A general solid phase method for the synthesis of sequence independent peptidyl-fluoromethyl ketones

Article abstract of DOI:10.1039/c2ob25096a

We present here a new, general, solid phase strategy for the synthesis of sequence independent peptidyl-fluoromethyl ketones using standard Fmoc peptide chemistry. Our method is based on the synthesis of bifunctional linkers which allows the incorporation of amino acid fluoromethyl ketone unit at the C-terminal end of peptide sequences. Application of this approach for the synthesis of activity based probes for SENPs is also described.

Full text of DOI:10.1039/c2ob25096a

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Organic &
Biomolecular
Chemistry
www.rsc.org/obc
Volume 10 | Number 23 | 21 June 2012 | Pages 4473–4628
ISSN 1477-0520
PAPER
Daniel P. Funeriu et al.
A general solid phase method for the synthesis of sequence independent
peptidyl-fluoromethyl ketones
1477-0520(2012)10:23;1-E

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PAPER
A general solid phase method for the synthesis of sequence independent
peptidyl-fluoromethyl ketones†
Gheorghe-Doru Roiban,a Mihaela Matache,b Niculina D. Hădade*a,c and Daniel P. Funeriu*a
Received 13th January 2012, Accepted 3rd April 2012
DOI: 10.1039/c2ob25096a
We present here a new, general, solid phase strategy for the synthesis of sequence independent peptidyl-
fluoromethyl ketones using standard Fmoc peptide chemistry. Our method is based on the synthesis of
bifunctional linkers which allows the incorporation of amino acid fluoromethyl ketone unit at the
C-terminal end of peptide sequences. Application of this approach for the synthesis of activity based
probes for SENPs is also described.
and racemisation of the C-terminal amino acid that may occur
during the coupling reaction. Lastly, the structure of sensitive
Introduction
Fluoromethyl ketones (FMKs) and, more often, peptidyl-fluoro-
methyl ketones (PFMKs) have emerged as important targets for
bioorganic and medicinal chemistry. Many of these compounds
have been designed as activity based probes for cysteine pro-
teases1 or potential drugs for treatment of certain diseases.2 In
addition, the FMK electrophilic group has been used to prepare
selective activity based probes for cathepsin B3 and G,4 cas-
pases,5 calpain I,6 SENPs7 or N-glycanase.8 Despite the number
of the PFMKs reported is continuously growing, a simple,
general and efficient/atom economy method to prepare FMK-
based peptides is still lacking. Their synthesis is usually per-
formed by solution coupling of the carboxy-terminated peptide
sequences with the corresponding fluoroalcohol4,6,9 followed by
the alcohol oxidation with K2Cr2O7,10 using a modified
Pfitzner–Moffat procedure11 or Dess–Martin reagent4,6,9a,12 to
provide the expected peptidyl-fluoromethyl ketones. However,
the solution coupling reaction is limited13 to peptide sequences
that do not contain cross-reacting side-chain groups (e.g. amino,
carboxy, etc.) or require orthogonal protection of the side chains
of amino acids (thus increasing costs and reducing yield of the
final compound); moreover, a major drawback is the low solubi-
lity of the fully protected peptide, as well as the low reaction rate
residues, in particular Cys, Met, Trp and Tyr, may be easily
altered during the final oxidation process, yielding a great series
of byproducts. Consequently, such routes cannot be generally
applicable and it is necessary to identify a general, sequence
independent, solid phase method for the synthesis of PFMKs.
Such a general and smooth method would also open the access
to peptide library synthesis, a field relatively less explored in the
chemistry of FMKs. To our knowledge synthetic supported solid
phase strategies are known for acyloxymethyl,14 aminomethyl
and thiomethyl ketones.15 However, this method yields very
often low-purity peptides (especially when long sequences are
prepared). To address the above mentioned issues, we present
here a new strategy to prepare PFMKs using a general, sequence
independent, Fmoc-based solid phase synthetic method.
Results and discussion
Beside the difficulties encountered in the obtaining of PFMKs,
the synthesis of FMKs themselves is challenging. Unlike the
trifluoromethyl ketones for which mild and efficient preparation
methods have been developed,16 the synthesis of mono α-fluori-
nated methyl ketone derivatives is less accessible.6a,16a,17 The
direct conversion of the acids into the corresponding FMKs is
usually reported, using magnesium fluoromalonate,18 although
the preparation of fluoromalonates is tedious and difficult. Reac-
tion of allene epoxides with tetrabutylammonium fluoride trihy-
drate in THF19 or dediazonative hydrofluorination of
diazoketones20 constitutes alternative strategies which proved
efficient for small scale synthesis, but rather inconvenient for
large scale preparations. Considering the importance of the
aspartic acid fluoromethyl ketone (D-FMK) as well as the
glycine fluoromethyl ketone (G-FMK) units as reactive groups in
the structure of various activity based probes for caspases1,5 and
sentrin proteases (SENPs)7 respectively, we turned our sights to
aDepartment of Chemistry, Marie Curie Excellence Team, Technical
University München, 4 Lichtenberg str. 85748, Garching, Germany.
E-mail: daniel.funeriu@ch.tum.de; Fax: (+) 49 89 28 914 512;
Tel: (+) 49 89 28 913 133
bDepartment of Chemistry, University of Bucharest, 90-92 Panduri str.,
050663 Bucharest, Romania
cFaculty of Chemistry and Chemical Engineering, “Babes-Bolyai”
University, 11 Arany Janos str., 400028 Cluj-Napoca, Romania.
E-mail: nbogdan@chem.ubbcluj.ro; Fax: (+) 40 264 590 818
†Electronic supplementary information (ESI) available: Experimental
details for synthesis of diols 9 and 10 as well as HPLC chromatograms
and MS (ES+) spectra of peptide products are given in Supporting Infor-
mation file. See DOI: 10.1039/c2ob25096a
4516 | Org. Biomol. Chem., 2012, 10, 4516–4523
This journal is © The Royal Society of Chemistry 2012

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encountered for solid phase peptide synthesis (SPPS), namely
TFA/H2O, 95 : 5.
With FMKs 3 and 8 in our hands, we proceeded to their incor-
poration in peptide sequences. In the particular case of the
D-FMK, the attachment to the resin can be easily performed by
means of the side chain carboxylic group of compound 4, and
peptide elongation can be carried out by standard Fmoc-SPPS
protocols. However, incorporation of FMKs derived from other
amino acids, such as G-FMK 7, at C-terminal of various peptide
sequences require a different approach as a result of their reverse
backbone polarity. Therefore, we focused on finding a bifunc-
tional linker that contain: (i) a group that is able to react with the
keto functionality of the FMK, is stable under the standard SPPS
conditions and can be easily removed under the acidic conditions
used for the peptide cleavage from the solid support; and (ii) a
group which ensures the attachment of the linker to the solid
support.
To do so, we initially considered a linker that contained a 1,3-
diol unit useful for the ketone protection (which can be easily
deprotected in acidic medium) and a carboxylic functionality
required to anchor the FMK onto the resin. In the design of our
linker, we took in consideration that the introduction of a car-
boxyl group in the structure of the 1,3-dioxane-protected FMK
could influence the acid catalyzed deprotection reaction since it
is known that the presence of a second withdrawing group
(COOH in our case) on the dioxane ring drastically reduce the
hydrolysis rate.24
Therefore, we based our synthesis on compound 9 (Scheme 2)
which has a long aliphatic chain to counterbalance the inducing
effect (−I) of the carboxyl. Moreover, the two methyl groups in
positions 4 and 6 of 1,3-dioxane ring in compound 11 favour the
hydrolysis reaction. Compound 9 was obtained in a three step
process. First, acetylacetone was treated with ethyl-6-bromohexa-
nanoate in presence of 1,8-diazabicyclo[5.4.0]undec-7-ene
(DBU) as base to give ethyl 7-acetyl-8-oxononanoate (com-
pound S1 in ESI†) which was further reduced with sodium boro-
hydride and the ester group was hydrolysed to yield 9 as a
mixture of diastereoisomers (see ESI† for details about the
Scheme 1 Synthesis of the FMKs used in this work. Reagents and
conditions: A: (a) i: iso-butylchloroformate, N-methylmorpholine, THF,
−10 °C, 30 min; ii: CH2N2/Et2O (in situ), 0 °C to r.t., 3 h, 84%; (b)
HBr/HOAc (33 wt%)/water, THF, 0 °C, 15 min, quantitative; (c) TBAF,
PTSA, THF, 4 h, reflux, 49%; (d) methanol, PTSA, toluene, 60 °C, 5 h,
77%. B: (a) i: iso-butylchloroformate; N-methylmorpholine, THF,
−10 °C, 30 min; ii: CH2N2/Et2O (in situ), 0 °C to r.t., 3h, 96%; (b) HBr/
HOAc (33 wt%)/water, THF, 0 °C, 10 min, quantitative; (c) H2SO4 0.25
M, 1,4-dioxane, 90 °C, 2 h, 69%; (d) TBAF, PTSA, THF, 4 h, reflux; (e)
perfluoro-1-butanesulfonyl fluoride (PBSF), Et3N × 3HF, Et3N, aceto-
nitrile, −30 °C to r.t., 3 h, 61%.
the synthesis of these compounds and their incorporation into
peptide sequences as C-terminal functionalities.
The synthetic routes to prepare the Fmoc protected D-FMK 3
and G-FMK 8 are described in Scheme 1. Briefly, Fmoc-Asp
(OtBu)-OH was transformed into the diazoketone derivative 1 in
a two step process involving the activation of the carboxylic acid
followed by reaction with diazomethane. Compound 1 was
treated with HBr/HOAc to give the bromomethyl ketone 2 which
was then reacted with tetra-n-butylammonium fluoride (TBAF)
in presence of p-toluenesulfonic acid (PTSA)21 to yield the
fluoromethyl ketone 3. In order to incorporate the D-FMK unit
in peptide sequences, the keto group in 3 was protected as
dimethylketal. Notably, the acetalization step affected the t-butyl
ester, compound
4 being prepared with a 77% yield
(Scheme 1A). Similarly, G-FMK 8 was initially attempted start-
ing from Fmoc-Gly-OH through diazoketone 5 and bromo-
methyl ketone 6 (Scheme 1B). However, the separation of
G-FMK 8 obtained by this method proved to be difficult. There-
fore, we chose to transform the diazoketone 5 into the hydroxy-
methyl ketone 7 by treatment with aqueous H2SO4 0.25 M,22
followed by fluorination of 7 with perfluoro-1-butanesulfonyl
fluoride (PBSA) and Et3N × 3HF complex23 to give the target
G-FMK 8 in good yield (61%).
Scheme 2 Synthesis of the protected G-FMKs. Reagents and con-
ditions: (a) i: trimethylsilyl chloride (TMSCl), Et3N, dry Et2O, (S3 in
ESI†); ii: 8, trimethylsilyl trifluoromethanesulfonate (TMSOTf),
−27 °C, dry DCM, 44%; (b) i: TMSCl, Et3N, dry Et2O, 91% (S4 in
ESI†); ii: 8, TMSOTf, dry DCM, −27 °C, 81%; (c) N3CH2COOH, Tris-
[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine (TBTA), CuSO4·5H2O,
ascorbic acid, H2O/t-BuOH (1 : 1), 10 h, 93%.
Ketal deprotection studies conducted on 4 and monitored by
TLC showed that complete hydrolysis occurs in less than 30 min
at room temperature using the acidic cleavage conditions usually
This journal is © The Royal Society of Chemistry 2012
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synthesis of 9). Compound 9 was then transformed into its
O-silylated derivative (S3 in ESI†) which was further reacted
with 8, using trimethylsilyl trifluoromethanesulfonate
(TMSOTf) as catalyst, to afford the protected fluoromethyl
ketone 11, in 44% yield after workup, as a complex mixture of
diastereoisomers as inferred from 1H and 19F NMR spectra.
Next, we studied the deprotection of 11 to the parent G-FMK
8 under acidic cleavage conditions, as well as its stability in the
SPPS conditions. Initial deprotection tests using a mixture of
TFA/H2O 95 : 5 showed partial cleavage of the dioxane with the
formation of the FMK 8. Fortunately, complete deprotection
could be achieved with HCl 37%.
Since all diastereoisomers are hydrolysed to compound 8, it
was of no great advantage to separate them. Very importantly, no
fluorine displacement was observed during the cleavage con-
ditions or in the presence of nucleophilic piperidine (used for
Fmoc deprotection during peptide synthesis) when compound 11
was treated with DMF/piperidine 4 : 1 for 12 hours at room
temperature.
The linker utility was demonstrated by the synthesis of the
peptidyl-fluoromethyl ketone 16 (Biotin-Teg-FQQQTG-8, Teg =
tetraethylenglycol based spacer25) which has been recently
shown to act as an activity based probe for SENPs.7 In order to
obtain peptide 16, starting from Rink resin, dioxane 11 and the
required amino acids were introduced by standard solid phase
peptide chemistry using O-benzotriazole-N,N,N′,N′-tetramethyl-
uronium hexafluorophosphate (HBTU)/N-hydroxybenzotriazole
(HOBt) as coupling agents. After biotin insertion, the peptide
was cleaved from the resin concomitant with the removal of the
side chain protecting groups using a mixture of TFA/TIS(triiso-
propylsilane)/H2O 95 : 2.5 : 2.5 for 3 hours at room temperature
(Scheme 3A). After precipitation, isolation and purification by
preparative reversed phase HPLC, a mixture of diastereomers of
the 1,3-dioxane protected peptide 15 as well as the expected pep-
tidyl-fluoromethyl ketone 16 were obtained (Scheme 3A). All
diastereoisomers 15 that resisted the action of the cleavage
mixture were completely hydrolysed to 16 using concentrated
HCl (37%) (see ESI†).
Despite the successful synthesis of 16, the low yield in prep-
aration of 11 and the large number of the resulted diastereo-
isomers constitute important drawbacks of this method. To
overcome these limitations, we envisioned another strategy that
makes use of the copper catalysed click chemistry coupling reac-
tion. Thus, we prepared the acetal 12 (Scheme 2) which can be
either directly attached to an azide modified resin (Scheme 3B)
or further functionalized with a carboxyl group containing sub-
strate to yield 13 (Scheme 2) which can be directly attached to a
commercial resin (Scheme 3C). Compared to the synthesis of 11,
preparation of 1,3-dioxane 12 involves fewer steps and occurs
with higher yields. Initial deprotection tests performed in TFA/
TIS/H2O (95 : 2.5 : 2.5) unexpectedly leaved dioxane 13 intact.
However, concentrated HCl (37%) could easily release FMK 8.
Compounds 12 and 13 were further used in the synthesis of
the peptide sequence 20 (Biotin-Teg-YQEQTG-8), derived from
the sequence of SUMO1 (small ubiquitin-like modifier – the
natural substrate of SENPs), which is expected to act as an
activity based probe for SENPs. It is to be noted that compound
20 cannot be obtained by direct solution coupling (without pro-
tection of Asp and Tyr side chain groups) or by using oxidative
methods. Firstly, alkyne 12 was attached to the azide functiona-
lized Rink resin 17, by click chemistry reaction (Scheme 3B).
Peptide elongation using standard Fmoc SPPS followed by clea-
vage from the resin (TFA/TIS/H2O: 95 : 2.5 : 2.5) gave peptide
19 as indicated by ES-MS spectra. Biotin-Teg-FYQETG-8
Scheme 3 Solid phase synthesis of PFMKs: A: Peptide sequence: Biotin-Teg-FQQQTG-8 (16) (a) SPPS using Fmoc strategy: i: Attachment of
11 : 11/HBTU/HOBt/DIPEA (1 equiv/1 equiv/1 equiv/3 equiv), DMF, r.t., 3 h; peptide elongation procedure: ii: deprotection: 20% piperidine in DMF,
2 × 10 min; iii: coupling: N-Fmoc-amino acid/HBTU/HOBt/DIPEA (4 equiv/4 equiv/4 equiv/12 equiv), DMF, r.t., 1 h; (b) peptide cleavage: TFA/
TIS/H2O (95 : 2.5 : 2.5) (15, 16); (c) HCl 37%, r.t., 2 h, (16). B: (d) TBTA, CuSO4·5H2O, ascorbic acid, H2O/t-BuOH (1 : 1), 20 h (18); C: (e) 13/
HBTU/HOBt/DIPEA (1 equiv/1 equiv/1 equiv/3 equiv), DMF, r.t., 3 h, (21); (f) i: SPPS in the same conditions as above (route A), ii: TFA/TIS/H2O
(95 : 2.5 : 2.5), (19, 22); (g) HCl 37%, r.t., 2 h, Biotin-Teg-YQEQTG-8 (20).
4518 | Org. Biomol. Chem., 2012, 10, 4516–4523
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peptide 20 could be obtained by hydrolysis of 19 with concen-
trated HCl.
time (Rt) is given in minutes with the gradient in percentage of
acetonitrile.
The same peptide 20 was prepared by direct attachment of the
dioxane 13 onto a commercial Rink resin followed by peptide
elongation, cleavage with TFA/TIS/H2O and hydrolysis of the
intermediate 1,3-dioxane protected peptide 22 with HCl 37%
(Scheme 3C).
General method for the synthesis of diazoketones 1 and 5
A modification of a described procedure14 was followed: to a
solution of N-Fmoc-protected amino acid [(Fmoc-Gly-OH or
Fmoc-Asp(OtBu)-OH), 5 mmol, 1 equiv) in dry THF (25 mL),
were sequentially added N-methylmorpholine (6.25 mmol, 1.25
equiv) and isobutyl chloroformate (5.75 mmol, 1.15 equiv) at
−10 °C. The reaction was stirred at −10 °C for 30 min. A solu-
tion of diazomethane in diethyl ether (15–20 mmol, generated
in situ from α-nitrosomethylurea26 and dried over potassium
hydroxide) was added to the reaction mixture at 0 °C. The reac-
tion was allowed to warm to room temperature over a period of
3 hours and then quenched with water (10 mL) and acetic acid
(0.5 mL). The reaction mixture was diluted with ethyl acetate
(50 mL) and washed with water, brine and saturated aqueous
sodium bicarbonate. The organic layer was dried over MgSO4
and the solvents were removed at the rotary evaporator. Crude
diazoketones 1 and 5 were purified by column chromatography
(silica gel, ethyl acetate/pentane = 2 : 3, Rf = 0.3 for 1 and ethyl
acetate/pentane = 1 : 1, Rf = 0.35 for 5) to give 1 (1.83 g, 84%)
and 5 (1.54 g, 96%), respectively.
Conclusions
We described a new, general and efficient solid phase synthetic
method for the preparation of peptidyl-fluoromethyl ketones
using standard Fmoc peptide chemistry as well as a synthetic
strategy to obtain glycine and aspartic acid fluoromethyl ketones.
This approach allows the facile introduction of amino acid based
fluoromethyl ketones at C-terminal end of any peptide sequence.
The utility of the solid phase method has been demonstrated by
the synthesis of the PFMK 16, which is known to act as an
activity based probe for SENPs, and by obtaining a new PFMK
20, which cannot be synthesised through previously reported
methods and that is also expected to behave as an activity based
probe for SENPs.
Current efforts are focused on the synthesis of PFMK libraries
and study of the biological properties of the synthesized
peptides.
tert-Butyl 3-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-5-
diazo-4-oxopentanoate (1). All analytical data for 1 were found
1
Experimental section
to be identical with those of the ref. 14. H-NMR (400 MHz,
2
CDCl3) δ (ppm): 1.44 (s, 9H, t-Bu), 2.60 (dd, J = 16.8 Hz,
General experimental information
3J = 4.9 Hz, 1H, CHHCOOtBu), 2.91 (dd, 2J = 16.8 Hz,
3
All air and/or water sensitive reactions were performed in anhy-
drous solvents and under a positive pressure of argon. Dry tetra-
hydrofurane (THF) and diethyl ether were obtained by
distillation under argon over sodium and benzophenone, while
CH2Cl2 was distilled over CaH2. All other solvents, reagents and
resins were purchased from commercial suppliers and used
without further purification. The NMR spectra were recorded at
r.t. on JEOL-Delta 400, Bruker DPX-500, Bruker DPX-360,
Bruker Advanced-300 or Bruker DPX-250 spectrometers.
Chemical shifts (δ) are reported in parts per million (ppm)
values using residual solvent peak as internal reference. Multipli-
cities are abbreviated as follows: br-broad; s-singlet; d-doublet;
t-triplet; q-quadruplet; and m-multiplet. The mass spectra were
obtained by electrospray ionization (ES) technique using a
ThermoFinnigan LCQ instrument. High resolution EI mass
spectra were measured on a Finnigan MAT 95S spectrometer
while analyses using ESI or APCI ionization techniques were
recorded on ThermoScientific LTQ-FT and LTQ XL spectro-
meters. Thin layer chromatography (TLC) was performed on
silica gel coated aluminium F254 plates from Merck. All plates
were visualized by UV irradiation at 254 nm and/or staining with
potassium permanganate. Preparative column chromatography
was performed on silica Merck 230. Reversed phase HPLC ana-
lyses and purifications of the peptide products were carried out
on a VARIAN Pro Star 210 system, equipped with UV detection,
using an analytical C18 VYDAC (300 Å, 4.6 mm × 150 mm,
5 μm) or a preparative C18 VYDAC (300 Å, 20 mm × 250 mm,
10 μm) columns, respectively and eluting with gradient mixtures
of water (containing 0.1% TFA) and acetonitrile. The retention
3J = 4.9 Hz, 1H, CHHCOOtBu), 4.21 [t, J = 6.4 Hz, 1H, CH
(Fmoc)] 4.22–4.93 [m, 2H, CH2(Fmoc)], 4.60–4.64 (m, 1H,
CH-NH), 5.34 (s, 1H, CHN2), 5.51 (br. s, 1H, NH), 7.32 [t,
3J = 7.6 Hz, 2H, HAr(Fmoc)], 7.39 [t, 3J = 7.6 Hz, 2H,
HAr(Fmoc)], 7.59 [d, 3J = 7.6 Hz, 2H, HAr(Fmoc)], 7.77 [d,
3J = 7.6 Hz, 2H, HAr(Fmoc)]; 13C-NMR (101 MHz, CDCl3)
δ (ppm): 28.2, 36.9, 47.5, 66.8, 82.0, 120.2, 124.0, 125.2,
127.3, 127.9, 141.5, 143.7, 143.9, 156.0, 170.8, 192.7.
(9H-Fluoren-9-yl)methyl
(3-diazo-2-oxopropyl)carbamate
(5). 1H-NMR (300 MHz, CDCl3) δ (ppm): 3.95 (s, 2H,
CH2NH), 4.22 [t, 3J = 6.6 Hz, 1H, CH(Fmoc)], 4.45 [d, 3J = 6.6
Hz, 2H, CH2(Fmoc)], 5.27 (s, 1H, CHN2), 5.51 (br. s, 1H, NH),
3
3
7.31 [t, J = 7.2 Hz, 2H, HAr(Fmoc)], 7.40 [t, J = 7.2 Hz, 2H,
HAr(Fmoc)], 7.60 [d, 3J = 7.2 Hz, 2H, HAr(Fmoc)], 7.76 [d, 3J =
7.2 Hz, 2H, HAr(Fmoc)]; 13C-NMR (75 MHz, CDCl3) δ (ppm):
43.7, 53.7, 67.3, 120.1, 124.9, 127.0, 127.8, 141.5, 143.9,
156.3, 156.7, 191.0; HRMS (ES+) calcd for C18H15N3NaO3
[M + Na]+: 344.1006; found: 344.1006.
General method for the synthesis of bromomethyl ketones 2 and 6
A previously reported procedure14 was followed: a solution of
diazoketone 1 (or 5) (2 mmol) in THF (15 mL) was cooled to
0 °C and a solution of HBr 33% in acetic acid and water (1 : 2),
(6 mL) was added dropwise. After the evolution of gas stopped
(10–15 min), the reaction mixture was diluted with ethyl acetate
and washed with water, brine and saturated aqueous sodium
bicarbonate. The organic phase was dried over MgSO4 and the
This journal is © The Royal Society of Chemistry 2012
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3-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)-5-fluoro-4,4-
dimethoxypentanoic acid (4). To a solution of 3 (100 mg,
0.23 mmol, 1 equiv) in toluene (10 mL), methanol (1 mL) and
p-toluenesulfonic acid (4 mg, 0.023 mmol, 0.1 equiv) were
added. The reaction mixture was heated at 60 °C for 5 hours and
the reaction was monitored by TLC. After cooling to room temp-
erature the reaction mixture was diluted with toluene (10 mL),
washed with an aqueous solution of sodium acetate 5% (15 mL),
brine (15 mL) and water (15 mL). Crude 4 was purified by
column chromatography (silica gel, ethyl acetate/pentane = 1 : 3,
Rf = 0.15) to yield 4 (74 mg, 77%). 1H-NMR (360 MHz,
CDCl3) δ (ppm): 2.86 (dd, 2J = 17.3 Hz, 3J = 4.0 Hz, 1H,
CHHCOOH), 3.07 (dd, 2J = 17.3 Hz, 3J = 4.0 Hz, 1H,
solvents were removed under vacuum. Bromomethyl ketones 2
and 6 were obtained as yellow (2) or white (6) solids in quanti-
tative yields and were used further without any purification.
tert-Butyl 3-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-5-
bromo-4-oxopentanoate (2). 1H-NMR (400 MHz, CDCl3) δ
2
3
(ppm): 1.42 (s, 9H, t-Bu), 2.72 (dd, J = 17.0 Hz, J = 4.8 Hz,
2
3
1H, CHHCOOtBu), 2.93 (dd, J = 17.0 Hz, J = 4.8 Hz, 1H,
CHHCOOtBu), 4.06 (s, 2H, CH2Br), 4.22 [t, J = 6.4 Hz, 1H,
3
2
3
CH(Fmoc)], 4.43 [dd, J = 10.6 Hz, J = 6.4 Hz, 1H. CHH-
(Fmoc)], 4.57 [dd, 2J = 10.6 Hz, 3J = 6.4 Hz, 1H. CHH(Fmoc)],
3
4.63–4.76 (m, 1H, CH-NH), 5.80 (d, J = 7.9, 1H, NH), 7.31 [t,
3J = 7.5 Hz, 2H, HAr(Fmoc)], 7.41 [t, 3J = 7.5 Hz, 2H,
HAr(Fmoc)], 7.58 [d, 3J = 7.5 Hz, 2H, HAr(Fmoc)], 7.75 [d, 3J =
7.6 Hz, 2H, HAr(Fmoc)]; 13C-NMR (100 MHz, CDCl3) δ
(ppm): 28.0, 31.2, 36.7, 47.3, 54.2, 67.0, 82.5, 120.1, 124.9,
125.0, 127.1, 127.9, 141.4, 143.5, 155.9, 170.3, 203.0; HRMS
(ES+) calcd for C24H26BrNNaO5 [M + Na]+: 510.0887; found:
510.0887.
3
CHHCOOH), 3.70 (s, 6H, OCH3), 4.21 [t, J = 6.5 Hz, 1H, CH
2
3
(Fmoc)], 4.45 [dd, J = 10.5 Hz, J = 6.5 Hz, 1H CHH(Fmoc)],
4.55 [dd, 2J = 10.5 Hz, 3J = 6.5 Hz, 1H CHH(Fmoc)],
4.61–4.77 (m, 1H, CH–NH), 4.98 (dd, 2(H,F)J = 44.9 Hz,
2(H,H)J = 14.4 Hz, 1H, CHHF), 5.10 (dd, 2(H,F)J = 44.7 Hz,
3
2(H,H)J = 14.4 Hz, 1H, CHHF), 5.73 (d, J = 7.8 Hz, 1H, NH),
7.33 [t, 3J = 7.3 Hz, 2H, HAr(Fmoc)], 7.42 [t, 3J = 7.3 Hz,
3
2H, HAr(Fmoc)], 7.58 [d, J = 7.3 Hz, 2H, HAr(Fmoc)], 7.77
(9H-Fluoren-9-yl)methyl
(3-bromo-2-oxopropyl)carbamate
3
[d, J = 7.3 Hz, 2H, HAr(Fmoc)]; 19F-NMR (376 MHz, CDCl3)
(6). 1H-NMR (300 MHz, CDCl3) δ (ppm): 3.92 (s, 2H, CH2Br),
δ (ppm): 170.92 [t (overlapped dd), 2(H,F)J = 2(H,F)J′ = 44.8 Hz];
13C-NMR (90 MHz, CDCl3) δ (ppm): 35.4, 47.4, 52.42, 54.2,
67.2, 84.2 (d, 1(F,C)J = 183.8), 120.2, 125.0, 125.1, 127.2, 128.0,
141.8, 143.5, 156.0, 171.7; HRMS (ES+) calcd for
C22H24FNO6K [M + K]+: 456.1219; found: 456.1218.
3
3
4.23 [t, J = 6.8 Hz, 1H, CH(Fmoc)], 4.35 (d, J = 5.1 Hz, 2H,
3
NH–CH2–CO), 4.43 [d, J = 6.8 Hz, 2H, CH2(Fmoc)], 5.40 (s,
1H, NH), 7.33 [t, 3J = 7.4 Hz, 2H, HAr(Fmoc)], 7.41 [t, 3J = 7.4
Hz, 2H, HAr(Fmoc)], 7.60 [d, 3J = 7.4 Hz, 2H, HAr(Fmoc)], 7.77
3
[d, J = 7.4 Hz, 2H, HAr(Fmoc)]; 13C NMR (75 MHz, CDCl3) δ
(ppm): 31.1, 47.2, 48.6, 67.4, 120.2, 125.2, 127.2, 127.9, 141.5,
143.8, 156.3, 198.0; HRMS (ES+) calcd for C18H16BrNNaO3
[M + Na]+: 396.0206; found: 396.0206.
(9H-Fluoren-9-yl)methyl (3-hydroxy-2-oxopropyl)carbamate
(7). A solution of diazoketone 5 (1.7 g, 5.29 mmol) in 1,4-
dioxane (5 mL) was added to a solution of aqueous H2SO4 0.25
M (22 mL) in 1,4-dioxane (10 mL) and the reaction mixture was
heated at 90 °C until the evolution of gas stopped (2 hours).
The reaction mixture was cooled to room temperature, basified
with solid sodium bicarbonate and extracted with ethyl acetate
(3 × 50 mL). Combined organic phases were washed succes-
sively with aqueous sodium bicarbonate (50 mL), water
(2 × 50 mL) and brine (2 × 50 mL), dried over MgSO4, concen-
trated in vacuo and purified by column chromatography (silica
gel, ethyl acetate/pentane = 1 : 1; Rf = 0.49) to give 7 as white
tert-Butyl 3-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-5-
fluoro-4-oxopentanoate (3). To a stirred solution of 2 (300 mg,
0.616 mmol, 1 equiv) and p-toluenesulfonic acid (318 mg,
1.84 mmol, 3 equiv) in THF (5 mL) was added dropwise a solu-
tion of tetra-n-butylammonium fluoride (TBAF, 964 mg,
3.69 mmol, 6 equiv) in THF (5 mL) and the reaction mixture
was heated to reflux. The reaction was monitored by TLC. After
4 hours the solvent was evaporated and the residue was dissolved
in ethyl acetate (30 mL), washed with water (2 × 20 mL) and
dried over MgSO4. Crude 3 was purified by column chromato-
graphy (silica gel, ethyl acetate/pentane = 1 : 4, Rf = 0.33) to
1
solid (1.14 g, 69%). H-NMR (400 MHz, CDCl3) δ (ppm): 2.83
3
(br. s, 1H, OH), 4.14 (br. s, 2H, CH2OH), 4.23 [t, J = 6.3 Hz,
1
1H, CH(Fmoc)], 4.34 (br. s, 2H, NH–CH2–CO), 4.44 [d,
3J = 6.3 Hz, 2H, CH2(Fmoc)], 5.39 (br. s, 1H, NH), 7.32 [t,
3J = 7.4 Hz, 2H, HAr(Fmoc)], 7.41 [t, 3J = 7.4 Hz, 2H,
HAr(Fmoc)], 7.59 [d, 3J = 7.4 Hz, 2H, HAr(Fmoc)], 7.77 [d, 3J =
7.4 Hz, 2H, HAr(Fmoc)]; 13C-NMR (75 MHz, CDCl3) δ (ppm):
47.2, 47.7, 66.7, 67.3, 120.1, 125.1, 127.2, 127.9, 141.4, 143.8,
156.5, 205.5; HRMS (ES+) calcd for C18H17NNaO4 [M + Na]+:
334.1050; found: 334.1049.
give 3 (130 mg, 49%). H-NMR (400 MHz, CDCl3) δ (ppm):
2
3
1.43 (s, 9H, t-Bu), 2.74 (dd, J = 16.9 Hz, J = 4.4 Hz, 1H,
CHHCOOtBu), 2.98 (dd, 2J = 16.9 Hz, 3J = 4.4 Hz, 1H,
3
CHHCOOtBu), 4.22 [t, J = 6.4 Hz, 1H, CH(Fmoc)], 4.43 [dd,
2J = 10.5 Hz, 3J = 6.8 Hz, 1H. CHH(Fmoc)], 4.54 [dd, 2J = 10.5
Hz, 3J = 6.8 Hz, 1H. CHH(Fmoc)], 4.58–4.67 (m, 1H, CH-NH),
4.98 (dd, 2(H,F)J = 48.3 Hz, 2(H,H)J = 15.7 Hz, 1H, CHHF), 5.10
(dd, 2(H,F)J = 48.1 Hz, 2(H,H)J = 15.7 Hz, 1H, CHHF), 5.74 (d, 3J
3
= 8.5 Hz, 1H, NH), 7.32 [t, J = 7.5 Hz, 2H, HAr(Fmoc)], 7.41
[t, 3J = 7.5 Hz, 2H, HAr(Fmoc)], 7.58 [d, 3J = 7.5 Hz, 2H,
(9H-Fluoren-9-yl)methyl
(3-fluoro-2-oxopropyl)carbamate
HAr(Fmoc)], 7.75 [d, J = 7.5 Hz, 2H, HAr(Fmoc)]; 19F-NMR
(8). Hydroxy ketone 7 (850 mg, 2.73 mmol, 1 equiv) was
placed in a dried flask and purged twice with argon. Dry aceto-
nitrile (25 mL) was added and the clear solution was cooled to
−30 °C. To this solution perfluoro-1-butanesulfonyl fluoride
(PBSF, 0.98 mL; 5.46 mmol, 2 equiv), triethylamine (1.52 mL,
10.92 mmol, 4 equiv) and Et3N × 3 HF complex (0.89 mL,
5.46 mmol, 2 equiv) were sequentially added. The reaction was
3
(376 MHz, CDCl3) δ (ppm): 170.89 (t (overlapped dd), 2(H,F)J =
2(H,F)J′ = 48.8 Hz); 13C-NMR (101 MHz, CDCl3) δ (ppm): 28.1,
36.8, 47.4, 57.4, 67.2, 82.5, 84.3 (d, 1(F,C)J = 183.3), 120.2,
125.0, 127.2, 128.0, 141.5, 143.7, 156.0, 170.4, 202.9
(d, 3(F,C)J = 18.2 Hz); HRMS (ES+) calcd for C24H26FNO5K
[M + K]+: 466.1427; found: 466.1418.
4520 | Org. Biomol. Chem., 2012, 10, 4516–4523
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allowed to warm to room temperature over a period of 3 hours,
when TLC showed complete conversion of the substrate 7. The
solvent was evaporated under vacuum and the solid residue was
purified by column chromatography (silica gel, ethyl acetate/
pentane = 1 : 3, Rf = 0.40) to give 8 as a white solid (0.52 g,
washed twice with water (20 mL) and the organic phase dried
over anhydrous MgSO4 and concentrated in vacuum. The
residue was loaded on a chromatographic column (silica gel,
ethyl acetate/pentane = 1 : 2, Rf = 0.62) to afford 12 as a mixture
of two diastereoisomers (cis and trans) in a ratio of 1 : 1 as deter-
1
3
1
1
61%). H-NMR (400 MHz, CDCl3) δ (ppm): 4.24 [t, J = 6.4
mined from the H-NMR spectrum (105 mg, 81%). H-NMR
3
Hz, 1H, CH(Fmoc)], 4.33 (d, 2H, J = 3.6 Hz, NH-CH2-CO),
(500 MHz, CDCl3) δ (ppm): 1.30–1.35 (overlapped peaks, 2H,
3
4
4.43 [d, J = 6.4 Hz, 2H, CH2(Fmoc)], 4.94 (d, 2(H,F)J = 47.2
CHCH2CCH cis and trans); 2.03 (t, J = 2.5 Hz, 2H, CH2CCH
3
Hz, 2H, CH2F), 5.42 (br. s, 1H, NH), 7.33 [t, J = 7.4 Hz, 2H,
cis and trans), 2.28 (dd, 3J = 6.9 Hz, 4J = 2.5 Hz, 2H,
3
3
3
4
HAr(Fmoc)], 7.41 [t, J = 7.4 Hz, 2H, HAr(Fmoc)], 7.61 [d, J =
CHCH2CCH cis or trans), 2.34 (dd, J = 7.3 Hz, J = 2.5 Hz,
2H, CHCH2CCH cis or trans), 3.57 (overlapped peaks, 4H,
3
7.4 Hz, 2H, HAr(Fmoc)], 7.77 [d, J = 7.4 Hz, 2H, HAr(Fmoc)];
19F-NMR (376 MHz, CDCl3) δ (ppm): 169.0 (t, 2(H,F)J = 47
Hz); 13C-NMR (101 MHz, CDCl3) δ (ppm): 47.2, 48.2, 67.3,
84.61 (d, C,FJ = 183.0 Hz), 120.1, 125.1, 127.2, 127.8, 141.4,
143.8, 156.3, 202.3; HRMS (ES+) calcd for C18H16FNNaO3
[M + Na]+: 336.1006; found: 336.1007.
NHCH2 cis and trans), 3.82 (dd, J = 11.7 Hz, J = 7.6 Hz, 2H,
2
3
2
3
Heq cis or trans), 3.88 (dd, J = 11.7 Hz, J = 6.4 Hz, 2H, Heq
cis or trans), 4.10 (td, 2J = 11.7 Hz, 3J = 4.0 Hz, 4H, Hax cis and
3
trans), 4.24 [t, J = 7.0 Hz, 2H, CH(Fmoc) cis and trans], 4.37
(d, 2(H,F)J = 47.0 Hz, 2H, CH2F cis or trans), 4.43 [d, J = 7.0
3
Hz, 4H, CH2(Fmoc) cis and trans], 4.47 (d, 2(H,F)J = 47.5 Hz,
2H, CH2F cis or trans), 5.00 (br. s, 2H, NH cis and trans), 7.32
6-(2-(((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)methyl)-2-
3
3
(fluoromethyl)-4,6-dimethyl-1,3-dioxan-5-yl)hexanoic
acid
[t, J = 7.4 Hz, 4H, HAr(Fmoc) cis and trans], 7.40 [t, J = 7.4
Hz, 4H, HAr(Fmoc) cis and trans], 7.60 [d, J = 7.4 Hz, 4H,
HAr(Fmoc) cis and trans], 7.77 [d, J = 7.4 Hz, 4H, HAr(Fmoc)
3
(11). A solution of G-FMK (8) (83 mg, 0.26 mmol, 1 equiv) in
dry DCM (5 mL) was added to a stirred solution of the silylated
alcohol (S3) in DCM (15 mL), under argon. Compound S3 (see
ESI†) was obtained in situ from diol 9 (240 mg, 1.10 mmol).
The solution was cooled to −27 °C and TMSOTf (20 μL) was
added; the reaction mixture was allowed to warm to room temp-
erature and was stirred for 12 hours. An additional amount of
TMSOTf (40 μL) was added and the reaction mixture was
allowed to stir for another 14 hours until TLC showed complete
conversion. The crude of reaction was washed with water, the
organic phase dried over anhydrous MgSO4 and concentrated in
vacuum. The residue was purified by column chromatography
(silica gel, ethyl acetate/pentane = 1 : 1 (Rf = 0.60) to afford 11
as mixture of diastereoisomers (59 mg, 44%). 1H-NMR
(400 MHz, CD3OD) δ (ppm): 0.87–1.59 (overlapped signals,
aliphatic H), 2.20–2.28 (overlapped signals, aliphatic H),
4.07–4.99 (overlapped signals, aliphatic H), 7.29 [t, 3J = 7.4 Hz,
3
cis and trans]; 19F-NMR (376 MHz, CDCl3) δ (ppm):
169.44 ppm (overlapped t, CH2F); 13C-NMR (63 MHz, CDCl3)
δ (ppm): 18.4, 18.6, 21.2, 21.5, 32.8, 33.1, 34.3, 41.7, 42.7,
47.4, 60.5, 64.0, 64.2, 67.1, 70.5, 70.6, 79.2–82.8 (5 peaks over-
lapped d, CH2F cis and trans and CCH), 96.8, 120.1, 125.2,
127.2, 127.8, 141.5, 144.1, 156.7; MS (ES+) m/z: 410.2 [M +
H]+, 432.5 [M + Na]+, 634.0 [(3M + H + K)/2]2+; HRMS (ES+)
calcd for C24H24FNNaO4 [M + Na]+: 432.1582; found:
432.1579.
2-(4-((2-(((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)methyl)-
2-(fluoromethyl)-1,3-dioxan-5-yl)methyl)-1H-1,2,3-triazol-1-yl)-
acetic acid (13). To a solution of azidoacetic acid (39 mg,
0.388 mmol, 2 equiv) and alkyne 12 (53 mg, 0.129 mmol,
1 equiv) in H2O/t-BuOH (1 : 1, 10 mL), tris[(1-benzyl-1H-1,2,3-
triazol-4-yl)methyl]amine (TBTA) (3.42 mg, 6.47 μmol, 5 mol
%), CuSO4·5H2O (1.62 mg, 6.47 μmol, 5 mol%) and a fresh sol-
ution of aqueous ascorbic acid (3.42 mg, 19.42 μmol, 15 mol%)
were added. The resulted solution was stirred at room tempera-
ture under argon atmosphere for 10 hours until TLC showed the
completion of the reaction. The mixture was diluted with ethyl
acetate (15 mL), washed with water (2 × 15 mL) and dried over
anhydrous MgSO4. The organic phase was evaporated to
dryness and the residue was washed with diethyl ether to remove
the unreacted azidoacetic acid. The expected dioxane 13 (cis and
trans isomers) precipitated as a white solid (61 mg, 93%).
1H-NMR (500 MHz, MeOD) δ (ppm): 2.00–2.22 (overlapped
3
2H, HAr(Fmoc)], 7.37 [t, J = 7.4 Hz, 2H, HAr(Fmoc)], 7.64 [d,
3J = 7.4 Hz, 2H, HAr(Fmoc)], 7.78 [d, 3J = 7.4 Hz, 2H,
HAr(Fmoc)]; 19F-NMR (376 MHz, CDCl3) δ (ppm): 166.66 (t,
2(H,F)J = 47.0 Hz, CH2F- diastereoisomer 1), 167.79 (t, 2(H,F)J =
46.8 Hz, CH2F- diastereoisomer 2), 168.49 (t, 2(H,F)J = 47.9 Hz,
CH2F- diastereoisomer 3), 168.56 (t, 2(H,F)J = 47.4 Hz, CH2F-
diastereoisomer 4), 169.06 ppm (t, 2(H,F)J = 46.8 Hz, CH2F- dia-
stereoisomer 5); 13C-NMR (101 MHz, CDCl3) δ (ppm): 13.95,
16.49, 18.58, 19.40, 20.82, 21.60, 24.69, 26.80, 29.52, 33.91,
46.03, 47.13, 60.55, 66.13, 66.73, 67.53, 69.96, 82.5–86.4
(overlapped d), 119.87, 124.94, 126.98, 127.62, 141.23, 143.79,
156.90, 174.60; MS (ES+): m/z: 514.3 [M + H]+, 536.6 [M +
Na]+, 1049.4 [2M + Na]+, 1065.4 [2M + K]+; HRMS (ES+)
calcd for C29H36FNNaO6 [M + H]+: 536.2419; found: 536.2414.
3
peaks, CHCH2-triazol cis and trans), 2.77 (d, J = 6.7 Hz, 1H,
3
CH2-triazol cis or trans), 2.85 (d, J = 7.1 Hz, 1H, CH2-triazol
cis or trans), 3.70–3.80 (overlapped peaks, 4H, Heq cis and
trans), 3.90 (br. s, 2H, NHCH2 cis and trans), 4.01–4.06 (over-
(9H-Fluoren-9-yl)methyl((2-(fluoromethyl)-5-(prop-2-yn-1-yl)-
1,3-dioxan-2-yl)methyl) carbamate (12). A solution of G-FMK
(8) (100 mg, 0.319 mmol, 1 equiv) in dry DCM (5 mL) was
added to a stirred solution of silylated diol S3 (154 mg,
0.638 mmol, 2 equiv) in dry DCM (15 mL) under argon. The
solution was cooled to −27 °C and TMSOTf (10 μL) was added.
The reaction mixture was allowed to warm to room temperature
and stirred for 48 h with TLC monitoring. After that it was
3
lapped peaks, 4H, Hax cis and trans), 4.23 [t, J = 6.8 Hz, 2H,
3
CH(Fmoc) cis and trans] 4.38 [d, J = 6.8 Hz, 2H, CH2(Fmoc)
3
cis or trans], 4.41 [d, J = 6.8 Hz, 2H, CH2(Fmoc) cis or trans],
4.43 (d, 2(H,F)J = 47.0 Hz, 2H, CH2F cis or trans), 4.50 (d,
2(H,F)J = 47.0 Hz, 2H, CH2F cis or trans), 5.18 (s, 4H,
CH2COOH, cis and trans), 5.66 (br. s, 2H, NH cis and trans),
7.26–7.33 [overlapped peaks, 5H, HAr(Fmoc) cis and trans and
This journal is © The Royal Society of Chemistry 2012
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CH(triazol)], 7.34–7.41 [overlapped peaks, 4H, HAr(Fmoc) cis
and trans], 7.67 [d, J = 7.4 Hz, 4H, HAr(Fmoc) cis and trans],
to 40% CH3CN within 16 min (yield 6 mg in total, 8%). Alter-
natively the crude mixture of peptides 15 and 16 was hydrolysed
using HCl 10 N (1 mL) to get peptide 16 which was purified by
RP-C18-HPLC using the above mentioned method. Biotin-Teg-
FQQQTG-11 (15): MS (ES+) m/z: 1439.4 [M + H]+, 1461.7
[M + Na]+; Biotin-Teg-FQQQTG-8 (16): (6 mg, 8% yield);
MS (ES+) m/z: 1240.5 [M + H]+, 1262.6 [M + Na]+.
3
7.78–7.81 [overlapped peaks, 4H, HAr(Fmoc) cis and trans];
19F-NMR (376 MHz, CDCl3) δ (ppm): 169.08–169.57 (over-
lapped peaks, CH2F, cis and trans); 13C NMR (63 MHz,
CDCl3): 24.2, 24.8, 33.4, 39.1, 40.6, 47.2, 48.0, 64.1, 64.3,
65.4, 66.6, 79.3 (d, 1(F,C)J = 167.5 Hz), 80.1 (d, 1(F,C)J = 170.0
Hz) 112.8, 119.7, 119.9, 124.9, 126.7, 127.4, 141.0, 143.7,
156.7, 176.2; MS (ES+) m/z: 511.4 [M + H]+, 1021.0 [2M +
H]+, 1043.3 [2M + Na]+, 1059.4 [2M + K]+; HRMS (ES+) calcd
for C26H28FN4O6 [M + H]+: 511.1987; found: 511.1986.
Preparation of resin 18
N3-Teg-COOH (147 mg, 0.53 mmol, 9.2 equiv) was linked on
Rink resin (82 mg, 0.7 mmol g−1 resin loading) using HBTU/
HOBt as coupling reagents. The unreacted sites were blocked
with acetic anhydride (10 equiv) and the resin was washed with
DMF to give resin 17. Compound 12 (37 mg, 0.090 mmol, 1.5
equiv) was dissolved in a mixture H2O/t-BuOH 1 : 1 (2 mL) and
added over the resin under nitrogen atmosphere. To this mixture,
TBTA (2.39 mg, 4 μmol), CuSO4·5H2O (1.12 mg, 4 μmol) and
fresh aqueous solution of ascorbic acid (2.68 mg, 0.013 mmol)
were added. The mixture was shaken at room temperature for
10 hours. t-BuOH (2 mL) and an additional amount of fresh
solution of ascorbic acid (2.68 mg, 0.013 mmol) were added and
mixture was stirred for another 10 hours. The resin 18 was then
washed with t-BuOH and DMF and kept wet until it was used in
the next step.
General solid phase peptide synthesis procedure
Unless otherwise stated, each building block was introduced
using N-Fmoc-amino acid/HBTU/HOBt/DIPEA (4 equiv/4
equiv/4 equiv/12 equiv with respect to the resin loading reported
by the supplier) in DMF (approximately 0.2 M) for 1–2 h at
room temperature. Fmoc protecting groups were removed with
piperidine/DMF 1 : 4 (2 × 10 min). The peptide was cleaved
from the resin using 1 mL of cleavage cocktail (95% TFA, 2.5%
H2O, 2.5% TIS) and allowed to shake for 4 hours. Ice cold ether
(40 mL) was used to precipitate the product. The resin was
washed with TFA (1.5 mL) and the solution was collected separ-
ately in ice cold ether. The solid was centrifuged, separated from
ether and dissolved in a minimal amount of acetonitrile/water
(0.1% TFA). The product was analyzed on a C18 reversed phase
high performance liquid chromatography (HPLC) column using
a gradient of water (0.1% TFA)/acetonitrile and purified on a cor-
responding preparative C18 column, using the same gradient.
Fractions containing the product were pooled, frozen and lyophi-
lized to dryness.
Preparation of resin 21
Compound 13 (35 mg, 0.68 mmol, 1 equiv) was attached to
Rink resin (96 mg, 0.7 mmol g−1 resin loading) using HBTU/
HOBt as coupling reagents. The reaction was shaken for 3 h, the
resin was washed with DMF and kept wet until it was used in
the next step (functionalized resin after loading approx. 23%).
Preparation of resin 14
Preparation of peptides 19 and 22
Compound 11 (30 mg, 0.058 mmol, 1 equiv) was attached to
Rink resin (83 mg, 0.7 mmol g−1 resin loading) using HBTU
(21.9 mg, 0.058 mmol, 1 equiv)/HOBt (7.8 mg, 0.058 mmol,
1 equiv) as coupling reagents. The unreacted resin amino groups
were then blocked by acetylation with acetic anhydride
(10 equiv). The resin was washed with DMF and used in further
step.
The functionalized resins 18 and 21 were elongated according to
general SPPS procedure to afford after the TFA-mediated clea-
vage from the resin, the crude peptides 19 and 22, which were
purified by reversed phase HPLC, as it follows 19: Rt = 10.6 min
and Rt = 12.6 min (two diastereoisomers), RP-C18, gradient:
2 min with 12% CH3CN, followed by a slope to 30% CH3CN
within 16 min; MS (ES+) m/z: 815.8 [(M + 2H)/2]2+, 1629.4
[M + H]+, 1651.6 [M + Na]+. 22: Rt = 10.0 min and
Rt = 10.9 min (two diastereoisomers), RP-C18, gradient: 2 min
with 12% CH3CN, followed by a slope to 50% CH3CN within
16 min; MS (ES+) m/z: 727.6 [M + 2H]2+, 1453.5 [M + H]+,
1475.5 [M + Na]+.
Preparation of Biotin-Teg-FQQQTG-11 (15) and
Biotin-Teg-FQQQTG-8 (16)
Functionalized resin 14 was further elongated using standard
SPPS procedure, as described above. After the TFA-mediated
peptide cleavage (TFA/TIS/H2O (95 : 2.5 : 2.5) a mixture of 15
and 16 was obtained. The peptides were separated by reversed
phase HPLC Rt = 9.6–12.5 min (different diastereoisomers) for
15 and Rt = 9.5 min for 16, using the following gradient: 20%
CH3CN, followed by a slope to 40% CH3CN within 30 min. A
solution of HCl 10 N (1 mL) was added to a solution of Biotin-
Teg-FQQQTG-11 (15) (4 mg/0.5 mL H2O) and stirred at room
temperature for 3 hours. The resulted crude peptide 16 was
purified by RP-C18-HPLC, Rt = 13.6 min, using the following
elution gradient: 2 min with 12% CH3CN, followed by a slope
Preparation of Biotin-Teg-YQEQTG-8 (20)
To a solution of peptide 19 (or peptide 22) dissolved in water
(0.5 mL) HCl 10 N (1.5 mL) was added, and the reaction
mixture was stirred at room temperature for 2 hours until com-
plete conversion of 19 (or 22) as inferred from HPLC. The solu-
tion was frozen, lyophilized and the crude 20 was purified by
RP-C18-HPLC, Rt = 7.7 min. Gradient: 2 min with 12%
CH3CN, followed by a slope to 30% CH3CN within 16 min
4522 | Org. Biomol. Chem., 2012, 10, 4516–4523
This journal is © The Royal Society of Chemistry 2012

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7 C. Dobrotă, D. Fasci, N. D. Hădade, G.-D. Roiban, C. Pop, V. M. Meier,
I. Dumitru, M. Matache, G. S. Salvesen and D. P. Funeriu, ChemBio-
Chem, 2012, 13, 80–84.
(yield: 12% starting from 19, and 14% starting from 22, respect-
ively). MS (ES+) m/z: 1257.4 [M + H]+, 1279.4 [M + Na]+.
8 M. D. Witte, C. V. Descals, S. V. P. de Lavoir, B. I. Florea, G. A. Van der
Marel and H. S. Overkleeft, Org. Biomol. Chem., 2007, 5, 3690–3697.
9 (a) H. Z. Zhang, H. Zhang, W. Kemnitzer, B. Tseng, J. Cinatl Jr.,
M. Michaelis, H. W. Doerr and S. X. Cai, J. Med. Chem., 2006, 49,
1198–1201; (b) L. Revesz, L. C. Briswalter, R. Heng, A. Leutwiler,
R. Mueller and H.-J. Wuethrich, Tetrahedron Lett., 1994, 35, 9693–9696.
10 B. Imperiali and R. H. Abeles, Tetrahedron Lett., 1986, 27, 135–138.
11 K. Fearon, A. Spaltenstein, P. B. Hopkins and M. H. Gelb, J. Med.
Chem., 1987, 30, 1617–1622.
Acknowledgements
Financial support by the European Commission (EXT Grant
25085) and the National Research Council (CNCS) of Romania
(project TE 314/2010) are gratefully acknowledged.
12 P. D. Edwards, J. A. Schwartz, M. M. Stein, D. A. Trainor and R.
A. Wildonger, U. S. Patent, 5 194 588, 1993.
13 F. Guzmán, S. Barberis and A. Illanes, Electron. J. Biotechnol., 2007, 10,
279–314.
Notes and references
14 D. Kato, K. M. Boatright, A. B. Berger, T. Nazif, G. Blum, C. Ryan, K.
A. Chehade, G. S. Salvesen and M. Bogyo, Nat. Chem. Biol., 2005, 1,
33–38.
15 (a) W. J. Wood, L. Huang and J. A. Ellman, J. Comb. Chem., 2003, 5,
869–880; (b) A. Lee, L. Huang and J. A. Ellman, J. Am. Chem. Soc.,
1999, 121, 9907–9914.
16 (a) S. Rozen and R. Filler, Tetrahedron, 1985, 41, 1111–1153; (b) R.
J. Linderman and D. M. Graves, Tetrahedron Lett., 1987, 28, 4259–4262;
(c) Y. Hanzawa, A. Yamada and Y. Kobayashi, Tetrahedron Lett., 1985,
26, 2881–2884.
17 D. E. McClure, B. H. Arison, J. H. Jones and J. J. Baldwin, J. Org.
Chem., 1981, 46, 2431–2434.
1 (a) B. F. Cravatt, A. T. Wright and J. W. Kozarich, Annu. Rev. Biochem.,
2008, 77, 383–414; (b) M. J. Evans and B. F. Cravatt, Chem. Rev., 2006,
106, 3279–3301; (c) J. C. Powers, J. L. Asgian, O. D. Ekici and
K. E. James, Chem. Rev., 2002, 102, 4639–4750.
2 (a) R. E. Esser, R. A. Angelo, M. D. Murphey, L. M. Watts,
L. P. Thornburg, J. T. Palmer, J. W. Talhouk and R. E. Smith, Arthritis
Rheum., 1994, 37, 236–247; (b) R. E. Esser, L. M. Watts, R. A. Angelo,
L. P. Thornburg, J. J. Prior and J. T. Palmer, J. Rheumatol., 1993, 20,
1176–1183.
3 (a) D. Rasnick, Anal. Biochem., 1985, 149, 461–465; (b) C. P. Lawrence,
A. Kadioglu, A. L. Yang, W. R. Coward and S. C. Chow, J. Immunol.,
2006, 177, 3827–3836; (c) N. K. Ahmed, L. A. Martin, L. M. Watts,
J. Palmer, L. Thornburg, J. Prior and R. E. Esser, Biochem. Pharmacol.,
1992, 44, 1201–1207.
4 N. P. Peet, J. P. Burkhart, M. R. Angelastro, E. L. Giroux, S. Mehdi,
P. Bey, M. Kolb, B. Neises and D. Schirlin, J. Med. Chem., 1990, 33,
394–407.
18 J. T. Palmer, U. S. Patent, 5 101 068, 1992.
19 M. M. Kabat, J. Fluorine Chem., 1989, 42, 435–439.
20 G. A. Olah and J. Welch, Synthesis, 1974, 896–497.
21 T. Matsumoto, M. Ohsaki, F. Matsuda and S. Terashima, Tetrahedron
Lett., 1987, 28, 4419–4422.
22 J. Haddow, C. J. Suckling and H. C. S. Wood, J. Chem. Soc., Perkin
Trans. 1, 1989, 1297–1304.
23 D. Zhou, K. E. Carlson, J. A. Katzenellenbogen and M. J. Welch, J. Med.
Chem., 2006, 49, 4737–4744.
24 P. G. M. Wuts and T. W. Greene, Greene’s Protective Groups in Organic
Synthesis, John Wiley & Sons, New Jersey, 2007.
25 N. D. Bogdan, M. Matache, V. M. Meier, C. Dobrota, I. Dumitru, G.
D. Roiban and D. P. Funeriu, Chem.–Eur. J., 2010, 16, 2170–2180.
26 (a) F. Arndt, Org. Synth., 1943, Coll. Vol. 2, 165–167; (b) F. Arndt, Org.
Synth., 1943, Coll. Vol. 2, 461–463.
5 (a) C. J. Van Noorden, Acta Histochem., 2001, 103, 241–251;
(b) Y. Zhao, T. L. Brown, H. Kohler and S. Muller, Apoptosis, 2003, 8,
631–637.
6 (a) S. Chatterjee, K. Josef, G. Wells, M. Iqbal, R. Bihovsky,
J. P. Mallamo, M. A. Ator, D. Bozyczko-Coyne, S. Mallya, S. Senadhi
and R. Siman, Bioorg. Med. Chem. Lett., 1996, 6, 1237–1240;
(b) S. Chatterjee, M. A. Ator, D. Bozyczko-Coyne, K. Josef, G. Wells,
R. Tripathy, M. Iqbal, R. Bihovsky, S. E. Senadhi, S. Mallya,
T. M. O’Kane, B. A. McKenna, R. Siman and J. P. Mallamo, J. Med.
Chem., 1997, 40, 3820–3828.
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