A Vinylogous Photocleavage Strategy Allows Direct Photocaging of Backbone Amide Structure

Article abstract of DOI:10.1021/jacs.8b04893

Side-chain modifications that respond to external stimuli provide a convenient approach to control macromolecular structure and function. Responsive modification of backbone amide structure represents a direct and powerful alternative to impact folding and function. Here, we describe a new photocaging method using histidine-directed backbone modification to selectively modify peptides and proteins at the amide N-H bond. A new vinylogous photocleavage method allows photorelease of the backbone modification and, with it, restoration of function.

Full text of DOI:10.1021/jacs.8b04893

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Communication
A vinylogous photocleavage strategy allows
direct photocaging of backbone amide structure
Alicia E. Mangubat-Medina, Samuel C. Martin, Kengo Hanaya, and Zachary T. Ball
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.8b04893 • Publication Date (Web): 20 Jun 2018
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A vinylogous photocleavage strategy allows direct photocag-
ing of backbone amide structure
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Alicia E. Mangubat-Medina, Samuel C. Martin, Kengo Hanaya, Zachary T. Ball*
Department of Chemistry, Rice University, Houston, Texas 77005, United States
Supporting Information Placeholder
alkenylation, are inaccessible by chemical synthesis. Furthermore,
ABSTRACT: Side-chain modifications that respond to external
it is not yet possible to engineer the few examples of enzymatic
backbone modification using general protein engineering tools.
Ribosomal incorporation of N-alkyl amino acids using unnatural
tRNA technology has also been demonstrated, but again, capabili-
ties are severely limited.20,21
stimuli provide a convenient approach to control macromolecular
structure and function. Responsive modification of backbone
amide structure represents a direct and powerful alternative to
impact folding and function. Here, we describe a new photocaging
method using histidine-directed backbone modification to selec-
tively modify peptides and proteins at the amide N–H bond. A
new vinylogous photocleavage method allows photorelease of the
backbone modification and with it, restoration of function.
Protein secondary structure derives primarily from specific back-
bone amide interactions, including requisite hydrogen bonding
that determines α-helix and β-sheet folds. Since folding deter-
mines function, modification of backbone structure is perhaps the
most direct way to alter or control polypeptide function, biasing
against or even disrupting natural folding altogether. In limited
instances, nature uses modification of the backbone structure to
control polypeptide function. For example, sulfenyl–amide S–N
bond formation reversibly inactivates protein tyrosine phospha-
tase 1B,1 and N-methylation in nonribosomal cyclic peptides al-
ters conformation, proteolytic stability, and other attributes.2 If
chemical backbone modification could be made responsive to an
external stimulus—such as light, redox potential, or metal ion
concentration—the approach could offer a direct and predictable
way to build responsive or switchable structure and function.
Photocaging backbone amide structure would be complementary
to exiting photocaging approaches, including intein caging3 and
other side-chain caging approaches,4–9 as well as whole photore-
sponsive protein domains.10,11 In some cases, such as photocaged
active-site residues, side-chain photocaging is an important and
effective way to deliver photoswitchable structures.12,13 But alter-
ing function with a side-chain photocaging agent can be rounda-
bout, difficult to predict, and require empirical approaches.14 De-
spite the rather obvious potential of directly photocaging a specif-
ic backbone amide bond, the approach has been little studied,
largely because access to peptides or proteins with backbone-
modified structures is extremely limited. Short backbone-
modified peptides can sometimes be accessed by solid-phase syn-
thesis with N-alkyl amino acids, which can potentially be inte-
grated into larger sequences by native chemical ligation14 or ex-
pressed protein ligation.15 However, solid-phase synthesis with N-
alkyl amino acids is plagued by low coupling efficiency, and
standard HATU/HOBt coupling agents fail in these demanding
applications.5,16–18 Quite recently, a photocaged backbone ap-
proach was used for photocontrol of β-sheet formation, a result
conveying both the significant potential of backbone photocon-
trol, but also the significant limitations of current methods.19
Many modifications of interest, including N-arylation and N-
Figure 1. Vinylogous photocleavable modification of backbone
N–H bonds. (a) Schematic depicting of structural perturbation
with a photoremoveable reagent. (b) A model photocleavage reac-
tion releasing N-methylacetamide (4) through the presumed in-
termediacy of C–H abstraction product 3. (inset) 1H NMR spectra
demonstrating release of amide 4 upon photoirradiation, follow-
ing peaks attributed to the N-methyl resonances (a) and (b) Con-
ditions: irradiation at 365 nm of
mine/trimethylamine HCl in 2:3 CD3OD/D2O soln.
a 35-mM trimethyla-
This report describes a photocleavable backbone N–H modifica-
tion that allows efficient photocaging of folding and function (Fig.
1a). Efforts to develop a backbone photocaging process stemmed
from our recent discovery of predictable access to backbone N–H
alkenylation and arylation with boronic acid reagents, directed by
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a neighboring histidine residue in the i + 1 position.22,23 We envi-
interactions are essential for the formation of the triple helix, and
substitutions of backbone N–H bonds at glycine completely dis-
rupts folding. The CMP peptide has a single histidine residue,
which enables backbone modification at the preceding glycine
residue. Consistent with prior understanding, this histidine-
containing peptide exhibits a characteristic polyproline type-II
secondary structure, with a circular dichroism (CD) spectrum
containing a maximum at 225 nm and a cooperative thermal un-
folding event (m.p. = 42 °C, Fig. S5). After modification at
Gly12 with boronate 1a, folding and trimerization was completely
disrupted: the CD spectra of the modified peptide lacked any fea-
ture at 225 nm (Fig. 2f). A CD thermal melt analysis contained no
cooperative unfolding event (Fig 2g). After three minutes irradia-
tion with 365-nm light, photocleavage of the modification was
observed, and the peptide regained the positive CD signature at
sioned that a well-designed boronic acid could conceivably deliv-
er a backbone modification capable of traceless photochemical
cleavage. However, traceless photochemical cleavage, in biologi-
cal contexts, typically involves cleavage of a C(sp3)–X of a 2-
nitrobenzyl derivative.24 In contrast, histidine-directed backbone
modification requires alkenyl- or aryl-boronic acids, delivering N-
alkenyl or N-aryl polypeptide products, and thus necessitating
photocleavage at a C(sp2)–N bond. Despite extensive literature on
photocleavage and photodeprotection,24 there is limited precedent
for C–X cleavage at sp2 carbon atoms, typically involving cleav-
age of N–acyl bonds producing (formally) hydrolysis prod-
ucts.25,26 In a new approach to this problem, we chose to examine
a vinylogous27–29 analogue of 2-nitrobenzyl cleavage (Fig. 1b). In
this case, putative benzylic hydrogen atom abstraction would
provide an extended conjugated system (3). We hypothesized that,
among several possible pathways, nucleophilic attack by water on
the -carbon of 3 would eventually release a "traceless" polypep-
tide product, together with an unsaturated aldehyde or decomposi-
tion product thereof. To explore this proposed pathway, a model
vinylogous 2-nitrobenzyl amide (2) was prepared by analogy to a
reported condensation.30 Upon treatment with 365-nm light in
water in the presence of triethylamine as a scavenger, the conver-
sion of caged amide 2 to N-methylacetamide (4) was monitored
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by H NMR, based on the N-methyl resonance shift from 3.16
ppm to 2.68 ppm upon photocleaveage.31 The clean and efficient
photocleavage observed provides a sufficient foundation for a
vinylogous approach to sp2 C–N bond cleavage in complex poly-
peptides.
In preparing an appropriate and effective boronic acid "photocag-
ing" reagent, we incorporated a 3,4-dimethoxy (6-nitroveratryl)
core for red-shifted absorption and improved photocleavage effi-
ciency,32 which ultimately led to the design of photocleavable
reagent 1a (Scheme 1). In the course of synthetic investigations, it
was determined that the 2-propargylnitrobenzene structure, as in
alkynes 6 and 7, is sensitive to basic conditions, necessitating
careful reaction sequencing, especially in more complex target
structures (Scheme 2). In the end, boronate 1a was effectively
prepared by reduction of alcohol 6 under acidic conditions, fol-
lowed by zirconium-catalyzed hydroboration.
Scheme 1. Synthesis of photocleavable reagent 1a.
Figure 2. Backbone modification and photorelease of peptides.
(a) Schematic depiction of modification and photocleavage reac-
tion, (b-e) MALDI–MS analysis of various peptides before (top,
black) and after (middle, blue) N–H modification with boronate
1a, and subsequent analysis after photocleavage (bottom, red). (f)
CMP modification-driven unfolding and photocleavage-driven
refolding. CD analysis of unmodified (black) and modified CMP
(sequence: Ac-(POG)4HOG(POG)3-NH2) before (blue) and after
(red) photocleavage with 365-nm light. (g) CD thermal melt (in-
tegrated, top and differential, bottom) of modified peptide before
and after irradiation. Peptide modification conditions: 100 μM
peptide, 1 mM boronate 1a, 10 mM buffer (varied pH); angioten-
sin I: 500 μM Cu(OAc)2, pH 8.5; CSP: 100 μM Cu(OAc)2, pH
6.5; leuprolide: 500 μM Cu(OAc)2, pH 8.5; CMP: 100 μM
Cu(OAc)2, pH 8.5. Size-exclusion filtration (700 MWCO) was
employed to remove small molecules prior to CD analyses.
We next evaluated the photocaging of a variety of peptides with
boronic acid 1a (Fig. 2). Upon treatment with boronic acid 1a in
the presence of a copper(II) salt, clean conversion of peptide to a
backbone-modified product was observed for angiotensin I, a
chymotrypsin substrate peptide (CSP), leuprolide, and a collagen
mimetic peptide (CMP) (Fig 2 b-e, blue spectra). Gratifyingly, in
each case the initial, uncaged peptide could be regenerated upon
irradiation with 365-nm light to remove the modification (red
spectra).
The CMP peptide allowed direct investigation of photocaged
folding. The CMP sequence consists of repeating Gly-Pro-Hyp
units (Hyp = hydroxyproline) and folds into a canonical polypro-
line type-II triple helix.33 Interstrand glycine hydrogen-bonding
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225 nm, consistent with triple-helix formation (Fig 2f). Further-
more, the unmasked CMP peptide exhibited a melting temperature
of 43 ºC (Fig 2g), indistinguishable from as-synthesized peptide.
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2
Modification of backbone structure is a direct approach to photo-
caged protease susceptibility of peptides, since proteolysis is quite
sensitive to backbone N–H substitution near the cleavage site.34
To test the potential for light-triggered peptide hydrolysis, we
synthesized an α-chymotrypsin substrate peptide (CSP, sequence:
Ac-SIINFGHKL-NH2 ), based a prior study of enzyme selectivi-
ty.35 Proteolysis with α-chymotrypsin cleaves this peptide be-
tween phenylalanine and glycine residues. Histidine-directed
modification with boronate 1a produced a peptide, photocaged at
glycine. Next, mixtures of caged and uncaged peptide were treat-
ed with α-chymotrypsin (Fig. 3). Subsequent HPLC and MS anal-
ysis indicated that the unmodified peptide was entirely degraded,
while the photocaged peptide remained intact. The α-
chymotrypsin was deactivated by a pH jump, and the peptide was
uncaged using 365-nm light, resulting in the formation of the
parent CSP. If the mixture is irradiated without α-chymotrypsin
deactivation, the CSP is consumed as it is formed, and thus no
full-length CSP is observed after photoirradiation (Fig. S4).
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Figure 4. Photocleavable modification of soybean trypsin inhibi-
tor (SBTI). (a) SBTI structure showing His71 (red). (b) MALDI
analysis of modified STBI before and after irradiation. (inset)
Anti-biotin western blot indicated loss of signal following photo-
cleavage. Protein modification conditions: 20 μM SBTI, 500 μM
Cu(OAc)2, 500 μM boronate 1b, 10 mM NMM buffer, pH 8. See
supporting information for full blot.
Scheme 2. Synthesis of photocleavable reagent 1b.
Figure 3. Photocaged control of a protease substrate, CSP. Prote-
ase activity was assessed by MALDI-MS and HPLC (inset). A
mixture of modified and unmodified CSP (left, green trace), was
treated with α-chymotrypsin, resulting in complete consumption
of the uncaged CSP peptide (blue trace). After inactivation of α-
chymotrypsin by a drop in pH, the parent CSP peptide was regen-
erated upon irradiation for 5 min at 365 nm. CSP modification
conditions: 200 μM CSP, 200 μM Cu(OAc)2, 2 mM boronate 1a,
10 mM NMM buffer, pH 6.5.
Finally, we examined modification and uncaging of a model pro-
tein, soybean trypsin inhibitor (SBTI), which contains a single
histidine residue (His 71) in an exposed loop region (Fig. 4a). To
facilitate work with larger proteins, we found it useful to develop
a bifunctional boronic acid reagent incorporating desthiobiotin36,37
as a convenient handle for purification and analysis (Scheme 2).
In designing this bifunctional probe, the core 6-nitroveratryl pho-
toactive component was preserved. A convergent synthesis in-
volving coupling of readily-available precursors 8 and 9 was car-
ried out, allowing formation of the target structure 1b through a
reduction–hydroboration route analogous to that of photocaging
agent 1a. In this case Zr-catalyzed hydroboration failed, and so
uncatalyzed hydroboration with catecholborane furnished the
alkenylboronic acid 1b, albeit with significant diminution in yield.
A vinylogous 2-nitrobenzylboronic acid enables, for the first
time, access to photocaged backbone N–H bonds by direct back-
bone modification of natural polypeptide structures. Furthermore,
the N-vinylamide photocaging structure significantly perturbs the
electronics of the amide bond, potentially allowing reversible
perturbation of amide carbonyl basicity or of the kinetics of cis-
trans amide interconversion. In contrast to side chain modifica-
tions, backbone modification presents a more direct, general, and
easily designed approach to caging the function of a protein or
polypeptide. Incorporation of a photocaging group is predictable,
and can work on natural substrates without sequence engineering.
ASSOCIATED CONTENT
Supporting Information
SBTI was modified with boronate 1b at Gly70. Affinity purifica-
tion produced modified protein of sufficient purity to test photo-
cleavage, and single modification was established by anti-biotin
western blot and by mass spectrometry. Gratifyingly, irradiation
at 365 nm for 30 minutes led to complete removal of the photo-
caging agent, as judged by mass spectrometry and anti-biotin
western blot (Fig. 4b).
The Supporting Information is available free of charge on the
ACS Publications website.
Experimental procedures, characterization, and additional HPLC
and CD spectra (PDF).
AUTHOR INFORMATION
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Corresponding Author
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*zb1@rice.edu
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Notes
The authors declare no competing financial interests.
ACKNOWLEDGMENT
We acknowledge support from the Robert A. Welch Foundation
Research Grant C-1680 (to Z.T.B.), from the National Science
Foundation under grant number CHE-1609654 (to Z.T.B.). We
thank Jeffery Hartgerink, I-Che Li, and Douglas Walker for use of
their peptide synthesizer for the collagen memetic peptide synthe-
sis.
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