Photorelease of tyrosine from α-carboxy-6-nitroveratryl (αCNV) derivatives

Article abstract of DOI:10.1039/c2pp05320a

The synthesis of photolabile tyrosine derivatives protected on the phenolic oxygen by the α-carboxy-6-nitroveratryl (αCNV) protecting group is described. The compounds undergo rapid photolysis at wavelengths longer than 300 nm to liberate the corresponding phenol in excellent yield (quantum yield for the deprotection of tyrosine = 0.19). Further protection of caged tyrosine is possible, yielding N-Fmoc protected derivatives suitable for direct incorporation of caged tyrosine in solid-phase peptide synthesis.

Full text of DOI:10.1039/c2pp05320a

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PAPER
Photorelease of tyrosine from α-carboxy-6-nitroveratryl (αCNV)
derivatives†‡
Alexander G. Russell,a Matthew J. Sadler,a Helen J. Laidlaw,a Agustín Gutiérrez-Loriente,a
Christopher W. Wharton,b David Carteau,c Dario M. Bassani*c and John S. Snaith*a
Received 29th September 2011, Accepted 30th November 2011
DOI: 10.1039/c2pp05320a
The synthesis of photolabile tyrosine derivatives protected on the phenolic oxygen by the α-carboxy-6-
nitroveratryl (αCNV) protecting group is described. The compounds undergo rapid photolysis at
wavelengths longer than 300 nm to liberate the corresponding phenol in excellent yield (quantum yield
for the deprotection of tyrosine = 0.19). Further protection of caged tyrosine is possible, yielding N-Fmoc
protected derivatives suitable for direct incorporation of caged tyrosine in solid-phase peptide synthesis.
Photolabile protecting groups have found application in a
number of different areas, such as the synthesis of complex
organic molecules,1 the preparation of spatially-addressable
arrays of macromolecules2 and microlithography.3 In particular,
the temporal and spatial resolution offered by the photorelease of
biomolecules has led to a very fertile area of research in which
biomolecules rapidly released from a ‘caged’ form are used to
initiate a biological reaction that may be studied by fast X-ray
diffraction,4 infrared spectroscopy,5 voltage clamp, patch clamp
or other physiological recording techniques.6 Photolabile protect-
ing groups have been available for some time for carboxylic
acids, phosphates and metal ions,7 while more recently attention
has turned to the photoliberation of phenols.8–13 This area is of
considerable interest, as it includes several families of biologi-
cally-active phenols including steroids, catecholamines14 such as
epinephrine and dopamine, as well as tyrosine and a host of tyro-
sine-containing peptides.15 For the latter, the development of
caged tyrosine derivatives compatible with automated peptide
synthesis that can be photo-deprotected in high yields and with
rapid kinetics using long-wavelength irradiation remains a
challenge.
photolysis kinetics of 2000 s−1 14a
, but its lower excitation wave-
length can lead to protein damage in biological samples.
The α-carboxy-6-nitroveratryl (αCNV) protecting group has
good kinetics for cleavage by light at wavelengths >350 nm and
it has been applied to the caging of amines,16 thiols17 and car-
boxylic acids.18 It has also been used to cage the phenol capsai-
cin for use in physiological experiments, although the kinetics
for capsaicin release have not been reported.19 Given the poten-
tial importance of caged tyrosine and tyrosine-containing pep-
tides, we set out to determine the applicability of αCNV to the
protection of tyrosine. For comparison, and to allow us to carry
out photolysis experiments in aqueous and organic media, we
studied the photoprotection of 4-tert-butylphenol in parallel.
To introduce the protecting group we used the previously pre-
pared bromide 1 (Scheme 1).18 The allyl ester has the advantage
that it may be removed by saponification or under the mild con-
ditions of palladium(0) catalysis. Alkylation of 4-tert-butylphe-
nol 2 and N-(tert-butoxycarbonyl)tyrosine methyl ester 3 by 1 in
Derivatives of the 2-nitrobenzyl group have been applied to
the caging of phenols. The 6-nitroveratryl protecting group may
be removed by photolysis at wavelengths up to 420 nm, but the
timescale for phenol release is only 220 s−1 14a
By contrast, the
.
α-carboxy-2-nitrobenzyl (αCNB) protecting group shows good
aSchool of Chemistry, University of Birmingham, Edgbaston,
Birmingham, U.K., B15 2TT. E-mail: j.s.snaith@bham.ac.uk
bSchool of Biosciences, University of Birmingham, Edgbaston,
Birmingham, U.K., B15 2TT
cISM CNRS UMR 5255, Université Bordeaux 1, 33405 Talence, France.
E-mail: d.bassani@ism.u-bordeaux1.fr
†This paper is part of a themed issue on photoremovable protecting
groups: development and applications.
‡Electronic supplementary information (ESI) available: 1H and 13C
NMR spectra for all compounds, details of photolysis kinetics and
quantum yield determinations. See DOI: 10.1039/c2pp05320a
Scheme 1 Synthesis of monoacids 6 and 7.
556 | Photochem. Photobiol. Sci., 2012, 11, 556–563
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THF with cesium carbonate as base proceeded smoothly, afford-
ing 4 and 5 in 93% and 86% yields, respectively, the latter as a
1 : 1 mixture of diastereoisomers. Ester saponification and, in the
case of 5, TFA treatment to remove the N-tert-butoxycarbonyl
group, gave good yields of the two target compounds 6 and 7.
The triacid derivatives 12 and 13 (Scheme 2) were also pre-
pared by a similar route. Trialkylation of 3,4-dihydroxyphenyl-
acetic acid by ethylbromoacetate, followed by nitration, gave 8
in 75% yield. Diazotransfer followed by treatment with HBr in
acetic acid proceeded in good yield to give 9, which smoothly
reacted with 2 and 3 in the presence of cesium carbonate to
provide 10 and 11 in 87% and 56% yields, respectively. Sapo-
nification of 10 gave 12, while saponification and further TFA
treatment in the case of 11 afforded the tyrosine derivative 13.
Fig. 1 Photolysis of 10 in acetone-d6 monitored by 1H NMR.
Table 1 Photolysis results
Entry Compound photolyzed Photolysis producta Photolysis yieldb
1
2
3
4
5
6
7
8
9
4
6
5
7
10
12
11
11
13
2
2
3
85%
79%
89%
90%
75%
88%
65%
80%e
92%
tyrosinec
2
2
3
3d
tyrosinec
a Photolysis performed in acetone using a 400 W high pressure mercury
lamp and a Pyrex filter, unless otherwise stated. b Yield determined by
1H NMR, using an internal standard. c Photolysis performed in D2O.
d Photolysis performed in acetonitrile. e Yield determined by HPLC.
The quantum yields for release of tyrosine from 7 and 13 in
water were determined by chemical actinometry to be 0.19 and
0.11, respectively. Laser flash photolysis (λex = 355 nm) of sol-
utions of 7 and 13 in water revealed the rapid formation of a
transient absorption with a maximum at 420 nm (see Fig. 2 for
the data from 13), assigned to the aci-nitro intermediate based on
its similarity with previously reported spectra. The rate of disap-
pearance of 7 was determined by monitoring the transient
absorption at 420 nm, which decayed monoexponentially on the
microsecond timescale (k = 2.4 × 104 s−1) towards the initial
absorbance level. The aci-nitro intermediate from 13 decayed at
Scheme 2 Synthesis of triacids 12 and 13.
Photolysis of esters 4, 5, 10 and 11 (in acetone) and acids 6, 7,
12 and 13 (in D2O or in acetone) was performed with the sample
in a 5 mm NMR tube using a Pyrex-filtered 400 W medium-
1
a similar rate of 2.3 × 104 s−1 23
These values are two orders of
.
pressure mercury lamp, with monitoring by H NMR or HPLC
(Table 1). The tyrosine derivatives exhibited good aqueous solu-
bility at pH 7.4, and in all cases the compounds were comforta-
bly soluble at the 30 mM concentration used for the photolysis
experiments. While the limits of solubility were not determined,
triacid 13 did not appear to show a significant solubility advan-
tage over 7, although the additional carboxylates may engender
improved solubility to peptides incorporating a caged tyrosine
residue. The photolyses proceeded smoothly, with typically 50%
product release within 20–30 min, and a plateau of between
80–90% released product being achieved after 1–2 h (Fig. 1).20
Although the products are cleanly released, prolonged photolysis
leads to darkening of the solution. It is assumed that the plateau
results from the production of UV-absorbing by-products that act
as light filters as it is known that the nitroso carbonyl compounds
that are released readily dimerize to form azodioxy com-
pounds,21 and these have been observed to reduce to diazo com-
pounds under photolytic conditions;22 both would be strongly
UV-absorbing.
magnitude faster than nitroveratryl-protected phenols and an
order of magnitude greater than CNB-protected phenols.14a
The decay of the aci-nitro transient has previously been
assumed to correlate with product release, and this has been
confirmed in the case of ATP release from ‘caged ATP’, the di-
sodium salt of adenosine-5′- triphosphate-[P3-(1-(2-nitrophenyl)
ethyl)] ester,24 for pH > 6.25 However, detailed investigations by
Corrie and co-workers26 and by Wirz and co-workers24 on the
release of alcohols from nitrobenzyl ether derivatives showed
that the hydrolysis of the hemi-acetal can be rate-limiting for
poor leaving groups, with the rate-limiting step being dependent
on solvent, pH and the nature of the leaving group. Although we
believe that the phenoxide anion can be considered a moderately
good leaving group for which the decay of the aci-nitro inter-
mediate may be correlated to product release,7e further exper-
iments are needed to corroborate this assumption. Thus, the
observed kinetic rates should be taken as an upper limit for the
rate of product release.
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Fig. 2 Transient absorption spectrum of 13 in water 10 μs after pulsed excitation at 355 nm and decay of the transient absorption monitored at
420 nm. The red line represents the best fit according to a mono-exponential decay with k = 2.3 × 104 s−1
.
Photolysis of the Mosher’s amide of 7 cleanly released the
Mosher’s amide of tyrosine. Comparison of the 19F NMR
spectra of this material with those from the Mosher’s amide of
racemic tyrosine indicated that, within the limits of detection, the
tyrosine released by photolysis was a single enantiomer.
The caging of tyrosine residues within peptides is of consider-
able potential interest, and so we set about the synthesis of two
orthogonally-protected, caged tyrosine building blocks, 19 and
20 (Scheme 3), that would be compatible with Fmoc solid-phase
peptide synthesis.
order of magnitude faster than αCNB-protected phenols,14a as
judged by decay of the aci-nitro intermediate. The methodology
should be applicable to the protection and release of neurotrans-
mitters such as catecholamines, while the orthogonally-pro-
tected, caged tyrosine building blocks 19 and 20 will allow for
the Fmoc solid-phase synthesis of caged tyrosine-containing
peptides.
Experimental
Allyl 2-(4-(t-butyl)phenoxy)-2-(4,5-dimethoxy-2-nitrophenyl)
acetate (4)
4-t-Butylphenol (0.068 g, 0.45 mmol) and Cs2CO3 (0.150 g,
0.46 mmol) were stirred in THF (20 mL) at 0 °C for 30 min
before 1 (0.150 g, 0.42 mmol) in THF (20 mL) was added. The
reaction was stirred at rt overnight before the THF was removed
in vacuo and the residue partitioned between water and EtOAc.
The organic phase was washed with 1 M NaOH and brine, dried
with MgSO4 and concentrated in vacuo. Purification by column
chromatography (hexane/EtOAc, 7 : 3) afforded ether 4 (0.180 g,
93%) as a yellow oil: Rf 0.50; νmax/cm−1 (film) 3099, 2963,
2868, 1751, 1649, 1609, 1583, 1524, 1512; δH (300 MHz,
CDCl3) 1.25 (9 H, s), 3.91 (3 H, s), 3.94 (3 H, s), 4.65 (2 H, dd,
J = 5.5, 1.2 Hz), 5.19 (1 H, dd, J = 10.7, 1.1 Hz), 5.25 (1 H, dd,
J = 17.3, 1.1 Hz), 5.77–5.92 (1 H, m), 6.62 (1 H, s), 6.92 (2 H,
d, J = 8.8 Hz), 7.27 (2 H, d, J = 8.8 Hz), 7.33 (1 H, s), 7.70 (1
H, s); δC (75 MHz, CDCl3) 31.4, 34.2, 56.4, 56.5, 66.3, 75.7,
108.2, 109.7, 115.5, 118.6, 126.5, 126.8, 131.3, 140.3, 145.2,
148.7, 153.8, 155.1, 168.1; m/z (electrospray) 452 (100%,
[M+Na]+); HRMS (electrospray) Calcd for C23H27NO7Na:
452.1685. Found: 452.1679.
Scheme 3 Synthesis of caged tyrosine derivatives 19 and 20.
Synthesis of tert-butyl ester 20 required alkylating agent 14.
Treatment of 3,4-dimethoxy-6-nitrophenylacetic acid with O-
tert-butyl-N,N-dicyclohexylisourea afforded the tert-butyl ester
in 97% yield. Diazo transfer and bromination as before gave 14,
which was used to alkylate N-trifluoroacetamidotyrosine methyl
ester 15 to give 17 in 93% yield. Basic hydrolysis of the methyl
ester and the trifluoroacetamide, followed by Fmoc protection of
the amine gave the orthogonally-protected tyrosine derivative 19
in 82% yield. In a similar fashion, N-Boc tyrosine tert-butyl
ester 16 was alkylated using the allyl ester 1, affording 18; treat-
ment with TFA, followed by Fmoc protection gave 20.
(2S)-Methyl 3-(4-(2-(allyloxy)-1-(4,5-dimethoxy-2-nitrophenyl)-
2-oxoethoxy)phenyl)-2-((t-butoxycarbonyl)amino)propanoate (5)
N-Boc-L-tyrosine methyl ester (0.271 g, 0.92 mmol) and
Cs2CO3 (0.300 g, 0.92 mmol) were stirred in THF (20 mL) at
0 °C for 30 min before 1 (0.299 g, 0.83 mmol) in THF (20 mL)
was added. The reaction was stirred at rt overnight before the
THF was removed in vacuo and the residue partitioned between
water and EtOAc. The organic phase was washed with 1 M
NaOH and brine, dried with MgSO4 and concentrated in vacuo.
In conclusion, we have developed a convenient route to
αCNV phenolic ethers, applicable to a range of phenols includ-
ing tyrosine derivatives. These compounds photolyse two orders
of magnitude faster than nitroveratryl-protected phenols and an
558 | Photochem. Photobiol. Sci., 2012, 11, 556–563
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Purification by column chromatography (hexane/EtOAc, 7 : 3)
afforded ether 5 (0.410 g, 86%) as a yellow solid: Rf 0.25; mp
49–52 °C; νmax/cm−1 (film) 3388, 3062, 2978, 2939, 2851,
1748, 1715, 1649, 1612, 1583, 1524, 1510; δH (300 MHz,
CDCl3) 1.32 (9 H, s), 2.82–3.04 (2 H, m), 3.62 (3 H, s), 3.85 (3
H, s), 3.89 (3 H, s), 4.41–4.50 (1 H, m), 4.60 (2 H, d, J = 5.5
Hz), 4.99 (1 H, d, J = 8.1 Hz), 5.14 (1 H, d, J = 11.4 Hz), 5.19
(1 H, d, J = 17.3 Hz), 5.72–5.87 (1 H, m), 6.56 (1 H, s), 6.86 (2
H, d, J = 8.3 Hz), 6.98 (2 H, d, J = 8.3 Hz), 7.23 (1 H, s), 7.65
(1 H, s); δC (75 MHz, CDCl3) 28.2, 37.4, 52.1, 54.4, 56.35,
56.44, 66.3, 75.4, 79.8, 108.2, 109.6, 115.9, 118.6, 126.3, 130.1,
130.5, 131.2, 140.2, 148.8, 153.7, 155.0, 156.3, 167.8, 172.2;
m/z (electrospray) 597 (100%, [M+Na]+); HRMS (electrospray)
Calcd for C28H34N2O11Na: 597.2060. Found: 597.2037.
(3.00 g, 17.8 mmol) were stirred in anhydrous DMF (80 mL) at
rt for 7 d. The mixture was poured into water, extracted with
Et2O and the organic phase was washed with saturated aqueous
NaHCO3, dried over MgSO4 and concentrated in vacuo to leave
a pale orange oil (6.56 g, 87%). The crude product was of suffi-
cient purity (>95%) to be used directly, but a portion was
purified by column chromatography (hexane/EtOAc, 3 : 2)
to give 2-(3,4-bis(ethoxycarbonylmethoxy)phenyl)acetic acid
ethoxycarbonylmethyl ester as a colorless oil: Rf 0.41; νmax
/
cm−1 (film) 2984, 2940, 1744, 1594, 1514; δH (300 MHz,
CDCl3) 1.10–1.18 (9 H, m), 3.53 (2 H, s), 4.00–4.17 (6 H, m),
4.49 (2 H, s), 4.58 (2 H, s), 4.60 (2 H, s), 6.65–6.80 (3 H, m);
δC (75 MHz, CDCl3) 13.8, 13.9, 39.8, 60.8, 60.9, 61.1, 66.4,
66.5, 115.5, 116.4, 123.0, 127.6, 147.0, 147.8, 167.3, 168.6,
168.7, 170.5; m/z (electrospray) 449 (100%, [M+Na]+); HRMS
(electrospray) Calcd for C20H26O10Na: 449.1424. Found:
449.1433.
2-(4-(t-Butyl)phenoxy)-2-(4,5-dimethoxy-2-nitrophenyl)acetic
acid (6)
2 M NaOH (10 mL) and 4 (0.184 g, 0.43 mmol) were stirred in
THF (20 mL) overnight before the THF was removed in vacuo.
The aqueous phase was acidified to pH 2 with 2 M HCl and
extracted with EtOAc. The organic phase was washed with
brine, dried with MgSO4 and evaporated to afford acid 6
(0.115 g, 69%) as a yellow oil: νmax/cm−1 (film) 3421, 3019,
1729, 1524; δH (300 MHz, acetone-d6) 1.26 (9 H, s), 3.91 (3 H,
s), 3.95 (3 H, s), 6.60 (1 H, s), 7.01 (2 H, d, J = 8.8 Hz), 7.33 (2
H, d, J = 8.8 Hz), 7.38 (1 H, s), 7.73 (1 H, s); δC (75 MHz,
acetone-d6) 31.7, 34.6, 56.6, 56.7, 76.0, 109.1, 110.9, 116.1,
127.19, 127.21, 141.5, 145.5, 149.9, 154.6, 156.2, 169.5; m/z
(electrospray) 412 (100%, [M+Na]+); HRMS (electrospray)
Calcd for C20H23NO7Na: 412.1372. Found: 412.1358.
2-(4,5-Bis(ethoxycarbonylmethoy-2-nitro)phenyl)acetic acid
ethoxycarbonylmethyl ester (8)
Fuming nitric acid (1.2 mL) and 2-(3,4-bis(ethoxycarbonyl-
methoxy)phenyl)acetic acid ethoxycarbonylmethyl ester (6.17 g,
14.5 mmol) were stirred in anhydrous CH2Cl2 (100 mL) at 0 °C
for 30 min. Saturated aqueous NaHCO3 was added until effer-
vescence ceased. The layers were separated and the organic
phase was washed with water, dried over MgSO4 and concen-
trated in vacuo to yield an off-white solid (5.88 g, 86%). The
crude product was of sufficient purity (>95%) to be used
directly, but a portion was purified by column chromatography
(hexane/EtOAc, 3 : 2) to give product 8 as a white solid: Rf 0.30;
mp 76–78 °C (from EtOAc); νmax/cm−1 (film) 2984, 2940,
1749, 1619, 1584, 1526; δH (300 MHz, CDCl3) 1.18–1.35 (9 H,
m), 4.06 (2 H, s), 4.16–4.31 (6 H, m), 4.63 (2 H, s), 4.76 (2 H,
s), 4.81 (2 H, s), 6.79 (1 H, s), 7.72 (1 H, s); δC (75 MHz,
CDCl3) 14.15, 14.18, 14.21, 39.4, 61.3, 61.6, 61.7, 61.8, 66.4,
66.6, 112.2, 117.9, 125.1, 141.8, 146.9, 152.0, 167.6, 167.8,
167.9, 169.5; m/z (electrospray) 494 (100%, [M+Na]+); HRMS
(electrospray) Calcd for C20H25NO12 Na: 494.1274. Found:
494.1269.
(1S)-1-Carboxy-2-(4-(carboxy(4,5-dimethoxy-2-nitrophenyl)
methoxy)phenyl)ethanaminium 2,2,2-trifluoroacetate (7)
2 M NaOH (10 mL) and 5 (0.144 g, 0.25 mmol) were stirred in
THF (20 mL) overnight before the THF was removed in vacuo.
The aqueous phase was acidified to pH 2 with 2 M HCl and
extracted with EtOAc. The organic phase was washed with
brine, dried with MgSO4 and evaporated to leave a yellow oil.
TFA (1 mL) was added and the solution was stirred for 24 h.
Removal of the TFA in vacuo afforded product 7 (0.097 g, 73%)
as a yellow solid: mp 137–141 °C; νmax/cm−1 (film) 2931, 2856,
1727, 1665, 1611, 1584, 1509; δH (300 MHz, acetone-d6,
mixture of diastereoisomers) 3.35–3.53 (2 H, m), 3.92 and 3.93
(3 H, 2 × s), 3.97 (3 H, s), 4.45–4.55 and 5.13–5.20 (1 H, 2 ×
m), 6.61 and 6.64 (1 H, 2 × s), 7.04–7.12 (2 H, m), 7.30–7.38 (3
H, m), 7.74 (1 H, s); δC (75 MHz, acetone-d6, mixture of diaster-
eoisomers) 35.4, 36.6, 54.9, 56.3, 56.4, 62.9, 75.6, 75.7, 108.8,
110.8, 116.5, 116.76, 116.82, 126.5, 126.6, 128.7, 129.6, 131.5,
131.6, 141.3, 149.6, 154.2, 157.5, 157.6, 168.9, 169.4, 170.4;
m/z (electrospray) 443 (18%, [M+Na]+), 421 (100%, [M+H]+);
HRMS (electrospray) Calcd for C19H20N2O9Na: 443.1067.
Found: 443.1070.
Diethyl 2,2′-((4-(1-diazo-2-(2-ethoxy-2-oxoethoxy)-2-oxoethyl)-5-
nitro-1,2-phenylene)bis(oxy))diacetate
DBU (0.79 mL, 5.3 mmol) was added to a solution of 8 (2.26 g,
4.8 mmol) in anhydrous acetonitrile (40 mL). After stirring for
5 min, methanesulfonyl azide (0.64 g, 5.3 mmol) was added and
the resulting mixture was stirred in the dark for 3 h. The solution
was concentrated in vacuo and the residue partitioned between
CH2Cl2 and water. The organic phase was dried over MgSO4
and evaporated in vacuo. Purification by column chromato-
graphy (hexane/EtOAc, 7 : 3) afforded diethyl 2,2′-((4-(1-diazo-
2-(2-ethoxy-2-oxoethoxy)-2-oxoethyl)-5-nitro-1,2-phenylene)
bis(oxy))diacetate (1.43 g, 60%) as a yellow solid: Rf 0.23; mp
92–96 °C; νmax/cm−1 (film) 3058, 2986, 2118, 1757, 1708,
1578, 1526; δH (300 MHz, CDCl3) 1.18–1.33 (9 H, m),
4.18–4.31 (6 H, m), 4.71 (2 H, s), 4.78 (2 H, s), 4.80 (2 H, s),
7.09 (1 H, s), 7.69 (1 H, s); δC (75 MHz, CDCl3) 14.1, 14.2,
2-(3,4-Bis(ethoxycarbonylmethoxy)phenyl)acetic acid
ethoxycarbonylmethyl ester
K2CO3 (7.64 g, 55.4 mmol), ethyl bromoacetate (6.14 mL,
9.25 g, 55.4 mmol) and 2-(3,4-dihydroxyphenyl)acetic acid
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61.2, 61.7, 61.8, 66.3, 66.5, 112.0, 115.8, 117.2, 140.5, 147.4,
151.8, 164.5, 167.6, 167.76, 167.77; m/z (electrospray) 520
(100%, [M+Na]+); HRMS (electrospray) Calcd for
C20H23N3O12Na: 520.1179. Found: 520.1172.
chromatography (hexane/EtOAc, 7 : 3) afforded ether 11
(0.310 g, 56%) as a yellow oil: Rf 0.36; νmax/cm−1 (film) 3430,
3061, 2984, 1753, 1714, 1611, 1585, 1526, 1510; δH (300 MHz,
CDCl3) 1.06–1.29 (9 H, m), 1.33 (9 H, s), 2.84–3.02 (2 H, m),
3.61 (3 H, s), 4.00–4.08 (4 H, m), 4.13 (2 H, q, J = 7.1 Hz),
4.40–4.50 (1 H, m), 4.55–4.69 (2 H, m), 4.73–4.79 (4 H, m),
4.96 (1 H, broad d, J = 7.7 Hz), 6.58 (1 H, s), 6.85 (2 H, d, J =
8.3 Hz), 6.96 (2 H, d, J = 8.3 Hz), 7.22 (1 H, s), 7.63 (1 H, s);
δC (75 MHz, CDCl3) 14.0, 14.1, 28.2, 37.3, 52.2, 54.4, 61.5,
61.60, 61.61, 66.0, 66.4, 74.9, 79.8, 111.8, 112.8, 116.0, 126.6,
130.2, 130.5, 140.8, 147.3, 152.3, 156.3, 166.7, 167.3, 167.52,
167.54, 167.8, 172.2; m/z (electrospray) 787 (100%, [M+Na]+);
HRMS (electrospray) Calcd for C35H44N2O17Na: 787.2538.
Found: 787.2552; Anal. calcd for C35H44N2O17: C, 54.97; H,
5.80; N, 3.66. Found: C, 54.86; H, 5.52; N, 3.29.
2-(4,5-Bis(ethoxycarbonylmethoxy)-2-nitrophenyl)-2-
bromoacetic acid ethoxycarbonylmethyl ester (9)
HBr (48% in acetic acid, 0.62 mL) was added to a solution of
diethyl 2,2′-((4-(1-diazo-2-(2-ethoxy-2-oxoethoxy)-2-oxoethyl)-
5-nitro-1,2-phenylene)bis(oxy))diacetate (1.30 g, 2.61 mmol) in
anhydrous CH2Cl2 (40 mL). After stirring for 30 min, the sol-
ution was washed with saturated aqueous NaHCO3, dried with
MgSO4 and concentrated in vacuo to afford bromide 9 (1.16 g,
81%) as a yellow oil: νmax/cm−1 (film) 2985, 1751, 1655, 1613,
1582, 1527; δH (300 MHz, CDCl3) 1.18–1.32 (9 H, m),
4.16–4.31 (6 H, m), 4.67 (2 H, s), 4.77 (2 H, s), 4.86 (2 H, s),
6.35 (1 H, s), 7.39 (1 H, s), 7.59 (1 H, s); δC (75 MHz, CDCl3)
13.78, 13.85, 13.88, 41.8, 61.74, 61.77, 61.78, 62.3, 66.3, 66.5,
111.8, 117.5, 126.2, 141.1, 148.3, 152.6, 167.5, 167.7, 168.1,
168.3; m/z (electrospray) 574 (90%, [M+Na]+), 572 (100%,
[M+Na]+); HRMS (electrospray) Calcd for C20H24NO12BrNa:
572.0380. Found: 572.0360.
2,2′-((4-((4-(t-Butyl)phenoxy)(carboxy)methyl)-5-nitro-1,2-
phenylene)bis(oxy))diacetic acid (12)
2 M NaOH (5 mL) and 10 (0.1 g, 0.16 mmol) in THF (10 mL)
were stirred overnight before the THF was removed in vacuo.
The aqueous phase was acidified to pH 2 with 2 M HCl and
extracted with EtOAc. The organic phase was washed with
brine, dried with MgSO4 and evaporated to leave acid 12
(0.050 g, 65%) as a yellow oil: νmax/cm−1 (film) 3023, 2964,
2870, 1755, 1584, 1521, 1292, 1192; δH (300 MHz, acetone-d6)
1.26 (9 H, s), 4.95 (2 H, s), 4.98 (2 H, s), 6.54 (1 H, s), 7.01 (2
H, d, J = 8.8 Hz), 7.33 (2 H, d, J = 8.8 Hz), 7.44 (1 H, s), 7.80
(1 H, s); δC (75 MHz, acetone-d6) 31.1, 34.0, 65.86, 65.92, 75.6,
111.8, 113.4, 115.7, 126.6, 127.4, 141.4, 145.0, 147.8, 152.5,
155.7, 168.7, 168.8, 169.1; m/z (electrospray) 500 (100%,
[M+Na]+); HRMS (electrospray) Calcd for C22H23NO11Na:
500.1169. Found: 500.1152.
Diethyl 2,2′-((4-(1-(4-(t-butyl)phenoxy)-2-(2-ethoxy-2-
oxoethoxy)-2-oxoethyl)-5-nitro-1,2-phenylene)bis(oxy))diacetate
(10)
4-t-Butylphenol (0.030 g, 0.2 mmol) and Cs2CO3 (0.065 g,
0.2 mmol) were stirred in THF (20 mL) at 0 °C for 30 min
before 9 (0.100 g, 0.18 mmol) in THF (20 mL) was added. After
stirring overnight at rt, the THF was removed in vacuo and the
residue partitioned between water and EtOAc. The organic phase
was washed with 1 M NaOH and brine, dried with MgSO4 and
concentrated in vacuo. Purification by column chromatography
(hexane/EtOAc, 11 : 9) afforded ether 10 (0.097 g, 87%) as a
yellow oil: Rf 0.58; νmax/cm−1 (film) 3064, 2965, 2908, 2871,
1756, 1608, 1584, 1527, 1511; δH (300 MHz, CDCl3) 1.06–1.32
(18 H, m), 4.04 (2 H, q, J = 7.1 Hz), 4.14 (2 H, q, J = 7.2 Hz),
4.23 (2 H, q, J = 7.1 Hz), 4.52–4.65 (2 H, m), 4.66–4.75 (4 H,
m), 6.62 (1 H, s), 6.88 (2 H, d, J = 8.8 Hz), 7.24 (2 H, d, J = 8.8
Hz), 7.28 (1 H, s), 7.68 (1 H, s); δC (75 MHz, CDCl3) 14.0,
14.1, 31.4, 34.1, 61.52, 61.56, 61.61, 61.62, 66.0, 66.4, 75.0,
111.9, 112.6, 115.3, 126.5, 127.0, 140.8, 145.3, 147.3, 152.3,
155.0, 166.8, 167.4, 167.6, 167.8; m/z (electrospray) 642 (100%,
[M+Na]+); HRMS (electrospray) Calcd for C30H37NO13Na:
642.2163. Found: 642.2169.
(1S)-2-(4-((4,5-Bis(carboxymethoxy)-2-nitrophenyl)(carboxy)
methoxy)phenyl)-1-carboxyethanaminium 2,2,2-trifluoroacetate
(13)
2 M NaOH (10 mL) and 11 (0.120 g, 0.16 mmol) in THF
(20 mL) were stirred overnight before the THF was removed
in vacuo. The aqueous phase was acidified to pH 2 with 2 M
HCl and extracted with EtOAc. The organic phase was washed
with brine, dried with MgSO4 and evaporated to leave a yellow
oil. TFA (1 mL) was added and the solution was stirred for 24 h.
Removal of the TFA in vacuo afforded product 13 (0.066 g,
68%) as an orange gum: νmax/cm−1 (film) 3430, 3055, 2987,
1732, 1511; δH (300 MHz, D2O) 2.83–3.06 (2 H, m), 4.02–4.10
(1 H, m), 4.54 (3 H, s), 4.60 (3 H, s), 6.23 (1 H, s), 6.71 (2 H, d,
J = 8.1 Hz), 6.88–6.97 (3 H, m), 7.43 (1 H, s); δC (75 MHz,
D2O) 34.0, 53.4, 64.6, 64.9, 74.2, 110.3, 112.0, 115.7, 125.3,
127.4, 130.1, 140.0, 145.8, 150.6, 155.1, 170.7, 170.8, 171.0,
171.1; m/z (electrospray) 531 (100%, [M+Na]+); HRMS (electro-
spray) Calcd for C21H20N2O13Na: 531.0863. Found: 531.0847.
Diethyl 2,2′-((4-(1-(4-((S)-2-((t-butoxycarbonyl)amino)-3-
methoxy-3-oxopropyl)phenoxy)-2-(2-ethoxy-2-oxoethoxy)-2-
oxoethyl)-5-nitro-1,2-phenylene)bis(oxy))diacetate (11)
N-Boc-L-tyrosine methyl ester (0.233 g, 0.79 mmol) and
Cs2CO3 (0.258 g, 0.79 mmol) were stirred in THF (20 mL) at
0 °C for 30 min before 9 (0.402 g, 0.73 mmol) in THF (20 mL)
was added. After stirring overnight the THF was removed in
vacuo and the residue partitioned between water and EtOAc. The
organic phase was washed with 1 M NaOH and brine, dried with
MgSO4 and concentrated in vacuo. Purification by column
t-Butyl 2-(4,5-dimethoxy-2-nitrophenyl)acetate
A
mixture of N,N-dicyclohexylcarbodiimide (35.40 g,
172 mmol), t-BuOH (20.1 mL, 15.6 g, 210 mmol) and CuCl
560 | Photochem. Photobiol. Sci., 2012, 11, 556–563
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(181 mg) was stirred at rt for 5 d to afford O-t-butyl-N,N-dicy-
clohexylisourea. The mixture was diluted with CH2Cl2 (50 mL)
and added to a solution of 2-(4,5-dimethoxy-2-nitrophenyl)
acetic acid (5.00 g, 20.75 mmol) in CH2Cl2 (150 mL). After stir-
ring at rt for 5 d the mixture was diluted with CH2Cl2 and the
precipitate removed by filtration and washed with EtOAc. The
organic phase was evaporated in vacuo. Purification by column
chromatography (hexane/EtOAc, 5 : 1) gave t-butyl 2-(4,5-
dimethoxy-2-nitrophenyl)acetate (6.16 g, 97%) as a colorless
crystalline solid: Rf 0.1; mp 78–81 °C (from hexane); νmax/cm−1
(film) 2977, 1731, 1618, 1524; δH (300 MHz, CDCl3) 1.42 (9
H, s), 3.86 (2 H, s), 3.92 (3 H, s), 3.94 (3 H, s), 6.69 (1 H, s),
7.70 (1 H, s); δC (75 MHz, CDCl3) 28.0, 41.4, 56.3, 81.5, 108.4,
114.6, 125.4, 141.0, 147.9. 153.1, 169.5; m/z (electrospray) 320
(100%, [M+Na]+); HRMS (electrospray) Calcd for
C14H19NO6Na: 320.1110. Found: 320.1098.
methyl ether 17 (1.60 g, 93%) as a yellow oil: Rf 0.1; νmax/cm−1
(film) 3338, 2980, 1727, 1612, 1584, 1510; δH (300 MHz,
acetone-d6) 1.40 (9 H, s), 3.04 (1 H, dd, J = 14.1, 9.6 Hz), 3.25
(1 H, dd, J = 14.1, 5.2 Hz), 3.70 (3 H, s), 3.90 (3 H, s), 3.95 (3
H, s), 4.69–4.78 (1 H, m), 6.42 (1 H, s), 7.03 (2 H, d, J = 8.5
Hz), 7.24 (2 H, d, J = 8.5 Hz), 7.31 (1 H, s), 7.72 (1 H, s), 8.70
(1 H, br s); δC (75 MHz, acetone-d6) 27.9, 36.4, 52.8, 55.0,
56.6, 76.90, 76.96, 82.9, 109.0, 110.6, 116.73 (q, J = 287 Hz),
116.74, 127.0, 131.0, 131.2, 141.3, 149.7, 154.5, 157.4 (q, J =
37 Hz), 157.5, 167.7, 171.1; m/z (electrospray) 609 (100%,
[M+Na]+); HRMS (electrospray) Calcd for C26H29N2O10F3Na:
609.1672. Found: 609.1680.
(2S)-t-Butyl 3-(4-(2-(allyloxy)-1-(4,5-dimethoxy-2-nitrophenyl)-2-
oxoethoxy)phenyl)-2-((t-butoxycarbonyl)amino)propanoate (18)
N-Boc-L-tyrosine t-butyl ester (0.152 g, 0.45 mmol) and K2CO3
(0.062 g, 0.45 mmol) were stirred in DMF (5 mL) for 30 min
before 1 (0.107 g, 0.30 mmol) in DMF (3 mL) was added. The
reaction was stirred overnight, after which time starting material
was still visible by TLC, and so a further portion of K2CO3
(0.035 g, 0.25 mmol) and allyl 2-bromo-2-(4,5-dimethoxy-2-
nitrophenyl)acetate (0.091 g, 0.25 mmol) were added and stir-
ring continued overnight. The reaction mixture was poured into
water and extracted with Et2O. The organic phase was washed
with 1 M NaOH and water, dried over MgSO4 and concentrated.
Purification by column chromatography (hexane/EtOAc, 2 : 1)
t-Butyl 2-bromo-2-(4,5-dimethoxy-2-nitrophenyl)acetate (14)
Mesyl azide (1.62 g, 13.4 mmol), DBU (2.00 mL, 2.04 g,
13.4 mmol) and t-butyl 2-(4,5-dimethoxy-2-nitrophenyl)acetate
(3.98 g, 13.4 mmol) were stirred in anhydrous acetonitrile
(50 mL) at 0 °C to rt for 3 h. Acetonitrile was removed in vacuo,
and the residue dissolved in EtOAc. The solution was washed
with water, dried over MgSO4 and concentrated in vacuo to give
a brown gum. Column chromatography (hexane/EtOAc, 7 : 3)
yielded t-butyl 2-diazo-2-(4,5-dimethoxy-2-nitrophenyl)acetate
(2.67 g) as a bright yellow solid that still contained impurities
(>90% pure) and so was used immediately in the next step.
Crude t-butyl 2-diazo-2-(4,5-dimethoxy-2-nitrophenyl)acetate
(2.67 g) in CH2Cl2 (100 mL) at 0 °C was treated with 45% HBr
in acetic acid (2.41 mL, 13.4 mmol). Vigorous effervescence
was observed and the bright yellow color quickly faded. The
reaction was warmed to rt and stirred for 20 min before being
poured into saturated aqueous NaHCO3. The phases were separ-
ated and the organic phase was washed with water, dried over
MgSO4 and concentrated in vacuo to yield a yellow oil which
was purified by column chromatography (hexane/EtOAc, 6 : 1)
to afford bromide 14 (2.73 g, 54% over 2 steps) as a colorless
oil: Rf 0.4; νmax/cm−1 (film) 1732, 1612, 1524; δH (300 MHz,
CDCl3) 1.45 (9 H, s), 3.93 (3 H, s), 3.98 (3 H, s), 6.06 (1 H, s),
7.39 (1 H, s), 7.57 (1 H, s); δC (75 MHz, CDCl3) 25.8, 43.4,
54.6, 54.7, 81.5, 106.0, 112.5, 123.6, 138.4, 147.2, 151.4, 164.9;
m/z (electrospray) 400 (85%, [M+Na]+), 398 (100%, [M+Na]+);
HRMS (electrospray) Calcd for C14H18NO6BrNa: 398.0215.
Found: 398.0218.
afforded ether 18 as a yellow oil (0.180 g, 65%): Rf 0.4; νmax
/
cm−1 (film) 2976, 2937, 1742, 1711, 1612, 1583, 1522, 1507;
δH (300 MHz, CDCl3) 1.37 (9 H, s), 1.396 (5 H, s), 1.404 (4 H,
s), 1.42 (2 H, s), 2.97 (2 H, d, J = 4.2 Hz), 3.91 (3 H, s), 3.96 (3
H, s), 4.38 (1 H, dd, J = 13.9, 6.0 Hz), 4.66 (2 H, dq, J = 5.6,
1.3 Hz), 4.92–5.00 (1 H, m), 5.17–5.32 (2 H, m), 5.85 (1 H, ddt,
J = 17.2, 10.5, 5.6 Hz), 6.63 (1 H, s), 6.90 (2 H, d, J = 8.6 Hz),
7.07 (2 H, d, J = 8.6 Hz), 7.28 (1 H, s), 7.70 (1 H, s); δC
(100 MHz, CDCl3) 28.03, 28.39, 30.42, 37.75, 54.96, 56.51,
56.58, 66.43, 75.49, 79.74, 82.08, 108.34, 109.78, 115.93,
118.78, 125.57, 126.52, 130.64, 130.89, 131.33, 140.42, 148.91,
153.90, 155.13, 156.34, 168.03, 170.93; m/z (electrospray) 639
(100%, [M+Na]+); HRMS (electrospray) Calcd for
C31H40N2O11Na: 639.2530. Found: 639.2527.
(1S)-2-(4-(2-(Allyloxy)-1-(4,5-dimethoxy-2-nitrophenyl)-2-
oxoethoxy)phenyl)-1-carboxyethanaminium 2,2,2-
trifluoroacetate
A solution of 18 (0.120 g, 0.195 mmmol) in TFA (2 mL) was
stirred at room temperature for 2 h. The TFA was removed in
vacuo and the residue was triturated with Et2O to afford (1S)-2-
(4-(2-(allyloxy)-1-(4,5-dimethoxy-2-nitrophenyl)-2-oxoethoxy)
phenyl)-1-carboxyethanaminium 2,2,2-trifluoroacetate (0.090 g,
87%) as a pale yellow solid: νmax/cm−1 (film) 2929, 1742, 1676,
1609, 1583, 1508; δH (300 MHz, acetone-d6) 2.99–3.15 (1 H,
m), 3.21–3.35 (1 H, m), 3.91 (3 H, s), 3.97 (3 H, s), 4.68 (2 H,
dt, J = 5.4, 1.5 Hz), 5.19 (1 H, ddd, J = 10.5, 2.8, 1.3 Hz), 5.28
(1 H, ddd, J = 17.2, 3.2, 1.6 Hz), 5.91 (1 H, ddt, J = 17.2, 10.7,
5.4 Hz), 6.58 (1 H, d, J = 1.3 Hz), 7.05 (2 H, d, J = 7.4 Hz),
7.29 (2 H, d, J = 8.6 Hz), 7.34 (1 H, s), 7.75 (1 H, s); m/z
(2S)-Methyl 3-(4-(2-(t-butoxy)-1-(4,5-dimethoxy-2-nitrophenyl)-
2-oxoethoxy)phenyl)-2-(2,2,2-trifluoroacetamido)propanoate (17)
N-Trifluoroacetyl-L-tyrosine methyl ester (0.94 g, 3.24 mmol)
and Cs2CO3 (1.06 g, 3.24 mmol) were stirred in THF (140 mL)
at rt for 30 min before 14 (1.11 g, 2.94 mmol) in THF (70 mL)
was added. After stirring for 36 h the reaction was quenched by
the addition of water and concentrated in vacuo. The residue was
partitioned between EtOAc and water. The organic phase was
dried with MgSO4, evaporated in vacuo and the residue purified
by column chromatography (Et2O/hexane, 1 : 1) to afford (2S)-
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(electrospray) 505 (25%, [M−2H+2Na]+), 483 (100%, [M
−H+Na]+), 280 (40%); HRMS (electrospray) Calcd for
C22H24N2O9Na: 483.1380. Found: 483.1376.
120.10, 125.07, 125.15, 126.40, 127.17, 127.85, 129.85, 130.82,
131.27, 140.33, 141.40, 143.71, 143.82, 148.89, 153.91, 155.87,
156.50, 168.16, 175.36; m/z (electrospray) 705 (100%,
[M+Na]+); HRMS (electrospray) Calcd for C37H34N2O11Na:
705.2060. Found: 705.2051.
(2S)-2-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)-3-(4-(2-(t-
butoxy)-1-(4,5-dimethoxy-2-nitrophenyl)-2-oxoethoxy)phenyl)
propanoic acid (19)
Acknowledgements
A solution of 20 (0.095 g, 0.162 mmol) and LiOH (0.010 g,
0.42 mmol) in THF/H2O (1 : 1, 2 mL) was stirred overnight
before being neutralized using 1 M HCl. To this solution was
added Na2CO3 (0.026 g, 0.245 mmol) and N-(9H-fluoren-9-
ylmethoxycarbonyloxy)succinimide (0.055 g, 0.162 mmol) and
the mixture was stirred overnight. The THF was removed in
vacuo and the residue acidified to pH 5 with 1 M HCl to afford a
white precipitate which was extracted into EtOAc. The organic
phase was dried with MgSO4 and the solvent removed in vacuo.
Purification by column chromatography (hexane/EtOAc/AcOH,
1 : 1 : 0.02) afforded product 19 (93 mg, 82%) as a pale yellow
oil: Rf 0.25; νmax/cm−1 (film) 3354, 3021, 2980, 2938, 1724,
1611, 1584, 1510; δH (300 MHz, CDCl3) 1.39 (9 H, s),
2.98–3.16 (2 H, m), 3.86–3.88 (3 H, m), 3.91–3.93 (3 H, m),
4.14–4.19 (1 H, m), 4.31–4.38 (1 H, m), 4.40–4.46 (1 H, m),
4.59–4.65 (1 H, m), 5.19 (1 H, d, J = 7.0 Hz), 6.50 (1 H, s),
6.92 (2 H, d, J = 8.3 Hz), 7.03 (2 H, d, J = 8.3 Hz), 7.21–7.30
(3 H, m), 7.32–7.39 (2 H, m), 7.48–7.55 (2 H, m), 7.62–7.67 (1
H, m), 7.70–7.76 (2 H, m); δC (75 MHz, CDCl3) 27.8, 36.8,
47.1, 54.5, 56.3, 56.5, 67.0, 75.6, 83.2, 108.2, 109.3, 116.1,
120.0, 124.9, 125.0, 126.6, 127.0, 127.7, 129.3, 130.6, 140.4,
141.3, 143.6, 143.7, 148.5, 153.6, 155.8, 156.5, 167.3, 175.3;
m/z (electrospray) 721 (100%, [M+Na]+); HRMS (electrospray)
Calcd for C38H38N2O11Na: 721.2373. Found: 721.2368.
We thank the Biotechnology and Biological Sciences Research
Council (grant BB/C50446X/1), the Engineering and Physical
Sciences Research Council (studentships to A.G.R. and M.J.S.),
the University of Birmingham, and the CNRS for financial
support and Mr Graham Burns for HPLC separations. The NMR
spectrometers used in this research were funded in part through
Birmingham Science City: Innovative Uses for Advanced
Materials in the Modern World (West Midlands Centre for
Advanced Materials Project 2), with support from Advantage
West Midlands (AWM) and part-funded by the European
Regional Development Fund (ERDF).
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(2S)-2-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)-3-(4-(2-
(allyloxy)-1-(4,5-dimethoxy-2-nitrophenyl)-2-oxoethoxy)phenyl)
propanoic acid (20)
(1S)-2-(4-(2-(Allyloxy)-1-(4,5-dimethoxy-2-nitrophenyl)-2-
oxoethoxy)phenyl)-1-carboxyethanaminium 2,2,2-trifluoroace-
tate (0.050 g, 0.088 mmol), N-(9H-fluoren-9-ylmethoxycarbony-
loxy)succinimide
(0.034
g,
0.1
mmol)
and
diisopropylethylamine (52 μL, 0.039 g, 0.3 mmol) were stirred
overnight in CH2Cl2 (3 mL). The solution was washed with 1 M
HCl, brine and dried (MgSO4). Removal of the solvent in vacuo
and purification of the residue by column chromatography
(CH2Cl2 then CH2Cl2/MeOH, 9 : 1) afforded product 19
(0.049 g, 82%) as a pale-yellow foam: Rf 0.2 (9 : 1 CH2Cl2/
MeOH); νmax/cm−1 (neat) 2936, 1743, 1719, 1611, 1583, 1507;
δH (400 MHz, CDCl3) 3.02 (1 H, dd, J = 12.9, 6.6 Hz), 3.13 (1
H, dd, J = 14.0, 4.8 Hz), 3.86 (1 H, s), 3.87 (1 H, s), 3.92 (1 H,
s), 3.93 (1 H, s), 4.13–4.20 (1 H, m), 4.28–4.37 (1 H, m),
4.40–4.49 (1 H, m), 4.56–4.70 (3 H, m), 5.16–5.25 (1 H, m),
5.25–5.32 (1 H, m), 5.84 (1 H, ddd, J = 21.8, 10.7, 5.5 Hz),
6.63 (1 H, d, J = 2.8 Hz), 6.91 (2 H, d, J = 7.5 Hz), 7.03 (1 H,
d, J = 8.2 Hz), 7.23–7.32 (3 H, m), 7.37 (2 H, dd, J = 13.8, 6.8
Hz), 7.49–7.57 (2 H, m), 7.68 (1 H, d, J = 5.5 Hz), 7.74 (2 H, d,
J = 7.5 Hz); δC (100 MHz, CDCl3) 37.00, 54.83, 56.51, 56.62,
66.57, 67.06, 75.41, 75.50, 108.34, 109.68, 116.15, 118.91,
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