Diazirine-functionalized mannosides for photoaffinity labeling: Trouble with FimH

Article abstract of DOI:10.3762/bjoc.14.163

Photoaffinity labeling is frequently employed for the investigation of ligand–receptor interactions in solution. We have employed an interdisciplinary methodology to achieve facile photolabeling of the lectin FimH, which is a bacterial protein, crucial fo

Full text of DOI:10.3762/bjoc.14.163

Diazirine-functionalized mannosides for photoaffinity labeling:
trouble with FimH
Femke Beiroth1, Tomas Koudelka2, Thorsten Overath2, Stefan D. Knight3,
Andreas Tholey2 and Thisbe K. Lindhorst*1
Full Research Paper
Open Access
Address:
Beilstein J. Org. Chem. 2018, 14, 1890–1900.
1Otto Diels Institute of Organic Chemistry, Christiana Albertina
University of Kiel, Otto-Hahn-Platz 3/4, 24118 Kiel, Germany,
2Systematic Proteomics & Bioanalytics, Institute for Experimental
Medicine, Christiana Albertina University of Kiel, Niemannsweg 11,
D-24105 Kiel, Germany and 3Department of Cell and Molecular
Biology, Uppsala University, Uppsala Biomedical Centre, P.O. Box
596, S-751 24 Uppsala, Sweden
doi:10.3762/bjoc.14.163
Received: 07 May 2018
Accepted: 27 June 2018
Published: 24 July 2018
Associate Editor: S. Flitsch
Email:
© 2018 Beiroth et al.; licensee Beilstein-Institut.
License and terms: see end of document.
Thisbe K. Lindhorst* - tklind@oc.uni-kiel.de
* Corresponding author
Keywords:
diazirines; docking; FimH; lectin ligands; mannosides; mass
spectrometry; photoaffinity labelling
Abstract
Photoaffinity labeling is frequently employed for the investigation of ligand–receptor interactions in solution. We have employed
an interdisciplinary methodology to achieve facile photolabeling of the lectin FimH, which is a bacterial protein, crucial for adhe-
sion, colonization and infection. Following our earlier work, we have here designed and synthesized diazirine-functionalized
mannosides as high-affinity FimH ligands and performed an extensive study on photo-crosslinking of the best ligand (mannoside 3)
with a series of model peptides and FimH. Notably, we have employed high-performance mass spectrometry to be able to detect ra-
diation results with the highest possible accuracy. We are concluding from this study that photolabeling of FimH with sugar
diazirines has only very limited success and cannot be regarded a facile approach for covalent modification of FimH.
Introduction
The investigation of the interactions between proteins and their recognition studies as they take molecular dynamics as well as
ligands, such as lectins and carbohydrates, is of fundamental solvent effects into consideration. In the latter respect, photoaf-
importance for the understanding of many biological processes. finity labeling has evolved as a useful tool for studies under
Several different methodologies are used for the elucidation of physiological conditions [1-4]. Photoaffinity labeling requires a
ligand–protein interactions. In addition to X-ray crystallogra- ligand equipped with a photolabile group, which can be con-
phy, studies in solution add valuable information in molecular verted into a highly reactive intermediate upon irradiation with
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light of an appropriate wavelength. This technique involves consolidate our projects on photoaffinity labeling of FimH, but
incubation of the photolabile ligand with the target protein (re- instead this methodology remained problematic in our hands.
ceptor) and irradiation of the ligand–protein complex to form an Until to date, no reliable ligands for photolabeling of FimH are
excited intermediate, which eventually leads to a covalently available.
crosslinked ligand–receptor conjugate, which has to be identi-
fied by mass spectrometry (Figure 1).
We reasoned that high performance mass spectrometry-based
proteomics could lead to more robust success in our approach to
Three widely used photoreactive groups are aryl azides, benzo- employ tailor-made sugar ligands for FimH labeling. Here, we
phenones and diazirines. They differ with respect to their steric report on the difficulties of FimH labeling even when computer-
properties, photochemistry, and reactivity. In order to be aided design and synthesis of photolabile FimH were combined
applied in a biological environment, the wavelength of the light with optimized photolabeling conditions and high-end mass
required for activation of the photolabile ligand has to be spectrometry.
biocompatible. Furthermore, small photophores are desirable.
Diazirines meet these requirements particularly well. The Results and Discussion
diazirine photophore is small and its photoactivation is possible FimH is a fimbrial lectin found at the tips of adhesive
at wavelengths around 350 nm and thus does not perturb pro- organelles (type 1 fimbriae), which are projecting from the sur-
tein structures. According to the literature, irradiation of a face of enterobacteriaceae such as E. coli. Fimbriae mediate
diazirine-functionalized ligand leads to a reactive carbene which firm attachment (adhesion) of bacteria to the glycosylated sur-
can insert into OH, NH, or CH groups of a protein in a fast reac- face of their target cells and constitute important virulence
tion [5,6]. Further advantages of the diazirine group are its factors in bacterial infection such as urinary tract infection
robustness at different pH values and its stability against [13-16]. Hence, FimH is an interesting target protein in medici-
nucleophiles. However, besides desired insertion reactions, nal chemistry and proteomics [17,18]. It is a two-domain pro-
carbenes can undergo unwanted side reactions, in particular tein comprising a lectin domain FimHL hosting the α-D-
olefin formation through abstraction of α-H atoms. mannose-specific carbohydrate binding site and a pilin domain
Therefore, aryl(trifluoromethyl)diazirines were introduced, FimHP connecting the protein to the fimbrial shaft (Figure 2).
which lack hydrogen atoms in α-position of the diazirine
function and consequently do not form olefins. Thus, aryl(tri- Complexation of α-D-mannopyranoside ligands involves the
fluoromethyl)diazirines have become reagents of first choice for entire mannoside glycon moiety whereas the aglycon portion
photolabeling studies [7,8].
sticks out of the binding site, undergoing interactions with the
protein surface, which add to affinity. Especially CH–π or
Over several years it has been our goal, to exploit diazirine- π–π interactions of a sugar ligand with the side chains of Y48
labeled mannopyranosides for protein labeling. In the course of and Y137, called the “tyrosine gate” [19,20], are known to
this work, we have gradually improved our target design and considerably increase the affinity of a specific mannoside for
synthetic procedures [9-11] with the aim to eventually address FimH. Consequently, α-D-mannopyranosides having an aromat-
the bacterial lectin FimH. Finally, back in 2010, we analyzed ic aglycon portion such as p-nitrophenyl α-D-mannopyranoside
photolabeling of the octapeptide angiotensin II with three differ- (1) and the squaric acid derivative 2 [19] (Figure 3) were identi-
ent sugar diazirines using mass spectrometry and then also fied as FimH ligands with relative high affinity (low μmolar
detected photolabeling of FimH with the same three manno- range). Based on this knowledge, we proposed the three
sides. However, we could not measure the labeling product with diazirine-labeled mannosides 3–5 as photolabile ligands of
full accuracy [11]. Although these results were even recognized FimH and evaluated their potential affinity by computer-aided
in a recent review [12], they unfortunately did not help to docking.
Figure 1: Principle of photoaffinity labeling of proteins with diazirine derivatives. Photolabile ligands are complexed with the receptor protein and the
following photochemical excitation of the complex leads to formation of a reactive carbene after extrusion of nitrogen and a crosslinked product after
insertion reaction; X = e.g., NH, O, CH2.
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hydrate binding site of FimH; mainly unspecific interactions
with the surface of FimH were predicted in this case. Thus, syn-
thesis of 5 was not undertaken. In contrast, the diazirine 3, with
scoring values of −34.7 (open gate) and −36.2 (closed gate), is
predicted as suitable FimH ligand with high affinity. Addition-
ally, docking suggests that the diazirine function of 3 is posi-
tioned in close proximity to the protein surface, making a spe-
cific insertion reaction of the carbene formed after irradiation
very likely (involving for example Y48 or T51, Figure 4).
Docking of mannoside 4 also delivered good scoring values and
therefore both photolabile ligands 3 and 4 were synthesized.
Synthesis of photolabile mannosides
The synthesis of the photolabile mannosides 3 and 4 started
from p-nitrophenyl α-D-mannopyranoside (1), which was first
reduced to the corresponding amine 6 [26,27] by catalytic
hydrogenation (Scheme 1). HATU-mediated peptide coupling
with Boc-protected glycine under basic conditions led to 7.
After removal of the Boc protecting group using trifluoroacetic
acid in water, the resulting crude product was subjected to a
subsequent peptide-coupling reaction employing the carboxy-
functionalized diazirine 8.
Figure 2: FimH crystal structure (pdb code 1KLF) with docked p-nitro-
phenyl α-D-mannopyranoside (1, pNPMan). FimH is a two-domain pro-
tein comprising a lectin domain (FimHL) with the carbohydrate binding
site (top) and a pilin domain (FimHP, bottom) that anchors the lectin
onto the fimbrial shaft. For photoaffinity labeling, a truncated version of
the protein (FimHtr) was used. The carbohydrate binding site is
depicted as Connolly surface and colored according to the electro-
static potential. The picture was generated with glide and rendered
with maestro, both implemented in Schrödinger software.
Diazirine 8 was prepared in a nine-step synthesis according to
the literature [28]. For the synthesis of ligand 4, mannoside 6
was first converted into the squaric acid monoester 10 employ-
ing squaric acid diester 9. The monoester 10 was reacted with
N-Boc-ethylendiamine to obtain the squaric acid diamide 11.
Then removal of the Boc protecting group with trifluoroacetic
acid followed by peptide coupling with the diazirine 8 led to
Docking studies
Mannosides 3–5 (Figure 3) were docked into the FimH carbo- target molecule 4. However, purification of the photolabile
hydrate binding site using FlexX as implemented in Sybyl 6.9 mannosides is problematic due to their poor solubility and the
[21-23]. Ligand structures were minimized using the Tripos light sensitivity of the diazirine moiety. Therefore, the products
force field and 30 different conformers of each ligand were often contain minor impurities after chromatography resulting
docked into two different X-ray structures of FimH (for details from light-induced activation of the diazirine and insertion reac-
see Supporting Information File 1). These two crystal struc- tion. This can be monitored by 19F NMR spectroscopy showing
tures differ in the conformation of the tyrosine gate, formed by the typical diazirine CF3 signal around −68 ppm [29]. Whereas
Y48 and Y137 positioned at the entrance of the carbohydrate mannoside 3 was received in good purity, compound 4 was
binding site. They are flexible and can be more distant to one always obtained with contaminations. Thus, 3 was the preferred
another (“open gate”) or closer together (“closed gate”) [20]. ligand for the following photolabeling experiments. Results ob-
Both conformations were considered for the docking studies. tained with mannoside 4 were less promising and are not re-
For each docked conformation, a scoring value is obtained that ported here. After all, also modeling had suggested that 3 has a
correlates with the affinity of the ligand to the carbohydrate higher affinity for FimH than 4.
binding site. A more negative value predicts better binding.
Scores obtained for mannosides 3 and 4 suggest a high affinity To test carbene formation, mannoside 3 was irradiated at
for FimH, surpassing that of pNPMan 1 for both protein confor- 345 nm in 1:1 acetonitrile/water as well as in 1:1 DMSO/water
mations tested (Table 1). (Diazirines cannot be tested in bacteri- mixtures with 4-hydroxybenzyl alcohol as the simplest tyrosine
al adhesion–inhibition assays due to their light sensitivity.)
mimic. Mass-spectrometric analysis indicated the desired cross-
linked product as well as insertion into water in both cases
The bivalent ligand 5, on the other hand, seems to be sterically (cf. Supporting Information File 1, Table S5). Hence, carbene
too demanding to allow good complexation with the carbo- formation upon irradiation of diazirine 3 was ensured and thus
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Figure 3: Based on the structure of the known FimH ligands 1 and 2, three photolabile α-D-mannosides, 3–5, were considered for FimH labeling.
In 3 and 4 the distance between the diazirine functional group and the anomeric center of the mannoside are varied. In 5 a bis-diazirine
functionalization is suggested to increase labeling probability.
Table 1: FlexX scoring values for photolabile mannosides 3–5 in comparison with methyl α-D-mannopyranoside (MeMan) and pNPMan 1 for the open
and closed gate crystal structure of FimH (PDB 1KLF and 1UWF, respectively).
Ligand
FlexX scoring value open gate
FlexX scoring value closed gate
MeMan
−22.5
−24.9
−34.7
−28.6
–
−23.3
−27.4
−36.2
−34.0
−18.8
1
3
4
5
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Figure 4: Connolly representation of photolabile α-D-mannoside 3 in the closed gate (A, PDB code 1UWF) and open gate crystal structure of FimH
(B, PDB code 1KLF) [24]. The surface is colored according to the lipophilic potential, where brown reflects more hydrophobic and blue more hydro-
philic amino acid residues [25]. Amino acid residues Y48 and Y137, forming the “tyrosine gate” are highlighted as well as T51, which is a good candi-
date for photolabeling.
Scheme 1: Synthesis of photolabile α-D-mannosides 3 and 4. a) H2, Pd-C, methanol, rt, 6 h, 94%; b) HATU, DIPEA, dry DMF, N2, rt, overnight,
quant.; c) 1. 50% TFA in water, rt, 3 h; 2. HATU, 8, DIPEA, dry DMF, N2, rt, 14 h, 19% (over 2 steps); d) dry methanol, rt, 16 h, 59%; e) Et3N, dry
methanol, rt, 15 h, 92%; f) 1. 50% TFA in water, rt, 3 h; 2. HATU, 8, DIPEA, dry DMF, N2, rt, overnight, crude product (68% over 2 steps).
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we continued further labeling studies with more complex sub- Also, we were interested to test, if threonine or tyrosine
strates.
residues, respectively, would lead to better labeling efficiency.
Because mannoside 3 is poorly water soluble, it was dissolved
in methanol and then added to the peptides in a 1:1 ratio. After
irradiation with UV light, two new signals were detected in
Irradiation of mannoside 3 and
model peptides
In analogy to our earlier work, we first used a series of model nano-LC–ESIMS experiments in comparison to the original
peptides (M2, M3, M7, M8, T3, and S17, cf. Table 2) for the peptide spectra (Table 2). It should be noted that whereas we
photolabeling experiments with mannoside 3. These model could monitor photodecomposition of the photolabels by
peptides were chosen rather arbitrarily as we have frequently 19F NMR spectroscopy (cf. [29]), we could not observe labeled
employed them to optimize mass spectrometric procedures. products by fluorine NMR. This is presumably due to the low
Table 2: ESIMS data of peptide labeling with ligand 3 (cf. Supporting Information File 1 for all details).
Peptide
Peptide mass Detected masses after labeling (m/z)
(M)
ILMEHIHKL (M2)
YLLPAIVHI (M3)
EIAMATVTALR (M7)
ETIGEILKK (M8)
EGHIARNCRA (T3)
1132.6499
1037.6274
1174.6380
1029.6071
1125.5462
531.1584 [M + H]+ a, 832.4043 [M + 2H]2+ b (Figure 5)
531.1584 [M + H]+ a, 784.8971 [M + 2H]2+ b (Figure S11, Supporting Information File 1)
531.1586 [M + H]+ a, 853.4019 [M + 2H]2+ b (Figure S14, Supporting Information File 1)
531.1587 [M + H]+ a, 780.8868 [M + 2H]2+ b (Figure S17, Supporting Information File 1)
531.1588 [M + H]+ a, no adduct observed (Figure S20, Supporting Information File 1)
531.1585 [M + H]+ a, 989.9307 [M + 2H]2+ b (Figure S21, Supporting Information File 1)
RPQYAEASWNAR (S17) 1447.6957
aInsertion product of 3 − N2 + H2O; bAdduct of 3 − N2 + H2O to the peptide.
Figure 5: ESIMS spectra of peptide M2 before (A) and after photoreaction (B) with mannoside 3.
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labeling efficiency as in the literature 19F NMR spectra were carbene, which is formed after irradiation, into OH, NH or
obtained with proteins having 19F-labeled amino acids incorpo- CH groups, respectively, in proximity of the lectin’s carbo-
rated [30].
hydrate binding site. We were hoping that after our initial
success in 2010 [11], high performance MS analysis would lead
Disappointingly, instead of crosslinking, we observed two dif- to results of higher accuracy. Indeed, we could detect FimHtr
ferent other products in all cases. As an example, for peptide labeling after irradiation with 3, however, photolabeling was not
M2 one peak at m/z 531.1584 ([M + H]+) could be assigned to at all efficient. Only after extensive and tedious optimization of
the ligand 3 that had reacted with a water molecule instead of reaction conditions, a new peak was observed by mass spec-
the peptide (Figure 5). The second signal of low intensity in this trometry on the intact protein level (Figure 6). For this, the
spectrum at m/z 832.4043 corresponds to the peptide mass plus photolabile mannoside 3 was incubated with FimH truncate
hydrolyzed carbene. However, MS/MS fragmentation of both (FimHtr) in buffer and 50% ACN or 10% DMSO, respectively,
the unmodified peptide (567.3281)2+ and the allegedly modi- and irradiated with UV light.
fied peptide (832.4043)2+ exhibited the same b- and y-ion series
(cf. Supporting Information File 1, Figure S10). Accordingly, The theoretically most abundant isotope peak of unbound
the low intensity peak is most likely the result of non-covalent FimHtr (1373.4608 Da, z = 13) fits to the most abundant
binding of hydrolyzed 3 to the peptide M2 rather than a cova- experimentally observed mass for unbound FimHtr with
lent modification at one of the peptide’s side chains. The same 1373.4616 Da (Figure 6A) and 1373.5375 Da (Figure 6B) with
situation was found for all other peptides irradiated with the small deviations. The presence of a new peak at 1412.8578 Da
photolabile ligand 3 (cf. Supporting Information File 1).
(z = 13, Figure 6C) and 1412.8551 Da (z = 13, Figure 6D), re-
spectively, corresponded very well with insertion of the photo-
labile mannoside 3. In this case, non-covalent bonding of the
photolabile mannoside 3 to the substrate (FimHtr) could be
Irradiation of mannoside 3 and
FimH-truncate
Whereas we first expected that it would be easier to photolabel ruled out, as this would have led to a significantly higher mass
peptides than FimH, after the failure with photolabeling of the according to the intact label 3 with a relative mass shift of ≈1.38
model peptides we were hoping that labeling of FimH would be (at z = 13). In addition, under the LC–ESIMS conditions applied
more successful, as our ligand was designed to bind to FimH. here, e.g., low pH and use of organic eluents in ion-paring
We reasoned that complexation of the photolabile mannoside 3 reversed-phase chromatography, as well as under the ESI condi-
and FimH could support the desired crosslinking reaction. Thus, tions employed, non-covalent bonding of the photolabile
the high affinity of 3 for FimH would facilitate insertion of the mannoside to FimH is unlikely to be observed. Due to the low
Figure 6: Intact protein ESIMS spectra of FimHtr labeling with diazirine derivative 3 with (A) 50% acetonitrile or (B) 10% DMSO additive. (C) Minor
peak at 1412.8578, as part of spectrum A, representing the covalently bound label. Zoom into the signal at charge state +13. Labeling yield calcu-
lated from peak intensities compared to the corresponding signal of unlabeled peptide: 1.41% (50% acetonitrile). (D) Minor peak at 1412.8551, as part
of spectrum B, representing the covalently bound label. Labeling yield calculated from peak intensities compared to corresponding signal of unla-
beled peptide: 1.55% (10% DMSO). The theoretically expected mass of labeled FimHtr is 1412.9333 Da. The fact that the most abundant isotope was
not 1412.9333, but 1412.8578 and 1412.8551 Da is simply due to the high signal to noise ratio of the spectra as a result of low labeling efficiency.
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labeling yield obtained, all our attempts to identify the specific 500 pmol of ligand solution (3 in 12.9 µL methanol) was added
position of FimH labeling by peptic digestion of the labeled and incubated for 10 min at 37 °C and 100 rpm. Subsequently,
protein and following MS/MS analysis were unsuccessful.
the solution was irradiated under ice cooling with a medium
pressure mercury vapor lamp for 10 min. Afterwards, 50 µL
double distilled water were added and 2 µL of this solution
Conclusion
In continuation of our earlier work, we have employed an ad- given to 20 µL of a 0.1% trifluoroacetic acid (TFA) solution,
vanced interdisciplinary approach to identify diazirine-labeled desalted with a C18 µZipTip and spotted on a MALDI-plate
mannoside ligands for facile and reliable photolabeling of the together with 5 mg/mL α-cyano-4-hydroxycinnamic acid
bacterial lectin FimH. We could indeed show photolabeling of (HCCA) matrix (70% ACN in 0.1% TFA solution in double
the protein with the designer mannoside 3 with much higher distilled water). MALDI–TOF MS was performed on a AB
accuracy than before. However, a great excess of ligand was 5800 MS (AB Sciex, Darmstadt, Germany) in positive ion
needed and only traces of labeled FimH were observed. Due to mode.
the high-performance mass spectrometry which was employed
here, this result can be considered specific. However, it was not FimHtr
possible to identify the site of labeling due to the low labeling FimHtr (3.36 nmol) was dissolved in PBS buffer (pH 7.8).
yield. Photolabeling of FimH with sugar diazirines remains Ligand 3 was dissolved in ACN or DMSO. The ratio ligand to
difficult and thus this approach is not very likely to be de- protein was 70:1. The total volume of the reaction was 11 µL
veloped into a standard method for FimH labeling. As we wish either with 50% ACN or 10% DMSO. Ligand and protein were
to eventually control bacterial adhesion by crosslinking of func- combined and incubated for 10 min at 37 °C and 100 rpm.
tional molecules to the adhesin (FimH), we will in the Afterwards the solution was irradiated under ice cooling with a
future utilize alternative labeling chemistry to efficiently target medium pressure mercury vapor lamp for 10 min. The
FimH.
protein solution was then investigated by ESI mass spectrome-
try.
Experimental
Peptides and proteins
LC–MS methods
For photolabeling experiments model peptides from Biosyn- Nano-LC–MS was performed on the U3000 nano-LC-UV
than (Berlin) were used: ILMEHIHKL (M2), YLLPAIVHI system (Dionex, Idstein, Germany) coupled online to a LTQ
(M3), EIAMATVTALR (M7), ETIGEILKK (M8), Orbitrap Velos mass spectrometer equipped with LTQ Tune
EGHIARNCRA (T3), and RPQYAEASWNAR (S17). Plus 2.7.0 and XCalibur 2.2 (all Thermo Fisher Scientific). The
Furthermore, FimH truncate (FimHtr) was used, which was column oven was set to 30 °C and the UV trace was monitored
expressed and purified in the laboratory of the S. D. Knight ac- at a wavelength of 214 nm. The modified FimHtr (10 pmol) was
cording to [31]. FimHtr has the following amino acid sequence: desalted on a C4 trap column (Acclaim PepMap C-4, 300 µm
FACKTANGTAIPIGGGSANVYVNLAPVVNVGQNL- i.d. × 5 mm, 5 µm, 300 Å, Thermo Fisher Scientific) using a
VVDLSTQIFCHNDYPETITDYVTLQRGSAYGGVLSNFSG- flow rate of 30 µL/min. After 5 min, the analytes were eluted
TVKYSGSSYPFPTTSETPRVVYNSRTDKPWPVALYLT- with a flow rate of 300 nL/min onto an analytical monolithic
PVSSAGGVAIKAGSLIAVLILRQTNNYNSDDFQF- column (ProSwift RP-4H, 100 µm × 250 mm, Thermo Fisher
VWNIYANNDVVVPTGGHHHHHH.
Scientific) using eluent A (0.05% formic acid (FA)) and eluent
B (80% ACN in 0.04% FA). A gradient from 5 to 95% B was
applied over 30 min followed by a 10 min washing step at 95%
Docking studies
Computer-aided docking studies were performed to evaluate the B and a 15 min equilibration step at 5% B. MS data were re-
binding affinity of FimH ligands using FlexX flexible docking corded in MS full scan mode between 400 and 2000 m/z using
and consensus scoring, both implemented in Sybyl 6.9, as de- 445.120025 as a lock mass. Data were recorded at a resolution
scribed earlier [19]. Two published X-ray structures of FimH of 60,000 in profile mode. To obtain better quality MS data, no
(PDB codes 1KLF and 1UWF) were considered. During MS/MS data were acquired. Raw files were opened in XCal-
docking, the receptor was held fixed whereas the ligand was ibur 2.2 and spectra during which FimHtr was observed were
allowed to change its conformation. All calculations were done averaged. Theoretical spectra of FimHtr were compared to the
with the Tripos force field.
experimental spectra using IDCalc (version 0.3). Experimental
spectra were calculated in profile mode using a charge state of
13 and at a resolution of 35,000 (the actual resolution observed
Peptides
To 500 pmol of a model peptide (cf. Table 1) dissolved in 5 µL at m/z of 13 for FimHtr) and an elemental composition of
double distilled water (18 MΩ) with 5% acetonitrile (ACN), truncated and his-tagged FimH with a disulfide bond
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(C803H1213O242N217S2). The relative peak abundances were 1H, H-2), 3.89 (dd, 3J3,2 = 3.4 Hz, 3J3,4 = 9.4 Hz, 1H, H-3),
extracted and compared to the theoretical spectra. A change in 3.76 (dd, 3J6,5 = 2.5 Hz, 2J6,6‘ = 11.9 Hz, 1H, H-6), 3.73 (mc,
molecular composition upon addition of the diazirine 3 1H, H-4), 3.71 (dd, 3J6‘,5 = 5.1 Hz, 2J6‘,6 = 11.9 Hz, 1H, H-6‘),
(C23H23F3N2O8) was appended to the elemental composition of 3.61 (ddd, 3J5,6 = 2.5 Hz, 3J5,6‘ = 5.1 Hz, 3J5,4 = 9.8 Hz, 1H,
FimHtr.
H-5) ppm; 13C NMR (126 MHz, MeOH-d4) δ 171.6 (1C, CN2),
169.5 (1C, C=O), 169.2 (1C, C=O), 154.7 (1C, Caryl-4), 136.7
(1C, Caryl-1‘), 134.1 (1C, Caryl-1), 133.3 (1C, Caryl-4‘), 129.3
Methods and materials for synthesis
TLC was performed on silica gel GF254 (Merck). Spots were (2C, CHaryl-2‘,6‘), 127.7 (2C, CHaryl-3‘,5‘), 122.9 (2C, CHaryl-
visualized under UV light and by charring with 10% sulfuric 2,6), 121.3 (1C, q, J = 274.9 Hz, CF3), 118.2 (2C, CHaryl-3,5),
acid in ethanol and subsequent heating. Flash column chromato- 100.6 (1C, C-1), 75.4 (1C, C-5), 72.4 (1C, C-3), 72.0 (1C, C-2),
graphy was performed on silica gel 60 (230–400 mesh, particle 68.4 (1C, C-4), 62.7 (1C, C-6), 44.4 (1C, CH2-glycine) ppm;
size 40–63 µm, Merck). All used solvents were distilled. NMR 19F NMR (471 MHz, MeOH-d4) δ −66.87 (s) ppm; ESIMS m/z:
spectra were recorded on Bruker DRX 500 or Bruker Avance 579.12 [M + K]+, 563.14 [M + Na]+, 535.13 [(M + Na) − N2]+;
600 instruments at 300 K. Chemical shifts are relative to the HRESIMS m/z: [M + H]+ calcd. for C23H23F3N4O8,
solvent peak of MeOD (3.35 ppm and 4.78 ppm for 1H, 49.3 for 541.15408; found, 541.15424.
13C). Assignments of the signals were done using
2D experiments (COSY, HSQC and HMBC). IR spectra were {N-[4-(α-D-Mannopyranosyloxy)phenyl]-N’-[2’-(4’’-(3-tri-
measured with a Perkin Elmer FT IR Paragon 1000 (KBr). fluoromethyl-3H-diazirin-3-yl)phenylamido)ethyl]}squaric
Optical rotation values were determined with a Perkin-Elmer acid diamide (4). The squaric acid diamide 11 (152 mg,
polarimeter (589 nm, length of cuvette: 1 dm). MS analysis of 290 µmol) was dissolved in distilled water which contained
synthetic products were carried out with a MALDI–TOF mass 50% TFA (9 mL). The solution was then stirred at room tem-
spectrometer Bruker Biflex III with 19 kV acceleration voltage perature for 3 h. After complete conversion, the solvent was re-
(matrix: 2,5-dihydroxybenzoic acid) and with a ESI–TOF MS moved under reduced pressure, codistilled with toluene and
spectrometer Applied Biosystems Mariner ESI–TOF 5280. UV neutralized with basic ion exchange resin in methanol. The resin
data were obtained with the Perkin-Elmer UV–vis spectrometer was filtered off and the solvent removed under reduced pres-
Lambda 14 at 25 °C. All reactions and purification steps of sure. The primary amine was then dried under vacuum together
diazirine compounds were performed in the dark.
with the diazirine 8 [28] (56.0 mg, 250 µmol) und HATU
(114 mg, 300 µmol) for 30 min. The substances were dissolved
N-{2-Oxo-2-[(4-(α-D-mannopyranosyloxy)phenyl)amino]- in dry DMF (10 mL) and DIPEA (20.0 µL, 180 µmol) was
ethyl}-4-(3-trifluoromethyl-3H-diazirin-3-yl)benzamide (3). added. The solution was stirred at room temperature for 16 h.
The glycoamino acid 7 (52.8 mg, 160 µmol) was dissolved in Subsequently, the solvent was removed under reduced pressure
distilled water which contained 50% TFA (5 mL). The solution and the crude product was purified by flash column chromato-
was then stirred at room temperature for 3 h. After complete graphy (dichloromethane/methanol 8:1→4:1). The product was
conversion, the solvent was removed under reduced pressure, obtained as a colorless solid (123 mg) containing minor
co-distilled with toluene and neutralized with basic ion amounts of side products which could not be separated (crude
exchange resin in methanol. The crude product was then dried yield 68%). 1H NMR (500 MHz, MeOH-d4) δ 7.96–7.92 (m,
together with diazirine 8 [28] (27.6 mg, 120 µmol) and HATU 2H, CHaryl-2‘,6‘), 7.39 (d, 3J = 8.8 Hz, 2H, CHaryl-2,6), 7.35 (d,
(83.7 mg, 220 µmol) for 1 h under vacuum. Subsequently this 3J = 8.2 Hz, 2H, CHaryl-3‘,5‘), 7.14–7.10 (m, 2H, CHaryl-3,5),
was mixture dissolved in dry DMF (5 mL) and DIPEA 5.47 (d, 3J1,2 = 1.8 Hz, 1H, H-1), 4.04 (dd, 3J2,1 = 1.8 Hz,
(20.0 µL, 140 µmol). The solution was stirred at room tempera- 3J2,3 = 3.4 Hz, 1H, H-2), 3.93 (dd, 3J3,2 = 3.4 Hz,
ture for 14 h. The solvent was removed under reduced pressure 3J3,4 = 9.4 Hz, H-3), 3.80 (dd, 3J6,5 = 2.6 Hz, 2J6,6‘ = 12.0 Hz,
and the residue purified by flash column chromatography (ethyl 1H, H-6), 3.78–3.73 (m, 2H, H-4, H-6‘), 3.70–3.66 (m, 2H,
acetate/methanol 5:1). The product was isolated as a colorless CH2-ethyl) 3.64 (ddd, 3J5,4 = 9.8 Hz, 3J5,6‘ = 4.5 Hz,
solid (17.2 mg, 30.0 µmol, 19%); Rf = 0.76 (ethyl acetate/meth- 3J5,6 = 2.6 Hz, 1H, H-5), 3.29–3.23 (m, 2H, CH2-ethyl) ppm;
anol 2:1); UV–vis (MeOH) λmax: 345 nm; FTIR (KBr) : 3317, 13C NMR (126 MHz, MeOH-d4, 300 K) δ 169.3, 165.6 (2C,
2927, 2503, 2093, 1671, 1637, 1550, 1511, 1224, 1015, 940, Csquaric acid), 154.6 (1C, Caryl-4), 137.0 (1C, Caryl-1‘‘), 134.6
681 cm−1; 1H NMR (500 MHz, MeOH-d4) δ 7.99 (d, (1C, Caryl-1), 133.2 (1C, Caryl-4’’), 129.1 (2C, CHaryl-2‘‘,6‘‘),
3J = 8.5 Hz, 2H, CHaryl-2‘,6‘), 7.48 (d, 3J = 9.1 Hz, 2H, CHaryl- 127.7 (2C, CHaryl-3‘‘,5‘‘), 121.5 (2C, CHaryl-2,6), 118.7 (2C,
2,6), 7.36 (d, 3J = 8.5 Hz, 2H, CHaryl-3‘,5‘), 7.08 (d, CHaryl-3,5), 100.6 (1C, C-1), 75.4 (1C, C-5), 72.4 (1C, C-3),
3J = 9.1 Hz, 2H, CHaryl-3,5), 5.43 (d, 3J1,2 = 1.7 Hz, 1H, H-1), 72.0 (1C, C-2), 60.3 (1C, C-4), 62.6 (1C, C-6), 43.8 (1C,
4.18 (s, 2H, CH2-Glycin), 3.99 (dd, 3J1,2 = 1.7 Hz, 3J2,3 = 3.4 Hz, CH2-ethyl), 42.1 (1C, CH2-ethyl) ppm; not visible owing to slow
1898

Beilstein J. Org. Chem. 2018, 14, 1890–1900.
relaxation: 2 squaric acid C=O), CF3; 19F NMR (471 MHz, 3J5,6 = 4.6 Hz, 3J5,4 = 9.4 Hz, 1H, H-5) ppm; 13C NMR
MeOH-d4) δ −66.84 (s) ppm; MALDI–TOF MS m/z: 620.72 (151 MHz, MeOH-d4, 300 K) δ 193.9, 193.6 (2C,
[M]+.
C=Osquaric acid), 179.8, 164.9 (2C, Csquaric acid), 154.4 (1C,
Caryl), 132.8 (1C, Caryl), 122.6 (2C, CHaryl), 118.5 (2C, CHaryl),
Nα-Boc-glycin-[p-(α-D-mannopyranosyloxy)]phenyl amide 100.5 (1C, C-1), 75.5 (1C, C-5), 72.4 (1C, C-3), 72.0 (1C, C-2),
(7). The aminophenyl mannoside 6 [26,27] (150 mg, 553 µmol) 68.4 (1C, C-4), 62.7 (1C, C-6), 59.8 (1C, OCH3) ppm;
was dried together with N-Boc-glycine (64.6 mg, 369 µmol) MALDI–TOF MS m/z: 404.46 [M + Na]+.
and HATU (280 mg, 738 µmol) for 45 min under vacuum.
Afterwards, this mixture was dissolved in dry DMF (8 mL), {N-[4-(α-D-Mannopyranosyloxy)phenyl]-N’-[(2‘-tert-butyl-
DIPEA (80.0 µL, 443 µmol) was added and the reaction mix- oxycarbonylamido)ethyl]}squaric acid diamide (11). The
ture stirred overnight at room temperature. The solvent was re- squaric acid monoamide 10 (90.0 mg, 240 µmol) was dissolved
moved under vacuum and purified with flash column chromato- in dry methanol (5 mL). Afterwards, N-Boc-ethylendiamine
graphy (ethyl acetate/methanol 6:1) leading to a colorless solid (46.0 mg, 465 µmol) and triethylamine (140 µL) were added.
(158 mg, 369 µmol, quant.); Rf = 0.15 (ethyl acetate/methanol The reaction solution was stirred at room temperature for 15 h.
6:1); mp 74 °C; [α]D21 = 91.8 (c 0.5, MeOH); FTIR (KBr) The solution was neutralized with an acidic ion exchange resin
: 3388, 2936, 1660, 1508, 1445, 1368, 1220, 1161, 1015, 978, (Amberlyst-A21), filtered and concentrated under reduced pres-
835, 556 cm−1; 1H NMR (600 MHz, MeOH-d4) δ 7.46 (d, sure. The crude product was purified by column chromatogra-
3J = 8.9 Hz, 2H, CHaryl-2,6), 7.08 (d, 3J = 8.9 Hz, 2H, CHaryl- phy (ethyl acetate/methanol 3:1), leading to a colorless solid
3,5), 5.43 (d, 3J1,2 = 1.8 Hz, 1H, H-1), 3.99 (dd, 3J2,1 = 1.8 Hz, (114 mg, 220 µmol, 92%); Rf = 0.44 (ethyl acetate/methanol
3J 2,3 = 3.4 Hz, 1H, H-2), 3.89 (dd, 3J3,2 = 3.4 Hz, 3:1); mp 176–185 °C (decomp.); [α]D25 = +72.1 (c 0.3,
3J3,4 = 9.4 Hz, 1H, H-3), 3.84 (s, 2H, CH2-Glycin), 3.76 (dd, CH3OH); FTIR (KBr) : 3359, 2970, 2933, 1798, 1689, 1650,
3J6‘,5 = 2.4 Hz, 2J6‘,6 = 11.9 Hz, 1H, H-6‘), 3.75–3.69 (m, 2H, 1609, 1550, 1514, 1454, 1285, 1118, 1021, 918, 881, 826,
H-6, H-4), 3.61 (ddd, 3J5,6‘ = 2.4 Hz, 3J5,6 = 5.1 Hz, 765 cm−1; 1H NMR (500 MHz, MeOH-d4) δ 7.34 (d,
3J5,4 = 9.4 Hz, 1H, H-5), 1.47 (s, 9H, C(CH3)3) ppm; 13C NMR 3J = 8.8 Hz, 2H, CHaryl-2,6), 7.13 (d, 3J = 8.8 Hz, 2H, CHaryl-
(151 MHz, MeOH-d4) δ 170.4 (1C, C=Oglycine), 158.6 (1C, 3,5), 5.44 (d, 3J1,2 = 1.9 Hz, 1H, H-1), 4.00 (dd, 3J2,1 = 1.9 Hz,
C=OBoc), 154.6 (1C, Caryl-4), 134.1 (1C, Caryl-1), 122.9 (2C, 3J2,3 = 3.5 Hz, 1H, H-2), 3.89 (dd, 3J3,2 = 3.5 Hz,
CHaryl-2,6), 118.1 (2C, CHaryl-3,5), 100.5 (1C, C-1), 80.7 (1C, 3J3,4 = 9.4 Hz, 1H, H-3), 3.78 (dd, 3J6,5 = 2.5 Hz,
C(CH3)3), 75.4 (1C, C-5), 72.4 (1C, C-3), 72.0 (1C, C-2), 68.4 2J6,6‘ = 11.9 Hz, 1H, H-6), 3.75–3.69 (m, 2H, Boc-
(1C, C-4), 62.7 (1C, C-6), 45.0 (1C, CH2-glycine), 28.7 (3C, NHCH2CH2), 3.72 (dd~t, 3J4,3 = 3J4,5 = 9.4 Hz, 1H, H-4), 3.71
C(CH3)3) ppm; MALDI–TOF MS m/z: 451.10 [M + Na]+; (dd, 3J6‘,5 = 5.4 Hz, 2J6‘,6 = 11.9 Hz, 1H, H-6‘), 3.62 (ddd,
ESIMS m/z: 448.3 [M + Na]+, 467.3 ([M + K]+.
3J5,6 = 2.5 Hz, 3J5,6‘ = 5.4 Hz, 3J5,4 = 9.4 Hz, 1H, H-5), 3.29
(mc, 2H, Boc-NHCH2CH2), 1.40 (s, 9H, C(CH3)3) ppm;
N-{[4-(α-D-Mannopyranosyloxy)phenyl]amido}squaric acid 13C NMR (126 MHz, MeOH-d4, 300 K) δ 185.0, 182.6 (2C,
methyl ester (10). To a solution of the aminophenyl manno- C=Osquaric acid), 170.7, 165.5 (2C, Csquaric acid), 154.7 (1C,
side 6 [26,27] (200 mg, 737 µmol) in dry methanol (15 mL), Caryl-4), 134.6 (1C, Caryl-1), 121.7 (2C, CHaryl-2,6), 118.8 (2C,
squaric acid dimethyl ester (9, 314 mg, 2.21 mmol) was added. CHaryl-3,5), 100.6 (1C, C-1), 80.4 (1C, C(CH3)3), 75.4 (1C,
The solution was stirred at room temperature for 16 h. Subse- C-5), 72.4 (1C, C-3), 72.0 (1C, C-2), 68.4 (1C, C-4), 62.7 (1C,
quently, the solvent was removed under reduced pressure and C-6), 45.3 (1C, Boc-NHCH2CH2), 42.4 (1C, Boc-NHCH2CH2),
the crude product was purified by flash column chromatogra- 28.7 (3C, C(CH3)3) ppm; MALDI–TOF MS m/z:
phy (ethyl acetate/methanol 3:1). The product was isolated as a 532.16 [M + Na]+, 548.14 [M + K]+; ESIMS m/z: 532.3
colorless solid (166 mg, 436 µmol, 59%); Rf = 0.43 (ethyl [M + Na]+.
acetate/methanol, 2:1); mp 198 °C (decomp.); [α]D21 = +88.0
(c 0.4, DMSO); FTIR (KBr) : 3260, 1797, 1698, 1624, 1586,
Supporting Information
1396, 1234, 1004, 811 cm−1; 1H NMR (200 MHz, MeOH-d4)
δ 7.30 (d, 3J = 9.0 Hz, 2H, CHaryl-2,6), 7.12 (d, 3J = 9.0 Hz,
Supporting Information File 1
2H, CHaryl-3,5), 5.45 (d, 3J1,2 = 1.8 Hz, 1H, H-1), 4.44 (s, 3H,
OCH3), 4.01 (dd, 3J2,1 = 1.8 Hz, 3J2,3 = 3.4 Hz, 1H, H-2), 3.89
(dd, 3J3,2 = 3.4 Hz, 3J3,4 = 9.4 Hz, 1H, H-3), 3.80 (dd,
3J6‘,5 = 2.8 Hz, 2J6‘,6 = 11.9 Hz, 1H, H-6‘), 3.73 (dd~t,
3J4,3 = 3J4,5 = 9.4 Hz, 1H, H-4), 3.71 (dd, 3J6,5 = 4.6 Hz,
2J6,6‘ = 11.9 Hz, 1H, H-6), 3.60 (ddd, 3J5,6‘ = 2.8 Hz,
NMR spectra of the synthetic compounds 3, 4, 7, and 11,
docking results obtained with 3 and 4, and MS and MS/MS
spectra of labeling experiments.
[https://www.beilstein-journals.org/bjoc/content/
supplementary/1860-5397-14-163-S1.pdf]
1899

Beilstein J. Org. Chem. 2018, 14, 1890–1900.
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Acknowledgements
Financial support by the DFG-Cluster of Excellence “Inflam-
mation at Interfaces” and by the DFG Collaborative Network
SFB 677 is gratefully acknowledged.
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