Irreversible Protein Labeling by Paal–Knorr Conjugation

Article abstract of DOI:10.1002/cbic.201700210

The application of new chemical reactions in a biological context has advanced bioconjugation methods for both fundamental research and commercial arenas. Recent adaptations of reactions such as Huisgen 1,3-dipolar or Diels–Alder cycloadditions have enabled the labeling of specific residues in biomolecules by the attachment of molecules carrying azides, alkynes, or strained alkenes. Although these are fundamental tools, there is a need for the discovery of reactions that can label native proteins. We report herein the adaptation of the Paal–Knorr reaction to label lysine residues in proteins via pyrrole linkages.

Full text of DOI:10.1002/cbic.201700210

Accepted Article
Title: Irreversible protein labeling via Paal-Knorr conjugation
Authors: Ramesh Dasari, James J La Clair, and Alexander Kornienko
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10.1002/cbic.201700210
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Irreversible protein labeling via Paal-Knorr conjugation
Ramesh Dasari,[a] James J. La Clair,*[b,c] and Alexander Kornienko*[a]
Abstract: The application of new chemical reactions in a biological
context has advanced bioconjugation methods for both fundamental
research and commercial arenas. Recent adaptations of reactions
such as Huisgen 1,3-dipolar or Diels-Alder cycloadditions have
enabled the labeling of specific residues in biomolecules by the
attachment of molecules carrying azides, alkynes, or strained
alkenes. Although these are fundamental tools, there is a need for
the discovery of reactions that can label native proteins. We now
report on the adaptation of the Paal-Knorr reaction to label lysine-
residues in proteins via pyrrole linkages.
products, such as that of roseophilin reported by Trost.[13]
New protein labeling methods, characterized by efficiency, short
reaction times, site-selective targeting, or the use of low
substrate concentrations, are in great demand.[1] Many reported
labeling reagents for native proteins[2] target strongly
nucleophilic cysteine residues.[3] Covalent modification of lysine
(Lys) is another important strategy, although the nucleophilicity
of Lys-NH2 is diminished by its protonated nature under
physiological conditions (pKa 10.5). Some recently reported Lys-
targeting methods include reductive alkylation using iridium-
catalyzed transfer hydrogenation,[4] coupling with diazonium
terephthalates,[5] Michael addition with allylic elimination of
sulphonamides,[6] sulfonylation with fluorosulfonylbenzoates,[7]
and nucleophilic aromatic substitution with dichlorotriazines.[8]
We now describe the advancement of a class of labels (Fig. 1)
that target Lys residues by the formation of irreversible pyrrole
linkages.
One of the oldest name reactions involving primary amines
is the classical Paal-Knorr pyrrole synthesis that was reported
independently by Paal[9] and Knorr[10] in 1884. The reaction, as
depicted in Scheme 1, involves the condensation of a primary
amine with 1,4-dicarbonyl compound 3 in the presence of an
acid catalyst to give pyrroles 5 through the intermediary
hemiaminal 4.[11]
Figure 1. Structures of Paal-Knorr probes 1 and 2. The 7-dimethylamino-4-
coumarin or immunoaffinity-fluorescent (IAF) tag and 1,4-diketone warhead
are colored in blue and red, respectively.
Scheme 1. The Paal-Knorr reaction. Diketone 3 condenses with an amine to
generate hemiaminal 4, which undergoes a facile double dehydration to afford
pyrrole 5. The 1,4-diketone and amine moieties are coloured in red and green,
respectively.
The Paal-Knorr reaction is also biologically relevant based
on its involvement in n-hexane-induced axonal atrophy within
the central nervous system. As shown in both in vitro and in vivo
systems, 2,5-hexanedione (R1=R2=Me, Scheme 1), a neurotoxic
As the Paal-Knorr reaction proceeds under mild reaction
conditions often at room temperature with the use of a mild acid,
it is a method of choice for sensitive functionality. This has been
well documented through a variety of synthetic applications,
such as the commercial preparation of the anticholesterolemic
drug atorvastatin (Lipitor),[12] or total synthesis of natural
metabolite of n-hexane, undergoes
a selective Paal-Knorr
reaction with Lys residues (R3NH2, Scheme 1) of axonal
cytoskeleton proteins, forming 2,5-dimethylpyrrole adducts
within specific regions of neurofilaments.[14] Based on this
biological precedent, it appeared to us that the Paal-Knorr
reaction would be an interesting addition to the arsenal of Lys-
labeling methods. We focused our efforts on demonstrating this
method through probe 1, as shown in Scheme 2.
[a]
R. Dasari, Prof. A. Kornienko
Department of Chemistry and Biochemistry
Texas State University
The synthesis of the Paal-Knorr probes 1 and 2 began with
the Wittig reaction of 5-methylfurfural with Ph3P=CHCO2Me to
afford the corresponding α,β-unsaturated ester, which was
hydrogenated to yield furan ester 9 (Scheme 3). Ester 9 was
subjected to an acid-catalyzed hydrolytic opening of the furan
ring with the concomitant ester hydrolysis to afford 4,7-
dioxooctanoic acid (10) in 62% yield (Scheme 3). The resulting
acid was coupled with immunoaffinity fluorescent (IAF) tag 12[15]
using EDAC and DMAP to afford the desired probe 1 in 86%
yield. The absence of any detectable aldol processes under both
acidic (9 to 10) and basic (10 to 1) reaction conditions attests to
the stability of such aliphatic 1,4-dicarbonyl compounds and
bodes well for their applications in a biological context.
San Marcos, TX 78666, United States
E-mail: a_k76@txstate.edu
Dr. J. J. La Clair
Department of Chemistry and Biochemistry
University of California at San Diego
La Jolla, CA 92093, United States
Dr. J. J. La Clair
[b]
[c]
Xenobe Research Institute
P. O. Box 3052
San Diego, CA 92163, United States
E-mail: i@xenobe.org
Supporting information for this article is given via a link at the end of
the document.
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mildly acidic conditions at 23 °C (Scheme 3). Similar methods
were then applied to prepare a chain elongated probe 2 from 10
(see Supplementary Information). This probe was prepared to
further explore the linker requirements between the IAF tag and
the reactive moiety. In addition, we also synthesized a mono-
ketone 14 (Scheme 4, see Supplementary Information) as a
control. Here, the presence of a single ketone in 14 would
prevent it from undergoing the Paal-Knorr reaction, but rather
lead to Schiff base formation (Scheme 4).
Scheme 2. A schematic representation of the Paal-Knorr labeling of a protein
with probe 1. The IAF tag, 1,4-diketone and amine are coloured blue, red and
green, respectively.
Scheme 4. A schematic representation of Schiff base formation with control
14. The IAF tag and amine are coloured blue and green, respectively.
We then examined the use of probes 1 and 2 to modify two
proteins (as depicted in Scheme 2), bovine serum albumin
(BSA) and keyhole limpet serum (KLH), commonly conjugated
via their lysine residues for antibody production.16 As shown in
Fig. 2a, probes 1 and 2 were able to label BSA and KLH. The
fact that both probes operated at similar efficacy (Fig. 2a)
indicated that both short linkers in 1 and longer in 2 were viable.
For both proteins, the reaction was strongest with probes 1 and
2, although a minor level of labeling was observed with the
mono-ketone control 14 (Fig. 2a). After further analysis we found
that this background labeling of BSA with 14 could be removed
by spin-dialysis prior to gel analysis, however traces of
fluorescence from 14, likely resulting from Schiff base formation,
were still observed with KLH. Comparative analyses17 of the
level of fluorescence in Fig. 2a with standards indicated that
1.4±0.1 and 2.4±0.2 tags were observed per protein for BSA and
KLH, respectively, after treatment with 50 eq. of probe in PBS
pH 7.2 for 24 h at 37 °C.
Scheme 3. Synthesis of Paal-Knorr probe 1. Reagents and conditions: a)
Ph3P=CHCO2Me, THF, reflux, 20 h, 92%; b) H2, Pd/C, EtOH, rt, 24 h, 78%; c)
H2SO4 (trace), 50% aq. HOAc, 110 °C, 15 h, 62%; d) 0.5 M HCl, 1,4-dioxane,
CH2Cl2, rt, 2 h, 98%; e) EDAC, DMAP, DMF, CH2Cl2, 20 h, 86%; f) AcOH, THF,
rt, 5 h, 86%. The IAF tag, 1,4-diketone and amine are coloured in blue, red
and green, respectively.
Experiments with other proteins revealed that the Paal-Knorr
conjugation was not unique to BSA and KLH. Of interest,
labeling of LC-8,
a
protein known to oligomerize,19 was
accompanied by increased levels of dimeric and trimeric
oligomers (Fig 2c). These oligomers were not reverted by the
addition of a reducing agent (BME, DTT, or TCEP), boiling the
sample prior to SDS PAGE analysis, or changing of the type of
gel, indicating that they may have arisen from covalent
In contrast, we found that probe 1 was highly reactive toward
primary amines as is evident by the facile condensation with
benzyl amine to form pyrrole 13 in an excellent 86% yield under
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modifications possibly involving crosslinking of the pyrrole units20
brought into proximity by LC-8 oligomerizaton. While attempts
were made to identify this by mass spectral analyses, the
complexity of finding trace levels of crosslinked peptides in this
sample proved too difficult. This result provides early evidence
that the Paal-Knorr technique may also be used to trap
oligomerization states within proteins or even act on protein
complexes.
pH did not seem to adjust the ability of 1 to label BSA (Fig. 3c).
Finally, we carefully checked the spectroscopic properties of gel
purified 1•BSA conjugate and found that its spectral properties
matched that of 1 (Fig. 3d).
Figure 3. Paal-Knorr labeling of BSA. a) The labelling of BSA can be detected
down to 0.5 eq. of probe 1 when scanned on a Typhoon laser scanner
(Amersham). b) Western blot analyses can be used to further reduce the
detection level of BSA labeled with 1. c) Evaluation of pH effects on the
labelling of BSA by probe 1. Concentrations are provided in µM and ~1 µg of
protein was loaded per lane. d) Fluorescence (Φ) spectra of 10 µM probe 1
and 15 BSA•1 prepared by treatment of 1 µM BSA with 5 µM probe 1 for 12 h
in PBS pH 7.2. TLC and LC-MS analysis indicated that samples of BSA•1
were free of unreacted probe 1.
Figure 2. The Paal-Knorr labeling of BSA, KLH and LC-8. a) The labeling of
BSA or KLH in PBS pH 7.2 with diketone probes 1 and 2 as compared to
monoketone control 14. The gel was visualized on a UV light at 280 nm (top)
and then silver stained (bottom). b) The limit of visual detection via SDS-
PAGE was approximately 1 µg of protein bearing > 1 label per protein.17 c)
The labeling of LC-8 with probe 1 at 1 h or 24 h. B, K, and L denote BSA, KLH
and LC-8, respectively. Concentrations are provided in µM. For each lane 1 µg
of protein was loaded per lane with the exception of the control lanes (right two
lanes in Fig. 2a) where 0.5 µg was loaded. Aggregate bands were observed
from BSA (Ba). KHL was obtained commercially and the addition bands
observed during silver staining are not atypical, as it is purified from natural
sources.
Next, we set out to confirm that protein labeling of BSA
occurs through the pyrrole forming reaction as illustrated in
Scheme 2. Using 1 h and 24 h treatments at 37 °C, we
conducted the labeling on 100 µg scale using 10 eq. of 1. Once
complete, we purified the resulting protein by SDS-PAGE.
Samples of the bands containing 1-labeled BSA were collected
under clean conditions and dried. These samples were
submitted to Trypsin-digestion and LC-MS/MS returning
excellent peptide coverage with multiple long peptides (Fig.
4b).22 We then conducted a detailed search of the identified
peptides with MS-GF+23 and MODA24 database search
algorithms that enable the identification of peptides with non-
natural modifications. Within this dataset, modified peptides
were identified, such as S310HCIAEVEKDAIPENLPPLTADFAE-
DK336DV338 (orange, Fig. 4). Samples treated for 1 h contained
We then tested the limits of the method. We found that
visualization by eye was limited by the treatment with a 10-fold
excess of probe 1 when loading 1 µg of protein per lane on a
typical SDS-PAGE gel (Fig. 2b). Using a Typhoon scanner, the
labeling of BSA could be visualized after treatments with 0.5 to
2.5 eq. of probe 1 (Fig. 3a). For both, visualization improved
after fixation with 10% AcOH in 30% aq. EtOH, as it removed
background from unreacted 1. The detection limit of the labeling
process was further reduced by Western blot analyses using an
anti-IAF monoclonal antibody (mAb).18 As illustrated in Fig. 3b,
we were able to detect labeling in bands containing greater than
1 ng of BSA treated with 5 eq. of 1.
peaks for both hemiaminal
4 (3567.6937 m/z observed,
3567.4706 m/z calculated) and pyrrole adduct 5 (M+407,
3532.6377 m/z observed, 3531.8295 m/z calculated). However,
the sample treated for 24 h only contained the peak due to the
pyrrole 5. While detection of this peptide was possible, the low
level and complexity of this detection did not allow for an
analytical evaluation of the site selectivity within this reaction.
We also conducted studies to identify the optimal labeling
conditions for BSA. We found that the reaction could be
observed within 1 h at 37 °C (Fig. 2b) and labeling was not
enhanced with longer treatment times, as indicated by SDS-
PAGE analysis (Fig. 2b). Thus, we typically allowed the reaction
to run for 6 h to 12 h to ensure completion of the pyrrole
formation (Scheme 1). We also observed that reducing the pH
did not influence the outcome of this reaction. While pH 4-6 is
often used to accelerate Paal Knorr reactions, a reduction in the
We then evaluated the stability of these conjugates.
Samples of 1-labeled BSA or 1-labeled KLH remained
fluorescent and intact at pH 4-9. Moreover, the tagged-protein
did not degrade after long-term storage, as samples of 1-labeled
BSA used in Fig. 4 were stored at 4 °C for over 1 month (limit of
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testing) or in lyophilized dry form at rt for over 1 month (limit of
testing) as evaluated by SDS-PAGE.
diketones are resistant to metabolism[28] and have demonstrated
Paal-Knorr reactivity in humans[14] indicate their potential for
further advance as next generation tools for chemical biological
applications.
Figure 4. The labeling of BSA with probe 1. a) Each molecule of BSA has 59
Lys residues, 30-35 of which have primary amines that are capable of being
conjugated21] b) Peptide coverage map for BSA labeled with detected peptides
in red or blue and overlapping regions of these peptides in magenta. LC-
MS/MS analysis confirmed the modification of Lys336 (orange dot) within an
observed peptide (highlighted in orange). The observation of the peptide in b)
does not suggest that labelling occurred on a specific Lys residue, rather, we
were able to detect labelling at this position.
Figure 5. Cell imaging of the uptake of 1-labeled BSA. a) An image of HCT-
116 cells stained with 1-labeled BSA (blue). HCT-116 cells grown to 105
cells/cm2 were incubated for 3 h in media containing 25 µM 1-labeled BSA.
After this period the cells were washed with media and imaged live. b) Image
of HCT-116 cells in a) after incubating with 2.5 µM LysoTracker Red DND-99
for 15 min prior to imaging.23 Images were collected with blue (405 nm laser
with emission filtered at 447±60 nm); green (543 nm laser and emission
filtered at 593±40 nm) and red (633 nm laser with emission filtered at 692±40
nm) fluorescence. Overlay image is provided at left and selected channels at
the right of each panel. We confirmed the presence of 1-labeled BSA in the
treated cells using Western blot analyses as shown in Supporting Fig. S1.
With stable materials in hand, we evaluated whether our
conjugates were viable for cellular work. Using confocal
microscopy, we observed rapid endocytosis and localization of
1-labeled BSA within viable HCT-116 cells (Fig. 5). Subsequent
staining with LysoTracker Red DND-99,25 a red-fluorescent
lysosomal organelle stain, confirmed that 1-labeled BSA was
observed in the lysosomes as previously reported for BSA
conjugates in HCT-116 cells.26 Using Western blot analyses
(Supporting Fig. S1), we were able to confirm the fact that 1-
labeled BSA remained intact within the cells during this process.
Acknowledgements
This work was generously supported by funding from the
Xenobe Research Institute (to J. J. L.) and the National Cancer
Institute grant CA186046 (to A. K.). We thank Majid Ghassemian
(UC San Diego) for protein digestion and LC-MS/MS analyses
and Nuno Bandeira (UC San Diego) for suggestions for the
MS/MS data searching and analyses.
In conclusion, we have demonstrated the application of the
Paal-Knorr reaction for protein labeling using Lys-rich proteins
as substrates (BSA, KLH or LC-8). There are several features to
this reaction that make its use for protein conjugation
applications and chemical biological studies attractive. First,
there are no reagents required or special media. In these
examples, labeling was readily observed at pH 7.2 in PBS at
ambient temperature. The labeling process occurred rapidly and
was completed within 24 h. Second, while we demonstrated the
labeling application using an IAF tag, the ease in the synthesis
of the warhead unit 10 (Scheme 3) allows tags to be appended
to a wide range of agents. Third, probes containing warhead 10
are not reactive to media like many of the other functional
handles such as acrylates, ɑ-haloamides, succinimidyl esters, or
sulfonyl halides, an issue that often creates significant batch-
based inhomogeneity, which has recently been addressed in
part with continuous-flow reactors.27 Finally, the fact that 1,4-
Keywords: protein labeling • Paal-Knorr reaction • Click
chemistry • biocompatible reactions • bioconjugation
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Entry for the Table of Contents
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The advance of a suite of
bioorthogonal chemical reactions has
profoundly advanced the tools
Ramesh Dasari, James J. La Clair,* and
Alexander Kornienko*
Page No. – Page No.
available to biochemists to study
proteins and other biomolecules.
Here, we describe the use of the Paal-
Knorr reaction to fluorescently-label
proteins. Akin to the needs of a
biocompatible method, the described
procedures operate without reagents,
catalysts, or organic solvents.
Irreversible protein labeling via Paal-
Knorr conjugation
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