Backbone-Fluorinated 1,2,3-Triazole-Containing Dipeptide Surrogates

Article abstract of DOI:10.1021/acs.joc.7b01744

The 1,2,3-triazole moiety can be incorporated as a peptide bond bioisostere to provide protease resistance in peptidomimetics. Herein, we report the synthesis of peptidomimetic building blocks containing backbone-fluorinated 1,4-disubstituted 1,2,3-triazole moieties. Synthetic protocols for the preparation of various Xaa-Gly dipeptide surrogates in the form of Xaa-ψ[triazole]-F₂Gly building blocks were established, and selected examples were introduced into the endogenous peptide opioid receptor ligand Leu-enkephalin as a model compound.

Full text of DOI:10.1021/acs.joc.7b01744

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Note
Backbone-Fluorinated 1,2,3-Triazole-Containing Dipeptide Surrogates
Jens Engel-Andreasen, Isabelle Wellhöfer, Kathrine Wich, and Christian Adam Olsen
J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.7b01744 • Publication Date (Web): 06 Oct 2017
Downloaded from http://pubs.acs.org on October 8, 2017
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Backbone-Fluorinated 1,2,3-Triazole-Containing Dipeptide
Surrogates
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Jens EngelꢀAndreasen,† Isabelle Wellhöfer, Kathrine Wich,‡ and Christian A. Olsen*
Center for Biopharmaceuticals & Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences,
University of Copenhagen, Universitetsparken 2, DKꢀ2100, Copenhagen, Denmark
Supporting Information Placeholder
ABSTRACT: The 1,2,3ꢀtriazole moiety can be incorporated as a peptide bond bioisostere to provide protease resistance in pepꢀ
tidomimetics. Herein, we report the synthesis of peptidomimetic building blocks containing backbone fluorinated, 1,4ꢀ
disubstitutedꢀ1,2,3ꢀtriazole moieties. Synthetic protocols for preparation of various XaaꢀGly dipeptide surrogates in the form of
Xaaꢀψ[triazole]ꢀF2Gly building blocks were established and selected examples were introduced into the endogenous peptide opiod
receptor ligand Leuꢀenkephalin as a model compound.
Peptides are pharmacologically active compounds that play
vital roles in cellular regulation. Naturally occurring peptides
have generally been evolved to exhibit high target affinity and
specificity, and may therefore offer attractive starting points
for development of novel therapeutics. However, peptides
suffer from poor metabolic stability as a result of proteolytic
susceptibility in vivo and are often poorly cell permeable.
Efforts to circumvent these disadvantages related to peptides
and proteins as drug candidates have resulted in extensive
research within unnatural oligomers mimicking biological
function.1 Substitution of amide bonds with bioisosteres in
biologically active peptides may provide access to new pepꢀ
tidomimetic structures with improved physicochemical and
pharmacokinetic properties. Examples of these modifications
include functional groups such as esters, thioesters, thioamꢀ
ides, hydrazides, and ureas2 as well as heterocycles such as
tetrazoles, oxazoles, and 1,2,4ꢀtriazoles.3 Over the last decade,
the application of 1,2,3ꢀtriazoles have attracted increased
in the periodic table and thus, the C–F bond is highly polarized
and displays significant ionic character. The large dipole moꢀ
ment of the C–F bond has a considerable effect on the conꢀ
formational behavior of organofluorine compounds.12 Fluorine
has been introduced extensively into drug molecules,13 here it
can engage in dipole–dipole and charge–dipole interactions14
in addition to the potential benefits of the altered physicoꢀ
chemical properties.11c Substitution of hydrogen atoms with
fluorine have also been reported for proteins15 and
peptidomimetics.16
βꢀpeptide
We envisioned that triazoleꢀbased amide bond isosteres
could be combined with a gemꢀdifluoromethylene moiety in
peptide backbones to combine their features and extend the
current arsenal of peptidomimetic architectures.17 While this
manuscript was in preparation, a communication describing
the same backbone modification was published.18 However,
the synthetic strategy developed in the current work is fundaꢀ
mentally different, since we rely on synthesis of dimer buildꢀ
ing blocks, rather than assembly of fragments via the click
reaction. Our protocols therefore complement the previous
work by extending the structural diversity to include amino
acid side chains.
attention, following the discovery of regioselective Cu(I
)ꢀ4 or
Ru(II)ꢀcatalyzed5 azide–alkyne cycloaddition, to furnish 1,4ꢀ
or 1,5ꢀdisubstituted 1,2,3ꢀtriazoles, respectively. Although
distances between side chains are slightly longer than for a
peptide bond, the 1,2,3ꢀtriazoles have shown promise as amide
bond bioisosteres.6 with efficient synthesis of their azide and
alkyne precursors from commercial amino acids by diazo
transfer7 and SeyferthꢀGilbert homologation8 using the Ohiraꢀ
9
Bestmann reagent,6j, respectively. Furthermore, macrocyꢀ
clization of peptidomimetics by “click chemistry” has gained
interest due to its synthetic efficiency.6j, 10
Figure 1. Generic structure of XaaꢀGly dipeptide (1) and a backꢀ
bone fluorinated 1,2,3ꢀtriazoleꢀcontaining dipeptide surrogate (2).
It has been acknowledged that hydrogen to fluorine substituꢀ
tion in organic compounds can provide useful pharmacokinetꢀ
ic properties such as enhanced stability and altered lipophiliciꢀ
ty.11 Furthermore, fluorine is the most electronegative element
Thus, we report the synthesis of a series of gemꢀ
difluorinated 1,2,3ꢀtriazole building blocks manifested as Xaaꢀ
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Gly dipeptide surrogates that may be incorporated to modify reaction conditions was difficult to determine due to the volaꢀ
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the properties of bioactive peptides (Figure 1). Finally, we
demonstrate the solidꢀphase peptide synthesis (SPPS) of anaꢀ
logs of the endogenous opioid receptor ligand Leuꢀenkephalin.
tile nature of intermediates 6 and 7, which precluded their
isolation. Thus, azide 7 was reacted with alkyne 5 directly
without purification or evaporation to dryness to give 8 in a
threeꢀstep procedure. Finally, the Cꢀterminal tꢀBu ester was
hydrolyzed by 50% trifluoroacetic acid (TFA) in CH2Cl2 to
give building block 9 (Scheme 1C).
The synthesis of gemꢀdifluoromethyleneꢀcontaining 1,2,3ꢀ
triazoles is scarcely described in literature.18ꢀ19 However, in
2011 Zhao et al. described the synthesis of 1,4ꢀdisubstituted
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9
1,2,3ꢀtriazoles by Cu(
I
)ꢀcatalyzed azide–alkyne click chemisꢀ
Next, a series of dipeptide surrogates containing proteinoꢀ
genic amino acid side chains in the alkyne half of the molecule
were prepared. The alkynes corresponding to phenylalanine
(a), tyrosine (b), methionine (c), lysine (d), and arginine (e)
were prepared from their corresponding Bocꢀprotected amino
acids to demonstrate functional group compatibility with the
building block synthesis (Scheme 2A). First, carbodiimideꢀ
mediated reaction with N,Oꢀdimethylhydroxylamine afforded
Weinreb amides 10a–e, which were reduced using LiAlH4 to
give Bocꢀprotected amino aldehydes 11a–e. Preformed Ohiraꢀ
Bestmann reagent (12) in acetonitrile was mixed with crude
aldehyde in methanol solution to provide the desired alkynes
13a–e (Scheme 2A).
try (CuAAC) from ethyl 2ꢀazidoꢀ2,2ꢀdifluoroacetate,19d which
potentially could be directly applied for the synthesis of Xaaꢀ
ψ[triazole]ꢀF2Gly building blocks. The azide substrate was
prepared by azidation of the commercially available ethyl 2ꢀ
bromoꢀ2,2ꢀdifluoroacetate. However, in our hands this azidaꢀ
tion reaction provided poor yields of a product containing
impurities that were difficult to separate, presumably due to
the reactivity of the ethyl ester. Instead, the N,Nꢀdiethylamideꢀ
protected derivative (3)20 provided compound 4 in good yield
(Scheme 1A). For initial testing of the cycloaddition, the
Fmocꢀprotected alkyne derivative of glycine (5) was selected,
which is readily available by Fmoc protection of propargyl
amine (Scheme 1B). Subjecting compounds 4 and 5 to the
conditions reported by Zhao et al. provided the fully protected
dipeptide surrogate in 27% yield.17d The subsequent hydrolysis
of the N,Nꢀdiethylamide was previously accomplished in reꢀ
fluxing 12 M aqueous HCl,21 and we hypothesized that milder
conditions could be applied due to the electron withdrawing
effect of the two alpha fluorine atoms. Unfortunately, milder
acidic conditions (1–6 M HCl) failed to hydrolyze the amide,
and basic hydrolysis was inappropriate because of the base
labile Fmoc group. Instead, we chose to prepare the tꢀBu ester
derivative 8, which was synthesized by cycloaddition of buildꢀ
ing blocks 5 and 7 (Scheme 1C).
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Scheme 2. Synthesis of protected Xaa-Gly dipeptide surro-
gates 14a–e
This method has been employed previously for selected
amino acids where the stereochemical integrity was investiꢀ
gated by Mosher’s method.23 It was therefore gratifying that
optical rotations of our products were in agreement with literaꢀ
ture in the cases of known compounds (13a,b,d). The Boc
protecting group was chosen because Fmoc is not compatible
with LiAlH4, and depending on the desired protecting groups
in one’s final building block, manipulation may be considered
at the amino alkyne stage. We decided to prepare Bocꢀ
Scheme 1. Synthesis of Fmoc protected Gly-Gly dipeptide
surrogate 9
In this reaction, we added the ligand TBTA (tris[(1ꢀbenzylꢀ
1Hꢀ1,2,3ꢀtriazolꢀ4ꢀyl)methyl]amine)22 for the Cu(
I) catalyst,
which resulted in an improved yield of 8, compared to the
initial reaction. Albeit, the isolated efficiency of the modified
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protected building blocks 14a–e, which can undergo Cꢀ
eties.24 Because it comprises two glycine residues, we could
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terminal deprotection under basic conditions or acidic cleavꢀ
age of the Boc group for solutionꢀphase peptide synthesis
(Scheme 2B).
prepare analogs containing either the GlyꢀGly or TyrꢀGly
dipeptide surrogate, 9 and 14b, respectively. Standard Fmocꢀ
based SPPS was applied for coupling the canonical amino
acids using HATU (1ꢀ[bisꢀ(dimethylamino)methylene]ꢀ1Hꢀ
1,2,3ꢀtriazolo[4,5ꢀb]pyridinium 3ꢀoxide hexafluorophosphate)
as the coupling reagent and 2,6ꢀlutidine as base. In the Leuꢀ
enkephalin analog, containing the GlyꢀGly dipeptide surrogate
(16), building block 9 was introduced using DIC (1,3ꢀ
diisopropylcarbodiimide) and HOBt (1ꢀhydroxybenzotriazole),
followed by coupling of Boc/tꢀBuꢀprotected tyrosine, which
enabled concomitant cleavage from the resin and deprotection
(Scheme 3).
The yields of compounds 14a–e ranged from good to excelꢀ
lent, which illustrate the compatibility of the fluorinated azide
4
with the Cu(I)ꢀcatalyzed azide–alkyne cycloaddition
(Scheme 2B). At this stage, the stereochemical integrities of
the building blocks were investigated by removal of the Boc
group and subsequent functionalization with Marfey’s reagent.
Analytical HPLC traces showed that the compounds contained
85–98% of the major stereoisomer (Supporting Figure S1).
We suspect that the partial epimerization occurred at the amiꢀ
no aldehyde stage (11a–e) and therefore underline the imꢀ
portance of careful handling of these intermediates to miniꢀ
mize potential epimerization.
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For synthesis of the analog containing the TyrꢀGly surrogate
(17), compound 14b was treated with LiOH in ethanol to
deprotect the Cꢀterminal carboxylic acid (15b, see the Experꢀ
imental Section for details). This building block was coupled
to 2ꢀchlorotrityl resinꢀbound GlyꢀPheꢀLeu using DIC and
HOBt, and again, simultaneous cleavage and deprotection
furnished crude Leuꢀenkephalin analog 17 (Scheme 3). Both
compounds were isolated by reversedꢀphase preparative HPLC
to give 9% and 25% of pure peptide, respectively.
In summary, we describe the design and synthesis of a series
of dipeptide surrogates encompassing
a
central 1,4ꢀ
disubstituted 1,2,3ꢀtriazole as amide bond bioisostere, comꢀ
bined with a difluoromethylene group in its 4ꢀposition. Substiꢀ
tution of the amide bond with a 1,2,3ꢀtriazole bioisostere was a
key feature because difluoroglycine itself is unstable and readꢀ
ily eliminates fluoride.25 Given the continued interest in incorꢀ
poration of fluorine atoms into the side chains of peptides and
proteins, the present work should be of pertinent interest as it
provides a means to introduce fluorine into the backbones of
peptidomimetics. This has previously been achieved for
βꢀ
peptides and recently for architectures mimicking the αꢀamino
acid topology by introduction of GlyꢀF2Gly surrogates.18 Our
study complements this work by including proteinogenic side
chains in the Nꢀterminal residue of the dipeptide surrogates.
We demonstrate the synthesis of examples of dipeptide surꢀ
rogates containing either Fmoc or Boc groups at their Nꢀ
termini and apply selected building blocks for incorporation
into analogs of the endogenous peptide Leuꢀenkephalin. We
envision that the novel peptidomimetic backbone motif introꢀ
duced in this communication may find applications in alteraꢀ
tion and fine tuning of the properties of biologically active
peptides.
EXPERIMENTAL SECTION
General methods and materials. All reagents and solvents were
of analytical grade and used without further purification as obtained
from commercial suppliers. All reactions under nitrogen or argon
atmosphere were performed in dry solvents. Dichloromethane, N,Nꢀ
dimethylformamide (DMF), tetrahydrofuran (THF), and toluene were
retrieved from a solvent purification system. All reactions were moniꢀ
tored by thinꢀlayer chromatography (TLC) using silica gel coated
plates (analytical SiO2ꢀ60, Fꢀ254). Vacuum liquid chromatography
(VLC) purification was performed on silica gel 60 (particle size
0.015−0.040 µm). Ultraꢀhighꢀperformance liquid chromatography
mass spectrometry (UPLCꢀMS) analyses were performed using a
gradient with eluent I (0.1% HCOOH in water) and eluent II (0.1%
HCOOH in MeCN) rising linearly from 0% to 95% of II during t =
0.00–2.50 min applied at a flow rate of 0.6 mL/min (gradient A) or
during t = 0.00–5.20 min (gradient B). Compounds which were not
obtained in sufficient purity by VLC, were purified by preparative
HPLC performed on a C18 column [250 mm × 20 mm, 5 ꢁm, 100 Å]
Scheme 3. Solid-phase peptide synthesis of Leu-enkephalin
analogs 16 and 17
Finally, we wanted to evaluate the Xaaꢀψ[triazole]ꢀF2Gly
dipeptide mimic as a suitable building block for incorporation
into peptides. As a model peptide, we chose Leuꢀenkephalin –
an endogenous opioid receptor ligand – which has the amino
acid sequence TyrꢀGlyꢀGlyꢀPheꢀLeu and has previously been
modified by substituting amide bonds with 1,2,3ꢀtriazole moiꢀ
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using a diode array UV detector. A gradient C with eluent III
stirred for 16 h, quenched with water, and extracted with CH2Cl2 (3×).
(95:5:0.1, water−MeCN−trifluoroacetic acid(TFA)) and eluent IV
(0.1% TFA in MeCN) rising linearly from 0% to 95% of IV during t =
5−45 min was applied at a flow rate of 20 mL/min. All final comꢀ
pound purities were determined by analytical HPLC analysis to be
>95% pure on a C18 column [150 mm × 4.6 mm, 3 µm)] using a
multi wavelength UV detector. The gradient consisted of eluent III
(95:5:0.1, water−MeCN−TFA) and eluent IV (0.1% TFA in MeCN)
rising linearly from 0% to 95% of IV during t = 2−20 min at a flow
rate of 1 mL/min (gradient C). 1D and 2D NMR spectra were recordꢀ
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The combined organic layers were washed with water (2×) and brine,
dried over MgSO4, and filtered. Most of the solvent was carefully
removed in vacuo and the material was used as a solution in the next
step. CAUTION: this compound has not been reported previously but
due to the ratio of nitrogen to carbon/oxygen, it may be a potential
explosive. We therefore performed the reaction behind a blast shield
in the fume hood and stopped evaporation of the solvent before comꢀ
plete dryness was reached. An IRꢀsignal at 2100 cmꢀ1 confirms the
presence of an azide. 1H NMR (400 MHz, CDCl3) δ 1.57 (s, 9H) ppm.
13C NMR (101 MHz, CDCl3) δ 158.6 (t, J = 38.3 Hz), 112.3 (t, J =
269.1 Hz), 87.0, 27.8.
1
ed on a 400 MHz spectrometer at 298 K. H NMR spectra were recꢀ
orded at 400 MHz and 13C NMR spectra at 100 MHz, respectively.
NMR chemical shifts are reported in ppm relative to deuterated solꢀ
vent peaks as internal standards (δH: CD3OD 3.31 ppm, CDCl3 7.26
ppm, and DMSOꢀd6 2.50 ppm, δC: CD3OD 49.00 ppm, CDCl3 77.16
ppm, and DMSOꢀd6 39.50 ppm). Coupling constants (J) are given in
hertz (Hz). Multiplicities of NMR signals are reported as follows: s,
singlet; br s, broad singlet; d, doublet; t, triplet; q, quartet; sept, septet;
m, multiplet. The HRMS spectra were recorded using either matrix
assisted laser desorption/ionization (MALDI) or electrospray ionizaꢀ
tion (ESI) as indicated for each compound with Fourier transform ion
cyclotron resonance (FTꢀICR) as mass analyzer.
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FmocꢀGlyꢀψ[triazole]ꢀF2GlyꢀOtꢀBu (8). 2,6ꢀLutidine (2 equiv) and
iꢀPr2NEt (2 equiv) were added to a solution of 5 (416 mg, 1.5 mmol)
was reacted with 7 (193 mg, 1 mmol) in THF (0.2 M with respect to
alkyne) and argon was bubbled through the solution for 2 min. CuI
(0.2 equiv) and TBTA (0.2 equiv) was added and argon bubbling was
repeated for 2 min. The mixture was stirred at room temperature for 2
h under argon and the reaction was quenched with water. The aqueous
phase was extracted with CH2Cl2 (×3) and the combined organic
layers were dried (MgSO4), filtered, and concentrated. The crude
residue was purified by VLC to yield a colorless oil (190 mg, 43%
over three steps). VLC eluent: 0–20% EtOAc in hexane. 1H NMR
(400 MHz, CDCl3) δ 7.83 (s, 1H), 7.68 (d, J = 7.5 Hz, 2H), 7.49 (d, J
= 7.4 Hz, 2H), 7.32 (t, J = 7.4 Hz, 2H), 7.22 (t, J = 7.4 Hz, 2H), 5.37
(s, 1H), 4.42 (d, J = 6.0 Hz, 2H), 4.37 (d, J = 6.8 Hz, 2H), 4.12 (t, J =
6.7 Hz, 1H), 1.50 (s, 9H) ppm. 13C NMR (101 MHz, CDCl3) δ 156.9
(t, J = 33.2 Hz), 156.3, 145.7, 143.7, 141.3, 127.7, 127.0, 125.0,
120.2, 120.1, 109.1 (t, J = 267.7 Hz), 87.8, 66.8, 47.2, 36.20, 27.6
ppm. UPLCꢀMS gradient A, tR = 2.45 min, m/z: [M+H]+ Calcd for
2ꢀAzidoꢀN,Nꢀdiethylꢀ2,2ꢀdifluoroacetamide (4). Ethyl bromodiꢀ
fluoroacetate (2.5 g, 12.5 mmol) was added to a solution of Et2NH
(3.65 g, 50 mmol) in DMF (15 mL) at 0 °C under argon. The mixture
was allowed to reach room temperature and stirred 18 h. The reaction
mixture was diluted with water (40 mL) and extracted with EtOAc (3
× 40 mL). The organic phase was washed with water (2 × 100 mL)
and 1 M HCl (2 × 100 mL), dried (MgSO4), filtered, and concentrated
to give compound 320 as a light yellow oil (2.75 g, 96%). Caution: this
product is volatile! Without further purification, a solution of 3 (460
mg, 2 mmol) in DMSO (2 mL) was stirred under argon and NaN3
(195 mg, 3 mmol) was added in small portions over 10 min. The
mixture was heated to 50 °C for 5 h before it was cooled to room
temperature, quenched with water (10 mL), and extracted with Et2O
(3 × 10 mL). The organic phase was dried (Na2SO4), filtered, and
concentrated in vacuo to yield a colorless oil (343 mg, 89%). Caution:
this product is volatile and was used without further purification. An
+
C24H25F2N4O4 471.2; Found 471.2. HRMS (MALDIꢀTOF) m/z:
[M+H]+ Calcd for C24H25F2N4O4+ 471.1838; Found 471.1846.
FmocꢀGlyꢀψ[triazole]ꢀGlyꢀOH (9). Compound 8 was dissolved in
CH2Cl2–TFA (1:1; 0.05 M) and the solution was stirred for 1 h. The
solvent was removed in vacuo and residual TFA coꢀevaporated with
CH2Cl2 (3×). The organic phase was dried (MgSO4) and concentrated
in vacuo. The crude was purified by VLC and the product obtained as
a colorless amorphous solid (92 mg, 94%). VLC eluent: CH2Cl2–
MeOH–AcOH (95:4.5:0.5–90:9:1). 1H NMR (600 MHz, DMSOꢀd6) δ
8.14 (s, 1H), 7.88 (d, J = 7.6 Hz, 3H), 7.69 (d, J = 7.5 Hz, 2H), 7.41
(t, J = 7.5 Hz, 2H), 7.33 (t, J = 7.5 Hz, 2H), 4.31 (d, J = 7.0 Hz, 2H),
4.28 (d, J = 5.9 Hz, 2H), 4.22 (t, J = 7.0 Hz, 1H). 13C NMR (151
MHz, DMSOꢀd6) δ 158.4 (t, J = 25.9 Hz), 156.2, 144.9, 143.8, 140.7,
127.6, 127.1, 125.2, 121.8, 120.1, 110.6 (t, J = 274.1 Hz), 65.6, 46.7,
1
IRꢀsignal at 2100 cmꢀ1 confirms the presence of an azide. H NMR
(400 MHz, CDCl3) δ 3.54–3.24 (m, 4H), 1.23–1.01 (m, 6H).
13C NMR (101 MHz, CDCl3) δ 158.8 (t, J = 32.9 Hz), 115.6 (t, J =
270.1 Hz), 42.3, 41.9, 14.1, 12.3. MS (ESI) m/z: [M+H]+ Calcd for
C6H11F2N4O+ 192; Found 192.
NꢀFmoc propargyl amine (5). FmocꢀOSu (1.35 g, 4 mmol) folꢀ
lowed by 4ꢀdimethylaminopyridine (50 mg, 0.4 mmol) and iꢀPr2NEt
(0.71 mL, 4 mmol) was added to a solution of propargylamine (0.26
mL, 4 mmol) in CH2Cl2 (13 mL) at 0 °C under argon. After stirring
for 4 h at room temperature the mixture was washed with 1 M HCl,
saturated NaHCO3, and brine. The organic phase was dried (Na2SO4),
flitered, and concentrated in vacuo to give a white solid (1.03 g,
+
35.6. HRMS (MALDIꢀTOF) m/z: [M+H]+ Calcd for C20H17F2N4O4
415.1212; Found 415.1222.
General procedure for Synthesis of Boc-protected alkyne de-
rivatives 13a–e. iꢀPr2NEt (2.0 equiv) was added to a solution of the
amino acid (1.0 equiv), N,Oꢀdimethylhydroxylamine·HCl (1.1 equiv)
and HOBt (1.1 equiv) in CH2Cl2 (0.3 M with respect to amino acid)
under nitrogen. EDC•HCl (1.1 equiv) was added to the mixture at
0 °C and stirring was continued for 3 h, after which it was allowed to
reach room temperature. The mixture was diluted with EtOAc (2 ×
volume of CH2Cl2), washed with 5% aq. KHSO4 (2×), 5% aq. Naꢀ
HCO3 (2×), and brine (2×). The organic phase was dried (MgSO4),
filtered, and concentrated in vacuo to give the crude Weinreb amide.
The crude product was dissolved in dry THF (0.25 M) and LiAlH4
(1.25 equiv) was added in one portion at 0 °C under nitrogen. After
stirring at 0 °C for 1 h the reaction was quenched with 5% aq. KHSO4
(SLOW ADDITION!). The mixture was diluted with EtOAc and
washed with 5% aq. KHSO4 (2×), 5% aq. NaHCO3 (2×), and brine
(2×). The organic phase was dried (MgSO4), filtered, and concentratꢀ
ed in vacuo to give the crude aldehyde, which was used directly
without further purification. K2CO3 (3.0 equiv) and pꢀ
toluenesulfonylazide (1.2 equiv) were suspended in MeCN (0.5 M
with respect to pꢀtoluenesulfonylazide) under nitrogen. Dimethylꢀ(2ꢀ
oxopropyl)ꢀphosphonate (1.2 equiv) was added and the mixture was
stirred for 4–8 h. A solution of the crude aldehyde in dry MeOH (1.6
M) was added. After stirring for 16 h, the solvents were removed in
vacuo and the crude residue was dissolved in EtOAc, washed with
1
91%). H NMR (400 MHz, CDCl3) δ 7.77 (d, J = 7.5 Hz, 2H), 7.59
(d, J = 7.5 Hz, 2H), 7.41 (t, J = 7.5 Hz, 2H), 7.32 (td, J = 7.5, 1.0 Hz,
2H), 4.98 (s, 1H), 4.44 (d, J = 6.9 Hz, 2H), 4.23 (t, J = 6.9 Hz, 1H),
4.00 (m, 2H), 2.26 (t, J = 2.4 Hz, 1H). 13C NMR (101 MHz, CDCl3) δ
155.9, 143.8, 141.3, 127.7, 127.1, 125.0, 120.0, 79.60, 71.7, 67.0,
47.1, 30.8. UPLCꢀMS gradient A, tR = 2.12 min, m/z: [M+Na]+ Calcd
for C18H15NNaO2+ 300.1; Found 300.0). The data is in agreement with
literature.26
tertꢀButyl 2ꢀazidoꢀ2,2ꢀdifluoroacetate (7). Potassium tertꢀbutoxide
(393 mg, 3.5 mmol) was added in two portions over 5 min to a soluꢀ
tion of ethyl bromodifluoroacetate (5 g, 25 mmol) in pentane
(200 mL) and tꢀBuOH (35 mL) at 0 °C under argon. After stirring for
1 h at room temperature the reaction was quenched with concentrated
aqueous HCl and washed with water (2 × 50 mL) and brine (50 mL).
The organic phase was dried (MgSO4) and filtered through a pad of
silica gel (4 × 5 cm) followed by elution with 20% Et2O in pentane
(100 mL). Most solvent was carefully removed by distillation to give
compound 627 as a colorless oil, which was used without further
purification. To a solution of 6 (462 mg, 2 mmol) in DMSO (4 mL),
NaN3 (195 mg, 3 mmol) was added under argon. The reaction was
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The Journal of Organic Chemistry
water (2×) and brine (2×), dried (MgSO4), filtered, and concentrated
1H NMR (400 MHz, CDCl3) δ 7.55 (s, 1H), 7.25–7.22 (m, 2H), 7.22–
1
2
3
4
5
6
7
8
in vacuo. The crude material was purified by VLC.
7.19 (m, 1H), 7.09–7.06 (m, 2H), 5.20 (br s, 1H), 5.16–5.08 (m, 1H),
3.49 (q, J = 7.1 Hz, 2H), 3.30–3.23(m, 3H), 2.21–2.14 (m, 1H), 1.40
(s, 9H), 1.23 (t, J = 7.1 Hz, 3H), 1.16 (t, J = 7.1 Hz, 3H). 13C NMR
(101 MHz, CDCl3) δ 157.2, 156.9, 155.2, 149.0, 136.9, 129.6, 128.6,
126.9, 119.5, 111.3, 80.1, 48.7, 42.7 (t, J = 4 Hz), 42.5, 41.5, 28.4,
BocꢀPheꢀalkyne (13a). The product was isolated as colorless solid
(206 mg, 45% over three steps). Mp: 106–111 °C. VLC eluent: 0–
1
20% EtOAc in heptane. H NMR (400 MHz, CDCl3) δ 7.33–7.26 (m,
5H, ArꢀH), 4.68 (br s, 2H, NH/αꢀCH), 3.03–2.91 (m, 2H, βꢀCH2),
2.27 (s, 1H, CCH), 1.43 (s, 9H, Bocꢀ(CH3)3). 13C NMR (101 MHz,
CDCl3) δ 154.7, 136.5, 129.9, 128.4, 127.0, 82.9, 80.2, 72.3, 44.0,
41.9, 28.5. UPLCꢀMS gradient B, tR = 2.23 min, m/z: [M+H]+ Calcd
14.0, 12.1. HRMS (MALDIꢀTOF) m/z: [M
+
H]+ Calcd for
C21H30F2N5O3+ 438.2311; Found 438.2317. [α]589.3: –15° (c = 0.1, 293
K, CHCl3).
+
BocꢀTyr(tꢀBu)ꢀψ[triazole]ꢀF2GlyꢀNEt2 (14b). Compound 13b (190
for C15H20NO2 246.1; Found 246.3). [α]589.3: –20° (c = 0.1, 293 K,
CHCl3), literature –10.6° (c = 1.01, CHCl3).28
mg, 0.6 mmol) was reacted with 4 (173 mg, 0.9 mmol) according to
the general procedure. The product was purified by VLC and obtained
as a colorless solid (304 mg, 99%). Mp: 93–97 °C. VLC eluent: 0–
9
BocꢀTyr(tꢀBu)ꢀalkyne (13b). The product was isolated as a colorꢀ
less gel (557 mg, 59% over 3 steps). VLC eluent: 20–40% EtOAc in
hexane. 1H NMR (400 MHz, CDCl3) δ 7.14 (d, J = 8.4 Hz, 2H), 6.92
(d, J = 8.4 Hz, 2H), 4.67 (2 × br s, 1H), 2.93 – 2.89 (m, 2H), 2.26 (d, J
= 2.1 Hz, 1H), 1.42 (s, 9H), 1.33 (s, 9H). 13C NMR (101 MHz,
CDCl3) δ 154.7, 154.5, 131.3, 130.3, 124.0, 83.1, 80.1, 78.4, 72.1,
44.0, 41.2, 29.0, 28.5. UPLCꢀMS gradient A, tR = 2.51 min, m/z:
[M+H]+ Calcd for C19H28NO3+ 318.2, found 318.1. HRMS (MALDIꢀ
TOF) m/z: [M+H]+ Calcd for C19H28NO3+ 318.2064; Found 318.2065.
[α]589.3: –4° (c = 1.0, 293 K, CHCl3), literature –0.44° (c = 3.18,
CHCl3).24
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
1
20% EtOAc in hexane. H NMR (400 MHz, CDCl3) δ 7.43 (s, 1H),
6.89 (d, J = 8.4 Hz, 2H), 6.80 (d, J = 8.4 Hz, 2H), 5.16 (s, 1H), 5.02
(d, J = 7.4 Hz, 1H), 3.42 (q, J = 7.4 Hz, 2H), 3.19 (m, 3H), 3.03 (m,
1H), 1.34 (s, 9H), 1.24 (s, 9H), 1.16 (t, J = 7.1 Hz, 3H), 1.10 (t, J =
7.1 Hz, 3H) ppm. 13C NMR (101 MHz, CDCl3) δ 157.0 (t, J = 29.1
Hz), 155.0, 154.1, 148.9, 131.6, 129.8, 124.1, 119.2, 111.1 (t, J =
266.6 Hz). 79.9, 78.3, 48.5, 42.6 (t, J = 4 Hz), 42.3, 40.7, 28.8, 28.3,
13.8, 11.9. UPLCꢀMS gradient A, tR = 2.51 min, m/z: [M+H]+ Calcd
+
for C25H38F2N5O4 510.3; Found 510.3. HRMS (MALDIꢀTOF) m/z:
BocꢀMetꢀalkyne (13c). The product was isolated as a colorless oil
(230 mg, 50% over three steps). VLC eluent: 0–10% EtOAC in hepꢀ
[M+H]+ Calcd for C25H38F2N5O4+ 510.2886; Found 510.2895. [α]589.3
:
–4° (c = 1.0, 293 K, CHCl3).
BocꢀMetꢀψ[triazole]ꢀF2GlyꢀNEt2 (14c). Compound 13c (150 mg,
1
tane. H NMR (400 MHz, CDCl3) δ 4.81 (br s, 1H, NH), 4.55 (br s,
1H, αꢀCH), 2.67–2.55 (m, 2H, γꢀCH2), 2.30 (d, J = 2.3 Hz, 1H, CCH),
2.11 (s, 3H, εꢀCH3), 2.00–1.89 (m, 2H, βꢀCH2), 1.45 (s, 9H, Bocꢀ
(CH3)3). 13C NMR (101 MHz, CDCl3) δ 154.8, 82.9, 80.2, 71.9, 42.3,
35.5, 30.2, 28.5, 15.6. UPLCꢀMS gradient B, tR = 2.05 min, m/z:
[M+H]+ Calcd for C11H20NO2S+ 230.1; Found 230.1. HRMS (ESIꢀ
TOF) m/z: [M+H]+ Calcd for C11H20NO2S+ 230.1209; Found
230.1216. [α]589.3: –60° (c = 0.1, 293 K, CHCl3).
BocꢀLys(Cbz)ꢀalkyne (13d). The product was isolated as a colorless
solid (533 mg, 46%). Mp: 84–87°C. VLC eluent: 0–20% EtOAC in
heptane. 1H NMR (400 MHz, CDCl3) δ 7.36–7.32 (m, 5H, ArꢀH),
5.09 (s, 2H, CBzꢀCH2), 4.81 (br s, 1H, εꢀNH), 4.73 (br s, 1H, αꢀNH),
4.38 (br s, 1H, αꢀCH), 3.20 (q, J = 6.3 Hz, 2H, εꢀCH2), 2.25 (d, J =
2.3 Hz, 1H, CCH), 1.74–1.62 (m, 2H, βꢀCH2), 1.58–1.51 (m, 2H, δꢀ
CH2) 1.49–1.44 (m, 11H, γꢀCH2/Bocꢀ(CH3)3)). 13C NMR (101 MHz,
CDCl3) δ 156.6, 155.0, 136.8, 128.6, 128.2, 128.2, 83.5, 80.1, 71.3,
0.65 mmol) was reacted with 4 (209 mg, 0.98 mmol) according to the
general procedure. The product was purified by VLC and obtained as
a colorless oil (270 mg, 98%). VLC eluent: 0–20% EtOAc in heptane.
1H NMR (400 MHz, CDCl3) δ 7.90 (s, 1H), 5.19 (br s, 1H), 5.03 (q, J
= 7.7 Hz, 1H), 3.48 (q, J = 7.1 Hz, 2H), 3.34 (q, J = 7.1 Hz, 2H),
2.59–2.46 (m, 2H), 2.31–2.16 (m, 2H), 2.09 (s, 3H), 1.42 (s, 9H),
1.25–1.16 (m, 6H). 13C NMR (101 MHz, CDCl3) δ 157.2 (t, J = 29.1
Hz), 155.3, 149.4, 119.6, 114.1, 111.5, 80.1, 46.2, 42.8 (t, J = 4.2 Hz),
42.5, 34.3, 30.4, 28.5, 15.5, 14.0, 12.0. UPLCꢀMS gradient B, tR
=
2.34 min, m/z: [M+H]+ Calcd for C17H30F2N5O3S+ 422.2; Found
422.2. HRMS (MALDIꢀTOF) m/z: [M+H]+ Calcd for C17H30F2N5O3S+
422.2032, found 422.2037. [α]589.3: –45° (c = 0.1, 293 K, CHCl3).
BocꢀLys(Cbz)ꢀψ[triazole]ꢀF2GlyꢀNEt2 (14d). Compound 13d (150
mg, 0.4 mmol) was reacted with 4 (133 mg, 0.6 mmol) according to
the general procedure. The product was purified by VLC and obtained
as a colorless oil (223 mg, 96%). VLC eluent: 55% EtOAc in heptane.
1H NMR (400 MHz, CDCl3) δ 7.86 (s, 1H), 7.37–7.27 (m, 5H), 5.12
(br s, 1H), 4.85 (br s, 2H), 3.48 (q, J = 7.1 Hz, 2H), 3.34 (q, J = 7.2
Hz, 2H), 3.24–3.13 (m, 2H), 2.01–1.93 (m, 1H), 1.89 (br s, 1H), 1.59–
1.48 (m, 2H), 1.42 (s, 9H), 1.25–1.14 (m, 6H). 13C NMR (101 MHz,
CDCl3) δ 157.3 (t, J = 29.2 Hz), 156.6, 155.5, 150.1, 136.8, 128.6,
128.2, 128.2, 119.3, 111.5 (t, J = 266.7 Hz), 80.0, 66.8, 47.0, 42.8 (t, J
= 4.2 Hz), 42.5, 40.7, 34.7, 29.6, 28.5, 22.9, 14.0, 12.0. UPLCꢀMS
66.7, 42.7, 40.9, 35.8, 29.5, 28.5, 22.8. UPLCꢀMS gradient B, tR
=
+
2.35 min, m/z: [M + H]+ Calcd for C20H29N2O4 361.2; Found 361.2.
[α]589.3: –25° (c = 0.1, 293 K, CHCl3) literature –23.1° (CHCl3).23
BocꢀArg(Pbf)ꢀalkyne (13e). The product was isolated as a colorless
solid (1.71 g, 68% over 3 steps). Mp: 85 °C. VLC eluent: CH2Cl2–
MeOH–NH4OH (99.5:0.45:0.05–98.5:1.35:0.15). 1H NMR (400
MHz, CDCl3) δ 6.29 (s, 2H), 4.91 (d, J = 8.6 Hz, 1H), 4.34 (br s, 1H),
3.35–3.09 (m, 2H), 2.95 (s, 2H), 2.57 (s, 3H), 2.51 (s, 3H), 2.26 (d, J
= 2.2 Hz, 1H), 2.09 (s, 3H), 1.75 (s, 1H), 1.73ꢀ 1.53 (m, 4H), 1.45 (s,
6H), 1.42 (s, 9H). 13C NMR (101 MHz, CDCl3) δ 159.0, 156.1, 155.5,
138.6, 132.6, 124.8, 117.71, 86.6, 83.0, 80.4, 71.7, 43.3, 40.9, 33.6,
+
gradient B, tR = 2.52 min, m/z: [M+H]+ Calcd for C26H39F2N6O5
553.3; Found 533.2. HRMS (MALDIꢀTOF) m/z: [M+H]+ Calcd for
C26H39F2N6O5+ 553.2945; Found 553.2950. [α]589.3: –50° (c = 0.1, 293
K, CHCl3).
28.7, 28.4, 28.3, 25.4, 19.5, 18.0, 12.6. UPLCꢀMS gradient A, tR
=
2.25 min, m/z: [M+H]+ Calcd for C25H39N4O5S+ 507.3; Found 507.3.
HRMS (MALDIꢀTOF) m/z: [M+H]+ Calcd for C25H39N4O5S+
507.2636, found 507.2668. [α]589.3: –8° (c = 1.03, 293 K, CHCl3).
General procedure for Synthesis of 14a–e through Cu(I)-
catalyzed azide–alkyne cycloaddition. The alkyne and the azide
were dissolved in dry THF (0.1 M with respect to alkyne). 2,6ꢀ
Lutidine (2.0 equiv) and iꢀPr2NEt (2.0 equiv) were added and nitrogen
was bubbled through the solution for 5 min while stirring. CuI
(0.2 equiv) and TBTA (0.2 equiv) were added and nitrogen bubbling
was repeated for 5 min. The mixture was stirred under nitrogen at
room temperature for 2 h. Subsequently, the reaction was quenched
with water and the aqueous phase was extracted with CH2Cl2 (3×).
The combined organic layers were dried (MgSO4), filtered, and conꢀ
centrated and the resulting residue was purified by VLC.
BocꢀArg(Pbf)ꢀψ(triazole)ꢀF2GlyꢀNEt2 (14e). Compound 13e (304
mg, 0.6 mmol) was reacted with 4 (96 mg, 0.5 mmol) according to the
general procedure. The product was purified by VLC and the product
obtained as a colorless gel (281 mg, 80%). VLC eluent: CH2Cl2–
MeOH–NH4OH (99.5:0.45:0.05–97:2.7:0.3). 1H NMR (400 MHz,
CDCl3) δ 7.98 (s, 1H), 5.47 (br s, 1H), 4.93–4.84 (m, 1H), 3.69 (s,
1H), 3.48 (q, J = 7.1 Hz, 2H), 3.36 (q, J = 7.1 Hz, 2H), 3.32ꢀ3.15 (m,
2H), 2.94 (s, 2H), 2.56 (s, 3H), 2.50 (s, 3H), 2.08 (s, 3H), 1.97–1.86
(m, 2H), 1.66–1.54 (m, 2H), 1.45 (s, 6H), 1.41 (s, 9H), 1.23–1.18 (m,
6H). 13C NMR (101 MHz, CDCl3) δ 158.8, 157.5 (t, J = 28.6 Hz),
156.3, 155.9, 150.1, 138.5, 133.1, 132.4, 129.2, 124.7, 119.6, 117.6,
111.6 (t, J = 266.8 Hz), 86.5, 80.2, 43.4, 42.9 (t, J = 4.0 Hz), 42.6,
41.0, 32.9, 28.7, 28.5, 25.6, 19.4, 18.0, 14.0, 12.6, 12.1. UPLC–MS
gradient A, tR = 2.35 min, m/z: [M+H]+ Calcd for C31H49F2N8O6S+
699.3; Found 699.3. HRMS (MALDIꢀTOF) m/z: [M + H]+ Calcd for
C31H49F2N8O6S+ 699.3468, found 699.3496. [α]589.3: –10° (c = 0.98,
293 K, CHCl3).
BocꢀPheꢀψ[triazole]ꢀF2GlyꢀNEt2 (14a). Compound 13a (100 mg,
0.4 mmol) was reacted with 4 (118 mg, 0.6 mmol) according to the
general procedure. The product was purified by VLC and obtained as
a colorless oil (140 mg, 78%). VLC eluent: 0–20% EtOAc in heptane.
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Page 6 of 7
BocꢀTyr(tBu)ꢀ
ψ[triazole]ꢀF2GlyꢀOH (15b). 1 M LiOH (10 equiv)
CH2Cl2–TFA–iꢀPr3SiH–H2O (50:47:1.5:1.5) mixture (2 × 20 min).
1
2
3
4
5
6
7
8
was added to a solution of 14b in EtOH (0.1 M). After stirring for 16
h the reaction was acidified with 2 M HCl (2.5 mL) and extracted
with CH2Cl2 (3×). The organic phase was dried (MgSO4) and concenꢀ
trated in vacuo. The crude was purified by VLC and the product
obtained as a colorless amorphous solid (143 mg, 78%). VLC eluent:
CH2Cl2–MeOH–AcOH (99:0.9:0.1–90:9:1). 1H NMR (600 MHz,
CD3OD) δ 8.09 (s, 1H), 7.12 (d, J = 8.0 Hz, 2H), 6.89 (d, J = 8.0 Hz,
2H), 5.05 (dd, J = 9.3, 5.9 Hz, 1H), 3.20 (dd, J = 13.8, 5.9 Hz, 1H),
3.00 (dd, J = 13.8, 9.3 Hz, 1H), 1.36 (s, 9H), 1.30 (s, 9H). 13C NMR
(151 MHz, CD3OD) δ 153.9 (t, J = 26.8 Hz), 148.0, 145.6, 141.1
124.8, 121.5, 115.6, 112.0, 102.7 (t, J = 271.3 Hz), 70.8, 70.0, 40.6,
32.1, 19.7, 19.2, 14.2. HRMS (MALDIꢀTOF) m/z: [M+Na]+ Calcd for
The solution was concentrated and the product precipitated from
Et2O, vacuumꢀdried, and purified by preparative HPLC to give a
white powder (23 mg, 25%). 1H NMR (400 MHz, DMSOꢀd6) δ 12.61
(br s, 1H, Leu COOH), 9.79 (t, J = 6.0 Hz, 1H, Gly amide NH), 9.33
(br s, 1H, Tyr OH), 8.70 (s, 1H, triazole 5ꢀCH), 8.58–8.47 (m, 2H,
Tyr amine NH2), 8.37 (d, J = 8.0 Hz, 1H, Leu amide NH), 8.30 (d, J =
8.4 Hz, 1H, Phe amide NH), 7.33–7.13 (m, 5H, Phe ArH), 6.94 (d, J =
8.4 Hz, 2H, Tyr ArH), 6.64 (d, J = 8.4 Hz, 2H, Tyr ArH), 4.75–4.68
(m, 1H, Tyr αꢀCH), 4.60 (td, J = 9.7, 4.0 Hz, 1H, Phe αꢀCH), 4.25–
4.18 (m, 1H, Leu αꢀCH), 3.94–3.86 (m, 1H, Gly αꢀCH2), 3.80–3.72
(m, 1H, Gly αꢀCH2), 3.15 (d, J = 8.2 Hz, 2H, Tyr βꢀCH2), 3.05 (dd, J
= 13.8, 4.0 Hz, 1H, Phe βꢀCH2), 2.75 (dd, J = 13.8, 9.7 Hz, 1H, Phe
βꢀCH2), 1.70ꢀ1.50 (m, 3H, Leu βꢀCH2 and γꢀCH), 0.90 (t, J = 5.7 Hz,
3H, Leu δꢀCH3), 0.84 (t, J = 5.7 Hz, 3H, Leu δꢀCH3). 13C NMR
(101 MHz, DMSOꢀd6) δ 173.9, 171.0, 166.7, 157.7, 156.3, 156.8,
144.2, 137.8, 137.6, 130.3, 129.3, 128.0, 126.3, 125.3, 123.5, 115.3,
110.1 (t, J = 268.0 Hz), 53.7, 50.3, 47.7, 41.9, 37.8, 37.5, 24.3, 22.9,
21.3. UPLC–MS gradient A, tR = 1.09 min, m/z: [M+H]+ Calcd for
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
+
C21H28F2N4NaO5 477.1920; Found 477.1929. [α]589.3: –6° (c = 1.0,
293 K, CHCl3).
General procedure for SPPS on 2-chlorotrityl polysterene res-
in. Loading of first residue and capping: Resin (1 equiv.) was washed
with CH2Cl2 (2×) and swelled in CH2Cl2 for 20 min. The resin was
drained and treated with Fmoc protected amino acid (4 equiv) and iꢀ
Pr2NEt (8 equiv) in CH2Cl2 (0.4 M with respect to amino acid). The
mixture was shaken for 4 h, drained, and washed (3 × CH2Cl2, 3 ×
MeOH, 3 × DMF). The unreacted sites on the resin were capped with
iꢀPr2NEt (10 equiv) in MeOH (10 min) and the resin washed (3 ×
CH2Cl2, 3 × MeOH, 3 × DMF). Fmoc deprotection and amino acid
coupling: A solution of piperidine–DMF (1:4, 2 mL per 100 mg resin)
was added to the resin and shaken for 20 min. The resin was drained
and the deprotection wasrepeated for 20 min. The resin was drained
and washed (3 × CH2Cl2, 3 × MeOH, 3 × DMF). Fmoc protected
amino acid (4 equiv) was preincubated with HATU (4 equiv) and 2,6ꢀ
lutidine (8 equiv) in DMF (0.4 M with respect to the amino acid) for
10 min. The solution was added to the resin and shaken for 2 h. The
resin was then drained and washed (3 × CH2Cl2, 3 × MeOH, 3 ×
DMF).
C29H36F2N7O6 616.3; Found 616.3. HRMS (ESIꢀTOF) m/z: [M+H]+
+
Calcd for C29H36F2N7O6+ 616.2690; Found 616.2704.
ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website.
Supporting Figure S1 as well as copies of 1H and 13C NMR specꢀ
tra (PDF)
AUTHOR INFORMATION
TyrꢀGlyꢀψ[triazole]ꢀF2GlyꢀPheꢀLeu (16). Dipeptide PheꢀLeu was
Corresponding Author
synthesized on resin according to the general SPPS procedure. A
mixture of 9 (53 mg, 0.128 mmol), HOBt (20 mg, 0.128 mmol) and
DIC (16 mg, 0.128 mmol) in DMF (1.5 mL) was shaken for 10 min
and added to the resinꢀbound PheꢀLeu (0.09 mmol). After shaking
overnight the resin was drained and washed (3 × CH2Cl2, 3 × MeOH,
3 × DMF). Fmoc deprotection and coupling to BocꢀTyr(tꢀBu)ꢀOH
was performed according to the general SPPS procedure. The resultꢀ
ing product was cleaved from the resin by treatment with a CH2Cl2–
TFA–iꢀPr3SiH–H2O (50:47:1.5:1.5) mixture (2 × 20min). The cleavꢀ
age solution was concentrated and the product precipitated from Et2O,
dried in vacuo, and purified by preparative HPLC to give a white
*Eꢀmail: cao@sund.ku.dk
ORCID
Christian A. Olsen: 0000ꢀ0002ꢀ2953ꢀ8942
Present Address
†J.E.ꢀA. is now holding a position as Research Scientist at Chr.
Hansen A/S, Hørsholm, Denmark.
‡K.W. is now at Qiagen A/S.
1
powder (4.1 mg, 9%). H NMR (400 MHz, DMSOꢀd6) δ 9.77 (d, J =
Author Contributions
8.4 Hz, 1H, Phe amide NH), 9.35 (s, 1H, Tyr OH), 8.98 (t, J = 5.5 Hz,
1H, Glyꢀψ[triazole] amide NH), 8.48 (d, J = 7.8 Hz, 1H, Leu amide
NH), 8.15 (s, 1H, triazole 5ꢀCH), 8.10 (br s, 2H, Tyr amine NH2),
7.39–7.15 (m, 5H, Phe ArH), 6.98 (d, J = 8.4 Hz, 2H, Tyr ArH), 6.68
(d, J = 8.4 Hz, 2H, Tyr ArH), 4.72–4.67 (m, 1H, Phe αꢀCH), 4.40 (2 ×
dd, J = 15.4, 5.5 Hz, 2H, Glyꢀψ[triazole] αꢀCH2), 4.31–4.20 (m, 1H,
Leu αꢀCH), 3.88 (m, 1H, Tyr αꢀCH), 3.15 (dd, J = 13.9, 3.5 Hz, 1H,
Phe βꢀCH2), 2.97 (dd, J = 13.9, 3.5 Hz, 1H, Phe βꢀCH2), 2.95 (dd, J =
14.1, 7.6 Hz, 1H), 2.83 (dd, J = 14.1, 7.6 Hz, 1H), 1.79–1.45 (m, 3H,
Leu βꢀCH2 and γꢀCH), 0.91 (d, J = 6.5 Hz, 3H, Leu δꢀCH3), 0.86 (d, J
= 6.4 Hz, 3H, Leu δꢀCH3). 13C NMR (101 MHz, DMSOꢀd6) δ 173.8,
169.8, 168.2, 158.1, 156.6, 144.7, 137.4, 130.4, 129.1, 128.2, 126.5,
124.7, 121.8, 115.3 (t, J = 297.7 Hz), 54.8, 53.8, 50.5, 39.7, 36.5,
36.2, 33.8, 24.3, 22.8, 21.3. UPLC–MS gradient A, tR = 2.28 min,
The manuscript was written through contributions from all auꢀ
thors. All authors have given approval to the final version of the
manuscript. Part of this work was performed by J.E.ꢀA. at the
Technical University of Denmark, Kgs. Lyngby, Denmark.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENT
This work was supported by the Danish Independent Research
Council  Technical and Production Sciences (Grant No. 6111ꢀ
00170), Danish Independent Research Council  Natural Sciences
(Steno Grant No. 10ꢀ080907), and The University of Copenhagen
(Center for Biopharmaceuticals grant).
+
m/z: [M+H]+ Calcd for C29H36F2N7O6 616.3; Found 616.3. HRMS
+
(ESIꢀTOF) m/z: [M+H]+ Calcd for C29H36F2N7O6 616.2690; Found
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