Phototriggered release of tetrapeptide AAPV from coumarinyl and pyrenyl cages

Article abstract of DOI:10.1007/s00726-017-2405-6

Ala–Ala–Pro–Val (AAPV) is a bioactive tetrapeptide that inhibits human neutrophil elastase, an enzyme involved in skin chronic inflammatory diseases like psoriasis. Caged derivatives of this peptide were prepared by proper N- and C-terminal derivatisation through a carbamate or ester linkage, respectively, with two photoactive moieties, namely 7-methoxycoumarin-2-ylmethyl and pyren-2-ylmethyl groups. These groups were chosen to assess the influence of the photosensitive group and the type of linkage in the controlled photo release of the active molecule. The caged peptides were irradiated at selected wavelengths of irradiation (254, 300, and 350?nm), and the photolytic process was monitored by HPLC–UV. The results established the applicability of the tested photoactive groups for the release of AAPV, especially for the derivative bearing the carbamate-linked pyrenylmethyl group, which displayed the shortest irradiation times for the release at the various wavelengths of irradiation (ca. 4?min at 254?nm, 8?min at 300?nm and 46?min at 350?nm).

Full text of DOI:10.1007/s00726-017-2405-6

Amino Acids
DOI 10.1007/s00726-017-2405-6
ORIGINAL ARTICLE
Phototriggered release of tetrapeptide AAPV from coumarinyl
and pyrenyl cages
1
1
1
Filipa C. Santos · Ana M. S. Soares · M. Sameiro T. Gonçalves
·
1
Susana P. G. Costa
Received: 14 October 2016 / Accepted: 28 February 2017
©
Springer-Verlag Wien 2017
Abstract Ala–Ala–Pro–Val (AAPV) is a bioactive tetra-
peptide that inhibits human neutrophil elastase, an enzyme
involved in skin chronic inflammatory diseases like pso-
riasis. Caged derivatives of this peptide were prepared by
proper N- and C-terminal derivatisation through a carba-
mate or ester linkage, respectively, with two photoactive
moieties, namely 7-methoxycoumarin-2-ylmethyl and
pyren-2-ylmethyl groups. These groups were chosen to
assess the influence of the photosensitive group and the
type of linkage in the controlled photo release of the active
molecule. The caged peptides were irradiated at selected
wavelengths of irradiation (254, 300, and 350 nm), and
the photolytic process was monitored by HPLC–UV. The
results established the applicability of the tested photoac-
tive groups for the release of AAPV, especially for the
derivative bearing the carbamate-linked pyrenylmethyl
group, which displayed the shortest irradiation times for
the release at the various wavelengths of irradiation (ca.
Introduction
The tetrapeptide Ala–Ala–Pro–Val (AAPV) has been evalu-
ated in the treatment of psoriasis, a chronic inflammatory
disease of the skin, as a competitive inhibitor of human neu-
trophil elastase (HNE). This enzyme relates physiologically
to immune processes and the extracellular matrix remodel-
ling after tissue injury (Wiedow et al. 1995; Vasconcelos
et al. 2011; Caccetta et al. 2006; Namjoshi et al. 2014; Mac-
donald et al. 1998; Hassal et al. 1985). In healthy condi-
tions, the proteolytic activity of this enzyme is controlled by
endogenous inhibitors; however, in inflammatory processes
an unbalance occurs between the levels of HNE, which is
produced in high levels, and its endogenous inhibitors, lead-
ing to an abnormal tissue destruction (Namjoshi et al. 2008;
Benson et al. 2003; Avlonitis et al. 2013).
Several derivatives of AAPV have been proposed with
modifications aiming at improving skin absorption and pas-
sage through the stratum corneum to allow efficient trans-
dermal delivery and be able to provide a therapeutic effect
with the least possible side effects. Reported modifications
include preparing peptide conjugates with PEG possessing
higher lipophilicity (Yingyuad et al. 2014), by coupling to
short chain lipoaminoacids (Caccetta et al. 2006; Namjoshi
et al. 2014; Benson et al. 2003; Toth et al. 1995; Rocco
et al. 2016; Benson and Namjoshi 2008), and encapsulating
the peptide in hydrophobic carriers improving its absorp-
tion through the s. corneum (Toth et al. 1995; Benson and
Namjoshi 2008). For example, improved efficacy of this
tetrapeptide as an inhibitor of HNE was demonstrated by
conjugating the N-terminus with one, two or three lipid
groups, wherein the inhibitory potency of the lipopeptides
in relation to the skin HNE increased about ten thousand
times compared with the unconjugated tetrapeptide (Cac-
cetta et al. 2006; Benson et al. 2003).
4
min at 254 nm, 8 min at 300 nm and 46 min at 350 nm).
Keywords AAPV · Caged peptide · Coumarin · Pyrene ·
Photorelease
Handling Editor: J. Bode.
*
Susana P. G. Costa
spc@quimica.uminho.pt
1
Centre of Chemistry, University of Minho, Campus de
Gualtar, 4710-057 Braga, Portugal
1
3

F. C. Santos et al.
Most peptides do not show ideal features to formulate
drugs due to their intrinsic physico-chemical and phar-
macokinetic profiles and often therapeutic peptides have
limitations such as low oral bioavailability, short half-life
due to rapid degradation by proteolytic enzymes and blood
plasma, rapid elimination from the systemic circulation by
hepatic and renal clearance, low capacity to cross physi-
ological barriers due to their hydrophilicity, high conforma-
tional flexibility which can cause a lack of selectivity for
the target to interact with different receptors, and immu-
nogenic risks. The strategy for chemical optimization of a
therapeutic peptide is usually based on structure–activity
relationship studies to improve bioavailability, reduce or
eliminate degradation and increase selectivity or affinity
for the corresponding receptor (Vlieghe et al. 2010). Prod-
rugs are, thus, inactive forms of a drug that, after efficient
transfer to the target site, suffer proper transformation to
release the active drug with control over space and time. It
is crucial to control the scission of chemical bonds between
the active drug and the moiety that masks the activity, and
among the various triggering stimuli that can be used, i.e.
enzymes, reducing or oxidising agents, temperature or pH,
the use of light represents a fast-developing methodology
for application in controlled drug delivery (Bildstein et al.
amines, butyric acid and 5-aminolevulinic acid (Fernandes
et al. 2007, 2008; Soares et al. 2010; Hungerford et al.
2010; Fonseca et al. 2010; Soares et al. 2012; Piloto et al.
2013; Fernandes et al. 2013; Piloto et al. 2015; Soares et al.
2015a, b, 2017). Considering our past experience in such
phototriggers, and although both photoactive moieties used
in this manuscript have been previously reported by us,
to the best of our knowledge the present work is the first
account on the application of 7-methoxycoumarin-2-yl-
methyl and pyren-2-ylmethyl groups in the preparation of
caged tetrapeptide AAPV (a bioactive peptide). The intro-
duction of lipophilic phototriggers may also enhance the
caged peptide’s lipophilicity and the study of its light-trig-
gered uncaging may lead, in future work, to a novel appli-
cation scope aided by light, for example, in topical skin
treatment of psoriasis and dermatitis.
Experimental section
General
All melting points were measured on a Stuart SMP3 appa-
ratus. TLC analyses were carried out on 0.25 mm thick
precoated silica plates (Merck Fertigplatten Kieselgel
60F254) and spots were visualised under UV light. Chro-
matography on silica gel was carried out on Merck Kiesel-
gel (230–240 mesh). Fluorescence spectra were collected
using a FluoroMax-4 spectrofluorometer (Horiba/Jobin–
Yvon). NMR spectra were obtained on a Bruker Avance
2
011; Kratz et al. 2008; Jin et al. 2014).
Photocontrolled triggering and delivery have known an
exponential growth in recent years, as it allows the cleav-
age of specific bonds within a structure with the consequent
removal of the photoactive moiety (linked covalently to the
active drug molecule) and the on-demand/on-site release of
an active molecule. Among the numerous examples of bio-
logical phototriggering applications, reports can be found
on caged capsaicin analogues for the in vitro activation of
the capsaicin receptor TRPV1 (Carr et al. 2006), short oli-
gonucleotide uracil prodrugs for the inhibition of cancer
cell proliferation (Fujimoto et al. 2014), protein biosynthe-
sis using a caged aminoacyl-tRNA (Akahoshi et al. 2014),
caged hydrogen sulphide for delivery to live cells (Fukush-
ima et al. 2015), photo-uncaging of nitric oxide for targeting
to mitochondria (Horinouchi et al. 2011), caged agonists for
light-activated gene patterning (Link et al. 2004), molecu-
lar release based on auto-degradable polymer-containing
organic–inorganic hybrids (Okada et al. 2014), photo-con-
trol of binding affinity to phosphopeptide-recognizing pro-
tein (Ebisuno et al. 2014), and light-switched inhibitors of
protein tyrosine phosphatase PTP1B (Wagner et al. 2015).
In recent years, our research has been focused on the
release of bioactive molecules mainly from heterocyclic
cages, specifically designed for being triggered by light,
to act as photoremovable protecting groups and phototrig-
gers. Coumarin, benzocoumarin and pyrene derivatives,
among other moieties, have been designed and applied in
the caging of amino acids, neurotransmitters, biogenic
1
III 400 at an operating frequency of 400 MHz for H and
1
3
100.6 MHz for C using the solvent peak as internal refer-
ence at 25 °C. All chemical shifts are given in ppm using
δ Me Si = 0 ppm as reference and J values are given in
H
4
Hz. Assignments were supported by bidimensional het-
eronuclear correlation techniques. Microwave-assisted
solid-phase peptide synthesis was carried out on a CEM
Discover SPS equipment. Peptide analysis was carried
out with analytical HPLC using a Licrospher 100 RP18
(5 μm) column in a JASCO HPLC system composed by
a PU-2080 pump and a UV-2070 detector with ChromNav
software. Mass spectrometry analyses were performed at
the “C.A.C.T.I.—Unidad de Espectrometria de Masas”,
at University of Vigo, Spain. 4-Chloromethyl-7-methox-
ycoumarin 1a was synthesised as reported elsewhere (Pil-
oto et al. 2006). 1-Chloromethylpyrene 1b was purchased
from TCI Chemicals, 1-pyrenemethanol 1c from Sigma-
Aldrich, Fmoc-Val-OH, Fmoc-Pro-OH, Fmoc-Ala-OH,
N,N′-diisopropylcarbodiimide (DIC), 6-chloro-N-hydroxy-
benzotriazole (6-Cl-HOBt) and 2-chlorotrityl chloride resin
(degree of functionalisation 84%) were purchased from
AAPPTec and N,N-diisopropylethylamine (DIPEA) was
purchased from ACROS Organics and used as received.
1
3

Phototriggered release of tetrapeptide AAPV from coumarinyl…
MW‑assisted solid phase synthesis
of Fmoc‑Ala‑Ala‑Pro‑Val‑OH 2a
The same procedure was followed twice for the coupling
of Fmoc-Ala-OH, but the final N-deprotection was not car-
ried out, in order to keep the Fmoc group at the N-terminal.
Coupling of Fmoc‑Val‑OH to resin
Cleavage from the resin
Fmoc-Val-OH (0.858 g, 2.53 mmol, 2 equiv) was dis-
solved in dry DMF (7 mL) and DIPEA was added (878 µL,
The filtered resin was swollen in dry DMF and a solution of
AcOH/TFE/DCM (1:1:3) (10 mL) was added. The suspen-
sion was stirred for 1 h at room temperature and then the
solvent mixture was collected by filtration. The resin was
washed with DMF (2 × 10 mL) and DCM (2 × 10 mL)
and the solvents were combined and evaporated to dry-
ness on a rotary evaporator without heating. Cold diethyl
ether was added and a colourless solid separated, which
was filtered. The purity of the peptide was checked by
analytical HPLC. The peptide Fmoc-Ala–Ala–Pro–Val–
5
.04 mmol, 4 equiv). The mixture was stirred for 10 min
and then it was added to the previously swollen 2-chlo-
rotrityl resin (1.51 g, 1.27 mmol) in dry DMF. The sus-
pension was then subjected to microwave irradiation for
3
0 min (power = 25 W, temperature = 38 °C). The resin
was filtered and washed with MeOH (2 × 10 mL), DMF
(
2 × 10 mL) and MeOH (2 × 10 mL) and this washing
cycle was repeated two times. The filtered resin was dried
in vacuum.
OH 2a was obtained as a colourless solid (0.290 g, 85%).
1
Removal of Fmoc protecting group
Mp = 94.0–95.2 °C. H NMR (CDCl , 400 MHz): δ 0.88–
3
0
.91 (6H, m, 2 × γ-CH -Val), 1.35–1.39 (6H, m, 2 × CH
3
3
The filtered resin was swollen in DMF for 15 min and sub-
jected to an initial deprotection to remove the Fmoc group
using a solution of 20% piperidine in DMF (7 mL/g resin)
for 30 s, under microwave irradiation (power = 28 W,
temperature = 75 °C). The resin was washed with DMF
Ala), 2.02–2.08 (2H, m, H-3 Pro), 2.18–2.38 (3H, m, β-CH
Val and H-4 Pro), 3.71–3.76 (2H, m, H-5 Pro), 4.22 (1H,
t, J 7.2 Hz, H-9 Fmoc), 4.37–4.41 (3H, m, α-CH Ala and
CH Fmoc), 4.52–4.55 (1H, m, α-CH Val), 4.69 (1H, d, J
2
6 Hz, α-CH Pro), 4.83 (1H, t, J 6.8 Hz, α-CH Ala), 5.57
(1H, d, J 6.8 Hz, NH), 7.31 (2H, t, J 7.6 Hz, H-2 and H-7
Fmoc), 7.42 (3H, t, J 7.2 and 8.4 Hz, NH, H-3 and H-6
Fmoc), 7.60 (2H, d, J 7.6 Hz, H-4 and H-5 Fmoc), 7.63
(1H, d, J 6.8 Hz, NH), 7.76 (2H, d, J 7.2 Hz, H-1 and H-8
(
2 × 10 mL), MeOH (2 × 10 mL) and DMF (2 × 10 mL).
A fresh solution of 20% piperidine in DMF was added to
the resin and allowed to react for 3 min under microwave
irradiation (power = 28 W, temperature = 75 °C). The
resin was washed with DMF (2 × 10 mL) and MeOH
1
3
Fmoc). C NMR (CDCl , 100.6 MHz): δ 17.56 (γ-CH
3
3
(
2 × 10 mL), and this washing cycle was repeated three
Val), 17.99 (CH Ala), 18.93 (γ-CH Val), 19.11 (CH Ala),
3 3 3
times. The loading after the coupling of the first amino
acid was determined to be 0.39 mmol/g, by taking a small
sample of the resin (≈10 mg), cleaving the Fmoc group as
stated and measuring the liberated dibenzofluvene by UV–
vis absorption spectroscopy at 290 nm (Chan and White
25.03 (C-4 Pro), 29.68 (C-3 Pro), 31.35 (β-CH Val), 46.64
(α-CH Ala), 47.11 (C-9 Fmoc), 47.61 (C-5 Pro), 50.31
(α-CH Ala), 57.24 (α-CH Val), 60.27 (α-CH Pro), 67.11
(CH Fmoc), 119.97 (C-1 and C-8 Fmoc), 125.11 (C-4 and
2
C-5 Fmoc), 127.07 (C-2 and C-7 Fmoc), 127.71 (C-3 and
C-6 Fmoc), 141.23 (C-4a and C-4b Fmoc), 143.75 (C-1a
Fmoc), 143.86 (C-8a Fmoc), 155.88 (C=O Fmoc), 170.79
2
000).
Coupling of the remaining amino acids
(C=O Pro), 172.37 (C=O Ala), 172.94 (C=O Ala), 174.42
+
(
C=O Val). HRMS (ESI): calcd for C H N O [M +H]:
3
1
39
4
7
Fmoc-Pro-OH (0.992 g, 2.94 mmol, 5 equiv) was dissolved
in dry DMF (6 mL/g resin) and DIC (512 µL, 2.94 mmol,
579.28207; found: 579.28174.
5
equiv) and 6-Cl-HOBt (0.397 g, 2.94 mmol, 5 equiv)
Synthesis of H–Ala–Ala–Pro–Val–OH 2b
dissolved in dry DMF (2 mL/g) were added. The reac-
tion mixture was allowed to stir at room temperature for
The Fmoc-protected peptide 2a (0.200 g, 0.35 mmol) was
treated with a solution of 20% piperidine in DMF (3 mL),
with stirring at room temperature for 3 h. The mixture
was treated with diethyl ether to precipitate the resulting
1
5 min and then was added to the resin, previously swollen
in DMF. The suspension was subjected to microwave irra-
diation for 5 min (power = 28 W, temperature = 75 °C).
The resin was washed with DMF (2 × 10 mL) followed by
MeOH (2 × 10 mL) and this washing cycle was repeated
three times. The filtered resin was swollen in dry DMF
and the N-terminal Fmoc group was removed as described
above.
peptide 2b (0.112 g, 90%) which was filtered and used
1
immediately in the following reactions. H NMR (CDCl ,
3
400 MHz): δ 0.94–0.98 (6H, m, 2 × γ-CH Val), 1.42
3
(3H, d, J 7.2 Hz, CH Ala), 1.50 (3H, d, J 7.2 Hz, CH
3
3
Ala), 2.00–2.20 (5H, m, β-CH Val, H-3 Pro and H-4 Pro),
1
3

F. C. Santos et al.
3
.70–3.75 (2H, m, H-5 Pro), 3.98–4.00 (1H, m, α-CH
Synthesis of Fmoc‑Ala‑Ala‑Pro‑Val‑OMcm 3a
Ala), 4.19–4.21 (1H, m, α-CH Val), 4.54–4.56 (1H, m,
α-CH Pro), 4.67–4.69 (1H, m, α-CH Ala), 5.10 (2H, br s,
The peptide Fmoc-Ala-Ala-Pro-Val-OH 2a (0.135 g,
0.23 mmol, 1 equiv) was added to a solution of 4-chlo-
romethyl-7-methoxycoumarin 1a (0.079 g, 0.35 mmol,
NH ), 8.00 (2H, br s, 2 × NH).
2
1
.5 equiv) and potassium fluoride (0.041 g, 0.70 mmol,
Synthesis of Boc‑Ala‑Ala‑Pro‑Val‑OH 2c
3 equiv) in dry DMF (3 mL). The mixture was stirred at
room temperature for 48 h. The solid was filtered and the
solvent was evaporated to dryness. The coumarin-labelled
The peptide H-Ala-Ala-Pro-Val-OH 2b (0.100 g,
0
.18 mmol) was dissolved in a 1,4-dioxane (3.6 mL),
peptide 3a was obtained as a light orange solid (0.174 g,
1
water (1.8 mL) and sodium hydroxide 1 M aqueous solu-
tion (1.8 mL), with stirring in an ice bath. t-Butyl pyro-
carbonate (0.044 g, 0.20 mmol, 1.1 equiv) was added and
the mixture stirred at room temperature for 1 day. The
volume was reduced by half in a rotary evaporator under
reduced pressure and ethyl acetate (2 mL) was added. The
mixture was acidified by addition of aqueous potassium
hydrogen sulphate 1 M solution and extracted with ethyl
acetate (3 × 5 mL), followed by washing of the organic
layer with water (3 × 5 mL). The organic layer was dried
with anhydrous magnesium sulphate and evaporated
97%). H NMR (CDCl , 400 MHz): δ 0.86–0.90 (6H, m,
3
2 × γ-CH Val), 1.36–1.38 (6H, m, 2 × CH Ala), 1.94–
3
3
2.01 (2H, m, H-4 Pro), 2.09–2.12 (2H, m, H-3 Pro), 2.19–
2.22 (1H, m, β-CH Val), 3.65–3.72 (2H, m, H-5 Pro), 3.85
(3H, s, OCH ), 4.32–4.38 (1H, m, α-CH Ala), 4.45 (1H, t,
3
J 6.8 Hz, H-9 Fmoc), 4.47–4.51 (1H, m, α-CH Val), 4.63–
4.67 (1H, m, α-CH Pro), 4.68–4.70 (3H, m, α-CH Ala and
CH Fmoc), 5.23 (2H, d, J 1.6 Hz, CH Mcm), 6.38 (1H,
2
2
s, H-3 Mcm), 6.78–6.82 (2H, m, H-6 and H-8 Mcm), 7.28
(3H, t, J 7.2 Hz, H-2 and H-7 Fmoc and NH), 7.35 (3H, t, J
7.2 Hz, H-3 and H-6 Fmoc and NH), 7.42 (1H, d, J 7.2 Hz,
H-5 Mcm), 7.67 (2H, d, J 7.2 Hz, H-4 and H-5 Fmoc), 7.71
(2H, d, J 7.6 Hz, H-1 and H-8 Fmoc), 7.61 (1H, br s, NH).
under reduced pressure, resulting in an oily off-white
solid (0.067 g, 82%). H NMR (CDCl , 400 MHz): δ
1
3
0
.84-0.88 (6H, m, 2 × γ-CH Val), 1.38 (3H, d, J 7.2 Hz,
3
CH Ala), 1.45 (3H, d, J 7.2 Hz, CH Ala), 1.48 (9H, s,
Synthesis of Fmoc‑Ala‑Ala‑Pro‑Val‑OPym 3b
3
3
C(CH ) ), 1.67–1.71 (2H, m, H-4 Pro), 1.83–2.05 (2H,
3
3
m, H-3 Pro), 2.23–2.31 (1H, m, β-CH Val), 3.53–3.61
The peptide Fmoc-Ala-Ala-Pro-Val-OH 2a (0.135 g,
0.23 mmol, 1 equiv) was added to a solution of 1-chlo-
romethylpyrene 1b (0.088 g, 0.35 mmol, 1.5 equiv) and
potassium fluoride (0.041 g, 0.70 mmol, 3 equiv) in dry
DMF (3 mL). The mixture was stirred at room temperature
for 48 h. The solid was filtered and the solvent was evapo-
rated to dryness. The crude solid was purified by silica gel
column chromatography using ethyl acetate/petroleum
ether (4:6) as eluent. The fractions containing the product
were combined and evaporated to dryness and the pyrene-
(
(
(
2H, m, H-5 Pro), 4.27 (1H, br s, α-CH Val), 4.43–4.46
1H, m, α-CH Pro), 4.65–4.67 (1H, m, α-CH Ala), 4.74
1H, q, J 7.2 Hz, α-CH Ala), 5.21 (1H, d, J 7.6 Hz, NH),
7
.30 (2H, br s, 2 × NH).
Synthesis of H‑Ala‑Ala‑Pro‑Val‑OMe 2d
The peptide H-Ala-Ala-Pro-Val-OH 2b (0.100 g,
0
.18 mmol) was added to a previously prepared solu-
labelled peptide 3b was obtained as a beige solid (0.118 g,
1
tion of thionyl chloride (14 µL, 0.18 mmol) in meth-
anol (3 mL), in an ice bath. The solution was stirred
for 8 h at room temperature and the solvent was evapo-
rated under reduced pressure without heating, yielding
peptide 2d in the form of hydrochloride salt as an off-
65%). H NMR (CDCl , 400 MHz): δ 0.78–0.83 (6H, m,
3
2 × γ-CH Val), 1.33–1.35 (6H, m, 2 × CH Ala), 1.90–
3
3
2.14 (4H, m, H-4 Pro and H-3 Pro), 2.33 (1H, m, β-CH
Val), 3.48–3.55 (2H, m, H-5 Pro), 4.53–4.60 (3H, m, α-CH
Val, α-CH Ala and H-9 Fmoc),4.64–4.71 (4H, m, α-CH
Pro, α-CH Ala and CH Fmoc), 5.79 (2H, d, J 6.8 Hz, CH
1
white crystalline solid (0.070 g, quantitative). H NMR
2
2
(
CDCl , 400 MHz): δ 0.88–0.91 (6H, m, 2 × γ-CH
Pym), 7.27–7.38 (4H, m, H-2, H-3, H-6 and H-7 Fmoc),
7.68–7.75 (4H, m, H-1, H-4, H-5 and H-8 Fmoc), 7.82
(1H, d, J 6.8 Hz, NH), 8.01–8.18 (11H, m, 9 × H-Pym and
2 × NH).
3
3
Val), 1.40 (3H, d, J 7.2 Hz, CH Ala), 1.47 (3H, d, J
3
7
2
3
.2 Hz, CH Ala), 1.69–1.72 (2H, m, H-4 Pro), 1.82–
3
.04 (2H, m, H-3 Pro), 2.20–2.30 (1H, m, β-CH Val),
.54–3.63 (2H, m, H-5 Pro), 3.70 (3H, s, OCH ), 4.25
3
(
4
1H, br s, α-CH Val), 4.43–4.45 (1H, m, α-CH Pro),
.65–4.67 (1H, m, α-CH Ala), 4.69–4.71 (1H, m, α-CH
Synthesis of H‑Ala‑Ala‑Pro‑Val‑OMcm 4a
Ala), 5.21 (1H, d, J 7.6 Hz, NH), 7.30 (1H, br s, NH),
The peptide Fmoc-Ala-Ala-Pro-Val-OMcm 3a (0.170 g,
0.22 mmol) was treated with a solution of 20% piperidine
+
8
.12 (3H, s, NH3 ).
1
3

Phototriggered release of tetrapeptide AAPV from coumarinyl…
+
in DMF (5 mL), stirring at room temperature for 3 h. The
mixture was concentrated by evaporation under vacuum
without heating and treated with diethyl ether to precipitate
Ala). HRMS (ESI): calcd for C H N O [M +H]:
3
3
39
4
5
571.29227; found: 571.29193.
the resulting peptide 4a as a light yellow solid (0.050 g,
Synthesis of Boc‑Ala‑Ala‑Pro‑Val‑OMcm 4c
1
4
1%). Mp = 256.5–258.0 °C. H NMR (CDCl , 400 MHz):
3
δ 0.95–1.02 (6H, m, 2 × γ-CH Val), 1.37–1.41 (6H, m,
The peptide Boc-Ala-Ala-Pro-Val-OH 2c (0.105 g,
0.23 mmol, 1 equiv) was added to a solution of 4-chlo-
romethyl-7-methoxycoumarin 1a (0.079 g, 0.35 mmol,
1.5 equiv) and potassium fluoride (0.041 g, 0.70 mmol, 3
equiv) in dry DMF (3 mL). The mixture was stirred at room
temperature for 48 h. The solid was filtered and the solvent
was evaporated to dryness. The crude solid was purified
by silica gel column chromatography using DCM/MeOH
(50:1) as eluent. The fractions were combined and evapo-
rated and the coumarin-labelled peptide 4c was obtained
3
2
× CH Ala), 1.88–1.93 (2H, m, H-4 Pro), 1.96–1.99 (2H,
3
m, H-3 Pro), 2.17–2.23 (1H, m, β-CH Val), 3.78–3.79 (3H,
m, H-5 Pro and α-CH Ala), 3.90 (3H, s, OCH ), 4.17 (1H,
3
br d, J 4.4 Hz, α-CH Val), 4.55–4.57 (1H, m, α-CH Pro),
4
.62–4.64 (1H, m, α-CH Ala), 5.42 (2H, s, CH Mcm), 6.36
2
(
1H, s, H-3 Mcm), 6.92–6.97 (1H, m, H-6 and H-8 Mcm),
7
.62 (1H, d, J 7.2 Hz, H-5 Mcm) (NH protons not visible).
C NMR (CDCl , 100.6 MHz): δ 18.37 (CH Ala), 18.64
1
3
3
3
(
(
CH Ala), 19.57 (γ-CH Val), 20.19 (γ-CH Val), 23.09
C-3 Pro), 23.76 (C-4 Pro), 31.59 (β-CH Val), 45.71 (C-5
3 3 3
as a light yellow solid (0.101 g, 83%). Mp = 137.9–
1
Pro), 56.39 (OCH ), 56.48 (α-CH Ala), 59.60 (α-CH Val),
138.3 °C. H NMR (CDCl , 400 MHz): δ 0.90–0.94 (6H,
3
3
6
1
1.04 (α-CH Pro), 61.76 (α-CH Ala), 63.02 (CH Mcm),
02.05 (C-8 Mcm), 108.62 (C-4a Mcm), 110.78 (C-3
m, 2 × γ-CH Val), 1.34–1.39 (6H, m, 2 × CH Ala), 1.44
2
3
3
(9H, s, C(CH ) ), 1.99–2.10 (2H, m, H-4 Pro), 2.15–2.21
3 3
Mcm), 111.83 (C-6 Mcm), 113.84 (C-5 Mcm), 156.77 (C-4
Mcm), 158.19 (C-8a Mcm), 162.88 (C-2 Mcm), 164.70
(1H, m, β-CH Val), 2.40–2.45 (1H, m, H-3 Pro), 3.51–3.66
(2H, m, H-5 Pro), 3.89 (3H, s, OCH ), 4.28 (1H, br s,
3
(
(
C-7 Mcm), 172.29 (C=O Pro), 173.22 (C=O Ala), 173.53
α-CH Ala), 4.56–4.59 (1H, m, α-CH Pro), 4.69–4.72 (2H,
m, α-CH Val), 4.77–4.81 (1H, m, α-CH Ala), 5.19 (1H, br
C=O Ala), 174.57 (C=O Val). HRMS (ESI): calcd for
+
C H N O [M +H]: 545.26131; found: 545.26174.
s, NH), 5.30–5.36 (2H, m, CH Mcm), 6.58 (1H, s, H-3
2
7
37
4
8
2
Mcm), 6.86–6.90 (2H, m, H-6 and H-8 Mcm), 7.41 (2H, d,
Synthesis of Ala–Ala–Pro–Val‑OPym 4b
J 8.4 and 6.8 Hz, H-5 Mcm and NH), 7.67 (1H, d, J 8.0 Hz,
1
3
NH). C NMR (CDCl , 100.6 MHz): δ 17.71 (CH Ala),
3
3
The peptide Fmoc-Ala-Ala-Pro-Val-OPym 3b (0.110 g,
18.58 (γ-CH Val), 18.89 (CH Ala), 19.09 (γ-CH Val),
3 3 3
0
.14 mmol) was treated with a solution of 20% piperidine
25.14 (C-4 Pro), 26.58 (C-3 Pro), 28.32 (C(CH ) ), 31.18
3 3
in DMF (5 mL), stirring at room temperature for 3 h. The
mixture was concentrated by evaporation under vacuum
without heating and treated with diethyl ether to precipi-
(β-CH Val), 46.65 (α-CH Ala), 47.40 (C-5 Pro), 50.06
(α-CH Ala), 55.82 (OCH ), 57.74 (α-CH Pro), 59.84 (α-CH
3
Val), 61.86 (CH Mcm), 79.90 (C(CH ) ), 101.29 (C-8
2
3 3
tate the resulting peptide 4b as a light yellow oil (0.050 g,
Mcm), 110.34 (C-3 Mcm), 110.48 (C-4a Mcm), 112.74
(C-6 Mcm), 124.37 (C-5 Mcm), 148.53 (C-4 Mcm), 154.86
(C8a Mcm), 155.53 (C=O Boc), 161.04 (C-2 Mcm),
162.95 (C-7 Mcm), 170.56 (C=O Val), 170.81 (C=O Pro),
172.26 (C=O Ala), 172.94 (C=O Ala). HRMS (ESI): calcd
1
6
2
1
2
3
4
3%). H NMR (CDCl , 400 MHz): δ 0.87–0.92 (6H, m,
3
× γ-CH Val), 1.31–1.34 (6H, m, 2 × CH Ala), 1.58–
3
3
.63 (2H, m, H-4 Pro), 1.82–1.87 (2H, m, H-3 Pro), 2.12–
.17 (1H, m, β-CH Val), 3.45–3.55 (2H, m, H-5 Pro), 3.58–
.60 (1H, m, α-CH Ala), 4.32–4.33 (1H, m, α-CH Val),
.38–4.41 (1H, m, α-CH Pro), 4.54–4.56 (1H, m, α-CH
+
for C H N O [M +H]: 645.31375; found: 645.31351.
3
2
45
4
10
Ala), 5.77 (2H, d, J 4.8 Hz, CH Pym), 8.04–8.22 (11H,
Synthesis of Poc‑Ala‑Ala‑Pro‑Val‑OMe 4d
2
m, 9 × H-Pym and 2 × NH) (NH protons not visible).
2
1
3
C NMR (CDCl , 100.6 MHz): δ 16.88 (CH Ala), 18.52
N′,N′-Carbonyldiimidazole (0.061 g; 0.38 mmol, 1 equiv)
was dissolved in dry DMF (2 mL) in an ice bath, then
1-pyrene methanol 1c (0.087 g; 0.38 mmol, 1 equiv) was
added slowly and the solution was left stirring for 1 h at
low temperature. The peptide H-Ala-Ala-Pro-Val-OMe 2d
in the form of hydrochloride (0.182 g; 0.38 mmol, 1 equiv)
was neutralised with triethylamine in dry DMF (2 mL).
The triethylammonium chloride was filtered and the solu-
tion was added to the previous mixture and left stirring at
room temperature for 12 h. The solvent was evaporated
to dryness and the crude residue was purified by silica gel
3
3
(
(
γ-CH Val), 19.47 (γ-CH Val), 19.91 (CH Ala), 25.65
C-4 Pro), 30.02 (C-3 Pro), 31.59 (β-CH Val), 45.74 (C-5
3 3 3
Pro), 50.81 (α-CH Ala), 59.66 (α-CH Ala), 60.87 (α-CH
Pro and α-CH Val), 66.16 (CH Pym), 128.17 (C-Pym),
2
1
1
1
1
28.34 (C-Pym), 128.38 (C-Pym), 128.76 (C-Pym),
28.80 (C-Pym), 128.96 (C-Pym), 129.10 (C-Pym),
29.23 (C-Pym), 129.33 (C-Pym), 129.37 (C-Pym),
29.85 (C-Pym), 130.00 (C-Pym), 130.85 (C-Pym), 132.03
(
C-Pym), 132.55 (C-Pym), 133.23 (C-Pym), 172.75 (C=O
Val), 173.12 (C=O Ala), 174.38 (C=O Pro), 175.33 (C=O
1
3

F. C. Santos et al.
column chromatography using ethyl acetate/petroleum
ether (4:6) as eluent. The fractions containing the product
were combined and evaporated to yield the pyrene-labelled
Results and discussion
Synthesis of caged AAPV derivatives 4a–d
1
peptide 4d as an off-white solid (0.024 g, 10%). H NMR
(
1
CDCl , 400 MHz): δ 0.87–0.88 (6H, m, 2 × γ-CH Val),
Coumarin and pyrene derivatives 1a–c were chosen as
photosensitive groups to assess their influence in the
uncaging of the model bioactive tetrapeptide Ala–Ala–
Pro–Val (AAPV), by introducing these groups at the N-
and C-terminals of the peptide through carbamate or ester
linkages, respectively.
3
3
.15–1.18 (6H, m, 2 × CH Ala), 1.87–1.89 (2H, m, H-4
3
Pro), 1.92–1.98 (1H, m, β-CH Val), 1.99–2.01 (2H, m, H-3
Pro), 3.38–3.41 (2H, m, H-5 Pro), 3.78 (3H, s, OCH3),
4
.25–4.26 (1H, m, α-CH Ala), 4.27–4.28 (1H, m, α-CH
Val), 4.30–4.31 (1H, m, α-CH Pro), 4.33–4.35 (1H, m,
α-CH Ala), 5.23 (1H, br s, NH), 5.74 (2H, d, CH Pym),
4-Chloromethyl-7-methoxycoumarin 1a was obtained
by a Pechmann reaction between 3-methoxyphenol and
ethyl 4-chloro-3-oxobutanoate, catalysed by acid, in 93%
yield, by a previously published procedure (Piloto et al.
2006). This heterocycle has been used previously by us
as caging group for amino acids with interesting photo-
sensitivity (Fonseca et al. 2007, 2010). Likewise, pyrene-
based photolabile groups were also applied by us in the
caging of neurotransmitter amino acids and biogenic
amines (Fernandes et al. 2007, 2008, 2013). So, 4-chloro-
methyl-7-methoxycoumarin 1a, 1-choloromethylpyrene
1b and 1-pyrenemethanol 1c were used in the derivatiza-
tion of AAPV at the C- and N-terminals.
2
1
3
7
(
.93–8.15 (11H, m, 9 × H-Pym and 2 × NH). C NMR
CDCl , 100.6 MHz): δ 16.90 (CH Ala), 18.51 (γ-CH
3
3
3
Val), 19.45 (γ-CH Val), 19.92 (CH Ala), 25.68 (C-4 Pro),
3
3
3
0.07 (C-3 Pro), 31.60 (β-CH Val), 45.75 (C-5 Pro), 50.85
(
α-CH Ala), 52.51 (OCH ), 59.75 (α-CH Ala), 60.78 (α-CH
3
Pro), 60.82 (α-CH Val), 61.76 (CH Poc), 128.19 (C-Poc),
2
1
(
1
(
28.37 (C-Poc), 128.41 (C-Poc), 128.72 (C-Poc), 128.75
C-Poc), 128.94 (C-Poc), 129.13 (C-Poc), 129.24 (C-Poc),
29.38 (C- Poc), 129.45 (C-Poc), 129.81 (C-Poc), 130.04
C-Poc), 130.80 (C-Poc), 132.05 (C-Poc), 132.59 (C-Poc),
1
1
33.21 (C-Poc), 155.62 (C=O Poc), 172.77 (C=O Val),
73.22 (C=O Ala), 174.44 (C=O Pro), 175.36 (C=O Ala).
+
HRMS (ESI): calcd for C H N O [M +H]: 629.29773;
The synthesis of Fmoc-AAPV-OH 2a was accom-
plished by a solid-phase synthesis protocol in an acid-
labile 2-chlorotritylchloride resin on a manual microwave-
assisted peptide synthesizer (CEM Discover) (Scheme 1).
The resin and the necessary Fmoc-amino acids were com-
mercially available and the resin had a functionalisation
degree of 0.84 mmol/g. Amino acid coupling reactions
were carried out in dry DMF in the presence of N,N′-
diisopropylcarbodiimide (DIC) and 6-chloro-N-hydroxy-
benzotriazole (6-Cl-HOBt) (a 5 equiv excess was used),
with a standard MW coupling protocol (5 min, 28 W,
75 °C). After each coupling, the Fmoc protecting group
was removed with 20% piperidine in DMF solution using
a standard MW protocol (30 s cycle plus a 3 min’ cycle,
28 W, 75 °C). The cleavage of the peptide 2a from the
resin was achieved by treatment with a AcOH/TFE/DCM
(1:1:3) cocktail and Fmoc-AAPV-OH 2a was isolated as
a solid in 85% yield. The purity was checked by analyti-
3
5
41
4
7
found: 629.29810.
Photolysis general procedure
A 1 × 10− M MeOH/HEPES buffer (80:20) or ACN/
HEPES buffer (80:20) solution of compounds 4a–d (5 mL)
was placed in a quartz tube and irradiated in a Rayonet
RPR-100 reactor at the desired wavelength. The lamps used
for irradiation were 254, 300, and 350 ± 10 nm. HEPES
buffer solution was prepared in distilled water with HEPES
4
(
(
(
(
4-(2-hydroxyethyl)-1-piperazine
ethanesulfonic
acid)
10 mM), sodium chloride (120 mM), potassium chloride
3 mM), calcium chloride (1 mM) and magnesium chloride
1 mM) and pH adjusted to 7.2 with aqueous 1 M sodium
hydroxide solution. Aliquots of 100 µL were taken at regular
intervals and analysed by RP-HPLC using a Licrospher 100
RP18 (5 μm) column in a JASCO HPLC system composed
by a PU-2080 pump and a UV-2070 detector with ChromNav
software. The eluent was ACN/water (3:1) at a flow rate of
1
13
cal HPLC and the peptide was characterised by H and
C
NMR spectroscopy and high-resolution mass spectrometry.
After removal of the Fmoc group from peptide 2a to give
free H-AAPV-OH 2b (in 90% yield), reaction with t-butyl
pyrocarbonate afforded Boc-AAPV-OH 2c in 82% yield,
while reaction with methanol and thionyl chloride resulted
in the respective methyl ester H-AAPV-OMe 2d in the
form of hydrochloride salt in quantitative yield (Scheme 1).
The chloromethyl precursors 1a,b were reacted
with Fmoc-AAPV-OH 2a in the presence of potassium
0
0
.8 mL/min for compounds 4c–d, or ACN/water (3:1) with
.1% TFA at a flow rate of 0.6 mL/min for compounds 4a–b,
previously filtered through a Millipore, type HN 0.45 µm fil-
ter and degassed by ultra-sound for 30 min. The chromato-
grams were traced by detecting UV absorption at the wave-
length of maximum absorption for each compound at the
corresponding solvent (retention time in minutes: 4a, 15.5;
4b, 17.2; 4c, 6.9; 4d, 8.5).
1
3

Phototriggered release of tetrapeptide AAPV from coumarinyl…
pyrenylmethoxycarbonyl group), it was obtained by
reaction of H-AAPV-OMe 2d with 1-pyrenemethanol 1c
mediated by N′,N′-carbonyldiimidazole (CDI). The prod-
uct was isolated after column chromatography in lower
yield (10%) . The purity of the C-and N-terminal caged
peptides 4a-d was checked by analytical HPLC and the
Cl
Fmoc-Val-OH, DIPEA
dry DMF
MW (30 min, 25 W, 38 °C)
Fmoc Val
1
13
peptides were characterised by H and C NMR spec-
troscopy and high-resolution mass spectrometry.
piperidine 20% DMF
MW (30 s + 3 min, 28 W, 75 °C)
1
H NMR spectra showed the characteristic signals of the
photosensitive groups in the aromatic zone and the methyl-
ene group, adjacent to the ester or carbamate link, was vis-
ible between δ 5.30–5.77 ppm. The newly formed ester or
carbamate linkages in caged peptides 4a–d were confirmed
by C NMR spectra signals of the carbonyl group, at about
δ 170.56–174.57 (for the esters) or δ 155.62 ppm (for the
carbamate).
H N Val
2
Fmoc-Pro-OH, 2 x Fmoc-Ala-OH
DIC, 6-Cl-HOBt, dry DMF
MW (5 min, 28 W, 75 °C)
1
3
piperidine 20% DMF
MW (30 s + 3 min, 28 W, 75 °C)
Fmoc Ala Ala Pro Val
AcOH/TFE/DCM (1:1:3)
Photophysical characterisation of caged peptides 4a–d
Given the highly conjugated structure of the photosensi-
tive groups, coumarin and pyrene, which are known for
displaying strong absorption and fluorescence in the UV/
vis, fundamental UV/vis absorption characterisation was
performed for the caged peptides 4a–d, also considering
that their photolysis would be monitored by HPLC with
UV detection. For the photolysis studies, it was decided
to evaluate the influence of a protic (methanol) and aprotic
Fmoc Ala Ala Pro Val OH
2a
i)
ii)
2a
H-AAPV-OH
Boc-AAPV-OH
2b
2c
iii)
H-AAPV-OMe
(
acetonitrile) solvent on the photolysis rates.
−4
2d
The absorption spectra of degassed 10 M solutions
in MeOH/HEPES buffer (80:20) and ACN/HEPES buffer
(80:20) solutions of caged peptides 4a–d were measured
and the absorption maxima (λmax), and molar absorption
coefficients (log ε) are reported in Table 1.
Scheme 1 Synthesis of AAPV derivatives 2a–d by MW-SPPS on a
-chlorotrityl chloride resin. Reaction conditions: i) 20% piperidine
in DMF, rt; ii) Boc O, aq. 1 M NaOH 1,4-dioxane, rt; iii) SOCl ,
2
2
2
MeOH, rt
From the data in Table 1, it can be seen that the nature
of the organic solvent did not influence significantly the
absorption maxima: the values were very similar in both
solvents, with the exception of peptide 4c, for which a
bathochromic shift of 5 nm was observed when comparing
the protic and the aprotic solvent. As for the nature of the
photosensitive group, the presence of the pyrene group led
to larger λmax (between 17 and 19 nm) in both solvents for
peptides 4a (bearing a C-terminal coumarin) and 4b (bear-
ing a C-terminal pyrene).
fluoride in DMF (Tjoeng and Heavner 1981) to afford
the corresponding ester conjugate peptides Fmoc-AAPV-
OMcm 3a (97%) and Fmoc-AAPV-OPym 3b (65%)
(
Mcm will be used to designate the methoxycoumarinyl-
methyl group whereas Pym will represent the pyrenyl-
methyl group) (Scheme 2; Table 1). Considering the aro-
matic nature of the Fmoc protecting group, which could
interfere with the photocleavage process by competitive
radiation absorption, it was decided to remove this group
prior to subjecting the peptides to irradiation. Therefore,
treatment with 20% piperidine in DMF solution afforded
the N-deprotected peptides H-AAPV-OMcm 4a (in 41%
yield) and H-AAPV-OPym 4b (in 63% yield).
Considering the fluorescent nature of the photosensitive
groups, the fluorescence of caged peptide 4c, as represent-
ative example, was studied in different organic solvents
and aqueous mixtures. Emission maxima (λ ) and rela-
em
tive fluorescence quantum yields (Φ ) are given in Table 2.
F
In a similar manner, chloromethylated coumarin
Relative fluorescence quantum yields were calculated
1
a was reacted with Boc-AAPV-OH 2c in the pres-
using 9,10-diphenylanthracene in ethanol (Φ 0.95) (Mor-
F
ence of potassium fluoride in DMF, resulting in Boc-
AAPV-OMcm 4c in 83% yield. As for peptide Poc-
AAPV-OMe 4d (Poc will be used to designate the
ris et al. 1976) as standard. For the Φ determination, the
fluorescence standard was excited at the maximum absorp-
tion wavelength, in each solvent, and in all fluorimetric
F
1
3

F. C. Santos et al.
Scheme 2 Synthesis of caged
AAPV derivatives 4a–d. Reac-
tion conditions: i) KF, DMF, rt;
ii) 20% piperidine in DMF, rt;
iii) CDI, DMF, rt
Table 1 UV/vis absorption data for caged peptides 4a–d in MeOH/
HEPES buffer (80:20) and ACN/HEPES buffer (80:20) solutions
The collected data revealed similar wavelengths of
maximum absorption and emission, but the relative fluo-
rescence quantum yield varied significantly being higher
in protic solvents/mixtures (Table 2). The overall trend
revealed that caged peptide 4c was more fluorescent in
aqueous mixtures, especially with methanol. This fact
is of great significance for future biological applications
involving fluorescence-based techniques, as it could max-
imize the response and, consequently, increase the sen-
sitivity of detection of the labelled peptide in aqueous
media (Fig. 1). The Stokes’ shifts varied between 69 and
Compound
UV/vis absorption
MeOH/HEPES
ACN/HEPES
(80:20)
(
80:20)
λ
(nm)
log ε
λ
(nm)
max
log ε
max
H-AAPV-OMcm
325
342
325
342
3.89
323
342
320
341
3.84
4
a
H-AAPV-OPym
4.39
4.29
4.38
4.43
4.07
4.30
4
b
Boc-AAPV-OMcm
8
0 nm and a large Stokes’ shift is an attractive charac-
4
c
teristic for a fluorescent probe in biological media that
allows an improved separation of the light inherent to the
matrix and the light dispersed by the sample.
Poc-AAPV-OMe
4
d
measurements the absorbance of the solution did not
exceed 0.1. The fluorescence displayed by the caged pep-
tides 4a–d is an interesting feature since it allows the mon-
itoring of the processes and dynamics in which these pep-
tides could participate by fluorescence spectroscopy.
Photolysis studies of caged peptides 4a–d
Evaluation of the 7-methoxycoumarinylmethyl and pyre-
nylmethyl groups as photosensitive units for the caging of
peptides, using AAPV as a model bioactive tetrapeptide,
Table 2 UV/vis absorption
and fluorescence data for caged
peptide Boc-AAPV-OMcm 4c
in different organic solvents and
aqueous mixtures
Solvent
UV/vis absorption
(nm)
Fluorescence
λ
log ε
λ
(nm)
Φ
F
Stokes’ shift (nm)
max
em
EtOH
325
319
319
325
320
4.05
4.11
4.24
4.29
3.54
394
399
393
400
397
0.34
0.58
0.12
0.76
0.56
69
80
74
75
77
MeOH
ACN
MeOH/HEPES (80:20)
ACN/HEPES (80:20)
1
3

Phototriggered release of tetrapeptide AAPV from coumarinyl…
2
1
1
6
0
,40E+06
,80E+06
,20E+06
,00E+05
,00E+00
was followed by reverse-phase HPLC with UV detec-
tion and the disappearance of the peptide conjugates and
the release of the corresponding peptide precursors were
monitored (Scheme 3). The plots of peak area (A) of the
starting material versus irradiation time were obtained
for each compound, at chosen wavelengths. The peak
areas were determined by HPLC, which revealed a grad-
ual decrease with time and were taken as the average of
EtOH
MeOH/HEPES (80:20)
ACN
MeOH
ACN/HEPES (80:20)
three runs. The irradiation time (t ) given represents the
irr
time necessary for the consumption of the starting mate-
rials until less than 5% of the initial area was detected
3
30
380
430
480
530
580
Wavelength (nm)
(
Table 3). The photochemical quantum yields (Φphot) were
calculated based on half-lives (t ), molar extinction coef-
1
/2
Fig. 1 Normalised fluorescence spectra of caged peptide 4c in differ-
ent solvents (C = 7.6 × 10 M)
−
6
ficients (ε) and the incident photon flux (I ), which was
0
determined by potassium ferrioxalate actinometry (Muller
et al. 2001). The rate constants (k) were calculated for a
short conversion (30%) of the initial conjugates.
hν
H-AAPV-OMcm
H-AAPV-OH
The data in Table 3 revealed that, by irradiation at
4a
2b
2
54 nm, the time necessary for the release of the bio-
hν
hν
hν
active peptide from cages 4a–c was larger in ACN/
HEPES, with this difference being more accentuated for
caged peptide 4b. For peptides 4a and 4c, the presence
of a N-butyloxycarbonyl protecting group resulted in a
slightly longer irradiation time for the N-protected pep-
tide (4c). When considering the different photosensitive
groups linked through an ester bond at this wavelength
of irradiation, there was no clear tendency in the irra-
diation times in the solvents tested: the pyrenylmethyl
group (Pym) cleaved faster in MeOH/HEPES while the
coumarinylmethyl group (Mcm) cleaved faster in ACN/
HEPES. In terms of the influence of the type of linkage
between the photosensitive group and the peptide, in the
case of pyrene it was observed that a carbamate link, as
in Poc-AAPV-OMe 4d, cleaved much faster (ca. 4 times)
than the corresponding counterpart with an ester link, as
in H-AAPV-OPym 4b. Irradiation at 300 nm afforded
similar observations: once again the carbamate caged
peptide 4d was the fastest to cleave and the presence of
H-AAPV-OPym
H-AAPV-OH
4b
2b
+
photo by-products
Boc-AAPV-OMcm
Boc-AAPV-OH
4c
2c
Poc-AAPV-OMe
H-AAPV-OMe
4d
2d
Scheme 3 Photolysis of caged peptides 4a–d by irradiation at 254,
00, and 350 nm in MeOH/HEPES buffer (80:20) and ACN/HEPES
buffer (80:20) solutions
3
was carried out. Accordingly, peptides 4a–d were irradi-
ated at 254, 300 and 350 nm in mixtures of methanol or
acetonitrile with aqueous HEPES buffer in 80:20 solu-
tions, in a Rayonet RPR-100 reactor, and kinetic data
were collected. The course of the photolytic reaction
Table 3 Irradiation times (tirr in
Solventa
Cpd.
254 nm
300 nm
350 nm
min), photochemical quantum
−
−
3
yields (Φphot, ×10 ) and rate
tirr
Φ
k
tirr
Φ
k
tirr
Φ
phot
k
phot
phot
1
contants (k, in min ) for the
photolysis of caged peptides
4a
A
B
A
B
A
B
A
B
40
46
18
81
57
74
0.335
0.400
0.162
0.056
0.087
0.125
0.083
0.106
0.113
0.068
0.076
0.040
41
0.019
0.148
0.075
0.042
0.012
0.038
0.114
0.081
0.129
0.115
0.025
0.017
352
1108
310
0.018
0.007
0.006
0.004
0.001
0.002
0.045
0.013
0.009
0.006
0.006
0.003
0.002
0.001
0.073
0.018
4
a–d by irradiation at 254, 300,
55
and 350 nm in MeOH/HEPES
buffer (80:20) and ACN/HEPES
buffer (80:20) solutions
4
4
4
b
32
53
389
c
347
163
2080
2169
46
d
4.4
b
1.050
b
0.806
b
8.3
b
0.274
b
0.404
b
162
a
A = MeOH/HEPES (80:20); B = ACN/HEPES (80:20)
Not determined
b
1
3

F. C. Santos et al.
Fig. 2 Plot of ln A versus irra-
diation time for the photolysis
of caged peptides 4a–d at the
various wavelengths of irradia-
tion in MeOH/HEPES buffer
15,0
14,0
13,0
4 b (MeOH/HEPES)
254 nm
4
4
4
4
b (ACN/HEPES)
a (MeOH/HEPES)
a (ACN/HEPES)
d (MeOH/HEPES)
(
(
80:20) and ACN/HEPES buffer
80:20) solution
12,0
11,0
10,0
4 c (MeOH/HEPES)
c (ACN/HEPES)
4
0
5
10
15
20
25
30
Irradiation time (min)
4
4
4
4
4
4
4
b (MeOH/HEPES)
b (ACN/HEPES)
a (MeOH/HEPES)
a (ACN/HEPES)
d (MeOH/HEPES)
c (MeOH/HEPES)
c (ACN/HEPES)
3
00 nm
15,0
14,0
13,0
12,0
11,0
0
10
20
30
40
50
60
70
80
90
100
Irradiation time (min)
4
b (MeOH/HEPES)
b (ACN/HEPES)
a (MeOH/HEPES)
a (ACN/HEPES)
d (MeOH/HEPES)
d (ACN/HEPES)
c (MeOH/HEPES)
c (ACN/HEPES)
1
5,0
4,0
3,0
2,0
1,0
0,0
350 nm
4
4
4
4
4
4
4
1
1
1
1
1
9,0
0
50
100
150
200
250
300
350
Irradiation time (min)
a N-Boc group led to a dramatic difference in irradiation
times, which occurred in peptide 4c (Boc-AAPV-OMcm),
in comparison with and 4a (H-AAPV-OMcm).
At 350 nm, the influence of the solvent was clear with
photolysis being faster in MeOH/HEPES for all caged
peptides. At this wavelength of irradiation, the obtained
irradiation times were too large to be of practical use,
except for carbamate caged peptide Pym-AAPV-OMe 4d
in MeOH/HEPES, which cleaved readily in ca. 46 min.
The relationship between the molar extinction coef-
ficient of the conjugates at the wavelengths of irradiation
and the photolysis rate constants was confirmed, as larger
rate constants were obtained when the molar extinction
coefficient was also larger, which was expected due to the
1
3

Phototriggered release of tetrapeptide AAPV from coumarinyl…
higher probability of absorbing radiation and undergoing
a photolytic pathway. For compound 4c, nevertheless, this
trend was not followed at 350 nm irradiation, which may be
related to its tendency to undergo a radiative pathway (as
seen by the high relative fluorescence quantum yields in the
studied solvent mixtures).
Overall, in the photolysis studies the time necessary
to release the active peptide was shorter when photolyses
were carried out in the more polar and protic solvent,
MeOH/HEPES (80:20) and when the photosensitive group
was linked to the peptide by a carbamate bond, as in pep-
tide 4d. Given the need to minimise cellular damage in
the uncaging of molecules with biological activity using
wavelengths longer than 350 nm, the most suitable for bio-
logical applications, only the data obtained by irradiation
at 350 nm for peptide 4d constitute a promising result for
future development.
of linkage in the controlled photorelease of the active mol-
ecule, which was monitored by HPLC–UV.
The obtained data confirmed the applicability of the tested
photoactive groups for the release of AAPV, especially for
the derivative bearing the carbamate-linked pyrenylmethyl
group, which displayed the shortest irradiation times for the
release at the various wavelengths of irradiation (ca. 4 min at
254 nm, 8 min at 300 nm and 46 min at 350 nm).
Acknowledgements Thanks are due to the Fundação para a Ciência
e Tecnologia (FCT, Portugal) for financial support to the portuguese
NMR network (PTNMR, Bruker Avance III 400-Univ. Minho), FCT
and FEDER (European Fund for Regional Development)-COMPETE-
QREN-EU for financial support through the Chemistry Research Cen-
tre of the University of Minho (Ref. UID/QUI/00686/2013 and UID/
QUI/0686/2016). A PhD grant to A.M.S. (SFRH/BD/80813/2011) is
also acknowledged.
Compliance with ethical standards
Nevertheless, the reported results reflect the uniqueness
of the experimental setup and this work intends to be a pre-
liminary assessment of the applicability of the tested pho-
toactive groups in the photorelease of AAPV. As a follow-
up of this work, the most promising AAPV cage 4d will
undergo a more detailed study using time-resolved fluores-
cence spectroscopy to characterise its photophysical prop-
erties. This kind of studies has been previously reported by
us, regarding other bioactive targets, in order to relate to
the conjugate, the ion pair intermediate and photocleaved
species, by the determination of decay-associated spectra
to enable both spectral (energetic) and decay kinetics to be
compared.
Conflict of interest The authors declare that they have no conflict of
interest.
Research involving human participants and/or animals This arti-
cle does not contain any studies with human participants or animals
performed by any of the authors.
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