Strategy for catch and release of azide-tagged biomolecules utilizing a photolabile strained alkyne construct

Article abstract of DOI:10.1039/c2ob25440a

A photolabile o-nitrobenzyl linker-cyclooctyne conjugate was prepared, immobilized on poly(methacrylate) beads and utilized as a trap for azide-functionalized compounds. These could be released by 365 nm UV light irradiation in high yield and purity. The reagent-free and time economic catch and release protocol was deemed useful for chemical proteomics applications.

Full text of DOI:10.1039/c2ob25440a

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Electronic Supporting Information
Strategy for catch and release of azide-tagged biomolecules utilizing a
photolabile strained alkyne construct
M
artin Golkowski, Carlo Pergola, Oliver Werz, and Thomas Ziegler*
Table of contents
:
1. General methods and Catch and release experiments
2. Synthesis and characterization of the compounds 5, 7, 9, 3a and 10, 11
3. References
2-3
3-8
8
4. NMR-Spectra of all compounds prepared
9-14

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1. General methods
All chemicals were used as received unless noted otherwise. Dry solvents were prepared
according to standard methods (DMF: P2O5; DIPEA: CaH2; THF: sodium/benzophenone;
MeOH: Mg), distilled and stored over molecular sieves 3 Å under an atmosphere of nitrogen
until used.
NMR-spectra were recorded on either a Bruker Avance 400 or a Bruker ACX 250
spectrometer and calibrated for TMS (0.0 ppm) or the solvent-signal (1H-CDCl3: 7.26 ppm;
1
1
13C-CDCl3: 77.16 ppm; H-MeOH-d4: 3.31 ppm; 13C-MeOH-d4: 49.00 ppm, H-DMSO-d6:
2.50 ppm; 13C-DMSO-d6: 39.52 ppm; H-benzene-d6: 7.16 ppm; 13C-benzene-d6: 128.39
1
ppm).
FT-ICR-MS spectra were recorded on a Bruker Apex II FT-ICR-MS (FAB) spectrometer and
FAB-spectra on a Finnigan model TSQ 70. Optical rotations were measured with a Perkin-
Elmer Model 341 polarimeter. Melting points were determined with a Büchi Melting Point
M-560 and are uncorrected. Elemental analysis was performed on a HEKAtech Euro EA
Analyzer. IR-spectra were recorded on a Bruker Tensor 27. HPLC analysis was performed
with either a Thermo Betasil C8 column or a ZORBAX Eclipse XDB-C8 column with a flow
rate of 1.5 ml/min. of methanol/KH2PO3 pH 2.3 buffer. TLC-analysis was performed with
Polygram SIL G/UV pre-coated polyester sheets (Macherey-Nagel). UV-spectra for
photometry were recorded on a Perkin-Elmer Lambda 25 UV-Vis spectrometer using 1 cm
quartz cuvettes. Absorbance was measured at 266.8 nm.
Dibenzocyclooctynol 217 and the corresponding pNP-carbonate 8,11b nitroaromatic 4,9
ethylene glycol linker 620 were prepared following the published procedures.
For irradiation of the photolabile samples a 150 W medium pressure mercury lamp from UV-
Consulting Peschl Model TQ 150 was used. For elimination of UV light <350 nm a filter
solution composed of 2 M aq. KNO3 containing 0.02 w% 5,7-dimethyl-3,6-dihydro-2H-1,4-
diazepinium perchlorate was placed between light source and sample.21 The filter solutions
layer thickness was adjusted to ~2 cm and the samples placed perpendicular and as close as
possible to the light bulb. The filter solution was replaced with a freshly prepared one every
12 h of irradiation.
Immobilization of 3 on TOYOPEARL-AF-650-amino: 1 ml of a TOYOPEARL-AF-650-amino
suspension in aq. EtOH was transferred to a 5 ml PP-syringe with inserted PE-frit (35 µm
pore size) and washed successively 3 times with H2O and 5 times with dry MeCN. After re-
suspending the beads in 3 ml dry MeCN, 21.4 mg of pNP-ester 3a (25 µmol) and 13.1 µl
DIPEA (75 µmol) were added and the mixture shaken at 1500 rpm and rt for 72 h.
Subsequently 94.5 µl Ac2O (1 mmol, 10 eq.) and 172.3 µl DIPEA (1 mmol, 10 eq.) were
added and the mixture shaken as described for additional 12 h at RT. Then the beads were
washed successively 3 times each with DMSO and water. For storage at 4°C the alkyne
functionalized beads were suspended in 20 % aqueous ethanol.
Capture of azide 10 onto the alkyne functionalized solid support: The alkyne functionalized
beads from above were washed 3 times with dioxane/PBS 2:3 v/v and then re-suspended in 3
ml of the named solvent mixture containing 31,3 mg of azide 10 (50 µmol, 2 eq.). The
resulting suspension was shaken at RT or 0°C and 1500 rpm for 3 h and the reaction progress
tracked photometrically. After 30 min. no more progression of the reaction was observed.

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Afterwards the beads were washed 5 times with dioxane/PBS 2:3 v/v, 3-times with water, re-
suspended in 20 % aqueous ethanol and stored at 4°C until used further. Quantitative analysis
of the UV-spectra and subtraction of a relative amount of 10 non-specifically bound to the
beads (see below) indicated an effective loading of the beads with 12.5 µmol of the
corresponding triazole (uncorrected 14.7 µmol).
Non-specific binding of azide 10 on 2-azidoethanol-blocked alkyne functionalized solid
support: The alkyne functionalized beads from above were washed 3 times with dioxane/PBS
2:3 v/v and then re-suspended in 3 ml of the named solvent mixture containing 100 mg of 2-
azidoethanol (1.15 mmol, 46 eq. based on 3a). The resulting suspension was shaken at rt and
1500 rpm for 12 h. Afterwards the beads were washed 5 times with dioxane/PBS 2:3 v/v, 3-
times with water. Then the beads were re-suspended in 3 ml dioxane/PBS 2:3 v/v containing
31,3 mg of azide 10 (50 µmol, 2 eq.). The resulting suspension was shaken at RT and 1500
rpm for 90 min. and the reaction progress tracked photometrically. Quantitative analysis
revealed approx. 2.2 µmol non-specific binding to the blocked beads. The assumption was
confirmed by subjecting the beads to the cleavage conditions described below. No 11 could be
detected in the supernatant as indicated by HPLC-analysis, precluding that 10 was
immobilized due to reaction with residual non-blocked 3b.
UV light induced liberation of the immobilized model compound 11: The triazole-loaded
beads from above were washed 5-times either with dioxane/PBS 2:3 v/v or dioxane/Na-
phosphate pH 6.3 buffer 3:2 v/v containing 10 mM DTT. Then the beads were re-suspended in
10 ml of one of the named solvent mixtures, transferred to a transparent 20 ml PP-tube and
subjected to irradiation >350 nm for 4 h at RT while being shaken at 1500 rpm. The reaction
was monitored photometrically and after 120 min. and 40 min. no more progression of the
cleavage reaction could be observed, respectively. Quantitative analysis of the supernatant
indicated 85% recovery of triazole 11 in an HPLC-purity of 98.2 %.
2. Synthesis and characterization of the compounds 5, 7, 9, 3 and 10, 11
Scheme 5: Synthesis of model compound 10.
General Procedure for HBTU-mediated amide coupling A: In a round bottom flask equipped
with a gas inlet and a stirring bar the corresponding free base or ammonium derivative was
dissolved in dry DMF at a concentration of 0.18 M under an atmosphere of nitrogen. After
cooling the solution to 0°C HOBt (1.5 eq.), DIPEA (1.5 eq. for free bases; 3 eq. for
ammonium derivatives), the free carboxylic acid (1 eq.) and subsequently HBTU (1.5 eq.)
were added. Stirring was continued for 2 h at 0°C and at rt for 14 h. Then, the solution was
diluted with ethyl acetate, transferred to a separatory funnel, the organic layer separated and
washed twice each with 1 M NaHSO4-soln. and sat. NaHCO3-soln. and once with brine. After
drying the organic layer with Na2SO4 the solvent was removed under reduced pressure and

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the residue subjected to column chromatography, eluting with the solvent mixtures indicated
below.
4-[2-Methoxy-5-nitro-4-(1-aminoethyl)phenoxy]butanoicacid methyl ester hydrochloride 5: In
a 250 ml round bottom flask equipped with a gas inlet and a stirring bar 8.2 g of
trifluoroacetamide 49 (20.1 mmol) were dissolved in 100 ml of a 0.36 M soln. of HCl in dry
MeOH22 under an atmosphere of nitrogen. The resulting mixture was refluxed for 36 h,
cooled to rt and the volatiles removed in a stream of air. The residual white solid was
triturated with diethyl ether, filtered and crystallized from ethanol to yield 6.77 g of pure title
compound 5 (19.4 mmol, 97 %) as wooly colorless crystals. mp: 191.5°C (EtOH). Anal. calcd
for C14H21ClN2O6: N, 8.03; C, 48.21; H, 6.07; found: N, 7.87; C, 48.25; H, 6.21. IR (NaBr):
3440, 2924, 1739, 1582, 1525, 1339, 1281, 1225, 1053 cm-1. FAB-MS: m/z 313 [M-Cl-]+, 296
[M-NH3-Cl-]+. 1H-NMR (400.1 MHz, MeOH-d4): δ 7.68 (s, 1H, aryl), 7.32 (s, 1H, aryl), 5.13
(q, 1H, J = 6.8 Hz, CHNH4Cl), 4.14 (t, 2H, J = 6.1 Hz, aryl-OCH2), 4.02 (s, 3H, CH3), 3.68
(s, 3H, CH3), 2.55 (t, 2H, J = 7.3 Hz, CH2COOMe), 2.16-2.08 (m, 2H, CH2), 1.73 (d, 3H,
CH3CHNH4Cl). 13C-NMR (100.6 MHz, MeOH-d4): δ 175.3 (COOMe), 155.6, 149.8, 142.6,
128.2, 110.8, 110.6 (aryl), 69.6 (aryl-OCH2), 57.3 (aryl-OCH3), 52.2 (COOCH3), 47.6
(CHN), 31.2 (Bu-CH2), 25.5 (Bu-CH2), 20.0 (NCHCH3).
4-{4-[14-(tert.-Butoxycarbonyl)amino-4-oxo-6,9,12-trioxa-3-azatetradec-2-yl]-2-methoxy-5-
nitrophenoxy}butanoic acid methyl ester 7: In a 50 ml round bottom flask equipped with a
stirring bar 922 mg of methyl ester 620 (2.87 mmol) were saponified with 1 M aq. NaOH in
methanol at rt for 1 h. The mixture was acidified with solid NaHSO4, transferred to a
separatory funnel and extracted 5 times with DCM. The combined organic layers were dried
with Na2SO4 and the solvent evaporated. The crude free acid was dissolved in 15 ml dry THF
and transferred to a vessel equipped with a gas inlet and a stirring bar. Then 504 mg HOBt
(3.73 mmol, 1.3 eq.) were added, and the solution cooled to -20°C. Subsequently, 770 mg
DCC (3.73 mmol, 1.3 eq.) were added and the solution stirred for 6 h at -20°C. Thereafter, 1 g
of nitroaromatic 5 (2.87 mmol, 1 eq.) was added together with 1.5 ml DIPEA (8.61 mmol, 3
eq.) and the mixture stirred for additional 14 h at rt. Then the precipitate of DC-urea was
filtered off, the mixture transferred to a separatory funnel and diluted with ethyl acetate. The
organic layer was separated and washed twice with 1 M NaHSO4-soln., once each with sat.
NaHCO3-soln. and brine, dried over Na2SO4 and the solvent removed under reduced pressure.
The residual viscous yellow oil was subjected to column chromatography eluting with CHCl3

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containing 1 v% MeOH yielding 1.52 g of pure title compound 7 (2.60 mmol, 91 %) as pale
yellow viscous oil. Rf: 0.32 (CHCl3+v1% MeOH). IR (neat): 3357, 2974, 2935, 2875, 1712,
1518, 1456, 1366, 1274, 1176, 870, 758 cm-1. FT-ICR-MS: m/z [M+Na]+ calcd for
1
C27H43N3O12Na: 624.2739 found: 624.2743. H-NMR (400.1 MHz, CDCl3): δ 7.63-7.57 (m,
1H, CONH), 7.52 (s, 1H, aryl), 6.96 (s, 1H, aryl), 5.65-5.56 (m, 1H, aryl-CHN), 4.96 (s,
broad, 1H, OCONH), 4.06 (t, 2H, J = 6.2 Hz, aryl-OCH2), 4.01 (d, 1H, J = -15.8 Hz,
OCH2CON), 3.89 (s, 3H, CH3), 3.89 (d, 1H, J = -15.8 Hz, OCH2CON), 3.74-3.58 (m, 8 H,
EG-chain-CH2), 3.66 (s, 3H, CH3), 3.48 (t, 2H, J = 5.2 Hz, EG-chain-CH2), 3.27-3.21 (m, 2H,
CH2NHBoc), 2.51 (t, 2H, J = 7.2 Hz, CH2COOMe), 2.18-2.10 (m, 2H, CH2), 1.53 (d, 3H, J =
6.9 Hz, CH3CHN), 1.40 (s, 9H, tBu). 13C-NMR (100.6 MHz, CDCl3): δ 173.4 (COOMe),
169.3 (CON), 156.0 (OCONH), 153.9, 147.0, 140.6, 134.3, 110.4, 109.9 (aryl), 79.3 (tBu),
71.1, 70.6, 70.4, 70.4, 70.3, 70.2 (EG-CH2), 68.3 (aryl-OCH2), 56.4 (aryl-OCH3), 51.7
(COOCH3), 46.1 (CHN), 40.4 (CH2NHBoc), 30.4 (Bu-CH2), 28.5 (tBu), 24.3 (Bu-CH2), 21.4
(aryl-NCHCH3).
4-{4-[14-(11,12-didehydro-5,6-dihydrodibenzo[a,e]cycloocten-5-oxycarbonylamino)-4-oxo-
6,9,12-trioxa-3-azatetradec-2-yl]-2-methoxy-5-nitrophenoxy}butanoic acid methyl ester 9: In
a 50 ml round bottom flask equipped with a stirring bar 1.45 g of nitroaromatic 7 (2.41
mmol) were N-Boc-deprotected using 20 v% TFA in dry DCM for 1 h and the volatiles
subsequently removed in a stream of air. After drying in vacuum over night the corresponding
ammonium derivative was dissolved in 10 ml dry DMF in a round bottom flask equipped with
a gas inlet and a stirring bar under an atmosphere of nitrogen. To the solution were added 1.26
ml DIPEA (7.23 mmol, 3 eq.) and 929 mg of activated carbonate 811b (2.41 mmol, 1 eq.) and
the mixture stirred at rt for 24 h. Thereafter the shiny yellow solution was diluted with ethyl
acetate, transferred to a separatory funnel, washed twice with 1 M NaHSO4-soln., three times
with 5% Na2CO3-soln. and once with brine, dried over Na2SO4 and the solvent removed under
reduced pressure. The residue was subjected to column chromatography eluting with CHCl3
containing 1.5 v% MeOH yielding 1.62 g of pure title compound 9 (2.17 mmol, 90%) as pale
yellow foam. Rf: 0.43 (CHCl3+v2% MeOH). IR (NaBr): 3365, 2934, 1729, 1672, 1520, 1336,
1273, 1216, 1104, 1029, 760 cm-1. FT-ICR-MS: m/z [M+Na]+ calcd for C39H45N3O12Na:
770.2896 found: 770.2894. 1H-NMR from the mixture of diastereomers (400.1 MHz, Benzol-
d6): δ 7.77 (t, 1H, J = 8.2 Hz, CONH), 7.71-7.62 (m, 1H, aryl), 7.34 (s, 1H, aryl), 7.30-7.11
(m, 4H, aryl), 7.08-6.91 (m, 4H, aryl), 6.41-6.34/5.83-5.74 (m/m, 1H, Bn-CHO), 6.35/5.91
(s/s, broad, 1H, OCONH), 6.05-5.94 (m, 1H, Bn-CHN), 4.10-2.71 (m, 22H), 3.90 (dd, 1H, J =
-15.7 Hz/7.3 Hz, Bn-CH2), 2.95 (dd, 1H, J = -15.7 Hz/3.8 Hz, Bn-CH2), 2.29-2.22 (m, 2H,
CH2COOMe), 1.88-1.79 (m, 2H, CH2), 1.51/1.56-1.37 (d/m, 3H, J = 6.6 Hz, CH3CHN). 13C-

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NMR from the mixture of diastereomers (100.6 MHz, Benzol-d6): δ 172.8 (COOMe), 169.3
(CON), 155.7 (OCONH), 154.1, 153.0, 151.6, 147.5, 141.4, 134.6, 130.4, 128.1, 127.9,
127.3, 127.2, 127.0, 126.5, 126.2, 124.3, 124.2, 121.9, 113.6, 110.8, 110.6, 109.6 (aryl), 77.8,
77.1 (benzyl-CHO), 70.9, 70.5, 70.1, 70.1, 70.0 (EG-CH2), 67.8 (aryl-OCH2), 55.8/55.8 (aryl-
OCH3), 51.1 (COOCH3), 46.8, 46.2 (CHN/benzyl-CH2), 41.1 (CH2NHBoc), 30.4 (Bu-CH2),
24.6 (Bu-CH2), 21.4 (CH3).
4-{4-[14-(11,12-Didehydro-5,6-dihydrodibenzo[a,e]cycloocten-5-oxycarbonylamino)-4-oxo-
6,9,12-trioxa-3-azatetradec-2-yl]-2-methoxy-5-nitrophenoxy}butanoic acid 4-nitophenyl ester
3a: In an 25 ml round bottom flask 1.54 g of nitroaromatic 9 (2.06 mmol) were dissolved in
4.5 ml THF/MeOH 1:1 v/v. To the solution were added 184 mg LiOH*H2O (4.39 mmol, 2.1
eq.) dissolved in 4.5 ml water and the mixture stirred for 90 min. at rt.23 Then the solution was
acidified by adding 1 M NaHSO4-soln. and transferred to a separatory funnel. The aqueous
layer was extracted five times with DCM, the combined organic layers dried with Na2SO4 and
the solvent evaporated under reduced pressure. Subsequently, in a 25 ml round bottom flask
equipped with a gas inlet and a stirring bar the crude acid was dissolved in 10 ml dry THF
under an atmosphere of nitrogen. To the solution were added 315 mg p-nitrophenol (2.27
mmol, 1.1 eq.) and after cooling to -20°C 638 mg DCC (3.09 mmol, 1.5 eq.). The mixture
was then stirred for 6 h at -20°C and additional 14 h at rt. After filtration of the precipitated
DC-urea the solution was diluted with ethyl acetate, transferred to a separatory funnel, washed
twice with 1M NaHSO4-soln. and once each with sat. NaHCO3-soln. and brine. After removal
of the solvent under reduced pressure, the residue was subjected to column chromatography
eluting with toluene/acetone 7:3 v/v yielding 1.05 g of pure title compound 9 (1.23 mmol,
60%) as pale yellow foam. Rf: 0.34 (toluene/acetone 7:3 v/v). IR (NaBr): 3407, 2933, 1763,
1723, 1673, 1522, 1346, 1273, 1211, 1130, 760 cm-1. FT-ICR-MS: m/z [M+Na]+ calcd for
1
C44H46N4O14Na: 877.2903 found: 877.2905. H-NMR from the mixture of diastereomers
(400.1 MHz, Benzene-d6): δ 7.84-7.75 (m, 3H, pNP/CONH), 7.66-7.55 (m, 1H, aryl), 7.38 (s,
1H, aryl), 7.28-7.09 (m, 4H, aryl), 7.06-6.89 (m, 4H, aryl), 6.79-6.73 (m, 2H, pNP) 6.38-
6.33/5.75-5.66 (m/m, 1H, Bn-CHO), 6.30/5.85 (s/s, broad, 1H, OCONH), 6.03-5.93 (m, 1H,
Bn-CHN), 4.08-2.70 (m, 19H), 3.88 (dd, 1H, J = -15.8 Hz/5.1 Hz, Bn-CH2), 2.92 (dd, 1H, J =
-15.8 Hz/3.8 Hz, Bn-CH2), 2.41 (t, 2H, J = 7.1 Hz, CH2COOMe), 1.94-1.80 (m, 2H, CH2),
1.50/1.56-1.34 (d/m, 3H, J = 6.6 Hz, CH3CHN). 13C-NMR from the mixture of diastereomers
(100.6 MHz, Benzene-d6): δ 170.2 (COOMe), 169.4 (CON), 155.8, 155.5, 154.1
(OCONH/aryl), 153.0, 151.6, 147.4, 145.5, 141.4, 135.1, 130.4, 128.1, 127.9, 127.4, 127.3,
126.6, 126.3, 125.1, 124.3, 124.2, 122.4, 121.9, 113.6, 110.7, 110.6, 109.7 (aryl), 77.8, 77.2
(benzyl-CHO), 70.9, 70.8, 70.6, 70.5, 70.1, 70.1 (EG-CH2), 67.7 (aryl-OCH2), 55.8 (aryl-

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OCH3), 46.7, 46.1 (CHN/benzyl-CH2), 41.2 (CH2NHBoc), 31.0 (Bu-CH2), 24.5 (Bu-CH2),
21.5 (CH3).
(S)-N-α-(9-Fluorenyl)methoxycarbonyl-N`-β-tert.-butoxycarbonyl-β-aminoalanyl-11-azido-
3,6,9-trioxaundecyl amide 10: In a 100 ml round bottom flask equipped with a gas inlet and a
stirring bar 5 g of α-Fmoc-β-Boc-L-β-aminoalanine (7.98 mmol) were dissolved in 50 ml dry
THF under an atmosphere of nitrogen. Then the solution was cooled to 0°C and 1.62 g PFP
(8.78 mmol, 1.1 eq.) were added followed by 2.15 g DCC (10.4 mmol, 1.3 eq.) and stirring
continued for 6 h at 0°C. Thereafter 1.92 g of 2-{2-[2-(Azidoethoxy)ethoxy]ethoxy}amine24
(8.78 mmol, 1.1 eq.) and 2.08 ml DIPEA (12.0 mmol, 1.5 eq.) were added and the slurry
stirred for additional 14 h at rt. After filtration from the DC-urea precipitate the solution was
diluted with ethyl acetate and transferred to a separatory funnel. The organic layer was
washed twice each with 1 M NaHSO4-soln. and sat. NaHCO3-soln., once with brine, dried
with Na2SO4 and the solvent removed under reduced pressure. The residue was purified by
column chromatography eluting with toluene/acetone 3:1-7:3 v/v yielding 3.2 g of pure title
20
compound 10 (5.11 mmol, 64%) as colorless resin. Rf: 0.29 (toluene/acetone 3:1 v/v). [α]D
=
-12.5 (c=1.0, CHCl3). IR (NaBr): 3319, 2871, 2108, 1689, 1661, 1536, 1450, 1307, 1163, 739
cm-1. FT-ICR-MS: m/z [M+Na]+ calcd for C31H42N6O8Na: 649.2956 found: 649.2950.1H-
NMR (400.1 MHz, CDCl3): δ 7.75 (d, 2H, J = 7.5 Hz, Fmoc), 7.62-7.57 (m, 2H, Fmoc), 7.39
(t, 2H, J = 7.5 Hz, Fmoc), 7.30 (dt, 2H, J = 7.5 Hz/0.9 Hz, Fmoc), 6.96 (s, broad, 1H, CONH),
6.32 (s, broad, 1H, CONH), 5.29 (s, broad, 1H, CONH), 4.44-4.24 (m, 2H, Fmoc-CH2O/α-
CH ), 4.20 (t, 1H, J = 7.2 Hz, Fmoc-CH), 3.65-3.40 (m, 16H, EG-chain-CH2/β-CH), 3.34 (t,
2H, J = 5.2 Hz, CH2N3), 1.44 (s, 9H, tBu). 13C-NMR (100.6 MHz, CDCl3): δ 170.2 (CONH),
157.2, 156.6 (OCONH), 143.9, 143.8, 141.4, 127.8, 127.2, 125.2, 120.1 (aryl), 80.1 (tBu),
70.7, 70.7, 70.6, 70.4, 70.0, 69.6 (EG-CH2), 67.3 (Fmoc-CH2O), 56.4 (α-CH), 50.7 (CH2N3),
47.2 (Fmoc-CH), 42.9, 39.5 (CH2N), 28.4 (tBu).
{12-[[[8,9-dihydro-1-[2-[2-[2-[[3-(tert.-butoxycarbonyl)amino-2-[(9-
fluorenyl)methoxycarbonyl]aminopropanamido]ethoxy]ethoxy]ethoxy]ethyl]-1H-
dibenzo[3,4:7,8]cycloocta[1,2-d]triazol-9-yl]oxy]carbonyl]amino}-3,6,10-trioxadodecanoic
acid amide and {12-[[[8,9-dihydro-3-[2-[2-[2-[[3-(tert.-butoxycarbonyl)amino-2-[(9-
fluorenyl)methoxycarbonyl]aminopropanamido]ethoxy]ethoxy]ethoxy]ethyl]-1H-
dibenzo[3,4:7,8]cycloocta[1,2-d]triazol-9-yl]oxy]carbonyl]amino}-3,6,10-trioxadodecanoic
acid amide 11: Triazole-loaded TOYOPEARL® was irradiated according to the procedure

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described above and the remaining solid support filtered off. Then dioxane was removed
under reduced pressure and the aqueous residue transferred to a separatory funnel. The aq.
layer was extracted five times with DCM, the combined organic layers dried over Na2SO4 and
the solvent removed under reduced pressure leaving 11 as colorless foam in analytical purity.
Rf: 0.34 (CHCl3+6v%MeOH). [α]D20= -5.3 (c=1.0, CHCl3). FT-ICR-MS: m/z [M+Na]+ calcd
1
for C56H70N8O14Na: 1101.4904 found: 1101.4900. HPLC: 8.65 min., 98.2 %. H-NMR from
the mixture of regioisomers/diastereomers (400.1 MHz, CDCl3): δ 7.74 (d, 2H, J = 7.6 Hz,
Fmoc), 7.62-7.46 (m, 4H, Fmoc/aryl), 7.45-7.34 (m, 2H, aryl), 7.38 (t, 2H, J = 7.4 Hz, Fmoc),
7.33-6.99 (m, 7H, Fmoc/aryl/CONH), 6.37 (s, broad, 1H, CONH), 6.25-6.15 (m, 1H,
CONH), 6.06/5.98-5.91 (m, 1H, CONH), 5.71-5.62/5.60-5.49 (m, 1H, OCONH), 4.76-
4.67/4.63-4.41 (m, 2H, Bn-CHO/α-CH), 4.41-4.24 (m, 3H, Fmoc-OCH2/CH2), 4.19 (t, 1H, J
= 7.2 Hz, Fmoc-CH), 4.05-3.84 (m, 4H, OCH2CONH2/Bn-CH2), 3.74-3.17/3.01 (m/t, 28H, J
= 12.1 Hz, EG-CH2/β-CH2), 1.42 (s, 9H, tBu). 13C-NMR from the mixture of
regioisomers/diastereomers (100.6 MHz, CDCl3): δ 173.4, 170.3 (CONH), 157.2, 156.7,
156.0, 155.4 (OCONH), 147.9, 146.3 (aryl), 143.9/143.9, 141.4 (Fmoc), 137.3, 135.8, 135.6,
134.6, 134.4, 133.6, 132.8, 132.2, 131.5, 130.3, 129.9, 129.6, 129.4, 129.1, 128.9, 128.7,
128.6 (aryl), 127.8 (Fmoc), 127.7, 127.3 (aryl), 127.2 (Fmoc), 126.8, 126.5, 125.3 (Fmoc),
124.9, 124.5 (aryl), 120.1 (Fmoc), 79.9 (tBu), 71.7, 71.1, 71.0, 70.7, 70.6, 70.4, 70.4, 70.3,
70.2, 70.2, 70.0, 69.7, 69.2 (EG-CH2), 67.3 (Fmoc-OCH2), 56.1 (α-CH), 48.6, 48.3, 48.2
(CH2N), 47.2 (Fmoc-CH), 43.3, 42.9, 40.9, 39.4, 37,9, 37.3 (CH2N), 28.4 (tBu).
4. References
(20) Gong, Y.; Luo, Y.; Bong, D. J. Am. Chem. Soc., 2006, 128 (45), 14430-14431.
(21) M. Montalti, A. Credi, L. Prodi, M. T. Gandolfi, Handbook of photochemistry, 3rd ed.,
Taylor & Francis, Boca Raton, FL, 2006, pp 595-600.
(22) King, S. B.; Ganem, B. J. Am. Chem. Soc., 1994, 116 (2), 562-570.
(23) Jayaprakasch, K. N.; Peng, C. G.; Butler, D.; Varghese, J. P.; Maier, M. A.; Rajeev, K.
G.; Manoharan, M. Org. Lett., 2010, 12 (23), 5410-5413.
(24) Schwabacher, A. W.; Lane, J. W.; Schiesher, M. W.; Leigh, K. M.; Johnson, C. W. J.
Org. Chem., 1998, 63, 1727-1729.

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