Synthesis of dopamine and serotonin derivatives for immobilization on a solid support

Article abstract of DOI:10.1021/jo2025477

The two important neurotransmitters dopamine and serotonin are synthesized with short PEG tethers and immobilized on a magnetic solid support. The tether is attached to the aromatic moiety of the neurotransmitters to conserve their original functional groups. This approach causes minimal alteration of the original structure with the aim of optimizing the immobilized neurotransmitters for aptamer selection by SELEX. For the dopamine derivative, the tether is attached to the aromatic core of a dopamine precursor by the Sonogashira reaction. For serotonin, a link to the indole core is introduced by a Claisen rearrangement from the allylated phenol moiety of serotonin. The tethers are azide-functionalized, which enables coupling to alkyne-modified magnetic beads. The coupling to the magnetic beads is quantified by UV spectroscopy using Fmoc-monitoring of the immobilized dopamine and serotonin derivatives.

Full text of DOI:10.1021/jo2025477

Article
pubs.acs.org/joc
Synthesis of Dopamine and Serotonin Derivatives for Immobilization
on a Solid Support
Erik Daa Funder, Anne Bjørnskov Jensen, Thomas Tørring, Anne Louise Bank Kodal,
Ane Rebolledo Azcargorta, and Kurt Vesterager Gothelf*
Danish National Research Foundation: Center for DNA Nanotechnology (CDNA) at Department of Chemistry and iNANO, Aarhus
University, Langelandsgade 140, DK-8000 Aarhus C, Denmark
S
* Supporting Information
ABSTRACT: The two important neurotransmitters dopamine
and serotonin are synthesized with short PEG tethers and
immobilized on a magnetic solid support. The tether is attached
to the aromatic moiety of the neurotransmitters to conserve their
original functional groups. This approach causes minimal
alteration of the original structure with the aim of optimizing
the immobilized neurotransmitters for aptamer selection by
SELEX. For the dopamine derivative, the tether is attached to
the aromatic core of a dopamine precursor by the Sonogashira
reaction. For serotonin, a link to the indole core is introduced by a
Claisen rearrangement from the allylated phenol moiety of
serotonin. The tethers are azide-functionalized, which enables coupling to alkyne-modified magnetic beads. The coupling to
the magnetic beads is quantified by UV spectroscopy using Fmoc-monitoring of the immobilized dopamine and serotonin
derivatives.
of reported aptamer selections, the ligands are immobilized on
a solid support, which, in the selection process, simplifies the
removal of nonbinding oligonucleotides.
INTRODUCTION
■
Dopamine and serotonin are essential neurotransmitters
controlling important aspects of human behavior. Imbalance
in the regulation of neurotransmitters plays a central role in
many major neurological diseases, for example, Parkinson’s
disease, schizophrenia, and depression.1 A variety of methods
have been developed to detect neurotransmitters in vivo, such
as microdialysis, fast-scan cyclic voltammetry, and positron
emission tomography.2,3 However, many of these methods
suffer from lack of specificity or modest temporal resolution.
An alternative method to detect neurotransmitters may be
aptamer-based detection, which can be extended to both an
optical or electrochemical readout.4−8 Here, we present the
design and synthesis of new dopamine and serotonin
derivatives optimized for the selection of aptamers that, in
turn, will be used for electrochemical detection of the two
neurotransmitters.
While aptamers for serotonin have not yet been reported,
Mannironi et al.11 have described the selection of an RNA
aptamer for dopamine. The aptamer binds to dopamine with a
Kd of 2.8 μM. The specificity of the aptamer was albeit low
compared to the binding of dopamine as it showed 58, 30, and
23% binding of norepinephrine, L-DOPA, and catechol,
respectively. We speculated that the relatively low specificity
of the aptamer originates from the dopamine immobilization
strategy used for the aptamer selection. Dopamine was linked
to the solid support via an amide bond (Figure 1). Since the
amide group is uncharged and has a different geometry than the
primary amine in dopamine, which is protonated at
physiological pH, the dopamine aptamer was primarily selected
for binding to the catechol moiety of dopamine.
The goal of the current study is to synthesize new dopamine
and serotonin derivatives for immobilization on solid supports
as improved targets for selection of aptamers by the SELEX
procedure. Prior to this study, all examples of dopamine and
serotonin immobilization onto a solid support have been via the
primary amine.11−14 In the case of serotonin, the structurally
closely related 5-hydroxytryptophan was immobilized via the
carboxylic acid group, thereby conserving the amino group in
the immobilized species (Figure 1).15
Aptamers are synthetic oligonucleotides or peptides that are
selected in vitro for binding to a specific target, hereby
representing an attractive alternative to antibodies.4 Oligonu-
cleotide aptamers are selected from a large library of
oligonucleotides for binding to a ligand by repeated selection
and amplification, a process called systematic evolution of
ligands by exponential enrichment (SELEX).9,10 The selected
aptamers bind to their target by forming a unique secondary
and tertiary structure with high specificity for the target.
Aptamers have been selected for binding to a variety of
different molecular targets ranging from small organic
molecules to proteins or even cell surfaces.4 In the majority
Received: December 14, 2011
Published: March 5, 2012
© 2012 American Chemical Society
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attached to the aromatic moiety, causing minimal interference
with the catechol and ethyleneamine moieties, and (ii) a useful
2-bromoaryl derivative 3 was readily available (Scheme 1).
Synthesis of Dopamine Derivative 1. The synthesis was
initiated by subjecting the benzylic position of 3 (Scheme 1) to
an SN2 attack from cyanide in DMF, affording 4 in an excellent
yield of 92%. The electron-rich nitrile compound was coupled
with propargylic alcohol by a Sonogashira cross-coupling, giving
5 in a poor yield of 27%. We have not been able to optimize
this step further, which mainly is accredited to the difficulty of
palladium(0) to initiate the catalytic cycle by oxidative addition.
Furthermore, the benzylic nitrile group can potentially
coordinate palladium in a cyclic system, hereby retarding the
efficiency of the catalyst.
Figure 1. Structures of dopamine and serotonin as well as selected
previous examples on immobilization of dopamine,11 serotonin,13 and
the structurally related 5-hydroxytryptophan.15
The propargylic alcohol moiety was then hydrogenated to
the aliphatic alcohol 6, using hydrogen gas and Pd/C at
atmospheric pressure. Notably, the nitrile group was stable
under these conditions. The primary alcohol was protected as
the acetyl ester 7, after which the nitrile group was reduced to
the amine and protected as the tert-butyl carbamate in a one-
pot nickel(II)-catalyzed reaction, yielding 8.20 The acetyl
protecting group was removed by methoxide, resulting in the
free primary alcohol 9. Reactions 5−9 proceeded according to
TLC and NMR analysis with full conversion. As a result, the
reactions only required aqueous work up and a final purification
step via flash column chromatography. The four steps resulted
in an overall yield of 48%, that is, an average yield of 83% per
step.
The previously reported methods share the disadvantage of
disrupting the electronic properties of the original primary
amine of the neurotransmitters. The immobilized derivates
may, therefore, not be optimal as targets in aptamer selection.
Here, we present new approaches for immobilization of the two
neurotransmitters in which the functional groups of native
dopamine and serotonin are conserved and instead a
polyethylene glycol (PEG) linker with a terminal azide has
been connected to the aryl skeleton of the neurotransmitters.
The two target derivatives of dopamine (1) and serotonin (2),
presented in Figure 2, have three important characteristics in
To introduce the PEG-azide linker, the primary alcohol was
mesylated using MsCl and triethylamine in dry diethyl ether,
giving 10 in a quantitative yield. Next, PEG-azide linker 11 was
treated with sodium hydride in DMF to generate the sodium
alkoxide salt. Subsequent slow addition of 10 facilitated the
crucial SN2 reaction, giving 12 in a 55% yield.
At this stage of the synthesis, it would be possible to convert
12 to the globally deprotected molecule; however, since our
intention was to install the Fmoc as a reporter group for
quantification of the subsequent immobilization, the Boc group
was cleaved using 4 M HCl in dioxane. The resulting
hydrochloric salt was subjected to Fmoc-protection conditions,
giving 13 in a yield of 60% over two steps. Thereafter, only the
acetal protection group was to be removed. However, this
turned out to be more troublesome than anticipated.
Treatment with hydrochloric acid in various concentrations
and at different temperatures, or use of different Lewis acids,
such as BBr3 and BCl3, turned out to be unsuccessful. Either the
acetal remained stable or the PEG linker was degraded.21
Eventually, the solution was to transform the stable acetal into
the much more labile ortho-ester.22 This was performed by
reacting 13 with Pb(OAc)4 in benzene, giving 14, which could
undergo acidic hydrolysis, generating the desired title
compound 1 in 30% yield over two steps.
Figure 2. Synthetic targets presented for dopamine analogue 1 and
serotonin analogue 2.
common. Attachment to the solid support is achieved through a
PEG linker. The PEG linker is chemically inert, water-soluble,
and known to reduce nonspecific binding to biomolecules.16,17
The immobilization onto the solid support proceeds via an
azide group situated at the terminal end of the PEG linker. By
the copper-catalyzed Huisgen−Meldal−Sharpless 1,3-dipolar
cycloaddition reaction between a terminal alkyne and an azide,
it is possible to react 1 and 2 with an alkyne-modified solid
support in an efficient and chemoselective manner.18,19
Furthermore, an Fmoc group has been introduced to function
as an indicator of immobilization efficiency. This can be
measured by cleaving the Fmoc group after immobilization.
The liberated 9-methylidenefluorene is detected by UV
spectroscopy and enables the facile quantification of 1 and 2
on the solid support.
Synthesis of Serotonin Derivative 2. For the synthesis of
a suitable serotonin derivative, we decided to start from
serotonin and introduce the linker to the 4-position of the
indole skeleton. A report from Macor et al.23 demonstrated that
an allyl group can be introduced in this position in high yields
via a Claisen rearrangement of the allylated phenol moiety
(Scheme 2).
First, the primary amine in serotonin hydrochloride 15 was
Boc-protected in 93% yield, followed by O-allylation of 16
using allyl iodide and Cs2CO3 in acetone to give 17 in 74%
yield (Scheme 2). Hereby, the stage was set for a central
RESULTS AND DISCUSSION
■
We decided to pursue the synthesis of dopamine derivative 1
(Figure 2), for the following two reasons: (i) it has the linker
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Scheme 1. Synthesis of the PEG-Azide Dopamine Derivative
Scheme 2. Synthesis of the PEG-Azide Serotonin Derivative
Claisen rearrangement in which a new carbon−carbon bond to
the indole moiety was formed and later utilized in the tether to
the solid support. The [3,3]-sigmatropic rearrangement
proceeded with full regioselectivity to form the 4-allylindole
derivative 18 in a satisfying yield of 64%. The liberated phenol
was then protected to avoid O-alkylation in subsequent steps. A
variety of protective groups were examined for protection of the
phenol. A screening showed that protection with tert-butyl
carbonate, TIPS, TBDPS, and MOM were either unsuccessful,
irreproducible, or the protected species was labile in the
subsequent alkylation reaction. Fortunately, the application of
THP as protection of the phenol was successful. The THP
ether was formed by using 3,4-dihydro-2H-pyran and PPTS in
dry DCM, giving 19 in 61% yield. The indole nitrogen has an
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Figure 3. Immobilization of neurotransmitter derivatives 1 and 2 on an alkyne-modified solid support.
acidity comparably to that of an aliphatic alcohol. As a
consequence, the indole nitrogen was Boc-protected to avoid
unwanted side reactions. After Boc protection, 20 was obtained
in 58% yield.
modified magnetic beads were obtained and set for further
modification.
The synthesized neurotransmitter derivatives were linked to
the alkyne-modified magnetic beads via the copper-catalyzed
Huisgen−Meldal−Sharpless 1,3 dipolar cycloaddition by
utilizing CuSO4, tris-(1-[3-hydroxypropyl]triazolyl-4-methyl)-
amine (THPTA),27 and (+)-sodium-L-ascorbate. As a result,
the neurotransmitter derivatives were linked to the solid
support by a 1,4-disubstituted 1,2,3-triazole, giving rise to a
solid support functionalized with dopamine derivative 1 and a
solid support functionalized with serotonin derivative 2. It was
investigated whether the catechol and 5-hydroxyindole, present
in the dopamine and serotonin derivatives, are stable under the
conditions applied for the click reactions. Dopamine and
serotonin were separately exposed to the click reaction
conditions, and subsequently, the product was analyzed by
1H NMR. It was found that the groups prone to oxidation were
stable to the experimental conditions (Supporting Informa-
tion). Therefore, we assume that the derivatives 1 and 2 are
also stable under the applied conditions.
To enable quantification of the amount of neurotransmitter
derivatives immobilized on the solid support, the Fmoc group
was cleaved and the UV (λ = 301 nm) absorption was
measured. Significant absorption by 9-methylidenefluorene was
measured, and an estimate of the amount of immobilized
neurotransmitter derivatives onto the solid support was
calculated by the Lambert−Beer law. Accordingly, a dop-
amine-functionalized solid support (30 nmol loading/8 × 108
beads) and a serotonin-functionalized solid support (29 nmol
loading/9 × 108 beads) were obtained.
Hydroboration of the terminal alkene in 20, followed by
oxidation, resulted in the primary alcohol 21 in 70% yield with
complete regioselectivity. The alcohol of 21 was converted into
an electrophile by mesylation to give 22 in 92% yield.
We attempted to react 22 with 11 to connect the PEG linker
to the serotonin skeleton via an ether, in analogy to the
synthesis of the dopamine derivative 1 (Scheme 1). However,
very poor yields were obtained from several attempts to
perform this reaction (<5%), and therefore, we speculated that
better results may be obtained by using a thiol nucleophile
instead of the alcohol 11. To test this, the thiol- and azide-
functionalized PEG linker 23 was prepared in four steps (see
the Experimental Section). Thiol 23 was coupled to the
serotonin skeleton by deprotonation of the thiol, followed by
substitution with the mesylate, to give 24a and 24b in a total
yield of 51%.
All acid labile protecting groups in 24a and 24b were
removed in a one-pot global deprotection by treatment with
methanol and 4 M HCl in dioxane. In the final step, 9-
fluorenylmethyl-N-succinimidyl carbonate and diisopropyl-
amine were used to Fmoc-protect the amine, giving the final
target molecule 2 in 34% yield over two steps.
Immobilization. For immobilization of the two linker-
functionalized neurotransmitter derivatives commercially avail-
able, amino-terminated magnetic beads were chosen. Such
magnetic beads are commonly used in the SELEX process24,25
because of their intrinsic magnetic properties, which ease
subsequent manipulations. To obtain an alkyne-modified solid
support, the amino-modified magnetic beads were reacted with
an alkyne NHS ester26 (Figure 3). The reaction between an
NHS-activated acid and an amine is normally a very high
yielding reaction; however, to functionalize as many amines as
possible, the reaction was repeated a total of three times. Any
remaining amines were capped as the acetyl ester using acetic
anhydride and diisopropylethylamine. Consequently, alkyne-
The beads 28 and 29, containing the immobilized neuro-
transmitters, were stored below 0 °C in a suspension containing
ascorbic acid as antioxidant to avoid oxidation of the
neurotransmitters.
CONCLUSION
■
PEG-azide derivatives of dopamine (12 steps) and serotonin
(10 steps) have been synthesized and successfully immobilized
onto a magnetic solid support via the Huisgen−Meldal−
Sharpless click reaction. Furthermore, the incorporated Fmoc
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2-(6-(3-Hydroxypropyl)benzo[d][1,3]dioxol-5-yl)acetonitrile
(6). In a 100 mL Schlenk flask, 5 (0.660 g, 3.07 mmol) was dissolved
in MeOH (10 mL) and EtOAc (10 mL). To the resulting mixture was
added Pd/C (65 mg, 0.55 mmol) from a batch containing a 10% w/w
loading. Subsequently, the reaction was purged with hydrogen gas
under vigorous magnetic stirring.
reporter group enabled the facile quantification of the
neurotransmitter derivatives immobilized onto the solid
support. Application of the immobilized neurotransmitters
described here for selection of aptamers toward serotonin and
dopamine is currently in progress.
After reacting overnight, the reaction was filtered through Celite
and evaporated to dryness in vacuo, affording crude 6 as a yellow oil
(100% conversion).
EXPERIMENTAL SECTION
■
General Experimental Details. For all water- and/or oxygen-
sensitive reactions, flame-dried glassware, which was purged with
argon and evacuated a total of three times, was used. All reactions were
monitored by thin-layer chromatography (TLC) analysis on Merck
silica gel 60 F254 TLC plates. The TLC plates were visualized by
exposure to UV (254 nm) or by staining with either a basic aq.
KMnO4 solution or a cerium−ammonium−molybdate stain. Flash
column chromatography was performed using Merck silica gel 60
(230−400 mesh). All solvents were of HPLC grade quality. For inert
reactions, the solvents THF and DCM were dried prior to use by the
use of a solvent purification system. Diethyl ether was dried with
sodium, and triethylamine was distilled from CaH2 before use. Unless
noted, all used reagents are commercially available. The reagents were
purchased at the highest possible quality and used without further
purification.
1H NMR (400 MHz, CDCl3): δ 6.81 (s, 1H), 6.69 (s, 1H), 5.93 (s,
2H), 3.81−3.45 (m, 4H), 2.74−2.47 (m, 2H), 2.06 (brs, 1H), 1.93−
1.65 (m, 2H).13C NMR (100 MHz, CDCl3): δ 147.8, 146.4, 133.6,
120.8, 118.4, 109.8, 109.2, 101.4, 61.5, 33.4, 28.7, 21.2. HRMS (ES)
m/z [M + Na]+ calcd for C12H13NO3Na: 242.0793. Found: 242.0788.
3-(6-(Cyanomethyl)benzo[d][1,3]dioxol-5-yl)propyl acetate
(7). To a 250 mL flask was added crude 6 (0.650 g, 2.96 mmol),
which was dissolved in pyridine (30 mL) and acetic anhydride (30
mL). The reaction was left to react overnight, after which the reaction
was quenched with 0.1 M hydrochloric acid (20 mL). The mixture was
transferred to a 250 mL separation funnel and then extracted with
EtOAc (3 × 60 mL). The combined organic fractions were washed
with NaHCO3 (2 × 50 mL), dried (MgSO4), filtered under suction,
and evaporated to dryness to afford crude 7 as a yellow oil (100%
conversion).
NMR spectra were recorded at 400 MHz (1H NMR) and at 100
MHz (13C NMR) and calibrated to the residual solvent peak.
Chemical shifts are reported in parts per million and coupling
1H NMR (400 MHz, CDCl3): δ 6.78 (s, 1H), 6.64 (s, 1H), 5.89 (s,
2H), 4.05 (t, J = 6.3, 2H), 3.58 (s, 2H), 2.65−2.48 (m, 2H), 2.02 (s,
3H), 1.93−1.75 (m, 2H).13C NMR (100 MHz, CDCl3): δ 171.1,
147.9, 146.5, 132.8, 120.7, 118.0, 109.7, 109.4, 101.4, 63.5, 29.4, 28.9,
21.2, 21.0. HRMS (ES) m/z [M + Na]+ calcd for C14H15NO4Na:
284.0899. Found: 284.0898.
3-(6-(2-((tert-Butoxycarbonyl)amino)ethyl)benzo[d][1,3]-
dioxol-5-yl)propyl Acetate (8). A solution of crude 7 (0.778 g, 2.98
mmol) in MeOH (25 mL) was cooled to 0 °C. Thereafter, Boc2O
(1.36 g, 6.23 mmol) and NiCl2(H2O)6 (0.100 g, 0.42 mmol) were
added. After 5 min of stirring, NaBH4 (0.788 g, 20.8 mmol) was added
portionwise, and the black solution was left to stir for 3 h, whereupon
NaBH4 (0.788 g, 20.8 mmol) again was added. Subsequently, the
reaction was left to react overnight.
The resulting white solution was diluted with EtOAc, washed twice
with NaHCO3, dried (MgSO4), filtered under suction, and evaporated
to dryness in vacuo, furnishing crude 8 (100% conversion).
1H NMR (400 MHz, CDCl3): δ 6.60 (s, 1H), 6.59 (s, 1H), 5.84 (s,
2H), 4.72 (brs, 1H), 4.04 (t, J = 6.5, 2H), 3.37−3.10 (m, 2H), 2.67 (t,
J = 7.2, 2H), 2.64−2.49 (m, 2H), 2.02 (s, 3H), 1.87−1.76 (m, 2H),
1.39 (s, 9H).13C NMR (100 MHz, CDCl3): δ 171.3, 156.0, 146.3,
146.1, 132.8, 129.8, 109.7, 109.5, 100.9, 79.3, 63.9, 41.7, 32.9, 30.3,
28.8, 28.5, 21.1. HRMS (ES) m/z [M + Na]+ calcd for C19H27NO6Na:
388.1736. Found: 388.1731.
tert-Butyl(2-(6-(3-hydroxypropyl)benzo[d][1,3]dioxol-5-yl)-
ethyl)carbamate (9). To MeOH (10 mL) in a 25 mL flask was
added Na (0.050 g, 2.0 mmol). The freshly generated solution of
MeONa/MeOH was then added directly to a solution of crude 8 (1.04
g, 2.84 mmol) in MeOH (15 mL), and the reaction was left to stir
overnight at room temperature.
1
constants are reported in hertz. In the interpretation of the H NMR
spectra, the following abbreviations are used: s, singlet; d, doublet; t,
triplet; q, quartet; qui, quintet; sext, sextet; m, multiplet; br, broad.
Melting points are uncorrected. High-resolution mass spectrometry
(HRMS) was conducted using electrospray ionization.
Compound 3 (CAS: 5434-47-9) was purchased from a chemical
supplier. Compound 11 is commercially available; however, it was
synthesized according to a literature procedure.28 Compound 15
(CAS: 153-98-0) was purchased from a chemical supplier, as were the
magnetic beads, Dynabeads M-270 amine.
2-(6-Bromobenzo[d][1,3]dioxol-5-yl)acetonitrile (4).29 To a
250 mL flame-dried flask under an argon atmosphere was added 3
(10.20 g, 34.70 mmol), which was dissolved in dry DMF. Thereafter,
sodium cyanide (6.82 g, 139 mmol) was added portionwise, and the
reaction was left to stir for 3 h.
The reaction was quenched by the addition of water and then
extracted three times with EtOAc. The combined organic fractions
were thoroughly washed with water, dried (MgSO4), filtered under
suction, and evaporated. The resulting yellow compound was purified
by flash chromatography (pentane/EtOAc 1:1). The product was
isolated as a white solid (7.5 g, 32.3 mmol, 92% yield).
1
mp(uncorr.) 61.8−66.1 °C. H NMR (400 MHz, CDCl3): δ 7.01
(s, 1H), 6.95 (s, 1H), 6.00 (s, 2H), 3.72 (s, 2H).13C NMR (100 MHz,
CDCl3): δ 148.5, 148.0, 122.7, 117.2, 114.3, 113.0, 109.5, 102.3, 24.7.
HRMS (ES) m/z [M + Na]+ calcd for C9H6BrNO2Na: 261.9480.
Found: 261.9484.
2-(6-(3-Hydroxyprop-1-yn-1-yl)benzo[d][1,3]dioxol-5-yl)-
acetonitrile (5). To a flame-dried Schlenk flask containing an argon
atmosphere was added 4 (4.070 g, 16.94 mmol), which was dissolved
in freshly distilled THF and Et3N (1:1). Propargyl alcohol (3.9 mL, 68
mmol) was added, and the mixture was degassed with argon for 30
min, after which Cl2Pd(II)(PPh3)2 (1.19 g, 1.69 mmol) and CuI
(0.161 g, 0.845 mmol) were added. The resulting yellow mixture was
heated to 80 °C and left to stir overnight.
The reaction was evaporated to dryness and purified via flash
column chromatography (pentane/EtOAc 6:4), affording pure 9
(0.482 g, 1.49 mmol, 48% after four steps).
1H NMR (400 MHz, CDCl3): δ 6.65 (s, 1H), 6.60 (s, 1H), 5.87 (s,
2H), 4.86 (brs, 1H), 3.66 (t, J = 5.6, 2H), 3.23 (q, J = 7.6, 2H), 2.72 (t,
J = 7.6, 2H), 2.67 (t, J = 8.0, 2H), 2.16 (brs, 1H), 1.85−1.70 (m, 2H),
1.42 (s, 9H). 13C NMR (100 MHz, CDCl3): δ 156.3, 146.2, 145.8,
133.8, 129.6, 109.7, 109.7, 100.8, 79.6, 62.1, 42.0, 34.8, 33.3, 28.9, 28.5.
HRMS (ES) m/z [M + Na]+ calcd for C17H25NO5Na: 346.1630.
Found: 346.1609.
The black mixture was filtered through Celite and evaporated to
dryness. Thereafter, the resulting yellow oil was purified by flash
column chromatography (pentane/EtOAc 1:1), and the product was
isolated as a white solid (1.00 g, 4.65 mmol, 27%).
mp(uncorr.) 133.2−134.3 °C. 1H NMR (400 MHz, CDCl3): δ 6.92
(s, 1H), 6.91 (s, 1H), 6.02 (s, 2H), 4.53 (d, J = 6.2, 2H), 3.82 (s, 2H),
1.76 (t, J = 6.2, 1H). 13C NMR (100 MHz, CDCl3): δ 148.9, 147.5,
126.7, 117.7, 115.4, 112.1, 108.9, 102.1, 92.4, 82.6, 51.6, 22.7. HRMS
(ES) m/z [M + Na]+ calcd for C12H9NO3Na: 238.0480. Found:
238.0491.
3-(6-(2-((tert-Butoxycarbonyl)amino)ethyl)benzo[d][1,3]-
dioxol-5-yl)propyl Methanesulfonate (10). To a solution of 9
(0.482 g, 1.49 mmol) in dry diethyl ether was added Et3N (0.41 mL,
3.0 mmol). Thereafter, the reaction mixture was cooled to 0 °C,
followed by the addition of MsCl (0.17 mL, 2.2 mmol). The reaction
was left to stir for 2 h, after which the resulting slurry was diluted with
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EtOAc and washed with water (×2) and brine. The combined water
and brine fraction was extracted once with EtOAc. Subsequently, the
combined organic fractions were dried (MgSO4), filtered under
suction, and evaporated to dryness.
1H NMR (400 MHz, CDCl3): δ 7.75 (d, J = 7.5, 2H), 7.64 (s, 1H),
7.57 (d, J = 7.4, 2H), 7.38 (t, J = 7.3, 2H), 7.30 (t, J = 7.4, 2H), 6.81 (s,
1H), 6.76 (s, 1H), 5.14 (brt, J = 5.5, 1H), 4.42 (d, J = 6.7, 2H), 4.20 (t,
J = 6.2, 1H), 3.68−3.57 (m, 8H), 3.58−3.53 (m, J = 4.7, 2H), 3.44 (t, J
= 5.8, 2H), 3.39−3.26 (m, 4H), 2.78 (t, J = 7.2, 2H), 2.65 (t, J = 7.6,
2H), 2.09 (s, 3H), 1.80 (qui, J = 6.8, 2H). 13C NMR (100 MHz,
CDCl3): δ 180.4, 169.2, 156.4, 144.0, 143.5, 143.1, 141.4, 134.9, 130.9,
127.7, 127.1, 125.0, 120.0, 112.7, 110.4, 110.2, 70.7, 70.6, 70.1, 70.0,
66.4, 50.7, 47.3, 42.1, 32.9, 31.2, 28.8, 21.1, 18.3. HRMS (ES) m/z [M
+ Na]+ calcd for C35H40N4O9Na: 683.2693. Found: 683.2703.
(9H-Fluoren-9-yl)methyl 2-(3-(2-(2-(2-azidoethoxy)ethoxy)-
ethoxy)propyl)-4,5-dihydroxyphenethylcarbamate (1). Crude
14 (31 mg, 0.048 mmol) was dissolved in a solution of 10% conc. HCl
in MeOH (10 mL). The mixture was reacted overnight, after which
the solution was evaporated to dryness in vacuo. Thereafter, the
product was purified by flash column chromatography (DCM/Et2O
7:3 to EtOAc/pentane 7:3), furnishing 1 (8.5 mg, 0.014 mmol, 30%
over two steps) as an oil.
The resulting yellow oil was purified via flash column chromatog-
raphy (pentane/EtOAc 13:7), affording 10 (590 mg, 1.47 mmol, 99%)
as a white yellow solid.
1
mp(uncorr.) 90.0−91.0 °C. H NMR (400 MHz, CDCl3): δ 6.60
(m, 2H), 5.85 (s, 2H), 4.77 (brs, 1H), 4.21 (t, J = 6.2, 2H), 3.21 (q, J =
8.0, 2H), 3.00 (s, 3H), 2.73−2.57 (m, 4H), 2.02−1.84 (m, 2H), 1.39
(s, 9H).13C NMR (100 MHz, CDCl3): δ 155.8, 146.2, 146.0, 131.8,
129.8, 109.7, 109.3, 100.8, 79.0, 69.3, 41.7, 37.2, 32.9, 30.7, 28.3, 28.1.
HRMS (ES) m/z [M + Na]+ calcd for C18H27NO7SNa: 424.1406.
Found: 424.1389.
tert-Butyl (2-(6-(3-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)-
propyl)benzo[d][1,3]dioxol-5-yl)ethyl)carbamate (12). A flame-
dried 50 mL Schlenk flask under an argon atmosphere was charged
with 11 (0.218 g, 1.25 mmol) dissolved in dry DMF (1 mL).
Thereafter, at 0 °C, NaH (45 mg, 1.1 mmol), from a 60% suspension
in mineral oil, was added portionwise. After 30 min, 10 (0.100 g, 0.250
mmol), dissolved in a minimum of dry DMF, was slowly added via a
syringe during a time period of 60 min. The reaction was stirred
overnight while gradually being allowed to reach room temperature.
The resulting reaction mixture was evaporated to dryness and
purified directly via flash column chromatography (pentane/EtOAc
7:3), yielding 12 (65.8 mg, 0.137 mmol, 55%) as a clear colorless oil.
1H NMR (400 MHz, CDCl3): δ 6.65 (s, 1H), 6.62 (s, 1H), 5.87 (s,
2H), 4.71 (brs, 1H), 3.73−3.61 (m, 8H), 3.57 (t, J = 5.2, 2H), 3.45 (t,
J = 5.8, 2H), 3.37 (t, J = 5.1, 2H), 3.33−3.14 (m, 2H), 2.71 (t, J = 6.9,
2H), 2.60 (t, J = 7.2, 2H), 1.79 (qui, J = 6.4, 2H), 1.42 (s, 9H). 13C
NMR (100 MHz, CDCl3): δ 155.9, 146.2, 145.8, 133.5, 129.8, 109.7,
109.6, 100.8, 79.2, 70.8, 70.8, 70.3, 70.2, 70.1, 50.8, 41.7, 32.9, 31.3,
28.8, 28.5. HRMS (ES) m/z [M + Na]+ calcd for C23H36N4O7Na:
503.2482. Found: 503.2495.
(9H-Fluoren-9-yl)methyl(2-(6-(3-(2-(2-(2-azidoethoxy)-
ethoxy)ethoxy)propyl)benzo[d][1,3]dioxol-5-yl)ethyl)-
carbamate (13). To a 20 mL flask containing 12 (0.045 g, 0.094
mmol) was added 10 mL of 4 M HCl in dioxane. The resulting
solution was left to react overnight and thereafter evaporated to
dryness in vacuo. The residue was then dissolved in DCM (1 mL) and
DIPEA (33 μL, 0.19 mmol), and then to the resulting mixture was
added 9-fluorenylmethyl N-succinimidyl carbonate (0.121 g, 0.377
mmol).
1H NMR (400 MHz, CDCl3): δ 7.76 (d, J = 7.3, 2H), 7.58 (d, J =
7.2, 2H), 7.40 (t, J = 7.2, 2H), 7.31 (t, J = 7.2, 2H), 6.83 (s, 1H), 6.66
(s, 1H), 6.42 (brs, 1H), 5.67 (brs, 1H), 5.03 (brt, J = 5.9, 1H), 4.41 (d,
J = 6.8, 2H), 4.21 (t, J = 7.6, 1H), 3.76−3.59 (m, 8H), 3.60−3.50 (m,
2H), 3.43−3.28 (m, 6H), 2.69 (t, J = 7.2, 2H), 2.61 (t, J = 7.2, 2H),
1.77 (m, 2H). 13C NMR (100 MHz, CDCl3): δ 156.7, 144.1, 142.3,
142.2, 141.5, 132.0, 128.7, 127.8, 127.2, 125.2, 120.1, 117.4, 116.4,
71.0, 70.7, 70.4, 70.1, 70.1, 69.4, 66.7, 50.7, 47.4, 42.2, 32.2, 30.5, 28.1.
HRMS (ES) m/z [M + Na]+ calcd for C32H38N4O7Na: 613.2638.
Found: 613.2640.
tert-Butyl(2-(5-hydroxy-1H-indol-3-yl)ethyl)carbamate
(16).30 Serotonin hydrochloride 15 (1.0 g, 4.7 mmol) was placed in a
flame-dried Schlenk flask and dissolved in methanol under an argon
atmosphere. Triethylamine (1.51 mL, 10.3 mmol) was added, and the
solution was cooled to 0 °C, whereupon Boc2O (1.54 g, 7.05 mmol)
was dissolved in methanol and added dropwise. After 3 h, the resulting
mixture was quenched with H2O, transferred to a separation funnel,
and extracted three times with DCM. The combined organic phases
were dried (Na2SO4), filtered under suction, and concentrated in
vacuo. The product was purified by flash column chromatography
(EtOAc/pentane 1:1) and isolated as a yellow solid (1.21 g, 4.38
mmol, 93%).
mp(uncorr.) 114.1−115.0 °C. 1H NMR (400 MHz, CDCl3): δ 8.11
(s, 1H), 7.17 (d, J = 8.6, 1H), 6.99 (d, J = 2.0, 1H), 6.92 (s, 1H), 6.80
(dd, J = 2.1, 8.6, 1H), 6.18 (brs, 1H), 4.73 (brs, 1H), 3.47−3.32 (m,
2H), 2.82 (t, J = 6.6, 2H), 1.45 (s, 9H). 13C NMR (100 MHz, CDCl3):
δ 156.4, 149.9, 131.7, 128.2, 123.3, 112.4, 112.2, 112.0, 103.4, 79.6,
40.9, 28.6, 26.0. HRMS (ES) m/z [M + Na]+ calcd for
C15H20N2O3Na: 299.1372. Found: 299.1369.
After 5 h, the reaction was quenched with 1 M HCl and evaporated
to dryness. The product was purified by flash column chromatography
(pentane/EtOAc first 7:3, then changed to 1:1 after isolating Fmoc
residues), affording 13 (34 mg, 0.056 mmol, 60% over two steps) as a
clear oil.
tert-Butyl(2-(5-(allyloxy)-1H-indol-3-yl)ethyl)carbamate (17).
Boc-protected serotonin 16 (3.33 g, 12.1 mmol) was placed in a dry
Schlenk flask under an argon atmosphere and dissolved in acetone.
Thereafter, Cs2CO3 (9.82 g, 30.1 mmol) was added, and after 10 min
of stirring, allyl iodide (2.75 mL, 30.1 mmol) was added. The resulting
yellow solution was stirred overnight, after which the reaction was
filtered under suction and evaporated to dryness in vacuo. The
compound was purified by flash column chromatography (EtOAc/
pentane 3:7), affording 17 as a yellow solid (2.82 g, 8.91 mmol, 74%).
1H NMR (400 MHz, CDCl3): δ 7.77 (d, J = 7.6, 2H), 7.59 (d, J =
7.5, 2H), 7.40 (t, J = 7.7, 2H), 7.32 (t, J = 7.0, 2H), 6.67 (s, 1H), 6.63
(s, 1H), 5.90 (s, 2H), 5.04 (brt, J = 5.4, 1H), 4.42 (d, J = 6.7, 2H), 4.22
(t, J = 6.8, 1H), 3.70−3.59 (m, 8H), 3.60−3.52 (m, 2H), 3.45 (t, J =
6.1, 2H), 3.39−3.29 (m, 4H), 2.75 (t, J = 7.2, 2H), 2.62 (t, J = 7.2,
2H), 1.81 (qui, J = 8.0, 2H). 13C NMR (100 MHz, CDCl3): δ 156.5,
146.3, 145.9, 144.1, 141.4, 133.6, 129.6, 127.8, 127.1, 125.1, 120.1,
109.7, 109.7, 100.9, 70.8, 70.7, 70.2, 70.2, 70.1, 66.5, 50.7, 47.4, 42.2,
32.9, 31.3, 28.8. HRMS (ES) m/z [M + Na]+ calcd for
C33H38N4O7Na: 625.2638. Found: 625.2637.
1
mp (uncorr.) 88.0−88.7 °C. H NMR (400 MHz, CDCl3): δ 8.05
(s, 1H), 7.25 (d, J = 8.8, 1H), 7.06 (d, J = 1.8, 1H), 7.00 (s, 1H), 6.90
(dd, J = 2.4, 8.8, 1H), 6.33−5.96 (m, 1H), 5.45 (dq, J = 1.6, 17.3, 1H),
5.29 (dq, J = 1.4, 10.5, 1H), 4.65 (brs, 1H), 4.59 (dt, J = 1.4, 5.3, 2H),
3.63−3.30 (m, 2H), 2.91 (t, J = 6.5, 2H), 1.44 (s, 9H). 13C NMR (100
MHz, CDCl3): δ 156.2, 152.7, 133.9, 131.8, 127.6, 123.1, 117.3, 112.6,
112.3, 112.0, 102.2, 79.2, 69.9, 40.9, 28.4, 25.7. HRMS (ES) m/z [M +
Na]+ calcd for C18H24N2O3Na: 339.1685. Found: 339.1680.
5-(2-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)ethyl)-6-
(3-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)propyl)benzo[d][1,3]-
dioxol-2-yl Acetate (14). Compound 13 (28 mg, 0.048 mmol) was
added to a flame-dried 50 mL flask under an argon atmosphere and
dissolved in benzene. Thereafter, Pb(OAc)4 (63 mg, 0.14 mmol) was
added and the temperature was raised to 80 °C. After 17 h, Pb(OAc)4
(21 mg, 0.048 mmol) was added, and the reaction was stirred for
another 15 h.
tert-Butyl(2-(4-allyl-5-hydroxy-1H-indol-3-yl)ethyl)-
carbamate (18). Compound 17 (3.33 g, 10.5 mmol) was dissolved in
o-xylene (100 mL) under an argon atmosphere and refluxed overnight.
The solvent was evaporated in vacuo, and thereafter, the product was
purified by flash column chromatography (EtOAc/pentane 4:6),
affording 18 as a yellow solid (2.12 g, 6.72 mmol, 64%).
The reaction was evaporated to dryness in vacuo, diluted with
EtOAc, washed with water (×3), and evaporated to dryness in vacuo,
affording crude 14.
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1
oxy)-1H-indole-1-carboxylate (22). To a 0 °C solution of 21
(0.144 g, 0.278 mmol) in dry Et2O (30 mL) were added Et3N (77 μL,
0.56 mmol) and MsCl (32 μL, 0.42 mmol). After 2 h, an additional
amount of Et3N (77 μL, 0.56 mmol) and MsCl (32 μL, 0.42 mmol)
was added, and the reaction was left to react overnight. Thereafter, the
reaction was diluted with water and extracted with EtOAc (×4). The
combined organic phases were evaporated to dryness, and then the
product was purified by flash column chromatography (EtOAc/
pentane 3:7), yielding 22 as an oil (152 mg, 0.254 mmol, 92%).
1H NMR (400 MHz, CDCl3): δ 7.99 (d, J = 9.2, 1H), 7.38 (s, 1H),
7.16 (d, J = 9.1, 1H), 5.37 (t, J = 3.2, 1H), 4.94 (bs, 1H), 4.34 (t, J =
6.1, 2H), 3.96−3.86 (m, 1H), 3.66−3.58 (m, 1H), 3.50−3.37 (m, 2H),
3.25−3.03 (m, 2H), 3.03−2.93 (m, 5H), 2.16−1.80 (m, 5H), 1.77−
1.65 (m, 2H), 1.65−1.60 (m, 10H), 1.45 (s, 9H). 13C NMR (100
MHz, CDCl3): δ 156.1, 151.0, 149.5, 131.8, 129.0, 124.9, 122.0, 117.7,
113.9, 112.7, 97.8, 83.5, 79.3, 70.4, 62.5, 41.1, 37.4, 31.0, 30.7, 28.5,
28.3, 28.1, 25.4, 22.6, 19.4. HRMS (ES) m/z [M + Na]+ calcd for
C29H44N2O9SNa: 619.2665. Found: 619.2679.
tert-Butyl(2-(4-(3-((2-(2-(2-azidoethoxy)ethoxy)ethyl)thio)-
propyl)-5-((tetrahydro-2H-pyran-2-yl)oxy)-1H-indol-3-yl)-
ethyl)carbamate (24a) and tert-Butyl-4-(3-((2-(2-(2-
azidoethoxy)ethoxy)ethyl)thio)propyl)-3-(2-((tert-
butoxycarbonyl)amino)ethyl)-5-((tetrahydro-2H-pyran-2-yl)-
oxy)-1H-indole-1-carboxylate (24b). To a 0 °C solution of 23
(98.4 mg, 0.514 mmol) in dry DMF (0.3 mL) under an argon
atmosphere was added NaH (13.2 mg, 0.329 mmol) from a 60%
suspension in mineral oil. After 40 min, 22 (61.4 mg, 0.103 mmol),
dissolved in a minimum amount of dry DMF, was added, and the
reaction was left to react overnight. Thereafter, the reaction was
quenched with water, extracted with EtOAc (×4), and evaporated to
dryness in vacuo.
mp (uncorr.) 120.9−121.4 °C. H NMR (400 MHz, CDCl3): δ
8.03 (s, 1H), 7.12 (d, J = 8.6, 1H), 6.96 (s, 1H), 6.79 (d, J = 8.6, 1H),
6.19−5.98 (m, 1H), 5.11 (d, J = 10.1, 1H), 5.01 (d, J = 17.3, 1H), 4.67
(brs, 1H), 3.78 (d, J = 5.3, 2H), 3.58−3.26 (m, 2H), 3.02 (t, J = 6.5,
2H), 1.47 (s, 9H). 13C NMR (100 MHz, CDCl3): δ 156.2, 147.9,
136.9, 132.6, 126.3, 123.7, 115.8, 113.1, 112.9, 110.2, 79.4, 41.5, 30.5,
28.6, 27.7. HRMS (ES) m/z [M + Na]+ calcd for C18H24N2O3Na:
339.1685. Found: 339.1662.
tert-Butyl(2-(4-allyl-5-((tetrahydro-2H-pyran-2-yl)oxy)-1H-
indol-3-yl)ethyl)carbamate (19). Compound 18 (0.412 g, 1.30
mmol) was dissolved in dry DCM (65 mL), after which pyridinium p-
toluenesulfonate (49.2 mg, 0.196 mmol) and dihydropyran (0.13 mL,
1.4 mmol) were added. After 3 h, the reaction was quenched by adding
water, extracted three times with EtOAc, dried (Na2SO4), and
evaporated to dryness in vacuo. The product was purified via flash
column chromatography (EtOAc/pentane first 2:8, then 3:7),
resulting in 19 (0.320 g, 0.800 mmol, 61%) as a light-sensitive oil.
1H NMR (400 MHz, CDCl3): δ 8.19 (s, 1H), 7.15 (d, J = 8.8, 1H),
7.11 (d, J = 8.8, 1H), 6.94 (s, 1H), 6.08 (m, 1H), 5.26 (t, J = 3.3, 1H),
4.99 (dq, J = 1.6, 10.1, 1H), 4.88 (dq, J = 1.8, 17.1, 1H), 4.69 (brs,
1H), 4.07−3.95 (m, 1H), 3.91−3.69 (m, 2H), 3.67−3.54 (m, 1H),
3.51−3.37 (m, 2H), 3.04 (t, J = 6.7, 2H), 1.95−1.82 (m, 2H), 1.74−
1.54 (m, 4H), 1.45 (s, 9H). 13C NMR (100 MHz, CDCl3): δ 156.1,
149.1, 138.2, 133.3, 126.2, 123.6, 120.6, 114.7, 113.3, 112.9, 109.7,
98.8, 79.3, 62.4, 41.5, 31.0, 30.5, 28.6, 27.5, 25.5, 19.4. HRMS (ES) m/
z [M + Na]+ calcd for C23H32N2O4Na: 423.2260. Found: 423.2252.
tert-Butyl-4-allyl-3-(2-((tert-butoxycarbonyl)amino)ethyl)-5-
((tetrahydro-2H-pyran-2-yl)oxy)-1H-indole-1-carboxylate (20).
To a dry flask were added Boc2O (0.104 g, 0.478 mmol), DMAP (83
mg, 0.68 mmol), and Et3N (0.19 mL, 1.3 mmol), and thereafter, the
compounds were dissolved in dry THF (8 mL). The resulting solution
was cooled to 0 °C, after which 19 (0.182 g, 0.455 mmol) was added
dropwise as a suspension in dry THF (8 mL). After 2 h, the reaction
was extrachted with DCM (×3), dried (Na2SO4), filtered under
suction, and evaporated in vacuo.
The resulting residue was purified via flash column chromatography
(gradient: first isolating 24a via pentane/EtOAc (7:3) and 24b in
pentane/EtOAc 6:4; any linker residues removed by second column in
acetone/pentane 2:8), resulting in 24a (22.8 mg, 0.0385 mmol, 37%)
and 24b (10.2 mg, 0.0147 mmol, 14%) as an oil, obtaining a combined
yield of 51%.
The product was purified by flash column chromatography
(EtOAc/pentane 1:9), affording 20 as an oil (0.133 g, 0.266 mmol,
58%).
1
24a: H NMR (400 MHz, CDCl3): δ 8.08 (brs, 1H), 7.13 (d, J =
8.8, 1H), 7.09 (d, J = 8.8, 1H), 6.97 (s, 1H), 5.28 (t, J = 3.3, 1H), 4.80
(brs, 1H), 4.04−3.92 (m, 1H), 3.70−3.56 (m, 9H), 3.49−3.41 (m,
2H), 3.41−3.30 (m, 2H), 3.20−2.96 (m, 4H), 2.78−2.64 (m, 4H),
2.11−1.83 (m, 5H), 1.73−1.57 (m, 3H), 1.45 (s, 9H). 13C NMR (100
MHz, CDCl3): δ 156.1, 148.9, 133.2, 125.8, 123.7, 122.8, 113.2, 112.4,
109.4, 98.5, 79.2, 71.1, 70.8, 70.4, 70.2, 62.5, 50.8, 41.7, 32.9, 31.6,
31.5, 31.2, 28.6, 27.9, 26.1, 25.5, 19.5. HRMS (ES) m/z [M + Na]+
calcd for C29H45N5O6SNa: 614.2988. Found: 614.2988.
1H NMR (400 MHz, CDCl3): δ 8.00 (d, J = 9.0, 1H), 7.35 (s, 1H),
7.18 (d, J = 9.1, 1H), 6.04 (ddt, J = 5.3, 10.3, 15.6, 1H), 5.33 (t, J = 3.1,
1H), 4.98 (dd, J = 1.6, 10.2, 1H), 4.80 (dd, J = 1.7, 17.2, 1H), 4.74 (bs,
1H), 4.00−3.87 (m, 1H), 3.86−3.65 (m, 2H), 3.64−3.52 (m, 1H),
3.51−3.36 (m, 2H), 2.98 (t, J = 6.8, 2H), 2.03−1.93 (m, 1H), 1.92−
1.83 (m, 2H), 1.72−1.55 (m, 12H), 1.44 (s, 9H). 13C NMR (100
MHz, CDCl3): δ 155.9, 151.0, 149.5, 137.6, 131.6, 129.4, 124.3, 120.7,
118.0, 114.8, 113.7, 113.0, 97.6, 83.2, 79.1, 62.0, 40.6, 30.7, 29.8, 28.4,
28.2, 27.5, 25.3, 19.0. HRMS (ES) m/z [M + Na]+ calcd for
C28H40N2O6Na: 523.2784. Found: 523.2787.
1
24b: H NMR (400 MHz, CDCl3): δ 8.05−7.86 (m, 1H), 7.36 (s,
1H), 7.16 (d, J = 9.1, 1H), 5.37 (t, J = 2.9, 1H), 4.77 (brs, 1H), 3.99−
3.84 (m, 1H), 3.74−3.55 (m, 9H), 3.54−3.30 (m, 4H), 3.17−2.91 (m,
4H), 2.73 (t, J = 7.0, 2H), 2.68 (t, J = 7.1, 2H), 2.14−1.77 (m, 5H),
1.75−1.60 (m, 12H), 1.45 (s, 9H). 13C NMR (100 MHz, CDCl3): δ
155.9, 150.8, 149.5, 131.6, 128.9, 124.6, 123.1, 117.7, 113.4, 112.6,
97.5, 83.3, 79.2, 71.0, 70.6, 70.3, 70.0, 62.2, 50.7, 40.9, 38.4, 32.7, 31.6,
31.3, 30.9, 28.4, 28.2, 25.5, 25.3, 19.2. HRMS (ES) m/z [M + H]+
calcd for C34H54N5O8S: 692.3693. Found: 692.3694.
tert-Butyl-3-(2-((tert-butoxycarbonyl)amino)ethyl)-4-(3-hy-
droxypropyl)-5-((tetrahydro-2H-pyran-2-yl)oxy)-1H-indole-1-
carboxylate (21). To a 0 °C solution of 20 (0.133 g, 0.266 mmol) in
dry THF was added BH3 (0.56 mL, 0.56 mmol), from a 1 M solution
in THF. After 3 h, NaOH (0.53 mL, 0.53 mmol) from a 1 M aq.
solution and H2O2 (0.12 mL, 1.2 mmol) from a 35% aq. solution were
added, and the resulting mixture was left to react for 3 h. Thereafter,
the reaction was diluted with water and extracted with Et2O (×4) and
DCM (×3). The combined organic phases were evaporated in vacuo,
and thereafter, the product was purified by flash column chromatog-
raphy (EtOAc/pentane 4:6), obtaining the product as a yellow oil
(0.097 g, 0.19 mmol, 70%).
(9H-Fluoren-9-yl)methyl (2-(4-(3-((2-(2-(2-azidoethoxy)-
ethoxy)ethyl)thio)propyl)-5-hydroxy-1H-indol-3-yl)ethyl)-
carbamate (2). To a solution of 24a (9.7 mg, 0.016 mmol) and 24b
(6.6 mg, 0.0095 mmol) in MeOH (10 mL) was added HCl (9.5 mL)
from a 4 M HCl solution in dioxane, and the mixture was reacted
overnight. Thereafter, the mixture was evaporated to dryness in vacuo.
The resulting residue was redissolved in toluene and evaporated to
dryness (repeated three times). Thereafter, the residue was dissolved
in dry DCM (6 mL), and DIPEA (9 μL, 0.026 mmol) was added,
followed by addition of 9-fluorenylmethyl N-succinimidyl carbonate
(16 mg, 0.047 mmol), which was added portionwise. After 3 h, the
reaction was quenched with 1 M HCl, evaporated to dryness in vacuo,
and purified via flash column chromatography (EtOAc/pentane 4:6),
affording 2 (5.5 mg, 0.0087 mmol, 34% over two steps).
1H NMR (400 MHz, CDCl3): δ 7.95 (d, J = 9.0, 1H), 7.36 (s, 1H),
7.15 (d, J = 9.1, 1H), 5.35 (t, J = 3.2, 1H), 4.99 (t, J = 5.4, 1H), 3.98−
3.87 (m, 1H), 3.79−3.50 (m, 3H), 3.46−3.33 (m, 2H), 3.28−2.88 (m,
4H), 2.06−1.76 (m, 5H), 1.75−1.57 (m, 12H), 1.44 (s, 9H). 13C
NMR (100 MHz, CDCl3): δ 156.4, 150.9, 149.6, 131.9, 129.0, 124.8,
123.8, 118.1, 113.4, 113.1, 98.0, 83.4, 79.6, 62.4, 41.7, 34.5, 30.9, 28.5,
28.3, 28.1, 27.9, 25.3, 22.6, 19.3. HRMS (ES) m/z [M + Na]+ calcd for
C28H42N2O7Na: 541.2890. Found: 541.2891.
1H NMR (400 MHz, CDCl3): δ 7.91 (s, 1H), 7.77 (d, J = 7.6, 2H),
7.59 (d, J = 7.2, 2H), 7.40 (t, J = 7.4, 2H), 7.31 (t, J = 7.6, 2H), 7.10
tert-Butyl-3-(2-((tert-butoxycarbonyl)amino)ethyl)-4-(3-
((methylsulfonyl)oxy)propyl)-5-((tetrahydro-2H-pyran-2-yl)-
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Article
(d, J = 8.6, 1H), 6.94 (s, 1H), 6.77 (d, J = 8.6, 1H), 5.02 (t, J = 7.5,
1H), 4.43 (d, J = 6.8, 2H), 4.22 (t, J = 6.6, 1H), 3.68−3.57 (m, 8H),
3.57−3.47 (m, 3H), 3.40−3.31 (m, 2H), 3.14−2.99 (m, 4H), 2.74 (t, J
= 6.7, 2H), 2.69 (t, J = 6.7, 2H), 2.03−1.91 (m, 2H). 13C NMR (100
MHz, CDCl3): δ 156.6, 147.7, 144.1, 141.5, 132.5, 127.8, 127.2, 125.9,
125.2, 123.7, 120.1, 118.3, 112.9, 112.8, 109.8, 71.1, 70.8, 70.5, 70.1,
66.6, 50.8, 47.5, 42.2, 32.3, 31.6, 30.5, 27.8, 24.8. HRMS (ES) m/z [M
+ Na]+ calcd for C34H39N5O5SNa: 652.2570. Found: 652.2572.
2,5-Dioxopyrrolidin-1-yl pent-4-ynoate (26).26 To a solution
of 4-pentynoic acid (0.100 g, 1.02 mmol) in DCM (5 mL) were added
N-hydroxysuccinimide (0.134 g, 1.17 mmol) and EDC (0.391 g, 2.04
mmol). After 2 h, the reaction was transferred to a separating funnel
and washed with a 2.5% aq. solution of NaHSO4 (×3). The organic
phase was subsequently dried (Na2SO4), filtered under suction, and
evaporated in vacuo to yield 26 as a white solid (0.191 g, 0.980 mmol,
96%).
DIPEA (50 μL) in DMF (950 μL). The acetylation reaction was
performed for 30 min, and the beads were washed with DMF (5 × 500
μL). Finally, the beads were resuspended in DMF (1 mL) and kept at
5 °C until further use.
Dopamine Derivative 1 Functionalized Dynabeads M-270
Amine (28). To an Eppendorff tube was transferred 0.250 mL from a
stock solution of 27 in DMF (1 mL). The supernatant was removed,
whereafter a solution of 1 (18 mM), CuSO4 (200 μM), THPTA31 (1.4
mM), and NaAsc (2 mM) in a water/DMF (1:4) mixture (500 μL)
was added. The reaction was shaken for 1 h at room temperature, and
then the supernatant was removed. The remaining beads were washed
with water/DMF (1:1) (2 × 500 μL), water (3 × 500 μL), water/
DMF (1:1) (1 × 500 μL), and DMF (3 × 500 μL). Thereafter, 50%
piperidine in DMF (500 μL) was added, and the beads were shaken
for 90 min. The supernatant was removed, and the beads were again
suspended in 50% piperidine in DMF. The supernatant was then
removed and combined with the first deprotection mixture. The
concentration of 9-methylidenefluorene was measured by a
determined max UV abs. between 301 and 298 nm (7800 M−1
cm−1),32 furnishing an estimated amount of 30 nmol. The beads
were stored in a freezer suspended in a 1 mM ascorbic acid, 150 mM
NaCl, 80 mM HEPES buffer (pH = 7.4) in order to avoid oxidation of
the neurotransmitter analogues.
Serotonin Derivative 2 Functionalized Dynabeads M-270
Amine (29). To an Eppendorff tube was transferred 1 mL from a
stock solution of 27 in DMF (1 mL). The supernatant was removed,
whereafter a solution of 2 (35 mM), CuSO4 (200 μM), THPTA31 (1.4
mM), and NaAsc (2 mM) in a water/DMF (1:4) mixture (500 μL)
was added. The reaction was shaken for 1 h at room temperature, and
then the supernatant was removed. The remaining beads were washed
with water/DMF (1:1) (2 × 500 μL), water (3 × 500 μL), water/
DMF (1:1) (1 × 500 μL), and DMF (3 × 500 μL). Thereafter, 50%
piperidine in DMF (500 μL) was added, and the beads were shaken
for 90 min. The supernatant was removed, and the beads were again
suspended in 50% piperidine in DMF. Thereafter, the supernatant was
removed and combined with the first deprotection mixture. The
concentration of 9-methylidenefluorene was measured by a
determined max UV abs. between 301 and 298 nm (7800 M−1
cm−1),32 furnishing an estimated amount of 98 nmol. The beads
were stored in a freezer suspended in a 1 mM ascorbic acid, 150 mM
NaCl, 80 mM HEPES buffer (pH = 7.4) in order to avoid oxidation of
the neurotransmitter analogues.
mp(uncorr.) 79.9−80.5 °C. 1H NMR (400 MHz, CDCl3): δ 2.92−
2.80 (m, 6H), 2.62 (dt, J = 2.7, 7.1, 2H), 2.05 (t, J = 2.7, 1H). 13C
NMR (100 MHz, CDCl3): δ 169.1, 167.1, 81.0, 70.1, 30.2, 25.5, 14.0.
HRMS (ES) m/z [M + Na]+ calcd for C9H9NO4Na: 218.0429. Found:
218.0429.
2-(2-(2-Azidoethoxy)ethoxy)ethyl Methanesulfonate (30).
To a 0 °C solution of 11 (0.500 g, 2.85 mmol) in dry Et2O (10
mL) were added Et3N (1.2 mL, 8.56 mmol) and, thereafter, MsCl
(0.33 mL, 4.28 mmol). The resulting white slurry was quenched with
water after 24 h and extracted with Et2O (×3). The combined organic
phases were dried (MgSO4), filtered under suction, and purified via
flash column chromatography (pentane/EtOAc 1:1), yielding 30
(0.600 g, 2.37 mmol, 83%) as a clear oil.
1H NMR (400 MHz, CDCl3): δ 4.35−4.21 (m, 2H), 3.73−3.65 (m,
2H), 3.63−3.53 (m, 6H), 3.31 (t, J = 5.2, 2H), 2.99 (s, 3H). 13C NMR
(100 MHz, CDCl3): δ 70.4, 70.4, 69.8, 69.3, 68.9, 50.5, 37.4. HRMS
(ES) m/z [M + H]+ calcd for C7H16N3O5S: 254.0811. Found:
254.0811.
S-(2-(2-(2-Azidoethoxy)ethoxy)ethyl) Ethanethioate (31). To
a solution of 30 (0.348 g, 1.37 mmol) in abs. EtOH (225 mL) was
added potassium thioacetate (0.863 g, 7.56 mmol). The resulting
mixture was heated to reflux overnight. The orange mixture was then
cooled to rt, quenched with water, and extracted with DCM (×3). The
combined organic phases were dried (MgSO4), filtered under suction,
and purified via flash column chromatography (EtOAc/pentane 2:8),
yielding 31 (0.288 g, 1.23 mmol, 90%) as a yellow oil.
1H NMR (400 MHz, CDCl3): δ 3.69−3.42 (m, 9H), 3.33 (t, J = 5.1,
2H), 3.03 (t, J = 6.4, 2H), 2.25 (s, 3H). 13C NMR (100 MHz, CDCl3):
δ 195.4, 70.5, 70.3, 70.0, 69.7, 50.6, 30.5, 28.7. HRMS (ES) m/z [M +
H]+ calcd for C8H16N3O3S: 234.0912. Found: 234.0917.
2-(2-(2-Azidoethoxy)ethoxy)ethanethiol (23). Sodium (0.05 g,
2.17 mmol) was added to MeOH (5 mL). After the generation of
NaOMe was complete, the mixture was added to a solution of 31
(0.253 g, 1.09 mmol) in MeOH (15 mL). The resulting mixture was
heated to 50 °C and reacted for 2 days (monitored by TLC).
Thereafter, the reaction was acidified by the addition of 0.1 M HCl,
brine was added, and the mixture was extracted with DCM (×3). The
combined organic phases were dried (MgSO4), filtered under suction,
and purified via flash column chromatography (EtOAc/pentane 3:7),
yielding 23 (0.175 mg, 0.915 mmol, 84%) as an oil.
ASSOCIATED CONTENT
■
S
* Supporting Information
1H NMR and 13C NMR spectra for all new compounds. This
material is available free of charge via the Internet at http://
pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*E-mail: kvg@chem.au.dk.
Notes
The authors declare no competing financial interest.
1H NMR (400 MHz, CDCl3): δ 3.74 (t, J = 6.7, 2H), 3.71−3.58 (m,
6H), 3.39 (t, J = 5.0, 2H), 2.89 (t, J = 6.7, 2H), 2.17 (s, 1H). 13C NMR
(100 MHz, CDCl3): δ 70.8, 70.5, 70.2, 69.8, 50.8, 38.5. HRMS (ES)
m/z [M + Na]+ calcd for C6H14N3O2SNa: 214.0626. Found:
214.0627.
Alkyne Functionalized Dynabeads M-270 Amine (27). The
supernatant of an aqueous solution of Dynabeads (M-270 amine,
Invitrogen, 1.5 mL, 2 × 109 beads/mL) was removed by magnetically
immobilizing the beads. Thereafter, the beads were washed with DMF
and added to a solution of 26 (50 mM), DIPEA (12.5 μL), and water
(112.5 μL) in DMF (375 μL). The beads were left to react for 30 min.
The coupling step was performed a total of three times. Thereafter, the
beads were washed with DMF (3 × 500 μL) and subsequently
acetylated by suspending the beads in acetic anhydride (20 mM) and
ACKNOWLEDGMENTS
■
We thank the The Danish Council for Strategic Research
(NABIIT), The Danish Council for Independent Research
(OChem Graduate School), and the Danish National Research
Foundation (CDNA and CFIN) for financial support. Addi-
tionally, we thank Dr. Henrik Helligsø Jensen for fruitful
discussions.
REFERENCES
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