Development of Spiroligomer-Peptoid Hybrids

Article abstract of DOI:10.1021/acs.joc.7b01956

Creating functional macromolecules that possess the diversity and functionality of proteins poses an enormous challenge, as this requires large, preorganized macromolecules to facilitate interactions. Peptoids have been shown to interact with proteins, and combinatorial libraries of peptoids have been useful in discovering new ligands for protein binding. We have created spiroligomer-peptoid hybrids that have a spirocyclic core that preorganizes functional groups in three-dimensional space. By utilizing spiroligomers, we can reduce the number of rotatable bonds between functional groups while increasing the stereochemical diversity of the molecules. We have synthesized 15 new spiroligomer monomer amines that contain two stereocenters and three functional groups (67-84% yields from a common hydantoin starting material) as well as a spiroligomer trimer 25 with six stereocenters and five functional groups. These 16 amines were used to synthesize five first-generation spiroligomer-peptoids hybrids.

Full text of DOI:10.1021/acs.joc.7b01956

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Article
Development of Spiroligomer-Peptoid Hybrids
Justin D Northrup, Giulia Mancini, Claire R. Purcell, and Christian E. Schafmeister
J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.7b01956 • Publication Date (Web): 21 Nov 2017
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DEVELOPMENT OF SPIROLIGOMERꢀPEPTOID HYBRIDS
‡
‡
‡
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Justin D. Northrup, Giulia Mancini,§ Claire R. Purcell, and Christian E. Schafmeister *
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‡
Department of Chemistry, Temple University, Philadelphia, Pennsylvania 19122, United States
of America
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§Department of Chemistry, University of the Sciences, Philadelphia, Pennsylvania 19104, United
States of America
*corresponding author: meister@temple.edu
Abstract
O
O
O
N
O
N
N
HN
N
O
CbzN
N
O
N
O
N
O
O
O
O
O
O
N
H
2N
N
N
O
O
N
O
N
N
H
N
N
O
O
O
HN
O
NH
O
N
O
NCbz
NH
O
N
CbzN
O
*
NH
N
N
O
N
O
O
O
N
N
O
CbzN
N
O
O
N
O
CO2tꢀBu
CbzN
O
O
N
N
NH
O
H
N
O
2
N
O
Creating functional macromolecules that possess the diversity and functionality of proteins poses
an enormous challenge, as this requires large, preorganized macromolecules to facilitate
interactions. Peptoids have been shown to interact with proteins, and combinatorial libraries of
peptoids have been useful in discovering new ligands for protein binding. We have created
spiroligomerꢀpeptoid hybrids that have a spirocyclic core that preorganizes functional groups in
threeꢀdimensional space. By utilizing spiroligomers, we can reduce the number of rotatable bonds
between functional groups, while increasing the stereochemical diversity of the molecules. We
have synthesized 15 new spiroligomer monomer amines that contain two stereocenters and three
functional groups (67ꢀ84% yields from a common hydantoin starting material), as well as a
spiroligomer trimer 25 with six stereocenters and five functional groups. These 16 amines were
used to synthesize five firstꢀgeneration spiroligomerꢀpeptoids hybrids.
Introduction
To create functional macromolecules with the protein binding, catalytic, and membrane
channel activities of folded proteins, it is necessary to create macromolecules that organize many
diverse functional groups in preꢀorganized threeꢀdimensional constellations. Many approaches to
this challenge have been developed including peptide synthesis, stapledꢀpeptides,1,2 betaꢀ and
gammaꢀpeptides,3,4 triazolamers,5,6 and Nꢀamino peptides,7,8 among many others. Two other
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approaches are spiroligomers and peptoids, which are both peptidomimetic oligomers
synthesized from readily available building blocks, with spiroligomers being synthesized from a
unique set of bisꢀamino acid monomers derived from transꢀ4ꢀhydroxyꢀL-proline,9,10 while peptoids
are typically synthesized from bromoacetic acid and a primary amine via a submonomer
method.11
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Peptoids have been studied for decades and have been utilized for a wide variety of
purposes: Peptoids have been demonstrated as biologically active molecules ranging from
protein secondary structure mimetics12ꢀ16 to antimicrobial agents17ꢀ19 and other therapeutics;20
they have been used to construct nanomaterials such as nanotubes21,22 and nanosheets;23,24 and
metal binding peptoids.25ꢀ28 Peptoids are assembled using a very convenient submonomer
approach, which allows for efficient synthesis of long oligomers with excellent yield and purity.
Compared to peptides, the side chain is moved from the αꢀcarbon to the backbone nitrogen
creating a tertiary amide linkage. This removes the backbone chirality, any interꢀ or intrachain
hydrogen bonding of the backbone, and leads to increased flexibility of the peptoid backbone
relative to peptides due to rotational freedom around the ω dihedral.29ꢀ31 To increase the degree
of preꢀorganization, Kirshenbaum,32 and others, have developed a macrocyclization strategy for
the peptoid core.18,19,33ꢀ38 These macrocycles range from only three residues in the peptoid core,33
to six, eight, or more residues.32 Peptoid containing macrocycles have been utilized for many
applications, including protein inhibitors,39,40 antimicrobials,18 and antifreeze agents.41
Spiroligomers are excellent at preꢀorganizing functional groups, as the fusedꢀspirocyclic
structures do not allow any free rotation throughout the backbone.9,42,43 Spiroligomers also benefit
from having a large pool of side chains to utilize, as almost any aldehyde, ketone, alkyl halide,
isocyanate, or amino acid can be incorporated to provide a functional group.44,45 Functionalized,
preorganized spiroligomers have been utilized for a variety of applications such as a
transesterification catalyst,46 a prolineꢀaldol catalyst,47 a spiroligomer that binds MDM2,48 and a
spiroligomer that forms supramolecular metal binding complexes,49 among many others.
The submonomer synthesis of peptoids allows the incorporation of a large variety of
functional groups as primary amines. The first peptoids that incorporated large side chains were
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glucoseꢀbased glycopeptide mimetics,50 with interest in these glycopeptoids ongoing.51,52 Large
side chains have also been incorporated into peptoids using a 3+2 Huisgen’s cyclization (a click
reaction),53 after the displacement step with propargyl amine.54ꢀ56
9
Herein, we investigate linking complex, stereochemicalyꢀpure spiroligomers that display
multiple functional groups through a peptoid backbone. This approach would provide new
molecules for combinatorial screening of proteinꢀprotein interaction inhibitors and catalysts. To
achieve this, we need to incorporate a protected primary amine in the spiroligomer synthesis, and
we need to develop efficient conditions for integrating these complex amines into peptoids. In
doing so, we can then combine any number of uniquely functionalized spiroligomers into any
sequence of a peptoid for a variety of applications.
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Figure 1. Three theoretical molecules displaying identical functional groups with increasing
preorganization. (A) A theoretical peptoid 15ꢀmer (B) A theoretical spiroligomerꢀpeptoid hybrid
5ꢀmer, incorporating five spiroligomers which each contain three functional groups and two
stereocenters (15 total groups, 10 total stereocenters) (C) A spiroligomerꢀpeptoid hybrid 3ꢀ
mer, incorporating three spiroligomers which each contain five functional groups and six
stereocenters (15 total groups, 18 total stereocenters). (D) Depiction of a peptoid showing
omega, phi, and psi dihedral angles.
As shown in Figure 1A, a prototypical linear peptoid 15ꢀmer has a very high ratio of
rotatable bonds to functional groups. Each peptoid residue has a φ, ψ, and ω rotatable bond
(Figure 1D), which means there are 42 rotatable bonds in the peptoid backbone. In comparison, a
spiroligomerꢀpeptoid hybrid like the one shown in Figure 1B incorporates fifteen functional groups
and ten stereoꢀcenters, yet it contains only 27 rotatable bonds in the peptoid backbone (a 35%
reduction compared to the prototypical peptoid). Spiroligomers similar in size and functional group
display to the amines for Figure 1B have previously been used by our group as small, organic
catalysts.47 If we were to incorporate a spiroligomer trimer into a peptoid (Figure 1C), fifteen
functional groups can be incorporated with just three spiroligomer trimers containing eighteen
total stereoꢀcenters, and only 21 rotatable bonds in the backbone (a 50% reduction compared to
the prototypical peptoid). For instance, between groups R1ꢀR5 on the peptoid there are 12
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rotatable bonds between residues in the peptoid backbone, whereas between groups R1ꢀR5 on
the spiroligomer, there are no rotatable bonds in the spiroligomer core. Spiroligomers similar in
size and functional group display to the amines for Figure 1C have been shown to bind in the P53
groove of HDM2.48 These hypothetical spiroligomerꢀpeptoid hybrids represent a wide range of
functional group and stereochemical diversity relative to flexibility, with increasing diversity and
increasing preꢀorganization as the size of the spiroligomer is increased. Each of these
functionalized spiroligomers approximates the structural complexity of a smallꢀmolecule fusedꢀ
ring natural product containing two, four, six or more fused rings and several functional groups in
precise threeꢀdimensional constellations. Three to five of these spiroligomer domains can be
displayed – in close proximity – on a peptoid chain and provide a molecule with a great deal of
preorganization that has a large surface area which could potentially interact with other
molecules, and at the same time display flexibility between the domains to accommodate different
protein surfaces. One could imagine molecules like these binding to multiple shallow grooves on
a protein surface through many nonꢀcovalent contacts. By incorporating the structural rigidity and
stereochemical diversity of spiroligomers with the modular linking chemistry provided by peptoid
synthesis, this should facilitate the rapid discovery of new molecules of interest for catalysis,
disrupting proteinꢀprotein interactions, and exploring large surface area hostꢀguest interactions.
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RESULTS AND DISCUSSION
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Synthesis of Spiroligomer Primary Amines
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To facilitate the synthesis of spiroligomerꢀpeptoid hybrids, we must first synthesize a
variety of spiroligomers displaying primary amines. The building blocks of spiroligomers are the
four stereoisomers of the proline hydantoin intermediate in spiroligomer bisꢀamino acid synthesis
that we routinely synthesize on a 600 mmol scale (230g of mixed diastereomers and we have
synthetic access to all four stereoisomers).10 We have recently developed chemistry that allows
us to directly functionalize the hydantoin by exploiting the varying reactivity between the imide
and amide nitrogen atoms of the hydantoin.45 As shown in Scheme 1, we can install a functional
Scheme 1: Synthesis of spiroligomer amine 5 from (2S, 4R) proline hydantoin 1. a) i. DMF,
K2CO3, 1ꢀiodoꢀ2ꢀmethylpropane; ii. Et2NH; b) 1. TFA; 2. i. HOAT, EDCI, DMF, DCM; ii.
benzylamine; c) 1. 1:1 HBr/DCM; 2. DMF, DCM, DIPEA, BocꢀGlyꢀOAt; d) i. DMF, K2CO3,
benzylbromide; ii. Et2NH
group onto the imide of hydantoin 1 via direct alkylation with either alkyl, allyl, or benzyl halides to
form 2. We then remove the tertꢀbutyl protecting group with TFA, and couple a primary amine to
form 3. This is followed by removal of the Cbz protecting group and coupling an NꢀBoc protected
amino acid to make 4. Finally, another alkylation with either an allyl or benzyl halide to install a
functional group on the amide of the hydantoin provides the final spiroligomer NꢀBoc protected
amine 5. We observe that the hydantoin amide is functionalized selectively in the presence of the
secondary amide and secondary carbamate on 4. When a small, highly reactive electrophile such
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as methyl iodide is used, we observe less than 3% alkylation to what is most likely the secondary
amide N11 (Scheme 2). The evidence that the alkylation is taking place selectively on the
1
hydantoin amide was determined from the H NMR for the alkylated products. If N6 were
9
unalkylated, we would expect to see a triplet for NH16 and a singlet for NH6, yet we see two
amide NH signals that show either a triplet for NH16 and a triplet for NH11 or a triplet for NH16
and a doublet for NH11, depending on whether the carbon attached to N11 is a methylene or
methyne, respectively. This chemistry was used with varying electrophiles and amines to form the
spiroligomer amines 6 to 18 shown in Table 1.
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Scheme 2. a) DMF, 2 equiv CH3ꢀI, 2 equiv K2CO3, 24 hours. Methylation of N6 is the only
alkylation that led to the formation of another product, which has the same mass as 18ꢀX and we
presume that it has the structure shown above (this byproduct was not recovered)
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Table 1. Spiroligomer amines synthesized from
proline hydantoin 1. Yield represents total yield of
the amine from 1.
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Testing the Minimum Requirements for Peptoid Chemistry
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To make efficient use of these complex and synthetically derived amines we carried out a
series of trials to determine the minimum amount of the amines required to achieve peptoid
couplings while minimizing crossꢀlinking (CꢀL) of the nascent peptiods on solid support. We varied
the number of equivalents of amine relative to the resin loading on solid support and the
concentration of amine required for peptoid chemistry to function reliably. We utilized
commercially available benzylamine to obtain the baseline conditions for peptoid chemistry
shown in Table 2, below. Varying the concentration of amine in the peptoid submonomer
synthesis,11,26 we determined that 3 equivalents of amine relative to resin loading (Rink Amide
polystyrene, 0.63 mmol/g) with one equivalent of exogenous base (DIPEA; extra base lead to
uncharacterized side products), at a concentration of 150 mM of benzylamine in DMF were the
minimum equivalents and concentration that achieved efficient peptoid coupling and suppressed
crossꢀlinking to below 2%. Crossꢀlinked products were observed and quantitated using HPLCꢀ
MS/UVꢀVIS. Any further decrease in equivalents or concentration resulted in significant crossꢀ
linking of the resin, whereby a secondary amine on resin would react with another bromoacetate
on resin before an amine in solution could react, thereby terminating the sequence (shown in
Scheme 3). On Rink Amide Tentagel® resin (loading 0.37 mmol/g), we found that we could lower
the required concentration of amine to 125 mM, but the minimum number of equivalents required
remained at 3 equivalents. A 2ꢀClꢀTrityl chloride polystyrene resin (loading 1.1 mmol/g) required
an increase in both the equivalents and concentration of benzylamine (5 equiv and 250 mM,
respectively) to ensure efficient coupling and suppress crossꢀlinking to less than 2%. These
results were achieved at room temperature with four hour couplings.
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Table 2. Minimum equivalents required for peptoid
couplings on various resins
Amine
Conc.
Resin
Loading
Equiv. Equiv.
Trial
% C-L (UV)
Amine
Base
(mM) (mmol/g)
9
1
2
20
3
0
2
2000
150
0.63
0.63
< 1%
Side
Product
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3
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3
2
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5
1
1
1
1
1
1
1
1
1
1
150
10
0.63
0.63
0.63
0.63
0.63
0.63
0.37
0.37
1.1
< 2%
6-8%
6-8%
2-3%
2-3%
2-3%
< 2%
6-8%
8-10%
< 2%
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30
6
100
100
150
125
75
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12
150
250
1.1
Scheme 3. Benzylamine is combined with the resin (A);
however, low concentration of amine in solution allows
for an amine on resin to displace a neighboring bromine
(B). This results in a crosslinked resin, and the
termination of two peptoid sequences (C). CꢀL(UV)
stands for crossꢀlinking as determined using HPLC with
UV detection.
Having determined the requirements of peptoid chemistry at sub molar concentrations,
we proceeded to synthesize several variants of spiroligomerꢀpeptoid hybrids. Any primary amine
we use would first need to be Boc deprotected, as shown in Scheme 4, which was achieved with
a 50% TFA / DCM mix, followed by concentration in vacuo. The free base of the amine 17 was
obtained by extracting the trifluoroacetate salt with EtOAc and 1M KOH, followed by two washes
with an equal volume of 1M KOH, which were back extracted with EtOAc. The Bocꢀprotected
amines are stable at room temperature for several years; however, if the free amine is left dry at
room temperature or in solution, we observed slow, spontaneous formation of the
diketopiperazine (DKP) 19. To avoid this, the amine freeꢀbase was concentrated under reduced
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pressure to yield a foamy solid, which is stable for over a year (dry, ꢀ20 °C). Unreacted amine in
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the peptoid coupling can be recovered, protected, and stored.
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Scheme 4. Generation of the spiroligomer free
amine 17a from 17. Spontaneous DKP formation of
spiroligomer free amines is possible at room
temperature in solution.
Synthesis of Spiroligomer Peptoid Hybrids
Scheme 5 demonstrates the synthesis of spiroligomerꢀpeptoid hybrid 1 (SPHꢀ1) on Rink Amide
polystyrene resin using standard submonomer synthesis and spiroligomer primary amines. This
Scheme 5. Peptoid synthesis of the spiroligomerꢀpeptoid hybrid, SPHꢀ1 (a) Bromoacetic acid,
DIC, DMF; (b) DMF, 150 mM 7, 1 equiv DIPEA (c) DMF, 150 mM 14, 1 equiv DIPEA; (d) DMF,
150 mM 15, 1 equiv DIPEA; (e) DMF, 150 mM 11, 1 equiv DIPEA; (f) DMF, 150 mM 16, 1
equiv DIPEA; (g) TFA, neat.
spiroligomerꢀpeptoid hybrid incorporates 15 functional groups and ten stereocenters on preꢀ
organized spiroligomer side chains. HPLC chromatrograms documenting each elongation of
SPHꢀ1 are highlighted in Figure 2. There is a solvent injection peak (denoted I) and a resin
impurity (denoted as II) that are present in each chromatogram (Figure 2AꢀD, test cleavages)
which do not change. The intended product III is the only other peak in the test cleavages. Figure
2E shows the crude HPLC chromatogram for SPHꢀ1, which indicates a high level of fidelity for
each coupling step. The peak around 2.8 min is from the final spiroligomer amine, which indicated
longer washing steps were needed to recover unreacted amine from the resin. The spiroligomerꢀ
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peptoid hybrid can then be purified to remove the small impurities present in the crude material
(Figure 2FꢀG).
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Figure 2. HPLC Chromatograms (AꢀF) following the synthesis of SPHꢀ1 (I = DMSO for AꢀD,
50% ACN/Water for EꢀG, II = resin impurity, III = desired product): (A) Test Cleavage of SPHꢀ0.1
(B) Test cleavage of SPHꢀ0.2 (C) Test cleavage of SPHꢀ0.3 (D) Test cleavage of SPHꢀ0.4 (E)
Full resin cleavage for SPHꢀ1, crude (F, G) HPLCꢀMS for purified SPHꢀ1.
To test the fidelity of a spiroligomerꢀpeptoid hybrid with increasing chain length, we
synthesized both a 9ꢀmer (SPHꢀ2, 58% yield) and a 12ꢀmer (SPHꢀ3, 57%) incorporating 3 and 6
different amines, respectively (Figure 3A, B). Other spiroligomerꢀpeptoid hybrids that we have
synthesized include custom linkers incorporating propargyl (postꢀpeptoid synthesis modification
potential) or bromobenzyl side chains (the bromobenzyl group provides an easily identified mass
spectroscopy signature due to the natural isotopic abundance of bromine). Installation of a cꢀ
terminal methionine residue to facilitate CNBr mediated cleavage from resin has been tested
successfully on various spiroligomerꢀpeptoid hybrids (Figure 3C, SPHꢀ4, 22% yield). These types
of hybrids would be most beneficial to those interested in onꢀresin screening for compounds, as it
allows for the deprotection of any side chains, while leaving the peptoid still attached to the resin.
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O
O
O
O
O
O
C
A
O
N
O
O
N
N
N
N
N
N
O
N
O
N
O
O
N
O
CF3
N
O
O
NH2
N
O
O
N
N
N
O
HN
N
HN
O
HN
HN
O
O
O
O
O
O
O
H
O
NHCbz
NHCbz
O
H
H
N
N
N
N
N
N
NH
N
N
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O
O
O
O
O
O
O
O
O
O
O
O
O
O
NH
N
N
N
N
O
N
N
O
N
N
N
HN
H2N
O
N
NH
O
O
H2N
F3C
O
O
N
O
O
H
N
NH2
O
O
NHCbz
O
N
NH
H
O
N
N
N
N
N
N
N
N
N
O
O
O
O
O
O
N
O
N
N
O
F3C
F
3C
O
CF3
CF3
F3
C
SPHꢀ4
SPHꢀ2
NHCbz
N
NHCbz
NHCbz
NHCbz
N
NHCbz
N
NHCbz
B
N
O
O
O
O
O
O
O
O
N
O
N
O
N
N
O
N
H
N
N
N
O
N
O
N
H2N
O
N
O
O
O
O
O
F3C
O
F3C
O
NH
O
H
N
O
NH
N
O
N
O
N
NH
N
NH
O
N
O
N
N
N
NH
O
O
O
O
O
N
N
N
N
O
O
N
O
N
N
O
O
O
O
CF3
SPHꢀ3
F3C
O
Figure 3. Three spiroligomerꢀpeptoid hybrids of varying length, functional groups, and resin
linker. (A) SPHꢀ2, a peptoid 9ꢀmer incorporating 6 spiroligomers (B) SPHꢀ3, a peptoid 12ꢀmer
incorporating 6 spiroligomers (C) SPHꢀ4, a hybrid peptoid containing 8 residues, four of which
are spiroligomers, and an initial methionine residue for CNBr mediated cleavage.
Synthesis of a spiroligomerꢀpeptoid hybrid containing a spiroligomer trimer
To further reduce the number of rotatable bonds in the peptoid backbone, we have
synthesized a spiroligomerꢀpeptoid hybrid containing three copies of a highly preorganized,
spiroligomer trimer. Spiroligomer trimers have been demonstrated within our lab as designed
catalysts46 or for protein binding.48 Incorporating large, structured macromolecules like these will
be used to create even more functional, preorganized macromolecules. The trimer 25 was
synthesized from a bifunctionalized proline hydantoin 20 and two bisꢀamino acids 21 and 23, as
shown in Scheme 6. The Cbz and tꢀBu protected, dialkylated proline hydantoin 20 was treated
with 1:1 DCM/(33% HBr/AcOH) for 30 minutes, after which the solvent was removed in vacuo,
and the molecule left on a high vacuum pump overnight. Bisꢀamino acid 21 was preactivated
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under inert conditions with EDC and HOAt in a 1:1 mix of anhydrous DMF/DCM for 1.5 h. This
preactivation is achieved without base so the bisꢀamino acid does not react with itself.
Deprotected compound 20 was dissolved in a minimal amount of anhydrous DMF, and added to
the preactivated bisꢀamino acid 21 along with DIPEA. After stirring overnight, more EDC was
added to the reaction to close the diketopiperazine ring to afford the spiroligomer dimer 22.
Spiroligomer dimer 22 is purified via flash chromatography (gradient from 0ꢀ100% ethyl acetate in
hexanes) in 46% yield.
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N
O
N
N
O
N
N
O
O
O
O
O
a
N
O
O
N
N
O
O
b
O
CbzN
c
O
N
O
N
N
N
N
N
tꢀBuO
N
N
O
O
O
O
O
20
CbzN
tꢀBuO
N
O
HO
HN
N
O
O
O
HO
HN
O
CO2tꢀBu
CbzN
O
22
CbzN
tꢀBuO
CO2tꢀBu
Cbz
23
N
25
O
21
24
Cbz
O
N
H
BocHN
Scheme 6. Synthesis of the spiroligomer trimer 25 (a) i. 1:1 DCM/(33% HBr/AcOH); ii.
(Preactivated 21: HOAT, EDC, 1:1 dry DMF/DCM, 1.5 h), DMF, DIPEA; (b) i. 1:1 DCM/(33%
HBr/AcOH); ii. (Preactivated 23: HOAT, EDC, 1:1 dry DMF/DCM, 1.5 h), DMF, DIPEA; (c) i.
95:4:1 TFA/H2O/TIPS, 1 h; ii. EDC, HOAT, 1:1 dry DMF/DCM, 1.5 h; iii. Nꢀbocꢀ
ethylenediamine
Dimer 22 was deprotected with 1:1 DCM/(33% HBr/AcOH) for 30 minutes, the solvent
removed in vacuo, and held under high vacuum overnight. Bisꢀamino acid 23 was preꢀactivated
under inert conditions with EDC and HOAt in a 1:1 mix of anhydrous DMF/DCM for 1.5 h. This
preactivation is achieved without base so the bisꢀamino acid does not react with itself. Dimer 22
was dissolved in a minimal amount of anhydrous DMF, and added along with DIPEA to the
preactivated bisꢀamino acid 23 to afford the spiroligomer trimer 24. Spiroligomer trimer 24 is then
purified via flash chromatography (gradient from 0ꢀ100% ethyl acetate in hexanes) in 21% yield.
Spiroligomer 24 can then be treated with a 95:4:1 TFA/H2O/TIPS mixture to remove the tꢀBu
protecting group, then following the removal of the solvent, this spiroligomer is preactivated using
EDC and HOAt in a 1:1 mix of dry DMF/DCM for 1.5 h. The preactivated spiroligomer was
combined with NꢀBocꢀethylenediamine to provide spiroligomer trimer 25, which was utilized to
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make SPHꢀ5 shown in Figure 4 (25% purified yield) using standard peptoid synthesis. This
spiroligomer trimer is, to the best of our knowledge, the largest amine utilized for the
displacement step of a peptoid synthesis, containing five unique functional groups and six
stereocenters per residue. This means the spiroligomerꢀpeptoid hybrid SPHꢀ5 contains 15
functional groups and 18 stereocenters, over all of which we have total control.
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Figure 4. Comparisons of theoretical peptoids PEPꢀ1 (15mer) and PEPꢀ2 (5mer) vs SPHꢀ5 and
amine 25. (A) Chemdraws for PEPꢀ1, PEPꢀ2, SPHꢀ5, and 25. (B) CANDO models with solventꢀ
excluded surface for PEPꢀ1 (surface area = 1743 Å2), PEPꢀ2 (surface area = 628.3 Å2), SPHꢀ5
(surface area = 2385 Å2), and 25 (surface area = 774.6 Å2).
By incorporating these large preorganized amines, we have successfully reduced the
number of peptoidꢀbackbone rotatable bonds by 50%. This was accomplished while increasing
the potential surface area available for interactions as shown in Figure 4B, which depicts the
solventꢀexcluded surface for two theoretical peptoids PEPꢀ1 and PEPꢀ2, as well as hybrid SPHꢀ5
and its component amine 25. When compared to a theoretical peptoid (PEPꢀ1) with the same
functional groups, SPHꢀ5 increases the total surface area by 33%. Similarly, when compared to a
theoretical peptoid (PEPꢀ2) with the same functional groups, amine 25 increases the total surface
area by 23%. This shows that preorganization of functional groups on a spiroligomerꢀpeptoid
hybrid will not only reduce the number of backbone rotatable bonds, but it will also increase the
potential surface area available for hostꢀguest or proteinꢀprotein interactions. These large
spiroligomer trimers, like the smaller monomers shown previously, can be used sequentially, or
interspersed with smaller units, indicating that they would make excellent candidate amines for
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future work. Furthermore, large spiroligomer trimers preorganize many more groups than the
spiroligomer monomer amines or regular peptoid amines, thus reducing any entropic penalty that
would be associated when binding a protein for example.
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Conclusion
We have successfully synthesized 16 new spiroligomers containing three to five functional
groups, two to six stereocenters, and a protected primary amine, which we have used to
incorporate the spiroligomers into peptoids of varying lengths. To the best of our knowledge, this
is the first reported synthesis which utilized large, preorganized, spirocyclic amines during the
displacement step of peptoid synthesis. Notably, this work featured the use of spiroligomer trimer
25, which organizes five functional groups on a spiroligomer backbone containing six
stereocenters and no interior rotatable bonds. This spiroligomer was used to make the
spiroligomerꢀpeptoid hybrid SPHꢀ5, which contains 18 stereocenters and 15 functional groups
across three peptoid residues. A linear peptoid with the same number of functional groups would
have 42 rotatable bonds in the backbone, whereas this hybrid has only 6 in the backbone and 15
in the linkers, a 50% reduction in the number of rotatable bonds for the same number of
functional groups. This preorganization of functional groups will help facilitate the rapid discovery
of novel structures for hostꢀguest interactions, proteinꢀprotein interactions, and catalysis. Now that
we can synthesize these new hybrid peptidomimetics, further work will be focused on developing
spiroligomerꢀpeptoid hybrids that display the appropriate side chains for these important
interactions.
Experimental Details
General Procedure 1: Mono alkylation of P4srZBHyd
To a stirred mixture of 1 in DMF [100 mM] was added 0.75ꢀ0.85 equiv of alkyl, allyl, or benzyl
halide (equiv dependent on salt content of the specific batch 1, which can be determined utilizing
an internal standard) along with 1.5 equiv of K2CO3. The reaction proceeded at room temperature
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for 2ꢀ24 h, dependent on electrophile, and the progress checked via LCMS. The reaction was
diluted with four times the reaction volume of EtOAc and washed with water, saturated
ammonium chloride solution, and brine. The organic layer was dried with Na2SO4, and
concentrated in vacuo to yield 26ꢀ29 as foamy offꢀwhite to yellow solids. 1H / 13C NMR spectra for
compounds 26ꢀ29 are available in the supporting information.
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Compound 26 ꢀ 7ꢀbenzyl 8ꢀ(tertꢀbutyl) (5R,8S)ꢀ3ꢀ(naphthalenꢀ2ꢀylmethyl)ꢀ2,4ꢀdioxoꢀ1,3,7ꢀ
triazaspiro[4.4]nonaneꢀ7,8ꢀdicarboxylate ꢀ Compound 26 was synthesized using General
Procedure 1, with 1 [(2S, 4R) 7.5 mmol] and 2ꢀ(bromomethyl)naphthalene [6 mmol (0.80 equiv)].
1
Recovered yield was quantitative and the product used without purification. H NMR (500 MHz,
CDCl3) 1.42 (9H, s, rotameric), 2.36 (1H, m), 2.48 (1H, m), 3.81 (2H, m), 4.45 (1H, m), 4.78 (1H,
s, rotameric), 5.07 (2H, m), 7.25ꢀ7.84 (13H), 9.08 (1H, s, rotameric); 13C NMR (125 MHz, CDCl3,
rotamers present) 27.8, 34.1, 39.6, 40.6, 42.8, 55.7, 58.4, 65.9, 66.8, 67.6, 82.4, 125.8, 126.2,
126.3, 126.4, 126.6, 126.8, 127.6, 127.8, 127.9, 128.0, 128.1, 128.2, 128.5, 128.6, 128.7, 132.9,
133.0, 133.2, 135.1, 135.8, 136.0, 154, 7, 156.3, 169.8, 172.9; HRMS (ESI/QꢀTOF) m/z: (M+K)+
calcd for C30H31N3O6K 568.1844, found 568.1840
Compound
27
ꢀ
7ꢀbenzyl
8ꢀ(tertꢀbutyl)
(5R,8S)ꢀ3ꢀbenzylꢀ2,4ꢀdioxoꢀ1,3,7ꢀ
triazaspiro[4.4]nonaneꢀ7,8ꢀdicarboxylate ꢀ Compound 27 was synthesized using General
Procedure 1, with 1 [(2S, 4R) 15 mmol] and benzyl bromide [12 mmol (0.80 equiv)]. Recovered
1
yield was quantitative and the product used without purification. H NMR (500 MHz, CDCl3) 1.43
(9H, s, rotameric), 2.31 (1H, dd, rotameric, J = 14.5, 8.1), 2.49 (1H, ddd, J = 18.0, 13.4, 9.2), 3.84
(2H, m), 4.43 (1H, dt, J = 24.4, 8.6), 4.63 (2H, s), 5.08 (2H, m), 7.30 (11H, m); 13C NMR (125
MHz, CDCl3, rotamers present) 27.9, 39.7, 40.7, 42.5, 55.7, 58.9, 65.5, 66.2, 67.6, 82.4, 127.7,
127.9, 128.1, 128.2, 128.5, 128.6, 128.8, 135.7, 136.0, 154.5, 155.9, 169.7, 171.7; HRMS
(ESI/QꢀTOF) m/z: (M+K)+ calcd for C26H29N3O6K 518.1688, found 518.1699
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Compound 28 ꢀ 7ꢀbenzyl 8ꢀ(tertꢀbutyl) (5R,8S)ꢀ3ꢀ(cyclopropylmethyl)ꢀ2,4ꢀdioxoꢀ1,3,7ꢀ
triazaspiro[4.4]nonaneꢀ7,8ꢀdicarboxylate ꢀ Compound 28 was synthesized using General
Procedure 1, with 1 [(2S, 4R) 7.5 mmol] and (bromomethyl)cyclopropane [6.0 mmol (0.80 equiv)].
1
Recovered yield was quantitative and the product used without purification. H NMR (500 MHz,
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CDCl3) 0.33 (2H, d, J = 5.8), 0.49 (2H, d, J = 7.8), 1.13 (1H, m), 1.45 (9H, s, rotameric), 2.40 (1H,
dd, rotameric, J = 14.3, 8.2), 2.54 (1H, ddd, J = 18.2, 13.4, 9.0), 3.35 (2H, dd, J = 7.2, 3.5), 3.81
(2H, m), 4.47 (1H, dt, J = 24.8, 8.3), 5.11 (2H, m), 7.32 (6H, m); 13C NMR (125 MHz, CDCl3,
rotamers present) 3.7, 10.2, 27.9, 40.7, 43.7, 55.8, 58.9, 66.1, 67.6, 82.4, 127.9, 128.2, 128.5,
136.0, 154.5, 156.8, 169.7, 172.2 HRMS (ESI/QꢀTOF) m/z: (M+K)+ calcd for C23H29N3O6K
482.1688, found 482.1693
Compound
29
ꢀ
7ꢀbenzyl
8ꢀ(tertꢀbutyl)
(5R,8S)ꢀ3ꢀisobutylꢀ2,4ꢀdioxoꢀ1,3,7ꢀ
triazaspiro[4.4]nonaneꢀ7,8ꢀdicarboxylate – Compound 29 was synthesized using General
Procedure 1, with 1 [(2S, 4R) 15 mmol] and 1ꢀiodoꢀ2ꢀmethylpropane [18 mmol (1.2 equiv)].
1
Recovered yield was quantitative and the product used without purification. H NMR (500 MHz,
CDCl3) 0.88 (6H, d, J = 6.7), 1.45 (9H, s, rotameric), 2.05 (1H, m), 2.38 (1H, m), 2.53 (1H, ddd, J
= 18.2, 13.3, 9.2), 3.31 (2H, dd, J = 7.3, 3.1), 3.78 (2H, m), 4.48 (1H, dt, J = 24.4, 8.6), 5.11 (2H,
m), 7.35 (6H, m); 13C NMR (125 MHz, CDCl3, rotamers present) 20.9, 27.8, 27.9, 39.8, 40.8,
46.1, 55.8, 56.0, 58.4, 58.9, 65.2, 65.9, 67.6, 82.4, 127.8, 127.9, 128.2, 128.5, 135.9, 136.0,
154.2, 154.5, 156.6, 156.8, 169.7, 169.9, 172.2, 172.4; HRMS (ESI/QꢀTOF) m/z: (M+K)+ calcd for
C23H31N3O6K 484.1844, found 484.1861
General Procedure 2: Deprotection of C2 tꢀBu group and coupling
Each of 26ꢀ29 was placed in a RB flask and then treated with 95% TFA / TIPS for 1 h, with the
reaction progress checked via LCMS. Upon successful deprotection of the tertꢀbutyl group, the
solvent was removed in vacuo, and placed on a high vacuum pump overnight. To an inert
atmosphere RB flask containing the free acid in dry DMF/DCM (1:1 ratio, [100 mM]) was added 1ꢀ
Ethylꢀ3ꢀ(3ꢀdimethylaminopropyl)carbodiimide hydrochloride (EDC, 1.5 equiv) and 1ꢀHydroxyꢀ7ꢀ
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azabenzotriazole (HOAT, 3 equiv), then stirred for 1 h. An amine (3 equiv) and DIPEA (2 equiv)
were then added, and the reaction proceeded for 2 h, at which point the progress was checked
via LCMS. Upon completion, the reaction was diluted with four times the reaction volume of
EtOAc and washed with saturated ammonium chloride solution, saturated sodium bicarbonate
solution, and brine. The organic layer was dried with Na2SO4, and concentrated in vacuo to yield
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1
30ꢀ35 as foamy yellow solids. H / 13C NMR spectra for compounds 30ꢀ35 are available in the
supporting information.
Compound 30 benzyl (5R,8S)ꢀ8ꢀ(isobutylcarbamoyl)ꢀ3ꢀ(naphthalenꢀ2ꢀylmethyl)ꢀ2,4ꢀdioxoꢀ
1,3,7ꢀtriazaspiro[4.4]nonaneꢀ7ꢀcarboxylate – Compound 30 was synthesized using General
Procedure 2 and compound 26, with HOAt (22.5 mmol), EDC (11.25 mmol), Isobutylamine (22.5
mmol), DIPEA (15 mmol), and 75 mL of 1:1 DMF/DCM ꢀ Recovered yield was quantitative and
the product used without purification. 1H NMR (500 MHz, CDCl3) 0.82 (6H, d, J = 6.4), 1.89 (1H,
m), 2.25 (1H, m), 2.64 (1H, m), 2.99 (1H, m), 3.05 (1H, sep, J = 6.5), 3.68 (1H, m), 3.84 (1H, d, J
= 11.3), 4.59 (1H, m), 4.78 (2H, s, rotameric), 5.05 (2H, m), 7.02 (1H, m), 7.21 (5H, m), 7.44 (3H,
m), 7.66 (1H, m), 7.78 (4H, m); 13C NMR (125 MHz, DMSOꢀd6, rotamers present) 20.0, 28.0,
28.1, 40.6, 41.5, 41.7, 46.0, 56.1, 56.5, 58.7, , 59.2, 65.1, 65.8, 66.2, 125.4, 125.5, 125.7, 125.8,
126.0, 126.3, 127.1, 127.5, 127.6, 127.7, 127.8, 128.2, 128.3, 132.2, 132.8, 134.0, 136.5, 136.6,
153.5, 152.7, 155.4, 155.5, 170.4, 170.7, 172.4; HRMS (ESI/QꢀTOF) m/z: (M+H)+ calcd for
C30H33N4O5 529.2445, found 529.2446
Compound 31
ꢀ
benzyl (5R,8S)ꢀ3ꢀbenzylꢀ8ꢀ(cyclopropylcarbamoyl)ꢀ2,4ꢀdioxoꢀ1,3,7ꢀ
triazaspiro[4.4]nonaneꢀ7ꢀcarboxylate ꢀ Compound 31 was synthesized using General
Procedure 2 and compound 27, with HOAt (22.5 mmol), EDC (11.25 mmol), cyclopropylamine
(22.5 mmol), DIPEA (15 mmol), and 75 mL of 1:1 DMF/DCM. Recovered yield was quantitative
and the product used without purification. 1H NMR (500 MHz, CDCl3) 0.30 (1H, m), 0.41 (1H, M),
0.58 (2H, dd, rotameric, J = 23.2, 7.1), 2.21 (1H, m), 2.33 (1H, m), 2.60 (1H, m), 3.64 (1H, dd,
rotameric, J = 15.5, 11.4), 3.81 (1H, d, J = 11.3), 4.30 (1H, m), 4.55 (2H, d, J = 12.5), 5.05 (2H,
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m), 7.30 (10H, m), 8.20 (1H, m), 9.16 (1H, m); 13C NMR (125 MHz, DMSOꢀd6, rotamers present)
5.4, 5.5, 5.6, 5.7, 22.3, 40.4, 41.3, 41.4, 56.0, 56.4, 58.6, 59.0, 65.1, 65.7, 66.2, 127.1, 127.2,
127.4, 127.5, 127.7, 127.8, 128.2, 128.4, 128.5, 128.6, 136.5, 136.6, 153.4, 153.6, 155.3, 155.4,
171.5, 171.8, 172.3; HRMS (ESI/QꢀTOF) m/z: (M+Na)+ calcd for C25H26N4O5Na 485.1795, found
485.1790
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Compound
32
ꢀ
benzyl
(5R,8S)ꢀ3ꢀbenzylꢀ8ꢀ(isobutylcarbamoyl)ꢀ2,4ꢀdioxoꢀ1,3,7ꢀ
triazaspiro[4.4]nonaneꢀ7ꢀcarboxylate ꢀ Compound 32 was synthesized using General
Procedure 2 and compound 27, with HOAt (22.5 mmol), EDC (11.25 mmol), Isobutylamine (22.5
mmol), DIPEA (15 mmol), and 75 mL of 1:1 DMF/DCM. Recovered yield was quantitative and the
product used without purification. 1H NMR (500 MHz, CDCl3) 0.76 (6H, d, rotameric, J = 6.7) 1.62
(1H, sep, rotameric, J = 6.7), 2.22 (1H, dt, J = 12.8, 10.0), 2.38 (1H, m), 2.87 (2H, m), 3.66 (1H,
dd, J = 17.0, 11.5), 3.84 (1H, d, rotameric, J = 11.5), 4.43 (1H, dd, rotameric, J = 10.0, 7.5), 4.56
(2H, d, J = 11.9), 5.05 (1H, d, J = 7.6), 5.07 (1H, d, J = 5.2), 7.30 (10H, m), 8.14 (1H, t, rotameric,
J = 5.9), 9.22 (1H, s, rotameric); 13C NMR (125 MHz, DMSOꢀd6, rotamers present) 19.42, 27.4,
27.5, 40.0, 40.9, 45.4, 55.5, 55.9, 58.1, 58.6, 64.5, 65.1, 65.6, 126.5, 126.6, 126.8, 126.9, 127.0,
127.2, 127.6, 127.8, 128.0, 135.9, 136.0, 152.9, 153.1, 154.8, 154.9, 169.8, 170.1, 171.8; HRMS
(ESI/QꢀTOF) m/z: (M+Na)+ calcd for C26H30N4O5Na 501.2108, found 501.2110
Compound
33
ꢀ
benzyl
(5R,8S)ꢀ3ꢀ(cyclopropylmethyl)ꢀ2,4ꢀdioxoꢀ8ꢀ((4ꢀ
(trifluoromethyl)benzyl)carbamoyl)ꢀ1,3,7ꢀtriazaspiro[4.4]nonaneꢀ7ꢀcarboxylate Compound
33 was synthesized using General Procedure 2 and compound 28, with HOAt (22.5 mmol), EDC
(11.25 mmol), 4ꢀ(trifluoromethyl)benzylamine (22.5 mmol), DIPEA (15 mmol), and 75 mL of 1:1
1
DMF/DCM. Recovered yield was quantitative and the product used without purification. H NMR
(500 MHz, CDCl3) 0.25 (2H, m), 0.43 (2H, m), 1.04 (1H, m), 2.26 (1H, ddd, J = 13.0, 10.0, 7.2),
2.38 (1H, m), 3.23 (2H, dd, J = 11.0, 7.3), 3.67 (1H, d, rotameric, J = 11.2), 4.33 (1H, dd, J =
12.5, 5.8), 4.40 (1H, t, J = 6.1), 4.50 (1H, dd, rotameric, J = 9.9, 7.5), 5.07 (2H, m), 7.34 (6H, m),
7.50 (2H, m), 7.65 (1H, d, J = 7.9), 8.83 (1H, m), 9.10 (1H, s, rotameric); 13C NMR (125 MHz,
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DMSOꢀd6, rotamers present) 3.4, 9.9, 40.3, 41.3, 41.7, 42.5, 56.1, 56.4, 58.9, 59.4, 64.9, 65.5,
66.3, 123.2, 125.0, 127.0, 127.3, 127.5, 127.6, 127.8, 128.2, 128.3, 136.5, 144.0, 153.7, 155.6,
155.7, 170.9, 171.1, 172.3; HRMS (ESI/QꢀTOF) m/z: (M+Na)+ calcd for C27H27F3N4O5Na
567.1826, found 567.1827
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Compound
34
ꢀ
benzyl
(5R,8S)ꢀ3ꢀisobutylꢀ2,4ꢀdioxoꢀ8ꢀ((4ꢀ
(trifluoromethyl)benzyl)carbamoyl)ꢀ1,3,7ꢀtriazaspiro[4.4]nonaneꢀ7ꢀcarboxylate Compound
34 was synthesized using General Procedure 2 and compound 29, with HOAt (22.5 mmol), EDC
(11.25 mmol), 4ꢀ(trifluoromethyl)benzylamine (22.5 mmol), DIPEA (15 mmol), and 75 mL of 1:1
1
DMF/DCM. Recovered yield was quantitative and the product used without purification. H NMR
(500 MHz, CDCl3) 0.83 (6H, d, J = 6.8), 1.94 (1H, m), 2.26 (1H, ddd, J = 13.0, 10.1, 8.1), 2.41
(1H, ddd, rotameric, J = 20.6, 13.1, 7.3), 3.18 (2H, dd, J = 10.2, 7.5), 3.65 (1H, d, rotameric, J =
11.4), 3.82 (1H, d, rotameric, J = 11.2), 4.33 (1H, dd, J = 10.8, 6.0), 4.40 (1H, t, J = 5.6), 4.48
(1H, dd, rotameric, J = 9.9, 7.5), 5.07 (2H, m), 7.34 (6H, M), 7.51 (2H, m), 7.66 (1H, d, rotameric,
J = 8.2), 8.83 (1H, m), 9.10 (1H, s, rotameric); 13C NMR (125 MHz, DMSOꢀd6, rotamers present)
19.8, 26.9, 40.4, 41.4, 41.7, 41.8, 45.3, 56.5, 58.9, 59.4, 64.8, 65.4, 66.3, 123.2, 123.3, 125.0,
125.1, 125.4, 127.0, 127.3, 127.6, 127.7, 127.9, 128.3, 128.4, 136.5, 136.6, 144.1, 144.3, 153.7,
153.8, 155.7, 155.8, 171.0, 171.2, 172.6; HRMS (ESI/QꢀTOF) m/z: (M+Na)+ calcd for
C27H29F3N4O5Na 569.1982, found 569.1986
Compound
35
ꢀ
benzyl
(5R,8S)ꢀ8ꢀ(benzylcarbamoyl)ꢀ3ꢀisobutylꢀ2,4ꢀdioxoꢀ1,3,7ꢀ
triazaspiro[4.4]nonaneꢀ7ꢀcarboxylate ꢀ Compound 35 was synthesized using General
Procedure 2 and compound 29, with 22.5 mmol HOAt, 11.25 mmol EDC, 22.5 mmol
benzylamine, 15 mmol DIPEA, and 75 mL of 1:1 DMF/DCM. Recovered yield was quantitative
1
and the product used without purification. H NMR (500 MHz, DMSOꢀd6) 0.82 (6H, dd, J = 6.7,
4.6), 1.94 (1H, m), 2.24 (1H, m), 2.39 (1H, m), 3.18 (2H, dd, J = 10.4, 7.3), 3.66 (1H, m), 3.81
(1H, m), 4.27 (2H, m), 4.46 (1H, ddd, J = 17.5, 8.9, 7.5), 5.07 (2H, m), 7.20 (10H, m), 8.72 (1H,
m), 9.10 (1H, s, rotameric); 13C NMR (125 MHz, DMSOꢀd6, rotamers present) 19.9, 26.9, 40.5,
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41.4, 42.0, 42.1, 45.3, 56.5, 58.9, 59.3, 64.8, 65.4, 66.3, 126.7, 127.0, 127.1, 127.6, 127.7, 127.9,
128.2, 128.3, 128.4, 136.6, 139.2, 153.6, 153.7, 155.7, 155.8, 170.7, 170.9, 172.6; HRMS
(ESI/QꢀTOF) m/z: (M+Na)+ calcd for C26H30N4O5Na 501.2108, found 501.2113
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General Procedure 3: Deprotection of Proline Cbz group
Each of 30ꢀ35 was added to a RB flask and reacted with 1:1 DCM/(33% HBr in AcOH) for 30 min,
and the reaction progress checked via LCMS. Upon successful deprotection of the Cbz group,
the solvent was removed in vacuo with the aid of toluene, and placed on a high vacuum pump
1
overnight to afford 36ꢀ41. H / 13C NMR spectra for compounds 36ꢀ41 are available in the
supporting information.
Compound
36
ꢀ
(5R,8S)ꢀNꢀisobutylꢀ3ꢀ(naphthalenꢀ2ꢀylmethyl)ꢀ2,4ꢀdioxoꢀ1,3,7ꢀ
triazaspiro[4.4]nonaneꢀ8ꢀcarboxamide ꢀ Compound 36 was synthesized using General
Procedure 3 and compound 30. Recovered yield was quantitative and the product used without
1
purification. H NMR (500 MHz, DMSOꢀd6) 08.4 (6H, d, J = 6.7), 1.72 (1H, m), 2.23 (1H, dd, J =
13.4, 11.9), 2.69 (1H, dd, J = 13.4, 6.7), 2.93 (1H, m), 3.02 (1H, m), 3.53 (1H, d, J = 12.8), 3.67
(1H, d, J = 12.8), 4.53 (1H, dd, J = 11.6, 6.7), 4.72 (2H, m), 7.41 (2H, m), 7.50 (1H, m), 7.76 (1H,
m), 7.90 (3H, m), 8.66 (1H, m), 9.01 (1H, s), 9.31 (1H, s), 9.91 (1H, s); 13C NMR (125 MHz,
DMSOꢀd6, rotamers present) 20.0, 27.9, 34.6, 40.7, 42.0, 46.4, 48.6, 52.5, 55.0, 59.3, 65.7,
125.6, 125.9, 126.1, 126.4, 127.6, 127.7, 128.3, 128.7, 129.3, 132.3, 132.8, 133.8, 155.2, 165.9,
172.7; HRMS (ESI/QꢀTOF) m/z: (M+H)+ calcd for C22H27N4O3 395.2078, found 395.2084
Compound 37 (5R,8S)ꢀ3ꢀbenzylꢀNꢀcyclopropylꢀ2,4ꢀdioxoꢀ1,3,7ꢀtriazaspiro[4.4]nonaneꢀ8ꢀ
carboxamide ꢀ Compound 37 was synthesized using General Procedure 3 and compound 31.
1
Recovered yield was quantitative and the product used without purification. H NMR (500 MHz,
DMSOꢀd6) 0.47 (2H, m), 0.66 (2H, m), 2.21 (1H, dd, J = 13.4, 11.6), 2.56 (1H, dd, J = 13.3, 6.9),
2.7 (1H, tq, J = 7.3, 3.9) 3.49 (1H, d, J = 13.1), 3.62 (1H, d, J = 12.8), 4.42 (1H, dd, J = 11.6, 6.7),
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4.55 (2H, s), 7.28 (5H, m), 8.73 (1H, d, J = 4.3), 8.96 (1H, s), 9.30 (1H, s), 9.88 (1H, s); 13C NMR
(125 MHz, DMSOꢀd6,) 5.6, 21.1, 22.6, 40.5, 41.7, 48.6, 52.5, 59.2, 65.5, 127.3, 127.5, 128.6,
136.2, 150.1, 166.9, 172.6 HRMS (ESI/QꢀTOF) m/z: (M+H)+ calcd for C17H21N4O3 329.1608,
found 329.1602
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Compound
38
(5R,8S)ꢀ3ꢀbenzylꢀNꢀisobutylꢀ2,4ꢀdioxoꢀ1,3,7ꢀtriazaspiro[4.4]nonaneꢀ8ꢀ
carboxamide ꢀ Compound 38 was synthesized using General Procedure 3 and compound 32.
1
Recovered yield was quantitative and the product used without purification. H NMR (500 MHz,
DMSOꢀd6) 0.86 (6H, d, J = 7.6), 1.74 (1H, m), 2.22 (1H, dd, J = 13.4, 11.6), 2.69 (1H, dd, J =
13.4, 7.0), 2.95 (1H, m), 3.03 (1H, m), 3.54 (1H, d, J = 12.8), 3.66 (1H, d, J = 13.1), 4.57 (2H, s),
4.58 (1H, dd, obscured, J = 11.7, 6.7), 7.31 (5H, m), 8.70 (1H, t, J = 5.8), 9.01 (1H, s), 9.29 (1H,
s), 9.96 (1H, s); 13C NMR (125 MHz, DMSOꢀd6) 20.0, 20.1, 27.9, 35.6, 40.7, 41.7, 46.4, 48.6,
52.4, 59.3, 65.6, 127.3, 127.5, 128.3, 128.6, 136.2, 155.1, 165.9, 172.7; HRMS (ESI/QꢀTOF) m/z:
(M+H)+ calcd for C18H25N4O3 345.1921, found 345.1920
Compound 39 ꢀ (5R,8S)ꢀ3ꢀ(cyclopropylmethyl)ꢀ2,4ꢀdioxoꢀNꢀ(4ꢀ(trifluoromethyl)benzyl)ꢀ1,3,7ꢀ
triazaspiro[4.4]nonaneꢀ8ꢀcarboxamide ꢀ Compound 39 was synthesized using General
Procedure 3 and compound 33. Recovered yield was quantitative and the product used without
purification. 1H NMR (500 MHz, DMSOꢀd6) 0.24 (2H, m), 0.43 (2H, m), 1.02 (1H, m), 2.27 (1H, dd,
J = 13.4, 11.6), 2.67 (1H, dd, J = 13.3, 6.9), 3.22 (2H, d, J = 7.0), 3.49 (1H, d, J = 13.1), 3.62 (1H,
d, J = 13.1), 4.46 (2H, m), 4.65 (1H, dd, J = 11.4, 6.9), 7.51 (2H, d, J = 8.2), 7.69 (2H, d, J = 7.9),
8.89 (1H, s), 9.36 (1H, t, J = 6.0), 9.46 (1H, s), 9.80 (1H, s); 13C NMR (125 MHz, DMSOꢀd6) 3.5,
9.8, 40.4, 42.1, 42.8, 48.6, 52.5, 59.4, 65.4, 125.3, 125.4, 127.7, 127.9, 128.1, 143.3, 155.4,
166.3, 172.7; HRMS (ESI/QꢀTOF) m/z: (M+H)+ calcd for C19H22F3N4O3 411.1639, found
411.1638
Compound
40
ꢀ
(5R,8S)ꢀ3ꢀisobutylꢀ2,4ꢀdioxoꢀNꢀ(4ꢀ(trifluoromethyl)benzyl)ꢀ1,3,7ꢀ
triazaspiro[4.4]nonaneꢀ8ꢀcarboxamide ꢀ Compound 40 was synthesized using General
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Procedure 3 and compound 34. Recovered yield was quantitative and the product used without
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purification. H NMR (500 MHz, CDCl3) 0.77 (6H, d, J = 6.4), 1.88 (1H, m), 2.49 (1H, m), 2.83,
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8
9
1H, m), 3.16 (2H, m), 3.29 (1H, m), 3.91 (2H, m), 4.36 (1H, d, J = 11.0), 4.56 (1H, d, J = 9.2),
5.36 (1H, m), 7.35 (2H, d, J = 7.2), 7.47 (2H, d, J = 7.6), 8.31 (1H, m), 8.68 (1H, m), 9.45 (1H, m);
13C NMR (125 MHz, CDCl3) 19.8, 27.3, 33.6, 43.4, 46.5, 60.2, 65.9, 122.9, 125.0, 125.5, 127.7,
127.9, 128.8, 129.0, 129.6, 129.8, 141.2, 156.3, 167.3, 172.2; HRMS (ESI/QꢀTOF) m/z: (M+H)+
calcd for C19H24F3N4O3 413.1795, found 413.1794
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Compound
41
(5R,8S)ꢀNꢀbenzylꢀ3ꢀisobutylꢀ2,4ꢀdioxoꢀ1,3,7ꢀtriazaspiro[4.4]nonaneꢀ8ꢀ
carboxamide ꢀ Compound 41 was synthesized using General Procedure 3 and compound 35.
1
Recovered yield was quantitative and the product used without purification. H NMR (500 MHz,
DMSOꢀd6) 0.82 (6H, d, J = 6.7), 1.92 (1H, m), 2.24 (1H, dd, J = 13.3, 11.7), 2.65 (1H, dd, J =
13.3, 6.9), 3.17 (2H, d, J = 7.3), 3.49 (1H, d, J = 13.1), 3.61 (1H, d, J = 12.8), 4.36 (2H, dd, J =
5.8, 3.1), 4.60 (1H, dd, J = 11.6, 7.0), 7.29 (6H, m), 8.87 (1H, s), 9.21 (1H, t, J = 5.8); 13C NMR
(125 MHz, DMSOꢀd6) 19.8, 26.9, 34.6, 40.5, 42.5, 45.5, 52.5, 59.3, 65.3, 127.1, 127.3, 128.4,
128.7, 129.3, 138.3, 155.4, 166.0, 172.9; HRMS (ESI/QꢀTOF) m/z: (M+H)+ calcd for C18H25N4O3
345.1921, found 345.1921
General Procedure 4: Coupling of BocꢀGlyꢀOH to 36ꢀ41
To an inert atmosphere RB flask containing 2 equiv of BocꢀGlyꢀOH in dry DMF/DCM (1:1 ratio,
[200 mM]) was added EDC (2 equiv) and HOAT (4 equiv) and stirred for 1 h. Afterwards, one of
36ꢀ41 in DMF [200 mM] and DIPEA (4 equiv) were added and the reaction stirred for another 2 h,
at which point the progress was checked via LCMS. Upon completion, the reaction was diluted
with four times the reaction volume of EtOAc and washed with saturated ammonium chloride
solution, saturated sodium bicarbonate solution, and brine. The organic layer was then dried with
Na2SO4, and concentrated in vacuo to yield 42ꢀ47 as foamy darkꢀyellow solids. These
compounds were then purified by normal phase flash chromatography (0ꢀ5% MeOH in DCM) to
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yield offꢀwhite foamy solids. H / 13C NMR spectra are available for compounds 42ꢀ47 in the
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supporting information.
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Compound 42 ꢀ tertꢀbutyl (2ꢀ((5R,8S)ꢀ8ꢀ(isobutylcarbamoyl)ꢀ3ꢀ(naphthalenꢀ2ꢀylmethyl)ꢀ2,4ꢀ
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dioxoꢀ1,3,7ꢀtriazaspiro[4.4]nonanꢀ7ꢀyl)ꢀ2ꢀoxoethyl)carbamate
–
Compound
42
was
synthesized using General procedure 4, BocꢀGlyꢀOH (15 mmol), HOAt (30 mmol), EDC (15
1
mmol), DIPEA (30 mmol), and compound 36. Purified yield 3.23 g (78%); H NMR (500 MHz,
CDCl3) 0.80 (6H, m), 1.21 (9H, s, rotameric), 1.65 (1H, m), 2.24 (1H, dd, J = 12.8, 7.6), 2.47 (2H,
m), 3.24 (1H, m), 3.73 (1H, dd, J = 17.1, 4.6), 3.95 (1H, m), 4.08 (1H, dd, J = 10.4), 4.43 (1H, m),
4.69 (1H, d, J = 14.6), 4.79 (1H, m), 4.82 (1H, d, J = 14.6), 5.77 (1H, m), 7.32 (1H, m), 7.46 (3H,
m), 7.76 (4H, m), 8.18 (1H, s); 13C NMR (125 MHz, CDCl3, rotamers present) 20.0, 20.1, 25.3,
28.1, 28.3, 28.4, 40.9, 43.0, 46.7, 54.5, 59.7, 64.3, 66.5, 79.6, 126.4, 126.5, 127.7, 127.8, 128.1,
128.6, 132.9, 133.0, 133.1, 155.5, 156.5, 169.6, 171.5; HRMS (ESI/QꢀTOF) m/z: (M+Na)+ calcd
for C29H37N5O6Na 574.2636, found 574.2633
Compound 43 ꢀ tertꢀbutyl (2ꢀ((5R,8S)ꢀ3ꢀbenzylꢀ8ꢀ(cyclopropylcarbamoyl)ꢀ2,4ꢀdioxoꢀ1,3,7ꢀ
triazaspiro[4.4]nonanꢀ7ꢀyl)ꢀ2ꢀoxoethyl)carbamate ꢀ Compound 43 was synthesized using
General procedure 4, BocꢀGlyꢀOH (15 mmol), HOAt (30 mmol), EDC (15 mmol), DIPEA (30
1
mmol), and compound 37 ꢀ Purified yield 2.94 g (81%); H NMR (500 MHz, CDCl3, rotamers
present) 0.44 (1H, m), 0.52 (2H, m), 0.67 (1H, m), 1.31 (9H, s, rotameric), 2.17 (1H, m), 2.43 (1H,
t, J = 11.7), 2.55 (1H, m), 3.76 (1H, dd, J = 17.1, 5.5), 3.84 (1H, d, J = 10.4), 4.06 (1H, d, J =
10.4), 4.22 (1H, m), 4.58 (1H, d, J = 14.6), 4.65 (1H, d, J = 14.6), 4.67 (1H, m), 5.80 (1H, s), 7.30
(5H, m), 7.45 (1H, m), 8.21 (1H, s); 13C NMR (125 MHz, CDCl3, rotamers present) 5.9, 6.7, 22.6,
28.2, 40.3, 42.7, 43.2, 54.9, 59.7, 66.4, 79.8, 128.1, 128.6, 128.8, 135.7, 155.6, 156.6, 171.1,
171.5; HRMS (ESI/QꢀTOF) m/z: (M+Na)+ calcd for C24H31N5O6 Na 508.2167, found 508.2165
Compound 44
ꢀ
tertꢀbutyl (2ꢀ((5R,8S)ꢀ3ꢀbenzylꢀ8ꢀ(isobutylcarbamoyl)ꢀ2,4ꢀdioxoꢀ1,3,7ꢀ
triazaspiro[4.4]nonanꢀ7ꢀyl)ꢀ2ꢀoxoethyl)carbamate ꢀ Compound 44 was synthesized using
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General procedure 4, BocꢀGlyꢀOH (15 mmol), HOAt (30 mmol), EDC (15 mmol), DIPEA (30
1
mmol), and compound 38. Purified yield 2.87 g (78%); H NMR (500 MHz, CDCl3) 0.85 (6H, m),
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1.29 (9H, s, rotameric), 1.70 (1H, m), 2.22 (1H, dd, J = 12.5, 7.6), 2.45 (1H, t, J = 11.9), 2.47 (1H,
m), 3.27 (1H, m), 3.75 (1H, dd, J = 17.1, 4.6), 3.99 (1H, m), 4.08 (1H, d, J = 9.8), 4.49 (1H, m),
4.55 (1H, d, J = 14.6), 4.67 (1H, d, J = 14.6), 4.82 (1H, dd, J = 10.1, 8.2), 5.79 (1H, m), 7.31 (6H,
m), 8.17 (1H, s); 13C NMR (125 MHz, CDCl3, rotamers present) 20.1, 20.2, 25.3, 28.2, 28.3, 28.4,
41.0, 42.8, 43.1, 46.7, 54.4, 59.7, 60.4, 66.5, 79.6, 128.2, 128.7, 128.8, 135.7, 155.5, 156.5,
169.5, 171.4 HRMS (ESI/QꢀTOF) m/z: (M+Na)+ calcd for C25H35N5O6 Na 524.2480, found
524.2487
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Compound 45
ꢀ
tertꢀbutyl (2ꢀ((5R,8S)ꢀ3ꢀbenzylꢀ8ꢀ(isobutylcarbamoyl)ꢀ2,4ꢀdioxoꢀ1,3,7ꢀ
triazaspiro[4.4]nonanꢀ7ꢀyl)ꢀ2ꢀoxoethyl)carbamate ꢀ Compound 45 was synthesized using
General procedure 4, BocꢀGlyꢀOH (15 mmol), HOAt (30 mmol), EDC (15 mmol), DIPEA (30
1
mmol), and compound 39 – Purified yield 3.64 g (86% Yield); H NMR (500 MHz, CDCl3) 0.25
(2H, d, J = 4.6), 0.44 (2H, m), 1.04 (1H, m), 1.33 (9H, s, rotameric), 2.07 (1H, m), 2.45 (1H, t, J =
11.9), 3.22 (1H, dd, J = 14.0, 7.3), 3.33 (1H, dd, J = 14.0, 7.0), 3.83 (1H, dd, J = 17.1, 5.2), 3.92
(1H, m), 4.11 (1H, m), 4.34 (2H, m), 4.62 (1H, dd, J = 15.4, 6.6), 4.89 (1H, m), 5.82 (1H, s), 7.42
(2H, d, J = 8.2), 7.54 (2H, d, J, = 8.2), 8.08 (1H, s), 8.30 (1H, s); 13C NMR (125 MHz, CDCl3,
rotamers present) 3.6, 4.1, 10.1, 21.1, 25.3, 28.2, 42.9, 43.2, 43.8, 54.8, 60.1, 60.4, 66.3, 79.9,
123.0, 125.2, 125.4, 125.5, 127.6, 129.4, 129.7, 142.2, 156.1, 156.6, 170.3, 171.5; HRMS
(ESI/QꢀTOF) m/z: (M+Na)+ calcd for C26H32F3N5O6 Na 590.2197, found 590.2196
Compound
46
ꢀ
tertꢀbutyl
(2ꢀ((5R,8S)ꢀ3ꢀisobutylꢀ2,4ꢀdioxoꢀ8ꢀ((4ꢀ
(trifluoromethyl)benzyl)carbamoyl)ꢀ1,3,7ꢀtriazaspiro[4.4]nonanꢀ7ꢀyl)ꢀ2ꢀoxoethyl)carbamate ꢀ
Compound 42 was synthesized using General procedure 4, BocꢀGlyꢀOH (30 mmol), HOAt (60
1
mmol), EDC (30 mmol), DIPEA (60 mmol), and compound 40 ꢀ Purified yield 6.82 g (91%); H
NMR (500 MHz, CDCl3) 0.82 (6H, d, rotameric, J = 6.7), 1.33 (9H, s, rotameric), 1.96 (1H, m),
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2.02 (1H, m), 2.44 (1H, t, J = 11.9), 3.12 (1H, dd, J = 13.1, 8.2), 3.29 (1H, dd, J = 13.4, 6.7), 3.78
(1H, dd, J = 17.1, 5.2), 3.92 (1H, m), 4.07 (1H, m), 4.17 (1H, m), 4.56 (1H, dd, J = 15.1, 7.2), 4.86
(1H, m), 5.81 (1H, s), 7.26 (5H, m), 7.85 (1H, m), 8.36 (1H, s); 13C NMR (125 MHz, CDCl3,
rotamers present) 19.7, 19.8, 25.3, 27.1, 28.2, 40.7, 43.1, 43.3, 45.8, 54.5, 59.9, 66.1, 79.7,
127.3, 127.3, 128.5, 138.0, 156.0, 156.5, 169.9, 170.7, 171.7; HRMS (ESI/QꢀTOF) m/z: (M+Na)+
calcd for C25H35N5O6 Na 524.2480, found 524.2487
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Compound 47
ꢀ
tertꢀbutyl (2ꢀ((5R,8S)ꢀ8ꢀ(benzylcarbamoyl)ꢀ3ꢀisobutylꢀ2,4ꢀdioxoꢀ1,3,7ꢀ
triazaspiro[4.4]nonanꢀ7ꢀyl)ꢀ2ꢀoxoethyl)carbamate ꢀ Compound 42 was synthesized using
General procedure 4, BocꢀGlyꢀOH (15 mmol), HOAt (30 mmol), EDC (15 mmol), DIPEA (30
1
mmol), and compound 41 – Purified Yield 3.50 g (82%); H NMR (500 MHz, CDCl3) 0.84 (6H, d,
rotameric, J = 6.7), 1.32 (9H, s), 2.00 (2H, m), 2.16 (1H, s), 2.46 (1H, t, J = 11.9), 3.17 (1H, dd, J
= 13.1, 7.9), 3.31 (1H, dd, J = 13.4, 7.0), 3.81 (1H, dd, J = 16.9, 5.2), 4.08 (1H, d, J = 10.7), 4.32
(2H, m), 4.57 (1H, dd, J = 15.6, 6.4), 4.88 (1H, m), 5.72 (1H, s), 7.4 (2H, d, J = 8.2), 7.54 (2H, d, J
= 8.2), 7.99 (1H, m), 8.30 (1H, s); 13C NMR (125 MHz, CDCl3, rotamers present) 19.7, 19.8, 27.2,
28.2, 31.0, 40.3, 42.9, 43.3, 46.0, 54.9, 60.1, 66.1, 80.0, 123.0, 125.1, 125.4, 125.5, 127.5, 129.5,
129.7, 142.2, 156.1, 156.6, 170.4, 171.5; HRMS (ESI/QꢀTOF) m/z: (M+Na)+ calcd for
C26H34F3N5O6 Na 592.2353, found 592.2354
General Procedure 5: Alkylation of 42ꢀ47 to Synthesize 2ꢀ15
To a stirred mixture of one of 42ꢀ47 in DMF [100 mM] was added 1.05 equiv of allyl or benzyl
halide along with 1.5 equiv of K2CO3. The reaction proceeded at room temperature for 14ꢀ24 h,
and the progress checked via LCMS. The reaction was diluted with four times the reaction
volume of EtOAc and washed with water, saturated ammonium chloride solution, and brine. The
organic layer was dried with Na2SO4, and concentrated in vacuo to yield 5ꢀ18 as tan to yellow
foamy solids. UHPLC (C18, 5ꢀ100% ACN in Water) and 1H / 13C NMR spectra are available in the
supporting information.
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Spiroligomer Amine 5 ꢀ tertꢀbutyl (2ꢀ((5R,8S)ꢀ1ꢀbenzylꢀ8ꢀ(benzylcarbamoyl)ꢀ3ꢀisobutylꢀ2,4ꢀ
dioxoꢀ1,3,7ꢀtriazaspiro[4.4]nonanꢀ7ꢀyl)ꢀ2ꢀoxoethyl)carbamate – Spiroligomer Amine 5 was
synthesized using General Procedure 5, compound 46, and benzyl bromide (1.05 equiv). Purified
yield 1.10 g (93%). 1H NMR (500 MHz, CDCl3) 0.94 (6H, d, J = 6.7), 1.39 (9H, s, rotameric), 2.16
(1H, m), 2.36 (1H, dd, J = 14.5, 9.0), 2.44 (1H, dd, J = 17.1, 4.3), 3.03 (1H, dd, J = 14.6, 8.5),
3.27 (1H, dd, J = 16.9, 5.3), 3.4 (1H, t, J = 3.5), 3.44 (2H, d, rotameric, J = 7.6), 3.80 (1H, d, J =
11.6), 4.05 (1H, d, J = 16.2), 4.37 (1H, d, rotameric, J = 5.5), 4.46 (1H, d, rotameric, J = 6.4), 4.76
(1H, t, J = 8.7), 5.00 (1H, t, J = 4.4), 5.04 (1H, d, J = 16.2), 7.07 (1H, t, J = 5.8), 7.18 (2H, d, J =
7.3), 7.25 (3H, m), 7.33 (5H, m); 13C NMR (125 MHz, CDCl3, rotamers present) 19.9, 20.0, 21.0,
24.8, 24.9, 27.3, 28.3, 36.5, 42.7, 43.7, 44.1, 46.8, 54.6, 60.0, 63.8, 69.3, 80.0, 80.8, 120.7,
127.1, 127.4, 127.5, 128.3, 128.7, 129.1, 129.3, 137.0, 137.8, 151.3, 156.1, 168.3, 169.0, 171.5 ;
HRMS (ESI/QꢀTOF) m/z: (M+Na)+ calcd for C32H41N5O6Na 614.2949, found 614.2955
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Spiroligomer Amine
6
ꢀ
tertꢀbutyl (2ꢀ((5R,8S)ꢀ3ꢀbenzylꢀ1ꢀ(3,4ꢀdichlorobenzyl)ꢀ8ꢀ
(isobutylcarbamoyl)ꢀ2,4ꢀdioxoꢀ1,3,7ꢀtriazaspiro[4.4]nonanꢀ7ꢀyl)ꢀ2ꢀoxoethyl)carbamate
ꢀ
Spiroligomer Amine 6 was synthesized using General Procedure 5, compound 44, and 3,4ꢀ
dichlorobenzyl bromide (1.05 equiv). Purified yield 1.16 g (88%); 1H NMR (500 MHz, CDCl3) 0.87
(6H, d, J = 6.7), 1.43 (9H, s, rotameric), 1.75 (1H, m), 2.24 (1H, ddd, J = 14.7, 9.1, 1.2), 2.96 (1H,
dd, J = 17.1, 4.6), 3.05 (3H, m), 3.45 (1H, dd, J = 17.1, 5.2), 3.52 (1H, d, J = 11.6), 3.84 (1H, d, J
= 11.9), 4.21 (1H, d, J = 16.2), 4.63 (1H, t, J = 8.5), 4.75 (3H, m), 5.14 (1H, t, J = 4.7), 6.70 (1H, t,
J = 5.5), 7.06 (1H, d, J = 6.4), 7.36 (7H, m); 13C NMR (125 MHz, CDCl3, rotamers present) 20.1,
28.3, 28.4, 36.0, 43.1, 43.2, 43.3, 47.2, 54.5, 59.7, 69.5, 80.2, 126.6, 128.3, 128.6, 128.7, 128.9,
129.1, 129.2, 130.0, 131.1, 132.6, 133.3, 135.2, 135.4, 137.2, 151.7, 155.7, 168.2, 168.6, 170.9 ;
HRMS (ESI/QꢀTOF) m/z: (M+Na)+ calcd for C32H39Cl2N5O6Na 682.2170, found 682.2161
Spiroligomer
Amine
7
ꢀ
tertꢀbutyl
(2ꢀ((5R,8S)ꢀ1ꢀallylꢀ3ꢀisobutylꢀ2,4ꢀdioxoꢀ8ꢀ((4ꢀ
(trifluoromethyl)benzyl)carbamoyl)ꢀ1,3,7ꢀtriazaspiro[4.4]nonanꢀ7ꢀyl)ꢀ2ꢀoxoethyl)carbamate ꢀ
Spiroligomer Amine 7 was synthesized using General Procedure 5, compound 47, and allyl
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bromide (1.05 equiv). Purified yield 804 mg (92%); H NMR (500 MHz, CDCl3) 0.80 (6H, d, J =
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8
6.7), 1.39 (9H, s, rotameric), 2.07 (1H, m), 2.44 (1H, dd, J = 14.3, 9.5), 2.95 (1H, dd, J = 14.5,
7.5), 3.36 (2H, d, rotameric, J = 7.3), 3.82 (4H, m), 3.97 (1H, d, J = 1.16), 4.14 (1H, dd, rotameric,
J = 16.5, 4.9), 4.46 (1H, d, rotameric, J = 5.8), 4.53 (1H, d, rotameric, J = 6.1), 4.83 (1H, t, J =
8.2), 5.18 (2H, m), 5.32 (1H, t, J = 4.6), 5.84 (1H, dddd, J = 16.9, 10.5, 6.5, 5.2), 7.32 (1H, t, J =
6.0), 7.40 (2H, d, J = 8.2), 7.57 (2H, d, J = 7.9); 13C NMR (125 MHz, CDCl3, rotamers present)
19.9, 27.3, 28.2, 36.1, 43.1, 43.6, 46.6, 54.3, 60.2, 68.8, 80.5, 118.1, 125.5, 125.6, 127.7, 133.4,
142.1, 156.3, 156.0, 168.7, 169.5, 172.1; HRMS (ESI/QꢀTOF) m/z: (M+Na)+ calcd for
C29H38F3N5O6Na 632.2666, found 632.2655
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Spiroligomer Amine 8 ꢀ tertꢀbutyl (2ꢀ((5R,8S)ꢀ1ꢀ(2ꢀaminoꢀ2ꢀoxoethyl)ꢀ3ꢀisobutylꢀ2,4ꢀdioxoꢀ8ꢀ
((4ꢀ(trifluoromethyl)benzyl)carbamoyl)ꢀ1,3,7ꢀtriazaspiro[4.4]nonanꢀ7ꢀyl)ꢀ2ꢀ
oxoethyl)carbamate ꢀ Spiroligomer Amine 8 was synthesized using General Procedure 5,
compound 47, and 4ꢀ(trifluoromethyl)benzyl bromide (1.05 equiv). Purified yield 770 mg (82%);
1H CDCl3 (500 MHz, CDCl3) 0.87 (6H, t, J = 6.7), 1.31 (9H, s, rotameric), 2.02 (1H, tq, J = 13.4,
6.6), 2.16 (1H, m), 2.47 (1H, dd, J = 13.3, 9.6), 3.32 (2H, d, J = 7.3), 3.81 (4H, m), 4.19 (1H, d, J
= 11.3), 4.42 (3H, m), 4.83 (1H, m), 5.88 (1H, s), 6.62 (1H, s), 7.36 (2H, d, J = 7.6), 7.51 (2H, d, J
= 7.9), 7.83 (1H, s, rotameric), 8.23 (1H, s, rotameric); 13C NMR (125 MHz, CDCl3, rotamers
present) 3.6, 9.9, 19.8, 25.3, 27.3, 28.2, 28.3, 37.2, 42.6, 42.9, 43.7, 46.8, 54.9, 60.1, 64.4, 69.1,
80.7, 122.0, 122.9, 125.1, 125.4, 127.5, 127.7, 129.6, 155.8, 157.0, 170.0, 170.3, 170.9; HRMS
(ESI/QꢀTOF) m/z: (M+Na)+ calcd for C28H37F3N6O7Na 649.2568, found 649.2566
Spiroligomer Amine 9 ꢀ tertꢀbutyl (2ꢀ((5R,8S)ꢀ1ꢀ(3,4ꢀdichlorobenzyl)ꢀ3ꢀisobutylꢀ2,4ꢀdioxoꢀ8ꢀ
((4ꢀ(trifluoromethyl)benzyl)carbamoyl)ꢀ1,3,7ꢀtriazaspiro[4.4]nonanꢀ7ꢀyl)ꢀ2ꢀ
oxoethyl)carbamate ꢀ Spiroligomer Amine 9 was synthesized using General Procedure 5,
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compound 47, and 3,4ꢀDichlorobenzyl bromide (1.05 equiv). Purified yield 971 mg (89%). H
NMR (500 MHz, CDCl3) 0.93 (6H, d, J = 7.0), 1.37 (9H, s, rotameric), 2.12 (1H, m), 2.35 (1H, dd,
J = 14.2, 9.6), 2.52 (1H, q, J = 7.3), 2.98 (1H, m), 3.43 (3H, m), 3.53 (1H, m), 3.90 (1H, d, J =
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