Synthesis and hydrolysis studies of a peptide containing the reactive triad of serine proteases with an associated linker to a dye on a solid phase support

Article abstract of DOI:10.1039/b302239k

The synthesis of a Tentagel-supported peptide incorporating the reactive triad of serine, histidine and aspartic acid, found within serine protease enzymes, is described.

Full text of DOI:10.1039/b302239k

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Synthesis and hydrolysis studies of a peptide containing the
reactive triad of serine proteases with an associated linker to a dye
on a solid phase support†
John M. Clough,a Ray V. H. Jones,b Hannah McCann,b David J. Morrisc and Martin Wills*c
a Syngenta, Jealott’s Hill Research Centre, Bracknell, Berkshire, UK RG42 6EY
b Syngenta, Earls Road, Grangemouth, Stirlingshire, Stirling, Scotland FK3 8XG
c Department of Chemistry, University of Warwick, Coventry, UK CV4 7AL.
E-mail: m.wills@csv.warwick.ac.uk; Fax: 024 7652 4112; Tel: 024 7652 3260
Received 25th February 2003, Accepted 7th March 2003
First published as an Advance Article on the web 1st April 2003
The synthesis of a Tentagel®-supported peptide incorporating the reactive triad of serine, histidine and aspartic acid,
found within serine protease enzymes, is described.
Introduction
Serine protease enzymes are characterised by the presence of a
uniquely reactive serine side chain. Its reactivity is associated
with the interaction of the side chains of three amino acid
residues: aspartic acid, histidine and serine. This is commonly
referred to as the ‘charge relay system’ or ‘reactive triad’ within
the enzyme (Scheme 1).1 A number of peptide-based synthetic
catalysts, both synthetic and based on antibodies, have been
prepared in an attempt to mimic the method by which serine
proteases work.2 However to date none of these systems have
proven capable of reproducing the high rate enhancements and
stereoselectivity achieved by enzymes. In this paper, we describe
the synthesis of a model system to probe the capacity for a
simple linear peptide, incorporating the reactive triad, to cleave
ester bonds via intramolecular hydrolysis. The model consists
of a highly coloured red azo dye, disperse red, connected to a
supported peptide through a glycol linker and the ester to be
hydrolysed (Fig. 1). We anticipate that the model can potentially
be utilised to analyse a combinatorial library of beads. Using
this technique, peptide sequences with hydrolytic activity would
be identified by observing the loss of colour from the individual
beads. In this way, we reasoned that we should be able to iden-
tify the optimum length of linker for intramolecular cleavage of
Scheme 1 Reactive triad in serine protease enzymes.
† Electronic supplementary information (ESI) available: Description of
preliminary qualitative hydrolysis studies using the materials prepared
and described in the main paper. See http://www.rsc.org/suppdata/ob/
b3/b302239k/
Fig. 1 Structure of synthetic esterase model 1.
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 1 4 8 6 – 1 4 9 7
T h i s j o u r n a l i s © T h e R o y a l S o c i e t y o f C h e m i s t r y 2 0 0 3
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the ester bond. With this information in hand, an investigation
could be carried out into enantioselective ester hydrolysis.
Results and discussion
Initially the sixteen residue peptide NH2–Asp–Pro–Asp–Glu–
Leu–Glu–His–Ala–Ala–Lys–Ser–Glu–Ala–Ala–Ala–Lys–OH
(DPDELEHAAKSEAAAK) was selected for incorporation
into our protease model 1. This peptide is a modified version of
a sequence reported to form an α-helical structure stabilised by
salt bridges and helix-promoting alanine residues. This peptide
has been demonstrated to hydrolyse DNA when covalently
linked to a rhodium intercalator in the presence of zinc.3 On
this basis it seemed that this sequence could represent a mini-
mal protease model. The histidine residue at position six was
replaced with a serine residue with the intention that, should
the peptide form the predicted α-helical structure, the three key
residues would be aligned in a favourable conformation for
co-operative catalysis of the hydrolysis of an ester bond.
Tentagel® was selected as the support in this application for
its favourable swelling properties in a range of solvents, high
loading and mechanical stability. In addition to the Tentagel®,
ca. 10% of a Rink acid-labile linker was employed in the solid
phase synthesis. This was employed in order to permit analysis
of the material on the beads using ESI-mass spectrometry,
and would serve to follow the progress of the functionalisation
process at several stages (see below).
The Fmoc-protected amino acids were purchased from
Novabiochem with side chains protected, where applicable,
with acid-labile groups. Couplings were mediated by benzo-
Scheme 2 Reagents and conditions: i) DEAD, Ph3P, toluene, 80 ЊC, 1 h;
triazole-1-yloxy-tris(pyrrolidino)phosphonium
hexafluoro-
ii) LiOH, THF, H2O, rt, 3 h; iii) DEAD, PH3P, DCM, rt, 3 h; iv) o-
(OH)C6H4CH2CH2CO2tBu 6, DEAD, Ph3P, toluene, 80 ЊC, 1 h; v) TFA,
TIS, DCM, 5 min, rt; vi) Tentagel®-supported peptide, PyBOP, HOBt,
DIPEA, DMF, rt, 1 h.
phosphate (PyBop), N-hydroxybenzotriazole (HOBt) and
diisopropylethylamine (DIPEA or Hunig’s base) in DMF.
Double couplings were used for all residues and complete con-
version was deduced by Kaiser, 2,4,6-trinitrobenzenesulfonic
acid (TNBS) and Chloroanil resin tests. Removal of the amino
Fmoc protection was effected with washes of 20% piperidine in
DMF. Partial cleavage of a portion of the peptide with con-
comitant deprotection of its side chains was effected with 2.5%
triisopropylsilane (TIS) in trifluoroacetic acid. The filtrate was
analysed by ESI-MS and exhibited the expected peptide.
The strongly red coloured and readily available azo dye, dis-
perse red, was chosen as our visual indicator. Five lengths of
linker and two simple esters were examined. The correct choice
of linker length was crucial. If too short, the serine hydroxy
group would not be able to reach the ester to effect the desired
intramolecular hydrolysis. However, if the length was too long
then it is likely that intermolecular hydrolysis would compete.
The parallel synthesis of ten dye-linkers was performed using a
common reaction sequence in moderate overall yield to eventu-
ally produce ten synthetic esterase systems upon coupling to the
peptide and side-chain deprotection (Scheme 2).
Initially, the primary alcohol of disperse red 2 was coupled
with the hydroxyphenyl of 3a/b via Mitsunobu alkylation to
give 4a/b. Saponification with lithium hydroxide afforded the
respective acids, which were again coupled via Mitsunobu
alkylation to a range of glycols to yield 5a/b (five derivatives of
each) in reasonable yields. Previous attempts to oxidise the
primary alcohol of disperse red (in order to perform reductive
amination) and displacement of its sulfonates had proved
troublesome. An acidic functionality was therefore introduced
via Mitsunobu alkylation with the hydroxyphenyl of the ter-
tiary butyl ester 6 to give esters 7a/b (a total of 10 derivatives).
Following deprotection with TFA, the individual ester deriv-
atives 7a/b were efficiently coupled to the bead-supported
peptide using a combination of PyBOP and HOBt. Deep red-
coloured beads were quickly afforded indicating a high level of
loading (Fig. 2 illustrates the contrast between functionalised
and unfunctionalised beads). Partial cleavage of the peptide
during the acidic side-chain deprotection stage allowed tandem
Fig. 2 a) unfunctionalised beads, b) functionalised beads.
ESI-MS analysis of the crude peptide–dye-linker derivatives to
be completed, indicating that the coupling had been realised
successfully.
In an alternative approach, the free amine of the supported
peptide was functionalised with 4-hydroxyphenylacetic acid to
terminal hydroxyphenyl functionality. This was used to attach
the dye linkers 5a/b via Mitsunobu alkylation. Unfortunately
the level of loading was extremely low giving light red beads
whereas in contrast the identical coupling with Tentagel® bear-
ing the same terminal hydroxyphenyl functionality but no
peptide, quickly yielded dark red beads, indicating a far higher
level of loading (Fig. 2).
In order to investigate if it was possible to observe if enantio-
selective control was being expressed during any hydrolysis pro-
cess, we prepared another supported peptidic DL52 system, i.e. 8
in which a chiral centre was incorporated α- to the carbonyl
group on the ester being hydrolysed (Scheme 3). It was envis-
aged that any enantioselective control during the hydrolysis
would be evaluated via chiral HPLC analysis.
The synthesis of the racemic chiral glycol dye-linker 8 was
initially undertaken by protecting the hydroxyphenyl group of 6
with tert-butyldimethylsilyl chloride to give 9 in 79% yield.
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 1 4 8 6 – 1 4 9 7
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Scheme 3 Reagents and conditions: i) TBDMSCl (1.1 eq.), Et3N (1.5 eq.), DMAP (catalytic), DCM, rt, 8 h; ii) LDA (1.1 eq.), HMPA (10 eq.),
MeI (10 eq.), THF, Ϫ78 ЊC, 6 h, rt, 12 h; iii) TBAF (1.0 eq.), THF, rt, 20 min; iv) DEAD (1.0 eq.), PPh3 (1.0 eq.), disperse red (1.0 eq.), DCM, rt, 3 h,
33%; v) TIS : TFA, 1 : 49, DCM, rt, 10 min, 81%; vi) Pentaglycol (1.3 eq.), DEAD (1.1 eq.), PPh3 (1.1 eq.), DCM, rt, 4 h, 56%; vii) DEAD (1.1 eq.),
PPh3 (1.1 eq.), p-(OH)C6H4CH2CH2CO2tBu 6 (1.0 eq.), DCM, rt, 2 h, 59%; viii) TIS : TFA, 1 : 49, DCM, rt, 10 min, 79%; ix) Tentagel®-supported
peptide (1.0 eq.), 12 (2.5 eq.), PyBOP (2.5 eq.), HOBt (2.5 eq.), DIPEA (5.0 eq.), DMF, rt, 2 h; x) TIS : TFA, 1 : 49, rt, 1 h.
Formation of the enolate of 9 via deprotonation α- to the carb-
onyl group with LDA at Ϫ78 ЊC, followed by quenching at Ϫ78
ЊC with a ten fold excess of methyl iodide in the presence of
HMPA afforded 10 in 60% yield. The HMPA was required to
reduce unwanted side products resulting from polyalkylation of
the enolate intermediate, and di-carbon and oxygen alkylation
during the methyl iodide quenching. The silyl protecting group
of 10 was then removed with TBAF in THF. The deprotection
was rapid and full conversion was observed within 3 minutes to
give 11 in 82% yield.
In conclusion, we have completed the synthesis of a bead-
supported peptide–linker–ester–dye conjugate which provides a
basis for the study of intramolecular cleavage of the ester by the
peptide sequence. This lays the foundations for a possible com-
binatorial chemistry study for the optimisation of this reaction
through the production of a large peptide library. We are con-
tinuing our studies in this area.
Experimental
The coupling of 11 to disperse red was successfully achieved
via Mitsunobu alkylation using DCM as solvent. Full conver-
sion was observed within 3 hours to give the adduct in 33%
yield. We found that a major side product was the dimer of
disperse red. The removal of the tert-butyl group was achieved
in 81% yield by dissolving the substrate in a minimum of DCM
and direct delivery to a solution of TIS : TFA, 1 : 49. It was
found if a higher concentration of TIS was present or too much
DCM was used to dissolve the substrate, efficient deprotection
was difficult to achieve. The acid was then converted to the ester
of pentaglycol to give 12 in 56% yield.
The coupling of 12 to 6 was also achieved via Mitsunobu
alkylation in a moderate yield of 59% using DCM as the sol-
vent and the final deprotection was again achieved by dissolving
the substrate in a minimum of dichloromethane followed by
addition to a solution of TIS : TFA, 1 : 49. The removal of the
tert-butyl group gave the acid in 79% yield. The acid was
coupled to the Tentagel®-supported protected peptide using
PyBOP. The solvent of choice was DMF and an excellent level
of loading was achieved after gentle stirring at rt for 2 h to give
a jet black resin. Side chain deprotection was afforded by gently
stirring the supported peptide (9 mg) at rt for 1 h in a solution
of TIS : TFA (1 : 49). After subjecting the resin to washes with a
series of solvents including TFA : methanol : dichloromethane
(1 : 10 : 39), the resin was found to retain a high level of loading,
giving the supported deprotected peptidic system 8 as a jet
black resin (Scheme 3).
General
All air and moisture sensitive reactions were performed under
an atmosphere of dry nitrogen in flame dried glassware. All
anhydrous solvents were used as supplied by Romil in HyDry
bottles except dimethylsulfoxide and dimethylformamide which
were supplied by Aldrich in Sure Seal bottles. Commercially
available starting materials were used without further purifi-
cation unless otherwise stated. Petroleum ether refers to that
fraction which boils in the range 40–60 ЊC and MgSO4 was used
as the drying agent for solutions.
The reactions were monitored by TLC using aluminium
backed silica gel (F254) plates, pre-coated with a layer of silica
(Merck), visualised by UV254nm and then 2,4-dinitrophenol-
hydrazine, phosphomolybdic acid, potassium permanganate or
ninhydrin solution. All organic layers were removed by rotary
evaporation on a Buchi rotary evaporator, the final traces
of solvents being removed on a static oil pump (0.2 mbar).
Column chromatography was carried out on silica gel 60
(40–63 µm).
Melting points were measured on a Stuart Scientific SMP1
instrument and are uncorrected. Infrared spectra (IR) were
recorded on a Perkin-Elmer 1310 FTIR spectrometer between
sodium chloride plates. Nuclear magnetic resonance spectra
(NMR) were recorded on either a Bruker AC 250 MHz or
300 MHz spectrometer. The chemical shift values were quoted
in values of ppm relative to the standard tetramethylsilane
1
Although we have not yet systematically investigated quanti-
tatively the use of any of our peptides in the intramolecular
ester hydrolysis process, a preliminary qualitative investigation
has been completed. This is described in the supporting data.†
(TMS) for H NMR and to the centre line of the chloroform
triplet, δ 77.0, for 13C NMR. The peak multiplicities were speci-
fied as singlet (s), doublet (d), doublet-doublet (dd), triplet (t),
quartet (q) or quintet (quint) and coupling constants (J) quoted
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 1 4 8 6 – 1 4 9 7
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in Hz. Mass spectra (MS) were obtained from the EPSRC Mass
Spectrometry Service Centre, Swansea, as were high resolution
determinations unless indicated, then mass spectra were
obtained with a Kratos analytical MS80 RFAO spectrometer.
Elemental analyses were performed with a Carlo Erba 1106
elemental analyser. Reverse phase HPLC was performed with a
C18 Anachem ODS2 analytical column or a C18 technology
ODS semi-preparative column using a Krontron HPLC pump
422 and 332 UV detector. The absorbance was measured at
254 nm.
3-(4-Hydroxyphenyl)propionic acid methyl ester 3b
To a solution of 3-(4-hydroxyphenyl)propionic acid (1.0 g,
6.0 mmol) dissolved in methanol (100 mL), 4 drops of concen-
trated sulfuric acid were carefully added. The reaction mixture
was refluxed for 3 hours after which TLC (ethyl acetate–hexane,
2 : 3) indicated complete conversion of the starting material.
The reaction mixture was allowed to cool to room temperature
and the methanol removed under reduced pressure. The prod-
uct was firstly precipitated from diethyl ether and hexane at
Ϫ78 ЊC and then further purified by careful recrystallisation at
Ϫ20 ЊC from diethyl ether, cyclohexane and hexane to give
white needles (594 mg, 3.3 mmol, 55%). Alternatively the prod-
uct could be isolated via reduced pressure distillation on a
larger scale at 148 ЊC (0.2 bar) to give a comparative yield,
(66%). Isolation of the product via flash column chromato-
graphy (ethyl acetate–hexane, 1 : 4) gave an improved yield
of 94%. Mp 36–37 ЊC (diethyl ether–cyclohexane); (Found:
C, 67.00; H, 6.77. C10H12O3 requires C, 66.65; H, 6.71%);
νmax(Nujol)/cmϪ1 3398, 1715, 1614; δH(300 MHz; CDCl3) 7.03
(2 H, d, J 8.6, ArCH), 6.75 (2 H, d, J 8.6, ArCH), 6.14 (1 H, s,
ArOH), 3.67 (3 H, s, CO2Me), 2.87 (2 H, t, J 7.8, ArCH2CH2),
2.60 (2 H, t, J 7.8, ArCH2CH2); δC(300 MHz; CDCl3) 174.7
(Ci), 154.7 (Ci), 132.6 (Ci), 129.8 (2 CH), 115.8 (2 CH), 52.3
(CH3), 36.5 (CH2), 30.5 (CH2); m/z (EI) 180 (Mϩ); (CI) 181
(MHϩ), 198 (MNH4ϩ); COSY spectra exhibited a good
correlation with the proposed structure.
[4-(2-{[4-(2-Chloro-4-nitrophenylazo)phenyl]ethylamino}-
ethoxy)phenyl]acetic acid methyl ester, 4a
To a solution of methyl-4-hydroxyphenyl acetate (4.8 g,
29 mmol), triphenylphosphine (7.6 g, 29 mmol) and diethyl
azodicarboxylate (4.6 mL, 29 mmol) in toluene (350 mL) at
80 ЊC was added disperse red (5 g, 14.3 mmol, 0.5 eq.). The
reaction mixture was stirred at 80 ЊC for 30 minutes and then
cooled to room temperature. This was then concentrated at
50 ЊC under reduced pressure, then firstly crystallised and
secondly recrystallised from ethyl acetate–hexane to give 4a as
dark purple fine needles (4.18 g, 8.41 mmol, 58%). Mp 109–
110 ЊC (ethyl acetate–hexane); (Found: C, 60.50; H, 5.09; N,
11.28. C25H25ClN4O5 requires C, 60.42; H, 5.07; N, 11.27%);
νmax(Nujol)/cmϪ1 1737, 1602, 1512, 1339; δH(300 MHz; CDCl3)
8.33 (1 H, d, J 2.5, ArCH), 8.09 (1 H, dd, J 9.0, 2.5, ArCH),
7.91 (2 H, d, J 9.2, ArCH), 7.74 (1 H, d, J 9.0, ArCH), 7.19
(2 H, d, J 8.8, ArCH), 6.84 (2 H, d, J 8.8, ArCH), 6.78 (2 H, d,
J 9.2, ArCH), 4.16 (2 H, t, J 5.8, ArOCH2CH2), 3.83 (2 H, t,
J 5.8, ArOCH2CH2), 3.67 (3 H, s, CO2Me), 3.60 (2 H, q, J 7.1,
CH2Me), 3.56 (2 H, s, ArCH2), 1.28 (3 H, t, J 7.1, CH2Me);
δC(300 MHz; CDCl3) 172.7 (Ci), 158.0 (Ci), 153.4 (Ci), 152.1
(Ci), 147.5 (Ci), 144.7 (Ci), 134.3 (Ci), 130.8 (2 CH), 127.4
(CH), 127.1 (Ci), 126.4 (CH), 123.0 (CH), 118.4 (2 CH), 115.0
(2 CH), 111.9 (2 CH), 65.7 (CH2), 52.4 (CH3), 50.3 (CH2), 46.7
3-[4-(2-{[4-(2-Chloro-4-nitrophenylazo)phenyl]ethylamino}-
ethoxy)phenyl]propionic acid methyl ester, 4b
To a solution of 3b (3 g, 16.7 mmol), triphenylphosphine
(4.37 g, 16.7 mmol) and diethyl azodicarboxylate (2.59 mL,
16.7 mmol) in toluene (250 mL) at 80 ЊC was added disperse red
(5.2 g, 15.0 mmol, 0.9 eq.). The reaction mixture was stirred at
80 ЊC for 6 hours after which time TLC (ethyl acetate–hexane,
4 : 1) indicated that the disperse red had been entirely con-
sumed. The toluene was then removed at 50 ЊC under reduced
pressure and the product isolated by flash column chromato-
graphy (ethyl acetate–hexane, 2 : 3) to give 4b as a purple
powder (4.43 g, 8.67 mmol, 52%). Mp 116–118 ЊC (ethyl
acetate–hexane); (Found: C, 61.09; H, 5.32; N, 10.92. C26H27-
ClN4O5 requires C, 61.11; H, 5.33; N, 10.96%); νmax(Nujol)/
cmϪ1 1734, 1599, 1512, 1336; δH(300 MHz; CDCl3) 8.37 (1 H, d,
J 2.4, ArCH), 8.13 (1 H, dd, J 8.9, 2.4, ArCH), 7.94 (2 H, d,
J 8.9, ArCH), 7.77 (1 H, d, J 8.9, ArCH), 7.11 (2 H, d, J 8.9,
ArCH), 6.81 (4 H, d, J 8.9, ArCH), 4.17 (2 H, t, J 5.7,
ArOCH2CH2), 3.85 (2 H, t, J 5.7, ArOCH2CH2), 3.72–3.59
(5 H, m, CO2Me, CH2Me), 2.89 (2 H, t, J 7.6, CH2CH2CO2),
2.59 (2 H, t, J 7.6, CH2CH2CO2), 1.29 (3 H, t, J 7.1, CH2Me);
δC(300 MHz; CDCl3) 173.3 (Ci), 156.8 (Ci), 153.0 (Ci), 151.6
(Ci), 147.0 (Ci), 144.3 (Ci), 133.9 (Ci), 133.2 (Ci), 129.3 (CH),
126.9 (CH), 126.0 (CH), 122.6 (CH), 118.0 (CH), 114.4 (CH),
111.5 (CH), 65.2 (CH2), 51.6 (CH3), 50.0 (CH2), 46.3 (CH2),
35.9 (CH2), 30.0 (CH2), 12.3 (CH3); m/z (EI) 510 (Mϩ), 512 (Mϩ
ϩ 2); (CI) 511 (MHϩ), 513 (MHϩ ϩ 2); COSY and HMQC
spectra both exhibited a good correlation with the proposed
structure.
(CH2), 40.6 (CH2), 12.7 (CH3); m/z (EI) 496 (Mϩ), 498 (Mϩ
ϩ
2); (CI) 497 (MHϩ), 499 (MHϩ ϩ 2); COSY and HMQC
spectra both exhibited a good correlation with the proposed
structure.
[4-(2-{[4-(2-Chloro-4-nitrophenylazo)phenyl]ethylamino}-
ethoxy)phenyl]acetic acid
To a solution of the protected disperse red derivative 4a (1.08 g,
2.17 mmol) in tetrahydrofuran (150 mL) and water (10 mL) was
added lithium hydroxide monohydrate (182 mg, 4.34 mmol,
2.0 eq.). The reaction mixture was stirred at 45 ЊC for 30 min-
utes after which time TLC (ethyl acetate–hexane, 4 : 1) indi-
cated that the starting material had been consumed. This was
then cooled to room temperature, concentrated at 30 ЊC under
reduced pressure, diluted with dichloromethane (40 mL),
washed with HCl (2 M, 3 × 30 mL), water (3 × 30 mL), dried
(MgSO4) and concentrated in vacuo to give the acid as a red
powder (828 mg, 1.71 mmol, 79%). Mp 192–193 ЊC (ethyl acet-
ate–hexane); (Found: C, 59.43; H, 4.82; N, 11.65. C24H23ClN4O5
requires C, 59.69; H, 4.80; N, 11.60%); νmax(Nujol)/cmϪ1 1695,
1601, 1511, 1337; δH(300 MHz; CDCl3) 12.26 (1 H, br s, CO2H),
8.37 (1 H, d, J 2.5, ArCH), 8.20 (1 H, dd, J 9.0, 2.5, ArCH),
7.84 (2 H, d, J 9.3, ArCH), 7.74 (1 H, d, J 9.0, ArCH), 7.17
(2 H, d, J 8.7, ArCH), 6.94 (2 H, d, J 9.3, ArCH), 6.88 (2 H, d,
J 8.7, ArCH), 4.17 (2 H, t, J 5.4, ArOCH2CH2), 3.86 (2 H, t,
J 5.4, ArOCH2CH2), 3.62 (2 H, q, J 7.0, CH2Me), 3.48 (2 H, s,
ArCH2), 1.19 (3 H, t, J 7.0, CH2Me); δC(300 MHz; CDCl3)
173.3 (Ci), 157.4 (Ci), 152.6 (Ci), 152.5 (Ci), 147.0 (Ci), 143.7
(Ci), 132.7 (Ci), 130.8 (2 CH), 127.7 (Ci), 127.1 (CH), 126.0
(CH), 123.6 (CH), 118.3 (2 CH), 114.5 (2 CH), 112.2 (2 CH),
65.7 (CH2), 49.6 (CH2), 45.8 (CH2), 40.1 (CH2), 12.4 (CH3); m/z
(EI) 482 (Mϩ), 484 (Mϩ ϩ 2); (CI) 483 (MHϩ), 485 (MHϩ ϩ 2);
COSY and HMQC spectra both exhibited a good correlation
with the proposed structure.
3-[4-(2-{[4-(2-Chloro-4-nitrophenylazo)phenyl]ethylamino}-
ethoxy)phenyl]propionic acid
To a solution of 4b (2 g, 3.92 mmol) in tetrahydrofuran
(150 mL) and water (5 mL) was added lithium hydroxide
monohydrate (247 mg, 5.88 mmol, 1.5 eq.). The reaction mix-
ture was stirred at room temperature for 12 hours and then
40 ЊC for 3 hours after which TLC (ethyl acetate–hexane, 4 : 1)
indicated that the starting material had been consumed. This
was then allowed to cool to room temperature, concentrated
at 30 ЊC under reduced pressure, diluted with dichloromethane
(40 mL), washed with HCl (2 M, 3 × 30 mL) and water
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(3 × 30 mL), dried (MgSO4) and concentrated in vacuo. The
product was eluted by flash column chromatography (ethyl
acetate–hexane, 3 : 2) to give a dark red oil which solidified on
standing (1.46 g, 2.94 mmol, 75%). A sample of this solid was
recrystallised from hexane–EtOAc. Mp 177–179 ЊC (ethyl
acetate–hexane); (Found: C, 60.33; H, 5.08; N, 11.13. C25H25-
ClN4O5 requires C, 60.42; H, 5.07; N, 11.27%); νmax(Nujol)/
cmϪ1 1698, 1603, 1511, 1341; δH(300 MHz; CDCl3) 8.37 (1 H, d,
J 2.5, ArCH), 8.18 (1 H, dd, J 9.0, 2.5, ArCH), 7.80 (2 H, d,
J 9.2, ArCH), 7.73 (1 H, d, J 9.0, ArCH), 7.06 (2 H, d,
J 8.6, ArCH), 6.91 (2 H, d, J 9.2, ArCH), 6.78 (2 H, d, J 8.6,
ArCH), 4.10 (2 H, t, J 5.4, ArOCH2CH2), 3.81 (2 H, t, J 5.4,
ArOCH2CH2), 3.56 (2 H, q, J 7.0, CH2Me), 2.68 (2 H, t, J 7.5,
CH2CH2CO2), 2.41 (2 H, t, J 7.5, CH2CH2CO2), 1.14 (3 H, t,
J 7.0, CH2Me); δC(300 MHz; CDCl3) 174.3 (Ci), 156.8 (Ci),
152.7 (Ci), 152.5 (Ci), 147.1 (Ci), 143.7 (Ci), 133.9 (Ci), 132.7
(Ci), 129.6 (2 CH), 127.1 (CH), 126.1 (CH), 123.7 (CH), 118.4
(2 CH), 114.5 (2 CH), 112.3 (2 CH), 65.7 (CH2), 49.6 (CH2),
45.8 (CH2), 36.6 (CH2), 30.2 (CH2), 12.4 (CH3); m/z (CI) 497
(MHϩ), 499 (MHϩ ϩ 2); COSY spectra exhibited a good
correlation with the proposed structure.
(1 H, d, J 9.0, ArCH), 7.20 (2 H, d, J 8.7, ArCH), 6.84 (2 H, d,
J 8.7, ArCH), 6.81 (2 H, d, J 9.2, ArCH), 4.25 (2 H, t, J 4.7,
CH2), 4.17 (2 H, t, J 5.7, ArOCH2CH2), 3.85 (2 H, t, J 5.7,
ArOCH2CH2), 3.74–3.68 (4 H, m, CH2), 3.66–3.58 (10 H, m,
CH2), 2.41 (1 H, s, OH), 1.29 (3 H, t, J 7.0, CH2Me); δC(300
MHz; CDCl3) 172.3 (Ci), 158.0 (Ci), 153.5 (Ci), 152.1 (Ci),
147.5 (Ci), 144.7 (Ci), 134.3 (Ci), 130.9 (2 CH), 127.4 (CH),
127.0 (Ci), 126.4 (CH), 123.0 (CH), 118.4 (2 CH), 114.9 (2 CH),
111.9 (2 CH), 72.9 (CH2), 70.9 (CH2), 70.7 (CH2), 69.5 (CH2),
65.7 (CH2), 64.2 (CH2), 62.1 (CH2), 50.4 (CH2), 46.7 (CH2),
40.6 (CH2), 12.7 (CH3); m/z (CI) 615 (MHϩ), 617 (MHϩ ϩ 2)
(Found: Mϩ, 614.2140. C30H3535ClN4O8 requires Mϩ, 614.2143);
HMQC spectra exhibited a good correlation with the proposed
structure.
[4-(2-{[4-(2-Chloro-4-nitrophenylazo)phenyl]ethylamino}ethoxy)-
phenyl]acetic acid 2-{2-[2-(2-hydroxyethoxy)ethoxy]ethoxy}-
ethyl ester, 5a, m ؍ 3 (DL41)
To a solution of 4a (200 mg, 0.41 mmol), triphenylphosphine
(162 mg, 0.62 mmol, 1.5 eq.) and diethyl azodicarboxylate
(108 mg, 0.62 mmol, 1.5 eq.) in toluene (20 mL) was added
tetraethylene glycol (120 mg, 0.62 mmol, 1.5 eq.). The reaction
mixture was stirred for 5 days at room temperature after which
TLC (methanol–ethyl acetate, 1 : 9) indicated that the acid had
been entirely consumed. The toluene was removed at 50 ЊC
under reduced pressure and the product eluted by gradient
flash column chromatography (ethyl acetate–hexane, 4 : 1;
methanol–ethyl acetate, 1 : 9) to give 5a, m = 3 as a dark red
oil (198 mg, 0.30 mmol, 73%). νmax(Nujol)/cmϪ1 3441, 1737,
1600, 1514, 1337; δH(300 MHz; CDCl3) 8.37 (1 H, d, J 2.5,
ArCH), 8.14 (1 H, dd, J 9.0, 2.5, ArCH), 7.94 (2 H, d, J 9.1,
ArCH), 7.77 (1 H, d, J 9.0, ArCH), 7.20 (2 H, d, J 8.6, ArCH),
6.84 (2 H, d, J 8.6, ArCH), 6.81 (2 H, d, J 9.1, ArCH), 4.25
(2 H, t, J 4.8, CH2), 4.18 (2 H, t, J 5.7, ArOCH2CH2), 3.86 (2 H,
t, J 5.7, ArOCH2CH2), 3.73–3.59 (18 H, m, CH2), 2.46 (1 H, s,
OH), 1.29 (3 H, t, J 7.1, CH2Me); δC(300 MHz; CDCl3) 172.2
(Ci), 157.9 (Ci), 153.5 (Ci), 152.1 (Ci), 147.5 (Ci), 144.8 (Ci),
134.3 (Ci), 130.9 (2 CH), 127.4 (CH), 127.1 (Ci), 126.4 (CH),
123.0 (CH), 118.4 (2 CH), 114.9 (2 CH), 112.0 (2 CH), 72.9
(CH2), 71.0 (CH2), 70.9 (2 CH2), 70.7 (CH2), 69.5 (CH2), 65.7
(CH2), 64.3 (CH2), 62.1 (CH2), 50.4 (CH2), 46.7 (CH2), 40.6
(CH2), 12.7 (CH3); m/z (EI) 658 (Mϩ), 660 (Mϩ ϩ 2); (CI) 659
(MHϩ), 661 (MHϩ ϩ 2) (Found: Mϩ, 658.2413. C32H3935ClN4O9
requires Mϩ, 658.2406); HMQC spectra exhibited a good
correlation with the proposed structure.
[4-(2-{[4-(2-Chloro-4-nitrophenylazo)phenyl]ethylamino}-
ethoxy)phenyl]acetic acid 2-(2-hydroxyethoxy)ethyl ester, 5a,
m ؍ 1 (DL21)
To a solution of 4a (200 mg, 0.41 mmol), triphenylphosphine
(162 mg, 0.62 mmol, 1.5 eq.) and diethyl azodicarboxylate
(108 mg, 0.62 mmol, 1.5 eq.) in toluene (20 mL), diethylene
glycol (66 mg, 0.62 mmol, 1.5 eq.) was added. The reaction
mixture was stirred for 5 days at room temperature after which
TLC (methanol–ethyl acetate, 1 : 9) indicated that the acid had
been entirely consumed. The toluene was removed at 50 ЊC
under reduced pressure and the product eluted by gradient
flash column chromatography (ethyl acetate–hexane, 4 : 1;
methanol–ethyl acetate, 1 : 9) to give 5a, m = 1 as a dark red oil
(190 mg, 0.33 mmol, 81%). νmax(Nujol)/cmϪ1 3451, 1731, 1599,
1514, 1335; δH(300 MHz; CDCl3) 8.38 (1 H, d, J 2.4, ArCH),
8.14 (1 H, dd, J 9.0, 2.4, ArCH), 7.95 (2 H, d, J 9.0, ArCH),
7.78 (1 H, d, J 9.0, ArCH), 7.21 (2 H, d, J 9.3, ArCH), 6.85
(2 H, d, J 9.0, ArCH), 6.82 (2 H, d, J 9.3, ArCH), 4.26 (2 H,
t, J 4.7, CH2), 4.18 (2 H, t, J 5.7, ArOCH2CH2), 3.86 (2 H, t,
J 5.6, ArOCH2CH2), 3.72–3.67 (4 H, m, CH2), 3.64–3.54
(6 H, m, CH2), 1.95 (1 H, s, OH), 1.29 (3 H, t, J 7.0, CH2Me);
δC(300 MHz; CDCl3) 172.2 (Ci), 158.0 (Ci), 153.5 (Ci), 152.0
(Ci), 147.5 (Ci), 144.8 (Ci), 134.3 (Ci), 130.8 (2 CH), 127.4
(CH), 127.0 (Ci), 126.4 (CH), 123.0 (CH), 118.4 (2 CH), 114.9
(2 CH), 112.0 (2 CH), 72.7 (CH2), 69.4 (CH2), 65.7 (CH2),
64.2 (CH2), 62.1 (CH2), 50.4 (CH2), 46.8 (CH2), 40.7 (CH2),
12.7 (CH3); m/z (CI) 571 (MHϩ), 573 (MHϩ ϩ 2) (Found:
MHϩ, 571.1947. C28H3135ClN4O7 requires MHϩ, 571.1960);
HMQC spectra exhibited a good correlation with the proposed
structure.
[4-(2-{[4-(2-Chloro-4-nitrophenylazo)phenyl]ethylamino}-
ethoxy)phenyl]acetic acid 2-(2-{2-[2-(2-hydroxyethoxy)ethoxy]-
ethoxy}ethoxy)ethyl ester, 5a, m ؍ 4 (DL51)
To a solution of 4a (200 mg, 0.41 mmol), triphenylphosphine
(162 mg, 0.62 mmol, 1.5 eq.) and diethyl azodicarboxylate
(108 mg, 0.62 mmol, 1.5 eq.) in toluene (20 mL) was added
pentaethylene glycol (148 mg, 0.62 mmol, 1.5 eq.). The reaction
mixture was stirred for 5 days at room temperature after which
TLC (methanol–ethyl acetate, 1 : 9) indicated that the acid had
been entirely consumed. The toluene was removed at 50 ЊC
under reduced pressure and the product eluted by gradient flash
column chromatography (ethyl acetate–hexane, 4 : 1; meth-
anol–ethyl acetate, 1 : 9) to give 5a, m = 4 as a dark red oil (225
mg, 0.32 mmol, 78%). (Found: C, 57.89; H, 6.17; N, 7.91.
C34H43ClN4O10 requires C, 58.07; H, 6.16; N, 7.97%); νmax(Nu-
jol)/cmϪ1 3466, 1731, 1599, 1518, 1336; δH(300 MHz; CDCl3)
8.37 (1 H, d, J 2.5, ArCH), 8.13 (1 H, dd, J 9.0, 2.5, ArCH),
7.94 (2 H, d, J 9.3, ArCH), 7.77 (1 H, d, J 9.0, ArCH), 7.21
(2 H, d, J 8.8, ArCH), 6.84 (2 H, d, J 8.8, ArCH), 6.81 (2 H, d,
J 9.3, ArCH), 4.24 (2 H, t, J 4.8, CH2), 4.17 (2 H, t, J 5.8,
ArOCH2CH2), 3.85 (2 H, t, J 5.8, ArOCH2CH2), 3.73–3.58
(22 H, m, CH2), 2.66 (1 H, s, OH), 1.29 (3 H, t, J 7.1, CH2Me);
δC(300 MHz; CDCl3) 172.2 (Ci), 157.9 (Ci), 153.5 (Ci), 152.1
[4-(2-{[4-(2-Chloro-4-nitrophenylazo)phenyl]ethylamino}-
ethoxy)phenyl]acetic acid 2-[2-(2-hydroxyethoxy)ethoxy]ethyl
ester, 5a m ؍ 2 (DL31)
To a solution of 4a (200 mg, 0.41 mmol), triphenylphosphine
(162 mg, 0.62 mmol, 1.5 eq.) and diethyl azodicarboxylate (108
mg, 0.62 mmol, 1.5 eq.) in toluene (20 mL) was added triethyl-
ene glycol (93 mg, 0.62 mmol, 1.5 eq.). The reaction mixture
was stirred for 5 days at room temperature after which TLC
(methanol–ethyl acetate, 1 : 9) indicated that the acid had been
entirely consumed. The toluene was removed at 50 ЊC under
reduced pressure and the product eluted by gradient flash col-
umn chromatography (ethyl acetate–hexane, 4 : 1; methanol–
ethyl acetate, 1 : 9) to give 5a, m = 2 as a dark red oil (180 mg,
0.29 mmol, 71%). νmax(Nujol)/cmϪ1 3439, 1736, 1599, 1513,
1336; δH(300 MHz; CDCl3) 8.37 (1 H, d, J 2.5, ArCH), 8.13
(1 H, dd, J 9.0, 2.5, ArCH), 7.93 (2 H, d, J 9.2, ArCH), 7.76
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 1 4 8 6 – 1 4 9 7
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(Ci), 147.5 (Ci), 144.8 (Ci), 134.3 (Ci), 130.9 (2 CH), 127.4
(CH), 127.1 (Ci), 126.4 (CH), 123.0 (CH), 118.4 (2 CH), 114.9
(2 CH), 112.0 (2 CH), 72.9 (CH2), 70.9 (5CH2), 70.7 (CH2), 69.4
(CH2), 65.7 (CH2), 64.3 (CH2), 62.1 (CH2), 50.4 (CH2), 46.7
(CH2), 40.6 (CH2), 12.7 (CH3); m/z (EI) 702 (Mϩ), 704 (Mϩϩ2);
(CI) 703 (MHϩ), 705 (MHϩϩ2); HMQC spectra exhibited a
good correlation with the proposed structure.
3-[4-(2-{[4-(2-Chloro-4-nitrophenylazo)phenyl]ethylamino}-
ethoxy)phenyl]propionic acid 2-[2-(2-hydroxyethoxy)ethoxy]-
ethyl ester, 5b, m ؍ 2 (DL32)
To a solution of 4b (200 mg, 0.40 mmol), triphenylphosphine
(158 mg, 0.60 mmol, 1.5 eq.) and diethyl azodicarboxylate
(104 mg, 0.60 mmol, 1.5 eq.) in toluene (20 mL), triethylene
glycol (91 mg, 0.60 mmol, 1.5 eq.) was added. The reaction
mixture was stirred for 5 days at room temperature after which
TLC (methanol–ethyl acetate, 1 : 9) indicated that the acid had
been entirely consumed. The toluene was removed at 50 ЊC
under reduced pressure and the product eluted by gradient
flash column chromatography (ethyl acetate–hexane, 4 : 1;
methanol–ethyl acetate, 1 : 9) to give 5b, m = 2 as a dark red oil
(175 mg, 0.28 mmol, 69%). νmax(Nujol)/cmϪ1 3456, 1736, 1599,
1514, 1337; δH(300 MHz; CDCl3) 8.36 (1 H, d, J 2.5, ArCH),
8.13 (1 H, dd, J 9.0, 2.5, ArCH), 7.93 (2 H, d, J 8.9, ArCH),
7.76 (1 H, d, J 9.0, ArCH), 7.12 (2 H, d, J 8.9, ArCH), 6.81
(4 H, d, J 8.9, ArCH), 4.23 (2 H, t, J 4.7, CH2), 4.16 (2 H, t,
J 5.7, ArOCH2CH2), 3.85 (2 H, t, J 5.7, ArOCH2CH2), 3.73–
3.58 (12 H, m, CH2), 2.89 (2 H, t, J 7.6, CH2CH2CO2), 2.63
(2 H, t, J 7.6, CH2CH2CO2), 2.41 (1 H, s, OH), 1.29 (3 H, t,
J 7.0, CH2Me); δC(300 MHz; CDCl3) 173.3 (Ci), 157.3 (Ci),
153.5 (Ci), 152.1 (Ci), 147.5 (Ci), 144.7 (Ci), 134.3 (Ci), 133.6
(Ci), 129.8 (2 CH), 127.4 (CH), 126.4 (CH), 123.0 (CH), 118.4
(2 CH), 114.8 (2 CH), 112.0 (2 CH), 72.9 (CH2), 70.9 (CH2),
70.7 (CH2), 69.5 (CH2), 65.7 (CH2), 63.8 (CH2), 62.1 (CH2),
50.4 (CH2), 46.7 (CH2), 36.4 (CH2), 30.4 (CH2), 12.7 (CH3); m/z
(EI) 628 (Mϩ), 630 (Mϩ ϩ 2); (CI) 629 (MHϩ), 631 (MHϩ ϩ 2)
(Found: Mϩ, 628.2296. C31H3735ClN4O8 requires Mϩ, 628.2300).
[4-(2-{[4-(2-Chloro-4-nitrophenylazo)phenyl]ethylamino}-
ethoxy)phenyl]acetic acid 2-[2-(2-{2-[2-(2-hydroxyethoxy)-
ethoxy]ethoxy}ethoxy)ethoxy]ethyl ester, 5a, m ؍ 5 (DL61)
To a solution of 4a (200 mg, 0.41 mmol), triphenylphosphine
(162 mg, 0.62 mmol, 1.5 eq.) and diethyl azodicarboxylate
(108 mg, 0.62 mmol, 1.5 eq.) in toluene (20 mL) was added
hexaethylene glycol (175 mg, 0.62 mmol, 1.5 eq.). The reaction
mixture was stirred for 5 days at room temperature after
which TLC (methanol–ethyl acetate, 1 : 9) indicated that the
acid had been entirely consumed. The toluene was removed
at 50 ЊC under reduced pressure and the product eluted by
gradient flash column chromatography (ethyl acetate–hexane,
4 : 1; methanol–ethyl acetate, 1 : 9) to give 5a, m = 5 as a
dark red oil (209 mg, 0.28 mmol, 68%). νmax(Nujol)/cmϪ1
3463, 1731, 1598, 1513, 1337; δH(300 MHz; CDCl3) 8.36 (1 H,
d, J 2.5, ArCH), 8.12 (1 H, dd, J 9.0, 2.5, ArCH), 7.93 (2 H,
d, J 9.2, ArCH), 7.76 (1 H, d, J 9.0, ArCH), 7.20 (2 H,
d, J 8.7, ArCH), 6.84 (2 H, d, J 8.7, ArCH), 6.81 (2 H, d,
J 9.2, ArCH), 4.24 (2 H, t, J 4.8, CH2), 4.17 (2 H, t, J 5.8,
ArOCH2CH2), 3.85 (2H, t, J 5.8, ArOCH2CH2), 3.73–3.58
(26 H, m, CH2), 2.76 (1 H, s, OH), 1.29 (3 H, t, J 7.1, CH2Me);
δC(300 MHz; CDCl3) 172.2 (Ci), 157.9 (Ci), 153.5 (Ci), 152.1
(Ci), 147.5 (Ci), 144.7 (Ci), 134.3 (Ci), 130.9 (2 CH), 127.4
(CH), 127.0 (Ci), 126.4 (CH), 123.0 (CH), 118.4 (2 CH), 114.9
(2 CH), 111.9 (2 CH), 72.9 (CH2), 71.0 (CH2), 70.9 (6CH2), 70.7
(CH2), 69.4 (CH2), 65.7 (CH2), 64.4 (CH2), 62.1 (CH2), 50.3
(CH2), 46.7 (CH2), 40.6 (CH2), 12.7 (CH3); m/z (EI) 746 (Mϩ),
748 (Mϩ ϩ 2); (CI) 747 (MHϩ), 749 (MHϩ ϩ 2) (Found: Mϩ,
746.2914. C36H4735ClN4O11 requires Mϩ, 746.2930); HMQC
spectra exhibited a good correlation with the proposed
structure.
3-[4-(2-{[4-(2-Chloro-4-nitrophenylazo)phenyl]ethylamino}-
ethoxy)phenyl]propionic acid 2-{2-[2-(2-hydroxyethoxy)ethoxy]-
ethoxy}ethyl ester, 5b, m ؍ 3 (DL42)
To a solution of 4b (200 mg, 0.40 mmol), triphenylphosphine
(158 mg, 0.60 mmol, 1.5 eq.) and diethyl azodicarboxylate
(104 mg, 0.60 mmol, 1.5 eq.) in toluene (20 mL) tetraethylene
glycol (117 mg, 0.60 mmol, 1.5 eq.) was added. The reaction
mixture was stirred for 5 days at room temperature after which
TLC (methanol–ethyl acetate, 1 : 9) indicated that the acid had
been entirely consumed. The toluene was removed at 50 ЊC
under reduced pressure and the product eluted by gradient
flash column chromatography (ethyl acetate–hexane, 4 : 1;
methanol–ethyl acetate, 1 : 9) to give 5b, m = 3 as a dark red oil
(190 mg, 0.28 mmol, 70%). νmax(Nujol)/cmϪ1 3457, 1731, 1599,
1519, 1337; δH(300 MHz; CDCl3) 8.35 (1 H, d, J 2.5, ArCH),
8.11 (1 H, dd, J 9.0, 2.5, ArCH), 7.92 (2 H, d, J 9.2, ArCH),
7.76 (1 H, d, J 9.0, ArCH), 7.12 (2 H, d, J 8.5, ArCH), 6.81
(2 H, d, J 8.5, ArCH), 6.80 (2 H, d, J 9.2, ArCH), 4.23 (2 H, t,
J 4.7, CH2), 4.16 (2 H, t, J 5.7, ArOCH2CH2), 3.84 (2 H, t, J 5.7,
ArOCH2CH2), 3.73–3.58 (16 H, m, CH2), 2.89 (2 H, t, J 7.7,
CH2CH2CO2), 2.62 (2 H, t, J 7.7, CH2CH2CO2), 2.61 (1 H, s,
OH), 1.29 (3 H, t, J 7.0, CH2Me); δC(300 MHz; CDCl3) 173.3
(Ci), 157.3 (Ci), 153.4 (Ci), 152.1 (Ci), 147.4 (Ci), 144.7 (Ci),
134.3 (Ci), 133.6 (Ci), 129.8 (2 CH), 127.4 (CH), 126.4 (CH),
123.0 (CH), 118.4 (2 CH), 114.8 (2 CH), 111.9 (2 CH),
72.9 (CH2), 71.0 (CH2), 70.9 (2CH2), 70.7 (CH2), 69.5 (CH2),
65.7 (CH2), 63.9 (CH2), 62.1 (CH2), 50.4 (CH2), 46.7 (CH2),
36.4 (CH2), 30.4 (CH2), 12.7 (CH3); m/z (EI) 672 (Mϩ),
674 (Mϩ ϩ 2); (CI) 673 (MHϩ), 675 (MHϩ ϩ 2) (Found: Mϩ,
672.2553. C33H4135ClN4O9 requires Mϩ, 672.2562).
3-[4-(2-{[4-(2-Chloro-4-nitrophenylazo)phenyl]ethylamino}-
ethoxy)phenyl]propionic acid 2-(2-hydroxyethoxy)ethyl ester, 5b,
m ؍ 1 (DL22)
To a solution of 4b (200 mg, 0.40 mmol), triphenylphosphine
(158 mg, 0.60 mmol, 1.5 eq.) and diethyl azodicarboxylate
(105 mg, 0.60 mmol, 1.5 eq.) in dichloromethane (20 mL) di-
ethylene glycol (64 mg, 0.60 mmol, 1.5 eq.) was added. The
reaction mixture was stirred for 3 hours at room temperature
after which TLC (methanol–ethyl acetate, 1 : 9) indicated that
the acid had been entirely consumed. The toluene was removed
at 50 ЊC under reduced pressure and the product eluted by
gradient flash column chromatography (ethyl acetate–hexane,
4 : 1; methanol–ethyl acetate, 1 : 9) to give 5b, m = 1 as a dark
red oil (174 mg, 0.30 mmol, 74%). νmax(Nujol)/cmϪ1 3443, 1731,
1599, 1513, 1336; δH(300 MHz; CDCl3) 8.36 (1 H, d, J 2.5,
ArCH), 8.13 (1 H, dd, J 9.0, 2.5, ArCH), 7.93 (2 H, d, J 9.1,
ArCH), 7.76 (1 H, d, J 9.0, ArCH), 7.11 (2 H, d, J 8.5, ArCH),
6.81 (2 H, d, J 8.5, ArCH), 6.80 (2 H, d, J 9.1, ArCH), 4.23
(2 H, t, J 4.6, CH2), 4.16 (2 H, t, J 5.6, ArOCH2CH2), 3.84 (2 H,
t, J 5.6, ArOCH2CH2), 3.73–3.55 (8 H, m, CH2), 2.89 (2 H, t,
J 7.6, CH2CH2CO2), 2.63 (2 H, t, J 7.6, CH2CH2CO2), 2.12
(1 H, s, OH), 1.29 (3 H, t, J 7.0, CH2Me); δC(300 MHz; CDCl3)
173.3 (Ci), 157.3 (Ci), 153.5 (Ci), 152.1 (Ci), 147.5 (Ci), 144.7
(Ci), 134.3 (Ci), 133.5 (Ci), 129.8 (2 CH), 127.4 (CH), 126.4
(CH), 123.0 (CH), 118.4 (2 CH), 114.8 (2 CH), 111.9 (2 CH),
72.7 (CH2), 69.5 (CH2), 65.7 (CH2), 63.9 (CH2), 62.1 (CH2),
50.4 (CH2), 46.7 (CH2), 36.4 (CH2), 30.4 (CH2), 12.7 (CH3); m/z
(CI) 585 (MHϩ), 587 (MHϩ ϩ 2) (Found: MHϩ, 585.2113.
C29H3335ClN4O7 requires MHϩ, 585.2116).
3-[4-(2-{[4-(2-Chloro-4-nitrophenylazo)phenyl]ethylamino}-
ethoxy)phenyl]propionic acid 2-(2-{2-[2-(2-hydroxyethoxy)-
ethoxy]ethoxy}ethoxy)ethyl ester, 5b, m ؍ 4 (DL52)
To a solution of 4b (200 mg, 0.40 mmol), triphenylphosphine
(158 mg, 0.60 mmol, 1.5 eq.) and diethyl azodicarboxylate
(104 mg, 0.60 mmol, 1.5 eq.) in toluene (20 mL), pentaethylene
glycol (144 mg, 0.60 mmol, 1.5 eq.) was added. The reaction
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 1 4 8 6 – 1 4 9 7
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mixture was stirred for 5 days at room temperature after
which TLC (methanol–ethyl acetate, 1 : 9) indicated that the
acid had been entirely consumed. The toluene was removed at
50 ЊC under reduced pressure and the product eluted by gradi-
ent flash column chromatography (ethyl acetate–hexane, 4 : 1;
methanol–ethyl acetate, 1 : 9) to give 5b, m = 4 as a dark red oil
(190 mg, 0.26 mmol, 66%). νmax(Nujol)/cmϪ1 3477, 1731, 1599,
1519, 1337; δH(300 MHz; CDCl3) 8.37 (1 H, d, J 2.5, ArCH),
8.13 (1 H, dd, J 9.1, 2.5, ArCH), 7.93 (2 H, d, J 8.8, ArCH),
7.77 (1 H, d, J 9.1, ArCH), 7.12 (2 H, d, J 8.8, ArCH), 6.81
(4 H, d, J 8.8, ArCH), 4.22 (2 H, t, J 4.8, CH2), 4.16 (2 H, t,
J 5.8, ArOCH2CH2), 3.85 (2 H, t, J 5.8, ArOCH2CH2), 3.73–
3.58 (20 H, m, CH2), 2.89 (2 H, t, J 7.7, CH2CH2CO2), 2.68 (1
H, s, OH), 2.62 (2 H, t, J 7.7, CH2CH2CO2), 1.29 (3 H, t, J 7.1,
CH2Me); δC(300 MHz; CDCl3) 173.3 (Ci), 157.3 (Ci), 153.5
(Ci), 152.1 (Ci), 147.5 (Ci), 144.7 (Ci), 134.3 (Ci), 133.6 (Ci),
129.8 (2 CH), 127.4 (CH), 126.4 (CH), 123.0 (CH), 118.4
(2 CH), 114.8 (2 CH), 111.9 (2 CH), 72.9 (CH2), 70.9 (5CH2),
70.7 (CH2), 69.5 (CH2), 65.7 (CH2), 64.0 (CH2), 62.1 (CH2),
50.4 (CH2), 46.7 (CH2), 36.4 (CH2), 30.4 (CH2), 12.7 (CH3); m/z
(CI) 717 (MHϩ), 719 (MHϩ ϩ 2) (Found: MHϩ, 717.2897.
C35H4335ClN4O10 requires MHϩ, 717.2902).
ArCH), 6.74 (2 H, d, J 8.5, ArCH), 2.82 (2 H, t, J 7.6,
ArCH2CH2), 2.51 (2 H, t, J 7.6, ArCH2CH2), 1.41 (9 H, s,
CiMe3); δC(300 MHz; CDCl3) 174.0 (Ci), 154.9 (Ci), 132.4 (Ci),
129.8 (2 CH), 115.8 (2 CH), 81.5 (Ci), 38.0 (CH2), 30.7 (CH2),
15.5 (CH3); m/z (CI) 240 (MNH4ϩ) (Found: MNH4ϩ, 240.1597.
C13H18O3 requires MNH4ϩ, 240.1600); COSY spectra exhibited
a good correlation with the proposed structure.
3-{4-[2-(2-{2-[4-(2-{[4-(2-Chloro-4-nitrophenylazo)phenyl]ethyl-
amino}ethoxy)phenyl]acetoxy}ethoxy)ethoxy]phenyl}propionic
acid tert-butyl ester, 7a, m ؍ 1
To a solution of 5a, m = 1 (144 mg, 0.25 mmol), triphenyl-
phosphine (98 mg, 0.38 mmol, 1.5 eq.) and diethyl azodicarb-
oxylate (66 mg, 0.38 mmol, 1.5 eq.) in dichloromethane (5 mL)
was added tert-butyl ester 6 (111 mg, 0.50 mmol, 2.0 eq.). The
reaction mixture was stirred at room temperature for 5 hours
after which time TLC (methanol–ethyl acetate, 1 : 9) indicated
that the starting material had been consumed. The dichloro-
methane was removed under reduced pressure and the product
isolated by gradient flash column chromatography (ethyl
acetate–hexane, 4 : 1; methanol–ethyl acetate, 1 : 9) to give 7a,
m = 1 as a dark red oil (102 mg, 0.13 mmol, 53%). νmax(Nujol)/
cmϪ1 1729, 1600, 1513, 1340; δH(300 MHz; CDCl3) 8.39 (1 H, d,
J 2.5, ArCH), 8.15 (1 H, dd, J 9.0, 2.5, ArCH), 7.95 (2 H, d,
J 9.2, ArCH), 7.78 (1 H, d, J 9.0, ArCH), 7.19 (2 H, d, J 8.7,
ArCH), 7.10 (2 H, d, J 8.7, ArCH), 6.84–6.79 (6 H, m, ArCH),
4.27 (2 H, t, J 4.8, ArOCH2CH2), 4.16 (2 H, t, J 5.8, ArO-
CH2CH2), 4.07 (2 H, t, J 4.8, OCH2CH2O2C), 3.87–3.74 (6 H,
m, CH2), 3.62 (2 H, q, J 7.2, CH2Me), 3.58 (2 H, s, ArCH2), 2.84
(2 H, t, J 7.6, CH2CH2CO2), 2.50 (2 H, t, J 7.6, CH2CH2CO2),
1.41 (9 H, s, CiMe3), 1.29 (3 H, t, J 7.2, CH2Me); m/z (FAB) 774
(MHϩ), 797 (MNaϩ) (Found: MHϩ, 775.3120. C41H4735ClN4O9
requires MHϩ, 775.3110); COSY spectra exhibited a good
correlation with the proposed structure.
3-[4-(2-{[4-(2-Chloro-4-nitrophenylazo)phenyl]ethylamino}-
ethoxy)phenyl]propionic acid 2-[2-(2-{2-[2-(2-hydroxyethoxy)-
ethoxy]ethoxy}ethoxy)ethoxy]ethyl ester, 5b, m ؍ 5 (DL62)
To a solution of 4b (200 mg, 0.40 mmol), triphenylphosphine
(158 mg, 0.60 mmol, 1.5 eq.) and diethyl azodicarboxylate
(104 mg, 0.60 mmol, 1.5 eq.) in toluene (20 mL), hexaethylene
glycol (170 mg, 0.60 mmol, 1.5 eq.) was added. The reaction
mixture was stirred for 5 days at room temperature after which
TLC (methanol–ethyl acetate, 1 : 9) indicated that the acid
had been entirely consumed. The toluene was removed at 50 ЊC
under reduced pressure and the product eluted by gradient
flash column chromatography (ethyl acetate–hexane, 4 : 1;
methanol–ethyl acetate, 1 : 9) to give 5b, m = 5 as a dark red oil
(184 mg, 0.24 mmol, 60%). νmax(Nujol)/cmϪ1 3455, 1732, 1600,
1519, 1337; δH(300 MHz; CDCl3) 8.37 (1 H, d, J 2.3, ArCH),
8.13 (1 H, dd, J 9.0, 2.3, ArCH), 7.94 (2 H, d, J 8.9, ArCH),
7.77 (1 H, d, J 9.0, ArCH), 7.12 (2 H, d, J 8.9, ArCH), 6.81
(4 H, d, J 8.9, ArCH), 4.22 (2 H, t, J 4.7, CH2), 4.17 (2 H, t,
J 5.7, ArOCH2CH2), 3.85 (2 H, t, J 5.7, ArOCH2CH2), 3.73–
3.58 (24 H, m, CH2Me), 2.89 (2 H, t, J 7.7, CH2CH2CO2), 2.79
(1 H, s, OH), 2.62 (2 H, t, J 7.7, CH2CH2CO2), 1.29 (3 H, t,
J 7.1, CH2Me); δC(300 MHz; CDCl3) 173.3 (Ci), 157.3 (Ci),
153.5 (Ci), 152.1 (Ci), 147.5 (Ci), 144.7 (Ci), 134.3 (Ci), 133.6
(Ci), 129.8 (2 CH), 127.4 (CH), 126.4 (CH), 123.0 (CH), 118.4
(2 CH), 114.8 (2 CH), 111.9 (2 CH), 73.0 (CH2), 71.0 (CH2),
70.9 (6CH2), 70.7 (CH2), 69.5 (CH2), 65.7 (CH2), 64.0 (CH2),
62.1 (CH2), 50.4 (CH2), 46.7 (CH2), 36.4 (CH2), 30.4 (CH2),
12.7 (CH3); m/z (CI) 761 (MHϩ), 763 (MHϩ ϩ 2) (Found: MHϩ,
761.3165. C37H4935ClN4O11 requires MHϩ, 761.3165).
3-(4-{2-[2-(2-{2-[4-(2-{[4-(2-Chloro-4-nitrophenylazo)phenyl]-
ethylamino}ethoxy)phenyl]acetoxy}ethoxy)ethoxy]ethoxy}-
phenyl)propionic acid tert-butyl ester, 7a, m ؍ 2
To a solution of 5a, m = 2 (168 mg, 0.27 mmol), triphenyl-
phosphine (108 mg, 0.41 mmol, 1.5 eq.) and diethyl azodicarb-
oxylate (71 mg, 0.41 mmol, 1.5 eq.) in dichloromethane (5 mL)
was added tert-butyl ester 6 (121 mg, 0.55 mmol, 2.0 eq.). The
reaction mixture was stirred at room temperature for 5 hours
after which time TLC (methanol–ethyl acetate, 1 : 9) indicated
that the starting material had been consumed. The dichloro-
methane was removed under reduced pressure and the product
isolated by gradient flash column chromatography (ethyl
acetate–hexane, 4 : 1; methanol–ethyl acetate, 1 : 9) to give 7a,
m = 2 as a dark red oil (123 mg, 0.15 mmol, 55%). νmax(Nujol)/
cmϪ1 1735, 1601, 1514, 1340; δH(300 MHz; CDCl3) 8.39 (1 H, d,
J 2.5, ArCH), 8.15 (1 H, dd, J 9.0, 2.5, ArCH), 7.95 (2 H,
d, J 9.2, ArCH), 7.78 (1 H, d, J 9.0, ArCH), 7.19 (2 H, d,
J 8.7, ArCH), 7.09 (2 H, d, J 8.7, ArCH), 6.85–6.79 (6 H,
m, ArCH), 4.24 (2 H, t, J 5.3, ArOCH2CH2), 4.17 (2 H, t,
J 5.7, ArOCH2CH2), 4.09 (2 H, t, J 5.1, OCH2CH2O2C), 3.87–
3.81 (4 H, m, CH2), 3.71–3.61 (8 H, m, CH2), 3.58 (2 H, s,
ArCH2), 2.83 (2 H, t, J 7.7, CH2CH2CO2), 2.49 (2 H, t, J 7.7,
CH2CH2CO2), 1.41 (9 H, s, CiMe3), 1.30 (3 H, t, J 7.1, CH2Me);
m/z (FAB) 819 (MHϩ), 841 (MNaϩ) (Found: MNaϩ, 841.3181.
C43H5135ClN4O10 requires MNaϩ, 841.3191); COSY spectra
exhibited a good correlation with the proposed structure.
3-(4-Hydroxyphenyl)propionic acid tert-butyl ester, 6
To a solution of 3-(4-hydroxyphenyl)propionic acid (3.32 g,
20 mmol) in DMF (20 mL) was carefully added carbonyl-
diimidazole (3.24 g, 20 mmol). The reaction mixture was stirred
at 40 ЊC for 2 hours. DBU (6.08 mL, 40 mmol, 2 eq.) and
tert-butanol (3.70 mL, 50 mmol, 2.5 eq.) were then added and
the reaction mixture stirred at 65 ЊC for 2 days after which time
TLC (ethyl acetate–hexane, 1 : 4) indicated that the starting
material had been consumed. The reaction mixture was cooled
to room temperature, water (40 mL) added and the product
extracted with diethyl ether (3 × 20 mL). The organic fraction
was dried (MgSO4), concentrated under reduced pressure and
the product isolated by flash column chromatography (ethyl
acetate–hexane, 1 : 4) to give 6 as a clear colourless oil (2.89 g,
13 mmol, 65%). νmax(Nujol)/cmϪ1 3400, 1700, 1601, 1516;
δH(300 MHz; CDCl3) 7.05 (1 H, s, ArOH), 7.01 (2 H, d, J 8.5,
3-[4-(2-{2-[2-(2-{2-[4-(2-{[4-(2-Chloro-4-nitrophenylazo)-
phenyl]-ethylamino}ethoxy)phenyl]acetoxy}ethoxy)ethoxy]-
ethoxy}ethoxy)phenyl]propionic acid tert-butyl ester, 7a,
m ؍ 3
To a solution of 5a, m = 3 (300 mg, 0.46 mmol), triphenyl-
phosphine (179 mg, 0.68 mmol, 1.5 eq.) and diethyl azodicarb-
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 1 4 8 6 – 1 4 9 7
1492

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oxylate (120 mg, 0.68 mmol, 1.5 eq.) in dichloromethane (5 mL)
was added tert-butyl ester 6 (202 mg, 0.91 mmol, 2.0 eq.).
The reaction mixture was stirred at room temperature for
5 hours after which time TLC (methanol–ethyl acetate, 1 : 9)
indicated that the starting material had been consumed. The
dichloromethane was removed under reduced pressure and the
product isolated by gradient flash column chromatography
(ethyl acetate–hexane, 4 : 1; methanol–ethyl acetate, 1 : 9) to
give 7a, m = 3 as a dark red oil (204 mg, 0.24 mmol, 52%).
νmax(Nujol)/cmϪ1 1733, 1601, 1513, 1266; δH(300 MHz; CDCl3)
8.38 (1 H, d, J 2.4, ArCH), 8.14 (1 H, dd, J 8.9, 2.4, ArCH),
7.94 (2 H, d, J 9.0, ArCH), 7.78 (1 H, d, J 8.9, ArCH), 7.19
(2 H, d, J 8.7, ArCH), 7.09 (2 H, d, J 8.7, ArCH), 6.86–6.79
(6 H, m, ArCH), 4.24 (2 H, t, J 5.3, ArOCH2CH2), 4.17 (2 H, t,
J 5.7, ArOCH2CH2), 4.09 (2 H, t, J 4.9, OCH2CH2O2C), 3.87–
3.56 (16 H, m, CH2), 2.83 (2 H, t, J 7.7, CH2CH2CO2), 2.49
(2 H, t, J 7.7, CH2CH2CO2), 1.41 (9 H, s, CiMe3), 1.30 (3 H, t,
J 7.1, CH2Me); m/z (FAB) 863 (MHϩ), 885 (MNaϩ) (Found:
MHϩ, 863.3619. C45H5535ClN4O11 requires MHϩ, 863.3634);
COSY spectra exhibited a good correlation with the proposed
structure.
7.19 (2 H, d, J 8.7, ArCH), 7.09 (2 H, d, J 8.7, ArCH),
6.86–6.79 (6 H, m, ArCH), 4.23 (2 H, t, J 4.8, ArOCH2CH2),
4.17 (2 H, t, J 5.7, ArOCH2CH2), 4.09 (2 H, t, J 4.9,
OCH2CH2O2C), 3.87–3.81 (4 H, m, CH2), 3.73–3.55 (22 H,
m, CH2), 2.83 (2 H, t, J 7.8, CH2CH2CO2), 2.49 (2 H, t,
J 7.8, CH2CH2CO2), 1.41 (9 H, s, CiMe3), 1.29 (3 H, t,
J 7.1, CH2Me); m/z (FAB) 951 (MHϩ), 973 (MNaϩ) (Found:
MHϩ, 951.4167. C49H6335ClN4O13 requires MHϩ, 951.4158);
COSY spectra exhibited a good correlation with the proposed
structure.
3-[4-(2-{[4-(2-Chloro-4-nitrophenylazo)phenyl]ethylamino}-
ethoxy)phenyl]propionic acid 2-{2-[4-(2-tert-butoxycarbonyl-
ethyl)phenoxy]ethoxy}ethyl ester, 7b, m ؍ 1
To a solution of 5b, m = 1 (178 mg, 0.30 mmol), triphenyl-
phosphine (120 mg, 0.46 mmol, 1.5 eq.) and diethyl azodicarb-
oxylate (78 mg, 0.45 mmol, 1.5 eq.) in dichloromethane (5 mL)
was added tert-butyl ester 6 (135 mg, 0.61 mmol, 2.0 eq.).
The reaction mixture was stirred at room temperature for
5 hours after which time TLC (methanol–ethyl acetate, 1 : 9)
indicated that the starting material had been consumed.
The dichloromethane was removed under reduced pressure
and the product isolated by gradient flash column chromato-
graphy (ethyl acetate–hexane, 4 : 1; methanol–ethyl acetate,
1 : 9) to give 7b, m = 1 as a dark red oil (138 mg, 0.18 mmol,
57%). νmax(Nujol)/cmϪ1 1735, 1600, 1513, 1339; δH(300 MHz;
CDCl3) 8.37 (1 H, d, J 2.4, ArCH), 8.13 (1 H, dd, J 9.0, 2.4,
ArCH), 7.94 (2 H, d, J 9.4, ArCH), 7.77 (1 H, d, J 9.0, ArCH),
7.11 (2 H, d, J 8.7, ArCH), 7.09 (2 H, d, J 8.9, ArCH), 6.83–
6.78 (6 H, m, ArCH), 4.24 (2 H, t, J 4.7, ArOCH2CH2), 4.16
(2 H, t, J 5.7, ArOCH2CH2), 4.08 (2 H, t, J 4.8, OCH2CH2O2C),
3.83 (2 H, t, J 5.7, ArOCH2CH2), 3.81 (2 H, t, J 4.7, ArO-
CH2CH2), 3.74 (2 H, t, J 4.8, OCH2CH2O2C), 3.61 (2 H, q,
J 7.1, CH2Me), 2.88 (2 H, t, J 7.8, CH2CH2CO2), 2.84 (2 H, t,
J 7.8, CH2CH2CO2), 2.61 (2 H, t, J 7.8, CH2CH2CO2), 2.49
(2 H, t, J 7.8, CH2CH2CO2), 1.41 (9 H, s, CiMe3), 1.28 (3 H, t,
J 7.1, CH2Me); m/z (FAB) 789 (MHϩ), 811 (MNaϩ) (Found:
MHϩ, 789.3252. C42H4935ClN4O9 requires MHϩ, 789.3266);
COSY spectra exhibited a good correlation with the proposed
structure.
3-{4-[2-(2-{2-[2-(2-{2-[4-(2-{[4-(2-Chloro-4-nitrophenylazo)-
phenyl]ethylamino}ethoxy)phenyl]acetoxy}ethoxy)ethoxy]-
ethoxy}ethoxy)ethoxy]phenyl}propionic acid tert-butyl ester, 7a,
m ؍ 4
To a solution of 5a, m = 4 (280 mg, 0.40 mmol), triphenyl-
phosphine (108 mg, 0.60 mmol, 1.5 eq.) and diethyl azodicarb-
oxylate (104 mg, 0.60 mmol, 1.5 eq.) in dichloromethane (5 mL)
was added tert-butyl ester 6 (177 mg, 0.80 mmol, 2.0 eq.).
The reaction mixture was stirred at room temperature for
5 hours after which time TLC (methanol–ethyl acetate, 1 : 9)
indicated that the starting material had been consumed. The
dichloromethane was removed under reduced pressure and the
product isolated by gradient flash column chromatography
(ethyl acetate–hexane, 4 : 1; methanol–ethyl acetate, 1 : 9)
to give 7a, m = 4 as a dark red oil (187 mg, 0.21 mmol,
53%). νmax(Nujol)/cmϪ1 1730, 1601, 1513, 1340; δH(300 MHz;
CDCl3) 8.37 (1 H, d, J 2.4, ArCH), 8.13 (1 H, dd, J 9.0,
2.4, ArCH), 7.94 (2 H, d, J 9.2, ArCH), 7.77 (1 H, d, J 9.0,
ArCH), 7.20 (2 H, d, J 8.9, ArCH), 7.09 (2 H, d, J 8.7,
ArCH), 6.86–6.79 (6 H, m, ArCH), 4.23 (2 H, t, J 4.8,
ArOCH2CH2), 4.17 (2 H, t, J 5.7, ArOCH2CH2), 4.09 (2 H,
t, J 5.0, OCH2CH2O2C), 3.87–3.81 (4 H, m, CH2), 3.73–3.58
(18 H, m, CH2), 2.83 (2 H, t, J 7.8, CH2CH2CO2), 2.49 (2 H, t,
J 7.8, CH2CH2CO2), 1.41 (9 H, s, CiMe3), 1.29 (3 H, t,
J 7.1, CH2Me); m/z (FAB) 907 (MHϩ), 929 (MNaϩ) (Found:
MHϩ, 907.3904. C47H5935ClN4O12 requires MHϩ, 907.3896);
COSY spectra exhibited a good correlation with the proposed
structure.
3-[4-(2-{[4-(2-Chloro-4-nitrophenylazo)phenyl]ethylamino}-
ethoxy)phenyl]propionic acid 2-(2-{2-[4-(2-tert-butoxycarbonyl-
ethyl)phenoxy]ethoxy}ethoxy)ethyl ester, 7b, m ؍ 2
To a solution of 5b m = 2 (767 mg, 1.22 mmol), triphenyl-
phosphine (480 mg, 1.83 mmol, 1.5 eq.) and diethyl azodicarb-
oxylate (318 mg, 1.83 mmol, 1.5 eq.) in dichloromethane (5 mL)
was added tert-butyl ester 6 (542 mg, 2.44 mmol, 2.0 eq.). The
reaction mixture was stirred at room temperature for 5 hours
after which time TLC (methanol–ethyl acetate, 1 : 9) indicated
that the starting material had been consumed. The dichloro-
methane was under reduced pressure and the product isolated
by gradient flash column chromatography (ethyl acetate–
hexane, 4 : 1; methanol–ethyl acetate, 1 : 9) to give 7b, m = 2 as a
dark red oil (386 mg, 0.46 mmol, 38%). νmax(Nujol)/cmϪ1 1736,
1600, 1512, 1339; δH(300 MHz; CDCl3) 8.37 (1 H, d, J 2.5,
ArCH), 8.13 (1 H, dd, J 9.0, 2.5, ArCH), 7.93 (2 H, d, J 9.4,
ArCH), 7.77 (1 H, d, J 9.0, ArCH), 7.11 (2 H, d, J 8.9, ArCH),
7.09 (2 H, d, J 8.9, ArCH), 6.83–6.79 (6 H, m, ArCH), 4.24
(2 H, t, J 4.7, ArOCH2CH2), 4.16 (2 H, t, J 5.7, ArOCH2CH2),
4.09 (2 H, t, J 4.9, OCH2CH2O2C), 3.86–3.81 (4 H, m, CH2),
3.75–3.58 (8 H, m, CH2), 2.88 (2 H, t, J 7.8, CH2CH2CO2), 2.83
(2 H, t, J 7.8, CH2CH2CO2), 2.61 (2 H, t, J 7.8, CH2CH2CO2),
2.49 (2 H, t, J 7.8, CH2CH2CO2), 1.41 (9 H, s, CiMe3), 1.29
(3 H, t, J 7.0, CH2Me); m/z (FAB) 833 (MHϩ), 855 (MNaϩ)
(Found: MHϩ, 833.3507. C44H5335ClN4O10 requires MHϩ,
833.3528); COSY spectra exhibited a good correlation with the
proposed structure.
3-(4-{2-[2-(2-{2-[2-(2-{2-[4-(2-{[4-(2-Chloro-4-nitrophenylazo)-
phenyl]ethylamino}ethoxy)phenyl]acetoxy}ethoxy)ethoxy]-
ethoxy}ethoxy)ethoxy]ethoxy}phenyl)propionic acid tert-butyl
ester, 7a, m ؍ 5
To a solution of 5a, m = 5 (245 mg, 0.33 mmol), triphenyl-
phosphine (129 mg, 0.49 mmol, 1.5 eq.) and diethyl azodicarb-
oxylate (86 mg, 0.49 mmol, 1.5 eq.) in dichloromethane (5 mL)
was added tert-butyl ester 6 (146 mg, 0.66 mmol, 2.0 eq.).
The reaction mixture was stirred at room temperature for
5 hours after which time TLC (methanol–ethyl acetate, 1 : 9)
indicated that the starting material had been consumed. The
dichloromethane was removed under reduced pressure and the
product isolated by gradient flash column chromatography
(ethyl acetate–hexane, 4 : 1; methanol–ethyl acetate, 1 : 9)
to give 7a, m = 5 as a dark red oil (184 mg, 0.19 mmol,
58%). νmax(Nujol)/cmϪ1 1731, 1601, 1514, 1340; δH(300 MHz;
CDCl3) 8.37 (1 H, d, J 2.4, ArCH), 8.13 (1 H, dd, J 9.1, 2.4,
ArCH), 7.94 (2 H, d, J 9.2, ArCH), 7.77 (1 H, d, J 9.1, ArCH),
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 1 4 8 6 – 1 4 9 7
1493

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3-[4-(2-{[4-(2-Chloro-4-nitrophenylazo)phenyl]ethylamino}-
ethoxy)phenyl]propionic acid 2-[2-(2-{2-[4-(2-tert-butoxy-
carbonylethyl)phenoxy]ethoxy}ethoxy)ethoxy]ethyl ester, 7b,
m ؍ 3
that the starting material had been consumed. The dichloro-
methane was removed under reduced pressure and the product
isolated by gradient flash column chromatography (ethyl
acetate–hexane, 4 : 1; methanol–ethyl acetate, 1 : 9) to give 7b,
m = 5 as a dark red oil (549 mg, 0.57 mmol, 56%). νmax(Nujol)/
cmϪ1 1727, 1601, 1513, 1340; δH(300 MHz; CDCl3) 8.37 (1 H, d,
J 2.5, ArCH), 8.13 (1 H, dd, J 9.0, 2.5, ArCH), 7.94 (2 H, d,
J 9.2, ArCH), 7.77 (1 H, d, J 9.0, ArCH), 7.11 (2 H, d, J 8.7,
ArCH), 7.09 (2 H, d, J 8.9, ArCH), 6.83–6.79 (6 H, m, ArCH),
4.21 (2 H, t, J 4.8, ArOCH2CH2), 4.16 (2 H, t, J 5.7, ArO-
CH2CH2), 4.09 (2 H, t, J 4.9, OCH2CH2O2C), 3.86–3.81 (4 H,
m, CH2), 3.73–3.56 (20 H, m, CH2), 2.89 (2 H, t, J 7.7,
CH2CH2CO2), 2.83 (2 H, t, J 7.7, CH2CH2CO2), 2.61 (2 H, t,
J 7.7, CH2CH2CO2), 2.49 (2 H, t, J 7.7, CH2CH2CO2), 1.31
(9 H, s, CiMe3), 1.29 (3 H, t, J 7.1, CH2Me); m/z (FAB) 965
(MHϩ), 987 (MNaϩ) (Found: MHϩ, 965.4323. C50H6535ClN4O13
requires MHϩ, 965.4315); COSY spectra exhibited a good
correlation with the proposed structure.
To a solution of 5b, m = 3 (470 mg, 0.70 mmol), triphenyl-
phosphine (275 mg, 1.05 mmol, 1.5 eq.) and diethyl azodicarb-
oxylate (183 mg, 1.05 mmol, 1.5 eq.) in dichloromethane (5 mL)
was added tert-butyl ester 6 (311 mg, 1.40 mmol, 2.0 eq.). The
reaction mixture was stirred at room temperature for 5 hours
after which time TLC (methanol–ethyl acetate, 1 : 9) indicated
that the starting material had been consumed. The dichloro-
methane was removed under reduced pressure and the product
isolated by gradient flash column chromatography (ethyl
acetate–hexane, 4 : 1; methanol–ethyl acetate, 1 : 9) to give 7b,
m = 3 as a dark red oil (291 mg, 0.33 mmol, 47%). νmax(Nujol)/
cmϪ1 1728, 1600, 1513, 1340; δH(300 MHz; CDCl3) 8.34 (1 H, d,
J 2.5, ArCH), 8.10 (1 H, dd, J 8.9, 2.5, ArCH), 7.92 (2 H, d,
J 9.2, ArCH), 7.75 (1 H, d, J 8.9, ArCH), 7.11 (2 H, d, J 8.7,
ArCH), 7.09 (2 H, d, J 8.7, ArCH), 6.84–6.77 (6 H, m, ArCH),
4.21 (2 H, t, J 4.8, ArOCH2CH2), 4.15 (2 H, t, J 5.7, ArO-
CH2CH2), 4.08 (2 H, t, J 4.9, OCH2CH2O2C), 3.85–3.80 (4 H,
m, CH2), 3.73–3.57 (12 H, m, CH2), 2.88 (2 H, t, J 7.7,
CH2CH2CO2), 2.83 (2 H, t, J 7.7, CH2CH2CO2), 2.61 (2 H, t,
J 7.7, CH2CH2CO2), 2.49 (2 H, t, J 7.7, CH2CH2CO2), 1.41
(9 H, s, CiMe3), 1.28 (3 H, t, J 7.1, CH2Me); m/z (FAB) 877
(MHϩ), 899 (MNaϩ) (Found: MHϩ, 877.3802. C46H5735ClN4O11
requires MHϩ, 877.3790); COSY spectra exhibited a good
correlation with the proposed structure.
General procedure for deprotection of 7a, m ؍ 1–5 and 7b,
m ؍ 1–5 to carboxylic acids
To a solution of TIS : TFA, 1 : 49 (10 mL) was added a solution
of 7a, m = 1 (200 mg, 258 µmol) in dichloromethane (1.5 mL).
The reaction mixture was stirred at room temperature for
10 minutes after which time TLC (ethyl acetate–hexane, 3 : 2)
indicated that the starting material had been consumed. The
reaction mixture was concentrated under reduced pressure and
the product isolated via flash column chromatography to give
the carboxylic acid as a red oil (158 mg, 220 µmol, 85%). The
acid derivative was then coupled directly to the supported
protected peptide as described below.
3-[4-(2-{[4-(2-Chloro-4-nitrophenylazo)phenyl]ethylamino}-
ethoxy)phenyl]propionic acid 2-{2-[2-(2-{2-[4-(2-tert-butoxy-
carbonylethyl)phenoxy]ethoxy}ethoxy)ethoxy]ethoxy}ethyl
ester, 7b, m ؍ 4
Synthesis of the protected and deprotected 16 residue peptide on
Tentagel® and the cleaved peptide
To a solution of 5b, m = 4 (420 mg, 0.59 mmol), triphenyl-
phosphine (231 mg, 0.88 mmol, 1.5 eq.) and diethyl azodicarb-
oxylate (154 mg, 0.89 mmol, 1.5 eq.) in dichloromethane
(5 mL) was added tert-butyl ester 6 (260 mg, 1.17 mmol,
2.0 eq.). The reaction mixture was stirred at room temperature
for 5 hours after which time TLC (methanol–ethyl acetate, 1 : 9)
indicated that the starting material had been consumed. The
dichloromethane was removed under reduced pressure and the
product isolated by gradient flash column chromatography
(ethyl acetate–hexane, 4 : 1; methanol–ethyl acetate, 1 : 9) to
give 7b, m = 4 as a dark red oil (235 mg, 0.26 mmol, 44%).
νmax(Nujol)/cmϪ1 1727, 1601, 1513, 1340; δH(300 MHz; CDCl3)
8.37 (1 H, d, J 2.5, ArCH), 8.13 (1 H, dd, J 9.0, 2.5, ArCH),
7.94 (2 H, d, J 9.2, ArCH), 7.77 (1 H, d, J 9.0, ArCH), 7.11 (2
H, d, J 8.7, ArCH), 7.09 (2 H, d, J 8.7, ArCH), 6.84–6.79 (6 H,
m, ArCH), 4.21 (2 H, t, J 4.8, ArOCH2CH2), 4.16 (2 H, t, J 5.7,
ArOCH2CH2), 4.09 (2 H, t, J 4.9, OCH2CH2O2C), 3.86–3.81
(4 H, m, CH2), 3.73–3.56 (16 H, m, CH2), 2.89 (2 H, t, J 7.7,
CH2CH2CO2), 2.83 (2 H, t, J 7.7, CH2CH2CO2), 2.61 (2 H, t,
J 7.7, CH2CH2CO2), 2.49 (2 H, t, J 7.7, CH2CH2CO2), 1.41
(9 H, s, CiMe3), 1.29 (3 H, t, J 7.1, CH2Me); m/z (FAB) 921
(MHϩ), 943 (MNaϩ) (Found: MHϩ, 921.4048. C48H6135ClN4O12
requires MHϩ, 921.4053); COSY spectra exhibited a good
correlation with the proposed structure.
The 16 residue peptide was sequentially synthesised via a
conventional manual solid phase Fmoc–TFA strategy on Ten-
tagel® polymer support. The peptide’s sequence was NH2–Asp–
Pro–Asp–Glu–Leu–Glu–His–Ala–Ala–Lys–Ser–Glu–Ala–
Ala–Ala–Lys–OH, where -lysine was the first amino acid
attached, and -aspartic acid was the last. The peptide was
prepared on a mixture of 90% Tentagel® and 10% acid labile
Rink linker.
The amino acids were all Fmoc protected at the amino
terminal, leaving the acid terminal unprotected. Additional
protecting groups were used for the side chains if necessary.
These were:
a) t-Butyl for aspartic acid, glutamic acid and serine.
b) t-Butyl carbamate for lysine.
c) Trityl for histidine.
The coupling reagent of choice was PyBOP, utilised with
DIPEA and HOBt in DMF. 2 g of Tentagel® with a loading of
0.60 mmol gϪ1 and 200 mg of Tentagel® Rink linker with a
loading of 0.40 mmol gϪ1 was used, giving a mixture with a
loading of 0.58 mmol gϪ1
.
The general amino acid coupling and Fmoc deprotection
procedures were:
a) The Tentagel® resin (1.0 eq.) was swollen for 30 minutes in
DMF (15 mL) in a peptide flask designed for nitrogen bubbling
and suction. Gentle mixing was afforded by bubbling nitrogen
through the suspension. The protected amino acid, (2.5 eq.),
PyBOP (2.5 eq.), HOBt (2.5 eq.) and DIPEA (5 eq.) were then
added and the mixing continued by bubbling nitrogen through
the solution for 3 hours. The solvent was then removed via
filtration and the support washed with DMF (2 × 20 mL), di-
chloromethane (2 × 20 mL), methanol (2 × 20 mL) and DMF
(1 × 20 mL). The conversion was verified by a negative Kaiser
and TNBS test. A double coupling was undertaken if necessary.
3-[4-(2-{[4-(2-Chloro-4-nitrophenylazo)phenyl]ethylamino}-
ethoxy)phenyl]propionic acid 2-(2-{2-[2-(2-{2-[4-(2-tert-butoxy-
carbonylethyl)phenoxy]ethoxy}ethoxy)ethoxy]ethoxy}ethoxy)-
ethyl ester, 7b, m ؍ 5
To a solution of 5b, m = 5 (767 mg, 1.01 mmol), triphenyl-
phosphine (397 mg, 1.51 mmol, 1.5 eq.) and diethyl azodicarb-
oxylate (264 mg, 1.51 mmol, 1.5 eq.) in dichloromethane (5 mL)
was added tert-butyl ester 6 (448 mg, 2.02 mmol, 2.0 eq.). The
reaction mixture was stirred at room temperature for 5 hours
after which time TLC (methanol–ethyl acetate, 1 : 9) indicated
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b) The resin functionalised with an Fmoc protected amino
acid was gently mixed in a solution of piperidine–DMF, 1 : 4,
by bubbling nitrogen through for 20 minutes. The solution was
then removed by filtration and the resin washed with DMF
(2 × 20 mL). The Kaiser and TNBS tests indicated successful
deprotection by yielding a positive result.
Upon coupling the last amino acid residue and removal
of its Fmoc protection, the bulk resin was washed with
DMF (2 × 20 mL), dichloromethane (2 × 20 mL) and
methanol (2 × 20 mL) and dried under reduced pressure to
give peptide on Tentagel® as a pale yellow resin (3.8 g, 71%).
The yield was based on the expected increase of the resin’s
weight.
Removal of the peptide’s side chain protecting groups and
partial cleavage of the peptide (i.e. from the 10% acid-cleavable
Rink linker) was performed on a small sample (200 mg) by
gently bubbling nitrogen through a suspension of the resin in a
solution of triisopropylsilane–TFA, 1 : 49 (2 mL) for 1 hour.
The solution was removed by filtration and the filtrate was
collected and dried under reduced pressure to give an off white
powder (6 mg). m/z (FAB) 1681 (Mϩ) (Found (ES) 561.2787
(MH33ϩ), 841.4056 (MH22ϩ), C70H114N21O27 requires (ES)
561.2809 (MH33ϩ), 841. 4175 (MH22ϩ)).
1, n = 2, m = 2; m/z (ES) 814 (MH33ϩ), 1221 (MH22ϩ), for
C110H156ClN25O36.
1, n = 2, m = 3; m/z (ES) 829 (MH33ϩ), 1243 (MH22ϩ), for
C112H160ClN25O37.
1, n = 2, m = 4; m/z (ES) 829 (MH33ϩ), 1243 (MH22ϩ), for
C114H164ClN25O38.
1, n = 2, m = 5; m/z (ES) 858 (MH33ϩ), 1287 (MH22ϩ), for
C116H168ClN25O39.
3-[4-(tert-Butyldimethylsilanyloxy)phenyl]propionic acid
tert-butyl ester, 94
To a solution of 6 (8.0 g, 36.0 mmol) in dichloromethane
(100 ml) was added dimethylaminopyridine (1–2 crystals), tri-
ethylamine (7.80 ml, 54.1 mmol, 1.5 eq.) and tert-butyl-
dimethylsilyl chloride (5.95 g, 39.6 mmol, 1.1 eq.). The reaction
was stirred at room temperature for 8 hours after which time
TLC (ethyl acetate–hexane, 1 : 9, and phosphomolybdic acid
(PMA) dip to visualise) indicated that the starting material had
been consumed. The reaction mixture was washed with water
(3 × 20 ml), dried (MgSO4) and concentrated under reduced
pressure. The product was isolated with flash column chrom-
atography (ethyl acetate–hexane, 1 : 9) to give 9 as a clear
colourless oil (9.60 g, 28.2 mmol, 79%). νmax(Nujol)/cmϪ1 1731,
1600, 1511; δH(300 MHz; CDCl3) 7.06 (2 H, d, J 8.5, ArCH),
6.77 (2 H, d, J 8.5, ArCH), 2.86 (2 H, t, J 7.7, ArCH2CH2), 2.52
(2 H, t, J 7.7, ArCH2CH2), 1.43 (9 H, s, CiMe3), 1.00 (9 H, s,
SiCiMe2), 0.20 (6 H, s, SiMe); δC(300 MHz; CDCl3) 172.7
(Ci), 154.3 (Ci), 133.8 (Ci), 129.6 (2 CH), 120.3 (2 CH), 80.6
(Ci), 37.7 (CH2), 30.8 (CH2), 28.5 (CH3), 26.1 (CH3), 18.6 (Ci),
Ϫ4.0 (CH3); m/z (EI) 336 (Mϩ); (CI) 354 (MNH4ϩ); COSY
spectra exhibited a good correlation with the proposed
structure.
The supported deprotected peptide was washed with DMF,
dichloromethane and methanol, and dried under reduced
pressure to give a pale yellow resin (160 mg).
Individual attachment of the dye-linkers 7a, m ؍ 1–5 and 7b,
m ؍ 1–5 to the supported 16 residue peptide
General preparation for attaching the ten dye-linkers to the
terminal amino group of the peptide, followed by deprotection
and partial cleavage of these systems from the polymer
supported was:
a) To a suspension of the peptide on Tentagel® (25 mg)
in DMF (1 mL) were added the dye-linker (20 mg,
21.0–27.9 µmol, excess), PyBOP (30 mg, 57.7 µmol, excess),
HOBt (30 mg, 222 µmol, excess) and DIPEA (3 drops,
∼30 mg, 233 µmol, excess) and gently mixed by bubbling
nitrogen through at room temperature for 3 hours. The
resin was then isolated by filtration and washed with DMF
(2 × 10 mL), dichloromethane (2 × 10 mL), methanol (2 ×
10 mL) and dried under reduced pressure to give the sup-
ported protected peptidyl dye-linker system as a black resin
(26 mg).
3-[4-(tert-Butyldimethylsilanyloxy)phenyl]-2-methylpropionic
acid tert-butyl ester, 105
A solution of 9 (4.5 g, 13.4 mmol) in tetrahydrofuran (25 ml)
was cooled to Ϫ78 ЊC and stirred for 30 minutes. To this
methyl iodide (8.2 ml, 132.4 mmol, 10 eq.), HMPA (16.2 ml,
132.4 mmol, 10 eq.) and LDA (2 M solution in THF), (7.3 ml,
14.6 mmol, 1.1 eq.) were added and stirred at Ϫ78 ЊC for
6 hours. The reaction mixture was then stirred at room
temperature overnight after which time TLC (diethyl ether–
hexane, 1 : 9) indicated that the starting material had been
consumed. The excess reagents and solvent were carefully
removed under reduced pressure and the product isolated via
flash column chromatography (ethyl acetate–hexane, 1 : 9)
to give 10 as a clear colourless oil (2.8 g, 8.0 mmol, 60%).
νmax(Nujol)/cmϪ1 1729, 1600, 1510; δH(300 MHz; CDCl3) 7.04
(2 H, d, J 8.4, ArCH), 6.76 (2 H, d, J 8.4, ArCH), 2.94–2.83
(1 H, m, ArCH2CH), 2.64–2.49 (2 H, m, ArCH2CH), 1.39 (9 H,
s, CiMe3), 1.12 (3 H, d, J 6.6, CHMe), 1.00 (9 H, s, SiCiMe3),
0.20 (6 H, s, SiMe2); δC(300 MHz; CDCl3) 172.7 (Ci), 154.3
(Ci), 133.8 (Ci), 129.6 (2 CH), 120.3 (2 CH), 80.6 (Ci), 42.9
(CH), 38.0 (CH2), 28.5 (CH3), 26.1 (CH3), 16.5 (CH3), 18.6 (Ci),
Ϫ4.0 (CH3); m/z (EI) 350 (Mϩ); (CI) 368 (MNH4ϩ) (Found:
MNH4ϩ, 368.2622. C20H34O3Si requires MNH4ϩ, 368.2621).
COSY spectra exhibited a good correlation with the proposed
structure.
b) The protected peptide dye-linker system (20 mg) was sus-
pended in triisopropylsilane–TFA, 1 : 49 (3 mL) and gently
mixed by bubbling nitrogen through at room temperature for
2 hours. The resin was isolated by filtration, washed with
methanol (2 × 10 mL), dichloromethane (2 × 10 mL) and dried
under reduced pressure to give the supported unprotected
peptide dye-linker system as a black resin (18 mg). The filtrate
was dried under reduced pressure to give a red powder (2 mg),
the result of cleavage of the 10% Rink linker. The peptide
dye-linker systems were identified from their filtrates by low
resolution electrospray mass spectrometry performed by the
EPSRC Swansea Mass Spectrometry Service as summarised
below.
1, n = 1, m = 1; m/z (ES) 795 (MH33ϩ), 1191 (MH22ϩ), for
C107H150ClN25O35.
1, n = 1, m = 2; m/z (ES) 809 (MH33ϩ), 1214 (MH22ϩ), for
C109H154ClN25O36.
3-(4-Hydroxyphenyl)-2-methylpropionic acid tert-butyl ester,
114
1, n = 1, m = 3; m/z (ES) 824 (MH33ϩ), 1236 (MH22ϩ), for
C111H158ClN25O37.
A solution of 10 (1.5 g, 4.29 mmol) in tetrahydrofuran (20 ml)
was cooled to Ϫ10 ЊC to which TBAF (1.34 g, 4.24 mmol) was
quickly added. The reaction mixture was stirred at room
temperature for 20 minutes after which time TLC (ethyl
acetate–hexane, 1 : 4 and PMA dip to visualise) indicated that
the starting material had been consumed. The reaction mix-
ture was diluted with diethyl ether (40 ml), washed with water
1, n = 1, m = 4; m/z (ES) 839 (MH33ϩ), 1258 (MH22ϩ), for
C113H162ClN25O38.
1, n = 1, m = 5; m/z (ES) 854 (MH33ϩ), 1280 (MH22ϩ), for
C115H166ClN25O39.
1, n = 2, m = 1; m/z (ES) 780 (MH33ϩ), 1199 (MH22ϩ), for
C108H152ClN25O35.
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(3 × 20 ml), dried (MgSO4) and concentrated under reduced
pressure. The product was isolated via flash column chromato-
graphy (ethyl acetate–hexane, 1 : 4) to give 11 as a clear
colourless oil (830 mg, 3.52 mmol, 82%). νmax(Nujol)/cmϪ1
3392, 1728, 1600, 1516; δH(300 MHz; CDCl3) 7.00 (2 H, d,
J 8.5, ArCH), 6.72 (2 H, d, J 8.5, ArCH), 6.53 (1 H, s,
ArOH), 2.89–2.80 (1 H, m, ArCH2CH), 2.64–2.49 (2 H, m,
ArCH2CH), 1.38 (9 H, s, CiMe3), 1.11 (3 H, d, J 6.6, CHMe);
δC(300 MHz; CDCl3) 177.0 (Ci), 154.3 (Ci), 132.6 (Ci), 131.5
(2 CH), 115.7 (2 CH), 81.0 (Ci), 43.2 (CH), 39.4 (CH2), 28.5
(CH3), 17.4 (CH3); m/z (EI) 236 (Mϩ); (CI) 254 (MNH4ϩ);
COSY spectra exhibited a good correlation with the proposed
structure.
3-[4-(2-{[4-(2-Chloro-4-nitrophenylazo)phenyl]ethylamino}-
ethoxy)phenyl]-2-methylpropionic acid 2-(2-{2-[2-(2-hydroxy-
ethoxy)ethoxy]ethoxy}ethoxy)ethyl ester, 126
To a solution of acid from the previous step (200 mg, 0.39
mmol) in dichloromethane (10 ml) was added triphenyl-
phosphine (113 mg, 0.43 mmol, 1.1 eq.), diethyl azodicarb-
oxylate (75 mg, 0.43 mmol, 1.1 eq.) and pentaglycol (0.11 ml,
0.51 mmol, 1.3 eq.). The reaction mixture was stirred at room
temperature for 4 hours after which time TLC indicated that the
starting material had been consumed. The reaction mixture was
diluted with further dichloromethane (20 ml), washed with
water (3 × 20 ml), dried (MgSO4) and concentrated under
reduced pressure. The product was isolated via flash column
chromatography to give 12 as a red oil (160 mg, 0.22 mmol,
56%). νmax(Nujol)/cmϪ1 1731, 1600, 1514, 1337; δH(300 MHz;
CDCl3) 8.39 (1 H, d, J 2.4, ArCH), 8.15 (1 H, dd, J 8.9, 2.4,
ArCH), 7.95 (2 H, d, J 9.3, ArCH), 7.78 (1 H, d, J 8.9, ArCH),
7.08 (2 H, d, J 8.7, ArCH), 6.81 (2 H, d, J 9.3, ArCH), 6.79
(2 H, d, J 8.7, ArCH), 4.24–4.15 (4 H, m, ArOCH2CH2), 3.86
(2 H, t, J 5.8, HOCH2CH2), 3.73–3.58 (20 H, m, CH2), 2.99–
2.93 (1 H, m, ArCH2CH), 2.75–2.58 (2 H, m, ArCH2CH),
1.30 (3 H, t, J 7.1, CH2Me), 1.13 (3 H, d, J 6.8, CHMe);
δC(300 MHz; CDCl3) 176.4 (Ci), 157.3 (Ci), 153.5 (Ci),
152.1 (Ci), 147.5 (Ci), 144.7 (Ci), 134.3 (Ci), 132.4 (Ci), 130.5
(2 CH), 127.4 (CH), 126.4 (CH), 123.0 (CH), 118.4 (2 CH),
114.6 (2 CH), 111.9 (2 CH), 72.9 (CH2), 71.0 (4 CH2), 70.7 (2
CH2), 69.5 (CH2), 65.7 (CH2), 63.8 (CH2), 62.1 (CH2), 50.4
(CH2), 46.7 (CH2), 41.9 (CH), 39.1 (CH2), 17.0 (CH3), 12.7
(CH3); m/z (FAB) 753 (MNaϩ), 755 (MNaHϩ ϩ 2) (Found:
MHϩ, 731.3053. C36H4735ClN4O10 requires MHϩ, 731.3059);
COSY spectra exhibited a good correlation with the proposed
structure.
3-[4-(2-{[4-(2-Chloro-4-nitrophenylazo)phenyl]ethylamino}-
ethoxy)phenyl]-2-methylpropionic acid tert-butyl ester6
To a solution of 11 (660 mg, 2.80 mmol) in dichloromethane
(10 ml) was added triphenylphosphine (734 mg, 2.80 mmol),
diethyl azodicarboxylate (0.44 ml, 2.80 mmol) and disperse red
(974 mg, 2.80 mmol). The reaction mixture was stirred at room
temperature for 3 hours after which time TLC (ethyl acetate–
hexane, 1 : 4) indicated that the starting material had been con-
sumed. The dichloromethane was removed under reduced pres-
sure and the product isolated via flash column chromatography
to give the product as a dark red oil (514 mg, 0.91 mmol, 33%).
νmax(Nujol)/cmϪ1, 1724, 1601, 1514, 1337; δH(300 MHz; CDCl3)
8.35 (1 H, d, J 2.5, ArCH), 8.11 (1 H, dd, J 8.9, 2.5, ArCH),
7.92 (2 H, d, J 9.0, ArCH), 7.75 (1 H, d, J 8.9, ArCH), 7.08
(2 H, d, J 9.0, ArCH), 6.79 (4 H, d, J 9.0, ArCH), 4.16 (2 H, t,
J 5.8, ArOCH2CH2), 3.84 (2 H, t, J 5.8, ArOCH2CH2), 3.61
(2 H, q, J 7.1, CH2Me), 2.94–2.80 (1 H, m, ArCH2CH), 2.63–
2.47 (2 H, m, ArCH2CH), 1.41 (9 H, s, CiMe3), 1.28 (3 H, t,
J 7.1, CH2Me), 1.09 (3 H, d, J 6.6, CHMe); δC(300 MHz;
CDCl3) 176.0 (Ci), 157.3 (Ci), 153.5 (Ci), 152.2 (Ci), 147.5
(Ci), 144.7 (Ci), 134.3 (Ci), 132.8 (Ci), 130.5 (2 CH), 127.4
(CH), 126.4 (CH), 123.0 (CH), 118.4 (2 CH), 114.6 (2 CH),
111.9 (2 CH), 81.5 (Ci), 65.7 (CH2), 50.4 (CH2), 46.7 (CH2),
42.9 (CH), 39.3 (CH2), 28.4 (CH3), 17.3 (CH3), 12.7 (CH3); m/z
(EI) 566 (Mϩ), 568 (Mϩ ϩ 2); (CI) 567 (MHϩ), 569 (MHϩ ϩ 2);
COSY spectra exhibited a good correlation with the proposed
structure.
3-[4-(2-{[4-(2-Chloro-4-nitrophenylazo)phenyl]ethylamino}-
ethoxy)phenyl]-2-methylpropionic acid 2-{2-[2-(2-{2-[4-(2-tert-
butoxycarbonylethyl)phenoxy]ethoxy}ethoxy)ethoxy]ethoxy}-
ethyl ester6
To a solution of 12 (105 mg, 144 µmol) in dichloromethane
was added triphenylphosphine (41.4 mg, 158 µmol, 1.1 eq.),
diethyl azodicarboxylate (28 mg, 158 µmol, 1.1 eq.) and 6
(32 mg, 144 µmol). The reaction mixture was stirred at room
temperature for 2 hours after which time TLC (ethyl acetate–
hexane, 1 : 4) indicated that the starting material had been
consumed. The reaction mixture was concentrated under
reduced pressure and the product isolated via flash column
chromatography (ethyl acetate–hexane, 1 : 4) to give the ester
(79 mg, 85 µmol, 59%); δH(300 MHz; CDCl3) 8.38 (1 H, d,
J 2.5, ArCH), 8.14 (1 H, dd, J 9.0, 2.5, ArCH), 7.95 (2 H, d,
J 9.2, ArCH), 7.78 (1 H, d, J 9.0, ArCH), 7.09 (2 H, d, J 8.5,
ArCH), 7.07 (2 H, d, J 8.7, ArCH), 6.82 (2 H, d, J 8.7,
ArCH), 6.80 (2 H, d, J 9.2, ArCH), 6.78 (2 H, d, J 8.5,
ArCH), 4.24–4.14 (4 H, m, ArOCH2CH2), 4.09 (2 H, t, J 4.9,
HOCH2CH2), 3.87–3.81 (4 H, m, CH2), 3.76–3.59 (16 H, m,
CH2), 2.99–2.92 (1 H, m, ArCH2CH), 2.83 (2 H, t, J 7.8,
ArCH2CH2), 2.75–2.57 (2 H, m, ArCH2CH), 2.49 (2 H, t,
J 7.8, ArCH2CH2), 1.41 (9 H, s, CiMe3), 1.29 (3 H, t, J 7.0,
CH2Me), 1.13 (3 H, d, J 6.8, CHMe); δC(300 MHz; CDCl3)
176.4 (Ci), 172.8 (Ci), 164.0 (Ci), 157.6 (Ci), 157.3 (Ci),
153.5 (Ci), 152.1 (Ci), 147.5 (Ci), 144.7 (Ci), 134.3 (Ci),
133.5 (Ci), 132.4 (Ci), 130.5 (2 CH), 129.6 (2 CH), 127.4
(CH), 126.4 (CH), 123.0 (CH), 118.4 (2 CH), 114.9 (2 CH),
114.6 (2 CH), 111.9 (2 CH), 80.7 (CH2), 71.2 (CH2), 71.0
(3CH2), 70.2 (CH2), 69.5 (CH2), 67.8 (CH2), 65.7 (CH2), 63.8
(CH2), 62.7 (CH2), 50.4 (CH2), 46.7 (CH2), 41.9 (CH), 39.1
(CH2), 37.7 (CH2), 30.7 (CH2), 28.5 (CH), 17.1 (CH3), 12.7
(CH3); m/z (FAB) 957 (MNaϩ), 959 (MNaϩϩ2) (Found:
MHϩ, 935.4216. C49H6335ClN4O12 requires MHϩ, 935.4209);
COSY spectra exhibited a good correlation with the proposed
structure.
3-[4-(2-{[4-(2-Chloro-4-nitrophenylazo)phenyl]ethylamino}-
ethoxy)phenyl]-2-methylpropionic acid7
To a solution of TIS : TFA, 1 : 49 (5 ml) was added a solution
of the t-butyl ester from the previous reaction (354 mg, 0.63
mmol) in dichloromethane (1 ml). The reaction mixture was
stirred at room temperature for 10 minutes after which time
TLC (ethyl acetate–hexane, 2 : 3) indicated that the starting
material had been consumed. The reaction mixture was concen-
trated under reduced pressure and the product isolated via flash
column chromatography to give the acid as a red oil (258 mg,
0.51 mmol, 81%). νmax(Nujol)/cmϪ1 1707, 1601, 1515, 1339;
δH(300 MHz; CDCl3) 8.35 (1 H, d, J 2.5, ArCH), 8.11 (1 H,
dd, J 8.9, 2.5, ArCH), 7.92 (2 H, d, J 9.0, ArCH), 7.75 (1 H, d,
J 8.9, ArCH), 7.08 (2 H, d, J 9.0, ArCH), 6.79 (4 H, d,
J 9.0, ArCH), 4.16 (2 H, t, J 5.8, ArOCH2CH2), 3.85 (2 H, t,
J 5.8, ArOCH2CH2), 3.61 (2 H, q, J 7.1, CH2Me), 3.01–2.93
(1 H, m, ArCH2CH), 2.74–2.58 (2 H, m, ArCH2CH), 1.29 (3 H,
t, J 7.1, CH2Me), 1.15 (3 H, d, J 6.6, CHMe); δC(300 MHz;
CDCl3) 176.0 (Ci), 157.3 (Ci), 153.5 (Ci), 152.2 (Ci), 147.5
(Ci), 144.7 (Ci), 134.3 (Ci), 132.8 (Ci), 130.5 (2 CH), 127.4
(CH), 126.4 (CH), 123.0 (CH), 118.4 (CH), 115.7 (CH), 114.7
(2 CH), 111.9 (2 CH), 65.7 (CH2), 50.3 (CH2), 46.7 (CH2), 41.8
(CH), 38.9 (CH2), 16.9 (CH3), 12.7 (CH3); m/z (EI) 510
(Mϩ), 512 (Mϩ ϩ 2); (CI) 511 (MHϩ), 513 (MHϩ ϩ 2) (Found
MHϩ, 511.1751. C26H2735ClN4O5 requires MHϩ, 511.1748);
COSY spectra exhibited a good correlation with the proposed
structure.
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3-[4-(2-{[4-(2-Chloro-4-nitrophenylazo)phenyl]ethylamino}-
ethoxy)phenyl]-2-methylpropionic acid 2-{2-[2-(2-{2-[4-(2-
carboxyethyl)phenoxy]ethoxy}ethoxy)ethoxy]ethoxy}ethyl
ester7
formamide (2 × 10 ml), dichloromethane (2 × 10 ml), methanol
(2 × 10 ml) and dried under reduced pressure to give a black
resin (11 mg). This was suspended in triisopropylsilane : TFA,
1 : 49 (3 ml) and gently mixed by bubbling nitrogen through at
room temperature for 2 hours. The resin was isolated by fil-
tration, washed with methanol (2 × 10 ml), dichloromethane
(2 × 10 ml) and dried under reduced pressure to give the sup-
ported unprotected peptide chiral dye-linker system 8 as a black
resin (7 mg). The filtrate was dried under reduced pressure to
give a red powder (1 mg), the result of cleavage of the 10% Rink
resin.
To a solution of TIS : TFA, 1 : 49 (3 ml) was added a solution
of ester from the previous step (20 mg, 21 µmol) in dichloro-
methane (0.5 ml). The reaction mixture was stirred at room
temperature for 10 minutes after which time TLC (ethyl acet-
ate–hexane, 2 : 3) indicated that the starting material had been
consumed. The reaction mixture was concentrated under
reduced pressure and the product isolated via flash column
chromatography to give the acid as a red oil (14.5 mg, 16.5
µmol, 79%); δH(300 MHz; CDCl3) 8.38 (1 H, d, J 2.5, ArCH),
8.14 (1 H, dd, J 9.0, 2.5, ArCH), 7.95 (2 H, d, J 9.2, ArCH),
7.78 (1 H, d, J 9.0, ArCH), 7.09 (2 H, d, J 8.5, ArCH), 7.07
(2 H, d, J 8.7, ArCH), 6.82 (2 H, d, J 8.7, ArCH), 6.80 (2 H, d,
J 9.2, ArCH), 7.68 (2 H, d, J 8.5, ArCH), 4.24–4.14 (4 H, m,
ArOCH2CH2), 4.09 (2 H, t, J 4.9, HOCH2CH2), 3.87–3.81 (4 H,
m, CH2), 3.76–3.59 (16 H, m, CH2), 2.99–2.92 (1 H, m,
ArCH2CH), 2.83 (2 H, t, J 7.8, ArCH2CH2), 2.75–2.57 (2 H, m,
ArCH2CH), 2.49 (2 H, t, J 7.8, ArCH2CH2), 1.29 (3 H, t, J 7.0,
CH2Me), 1.11 (3 H, d, J 6.8, CHMe); δC(300 MHz; CDCl3)
176.4 (Ci), 172.8 (Ci), 157.6 (Ci), 157.3 (Ci), 153.5 (Ci), 152.1
(Ci), 147.5 (Ci), 144.7 (Ci), 134.3 (Ci), 133.5 (Ci), 132.4 (Ci),
130.5 (2 CH), 129.6 (2 CH), 127.4 (CH), 126.4 (CH), 123.0
(CH), 118.4 (2 CH), 114.9 (2 CH), 114.6 (2 CH), 111.9 (2 CH),
80.7 (CH2), 71.2 (CH2), 71.0 (3CH2), 70.2 (CH2), 69.5 (CH2),
67.8 (CH2), 65.7 (CH2), 63.8 (CH2), 62.7 (CH2), 50.4 (CH2),
46.7 (CH2), 41.9 (CH), 39.1 (CH2), 37.7 (CH2), 30.7 (CH2), 17.1
(CH3), 12.7 (CH3); m/z (FAB) 901 (MNaϩ), 903 (MNaϩ ϩ 2)
(Found: MHϩ, 879.8577. C45H5535ClN4O12 requires MHϩ,
879.3583); COSY spectra exhibited a good correlation with the
proposed structure.
Acknowledgements
We thank the EPSRC and Syngenta for a CASE award (to
D. J. M.), and Professor D. Games and Dr B. Stein of the
Swansea EPSRC Mass Spectrometry service for the analysis of
peptide derivatives. We wish to acknowledge the use of the
EPSRC Chemical Database Service at Daresbury.8
References
1 (a) T. D. H. Bugg, An Introduction to Enzyme and Coenzyme
Chemistry, 1st edn., Blackwell Science Ltd, Oxford, 1997;
(b) A. Fersht, Structure and Mechanism in Protein Science,
W. H. Freeman and Company, New York, 1999; (c) J. Kraut,
Ann. Rev. Biochem., 1977, 46, 331.
2 (a) A. J. Kirby, Angew. Chem., Int. Ed. Engl., 1996, 35, 707;
(b) Y. Fukushima, Bull. Chem. Soc. Jpn., 1996, 69, 2269; (c) J. Nilsson
and L. Baltzer, Chem. Eur. J., 2000, 6, 2214; (d ) K. S. Broo,
L. Brive, P. Ahlberg and L. Baltzer, J. Am. Chem. Soc., 1997, 119,
11362; (e) B. W. Matthews, C. S. Craik and H. Neurath, Proc. Natl.
Acad. Sci. U. S. A., 1994, 91, 4103; ( f ) M. Z. Atassi and
T. Manshouri, Proc. Natl. Acad. Sci. U. S. A., 1993, 90, 8282;
(g) V. T. D’Sousa and M. L. Bender, Acc. Chem. Res., 1987, 20, 146;
(h) S. J. Pollack, P. Hsiun and P. G. Schultz, J. Am. Chem. Soc., 1989,
111, 5961; (i) R. Breslow, P. J. Duggan and J. P. Light, J. Am. Chem.
Soc., 1992, 114, 3982.
Synthesis of the peptidyl chiral dye-linker systems, 8
3 M. P. Fitzsimons and J. K. Barton, J. Am. Chem. Soc., 1997, 119,
3379.
The method for attaching the chiral dye-linker to the terminal
amino group of the peptide, followed by deprotection and par-
tial cleavage of the system from the polymer support was as
follows: to a suspension of the peptide on Tentagel® (10 mg) in
dimethylformamide (1 ml) was added the dye-linker from the
previous experiment (10 mg, 11.4 µmol, excess), PyBOP (30 mg,
57.7 µmol, excess), HOBt (30 mg, 222 µmol, excess) and DIPEA
(3 drops, ∼30 mg, 233 µmol, excess) and gently mixed by bub-
bling nitrogen through at room temperature for 3 hours. The
resin was then isolated by filtration and washed with dimethyl-
4 E. J. Corey and A. Venkateswarlu, J. Am. Chem. Soc., 1972, 94, 6190.
5 R. Noyori, Y. Morita and M. Suzuki, J. Org. Chem., 1989, 54, 1785.
6 (a) O. Mitsunobu, Synthesis, 1981, 1; (b) M. L. Maddox, M. Varasi
and K. A. M. Walker, J. Org. Chem., 1987, 52, 4235; J. J. Bergan,
E. J. J. Grabowski, D. L. Hughes and R. A. Reamer, J. Am. Chem.
Soc., 1988, 110, 6487.
7 M. L. Baker, M. Blanchette, C. A. Guindon and D. A. Pearson,
Tetrahedron Lett., 1989, 30, 2739.
8 D. A. Fletcher, R. F. McMeeking and D. Parkin, J. Chem. Inf.
Comput. Sci., 1996, 36, 746.
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 1 4 8 6 – 1 4 9 7
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