A concise preparation of the fluorescent amino acid L-(7-hydroxycoumarin-4- yl) ethylglycine and extension of its utility in solid phase peptide synthesis

Article abstract of DOI:10.1016/j.bmc.2012.10.055

Incorporation of the unnatural amino acid L-(7-hydroxycoumarin-4-yl) ethylglycine (7-HC) is a powerful and reliable approach for the preparation of fluorescently labeled proteins. The growing popularity of this valuable amino acid prompted us to pursue an

Full text of DOI:10.1016/j.bmc.2012.10.055

Bioorganic & Medicinal Chemistry 21 (2013) 553–559
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Bioorganic & Medicinal Chemistry
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A concise preparation of the fluorescent amino acid L-(7-hydroxycoumarin-4-yl)
ethylglycine and extension of its utility in solid phase peptide synthesis
⇑
Timo Koopmans, Matthijs van Haren, Linda Quarles van Ufford, Jeffrey M. Beekman, Nathaniel I. Martin
Department of Medicinal Chemistry & Chemical Biology, Utrecht Institute for Pharmaceutical Sciences, University of Utrecht, 3584 CG Utrecht, The Netherlands
a r t i c l e i n f o
a b s t r a c t
Article history:
Incorporation of the unnatural amino acid L-(7-hydroxycoumarin-4-yl)ethylglycine (7-HC) is a powerful
Received 3 September 2012
Revised 18 October 2012
Accepted 25 October 2012
Available online 21 November 2012
and reliable approach for the preparation of fluorescently labeled proteins. The growing popularity of this
valuable amino acid prompted us to pursue an improved protocol for its synthetic preparation. The opti-
mized procedure here described provides ready access to multi-gram quantities of 7-HC. Also reported is
an extension of the utility of 7-HC in the generation of a protected building block suitable for use in solid
phase peptide synthesis. The building block was successfully incorporated at various positions in a series
of model peptides, including analogues of the cell penetrating HIV-Tat peptide, further illustrating the
utility of this unique amino acid.
Keywords:
Fluorescent amino acid
L
-(7-hydroxycoumarin-4-yl)ethylglycine
Ó 2012 Elsevier Ltd. All rights reserved.
Solid phase peptide synthesis
HIV-Tat peptide
O
1. Introduction
OH
NH2
O
The expansion of the genetic code to allow for the site-specific
incorporation of unnatural amino acids (UAAs) is a hallmark of
modern protein chemistry. The ability to introduce functionalities
beyond those contained within the twenty regular amino acids
now makes it possible to probe and tune the properties of proteins
expressed in bacterial, fungal, or mammalian cells.1–3 To date, mul-
tiple UAAs bearing structurally diverse, chemically reactive, photo-
reactive, and fluorescent side chains have been successfully
incorporated into a variety of proteins.4–6 Fluorescently labeled
proteins are of particular value in molecular and chemical biology
investigations and are often produced as fusions with other inher-
ently fluorescent proteins. As an alternative, the use of a fluores-
cent UAA offers the advantage of providing greater flexibility
with respect to the site of incorporation as well as a significantly
smaller perturbation of the size and shape of the protein under
investigation. To this end, Schultz and co-workers were the first
to successfully incorporate the small coumarin-based fluorescent
HO
O
7-HC (1)
Figure 1. Structure of L-(7-hydroxycoumarin-4-yl) ethylglycine (7-HC) 1.
biosensor.20,21 Specifically, Wang and co-workers showed that by
incorporating 7-HC in close proximity to a known site of protein
phosphorylation, it is possible to directly detect phosphorylation
owing to the measurable change it induces in the 7-HC fluorescent
signal.20 This effect is ascribed to an increase in the local pH result-
ing from addition of a phosphate group. Increases in pH are known
to red-shift the maximum excitation peak of 7-HC along with an in-
crease in fluorescence emission intensity at 450 nm.21
Despite its clear value, the methods reported to date for the
preparation of 7-HC 1 in significant quantities (multi-gram) are
limited. The approach most often employed, derives from a proce-
dure originally developed by Garbay and co-workers for the
synthesis of coumarin-bearing fluorescent amino acids.7,22 While
direct and straightforward, the route as described makes use of a
final purification step employing preparative reverse phase high
performance liquid chromatography. Recently, an alternative syn-
thesis of 1, avoiding HPLC purification, has also been reported,
however this procedure yields 1 as a racemate.23 To address these
issues we chose to further investigate the synthesis of 1 with the
aim of optimizing its preparation to allow for an efficient prepara-
tion on the multi-gram scale. Furthermore, with access to larger
amino acid
L-(7-hydroxycoumarin-4-yl) ethylglycine (7-HC 1,
Fig. 1) into a model protein.7 While other fluorescent UAAs have also
been described8–11 and in some cases successfully incorporated into
proteins,12–16 7-HC remains a preferred choice for many applica-
tions due to its relatively large Stokes shift and the sensitivity of
its fluorescent intensity to both pH and solvent polarity.7,17–19 Of
particular note is the recent application of 7-HC in what can be
considered the smallest genetically encoded phosphorylation
⇑
Corresponding author.
E-mail address: n.i.martin@uu.nl (N.I. Martin).
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.10.055

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T. Koopmans et al. / Bioorg. Med. Chem. 21 (2013) 553–559
O
O
O
O
O
O
O
CDI
OH
O
B
HO
HO
EtO
OBn
NHCbz
OBn
NHCbz
N
O
9-BBN dimer
MeOH,
N
THF
NH2
O
H2N
O
RT, 2 hr
2
HO
O
1
HO
O
OEt
4
O
O
O
(76%)
+
Mg2
O
O
O
O
O
MOM-Cl
O
resorcinol
OH
K2CO3
MeOH/CHCl3
48 hr
OBn
O
H2N
O
B
MeSO3H
0 oC to RT
5 hr
(prepared fresh)
NHCbz
Acetone
0 oC to RT
4 hr
3
(90% over 2-steps)
MOMO
O
O
5
OH
(73%)
NH2
O
O
O
after crystallization)
FmocOSu
NaHCO3
OH
OH
HO
O
1
NH2
NHFmoc
H2O:Acetone
16 hr
Scheme 1. Synthetic route optimized for the preparation of
4-yl) ethylglycine 1.
L
-(7-hydroxycoumarin-
MOMO
O
6
O
MOMO
O
7
O
(61% over 2-steps
from compound 5)
quantities of 1, a protecting group strategy was also devised so as
to allow for its ready incorporation into synthetic peptides via
standard Fmoc solid phase peptide synthesis (SPPS) approaches.
Scheme 2. Preparation of protected SPPS building block 7.
of 1 and the cross reactivity of the amino acid functionalities. To
simultaneous address both of these issues we turned to a known
9-borabicyclononane (9-BBN)-based strategy previously developed
2. Results and discussion
for the protection of a
-amino acids.25 Treatment of 1 with a slight
The most direct route described in the literature for the prepa-
ration of 1 offers the advantage of starting from a commercially
available protected glutamic acid building block 2 (Scheme 1).7,22
The conversion of 2 into b-ketoester 3 has previously been
achieved in moderate-to-good yield by pre-activation of the side
chain carboxyl group of 2 with carbonyldiimidazole, followed by
condensation with ethyl magnesium malonate. In optimizing this
step we found that a large enhancement in the yield of 3 (up to
90%) could be attained by using freshly prepared magnesium
monoethylmalonate. The direct conversion of b-ketoester 3 to the
desired amino acid product is then accomplished by condensation
with resorcinol in neat methanesulfonic acid.7 Under these
strongly acidic conditions, the von Pechmann condensation leads
to formation of the coumarin moiety while the excess resorcinol
employed serves as a scavenger leading to concomitant removal
of both the benzyl ester and benzyl carbamate. Following optimi-
zation, purified yields of up to 64% were achieved for this transfor-
mation, providing 1 in higher yield than previously reported. The
most significant improvement in this particular step relates to
the purification protocol employed. Previous approaches to purify-
ing 1 have relied on the use of extensive RP-HPLC severely limiting
the practicality and ease of large-scale preparation. Thus, we were
pleased to find that a straightforward acid–base titration/crystalli-
zation approach could be applied to provide compound 1 as a dark
red crystalline material in high purity. In this manner multi-gram
quantities of 1 can be rapidly prepared in routine fashion.
With ready access to gram quantities of 1, we next turned our
attention to developing a protecting group strategy that would al-
low for incorporation of the amino acid into synthetic peptides.
While there are reports of using an Fmoc-protected analogue of
1, without protection of the phenol group, the yields of peptides
obtained via this approach were low.24 Furthermore, we found that
unprotected 1 decomposes under the basic conditions typically
utilized for Fmoc protection. We therefore chose to pursue a fully
protected variant of 1 wherein the 7-hydroxy group of the couma-
rin is masked with an acid labile group and the amine with the
standard Fmoc carbamate. Initial attempts at directly protecting
the phenolic group were unsuccessful due to the limited solubility
excess of 9-BBN dimer led to formation of complex 4 which exhib-
ited good solubility in organic solvent (Scheme 2). Compound 4
was next treated with chloromethyl methyl ether to provide
MOM-protected intermediate 5. Decomplexation of the 9-BBN
moiety was achieved using very mild conditions whereby com-
pound 5 was treated with a 1:6 mixture of methanol and chloro-
form to yield the free amino acid 6.26,27 Standard conditions
were then employed to convert compound 6 directly into the
Fmoc-protected building block 7 in good yield.
To demonstrate the utility of building block 7 in SPPS, a small
model peptide based upon the Leu-enkephalin pentapeptide
HO
O
O
H
N
Gly-Gly-Phe-Leu
CO2H
H2N
O
8
HO
O
O
H
N
Tyr-Gly
H2N
Phe-Leu
CO2H
N
H
O
9
HO
O
O
Tyr-Gly-Gly-Phe
10
OH
H2N
N
H
O
Figure 2. Leu-Enkephalin analogues 8–10 prepared with incorporation of 7-HC
building block 7.

T. Koopmans et al. / Bioorg. Med. Chem. 21 (2013) 553–559
555
Figure 3. Fluorescence excitation spectra, emission recorded at 450 nm (left) and fluorescence emission spectra, excitation at 363 nm (right) at pH 6.5, 7.5, and 8.5 for
peptides 8 (A), 9 (B), and 10 (C).
(YGGFL) was employed. Three different enkephalin analogues were
prepared whereby 7 was incorporated at either the C-terminus,
N-terminus or in the center of the peptide (Fig. 2). In all cases 7
was incorporated with ease using standard SPPS coupling and Fmoc
deprotection conditions to provide the desired peptide as the major
product after global deprotection and cleavage from the resin.

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T. Koopmans et al. / Bioorg. Med. Chem. 21 (2013) 553–559
HO
O
O
O
Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-Pro-Pro-Gln
H2N
OH
N
H
11
HO
O
O
H
N
Gly-Arg-Lys-Lys-Arg-Arg
H2N
Arg-Arg-Arg-Pro-Pro-Gln
CO2H
N
H
O
12
Figure 4. Analogues of the HIV-Tat48–60 peptide prepared with addition of 7-HC at the C-terminus (compound 11) or substitution for Gln54 (compound 12).
Figure 5. HeLa cell internalization of fluorescent HIV-Tat48–60 analogues visualized by confocal microscopy: (A) Analogue 11, (B) Analogue 12, and (C) Negative control (no
peptide added). For each panel: (i) differential interference contrast image, (ii) confocal fluorescence image (grayscaled for clarity) and (iii) overlay (native colors).
Peptides 8–10 were further characterized by fluorescence spec-
troscopy over a range of pH values to provide the excitation and
emission spectra illustrated in Figure 3. Of particular note in Fig-
ure 3 is the clear pH dependence of the fluorescence exhibited by
each peptide. While the fluorescence excitation spectrum of each
peptide is essentially identical at both pH 6.5 and pH 8.5, at pH
7.5 there are differences. In the case of peptide 10, the spectrum
obtained at pH 7.5 indicates a more red-shifted excitation maxi-
mum which may suggest an equilibrium laying further towards
the deprotonated coumarin species. By comparison, the spectral
shift observed for peptide 8 at pH 7.5 is significantly less while
the spectrum obtained for peptide 9 at pH 7.5 appears to fall in
between that obtained for 8 and 10 (for comparative purposes,
Supplementary Fig. S1 provides an overlay of the fluorescence exci-
tation spectra obtained for peptides 8–10 at pH 7.5). These obser-
vations can be rationalized based upon the proximity of the 7-HC
residue to the C-terminus and the impact this likely has on the
effective pKa of the 7-hydroxy coumarin moiety (the approximate
pKa of 7-hydroxy coumarin is reported to be 7.8).28
To further establish the utility of building block 7 in the prepa-
ration of more challenging and biologically relevant constructs,
analogues of the HIV-Tat peptide were next investigated. It is well
established that truncated peptides comprising the arginine rich
motif of the HIV-Tat protein are able to cross membranes and as

T. Koopmans et al. / Bioorg. Med. Chem. 21 (2013) 553–559
557
such can also be used to deliver various molecular cargos to the
interior of cells.29,30 In this regard, one of the more commonly em-
ployed HIV-Tat peptide derived sequences, GRKKRRQRRRPPQ, is a
13-mer spanning residues 48–60 of the full-length Tat protein.31,32
To generate two fluorescent analogues of HIV-Tat48–60, building
Merck type 60, 230–400 mesh silica gel. High resolution mass
spectrometry (HRMS) analysis was performed using an ESI-TOF
LC/MS instrument. 1H NMR spectra were recorded at 300 MHz
with chemical shifts reported in parts per million (ppm) downfield
relative to tetramethylsilane (TMS). 1H NMR data are reported in
the following order: multiplicity (s, singlet; d, doublet; t, triplet;
q, quartet; qn, quintet and m, multiplet), number of protons, cou-
pling constant (J) in Hertz (Hz) and assignment. When appropriate,
the multiplicity is preceded by br, indicating that the signal was
broad. 13C NMR spectra were recorded at 75 MHz with chemical
shifts reported relative to the residual carbon resonance of the sol-
vent used. 13C NMR spectra were recorded using the attached pro-
ton test (APT) sequence. All literature compounds had 1H NMR, and
mass spectra consistent with the assigned structures.
block
7 was incorporated, again using standard Fmoc-SPPS
approaches. In analogue 11 the fluorescent amino acid was
included at the C-terminus of the peptide while in analogue 12
the central glutamine residue was replaced with 7-HC (Fig. 4).
To assess both the cell permeability and fluorescent properties of
Tat analogues 11 and 12, each peptide was incubated with HeLa cells
after which internalization was visualized using confocal micros-
copy. As is seen in Figure 5, those cells treated with either 11 or 12
exhibit a clear and evenly distributed fluorescence indicative of
cellular uptake of the peptides. While internalization of analogue
11 was expected, given that it retains the native HIV-Tat48–60 se-
quence, the observation that analogue 12 is also able to effectively
enter cells is of note. In analogue 12 the glutamine residue at posi-
tion 54 is replaced by 7-HC to generate a fluorescent mutant of
HIV-Tat48–60. While it is has been reported that the substitution of
alanine for glutamine at position 54 is tolerated,33 the inclusion of
larger or unnatural amino acids such as 7-HC at this position, and
the impact this might have on cell permeability, has not been
described.
In general, the preparation of fluorescently labeled peptides of-
ten requires the initial synthesis of the peptide of interest by SPPS
after which the fluorophore is introduced, typically as a conjugate
containing either an amino- or thiol-reactive moiety. Aside from
requiring an additional reaction step, this approach can be prob-
lematic in peptides bearing more than one amino or thiol group,
presenting the possibility of generating multiply labeled species
or mixtures bearing different labeling patterns. By comparison,
the use building block 7 allows for the direct and site-specific
introduction of the fluorescent moiety as an integrated part of
the SPPS process. These advantages are clearly illustrated in the
preparation of the fluorescent Leu-enkephalin analogues 8–10
and HIV-Tat analogues 11 and 12 which were synthesized in direct
fashion using conventional SPPS.
4.2. Preparative details and analytical data for compounds
synthesized
4.2.1. (S)-1-Benzyl-7-ethyl-2-(benzyloxycarbonylamino)-5-
oxoheptanedioate (3)
N-Cbz-L-Glu-O-Bn (10.0 g, 27 mmol, 1.0 equiv) was dissolved in
dry THF (100 mL) and treated with 1,10-carbonyldiimidazole
(5.69 g, 35 mmol, 1.3 equiv) and the mixture was stirred at room
temperature for 2 h. Separately, potassium monoethylmalonate
(6.8 g, 40 mmol) was dissolved in water (25 mL) and treated with
concentrated hydrochloric acid (6 mL) and NaCl (11 g). The mix-
ture was extracted with EtOAc (5 ꢀ 25 mL) and the combined
EtOAc layers dried over Na2SO4, filtered and evaporated. The free
acid of monoethylmalonate thus obtained was then directly dis-
solved in dry THF (80 mL), treated with magnesium ethoxide
(2.9 g, 25 mmol), and stirred at room temperature for 1 h to
generate magnesium monoethylmalonate. The fresh solution of
magnesium monoethylmalonate in THF was then directly added
to CDI-activated glutamatic acid species and the mixture stirred
at room temperature for 16 h. The volume of the reaction mixture
was next reduced to ca. 50 mL after which Et2O (300 mL) and 1 M
HCl (100 mL) were slowly added. The layers were separated and
the Et2O layer washed with 1 M HCl (1 ꢀ 100 mL), 5% NaHCO3
(3 ꢀ 100 mL), dried over Na2SO4, and evaporated to yield a yellow
oil. This material was applied to a silica column and eluted using a
gradient of 1:3 ? 2:3 EtOAc/hexane to yield compound 3 as a light
yellow oil that solidified upon storage (10.76 g, 24.4 mmol, 90%).
Analytical data: Rf 0.15 (1:3 EtOAc/hexane); mp: 55–57 °C; 1H
NMR (300 MHz, CDCl3) d 7.34–7.33 (m, 10H), 5.48 (br d, 1H,
J = 7.9 Hz), 5.15 (s, 2H), 5.09 (s, 2H), 4.43–4.36 (m, 1H), 4.15 (q,
2H, J = 7.1 Hz), 3.35 (s, 1H), 2.63–2.50 (m, 1H), 2.22–2.15 (m, 1H),
2.00–1.88 (m, 1H), 1.24 (t, 3H, J = 7.1 Hz); 13C NMR (75 MHz,
CDCl3) d 201.5, 171.7, 167.0, 156.0, 136.2, 135.2, 128.7, 128.5,
128.4, 128.2, 128.1, 67.4, 67.1, 61.4, 53.2, 49.2, 38.6, 26.2, 14.1.
HRMS (ESI) Calcd for C24H27NO7Na [M+Na]+, 464.1685. Found
464.1673.
3. Conclusions
In summary, an optimized protocol for the concise and efficient
preparation of the fluorescent amino acid L-(7-hydroxycoumarin-
4-yl) ethylglycine 1 on the multi-gram scale has been developed.
This amino acid itself is of great value, having gained popularity
as one of the smallest fluorescent labels that can be selectively
incorporated into proteins via ‘expanded genetic code’ technolo-
gies. Furthermore, amino acid 1 was successfully converted into
building block 7, suitably protected for use in SPPS. Using standard
Fmoc SPPS approaches, building block 7 was incorporated into a
series of model peptides based upon the Leu-enkephalin (5-mer)
and the HIV-Tat (13-mer) peptides. When used as an Fmoc SPPS
building block, compound 7 requires no special handling and in
all cases examined was readily incorporated into the synthetic
peptides prepared. Access to such building blocks expands the
repertoire of available tools for use in future explorations requiring
access to proteins and peptides bearing small fluorescent labels.
4.2.2. L-(7-Hydroxycoumarin-4-yl) ethylglycine (1)
Resorcinol (6.98 g, 63.4 mmol, 5.0 equiv) was dissolved in
methanesulfonic acid (50 mL) with vigorous stirring. Once homog-
enous, the clear solution was cooled on ice and compound 3 (5.6 g,
12.7 mmol, 1.0 equiv) added. After 20 min, the ice batch was re-
moved and the mixture stirred vigorously under N2 for 4 h at room
temperature. The mixture was then poured into a 500 mL volume
of Et2O and placed on ice for 30 min. The Et2O was decanted and
the remaining precipitate dissolved in 2 M HCl (200 mL). The HCl
layer was washed with EtOAc (2 ꢀ 100 mL) after which the pH of
the aqueous layer was adjusted to 3.5 by slow addition of 6 M
NH4OH. Upon overnight storage at 4 °C the desired product crystal-
lized from solution. The crystals were collected by filtration,
washed with Et2O, and dried under vacuum to yield compound 1
4. Experimental
4.1. General
All reagents employed were of American Chemical Society (ACS)
grade or finer and were used without further purification unless
otherwise stated. Flash chromatography was performed using

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T. Koopmans et al. / Bioorg. Med. Chem. 21 (2013) 553–559
as a dark red solid (2.13 g, 8.1 mmol, 64%). Analytical data: Mp:
251 °C (decomp.); 1H NMR (300 MHz, 0.1 M KOD/D2O) d 7.37 (d,
1H, J = 9.0 Hz), 6.52–6.48 (m, 1H), 6.50 (ddd, 1H, J1 = 0.6 Hz,
J2 = 2.4 Hz, J3 = 8.9 Hz), 6.31 (dd, 1H, J1 = 0.7 Hz, J2 = 2.4 Hz), 5.79
(s, 1H), 3.22 (t, 1H, J = 6.2 Hz), 2.62 (t, 2H, J = 8.3 Hz), 1.87–1.69
(m, 2H); 13C NMR (75 MHz, 0.1 M KOD/D2O) d 177.3, 171.6,
165.9, 159.0, 156.0, 125.5, 118.0, 107.9, 104.5, 104.3, 54.9, 31.1,
27.3. HRMS (ESI) Calcd for C13H14NO5 = [M+H]+, 264.0872. Found
264.0880.
(decomp.) 1H NMR (300 MHz, CD3OD) d 7.75–7.71 (m, 2H), 7.67–
7.58 (m, 2H), 7.37–7.23 (m, 4H), 6.97–6.93 (m, 2H), 6.14 (s, 1H),
5.24–5.21 (m, 2H), 4.38–4.35 (m, 2H), 4.30–4.25 (m, 1H), 4.19–
4.14 (m, 1H), 3.43 (s, 3H), 2.83–2.78 (m, 2H), 2.58–2.15 (m, 1H),
2.08–1.98 (m, 1H); 13C NMR (75 MHz, CD3OD) d 173.7, 161.9,
160.4, 157.2, 156.4, 155.0, 143.9, 143.7, 141.2, 127.4, 126.8,
125.6, 124.8, 119.5, 113.4, 113.1, 110.7, 103.2, 94.1, 66.4, 55.2,
53.4, 30.2, 27.7. HRMS (ESI) Calcd for
530.1815. Found 530.1802.
C
30H28NO8 [M+H]+,
4.2.3. (1R,40S,5S)-40-(2-(7-(methoxymethoxy)-2-oxo-2H-
chromen-4-yl)ethyl)-50-oxospiro[bicyclo[3.3.1]-nonane-9,20-
[1,3,2]oxazaborolidin]-30-ium-11-uide (5)
4.3. Peptide synthesis
Enkephalin analogues 8, 9 and 10 were prepared using an auto-
9-BBN-H dimer (2.39 g, 9.78 mmol, 1.1 equiv) was dissolved in
methanol (100 mL) while stirring vigorously under N2 at reflux
mated peptide synthesizer. Peptides were assembled on 2-chloro-
trityl resin, on
a 0.25 mmol scale. Peptide couplings were
temperature for 30 min. Amino acid
1
(2.25 g, 8.55 mmol,
performed using 4.0 equiv of Fmoc-protected amino acid, 4.0 equiv
of HBTU, 4.0 equiv of HOBt and 8.0 equiv of DIPEA in an approxi-
mate total volume of 10 mL NMP at room temperature for 1 h. In
the case of the 7-hydroxycoumarin building block (7), 2.0 equiv
of the 7-hydroxycoumarin building block (7) were used, keeping
all other reagents and reaction times equal. Upon completion of
SPPS, peptides were cleaved from the resin and the side chains
deprotected using TFA/TIS/H2O (95:2.5:2.5), followed by precipita-
tion in MTBE/hexanes (1:1) and washing twice with MTBE, to yield
the crude peptides. Each peptide was purified to homogeneity
using RP-HPLC, employing a C18 column (250 ꢀ 22 mm, 300 Å,
1.0 equiv) was added to the yellow solution and the mixture stirred
under reflux conditions for 2 h during which time the mixture was
homogenous. The mixture was then concentrated under vacuum
and re-dissolved in CHCl3. Silica gel was added directly to the
CHCl3 solution after which the solvent was evaporated leaving a
mixture of the crude intermediate 4 adsorbed on silica. This silica
mixture was applied to the top of a silica column and rapidly
eluted using a gradient of 4:1 EtOAc/hexane ? 4:1 acetone/hexane
to yield compound 4 in a semi-pure form (2.5 g, 6.5 mmol, 76%).
This material was then directly dissolved in acetone (130 mL)
and treated with K2CO3 (1.8 g, 13 mmol, 2.0 equiv) while stirring
under N2. This suspension was vigorously stirred for 15 min, cooled
10 lm) with a gradient of 5–95% acetonitrile (0.1% TFA) in
90 min at a flow rate of 12.0 mL minꢁ1. Peptide purity was con-
firmed by analytical HPLC (see Supplementary data sections for
HPLC traces) Analytical data peptide 8: 1H NMR (300 MHz, CD3OD)
d 7.66 (d, 1H, J = 8.8 Hz), 7.23–7.13 (m, 5H) 6.81 (d, 1H, J = 8.8 Hz),
6.71 (s, 1H), 6.14 (s, 1H), 4.72–4.67 (m, 1H), 4.42–4.37 (m, 1H),
4.15–4.11 (m, 1H), 4.05–3.72 (m, 4H), 3.18–3.12 (m, 2H), 2.99–
2.88 (m, 4H), 2.27–2.23 (m, 1H), 1.67–1.59 (m, 2H), 0.90 (dd, 6H,
on ice and then chloromethyl methyl ether (743 lL, 9.8 mmol,
1.5 equiv) was added. The ice bath was removed and the mixture
was stirred at room temperature for 4 h. The volume was then re-
duced to ca. 25 mL and EtOAc (250 mL) added. The EtOAc layer was
washed with brine (1 ꢀ 250 mL), dried over Na2SO4, filtered, and
concentrated. The residue was applied to a silica column and elut-
ing with a gradient of 1:1 ? 3:2 EtOAc/hexane to yield compound
5 in pure form as an off-white solid (2.0 g, 4.7 mmol, 73%). Analyt-
ical data: Mp: 129 °C (decomp.) 1H NMR (300 MHz, CD3OD) d 7.75
(d, 1H, J = 8.9 Hz), 7.00 (dd, 1H, J1 = 2.6 Hz, J2 = 8.9 Hz), 6.94 (d, 1H,
J = 2.4 Hz), 6.54–6.48 (m, 1H), 6.19 (s, 1H), 5.99–5.93 (m, 1H), 5.25
(s, 2H), 3.88–3.83 (m, 1H), 3.46 (s, 3H), 3.12–2.97 (m, 2H), 2.35–
2.26 (m, 1H), 2.15–2.06 (m, 1H), 1.94–1.46 (m, 12H), 0.61 (s, 2H);
13C NMR (75 MHz, CD3OD) d 176.7, 163.3, 161.8, 157.6, 156.3,
127.0, 114.9, 114.5, 111.8, 104.5, 95.5, 56.6, 55.6, 32.6, 32.5, 32.4,
32.2, 30.6, 29.2, 25.6, 25.2. HRMS (ESI) Calcd for C23H31BNO6
[M+H]+, 428.2244. Found 428.2234.
J1 = 5.8 Hz, J2 = 12.9 Hz); 13C NMR (75 MHz, CD3OD)
d 178.2,
176.2, 173.9, 173.7, 173.2, 166.2, 165.6, 159.8, 159.4, 140.8,
132.9, 131.9, 130.2, 129.7, 117.1, 115.1, 113.1, 106.3, 58.2, 56.7,
54.7, 46.3, 45.8, 44.0, 41.5, 33.7, 30.4, 25.9, 24.4; HRMS (ESI) Calcd
for C32H40N5O9 [M+H]+, 638.2826. Found 638.2825. Analytical data
peptide 9: 1H NMR (300 MHz, CD3OD) d 7.58 (d, 1H, J = 8.8 Hz),
7.30–7.20 (m, 4H), 7.15–7.10 (m, 3H), 6.83–6.76 (m, 3H), 6.72–
6.71 (m, 1H), 6.06 (s, 1H), 4.75–4.70 (m, 1H), 4.40 (q, 2H,
J = 6.3 Hz), 4.09–4.04 (m, 1H), 4.00–3.95 (m, 1H), 3.81–3.76 (m,
1H), 3.21–3.14 (m, 2H), 3.01–2.91 (m, 2H), 2.71–2.66 (m, 2H),
2.03–1.86 (m, 2H), 1.71–1.59 (m, 3H), 0.87 (dd, 6H, J1 = 6.1 Hz,
J2 = 17.2 Hz); 13C NMR (75 MHz, CD3OD) d 178.2, 176.1, 175.5,
173.7, 173.3, 166.4, 165.5, 160.9, 159.4, 140.9, 134.1, 132.9,
132.0, 130.3, 130.0, 128.5, 119.4, 117.0, 115.3, 113.2, 106.2, 58.6,
58.2, 56.8, 54.7, 46.2, 44.1, 41.2, 40.3, 34.5, 31.3, 25.9, 24.4; HRMS
(ESI) Calcd for C39H46N5O10 [M+H]+, 744.3245, Found 744.3231.
Analytical data peptide 10: 1H NMR (300 MHz, CD3OD) d 7.61 (d,
1H, J = 8.6 Hz), 7.28–7.24 (m, 3H), 7.20–7.18 (m, 2H), 7.13–7.10
(m, 2H), 6.84–6.77 (m, 3H), 6.71 (s, 1H), 6.07 (s, 1H), 4.73–4.68
(m, 1H), 4.46–4.42 (m, 1H), 4.19–4.14 (m, 1H), 4.00–3.94 (m,
2H), 3.82–3.74 (m, 2H), 3.21–3.14 (m, 2H), 3.05–2.95 (m, 2H),
2.87–2.77 (m, 2H), 2.26–2.23 (m, 1H), 2.09–2.04 (m, 1H); 13C
(75 MHz, CD3OD) d 173.0, 172.4, 170.2, 169.9, 169.8, 162.6,
161.5, 156.9, 156.6, 155.4, 136.8, 130.2, 129.0, 128.1, 126.4,
125.9, 124.6, 115.5, 113.2, 109.8, 102.4, 54.8, 54.7, 51.5, 42.7,
41.9, 37.2, 36.3, 30.0, 27.7; HRMS (ESI) Calcd for C35H38N5O10
[M+H]+, 688.2619. Found 688.2622.
4.2.4. (S)-2-(((9H-Fluoren-9-yl)methoxy)carbonylamino)-4-(7-
(methoxymethoxy)-2-oxo-2H-chromen-4-yl)butanoic acid (7)
Compound 5 (2.8 g, 6.6 mmol) was dissolved in a mixture of
methanol (8 mL) and chloroform (40 mL) and stirred at 40 °C (with
a condenser attached) for 48 h. The mixture was concentrated and
the residue triturated first with hot hexanes (2 ꢀ 50 mL) and then
with diethyl ether (2 ꢀ 50 mL). The residue was then dried under
vacuum to yield intermediate 6 as an off-white solid (2.03 g,
6.6 mmol) that was used directly in the following step. To a mix-
ture of compound 6 (2.03 g, 6.6 mmol, 1.0 equiv) and NaHCO3
(0.61 g, 7.3 mmol, 1.1 equiv) in water (42 mL), was added a solu-
tion of FmocOSu (2.67 g, 7.9 mmol, 1.2 equiv) in acetone (34 mL).
The reaction mixture was stirred overnight at room temperature
after which it was concentrated under vacuum to remove the ace-
tone. A solution of 0.2 M HCl (100 mL) was then added and the
aqueous layer was extracted with EtOAc (3 ꢀ 100 mL). The organic
layers were combined, dried over Na2SO4, filtered, and concen-
trated. The residue was applied to a silica column and eluted using
a gradient of 1:1 ? 4:1 EtOAc/hexane to yield compound 7 as a
white foam (2.14 g, 4.0 mmol, 61%). Analytical data: Mp: 80 °C
HIV-Tat48–60 analogues 11 and 12 were prepared via standard
Fmoc SPPS on 2-chlorotrityl resin, on a 0.25 mmol scale. Peptide
couplings were performed using 4.0 equiv of Fmoc-protected ami-
no acid, 4.0 equiv of HBTU, 4.0 equiv of HOBt and 8.0 equiv of DI-
PEA in an approximate total volume of 10 mL NMP at room

T. Koopmans et al. / Bioorg. Med. Chem. 21 (2013) 553–559
559
temperature for 1 h. In the case of the 7-hydroxycoumarin building
block (7), 2.0 equiv of the 7-hydroxycoumarin building block (7)
were used, keeping all other reagents and reaction times equal.
Upon completion of SPPS, peptides were cleaved from the resin
and the side chains deprotected using TFA/TIS/H2O (95:2.5:2.5)
for 4 h, followed by precipitation in MTBE/hexanes (1:1) and wash-
ing twice with MTBE, to yield the crude peptides. Each peptide was
purified to homogeneity using RP-HPLC, employing a semi-pre-
Institute for Pharmaceutical Sciences and The University of Utrecht
are also gratefully acknowledged for their support.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2012.10.055.
parative C18 column (250 ꢀ 10 mm, 300 Å, 10
lm) with a gradient
References and notes
of 5–33% acetonitrile (0.1% TFA) in 90 min at a flow rate of
6.0 mL minꢁ1. Peptide purity was confirmed by analytical HPLC
(see Supplementary data sections for HPLC traces) and identity
confirmed by MALDI-MS: Peptide 11 LRMS (MALDI) Calcd for
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60 lM solutions of peptides 8, 9, and 10 were prepared in
Britton–Robinson buffer at pH 6.5, 7.5 and 8.5. Excitation and
emission spectra were recorded on a Horiba Fluorolog-3 spectro-
fluorometer. Emission spectra were excited at 363 nm and the
emission recorded from 400 to 550 nm. Excitation spectra were re-
corded from 280 to 400 nm with the emission set at 450 nm. The
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Acknowledgements
This work was supported by The Netherlands Organization for
Scientific Research (NWO-VIDI fellowship to N.I.M.). The Utrecht