Directed C(sp3)-H arylation of tryptophan: Transformation of the directing group into an activated amide

Article abstract of DOI:10.1039/c9sc03440d

The 8-aminoquinoline (8AQ) directed C(sp³)-H functionalization was applied in the synthesis of β-arylated tryptophan derivatives. The laborious protecting group reorganization towards α-amino acids compatible for solid phase peptide synthesis (SPPS) was cut short by the transformation of the directing group into an activated amide, which was either used directly in peptide coupling or in the gram scale synthesis of storable Fmoc-protected amino acids for SPPS. In this work, directed C-H activation and nonplanar amide chemistry complement each other for the synthesis of hybrids between phenylalanine and tryptophan with restricted side chain mobility.

Full text of DOI:10.1039/c9sc03440d

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Directed C(sp3)–H arylation of tryptophan:
transformation of the directing group into an
Cite this: DOI: 10.1039/c9sc03440d
activated amide†
All publication charges for this article
have been paid for by the Royal Society
of Chemistry
*
Lennart Nicke, Philip Horx, Klaus Harms
and Armin Geyer
The 8-aminoquinoline (8AQ) directed C(sp3)–H functionalization was applied in the synthesis of b-arylated
tryptophan derivatives. The laborious protecting group reorganization towards a-amino acids compatible
for solid phase peptide synthesis (SPPS) was cut short by the transformation of the directing group into
an activated amide, which was either used directly in peptide coupling or in the gram scale synthesis of
storable Fmoc-protected amino acids for SPPS. In this work, directed C–H activation and nonplanar
amide chemistry complement each other for the synthesis of hybrids between phenylalanine and
tryptophan with restricted side chain mobility.
Received 11th July 2019
Accepted 3rd August 2019
DOI: 10.1039/c9sc03440d
rsc.li/chemical-science
challenging endeavor. The construction of b,b-diaryl a-amino
acids with different aryl substituents, ensuring full control over
Introduction
two vicinal stereocenters, has stimulated numerous synthetic
studies in the eld of asymmetric alkylation/conjugate addi-
tion,10 asymmetric hydrogenation11 and directed C–H activa-
tion.12 Most of these methods focus on the efficient
construction of the carbon skeletons and offer remarkable
stereocontrol, but neglect the use of versatile removable
directing groups to give adequately protected amino acids for
peptide synthesis based on the approach of Fmoc/Boc strategy.
Thus, to the best of our knowledge, there is no example of
a synthetic peptide bearing a nonsymmetrical b,b-diaryl alanine
motif.13,14 Inspired by the seminal work of Corey,12a as well as
Daugulis,12b Yu12c,d and Chen,15 we envisioned that diastereo-
meric b-phenyl tryptophans could be accessed employing
sequential C–H activation of alanine. In necessity of a strong,
bidentate directing group, we chose 8-aminoquinoline (8AQ).
Phth-Ala-8AQ and an N-protected 3-iodoindole could be starting
materials in a palladium-catalyzed, directed C–H activation to
give access to an enantiomerically enriched tryptophan deriva-
tive.15b These compounds have been shown to be competent
substrates in b-alkynylation16 and might also undergo arylation.
Given the sensitivity of indoles towards oxidative conditions,12d
we evaluated a mild method of 8-aminoquinoline cleavage. This
effort has proven to be an unnerving challenge17 and the prac-
titioner must choose from protocols only suitable for a limited
class of substances. In spite of numerous reactions employing
aminoquinoline-directed C–H activation chemistry,18 examples
of its use in natural product or functional molecule synthesis
are rare.19 The removal of 8-aminoquinoline oen demands
harsh reaction conditions, which represents a major drawback
in synthetic applications.20,12b Studies addressing this concern
have been published by Maulide, using an ozonolysis
Unnatural b-branched a-amino acids are promising tools for
the synthesis of peptide ligands with conformational constric-
tion in a topologically designed structure.1 The increased
hydrophobic surface of b-branched a-amino acids can mediate
molecular recognitions processes between bioactive peptides
and their target receptors.2 Full control of c-space3 is achieved
in bicyclic peptidomimetics, which strongly inuence the
properties of the target peptides and proteins.4 The conforma-
tional design of open-chain b-branched a-amino acids,
however, serves as a valuable platform in peptide ligand
design.2 Consequently, the investigation of reliable synthetic
routes towards b-branched a-amino acids has experienced
a renaissance in the past couple of years.5 In particular, great
attention has been payed to the synthesis of b,b-diaryl a-amino
acids due to unique hydrophobic interaction patterns6 and
promising medicinal applications of therapeutics containing
these structural motifs (Fig. 1).7
In our investigation of peptide-receptor interactions,
focusing on the inuence of altered indole presentation on
biological activity, we were in requirement of a c1-constricted,
conformationally anchored tryptophan building block for
standard Fmoc-SPPS. We assumed that b-phenylated trypto-
phan will be suitable for this purpose. While the synthesis of
b,b-diaryl a-amino acids bearing two identical aromatic groups
was widely elaborated in earlier studies,9 the synthesis of these
amino acids containing different aromatic groups is a more
¨
Philipps-Universitat Marburg, Fachbereich Chemie, Hans Meerwein Straße, 35032
Marburg, Germany. E-mail: geyer@staff.uni-marburg.de
† Electronic supplementary information (ESI) available. CCDC 1903900 and
1903901. For ESI and crystallographic data in CIF or other electronic format see
DOI: 10.1039/c9sc03440d
approach,21 Ohshima, using
a
Nickel-chelate assisted
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steps using hazardous TfN3 to achieve amide activation and
directing group removal.26 From our point of view, an applicable
method for directing group removal has to full the following
premises:
- high functional group tolerance
- preservation of stereochemical integrity
- compatibility with standard SPPS protecting groups
- useful yields in large-scale applications
Hence, we were intrigued by the structural similarity of 8-
aminoquinoline amides and the widely used Dawson linker for
C-terminal diversication of resin-bound peptides.27 A similar
amide activation strategy could be feasible for 8-aminoquino-
line amides, that opens new possibilities towards C-terminal
amino acid modications (Scheme 1).
Results and discussion
Fig. 1 (A) Aromatic mobility and conformational design of Phe, Tic
(L-tetrahydroisoquinoline carboxylate) and Dip (L-diphenyl alanine)
amino acids.8 (B) Therapeutic applications of the b,b-diaryl alanine
motif.7
To prove this concept, we subjected the common starting
material Phth-Phe-8AQ to hydrogenation conditions to generate
a 1,2,3,4-tetrahydroquinoline species. 5 mol% PtO2-catalyzed
hydrogenation in DCM/AcOH gave rise to reduction product 1
in irreproducible yields with concomitant formation of non-
identiable byproducts (Scheme 2).
However, enough starting material was generated to perform
a triphosgene amide activation, which has previously been used
in custom-made directing group removal.28 Treatment of tetra-
hydroquinoline 1 with triphosgene in DCM in the presence of
methanolysis22 and Mashima, utilizing epoxide opening for
tandem esterication.23 A recent report by Chen et al. utilized
IBX in an oxidative protocol for 8AQ cleavage to give various a-
amino acids as amides in high yields, however, Trp formed
a spiro-fused compound and could not be deprotected accord-
ingly.24 Standard procedures of amide activation using Boc2O to
generate a labile imide, show limited applicability, because they
only tolerate substrates with low steric hindrance.25 More
sterically demanding substrates usually require strain-releasing
¨
Hunig's base provided the acylurea 2 in 75% yield. In analogy to
Dawson's linker (MeNbz: N-acyl benzimidazolinone) for C-
terminal peptide modication, we suggest Nbzcyc (activated
urea) as abbreviation to indicate a similar, cyclic (index: cyc)
arrangement. We were pleased to nd that exposure of the urea
compound 2 to standard LiOH/H2O2 hydrolysis conditions gave
a clean formation of Phth-Phe-OH without opening of the
phthalimide protecting group or decarbonylation of the Nbzcyc
group. As a bonus, simple acid/base extraction was enough to
separate the pure free carboxylic acid from the cleaved urea 3.
With this valid concept in hand, we started to develop an
improved, chemoselective and high yielding procedure for
quinoline reduction under conditions described by Rueping for
the reduction of simpler quinolines using (chiral) phosphoric
acids and Hantzsch ester as reducing agents.29 This hydroge-
nation method shows a high functional group tolerance which
was displayed by the successful use of peptide catalysts for
stereoinduction on substituted 8-aminoquinolines.30 Prelimi-
nary trials of reducing the 8-aminoquinoline moiety of Phth-
Wrf(Boc)-8AQ31 4 (synthesis vide infra) with 2.4 eq. Hantzsch's
ethyl ester (HEEt) in the presence of 5 mol% diphenylphosphate
ꢀ
(DPP) in toluene at 60 C for 12 h gave 42% conversion of the
starting material to the corresponding transfer hydrogenation
product. Encouraged by these results, we supposed that raising
the reaction temperature might lead to higher conversion.
Contradictory to our hypothesis, executing this reaction at 80 ꢀC
gave only 22% conversion of the starting material aer full
consumption of HEEt, whereas room temperature led to 94%
conversion of the starting material aer three weeks. With these
surprising results in hand, we took an in-depth look in the
Scheme 1 (A) Sequential C(sp3)–H activation.12a,b,13 (B) Linker design
for C-terminal peptide modification.27 This work: Rationale of amidic
weakening strategy applied for a-amino acids prepared by directed
C–H activation.
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Scheme 2 (A) Validation of 8-aminoquinoline cleavage by amidic weakening strategy. (B) Reaction outcome using Rueping's conditions.
reaction products, suspecting non-productive pathways to be
As shown in Table 1, the use of LiBr dramatically increases
present, resulting in consumption of reducing agent. Indeed, the conversion (entry 1 and 2). The use of weaker Brønsted acid
high reaction temperatures favoured the progression of HCOOH increases the conversion up to 86% (entry 3), but is not
a Hantzsch ester disproportionation reaction, giving rise to capable of catalyzing the reaction without Lewis acid (entry 4).
a diastereomeric mixture of tetrahydropyridine 6 (compare As expected, using Lewis acid alone or omitting the Hantzsch
scheme 2B) and the oxidized pyridine derivative, respectively. ester shuts down the reaction completely (entry 5 and 6). Using
This side reaction was the underlying cause of rapid LiCl or LiI did not result in an improvement (entry 7 and 8).
consumption of reducing agent, which was not observed in the Optimal results were obtained when 2.4 eq. HEtBu were used35 in
reactions investigated by Rueping.32 We hypothesized, that the THF in the presence of ꢀ1.0 eq. of lithium bromide and formic
formation of the tetrahydropyridine is facilitated by the strong acid, respectively, at 40 C.
acid DPP, acting in a C-protonation step,33 followed by iminium
With optimized conditions for 8AQ reduction in hand, the
reduction with another molecule of Hantzsch ester.34 This arylation of alanine to generate a suitably protected tryptophan
intriguing alternative reaction pathway prompted us to carry was undertaken. Owing to the high steric demand of N-
out a competition reaction between quinaldine, a commonly protected 3-iodoindoles, C–H arylation was deemed
used substrate in Hantzsch ester mediated transfer hydroge-
nation, and Phth-Phe-8AQ (Scheme 3).
Only quinaldine was reduced in this competition experi-
Table 1 Reaction screening of 8AQ reduction. The reactions were
conducted in a 50 mg scale in THF. 2.4 eq. of HEtBu was used. 1.0 eq. of
Lewis acid and Brønsted acid were used unless otherwise stated
ment, so that 8-aminoquinoline amides were identied as
a substrate with signicantly lower reactivity. We reasoned that
this nding is due to (1) the lack of benecial effects of the
methyl group participating in the stabilization of cationic
intermediates and (2) the electron-donating properties of the
amide nitrogen atom. We assumed that these electron-donating
effects could potentially be lowered using oxygenophilic Lewis
acids. Furthermore, the use of weaker Brønsted acids might
decrease the Hantzsch ester's tendency to undergo dispropor-
tionation. The results of the reaction screening are surveyed in
Table 1.
Lewis
acid
Brønsted
acid
Conversion
(NMR)
Entry
Temp.
Time
1
2
3
4
—
DPP
DPP
HCOOH
HCOOH
—
HCOOH
HCOOH
HCOOH
HCOOHb
HCOOHb
HCOOH
HCOOH
HOAc
60 ꢀ
60 ꢀ
60 ꢀ
60 ꢀ
60 ꢀ
60 ꢀ
60 ꢀ
60 ꢀ
60 ꢀ
60 ꢀ
60 ꢀ
40 ꢀ
C
C
C
C
C
C
C
C
C
C
C
C
12 h
12 h
12 h
24 h
24 h
24 h
12 h
12 h
12 h
12 h
12 h
16 h
16 h
18%
78%
86%
n. r.
n. r.
n. r.
85%
72%
27%
85%
90%
99%
27%
LiBr
LiBr
—
LiBr
LiBr
LiCl
LiI
5
6a
7
8
9
10
11
12
13
LiBrb
LiBrc
LiBrc
LiBr
LiBr
40 ꢀC
a
no Hantzsch ester was used. b 0.25 eq. was used. c 2.4 eq. was used. n.
Scheme 3 Competition experiment between quinaldine and Phth-
r. ¼ no reaction.
Phe-8AQ.
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challenging. However, aer screening of silver additives (AgTFA, the majority of unconsumed halide could be reisolated (98%)
AgOAc, AgBF4, Ag2CO3 and Ag2CO3/(BnO)2PO2H), we found that and used in further reactions without any loss of efficiency.
AgTFA gave a clean indoylation at 60 ꢀC using 10 mol% More importantly, the arylated amino acid 4 could be isolated in
Pd(OAc)2 in tert-amyl alcohol to provide 83% of the desired 65% yield, again with excellent diastereoselectivity (dr > 25 : 1)
tryptophan 7 with complete retention of the a-stereocenter and no loss in enantiomeric excess (97% ee). The next steps
(99% ee). The methylene arylation proceeded in a substrate- were conducted in the same manner as for diastereomer 11
controlled, highly diastereoselective fashion (dr > 25 : 1) and shown in Scheme 4 giving yields in comparable ranges. Notably,
good yields (71%). The presence of an Na-phthalimide pro- for this respective diastereomer, the tetrahydroquinoline
tecting group signicantly increased a-epimerization in subse- product was isolated by ltration directly from the reaction
quent reactions. Generally, the manageability of the large-scale mixture in 72% yield. Again, the nal hydrolysis step yielded
synthesis was improved, when the phthalimide protecting excellent 97% of the SPPS building block Fmoc Wrf(Boc) OH
group was removed directly aer C–H arylation. The phthali- (12) (Scheme 5).
mide deprotection was accomplished using excess ethylenedi-
With both building block syntheses completed, we were
amine to give the free amine in 82% yield. Treatment of the interested in elucidating the origin of amidic weakening by our
amine with FmocCl resulted in the formation of the Fmoc developed method. In a rapidly growing eld of cross-coupling36
carbamate 9 in nearly quantitative yield. The following reduc- and transamidation chemistry25a,37 using activated, nonplanar
tion of the quinoline directing group was accomplished using amides as valuable synthons, we suspected the Nbzcyc to exhibit
our optimized reaction conditions gave 66% of the tetrahy- comparable features. Fortunately, we were able to crystallize
droquinoline, which was treated with triphosgene to generate a racemic sample of Phth-Wrf(Boc)-8AQ (rac-4) and D-congured
the acylurea 10 in 63% yield. With the activated amide in hand, Phth-ala-Nbzcyc (13). X-ray analysis of these single crystals (Fig. 2)
LiOH/H2O2 mediated hydrolysis was carried out. We were revealed distinct Winkler–Dunitz parameters38 regarding an
rewarded with the clean amide hydrolysis needing as little as increased amide twist s of the activated urea (s ¼ 6.9ꢀ) compared
ve minutes to give 96% of the target SPPS building block 11 to the amino-quinoline amide (s ¼ 3.3ꢀ). Nitrogen pyramidaliza-
without noteworthy cleavage of the Fmoc protecting group. tion (cN) was found to rise to 11.7ꢀ in the activated urea form,
Remarkably, all the reaction steps of the sequence shown in whereas cN was found to be 2.3ꢀ for the 8AQ amide rac-4.
Scheme 4 could be conducted in gram scale, so that in the end, Furthermore, the respective C–N bond length between the carbon
1.10 g (1.83 mmol) of the building block was obtained as a white atom of the carbonyl and the nitrogen atom of the directing group
solid, easily storable and ready to use for peptide synthesis.
For the synthesis of the b-diastereomer, it was necessary to
reverse the order of introduction of the aromatic residues,15b
which was achieved by starting from phenylalanine. The b-
indoylation of Phth-Phe-8AQ was expected to be a challenging
step, because the aryl halide decomposes at elevated tempera-
ture. However, conducting the reaction at temperatures not
higher than 80 ꢀC favoured the C–H arylation pathway. At these
temperatures, the reaction rate is only moderate and was
elevated using as much as ve equivalents of the halide. Luckily,
Scheme 5 Key arylation and completion of Fmoc-Wrf(Boc)-OH (12)
synthesis.
Fig. 2 The crystal structures of rac-4 and Phth-ala-Nbzcyc (13) show
distinct differences concerning amide twist, C–N bond length and
Scheme 4 Building block synthesis of Fmoc-Wsf(Boc)-OH (11).
nitrogen pyramidalization, explaining the easy removal of Nbzcyc
.
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˚
˚
was enlarged from 1.35 A to 1.40 A in the activated urea form,
resulting in a lowered efficiency of amidic resonance.
To gain a profound insight in the induced amidic weak-
ening, theoretical investigations were applied. Szostak et al.
showed that the resonance energy (ER) of amides correlates to
their reactivity in nucleophilic substitution reactions.37,39 We
therefore calculated the resonance energy for 8AQ amide rac-4
and urea 13, using crystal structures (Fig. 2) as starting points
for calculations. Following the established COSNAR method,40
which is readily applied for nonplanar amides,41 a decrease of
9.6 kcal molꢁ1 in comparison to the parent aminoquinoline was
observed. This decline in resonance energy correlates well to the
increase in reactivity towards nucleophilic substitution. Addi-
tionally, the bond length from C–N increases while the C]O
bond contracts, further supporting the weakening in amidicity.
The Winkler–Dunitz distorsion parameters of the calculated
structures are compared to the X-ray structures in Table 2.
To prove the overall applicability of the concept, we synthe-
sized the chimeric amino acid Boc-Wsy(Boc,Me)-Nbzcyc (15) and
probed the urea cleavage using nucleophiles for common
synthetic precursors. Considering the utility of Dawson's link-
er,27b,c Nbzcyc ureas could provide rapid access to several
carboxylic acid derivatives. The esterication of activated amide
15 using K2CO3 in MeOH led to a loss of stereointegrity at the
acidic a-position (dr 4 : 1), making a milder approach using
Scheme 6 Diverse synthetic applications.
used as a tryptophan substitute to replace the buried Trp6 residue
in standard automated SPPS to generate two modied 20mer Trp
cage (TC5b) mutants 21 and 22 (Scheme 7B).43
The Trp cage miniprotein was chosen because the side chain
rotamer of the buried Trp6 indole group is similar to Wrf.
Therefore, we hypothesized that the incorporation of Wrf into
TC5b would lead to less disturbance of the densely arranged cage
fold. The indole presentation in Wsf, however, should destabilize
the cage fold, because the antiperiplanar orientation between Ca–
Cb would force the phenyl ring to occupy the indole cavity in the
Trp cage motif. Both diastereomeric peptides 21 and 22 were
studied by NMR spectroscopy and compared to the native Trp
cage miniprotein TC5b. The chemical shi deviations of key
residues for cage fold indication44 were used to determine the
folded fraction of the two modied miniproteins (Fig. 3).
NOE patterns suggest that the N-terminal a-helical environ-
ment is retained for both diastereomers of the peptide.
However, according to the CSD values only the Wrf6 cage mutant
21 assumes a folded state (75%), whereas the Wsf6 mutant 22
shows a Trp cage folding population of only 28%. The strong
inuence of the restricted side chain mobility and the preferred
¨
Hunig's base imperative to give 90% of the methyl ester 16 with
no detectable erosion42 of the a-stereocenter. Next, we examined
the potential of the activated amide 15 to function in an active
ester fashion for coupling of dipeptides. The formation of
a dipeptide 17 was achieved using glycine methyl ester as the
ꢀ
¨
nucleophile in DMF with the aid of Hunig's base at 50 C for
24 h, which was isolated in 82% yield. Finally, treatment of the
activated amide with NaBH4 resulted in the clean formation of
the protected amino alcohol 18 in 91% yield (Scheme 6).
Based on the results of dipeptide formation, that proved the
ureas capability of acylating primary amines, we performed the
acylation of a resin-bound Leu-enkephalin precursor 19. Treat-
ment of the resin-bound peptide with Boc-Wsy(Boc,Me)-Nbzcyc led
to the formation of the Leu-enkephalin derivate 20 (Scheme 7A).
Furthermore, Fmoc-Wrf(Boc)-OH and Fmoc-Wsf(Boc)-OH were
Table
2 Comparison of Winkler–Dunitz parameters of Phth-
Wrf(Boc)-8AQ (rac-4) and Phth-ala-Nbzcyc (13), as well as resonance
energies and relative amidicity
rac-4
(X-ray)
rac-4
(Calcd)
Urea 13
(X-ray)
Urea 13
(Calcd)
Parameter
s [ꢀ]
3.3
2.3
3.1
5.6
1.22
1.35
—
3.0
0.3
3.4
3.3
1.22
1.37
16.6
91%
6.9
11.7
1.7
18.6
1.21
1.40
—
8.4
6.3
0.9
14.7
1.21
1.41
7.0
cN [ꢀ]
cC [ꢀ]
s + cN [ꢀ]
˚
C]O [A]
˚
C–N [A]
ER [kcal molꢁ1
]
Amidicitya
—
—
38%
a
Relative to dimethyl acetamide.
Scheme 7 Applications in peptide synthesis.
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Notes and references
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Conclusion
In conclusion, we showed that the close-to-inert amide bond of
the widely used directing group 8-aminoquinoline for C–H
activation could be addressed via amidic weakening through
a two-step synthetic procedure, including a highly chemo-
selective Hantzsch ester mediated reduction of the pyridyl
moiety, followed by urea cyclization. These ndings paved the
way to the gram-scale synthesis of highly sterically congested b-
branched a-amino acids in an orthogonally protected fashion
for solid phase peptide synthesis, bridging the rapidly growing
elds of directed C–H activation and nonplanar amide chem-
istry. The altered amide geometry was quantied through X-ray
and computational analysis of respective 8-aminoquinoline
amides and derived urea compounds to give a better under-
standing of the induced reactivity in nucleophilic substitution
and addition reactions. Ultimately, the generated chimeric
amino acids Wrf, Wsf and Wsy were used in solid phase peptide
synthesis to give derivatives of Leu-enkephalin and the mini-
protein Trp cage, posing the rst example of incorporating the
optically pure, nonsymmetrical b,b-diaryl alanine motif in
peptidic environments. Further investigations on the inuence
of these amino acids concering the folding and biological
activity of peptides are currently under way in our laboratory.
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Conflicts of interest
There are no conicts to declare.
´
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Acknowledgements
We thank Paultheo von Zezschwitz for access to chiral HPLC
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