Total Synthesis and Stereochemical Assignment of Streptide

Article abstract of DOI:10.1021/jacs.9b09067

Streptide (1) is a peptide-derived macrocyclic natural product that has attracted considerable attention since its discovery in 2015. It contains an unprecedented post-translational modification that intramolecularly links the β-carbon (C3) of a residue 2 lysine with the C7 of a residue 6 tryptophan, thereby forming a 20-membered cyclic peptide. Herein, we report the first total synthesis of streptide that confirms the regiochemistry of the lysine-tryptophan cross-link and provides an unambiguous assignment of the stereochemistry (3R vs 3S) of the lysine-2 C3 center. Both the 3R and the originally assigned 3S lysine diastereomers were independently prepared by total synthesis, and it is the former, not the latter, that was found to correlate with the natural product. The approach enlists a powerful Pd(0)-mediated indole annulation for the key macrocyclization of the complex core peptide, utilizes an underdeveloped class of hypervalent iodine(III) aryl substrates in a palladium-catalyzed C-H activation/β-arylation reaction conducted on a lysine derivative, and provides access to material with which the role of streptide and related natural products may be examined.

Full text of DOI:10.1021/jacs.9b09067

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Article
Total Synthesis and Stereochemical Assignment of Streptide
Nicholas A. Isley, Yusuke Endo, Zhi-Chen Wu, Brett C. Covington,
Leah B. Bushin, Mohammad R. Seyedsayamdost, and Dale L. Boger
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.9b09067 • Publication Date (Web): 02 Oct 2019
Downloaded from pubs.acs.org on October 5, 2019
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Journal of the American Chemical Society
Total Synthesis and Stereochemical Assignment of Streptide
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Nicholas A. Isley, Yusuke Endo, Zhi-Chen Wu, Brett C. Covington, Leah B. Bushin, Mohammad R.
‡
†,
Seyedsayamdost, and Dale L. Boger *
†
Department of Chemistry and Skaggs Institute for Chemical Biology, The Scripps Research Institute,
10550 N. Torrey Pines Road, La Jolla, CA 92037, USA
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‡
Department of Chemistry, Princeton University, Princeton, NJ 08544, USA
ABSTRACT. Streptide (1) is a peptide-derived macrocyclic natural product that has attracted
considerable attention since its discovery in 2015. It contains an unprecedented post-translational
modification that intramolecularly links the β-carbon (C3) of a residue 2 lysine with the C7 of a residue
6
tryptophan, thereby forming a 20-membered cyclic peptide. Herein, we report the first total synthesis
of streptide that confirms the regiochemistry of the lysine-tryptophan crosslink and provides an
unambiguous assignment of the stereochemistry (3R vs 3S) of the lysine-2 C3 center. Both the 3R and
the originally assigned 3S lysine diastereomers were independently prepared by total synthesis and it
is the former, not the latter, that was found to correlate with the natural product. The approach enlists
a powerful Pd(0)-mediated indole annulation for the key macrocyclization of the complex core peptide,
utilizes an underdeveloped class of hypervalent iodine(III) aryl substrates in a palladium-catalyzed C–
H activation/β-arylation reaction conducted on a lysine derivative, and provides access to material with
which the role of streptide and related natural products may be examined.
INTRODUCTION
Streptide (1) is a peptide-derived macrocyclic natural product that was first isolated from
Streptococcus thermophilus (Figure 1). Its production is controlled by a quorum sensing system,
consisting of a short hydrophobic peptide signal and a cognate transcriptional regulator, that is common
to many streptococci, including pathogenic organisms.1,2 Streptide biosynthesis commences with the
ribosomal production of a 30 amino acid peptide that is post-translationally modified by
macrocyclization and further processed by proteolysis to deliver the mature nine amino acid cyclic
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peptide.1-3 The structure of streptide and the biosynthetic origin of the macrocycle were disclosed in
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2015 through a combination of spectroscopic and enzymatic studies. NMR spectroscopic analyses
located the crosslink to the β-carbon (C3) of the residue 2 lysine and C7 of the residue 6 tryptophan,
giving rise to a 20-membered cyclic peptide. The α-carbon configurations were experimentally
determined to be L while computational studies in conjunction with NMR NOESY constraints suggested
3
a lysine-2 (S)-C3 configuration at the new chiral center. Subsequent biosynthetic investigations
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showed that the crosslink is installed in a single step by StrB, a radical SAM metalloenzyme, via radical-
mediated chemistry. More recently, homologs of StrB have been identified in pathogenic streptococci.4,5
2
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They have been shown to catalyze the same crosslinking reaction on a common AK GDGW peptide
core that contains the identical leader sequence but altered C-termini, depending on the producing
strain, indicating that streptide is the first characterized member of a growing family of such post-
6
translationally modified macrocyclic peptides. At present, the role of streptide and its congeners upon
secretion remains undefined.
Figure 1. Assigned lysine (3S) and revised (3R) structure of streptide (1).
7
In efforts that culminated in the total synthesis of the chloropeptins and complestatins, we
introduced an intramolecular palladium(0)-mediated indole annulation for macrocyclization of their
strained biaryl ring systems that contain crosslinks to either C6 or C7 of the indole of a (R)-tryptophan
embedded within their peptide cores.7,8 Not only did this reaction permit synthesis of their strained 16-
or 17-membered macrocyclic rings, but it did so in the context of fully functionalized peptides, providing
a powerful new macrocyclization reaction that was more effective than conventional alternatives.9
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Given the unusual structure of streptide, its unprecedented crosslink, its unknown function, and its low
natural abundance (1.7 mg/20 L production culture), we embarked on a total synthesis of streptide that
would enlist this palladium(0)-mediated macrocyclization reaction as a key step in the assembly of the
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0-membered cyclic peptide core. This approach would afford a total synthesis of 1 amenable to the
preparation of analogues or congeners, and at the same time provide a formidable test of the generality
of the indole annulation macrocyclization. Thus, key to our approach was the use of this intramolecular
Larock indole synthesis to close the 20-membered cyclic peptide, enlisting conditions that permit the
use of a 2-bromoaniline and its reaction with an alkyne that contains a removable large terminal
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substituent (–SiEt
) to control the indole cyclization regioselectivity (Figure 2).10 Not only would this
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cyclization provide the fully functionalized ring system of 1, but its use would represent only the second
reported application of what has proven to be a powerful macrocyclization reaction for the synthesis of
complex natural products. Additionally, a palladium-catalyzed C–H activation reaction11 on a lysine
derivative was planned for diastereoselective installation of a suitably functionalized β-aryl substituent
needed for implementation of the indole annulation.
Figure 2. Key retrosynthetic steps for the total synthesis of streptide.
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The efforts detailed herein culminated in the first total synthesis of streptide and provided an
unambiguous assignment of the regiochemistry and stereochemistry (3R vs 3S) of the lysine C3 center.
Both the 3R and the originally proposed 3S lysine diastereomers were independently prepared by total
synthesis and subsequent NMR and HPLC analysis demonstrated that the 3R diastereomer correlates
with the natural product. In addition to demonstrating the generality of the indole annulation
macrocyclization reaction, the approach led to the use of an underutilized class of hypervalent iodine(III)
aryl substrates in the palladium-catalyzed C–H activation/β-arylation functionalization of a lysine
derivative. With an approach at hand to synthesize streptide and related analogues, the function of this
natural product and its congeners can now be explored.
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RESULTS AND DISCUSSION
β-Arylation of lysine derivatives by palladium-catalyzed C–H activation. Since the initial
12
13
report by Corey utilizing Daugulis’ bidentate 8-aminoquinoline directing group, additional advances
in Pd-catalyzed β-functionalization of amino acids have been reported,14 including recent
15
functionalizations reported by Yu without added directing groups. Despite these reports, Pd-catalyzed
β-aryl functionalization of amino acids at non-benzylic secondary alkyl centers possessing β-hydrogens
16
is still underdeveloped. This challenge is compounded when intercepting an intermediate Pd(II) alkyl
species with a sterically demanding aryl halide coupling partner as required for our work (i.e. ortho-
substitution).17 To effectively install the appropriately functionalized aryl unit for the key indole
annulation macrocyclization reaction, we found that the use of a diaryliodonium salt allowed introduction
of a sterically hindered 3-bromo-2-acetamidophenyl group without competitive reactions of the requisite
aryl bromide in a reaction in which the empirically-derived conditions also proved important, perhaps
accelerating the reaction or suppressing competitive β-hydride elimination and C–Pd homolysis.18,19
This β-arylation reaction was efficient, scalable, diastereoselective and conducted with an
unsymmetrical diaryliodonium salt, bearing a non-transferable sterically large mesityl group.18 Thus,
20
treatment of 4, prepared from L-lysine (Supporting Information), and the iodonium salt 3, prepared in
two steps (86%) from commercially available 6-bromo-2-iodoaniline, with Pd(OAc) (0.2 equiv) and
2
AgOAc (2.0 equiv) in t-BuOH (0.1 M) provided 5 in good yield and diastereoselectivity (60%, 4–6:1 dr)
under mild reaction conditions (80 °C, 18 h). This was observed without competitive α-epimerization of
the product (>99% ee), intramolecular trap of the intermediate Pd(II)-palladacycle by the distal
carbamate, or β-hydride elimination (Scheme 1). The outcome of the reaction allowed us to carry both
the 3R (major) and 3S (minor) diastereomers forward to complete the synthesis of both streptide
stereoisomers. Fortuitously, though unanticipated, it was the major 3R diastereomer 5, not the minor
3S diastereomer 6, of this reaction that afforded streptide.
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1
Journal of the American Chemical Society
Scheme 1
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Initial efforts to install a functionalized aryl group at the β-position of lysine were conducted with
N-acetyl-6-bromo-2-iodoaniline (2b) and N-acetyl-2,6-dibromoaniline and were only modestly
15
successful. The native directing group ability of the carboxylic acid was explored as well as removable
carboxylic acid-derived directing groups (e.g., -CONHOMe and -CONHC carboxamides), albeit
F
6 5
11
unsuccessfully. Notably, when 4 was employed in early studies, the intermediate Pd(II) C–H insertion
product was often intercepted in an intramolecular amination reaction by the distal carbamate (NHBoc)
to give a pyrrolidine. Thus, a screen was conducted with the bis-phthalimide protected lysine derivative
1
0, lacking a distal carbamate NH and incorporating the strongly coordinating 8-aminoquinoline
13
directing group. The screen, which enlisted 2b as the coupling partner, was conducted with varied
solvents, palladium sources, bases, and additives, leading to confirmed product formation. However,
even with extensive optimization, product 11 was only observed at near stoichiometric loadings of
Pd(OAc)
2
(0.4 equiv) with excess aryl iodide (4 equiv), and then only afforded 11 in low yield (27%, 0.4
, 1.5 equiv of AgOAc, 0.07 M t-BuOH, 110 °C, 48 h) (Figure 3). As a result, efforts
equiv of Pd(OAc)
2
were refocused on alternatives to the coupling partner 2b, expecting that a stronger oxidizing reagent
may better access the needed Pd(IV) oxidative insertion intermediate than an aryl iodide with our
hindered substrate. Unsymmetrical diaryliodonium salts such as 3, bearing a nontransferable aryl
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group, were explored as coupling partners. Although such reagents have been used effectively,18,19
Sanford’s early mechanistic studies indicated that their participation in oxidative addition at Pd(II) is still
the rate-determining step in a PdII/IV cycle. This likely has discouraged their subsequent exploration in
couplings that might suffer competing reactions of the intermediate Pd(II) palladacycle. Treatment of
1
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1
0 even with near stoichiometric (vs excess) amounts of the iodonium salt 3 (1.2 equiv, 0.2 equiv of
Pd(OAc) , 1.2 equiv of K CO ) provided 11 in good yield (60%) with excellent diastereoselectivity (>20:1
2
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dr) under mild reaction conditions (110 °C, 16 h) without the need for other heavy metal additives (e.g.,
Ag(I)) (Figure 3). In the optimization of the reaction of 10 with 3, no reaction was observed at 25–60
°
C, an increased loading of base did not further improve conversions and use of toluene versus
ClCH CH Cl led to competitive β-hydride elimination. The extension of these observations to 4, as
2
2
shown in Scheme 1, now proved possible and provided 5 under mild conditions (t-BuOH, 80 °C, 18 h)
but required inclusion of AgOAc in the reaction mixture to avoid distal carbamate interception of the
intermediate Pd(II) C–H insertion product.
Figure 3. Initial key observations on C–H activation and subsequent arylation of a lysine derivative.
12
The stereochemistry of 5 and 6 was assigned initially based on the original work of Corey for
Pd-catalyzed β-arylation of phthaloyl-protected L-amino acids that contain the 8-aminoquinolyl directing
group (>20–4:1 dr favoring 2S,3R), in which the initial C–H insertion preferentially forms the sterically
favored intermediate 5-membered trans-palladacycle. In Corey’s work, the stereochemical
assignments were unambiguously established by X-ray structures of several of the major products,12
and such work subsequently has been extended to crystallographic characterization of the intermediate
trans-palladacycles themselves by Yu and Chen.14f,g Our initial assignments based on this work were
confirmed with the single crystal X-ray structure determinations of 12 and 13, derived from the major
and minor diastereomers 5 and 6, respectively (Figure 4).20
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Journal of the American Chemical Society
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Figure 4. Relative and absolute stereochemistry of 5 and 6 established by X-ray diffraction analysis.20
The major diastereomer 5 and its accompanying minor isomer 6 were not easily separated by
chromatography at this stage but were found to be readily separated after installation of the protected
N-terminal alanine residue. Thus, phthalimide deprotection of the diastereomeric mixture of 5 and 6 (4–
6
:1) with 1,2-diaminoethane (EtOH, 0–25 °C, 18 h) provided the corresponding free amines (99%),
which were immediately coupled with BocHN-Ala-OH (1.2 equiv, 1.2 equiv of 1-cyano-2-ethoxy-2-
oxoethylidenaminooxy)-dimethylamino-morpholino-carbenium hexafluorophosphate (COMU), 1.0
equiv of N-methylmorpholine (NMM), DMF, 0–25 °C, 18 h, 75–87% combined yield) to provide the
alanine-linked lysine-2 2S,3R (7) and 2S,3S (8) diastereomers (Scheme 1). At this point, the lysine C3
diastereomers were easily separated by column chromatography. Removal of the 8-aminoquinoline
directing group from the major isomer 7 was accomplished by mild oxidative deprotection (via
21
ozonolysis), which cleaves the quinolyl group to a reactive imide intermediate that we found can be
hydrolyzed selectively to provide not only a carboxamide (NH OH, THF, 25 °C, 18 h, 67%), but also
the carboxylic acid. Thus, ozonolysis of 7 in the presence of pyridine (3 equiv) at –78 °C (CH
0 min) followed by treatment of the isolated reaction product with H –LiOH (THF/H O, 0 °C, 1 h)
4
22
2
Cl , 5–
2
1
2
O
2
2
directly provided the carboxylic acid 9 in good yield (63%, Scheme 1). Whereas ozonolysis in absence
of pyridine led to substantial amounts of carboxamide product upon hydrolysis, its use as an ozonolysis
22
additive tempered the oxidation reaction and thereby helped suppress the competitive carboxamide
formation in the subsequent intermediate imide hydrolysis.
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Assembly of the N-terminal core hexapeptide and Larock macrocyclization. The linear
peptide 19, incorporating 9 and the partner triethylsilyl alkyne that serve as the precursors for the
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crosslinked tryptophan, was prepared by coupling H N-Gly-OBn with FmocHN-(OtBu)Asp-OH
2
promoted by N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride and 1-hydroxy-7-
azabenzotriazole (EDCI–HOAt, DMF) to afford the dipeptide 14 in 89% yield (Scheme 2). Deprotection
of 14 (1 equiv of 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), CH
2
Cl , 0 °C, 1.5 h) under conditions found
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to suppress cyclization followed by coupling of the released free amine in the same vessel with
FmocHN-Gly-OH (EDCI–HOAt, 16 h) provided 15 in 85% yield. Benzyl ester removal by hydrogenolysis
7a
(
H , Pd/C, THF, 23 °C, 16 h) afforded the tripeptide 16 (90%), which was coupled with 17 (preparation
2
supplied in Supporting Information, 1.07 equiv of HBTU, 1.07 equiv of NMM, DMF, 0–23 °C, 16 h) to
provide 18. (2-(1H-Benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU) was
found to be the optimal coupling reagent of those examined, providing 18 in good yield (84%) and under
conditions that do not result in α-epimerization adjacent to the methyl ester. After in situ Fmoc
deprotection of 18 (DBU, CH
was accessed by coupling the liberated amine with 9 (1.2 equiv of COMU, 1.0 equiv of NMM, CH
–25 °C, 18 h, 83%).
2
Cl
2
, 0 °C, 1 h), the linear hexapeptide 19 of the streptide macrocyclic core
2
Cl ,
2
0
Scheme 2
Macrocyclization of 19 was first examined under conditions we previously introduced (1.1 equiv
of Pd(OAc)
2
, 1.3 equiv of 1,1’-bis(di-tert-butylphosphino)ferrocene (dtbpf), 1.3 equiv of Et N,
3
7
toluene/MeCN (1:1), 100 °C, 1 h), permitting use of an aryl bromide. The reaction provided 20 in good
albeit variable yield (50%, Figure 5). The addition of Bu NBr (1 equiv), which is thought to increase
4
catalyst stability by preventing Pd black formation,23 maintained but did not improve the yield of 20
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(
45%). However, the reaction remained variable, especially on small scale, complicating its optimization
and scale up. The use of other ammonium salts or additives (e.g., LiCl) did not improve this further.
Whereas other Pd catalysts such as PdCl (MeCN) , Pd (dba) , PdCl (dtbpf), and (o-tol P) Pd failed to
provide the macrocyclization product or did so poorly as noted in our previous work,7,8 (t-Bu
P) Pd also
1
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1
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2
2
2
3
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3
3
3
2
proved to be effective and provided consistent conversions as reported by Reisman.24 Further
optimization focused on the use of this catalyst (Figure 5). Macrocycle 20 was obtained in 60% yield
0
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under even milder reaction conditions (80 °C, 0.001 M MeCN, 1 h), using Et
3
N or dicyclohexyl
methylamine (Cy NMe, 1.3 equiv) and (t-Bu P) Pd (1.1 equiv) (entries 4 and 8). Although Et
2
3
2
3
N could
be used as the added base (entry 4), its lower boiling point led to more variable yields on small scale
(entry 4 vs 3), and this is likely the source of the variable conversions we initially observed with its use
with Pd(OAc)
the desired product in comparable, but not improved, yields (entries 7 and 9). Lastly, a catalyst loading
of 0.6 equiv of (t-Bu P) Pd matched the conversion of its stoichiometric use (58%, 2 h, entry 6 vs 8 and
), whereas use of 0.2 equiv of (t-Bu P) Pd also provided 20, albeit in lower yield (34%, 4 h, entry 5).
Because such reactions work effectively with catalytic Pd(OAc) or (t-Bu P) Pd on simpler non-peptide
2
. Prolonged reaction times beyond 1 h in addition to increased base loading also provided
3
2
9
3
2
2
3
2
substrates,8,24 it is likely that the precursor 19 or the macrocycle 20 act to sequester the catalyst,
requiring the higher catalyst loading.7,8 However, further efforts to optimize the reaction for sub-
stoichiometric use of the Pd catalyst were not pursued as it was not central for the work detailed herein.
Nonetheless, it is remarkable that the Larock cyclization, adapted for use with an aryl bromide, performs
effectively for formation of a 20-membered macrocycle on a complex substrate that contains 6 amides,
2 carbamates and 2 esters, one of which is prone to epimerization.
Figure 5. Representative results of the macrocyclization optimization.
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With 20 in hand, its selective or exhaustive deprotections were examined. This not only set the
stage for completion of the total synthesis of streptide, but it also permitted the examination of the
conformational properties of 1 and the core macrocycle. TES removal under mild conditions (3 equiv
1
2
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8
9
1
1
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of Bu
of 20 with LiOH or related reagents was challenging due to competing epimerization and partial indole
N-acetyl deprotection, the hydrolysis to provide 22 was accomplished cleanly with Me SnOH (88–
3%). Finally, studies on the single-step global deprotection of 22 provided complex mixtures unless
4
NF, THF, 0 °C, 78%) provided 21 (Figure 6). Whereas hydrolysis of the C-terminal methyl ester
3
0
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25
9
a trialkylsilane was included in the reaction mixture, acting to trap cationic intermediates arising from
the deprotections. Thus, both 23 (71%) and 24 (94%) were obtained in excellent yields from 22 following
HCl or trifluoroacetic acid (TFA) treatment and are indicative of clean TES removal along with cleavage
of the t-butyl carbamates and t-butyl ester and a slower hydrolysis of the N-acetylindole.
Figure 6. Deprotection studies on the macrocyclic core.
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Macrocycle 20 and each of the partial or complete deprotection products 21–24 displayed
1
2
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5
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7
8
9
1
1
1
1
1
1
1
1
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2
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6
relatively sharp H NMR spectra consistent with adoption of rigid or rapidly interconverting
conformations. At 1 °C but not at 25 °C, only 20 displayed a limited number of distinct (e.g., indole C4
1
CH) and sharp H NMR signals (CD
3
OD) attributable to two (major and minor) conformations
(Supporting Information Figures S1–S6). Although not examined in detail, these comparisons of 20 and
21 suggest that 20, as well as 21, do not display atropisomeric behavior at room temperature.
Completion of the total synthesis of streptide. As detailed above, selective hydrolysis of the
0
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0
methyl ester 20 to provide 22 was achieved with use of Me
.5–2 h, 88–93%), leaving the indole N-acetyl group untouched and avoiding detectable epimerization
(Figure 6). Typical hydrolysis reactions mediated by LiOH, with or without added H , either led to
3
SnOH (4–10 equiv, ClCH
2
CH Cl, 80 °C,
2
4
2
O
2
apparent competitive epimerization or partial deprotection of the indole N-acetyl group. Fmoc
deprotection of the tripeptide 25 (2 equiv, synthesis in Supporting Information) with DBU (1.5 equiv, 0
°C, 1 h) followed by coupling of the free amine with 22 (2 equiv of HBTU, 1 equiv of NMM, DMF/CH
–25 °C, 18 h) provided 26 (75%, >99:1 dr), constituting a fully protected streptide (Scheme 3).
Although not investigated in detail, alternative amide coupling reagents such as COMU provided only
trace product formation, whereas use of EDCI–HOAt (CH Cl ) and HATU (DMF) led to apparent
2 2
Cl ,
0
2
2
competitive α-epimerization but provided the products in good yield (56% and 67%, respectively). A
single-step global deprotection of 26 to provide streptide (1) with removal of 7 protecting groups was
achieved by treatment with aqueous 5 N HCl/Et SiH/1,2-ethanedithiol (8:1:1) at room temperature. This
3
smoothly and cleanly provided streptide (1) in high yield (82–94%) after purification by reverse-phase
HPLC, completing the first reported total synthesis of streptide.
Scheme 3
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Comparison of the spectroscopic properties of synthetic 1 with the H and 13C NMR spectra
reported for authentic streptide provided a near identical match. In the course of the initial
characterization of synthetic 1, we found that trace amounts of acid (TFA) residual from our HPLC
purification can impact the chemical shifts observed in the NMR spectra and it is possible concentration
dependent distinctions were also observed. This initial and unexpected correlation with natural streptide
1
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
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6
1
was particularly strong for the H NMR spectrum, whereas there were a few larger chemical shift
0
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0
differences in the 13C NMR. It was not clear whether the unexpected spectroscopic correlation of
synthetic 1, containing the lysine 3R (not the proposed 3S) stereochemistry, with natural streptide
represented a true correlation or one that was fortuitously close. Consequently, and as originally
planned, the lysine 3S-diastereomer of streptide was also prepared.
Total synthesis of the lysine-2 3S-diastereomer of streptide. The lysine-2 C3 configuration
in streptide could not be unambiguously established by spectroscopic methods in the original structure
3
determination studies. Instead, a computational approach was employed to assess correspondence
between the computed streptide structures and constraints derived from NOESY correlations using the
CYANA algorithm. The diastereomer that satisfies more constraints would give a lower target function
1
(
f). Both S and R C3 configurations could be acceptably fit to the H NMR data (e.g.; S vs R: f = 0.042
vs 0.054, rmsd = 1.62 vs 1.72 Å, and accounting for 29 vs 27 observed NOEs between calculated and
3
observed values) with the S-diastereomer providing a slightly better fit. In addition, the original
characterization of the natural product and its spectroscopic data were collected on limited amounts of
sample. To unambiguously assign the configuration at the lysine C3, the lysine 3S-diastereomer 8,
arising from the minor diastereomer 6 generated in the C–H functionalization reaction, was carried
through the identical synthetic sequence (Scheme 4). Notably, the Pd(0)-promoted macrocyclization of
2
8 provided 29 in conversions (60%) identical to that observed with the diastereomer 19 without
alteration of the reaction conditions (80 °C, 0.001 M CH CN, 1 h). This synthesis provided 32, the
3
lysine-2 C3 S-diastereomer.
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Scheme 4
1
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7
8
9
1
1
1
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0
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Reisolation of and comparison with natural streptide. To facilitate the comparison of the
synthetic streptide diastereomers with the authentic natural product, streptide was isolated from 20 L
3
of S. thermophilus production cultures using previously established procedures, yielding approximately
1
.7 mg of pure material. After completion of the synthetic work and with the benefit of having freshly
isolated streptide available, detailed NMR and HPLC-MS comparisons were conducted. Upon side-by-
side examination, natural streptide and the synthetic lysine-2 3R diastereomer clearly correlated. The
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1H and 13C NMR data were essentially identical, whereas the 3S diastereomer was easily
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
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2
2
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2
2
2
2
3
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3
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3
3
3
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4
4
4
4
4
4
4
4
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5
5
5
5
5
5
5
6
1
distinguishable with divergent chemical shifts at the lysine-2 α- H (4.81 ppm for the 3R diastereomer
1
and authentic streptide, 5.02 for the 3S diastereomer) and lysine-2 β- H (3.52 ppm for the 3R
diastereomer and authentic streptide, 4.03 ppm for the 3S diastereomer), among others (Supporting
1
13
Information Figure S20–S23). Moreover, we were able to demonstrate that the H NMR and C NMR
spectra of 1 display subtle condition-dependent shifts and changes largely centered within or adjacent
0
1
2
3
4
5
6
7
8
9
0
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5
to the acidic residues (Met , Asp , Gly and Gly ), accounting for the uncertainty in our initial correlation
with the original published spectra (Supporting Information Figures S17–S19 and accompanying
1
2
6
discussion). However, changes in the chemical shifts for Ala , Lys and Trp were not observed (< 0.01
ppm differences), indicating a lack of any impact of either the protonation state or any major
conformational change of the macrocycle under the conditions examined. In agreement with the NMR
comparisons, high-resolution HPLC-MS analysis unambiguously demonstrated co-elution of authentic
streptide with the 3R diastereomer 1, but not with the 3S diastereomer 32, which could be separated
from both when injected as mixtures (Figure 7). Together, these results confirm the first chemical
synthesis and establish the full structural assignment of streptide.
Figure 7. HR-HPLC-MS analysis of authentic and synthetic streptides. The sample loaded is labeled
on top of the resulting elution profile. Streptide elution was monitored by absorbance at 280 nm (not
shown) and by MS total ion count. Shown is the HR-MS-extracted ion chromatogram for streptide (m/z
9
3
89.489) in each trace. Authentic streptide co-elutes with lysine-2 C3 diastereomer 1 (3R) but not with
2 (3S). See Supporting Information for details.
With knowledge of the absolute configuration of the lysine-2 C3, we generated a computational
model of streptide in CYANA using constraints derived from NOESY data that were collected on
authentic 1.3,26 The crosslinked Lys/Trp residues were added manually to the CYANA residue library
and the configuration of the lysine-2 C3 was explicitly defined as R. The calculation provided possible
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solution conformers, with the conformer that had the lowest target function, and therefore exhibited the
best agreement with the experimental constraints, depicted in Figure 8. It features an indole-based
cyclophane, a (3,7)indolophane, in which the encircling peptide adopts a rigid conformation enforced
by the amide bonds within the macrocycle and the bridging indole. The three-dimensional model of 1
facilitates future docking studies to possible target sites.
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
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8
9
0
Figure 8. Computational model of the three-dimensional structure of streptide.
CONCLUSION
Herein, we report the first total synthesis of streptide that provided confirmation of the site
(regiochemistry) and established the configuration (stereochemistry, 3R vs 3S) of the residue 2 lysine
C3 central to the unusual crosslink in this natural product. Both the 3R and the initial computationally
assigned 3S lysine diastereomers were independently prepared by total synthesis, involving a longest
linear synthetic sequence of 11 steps, and it was the 3R diastereomer that was found to correlate with
the natural product. The approach utilized a powerful Pd(0)-mediated indole annulation for
macrocyclization of the core 20-membered cyclic peptide, enlisted an underutilized hypervalent
iodine(III) aryl substrate in a key palladium-catalyzed C–H activation and -arylation reaction conducted
on a lysine derivative, and provides access to material with which the role of streptide and related
natural products may be examined.
ASSOCIATED CONTENT
Supporting Information
Full experimental details. The Supporting Information is available free of charge on the ACS
Publications website at DOI: XX.
AUTHOR INFORMATION
Corresponding Author
*Email: dale.boger@outlook.com
N.A.I. and Y.E. contributed equally to this work as first authors
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Notes
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9
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1
1
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6
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
We gratefully acknowledge financial support from the NIH (CA041101 to D.L.B. and postdoctoral
fellowship F32 GM114948 to N.A.I.), Kaken Pharmaceutical Co., Ltd. (to Y.E.), the Burroughs
Wellcome Fund (PATH Investigator Award to M.R.S.), and the NSF (NSF GRF Award to L.B.B.). We
thank Milan Gembicky, C. Moore and A. Rheingold of the Crystallography Facility at the University of
California, San Diego for X-ray structure determinations of 3, 12 and 13.
0
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(
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0
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