Structural and Kinetic Considerations for the Application of the Traceless Staudinger Ligation to Future 18F Radiolabeling Using XRD and 19F NMR

Article abstract of DOI:10.1002/kin.21137

A 4-fluorobenzoate-functionalized phosphane was synthesized and reacted with different azides using the traceless Staudinger ligation as a representative sample reaction for future radiolabeling purposes with short-lived radionuclides like fluorine-18. For this purpose, the reaction rate was evaluated at different temperatures. The effect of starting material concentrations and the influence of the steric effect coming from the applied azides were investigated. ¹⁹F NMR was used to determine the reaction half-live (τ_1/2) and the reaction rate constant (k_obs) of this ligation under mild reaction conditions in a water–acetonitrile mixture. Furthermore, the phosphane key compound 1 (orthorhombic, space group Pna2₁, a = 18.6363(9), b = 8.3589(4), c = 18.5480(9) ?, V = 2889.4(2) ?³, Z = 8, D_obs = 1.277 g/cm³), which acts as starting material for all subsequent syntheses, and the fluorine-containing phosphane 3 (monoclinic, space group P2₁/c, a = 8.321(2), b = 16.160(4), c = 14.940(4) ?, β = 99.51(1)°, V = 1981.4(8) ?³, Z = 4, D_obs = 1.342 g/cm³) were analyzed by single-crystal XRD.

Full text of DOI:10.1002/kin.21137

Structural and Kinetic
Considerations for the
Application of the Traceless
Staudinger Ligation to
Future 18F Radiolabeling
Using XRD and 19F NMR
1
2
¨
MARTIN KOCKERLING, CONSTANTIN MAMAT
1Institut fu¨r Chemie, Anorganische Festko¨rperchemie, Universita¨t Rostock, D-18059, Rostock, Germany
2Institut fu¨r Radiopharmazeutische Krebsforschung, Helmholtz-Zentrum Dresden-Rossendorf, D-01328, Dresden, Germany
Received 4 March 2017; revised 19 October 2017; accepted 19 October 2017
DOI 10.1002/kin.21137
Published online in Wiley Online Library (wileyonlinelibrary.com).
ABSTRACT: A 4-fluorobenzoate-functionalized phosphane was synthesized and reacted with
different azides using the traceless Staudinger ligation as a representative sample reaction for
future radiolabeling purposes with short-lived radionuclides like fluorine-18. For this purpose,
the reaction rate was evaluated at different temperatures. The effect of starting material concen-
trations and the influence of the steric effect coming from the applied azides were investigated.
19F NMR was used to determine the reaction half-live (τ1/2) and the reaction rate constant
(kobs) of this ligation under mild reaction conditions in a water–acetonitrile mixture. Further-
more, the phosphane key compound 1 (orthorhombic, space group Pna21, a = 18.6363(9), b =
3
8.3589(4), c = 18.5480(9) A, V = 2889.4(2) A , Z = 8, Dobs = 1.277 g/cm3), which acts as starting
˚
˚
material for all subsequent syntheses, and the fluorine-containing phosphane 3 (monoclinic,
3
˚
˚
space group P21/c, a = 8.321(2), b = 16.160(4), c = 14.940(4) A, β ꢀ=C 99.51(1)°, V = 1981.4(8) A ,
Z = 4, Dobs = 1.342 g/cm3) were analyzed by single-crystal XRD.
2017 Wiley Periodicals, Inc.
Int J Chem Kinet 1–10, 2017
INTRODUCTION
Correpondence to: Constantin Mamat; e-mail: c.mamat@
hzdr.de.
Both variants of Staudinger ligation [1] belong to
the most important bioorthogonal conjugation reac-
tions for site-selective labeling purposes [2,3] and
Supporting Information is available in the online issue at
www.wileyonlinelibrary.com.
ꢀC
2017 Wiley Periodicals, Inc.

¨
KOCKERLING AND MAMAT
2
NMR measurements in the same manner as 1H or 31P.
No supplemental introduction or enrichment of NMR
sensitive nuclides into one or both of the reactants is
mandatory in contrast to 12C/13C measurements. In the
past, 19F NMR has been used as an excellent tool for
kinetic studies on chemical reactions [38,39].
Our first objective was the structure elucidation of
fluorine-containing phosphane, which acts as starting
material for the traceless Staudinger ligation to intro-
duce the 4-fluorobenzoate moiety. Our second objec-
tive was to develop a convenient method to determine
the reaction rate of this ligation reaction via 19F NMR.
The obtained results give an impression to make a pre-
diction for future applications of this ligation method
for mild radiolabeling with the β+-emitter fluorine-18.
proceed without any catalyst in contrast to other re-
actions (e.g., the Huisgen click reaction) [4]. The ab-
sence of a metal catalyst (e.g., Cu) is important es-
pecially for in vitro and in vivo applications, because
Cu is known to be cytotoxic [5]. Therefore, the trace-
less variant of the Staudinger ligation [6–8] indepen-
dently developed by Raines et al. [9] and Bertozzi et
al. [10] is employed for several applications in chem-
istry, biochemistry, and (radio)pharmacy. The connec-
tion of proteins [11,12], peptides, and peptide frag-
ments [9,13], the glycosylation [14] of amino acids
and peptides [15,16], the preparation of large-sized lac-
tams [17,18], the functionalization of polymers [19],
and the introduction of radiolabels [20–23] as well as
fluorescence dyes [24–26] makes this reaction widely
applicable. Both the chemical modification of the bi-
ological active molecule and the preparation of the
labeling building block are also quite facile. In gen-
eral, one of the reactants is modified with an azide
function [27,28] and the other is functionalized with a
phosphane moiety [29,30].
EXPERIMENTAL
General
Knowledge about the reaction rate of the trace-
less Staudinger ligation in particular for radiolabeling
purposes is beneficial, since the applied radionuclides
often suffer from short half-live (e.g., 11C: 20 min,
18F: 110 min, 99mTc: 6 h) [31]. Thus, a short reaction
time under mild reaction conditions (e.g., low reac-
tion temperature, aqueous reaction medium) is crucial
especially for the introduction of radiolabels into sen-
sitive bio(macro)molecules like proteins or antibod-
ies [32,33].
In the past, NMR was used to study the mechanism
and the reaction rate of the Staudinger ligation [34,35],
but costly 13C-labeled starting materials were used to
quantitatively determine the spectra in a reasonable
time span. To avoid performing circumstantial synthe-
ses for the preparation of the 13C-enriched starting ma-
terials and/or long-lasting measurements, we focused
on the application of 19F NMR, since 19F-containing
derivatives were mandatory to prepare as reference
compounds for the identification of the corresponding
18F radiotracers and, therefore, they were already avail-
able [36]. Advantageously, 19F NMR measurements
are simply to perform nowadays, and the progress of
the reaction can easy be monitored, because the 19F nu-
cleus is present in the starting material and also in the
ligation product. In our case, the fluorine-containing
phosphane 3 acts as starting material and gives amides
7–9 as ligation products.
Phosphanes 1, 3, 5 [29] and azide 4 [40] were syn-
thesized as reported in the literature. NMR spectra of
all compounds were recorded on an Agilent DD2-600
MHz NMR spectrometer (ProbeOne) at 25 and 45°C
(Agilent Technologies, Waldronn, Germany). Chemi-
cal shifts of the 1H, 13C, and 31P spectra were reported
in parts per million (ppm) using TMS as internal stan-
1
dard for H and 13C, CFCl3 for 19F, and H3PO4 for
31P spectra. The assignment was further verified by
two-dimensional measurements (H,H-COSY, HSQC).
Deuterated solvents (C6D6, CD3CN, D2O) were pur-
chased from Deutero (Kastellaun, Germany). For the
first kinetic NMR experiment, stock solutions of phos-
phane 3 were prepared with a concentration of 71 mM
and azide 4 with final concentrations of 71 mM and
710 mM, respectively, using a mixture of CD3CN
and D2O (v:v = 10:1). 300 µL of each solution was
combined into an NMR tube to reach a final solvent
amount of 600 µL. The reaction of 3 with fructose
derivatives 5 and 6 was done according to the same
procedure but with lower concentrations due to sol-
ubility reasons (3: 17.7 mM, 5: 187.4 mM, 6: 176.4
mM). At the beginning of the reaction, 19F and 31P
NMR spectra were recorded all 3–15 min, later all 30–
60 min. In all experiments, 16 scans were recorded
for the recording of the 19F and 31P spectra. Mea-
surements were accomplished using the standard pulse
sequence from OpenVnmrJ software. Determined sig-
nals were integrated, and the values were plotted ver-
sus time. Logarithmic plotting was used to determine
the reaction half-life τ1/2 and the rate constant kobs
of the reaction. The concentration dependence of the
OH group in 3 was determined using a stock solution
Fluorine-19 has favorable properties for NMR mea-
surements like a nuclear spin of 1/2, a high gyromag-
1
netic ratio (0.94 × of H), and it is 100% naturally
enriched as 19F (monoisotopic) [37], which makes
this isotope highly responsive for fast and quantitative
International Journal of Chemical Kinetics DOI 10.1002/kin.21137

KINETIC CONSIDERATION OF THE TRACELESS STAUDINGER LIGATION VIA 19F NMR
3
(72.3 mM) of 3 in C6D6, which was diluted using 300
1-(4-Fluorobenzamido)-1-deoxy-2,3:4,5-di-O-
µL stock solution and 300 µL C6D6 (except the last:
300 µL stock solution + 900 µL C6D6). All NMR
experiments were performed in triplicate. Diffraction
data were collected with a Bruker-Nonius-Apex-II-
diffractometer using graphite-monochromated Mo Kα
isopropylidene-β-D-fructopyranose (8). Compounds
3 (50 mg, 0.12 mmol) and 5 (36 mg, 0.12 mmol) were
dissolved in a mixture of acetonitrile and water (1.1
mL, 10:1), and the resulting solution was heated at
40°C for 6 h. Afterwards, the solvent was removed
and the crude product was purified via column
chromatography (petroleum ether: ethyl acetate =
10:1) to yield 8 (40 mg, 84%) as colorless syrup. Rf =
˚
radiation (λ = 0.71073 A). All diffraction measure-
ments were done at –150°C. The unit cell dimen-
sions were recorded and refined using the angular set-
tings of 7238 (1), and 9338 (3) reflections, and the
structures were solved by direct methods and refined
against F2 on all data by full-matrix least-squares us-
ing the program suits from Sheldrick [41–43]. All
nonhydrogen atoms were refined anisotropically; all
hydrogen atoms were placed on geometrically cal-
culated positions and refined using a riding model.
CCDC 1482485 (1) and CCDC 1482528 (3) con-
tain the supplementary crystallographic data for this
paper. These data can be obtained free of charge
from The Cambridge Crystallographic Data Centre via
www.ccdc.cam.ac.uk/data request/cif.
1
0.6 (petroleum ether: ethyl acetate = 3:2); H NMR
(CDCl3): δ 1.30 (s, 3H, CH3), 1.32 (s, 3H, CH3), 1.40
(s, 3H, CH3), 1.53 (s, 3H, CH3), 3.68–3.77 (m, 2H,
H-1a, H-6a), 3.82–3.92 (m, 2H, H-1b, H-6b), 4.21
(dd, 3J = 7.8 Hz, 4J = 1.7 Hz, 1H, H-5), 4.36 (d, 3J =
2.5 Hz, 1H, H-3), 4.58 (dd, 3J = 2.5 Hz, 3J = 7.8 Hz,
3
3
1H, H-4); 6.69 (br s, 1H, NH), 7.08 (t, Jo,m = JH,F
= 8.6 Hz, 2H, Hm), 7.80 (dd, 4JH,F = 5.4 Hz, 3Jo,m
=
8.6 Hz, 2H, Ho); 13C (400 MHz, CDCl3): δ 24.1, 25.1,
26.0, 26.2 (4 × CH3), 47.6 (C-1), 61.6 (C-6), 65.4
(C-1), 70.4 (C-4), 70.6 (C-5), 72.1 (C-3), 102.8 (C-2),
2
108.7 (C-q), 109.2 (C-q), 115.5 (d, JC,F = 21.9 Hz,
3
1
Cm), 129.6 (d, JC,F = 8.7 Hz, Co), 164.7 (d, JC,F
=
250.3 Hz, Cp), 166.9 (Ci + C=O); MS (ESI+) m/z:
382 [M++H]; Anal. calcd for C19H24FNO6 (381.40):
C, 59.83; H, 6.34; N, 3.67. Found: C, 60.01; H, 6.23;
N, 3.71.
Chemistry
1-(6-Azidohexanamido)-1-deoxy-2,3:4,5-di-
O-isopropylidene-β-D-fructopyranose
(6). 6-
Azidohexanoic acid (197 mg, 1.25 mmol), 2,3:4,5-
di-O-isopropylidene-β-D-fructopyranose (398 mg,
1.52 mmol), EDC·HCl (347 mg, 1.81 mmol), and
N,N-dimethylaminopyridine in catalytic amounts
were dissolved in anhydrous THF (8 mL), and the
mixture was heated under stirring at 50°C for 6 h.
After TLC control, the solids were filtered, the solvent
was removed, and the crude product was purified
via column chromatography (petroleum ether: ethyl
acetate = 10:1) to yield 6 (249 mg, 50%) as colorless
syrup. Rf = 0.6 (petroleum ether: ethyl acetate = 3:2);
1H NMR (CDCl3): δ 1.33 (s, 3H, CH3), 1.36–1.45 (m,
5H, CH2+CH3), 1.47 (s, 3H, CH3), 1.53 (s, 3H, CH3),
1-(6-(4-Fluorobenzamido)hexanamido)-1-deoxy-
2,3:4,5-di-O-isopropylidene-β-D-fructopyranose (9).
Compounds 3 (47 mg, 0.12 mmol) and 6 (47 mg,
0.12 mmol) were dissolved in a mixture of acetonitrile
and water (1.1 mL, 10:1), and the resulting solution
was heated at 40°C for 6 h. Afterwards, the solvent
was removed and the crude product was purified
via column chromatography (petroleum ether: ethyl
acetate = 2:1) to yield 9 (50 mg, 86%) as colorless
syrup. Rf = 0.6 (petroleum ether: ethyl acetate = 3:2);
1H NMR (400 MHz, CDCl3): δ 1.33 (s, 3H, CH3),
1.36–1.45 (m, 5H, CH2+CH3), 1.47 (s, 3H, CH3),
1.53 (s, 3H, CH3), 1.60–1.74 (m, 4H, CH2), 2.38 (t,
3
1.56–1.72 (m, 4H, CH2), 2.37 (t, J = 7.5 Hz, 2H,
CH2C=O), 3.26 (t, 3J = 6.8 Hz, 2H, CH2N3), 3.75 (d,
3J = 13.0 Hz, 1H, H-6a), 3.90 (dd, 3J = 13.0 Hz, 4J =
1.5 Hz, 1H, H-6b), 4.03 (d, 3J = 11.8 Hz, 1H, H-1a),
3J = 7.1 Hz, 2H, CH2C=O), 3.45 (dt, J = 6.7 Hz,
3
2H, CH2NH), 3.76 (d, 3J = 13.2 Hz, 1H, H-6a), 3.90
3
4
(dd, J = 13.2 Hz, J = 1.5 Hz, 1H, H-6b), 4.03 (d,
3
4
3
4
4.23 (dd, J = 7.9 Hz, J = 1.5 Hz, 1H, H-5), 4.29
(d, 3J = 2.6 Hz, 1H, H-3), 4.40 (d, 3J = 11.8 Hz, 1H,
H-1b), 4.59 (dd, 3J = 2.6 Hz, 3J = 7.9 Hz, 1H, H-4);
13C NMR: δ 24.2 (CH3), 24.4 (CH2), 25.4 (CH3),
26.0 (CH3), 26.4 (CH2), 26.6 (CH3), 28.7 (CH2), 34.0
(CH2C=O), 51.3 (CH2N3), 61.4 (C-6), 65.4 (C-1),
70.2 (C-4), 70.7 (C-3), 70.9 (C-5), 101.7 (C-2), 108.8
(C-q), 109.3 (C-q), 172.8 (C=O); MS (ESI+) m/z:
399 [M++H]; Anal. calcd for C18H30N4O6 (398.22):
C, 54.26; H, 7.59; N, 14.06. Found: C, 53.99; H, 7.55;
N, 14.19.
3J = 11.6 Hz, 1H, H-1a), 4.23 (dd, J = 7.7 Hz, J
3
= 1.5 Hz, 1H, H-5), 4.29 (d, J = 2.4 Hz, 1H, H-3),
3
3
4.41 (d, J = 11.6 Hz, 1H, H-1b), 4.60 (dd, J = 2.4
Hz, 3J = 7.7 Hz, 1H, H-4), 6.24 (br s, 1H, NH), 7.09
3
3
4
(t, Jo,m = JH,F = 8.7 Hz, 2H, Hm), 7.78 (dd, JH,F
= 5.3 Hz, Jo,m = 8.7 Hz, 2H, Ho); 13C NMR (400
3
MHz, CDCl3): δ 24.2 (CH3), 24.3 (CH2), 25.4 (CH3),
26.0 (CH3), 26.5 (CH2), 26.6 (CH3), 29.3 (CH2),
34.0 (CH2C=O), 39.9 (CH2NH), 61.4 (C-6), 65.4
(C-1), 70.2 (C-4), 70.7 (C-3), 70.9 (C-5), 101.7 (C-2),
2
108.9 (C-q), 109.3 (C-q), 115.7 (d, JC,F = 21.8 Hz,
International Journal of Chemical Kinetics DOI 10.1002/kin.21137

¨
4
KOCKERLING AND MAMAT
3
1
RESULTS AND DISCUSSION
Cm), 129.3 (d, JC,F = 9.3 Hz, Co), 164.8 (d, JC,F
=
251.3 Hz, Cp), 166.6 (Ci), 173.0 (C=O); 19F NMR
(376 MHz, CDCl3): δ –108.6; MS (ESI+) m/z: 495
[M++H]; Anal. calcd for C25H35FN2O7 (494.56): C,
60.72; H, 7.13; N, 5.66. Found: C, 60.61; H, 7.09; N,
5.45.
Synthesis and X-Ray Determination
Independent on the variant of the Staudinger ligation
(traceless or nontraceless), functionalized phosphanes
act as starting material in general [6–8]. Furthermore,
the traceless ligation can be divided in two approaches.
In the direct approach, the imaging probe (radionu-
clide, fluorescent dye, etc.) is connected to the phos-
phane moiety whereas the imaging probe is connected
to the azide and the phosphane is connected to the
molecule to be labeled in the indirect approach.
Especially for the traceless variant, phosphanes like
3 with an electrophilic center (e.g., ester, thioester,
amide) in close proximity to the phosphorus are
required. For this purpose, phosphane 3 [29] was
prepared by the reaction of phosphane 1 with 4-
fluorobenzoyl chloride (2) under basic conditions
[44,45]. The reaction is shown in Scheme 1.
Phosphane 1 acts as a basic starting compound to
prepare further functionalized derivatives for ligation
purposes. Single crystals of 1 and 3 were obtained dur-
ing the purification process and were characterized by
single crystal X-ray analysis [41–43]. The molecular
structures of both phosphanes are illustrated in Figs. 1
and 2.
2-(Diphenylphosphoryl)phenyl
4-fluorobenzoate
(11). Compound 3 (100 mg, 0.25 mmol) and NaN3
(32 mg, 0.5 mmol) were dissolved in a mixture of
acetonitrile and water (300 µL, 10:1), and the resulting
solution was heated at 40°C for 6 h. Afterwards,
the solvent was removed and the crude product was
purified via column chromatography (petroleum
ether: ethyl acetate = 10:1) to yield 11 (88 mg, 85%)
as colorless syrup. Rf = 0.3 (petroleum ether:ethyl
1
acetate = 3:1); H NMR (400 MHz, C6D6): 6.56 (t,
3Jo,m = JH,F = 8.8 Hz, 2H, Hm(F)), 6.76 (t, J = 7.5
3
3
3
Hz, 1H, H-4), 6.87–6.99 (m, 6H, H-Ar), 7.08 (t, J
3
= 7.7 Hz, 1H, H-5), 7.22 (dd, J = 4.3 Hz, J = 8.4
3
Hz, 1H, H-6), 7.37 (dd, J = 7.5 Hz, J = 12.9 Hz,
4
1H, H-3), 7.69–7.77 (m, 4H, Ar), 7.87 (dd, JH,F
=
5.6 Hz, 3Jo,m = 8.8 Hz, 2H, Ho(F)); 13C NMR (C6D6):
δ 115.4 (d, 3JC,F = 22.4 Hz, C o(F)), 124.5 (d, 3JC,P
=
3
5.7 Hz, C-6), 125.8 (d, JC,P = 11.2 Hz, C-4), 127.0
(d, 1JC,P = 100.1 Hz, C-2), 128.5 (d, 3JC,P = 12.2 Hz,
Cm(P)), 131.6 (d, 4JC,P = 2.5 Hz, Cp(P)), 132.1 (d, 2JC,P
= 9.7 Hz, Co(P)), 133.3 (d, 4JC,P = 2.2 Hz, C-5), 133.5
(d, 2JC,F = 9.7 Hz, Cm(F)), 134.2 (d, 1JC,P = 104.5 Hz,
O
O
OH
Et3N, THF
PPh2
2
+
Cl
Ci(P)), 134.5 (d, JC,P = 8.6 Hz, C-3), 153.6 (Ci(F)),
O
162.9 (C=O), 166.2 (d, JC,F = 254.3, Cp(F)); 19F
1
PPh2
F
F
1
2
3
NMR (376 MHz, C6D6): δ –105.1; 31P NMR (C6D6):
23.3; MS (ESI+) m/z: 417 [M++H]; Anal. calcd for
C25H18FO3P (416.39): C, 72.11; H, 4.36. Found: C,
72.17; H, 4.29.
Scheme 1 Synthesis of the fluorine containing phosphane
building block 3 by the reaction of phosphane 1 and 4-
fluorobenzoyl chloride (2).
Figure 1 Structure of the two symmetry independent molecules of the phosphane 1 (ORTEP plot, 50% probability level). [Color
figure can be viewed at wileyonlinelibrary.com]
International Journal of Chemical Kinetics DOI 10.1002/kin.21137

KINETIC CONSIDERATION OF THE TRACELESS STAUDINGER LIGATION VIA 19F NMR
5
The yields ranged from 78% to 86%. In general, phos-
phane oxide 10 was always obtained as a by-product in
all reactions. To check other possible oxidation prod-
ucts, which could be formed during the Staudinger
ligation, fluorinated phosphane oxide 11 was prepared
from phosphane 3 which was treated with NaN3. An
overview of all reactions is given in Scheme 2.
Determination of kobs via 19F NMR
Studies on the reaction rate were performed to ensure
that the reaction time of the traceless Staudinger liga-
tion is suitable to apply for future 18F radiolabeling. For
analysis of the reaction times, it is necessary to distin-
guish between starting phosphane 3 and the Staudinger
products 7–9, based on their different 19F chemical
shifts pointed out in Fig. 4. The fluorine signal of start-
ing material 3 was determined at δ = –106.1 ppm and
the signal of the Staudinger product 7 at δ = –110.4
ppm in the 19F NMR using a mixture of acetonitrile-d6
and D2O (ratio: v:v = 10:1). For comparison, the re-
Figure 2 Molecular structure of the fluorine-containing
phosphane 3 (ORTEP plot, 50% probability level). [Color fig-
ure can be viewed at wileyonlinelibrary.com]
Crystals of 1 are composed of two symmetry in-
dependent molecules. The OH group in the molecule
around P2 is disordered on two sites, labeled O2A and
O2B. In both compounds, the residues around the P-
atoms are arranged in a trigonal-pyramidal fashion with
average C−P−C angles slightly smaller than the ideal
tetrahedral value, i.e., for 1, P1: 102.5°, P2: 102.7°
and for 3, P1: 102.8°. Whereas in 3 classical hydrogen
bonding is not observed, in 1 one O−HꢀꢀꢀO contact
(O2B−H2BꢀꢀꢀO1) with a donor−acceptor distance of
actions were additionally monitored by “indirect” 31
P
NMR measurements. The product 7 does not contain
phosphorus, so the by-product 10 (δ = +35 ppm) was
detected instead in addition to the starting phosphane
3 (δ = –15 ppm).
Our initial study comprised of three experiments.
The first two were based on the equimolar reaction
of both starting materials 3 and 4 at temperatures of
25 and 45°C, respectively. The low temperatures were
chosen in consideration of future labeling reactions.
Mild conditions are essential especially for labeling
proteins or antibodies, which will denature or will be
destroyed at elevated temperatures. For the third ex-
periment, the reaction was performed with 10-fold ex-
cess of the starting azide 4 at 25°C to simulate condi-
tions observed in radiolabeling procedures. Generally,
the precursor (nonradioactive starting material) is al-
ways in high excess (100:1 to 1000:1) compared to
the compound containing the radionuclide (radiolabel-
ing building block) [31]. This leads to pseudo–first-
order conditions for every radiolabeling reaction with
noncarrier-added radionuclides (e.g., 18F, 11C, 99mTc).
Owing to the azide 4 (as a prospective precursor) being
10-fold higher in concentration compared to the phos-
phane 3, the pseudo–first-order conditions can also be
supposed [50] and gives a good impression for the
transfer to noncarrier-added radiolabeling conditions.
All NMR experiments were carried out in a mix-
ture of acetonitrile-d3 and D2O (ratio: v:v = 10:1). A
stock solution of phosphane 3 was prepared with a
concentration of 71 nM. Solutions with azide 4 were
prepared with final concentrations of 71 and 710 nM,
respectively. At the beginning of the reaction, 19F and
˚
3.16(2) A and a bond angle of 162.9° is observed. This
is in accordance with the behavior of the compound 1 in
solution. A broad band at 3227 cm−1 and a sharp band
at 3515 cm-1 was found in the IR spectrum of com-
pound 1 [23], indicating an intramolecular hydrogen
bond and the occurrence of free OH group, respec-
1
tively [46]. Additionally, data obtained from the H
NMR spectrum of 1 at a concentration of 72.33 mM
show a broad signal for the proton of the OH group at
δ = 6.07 ppm. Further measurements were done with
half of the concentration showing a movement of the
OH signal [47,48]. At a concentration of c = 2.26 mM,
the OH group was found to be split to a doublet with
coupling constant of J = 6.0 Hz at a chemical shift of
δ = 6.01 ppm. This is due to the coupling to the phos-
phorus. No cross peak of the OH-group to the respec-
tive proton in para position was found in the H,H-
COSY. The results are outlined in Fig. 3 and in the
Supporting Information.
To apply the ligation, phosphane 3 was reacted
with three different azides 4–6 to obtain the desired
Staudinger products 7–9. Benzyl azide (4) gave the
desired Staudinger product 7 [49], and the azide-
functionalized glucose 5 as well as the amino acid
6 were reacted with 3 to yield 8 and 9, respectively.
International Journal of Chemical Kinetics DOI 10.1002/kin.21137

¨
KOCKERLING AND MAMAT
6
Figure 3 1H NMR spectra using different concentrations (spectra recorded in C6D6) and movement of the chemical shift
of the OH group of compound 1 as a function of different concentrations (small figure). [Color figure can be viewed at
wileyonlinelibrary.com]
31P NMR spectra were recorded all 3–10 min, later all
30 min. A representative record of 19F and 31P NMR
spectra for the reaction of 3 with 4 in 10-fold excess of
4 is displayed in Figs. 5 and 6.
Under the assumption that the reaction of phos-
phane 3 with a 10-fold excess of azide 4 behaves like a
pseudo–first-order reaction, the rate constant kobs was
calculated according to the below equation [51]. With
a concentration of 710 nM of azide 4, kobs is calculated
To give a first impression on the Staudinger liga-
tions reaction rate, the reaction half-live τ1/2 was de-
termined. A summary of these data is found in Table I.
When comparing the dependence on the temperature,
the reaction rate at 45°C is higher. Thus, τ1/2 = 41.1
min was found for the reaction at 45°C in contrast to
τ1/2 = 203.8 min for the reaction at 25°C. The same
result is found when comparing the reaction (1:1 ratio)
with the reaction (10:1 ratio) at 25°C. For the reaction
with 10-fold excess of azide 4, the shortest reaction
half-life of τ1/2 = 22.2 min was calculated. No starting
material 3 was found after 92 min when using 10-fold
excess of azide 4, whereas starting material 3 from the
equimolar reaction was still detectable after 350 min.
The results are visualized in Fig. 7. Only slight differ-
ences between the results of the 19F and the 31P NMR
measurements were observed.
with a value of 0.7 × 10−3 M−1 s−1
.
ln2
kꢁ
τ1/2
=
with: kꢁ = kobs · [azide]
Additionally, carbohydrate 5 was chosen, which
contains a more sterically demanding N3 group and
reacted with phosphane 3 to give the Staudinger prod-
uct 8. The following NMR investigations were accom-
plished at 25 and 40°C with the 10-fold excess of azide
5, indicating a slower reaction rate compared to the first
reaction of 3 with 4. A higher value for τ1/2 was found
(53.7 min at 25°C and 20.2 min at 40°C), and the rate
constant was calculated with kobs = 1.1 × 10−3 M−1
s−1 (25°C) and kobs = 3.1 × 10−3 M−1
s
−1 (40°C).
Based on these results, the fructose derivative 6 with
a spacer group between the fructose core and the azide
International Journal of Chemical Kinetics DOI 10.1002/kin.21137

KINETIC CONSIDERATION OF THE TRACELESS STAUDINGER LIGATION VIA 19F NMR
7
Scheme 2 Traceless Staudinger ligation of 3 with azides 4–6 to amide 7 and fructose derivatives 8 and 9.
Figure 4 Chemical shifts of starting compound 3 and the products 7–10 in the 19F and 31P NMR (all samples measured in
acetonitrile-d3:D2O = 10:1).
function was additionally prepared and reacted with
phosphane 3 to give 9 under the aforementioned con-
ditions. The reaction half-life τ1/2 was determined with
139 min at 25°C and 43 min at 40°C, and the reaction
rate was calculated to be kobs = 0.5 × 10−3 M−1 s−1
(25°C) and kobs = 1.5 × 10−3 M−1 s−1 (40°C). Results
are summarized in Table I.
All determined values for kobs are in the simi-
lar range and in good agreement with other results
found for the Staudinger ligation [34]. Although other
bioorthogonal (click) reactions proceed faster [32], the
traceless Staudinger ligation is readily suitable for ra-
diolabeling purposes with fluorine-18 under mild reac-
tion conditions.
CONCLUSION
X-ray structure determinations of the starting phos-
phanes and kinetic measurements of the reaction rate
between fluorinated phosphane and various azides
were accomplished under different reaction conditions
to transfer and apply the traceless Staudinger ligation
for future radiolabeling with short-lived β+-emitter
fluorine-18 and other radionuclides. For this purpose,
19F NMR was used due to the presence of 19F in the
starting material (phosphane 3) as well as in the corre-
sponding products (7–9). The results of the measure-
ments clearly indicate that it is possible to apply the
traceless Staudinger ligation for radiolabeling purposes
International Journal of Chemical Kinetics DOI 10.1002/kin.21137

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KOCKERLING AND MAMAT
8
Figure 5 Representative set of 19F NMR spectra recorded on different time points to determine the reaction half-life τ1/2 and the
rate constant for the reaction of 3 with 10-fold excess of azide 4 at 25°C. [Color figure can be viewed at wileyonlinelibrary.com]
Figure 6 Representative set of 31P-NMR spectra recorded on different time points for the reaction of 3 with 10-fold excess of
azide 4 at 25°C. [Color figure can be viewed at wileyonlinelibrary.com]
Table I Summary of Results from NMR Measurements
Molar ratio of Starting Materials
Temperature
Reaction Half-Life τ1/2
kobs
Phosphane 3: azide 4 = 1:1
Phosphane 3: azide 4 = 1:1
Phosphane 3: azide 4 = 1:10
Phosphane 3: azide 5 = 1:10
Phosphane 3: azide 5 = 1:10
Phosphane 3: azide 6 = 1:10
Phosphane 3: azide 6 = 1:10
25°C
45°C
25°C
25°C
40°C
25°C
40°C
203.8
41.1
22.2
53.7
20.2
129.4
43.8
3.4 min
1.2 min
0.1 min
0.9 min
0.9 min
1.1 min
0.38 min
n.d.
n.d.
0.7 × 10−3
1.1 × 10−3
3.1 × 10−3
0.5 × 10−3
1.5 × 10−3
0.04 × 10−3
0.02 × 10−3
0.13 × 10−3
M
M
M
M
M
−1s−1
−1s−1
−1s−1
−1s−1
0.004 × 10−3
−1s−1
0.01 × 10−3
International Journal of Chemical Kinetics DOI 10.1002/kin.21137

KINETIC CONSIDERATION OF THE TRACELESS STAUDINGER LIGATION VIA 19F NMR
9
80
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c / mM
60
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