
D
D
D
D
D
D
D
D
70:30
41:59
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
55:45
52:48
66:34
60:40
59:41
58:42
58:42
66:34
62:38
Me
benzyl
benzyl
Me
Me
benzyl
benzyl
Me
Fmoc-Ala
Fmoc-Ala
NH-Ala-OMe
NH-Ala-OMe
Me
a
Material released from resin was collected after 24 h at rt, then after successive 24 h/55 ◦C periods.
The diastereomeric ratio (dr) cited is that from the most productive, the first 24 h/55 ◦C period;
is defined
as R1 “up” (wedge bond), b in methanol-d4, c number of steps from Boc-Gly(allyl)-Merrifield, d an additional 3%
was obtained as a mixture of the two diastereomers. Method A: Acetal resin 25c, 2.0–2.5 equiv. Cys(OEt) HCl or
Cys-Gly, 5–7 equiv.KOAc, acetic acid, 90 ◦C/24 h. Method B: Aldehyde resin (R1 = Me, R2 = 4-ClC6H5), 2.0 equiv.
Cys(OEt)
HCl, 6 equiv KOAc, AcOH, rt/24h, filter then replace with PhCl/6 equiv DIEA/55–80 ◦C/88 h. Method
C: Aldehyde resin HCl or Cys-AA-OMe HCl or
(R1 = H, R2 = 4-ClC6H5 or 4-FC6H5), 0.8–0.9 equiv. Cys(OEt)
TFA, 1.4–1.7 equiv poly-4-vinylpyridine (PVP), AcOH/rt-55 ◦C/66 h. Method D: Aldehyde resin
, 0.9–1.3 equiv.
Cys(OEt) HCl or Cys-AA-OMe
TFA, 6 equiv KOAc, AcOH, rt then 55 ◦C. Method E: Conditions of Method B
followed by methoxide cleavage (22 equiv sodium methoxide in methanol/THF/3 h) to give the acid which was
β
·
7
·
7
·
·
7
·
·
then esterified (DBU, EtI/THF). Method F: Aldehyde resin
7
(R1 = Me, R2 = 4-ClC6H5), 2.0 equiv. Cys(OEt)
HCl,
·
6 equiv KOAc, AcOH, rt/24 h, filter then replace with PhCl/6 equiv DIEA/55 ◦C/24 h, filter then replace with
AcOH/75–90 ◦C/66 h.
Preparation of all the above scaffold
3 compounds and the others presented in Table 2 from Fmoc
acetal resins 13a resulted in the formation of two predominating diastereomers in each case, and their
–c
diastereomeric ratios (dr) are given. The isolation of two major stereoisomers is a consequence of
the achiral alkylation of 39; this introduces the allyl side chain, which is the precursor to the acetal
moiety of 13a–c (Scheme 13). Although two additional diastereomers were observed in cases where
R1 = H and Me, they accounted for less than 10% of the product mixture. These two additional
diastereomers are not represented in Table 2, and no effort was made to isolate and characterize
them. Standard reverse-phase chromatography was used to separate the diastereomers. The overall
yields (1–29%) from the commercially available starting resins, based on advertised loadings, are also
provided with the number of steps (5–13) recorded in parentheses. The overall yields represent an
average of 76–87% per step.

Molecules 2018, 23, 1762
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Scheme 13. Origin of racemic stereochemistry at C-terminal α-carbon of 13.
Similar to the case with the preparation of 22c (Scheme 6), when the R1 of 13 is methyl, coupling
with Fmoc-Ala-OH using hexafluorophosphate benzotriazole tetramethyl uronium (HBTU) was
found to be incomplete, resulting in the isolation of a small amount of the deletion peptidomimetic
35b along with the desired 36b (Scheme 14). Problematic coupling was also observed with
13c (R1 = Bn). These two examples (R1 = Me and Ph) represent difficult couplings [27
in peptide synthesis in which one or both fragments possess a dialkylated -carbon adjacent
to their amine and carboxyl reaction centers such as the aminoisobutyryl (Aib) moiety [28 30].
–30]
α
–
Such couplings, for example, have been carried out most efficiently using the highly-reactive
Fmoc acid fluorides [39], urethane-protected amino acid N-carboxyanhydrides (UNCAs) [40],
and symmetrical anhydrides [41–44] as the activated carboxyl contributor. Use of the coupling
reagents hexafluorophosphate azabenzotriazole tetramethyl uronium (HATU) [29], the benzotriazole
(1H-1,2,3-benzotriazol-1-yloxy)-tris(pyrrolidino)-phosphonium hexafluorophosphate (PyBOP) [30],
and the phosphonium bromide bromo-tris(pyrrolidino)-phosphonium hexafluorophosphate
(PyBroP) [30] have also led to efficient coupling involving the highly-hindered Aib. Four methods
were explored in the search for a more efficient method to couple Fmoc-Ala-OH with 13b
(a) Fmoc-Ala-OH/HBTU/diisopropylethylamine [41 42]; (b) Fmoc-Ala-OH/HOBt/DIC [41 43]; (c) the
symmetrical anhydride [41 44] (Fmoc-Ala)2O and (d) the acid chloride [41] Fmoc-Ala-Cl [45]. Use of
,c:
,
,
,
the symmetrical anhydride (method c) was found to be the superior method in this application.
Scheme 14. Problematic coupling leading to the deletion of peptide 35b.
2.3. Aspects of Bicyclic Thiazolidine Lactam NMR Spectra
2.3.1. Use of the Nuclear Overhauser Enhancement to Assign Stereochemistry
It is assumed that epimerization at the alpha carbon originating with the cysteine reagent
had not taken place under the mildly acidic conditions for cyclitive cleavage [46]. To determine

Molecules 2018, 23, 1762
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the configurations at the other two stereocenters of
3
, both one-dimensional and two-dimensional
(2D-NOESY) nuclear Overhauser enhancement (nOe) experiments were performed. The results from
diastereomers 30b and 30b are illustrative. Irradiation of the methyl protons (H-18) of 30b
α-
β
-
β-
resulted in the enhancement of the bridgehead proton H-10, and the enhancement was reciprocated
upon irradiation of H-10 (Figure 2). In addition, the irradiation of H-10 gave an enhancement at amide
proton H-21. These enhancements were observed in both the one-dimensional and two-dimensional
experiments, and are consistent with the methyl group H-18, the bridgehead proton H-10, and the
amide proton H-21 oriented syn to one another. In contrast, no nOe to H-10 is observed upon irradiation
of the methyl group of α-30b, nor in the reverse direction. Also, for α-30b, reciprocal enhancements
are observed between protons H-10 and H-22 upon irradiation of either proton establishing their syn
relationship to one another.
Figure 2. Selected Nuclear Overhauser enhancement (nOe) results from irradiation of diastereomeric 30b
.
2.3.2. Identification of Chemical Shift Differences to Assign Stereochemistry
Table 3 compiles the chemical shifts for the ring fusion proton and the two diastereotopic protons
on the adjacent carbon atom (labeled as Hx, Hy, and Hz, respectively) for the Scaffold 3 compounds
described in Table 2, and illustrates a predictive aspect of stereoisomer assignment. The compounds are
arranged in three groups according to their R1 substituent (H, Me, or Bn). Also provided as ∆δyz (ppm)
is the difference in chemical shift between the two diastereotopic protons of each pair of diastereomers.
Listed next to this column is the nOe-assigned stereochemistry of the R1 group, which is described
as
Comparison of the R1 = benzyl series shows without exception that the ∆δ is significantly larger for
the isomer. The ∆δ is even larger with the isomer
isomer for the R1 = H series; however, it is the
α β
(R1 anti to cysteine carbonyl group or down) or (R1 syn to cysteine carbonyl group or up).
β
β
α
in the R1 = Me series that displays the larger ∆δ values. Baldwin et al. [47] observed the same trend in
the R1 = H series with diastereomers 42 and 43 (Figure 3), with the
β isomer 43 showing the larger ∆δ
value between the chemical shifts of Ha and Hb.
Figure 3. α and β diastereomers 42 and 43.

Molecules 2018, 23, 1762
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2.3.3. Use of the Nuclear Overhauser Enhancement to Assess Molecular Shape
Table 3. Chemical shifts of Hx, Hy, and Hz and the chemical shift differences between Hy and Hz in
diastereomeric pairs.
R1 δ (Hx)
δ (Hy)
δ (Hz)
∆δ
Isomer
yz
α-33a
β-33a
α-34a
β-34a
α-35a
β-35a
α36a
5.24
5.21
5.31
5.11
5.32
5.13
5.29
5.06
5.34
5.21
5.26
5.13
5.26
5.11
5.26
5.15
5.21
5.06
5.39
4.75
5.25
4.25
5.23
4.09
2.49
3.35
2.56
3.35
2.56
3.35
2.48
3.14
3.10
3.03
2.88
2.99
2.79
2.79
2.80
2.82
2.75
2.78
2.93
3.32
2.62
3.27
2.54
3.02
2.56
3.06
2.86
2.11
2.65
2.14
2.65
2.14
2.56
2.07
2.51
2.67
2.34
2.73
2.26
2.63
2.28
2.65
2.24
2.62
2.71
2.61
2.68
2.69
2.54
2.58
2.56
2.59
0.37
1.24
0.09
1.21
0.09
1.21
0.08
1.07
0.59
0.36
0.54
0.26
0.52
0.16
0.52
0.17
0.51
0.16
0.22
0.71
0.06
0.58
0.00
0.44
0.00
0.47
α
β
α
β
α
β
α
β
α
β
α
β
α
β
α
β
α
β
α
β
α
β
α
β
α
β
H
H
H
H
β-36a
α-33b
β-33b
α-35b
β-35b
α-36b
β-36b
α-30b
β-30b
α-37b
β-37b
α-33c
β-33c
α-35c
β-35c
α-36c
β-36c
α-30c
β-30c
Me
Me
Me
Me
Me
Bn
Bn
Bn
5.25
4.14
Bn
The
when suitably substituted with amino acid residues, the resulting peptidomimetic can adopt
-turn secondary structure [20 22]. Typically, this reverse-turn involves a tetrapeptide segment
γ-bicyclic thiazolidine lactam core imparts conformational restriction [15–19] and
a
β
–
and is observed in optical rotatory dispersion (ORD) and circular dichroism (CD) spectra.
From one-dimensional nOe analyses of the diastereomers of 30b, a small enhancement (0.1%) was
observed at the aromatic protons H-8 and H-9 upon irradiation of the methyl protons, H-13, of the
α
-isomer,
upon irradiation of H-8. No such enhancements were observed with
with an approach of the termini of 30b, and may reflect the adoption of a
by this stereoisomer in chloroform solution.
α
-
30b (Figure 4). A reciprocated enhancement was observed at the ester methyl protons
30b. These results are consistent
-turn secondary structure
β
-
α-
β

Molecules 2018, 23, 1762
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Figure 4. Remote nOe at H-8 and H-9 from irradiation at H-13 of α-30b.
3. Materials and Methods
All of the reagents were purchased from Acros Organics (Geel, Belgium), Advanced Chemtech
(Louisville, KY, USA), EMD Millipore (Burlington, MA, USA), or Sigma-Aldrich Chemical (St. Louis,
MO, USA), and were used without further purification. Boc-protected amino acids on Merrifield resin
were purchased from Polymer Laboratories, Amherst, MA, USA (now Agilent Technologies, Santa Clara,
California, USA). Solid-phase peptide synthesis (SPPS) vessels were purchased from Chemglass (Vineland,
NJ, USA). 1H-NMR and 13C-NMR spectra were recorded on a Bruker Avance III 500 and 400 spectrometers
(1H, 500.13 MHz; 13C, 125.76 MHz) (Billerica, MA, USA) using tetramethylsilane as internal standard
in CDCl3 or unless otherwise indicated. Chemical shifts are reported in parts per million (ppm,
δ
units). Coupling constants, J, are reported in Hertz (Hz). Splitting patterns are designated as s:
singlet, br s: broad singlet, d: doublet, dd: doublet of doublets, ddd: doublet of doublets of doublets,
q: quartet, and m: multiplet. High-resolution mass spectra (HRMS) were obtained using a Thermo
Electron Corporation MAT 95XP-Trap (Waltham, MA, USA) or an Agilent 1200 HPLC-6130 MSD Mass
Spectrometer (Santa Clara, CA, USA) in the electrospray ionization (ESI) or in the chemical ionization (CI)
modes at the Mass Spectrometry Facility of Indiana University, Bloomington, IN. High performance liquid
chromatography-mass spectrometry (HPLC/MS) was performed on an Agilent 1100 Series LC/MSD
(Santa Clara, CA, USA) in the electrospray ionization positive mode using a 4.6
×
150 mm Agilent Eclipse
XDB five-micron C18 reverse-phase column. Nuclear Overhauser enhancement (nOe) 1-D difference
spectroscopy was conducted using the Bruker pulse sequence SELNOGP (Billerica, MA, USA) with the
following acquisition parameter changes: Spectral width, SW = 10.0 ppm, Transmitter frequency offset,
O1P = 4.0 ppm, Duration delay, D[8] = 0.6 s.
α-Allyl-α-R1-N-(fluorenylmethyloxycarbonyl)glycine on Merrifield resins (11a–c, R1 = H, Me,
Bn). The amine resin 9a
was washed with 4 10 mL of N-methyl pyrrolidinone (NMP). To the resin was then added 40 mL of
–c 3], contained in a 500-mL SPPS vessel,
(R1 = H, Me, Bn, 7.60–8.50 mmol) [
×
NMP, followed by 3.8 equivalents of diisopropylethylamine (DIEA). The vessel was swirled gently to
mix the contents, and 3.0 equivalents of Fmoc chloride in 60–70 mL of NMP was added in one portion.
The vessel was rocked on an orbital shaker and after 24 h the vessel was drained, and the resin was
washed 3
×
45 mL each with NMP, 1:1 THF:EtOH, THF, and dichloromethane (DCM) to give resins
11a–c (R1 = H, Me, Bn). The resin was then dried under a slow stream of dry nitrogen gas overnight.

Molecules 2018, 23, 1762
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α-(2-Oxoethyl)-α-R1-N-(fluorenylmethyloxycarbonyl)glycine on Merrifield resins (12a–c,
R1 = H, Me, Bn). A 250-mL, three-neck, round-bottomed flask was charged with 1.80–3.20 mmol of
resin 11a–c
(R1 = H, Me, Bn), a trace of Sudan III red dye, and 40 mL of DCM under dry argon gas.
The contents were cooled in a dry ice/acetone bath, and the argon flow was replaced with a subsurface
flow of oxygen gas at a rate of 0.6–0.8 L/min. Ozonolysis using an ozone generator was performed at
a current of 1.0 ampere until the red dye was rendered colorless (2–3 h). The current was then reduced to
zero while oxygen bubbling continued for 10 min. Diethyl sulfide (1.0 mL, 9.3 mmol) was added, and the
solution was allowed to gradually warm to room temperature and stir overnight. The resin was collected
by filtration into a 50-mL SPPS vessel rinsing over with DCM and THF. The resin was dried under a slow
stream of dry argon gas and then under high vacuum to give 12a–c (R1 = H, Me, Bn).
α-(2,2-Dimethoxyethyl)-α-R1-N-(fluorenylmethyloxycarbonyl)glycine on Merrifield resins
(13a–c, R1 = H, Me, Bn). To 1.80–3.20 mmol of resin 12a (R1 = H, Me, Bn) contained in a 50-mL
–c
SPPS vessel, 12–28 mL of absolute methanol, 4.4 equivalents of 1.0 M trimethylsilyl chloride
in THF, and 25 equivalent of trimethylorthoformate were added under dry argon. The vessel
was rocked overnight, and was then drained to leave a small volume of liquid over the resin.
Diisopropylethylamine in absolute methanol (30 mL of a 3% v/v solution) was added. After shaking
for 15 min, the vessel was drained, and the resin was washed with 3
×
20 mL of 3% DIEA in MeOH,
3
× 15 mL of NMP, and 8 × 15 mL of DCM and was dried overnight under a slow stream of dry
nitrogen gas and then under high vacuum to give resin 13a–c (R1 = H, Me, Bn).
2-(Furan-2-carboxamido)-4,4-dimethoxy-2-R1-butanoic acid on Merrifield resins (16a–c,
R1 = H, Me, Bn). Fmoc acetal resin 13a
–
c
(R1 = H, Me, Bn, 100–157
µ
mol) contained in a 3.5-mL
or 5-mL SPPS vessel was treated with 20% piperidine in NMP, and the vessel was gently agitated for
40–50 minutes at room temperature. The vessel was drained, and the resin was washed with 5 2–3 mL
×
of NMP. To the deprotected resin was then added 5.2 eq of a 0.50-M solution of diisopropylethylamine
(DIEA) in NMP, followed by 4.4 eq of a 0.50 M solution of 2-furoyl chloride in NMP. After 16–18 h,
the vessel was drained, and the resin was washed twice each with NMP, 1:1 THF/MeOH (EtOH),
and THF, and four times with DCM to give resin 16a–c (R1 = H, Me, Bn).
2-(Furan-2-carboxamido)-2-R1-4-oxobutanoic acid on Merrifield resins (17a–c, R1 = H, Me, Bn).
To 100–157 µmol of DCM-swelled resin 16a–c
(R1 = H, Me, Bn), 3 mL of TFA:DCM:water (4:4:1) was
added. The mixture was gently agitated for 35 min at room temperature. The SPPS vessel was drained,
and the resin was washed with 3 × 2 mL each with DCM and then THF to give aldehyde resin 17a–c
(R1 = H, Me, Bn).
2-(Furan-2-carboxamido)-4-hydroxy-2-R1-butanoic acid on Merrifield resins (18a–c, R1 = H,
Me, Bn). To 100–157 µmol of resin 17a–c
(R1 = H, Me, Bn), 6 eq of a 0.90-M solution of sodium
cyanoborohydride in 0.50 M of acetic acid in THF (for R1 = H, Me) or 7.5 eq of a suspension of sodium
triacetoxyborohydride in 0.50 M of acetic acid in THF (for R1 = Bn) was added. The mixture was gently
agitated for 6 h at room temperature. The vessel was drained, and the resin was washed with 3 × 2 mL
each with THF, 30% aqueous THF, and THF to give the hydroxyl resin (18a–c, R1 = H, Me, Bn).
N-(3-R1-2-oxotetrahydrofuran-3-yl)furan-2-carboxamide (19a–c, R1 = H, Me, Bn). Resin 18a
–c
(100–157
µ
mol, R1 = H, Me, Bn) was washed with 2
×
2 mL of chlorobenzene, and was t◦hen treated
with 2 mL of chlorobenzene followed by 8 eq of DIEA. The mixture was heated at 75 C for 16 h.
After cooling, the vessel was drained, and the resin was washed with 2
×
2 mL of DCM. The combined
filtrates were evaporated to dryness to give a residue that was purified by silica gel chromatography:
N-(2-oxotetrahydrofuran-3-yl)furan-2-carboxamide (19a, R1 = H), dichloromethane and
dichloromethane/ethyl acetate mobile phases (94/6, 9/1); 5.7 mg (19% over nine steps) of 19a as
a white solid; 1H-NMR
δ 2.28 (dq, J = 11.5 and 8.9 Hz, 1H), 2.92 (dddd, J = 12.6, 8.6, 5.8, and 1.0 Hz,
1H), 4.34 (ddd, J = 11.2, 9.2, and 5.8 Hz, 1H), 4.52 (dt, J = 9.7 and 0.8 Hz, 1H), 4.74 (ddd, J = 11.7, 8.6,
and 6.3 Hz, 1H), 6.52 (dd, J = 3.5 and 1.8 Hz, 1H), 6.89 (br d, J = 3.9 Hz, 1H), 7.16 (dd, J = 3.5 and 0.5 Hz,
1H), 7.47 (dd, J = 1.6 and 0.6 Hz, 1H); 13C-NMR
δ 30.6, 48.1, 66.1, 112.3, 115.3, 144.6, 146.9, 158.6, 175.1;

Molecules 2018, 23, 1762
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HRMS (TOF ES+) m/z calculated for C9H9NO4 (M + Na) 218.0429; found 218.0429 (As shown in the
Supplementary Materials).
N-(3-methyl-2-oxotetrahydrofuran-3-yl)furan-2-carboxamide (19b, R1 = Me), dichloromethane
and dichloromethane/ethyl acetate mobile phases (9/1, 8/2) as mobile phases; 5.2 mg (25% over nine
steps) of 19b as a white solid; 1H-NMR
δ
1.63 (s, 3H), 2.54 (ddd, J = 13.0, 7.0, and 2.3 Hz, 1H), 2.83 (dt,
J = 12.9 and 9.7 Hz, 1H), 4.32 (dt, J = 9.6 and 7.0 Hz, 1H), 4.56 (dt, J = 9.3 and 2.3 Hz, 1H), 6.51 (dd,
J = 3.5 and 1.7 Hz, 1H), 6.75 (br s, 1H), 7.13 (d, J = 3.5 Hz, 1H), 7.46 (d, J = 1.6 Hz, 1H); 13C-NMR
δ 22.5,
35.1, 55.7, 65.6, 112.3, 115.1, 144.3, 147.2, 157.7, 177.3; HRMS (TOF ES+) m/z calculated for C10H12NO4
(M + H) 210.0761; found 210.0762.
N-(3-benzyl-2-oxotetrahydrofuran-3-yl)furan-2-carboxamide (19c, R1 = Bn), dichloromethane
and dichloromethane/ethyl acetate (96/4, 9/1) as mobile phases; 4.3 mg (14% over nine steps) of 19c
as a white film; 1H-NMR
δ 2.74–2.79 (m, 2H), 3.23 (d, J = 13.2 Hz, 1H), 3.29 (d, J = 13.2 Hz, 1H), 3.49 (dt,
J = 9.2 and 7.6 Hz, 1H), 4.31 (dt, J = 8.9 and 3.2 Hz, 1H), 6.51 (dd, J = 3.5 and 1.7 Hz, 1H), 6.83 (br s, 1H),
7.14 (dd, J = 3.5 and 0.7 Hz, 1H), 7.26–7.28 (m, 2H), 7.32–7.35 (m, 3H), 7.45 (dd, J = 1.6 and 0.7 Hz, 1H),
13C-NMR
δ 33.0, 42.0, 59.8, 65.9, 112.3, 115.2, 127.9, 128.9, 130.0, 133.6, 144.4, 147.2, 157.7, 179.8; HRMS
(TOF ES+) m/z calculated for C16H15NO4Na (M + Na) 308.0899; found 308.0907.
2-Benzyl-4,4-dimethoxy-2-(3,4,5-trimethoxybenzamido)butanoic acid on Merrifield resin (20c).
The Fmoc acetal resin 13c (320 mg, 0.298 mmol) was placed in a 5-mL SPPS vessel, washed with DMF
(2 × 3 mL), and then slowly washed with 20% piperidine/DMF (6
×
4 mL
×
5 min), then with DMF
(4 × 4 mL), with CH2Cl2 (3
×
4mL), and again with DMF (3 4 mL). The vessel was drained with
×
a stream of argon, and the resin was treated with a freshly prepared solution of 3,4,5-trimethoxybenzoic
acid (191 mg, 0.9 mmol, 3 eq) and HOBt-H2O (122 mg, 0.9 mmol, 3 eq) in 3 mL of a 1:1 CH2Cl2-DMF
mixture. This was followed by the addition of diisopropylcarbodiimide (DIC, 140
and DIEA (78 L). The vessel was capped and rotated at room temperature (RT) for 24 h. The solution
was then drained from the vessel, and the resin was washed with DMF (3 4 mL), and CH2Cl2
µL, 114 mg, 0.9 mmol)
µ
×
(4 × 4 mL), and then dried with a stream of argon for 5 min to afford resin 20c.
Ethyl(3R,6R,S,7aS)-6-benzyl-5-oxo-6-(3,4,5-trimethoxybenzamido)hexahydropyrrolo[2,1-b]-
thiazole-3-carboxylate (21c). To 0.298 mmol of resin 20c, a solution of KOAc (147.5 mg, 1.5 mmol,
5 eq) and L-Cys-OEt-HCl (111.4 mg, 0.6 mmol, 2 eq) in glacial AcOH (3 mL) was added. The vessel
◦
was capped, rotated for 15 min, and then transferred to an oven preheated to 90 C. After 24 h,
the mixture was cooled to RT, the solution was drained from the resin, and the resin was washed
with THF (2 × 3 mL) and then with CH2Cl2 (3
×
3 mL). The combined filtrates were transferred to
a separatory funnel containing brine (50 mL), water (50 mL), and CH2Cl2 (60 mL). After extraction,
the phases were separated, and the organic phase was extracted with 10% KHCO3/H2O (80 mL).
The organic layer was dried over Na2SO4, filtered, and concentrated in vacuo to afford a yellow oil
(206 mg). This sample was purified by flash chromatography on silica gel (4.5 g; 45% EtOAc/hexanes).
This gave the product diastereomeric thiazolidines 21c as a colorless solidifying oil (41 mg; 27%).
A portion (12.5 mg) was chromatographed on a 500-mg HyperSep SI column of silica gel to give
4.6 mg of 21c as a 2:1 mixture of
2.64, (dd, 0.65H, J = 13.3 Hz and 7.3 Hz), 2.71 (dd, 0.37H, J = 14.6 Hz and 2.2 Hz), 2.93 (dd, 0.39H,
J = 14.6 Hz and 7.7 Hz ), 3.21–3.49 (m, 5.14H), 3.86–3.87 (s, 3H), 3.86–3.87 (s, 2 3H), 4.20–4.31 (m, 2H),
β:α δ 1.31 and 1.32 (2t, 3H, J = 7.2 Hz and 7.2 Hz,),
; 1H-NMR (CDCl3)
×
4.78 (t, 0.68H, J = 6.9 Hz), 5.10 (dd, 0.38H, J = 8.1 Hz and 3.7 Hz 1H), 5.14 (dd, 0.64H, J = 7.2 Hz and
3.7 Hz), 5.41 (dd, 0.36H, J = 7.7 Hz and 2.2 Hz), 6.44 (bs, 0.39H), 6.60 (bs, 0.60H), 6.90 (s, 0.73H), 6.92
(s, 1.26H), 7.23–7.39 (m, 5.05H); 13C-NMR (CDCl3)
δ 14.1, 14.2, 33.7, 35.4, 36.5, 41.7, 42.7, 42.9, 56.3, 56.4,
58.2, 58.4, 60.9, 62.06, 62.09, 63.0, 63.1, 65.4, 77.2, 104.47, 104.53, 127.5, 127.8, 128.5, 128.9, 129.3, 130.4,
130.6, 134.6, 135.0, 141.4, 153.20, 153.24, 166.1, 166.4, 168.8, 169.3, 173.0, 174.6. The diastereomers were
separated on a 2.1
35% 0.1% formic acid, and 5% acetonitrile at a flow rate of 0.3
×
100 mm Agilent Eclipse XDB-C18 column using the mobile phase 65% MeOH,
L/min, and their accurate masses
µ
were determined: 5.19 min, calculated for C26H31N2O7S (M + H) 515.1846; found 515.1848; 6.07 min,
calculated for C26H31N2O7S (M + H) 515.1846; found 515.1848.

Molecules 2018, 23, 1762
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2-((S)-2-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)propanamido)-2-benzyl-4,4-dimethoxybutanoic
acid on Merrifield resin (22c). Resin 13c (87.1 mol) was treated with 2.5 mL of 20% piperidine in
NMP for 30 min with gentle agitation. The vessel was drained, and the deprotected resin was washed
with 5 3 mL of NMP. To this resin 262 mg (5.00 eq) of Fmoc-Ala anhydride in NMP in 3 mL of NMP
µ
×
was added. After 48 h, the vessel was drained, and the resin was washed with 4
×
3 mL each of NMP,
THF, and NMP to give resin 22c.
2-Benzyl-2-((S)-2-(4-chlorobenzamido)propanamido)-4,4-dimethoxybutanoic acid on Merrifield
resin (23c Resin 22c (87.1 mol) was treated with 2.5 mL of 20% piperidine in NMP for 30 min with gentle
agitation. The vessel was drained, and the deprotected resin was washed with 6 3 mL of NMP. The resin
was then treated with 4.4 eq of 1.0 M solutions of 4-chlorobenzoyl chloride followed by 5.2 eq of DIEA.
).
µ
×
After 18 h, the vessels were drained, and the resin was washed with 4
THF, and DCM to give resin 23c.
×
3 mL each of NMP, 1:1 THF:MeOH,
N-((2S)-1-((3-Benzyl-2-oxotetrahydrofuran-3-yl)amino)-1-oxopropan-2-yl)-4-chlorobenz-amide
(24c). Resin 23c (87.1 mol) was treated with 3 mL of 4:4:1 TFA:DCM:water for 35 min with gentle
agitation. The vessel was drained, and the resin was washed with 5 3 mL of DCM and 3 3 mL of
µ
×
×
THF to afford the aldehyde resin. The resin was then treated with 3 mL of 0.50 M acetic acid in THF for
10–15 min. The vessel was drained, and the resin was treated with 1 mL of 0.50 M acetic acid in THF
followed by 1.04 mL (6.3 eq) of a 0.53-M solution of sodium cyanoborohydride in 0.50 M of acetic acid
in THF. The vessel was gently agitated for 6 h at room temperature; then, it was drained, and the resin
was washed with 3
×
3 mL each of THF, 30% aqueous THF, and 4
×
3 mL of THF. The resin was dried
under a stream of nitrogen gas, and was then washed with 3 mL of chlorobenzene. Chlorobenzene
(3 mL) was then added, followed by 7.9 eq (690
◦
µmol) of DIEA. The resin was then heated at 75 C for
16 h. After cooling, the vessel was drained, and the filtrate was evaporated to dryness to yield 4.6 mg
of crude 24c. Separation of the diastereomers by reverse-phase HPLC chromatography gave 1.4 mg of
1
the stereoisomer with the earlier retention time: H-NMR (methanol-d4)
δ 1.49 (d, 3H, J = 7.2 Hz), 2.51
(ddd, 1H, J = 13.3 Hz, 8.2 Hz, and 3.0 Hz), 2.65–2.73 (m, 2H), 3.10 (d, 1H, J = 13.0 Hz), 3.15–3.21 (m,
1H), 3.20 (d, 1H, J = 13.0 Hz), 4.18 (ddd, 1H, J = 10.4 Hz, 8.9 Hz, and 3.0 Hz), 4.57 (q, 1H, J = 7.2 Hz),
7.30–7.35 (br m, 5H), 7.49 (d, 2H, J = 8.7 Hz), 7.87 (d, 2H, J = 8.8 Hz); 13C-NMR (methanol-d4)
δ 16.2,
31.0, 41.5, 49.3, 60.0, 65.1, 127.4, 128.28, 128.33, 128.9, 130.0, 132.4, 133.6, 137.6, 167.6, 173.2, 177.7;
HRMS (ES+) m/z calculated for C21H21ClN3O4S (M + Na) 423.1082; found 423.1085, and 1.5 mg
1
of the stereoisomer with the later retention time: H-NMR (CDCl3)
δ 1.46 (d, 3H, J = 7.0 Hz), 2.59
(ddd, 1H, J = 13.2 Hz, 7.6 Hz, and 4.9 Hz), 2.64–2.72 (m, 1H), 3.07 (d, 1H, J = 13.3 Hz), 3.16 (d, 1H,
J = 13.2 Hz), 3.51 (m, 1H), 4.30 (dt, 1H, J = 9.5 Hz and 2.8 Hz), 4.67 (quintet, 1H, J = 7.1 Hz), 6.78 (br d,
1H, J = 7.3 Hz), 6.92 (br s, 1H), 7.21–7.24 (m, 2H), 7.28–7.31 (m, 3H), 7.42 (d, 2H, J = 8.6 Hz), 7.75 (d,
2H, J = 8.6 Hz); 13C-NMR (CDCl3) 17.6, 32.3, 41.9, 49.0, 59.8, 65.5, 128.0, 128.6, 128.90, 128.95, 130.1,
132.0, 133.4, 138.3, 166.6, 171.7, 176.7; HRMS (ES+) m/z calculated for C21H21ClN3O4S (M + Na)
423.1082; found 423.1085.
2-Benzyl-4,4-dimethoxy-2-(4-methylbenzamido)butanoic acid on Merrifield resin (25c):
Resin 13c (791
4 × 25 mL × five minutes of 20% piperidine in NMP and then with 3
deprotected resin was added 20 mL of NMP, 722 L (5.24 eq) of DIEA, and then 542 mg (4.4 eq) of
p-toluoyl chloride. The vessel was placed on an orbital shaker, and after 40 h was drained, and the resin
was washed with 3 20 mL of NMP, 2 20 mL of 1:1 THF:MeOH, 2
µ
mol) in a 50-mL SPPS vessel was washed with 3
×
20 mL of NMP, followed by
×
20 mL of NMP. To the
µ
×
×
× 20 mL of THF, and 3 × 20 mL
of DCM to give resin 25c, which was dried under vacuum to afford a mass of 1.19 g.
2-((3R)-6-Benzyl-6-(4-methylbenzamido)-5-oxohexahydropyrrolo[2,1-b]thiazole-3-carboxamido)-acetic
acid (β-26c): Resin 25c (118
µmol), 56 mg (570 µmol, 4.8 eq) of potassium acetate and 42.9 mg
(241 mol, 2.0 eq) of Cys-Gly-OH in 1.5 mL of acetic acid contained in a 3.5-mL reaction vessel
µ
◦
was heated at 90 C for 24 h. After cooling, the vessel was drained, and the resin was washed
with 3 × 2 mL of dichloromethane. The combined filtrates were evaporated, and the residue was
partitioned between 25 mL of dichloromethane and 25 mL of 1.0 N HCl. After separation, the aqueous

Molecules 2018, 23, 1762
18 of 30
phase was extracted with 10 mL of dichloromethane, and the combined organics were washed with
20 mL of 1.0 N HCl and were dried (Na2SO4). Concentration gave 9.7 mg, which was triturated under
1
250
µL of dichloromethane to afford 3.7 mg (7% over 10 steps) of
β
-
26c as a white solid; H-NMR
(CD3OD)
δ
2.64 (dd, J = 13.2 Hz and 6.6 Hz, 1H),
δ
2.90 (dd, J = 13.3 Hz and 7.3 Hz, 1H), 3.15 (m, 2H),
3.28 (dd, J = 10.8 Hz and 3.4 Hz, 1H), 3.32 (10.8 Hz and 6.5 Hz, 1H), 3.76 (m, 2H), 4.10 (t, J = 7.0 Hz,
1H), 4.91 (dd, J = 6.5 Hz and 3.4 Hz, 1H), 6.55 (br t, J = 5.8 Hz, 1H), 7.19 (d, J = 8.0 Hz, 2H), 7.21–7.30
(m, 5H), 7.63 (d, J = 8.2 Hz, 2H), 8.37 (br s, 1H); 13C-NMR (CD3OD)
δ 21.5, 34.6, 41.5, 41.8, 43.0, 61.7,
62.1, 67.4, 128.6, 128.8, 129.8, 130.2, 131.6, 132.3, 135.9, 143.8, 169.7, 170.9, 172.3, 176.2; HRMS (TOF ES+)
m/z calculated for C24H26N3O5S (M + H) 468.1593; found 468.1609. Recrystallization from ethanol
gave a sample for x-ray analysis.
2-((S)-2-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)propanamido)-4,4-dimethoxy-2-methylbutanoic
acid on Merrifield resin (28b). A 25-mL SPPS vessel was charged with 773 mg (363 µmol) of resin
13b, and was swelled in NMP for 30 minutes. The vessel was drained, and the resin was treated
with 5 mL of 20% piperidine in NMP for 5 min. The vessel was drained, the resin was treated with
10 mL of 20% piperidine, and the vessel was rocked on an orbital shaker for 40 min. Then, it was
drained, and the resin was washed with 5
×
10 mL of NMP. The deprotected resin was then treated
with 859 mg (1.11 mmol, 3.05 eq) of Fmoc-Ala anhydride in 4.5 mL of NMP. The vessel was rocked for
42 h, drained, and the resin was washed with 6 × 15 mL of NMP to give resin 28b.
4,4-Dimethoxy-2-methyl-2-((S)-2-(4-nitrobenzamido)propanamido)butanoic acid on Merrifield
resin (29b). To 363
added. After five min, the vessel was drained, and the resin was treated with 15 mL of 20% piperidine.
The vessel was rocked for 40 min, drained, and the resin was washed with 6 15 mL of NMP. To the
µmol of resin 28b in a 25-mL SPPS vessel, 5 mL of 20% piperidine in NMP was
×
deprotected resin, 3.80 mL (1.90 mmol, 5.2 eq) of a 0.50-M solution of DIEA in NMP was added, followed
by 3.21 mL (1.60 mmol, 4.4 eq) of a 0.50-M solution of 4-nitrobenzoyl chloride in NMP. The vessel was
rocked for 18 h, drained, and the resin was washed with 2
3 × 15 mL of THF, and 5 × 15 mL of DCM to give resin 29b.
×
15 mL of NMP, 3
×
15 mL of 1:1 THF:MeOH,
Fmoc-Ala Anhydride, (Fmoc-Ala)2O. A variation of the method of Izdebski and Pawlak [48] is
described here. A 50-mL, three-neck, round-bottomed flask under argon was charged with 1.25 g
(4.00 mmol) of Fmoc-Ala-OH. The flask was fitted with a thermometer and two rubber septa, and 11 mL
of DCM was added via syringe. The mixture was treated with 1 mL of anhydrous DMF, and then
◦
cooled to 3 C. To the mixture, 1.69 mL (252 mg, 2.00 mmol) of 1.0 M diisopropylcarbodiimide
(DIC) in DCM was added dropwise via syringe over a two-minute period. After stirring at 3 ◦C for
30 minutes, the mixture was allowed to warm to room temperature, and was stirred an additional
10 minutes. The contents were filtered to afford 469 mg of crude anhydride (70 mol%) containing
30 mol% diisopropylurea (DIU), 1H-NMR 1.14 (d, 12H, J = 6.5 Hz, DIU), 1.49 (d, 6H, J = 7.0 Hz), 3.83
(octet, 2H, J = 6.6 Hz, DIU), 3.99 (br s, 2H, DIU), 4.21 (t, 2H, J = 7.0 Hz), 4.38–4.49 (2m, 6H), 5.26 (m,
2H), 7.31 (t, 4H, J =7.2 Hz), 7.40 (t, 4H, J =7.5 Hz), 7.58 (br t, 4H, J =6.1 Hz), 7.76 (d, 4H, J =7.6 Hz).
Further drying gave 288 mg. The mother liquor was concentrated to a small volume and afforded an
additional 463 mg (81 mol% anhydride) after drying. The combined yield of (Fmoc-Ala)2O was 56%.
Methyl((3R,7aS)-6-methyl-6-((S)-2-(4-nitrobenzamido)propanamido)-5-oxohexahydropyrrolo
[2,1-b]thiazole-3-carbonyl)-L-alaninate (30b). Hydrolysis of dimethyl acetal functional group: Resin 29b
(363
The SPPS vessel was drained, and the resin was washed with 6
aldehyde resin. Preparation of Cys-Ala-OMe TFA: Boc-Cys(Trt)-Ala-OMe (262 mg, 478
was treated with 5 mL of trifluoroacetic acid (TFA): triethylsilane (TES) (97.5:2.5) for two hours at
room temperature. The volatiles were removed in vacuo, and the residue was treated with 5 mL of
1:1 diethyl ether:hexanes, and then decanted. This was repeated again with 5 mL of 1:1 diethyl ether:
hexanes. The residue was treated with 5 mL of diethyl ether to induce solidification, and then the
ether was evaporated to give the TFA salt of Cys-Ala-OMe. This material was dissolved in 3 mL of
acetic acid, and was added to the pre-formed aldehyde resin described above followed by 294 mg
µ
mol) was treated with 13 mL of TFA:DCM:water (4:4:1) for 35 min at room temperature.
15 mL of DCM to give the
mol, 1.3 eq)
×
·
µ

Molecules 2018, 23, 1762
19 of 30
(3.00 mmol, 8.3 eq) of potassium acetate. The vessel was rocked overnight at room temperature
and then drained. The resin was then washed with 2 5 mL of acetic acid. Acetic acid (5 mL) was
added, and the vessel was heated at 55–60 ◦C for 48 h. After cooling, the vessel was drained, and the
resin was washed with 2 5 mL of acetic acid. The three filtrates were combined and evaporated to
×
×
dryness to give 65.6 mg of crude 30b. The crude material was chromatographed on 2.0 g of normal
phase silica gel 60 slurried in DCM. Elution with DCM, 98/2 DCM/MeOH, and 95/5 DCM/MeOH
afforded 43.4 mg, which was then separated into its two major diastereomers by reverse-phase
chromatography on a 5-micron, 21.4
×
250 mm, C18 column using 50/50 1:1 MeOH/MeCN (5 mM
1
NH4OAc)/water (5 mM NH4OAc) to give 12.5 mg (7% over 13 steps) of α-30b: H-NMR 1.45 (d,
J = 6.8 Hz, 3H), 1.45 (d, J = 7.2 Hz, 3H), 1.51 (s, 3H), 2.28 (dd, J = 14.3 Hz and 4.3 Hz, 1H), 2.80 (dd,
J = 14.3 and 7.8 Hz, 1H), 3.56–3.64 (m, 2H), 3.63 (s, 3H), 4.66 (quintet, J = 7.6 Hz, 1H), 4.79–4.82 (m, 2H),
5.26 (dd, J = 7.8 Hz and 4.3 Hz, 1H), 7.25 (br s, 1H), 7.57 (br d, J = 7.8 Hz, 1H), 7.67 (br d, J = 8.2 Hz,
1H), 8.01 (d, J = 8.8 Hz, 2H), 8.26 (d, J = 8.8 Hz, 2H); 13C-NMR
δ 17.0, 18.4, 25.6, 36.7, 38.7, 47.9, 49.0,
52.4, 57.5, 60.8, 62.9, 123.7, 128.6, 139.4, 149.8, 166.2, 168.3, 172.2, 172.6, 173.5; HRMS (TOF ES+) m/z
calculated for C22H27N5O8SNa (M + Na) 544.1478; found 544.1493 and 16.2 mg (9% over 13 steps) of
α-30b; 1H-NMR
δ 1.38 (d, J = 7.2 Hz, 3H), 1.52 (d, J = 6.9 Hz, 3H), 1.60 (s, 3H), 2.65 (dd, J = 12.9 Hz
and 7.3 Hz, 1H), 2.82 (dd, J = 12.9 Hz and 6.6 Hz, 1H), 3.48 (dd, J = 11.7 Hz and 7.4 Hz, 1H), 3.67
(dd, J = 11.8 Hz and 5.8 Hz, 1H), 3.76 (s, 3H), 4.50 (quintet, J = 7.1 Hz, 1H), 4.83 (quintet, J = 7.0 Hz,
1H), 4.95 (t, J = 6.6 Hz, 1H), 5.15 (t, J = 7.0 Hz, 1H), 7.06 (br s, 1H), 7.35 (br d, J = 8.6 Hz, 1H), 7.37
(br d, J = 8.0 Hz, 1H), 7.97 (d, J = 8.6 Hz, 2H), 8.27 (d, J = 8.6 Hz, 2H); 13C-NMR
δ 18.0, 19.1, 23.6, 35.4,
41.7, 48.4, 49.3, 52.6, 58.8, 61.9, 62.6, 123.8, 128.4, 139.1, 149.8, 165.0, 168.0, 171.7, 172.9, 174.4; HRMS
(TOF ES+) m/z calculated for C22H27N5O8SNa (M + Na) 544.1478; found 544.1478.
Boc-Cys(Trt)-Ala-OMe (31). A solution of 1.28 g (2.76 mmol) of Boc-Cys(Trt)-OH and 303 µL
(2.76 mmol) of N-methylmorpholine in 7 mL of anhydrous DMF was prepared in a 20-mL scintillation
vial fitted with a rubber septum and under dry argon gas. This solution was transferred via syringe
to a 50-mL, three-neck, round-bottomed flask also under argon. The solution was then cooled in an
ice/acetone bath (
over a 30 s interval. After three minutes, a solution of 385 mg (2.76 mmol) alanine methyl ester
hydrochloride and 303 L (2.76 mmol) of N-methylmorpholine in 7 mL of anhydrous DMF cooled in
ice/acetone was added via syringe. The mixture was stirred at
9 ◦C for one hour, and then at room
−
9 ◦C) and treated via syringe with 358
µL (2.76 mmol) of isobutyl chloroformate
µ
−
temperature for one hour. The reaction mixture was transferred to a 250-mL beaker and was evaporated
with a stream of nitrogen overnight. The residue was partitioned between 75 mL of ethyl acetate
and 30 mL of pH 2 buffer. The layers were separated, and the organic phase was washed with pH 2
buffer, saturated sodium bicarbonate, twice with water (to pH 7), and then dried over sodium sulfate.
Concentration gave 1.38 g (89%) of 31. LC/MS analysis (4.6
SB-C18 column, 70–100% solvent B over 20 min at 0.5 mL/min (solvent B: 1/1 MeCN/MeOH w 5 mM
NH4OAc—solvent A: 5 mM NH4OAc) showed that no epimerization had occurred (Rt = 17.0 min,
×
75 mm, 3.5-micron, Agilent Zorbax
M + 59 = 607, 100%). A portion (518 mg) was recrystallized from 7 mL of methanol to afford 346 mg
1
of 31. H-NMR
δ 1.35 (d, 3H, J = 7.1 Hz), 1.42 (s, 9H), 2.53 (br dd, 1H, J = 13.0 Hz and 5.0 Hz), 2.74
(br m, 1H), 3.70 (s, 3H), 3.82 (br s, 1H), 4.49 (quintet, 1H, J = 7.2 Hz), 4.77 (br s, 1H), 6.52 (br d, 1H,
J = 5.6 Hz), 7.22 (t, 3H, J = 7.2 Hz), 7.29 (t, 6H, J = 7.5 Hz), 7.42 (d, 6H, J = 7.3 Hz); 13C-NMR
δ
18.4, 28.3,
33.7, 48.1, 52.4, 53.5, 67.2, 80.3, 126.9, 128.1, 129.6, 144.4, 155.3,170.0, 172.8; HRMS (ESI) m/z calculated
for C31H36N2O5SNa (M + Na) 571.2237; found 571.2239.
Cys-Ala-OMe, trifluoroacetic acid salt (32). Boc-Cys(Trt)-Ala-OMe (411 mg, 750 µmol) was
treated with 6 mL of trifluoroacetic acid/triethylsilane (97.5/2.5) solution. The mixture was stirred at
room temperature for two hours, and was then concentrated to a residue that was treated with 8 mL of
1:1 hexane:diethyl ether, and then decanted from the insoluble oil. This decantation was performed
two additional times using 4 mL of 1:1 hexane:diethyl ether. The oil was then triturated under diethyl
ether to induce solidification. The mixture was then evaporated to give 32 as a white solid, which was
immediately used in the cyclitive cleavage.

Molecules 2018, 23, 1762
20 of 30
Compounds 33a
–
c, 34a, and 35a
–
c
were prepared according to the methods outlined in
Schemes 11 and 12:
(3R)-Ethyl 6-(4-chlorobenzamido)-5-oxohexahydropyrrolo[2,1-b]thiazole-3-carboxylate (α-33a
and β-33a): A mixture of 242
µ
mol of 15 (R1 = H, R2 = 4-ClPh) in 10 mL of dichloromethane contained
in a 25-mL glass reaction vessel for 15 minutes was drained, and the resin was then treated with 10 mL
of 4:4:1 TFA:CH2Cl2:water. The vessel was rocked for 35 minutes, drained, and the resin was washed
with 5
resin
(1.40 equiv.) of polyvinylpyridine, 193
×
3 mL of dichloromethane, and then with 2
(R1 = H, R2 = 4-ClPh) was then converted to 33a using Method C by treatment with 340
mol of cysteine ethyl ester hydrochloride, and 5 mL of acetic
×
3 mL of acetic acid. The now-formed aldehyde
7
µmol
µ
acid. The vessel was rocked overnight at room temperature. LC/MS analysis indicated product
formation. The contents were then heated/rocked at 55 ◦C for 24 h. The vessel was drained, and the
filtrate was evaporated to dryness affording 41.5 mg, which was chromatographed on 3.0 g of silica gel
eluting with toluene and 80/20 toluene/ethyl acetate to afford 5.9 mg (8% over 11 steps) of
α-33a as
an oil; 1H-NMR
δ
1.32 (t, J = 7.2 Hz, 3H), 2.49 (dt, J = 14.9 Hz and 7.4 Hz, 1H), 2.86 (ddd, J = 14.2 Hz,
9,3 Hz, and 1.2 Hz, 1H), 3.40 (dd, J = 11.5 Hz and 4.6 Hz, 1H), 3.54 (dd, J = 11.5 Hz and 8.6 Hz, 1H), 4.26
(q, J = 7.1 Hz, 2H), 4.77 (ddd, J = 9.2 Hz, 8.3 Hz, and 6.3 Hz, 1H), (dd, J = 8.7 Hz and 4.6 Hz, 1H), 5.24
(dd, J = 7.1 Hz and 1.3 Hz, 1H), 6.92 (br d, J = 6.2 Hz, 1H), 7.40 (d, J = 8.5 Hz, 2H), 7.74 (d, J = 8.6 Hz,
2H); 13C-NMR
δ
14.1, 30.1, 37.2, 51.6, 58.6, 62.3, 64.4, 128.6, 128.9, 131.6, 138.3, 166.6, 169.7, 175.1; HRMS
(TOF ES+) m/z calculated for C16H18ClN2O4S (M + H) 369.0670; found 369.0668. Compound
β
-
33a
was eluted afterwards with 80/20 toluene/ethyl acetate to afford 5.6 mg (8% over 11 steps) as an
1
oil; H-NMR
δ 1.31 (t, J = 7.2 Hz, 3H), 2.11 (ddd, J = 12.7 Hz, 10.6 Hz, and 7.6 Hz, 1H), 3.35 (ddd,
J = 12.8 Hz, 8.1 Hz, and 6.2 Hz, 1H), 3.42 (d, J = 5.7 Hz, 2H), 4.25 (m, 2H), 5.00 (dd, J = 10.5 Hz, 8.2 Hz,
and 5.7 Hz, 1H), 5.17 (t, J = 5.4 Hz, 1H), 5.21 (dd, J = 7.4 Hz and 6.4 Hz, 1H), 6.95 (br d, J = 5.5 Hz, 1H),
7.38 (d, J = 8.6 Hz, 2H), 7.72 (d, J = 8.6 Hz, 2H); 13C-NMR
δ 14.1, 35.3, 38.6, 54.0, 57.8, 62.2, 62.3, 128.6,
128.9, 131.6, 138.3, 166.4, 168.9, 171.5; HRMS (TOF ES+) m/z calculated for C16H18ClN2O4S (M + H)
369.0670; found 369.0672.
(3R)-Ethyl6-(4-chlorobenzamido)-6-methyl-5-oxohexahydropyrrolo[2,1-b]thiazole-3-carboxylate
(β-33b and α-33b): A mixture of 133.5
TFA:CH2Cl2:water contained in a 5-mL glass reaction vessel was rotated for 35 minutes, drained, and the
resin was washed with 6 1.5 mL of dichloromethane. The resin was then dried under a stream of
nitrogen, and then under vacuum. The now-formed aldehyde resin
(R1 = Me, R2 = 4-ClPh) was
then converted to 33b using Method B by treatment with 270 mol (2.0 equiv) of cysteine ethyl ester
hydrochloride in 2 mL of NMP, followed by 927 mol (6.9 equiv.) of potassium acetate in 0.6 mL
µ
mol of 15 (R1 = Me, R2 = 4-ClPh) and 2.4 mL of 4:4:1
×
7
µ
µ
of acetic acid. The vessel was rotated for 24 h at room temperature, drained, and the resin was
washed with 2 × 3 mL each of NMP, 5% DIEA in NMP, 5% DIEA in dichloromethane, and 3 × 3 mL
of dichloromethane. The resin was dried under a stream of argon, and then treated with 2 mL of
◦
chlorobenzene. The contents were heated at 60 C for 67 h, cooled, the vessel was drained, and the
resin was washed with 3
×
4 mL of dichloromethane. The combined filtrates were evaporated to afford
1.7 mg. The resin was then heated at 75 ◦C in 3 mL of chlorobenzene for 50 h. The vessel was drained,
and the resin was washed with 3
×
4 mL of dichloromethane. The combined filtrates were evaporated to
afford 4.1 mg. This process was repeated at 75 ◦C for 40 h to afford 2.2 mg. The combined materials
(1.7 mg, 4.1 mg, and 2.2 mg) were chromatographed on a Dynamax Microsorb 5-micron C18 column
(21.4
acetate/water with 5.0 mM ammonium acetate to afford 2.5 mg (5% over 11 steps) of
1H-NMR
1.31 (t, J = 7.2 Hz, 3H), 1.69 (s, 3H), 2.67 (dd, J = 13.1 Hz and 7.4 Hz, 1H), 3.03 (dd, J = 13.0 Hz
×
250 mm) using a step gradient beginning with 6/4 1:1 MeCN/MeOH with 5.0 mM ammonium
β-33b as an oil;
δ
and 6.5 Hz, 1H), 3.40 (dd, J = 11.1 Hz and 3.0 Hz, 1H), 3.50 (dd, J = 11.2 Hz and 7.3 Hz, 1H), 4.24 (m,
2H), 5.19 (dd, J = 7.2 Hz and 3.0 Hz, 1H), 5.21 (t, J = 7.0 Hz, 1H), 6.63 (br s, 1H), 7.40 (d, J = 8.6 Hz,
2H), 7.72 (d, J = 8.6 Hz, 2H); 13C-NMR
δ 14.1, 23.1, 35.3, 43.7, 57.9, 61.6, 61.7, 62.1, 128.5, 128.8, 132.1,
138.2, 165.5, 169.0, 173.9; HRMS (TOF ES+) m/z calculated for C17H19ClN2O4SNa (M + Na) 405.0652;
found 405.0642. The resin was subjected to methoxide cleavage conditions by treating with 1.75 mL

Molecules 2018, 23, 1762
21 of 30
of anhydrous THF followed by 700
µL (660 mg, 3.00 mmol, 22 equiv.) of 25% sodium methoxide in
methanol for 3 h. The vessel was drained under positive argon pressure while the resin was washed
with 4 mL of absolute methanol. The combined filtrates were added to a rapidly stirred, cold mixture
of 20 mL of dichloromethane and 20 mL of 1 N HCl. The layers were separated, the aqueous phase
was extracted once with 10 mL of dichloromethane, and the combined organics were dried (Na2SO4).
Concentration gave 13.9 mg of a mixture consisting primarily of the carboxylic acid of 33b, which was
esterified with ethyl iodide (300 mg) and 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) (648 µmol) overnight
at room temperature. The mixture was concentrated to a residue that was partitioned between ethyl
acetate/5% citric acid/brine. The organic phase was washed with 5% citric acid and dried (MgSO4).
Concentration gave a wet residue that was diluted with dichloromethane and then dried with sodium
sulfate. Concentration gave 8.7 mg, which was chromatographed on a Dynamax Microsorb 5-micron
C18 column (21.4
ammonium acetate/water with 5.0 mM ammonium acetate to afford 2.1 mg (4% over 13 steps) of
as an oil; 1H-NMR
1.33 and 1.34 (2t, J = 7.2 Hz and 7.2 Hz, 3H), 1.65 (s, 3H), 2.51 (d, J = 14.5 Hz, 1H),
×
250 mm) using a step gradient beginning with 1/1 1:1 MeCN/MeOH with 5.0 mM
α-33b
δ
3.10 (dd, J = 14.4 Hz and 7.8 Hz, 1H), 3.41 (dd, J = 11.4 Hz and 4.3 Hz, 1H), 3.48 (dd, J = 11.3 Hz and
8.4 Hz, 1H), 4.26 (m, 2H), 5.07 (dd, J = 8.2 Hz and 4.2 Hz, 1H), 5.34 (d, J = 8.7 Hz, 1H), 6.40 (br s, 1H), 7.40
(d, J = 8.6 Hz, 2H), 7.71 (d, J = 8.6 Hz, 2H); 13C-NMR
δ 14.1, 25.8, 36.2, 36.5, 58.3, 59.6, 62.1, 63.3, 128.5,
128.9, 132.0, 138.2, 165.8, 169.4, 176.0; HRMS (TOF ES+) m/z calculated for C17H19ClN2O4SNa (M + Na)
405.0652; found 405.0667.
(3R)-Ethyl6-benzyl-6-(4-chlorobenzamido)-5-oxohexahydropyrrolo[2,1-b]thiazole-3-carboxylate
(β-33c and α-33c): A mixture of 260
TFA:CH2Cl2:water contained in a 5-mL glass reaction vessel was rotated for 35 minutes, drained, and the
resin was washed with 6 1.5 mL of dichloromethane. The resin was then dried under a stream of
nitrogen, and then under vacuum. The now-formed aldehyde resin
(R1 = benzyl, R2 = 4-ClPh) was
µ
mol of 15 (R1 = benzyl, R2 = 4-ClPh) and 2.4 mL of 4:4:1
×
7
then converted to 33c using Method F by treatment with 1.56 mL (1.56 mmol, 6.00 equivalent) of 1.0 M
potassium acetate in acetic acid followed by 1.04 mL (0.520 mmol, 2.00 equivalent) of cysteine ethyl ester
hydrochloride in acetic acid. After rotation at room temperature for 18 h, the vessel was drained, and the
resin was washed once with 3 mL of THF, and then with 4
×
2.5 mL of 5% DIEA in dichloromethane,
and then with 4 2 mL of dichloromethane. The resin was dried in vacuo, and then treated with 3 mL of
×
chlorobenzene followed by 0.270 mL (200 mg, 1.55 mmol, 6.0 equiv.) of DIEA. The mixture was heated
◦
at 55 C for 24 h, the vessel was drained, and the resin washed with 2
×
3 mL of dichloromethane.
Evaporation of the combined filtrate◦s gave only 5.0 mg. The resin was then treated with 3 mL of acetic
acid, and the vessel was heated at 75 C for 40 h. After cooling, the vessel was drained, and the resin was
washed with 2
triturated under 500
solid; 1H-NMR
1.31 (t, J = 7.2 Hz, 3H), 2.61 (dd, J = 13.4 Hz and 7.4 Hz, 1H), 3.23 (d, J = 13.3 Hz, 1H), 3.32
×
2 mL of acetic acid. The combined filtrates were evaporated to give 46.6 mg, which was
µ
L of warm acetonitrile to afford 15.6 mg (13% over 11 steps) of 33c as a white
β-
δ
(dd, J = 13.4 Hz and 6.4 Hz, 1H), 3.37 (dd, J = 11.3 Hz and 3.6 Hz, 1H), 3.44 (dd, J = 11.3 Hz and 7.5 Hz,
1H), 3.45 (d, J = 13.4 Hz, 1H), 4.25 (m, 2H), 4.75 (t, J = 6.8 Hz, 1H), 5.14 (dd, J = 7.1 Hz and 3.6 Hz, 1H), 6.66
(br s, 1H), 7.21 (m, 2H), 7.28–7.31 (m, 3H), 7.39 (d, J = 8.6 Hz, 2H), 7.67 (d, J = 8.5 Hz, 2H); 13C-NMR
δ 14.2,
35.3, 41.5, 42.9, 58.1, 62.06, 62.07, 65.5, 127.5, 128.4, 128.5, 128.9, 130.2, 132.2, 134.4, 138.2, 165.6, 168.8, 172.9;
HRMS (TOF ES+) m/z calculated for C23H24ClN2O4S (M + H) 459.1140; found 459.1147. The filtrate was
evaporated to a solid that was taken up in 650
filtrate was diluted with 350 L of water. The mixture was filtered through a 0.45 micron filter, and was
injected onto a Dynamax Microsorb 5-micron C18 column (21.4 250 mm) and chromatographed using
µL of 1:1 MeCN/MeOH, decanted with syringe, and the
µ
×
a step gradient beginning with 65/35 1:1 MeCN/MeOH with 5.0 mM ammonium acetate/water with
1
5.0 mM ammonium acetate to afford 1.3 mg (1% over 11 steps) of
α-
33c as a film; H-NMR
δ
1.31 (t,
J = 7.1 Hz, 3H), 2.70 (dd, J = 14.6 Hz and 2.3 Hz, 1H), 2.93 (dd, J = 14.6 Hz and 7.7 Hz, 1H), 3.19 (d,
J = 11.5 Hz, 1H), 3.21 (d, J = 11.2 Hz, 1H), 3.31 (d, J = 11.5 Hz, 1H), 3.33 (dd, J = 11.0 Hz and 3.6 Hz, 1H),
4.25 (m, 2H), 5.08 (dd, J = 8.1 Hz and 3.5 Hz, 1H), 5.39 (dd, J = 7.8 Hz and 2.3 Hz, 1H), 6.47 (br s, 1H),
7.27–7.34 (m, 5H), 7.39 (d, J = 8.5 Hz, 2H), 7.63 (d, J= 8.6 Hz, 2H); 13C-NMR
δ 14.1, 33.6, 36.4, 42.8, 58.2,

Molecules 2018, 23, 1762
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62.1, 63.0, 63.2, 127.8, 128.4, 128.9, 129.0, 130.6, 132.0, 134.7, 138.2, 165.5, 169.3, 174.3; HRMS (TOF ES+) m/z
calculated for C23H24ClN2O4S (M + H) 459.1140; found 459.1146.
(2S)-Methyl2-((3R)-6-(4-fluorobenzamido)-5-oxohexahydropyrrolo[2,1-b]thiazole-3-carboxamido)
-4-methylpentanoate (β-34a and α-34a): Prepared using Method C and Scheme 12 Preparation of
Cys-Leu-OMe. Fmoc-Leu-Wang resin (559
µmol) contained in an SPPS vessel was washed four
times with NMP, and was then treated with 6
×
8 mL 5 min 20% (v/v) piperidine in NMP.
×
To the deprotected resin was then added a mixture of Boc-Cys(Trt)-OH (3.0 equivalent), DIEA
(6.0 equivalent), N,N,N0,N0-tetramethyl-O-(1H-benzotriazol-1-yl)uronium hexafluorophosphate,
and O-(benzotriazol-1-yl)-N,N,N0,N0-tetramethyluronium hexafluorophosphate (HBTU, 3 equivalent)
in 6 mL of 85/15 DCM/DMF. The vessel was rocked for 20 h, drained, and the resin was washed three
times each with DMF and 1:1 THF:MeOH, and then four times with methanol to give Boc-Cys(Trt)-Leu
on Wang resin. The resin was then treated with 12 mL 30% (v/v) triethylamine in methanol, and the
vessel was placed in an oven at 55–60 ◦C for 48 h. The contents were allowed to cool, the vessel was
drained, and the resin was washed with 2
×
20 mL of methanol. The combined filtrates were then
0.89 (d, 3H, J = 6.2 Hz), 0.90 (d,
concentrated to give 287 mg of crude Boc-Cys(Trt)-OMe; 1H-NMR
δ
3H, J = 6.1 Hz), 1.42 (s, 9H), 1.51 (m, 1H), 1.57–1.63 (m, 2H), 2.50 (dd, 1H, J = 13.0 Hz and 5.0 Hz),
2.75 (br m, 1H), 3.68 (s, 3H), 3.82 (br m), 4.54 (br m), 4.74 (br s), 6.41 (br s), 7.22 (t, 3H, J = 7.2 Hz), 7.30
(t, 6H, J = 7.7 Hz), 7.42 (d, 6H, J = 7.7 Hz). Chromatography on 2.0 g of silica gel afforded 227.5 mg
(57%) of purified Boc-Cys(Trt)-OMe. To 7 mL of trifluoroacetic acid (TFA):triethylsilane (TES) (97.5:2.5)
under argon was added 175 mg (297 µmol) of purified Boc-Cys(Trt)-OMe. The mixture was stirred at
35 ◦C for one hour, and was then concentrated to a residue that was triturated twice with 15 mL of
1:1 hexanes:diethyl ether and decanted. The residue was then dried under vacuum to give a white
powder that was dissolved in 2.7 mL of acetic acid and used immediately in the cyclitive cleavage,
which is described as follows.
A 3.5-mL reaction vessel was charged with 106
mol, 1.7 equivalent) of polyvinylpyridine, 0.8 mL (85
of Cys-Leu-OMe in acetic acid, and 0.8 mL of acetic acid, and heated at 55 ◦C for 20 h. The vessel
µmol of resin
7
(R1 = H, R2 = 4-FPh), 19 mg
(181
µ
µmol, 0.80 equivalent) of a 0.106 M solution
was drained, the resin was washed with 2
evaporated to give 23 mg. Further exposure (72 h) to 55 C gave an additional 4.1 mg. Both quantities
×
2 mL of ◦acetic acid, and the combined filtrates were
were combined and chromatographed on 1.0 g of silica gel. The higher Rf material,
with 1/1 hexanes/ethyl acetate to afford 5.7 mg, while the lower Rf material, 34a, was eluted with
1/2 hexanes/ethyl acetate to afford 6.4 mg. Each of these materials was separately further purified
by reverse-phase HPLC on a Dynamax Microsorb 5-micron C18 column (21.4 250 mm) using step
α-34a, was eluted
β
-
×
gradients of 1:1 MeCN/MeOH with 5.0 mM ammonium acetate/water with 5.0 mM ammonium
1
acetate to afford 4.6 mg (12% over seven steps) of
α-
34a as a film; H-NMR
δ
0.92 and 0.93 (2d,
J = 6.3 Hz and 6.3 Hz, 6H), 1.54–1.64 (m, 2H), 1.65–1.69 (m, 1H), 2.14 (ddd, J = 12.8 Hz, 10.8 Hz,
and 7.6 Hz, 1H), 3.35 (ddd, 12.7 Hz, 8.4 Hz, and 6.3 Hz, 1H), 3.36 (dd, J = 12.0 Hz and 6.3 Hz, 1H),
3.75 (s, 3H), 3.79 (dd, J = 12.0 Hz and 6.4 Hz, 1H), 4.55–4.60 (m, 1H), 4.88 (t, J = 6.8 Hz, 1H), 5.09 (dd,
J = 8.4 Hz and 6.1 Hz, 1H), 5.11 (dd, J = 7.3 Hz and 6.5 Hz, 1H), 6.84 (br d, J = 5.9 Hz, 1H), 6.95 (br d,
J = 7.9 Hz, 1H), 7.12 (t, J = 8.6 Hz, 2H), 7.82 (dd, J = 8.7 Hz and 5.2 Hz, 2H); 13C-NMR
δ 22.0, 22.7, 25.0,
34.8, 37.7, 41.3, 51.3, 52.5, 54.1, 59.3, 63.1, 115.7 (d, 2JCF = 22.0 Hz), 129.3 (d, 4JCF = 3.1 Hz), 129.5 (d,
3JCF = 9.1 Hz), 165.0 (d, 1JCF = 253 Hz), 166.4, 167.6, 172.8, 173.1; HRMS (TOF ES+) m/z calculated for
C21H27FN3O5S (M + H) 452.1650; found 452.1654. β- δ 0.97
34a: 4.7 mg (12% over 7 steps); 1H-NMR
and 0.98 (2d, J = 6.7 Hz and 6.6 Hz, 6H), 1.72–1.79 (m, 3H), 2.56 (ddd, J = 14.3 Hz, 7.5 Hz, and 4.7 Hz,
1H), 2.65 (ddd, J = 14.5 Hz, 10.5 Hz, and 4.2 Hz, 1H), 3.60 (dd, J = 11.6 Hz and 8.6 Hz, 1H), 3.69 (dd,
J = 11.7 Hz and 4.9 Hz, 1H), 3.76 (s, 3H), 4.36 (ddd, J = 11.1 Hz, 6.7 Hz, and 4.9 Hz, 1H), 4.53–4.57 (m,
1H), 4.93 (dd, J = 8.5 Hz and 4.8 Hz, 1H), 5.31 (dd, J = 7.6 Hz and 4.2 Hz, 1H), 6.98 (t, J = 8.6 Hz, 2H), 7.47
(br d, J = 7.6 Hz, 1H), 7.63 (dd, J = 8.7 Hz and 5.2 Hz, 2H), 8.16 (br d, J = 6.7 Hz, 1H); 13C-NMR δ 21.8,
22.8, 25.0, 32.1, 36.5, 40.6, 51.4, 52.3, 54.8, 57.8, 64.7, 115.5 (d, 2JCF = 22.0 Hz), 128.3 (d, 4JCF = 3.0 Hz),

Molecules 2018, 23, 1762
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3
1
129.5 (d, JCF = 9.2 Hz), 165.0 (d, JCF = 253 Hz), 166.4, 168.8, 172.6, 172.9; HRMS (TOF ES+) m/z
calculated for C21H27FN3O5S (M + H) 452.1650; found 452.1656.
(2S)-Methyl2-((3R)-6-(4-chlorobenzamido)-5-oxohexahydropyrrolo[2,1-b]thiazole-3-carboxamido)
propanoate (α-35a and β-35a): Separated by reverse-phase HPLC on a Dynamax Microsorb 5-micron
C18 column (21.4
×
250 mm) using a step gradient beginning with 60/40 of 1:1 MeCN/MeOH with
5.0 mM of ammonium acetate/water with 5.0 mM ammonium acetate to afford 6.3 mg (6% over
1
11 steps) of
α-
35a; H-NMR
δ
1.53 (d, J = 7.3 Hz, 3H), 2.56 (ddd, J = 14.3 Hz, 7.6 Hz, and 4.9 Hz,
1H), 2.65 (ddd, J = 14.5 Hz, 10.5 Hz, and 4.1 Hz, 1H), 3.60 (dd, J = 11.6 Hz and 8.6 Hz, 1H), 3.69 (dd,
J = 11.6 Hz and 4.7 Hz, 1H), 3.80 (s, 3H), 4.37 (ddd, J = 10.8 Hz, 6.7 Hz, and 4.9 Hz, 1H), 4.56 (quintet,
J = 7.2 Hz, 1H), 4.95 (dd, J = 8.7 Hz and 4.8 Hz, 1H), 5.32 (dd, J = 7.6 Hz and 4.1 Hz, 1H), 7.26 (d,
J = 8.6 Hz, 2H), 7.50 (d, J = 8.6 Hz, 2H), 7.58 (br d, J = 7.1 Hz, 1H), 8.42 (br d, J = 6.8 Hz, 1H); 13C-NMR
δ
17.5, 31.8, 36.4, 48.6, 52.5, 54.7, 57.8, 64.6, 128.5, 128.7, 130.3, 138.2, 166.5, 168.5, 172.8, 172.9; HRMS
(TOF ES+) m/z calculated for C18H20ClN3O5SNa (M + Na) 448.0710; found 448.0725 and 4.6 mg (4%
over 11 steps) of
35a; 1H-NMR 1.42 (d, J = 7.2 Hz, 3H), 2.14 (ddd, J = 12.9 Hz, 10.7 Hz, and 7.5 Hz,
β
-
1H), 3.35 (dd, J = 8.3 Hz and 6.3 Hz, 1H), 3.38 (dd, J = 12.0 Hz and 7.2 Hz, 1H), 3.77 (s, 3H), 3.78 (dd,
J = 12.1 Hz and 6.4 Hz, 1H), 4.54 (quintet, J = 7.1 Hz, 1H), 4.86 (t, J = 6.8 Hz, 1H), 5.08 (ddd, J = 10.6 Hz,
8.5 Hz and 5.9 Hz, 1H), 5.13 (t, J = 6.8 Hz, 1H), 6.74 (br d, J = 5.6 Hz, 1H), 7.10 (br d, J = 6.9 Hz, 1H), 7.42
(d, J = 8.5 Hz, 2H), 7.74 (d, J = 8.5 Hz, 2H); 13C-NMR
δ 18.2, 34.9, 37.6, 48.6, 52.7, 54.2, 59.3, 63.1, 128.6,
129.0, 131.5, 138.4, 166.4, 167.5, 172.8, 173.0; HRMS (TOF ES+) m/z calculated for C18H20ClN3O5SNa
(M + Na) 448.0710; found 448.0710.
(2S)-Methyl2-((3R)-6-(4-chlorobenzamido)-6-methyl-5-oxohexahydro-pyrrolo[2,1-b]thiazole-
3carboxamido)propanoate (β-35b and α-35b): Separated on 2.0 g of silica gel 60 using hexanes/ethyl
1
acetate eluents (40/60 and 1/2) to afford 10.3 mg (9% over 11 steps) of β-35b; H-NMR
δ 1.41 (d,
J = 7.1 Hz, 3H), 1.69 (s, 3H), 2.73 (dd, J = 13.1 Hz and 7.3 Hz, 1H), 2.99 (dd, J = 13.1 Hz and 6.6 Hz, 1H),
3.47 (dd, J = 12.9 Hz and 7.3 Hz, 1H), 3.73 (dd, J = 11.9 Hz and 6.0 Hz, 1H), 3.76 (s, 3H), 4.51 (quintet,
J = 7.1 Hz, 1H), 4.89 (dd, J = 6.9 Hz and 6.3 Hz, 1H), 5.13 (t, J = 6.9 Hz, 1H), 6.71 (br s, 1H), 7.22 (br
d, J = 6.8 Hz, 1H), 7.40 (d, J = 8.6 Hz, 2H), 7.73 (d, J = 8.6 Hz, 2H); 13C-NMR
δ 19.2, 23.5, 35.0, 42.2,
48.5, 52.6, 59.2, 62.0, 62.7, 128.5, 128.9, 131.9, 138.3, 165.5, 167.8, 172.9, 175.4; HRMS (TOF ES+) m/z
calculated for C19H23ClN3O5S (M + H) 440.1041; found 440.1039 and 10.1 mg (9% over 11 steps) of
α-35b; 1H-NMR
δ 1.51 (d, J = 7.3 Hz, 3H), 1.64 (s, 3H), 2.34 (dd, J = 14.3 Hz and 3.8 Hz, 1H), 2.88 (dd,
J = 14.3 and 7.9 Hz, 1H), 3.55 (dd, J = 11.6 Hz and 8.6 Hz, 1H), 3.68 (dd, J = 11.7 Hz and 4.8 Hz, 1H),
3.69 (s, 3H), 4.54 (quintet, J = 7.2 Hz, 1H), 4.93 (dd, J = 8.6 Hz and 4.8 Hz, 1H), 5.26 (dd, J = 7.9 Hz and
3.8 Hz, 1H), 6.59 (br s, 1H), 7.40 (d, J = 8.6 Hz, 2H), 7.62 (br d, J = 7.1 Hz, 1H), 7.67 (d, J = 8.6 Hz, 2H);
13C-NMR δ 17.6, 26.2, 36.0, 38.7, 48.5, 52.4, 57.8, 60.5, 62.5, 128.5, 129.0, 131.4, 138.5, 166.4, 168.7, 172.5,
173.1; HRMS (TOF ES+) m/z calculated for C19H23ClN3O5S (M + H) 440.1041; found 440.1044.
(2S)-Methyl2-((3R)-6-benzyl-6-(4-chlorobenzamido)-5-oxohexahydro-pyrrolo[2,1-b]thiazole-
3carboxamido)propanoate (β-35c and α-35c): Separated on two 2.0 g of silica gel 60 using
hexanes/ethyl acetate eluents (70/30, 60/40, 55/45, 1/1) to afford 10.3 mg (8% over 11 steps) of β-35c
;
1H-NMR
δ
1.43 (d, J = 7.2 Hz, 3H), 2.69 (dd, J = 13.7 Hz and 7.1 Hz, 1H), 3.27 (dd, J = 13.7 Hz and
6.7 Hz, 1H), 3.30 (d, J = 12.9, 1H), 3.34 (dd, J = 8.6 Hz and 7.5 Hz, 1H), 3.35 (d, J = 13.1 Hz, 1H), 3.64
(dd, J = 11.8 Hz and 5.8 Hz, 1H), 3.80 (s, 3H), 4.25 (t, J = 6.8 Hz, 1H), 4.59 (quintet, J = 7.3 Hz, 1H),
4.85 (t, J = 6.4 Hz, 1H), 6.73 (br s, 1H), 6.94 (br d, J = 7.4 Hz, 1H), 7.23–7.25 (m, 2H), 7.29–7.31 (m, 3H),
7.41 (d, J = 8.5 Hz, 2H), 7.71 (d, J = 8.5 Hz, 2H); 13C-NMR
δ 18.2, 34.6, 41.5, 42.2, 48.3, 52.6, 60.1, 62.7,
66.0, 127.9, 128.5, 128.7, 128.9, 130.1, 132.0, 134.3, 138.3, 165.5, 167.3, 172.7, 174.9; HRMS (TOF ES+)
m/z calculated for C25H26ClN3O5SNa (M + Na) 538.1179; found 538.11 and 5.1 mg (3% over 11 steps)
of α-35c (90% by-NMR); 1H-NMR
δ 1.50 (d, J = 7.2 Hz, 3H), 2.62 (dd, J = 14.3 and 7.6 Hz, 1H), 2.68
(dd, J = 14.3 and 4.5 Hz, 1H), 3.19 (d, J = 13.6 Hz, 1H), 3.27 (d, J = 13.5 Hz, 1H), 3.35 (dd, J = 11.3 Hz
and 8.4 Hz, 1H), 3.63 (dd, J = 11.4 Hz and 4.3 Hz, 1H), 3.71 (s, 3H), 4.53 (quintet, J = 7.2 Hz, 1H), 4.94
(dd, J = 8.4 Hz and 4.3 Hz, 1H), 5.25 (dd, J = 7.6 Hz and 4.5 Hz, 1H), 6.56 (br s, 1H), 7.31–7.33 (m, 2H),
7.37–7.43 (m, 5H), 7.56 (d, J = 8.6 Hz, 2H), 7.68 (br d, J = 7.2 Hz, 1H); 13C-NMR
δ 17.5, 35.8, 37.0, 43.5,

Molecules 2018, 23, 1762
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48.6, 52.3, 57.9, 62.5, 63.9, 128.2, 128.3, 129.0, 129.3, 130.2, 131.6, 134.0, 138.6, 165.9, 168.5, 171.8, 172.5;
HRMS (TOF ES+) m/z calculated for C25H27ClN3O5S (M + H) 516.1354; found 516.1355.
General procedure for the preparation of 36a
Scheme 10, 250 mol of resin 13a contained in a SPPS vessel was treated with 20% piperidine in
NMP at room temperature for 40 minutes with gentle agitation. The vessel was drained, and the
resin was washed with 6 4 mL of NMP. To the deprotected resin, Fmoc-Ala, HOBt, and DIC (5 eq
each) were added in NMP for R1 = H or (Fmoc-Ala)2O (3 eq) in NMP for R1 = Me and Bn. The vessel
was agitated for 18–42 h, drained, and the resin was washed with 6 6 mL of NMP to give resin
22a . Resin 22a was treated with 20% piperidine in NMP at room temperature for 40 minutes with
gentle agitation. The vessel was drained, and the resin was washed with 6 4 mL of NMP. To the
deprotected resin 4-chlorobenzoyl chloride (4.4 eq) in NMP was added, followed by DIEA (5.2 eq) in
NMP. The vessel was rocked for 18 h to 24 h, drained, and the resin was washed with 3 6 mL of NMP,
6 mL of 1:1 THF:MeOH, 3 6 mL of THF, and 5 6 mL of DCM. The resin was treated with 5 mL
of 4:4:1 TFA:CH2Cl2:water for 35 min at room temperature. The mixture was filtered, and the resin
was washed with 5 2 mL of dichloromethane, and then dried under a stream of nitrogen. To the
resulting resin 38a , 1.0–1.3 equivalents of a 0.25-M solution of 32 in acetic acid was added, followed
–c and 30b,c (Method D cleavage). According to
µ
–c
×
×
–
c
–c
×
×
3
×
×
×
×
–
c
by 6.2–8.6 equivalents of a 1.6-M solution of potassium acetate in acetic acid. The vessel was rotated at
room temperature overnight (16–22 h) and was then drained, and the resin was washed with acetic
acid. The combined filtrates were evaporated to a residue that was taken up in dichloromethane and
washed two times with saturated sodium bicarbonate and dried (MgSO4). Concentration gave a crude
sample that was analyzed by LC/MS and quantitative NMR. The resin was then treated with 3 mL of
◦
acetic acid, and was heated at 55 C for 24 h. The vessel was drained, the resin was washed with acetic
acid, and the combined filtrates were evaporated to a residue. A further exposure of the resin/acetic
acid to 55 ◦C for 24 h resulted in a minor amount of cyclitive cleavage product. The residues were
combined, and the diastereomers were separated by normal phase or reverse-phase chromatography.
(2S)-Methyl2-((3R)-6-((S)-2-(4-chlorobenzamido)propanamido)-5-oxohexahydropyrrolo[2,1-b]
thiazole-3-carboxamido)propanoate (β-36a and α-36a): Separated by chromatography on 2.0 g of
silica gel 60 using CH2Cl2 and CH2Cl2/MeOH (98/2, 97/3, and 95/5) to afford 4.1 mg (4% over
13 steps) of β-36a; 1H-NMR
δ 1.37 (d, J = 7.2 Hz, 3H), 1.49 (d, J = 7.0 Hz, 3H), 2.07 (ddd, J = 12.8 Hz,
10.8 Hz, and 7.4 Hz, 1H), 3.14 (ddd, J = 12.9 Hz, 8.5 Hz, and 6.3 Hz, 1H), 3.33 (dd, J = 11.8 Hz and
7.2 Hz, 1H), 3.71 (dd, J = 11.8 Hz and 5.8 Hz, 1H), 3.74 (s, 3H), 4.51 (quintet, J = 7.2 Hz, 1H), 4.78
(quintet, J = 7.2 Hz, 1H), 4.84–4.89 (m, 2H), 5.06 (t, J = 6.8 Hz, 1H), 6.95 (br d, J = 7.3 Hz, 1H), 7.14 (br d,
J = 6.3 Hz, 1H), 7.20 (br d, J = 7.1 Hz, 1H), 7.41 (d, J = 8.5 Hz, 2H), 7.75 (d, J = 8.5 Hz, 1H); 13C-NMR
δ
18.0, 18.3, 34.8, 37.0, 48.5, 49.0, 52.6, 53.7, 59.1, 62.7, 128.6, 128.9, 132.0, 138.3, 166.4, 167.7, 172.5, 172.6,
172.9; HRMS (TOF ES+) m/z calculated for C21H26ClN4O6S (M + H) 497.1256; found 497.1260 and
2.7 mg (3% over 13 steps) of α-36a; 1H-NMR
δ 1.44 (d, J = 7.1 Hz, 3H), 1.46 (d, J = 7.3 Hz, 3H), 2.48
(ddd, J = 14.3 Hz, 7.3 Hz, and 4.8 Hz, 1H), 2.56 (ddd, J = 14.4 Hz, 10.2 Hz, and 4.3 Hz, 1H), 3.59 (d,
J = 7.1 Hz, 2H), 3.67 (s, 3H), 4.20 (ddd, J = 11.7 Hz, 7.1 Hz, 4.8 Hz, 1H), 4.58 (quintet, J = 7.4 Hz, 1H),
4.72 (quintet, J = 7.2 Hz, 1H), 4.76 (t, J = 7.1 Hz, 1H), 5.29 (dd, J = 7.3 Hz and 4.3 Hz, 1H), 7.27 (br d,
J = 7.7 Hz, 1H), 7.41 (d, J = 8.5 Hz, 2H), 7.56 (br d, J = 7.6 Hz, 1H), 7.75 (br d, J = 7.0 Hz, 1H), 7.79 (d,
J = 8.6 Hz, 2H); 13C-NMR
δ 17.4, 17.8, 31.7, 36.9, 48.2, 49.4, 52.4, 54.3, 58.0, 64.9, 128.78, 128.85, 131.8,
138.3, 166.7, 168.5, 171.9, 173.3, 173.6; HRMS (TOF ES+) m/z calculated for C21H26ClN4O6S (M + H)
497.1256; found 497.1253.
(2S)-Methyl2-((3R)-6-((S)-2-(4-chlorobenzamido)propanamido)-6-methyl-5-oxohexahydropyrrolo-
[2,1-b]thiazole-3-carboxamido)propanoate (α-36b and β-36b): Separated by reverse-phase HPLC on
a Dynamax Microsorb 5-micron C18 column (21.4
60/40 of 1:1 MeCN/MeOH with 5.0 mM ammonium acetate/water with 5.0 mM ammonium acetate
to afford 4.2 mg (4% over 13 steps) of α-36b; 1H-NMR
1.38 (d, J = 7.1 Hz, 3H), 1.49 (d, J = 7.0 Hz,
×
250 mm) using a step gradient beginning with
δ
3H), 1.59 (s, 3H), 2.63 (dd, J = 13.0 Hz and 7.4 Hz, 1H), 2.79 (dd, J = 13.0 Hz and 6.6 Hz, 1H), 3.47 (dd,
J = 11.9 Hz and 7.5 Hz, 1H), 3.69 (dd, J = 11.9 Hz and 5.9 Hz, 1H), 3.75 (s, 3H), 4.50 (quintet, J = 7.1 Hz,

Molecules 2018, 23, 1762
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1H), 4.78 (quintet, J = 7.0 Hz, 1H), 4.94 (dd, J = 7.0 and 6.2 Hz, 1H), 5.11 (t, J = 7.0 Hz, 1H), 6.97 (br d,
J = 7.2 Hz, 1H), 7.02 (br s, 1H), 7.35 (br d, J = 6.9 Hz, 1H), 7.41 (d, J = 8.5 Hz, 2H), 7.74 (d, J = 8.5 Hz,
2H); 13C-NMR
δ
18.0, 18.9, 23.6, 35.2, 41.7, 48.4, 49.1, 52.6, 58.8, 61.7, 62.5, 128.5, 128.9, 131.9, 138.2,
166.0, 168.1, 171.7, 172.9, 174.5; HRMS (TOF ES+) m/z calculated for C22H28ClN4O6S (M + H) 511.1413;
found 511.1416 and 4.1 mg (4% over 13 steps) of β-36b; 1H-NMR
1.42 (d, J = 6.9 Hz, 3H), 1.45 (d,
δ
J = 7.2 Hz, 3H), 1.50 (s, 3H), 2.26 (dd, J = 14.2 Hz and 4.3 Hz, 1H), 2.78 (dd, J = 14.3 Hz and 7.8 Hz, 1H),
3.55–3.62 (m, 2H), 3.64 (s, 3H), 4.63 (quintet, J = 7.5 Hz, 1H), 4.77 (quintet, J = 7.2 Hz, 1H), 4.81 (dd,
J = 8.1 Hz and 5.8 Hz, 1H), ), 5.26 (dd, J = 7.8 Hz and 4.4 Hz, 1H), 7.21 (br s, 1H), 7.26 (br d, J = 6.8 Hz,
1H), 7.38 (d, J = 8.4 Hz, 2H), 7.66 (br d, J = 7.9 Hz, 1H), 7.77 (d, J = 8.5 Hz, 2H); 13C-NMR
δ 16.9, 18.2,
25.6, 36.6, 38.8, 47.9, 48.8, 52.3, 57.5, 60.7, 62.9, 128.7, 128.8, 132.1, 138.2, 167.1, 168.4, 172.2, 172.8, 173.3;
HRMS (TOF ES+) m/z calculated for C22H28ClN4O6S (M + H) 511.1413; found 511.1411.
(2S)-Methyl2-((3R)-6-benzyl-6-((S)-2-(4-chlorobenzamido)propanamido)-5-oxohexahydropyrrolo-
[2,1-b]thiazole-3-carboxamido)propanoate (α-36c and β-36c): Compound α-36c was separated from
β-36c by chromatography on 1.5 g of silica gel. It was further purified by reverse-phase HPLC on
a Dynamax Microsorb 5-micron C18 column (21.4
70/30 of 1:1 MeCN/MeOH with 5.0 mM of ammonium acetate/water with 5.0 mM of ammonium
acetate to afford 2.1 mg (1% over 13 steps) of α-36c; H-NMR
×
250 mm) using a step gradient beginning with
1
δ 1.37 (d, J = 6.9 Hz, 3H), 1.44 (d,
J = 7.2 Hz, 3H), 2.54 (d, J = 6.2 Hz, 2H), 3.02 (d, J = 13.6 Hz, 1H), 3.10 (d, J = 13.6 Hz, 1H), 3.42 (dd,
J = 11.6 Hz and 8.6 Hz, 1H), 3.50 (dd, J = 11.6 Hz and 5.2 Hz, 1H), 3.67 (s, 3H), 4.63 (quintet, J = 7.6 Hz,
1H), 4.72 (quintet, J = 7.4 Hz, 1H), 4.78 (dd, J = 8.5 Hz and 5.2 Hz, 1H), 5.23 (t, J = 6.2 Hz, 1H), 7.11
(br d, J = 8.4 Hz, 1H), 7.12 (br s, 1H), 7.24–7.31 (m, 5H), 7.42 (d, J = 8.5 Hz, 2H), 7.63 (br d, J = 8.0 Hz,
1H), 7.79 (d, J = 8.5 Hz, 2H); 13C-NMR
δ 16.5, 18.2, 36.0, 36.4, 42.9, 47.9, 48.8, 52.3, 57.5, 63.1, 64.6, 127.8,
128.7, 128.86, 128.93, 130.3, 132.0, 133.9, 138.2, 167.1, 168.2, 171.2, 172.6, 173.3; HRMS (TOF ES+) m/z
calculated for C28H31ClN4O6SNa (M + Na) 609.1551; found 609.1565. Compound β-36c was further
purified by reverse-phase HPLC on a Dynamax Microsorb 5-micron C18 column (21.4 × 250 mm)
using a step gradient beginning with 70/30 of 1:1 MeCN/MeOH with 5.0 mM of ammonium
acetate/water with 5.0 mM of ammonium acetate to afford 3.2 mg (2% over 13 steps) of β-36c; 1HNMR
δ
1.39 (d, J = 7.3 Hz, 3H), 1.50 (d, J = 6.9 Hz, 3H), 2.57 (dd, J = 13.5 Hz and 7.0 Hz, 1H), 3.02 (dd,
J = 13.5 Hz and 6.8 Hz, 1H), 3.09 (d, J = 13.0 Hz, 1H), 3.24 (d, J = 13.0 Hz, 1H), 3.32 (dd, J = 11.6 Hz and
6.9 Hz, 1H), 3.61 (dd, J = 11.7 Hz and 5.5 Hz, 1H), 3.79 (s, 3H), 4.09 (t, J = 6.9 Hz, 1H), 4.57 (quintet,
J = 7.3 Hz, 1H), 4.69 (quintet, J = 7.1 Hz, 1H), 4.85 (t, J = 6.2 Hz, 1H), 6.70 (br d, J = 7.2 Hz, 1H), 6.84 (br
s, 1H), 6.88 (br d, J = 7.6 Hz, 1H), 7.21–7.22 (m, 2H), 7.27–7.28 (m, 3H), 7.42 (d, J = 8.5 Hz, 2H), 7.73 (d,
J = 8.5 Hz, 2H); 13C-NMR
δ 18.2, 18.3, 34.4, 40.6, 42.4, 48.2, 49.2, 52.6, 60.1, 62.1, 65.7, 128.0, 128.5, 128.7,
128.9, 130.1, 131.9, 133.9, 138.3, 166.1, 167.4, 171.5, 172.7, 174.3; HRMS (TOF ES+) m/z calculated for
C28H31ClN4O6SNa (M + Na) 609.1551; found 609.1572.
(2S)-Methyl2-((3R)-6-benzyl-6-((S)-2-(4-nitrobenzamido)propanamido)-5-oxohexahydropyrrolo
[2,1-b]thiazole-3-carboxamido)propanoate (β-30c and α-30c): Partial separation was achieved
by chromatography on 1.0 g of silica gel using CH2Cl2 and CH2Cl2/EtOAc mobile phases.
Compound β-30c was then purified by reverse-phase HPLC on a Dynamax Microsorb 5-micron,
C18 column (21.4
acetate/water with 5.0 mM of ammonium acetate to afford 9.1 mg (4% over 13 steps) of β-30c
1H-NMR
1.40 (d, J = 7.2 Hz, 3H), 1.54 (d, J = 7.0 Hz, 3H), 2.59 (dd, J = 13.4 Hz and 7.0 Hz, 1H),
×
250 mm) using 65/35 of 1:1 MeCN/MeOH with 5.0 mM of ammonium
;
δ
3.06 (dd, J = 13.5 Hz and 6.8 Hz, 1H), 3.11 (d, J = 13.1 Hz, 1H), 3.25 (d, J = 13.1 Hz, 1H), 3.32 (dd,
J = 11.7 Hz and 7.0 Hz, 1H), 3.60 (dd, J = 11.7 Hz and 5.6 Hz, 1H), 3.79 (s, 3H), 4.14 (t, J = 6.9 Hz, 1H),
4.57 (quintet, J = 7.3 Hz, 1H), 4.75 (quintet, J = 7.0 Hz, 1H), 4.85 (t, J = 6.3 Hz, 1H), 6.84 (br s, 1H), 6.92
(br d, J = 7.6 Hz, 1H), 7.07 (br d, J = 7.1 Hz, 1H), 7.21–7.23 (m, 2H), 7.26–7.29 (m, 3H), 7.97 (d, J = 8.6 Hz,
2H), 8.29 (d, J = 8.7 Hz, 2H); 13C-NMR
δ 18.2, 18.7, 34.6, 40.7, 42.4, 48.3, 49.4, 52.6, 60.0, 62.2, 65.8, 123.8,
128.0, 128.4, 128.8, 130.1, 133.8, 139.1, 149.8, 165.0, 167.4, 171.5, 172.7, 174.1; HRMS (TOF ES+) m/z
calculated for C28H32N5O8SNa (M + H) 598.1966; found 598.1957. Compound α-30c was purified by
reverse-phase HPLC on a Dynamax Microsorb 5-micron, C18 column (21.4
×
250 mm) using 65/35

Molecules 2018, 23, 1762
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of 1:1 MeCN/MeOH with 5.0 mM of ammonium acetate/water with 5.0 mM ammonium acetate
to afford 4.7 mg (2% over 13 steps) of α-30c; 1H-NMR
1.39 (d, J = 6.9 Hz, 3H), 1.45 (d, J = 7.2 Hz,
δ
3H), 2.56 (d, J = 6.2 Hz, 2H), 3.03 (d, J = 13.7 Hz, 1H), 3.12 (d, J = 13.7 Hz, 1H), 3.44 (dd, J = 11.6 Hz
and 8.4 Hz, 1H), 3.48 (dd, J = 11.7 Hz and 5.4 Hz, 1H), 3.65 (s, 3H), 4.67 (quintet, J = 7.2 Hz, 1H),
4.74–4.77 (m, 2H), 5.25 (t, J = 6.1 Hz, 1H), 7.09 (br s, 1H), 7.25–7.26 (m, 2H), 7.29–7.34 (m, 3H), 7.43 (br d,
J = 7.9 Hz, 1H), 7.62 (br d, J = 8.2 Hz, 1H), 8.04 (d, J = 8.7 Hz, 2H), 8.29 (d, J = 8.8 Hz, 2H); 13C-NMR
δ
16.3, 18.4, 35.7, 36.6, 42.9, 47.8, 49.0, 52.4, 57.4, 63.2, 64.8, 123.7, 127.9, 128.6, 129.0, 130.3, 133.9, 139.4,
149.8, 166.3, 168.1, 171.1, 172.5, 173.6; HRMS (TOF ES+) m/z calculated for C28H32N5O8SNa (M + H)
598.1966; found 598.1960.
(2S)-Methyl2-((3R)-6-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-propanamido)-6-
methyl-5-oxohexahydropyrrolo[2,1-b]thiazole-3-carboxamido)-propanoate (α-37b and β-37b):
To 252 µ
mol of resin 13b (R1 = Me) swelled with NMP contained in a 3.5-mL reaction vessel, 2 mL of
20% piperidine in NMP was added. The vessel was drained, charged with 3 mL of the 20% piperidine
solution, and rotated at room temperature for 45 minutes. The vessel was drained, and the resin
was washed with 7
×
3 mL of NMP to remove all of the piperidine. The resin was then treated with
a solution of 1.25 mmol (5 equiv.) each of HBTU and Fmoc-Ala-OH and 2.50 mmol (10 equiv.) of DIEA
in 3.75 mL of NMP. The vessels were rotated at room temperature for six days, drained, and the resins
were washed with 3
×
3 mL each of NMP, 1;1 THF:MeOH, THF, and 4
×
3 mL of dichloromethane to
give acetal resin 22b (R1 = Me), which was hydrolyzed using 3 mL of 4:4:1 TFA:CH2Cl2/water over
35 minutes at room temperature. The vessel was drained, and the resulting aldehyde resin was washed
six times with dichloromethane. To this resin, a solution of 32 in 1 mL of acetic acid that was generated
from 268
potassium acetate in acetic acid. After exposure at room temperature for 18 h, the vessel was drained
into a tared collection vial. The resin was washed with 2 2 mL of acetic acid, and the filtrates were
combined. The resin was treated with 3 mL of acetic acid, and was placed in an oven heated at
45–52 ◦C for 66 h. The vessels were drained, and the resin was washed with 2
2 mL of acetic acid.
µmol (1.07 equiv.) of 31 was then added, followed by a solution of 2.0 mmol (8.0 equiv.) of
×
×
This filtrate was evaporated to give 25.3 mg of α-37b and β-37b. The diastereomers were separated by
reverse-phase HPLC on a Dynamax Microsorb 5-micron, C18 column (21.4 × 250 mm) using a step
gradient beginning with 75/25 of 1:1 MeCN/MeOH with 5.0 mM of ammonium acetate/water
with 5.0 mM of ammonium acetate to afford 3.7 mg (3% over 11 steps) of α-37b; 1H-NMR
δ 1.35 (d,
J = 6.6 Hz, 3H), 1.45 (d, J = 7.2 Hz, 3H), 1.49 (s, 3H), 2.24 (br dd, J = 14.0 Hz and 3.2 Hz, 1H), 2.75 (br dd,
J = 13.1 Hz and 7.4 Hz, 1H), 3.55 (dd, J = 11.7 Hz and 8.7 Hz, 1H), 3.62 (dd, J = 11.7 Hz and 5.0 Hz, 1H),
3.69 (s, 3H), 4.21 (t, J = 7.0 Hz, 1H), 4.26 (br m, 1H), 4.39–4.47 (br m, 2H), 4.56 (quintet, J = 7.3 Hz, 1H),
4.86 (dd, J = 8.6 Hz and 5.1 Hz, 1H), 5.21 (br m, 1H), 5.58 (br d, J = 6.0 Hz, 1H), 6.75 (br s, 1H), 7.30
(q, J = 7.6 Hz, 2H), 7.40 (t, J = 7.4 Hz, 2H), 7.58 (d, J = 7.5 Hz, 2H), 7.59 (br d, J = 6.0 Hz, 1H), 7.76 (d,
J = 7.6 Hz, 2H); 13C-NMR
125.04, 127.06, 127.12, 127.8, 141.3, 143.6, 143.7, 156.5, 168.5, 172.5, 172.6, 172.8; HRMS (TOF ES+) m/z
calculated for C30H35N4O7S (M + H) 595.2221; found 595.2219 and 5.5 mg (4% over 11 steps) of β-37b
1H-NMR
1.39 (d, J = 7.2 Hz, 6H), 1.56 (s, 3H), 2.62 (br dd, J = 12.6 Hz and 7.3 Hz, 1H), 2.78 (br dd,
δ 17.8, 25.7, 36.3, 38.9, 47.1, 48.3, 50.4, 52.4, 57.7, 60.3, 62.6, 67.2, 120.0, 124.96,
:
δ
J = 12.8 Hz and 6.5 Hz, 1H), 3.42 (dd, J = 11.7 Hz and 7.3 Hz, 1H), 3.71 (dd, J = 11.8 Hz and 6.1 Hz, 1H),
3.75 (s, 3H), 4.22 (quintet, J = 7.1 Hz, 1H), 4.25 (br m, 1H), 4.40 (br d, J = 6.8 Hz, 2H), 4.51 (quintet,
J = 7.1 Hz, 1H), 4.84 (t, J = 6.6 Hz, 1H), 5.06 (t, J = 7.1 Hz, 1H), 5.33 (br d, J = 6.5 Hz, 1H), 6.61 (br s, 1H),
7.19 (br d, J = 6.2 Hz, 1H), 7.32 (t, J = 7.6 Hz, 2H), 7.41 (t, J = 7.0 Hz, 2H), 7.58 (d, J = 7.4 Hz, 2H), 7.76 (d,
J = 7.6 Hz, 2H); 13C-NMR
δ 18.1, 23.5, 34.9, 41.6, 47.1, 48.4, 50.3, 52.6, 59.1, 61.5, 62.4, 67.2, 120.0, 125.03,
125.06, 127.1, 127.8, 141.3, 143.7, 156.0, 167.8, 171.6, 172.9, 174.9; HRMS (TOF ES+) m/z calculated for
C30H34N4O7SNa (M + Na) 617.2046; found 617.2043.

Molecules 2018, 23, 1762
27 of 30
4. Summary and Conclusions
4.1. Synthesis and Use of Key Intermediate 13
The synthesis of Fmoc acetal resin 13 from the advanced intermediate amino resin
9 is
described (Scheme 3). Since ozonolysis of the allyl group precedes introduction of the R2CO group,
the strategy enables the incorporation of ozone-labile moieties such as furan and trimethoxyphenyl
at R2 for a variety of peptidomimetic and biomimetic scaffolds
1–5. Resin 13 also features two
orthogonally-related Fmoc and acetal protecting groups, which can be selectively removed under basic
and acidic conditions, respectively, thereby providing an Fmoc-based, SPPS approach to unnatural
peptides and peptidomimetics
syntheses of homoserine lactones 19a
are examples that contain ozone-labile substructures. Homoserine lactone 24c and many of the bicyclic
thiazolidine compounds listed in Table 2 offer examples of scaffolds and containing amino acid
residues. Most of the bicyclic thiazolidines were synthesized in 10–13 steps from commercially
1
–
5. The versatility of Fmoc acetal resin 13 is illustrated with the
–
c
(scaffold ) and the bicyclic thiazolidine 21c (scaffold ), which
1
3
1
3
3
available Boc-protected glycine, alanine, and phenylalanine on Merrifield resin. However, access to
the multiple scaffolds from the key orthogonally-protected intermediate 13 was often accomplished
in four or fewer steps. Several examples (36a–c, 30b–c, and 37b) represent the successful application
of peptide fragment condensation in which Cys-Ala-OMe is condensed with the Fmoc-Ala extended
analog of 13a–c.
4.2. NMR Characterization: Stereochemistry and Possible Secondary Structure
The cyclitive cleavage process in acetic acid at elevated temperatures using cysteine-based
nucleophiles to generate scaffold
3 compounds proceeds to afford primarily two diastereomers
in ratios from 1:1 to 4:1. These stereoisomers were separated by normal-phase or reverse-phase
chromatography, and their relative configurations were determined by one and two-dimensional nOe
studies. The difference in chemical shifts (∆δ) between diastereotopic protons at C-6 (the methylene
group of the lactam ring) is seen to be diagnostic. For the R1 = Bn and H series, ∆δ is significantly larger
for the
β α isomer that displays
isomer (major diastereoisomer), whereas for the R1 = Me series, it is the
the larger values. Thus, knowledge of the C-6 proton ∆δ values of the two major diastereomers in
the three series (R1 = H, Me, Bn) affords a predictive value in the assignment of the stereochemistry
of future compounds. Nuclear Overhauser enhancement studies also reveal a small enhancement of
signals from remote protons located on opposite ends of the four-residue peptidomimetic sequence of
α-30b, and may suggest that the molecule adopts a β-turn secondary structure in chloroform.
Supplementary Materials: The following are available online at http://www.mdpi.com/1420-3049/23/7/1762/
s1, Figure S1. Proton NMR Spectrum of 19a in CDCl3, Figure S2. Carbon-13 NMR Spectrum of 19a in CDCl3,
Figure S3. Proton NMR Spectrum of 19b in CDCl3, Figure S4. Carbon-13 NMR Spectrum of 19b in CDCl3,
Figure S5. Proton NMR Spectrum of 19c in CDCl3, Figure S6. Carbon-13 NMR Spectrum of 19c in CDCl3,
Figure S7. Proton NMR Spectrum of
α-21c and β-21c in CDCl3, Figure S8. Carbon-13 NMR Spectrum of α-21c
and 21c in CDCl3, Figure S9. Proton NMR Spectrum of 24c (earlier Rt) in CD3OD, Figure S10. Carbon-13 NMR
β
-
Spectrum of 24c (earlier Rt) in CD OD, Figure S11. Proton NMR Spectrum of 24c (later Rt) in CDCl3, Figure S12.
Carbon-13 NMR Spectrum of 24c3 (later Rt) in CDCl , Figure S13. Proton NMR Spectrum of
β-26c in CD3OD,
Figure S14. Carbon-13 NMR Spectrum of
Figure S16. Carbon-13 NMR Spectrum of 31 in CDCl3, Figure S17. Proton NMR Spectrum of α-33a in CDCl3,
β
-
26c in C3D3OD, Figure S15. Proton NMR Spectrum of 31 in CDCl3,
Figure S18. Carbon-13 NMR Spectrum of
Figure S20. Carbon-13 NMR Spectrum of
Figure S22. Carbon-13 NMR Spectrum of
Figure S24. Carbon-13 NMR Spectrum of
Figure S26. Carbon-13 NMR Spectrum of
Figure S28. Carbon-13 NMR Spectrum of
Figure S30. Carbon-13 NMR Spectrum of
Figure S32. Carbon-13 NMR Spectrum of
Figure S34. Carbon-13 NMR Spectrum of
Figure S36. Carbon-13 NMR Spectrum of
Figure S38. Carbon-13 NMR Spectrum of
Figure S40. Carbon-13 NMR Spectrum of
α
-
33a in CDCl3, Figure S19. Proton NMR Spectrum of
β
-
33a in CDCl3,
33b in CDCl3,
α-33b in CDCl3,
β
-
33a in CDCl3, Figure S21. Proton NMR Spectrum of β-
β
α
β
α
-
33b in CDCl3, Figure S23. Proton NMR Spectrum of
33b in CDCl3, Figure S25. Proton NMR Spectrum of
-
-
β
α-
-
33c in CDCl3,
33c in CDCl3,
34a in CDCl3,
34a in CDCl3,
35a in CDCl3,
35a in CDCl3,
33c in CDCl3, Figure S27. Proton NMR Spectrum of
33c in CDCl3, Figure S29. Proton NMR Spectrum of
-
β
α
α
β
-
β
α
α
-
-
-
34a in CDCl3, Figure S31. Proton NMR Spectrum of
34a in CDCl3, Figure S33. Proton NMR Spectrum of
35a in CDCl3, Figure S35. Proton NMR Spectrum of
-
-
-
β
-
35a in CDCl3, Figure S37. Proton NMR Spectrum of 35b in CDCl3,
β
-
β
-
35b in CDCl3, Figure S39. Proton NMR Spectrum of α-35b in CDCl3,
α-
35b in CDCl3, Figure S41. Proton NMR Spectrum of β-35c in CDCl3,

Molecules 2018, 23, 1762
28 of 30
Figure S42. Carbon-13 NMR Spectrum of
Figure S44. Carbon-13 NMR Spectrum of
β
-
35c in CDCl3, Figure S43. Proton NMR Spectrum of
35c in CDCl3, Figure S45. Proton NMR Spectrum of
α
-
35c in CDCl3,
α-
β
-
36a in CDCl3,
Figure S46. Carbon-13 Spectrum of
β
-
36a in CDCl3, Figure S47. Proton NMR Spectrum of
α
β
-
36a in CDCl3,
Figure S48. Carbon-13 NMR Spectrum of
Figure S50. Carbon-13 NMR Spectrum of
Figure S52. Carbon-13 NMR Spectrum of
Figure S54. Carbon-13 NMR Spectrum of
Figure S56. Carbon-13 NMR Spectrum of
Figure S58. Carbon-13 NMR Spectrum of
Figure S60. Carbon-13 NMR Spectrum of
Figure S62. Carbon-13 NMR Spectrum of
Figure S64. Carbon-13 NMR Spectrum of
Figure S66. Carbon-13 NMR Spectrum of
α
-
36a in CDCl3, Figure S49. Proton NMR Spectrum of 36b in CDCl3,
-
β
α
-
36b in CDCl3, Figure S51. Proton NMR Spectrum of α-36b in CDCl3,
-
36b in CDCl3, Figure S53. Proton NMR Spectrum of
α
-
36c in CDCl3,
36c in CDCl3,
30b in CDCl3,
α
-
36c in CDCl3, Figure S55. Proton NMR Spectrum of
36c in CDCl3, Figure S57. Proton NMR Spectrum of
30b in CDCl3, Figure S59. Proton NMR Spectrum of
β-
β
-
α
β
β
-
α
β
β
-
-
30b in CDCl3,
-
30b in CDCl3, Figure S61. Proton NMR Spectrum of
-
30c in CDCl3,
30c in CDCl3,
37b in CDCl3,
-
30c in CDCl3, Figure S63. Proton NMR Spectrum of
30c in CDCl3, Figure S65. Proton NMR Spectrum of
37b in CDCl3, Figure S67. Proton NMR Spectrum of
α-
α
-
α
β
-
α-
-
37b in CDCl3,
Figure S68. Carbon-13 NMR Spectrum of β-37b in CDCl3. Figure S69. Proton NMR Spectrum of (Fmoc-Ala)2O
in CDCl3 with Diisopropylurea (DIU), Figure S70. Proton NMR Spectrum of Boc-Cys(Trt)-Leu-OMe in CDCl3,
Figure S71. Carbon-13 NMR Spectrum of Boc-Cys(Trt)-Leu-OMe in CDCl3.
Author Contributions: Conceptualization, M.O. and W.S.; Formal analysis, G.S. and J.M.; Funding acquisition,
M.O. and W.S.; Investigation, G.S. and J.M.; Methodology, G.S. and J.M.; Supervision, M.O. and W.S.;
Writing–original draft, G.S.; Writing–review & editing, M.O. and W.S.
Funding: This research was funded by The National Institutes of Health [RO1-GM28193].
Acknowledgments: We are very grateful to Angela M. Hansen and Jonathan A. Karty of the Mass Spectrometry
Facility of Indiana University and Karl Dria for the high-resolution mass spectral measurements and to Maren
Pink of the Indiana University Molecular Structure Center for the x-ray crystal structure analysis and report for
compound β-26c.
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of compounds of Scaffolds
available from the authors.
2 and 4 and advanced intermediate resins 11a–c are
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