Chemoselective Probe Containing a Unique Bioorthogonal Cleavage Site for Investigation of Gut Microbiota Metabolism

Article abstract of DOI:10.1002/anie.201804828

While metabolites derived from gut microbiota metabolism have been linked to disease development in the human host, the chemical tools required for their detailed analysis and the discovery of biomarkers are limited. A unique and multifunctional chemical probe for mass spectrometric analysis, which contains p-nitrocinnamyloxycarbonyl as a new bioorthogonal cleavage site has been designed and synthesized. Coupled to magnetic beads, this chemical probe allows for straightforward extraction of metabolites from human samples and release under mild conditions. This isolation from the sample matrix results in significantly reduced ion suppression, an increased mass spectrometric sensitivity, and facilitates the detection of metabolites in femtomole quantities. The chemoselective probe was applied to the analysis of human fecal samples, resulting in the discovery of four metabolites previously unreported in this sample type and confirmation of the presence of medically relevant gut microbiota-derived metabolites.

Full text of DOI:10.1002/anie.201804828

A Journal of the Gesellschaft Deutscher Chemiker
Angewandte
International Edition Chemie
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Accepted Article
Title: Chemoselective Probe Harboring a Unique Bioorthogonal
Cleavage Site for Investigation of Gut Microbiota Metabolism
Authors: Neeraj Garg, Louis P. Conway, Caroline Ballet, Mário S.P.
Correia, Frida K.S. Olsson, Miroslav Vujasinovic, J.-Matthias
Löhr, and Daniel Globisch
This manuscript has been accepted after peer review and appears as an
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of the final Version of Record (VoR). This work is currently citable by
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.201804828
Angew. Chem. 10.1002/ange.201804828
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Chemoselective Probe Harboring a Unique Bioorthogonal Cleavage Site
for Investigation of Gut Microbiota Metabolism
+
+
Neeraj Garg , Louis P. Conway , Caroline Ballet, Mario S. P. Correia, Frida K. S. Olsson, Miroslav Vujasinovic,
J.-Matthias Löhr, and Daniel Globisch*
Abstract: While metabolites derived from gut microbiota metabolism have
been linked to disease development in the human host the chemical tools
required for their detailed analysis and the discovery of biomarkers are
limited. We have designed and synthesized a unique and multifunctional
chemical probe for mass spectrometric analysis, which contains
p-nitrocinnamyloxycarbonyl as a new bioorthogonal cleavage site. Coupled
to magnetic beads, our chemical probe allows for straightforward extraction
of metabolites from human samples and release under mild conditions. This
isolation from the sample matrix results in significantly reduced ion
suppression, an increased mass spectrometric sensitivity and facilitates the
detection of metabolites in femtomole quantities. We have applied our
chemoselective probe to the analysis of human fecal samples, identified four
previously unreported in this sample type and confirmed the presence of
medically relevant gut microbiota-derived metabolites.
One of the most exciting scientific developments in the past
decade has been the understanding that gut microbiota
[
1]
profoundly impact human physiology. This complex consortium
of trillions of microbes outnumbers the cells of the human host,
possesses a diverse range of biochemical and metabolic activities,
and has been referred to as an additional organ.[2] These
microbes influence numerous metabolic pathways, including
nutrition, detoxification, metabolism, hormonal homeostasis,
immune tolerance, and inflammation.[2] Mounting evidence
demonstrates that a dysregulated gut microflora significantly
impacts disease development in humans. Diverse metabolite
classes derived from microbiota metabolism toxic to the human
body have been identified.[3] Detailed analysis of metabolic
interactions of microbial communities with their host is required to
discover unknown links to disease development, new drug targets
and biomarkers.
Figure 1. a) Chemical probe design. The reactive site bearing an amine-specific
moiety (green) is connected via a multifunctional linker (purple) to the Noc-
cleavage site (red). This probe is conjugated to magnetic beads (orange) via a
spacer (black); b) The general workflow for analysis of metabolic amines using
the chemical probe.
biology are required for selective and enhanced analysis of
microbiota-derived metabolites. Metabolites at low concentrations
are largely omitted from metabolomics analyses as tools for their
detection
and
structural
determination
are
lacking.
Analysis of metabolites in any sample type has been termed
metabolomics, which is the newest major ‘omics’ research field in
the postgenomic era.[4] Due to its unparalleled dynamic range and
sensitivity, mass spectrometry is the principal technique
employed for simultaneous investigation of the complex mixture
of metabolites in human samples.[5] Despite perpetual
improvement, mass spectrometric analysis of metabolites has its
limitations and new methods at the interface of chemistry and
Chemoselective modification strategies for targeted metabolite
analysis have been developed to facilitate detection and
identification; however, methods described so far have to contend
with interference from the sample background.[6] Advanced
chemoselective methods for the modification of metabolites are
scarce.[7] Other ‘omics’ research fields, e.g. proteomics and
genomics, have in contrast benefitted from a diverse range of
chemical biology tools and chemical methods, which have played
key roles in major biologically and medically relevant
discoveries.[8]
[
a]
Dr. N. Garg,[+] Dr. L. P. Conway,[+] Dr. C. Ballet, M. Sc. M. S. P.
Correia, F. K. S. Olsson, Prof. Dr. D. Globisch
Department of Medicinal Chemistry, Science for Life Laboratory
Uppsala University
Herein, we report the design, synthesis and application of a
new type of chemical probe for simplified metabolite analysis in
human fecal samples. This probe has been designed to
chemoselectively derivatize amines, as a large percentage of gut
microbiota-derived metabolites linked to disease development or
prevention in humans possess primary or secondary amines.[
Furthermore, amines are involved in the majority of human
metabolic and biosynthetic pathways. The scaffold of chemical
probe 1 comprises five major components as depicted in Figure
Box 574, SE-75123 Uppsala, Sweden
E-mail: Daniel.globisch@scilifelab.uu.se
[
b]
Dr. M. Vujasinovic, Prof. Dr. J. Matthias Löhr
Department of Clinical Science, Intervention and Technology
9]
(CLINTEC) and Department for Digestive Diseases
Karolinska Institute and Karolinska University Hospital
Stockholm, Sweden
These authors contributed equally to this work.
[
+]
1
a. The probe is immobilized on magnetic beads (orange)
Supporting information for this article is given via a link at the end of
the document.
connected via a spacer (black). The new bioorthogonal cleavage
site (red) is connected by a multifunctional amino-linker (purple)
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COMMUNICATION
modification of labile analytes and would prevent identification of
their correct structure.
The design of chemical probe 1 required modification of the
original p-nitrocinnamyl scaffold by incorporating a carboxylic acid
as a synthetic handle. This modification permits the insertion of a
spacer for coupling of the chemical probe to magnetic beads.
While magnetic beads are widely used for analysis of
macromolecules such as enzymes, aptamers and antibodies,[
to the best of our knowledge there have been no reports of their
comprehensive application for the analysis of metabolites.
Furthermore, the probe also contains a multifunctional linker,
which connects the cleavage and the reactive sites while also
significantly enhancing mass spectrometric sensitivity of the
cleaved products.
13]
The chemical probe was prepared using a straightforward and
convergent synthetic procedure (Schemes S1/S2). We used
simplified synthetic intermediate 3 to test the cleavage properties
of this modified Noc-scaffold as we inserted a carboxylic acid as
a handle for further chemical synthesis and subsequent coupling
to magnetic beads. These cleavage experiments confirmed that
the Noc-scaffold is rapidly and quantitatively cleaved after a
reaction time of only 10 min by UPLC-MS as well as HPLC
analyses (Figures 2a/S1). This result demonstrates that the
electron-withdrawing ester moiety does not adversely affect the
properties of the cleavage site compared to the unmodified Noc-
group. The quantitative cleavage along with a short cleavage time
is crucial for application of our probe to metabolite analysis in
complex human samples and advantageous over other metal-
catalyzed bioorthogonal cleavage sites, which require long
cleavage times and generally do not proceed to completion. [
Figure 2. a) HPLC-chromatograms of the cleavage of simplified Noc-based
compound 3; b) Probe activation on magnetic beads for amine-specific
capturing and cleavage to release modified metabolites 2. R = captured amines.
to the reactive site containing the commonly used amine-selective
sulfo-N-hydroxysuccinimide (sulfo-NHS) moiety (green). This
probe design permits the capture of metabolic amines, facile
isolation from the sample matrix and subsequent release of the
captured metabolites 2 under mild conditions (Figure 1b). This
multifaceted procedure leads to enhanced ionization properties of
selectively captured metabolites and simplified mass spectra with
no matrix interference, resulting in improved mass spectrometric
sensitivity using ultra-performance liquid chromatography-mass
spectrometry (UPLC-MS).
12, 14]
Chemical probe 5 immobilized to carboxylate functionalized
magnetic beads was prepared by coupling the full-length probe
using standard conditions (Figure 2b and Scheme S3). The
coupling efficacy was determined in three independent
experiments to be 26 ± 4 µmol/g (probe / beads) using
quantitative HPLC assay (Figure S2). The Boc-protecting group
of immobilized chemical probe was removed using
a
5
trifluoroacetic acid to yield a terminal arylamine, which was
subsequently treated with glutaric anhydride followed by
activation of the resulting carboxylic acid to form the amine-
specific chemoselective sulfo-NHS ester and obtain immobilized
compound 1. The NHS moiety has found wide application in
The key moiety of the chemical probe is the bioorthogonal
cleavage site. We are introducing para-nitrocinnamyloxycarbonyl
(
Noc) as a new cleavage site, which we believe can find broad
[10]
application throughout the chemical biology research field. This
group was originally developed by H. Kunz in 1988 as the “fourth
dimension of protecting group” chemistry for peptide synthesis
[6, 15]
chemoselective analysis of amines in human samples.
While
conjugation of sulfo-NHS esters with free thiols has been
described, they are mainly present as disulfides in human
due to its stability under acidic and basic conditions as well as
Alloc-cleavage conditions.[10] Since then, the Noc-group has only
been used as a protecting group in peptide chemistry and in total
synthesis.[11] The major advantage of the Noc-moiety is the mild
cleavage conditions using palladium(0) and is thus orthogonal to
all biological functionalities. In contrast, commonly used cleavage
sites in chemical biology require harsh conditions such as
oxidizing/reducing reagents, nucleophilic/basic conditions,
electrophilic/acidic conditions, and photo-irradiation.[12] While
samples, which require pretreatment by a reducing agent to be
captured.[
16]
Furthermore, multistep chemical synthesis on
magnetic beads beyond simple conjugation chemistry is
uncommon. We thus optimized each step using a simplified probe,
which was required as assessing the completion of each synthetic
step after immobilization on magnetic beads is not possible using
standard analytical methods (Scheme S4). Test cleavage
experiments were also performed for each step to confirm
success of chemical reactions.
these cleavage sites can be safely employed for proteomics
research, the application of these harsh conditions to functionally
diverse metabolites can cause degradation or unexpected
We tested the selectivity and comprehensive application
properties of our activated probe 1 with a selection of different
amine classes including amino acids as well as primary,
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COMMUNICATION
Figure 3. Application of the chemical probe. Positive mode UPLC-MS analysis of a) conjugated and unconjugated lysine solution (500 nM), b) conjugated and
+
unconjugated glycine extracted from fecal samples ([M+H] ); c) Workflow of our advanced analysis of human fecal samples: i) Probe activation; ii) Sample
treatment; iii) Magnetic separation of captured metabolites; iv) Bioorthogonal cleavage; v) UPLC-MS analysis and selective peak detection.
secondary and aromatic amines. Captured metabolic amines 6
were extracted using magnetic separation and then quantitatively
cleaved from magnetic beads (Figure 2b). This mass
spectrometric experiment revealed that all selected compound
classes were captured (Figure S3). We demonstrated signal
amplification of lysine at a concentration of 500 nM using our
probe by obtaining a clear mass spectrometric peak. In contrast,
no signal was detected after direct analysis of the untreated lysine
solution (Figure 3a).
were more abundant in the treated sample (Figure S4). The
obtained list was reduced to 381 realistic metabolite features after
eliminating features a) with a mass smaller than that of the probe
adduct (320.1605 Da); b) more abundant in the control sample, or
c) less than fivefold more abundant in the feces-derived sample.
The masses of candidate features were compared with the
Human Metabolome Database and METLIN for determination of
their chemical structures.
Using this procedure, we were able to identify different
metabolites (Table 1), including amino acids (glycine, lysine),
In the next step, we examined amines in fecal samples. The
gut carries the most metabolically active microbiota in the entire
human body, and so feces are a rich source of microbiota-derived
metabolites, which have not yet been absorbed by the human
host. Intestinal microbes convert undigested dietary compounds
and also exchange metabolites with each other to coordinate and
compete within the human host.[17] It is noteworthy that only a low
number of studies on metabolite analysis in human fecal samples
monoamines
(tyramine,
phenethylamine),
polyamines
(putrescine, cadaverine and their monoacetamides), cyclic
secondary amines (piperidine), and small molecules
(methylamine, ammonia/ammonium). Of particular interest were
ammonia, 2-amino-3-methylbenzoic acid, 1-aminoacetone, and
1-aminopropan-2-ol, which are four metabolites previously
undiscovered in feces according to the Human Metabolome
compared to blood (serum/plasma) or urine samples are available. Database (HMDB).[19] Most listed metabolites were not detected
New techniques are required given the analytical challenges and
limitations inherent to this complex sample type as human fecal
using standard analysis conditions. After applying our procedure,
we predominantly observed increased signal intensities and
reduced background in the resulting mass spectrometric
chromatograms (e.g. glycine, Figure 3b). Additional examples are
depicted in Figures S5/S6.
samples present
a great opportunity to identify unknown
[18]
metabolites linked to pathogenesis.
We applied our activated chemical probe 1 to soluble
metabolite fractions obtained from fecal samples collected from
three different subjects (details are presented in the SI). Probe
activation was performed directly before sample treatment and
each fecal sample was incubated for 18 h at 20 C under vigorous
shaking. The beads, along with the captured metabolites, were
then separated from the sample using a magnet, rinsed, and
transferred into a new tube (Figure 3c). Derivatized metabolites
were cleaved from the magnetic beads using optimized Noc-
deprotection conditions, followed by separation from the beads.
Resulting metabolite conjugates 2 and control samples were
analyzed by UPLC-MS. Data processing was performed with the
XCMS metabolomics framework by comparing six UPLC-MS
analyses of each fecal sample extract and each control sample.
In total, 15,108 features were detected by the unique combination
of chemoselective metabolite modification and bioinformatic data
analysis. As expected, the majority of the most significant features
A straightforward synthesis to obtain authentic synthetic
standards was developed to validate each chemical structure of
these molecules (Figure 4a). Chemical structures were confirmed
by spiking experiments of a synthetic internal standard produced
in a two-step synthetic strategy. Amine conjugates of the general
structure 2 were obtained from intermediate 7 upon activation and
treatment with each metabolite of interest followed by Fmoc-
deprotection. One striking example is depicted in Figure 4b
validating the presence of 1-amino-2-propanol and distinguishing
it from two possible regioisomers, 3-amino-1-propanol and 2-
(methylamino)ethanol. Synthetic probe conjugates 2c-e of each
metabolite were analyzed using UPLC-MS analysis and co-
injected with the fecal sample extracts to identify 1-amino-2-
propanol as the fecal metabolite. Further examples are presented
in Figure S7 and Table S1. We also synthesized reference glycine
and phenethylamine conjugates 2b/f to perform level-of-detection
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(
2
LOD) analyses and determined the LOD to be as low as 190 and
00 fmol, respectively (Figures S8/S9). The LOD for unmodified
glycine was measured to be 500 pmol using the exact same
UPLC-MS analysis conditions demonstrating signal
a
amplification factor of 2,600. Considering that the LOD for glycine
in human samples is even higher as a result of matrix effects, the
presented procedure enables the investigation of metabolites in
the low fmol range in complex biological samples in greater detail.
Our procedure facilitates efficient analysis of metabolites of
diverse chemical structures using a single UPLC-MS setup. The
increase in molecular weight of 320.1605 Da resulting from our
modification procedure unexpectedly enables analysis of small
and volatile metabolites such as methylamine and ammonia.
These volatile metabolites usually require gas chromatography-
MS (GC-MS) analysis. Importantly, several amine metabolites
detected in human fecal samples are linked to microbial
metabolism or disease development (Table 1).[19] Polyamines
such as cadaverine and putrescine, the monoamines tyramine
and phenethylamine, and piperidine are derived from
decarboxylation of amino acids by colonic bacteria.[20] For
example, acetylcadaverine and phenethylamine have been linked
to inflammatory bowel disease,[21] while 1-aminoacetone has
been associated with diabetes mellitus.[22]
In summary, we report the chemical synthesis and application
of an advanced chemical probe with the first utilization of the Noc-
moiety as a bioorthogonal cleavage site in chemical biology and
mass spectrometric analysis. Application of this probe to human
fecal samples resulted in simplified UPLC-MS chromatograms
with no matrix background allowing for improved identification of
a large number of microbial and medically-relevant metabolites
among the captured compounds. Furthermore, the detection of
four previously unknown metabolites in fecal samples
Figure 4: Metabolite structure validation. a) Chemical synthesis of
conjugated standard metabolites. b) Structure validation of 1-amino-2-
propanol using synthetic internal standard 2d for mass spectrometric co-
injection experiments in positive mode.
Table 1: List of validated metabolites in fecal samples and link to disease
development. Highlighted in bold are metabolites previously not detected
in fecal samples and the (*) indicates bismodified metabolites.
demonstrates the advantages of this chemical probe for mass
spectrometric analysis. Future application of this new metabolite-
investigation strategy represents a tremendous potential for the
discovery of unknown biomarkers of pathology and detailed study
of the metabolic interplay of microbiota and their human host.
Fecal metabolites[20]
N-Acetylcadaverine
N-Acetylputrescine
Association with disease[19, 21-22]
Crohn's disease / IBD
Lung cancer
5
-Aminovaleric acid
Ammonia
Bacterial virulence
Lung cancer
Rectal cancer
Acknowledgements
Cadaverine
Methylamine
Phenethylamine
Piperidine
This study was supported by the Swedish Research Council (VR
2016-04423), the Carl Tryggers Foundation (CTS16:155) and the
Science for Life Laboratory to D.G.
Crohn's disease / IBD
Putrescine*
Tyramine
Lung cancer
1
,3-Diaminopropane*
Lung and bladder cancer
Conflict of interest
Ethanolamine
Glycine
Histamine
L-(Iso)leucine
L-Lysine
The authors declare no conflict of interest.
Keywords: Cleavage reactions • microbiota • metabolism • mass
spectrometry • magnetic beads
L-Alanine
1
1
2
-Aminoacetone
-Aminopropan-2-ol
-Amino-3-methylbenzoic
Diabetes
acid
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5
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COMMUNICATION
Entry for the Table of Contents
COMMUNICATION
+
+
Unique
chemoselective
probe
N. Garg , L.P. Conway , C. Ballet,
M.S.P. Correia, F.K.S. Olsson, M.
Vujasinovic, J.-M. Löhr, and D. Globisch
immobilized to magnetic beads
facilitates extraction of metabolic
amines from human fecal samples. A
novel, bioorthogonal cleavage site Noc
allows the captured metabolites to be
released under mild conditions. The
increased sensitivity of the probe
conjugates under UPLC-MS analysis,
reduced ion suppression and no matrix
*
Page No. – Page No.
Chemoselective Probe Harboring a
Unique Bioorthogonal Cleavage Site
for Investigation of Gut Microbiota
Metabolism
effects
significantly
improves
identification of metabolites.
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