Heating and Microwave assisted SPPS of C-terminal
acid peptides on trityl resin: The truth behind the
yield
Cécile Echalier a†• Soultan Al-Halifaa • Aude Kreiter†• Christine Enjalbal • Pierre Sanchez •
Luisa Ronga †• Karine Puget †• Pascal Verdié • Muriel Amblard • Jean Martinez • Gilles
Subra
Table of Contents
Experimental Conditions
Analytical HPLC analyses
S2
LC/MS analyses
S2
RP-Preparative HPLC
S2
First amino acid anchoring on hydroxymethyl phenoxy PS resin (Wang)
S3
First amino acid anchoring on Fmoc-Rink amide aminomethyl PS
S3
resin
Synthesis of compound 8 c[Phe-Ala] DKP
S3
Titration of Fmoc-Ala-OH 4’ (HPLC =214 nm)
S5

Calibration curve of Fmoc-Ala-OH 4’
S5

Titration of released Fmoc-Ala-OH from the resin at room temperature in DMF
S5

Titration of released Fmoc-Ala-OH from the resin at 70°C in DMF
S6
Titration of Fmoc-Phe-Ala-OH 5’ (HPLC =214 nm)
S6

Calibration curve of peptide 5’
S6

Titration of released peptide 5’ from the resin at room temperature in DMF
S7

Titration of released peptide 5’ from the resin at 70°C in DMF
S7
Titration of Fmoc-Leu-Phe-Ala-OH 7’ (HPLC =214 nm)
S8

Calibration curve of peptide 7’
S8

Titration of released peptide 7’ from the resin at room temperature in DMF
S8

Titration of released peptide 7’ from the resin at 70°C in DMF
S9
Calibration curve of DKP 8 (LC/MS, SIR MRM m/z 219.2)
S9
Calibration curve of protected peptide 1’ (HPLC =214 nm)
S10
LC/MS analyses of crude peptide 1’
S10
LC chromatogram of purified peptide 1’
S11
Titration of released protected peptide 1’
S12
LC/MS analyses of Crude Peptide 1 H-Glu-Lys-Arg-Tyr-Cys-Ser-OH
S13
Determination of stereochemistry of released peptide from resin 7
S15
Synthesis scheme of R2 with resin weighting and calculations
S17
Synthesis scheme of R3 with resin weighting and calculations
S17
LC/MS Analysis of peptide 2
S18
S1
LC/MS Analysis of peptides 3
S19
Analytical HPLC analyses
Conditions A
Analytical HPLC were run on a Beckman instrument, equipped with a photodiode array and Chromolith
SpeedROD (50 – 4.6 mm) reversed-phase column (RP-18e). Standard conditions were eluent system A
(water/0.1% TFA), system B (acetonitrile/0.1% TFA). A flow rate of 5 mL/min and a gradient of (0–100)% B
over 3 min were used, detection at 214 nm, unless otherwise stated. Retention times (Rt(A)) are given in
minutes.
Conditions B
Analytical HPLC were run on an Agilent 1100 instrument, equipped with a photodiode array and XBridge (150
– 3 mm) reversed-phase column (C18). Standard conditions were eluent system A (H2O/ACN/MSA 98/2/0.1%), system B (ACN/H2O/MSA - 98/2/0.1%). A flow rate of 5 mL/min and a gradient of (0–50)% B for 2
min, (50–100)% B for 5 min were used, detection at 214 nm, unless otherwise stated. Retention times (Rt(B)) are
given in minutes.
Conditions C
LC analyses were run on a Waters Alliance 2695 HPLC, equipped with monochromatic detector array and
Chromolith High resolution (25 – 4.6 mm) reversed-phase column (RP-18e). A flow rate of 3 mL/min and a
gradient of (0–100) % B over 2.5 min (or over 15 min) were used. Eluent A: water/0.1% HCO 2H; eluent B:
acetonitrile/0.1% HCO2H. Retention times (Rt(C)) are given in minutes
Conditions D (for separation of diastereoisomers of 7’)
LC analyses were run on a Beckman instrument, equipped with a monochromatic detector and Onyx monolithic
HD (50 – 4.6 mm) reversed-phase column (C18). Standard conditions were eluent system A (water/0.1% TFA),
system B (acetonitrile/0.1% TFA). A flow rate of 5 mL/min and a gradient of (32–34)% B over 30 min were
used, detection at 214 nm. Retention times (Rt(D)) are given in minutes.
LC/MS analyses
LC/MS analyses – samples were prepared in acetonitrile/water (50/50 v/v) mixture, containing 0.1% TFA. The
LC/MS system consisted of a Waters Alliance 2695 HPLC, coupled to a Micromass (Manchester, UK) ZQ
spectrometer (electrospray ionization mode, ESI+). All the analyses were carried out using a Merck Chromolith
Speed rod C18, 25 × 4.6 mm reversed-phase column. A flow rate of 3 mL/min and a gradient of (0–100) % B
over 2.5 min (or over 15 min) were used. Eluent A: water/0.1% HCO 2H; eluent B: acetonitrile/0.1% HCO2H.
Retention times (RT) are given in minutes. Positive ion electrospray mass spectra were acquired at a solvent
flow rate of 200 mL/min. Nitrogen was used for both the nebulizing and drying gas. The data were obtained in a
scan mode ranging from 100 to 1000 m/z in 0.1 s intervals; 10 scans were summed up to get the final spectrum.
MRM analysis of compound 8 c[Phe-Ala]
S2
Diketopiperazine c[Phe-Ala] analysis was perform using MRM acquisition mode (multiply reaction monitory)
on a Waters Alliance 2695 coupled to a Micromass Quatro Micro. A flow rate of 3 mL/min and a gradient of (0–
100) % B over 2.5 min were used. Eluent A: water/0.1% HCO2H; eluent B: acetonitrile/0.1% HCO2H. Retention
times (RT) are given in minutes. Detection of DKP was done at 214 nm using the same acquisition (ES+) and
the following parameters: Cone voltage: 20 V; Collision energy: 12 eV.
RP-Preparative HPLC
RP-preparative HPLC purification was performed on a Waters HPLC 4000 instrument, equipped with a UV
detector 486 and Waters Delta-Pack 40×100 mm, 15 Å, 100 µm, reversed-phase column. Eluent system A
(water/0.1% TFA), system B (acetonitrile/0.1% TFA). A flow rate of 50 mL/min and a gradient of (10–70)% B
over 30 min were used, detection at =214 nm.
First amino acid anchoring on hydroxymethyl phenoxy PS resin (Wang), example of Fmoc-Ala-OH.
The hydroxymethyl phenoxy PS resin (1g, 0.7 mmol, initial theoretical loading = 0.7 mmol/g) was conditioned
for 15 min in DCM. Then, 10 mL of DMF solution of Fmoc-Ala-OH (3 eq., 2.1 mmol, 654 mg) containing DIC
(1.5 eq, 1.05 mmol, 165 µl), DMAP (0.15 eq, 105 µmole, 13 mg) was poured unto the resin and stirred
overnight. The resin was filtered and then washed with DMF (2×), DCM/MeOH 1/1 v/v (2×), DMF (2×), DCM
(2×) and dried in a desiccator overnight.
First amino acid anchoring on Fmoc-Rink amide aminomethyl PS, example of Fmoc-Ala-OH.
The Fmoc rink amide aminomethyl PS resin (1g, 0.96 mmol, initial theoritical loading 0.96 mmol/g) was
conditioned for 15 min in DCM. The Fmoc protecting group was removed with a standard deprotection cycle
using 80/20 v/v DMF/pip solution (see main article Material and Methods). After washing step, the first
aminoacid was loaded on the resin through a standard coupling cycle (see main article Material and Methods)
using HBTU and NMM.
Synthesis of compound 8 c[Phe-Ala] DKP. (3R,6S)-3-benzyl-6-methylpiperazine-2,5-dione
O
O
N
H
OH
O
HBTU (1.1 eq)
NMM (2 eq)
NH2
O
+
O
O
DMF, RT 2hours
O
O
H
N
N
H
O
O
TFA
DCM
(3R,6S)-3-benzyl-6-methylpiperazine-2,5-dione
O
DCM reflux
HN
DIEA (10 eq) 6 hours
NH
8
O
S3
H
N
H2N
O
O
O
Boc-Ala-OH (1.1 eq) was dissolved in a solution of HBTU (1.1 eq), N-Methylmorpholine (NMM; 2 eq) in N,Ndimethylformamide (DMF), and the resulting solution was added to the H-Phe-OMe dissolved in DMF. The
reaction mixture was stirred at room temperature for 2 h and then DMF was evaporated under vacuum. The
residue was dissolved in AcOEt and washed with KHSO4 (3 times), NaHCO3 (3 times), NaCl (3 times) and then
dried over anhydrous sodium sulfate and evaporated to give an oily residue (compound 1). The compound 1 was
dissolved in trifluoroacetic acid (TFA) and stirred for 1 hour, followed by removal of the solvent by evaporation
under vacuum to give compound 2. This compound 2 was refluxed in DCM for 6 hours and DKP (compound 8)
precipitates directly into the flask. The precipitate was washed with DCM, Et 2O, frozen and lyophilized in
water/acetonitrile (50/50).
MS (ESI) calcd for C12H14N2O2, 218.11; [M + H]+m/z found 219.20 , Rt(C): 0.82 min.
TIC/LC and LC/MS analyses are presented in the following figures
S4
Titration of Fmoc-Ala-OH 4’ (HPLC =214 nm)

Calibration curve of Fmoc-Ala-OH 4’
A solution of Fmoc-Ala-OH (stock solution, 10-3 M) was prepared in 4 mL of DMF. Stock solution was
used to make a dilution range of 10-3 M to 10-6 M and 10 µl of each solution was injected on an
analytical HPLC (conditions A). Data are summarized in the following table.
Titration (M)
0
6.2x10-5
7.75x10-5
1.03x10-4
1.55x10-4
Areas
0
261479
453235
543635
730797
S5
HPLC 214nm
Peak area= f[Fmoc-Ala-OH]
800000
y = 4.96E+09x
R² = 9.83E-01
600000
400000
200000
Conc (M)
0
0.00E+00

5.00E-05
1.00E-04
1.50E-04
2.00E-04
Titration of released Fmoc-Ala-OH from the resin at room temperature in DMF
100 mg of resin 4 were weighted and placed in a test tube. 4 mL of DMF were added and kinetics was
carried out at room temperature on an analytic HPLC (Conditions A) for 1200 min. Data are
summarized in the following table.
Time (min)
0
60
150
240
360
1200
Area
73117
90434
298272
271477
199759
203332
Titration (M)
1.25 x10-5
1.61 x10-5
5.89 x10-5
5.23 x10-5
3.86 x10-5
3.93 x10-5
HPLC 214 nm
[Fmoc-Ala]= f(time)
1.00E-04
8.00E-05
6.00E-05
4.00E-05
2.00E-05
5.00E-19
0
500
1000
1500
Time (min)
-2.00E-05

Titration of released Fmoc-Ala-OH from the resin at 70°C in DMF
In a closed vessel containing 100 mg of resin 4, 4 mL of DMF were added and the mixture was heat till
70°C. Kinetics was carried out on an analytic HPLC (conditions A) for 400 min. Data are summarized
in the following table.
Time (min)
0
60
150
240
400
Area
11407110
32217440
49795400
11987300
0
Titration (M)
2.349 x10-3
6.640 x10-3
1.026 x10-3
2.469 x10-3
0
S6
HPLC 214 nm
[Fmoc-Ala-OH] = f(time)
1.20E-02
1.00E-02
Turbi dirty
8.00E-03
6.00E-03
4.00E-03
2.00E-03
Time (min)
0.00E+00
0
100
200
300
400
500
Titration of Fmoc-Phe-Ala-OH 5’ (HPLC =214 nm)

Calibration curve of peptide 5’
A solution of Fmoc-Phe-Ala-OH (stock solution, 10-3 M) was prepared in 4 mL of DMF. Stock solution
was used to make a dilution range of 10-3 M to 10-5 M and 10 µl of each solution was injected on an
analytical HPLC (Conditions A). Data are summarized in the following table.
Titration (M)
2.10-5
1.10-4
2.10-4
3.10-4
4.10-4
Areas
43035
237928
469037
797828
963521
HPLC 214
Peak area = f[Fmoc-Phe-Ala-OH]
1200000
1000000
800000
y = 2,46E+09x
R² = 9,92E-01
600000
400000
200000
0
0.00E+00

Conc (M)
1.00E-04
2.00E-04
3.00E-04
4.00E-04
5.00E-04
Titration of released peptide 5’ from the resin at room temperature in DMF
100 mg of resin 5’ were weighted and placed in a test tube. 4 mL of DMF were added and kinetics was
carried out at room temperature on an analytic HPLC (Conditions A) for 400 min. Data are summarized
in the following table.
Time (min)
0
Area
34564
Titration (M)
1.41 x10
67
132
184223
-5
7.49 x10
-5
206
243800
9.91 x10
S7
315
215285
-5
8.75 x10
-5
388
229580
9.33 x10
258306
-5
1.05 x10-4
HPLC 214 nm
[Fmoc-Phe-Ala-OH] = f(time)
1.20E-04
1.00E-04
8.00E-05
6.00E-05
4.00E-05
2.00E-05
Time(min)
0.00E+00
0

100
200
300
400
500
Titration of released peptide 5’ from the resin at 70°C in DMF
In a closed vessel containing 100 mg of resin 5’, 4 mL of DMF were added and the mixture was heat till
70°C. Kinetics was carried out on an analytic HPLC (Conditions A) for 400 min. Data are summarized
in the following table.
Time (min)
5
75
137
215
302
390
Area
691751
3676338
1353920
2037955
1669751
1303901
Titration (M)
2.81 x10-4
1.49 x10-3
5.50 x10-3
8.28 x10-3
13.6 x10-3
15.9 x10-3
HPLC 214 nm
[C] = f[time]
1.80E-02
1.60E-02
1.40E-02
1.20E-02
1.00E-02
8.00E-03
6.00E-03
4.00E-03
2.00E-03
Time (min)
0.00E+00
0
100
200
300
400
500
Titration of Fmoc-Leu-Phe-Ala-OH 7’ (HPLC =214 nm)

Calibration curve of peptide 7’
A solution of Fmoc-Phe-Ala-OH (stock solution, 10-2 M) was prepared in 4 mL of DMF. Stock solution
was used to make a dilution range of 10-2M to 10-5 M and 10 µl of each solution was injected on an
analytical HPLC (Conditions A). Data are summarized in the following table.
Titration (M)
Areas
1.67 x10-4
3.33 x10-4
6.66 x10-4
1 x10-3
1.5 x10-3
2.25 x10-3
3.38 x10-3
5.07 x10-3
76041
174998
352007
626643
829560
1180207
1730425
2417291
S8
HPLC 214 nm
Peak area = f[Fmoc-Leu-Phe-Ala-OH]
3000000
2500000
y = 4.98E+08x
R² = 9.91E-01
2000000
1500000
1000000
500000
Conc (M)
0
0.00E+00 1.00E-03 2.00E-03 3.00E-03 4.00E-03 5.00E-03 6.00E-03

Titration of released peptide 7’ from the resin at room temperature in DMF
100 mg of resin 7’ were weighted and placed in a test tube. 4 mL of DMF were added and kinetics was
carried out at room temperature on an analytic HPLC (Conditions A) for 400 min. Data are summarized
in the following table.
Time (min)
0
65
129
209
313
391
Area
34564
184223
243800
215285
229580
258306
Titration (M)
1.20 x10-5
1.14 x10-4
1.35 x10-4
1.42 x10-4
1.75 x10-4
1.64 x10-4
HPLC 214 nm
[Fmoc-Leu-Phe-Ala-OH] = f[time]
2.00E-04
1.50E-04
1.00E-04
5.00E-05
Time (min)
0.00E+00
0

100
200
300
400
500
Titration of released Peptide 7’ from the resin at 70°C in DMF
In a closed vessel containing 100 mg of resin 5’, 4 mL of DMF were added and the mixture was heat till
70°C. Kinetics was carried out on an analytic HPLC (Conditions A) for 400 min. Data are summarized
in the following table.
Time (min)
10
89
149
225
318
400
Area
1109903
3631953
2184003
1135121
1441678
852544
Titration (M)
3.4 x10-4
1.12 x10-3
3.38 x10-3
7.03 x10-3
8.93 x10-3
7.92 x10-3
S9
HPLC 214 nm
[Fmoc-Leu-Phe-Ala-OH] = f(time)
1.00E-02
8.00E-03
6.00E-03
4.00E-03
2.00E-03
Time (min)
0.00E+00
0
100
200
300
400
500
Calibration curve of DKP 8 (LC/MS, SIR MRM m/z 219.2)
A solution of DKP (stock solution, 10-2 M) was prepared using 2.18 mg of DKP in 1 mL of water/acetonitrile
(50/50). Stock solution was used to make a dilution range of 10 -2 M to 5.10-6 M and 1 µl of each solution was
injected on a mass spectrometer (Mass lynx power). Data are summarized in the following table.
Titration (M)
1 x10-3
5 x10-4
1 x10-4
5 x10-5
1 x10-5
5 x10-6
areas
7859
3700
791
374.21
59
51.54
HPLC 214 nm
Peak area = f[DKP]
8000
y = 8E+06x
R² = 0,9991
6000
4000
2000
0
0.00E+00
Conc (M)
5.00E-04
1.00E-03
1.50E-03
Calibration curve of protected peptide 1’ (HPLC =214 nm): Boc-Glu(OtBu)-Lys(Boc)-Arg(Pbf)-Tyr(tBu)Cys(trt)-Ser(tBu)-OH MW = 1646.8 g/mol.
Protected peptide 3 was synthesized on a LibertyTM Microwave Peptide Synthesizer (CEM Corporation,
Matthews, NC) providing MW irradiation at 2450 MHz using Fmoc/tBu SPPS strategy in the conditions
described in Material and methods section of the article. The crude peptide 3 was released from the resin using a
solution of HFIP for 45 min in order to keep the side chain protecting groups. LC/MS (conditions C) analyses
are presented below.
S10
[M-trt+H]+
[M+H]+
[M+H+Na]2+
[M+2Na]2+
[M-trt+Na]+
[M+Na]+
The crude peptide was purified on an autopurification system Waters Micromass equipped with an
injector/collector sample manager (Waters 2767), a pump (Waters 2767), a photodiode array detector (Waters
2996) and a mass spectrometer ZQ (Waters). LC chromatogram (conditions C) of purified 1’ is presented below.
S11
A calibration curve of the pure peptide (stock solution, 10-3 M) was prepared in 4 mL of DMF. Stock solution
was used to make a dilution range of 10-3M to 10-6 M and 10 µl of each solution was injected on an analytical
HPLC (Conditions B). Data are summarized in the following table.
Titration (M)
7.80 x10-6
1.56 x10-5
3.12 x10-5
6.25 x10-5
1.25 x10-4
2.50 x10-4
5.00 x10-4
7.60 x10-4
Area
514.5
562.1
746.9
1046
1697
2625
4725
6543
HPLC 214
Peak area = f[Protected peptide 1']
8000
y = 9E+06x
R² = 0,9589
6000
4000
2000
0
0.00E+00
Conc (M)
2.50E-04
5.00E-04
7.50E-04
1.00E-03
Titration of released Peptide 1’ from the resin at 70°C in DMF
In a closed vessel containing 100 mg of resin R1’, 4 mL of DMF were added and the mixture was heat till 70°C.
Aliquots were analyzed (conditions A) for 400 min. Data are summarized in the following table.
Time
(hour)
Peak area
Titration (M)
S12
1
1678
2.80 x10-4
2
2727
4.60 x10-4
3
3929
6.60 x10-4
4
6791
1.10 x10-3
5
4001
1.35 x10-3
6
4313
1.45 x10-3
7
4853
1.60 x10-3
8
3161
1.60 x10-3
9
4424
2.20 x10-3
10
3055
2.10 x10-3
11
3257
2.20 x10-3
12
3896
2.60 x10-3
13
4173
2.80 x10-3
14
4517
3.00 x10-3
15
4628
3.10 x10-3
16
4086
3.40 x10-3
17
4050
3.40 x10-3
18
4752
4.00 x10-3
19
5293
4.50 x10-3
LC/MS analysis of Crude Peptide 1 H-Glu-Lys-Arg-Tyr-Cys-Ser-OH, MW = 784.8 g/mol
S13
[M+2H]2+
[M+H]+
S14
Determination of stereochemistry of released peptide from resin 7
Chromatogram of 7’ Fmoc-Leu-Phe-Ala-OH (conditions D)
Fmoc
H
N
O
N
H
H
N
O
(S)
OH
O
Chromatogram of 7’(D) Fmoc-Leu-Phe-(D)Ala-OH (conditions D)
Fmoc
H
N
O
N
H
H
N
O
(R)
OH
O
S15
Coinjection of 7’(D) and 7’ (conditions D)
7’
Fmoc
H
N
O
N
H
H
N
7’(D)
O
(S)
OH
O
Fmoc
Released peptide from resin 7 after 6 hours heating (70°C)
Dibenzofulvene
Released peptide
DMF
S16
H
N
O
N
H
H
N
O
O
(R)
OH
Coinjection of released peptide after 6 hours heating (70°C) and 7’(D) (Fmoc-Leu-Ala-(D)Phe-OH)
7’(D) (Fmoc-Leu-Ala-(D)Phe-OH)
Released peptide
Dibenzofulvene
S17
Synthesis scheme of R2 with resin weighting and calculations
starting resin R2
0.76 mmol/g
0.1 mmol scale
2 batches (a and b) of 132 mg
R2'
Fmoc SPPS
Fmoc
H
N
O
H-Gly-Ser(tBu)-Asn(trt)-Lys(Boc)-Gly-Ala-Ile-Ile-Gly-Leu-Met O
O
Cl
a: MW 70°C
or
b: RT
S
0.1 mmol scale
MW increment / resin R2= +1087.6
theoretical weight : 240.7 mg
R2'a: 100.9 mg
R2'b: 226.0 mg
TFA/TIS/H2O
95/2.5/2.5 v/v/v
1 hour, RT
H-Gly-Ser-Asn-Lys-Gly-Ala-Ile-Ile-Gly-Leu-Met-OH
2
peptide 2 weight
0.1 mmol scale
theoretical :106.0 mg
1a: 2.8 mg
1b: 100.8 mg
Synthesis scheme of R3 with resin weighting and calculations
starting resin R3
0.72 mmol/g
0.1 mmol scale
2 batches (a and b) of 139 mg
R3'
Fmoc SPPS
Fmoc
H
N
O
O
H-Trp(Boc)-His(trt)-Trp(Boc)-Leu-Gln(trt)-Leu-Lys(Boc)-Pro-Gly-Gln(trt)-Pro-Met-Tyr(tBu)
O
Cl
a: MW 70°C
or
b: RT
O
0.1 mmol scale
MW increment / resin R3= +2308g/mol
theoretical weight : 369.8 mg
R3'a: 365.0 mg
R3'b: 94.2mg
TFA/TIS/H2O
95/2.5/2.5 v/v/v
1 hour, RT
H-Trp-His-Trp-Leu-Gln-Leu-Lys-Pro-Gly-Gln-Pro-Met-Tyr-OH 3
peptide 2 weight
0.1 mmol scale
theoretical 168.4mg
1a: 0.8 mg
1b: 152.2 mg
S18
LC/MS analysis of peptide 2b
[M+H]+
[M+2H]2+
S19
LC/MS analysis of peptide2+3b
[M+2H]
[M+2H]2+
[M+2H]2+
S20