Supporting information
for the article entitled
“Generation of peptide-MHC class I complexes through UV-mediated
ligand exchange.”
Boris Rodenko,1 Mireille Toebes,2 Sine Reker Hadrup,2 Wim J. E. van Esch,3
Annemieke M. Molenaar,3 Ton N. M. Schumacher2 & Huib Ovaa1
1
Division of Cellular Biochemistry, The Netherlands Cancer Institute, Plesmanlaan
121, 1066 CX, Amsterdam, The Netherlands.
2
Division of Immunology, The Netherlands Cancer Institute, Plesmanlaan 121, 1066
CX, Amsterdam, The Netherlands.
3
Sanquin, Plesmanlaan 125, 1066 CX, Amsterdam, The Netherlands.
Correspondence should be addressed to T.N.M.S. (t.schumacher@nki.nl) or H.O.
(h.ovaa@nki.nl)
Synthesis of N-fluorenylmethyloxycarbonyl-(2-nitro)phenylglycine
The title compound is obtained in five steps from commercially available 2nitrophenylacetic acid 3 (Figure S-1).
2-Nitrophenylacetic acid methyl ester (4). This compound was synthesised
according to a modified literature procedure.1 To a solution of 2-nitrophenylacetic
acid 3 (45 g, 0.25 mol) in 500 mL of methanol was added thionyl chloride (1.9 mL;
25 mmol) over 30 min while maintaining the temperature at 0–4 oC. The reaction
mixture was stirred at 0 oC for 30 min and then at room temperature for 24 h. After
removal of the solvent in vacuo, the residue was partitioned between ethyl acetate
(500 mL) and water (250 mL). The organic phase was washed with another 250 mL
of water and then dried over anhydrous Na2SO4 and filtered. Removal of ethyl acetate
in vacuo gave 50 g of 2-nitrophenylacetic acid methyl ester 4 as a yellow oil in
quantitative yield. 1H NMR (400MHz, CDCl3)  8.10 (dd, 1H, J = 1.1 and 7.6 Hz)
7.59 (dt, 1H, J = 1.1 and 7.6 Hz) 7.47 (dt, 1H, J = 1.1 and 7.6 Hz) 7.35 (d, 1H, J = 7.6
Hz), 4.02 (s, 2H), 3.70 (s, 3H). MS(ESI): [M+H]+ calculated: 196.06, found: 196.03.
1
1’-Bromo-2-nitrophenylacetic acid methyl ester (5). This compound was
synthesised according to a modified literature procedure.2 NBS (19.7 g, 0.11 mol) was
added to a solution of 2-nitrophenylacetic acid methyl ester (18.0 g, 0.092 mol) in 200
mL of CCl4 in a reaction flask equipped with a reflux condenser. The vigorously
stirred solution was irradiated with a 100 W tungsten lamp. The flask and lamp set-up
was wrapped in aluminum foil to ensure efficient heating of the reaction mixture.
After 6 h the white succinimide precipitate was filtered off and the filtrate was
concentrated in vacuo yielding an orange solid. Recrystallisation with ethanol and
washing of the off-white crystals with cold ethanol furnished 14.0 g (56 % yield) of
brominated product 5. 1H NMR (400MHz, CDCl3)  8.01 (dt, 2H, J = 1.2 and 7.8 Hz)
7.70 (dt, 1H, J = 1.2 and 7.8 Hz) 7.54 (dt, 1H, J = 1.2 and 7.8 Hz), 6.10 (s, 1H), 3.82
(s, 3H). MS(ESI): [M+H]+ calculated: 274.0 and 276.0, found: 274.0 and 276.0.
1’-Phtalimido-2-nitrophenylacetic acid methyl ester (6). This compound was
synthesised according to a literature procedure.3 A mixture of 1’-bromo-2nitrophenylacetic acid methyl ester (8.6g, 0.031 mol) and potassium phthalimide (5.8
g, 0.031 mol) in 100 ml of DMF was stirred at rt for 6 hr. The KBr was removed by
filtration, and the filtrate was partitioned between CH2Cl2 (80 mL) and water (200
mL). The CH2Cl2 layer was separated and the aqueous layer was extracted twice with
80 mL portions of CH2C12. The CH2Cl2 extracts were combined, dried over
anhydrous Na2SO4, and taken to dryness in vacuo to give a dark-red oil. Treatment of
the oil with hot EtOH effected the separation of a white solid. The precipitate was
filtered, washed with cold H2O, and dried in vacuo to yield 7.8 g (74 %) of product 6.
1
H NMR (400MHz, CDCl3)  8.16 (dd, 1H, J = 1.4 and 7.8 Hz), 7.93 (dd, 2H, J = 3.3
and 5.3 Hz), 7.80 (dd, 2H, J = 3.3 and 5.3 Hz), 7.60 (dt, 1H, J = 1.5 and 7.8 Hz), 7.53
(dt, 1H, J = 1.5 and 7.8 Hz), 7.43 (dd, 1H, J = 1.5 and 7.8 Hz), 6.93 (s, 1H), 3.78 (s,
3H). MS(ESI): [M+H]+ calculated: : 341.1, found: 341.0.
N-Fluorenylmethyloxycarbonyl-(2-nitro)phenylglycine (8). A solution of 7.8 g
(0.023 mol) of 1’-phtalimido-2-nitrophenylacetic acid methyl ester was hydrolysed in
50 mL of refluxing concentrated HCl-AcOH (3:2) for 5 hr. The phthalic acid was
removed by filtration and the filtrate was taken to dryness in vacuo, yielding crude (2-
2
nitro)phenylglycine-HCl salt 7 as a yellow foam (5.4 g; quant.), which was used
without further purification in the next reaction. Fluorenylmethylchloroformate (5.7 g,
22 mmol) dissolved in dioxane (75 mL) was slowly added over a 10 min period to a
cooled (4 oC) and rapidly stirring suspension of crude (2-nitro)phenylglycine-HCl salt
(4.6 g, 20 mmol) in 10% (w/w) aqueous sodium carbonate (75 mL) in dioxane (75
mL). The solution was allowed to warm to room temperature and stirred for an
additional 2 h. The reaction mixture was poured into 600 mL of water and extracted
with Et2O (200 mL, 2x). The aqueous layer was cooled (4 oC) and the pH was
adjusted to 2 by the addition of 2N aqueous HCl. The acidified aqueous layer was
extracted with Et2O (3x150 mL). The combined organic layers were dried with
Na2SO4 and concentrated in vacuo, yielding the crude product as a yellow foam. Flash
chromatography over silica gel using a gradient of ethylacetate-hexane (4:1) to
ethylacetate to ethylacetate-methanol (4:1) yielded racemic title compound 8 as an
off-white powder (4.6 g, 56 % yield over 2 steps). This compound is stored in the
dark at –20 oC. MS(ESI): [M+H]+ calculated: 419.1 found 419.0 1H-NMR (400 MHz,
d6-DMSO)  12.12 (bs, 1H), 8.22 and 7.91 (2 x d, 1H, J = 7.8 Hz, rotamers), 7.88 (d,
2H, J = 7.4 Hz), 7.84 (d, 2H, J = 7.4 Hz), 7.76-7.58 (m, 2H), 7.43-7.31 (m, 6H), 6.28
(s, 1H), 4.27-4.16 (m, 3H, rotamers).
CRITICAL. Fmoc-(2-nitro)phenylglycine was found to be unstable in NMP and
DMF, solvents routinely used in automated peptide synthesis. We therefore
recommend the use of dichloromethane during peptide coupling procedures.
3
Expression and purification of biotin ligase
Materials
Equipment

Incubator/orbital shaker

Centrifuge

250 mL – 1 L buckets

Spectrophotometer (for measuring OD at 600 nm and 280 nm)

Plastic cuvettes

Quartz cuvette

Sonicator
Reagents

Wash buffer: 0.5 % Triton X-100, 1 mM EDTA

LB medium

LB agar plates + antibiotics

Expression construct: BirA biotin ligase was cloned in a pET21B vector
(amino acids 1- 321, in frame with a His-tag). Mw 36,4 kDa

Bacterial strain BL21 (DE3) pLysS (cat. # 69451, Novagen)

Carbenicillin (cat. #: C1389, Sigma-Aldrich)
http://www.sigmaaldrich.com/catalog/search/ProductDetail/SIAL/C1389

Chloramphenicol (cat. #: C0378, Sigma-Aldrich)
http://www.sigmaaldrich.com/catalog/search/ProductDetail/SIGMA/C0378

1-S-Isopropyl--(D)-thiogalactopyranoside (IPTG) (cat #: 11 411 446001,
Roche)
http://www.roche-applied-science.com

Talon Co2+-resin (cat. #8901-2, Clontech)
http://www.clontech.com/AIT/Ecommerce/Clontech/ProductCatalog.aspx?enc
=true&idcategory=&idsearch=|242|188|26|67|28|101|212
Procedure
1
Transform biotin ligase expression plasmid into BL21(DE3)pLysS. Plate on
LB plates containing 50 µg/mL carbenicillin and 34 µg/mL chloramphenicol.
2
Pick colony and grow a 10 mL culture (LB + antibiotics) for approx. 6 hrs.
4
3
PAUSE POINT. Store culture at 4 oC overnight.
4
Next day, inoculate 1 L with 10 mL culture and grow at 37 oC until OD600nm=
0.5. Allow the culture to cool down to 30 oC before proceeding with the next
step.
5
Add IPTG (0.4 mM final concentration) and grow culture overnight at 30 oC!
6
Spin at 4 oC, 4,000 rpm for 15-20 min.
7
Resuspend pellet in 80 mL 20 mM Tris pH 8, 100 mM NaCl, divide over two
50 mL falcon tubes and sonicate (4 times 10 sec). Minimize sonication time,
as prolonged sonication may destroy protein functionality.
8
Spin samples at 12,000g for 10 min at 4 °C to pellet all insoluble material.
9
From here Clontech protocol B for Co2+-batch/gravity column purification is
followed (starting page 30):
http://www.clontech.com/clontech/techinfo/manuals/PDF/PT1320-1.pdf
10 Prepare 2 mL of Co2+ beads, which is sufficient for approx. 5 mg of protein.
11 Use sonicated supernatant for Co2+-batch/gravity column purification step 8
page 30.
12 Wash the resin with 10 bed volumes of wash buffer (page 31, step 12-14
Clontech) and repeat this step.
13 Prepare a column (page 31, step 17).
14 Wash with 5 bed volumes of wash buffer.
15 Wash the protein by adding 3 bed volumes of 5 mM imidazole, 20 mM Tris
pH 8, 100 mM NaCl. Collect 1 mL fractions and analyze by SDS-PAGE.
16 Repeat step 15 with 10 mM imidazole, 20 mM Tris pH 8, 100 mM NaCl.
Collect 1 mL fractions and analyze by SDS-PAGE.
17 Elute the protein by adding 6 bed volumes of 50 mM imidazole/ 20 mM Tris
pH 8, 100 mM NaCl. Collect 1 mL fractions and analyze by SDS-PAGE. Most
protein will be in fraction 1 and 2.
18 Add β-mercaptoethanol to 5 mM and glycerol to 5 %. Measure OD280nm (ε =
47,440) and make 20 μL aliquots of 100 μM. Mw 36.4 kDa. One aliquot is
sufficient for the biotinylation of 3 μM heavy chain in 20 mL.
PAUSE POINT. Store the protein at –20 oC.
19 Optionally, the activity of the purified BirA enzyme can be tested using the
following assay. To a 1.5 mL Sarstedt polypropylene microtube add:
-
x L BirA substrate (GGGLNDIFEAQKIEWH, Mw 1813) 50 M
5
final concentration.
-
50 L 10X ligase buffer (200 mM Tris pH 7.5, 50 mM MgCl2)
-
x L purified BirA (approx. 2 M BirA is needed for 50 M peptide)
-
10 L biotin (5 mM stock)
-
10 L ATP (500 mM stock)
-
x L H2O
500 L end volume
20 Incubate overnight at RT.
21 Add TFA to samples to 0.1 % final concentration. Analyze by reverse phase
HPLC (e.g. Waters Delta-Pak, C18, 100 Å, 15 m, 3.9 X 300 mm). As a
reference, analyze peptide without BirA exposure. Run a 60 minute gradient:
H2O: CH3CN (80:20), 0.1% TFA to H2O:CH3CN (65:35), 0.1 % TFA.
Anticipated results
The expected yield of BirA enzyme from 1 L of bacterial culture is 2 mL of a 100 μM
solution.
Timeline
(4 to 5 days)
Step 1
1 day
Step 2
6h
Step 3
16 h (overnight)
Step 4
6h
Step 5
16 h (overnight)
Step 6-18
3h
Step 19
30 min
Step 20
16 h (overnight)
Step 21
2.5 h
6
ELISA titration curve
References
1. Liu, B. & Hu, L. 5’-(2-Nitrophenylalkanoyl)-2’-deoxy-5-fluorouridines as
potential prodrugs of FUDR for reductive activation. Bioorg. Med. Chem. 11,
3889–3899 (2003).
2. Zhu, Q., Girish, A., Chattopadhaya, S.& Yao, S. Q. Developing novel activitybased fluorescent probes that target different classes of proteases. Chem.
Commun. 1512-1513 (2004).
3. Davis, A. L., Smith, D. R. & McCord, T. J. Synthesis and microbiological
properties of 3-amino-1-hydroxy-2-indolinone and related compounds. J. Med.
Chem. 16, 1043-1045 (1973).
7