A modular system for the preparation of diazirine-labeled mannose derivatives using thiourea bridging

Article abstract of DOI:10.1055/s-2006-926358

The protein FimH is a bacterial lectin, which is utilized by Escherichia coli to adhere to the glycocalyx of potential host cells. FimH has specificity for α-mannosyl residues, as revealed by biological as well as X-ray studies. To further investigate the

Full text of DOI:10.1055/s-2006-926358

952
PAPER
A Modular System for the Preparation of Diazirine-Labeled Mannose
Derivatives Using Thiourea Bridging
A
M
odula
r
Approach
t
a
o
D
iazirine
r
Labelin
k
Walter, Thisbe K. Lindhorst*
Otto Diels Institute of Organic Chemistry, Christiana Albertina University of Kiel, Otto-Hahn-Platz 3, 24098 Kiel, Germany
Fax +49(431)8807410; E-mail: tklind@oc.uni-kiel.de
Received 31 August 2005; revised 27 October 2005
offers an option to improve receptor binding. Binding of
a-mannosides such as methyl a-D-mannoside (MeMan) to
FimH is weak with IC50 values in the millimolar range. By
incorporation of aryl aglycon moieties, as found in p-ni-
Abstract: The protein FimH is a bacterial lectin, which is utilized
by Escherichia coli to adhere to the glycocalyx of potential host
cells. FimH has specificity for a-mannosyl residues, as revealed by
biological as well as X-ray studies. To further investigate the mo-
lecular details of carbohydrate binding to FimH, photolabile man- trophenyl-a-D-mannoside (pNPMan), carbohydrate bind-
ing to FimH is improved by approximately 100 times.8
nose derivatives are important tools to covalently mark
carbohydrate binding sites on FimH. Here, a modular approach for
Consequently, we envisaged a synthesis of photolabile
the synthesis of photolabile diazirine-labeled mannosides and man-
mannose derivatives which is compatible with fine-tuning
nosyl clusters is reported. The convergent synthesis utilizes thiourea
of binding potencies such as the appropriate incorporation
of phenyl rings.
bridging of the amino-functionalized diazirine 4 with NCS-func-
tionalized carbohydrates.
Thus, our work on the synthesis of diazirine-labeled car-
bohydrates was based on a convergent approach involving
an amino-functionalized diazirine and an isothiocyanato-
functionalized ligand, which can be combined by the thio-
urea bridging strategy9 (Equation 1).
Key words: carbohydrates, glycoclusters, photolabeling, diazir-
ines, thiourea
Escherichia coli bacteria possess protein appendages,
called fimbriae or pili, to adhere to the glycocalyx of their
potential host cells.The so-called type 1 fimbriae are man-
nose-specific and can interact with terminal a-mannosyl
residues of the glycocalyx.1 Type 1 fimbriae are known to
be critical factors for the ability of E. coli to colonize the
human bladder and persist in the urinary tract.2 The lectin
domain of type 1 fimbriae is formed by a protein named
FimH.3 X-ray studies4 have revealed a carbohydrate rec-
ognition domain (CRD) at the tip of the protein which can
accommodate one a-mannosyl residue together with one
water molecule. Additional carbohydrate binding sites on
FimH have not been identified, whereas testing results
with multivalent cluster mannosides5 as well as theoreti-
cal studies6 suggest, that there could be more than just one
mannose-interacting domain on FimH.
O
(RO)n
O
NH
N
N
(RO)n
+
S
NH2
NCS
N
H
N
N
Equation 1 Convergent synthetic strategy for the preparation of
diazirine-functionalized sugar derivatives by thiourea bridging.
Synthesis of the amino-functionalized diazirine 4 started
with ( )-1-aminopropan-2-ol (1) (Scheme 1). After the
amino group was reacted with Boc anhydride,10 the pro-
tected product was instantaneously oxidized in a Swern
reaction11 leading to the ketone 2 in an overall yield of
93%. A keto group can be turned into a diazirine ring us-
ing hydroxylamine-O-sulfonic acid in liquid ammonia,
followed by oxidative treatment of the intermediate diaz-
iridine with iodine.12 This procedure delivered the Boc-
protected diazirine 3 in 70% yield, which could be easily
deprotected with hydrochloric acid to give the amino hy-
drochloride 4. The diazirine 4 is a photoactive group to be
introduced into isothiocyanato-functionalized molecules
via thiourea bridging.
To further investigate carbohydrate binding to the bacteri-
al adhesin FimH, we decided to use the well-known pho-
tolabeling methodology.7 Therefore, mannose derivatives
were targeted, which carry a photolabile diazirine group.
Ideally, incubation of such functionalized sugar ligands
with specific lectins, such as FimH, leads to covalent at-
tachment of the sugar moiety to the CRD of the lectin
upon irradiation.
As it is unknown at which position and distance of the
sugar ring a diazirine group should ideally be attached, a
synthetic pathway allowing a maximum of structural flex-
ibility is advantageous. Such a modular approach for the
synthesis of diazirine-labeled mannose derivatives also
To explore the feasibility of thiourea bridging with the
amino-functionalized diazirine 4, two isothiocyanato-
functionalized sugars were used, the mannosyl isothiocy-
anate 513 and the NCS-functionalized phenyl mannoside
8,14 respectively, which were both prepared according to
literature-known procedures (Scheme 2). The hydrochlo-
ride 4 was converted into the free amine by adding an ex-
cess of diisopropylethyl amine (DIPEA) and was then
stirred with the carbohydrate components at room temper-
SYNTHESIS 2006, No. 6, pp 0952–0958
x
x
.
x
x
.2
0
0
6
Advanced online publication: 27.02.2006
DOI: 10.1055/s-2006-926358; Art ID: T11805SS
© Georg Thieme Verlag Stuttgart · New York

PAPER
A Modular Approach to Diazirine Labeling
953
O
philic displacement to give the 6-azido-6-deoxy deriva-
tives, which were finally converted into the 6-deoxy-6-
isothiocyanato mannosides 11 and 14 (Scheme 3) via a
Staudinger-type reaction with carbon disulfide and trieth-
yl phosphite.18 Indeed, thiourea bridging with the amino-
functionalized diazirine 4 under the above mentioned
standard conditions again proceeded in very good yields
to give the photolabile mannosides 13 and 16 after
deacetylation.
OH
H
i, ii
O
N
NH2
O
2
1
iii, iv
N
N
N
N
H
v
O
N
NH2⋅HCl
O
4
3
S
Scheme 1 Synthesis of the amino-functionalized diazirine 4.
Reagents and conditions: (i) Boc2O, MeOH, r.t., 1 h; (ii) oxalyl chlo-
ride, DMSO, anhyd CH2Cl2, –55 °C, Et3N, 15 min, 93% over two
steps; (iii) NH3, H2NOSO3H, MeOH–CH2Cl2, –50 → –20 °C → r.t.,
16 h; (iv) 10% I2/MeOH, Et3N, 0 °C, 70% over two steps; (v) HCl,
Et2O, r.t., 1 h, 99%.
NCS
HN
OAc
O
N
OR
O
H
i
AcO
AcO
N
N
RO
RO
OCH3
11
OCH3
12 R = Ac
13 R = H
ii
ature for 2 h. This delivered the protected diazirine-la-
beled mannose derivatives 6 and 9, respectively
(Scheme 2), which could be de-O-acetylated according to
Zemplén15 at 0 °C to give the unprotected photolabile
ligands 7 and 10 in quantitative yields.
S
NCS
HN
N
OR
O
OAc
H
O
i
N
N
AcO
AcO
RO
RO
O
O
14
15 R = Ac
16 R = H
OR
OR
OAc
OAc
ii
i
NO2
NO2
O
O
RO
RO
AcO
AcO
N
N
Scheme 3 Synthesis of the photoactive mannose derivatives 13 and
16. Reagents and conditions: (i) 4, DIPEA, r.t., 2 h, CH2Cl2, 62% for
12; quant for 15; (ii) 1 M NaOMe/MeOH (0.1 equiv/OH), 0.5 h, r.t.,
98% for 13; 91% for 16.
H
HN
N
NCS
5
6
7
R = Ac
R = H
S
ii
In extension of our work, it became our goal to apply this
approach for the synthesis of multivalent carbohydrate
ligands by thiourea bridging.9 Recently, we have reported
about the graded reactivity of differently isothiocyanato-
functionalized acetyl-protected mannose derivatives in
thiourea bridging.19 The difference in reactivity was most
pronounced when mannosyl isothiocyanates were com-
pared to 6-deoxy-6-isothiocyanato-mannosides. There-
fore we planned to synthesize the di-NCS-functionalized
mannose derivative 20 (Scheme 4) to eventually address
OAc
OAc
OR
OR
i
9
R = Ac
O
O
AcO
AcO
ii
RO
RO
10 R = H
O
8
O
S
NCS
N
N
H
H
N
N
Scheme 2 Synthesis of the photoactive mannose derivatives 7 and
10. Reagents and conditions: (i) 4, DIPEA, r.t., 2 h, CH2Cl2, 83% for
6; 62% for 9; (ii) 1 M NaOMe/MeOH (0.1 equiv/OH), 0.5 h, 0 °C,
97% for 7; quant for 10.
NCS
R
OAc
OAc
O
O
ii
AcO
AcO
AcO
AcO
Interestingly, when the deprotection reaction was carried
out at room temperature, 7 was obtained as an anomeric
mixture16 with an a/b ratio of 4:1. The anomeric configu-
ration was assigned on the basis of the values of JC-1,H-1 as
measured in the 13C gated-decoupling NMR experiment.
When deprotection was carried out at 0 °C, no anomeriza-
tion occurred.
19
OAc
Br
17 R = N3
i
iii
18 R = NCS
NCS
OAc
O
NCS
iv
OAc
AcO
O
AcO
N
N
AcO
AcO
H
N
21
HN
After thiourea bridging employing the amine 4 had been
successful, the scope of this reaction pathway was further
evaluated. Two mannosides, one with an alkyl, the other
one with an aryl aglycon, were selected and functional-
ized at the 6-position following known procedures.14,17
Thus, the respective mannoside was first regioselectively
6-O-tosylated, acetylated and then subjected to nucleo-
20
NCS
S
Scheme 4 Synthesis of di-NCS-functionalized sugar 20 and its re-
gioselective reaction to 21. Reagents and conditions: (i) CS2, P(OEt)3,
toluene, 8 h, 80 °C, 76%; (ii) 33% HBr–AcOH, Ac2O, 1.5 h, r.t., 68%;
(iii) KSCN (10 equiv), 190 °C, 7 min, 74%; (iv) 4, DIPEA, r.t.,
CH2Cl2, 88%.
Synthesis 2006, No. 6, 952–958 © Thieme Stuttgart · New York

954
M. Walter, T. K. Lindhorst
PAPER
both NCS functions with a branched oligoamine for clus- by thiourea bridging within a wide scope; and (ii) this
tering and the diazirine-functionalized amine 4 for photo- chemistry can be combined with carbohydrate clustering
activation in subsequent reactions.
by utilizing the graded reactivity of the different NCS
groups in diisothiocyanato-functionalized carbohydrates
such as 20 to give rise to photolabeled glycoclusters such
as 24.
Synthesis of 20 started with the 6-azido-6-deoxy mannose
derivative 17,20 which was converted into the correspond-
ing isothiocyanate 18 using carbon disulfide and triethyl
phosphite (Scheme 4). Then, the anomeric acetyl group Photolabeling experiments with the herein reported diaz-
was substituted in HBr–AcOH to deliver the functional- irines will eventually be carried out in analogy to the liter-
ized a-mannosyl bromide 19 in good yield with the 6- ature, which has reported successful protein labeling with
isothiocyanato group surviving the rather harsh reaction diazirine-functionalized sugars.22 Our first photolabeling
conditions. Melting of 19 with an excess of potassium studies show, that a thiourea function is compatible with
thiocyanate gave the 1,6-diisothiocyanato mannose deriv- photoactivation of diazirines.
ative 20 in over 70% yield. In the 13C NMR spectrum of
20 the peaks corresponding to both NCS-carbon atoms
In conclusion, the modular synthetic approach presented
in this paper offers a general possibility for the introduc-
tion of a diazirine function into various types of biologi-
cally interesting molecules by thiourea bridging. The
can be easily distinguished from one another, 1-NCS res-
onating at 144.7 and 6-NCS at 135.1 ppm.21 When 20 was
stirred with the diazirine-labeled amine 4 the NMR signal
method is orthogonal to ligand clustering based on the
at 144.7 ppm disappeared indicating that only the anomer-
graded reactivity of different NCS groups and thus also al-
ic isothiocyanato function reacted as expected19 to deliver
lows to investigate multivalency effects using photola-
beled conjugates.
the thiourea derivative 21 in excellent yield.
Finally, 21 was treated with tris(2-aminoethyl)amine (22)
leading to the trivalent mannosyl cluster 23, which was
Optical rotations were measured with a Perkin-Elmer model 241
polarimeter (20 °C, 589 nm, length of cuvette: 1 dm). Reactions
deprotected according to Zemplén15 giving rise to 24
(Scheme 5). The somewhat low yield in the coupling step
is due to difficulties in separating the product from side
products.
were monitored by TLC on silica gel GF254 (Merck) with detection
under UV and by charring with 10% H2SO4 in EtOH and subse-
quent heating. Flash column chromatography was performed on sil-
ica gel 60 (40–63 mm, Merck). NMR spectra were recorded on
Bruker AMX 400 or Bruker DRX 500 instruments. Chemical shifts
This contribution has two essential take home messages:
1
(i) the amino-functionalized diazirine 4 is suited for the
are relative to TMS or the solvent peaks of CDCl3 (7.24 ppm H,
introduction into NCS-functionalized carbohydrates, e.g. 77.0 ppm 13C) or acetone-d6 (2.04 ppm H, 29.3 ppm 13C). Where
1
necessary, assignments were based on COSY and HSQC experi-
NCS
OAc
ments. IR spectra were taken with a Perkin Elmer FT-IR Paragon
1000 (KBr). MALDI-TOF mass spectra were measured with a
Bruker Biflex III with 19 kV acceleration voltage. DHB (c = 10 mg/
mL in 40% MeCN–H2O, 0.1% TFA) was used as the matrix. Ioniza-
tion was effected with a nitrogen laser at 337 nm. ESI mass spectra
were measured with an Applied Biosystems Mariner ESI-TOF
5280.
O
AcO
AcO
N
N
H
21
HN
N
S
N
N
HN
O
S
NH2
N
(2-Oxopropyl)carbaminic Acid tert-Butyl Ester (2)
i
22
NH
Di-tert-butyl dicarbonate (6.0 g, 28 mmol) was dissolved in MeOH
(20 mL) and ( )-1-aminopropan-2-ol (1; 1.8 mL, 1.72 g, 23 mmol)
was added slowly. The mixture was stirred for 1 h at r.t. Then, the
solvent was removed in vacuo and the residue taken up in CH2Cl2,
washed with conc. NaHCO3 solution and dried (Na2SO4). The crude
product was directly used in the following Swern oxidation. Oxalyl
chloride (2.2 mL, 3.2 g, 25 mmol) was dissolved in anhyd CH2Cl2
(25 mL) at –55 °C and a solution of DMSO (3.6 mL, 3.9 g, 51
mmol) in anhyd CH2Cl2 (6 mL) was slowly added. After 5 min, a
solution of the crude Boc-protected alcohol obtained from 1 in
CH2Cl2 (10 mL) was added dropwise to the reaction mixture which
was then stirred for 15 min at 0 °C. After the addition of Et3N (8
mL), the solution was warmed to r.t., washed with conc. NaHCO3
solution, dried (Na2SO4) and filtered. Flash column chromatogra-
phy (cyclohexane–EtOAc, 3:1) delivered the product as a yellowish
solid; yield: 3.7 g (93%); mp 48–49 °C.
NH2
RO
H2N
RO
NH
OR
S
N
NH
N
S
N
N
H
HN
NH
O
RO
HN
N
S
H
N
H
RO
RO
S
RO
RO
OR
O
23 R = Ac
24 R = H
ii
HN
S
1H NMR (300 MHz, CDCl3): d = 5.26 (br s, 1 H, NH), 4.04 (d,
JCHHNH = 4.8 Hz, 2 H, CH2), 2.19 (s, 3 H, CH3), 1.45 (s, 9 H, t-C4H9).
NH
N
N
13C NMR (75.47 MHz, CDCl3): d = 203.4 (C=O), 155.5
[OC(O)NH], 79.8 [C(CH3)3], 50.9 (CH2), 28.3 [C(CH3)3], 27.1
(CH3).
Scheme 5 Synthesis of the photoactive mannosyl cluster 24.
Reagents and conditions: (i) CH2Cl2, 2 h, 0 °C, 30%; (ii) 1 M
NaOMe/MeOH (0.1 equiv/OH), 1 h, 0 °C, 69%.
MS (EI): m/z = 173.1 [M]+ (calcd m/z = 173.11) for C8H15NO3.
Synthesis 2006, No. 6, 952–958 © Thieme Stuttgart · New York

PAPER
A Modular Approach to Diazirine Labeling
955
MS (CI): m/z = 174.1 [M + H]+ (calcd m/z = 174.12) for C8H16NO3.
62.0 (C-6), 47.9 (NHCH2), 25.3 [C(NN)], 20.8, 20.8, 20.7, 20.7 [4
C(O)CH3], 18.3 [C(NN)CH3].
Anal Calcd for C8H15NO3: C, 55.47; H, 8.73; N, 8.09. Found: C,
55.39; H, 8.79; N, 8.11
MS (CI): m/z = 475.1 [M
+
H]+ (calcd m/z = 475.14) for
C18H27N4O9S; m/z = 447.1 [M + H – N2]+ (calcd m/z = 447.13) for
(2-Azipropyl)carbaminic Acid tert-Butyl Ester (3)
C18H27N2O9S.
Ketone 2 (3.6 g, 21 mmol) was dissolved in a 5:1 mixture of anhyd
MeOH and CH2Cl2 (30 mL) and cooled to –50 °C. Ammonia (100
mL) was condensed into the solution and the mixture was stirred for
4 h at –20 °C. Then, a solution of hydroxylamine-O-sulfonic acid
(2.85 g, 25 mmol) in MeOH (10 mL) was added at –50 °C. After 1
h, the mixture was allowed to warm to r.t. and was stirred overnight.
Solids were removed by filtration and the filtrate was concentrated
and taken up in MeOH (20 mL). After the addition of Et3N (10 mL),
I2 (10% in MeOH) was added until the solution remained yellow.
Once more, the solution was concentrated and taken up in Et2O and
H2O. The aqueous phase was extracted with Et2O (2 ×) and the com-
bined organic phases were washed with Na2S2O3 solution and H2O.
After drying (Na2SO4) and filtration, column chromatography (cy-
clohexane–EtOAc, 8:1) delivered the product as a colorless solid;
yield: 2.6 g (70%).
IR (KBr): 1686 (C=O), 1534 cm–1 (N=N).
1H NMR (300 MHz, CDCl3): d = 4.47 (br s, 1 H, NH), 3.13 (d,
JCHHNH = 6.4 Hz, 2 H, CH2), 1.44 (s, 9 H, t-C4H9), 1.06 (s, 3 H, CH3).
13C NMR (300 MHz, CDCl3): d = 155.6 [OC(O)NH], 79.9
[C(CH3)3], 43.9 (CH2), 28.3 [C(CH3)3], 25.6 [C(NN)], 17.8 (CH3).
N-(2-Azipropyl)-N¢-[p-(2,3,4,6-tetra-O-acetyl-a-D-mannopy-
ranosyloxy)phenyl]thiourea (9)
Yield: 62%; fine yellowish amorphous solid; Rf 0.37 (cyclohexane–
EtOAc, 1:1); [a]D20 +57.1 (c = 0.86, CHCl3).
1H NMR (300 MHz, CDCl3): d = 7.92 (s, 1 H, ArNH), 7.17 (s, 4 H,
Harom), 5.65 (br t, 1 H, NHCH2), 5.58–5.52 (m, J1,2 = 1.8 Hz, 2 H, H-
1, H-3), 5.44 (dd, J2,3 = 3.5 Hz, 1 H, H-2), 5.39 (t, J3,4 = 10.1 Hz,
J4,5 = 10.1 Hz, 1 H, H-4), 4.30 (dd, J5,6 = 5.5 Hz, 1 H, H-6), 4.14–
4.05 (m, J5,6¢ = 2.4 Hz, J6,6¢ = 13.0 Hz, 2 H, H-5, H-6¢), 3.74 (d,
JNHCHH = 6.2 Hz, 2 H, NHCH2), 2.22, 2.07, 2.06, 2.05 [4 s, 4 × 3 H,
4 × C(O)CH3], 1.11 [s, 3 H, C(NN)CH3].
13C NMR (75.47 MHz, CDCl3): d = 181.9 (C=S), 170.5, 170.0,
170.0, 169.7 (C=O), 155.0 (man-OCarom), 130.3 (NHCarom), 127.7
[NHCarom(Cx,x¢)], 118.1 [man-OCarom(Ca,a¢)], 95.9 (C-1), 69.3, 69.2,
68.6, 65.8 (C-2 to C-5), 62.1 (C-6), 47.7 (NHCH2), 25.5 [C(NN)],
20.9, 20.7, 20.7, 20.7 [4 C(O)CH3], 18.2 [C(NN)CH3].
MS (CI): m/z = 539.2 [M + H – N2]+ (calcd m/z = 539.17) for
C24H31N2O10S.
Methyl 2,3,4-Tri-O-acetyl-6-(2¢-azipropylthioureido)-6-deoxy-
a-D-mannopyranoside (12)
MS (CI): m/z = 186.1 [M + H]+ (calcd m/z = 186.1) for C8H16N3O2.
MS (ESI): m/z = 186.1213 [M + H]+ (calcd m/z = 186.1164) for
Yield: 62%; slightly yellow amorphous solid; Rf 0.29 (cyclohex-
ane–EtOAc, 1:1); [a]D20 +60.8 (c = 0.72, CDCl3).
C8H15N3O2 + H.
1H NMR (300 MHz, CDCl3): d = 6.42 (br s, 1 H, NH), 6.34 (br s, 1
H, NH), 5.35 (dd, J2,3 = 3.3 Hz, J3,4 = 10.1 Hz, 1 H, H-3), 5.25 (dd,
J = 1.7, 3.3 Hz, 1 H, H-2), 5.16 (dd ~t, J = 10.1 Hz, 1 H, H-4), 4.78
(d, J1,2 = 1.7 Hz, 1 H, H-1), 3.90 (ddd, J5,6 = 5.5 Hz, J5,6¢ = 2.0 Hz, 1
H, H-5), 3.83–3.65 (m, J6,6¢ = 12.0 Hz, 2 H, H-6, H-6¢), 3.58 (dd,
JCHHNH = 5.7 Hz, JCHH = 15.3 Hz, 1 H, CHHNH), 3.44 (s, 3 H,
OCH3), 3.41–3.29 (m, 1 H, CHHNH), 2.17, 2.09, 2.00 [3 s, 3 × 3 H,
3 × C(O)CH3], 1.12 (s, 3 H, CH3).
13C NMR (75.47 MHz, CDCl3): d = 184.4 (C=S), 170.4, 170.1,
169.9 (C=O), 98.6 (C-1), 69.6, 69.2, 68.53, 67.0 (C-2 to C-5), 55.6
(OCH3), 47.7 (CH2), 45.0 (C-6), 25.47 [C(NN)], 20.9, 20.8, 20.7 [3
C(O)CH3], 18.1 (CH3).
MS (EI): m/z = 418.1 [M – N2]+ (calcd m/z = 418.15) for
C17H26N2O8S.
MS (CI): m/z = 419.1 [M + H – N2]+ (calcd m/z = 419.15) for
C17H27N2O8S; m/z = 447.1 [M + H]+ (calcd m/z = 447.15) for
C17H27N4O8S.
1-Amino-2-azipropane Hydrochloride (4)
The diazirine 3 (150 mg, 0.8 mmol) was dissolved in Et2O (1 mL),
conc. HCl (0.5 mL) was added and the mixture was stirred for 30
min at r.t. Then the solution was concentrated and lyophilized;
yield: 94 mg (99%).
1H NMR (300 MHz, D2O): d = 2.94 (s, 2 H, CH2), 1.13 (s, 3 H,
CH3).
13C NMR (75.47 MHz, D2O): d = 47.6 (CH2), 26.9 [C(NN)], 20.5
(CH3).
Diazirine-Labeled Mannosyl Derivatives 6, 9, 12, 15 by Thio-
urea Bridging; General Procedure
To a solution of the isothiocyanato-functionalized mannose deriva-
tive (1.1 equiv) in anhyd CH2Cl2 (10 mL), was added dropwise a so-
lution of 1-amino-2-azipropane hydrochloride 4 (1 equiv) in DIPEA
(50 mL) and anhyd CH2Cl2 (1 mL). The course of the reaction was
monitored by TLC (cyclohexane–EtOAc). After completion of the
reaction (2 h), the solvent was removed in vacuo and the crude prod-
uct was purified by flash column chromatography with the eluents
described.
4-Nitrophenyl 2,3,4-Tri-O-acetyl-6-(2¢-azipropylthioureido)-6-
deoxy-a-D-mannopyranoside (15)
Yield: quantitative; yellowish amorphous solid; Rf 0.28 (cyclohex-
N-(2-Azipropyl)-N¢-(2,3,4,6-tetra-O-acetyl-a-D-mannopyran-
osyl)thiourea (6)
ane–EtOAc, 1:1); [a]D20 +93.9 (c = 0.82, CHCl3).
1H NMR (300 MHz, CDCl3): d = 8.24 (d, J = 9.3 Hz, 2 H, Hx,x¢),
7.23 (d, Ja,x = Ja¢,x¢ = 9.3 Hz, 2 H, Ha,a¢), 6.32 (br t, 1 H, C6-NH), 6.01
(CH2NH), 5.71 (d, J1,2 = 1.9 Hz, 1 H, H-1), 5.57 (dd, J2,3 = 3.4 Hz,
J3,4 = 10.1 Hz, 1 H, H-3), 5.47 (dd, J = 1.9, 3.4 Hz, 1 H, H-2), 5.28
(t, J4,5 = 10.1 Hz, 1 H, H-4), 4.05–3.86 (m, 2 H, H-6, H-6¢), 3.77–
3.58 (m, 1 H, H-5), 3.55–3.30 (m, JCHHNH = 5.7 Hz, 2 H, CH2), 2.23,
2.12, 2.06 [3 s, 3 × 3 H, 3 × C(O)CH3], 1.08 (s, 3 H, CH3).
13C NMR (75.47 MHz, CDCl3): d = 183.9 (C=S), 170.5, 169.99,
170.0 (C=O), 160.0 (man-OCarom), 143.1 (O2NCarom), 126.0
[O2NCarom(Cx,x¢)], 116.5 [man-OCarom(Ca,a¢)], 95.4 (C-1), 71.0, 68.8,
68.0, 66.5 (C-2 to C-5), 47.2 (CH2NH), 44.6 (C-6), 25.53 [C(NN)],
20.8, 20.8, 20.7 [3 C(O)CH3], 18.0 (CH3).
Yield: 83%; fine colorless amorphous solid; Rf 0.27 (cyclohexane–
EtOAc, 1:1); [a]D20 +58.9 (c = 0.95, CHCl3).
1H NMR (300 MHz, CDCl3): d = 7.26 (br s, 1 H, man-NH), 6.71 (br
t, 1 H, NHCH2), 5.32–5.16 (m, J1,2 = 1.5 Hz, J3,4 = 9.3 Hz, 4 H, H-
1 to H-4), 4.29 (dd, J5,6 = 5.7 Hz, 1 H, H-6), 4.11 (dd, J5,6¢ = 2.6 Hz,
J
6,6¢ = 12.5 Hz, 1 H, H-6¢), 4.01 (ddd, J4,5 = 9.3 Hz, H, H-5), 3.65
2
3
(dq, JCHH¢ = 15.0, JNHCHH¢ = 5.7 Hz, 2 H, NHCH2), 2.12, 2.06,
2.02, 1.98 [4 s, 4 × 3 H, 4 × C(O)CH3].
13C NMR (75.47 MHz, CDCl3): d = 184.8 (C=S), 171.3, 170.2,
169.9, 169.6 (C=O), 80.6 (C-1), 69.8, 68.6, 68.1, 65.7 (C-2 to C-5),
Synthesis 2006, No. 6, 952–958 © Thieme Stuttgart · New York

956
M. Walter, T. K. Lindhorst
PAPER
MS (EI): m/z = 525.1 [M – N2]+ (calcd m/z = 525.15) for
C22H27N3O10S.
4-Nitrophenyl-6-deoxy-6-(2¢-azipropylthioureido)-a-D-man-
nopyranoside (16)
Yield: 91%; colorless amorphous solid; Rf 0.72 (EtOAc–MeOH,
MS (CI): m/z = 526.1 [M + H – N2]+ (calcd m/z = 526.15) for
C22H28N3O10S.
1:1); [a]D20 +84.5 (c = 1.01, MeOH).
1H NMR (500 MHz, CD3OD): d = 8.22 (d, J = 9.3 Hz, 2 H, Hx,x¢),
7.25 (d, J = 9.3 Hz, 2 H, Ha,a¢), 5.65 (d, J1,2 = 1.9 Hz, 1 H, H-1), 4.05
(dd, J = 1.9, 3.3 Hz, 1 H, H-2), 3.90 (dd, J2,3 = 3.3 Hz, J3,4 = 9.0 Hz,
1 H, H-3), 3.86–3.57 (m, 4 H, H-4, H-5, H-6, H-6¢), 3.55–3.38 (m,
2 H, CH2), 0.99 (s, 3 H, CH3).
13C NMR (125.75 MHz, CD3OD): d = 185.1 (C=S), 162.5 (man-
OCarom), 143.9 (O2NCarom), 126.8 [O2NCarom(Cx,x¢)], 117.85 [man-
OCarom(Ca,a¢)], 99.9 (C-1), 74.1, 71.7, 71.4, 69.1 (C-2 to C-5), 48.0
(CH2), 46.2 (C-6), 26.7 [C(NN)], 18.2 (CH3).
Deacetylation of Thiourea-Bridged Diazirine-Labeled Manno-
syl Derivatives 7, 10, 13, 16; General Procedure
To a solution of the protected sugar (1 equiv), was added NaOMe
(1 M, 0.1 equiv per hydroxy group) and the mixture was stirred for
0.5 h at r.t. or 0 °C. After complete removal of the acetyl groups, the
solution was neutralized with Amberlite ion exchange resin IR 120.
It was filtered, washed with MeOH and the solvent removed in vac-
uo. Wherever necessary, the crude product was purified by HPLC.
ESI-MS: m/z = 450.1054 [M + Na]+ (calcd m/z = 450.1054) for
C16H21N5O7S + Na.
N-(2-Azipropyl)-N¢-(a-D-mannopyranosyl)thiourea (7)
Yield: 97%; colorless amorphous solid; Rf 0.64 (EtOAc–MeOH,
1:1); [a]D20 +82.6 (c = 1.13, MeOH).
1H NMR (500 MHz, CD3OD): d = 5.50 (br s, 1 H, H-1), 3.92 (br t,
1 H, H-2), 3.85 (dd, J6,6¢ = 11.9 Hz, 1 H, H-6¢), 3.79 (dd, J = 6.4,
11.9 Hz, 1 H, H-6), 3.73 (dd, J2,3 = 3.1 Hz, J3,4 = 9.0 Hz, 1 H, H-3),
3.70–3.62 (m, 3 H, H-4, CH2), 3.50 (ddd, J4,5 = 9.0 Hz, J5,6 = 6.4 Hz,
1,2,3,4-Tetra-O-acetyl-6-deoxy-6-isothiocyanato-D-mannopy-
ranose (18)
To a solution of the azide 17 (4.0 g, 0.01 mol) in anhyd toluene (30
mL), were added CS2 (25.9 mL, 32.6 g, 0.43 mol) and P(OEt)3 (7.4
mL, 7.1 g, 0.04 mol) and the mixture was stirred under reflux at
80 °C for 8 h. Then it was quenched with H2O (10 mL) and the mix-
ture was left in the refrigerator overnight. Then, the aqueous phase
was extracted with CH2Cl2 (2 ×) and the combined organic phases
were washed with H2O (2 ×). After drying (Na2SO4) and filtration,
column chromatography (cyclohexane–EtOAc, 3:2) delivered the
product as a colorless syrup; yield: 3.16 g (76%).
1H NMR (300 MHz, CDCl3): d = 6.12 (d, J1,2 = 2.0 Hz, 1 H, H-1a),
5.80 (d, J1,2 = 1.2 Hz, 1 H, H-1b), 5.48 (dd, J = 1.2, 3.1 Hz, 1 H, H-
2b), 5.38–5.33 (m, 2 H, H-2a, H-4a), 5.27–5.22 (m, 1 H, H-3a), 5.14
(dd, J2,3 = 3.1 Hz, J3,4 = 9.9 Hz, 1 H, H-3b), 4.00 (ddd, J4,5 = 9.3 Hz,
J5,6 = 4.5 Hz, J5,6¢ = 3.5 Hz, 1 H, H-5a), 3.74 (ddd, J = 3.4, 4.4, 9.3
Hz, 1 H, H-5b), 3.71 (dd, J6,6¢ = 15.1 Hz, 1 H, H-6¢a), 3.58 (dd, J =
4.5, 15.1 Hz, 1 H, H-6a), 2.20, 2.18, 2.09, 2.03 [4 s, 4 × 3 H, 4 ×
C(O)CH3,a], 2.23, 2.12, 2.04, 2.02 [4 s, 4 × 3 H, 4 × C(O)CH3,b];
anomeric ratio: a:b = 9:1.
J5,6¢ = 2.7 Hz, 1 H, H-5), 1.11 (s, 3 H, CH3).
13C NMR (125.75 MHz, CD3OD): d = 186.0 (C=S), 83.7 (C-1),
76.0 (C-5), 72.5 (C-3), 71.2 (C-2), 68.8 (C-4), 62.6 (C-6), 48.2
(CH2), 26.6 [C(NN)], 18.3 (CH3); JC-1,H-1 = 163.4 Hz.
ESI-MS: m/z = 279.1029 [M + H – N2]+ (calcd m/z = 279.1015) for
C10H19N2O5S; m/z = 329.0951 [M + Na]+ (calcd m/z = 329.0890)
for C10H18N4O5S + Na.
N-(2-Azipropyl)-N¢-[4-(a-D-mannopyranosyloxy)phenyl]thio-
urea (10)
Yield: quantitative; colorless amorphous solid; Rf 0.68 (EtOAc–
MeOH, 2:1); [a]D20 +74.5 (c = 1.28, MeOH).
1H NMR (500 MHz, CD3OD): d = 7.22 (d, Ja,x¢ = Ja¢,x¢ = 9.1 Hz, 2 H,
Ha,a¢), 7.18 (d, J = 9.1 Hz, 2 H, Hx,x¢), 5.52 (d, J1,2 = 1.8 Hz, 1 H, H-
1), 4.05 (dd, J = 1.8, 3.4 Hz, 1 H, H-2), 3.94 (dd, J2,3 = 3.4 Hz,
J3,4 = 9.6 Hz, 1 H, H-3), 3.81 (dd, J6,6¢ = 12.0 Hz, 1 H, H-6¢), 3.80–
3.73 (m, 2 H, H-4, H-6), 3.66 (s, 2 H, CH2) 3.63 (ddd, J4,5 = 9.6 Hz,
J5,6 = 5.3 Hz, J5,6¢ = 2.5 Hz, 1 H, H-5), 1.10 (s, 3 H, CH3).
13C NMR (75.47 MHz, CDCl3): d = 170.0, 169.9, 169.6, 168.0
(C=O), 90.2 (C-1), 70.9, 68.3, 68.1, 66.4 (C-2 to C-5), 45.8 (C-6),
20.8, 20.8, 20.6, 20.6 [4 C(O)CH3].
13C NMR (125.75 MHz, CD3OD): d = 183.4 (C=S), 156.2 (man-
OCarom), 133.3 (NHCarom), 128.0 [NHCarom(Cx,x¢)], 118.5 [man-
OCarom(Ca,a¢)], 100.2 (C-1), 75.4, 72.3, 71.9, 68.3 (C-2 to C-5), 62.6
(C-6), 47.8 (CH2), 26.9 [C(NN)], 18.4 (CH3).
ESI-MS: m/z = 371.1529 [M + H – N2]+ (calcd m/z = 371.1277) for
C16H23N2O6S; m/z = 421.1438 [M + Na]+ (calcd m/z = 421.1152)
for C16H22N4O6S + Na.
2,3,4-Tri-O-acetyl-6-deoxy-6-isothiocyanato-a-D-mannopyra-
nosyl bromide (19)
Mannose derivative 18 (3.0 g, 8 mmol) was dissolved in HBr–
AcOH (33%, 20 mL) and stirred with Ac2O (2 mL) for 1.5 h. Then,
the mixture was diluted with toluene (150 mL) and poured onto ice
(200 g). The phases were separated, the aqueous phase was extract-
ed with CH2Cl2 (2 ×) and the combined organic phases were washed
with aq sat. NaHCO3 solution (2 ×) and dried (Na2SO4). After filtra-
tion and removal of the solvent in vacuo, the title compound was ob-
tained as brown syrup. The product was moisture and light sensitive
and therefore used in the next step without further purification;
yield: 2.15 g (68%); [a]D20 +123.3 (c = 1.06, CHCl3).
Methyl 6-(2¢-Azipropylthioureido)-6-deoxy-a-D-mannopyrano-
side (13)
Yield: 98%; colorless amorphous solid; Rf 0.66 (EtOAc–MeOH,
1:1); [a]D20 +13.1 (c = 0.60, MeOH).
1H NMR (500 MHz, CD3OD): d = 4.67 (br s, 1 H, H-1), 3.95–3.83
(br m, 1 H, H-6), 3.83 (dd, J1,2 = 1.6 Hz, 1 H, H-2), 3.70 (dd,
J2,3 = 3.3 Hz, J3,4 = 9.5 Hz, 1 H, H-3), 3.68–3.53 [m, J4,5 = 9.5 Hz, 5
H, H-4, H-5, H-6¢, CH2C(NN)], 3.40 (s, 3 H, OCH3), 1.09 (s, 3 H,
CH3).
13C NMR (125.75 MHz, CD3OD): d = 102.9 (C-1), 72.7 (C-5), 72.0
(C-3), 71.9 (C-2), 69.4 (C-4), 55.3 (OCH3), 48.2 [CH2C(NN)], 26.7
[C(NN)], 18.2 (CH3).
1H NMR (300 MHz, CDCl3): d = 6.32 (d, J1,2 = 1.7 Hz, 1 H, H-1),
5.75 (dd, J2,3 = 3.4 Hz, J3,4 = 10.1 Hz, 1 H, H-3), 5.47–5.36 (m, 2 H,
H-2, H-4), 4.16 (ddd, J4,5 = 10.1 Hz, J5,6 = 4.4 Hz, J5,6¢ = 3.3 Hz, 1
H, H-5), 3.77 (dd, J6,6¢ = 15.3 Hz, 1 H, H-6¢), 3.63 (dd, J = 4.4, 15.3
Hz, 1 H, H-6), 2.21, 2.11, 2.03 [3 s, 3 × 3 H, 3 × C(O)CH3].
13C NMR (75.47 MHz, CDCl3): d = 169.71 169.7, 169.6 (C=O),
135.2 (NCS), 82.4 (C-1), 72.7, 71.8, 67.6, 66.1 (C-2 to C-5), 45.2
(C-6), 20.8, 20.7, 20.6 [C(O)CH3].
ESI-MS: m/z = 343.1046 [M + Na]+ (calcd m/z = 343.1047) for
C11H20N4O5S + Na.
2,3,4-Tri-O-acetyl-6-deoxy-6-isothiocyanato-a-D-mannopyra-
nosyl Isothiocyanate (20)
The mannosyl bromide 19 (1.51 g, 3.7 mmol) was dissolved in
CH2Cl2, KSCN (1.2 g, 0.01 mol, 10 equiv) was added and then
Synthesis 2006, No. 6, 952–958 © Thieme Stuttgart · New York

PAPER
A Modular Approach to Diazirine Labeling
957
20
CH2Cl2 was evaporated in vacuo. The remaining mixture was heat-
ed to 190 °C for 8 min. The cooled melt was dissolved in a 1:1 mix-
ture of H2O and CH2Cl2 (60 mL) and the aqueous phase was
extracted with CH2Cl2 (2 ×). The combined organic phases were
washed with H2O (2 ×). After drying (Na2SO4) and filtration, col-
umn chromatography (cyclohexane–EtOAc, 3:1) afforded the prod-
uct as an amorphous colorless solid; yield: 1.06 g (74%); [a]D
+116.6 (c = 0.9, CHCl3).
IR (KBr): 2069, 1990 cm–1 (2 NCS).
amorphous solid; yield: 23.5 mg (30%); [a]D +99.8 (c = 1.15,
CHCl3).
1H NMR (500 MHz, acetone-d6): d = 8.16 (br s, 3 H, 3 man-NH),
7.45, 7.29, 6.95, (3 br s, 3 × 3 H, 9 × NH), 6.02 (br s, 3 H, 3 H-1),
5.39–5.22 (m, 6 H, 3 H-2, 3 H-3), 5.08 (t, J = 7.3 Hz, 3 H, 3 H-4),
4.24–4.14 (br m, 3 H, 3 H-6¢), 4.09–4.01 (br m, J3,4 = 7.3 Hz,
J4,5 = 7.3 Hz, 3 H, 3 H-5), 3.81–3.42 [br m, 15 H, 3 H-6, 3
NHCH2CH2N, 3 CH2C(NN)], 2.84–2.60 (br m, 6 H, 3 NCH2), 2.12,
2.11, 2.04 [3 s, 3 × 9 H, 9 × C(O)CH3], 1.08 (s, 9 H, 3 CH3).
20
1H NMR (300 MHz, CDCl3): d = 5.6 (d, J1,2 = 2.1 Hz, 1 H, H-1),
5.4–5.3 (m, 3 H, H-2, H-3, H-4), 4.1 (ddd, J4,5 = 8.3 Hz, J5,6 = 4.6
Hz, J5,6¢ = 3.3 Hz, 1 H, H-5), 3.8 (dd, J6,6¢ = 15.1 Hz, 1 H, H-6¢), 3.6
(dd, J = 4.6, 15.1 Hz, 1 H, H-6), 2.2, 2.1, 2.0 [3 s, 3 × 3 H, 3 ×
C(O)CH3].
13C NMR (125.75 MHz, acetone-d6): d = 185.5, 184.1 (6 C=S),
170.3, 170.2, 170.1 (9 C=O), 79.5 (3 C-1), 73.3 (3 C-5), 69.4 (3 C-
2), 68.1 (3 C-3), 68.8 (3 C-4), 53.9 (3 NCH2), 48.0 [3 CH2C(NN)],
45.0 (3 C-6), 42.9 (3 NCH2CH2), 26.4 [3 C(NN)], 20.9, 20.9, 20.6,
[9 C(O)CH3], 18.3 (3 CH3).
13C NMR (300 MHz, CDCl3): d = 169.9, 169.7, 169.6 (C=O), 144.7
(1-NCS), 135.1 (6-NCS), 82.6 (JC-1,H-1 = 175.2 Hz, C-1), 71.5, 69.4,
68.0, 66.1 (C-2 to C-5), 45.6 (C-6), 20.8, 20.7, 20.6 [3 C(O)CH3].
ESI-MS: m/z = 1588.4900 [M + Na] + (calcd m/z = 1588.4540) for
C57H87N19O21S6 + Na.
Tris-{2-[3¢-(1¢¢,6¢¢-dideoxy-1¢¢-(2¢¢¢-azipropylthioureido)-a-D-
mannopyranos-6¢¢-yl)thioureido]ethyl}amine (24)
MS (EI): m/z = 330.0 [M – NCS]+ (calcd m/z = 330.0) for
C13H16NO7S.
The protected glycocluster 23 (23 mg, 15 mmol) was dissolved in
anhyd MeOH (500 mL) and treated with NaOMe (1 M, 14 mL, 0.1
equiv per hydroxy group) at 0 °C. The mixture was stirred at this
temperature for 1 h, then Amberlite ion exchange resin IR 120 was
added for neutralization. The mixture was filtered and after removal
of the solvent in vacuo, the unprotected title compound remained as
a colorless amorphous solid, which was pure according to its NMR
MS (CI): m/z = 388.9 [M
+
H]+ (calcd m/z = 389.0) for
C14H17N2O7S2.
Anal. Calcd for C14H16N2O7S2: C, 43.29; H, 4.15; N, 7.21. Found:
C, 43.69; H, 4.25; N, 7.22
N-(2-Azipropyl)-N¢-(2,3,4-tri-O-acetyl-6-deoxy-6-isothiocy-
anato-a-D-mannopyranosyl)thiourea (21)
20
spectra and not further purified; yield: 12 mg (69%); [a]D +7.0
(c = 0.63, MeOH).
The di-NCS functionalized sugar 20 (210 mg, 0.54 mmol) was dis-
solved in anhyd CH2Cl2 (3 mL) and a solution of the diazirine 4 (66
mg, 0.54 mmol) in a mixture of anhyd CH2Cl2 (1 mL) and DIPEA
(80 mL) was added dropwise. The mixture was stirred for 2 h at r.t.,
then the solvent was removed in vacuo and the residue was purified
by flash chromatography (cyclohexane–EtOAc, 1:1) to give a yel-
lowish solid; yield: 224 mg (88%); [a]D20 +51.5 (c = 2.28, CHCl3).
1H NMR (500 MHz, CD3OD): d = 5.72 (br s, 3 H, 3 H-1), 3.94 (t =
approx. dd, J2,3 = 2.9 Hz, 3 H, 3 H-2), 3.82–3.52 [m, 27 H, 3 H-3, 3
H-4, 3 H-5, 3 H-6, 3 H-6¢, 3 NCH2CH2, 3 CH2C(NN)], 2.90–2.76
(br m, 6 H, 3 NCH2), 1.12 (s, 9 H, CH3).
13C NMR (125.75 MHz, CD3OD): d = 185.9 (6 C=S), 83.8 (3 C-1),
74.7 (3 C-5), 71.9 (3 C-3), 71.3 (3 C-2), 69.0 (3 C-4), 54.6 (3
NCH2), 48.1 [3 CH2C(NN)], 45.8 (3 C-6), 43.8 (3 NCH2CH2), 26.8
[3 C(NN)], 18.5 (3 CH3).
IR (KBr): 2118 (NCS), 1543 cm–1 [C(NN)].
1H NMR (500 MHz, CDCl3): d = 7.27 (br s, 1 H, man-NH), 6.64 (br
t, 1 H, CH2NH), 5.38–5.33 (m, J1,2 = 1.6 Hz, 2 H, H-1, H-2), 5.31
(dd, J2,3 = 3.0 Hz, J3,4 = 9.7 Hz, 1 H, H-3), 5.25 (t, J4,5 = 9.7 Hz, 1
H, H-4), 4.02 (dt, J = 4.6, 9.7 Hz, 1 H, H-5), 3.88 (dd, JCHHNH = 5.9
Hz, JCHH = 15.0 Hz, 1 H, CHHNH), 3.79 (dd, J = 5.9, 15.0 Hz, 1 H,
CHHNH), 3.74 (d, J5,6 = 4.6 Hz, 2 H, H-6, H-6¢), 2.21, 2.11, 2.05 [3
s, 3 × 3 H, 3 × C(O)CH3], 1.15 (s, 3 H, CH3).
MS (ESI): m/z = 1188.3686 [M + H]+ (calcd m/z = 1188.3770) for
C39H70N19O12S6; m/z = 1210.3621 [M + Na] + (calcd m/z =
1210.3590) for C39H69N19O12S6 + Na.
Acknowledgment
13C NMR (125.75 MHz, CDCl3): d = 184.6 (C=S), 170.3, 169.9,
169.8 (C=O), 134.8 (NCS), 80.8 (C-1), 69.5, 68.3, 68.0, 66.6 (C-2
to C-5), 47.6 (CH2NH), 45.8 (C-6), 25.5 [C(NN)], 20.8, 20.7, 20.7
[3 C(O)CH3], 18.4 (CH3).
MS (EI): m/z = 445.1 [M – N2]+ (calcd m/z = 445.10) for
C17H23N3O7S2.
MS (CI): m/z = 446.2 [M + H – N2]+ (calcd m/z = 446.11) for
C17H24N3O7S2; m/z = 474.2 [M + H]+ (calcd m/z = 474.11) for
C17H24N5O7S2.
MS (ESI): m/z = 496.0855 [M + Na]+ (calcd m/z = 496.0931) for
C17H23N5O7S2 + Na.
This work was supported by the DFG (SFB 470).
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Synthesis 2006, No. 6, 952–958 © Thieme Stuttgart · New York

958
M. Walter, T. K. Lindhorst
PAPER
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Synthesis 2006, No. 6, 952–958 © Thieme Stuttgart · New York