En route to photoaffinity labeling of the bacterial lectin FimH

Article abstract of DOI:10.3762/bjoc.6.91

Mannose-specific adhesion of Escherichia coli bacteria to cell surfaces, the cause of various infections, is mediated by a fimbrial lectin, called FimH. X-ray studies have revealed a carbohydrate recognition domain (CRD) on FimH that can complex α-D-mannosides. However, as the precise nature of the ligand-receptor interactions in mannose-specific adhesion is not yet fully understood, it is of interest to identify carbohydrate recognition domains on the fimbrial lectin also in solution. Photoaffinity labeling serves as an appropriate methodology in this endeavour and hence biotin-labeled photoactive mannosides were designed and synthesized for photoaffinity labeling of FimH. So far, the photo-crosslinking properties of the new photoactive mannosides could be detailed with the peptide angiotensin II and labeling of FimH was shown both by MS/MS studies and by affino dot-blot analysis.

Full text of DOI:10.3762/bjoc.6.91

En route to photoaffinity labeling of the
bacterial lectin FimH
Thisbe K. Lindhorst*1, Michaela Märten1, Andreas Fuchs1
and Stefan D. Knight2
Full Research Paper
Open Access
Address:
Beilstein J. Org. Chem. 2010, 6, 810–822.
1Christiana Albertina University of Kiel, Otto Diels Institute of Organic
Chemistry, Otto-Hahn-Platz 4, D-24098 Kiel, Germany, Fax: +49 431
8807410 and 2Swedish University of Agricultural Sciences,
Department of Molecular Biology, Uppsala Biomedical Center,
SE-75124 Uppsala, Sweden
doi:10.3762/bjoc.6.91
Received: 20 May 2010
Accepted: 21 July 2010
Published: 26 August 2010
Email:
Editor-in-Chief: J. Clayden
Thisbe K. Lindhorst* - tklind@oc.uni-kiel.de
© 2010 Lindhorst et al; licensee Beilstein-Institut.
License and terms: see end of document.
* Corresponding author
Keywords:
diazirines; FimH; lectins; MS/MS analysis; photoactive mannoside
ligands; photoaffinity labeling
Abstract
Mannose-specific adhesion of Escherichia coli bacteria to cell surfaces, the cause of various infections, is mediated by a fimbrial
lectin, called FimH. X-ray studies have revealed a carbohydrate recognition domain (CRD) on FimH that can complex α-D-manno-
sides. However, as the precise nature of the ligand–receptor interactions in mannose-specific adhesion is not yet fully understood, it
is of interest to identify carbohydrate recognition domains on the fimbrial lectin also in solution. Photoaffinity labeling serves as an
appropriate methodology in this endeavour and hence biotin-labeled photoactive mannosides were designed and synthesized for
photoaffinity labeling of FimH. So far, the photo-crosslinking properties of the new photoactive mannosides could be detailed with
the peptide angiotensin II and labeling of FimH was shown both by MS/MS studies and by affino dot–blot analysis.
Introduction
Photoaffinity labeling is a technique by which ligand binding It has become our goal to utilize this methodology for the
sites of a receptor protein can be identified in solution. It investigation of carbohydrate binding of the bacterial lectin
requires a photoactive ligand derivative, which can form a reac- FimH. The protein FimH is found on the tips of type 1 fimbriae,
tive species upon photo-excitation. Thus, incubation of the long adhesive filamentous appendages on the surface of many
photoprobe with a protein followed by irradiation can result in a bacteria, such as Escherichia coli [2-5]. In X-ray studies a
photo-crosslinked product, that provides structural information carbohydrate recognition domain (CRD), which can complex
on the binding site of the protein (Figure 1) [1].
one α-D-mannosyl residue, has been clearly identified [6-8].
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Beilstein J. Org. Chem. 2010, 6, 810–822.
Figure 1: The photoaffinity technique allows the identification of ligand binding sites of a receptor protein after photo-crosslinking, proteolysis and
affinity chromatography.
However, other binding experiments performed with a multi- the binding pocket. The entrance of the CRD is flanked by the
tude of synthetic as well as natural mannosides and oligo- tyrosine residues Tyr48 and Tyr137 (‘tyrosine gate’) that can
mannosides are not in complete agreement with just one mono- form favorable interactions with appropriate mannosidic agly-
valent carbohydrate binding site on the FimH protein [9-13]. cone moieties, such as π-π-stacking with the phenyl group of
Thus, we decided to employ photoactive ligands to probe carbo- benzyl mannosides [14]. In a preceding work, we synthesized
hydrate binding to the known CRD in solution and to identify the three corresponding photoactive α-D-mannosides, 1, 2, and
possibly auxiliary, so far unknown, binding sites on bacterial 3 (Figure 2a) for photolabeling of the bacterial lectin FimH
lectin FimH [2].
[15]. They differ in their photoactive functional groups, which
are part of the aglycone moiety. Upon irradiation, the aryl azide
The known FimH CRD was taken as the lead structure for the 1 and the diazirine 2 expel nitrogen to yield a reactive nitrene
design of an appropriate photoactive ligand. Inspection of the and a carbene intermediate, respectively. Irradiation of the
available X-ray data clearly shows that α-D-mannosides are benzophenone 3, on the other hand, delivers a reactive triplet
complexed in the CRD with the aglycone moiety sticking out of diradical.
Figure 2: a) Three known photolabile α-D-mannosides that differ in the nature of the photoactive functional group (in red); b) the known bifunctional
mannoside ligand 5 is based on the orthogonally protected glycoamino acid 4 and well suited for photoaffinity labeling.
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Beilstein J. Org. Chem. 2010, 6, 810–822.
In addition, in order to combine a photoactive functional group in a series of tested ligands can be verified with high
with an affinity label within the same mannoside, the ortho- reproducibility. Hence, the inhibitory potencies of the tested
gonally protected glycoamino acid scaffold 4 was synthesized ligands were referenced to an internal standard inhibitor,
and used for the preparation of the biotin-labeled photophore 5, MeMan (inhibitory potency ≡ 1), to deduce relative inhibitory
which is well suited for the streptavidine-based photoaffinity potencies, so-called RIP-values (Table 1). This uniform refer-
labeling, relying on the extraordinary high affinity of the protein encing shows, that all photoactive mannosides surpass the
streptavidine for biotin (Figure 2b) [16].
inhibitory potency of MeMan, and that mannosides 1 and 2 are
more powerful inhibitors than 3 and 5. Consequently, the syn-
thetic photoactive mannosides are suited as ligands for the
Results and Discussion
In order to learn more about the ligand properties of the bacterial lectin FimH. Comparison of the measured IC50-values
previously prepared photoactive mannosides 1, 2, 3, and 5, we with the theoretical docking results, discloses a somewhat
have performed computer-aided docking studies using FlexX limited value of the computer-aided predictions in this case.
[17-19] to get an idea about their binding to the bacterial lectin Docking suggested that mannosides 2, 3 and 5 are the most
FimH, as reported earlier [20]. FlexX produces so-called potent ligands of FimH in the tested series, which was not
scoring values for each docked ligand, which can be regarded as confirmed experimentally. On the other hand, it has to be kept
a rough estimate of its free binding energy. Low (more in mind that the employed ELISA is not based on pure FimH,
negative) scores correlate with high affinities, whilst higher but rather on type 1-fimbriated bacteria, a dissimilar more com-
scores reflect diminished binding potency (Table 1). Docking plex system.
was based on two different X-ray structures. In the first case,
crystals with an open tyrosine gate were taken as the basis [7], The binding studies showed that mannosides with a typical
whilst in the second case, a closed-gate conformation of FimH photolabel in the aglycone moiety serve as ligands for FimH,
was used for the modeling [8]. This led to somewhat different and that even the more complex glycoamino acid derivative 5 is
predictions, as expected, however, the same trends were a suitable ligand. Encouraged by these results, we set out to
revealed for ligands 1–3 and 5 in comparison with the well- improve the photochemical properties of 5 in order to facilitate
known standard ligands methyl α-D-mannoside (MeMan) and later photoaffinity-labeling of FimH. Our earlier work
p-nitrophenyl α-D-mannoside (pNPMan), respectively suggested that diazirines are more useful photoactive groups
(Table 1).
than aryl azides and benzophenones [15]. Therefore, the syn-
thesis of a biotin-labeled daizirine-functionalized mannoside
When these mannosides were tested as inhibitors of type 1 was our next target. In this synthesis, aspartic acid was utilized
fimbriae-mediated bacterial adhesion to a mannan-coated as the scaffold molecule in two different ways in order to allow
surface in an ELISA [21,22], IC50-values were obtained, which fine-tuning of the spacing between the mannoside ligand and
reflect the concentration of the derivative employed, that the photoactive group. Thus, the amino acid derivatives Fmoc-
leads to 50% inhibition of bacterial adhesion. Three Asp-OtBu and Fmoc-Asp(OtBu)-OH were employed in two
independent measurements resulted in high standard deviations, analogous synthetic pathways, starting with peptide coupling to
a typical characteristic of this assay, whereas the relative trend the known 2-aminoethyl mannoside 6 (Scheme 1) [23]. This led
Table 1: Binding of mannoside ligands to FimH was predicted by computer-aided docking (FlexX) and measured by ELISA utilizing type 1-fimbriated
E. coli. IC50-values are averaged from three independent experiments.
mannoside
FlexX Scores
“open-gate”a
FlexX Scores
“closed-gate”b
IC50 (SD)c
[mmolar]
RIPd
MeMan
−22.5
−24.9
−23.6
−28.3e
−30.2
−31.9
−23.3
−27.4
−24.0
−29.3e
−29.8
−36.0
1.84 (1.32)
0.04 (0.01)
0.06 (0.01)
0.12 (0.09)
0.20 (0.10)
0.63 (0.42)
1
46
31
15
9
pNPMan
1
2
3
5
3
abased on open-gate structure of FimH, PDB ID: 1KLF [7].
bbased on closed-gate structure of FimH, PDB ID: 1UWF [8].
cSD: standard deviation.
dRIP: relative inhibitory potency.
eTo facilitate docking, the diazirine ring was substituted by a cyclopropyl ring.
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Beilstein J. Org. Chem. 2010, 6, 810–822.
Scheme 1: Synthesis of photoactive glycoamino acids 11 and 12. i) Fmoc-Asp-OtBu (for 7), Fmoc-Asp(OtBu)-OH (for 8), HATU, DIPEA, dry DMF,
N2, RT, 95% for 7 and 96% for 8; ii) 80% aq TFA, RT; iii) (+)-biotinylamidopropylammonium trifluoroacetate, HATU, DIPEA, dry DMF, N2, RT, 48%
(two steps) for 9 and 26% (two steps) for 10; (iv) 20% piperidine, dry DMF, RT; v) HATU, DIPEA, dry DMF, Ar, RT, 81% (two steps) for 12 and 98%
(two steps) for 13.
to the orthogonally protected mannoside amino acid tert-butyl first employed. It was irradiated with the three different
esters 7 and 8. The tert-butyl ester groups were then cleaved diazirine-functionalized mannosides 2, 12, and 13. From our
under acidic conditions and the resulting acids ligated with the earlier work [15] it was known that irradiation of the diazirine-
biotin derivative biotinylamidopropylammonium trifluoro- functionalized mannoside 2 with angiotensin II led to a photo-
acetate. These two steps gave the Fmoc-protected biotin-labeled crosslinked product with m/z = 698.82. This double positively
glycoamino acids 9 and 10, respectively. Fmoc-cleavage and charged ion correlates with a 1:1-photoaddition product of
peptide coupling with the diazirine 11 led to the target mole- peptide and photolabel with the molecular formula
cules 12 and 13 in good yield.
C65H90F3N13O182+ and a monoisotopic mass of 1397.47 Da.
We have now carried out MS/MS experiments to analyze the
To test the prepared photoactive mannosides in crosslinking structure of this photo-crosslinked adduct and unequivocally
reactions, the model peptide angiotensin II (DRVYIHPF) was shown that insertion of the carbene, which results after irradi-
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Beilstein J. Org. Chem. 2010, 6, 810–822.
ation of 2, occurs at the hydroxyl group in the side chain of the The photo-crosslinking experiments with angiotensin II demon-
angiotensin tyrosine (Y) (see Experimental Part). This strated, that the synthesized diazirines are well suited as photo-
crosslinking reaction leads to the ether 14 (Scheme 2). Analo- probes for the labeling of this peptide, with a preference for the
gously, the photo-crosslinked products, obtained from irradi- tyrosine side chain. Thus, the photoactive mannosides were next
ation of angiotensin II with either 12 or 13, can be correlated investigated with the bacterial lectin FimH. For the irradiation
with the structures of 15 and 16, respectively, both showing a experiments a FimH truncate, FimHtr, which resembles the
peak at m/z = 1851.1 in their mass spectra, corresponding to a adhesin domain of the complete FimH, was used [24]. FimHtr
molecular formula of C84H119F3N19O23S+ (monoisotopic mass comprises of amino acids 1-160 of FimH and is terminated by a
M = 1850.84 Da).
histidine tag His6. FimHtr has the same carbohydrate binding
Scheme 2: Photo-crosslinking experiments with the model peptide angiotensin II and the photoactive mannosides 2, 12, and 13, respectively (see
Experimental Part).
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Beilstein J. Org. Chem. 2010, 6, 810–822.
properties as FimH. Mass spectrometric analysis of FimHtr
revealed m/z = 17839 (calcd. m/z = 17845). Solutions of FimHtr
and the photophores 2, 12, and 13 were applied in a ratio of 5:1.
Samples were incubated at 37 °C to allow formation of the
lectin-ligand complex and then irradiated at λ ≥ 320 nm. This
led to 1:1-photo-crosslinked products with the corresponding
masses (Table 2).
In addition to the mass spectrometric analysis, dot–blots were
performed with FimHtr and the photoactive mannosides.
Affinity staining was carried out with a streptavidine–HRP
conjugate and the chromogene 3,3’-diaminobenzidine (DAB).
The biotin-labeled mannosides 12 and 13 gave violet spots on
the nitrocellulose membrane when tested. Affino dot–blot with
mannoside 2, that does not contain a biotin moiety, was
negative, as predicted. In addition, control experiments were
carried out, leading to the expected results in all cases.
Interestingly, photoaffinity probes 12 and 13 seem to exhibit
unequal affinity to FimHtr as suggested by the different inten-
sity of color of the respective precipitates (Figure 3). Affinity
staining using western blots led to analogous results (not
shown).
Figure 3: Affino dot–blot with FimHtr and photoactive mannosides
applied to nitrocellulose disks. It was irradiated, incubated with strepta-
vidine–HRP conjugate and stained by addition of the chromogene 3,3’-
diaminobenzidine. Photoaffinity probes 12 (lane 4) and 13 (lane 2) led
to different color intensities, suggesting that ligand 12 is bound more
tightly to FimHtr than ligand 13.
Conclusion
In conclusion, we have demonstrated the synthesis of new
biotin-labeled photoactive mannosides for photoaffinity-
labeling of the bacterial lectin FimH. The target molecules 12
and 13 were selected after docking studies based on the struc-
ture of FimH and according to binding studies employing type
1-fimbriated E. coli. Photo-crosslinking was tested with the
model peptide angiotensin II and the regiochemistry of the
insertion reaction could be solved by MS/MS studies. Further-
more, photoaffinity-labeling of FimHtr was successful and
could be demonstrated by mass spectrometric studies as well as
dot–blot analysis.
The overall goal of this study is to identify mannose binding
sites on the bacterial lectin FimH in solution. After photo-
crosslinking of the lectin with photoactive, biotin-labeled
mannosidic ligands, proteolytic digestion of the products of
photo-crosslinking, followed by affinity chromatography and
mass-spectrometric analysis of the fragments, should allow the
identification of the critical amino acid residues on FimH,
according to the photoaffinity methodology. However, so far we
Table 2: Results of mass spectrometric analysis of photo-crosslinked products, obtained from irradiation of FimHtr with photoactive mannoside
ligands 2, 12, and 13.
FimHtra
incubated with
FimHtrb
measured mass
m/z
crosslinked product
measured mass
m/z
Δ mass
m/z (FimHtr) - m/z
(crosslinked product)
Δ mass
expected
2
17858 [M + H]+
17905 [M + H]+
17697 [M + H]+
18200c [M + H]+
18672d [M + H]+
18500d [M + H]+
342
767
803
350e
804f
804f
12
13
aFor control experiments FimHtr was irradiated similarly without addition of photophore. Mass spectrometric analysis revealed that the protein FimH
survived the conditions of irradiation without any damage.
bCalcd. mass M = 17845 Da; measured values are acceptable within accuracy of the measurement.
cMeasured on 4700 Proteomics Analyzer (Applied Biosystems).
dMeasured on Bruker Biflex III.
eBased on the carbene resulting from irradiation of 2: C15H17F3O6 (M = 350 Da); measured Δm-values acceptable within accuracy of the measure-
ment.
fBased on the carbene resulting from irradiation of 12 and 13, respectively: C34H47F3N6O11S (M = 804 Da); measured Δm-values acceptable within
accuracy of the measurement.
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Beilstein J. Org. Chem. 2010, 6, 810–822.
have not been successful in an unequivocal mass-spectrometric sulfuric acid in ethanol or using anisaldehyde and subsequent
analysis of any proteolytic digest, we have obtained so far. heating. Flash column chromatography was performed on silica
Thus, this study is currently continued in our laboratory.
gel 60 (40–63 µm, Merck) and for RP-MPLC a Merck Licro-
prep RP-18 column (Büchi) was used. Preparative HPLC was
accomplished on a Shimadzu LC-8a machine (LiChrosorb
RP-8, HIBAR). NMR spectra were recorded on Bruker AMX
Experimental
Docking studies
Computer-aided modeling to predict binding of the various 400, Bruker DRX 500 or Bruker Avance 600 instruments.
FimH ligands was carried out using FlexX flexible docking and Chemical shifts are relative to TMS or the solvent peaks of
consensus scoring as implemented in Sybyl 6.8 as described CDCl3 (7.24 ppm for 1H, 77.0 ppm for 13C) or MeOD
earlier [20]. Docking was based on published X-ray structures (3.35 ppm and 4.78 ppm for 1H, 49.3 ppm for 13C). Where
of the FimH CRD. This CRD was held fixed during the mini- necessary, assignments were based on 2D experiments (COSY,
mization, whereas the sugar ligand was allowed to change its HSQC, HMBC or NOESY). IR spectra were taken with a
conformation freely under the influence of the force field.
Perkin Elmer FT IR Paragon 1000 (KBr). Optical rotations were
measured with a Perkin-Elmer polarimeter (22 °C, 589 nm,
length of cuvette: 1 dm). For MS analysis of the synthetic prod-
ELISA
ELISAs to determine IC50-values of the various FimH ligands ucts MALDI-TOF mass spectra were measured with a Bruker
were carried out with E. coli bacteria of strain HB101pPKL4 Biflex III with 19 kV acceleration voltage. 4-Hydroxy-α-
and mannan-coated microtiter plates as described earlier cyanocinnamic acid (HCCA) was used as matrix, either as a
[21,22].
saturated solution in a solvent mixture (33% MeCN/ double
distilled water and 0.1% TFA) or as saturated solution in
acetone. Ionisation was effected with a nitrogen laser at 337 nm.
Mass spectrometry
For mass spectrometric analyses of the photo-crosslinked prod- ESI-MS spectra of the synthesized derivatives were measured
ucts, a Bruker Biflex III instrument (MALDI-TOF-MS, Prof. with an Applied Biosystems Mariner ESI-TOF 5280 and milli-
Th. K. Lindhorst, CAU), or a Bruker Biflex II instrument (ESI- pore C18-pipette tips were used for ZipTipping®.
FT-ICR-MS/MS, group of PD Dr. B. Lindner at the Research
Center Borstel), or the 4700 Proteomics Analyzer mass N-(Fluoren-9-ylmethoxycarbonyl)-4-[2-(α-D-manno-
spectrometer (Applied Biosystems MALDI-TOF/TOF-MS, pyranosyloxy)ethylamido]-L-aspartic acid tert-butyl
group of Prof. M. Leippe, CAU) were used. Mass spectra were ester (7)
acquired using standard experimental sequences as provided by The aminoethyl mannoside 6 (200 mg, 0.90 mmol), HATU
the manufacturer. Analysis of photo-crosslinking with (320 mg, 0.90 mmol) and Fmoc-Asp-OtBu (400 mg,
angiotensin II is exemplified with diazirine 2 and presented in 0.98 mmol) were dried under vacuum for 10 min and then
Figure 4.
dissolved in dry DMF (12 mL) under a nitrogen atmosphere.
DIPEA (300 μL, 2.25 mmol) was added and the reaction mix-
ture stirred for 18 h at RT. The solvent was removed in vacuo
FimH truncate
The amino acid sequence of the FimH truncate [24], FimHtr, and the residue purified by flash chromatography (ethyl
used in the photoaffinity labeling studies is as follows:
acetate:MeOH:H2O = 6:2:1). Pure product fractions were
pooled, filtered, concentrated and the residual taken up in water.
FACKTANGT AIPIGGGSAN VYVNLAPVVN VGQN- Lyophylisation gave the title compound (531 mg, 0.86 mmol,
LVVDLS TQIFCHNDYP ETITDYVTLQ RGSAYGGVLS 95%); Rf = 0.86 (ethyl acetate:MeOH:H2O = 6:2:1).
NFSGTVKYSG SSYPFPTTSE TPRVVYNSRT DKPWP-
VALYL TPVSSAGGVA IKAGSLIAVL ILRQTNNYNS 1H NMR (600 MHz, D4-MeOH): δ = 7.84 (d, 2H, J = 7.6 Hz,
DDFQFVWNIY ANNDVVVPT GGHHHHHH
aryl-Fmoc), 7.71 (d, 2H J = 7.5 Hz, aryl-Fmoc), 7.43 (t, 2H, J =
7.5 Hz, aryl-Fmoc), 7.35 (t, 2H, J = 7.5 Hz, aryl-Fmoc), 4.81 (d,
1H, J = 1.5 Hz, H-1man), 4.50 (dd, 1H, J = 7.2 Hz, J = 5.6 Hz,
Affino dot–blot
Samples (2 μL, 1.15 mmolar) were applied on a nitrocellulose Hα-asp), 4.40 (dd, 1H, J = 10.5 Hz, J = 7.2 Hz, Fmoc-CHH),
membrane, incubated with streptavidine-HRP conjugate and 4.35 (dd, 1H, J = 10.5 Hz, J = 7.0 Hz, Fmoc-CHH), 4.28 (t, 1H,
stained with 3,3’-diaminobenzidine.
J = 7.0 Hz, Fmoc-CH-CH2), 3.88 (dd, 1H, J = 11.6 Hz, J =
2.2 Hz, manOCH2CHH), 3.87 (dd, 1H, J = 3.5 Hz, J = 1.8 Hz,
H-2man), 3.79 (dd, 1H, J = 10.5 Hz, J = 4.5 Hz,
Methods and materials for synthesis
Reactions were monitored by TLC on silica gel GF254 (Merck) manOCHHCH2), 3.75 (dd, 1H, J = 11.7 Hz, J = 5.9 Hz,
with detection under UV light and by charring with 10% manOCH2CHH), 3.74 (dd, 1H, J = 9.3 Hz, J = 3.4 Hz,
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Beilstein J. Org. Chem. 2010, 6, 810–822.
Figure 4: MS/MS spectra of angiotensin II (a) and of angiotensin II, photo-crosslinked with diazirine 2 (b), recorded on 4700 Proteomics Analyzer
mass spectrometer (Applied Biosystems); (c) fragments of angiotensin II and photo-crosslinked angiotensin II according to Biemann’s nomenclature
(Table 3).
H-3man), 3.65 (t, 1H, J = 9.5 Hz, H-4man), 3.58 (ddd, 1H, J = 170.92 (Fmoc-C=O), 170.63 (OtBu-C=O), 156.92 (C-γ),
9.7 Hz, J = 5.7 Hz, J = 2.1 Hz, H-5man), 3.57 (dd, 1H, J = 143.81, 141.15, 127.40, 126.79, 124.88, 119.54 (aryl-Fmoc),
10.2 Hz, J = 4.3 Hz, manOCHHCH2), 3.47 (dd, 1H, J = 100.28 (C-1man), 81.63 (OC(CH3)3), 74.78 (C-5man), 72.53
14.2 Hz, J = 6.2 Hz, H-6man), 3.04 (dd, 1H, J = 14.2 Hz, J = (C-3man), 72.05 (C-2man), 68.64 (C-4man), 68.12 (Fmoc-
4.7 Hz, H-6’man), 2.78 (dd, 1H, J = 15.1 Hz, J = 5.4 Hz, CH2), 67.17 (manOCH2CH2), 66.71 (manOCH2CH2), 51.63
Hβ-asp), 2.69 (dd, 1H, J = 15.2 Hz, J = 7.3 Hz, Hβ’-asp), 1.49 (C-α), 48.30 (CH-Fmoc), 40.35 (C-6), 38.55 (C-β), 28.21
(s, 9H, OtBu) ppm; 13C NMR (150.9 MHz, D4-MeOH): δ = (OC(CH3)3) ppm.
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Beilstein J. Org. Chem. 2010, 6, 810–822.
Table 3: Assignment of peptide fragment ion pattern according to Biemann [25]; left two columns: angiotensin; right two columns photo-crosslinked
product of angiotensin and diazirine 2 (Figure 4).
MS-MS experiment with
angiotensin II
m/z
corresponding
fragment
photo-crosslinking of
angiotensin II and 2;
MS-MS experiment
m/z
corresponding
fragment
1046.90
931.59
899.56
881.54
802.48
784.48
767.47
756.48
619.41
534.32
517.28
400.23
354.21
343.25
272.18
263.16
255.15
235.12
166.10
136.11
110.10
87.11
[MH]+
1397.07
1281.47
1046.68
969.50
[MH]+
y7
[MH – D]+
b7 – H2O
[MH – carbene]+
[a5 + carbene]
b7
b6 – H2O
b6
784.47
756.45
619.40
517.30
486.54
400.64
b6
b6 – NH3
a6
a5
a6
a5
b4 – NH3
[Y + carbene]
y3
b4
b4 – NH3
y3
b3 – NH3
354.21
343.26
b3 – NH3
a3
a3
b2
y2
263.17
255.15
y2
b2 – NH3
b2 – NH3
HP
y1
Y
136.10
110.11
72.12
Y
H
V
P
H
R
70.09
P
70.01
MALDI-TOF-MS: m/z = 639.2 [M + Na]+, 656.2 [M + K]+; 7.42 (dd, 2H, J = 7.5 Hz, J = 3.4 Hz, aryl-Fmoc), 7.34 (dd, 2H,
ESI-MS: m/z = 639.26 [M + Na]+ (616.66 calcd. for J = 6.9 Hz, J = 1.1 Hz, aryl-Fmoc), 4.81 (d, 1H, J = 1.4 Hz,
C31H40N2O11).
H-1man), 4.52 (dd, 1H, J = 8.7 Hz, J = 5.2 Hz, Hα-asp), 4.48
(dd, 1H, J = 10.5 Hz, J = 7.0 Hz, Fmoc-CHH), 4.39 (dd, 1H, J =
10.7 Hz, J = 6.6 Hz, Fmoc-CHH), 4.27 (t, 1H, J = 7.0 Hz,
Fmoc-CH), 3.88 (dd, 1H, J = 12.1 Hz, J = 2.2 Hz,
manOCH2CHH), 3.86 (dd, 1H, J = 1.4 Hz, J = 3.0 Hz,
N-(Fluoren-9-ylmethoxycarbonyl)-4-(tert-butyl
ester)-L-aspartic acid [2-(α-D-mannopyranosyloxy)
ethyl]-amide (8)
The aminoethyl mannoside 6 (200 mg, 0.90 mmol), HATU H-2man), 3.79 (dd, 1H, J = 10.7 Hz, J = 6.4 Hz,
(320 mg, 0.90 mmol) and Fmoc- Fmoc-Asp(OtBu)-OH manOCHHCH2), 3.73 (dd, 1H, J = 8.8 Hz, J = 3.0 Hz,
(400 mg, 0.98 mmol) were dried under vacuum for 10 min and H-3man), 3.75 (dd, 1H, J = 11.8 Hz, J = 5.8 Hz,
then dissolved in dry DMF (12 mL) under a nitrogen manOCH2CHH) 3.65 (t, 1H, J = 9.6 Hz, H-4man), 3.57 (ddd,
atmosphere. DIPEA (300 μL, 2.25 mmol) was added and the 1H, J = 10.3 Hz, J = 7.1 Hz, J = 4.7 Hz, H-5man), 3.56 (dd, 1H,
reaction mixture stirred for 18 h at RT. The solvent was J = 10.8 Hz, J = 5.8 Hz, manOCHHCH2), 3.48 (dd, 1H, J =
removed in vacuo and the residue purified by flash chromatog- 13.9 Hz, J = 7.6 Hz, H-6man), 3.42 (dd, 1H, J = 13.9 Hz, J =
raphy (ethyl acetate:MeOH:H2O = 6:2:1). Pure product 5.0 Hz, H-6’man), 2.82 (dd, 1H, J = 16.1 Hz, J = 5.1 Hz,
fractions were pooled, filtered, concentrated and the residue Hβ-asp), 2.60 (dd, 1H, J = 16.1 Hz, J = 8.9 Hz, Hβ’-asp), 1.47
taken up in water. Lyophylisation gave the title compound (536 (s, 9H, OC(CH3)3) ppm; 13C NMR (150.92 MHz, D4-MeOH): δ
mg, 0.87 mmol, 96%); Rf = 0.75 (ethyl acetate:MeOH:H2O = = 173.48 (OtBu-C=O), 172.48 (Fmoc-C=O), 171.39 (NH-
6:2:1).
(C=O)asp), 145.22, 142.59, 128.79, 128.17, 126.23, 120.91
(aryl-Fmoc), 101.64 (C-1man), 82.43 (OC(CH3)3), 74.67
1H NMR (500 MHz, D4-MeOH): δ = 7.82 (dd, 2H, J = 7.6 Hz, (C-5man), 72.52 (C-3man), 72.05 (C-2man), 68.67 (C-4man),
J = 3.5 Hz, aryl-Fmoc), 7.71 (t, 2H, J = 7.7 Hz, aryl-Fmoc), 68.19 (Fmoc-CH2), 67.06 (manOCH2CH2), 62.84
818

Beilstein J. Org. Chem. 2010, 6, 810–822.
(manOCH2CH2), 53.32 (C-α), 48.34 (CH-Fmoc), 40.42 13.9 Hz, J = 7.8 Hz, HN-CHH-CH2-CH2-NH), 2.56 (dd, 1H, J
(C-6man), 38.74 (C-β), 28.33 (C(CH3)3) ppm.
= 14.2 Hz, J = 7.2 Hz, HN-CH2-CH2-CHH-NH), 2.53 (dd, 1H,
J = 14.0 Hz, J = 6.8 Hz, HN-CH2-CH2-CHH-NH), 2.15 (t, 1H,
MALDI-TOF-MS: m/z = 639.2 [M + Na]+; ESI-MS: m/z = J = 7.3 Hz, CH-Fmoc), 2.13 (d, 1H, J = 6.8 Hz, Hβ-asp), 2.11
639.26 [M + Na]+ (616.66 calcd. for C31H40N2O11).
(d, 1H, J = 6.5 Hz, Hβ’-asp), 1.66 (dd, 1H, J = 14.3 Hz, J =
7.6 Hz, NH(C=O)CH2-CH2-CH2-CHH-biotin), 1.64 (dd, 1H, J
= 13.9 Hz, J = 7.0 Hz, HN-CH2-CHH-CH2-NH), 1.62 (dd, 1H,
J = 14.0 Hz, J = 7.2 Hz, HN-CH2-CHH-CH2-NH), 1.59 (dd,
1H, J = 14.5 Hz, J = 7.0 Hz, NH(C=O)CH2-CH2-CH2-CHH-
N-(Fluoren-9-ylmethoxycarbonyl)-4-[2-(α-D-manno-
pyranosyloxy)ethyl]-1-[(+)-biotinylamidopropyl]-L-
aspartic acid diamide (9)
The glycoamino acid 7 (220 mg, 0.36 mmol) was dissolved in biotin), 1.54 (dd, 1H, J = 15.3 Hz, J = 8.0 Hz, NH(C=O)CH2-
80% aq TFA (5 mL) and stirred at RT for 60 min to cleave the CH2-CHH-CH2-biotin), 1.49 (dd, 1H, J = 15.3 Hz, J = 8.1 Hz,
tert-butyl ester. When the deprotection reaction was complete NH(C=O)CH2-CH2-CHH-CH2-biotin), 1.33 (dd, 1H, J =
(TLC control in ethyl acetate:MeOH:H2O = 6:2:1) the solvent 15.2 Hz, J = 7.5 Hz, NH(C=O)CH2-CHH-CH2-CH2-biotin),
was removed in vacuo and the residue suspended in MeOH, 1.28 (dd, 1H, J = 15.0 Hz, J = 7.3 Hz, NH(C=O)CH2-CHH-
filtered and the filtrate concentrated under reduced pressure. CH2-CH2-biotin) ppm; 13C NMR (125.75 MHz; CD3CN:D2O =
The resulting crude product was combined with HATU 1:1): δ = 180.86 (C-γ), 176.52 (Casp(O)-NH-propyl), 173.26
(150 mg, 0.43 mmol), (+)-biotinylamidopropylammonium tri- (Cbiotin(O)-NH-propyl), 172.17 ((NH)2C=O), 165.62 (Fmoc-
fluoroacetate (170 mg, 0.41 mmol) and dried for 30 min under C=O), 144.81, 142.0, 128.93, 128.36, 126.25 (aryl-Fmoc),
vacuum. DMF (5 mL) and DIPEA (350 μL, 2.63 mmol) were 100.73 (C-1man), 73.82 (C-5man), 71.68 (C-2man), 71.12
then added under a nitrogen atmosphere. The reaction mixture (C-3man), 67.86 (Fmoc-CH2), 67.69 (C-4man), 66.75
was stirred overnight at RT, the solvent removed in vacuo and (manOCH2CH2), 63.0 (biotin-NHCHCHalkyl), 62.0 (C-6man),
the residue purified by HPLC (MeCN-H2O gradient: 0–5 min 61.07 (biotin-NHCHCH2S), 56.30 (biotin-NHCHCHalkyl),
100% H2O, 5–20 min 60% H2O, 20–30 min 40% H2O, 53.14 (C-α) 47.81 (CH-Fmoc), 40.79 (biotin-CH2), 39.89
30–40 min 0% H2O, 40–50 min 0% H2O, 50–60 min (manOCH2CH2), 38.45, 37.64 (HN-CH2-CH2-CH2-NH), 37.59
100% H2O). The title compound was obtained after lyophyl- (C(O)-CH2-CH2-CH2-CH2-biotin), 36.52 (C-β), 29.29 (HN-
isation (145 mg, 0.17 mmol, 48%); Rf = 0.47 (ethyl CH2-CH2-CH2-NH), 29.09 (C(O)-CH2-CH2-CH2-CH2-biotin),
acetate:MeOH:H2O = 6:2:1).
28.80 (C(O)-CH2-CH2-CH2-CH2-biotin), 26.27 (C(O)-CH2-
CH2-CH2-CH2-biotin) ppm.
1H NMR (500 MHz, CD3CN:D2O = 1:1): δ = 7.85 (d, 2H, J =
7.6 Hz, aryl-Fmoc), 7.71 (dd, 2H, J = 6.8 Hz, J = 4.2 Hz, aryl- MALDI-TOF-MS: m/z = 843.5 [M + H]+, 865.4 [M + Na]+,
Fmoc), 7.45 (t, 2H, J = 7.4 Hz, Fmoc-H), 7.37 (t, 2H, J = 881.4 [M + K]+, (842.96 calcd. for C40H54N6O12S);
7.5 Hz, J = 4.1 Hz, Fmoc-H), 4.80 (d, 1H, J = 1.6 Hz, H-1man), ESI-MS: m/z = 865.44 [M + Na]+ (842.96 calcd. for
4.51 (dd, 1H, J = 7.6 Hz, J = 5.8 Hz, biotin-NHCHCH2S), 4.42 C40H54N6O12S).
(dd, 1H, J = 11.8 Hz, J = 6.8 Hz, manOCHHCH2), 4.38 (dd,
1H, J = 11.8 Hz, J = 7.3 Hz, manOCHHCH2), 4.27 (d, 1H, J = N-(Fluoren-9-ylmethoxycarbonyl)-4-[(+)-biotinylami-
7.2 Hz, biotin-NHCHCHalkyl), 4.24 (t, 1H, J = 6.8 Hz, dopropyl]-1-[2-(α-D-mannopyranosyloxy)ethyl]-L-
Hα-asp), 3.86 (dd, 1H, J = 3.4 Hz, J = 1.7 Hz, H-2man), 3.77 aspartic acid diamide (10)
(dd, 1H, J = 12.1 Hz, J = 2.2 Hz, H-6man), 3.69 (dd, 1H, J = The glycoamino acid 8 (250 mg, 0.41 mmol) was dissolved in
9.6 Hz, J = 3.9 Hz. H-3man), 3.68 (dd, 1H, J = 12.2 Hz, J = 80% aq TFA (6 mL) and stirred at RT for 60 min to cleave the
3.6 Hz, H-6’man), 3.65 (dd, 1H, J = 11.9 Hz, J = 6.9 Hz, Fmoc- tert-butyl ester. When the deprotection reaction was complete
CHH), 3.59 (t, 1H, J = 9.6 Hz, H-4man), 3.53 (ddd, 1H, J = (TLC control in ethyl acetate:MeOH:H2O = 6:2:1) the solvent
9.9 Hz, J = 5.5 Hz, J = 2.2 Hz, H-5man), 3.50 (dd, 1H, J = was removed in vacuo and the residue suspended in MeOH,
11.2 Hz, J = 6.7 Hz, Fmoc-CHH), 3.41 (dd, 1H, J = 14.1 Hz, J filtered and the filtrate concentrated under reduced pressure.
= 5.3 Hz, manOCH2CHH), 3.28 (ddd, 1H, J = 14.2 Hz, J = The resulting crude product was combined with HATU
5.7 Hz, manOCH2CHH), 3.16 (dd, 1H, J = 9.3 Hz, J = 6.9 Hz, (98.1 mg, 0.28 mmol) and (+)-biotinylamidopropylammonium
NH(C=O)CHH-CH2-CH2-CH2-biotin), 3.15 (t, 1H, J = 7.4 Hz, trifluoroacetate (109 mg, 0.26 mmol), and dried for 30 min
biotin-NHCHCHalkyl), 3.12 (dd, 1H, J = 9.5 Hz, J = 6.6 Hz, under vacuum. DMF (4 mL) and DIPEA (220 μL, 1.65 mmol)
NH(C=O)CHH-CH2-CH2-CH2-biotin), 2.87 (dd, 1H, J = 12.8 were then added under a nitrogen atmosphere. The reaction
Hz, J = 5.1 Hz, biotin-NHCHCHHS), 2.69 (dd, 1H, J = 12.9 mixture was stirred overnight at RT, the solvent removed in
Hz, J = 4.9 Hz, biotin-NHCHCHHS), 2.67 (dd, 1H, J = 13.7 vacuo and the residue purified by HPLC (MeCN-H2O gradient:
Hz, J = 7.5 Hz, HN-CHH-CH2-CH2-NH), 2.59 (dd, 1H, J = 0–5 min 100% H2O, 5–20 min 60% H2O, 20–30 min 40% H2O,
819

Beilstein J. Org. Chem. 2010, 6, 810–822.
30–40 min 0% H2O, 40–50 min 0% H2O, 50–60 min N-[p-(Trifluoromethyl-diazirinyl)-benzoyl]-4-[2-(α-D-
100% H2O). The title compound was obtained after lyophyl- mannopyranosyloxy)ethyl]-1-[(+)-biotinylamido-
isation (90.4 mg, 0.11 mmol, 26%); Rf = 0.54 (ethyl propyl]-L-aspartic acid diamide (12)
acetate:MeOH:H2O = 6:2:1).
The glycoamino acid derivative 9 (25.0 mg, 29.7 μmol)
was dissolved in piperidine (20% in DMF, 3.0 mL) and stirred
1H NMR (600 MHz, CD3CN:D2O, 1:1): δ = 7.85 (d, 2H, J = at RT for 60 min to remove the Fmoc protecting
7.6 Hz, Fmoc-H), 7.67 (d, 2H, J = 7.5 Hz, Fmoc-H), 7.45 (t, group. The solvent was removed in vacuo and the resulting
2H, J = 7.4 Hz, Fmoc-H), 7.38 (td, 2H, J = 7.5 Hz, J = 0.9 Hz, crude product combined with HATU (11.3 mg, 29.7 μmol)
Fmoc-H), 4.77 (d, 1H, J = 1.7 Hz, H-1man), 4.45 (dd, 1H, J = and the diazirine 11 (7.2 mg, 31.0 μmol), and dry
6.7 Hz, J = 4.5 Hz, biotin-NHCHCH2S), 4.43 (t, 1H, J = 6.8 Hz, DMF (1.5 mL) added under a nitrogen atmosphere. DIPEA
Hα-asp), 4.40 (dd, 1H, J = 12.2 Hz, J = 5.5 Hz, Fmoc-CHH), (0.03 mL, 87.0 μmol) was then added and the reaction mixture
4.37 (dd, 1H, J = 12.0 Hz, J = 5.3 Hz, Fmoc-CHH), 4.29 (dd, stirred overnight at RT. The solvent was removed under
1H, J = 5.5 Hz, J = 4.4 Hz, CH-Fmoc), 4.27 (dd, 1H, J = reduced pressure and the residue purified by flash chromatog-
6.5 Hz, J = 4.7 Hz, biotin-NHCHCHalkyl), 3.83 (dd, 1H, J = raphy with the exclusion of light (ethyl acetate:MeOH:H2O =
3.2 Hz, J = 1.6 Hz, H-2man), 3.77 (dd, 1H, J = 12.2 Hz, J = 6:2:1) to yield, after lyophilisation, the title compound (20.1
6.5 Hz, manOCHHCH2), 3.69 (dd, 1H, J = 9.7 Hz, J = 3.2 Hz, mg, 24.1 μmol, 81%); Rf = 0.51; [α]D20 +23.0 (c 0.12 mM,
H-3man), 3.66 (dd, 1H, J = 12.1 Hz, J = 6.9 Hz, MeCN:H2O = 1:1); UV–Vis (c 0.12 mM, MeCN:H2O = 1:1):
manOCHHCH2), 3.64 (dd, 1H, J = 12.0 Hz, J = 5.3 Hz, λmax(1) = 353.3 nm, ε(1) = 6000 Lmol−1cm−1; λmax(2) = 303.5
H-6man), 3.58 (t, 1H, J = 9.8 Hz, H-4man), 3.51 (ddd~dd, 1H, J nm, ε(2) = 10000 Lmol−1cm−1; λmax(3) = 285.2 nm, ε(3) =
= 9.8 Hz, J = 5.5 Hz, H-5man), 3.50 (dd, 1H, J = 11.9 Hz, J = 24000 Lmol−1cm−1; λmax(4) = 251.6 nm, ε(4) = 37000
5.6 Hz, H-6’man), 3.43 (dd, 1H, J = 14.4 Hz, J = 6.5 Hz, Lmol−1cm−1; FT-IR (KBr): = 3447.5 cm−1, 2928.6 cm−1,
manOCH2CHH), 3.33 (dd, 1H, J = 13.9 Hz, J = 6.1 Hz, 2381.0 cm−1, 1654.2 cm−1, 1560.1 cm−1, 1267.9 cm−1,
manOCH2CHH), 3.17 (td, 1H, J = 7.1 Hz, J = 6.9 Hz, biotin- 1136.9 cm−1, 1101.9 cm−1, 1059.5 cm−1, 767.9 cm−1.
NHCHCHalkyl), 3.12, 3.10 (each dd, each 1H, HN-CH2-CH2-
CH2-NH), 3.09, 3.07 (each dd, each 1H, NH(C=O)CH2-CH2- 1H NMR (600 MHz, CD3CN:D2O = 1:1): δ = 7.90 (dt, 2H, J =
CH2-CH2-biotin), 2.66, 2.54 (each dd, each 1H, HN-CH2-CH2- 8.7 Hz, J = 2.0 Hz, aryl-H), 7.39 (d, 2H, J = 8.2 Hz, aryl-H),
CH2-NH), 2.11, 2.09 (each dd, each 1H, 2Hβ-asp), 1.61, 1.58 4.81 (dd, 1H, J = 7.7 Hz, J = 6.1 Hz, Hα-asp), 4.75 (d, 1H, J =
(each dd, each 1H, HN-CH2-CH2-CH2-NH), 1.52 (dd, 1H, J = 1.6 Hz, H-1man), 4.50 (dd, 1H, J = 7.9 Hz, J = 4.9 Hz, biotin-
13.9 Hz, J = 6.7 Hz, NH(C=O)CH2-CH2-CH2-CHH-biotin), NH2CHCHalkyl), 4.31 (dd, 1H, J = 7.9 Hz, J = 4.5 Hz, biotin-
1.50 (dd, 1H, J = 14.0 Hz, J = 6.8 Hz, NH(C=O)CH2-CH2- NH2CHCH2S), 3.82 (dd, 1H, J = 3.4 Hz, J = 1.7 Hz, H-2man),
CHH-CH2-biotin), 1.48 (dd, 1H, J = 13.9 Hz, J = 7.2 Hz, 3.76 (dd, 1H, J = 12.1 Hz, J = 2.2 Hz, manOCHHCH2), 3.69
NH(C=O)CH2-CH2-CH2-CHH-biotin), 1.45 (dd, 1H, J = (dd, 1H, J = 9.3 Hz, J = 3.4 Hz, H-3man), 3.67 (dd, 1H, J =
14.2 Hz, J = 7.0 Hz, NH(C=O)CH2-CH2-CHH-CH2-biotin), 12.0 Hz, J = 2.4 Hz, manOCHHCH2), 3.58 (t, 1H, J = 9.8 Hz,
1.30, 1.27 (each dd, each 1H, NH(C=O)CH2-CH2-CH2-CH2- H-4man), 3.52 (ddd, 1H, J = 9.8 Hz, J = 5.3 Hz, J = 2.2 Hz,
biotin) ppm; 13C NMR (150.92 MHz, CD3CN:D2O = 1:1): δ = H-5man), 3.49 (dd, 1H, J = 5.5 Hz, J = 4.2 Hz, H-6man), 3.42
175.93 (NH-(C=O)asp), 172.84 (NH-C(O)-(CH2)4-biotin), (dd, 1H, J = 5.7 Hz, J = 2.3 Hz, H-6’man), 3.39 (dd, 1H, J =
171.53 (C-γ), 165.08 ((NH2)2C=O), 157.25 (Fmoc-C=O), 6.7 Hz, J = 12.3 Hz, manOCH2CHH), 3.30 (dd, 1H, J = 6.4 Hz,
144.24, 144.19, 129.51, 128.51, 128.40, 127.83 (aryl-Fmoc), J = 12.3 Hz, manOCH2CHH), 3.27, 3.24 (each dd, each 1H,
100.1 (C-1man), 73.22 (C-5man), 71.12 (C-3man), 68.07 HN-CH2-CH2-CH2-NH), 3.20 (dd, 1H, J = 7.7 Hz, J = 6.8 Hz,
(C-2man), 67.38 (Fmoc-CH2), 67.13 (C-4man), 66.01 biotin-NH2CHCHalkyl), 3.13, 3.12 (each dd, each 1H,
(C-6man), 62.25 (biotin-NHCHCHalkyl), 61.36 NH(C=O)CH2-CH2-CH2-CH2-biotin), 2.91 (dd, 1H, J =
(manOCH2CH2), 60.50 (biotin-NHCHCH2S), 57.67 (biotin- 13.0 Hz, J = 5.0 Hz, biotin-NH2CHCHHS), 2.82, 2.72 (each dd,
NHCHCHalkyl), 52.61 (C-α), 47.22 (Fmoc-CH), 39.35 (biotin- each 1H, HN-CH2-CH2-CH2-NH), 2.70 (dd, 1H, J = 13.1 Hz, J
NHCHCH2S), 38.36 (manOCH2CH2), 37.07, 36.84 (HN-CH2- = 6.4 Hz, biotin-NH2CHCHHS), 2.16, 2.15 (each dd, each 1H,
CH2-CH2-NH), 35.95 (C(O)-CH2-CH2-CH2-CH2-biotin), 28.69 NH(C=O)CH2-CH2-CH2-CH2-biotin), 1.68, 1.67 (each dd, each
(C-β), 28.52 (HN-CH2-CH2-CH2-NH), 28.22 (C(O)-CH2-CH2- 1H, NH(C=O)CH2-CH2-CH2-CH2-biotin), 1.63 (dd, 1H, J =
CH2-CH2-biotin), 25.66 (C(O)-CH2-CH2-CH2-CH2-biotin), 13.3 Hz, J = 6.7 Hz, NH(C=O)CH2-CHH-CH2-CH2-biotin),
25.53 (C(O)-CH2-CH2-CH2-CH2-biotin) ppm.
1.57 (dd, 1H, J = 15.1 Hz, J = 7.4 Hz, HN-CH2-CHH-CH2-
NH), 1.53 (dd, 1H, J = 13.1 Hz, J = 6.9 Hz, NH(C=O)CH2-
MALDI-TOF-MS: m/z = 865.3 [M + Na]+; ESI-MS: m/z = CHH-CH2-CH2-biotin), 1.51 (dd, 1H, J = 13.9 Hz, J = 6.8 Hz,
865.34 [M + Na]+ (842.96 calcd. for C40H54N6O12S).
Hβ-asp), 1.36 (dd, 1H, J = 15.5 Hz, J = 7.2 Hz, HN-CH2-CHH-
820

Beilstein J. Org. Chem. 2010, 6, 810–822.
CH2-NH), 1.30 (dd, 1H, J = 13.4 Hz, J = 6.9 Hz, Hβ’-asp) ppm; manOCHHCH2), 3.69 (dd, 1H, J = 11.0 Hz, J = 4.3 Hz,
13C NMR (150.90 MHz, CD3CN:D2O = 1:1): δ = 176.60 (aryl- H-6man), 3.68 (dd, 1H, J = 9.7 Hz, J = 3.2 Hz, H-3man), 3.66
C=O), 172.78 (NH-(C=O)asp), 172.37 (C-γ), 168.61 (NH-C(O)- (dd, 1H, J = 12.2 Hz, J = 3.3 Hz, manOCHHCH2), 3.59 (t, 1H,
CH2-(CH2)3-biotin), 165.66 ((NH2)2C=O), 135.57, 133.14, J = 9.8 Hz, H-4man), 3.52 (dd, 1H, J = 10.6 Hz, J = 4.4 Hz,
129.11, 127.73 (aryl-C), 124.44 (q, JC,F = Hz, CF3), 100.72 H-6’man), 3.49 (dd, 1H, J = 10.0 Hz, J = 4.1 Hz, H-5man), 3.44
(C-1man), 87.77 (N=N-CCF3) 73.82 (C-5man), 71.68 (dd, 1H, J = 6.8 Hz, J = 4.1 Hz, manOCH2CHH), 3.32 (dd, 1H,
(C-3man), 71.10 (C-2man), 67.70 (C-4man), 66.73 (C-6man), J = 6.1 Hz, J = 4.0 Hz, manOCH2CHH), 3.20 (dd, 1H, J =
62.81 (biotin-NH2CHCHalkyl), 61.95 (manOCH2CH2), 61.09 8.8 Hz, J = 5.4 Hz, biotin-NHCHCHalkyl), 3.19 (dd, 1H, J =
(biotin-NH2CHCH2S), 56.29 (biotin-NH2CHCHalkyl), 52.36 13.7 Hz, J = 6.9 Hz, HN-CHH-CH2-CH2-NH), 3.16, 3.10 (each
(C-α), 40.78 (biotin-NH2CHCH2S), 39.89 (manOCH2CH2), dd, each 1H, NH(C=O)CH2-CH2-CH2-CH2-biotin), 3.05 (dd,
38.13 (HN-CH2-CH2-CH2-NH), 37.68 (HN-CH2-CH2-CH2- 1H J = 13.4 Hz, J = 7.0 Hz, HN-CHH-CH2-CH2-NH), 2.90 (dd,
NH), 37.36 (NH-C(O)-CH2-(CH2)3-biotin), 36.49 (C-β), 29.71 1H, J = 13.0 Hz, J = 5.1 Hz, biotin-NHCHCHHS), 2.77 (dd,
(HN-CH2-CH2-CH2-NH), 29.24 (NH-C(O)-(CH2)3-CH2- 1H, J = 14.7 Hz, J = 5.3 Hz, HN-CH2-CH2-CHH-NH), 2.69
biotin), 28.78 (NH-C(O)-CH2-CH2-CH2-CH2-biotin), 26.24 (dd, 1H, J = 12.6 Hz, J = 4.8 Hz, biotin-NHCHCHHS), 2.68
(NH-C(O)-CH2-CH2-CH2-CH2-biotin) ppm.
(dd, 1, J = 14.9 Hz, J = 5.8 Hz, HN-CH2-CH2-CHH-NH), 2.16
(dd, 1H, J = 15.3 Hz, J = 7.7 Hz, Hβ-asp), 2.12 (dd, 1H, J =
MALDI-TOF-MS: m/z = 827.3 [M – N2 + Na]+; ESI-MS: m/z = 14.9 Hz, J = 7.5 Hz, Hβ’-asp), 1.66 (ddd, 1H, J = 13.8 Hz, J =
855.29 [M + Na]+, 827.29 [M – N2 + Na]+ (832.85 calcd. for 7.1 Hz, J = 6.2 Hz, NH(C=O)CH2-CHH-CH2-CH2-biotin), 1.64
C34H47F3N8O11S).
(dd, 1H, J = 15.3 Hz, J = 6.2 Hz, NH(C=O)CH2-CH2-CHH-
CH2-biotin), 1.57 (ddd, 1H, J = 13.3 Hz, J = 6.8 Hz, J = 6.3 Hz,
NH(C=O)CH2-CHH-CH2-CH2-biotin), 1.55 (dd, 1H, J =
N-[p-(Trifluoromethyl-diazirinyl)-benzoyl]-4-[(+)-
biotinylamidopropyl]-1-[2-(α-D-mannopyranosyloxy)- 15.5 Hz, J = 6.8 Hz, NH(C=O)CH2-CH2-CHH-CH2-biotin),
ethyl]-L-aspartic acid diamide (13) 1.53, 1.52 (each dd, each 1H, NH(C=O)CH2-CH2-CH2-CH2-
The glycoamino acid derivative 10 (19.6 mg, 23.2 μmol) biotin), 1.34, 1.27 (each dd, each 1H, J = 15.3 Hz, J = 7.0 Hz,
was dissolved in piperidine (20% in DMF, 1.5 mL) and stirred HN-CH2-CH2-CH2-NH) ppm; 13C NMR (150.90 MHz,
at RT for 60 min to remove the Fmoc protecting CD3CN:D2O = 1:1): δ = 176.86 (C-γ), 172.58 (NH-(C=O)asp),
group. The solvent was removed in vacuo and the resulting 171.47 (NH(C=O)-(CH2)4-biotin), 168.28 (aryl-C=O), 165.16
crude product combined with HATU (14.0 mg, 36.8 μmol) ((NH2)2C=O), 149.11, 139.43 (aryl-C), 136.19 (q, JC,F = Hz,
and the diazirine 11 (7.2 mg, 30.9 μmol), and dry CF3), 129.08, 128.99 (aryl-C), 100.67 (C-1man), 84.21 (N=N-
DMF (1.5 mL) added under a nitrogen atmosphere. DIPEA CCF3), 73.78 (C-5man), 71.69 (C-2man), 71.12 (C-3man),
(0.05 mL, 145 μmol) was then added and the reaction mixture 67.74 (C-4man), 66.57 (C-6man), 62.85 (biotin-
stirred overnight at RT. The solvent was removed under NHCHCHalkyl), 61.92 (manOCH2CH2), 61.09 (biotin-
reduced pressure and the residue purified by flash chromatog- NHCHCH2S), 56.32 (biotin-NHCHCHalkyl), 52.34 (C-α),
raphy with the exclusion of light (ethyl acetate:MeOH:H2O = 40.78 (biotin-NHCHCH2S), 39.97 (manOCH2CH2), 38.47,
6:2:1) to yield, after lyophilisation, the title compound (19 mg, 37.63 (HN-CH2-CH2-CH2-NH), 37.39 (NH(C=O)CH2-(CH2)3-
22.8 μmol, 98%); Rf = 0.33; [α]D20 +30.0 (c 0.12 mM, biotin), 36.50 (C-β), 29.21 (C- HN-CH2-CH2-CH2-NH), 29.08
MeCN:H2O = 1:1); UV-Vis (c 0.12 mM, MeCN:H2O = 1:1): (NH(C=O)-(CH2)3-CH2-biotin), 28.79, 26.23 (NH(C=O)-CH2-
λmax(1) = 335.9 nm, ε(1) = 9833.3 Lmol−1cm−1; λmax(2) = CH2-CH2-CH2-biotin) ppm.
278.2 nm, ε(2) = 18333.3 Lmol−1cm−1; λmax(3) = 233.5 nm,
ε(3) = 23333.3 Lmol−1cm−1; FT-IR (KBr): = 3447.7 cm−1, MALDI-TOF-MS: m/z = 855.3 [M + Na]+; ESI-MS: m/z =
2940.5 cm−1, 2380.9 cm−1, 1684.7 cm−1, 1654.2 cm−1, 827.31 [M – N2 + Na]+; 855.31 [M + Na]+ (832.85 calcd. for
1636.6 cm−1, 1560.1 cm−1, 1438.3 cm−1, 1400.2 cm−1, C34H47F3N8O11S).
1279.8 cm−1, 1202.4 cm−1, 1113.1 cm−1, 761.9 cm−1.
Acknowledgements
1H NMR (600 MHz, CD3CN-D2O, 1:1): δ = 8.23 (dd, 2H, J = We are thankful to PD Dr. Buko Lindner (Research Center
8.5 Hz, J = 1.4 Hz, aryl-H), 7.38 (dd, 2H, J = 8.8 Hz, J = Borstel, Center for Medicine and Biosciences) and Prof. Dr.
1.3 Hz, aryl-H), 4.86 (dd, 1H, J = 8.6 Hz, J = 5.3 Hz, Hα-asp), Matthias Leippe and Dr. Christoph Gelhaus (Zoological Insti-
4.76 (d, 1H, J = 1.5 Hz, H-1man), 4.49 (dd, 1H, J = 7.7 Hz, J = tute at CAU) for making the MS analysis of photo-crosslinked
4.4 Hz, biotin-NHCHCH2S), 4.30 (dd, 1H, J = 8.0 Hz, J = products possible, and to Christiana Albertina University
4.4 Hz, biotin-NHCHCHalkyl), 3.80 (dd, 1H, J = 3.3 Hz, J = (CAU) for financial support.
1.7 Hz, H-2man), 3.73 (dd, 1H, J = 12.2 Hz, J = 2.3 Hz,
821

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