A latent reaction in a model GFP chromophore revealed upon confinement: Photohydroxylation of ortho -halo benzylidene-3-methylimidazolidiones via an electrocylization process

Article abstract of DOI:10.1021/ol5013058

Excited state behavior of halogen substituted model GFP chromophores was investigated in an acetonitrile solution and in a confined environment provided by an octa acid capsule in water. Of the ortho, meta, and para halogen substituted GFP chromophores on

Full text of DOI:10.1021/ol5013058

Letter
pubs.acs.org/OrgLett
A Latent Reaction in a Model GFP Chromophore Revealed upon
Confinement: Photohydroxylation of ortho-Halo Benzylidene-3-
methylimidazolidiones via an Electrocylization Process
Shampa R. Samanta,†,∥ Jose P. Da Silva,§ Anthony Baldridge,‡,∥ Laren M. Tolbert,*,‡
́
and V. Ramamurthy*,†
†Department of Chemistry, University of Miami, Coral Gables, Florida 33146, United States
‡School of Chemistry and Biochemistry, Georgia Institute of Technology, 901 Atlantic Drive, Atlanta, Georgia 30332-0400, United
States
§Centro de Investigaca
̧ o em Química do Algarve, FCT, Universidade do Algarve, Campus de Gambelas, 8005-139 Faro, Portugal
̃
S
* Supporting Information
ABSTRACT: Excited state behavior of halogen substituted model GFP chromo-
phores was investigated in an acetonitrile solution and in a confined environment
provided by an octa acid capsule in water. Of the ortho, meta, and para halogen
substituted GFP chromophores only the ortho compounds gave a new product
resulting from an unprecedented photosubstitution of halogens by the hydroxyl group.
This unusual reaction highlights the importance of confined spaces in bringing about
some unattainable photoreactions.
uring the past decade there has been considerable interest
in manipulating the excited state behavior of organic
confined OA capsule to prompt the enhanced fluorescence.
D
BMIs when irradiated within the OA capsule preferred the trans
geometry at the photostationary state (up to 98%) while in
solution nearly equal amounts of both the cis and trans isomers
were present.6 Similar to dicis hexatriene, the trans isomer of
BMI could undergo intramolecular 6e cyclization, a reaction
hitherto not reported for BMIs.8 Such a possibility was revealed
upon examination of the excited state behavior of halogen (F,
Cl, and Br) substituted BMIs 2−7 (Scheme 1) included within
OA. The results of this study are presented below. Of the ortho,
meta, and para halogen substituted BMIs 2−7 only the ortho
ones (2 and 5) gave a new product resulting in substitution of
halogen by the OH group (8 in Scheme 1). To our knowledge,
such a photosubstitution has not been reported in the case of
any BMI.
molecules.1,2 Octa acid (OA, 1, Scheme 1) is one of the hosts
Scheme 1. Chemical Structure of Octa Acid (1) and Guest
Molecules Studied
The photochemistry of a number of BMIs (ortho, meta, and
para halogen substituted at the aryl ring and N-methyl and N-
propyl derivatives)9,10 was examined in acetonitrile and as OA
complexes in borate buffer solution. In the text we illustrate
their photochemistry with the Cl/N-Me derivative as an
exemplar. The data on fluoro and bromo derivatives mimicking
the behavior of chloro derivatives are summarized in the
Supporting Information (SI). Host−guest complexes of these
BMIs were prepared by stirring an aliquot of DMSO-d6 stock
(see SI for details) solution of the guest with host OA in a
borate buffer D2O solution. 1H NMR spectra of 1:2 OA
complexes of 2 and free OA are presented in Figure 1. The
employed by us to control the excited state behavior of organic
molecules in water through confinement and weak inter-
actions.3 Generally, OA forms 1:2 or 2:2 (guest to host)
capsular assemblies with neutral organic molecules in sodium
tetraborate buffered water (pH 8.7).4,5 Recently, we demon-
strated a strong emission of substituted cis-benzylidene-3-
methylimidazolidiones (BMIs; Scheme 1), molecules having
the basic skeleton of the chromophore (para hydroxy-
benzylidene-imidazolidone) responsible for the fluorescence
of green fluorescent protein (GFP), within the OA capsule
compared to that in solution.6,7 We surmised this to be due to
the arrest of single bond rotation (connecting aryl to the double
bond; see the red colored bond in Scheme 1) within the
Received: May 7, 2014
© XXXX American Chemical Society
A
dx.doi.org/10.1021/ol5013058 | Org. Lett. XXXX, XXX, XXX−XXX

Organic Letters
Letter
case of ortho-Cl BMI 2 is presented in Figure 2 (for results on
ortho-F and ortho-Br derivatives, see Figures S13 and S14 in SI).
1
Figure 1. H spectra of (i) octa acid, and capsular assembly of (ii)
ortho-F/Me, (iii) ortho-Cl/Me, and (iv) ortho-Br/Me along with the
assignment of the aliphatic protons of guests ([OA] = 1 mM in 10
mM borate buffer in D2O and [guest] = 0.5 mM). Signals of octa acid
“a−g” are assigned according to Scheme 1.
changes in the chemical shifts of the signals due to OA and the
upfield shift of the signals due to the guest’s alkyl groups
indicated the inclusion of the guest within OA.4,11,12 The
inclusion is more apparent in the case of N-propyl derivatives
where the methylene and methyl signals are shifted to below δ
0 ppm (Figures S1 and S2 in SI). When the guest amount
exceeded the guest−host ratio of 1:2 the cloudy solution
resulted in additional 1H NMR signals due to free uncomplexed
BMIs. These spectral observations suggested that the BMIs
formed 1:2 capsular assemblies with OA. The single set of
upfield shifted signals for guests during titration experiments
suggested that there was no equilibrium between free and
complexed guest molecules in solution and the BMIs remained
complexed to OA in aqueous borate buffer solution under our
experimental conditions.
1
Figure 2. H NMR spectra of photochemical reaction (>320 nm) of
ortho-Cl/N-Me@OA2 in aerated solution of borate buffer in D2O
([OA] = 1 mM in 10 mM borate buffer in D2O and [guest] = 0.5
mM) (i) cis-ortho-Cl/N-Me@OA2, (ii) upon photolysis of (i) for 2
min, (iii) upon photolysis of (i) for 30 min, and (iv) cis-ortho-OH/N-
Me@OA2.
The signals due to the methyl groups changed from the initial
two due to the cis isomer (2) to two new ones, identified by
comparison with authentic samples, to be due to the trans 2.
Further irradiation led to the appearance of two additional
signals with concomitant less intense signals due to the trans
and the cis isomers.
Comparison of the 1H NMR spectra of the new signals with
an authentic sample confirmed the final photoproduct to be
ortho-OH BMI 8 (Scheme 2).13,14 It is clear from Figure 3 (plot
Irradiation (450 W medium pressure mercury lamp with
Corning filter 4-69; >320 nm) of OA complexes of ortho, meta,
or para halogen substituted BMIs (see SI for details) in
aqueous borate buffer resulted in the corresponding trans
1
isomer (Scheme 2; for LC-MS traces and H NMR spectra of
Scheme 2. Photoreactions of ortho, meta, and para
Halogenated BMIs in Acetonitrile Solution and as Host−
guest Complexes with OA in Water
Figure 3. Phototransformation of cis-ortho-Cl/N-Me@OA2 (100
μM:200 μM) in borate aqueous solution (10 mM) at pH 9, and
formation of trans-ortho-Cl/N-Me and cis-ortho-OH/N-Me.
of concentration of products with respect to duration of
irradiation as monitored by LC-MS) that the formation of BMI
8 requires accumulation of the trans isomer suggesting that the
trans-ortho halogen substituted BMIs 2 are the precursors of
BMI 8. The identical product distribution inferred from LC-MS
traces of irradiations conducted under degassed and aerated
conditions in Figure S15 rule out any role for oxygen in the
formation of 8 from 2.15−17
selected BMIs, see Figures S3−S12 in SI). The photoreaction
that culminated with the formation of the corresponding trans
isomers in the case of meta and para halogen substituted BMIs
surprised us when a new product was formed, as evident from
1
both the H NMR spectra and LC-MS traces in the case of
ortho halogen substituted N-Me and N-Pr BMIs (2 and 5).
The photochemical behavior of BMIs in acetonitrile (in the
absence of OA) was probed to infer the confining role of OA.
1
Progress of the photoreaction as followed by H NMR in the
B
dx.doi.org/10.1021/ol5013058 | Org. Lett. XXXX, XXX, XXX−XXX

Organic Letters
Letter
BMIs 2 −7 (100 mM) resulted in the conversion of the cis to
the trans isomer (Scheme 2) (for LC-MS traces of selected
samples, see Figures S3−S10 and S16−S17 in SI). In solution6,7
at the photostationary state the amount of the trans isomer
varied between 60% and 40% and, as expected, the ratio of the
trans to the cis isomer varied with the structure of the BMI. The
product distribution against time of irradiation in acetonitrile
solution shown in Figure S18 in SI for the para-Cl derivative
confirms that prolonged irradiation resulted in cis and trans
isomers only. In the current context it is important to note that
uncomplexed ortho-halogen substituted BMIs underwent only
geometric isomerization upon irradiation in acetonitrile.
Scheme 3. Plausible Mechanism for the Formation of
Hydroxylated Product from ortho-Halogen-BMIs Included
within Octa Acid Capsule
The above photoreaction could also be probed by the
fluorescence measurements of the reactant and products. While
the cis BMIs@OA (representing guest BMI included within the
host OA) showed fluorescence in the region 390−540 nm, the
trans isomer showed weak to no fluorescence. The noticeable
fluorescence, in the region 540−700 nm, of the hydroxyl
substituted BMI 8 within the OA capsule was weak to none
when free in borate buffer solution (Figure 4 insert).13,14 The
environment results in ring opening and single bond rotation to
yield the isolated product, cis 8. Apparently, 8 prefers to exist as
a cis rather than trans isomer within OA. The photostable cis
8@OA upon irradiation did not isomerize while trans 8@OA
quantitatively isomerized to the cis isomer. We have already
demonstrated that the oxygen of the OH did not come from
atmospheric oxygen. Irradiation in D2O resulted in OD instead
of OH as per mass spectrometry. This suggested that
hydroxylation occurs by the addition of the solvent water.
It is important to note that the time for quantitative
conversion of cis 2 to cis 8 under identical irradiation conditions
was dependent on the nature of the halogen at the ortho
position and the N-alkyl group: 100% conversion was reached
when 2:1 OA complexes of ortho bromo, chloro, and fluoro
substituted 2 were irradiated, in 1 h 15 min, 2 h 30 min, and 6
h, respectively. As for the role of the alkyl group in influencing
the rate of conversion, in the case of chloro derivative N-Me
(2) 2 h 30 min were required while N-Pr (5) took only 25 min
for quantitative conversion. It is quite likely the larger size of
the ortho halogen (bromo vs fluoro) and N-alkyl (propyl vs
methyl) groups favor the cyclization process. Our recent report
of enhancement of the fluorescence with the bulkiness of the
N-alkyl group and the ortho substituents in BMIs within the OA
capsule and the similarity observed here prompts us to wonder
if the “volume” effect is in play in the cyclization process as
well.6,7
In summary, we have established the unusual behavior of the
ortho-halogen substituted BMIs within the OA capsule while
meta and para counterparts behave no differently than in
organic solvents (cis−trans isomerization) (Scheme 2). The
unprecedented photosubstitution of ortho-halogens by the
hydroxyl group of BMIs within the OA capsule is an added
feature of the role of confined spaces in bringing about some
unattainable photoreactions. Photosubstitution of aromatics
proceeds by a number of different mechanisms,20−24 and the
one we have proposed here is different from the previous ones
and probably is restricted to confined spaces. The present
studies thus herald the value of confinement in revealing a new
photochemical facet of a GFP chromophore.
Figure 4. Emission spectra recorded at various times of photolysis
(>320 nm) of cis-ortho-Cl/N-Me@OA2 in aerated borate buffered
water ([guest] = 10−5 M, [OA] = 2 × 10−5 M). λex 370 nm, and
(inset) emission spectra of ortho-OH/N-Me ([guest] = 10−4 M) in
OA (red, [OA] = 2 × 10−4 M) and in borate buffer solution of H2O
(blue).
emission spectra of cis-ortho Cl N-methyl BMI (2) at different
stages of irradiation presented in Figure 4 shows a decrease in
the emission due to the cis-isomer with progress of irradiation
and new emission appearing in the region 540−700 nm, the
same region of the authentic BMI 8’s fluorescence. A similar
observation was made with cis-ortho F and cis-ortho Br isomers
(Figures S19 and S20 in SI). These cis-ortho halogen BMIs were
weakly fluorescent in benzene solution, and upon irradiation
the intensity decreased and no new emissions appeared
(Figures S21 and S22 in SI) consistent with the lack of
formation of BMI 8 in benzene solution. Thus, from 1H NMR,
LC-MS, and fluorescence results we conclude that ortho
halogen substituted BMIs exhibit unique behavior within the
OA capsule.
A plausible mechanism for formation of 8 from 2 is
presented in Scheme 3. We visualize the reaction sequence as
follows: (a) Isomerization of cis 2 to trans 2 upon excitation
within OA. (b) The trans isomer then adopts a conformation
suitable for 6e cyclization and readily gives 9 upon excitation.
(c) Aromatization of 9 leads to 10 via loss of the halogen
substituent. (d) Exposure of 10 to water within the dynamic
capsule18,19 leads to addition of OH− or H2O to yield the
intermediate 11. (d) The unstable hemiacetal 11 in a basic
ASSOCIATED CONTENT
* Supporting Information
■
S
Sample preparation and irradiation procedures, isolation and
characterization of products, and additional NMR, LC-MS
traces, and fluorescence spectra. This material is available free
of charge via the Internet at http://pubs.acs.org.
C
dx.doi.org/10.1021/ol5013058 | Org. Lett. XXXX, XXX, XXX−XXX

Organic Letters
Letter
AUTHOR INFORMATION
Corresponding Authors
■
*E-mail: murthy1@miami.edu.
*E-mail: tolbert@chemistry.gatech.edu.
Present Address
∥S.R.S. is currently at the Department of Chemistry, University
of Pennsylvania, PA, and A.B. is at the Department of
Chemistry, University of California, Berkeley, CA.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
V.R. and L.T. thank the National Science Foundation, USA for
generous support.
■
REFERENCES
■
(1) Molecular Encapsulation; Brinker, U. H., Mieusset, J.-L., Eds.;
John Wiley & Sons, Ltd.: Chichester, U.K., 2010.
(2) Supramolecular Photochemistry; Ramamurthy, V., Inoue, Y., Eds.;
John Wiley: Hoboken, NJ, 2011.
(3) Gibb, C. L. D.; Gibb, B. C. J. Am. Chem. Soc. 2004, 126, 11408−
11409.
(4) Porel, M.; Jayaraj, N.; Kaanumalle, L. S.; Maddipatla, M. V. S. N.;
Parthasarathy, A.; Ramamurthy, V. Langmuir 2009, 25, 3473−3481.
(5) Jayaraj, N.; Zhao, Y.; Parthasarathy, A.; Porel, M.; Liu, R. S. H.;
Ramamurthy, V. Langmuir 2009, 25, 10575−10586.
(6) Baldridge, A.; Samanta, S. R.; Jayaraj, N.; Ramamurthy, V.;
Tolbert, L. M. J. Am. Chem. Soc. 2010, 132, 1498−1499.
(7) Baldridge, A.; Samanta, S. R.; Jayaraj, N.; Ramamurthy, V.;
Tolbert, L. M. J. Am. Chem. Soc. 2011, 133, 712−715.
(8) Turro, N. J.; Ramamurthy, V.; Scaiano, J. C. Modern Molecular
Photochemistry of Organic Molecules; University Science Books:
Sausalito, CA, 2010; pp 736−743.
(9) Baldridge, A.; Kowalik, J.; Tolbert, L. M. Synthesis 2010, 2424−
2436.
(10) Tolbert, L. M.; Baldridge, A.; Kowalik, J.; Solntsev, K. M. Acc.
Chem. Res. 2012, 45, 171−181.
(11) Kaanumalle, L. S.; Gibb, C. L. D.; Gibb, B. C.; Ramamurthy, V.
J. Am. Chem. Soc. 2005, 127, 3674−3675.
(12) Gibb, C. L. D.; Sundaresan, A. K.; Ramamurthy, V.; Gibb, B. C.
J. Am. Chem. Soc. 2008, 130, 4069−4080.
(13) Chen, K.-Y.; Cheng, Y.-M.; Lai, C.-H.; Hsu, C.-C.; Ho, M.-L.;
Lee, G.-H.; Chou, P.-T. J. Am. Chem. Soc. 2007, 129, 4534−4535.
(14) Hsieh, C.-C.; Chou, P.-T.; Shih, C.-W.; Chuang, W.-T.; Chung,
M.-W.; Lee, J.; Joo, T. J. Am. Chem. Soc. 2011, 133, 2932−2943.
(15) Grabner, G.; Richard, C.; Koehler, G. J. Am. Chem. Soc. 1994,
116, 11470−11480.
(16) Da Silva, J. P.; Jockusch, S.; Turro, N. J. Photochem. Photobiol.
Sci. 2009, 8, 210−216.
(17) Da Silva, J. P.; Vieira Ferreira, L. F.; Machado, I. F.; Da Silva, A.
M. J. Photochem. Photobiol., A 2006, 182, 88−92.
(18) Jayaraj, N.; Jockusch, S.; Kaanumalle, L. S.; Turro, N. J.;
Ramamurthy, V. Can. J. Chem. 2011, 89, 203−213.
(19) Tang, H.; Oliveira, C. S. d.; Sonntag, G.; Gibb, C. L. D.; Gibb, B.
C.; Bohne, C. J. Am. Chem. Soc. 2012, 134, 5544−5547.
(20) Terrier, F.: Nucleophilic Aromatic Photosubstitutions. In
Nucleophilic Aromatic Displacement; Terrier, F., Ed.; VCH: New
York, 1991; pp 321−365.
(21) Cava, M. P.; Mitchell, M. J.; Havlicek, S. C.; Lindert, A.;
Spangler, R. J. J. Org. Chem. 1970, 35, 175−179.
(22) Cornelisse, J.: Photochemical Aromatic Substitution. In CRC
Handbook of Photochemistry and Photobiology; CRC Press, Inc.: 1995;
pp 250−265.
(23) Cornelisse, J.; E, H. Chem. Rev. 1975, 75, 353−388.
(24) Cornelisse, J.; Lodder, G.; Havinga, E. Rev. Chem. Intermed.
1979, 2, 231−265.
D
dx.doi.org/10.1021/ol5013058 | Org. Lett. XXXX, XXX, XXX−XXX