Peptide release upon photoconversion of 2-nitrobenzyl compounds into nitroso derivatives

Article abstract of DOI:10.1111/j.1751-1097.2011.00957.x

The photoinduced conversion via the aci-nitro into the nitroso form was studied for 4,5-dimethoxy-2-nitrobenzyl alcohols attached to various leaving groups: amino acids histidine (NHis) and aspartate (NAsp) as well as their fluorenylmethoxycarbonyl derivatives (FHis) and (FAsp). In addition, two peptides containing either of the two amino acids were studied, carrying the photoreactive group attached to a histidine (PHis), or to an aspartate (PAsp). The aci-nitro forms with maximum at λ_aci = 420 nm were observed for FHis and FAsp after the decay of a triplet-triplet absorption, analogous to those of other 2-nitrobenzyl type compounds. For both FHis and FAsp the quantum yield of photoconversion δ∥_p is 0.03 and for the peptides PHis and PAsp ca 0.01 and 0.005, respectively. Photocaged amino acids, histidine and aspartate were incorporated by chemical synthesis into peptides. The peptides show sequence motifs of proteins from the bacterial two-component signaling system. The light-labile protection group, 2-nitro-4,5-dimethoxybenzyl, could be removed by ultraviolet irradiation, releasing the active peptides. In both cases, the triplet forms and the aci-nitro species were generated transiently and yielded the 2-nitroso-4,5-dimethoxybenzaldehyde.

Full text of DOI:10.1111/j.1751-1097.2011.00957.x

Photochemistry and Photobiology, 2011, 87: 1031–1035
Peptide Release upon Photoconversion of 2-Nitrobenzyl Compounds into
Nitroso Derivatives
¨
¨
Koji Nakayama, Inge Heise, Helmut Gorner and Wolfgang Gartner*
Max-Planck-Institut fu¨r Bioanorganische Chemie, Mu¨lheim an der Ruhr, Germany
Received 22 April 2011, accepted 06 June 2011, DOI: 10.1111/j.1751-1097.2011.00957.x
other processes is often diffusion limited and thus can obscure
ABSTRACT
relevant kinetical processes. Herein, we present studies on two
peptides carrying the photocaged amino acids: histidine and
aspartate; in addition, these amino acids were also studied in
free form and as their Fmoc-(fluorenylmethoxycarbonyl)
derivatives, which is the functionalized form of amino acids
for peptide synthesis. A characterization of the photochemical
properties of both amino acids, either in free form or
incorporated into two peptides, is important, as these amino
acids, as well as the studied peptides represent sequence motifs
of two-component signaling (TCS) processes. Many prokary-
otes employ this TCS system for responding to environmental
stimuli (22).
Receptors active in the TCS signaling pathway employ
histidine kinases (HK) for signal propagation that are phos-
phorylated at a conserved histidine residue and transfer the
phosphate group via protein–protein interaction to an aspar-
tate residue in a so-called response regulator (RR) protein.
Phototriggered reactions of caged peptides have been studied
previously (23–28). Herein, we describe the photochemically
initiated release of peptides with sequences deriving from a
cyanobacterial two-component system, the HK CphA and its
cognate response regulator RcpA (29).
To understand these reactions in detail, 4,5-dimethoxy-2-
nitrobenzyl derivatives containing two amino acids, histidine
(NHis) and aspartate (NAsp), the corresponding derivatives
with Fmoc substitution (FHis) and (FAsp) for solid phase
peptide synthesis as well as two peptides carrying the
photocaged amino acids (PHis and PAsp, respectively) were
synthesized (Scheme 1). As the amino acids show best
solubility in acetonitrile ⁄ water, the photoconversion into
the nitroso products and related photochemical properties
of all compounds were studied in the same solvent for all
samples.
The photoinduced conversion via the aci-nitro into the nitroso
form was studied for 4,5-dimethoxy-2-nitrobenzyl alcohols
attached to various leaving groups: amino acids histidine (NHis)
and aspartate (NAsp) as well as their fluorenylmethoxycarbonyl
derivatives (FHis) and (FAsp). In addition, two peptides con-
taining either of the two amino acids were studied, carrying the
photoreactive group attached to a histidine (PHis), or to an
aspartate (PAsp). The aci-nitro forms with maximum at
kaci = 420 nm were observed for FHis and FAsp after the
decay of a triplet–triplet absorption, analogous to those of other
2-nitrobenzyl type compounds. For both FHis and FAsp the
quantum yield of photoconversion Fp is 0.03 and for the peptides
PHis and PAsp ca 0.01 and 0.005, respectively.
INTRODUCTION
The photoconversion of 2-nitrobenzyl (ArNO2) into nitroso
(OArNO) compounds is the subject of intensive studies (1–21),
due to their capability to deliver reactive molecules upon
irradiation. Various protecting groups have been developed for
these ‘‘caged’’ compounds, which are rapidly released for time-
resolved studies of chemical and biological processes (1).
Besides the parent compound, 2-nitrobenzyl alcohol, also its
4,5-dimethoxy derivative (N1) showing a more bathochromic
absorbance, has been investigated in greater detail. Such
investigations especially address the effects of solvent polarity,
temperature and quenching by water on the decay of the
intermittently generated aci-nitro forms (19). In contrast to the
parent compound 2-nitrobenzyl alcohol, its dioxy-derivative
(N1) and the corresponding 4,5-methylenedioxy-2-nitrobenzyl
alcohol form a triplet state with CT character in 308 nm flash
photolysis experiments, whereas the other photochemical
properties are very similar (19,20). Yet, the population of a
CT triplet state due to a 4,5-methylenedioxy group or two
4,5-dimethoxy substituents in 2-nitrobenzyl compounds was
found to be independent of the photoconversion into the
nitroso compounds (19).
MATERIALS AND METHODS
Materials. N1 (Aldrich-Chemie), 4,5-dimethoxy-2-nitrophenyl
bromide and the solvents (Merck), e.g. acetonitrile (Uvasol quality),
were used as received. Water was purified by a milliQ system.
Synthesis of photocaged amino acids. Both amino acids were
synthesized as their Fmoc derivatives; for study of the free photocaged
amino acids, the Fmoc group was removed by treatment with 25%
piperidine in dimethylformamide (DMF).
Of particular interest is the light-induced release of photo-
caged compounds in biological systems, where activation by
Histidine: Histidine was purchased as Fmoc derivative, carrying an
N-im-trityl group. The carboxy group was converted into the allyl ester
before removing the trityl group by treatment with trifluoroacetic acid
(TFA) in CH2Cl2 (Scheme 1, top). The photocaged moiety is intro-
duced by the reaction with 4,5-dimethoxy-2-nitrobenzyl bromide in the
*Corresponding author email: gaertner@mpi-muelheim.mpg.de (Wolfgang
Gartner)
¨
ꢀ 2011 The Authors
Photochemistry and Photobiology ꢀ 2011 The American Society of Photobiology 0031-8655/11
1031

1032 Koji Nakayama et al.
Scheme 1. Chemical synthesis of Fmoc-photocaged amino acids: (top) histidine and (bottom) aspartate (for detailed description of the chemical
reactions and the analytical characterization—1H NMR and ESI-MS—see Supporting Information).
presence of potassium carbonate. After this reaction, the allyl group is
removed with catalytic reaction using tetrakis(triphenylphosphine)pal-
ladium(0) and phenylsilane (for details see Data S1 Supporting
information).
Aspartate: N-a-Fmoc-c-4,5-dimethoxy-2-nitrobenzyl aspartate was
synthesized by introducing the photocaged moiety (4,5-dimethoxy-2-
nitrobenzyl alcohol) into the N-c-Fmoc-aspartate tert-butyl ester in the
presence of DIC and DMAP in DMF (Scheme 1, bottom). The tert-
butyl group of the compound is removed by TFA to yield the target
compound (for details see Data S1).
Solid phase peptide synthesis. All chemicals for peptide synthesis
were purchased from Iris Biotech and used without further
purification. Synthesis was performed by an Advanced Chemtec
348W peptide synthesizer using a 2-chlorotrityl alcohol resin, which
was swollen twice for 30 min in DMF. Side chain protection groups
were as follows: tert-butyl for: Asp, Glu, Ser, Thr, Tyr; tert-
butyloxycarbonyl (Boc) for: Lys, Trp; Trityl: Asn, Gln, His, Cys;
2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl (Pbf) for Arg.
Amino acids Ala, Gly, Ile, Leu, Met, Phe and Pro were employed
without protection groups. For synthesis, the amino acids and
coupling reagents (benzotriazol-1-yloxy)tripyrrolidinophosphonium
solution of dichloromethane, containing 25% of TFA and 5% of
triethyl silane. To ensure complete deprotection, the solution was
kept at ambient temperature overnight. The cleavage solution was
poured into diethylether, the formed precipitate was collected by
filtration, washed with diethyl ether, dissolved in water and freeze-
dried.
Spectroscopy. The absorption spectra were recorded on a UV ⁄ vis
diode array spectrophotometer (HP, 8453). For photoconversion the
366 nm line of a 250 W Hg lamp was applied. Relative yields were
obtained using optically matched solutions (A366 = 1 or 2). Flash
photolysis was performed at kexc = 355 nm (Nd-YAG laser, GWU
Lasertechnik), time-resolved absorption changes were measured by a
Luzchem system. Typically, a pathlength of 1 cm and absorbances
of 0.5–1 at k355 were used. The results refer to air-saturated solution
and room temperature unless otherwise indicated.
RESULTS AND DISCUSSION
Materials
Photocaged histidine and aspartate were synthesized chem-
ically and incorporated into peptides by solid phase synthesis.
These peptides were part of a histidine kinase (HK), CphA,
and a response regulator protein (RR), RcpA, from the
cyanobacterium Tolypothrix PCC7601 with the following
sequences: CphA, G-S-C-H(X)-D-L-Q-E-N-Y, RcpA, C-R-
P-E-D(X)-L-N-L-P-K-K-D-G-R-E-K (the photocaged amino
acids are indicated as H(X) and D(X), respectively).
hexafluorophosphate
(PyBOP),
O-(7-azobenzotriazol-1-yl-)-
N,N,N¢N¢-tetramethyluronium hexafluorophosphate (HATU) and
N,N-diisopropylethylamine (DIPEA) were dissolved in N-methyl-2-
pyrrolidone (NMP) containing 20% DMSO. The resulting mixture
and DIPEA were applied on the resin in a ratio of 1:5:5:10 (resin:
amino acid: coupling reagent: DIPEA). Each coupling cycle was
performed two or three times with reaction times between 1 and 3 h.
The Fmoc group was removed by applying a solution of 25%
piperidine in DMF. After each coupling and deprotection step, the
resin was washed six times with a DMF ⁄ diethyl ether solution. The
final deprotection of the N-terminus was performed twice for 15 min
each. After completion of the synthesis, the resin was washed six
times with a DMF ⁄ diethyl ether solution, twice with methanol and
five times with dichloromethane. After drying the resin 2 h under
argon, peptides were cleaved and fully deprotected by addition of a
Irradiation
The absorption spectrum of FAsp in argon-saturated aceto-
nitrile–water (7:3 vol) has a maximum at 260 nm and a second,

Photochemistry and Photobiology, 2011, 87 1033
less intense one at 350 nm. Upon irradiation at 355 or 366 nm,
the absorption at 350 nm decreases and A400 increases,
characteristic for the formation of the nitroso-photoproduct
(Fig. 1). Two isosbestic points at 300 and 380 nm indicate that
secondary photolysis is negligible. Plots of A350 and A420 show
the time dependence (Fig. 1, inset). The quantum yield of
photoconversion (Fp) was determined using the unsubstituted
dimethoxy derivative, N1, in acetonitrile with Fp = 0.8 as
reference (19), yielding for FHis and FAsp Fp = 0.03
(Table 1). The ca 30-fold smaller Fp for FHis and FAsp with
respect to unsubstituted 2-nitrobenzyl alcohols or to N1
indicates that amino acids strongly compromise the photore-
action. One has to keep in mind, however, that the attachment
of the amino acids via the ester group (aspartate) or through
the imidazole ring (histidine) induces a different photochemical
reactivity that should not be directly compared with the parent
compound. In principle, a portion of the incident light is
absorbed by the Fmoc group, which acts as an internal filter.
However, this can account for a reduction of Fp by only up to
ca 30%. Fp = 0.04 for NHis and 0.02 for NAsp. The
additional reduction in quantum yield is clearly ascribed to
the different type of attachment.
For PHis in acetonitrile–water (Fig. 2), A280 and A420
increase and A350 decreases in a similar manner as found with
the unsubstituted reference compound 2-nitrobenzyl alcohol.
But also for this compound, the quantum yield Fp is further
reduced than for the Fmoc cases. The photoinduced
absorption changes of PHis can again be ascribed to the
conversion of the 2-nitrobenzyl moiety into the nitroso
derivative, i.e. a possible photodamage can be neglected. The
much smaller Fp of PHis compared with the unsubstituted
parent compound indicates a strongly lowered reactivity when
the photocaged amino acid is incorporated into the polypep-
tide moiety, yet the absence of photodegradation would still
allow employing these peptides by applying a higher light
intensity or accumulating experiments. The photochemical
behavior of the Asp-containing peptide is comparable (Fig. 3
and Table 1). A recently reported literature value for a peptide
is Fp = 0.03 (27); compared with this experiment, Fp of PHis
and PAsp are markedly lower (Table 1). The reasons are not
yet understood and would probably demand further investi-
gations.
Figure 2. Plots of A280 (s), A350 (D) and A420 (n) of PHis in argon-
saturated acetonitrile–water (1:1 vol), inset: absorption spectra at 0, 20
and 100 min irradiation at 366 nm, 1–3, respectively.
Figure 1. Absorption spectra of FAsp in argon-saturated acetonitrile–
water (7:3 vol) prior to irradiation at 366 nm and at 120 and 300 s,
1–3, respectively; inset: plots of A260 (s), A350 (D) and A420 (h).
Table 1. Quantum yield of photoconversion, lifetimes of the triplet
state and aci-nitro form and rate constant for triplet quenching by
oxygen*.
Compound
Fp
sT (ls)
kox (109 · M)1s)1
)
saci (ms)
N1
0.80
2
–
–
1
1
–‡
0.3
0.2
–
–
0.2
>0.1
–
0.2†
–
–
0.1
0.1
–
NHis
NAsp
FHis
FAsp
PHis
PAsp
0.04*
0.02†
0.03
0.025
0.01
0.005
<1
–
*In acetonitrile–water (1:1 vol) solution at 25ꢁC, kexc = 308 nm;
†Secondary transient, mono-exponential fit; ‡DA is too low for precise
quantitation.
Figure 3. Plots of A270 (s) and A420 (n) of PAsp in argon-saturated
acetonitrile–water (1:1 vol), inset: absorption spectra at 0, 12, 30 and
90 min irradiation at 366 nm, 1–4, respectively.

1034 Koji Nakayama et al.
Transients
The time-resolved absorption spectra of N1 in argon-saturated
acetonitrile and of FAsp in acetonitrile–water (7:3 vol) after
the 355 nm pulse are shown in Figs. 4 and 5, respectively.
Three components can be identified, a short-lived and longer
lived intermediate (insets Figs. 4 and 5) and a permanent
component. The first signal at 420–500 nm is ascribed to a
triplet state, as can be expected for oxygen-substituted
compounds as the 4,5-dimethoxy derivatives (19,20). The
transient is formed within the pulse width of the laser, decays
with first-order kinetics, and is quenched by oxygen, indicative
for a triplet state with a triplet lifetime (sT) in the 1–2 ls range
(Table 1). For the reference compound, N1, the aci-nitro form
is detected as an intermediate on the pathway into the nitroso
product. Photolysis of N1 (and other alkoxy-substituted
2-nitrobenzyl alcohols [19,20]), however, reveals an additional
transient with a much broader spectrum and a larger DA value
than DAaci, very much akin to the above mentioned triplet
state.
Figure 6. Transient absorption spectra of PAsp in argon-saturated
acetonitrile–water (1:1 vol) at 50 ns (s), 0.6 ls (D) and 2 ls (n) after
the 355 nm pulse, inset: kinetics at 430 nm.
The transient absorption spectra and the triplet yields of
FHis and FAsp are similar. The rate constant for triplet
quenching of FAsp in acetonitrile by oxygen is kox = 2 ·
M
)1
108
s)1. The relative yields DAaci are consistent with the
determined 3% value of Fp for FHis or FAsp in relation to
that of N1. The signal at 420 nm after triplet decay is due to
the intermediate aci-nitro form on the pathway into the nitroso
product. The charge transfer, induced by the electron donating
methoxy groups in positions 4 and 5, shifts also the absorption
maximum (ca 350 nm), probably also lowering the specific
triplet energy level. For the unsubstituted parent compound
and its dimethoxy derivative N1 the lifetimes are similar in
each solvent, ranging from saci = 5–6 ls in carbon tetrachlo-
ride to 0.2–2 ms in DMSO (19). The triplet characteristics of
PAsp are shown in Fig. 6. Note that DAaci of PHis was found
to be too low to be detected.
It might be argued that the low quantum yield might impair
any application of these photocaged peptides in biological
assays. However, the straight-forward photochemistry, docu-
mented by the clearly identifiable isosbestic points and the
proof that the peptides do not undergo any photochemical
damage (controlled after irradiation), indicate that the low
quantum yield can readily be overcome by an increased light
intensity or by repetitive (accumulating) irradiation.
Figure 4. Transient absorption spectra of N-1 in argon-saturated
acetonitrile at 1 (s), 15 (m), 40 (r) and 90 (h) ls after the 355 nm
pulse, inset: kinetics at 420 nm.
CONCLUSION
The photochemical reactions of 2-nitrobenzyl compounds
carrying amino acids and also peptides containing the respec-
tive amino acids as protecting groups (FHis, FAsp, PHis and
PAsp) were studied. The alkoxy groups promote the formation
of a triplet state of CT character in this reaction (as also for
N1) which is, however, not part of the route into the photolysis
products (2-nitrosobenzaldehydes). The additional transient
can be ascribed to the aci-nitro form, which is part of the
photoconversion, resulting in the release of an appropriate
His- or Asp-substrate, either in free form or as residue in a
chemically synthesized peptide. The quantum yield of photo-
conversion Fp is 0.03 for FHis ⁄ FAsp and three to six times
smaller for the polypeptides. This low efficiency does not
Figure 5. Transient absorption spectra of FAsp in argon-saturated
acetonitrile–water (7:3 vol) at 20 ns (s), 1 ls (m) and 50 ls ( ) after
the 355 nm pulse, inset: kinetics at 420 nm.
n

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