Peptide Macrocyclization Inspired by Non-Ribosomal Imine Natural Products

Article abstract of DOI:10.1021/jacs.7b01624

A thermodynamic approach to peptide macrocyclization inspired by the cyclization of non-ribosomal peptide aldehydes is presented. The method provides access to structurally diverse macrocycles by exploiting the reactivity of transient macrocyclic peptide imines toward inter- and intramolecular nucleophiles. Reactions are performed in aqueous media, in the absence of side chain protecting groups, and are tolerant of all proteinogenic functional groups. Macrocyclic products bearing non-native and rigidifying structural motifs, isotopic labels, and a variety of bioorthogonal handles are prepared, along with analogues of four distinct natural products. Structural interrogation of the linear and macrocyclic peptides using variable-temperature NMR and circular dichroism suggests that preorganization of linear substrates is not a prerequisite for macrocyclization.

Full text of DOI:10.1021/jacs.7b01624

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Article
Peptide Macrocyclization Inspired by Non-Ribosomal Imine Natural Products
Lara R. Malins, Justine Nicole deGruyter, Kevin J. Robbins, Paul
M. Scola, Martin Eastgate, M. Reza Ghadiri, and Phil S. Baran
J. Am. Chem. Soc., Just Accepted Manuscript • Publication Date (Web): 22 Mar 2017
Downloaded from http://pubs.acs.org on March 22, 2017
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Peptide Macrocyclization Inspired by Non-Ribosomal Imine Natural
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Lara R. Malins,1‡ Justine N. deGruyter,1‡ Kevin J. Robbins,2 Paul M. Scola,2 Martin D. Eastgate,3 M.
Reza Ghadiri,1 and Phil S. Baran1*
1Department of Chemistry, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037
2Department of Discovery Chemistry, BristolꢀMyers Squibb Research and Development, 5 Research Parkway, Wallingford,
Connecticut 06492
3Chemical Development, BristolꢀMyers Squibb, One Squibb Drive, New Brunswick, New Jersey 08903
ABSTRACT: A thermodynamic approach to peptide macrocyclization inspired by the cyclization of nonꢀribosomal peptide aldeꢀ
hydes is presented. The method provides access to structurally diverse macrocycles by exploiting the reactivity of transient macroꢀ
cyclic peptide imines toward interꢀ and intramolecular nucleophiles. Reactions are performed in aqueous media, in the absence of
sideꢀchain protecting groups, and are tolerant of all proteinogenic functional groups. Macrocyclic products bearing nonꢀnative and
rigidifying structural motifs, isotopic labels, and a variety of bioorthogonal handles are prepared, along with analogues of four disꢀ
tinct natural products. Structural interrogation of the linear and macrocyclic peptides using variable temperature NMR and circular
dichroism suggests that preorganization of linear substrates is not a prerequisite for macrocyclization.
Introduction
The quest to develop potent and selective new therapies has
propelled pharmaceutical companies toward underexplored
areas of chemical space, including the privileged intermediary
between traditional smallꢀmolecule drugs (< 500 Da) and
highꢀvalue biologics (> 5000 Da).1 The rise of protein–protein
interactions (PPIs) as promising targets for therapeutic interꢀ
vention has further contributed to the increasing molecular
weight and complexity of drug leads, with mediumꢀsized pepꢀ
tides and peptidomimetics emerging as able mimics of the
protein surfaces and peptide epitopes involved in protein–
protein binding.2 Nevertheless, most peptide leads suffer from
low proteolytic stability and are historically poor drug candiꢀ
dates, rendering new synthetic strategies (e.g., macrocyclizaꢀ
tion, backbone and side chain modifications) aimed to enhance
their drugꢀlike properties3 in exceedingly high demand in the
pharmaceutical industry. As part of an onꢀgoing collaboration
with BristolꢀMyers Squibb (BMS) on the development of PPI
inhibitors, a synergistic academic–industrial collaboration4
was initiated to guide the development of a versatile peptide
macrocyclization strategy. The goals were twoꢀfold: 1) proꢀ
vide access to structurally diverse peptide products with imꢀ
mediate relevance to drug discovery, including access to nonꢀ
natural and rigidifying motifs at the ring junction; and 2) facilꢀ
itate macrocyclization through an unconventional, thermodyꢀ
namic pathway. Such a method would complement other reꢀ
cent chemical strategies for peptide stapling and macrocyclizaꢀ
tion,5 including pioneering work by Yudin and coworkers on
multicomponent peptide macrocyclizations.6
Inspiration for the design of a versatile macrocyclization
could be found in the diversity of peptide macrocycles proꢀ
Figure 1. Peptide macrocyclization inspired by reductively reꢀ
leased nonꢀribosomal peptide natural products.
duced by the nonꢀribosomal peptide synthetase (NRPS) enzyꢀ
matic machinery (Figure 1A).7 Following chain elongation on
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Scheme 1. Synthesis of linear peptide amino aldehydes. A) Side-chain incorporation of aminoacetaldehyde dimethyl acetal;
B) Loading of Fmoc-amino aldehydes onto Rink TG resin.
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the nonꢀribosomal assembly line, release of the mature peptide
from the enzyme complex can occur with concomitant macroꢀ
cyclization catalyzed by a thioesterase domain to generate
structurally complex macrolactones and macrolactams.8 Reꢀ
cently, headꢀtoꢀtail macrocyclization through an intriguing
imino linkage was shown to occur in certain bacteria followꢀ
ing the reductive release of a peptide aldehyde mediated by a
rare NAD(P)Hꢀdependent reductase domain (Figure 1A).9 The
nostocyclopeptide class of macrocyclic imines was the first
group of natural products shown to be reductively cleaved
probed using NMR techniques and circular dichroism, which
demonstrate that macrocyclization efficiency is not predicated
on substrate preorganization.
Synthesis of Linear Amino Aldehydes
Our investigations began with the synthesis of linear peptide
amino aldehydes. A number of strategies exist for the preparaꢀ
tion of peptide aldehydes,17 owing in part to their importance
in bioconjugation18 and as potent protease inhibitors.19 Herein,
two distinct solidꢀphase approaches20 were employed to asꢀ
semble a variety of structurally diverse linear precursors
(Scheme 1A and B, see Supporting Information for additional
details). The first method entailed incorporation of the key
aldehyde unit by direct loading of commercially available
aminoacetaldehyde dimethyl acetal onto the sideꢀchain of a
resinꢀbound glutamic acid residue (Scheme 1A). Acidic cleavꢀ
age from the resin facilitated global protecting group removal
and release of a sideꢀchain appended glycinal unit following
acetal hydrolysis. A second approach was employed to faciliꢀ
tate the synthesis of headꢀtoꢀtail macrocycles and accommoꢀ
date the incorporation of aldehydes bearing αꢀchirality. To this
end, preꢀformed Fmocꢀamino aldehydes were prepared21 and
loaded onto threonine–glycine (TG) functionalized Rink amꢀ
ide resin (Scheme 1B). This resin linkage, first reported by
Ede and Bray,22 relies on reversible oxazolidine formation
between the aldehyde building block and the threonine 1,2ꢀ
amino alcohol motif. The oxazolidine tether is stable to the
reaction conditions employed in standard FmocꢀSPPS, but is
readily hydrolyzed upon treatment with aqueous acid to unveil
a Cꢀterminal peptide aldehyde. The assembled aldehyde seꢀ
quences (28 examples in total) range from 5–10 amino acids in
length and encompass all proteinogenic functional groups. To
further increase the diversity of linear peptide amino aldeꢀ
hydes and limit the possibility of selecting substrates biased
toward cyclization, a number of peptide sequences were deꢀ
signed using a random sequence generator (see Supporting
Information for details).
from the NRPS9 and undergo spontaneous selfꢀassembly.10
A
number of additional imines—including scytonemide A,11
nostocyclopeptide M1,12 and koranimine13—have since been
reported, with biosynthetic investigations into the latter sugꢀ
gesting the involvement of a reductive release mechanism.
The biosynthesis of the promising antibiotic lugdunin,14 a thiaꢀ
zolidine macrocyclic peptide natural product isolated in 2016,
also implicates a terminal reductase domain. Formation of the
unique heterocyclic linkage is thought to occur via intramoꢀ
lecular trapping of a macrocyclic imine formed spontaneously
upon reductive release from the NRPS complex.
These rare but structurally intriguing nonꢀribosomal peptide
natural products prompted an exploration of imino macrocyꢀ
clization as a unique and innately diversifiable mode of pepꢀ
tide cyclization. The formation of a reversible macrocyclic
linkage sets the stage for a tunable, thermodynamic cyclization
event, in contrast to reported modes of irreversible macrocyꢀ
clization.15 Indeed, the reversible ring–chain equilibrium of
imino macrocycles has previously been exploited by Marahiel
and coworkers to gain a rare glimpse into the thermodynamics
of macrocyclization.16 In the context of drug discovery, the
trapping of a reactive imine intermediate with a variety of
interꢀ or intramolecular nucleophiles would provide rapid acꢀ
cess to structurally unique peptide macrocycles incorporating
nonꢀnative motifs at the site of ring closure (Figure 1B). Due
to the selective and reversible condensation of amines and
aldehydes, diversification could proceed at a late stage with
unprotected peptides, abrogating the need for preactivation
and sideꢀchain protection. Herein, this NRPSꢀinspired apꢀ
proach to macrocyclization is delineated along with applicaꢀ
tions to the preparation of diverse peptide macrocycles, inꢀ
cluding natural products and associated analogues. The soluꢀ
tion structures of select linear and cyclic peptides are also
Intermolecular Imine Traps
Unlike the spontaneous cyclization implicated in the formation
of the macrocyclic imine natural products, a variable propensiꢀ
ty toward cyclization was observed for the synthetic amino
aldehyde substrates. For some peptides, cyclization to the
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Scheme 2. Substrate scope for imine macrocyclization employing intermolecular imine traps. A) Strecker macrocyclization;
B) Reductive amination; C) Selective cyclization of substrates bearing internal lysine residues.
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imine was observed directly upon LCꢀMS analysis of the
cleaved peptide or upon incubation of the linear substrate in
the presence of various additives (see Supporting Information
for details), while others persisted in linear form. Reasoning
that a suitable imine trap might drive the equilibrium toward
cyclization, the reactivity of the peptide substrates toward
external nucleophiles was examined. Cyanide was quickly
identified as a potent imine trap capable of facilitating effiꢀ
cient macrocyclization (Scheme 2A).
not generally observed at 1 mM concentration—a possible
consequence of reversible imine condensation, wherein linear
oligomers may equilibrate to the target cyclic monomer prior
to trapping. The reaction was tolerant of various functional
groups—including carboxylic acids (3 and 5), primary amides,
His imidazoles (5), phenols (6), and disulfides (7)—and proꢀ
ceeded in excellent yields with Nꢀmethylated derivatives (1
and 3), in contrast to conventional macrolactamization methꢀ
ods in which Nꢀalkyl amines are often prohibitively sluggish.
The enhanced reactivity for Nꢀmethylated peptides potentially
lies in the formation of a highly reactive iminium intermediate
upon reversible condensation of the Nꢀmethyl amine with the
terminal aldehyde. Reactions typically afforded diastereomeric
The resulting Strecker reaction proceeded in aqueous soluꢀ
tion (1 mM in peptide) at rt with a slight excess of KCN (1.2
equiv.) to afford macrocyclic αꢀaminonitriles23 (1–7) in good
isolated yields. Interestingly, oligomerization products were
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Scheme 3. Late-stage peptide diversification through the introduction of isotopic labels and bioorthogonal handles.
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mixtures of αꢀaminonitriles which were inseparable by reꢀ
verseꢀphase HPLC. In cases where quantification was possiꢀ
ble, cyanide addition generally resulted in a 1:1 mixture of
diastereomers, with the exception of disulfideꢀcontaining macꢀ
rocycle 7 (3:1 dr). In this case, the steric bulk of the proximal
StBu group may influence the approach of the nucleophile
toward the cyclic imine. Substrates of various ring sizes (7 to
10 residues) and structural motifs were also amenable to cyꢀ
clization, and a linear precursor bearing a randomly generated
sequence of amino acids reacted smoothly to afford macrocyꢀ
cle 5 (86% yield). In some cases, slow macrocyclizations (e.g.
7) were accompanied by addition of cyanide to the openꢀchain
aldehyde, affording a linear cyanohydrin. The inherent reversꢀ
ibility of this addition, however, allows gradual equilibration
to the more stable cyclic αꢀaminonitrile. In contrast to convenꢀ
tional macrocyclizations, in which the yields of recalcitrant
substrates are generally limited by the hydrolysis of an actiꢀ
vated ester, this thermodynamic macrocyclization presents a
viable approach to challenging cyclic targets. Importantly, the
αꢀaminonitriles formed from the Strecker protocol were also
amenable to further diversification, including nitrile hydrolysis
to the corresponding primary amide (8).
In another powerful example of the versatility of the reducꢀ
tive macrocyclization, we demonstrated the cyclization of the
all ꢀpentapeptide (17), which resisted conventional macrolacꢀ
tamization entirely at the same ꢀLeu/
ꢀPro junction.5a,26 Based
L
L
L
on hydrogenꢀbonding networks identified using NMR techꢀ
niques, the authors of the original study concluded that the
linear substrate is not conformationally predisposed to cyclize
at this junction,26 leading instead to exclusive hydrolysis of the
activated Cꢀterminal pentafluorophenyl ester. Although partial
aldehyde reduction was observed herein as a consequence of
reductive cyclization at the same conformationally disfavored
junction, this competitive process did not preclude the desired
cyclization pathway, with macrocycle 17 obtained in 36%
isolated yield. In an effort to probe the influence of αꢀchirality
on the cyclization of 17 and address the possibility of αꢀ
epimerization, a linear pentapeptide analogue bearing the corꢀ
responding Cꢀterminal
Cyclization of the ꢀvariant occurred to form a distinct peptide
product (18), indicating configurational stability in the macroꢀ
cyclization event. Interestingly, the ꢀLeu variant cyclized
rapidly and in higher yield than the ꢀLeu analogue, consistent
DꢀLeu aldehyde was also prepared.
D
D
L
with prior reports of improved cyclization efficiencies in linear
peptides with configurationally mismatched termini.5a,27 These
results highlight that while the reductive aminationꢀ
macrocyclization is responsive to conformational bias, in this
example, cyclic predisposition is not a strict prerequisite.
Linear amino aldehydes were also efficiently trapped with
NaBH3CN, establishing
a
robust reductive aminationꢀ
macrocyclization protocol (Scheme 2B). Though previously
explored for the purpose of peptide modification,24 reductive
amination has not been widely probed as a means of peptide
macrocyclization.25 We found that in aqueous NaOAc buffer,
reductive amination was efficient and highly functional group
tolerant (9–18), leaving primary amides, disulfides (11 and
15), alcohols (13 and 14), acids (16), phenols (13 and 14),
imidazoles (16) and sulfoxides (13) unscathed. Cyclic prodꢀ
ucts ranging in size from 5 to 10 residues (15 to 32 atoms)
were efficiently prepared. Although reactions were preferenꢀ
tially carried out at 1 mM concentration to minimize tendenꢀ
cies toward oligomerization, a thorough analysis of the effect
of concentration on the cyclization yield of Nꢀmethylated pepꢀ
tide 10 revealed a tolerance for concentrations up to 10 mM
(see Supporting Information).
Probing the Impact of Internal Lysine Residues
The reversible aldehyde–imine equilibrium provides an inꢀ
triguing platform for the evaluation of functional group speciꢀ
ficity. In particular, the pKa difference between the peptide αꢀ
amine (pKa = 8.9) and the Lys εꢀamine (pKa = 10.5) prompted
an investigation into the possibility of αꢀamine selective cycliꢀ
zations in the presence of unprotected Lys residues (Scheme
2C). Such selectivity is rare in baseꢀmediated macrolactamizaꢀ
tion methods. Several linear amino aldehydes bearing unproꢀ
tected Lys residues were prepared and cyclized using the
Strecker (19 and 21) and reductive amination (22 and 24) macꢀ
rocyclization methods. In each case, the major isolated cyclic
products corresponded to αꢀamineꢀlinked macrocycles (see
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Scheme 4. Substrate scope for imine macrocyclization employing intramolecular imine traps. A) Pictet-Spengler cyclization;
B) Thia-/selenazolidine macrocyclization.
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Supporting Information for NMR characterization). Importantꢀ
ly, when the sideꢀchain Lys group was masked with an Alloc
protecting group (e.g., substrates 20 and 23), nearly identical
yields of the cyclic products were obtained, suggesting that in
these cases, minor amounts of Lysꢀcyclized regioisomers were
not removed during the course of HPLC purification. Altꢀ
hough selectivity resulting from a conformational predisposiꢀ
tion (rather than a difference in amine pKa) cannot be definiꢀ
tively excluded, the incorporation of two substrates with ranꢀ
domly generated amino acid sequences (21 and 24) and a deꢀ
tailed structural interrogation of the linear precursor to macroꢀ
cycles 19 and 22 using NMR techniques and circular dichroꢀ
ism (vide infra) support the argument that a conformational
predisposition of the linear precursor is not crucial to the obꢀ
served selectivity.
ly). Such tandem modifications enable rapid substrate diversiꢀ
fication and have potential applications in bioconjugation
studies.
Intramolecular Imine Traps
Analogous to the intramolecular trapping implicated in the
biosynthesis of the macrocyclic natural product lugdunin (Figꢀ
ure 1), tethered nucleophiles were next evaluated as imine
traps (Scheme 4). Aromatic rings, including indoles (29 and
31) and imidazoles (32), engaged in a PictetꢀSpengler macroꢀ
cyclization event29 to afford cyclic peptides with an embedded
piperidine ring. Tethered thiol and selenol nucleophiles led to
the corresponding thiaꢀ (33 and 35) and selenazolidines (36),30
the latter of which has garnered recent interest as a potential
prodrug for selenocysteine.31 Following cyclization, these
heterocyclic intermediates could be efficiently oxidized to
introduce conformational rigidity as in the βꢀcarboline (30)
and thiazole variants (34). Incorporation of nonꢀnative, rigidiꢀ
fying motifs would typically require the synthesis of protected
amino acid building blocks and laborious de novo peptide
synthesis. The lateꢀstage diversification herein is an attractive
alternative to enable rapid analogue synthesis.
Late-Stage Functionalization and Introduction of
Bioorthogonal Handles
In addition to reliable functional group selectivity, the poꢀ
tential for lateꢀstage substrate diversification is critical to the
rapid development of new peptideꢀbased drugs. From a single
linear precursor, a variety of target macrocycles should ideally
be accessible through targeted modification of the ring juncꢀ
tion. Scheme 3 depicts a number of lateꢀstage modifications
facilitated by the imine macrocyclization protocol. Specificalꢀ
ly, isotopic labeling for potential applications in metabolic
studies was accomplished through Strecker macrocyclization
with K13CN (Scheme 3A). Harnessing the synthetic power of
native chemical ligation,28 mild and chemoselective acylation
of 1,2ꢀamino thiolꢀbased macrocycles (e.g., 15) with peptide
thioesters (e.g., S19) facilitates the rapid construction of comꢀ
plex peptide architectures (Scheme 3B). In addition, secondary
amines formed through reductive amination can be Nꢀacylated
or Nꢀalkylated with bioorthogonal handles, including biotin
(27) or an alkyne moiety (28) (Scheme 3C and D, respectiveꢀ
Synthesis of Natural Products and Cyclic Analogues
In a final display of the versatility of the macrocyclization,
lateꢀstage diversification of nonꢀribosomal imine macrocycles
to afford the natural products and associated analogues of
scytonemide A (37),11 koranimine (38),13 and lugdunin (39 and
40)14 (Scheme 5) was pursued. Interestingly, the propensity of
these cyclic imino natural products to spontaneously cyclize
upon resin cleavage or LCꢀMS analysis was highly substrate
dependent. The linear precursor to scytonemide A did not selfꢀ
assemble, but a gradual titration of an amine base in organꢀ
icsolvent allowed for conversion to the cyclic imine (see Supꢀ
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Scheme 5. Synthesis of natural products and structural analogues from linear amino aldehyde precursors.
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8
iPr
iPr
H
N
H
N
N
O
H
N
O
HN
HN
OH
OH
NH2
HN
HN
OH
Ph
Ph
H
O
OH
O
H
O
O
N
O
O
N
O
O
O
N
N
O
O
O
O
H
O
O
H
O
HN
HO
H
H
O
O
HN
HO
HN
HN
HN
HN
O
H
HN
HN
O
H
H
H
N
N
N
N
N
H
N
H
N
H
Ph
N
H
NH2
O
NH2
O
O
O
Ph
O
37, 66%
koranimine (S27)
scytonemide A (S26)
38, 71%
linear amino aldehyde
precursors
9
X
N
X
X
R
O
O
O
10
11
12
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H
O
N
H
N
HN
HN
HN
O
NH
NH
O
39: X = S; R = H, 31%
X
HN
NH
lugdunin
H
O
O
O
NH
NH
NH2
O
N
N
O
O
O
O
O
40: X = O; R = CH3, 37%
O
HN
O
NH
H
N
Thr-lugdunin
Ph
N
N
Ph
N
H
N
H
[yields over 14 steps
from resin loading]
O
O
43: X = S, 30% (59% brsm)
41: X = S, 82% (4 diastereomers)
42: X = Se, 63% (4 diastereomers)
HN
sanguinamide A
ꢀporting Information for NMR studies). In aqueous solution,
the transient imino linkage could be effectively trapped with a
hydride to afford macrocycle 37 in 66% isolated yield.
Structural Interrogation of Linear and Cyclic Peptides
With an aim to investigate the effect of peptide structure on
the efficiency of macrocyclization, select amino aldehyde subꢀ
strates and linear precursors to the nonꢀribosomal natural
products were interrogated using variable temperature NMR
and circular dichroism (CD). Peptides were first dissolved in
aqueous acetonitrile and analyzed using 1H and TOCSY NMR
at 282, 291, 300, and 309 K (see Supporting Information for
details). The shift of each N–H bond as a function of temperaꢀ
ture was evaluated and correlated to the extent of engagement
in hydrogenꢀbonding interactions (Figure 2). Based on this
analysis, linear peptide substrates S2 and S13 (which bears an
internal Lys residue) did not exhibit any evidence of internal
hydrogenꢀbonding (Figure 2A). Nꢀterminal Trp peptide S20
exhibited shielding at Phe4, possibly resulting from an interacꢀ
tion with the Trp indole moiety. Importantly, the success of
these substrates in imine macrocyclization chemistry was not
strongly correlated to the presence of hydrogenꢀbonding interꢀ
actions, with comparable yields for macrocyclization (54%,
57%, and 75% for the corresponding macrocyclic peptides 9,
22 and 29, respectively) obtained in all cases. Although this
analysis does not rule out the influence of hydrophobic effects
on cyclization efficiency, a qualitative link can be established
between hydrogenꢀbonding interactions and the degree of
structural organization, with macrocyclic products generally
exhibiting smaller N–H shifts in response to temperature varꢀ
iation than their more flexible linear precursors. This observaꢀ
tion was particularly evident for Lysꢀcontaining macrocycle
22, which exhibited a strong hydrogenꢀbonding interaction at
Val5 as a result of cyclization.
Koranimine was observed as an equilibrium mixture of lineꢀ
1
ar and cyclic forms following resin cleavage. Curiously, H
NMR analysis of the peptide did not reveal the characteristic
imine peak (~6.8 ppm), an observation echoed in the original
isolation paper.13 This behavior prompted a hypothesis that the
Nꢀterminal threonine residue may engage the imine intramoꢀ
lecularly to form the corresponding oxazolidine linkage at the
ring junction. Such intramolecular trapping is supported by
preliminary NMR data (see Supporting Information). Neverꢀ
theless, the presence of macrocyclic imine as a contributor to
the equilibrium mixture is evidenced by successful reduction
to afford macrocycle 38 in excellent isolated yield.
The natural product lugdunin (39), isolated from the humanꢀ
associated bacteria Staphylococcus lugdunensis in 2016 and
the first example of a cyclic peptide thiazolidine natural prodꢀ
uct,14 exhibited a strong preference for spontaneous cyclization
upon resin cleavage. An oxoꢀanalogue of lugdunin (40) bearꢀ
ing an Nꢀterminal threonine residue was isolated as an interꢀ
converting mixture of macrocyclic oxazolidines and the correꢀ
sponding linear amino aldehyde. In this case, macrocyclization
occurred at the analogous LꢀVal/LꢀThr ring junction present in
koranimine, lending further credence to the hypothesis that
spontaneous oxazolidine formation may be a contributing
mode of macrocyclization in nonꢀribosomal imine natural
products.
In addition to the nonꢀribosomal natural products described
above, sanguinamide A (43),32 a ribosomallyꢀsynthesized and
postꢀtranslationally modified peptide natural product was also
prepared. The key thiazole moiety was constructed using a
thiazolidine cyclization–oxidation sequence. Interestingly,
thermodynamic cyclization of the sanguinamide linear precurꢀ
sor as well as a selenoꢀanalogue resulted in epimerization at
the αꢀIle aldehyde (41 and 42); such configurational instability
at the αꢀposition was not observed upon functionalization of
the nonꢀribosomal imine natural products, a possible conseꢀ
quence of the thermodynamic selfꢀassembly10 implicated in
the biosynthesis of these molecules. Nevertheless, oxidation of
the preꢀsanguinamide diastereomeric thiazolidine mixture (41
to 43) kinetically favored the native αꢀconfiguration, thus afꢀ
fording sanguinamide A in 16% overall yield from the original
resin loading.33
In contrast to the generally disordered linear substrates exꢀ
amined in Figure 2A, the precursors to the imine natural prodꢀ
ucts exhibited varying degrees of structural organization (Figꢀ
ure 2B). The variable temperature NMR data agrees with qualꢀ
itative observations about the propensity of the linear natural
product precursors to cyclize upon resin cleavage (vide supra).
For example, linear scytonemide A S26, experimentally charꢀ
acterized as the least prone toward cyclization, showed miniꢀ
mal hydrogenꢀbonding interactions. In contrast, linear koranꢀ
imine S27 engaged in a number of hydrogenꢀbonding interacꢀ
tions, and lugdunin, which cyclized spontaneously upon resin
cleavage, had the most extensive hydrogenꢀbonding network.
In the nonꢀribosomal peptides examined, a higher degree of
sequence
hydrophobicity
(e.g.,
the
presence
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Figure 2. Structural interrogation of linear and cyclic peptides. Variable temperature NMR studies on A) model aldehydes and asꢀ
sociated macrocycles, and B) natural products and linear precursors; C) Circular dichroism studies.
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of numerous Phe, Leu, and Val residues, as in peptides S27
and 39) tended to correlate to an increased number of observed
hydrogenꢀbonding interactions, suggesting that both hydroꢀ
phobic effects and hydrogenꢀbonding interactions may conꢀ
tribute to the spontaneous cyclization of imine natural prodꢀ
ucts. Nevertheless, the macrocyclization efficiency of linear
substrates S26 and S27 was independent of their disparate
structural properties and amino acid sequences; similar yields
were obtained for the reductive amination of both substrates
(S26 to 37, 66% and S27 to 38, 71%) and the cyclization effiꢀ
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ciency was comparable to the model peptides examined in
AUTHOR INFORMATION
1
2
3
figure 2A. Notably, the natural productꢀderived macrocycles
37 and 38 also displayed a higher degree of intramolecular
hydrogenꢀbonding than their linear precursors.
Corresponding Author
* Eꢀmail: pbaran@scripps.edu (P.S.B.).
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Variable temperature NMR data was further supported by
structural information obtained from CD analysis of the linear
peptides (Figure 2C). Devoid of persistent hydrogenꢀbonding
interactions, model aldehydes S2, S13 and scytonemide A
aldehyde S26 afforded spectra consistent with a random coil
structure. In contrast, aldehyde S27 and lugdunin (39) exhibitꢀ
ed more complex structural elements associated with ordered
peptides. In the context of macrocyclization, these findings
collectively highlight the versatility of imine macrocyclizaꢀ
tion, whereby the accessibility of a cyclic architecture is not
inextricably wedded to a conformational preference in the
linear substrate. Indeed, trapping of a minor component of a
complex equilibrium mixture may be sufficient to drive a
highꢀyielding macrocyclization reaction. Further studies probꢀ
ing the effect of structural and conformational elements on
cyclization efficiency, including the role of sequence hydroꢀ
phobicity, are currently underway.
Author Contributions
‡These authors contributed equally.
ACKNOWLEDGMENT
Financial support for this work was provided by BristolꢀMyers
Squibb, NIH (F32GM117816 postdoctoral fellowship to L. R. M.
and GMꢀ118176), and NSF GRFP (J. N. D.). We thank Dr. David
Langley and Dr. Claudio Mapelli (BMS) for helpful discussions
and Dr. Brad Maxwell (BMS) for a sample of K13CN. We are
grateful to Dr. DeeꢀHua Huang and Dr. Laura Pasternack (The
Scripps Research Institute) for assistance with nuclear magnetic
resonance (NMR) spectroscopy.
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ASSOCIATED CONTENT
Supporting Information
Detailed experimental procedures and analytical data for linear
and cyclic peptides. The Supporting Information is available free
of charge on the ACS Publications website.
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