On-resin N-formylation of peptides: a head-to-head comparison of reagents in solid-phase synthesis of ligands for formyl peptide receptors

Article abstract of DOI:10.1002/psc.2998

General conditions for efficient on-resin N-formylation of peptides were identified by screening of a number of reagents comprising aliphatic formates (ethyl formate, 2,2,2-trifluoroethyl formate, and cyanomethyl formate), aromatic esters (phenyl formate and p-nitrophenyl formate), and N-formylimidazole and in situ activation of formic acid with the coupling reagent 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide. Initially, reaction time and influence of solvent were examined for the formylation of a short model peptide. The most efficient reagents were examined further by using different linkers and solid supports in the synthesis of an array of longer formyl peptide ligands. For p-nitrophenyl formate and N-formylimidazole, almost complete conversion was reached within 2?h, albeit longer peptides attached to Tentagel resins via different linkers required an extended reaction time. Overall, the commercially available activated ester p-nitrophenyl formate proved to be most convenient and versatile as high formylation degrees were obtained after 1–3?h at room temperature, while either conventional or microwave-assisted heating allowed reduction of the formylation time to 20?min. Copyright

Full text of DOI:10.1002/psc.2998

Research Article
Received: 16 January 2017
Revised: 2 March 2017
Accepted: 3 March 2017
Published online in Wiley Online Library
(wileyonlinelibrary.com) DOI 10.1002/psc.2998
On-resin N-formylation of peptides: a
head-to-head comparison of reagents in
solid-phase synthesis of ligands for
formyl peptide receptors
Simon Bendt Christensen, Anna Mette Hansen and Henrik Franzyk*
General conditions for efficient on-resin N-formylation of peptides were identified by screening of a number of reagents
comprising aliphatic formates (ethyl formate, 2,2,2-trifluoroethyl formate, and cyanomethyl formate), aromatic esters (phenyl
formate and p-nitrophenyl formate), and N-formylimidazole and in situ activation of formic acid with the coupling reagent 1-
ethyl-3-(3-dimethylaminopropyl)carbodiimide. Initially, reaction time and influence of solvent were examined for the formylation
of a short model peptide. The most efficient reagents were examined further by using different linkers and solid supports in the
synthesis of an array of longer formyl peptide ligands. For p-nitrophenyl formate and N-formylimidazole, almost complete
conversion was reached within 2 h, albeit longer peptides attached to Tentagel resins via different linkers required an extended
reaction time. Overall, the commercially available activated ester p-nitrophenyl formate proved to be most convenient and
versatile as high formylation degrees were obtained after 1–3 h at room temperature, while either conventional or microwave-
assisted heating allowed reduction of the formylation time to 20 min. Copyright © 2017 European Peptide Society and John Wiley
& Sons, Ltd.
Keywords: formylation; solid-phase synthesis; formylating agents; peptides; heating
obtain an isonitrile-functionalized resin [11]. In another protocol,
formic acid was activated with 2-ethoxy-1-ethoxycarbonyl-1,2-
Introduction
Formyl peptides are recognized by the innate immune system as
bacteria-derived pathogen-associated molecular patterns or
mitochondria-derived danger-associated molecular patterns in
response to infection and cell damage, respectively [1,2]. Important
receptors involved in these processes comprise the formyl peptide
receptors that recognize peptides with an N-terminal formyl-
methionine residue thereby mediating activation and chemotaxis
of neutrophils [1–4]. Also, N-formylation of lysine constitutes an
epigenetic modification of histone tails, which has been linked to
the pathogenesis of oxidative stress [5]. Recently, it was elucidated
how N-formylation domains in nonribosomal peptide synthetases
are critical in the multistep biosynthesis of biologically active
peptides [6], for example, the gramicidins carrying an essential N-
terminal formyl-valine or formyl-isoleucine residue [7].
Moreover, as part of an ongoing study on ligands targeting
formyl peptide receptors, a general efficient solid-phase protocol
for obtaining N-terminally formylated peptides was required. Yet,
although several formylating reagents and solution-phase
protocols have been described [8–10], no systematic and
comprehensive investigation of on-resin N-formylation appears to
be reported so far.
dihydroquinoline, and in this case, the linker should withstand the
formic acid pre-treatment conditions (i.e. lack of compatibility with
2-chlorotrityl resins) [9,12]. Recently, a method involving pre-
activation of formic acid with dicyclohexylcarbodiimide in diethyl
ether for 4 h at 0 °C followed by overnight treatment of the resin-
bound peptide at 4 °C was reported [13]. Also, ethyl formate (1)
has been utilized, but only a poor yield was obtained for
formylation of longer peptides [14]. Finally, p-nitrophenyl formate
(2) was reported to furnish formylation in SPPS, but experimental
details were absent [15,16]. Thus, development of a convenient
simple protocol utilizing a commercially available reagent with
broad compatibility to Fmoc/tBu-based SPPS was clearly warranted.
Here, we present a comparative study comprising several well-
known formylating agents (Figure 1) that were examined for their
efficiency in N-terminal formylation of resin-bound peptides under
various conditions in order to develop a protocol with general
applicability to different resins, linkers, and peptides of different
length, prepared manually or by automated synthesizers.
*
Correspondence to: Henrik Franzyk, Department of Drug Design and
Pharmacology, University of Copenhagen, Jagtvej 162, DK-2100, Copenhagen,
Denmark. E-mail: henrik.franzyk@sund.ku.dk
Previously, several different methods have been applied
sporadically in solid-phase peptide synthesis (SPPS) of N-formyl
peptides. Formic acid in combination with diisopropylcarbodiimide
and pyridine was utilized to formylate a Rink amide resin in order to
Department of Drug Design and Pharmacology, Faculty of Health and Medical
Sciences, University of Copenhagen, Jagtvej 162, DK-2100, Copenhagen, Denmark
J. Pept. Sci. 2017; 23: 410–415
Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.

ON-RESIN N-FORMYLATION OF PEPTIDES
MicroTOF-Q II Quadrupol MS detector (Bruker Corporation, Billerica,
MS, USA).
N-formylimidazole (8)
was prepared in situ in the solvent used for the formylation by slow
addition (over 15 min) of formic acid (60 mg; 49 μl; 1.3 mmol; in
2.95 ml of the appropriate dry solvent) to a suspension of N,
N0-carbonyldiimidazole (115.3 mg; 1.2 mmol) in the appropriate
dry solvent (7 ml). The mixture was stirred for 1 h at room
temperature prior to use, and then 1 ml of this solution was used
in the formylation of the model peptide (0.12 mmol) [17].
Figure 1. Formylating agents included in the initial screening.
Resin-bound H-MFKFKF-NH2 (3a)
Materials and Methods
was assembled by using standard Fmoc-based SPPS. In brief, Rink
amide polystyrene resin (0.6 mmol; 1.25 g) was swelled in CH2Cl2
(10 ml) for 2 min, and upon draining, in DMF (10 ml) while shaking
for 25 min. Fmoc deprotection was performed by treatment with
20% piperidine in DMF (2 × 10 min, each 10 ml) followed by
successive washing with DMF, MeOH, and CH2Cl2 (15 ml, each for
3 × 3 min) with subsequent draining. Activation of Fmoc-Phe-OH
(2 equiv; 1.2 mmol; 465 mg) was performed with HBTU (1.95 equiv;
1.17 mmol; 444 mg) and DIPEA (4 equiv; 2.4 mmol; 418 μl), and after
10 min the mixture was added to the resin. Loading was continued
for 2 h, followed by washing as earlier and capping with Ac2O–
DIPEA–(N-Methyl-2-pyrrolidone) (1:2:3; 10 ml) for 10 min. Each
coupling cycle comprised washing as earlier, Fmoc removal,
washing, and addition of the next amino acid building block (4
equiv; 2.4 mmol) together with HBTU (3.9 equiv; 2.34 mmol;
444 mg) and DIPEA (8 equiv; 4.8 mmol; 840 μl) in dry DMF with
subsequent shaking for at least 2 h. After introduction of the last
amino acid and deprotection, the resin was washed as earlier and
then with hexane for 10 min. Subsequent lyophilisation for 16 h
gave the dry resin (1.85 g; loading: ~0.33 mmol/g. A portion of this
resin (75 mg) was cleaved with TFA–H2O (95:5) and the crude
purified by preparative HPLC (gradient elution: 10 → 45% B over
20 min) to provide a sample of the non-formylated model peptide.
Analytical HPLC (0 → 50% B): tR = 7.15 min. HR-MS (m/z): [M + 3H]3+
calcd for C44H63N9O6S, 282.8271; found 282.8274; ΔM = 1.06 ppm.
1H NMR (600 MHz, methanol-d4): δ = 7.28–7.15 (m, 15H, 3 × Ph),
4.63–4.55 (m, 3H, HPhe-α), 4.26–4.21 (m, 2H, HLys-α), 3.86 (t, J = 6.3 Hz,
1H, HMet-α), 3.17–3.04 (m, 6H, HPhe-β), 2.98–2.84 (m, 4H, HLys-β), 2.54
(m, 2H, HMet-β), 2.09 (s, 3H, S-CH3), 2.08–2.01 (m, 4H, HLys-ε),
1.77–1.56 (m, 8H, HLys-β and HLys-δ), 1.42–127 (m, 4H, HLys-γ); see
copy of spectrum on p. S35 in Supporting Information. 13C NMR
(600 MHz, methanol-d4): δ = 175.8, 173.6, 173.5, 173.3, 173.2,
138.3, 138.2, 138.0, 130.4, 130.2, 129.6*, 129.5, 128.0, 127.9, 56.3,
55.9, 55.7, 54.7, 54.6, 53.6, 40.5*, 38.9, 38.7, 38.6, 32.4, 32.3, 29.7,
28.1, 28.1, 23.7, 23.5, 15.1. *Denotes two close signals.
Fmoc-protected amino acid building blocks, Fmoc-protected Rink
amide ChemMatrix resin (loading: 0.48 mmol/g), 2-chlorotrityl
polystyrene resin (loading: 1.60 mmol/g), Wang ChemMatrix resin
(loading: 0.51 mmol/g), piperidine and trifluoroacetic acid were
obtained from IRIS Biotech (Marktredwitz, Germany). Fmoc-
protected Rink amide polystyrene resin (loading: 0.74 mmol/g), p-
nitrophenyl formate, cyanomethyl formate, 2,2,2-trifluoroethyl
formate and N,N0-carbonyldiimidazole were purchased from
Sigma-Aldrich (Munich, Germany). Fmoc-Lys(Boc)-preloaded
NovaSyn TG resin (loading: 0.18 mmol/g) and Nova Biochem PEGA
Rink amide resin (loading: 0.43 mmol/g) were purchased from
MerckMillipore (Darmstadt, Germany). Automated SPPS was
performed on a CEM LIBERTY CEM Corporation, Matthews, NC,
USA system with a DISCOVER microwave unit (CEM Corporation,
Matthews, NC, USA). 1H and 13C nuclear magnetic resonance
(NMR) spectra were acquired on a 600 MHz Bruker Avance III HD
(Bruker Corporation, Billerica, MS, USA) equipped with
a
1
cryogenically cooled 5 mm dual probe optimized for H and 13C.
Samples were dissolved in methanol-d4 (Cambridge Isotope
Laboratories) and analysed at 300 K. The residual solvent peak
was used as internal reference (CD3OD: δC = 49.00; δH = 3.31 or
(CD3)2SO: δC = 39.52 ppm; δH = 2.50 ppm). Coupling constants (J
values) are given in hertz (Hz). Multiplicities are reported as follows:
singlet (s), doublet (d), triplet (t), quartet (q), and multiplet (m).
Water used for analytical and preparative HPLC was filtered through
a 0.22 μm membrane filter. Analytical HPLC was performed on a
Shimadzu Nexera UHPLC System (Shimadzu Corporation, Kyoto,
Japan) comprising two LC-30AD pumps, an SIL-30AC auto-sampler,
CTO-20AC oven, an SPD-M20A PDA-Detector, and a DGU-20A5R
Controller; a Phenomenex Luna 2.5 μ C18(2) HST column
(3.0 × 100 mm) was used. Preparative HPLC was performed on a
Shimadzu Prominence (Shimadzu Corporation, Kyoto, Japan)
system comprising a CBM-20A controller, two LC-20AP pumps, an
SIL-20AHT, an SPD-M20A diode array detector, by using a
Phenomenex Luna 5 μ C18(2) column (30 × 250 mm) or
(21.2 × 250 mm) (Phenomenex Inc., Torrence, CA, USA). For both
systems, binary mixtures of eluent A (5% MeCN in H2O with 0.1%
TFA added) and eluent B (95% MeCN in H2O with 0.1% TFA added)
were used. Peak identity was confirmed by MALDI-TOF
spectrometry on a Bruker Microflex LRF (Bruker Corporation,
Billerica, MS, USA). Liquid chromatography-high-resolution mass
spectrometry was performed with a Phenomenex Luna C18 (2)
(3 μm) column (4.6 mm × 150 mm) using binary mixtures of eluent
C (water–MeCN–formic acid 95:5:0.1) and eluent D (water–MeCN–
formic acid 5:95:0.1); elution was performed with a linear gradient
of 0 → 100% D during 30 min at a flow rate of 0.5 ml/min. High-
resolution mass spectroscopy (HR-MS) was performed on a Bruker
f-MFKFKF-NH2 (3b)
was obtained by formylation of resin-bound 3a. The resin
(0.024 mmol; 75 mg) was swelled in CH2Cl2 for 10 min, upon which
a mixture of the formylating agent (5 equiv; 0.12 mmol) was added
along with DIPEA (0.24 mmol; 4.2 μl) in 1 ml of solvent. Resin
samples were removed from the formylation mixture after 1 and
2 h, while the remaining resin was continuously formylated for
16 h. The resin samples were immediately washed as earlier (2 ml
each), and cleaved with TFA–H2O (95:5) (2 × 1 h, 1 ml each). The
formylated model peptide was purified by preparative HPLC
(gradient elution: 10 → 45% B over 20 min). Analytical HPLC
J. Pept. Sci. 2017; 23: 410–415
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S. B. CHRISTENSEN, A. M. HANSEN AND H. FRANZYK
(0 → 50% B): tR = 8.03 min. HR-MS (m/z): [M + 2H]2+ calcd for
C45H63N9O7S, 437.7353; found 437.7357; ΔM = 0.9 ppm.
[M + 3H]3+ calcd for C110H185N25O26S, 796.1203; found, 769.1207;
ΔM = 0.5 ppm.
1H NMR (600 MHz, methanol-d4): δ = 8.10 (d, 1H, CHO), 7.30–7.16
(m, 15H, 3 × Ph), 4.62 (dd, J = 5.5 and 3.9 Hz, 1H, HPhe-α), 4.57 (m, 2H,
N-formyl Humanin (12)
H
Phe-α), 4.37 (dd, J = 5.7 and 2.4 Hz, 1H, HMet-α), 4.22 (dd, J = 6.0 and
was synthesized on a CEM Liberty synthesizer using a commercial
Fmoc-Ala-preloaded Wang TentaGel resin, and the resulting resin-
bound peptide was divided into two portions that were formylated
for 2 h with 8 in DMF and NMP, respectively. Another batch of 12
was prepared on an Fmoc-Ala-preloaded 2-chlorotrityl resin
(0.2 mmol; 68 mg), obtained as described in the synthesis of 9,
while peptide assembly was performed using an improved
microwave (MW)-assisted method (see protocols on p. S41 and
S42 in Supporting Information). Repeated formylation with 2 in
DMF was performed for 2 and 1 h, followed by washing and
cleavage (as described for 9). Purification by preparative HPLC
was achieved with a 20 → 60% B gradient over 20 min. Analytical
HPLC (20 → 60% B): tR = 6.82 min. HR-MS (m/z): [M + 5H]5+ calcd
for C120H204N34O33S2, 543.7110; found, 543.7086; ΔM = 4.4 ppm.
2.0 Hz, 1H, HLys-α), 4.15 (dd, J = 5.6 and 3.0 Hz, 1H, HLys-α), 3.16
(m, 3H, HPhe-β), 2.95 (m, 3H, HPhe-β), 2.86 (dt, J = 2 × 7.5 and
7.0 Hz, 4H, HLys-ε), 2.41 (m, 2H, HMet-γ), 2.03 (s, 3H, S-CH3), 1.94–
1.81 (m, 2H, HMet-β), 1.74–1.55 (m, 8H, HLys-β and HLys-δ), 1.37–1.25
(m, 4H, HLys-γ); see copy of spectrum on p. S36 in Supporting
Information. 13C NMR (600 MHz, methanol-d4): δ = 175.9, 174.2,
173.9, 173.7, 173.5, 164.5, 138.5, 138.3, 130.4, 130.4, 130.2, 129.7,
129.6, 129.5, 128.0, 127.9, 127.8, 56.7, 54.5, 55.8, 55.5, 54.8, 53.6,
40.5, 38.8, 38.1, 37.6, 32.0*, 31.7, 30.8, 28.0, 27.9, 23.6, 23.5, 15.2.
*Denotes two close signals.
f-MIFL-PSM5–12 (9)
To obtain an Fmoc-Lys(Boc)-preloaded 2-chlorotrityl chloride (CTC)
resin, a sample of 2-CTC resin (0.1 mmol) was treated with 10%
DIPEA in dry CH2Cl2 (3 ml) for 3 min followed by washing twice with
dry CH2Cl2 (5 ml; 2 × 2 min), and then Fmoc-Lys(Boc)-OH (2 equiv;
0.2 mmol) and DIPEA (0.4 mmol; 67 μl) in dry CH2Cl2 (3 ml) were
added to the resin, which subsequently was shaken for 2 h, and
then washed successively with DMF, MeOH, and CH2Cl2 (5 ml, each
for 3 × 3 min). After the remaining sequence was assembled on a
CEM Liberty synthesizer (see protocol on p. S38 in Supporting
Information), the resin was washed as earlier and divided into three
portions (each ~0.033 mmol), two of which were formylated with
either 2 or 8 (5 equiv) and DIPEA (10 equiv) in DMF for 2 h, while
the third portion was cleaved directly for comparison. Finally, the
formylated resins were washed as earlier followed by cleavage with
TFA–H2O–TIS (95:2.5:2.5) (2 × 1 ml) for 1 h. Purification was
performed by preparative HPLC using a 10 → 80% B gradient over
20 min. Analytical HPLC (10 → 80% B): tR = 7.94 min. HR-MS (m/z):
[M + 2H]2+ calcd for C71H116N14O14S, 711.4258; found, 711.4261;
ΔM = 0.4 ppm.
f-MIFL-OH (13)
A large batch of Fmoc-Leu-preloaded Wang resin was obtained
from Wang resin (1.0 mmol; 1.96 g) to which Fmoc-Leu-OH (5 mmol;
1.76 g) in DMF–CH2Cl2 (1:1; 4 ml) and then DIC (5 mmol; 0.77 ml)
followed by DMAP (1 mmol; 122 mg) were added. Subsequently,
the mixture was shaken for 24 h, and then the resin was washed
successively with DMF, MeOH, and CH2Cl2 (5 ml, each for
3 × 3 min). A sample of this resin (0.1 mmol) was used for assembly
of the remaining part of the sequence, including the formylation,
on a CEM Liberty synthesizer (see protocol on p. S43 and S44 in
Supporting Information). In addition, the formylation step was
performed manually using 5 equiv of formylating agent 2 in DMF
in a preheated heating block at 50 °C for 20 min. Finally, cleavage
with TFA–CH2Cl2 (1:1) for 2 × 1 h was followed by purification by
preparative HPLC using a 40 → 65% B gradient over 20 min.
Analytical HPLC (20 → 100% B): tR = 9.99 min.
1H NMR (600 MHz, DMSO-d6): δ = 8.30 (d, 1H, J = 8.3 Hz, NHMet),
8.18 (d, J = 8.0 Hz, 1H, NHLeu), 8.07 (d, J = 8.5 Hz, 1H, NHPhe), 8.00 (s,
1H, CHO), 7.85 (d, J = 8.8 Hz, 1H, NHIle), 7.24–7.21 (m, 4H, Ph), 7.18–
7.14 (m, 1H, Ph), 4.61–4.56 (m, 1H, HPhe-α), 4.45–4.41 (dt, J = 5.2 and
8.3 Hz, 1H, HMet-α), 4.25–4.21 (m, 1H, HLeu-α), 4.15 (dd, J = 7.4 and
8.8 Hz, 1H, HIle-α), 2.99 (dd, J = 4.3 and 14.2 Hz, 1H, HPhe-β), 2.77
(dd, J = 9.6 and 14.2 Hz, 1H, HPhe-β), 2.41–2.31 (m, 2H, HMet-γ),
2.00 (s, 3H, HMet-δ), 1.83–1.76 (m, 1H, HMet-β), 1.74–1.68 (m, 1H,
HMet-β), 1.66–1.59 (m, 2H, HIle-β and HLeu-γ), 1.56–1.46 (m, 2H,
PSMα3 (10)
was synthesized from an Fmoc-Asp(Trt)-preloaded resin, obtained
from Fmoc-Asp(Trt)-OH (0.2 mmol; 119.3 mg) and 2-CTC resin
(1.60 mmol/g; 62.5 mg; 0.1 mmol) as described in the synthesis of
9. Assembly of the remaining sequence was performed on a CEM
Liberty (see protocol on p S39 in Supporting Information). Upon
transfer to a teflon reactor, the resin was washed (as described for
9) and then subjected to formylation for 2 h followed by wash with
DMF and treatment with fresh reagent for another 1 h; then
cleavage was performed as described for 9 followed by purification
by preparative HPLC using a 30 → 100% B gradient over 20 min.
Analytical HPLC (30 → 100% B): tR = 6.98 min. HRMS (ESI): m/z
[M + 4H]4+ calcd for C128H192N28O30S, 659.3593; found, 659.3603;
ΔM: 1.5 ppm.
HLeu-β), 1.35–1.28 (m, 1H, HIle-γ), 1.02–0.94 (m, 1H, HIle-γ), 0.88
(d, J = 6.7 Hz, 3H, HLeu-δ), 0.83 (d, J = 6.5 Hz, 3H, HLeu-δ), 0.75
(t, J = 7.3 Hz, 3H, HIle-δ), 0.72 (d, J = 6.9 Hz, 3H, HIle-γ). 13C NMR
(600 MHz, DMSO-d6): δ 174.4, 171.4, 171.0, 170.8, 161.4, 138.1,
129.5 (2C), 128.4 (2C), 126.7, 57.1, 53.7, 50.8, 50.6, 40.5, 37.9, 37.3,
32.6, 29.9, 24.6, 24.5, 23.4, 21.7, 15.6, 15.1, 11.5. See copies of spectra
on p. S37 in Supporting Information.
PSMα2 (11)
Results and Discussion
was synthesized on a CEM Liberty synthesizer (see protocol on p.
S40 in Supporting Information) by using a commercial preloaded
Fmoc-Lys(Boc)-NovaSyn Tentagel Resin (0.1 mmol; 556 mg). Upon
completion of the sequence the resin was divided into smaller
portions that were formylated with either 2 or 8 for 2 h, followed
by cleavage as described earlier for 9. The crude product was
purified by preparative HPLC using a 30 → 100% B gradient over
20 min. Analytical HPLC (10 → 80% B): tR = 8.95 min. HR-MS (m/z):
Initially, a short model peptide, H-MFKFKF-NH2 (3a; obtained via
standard manual Fmoc-based SPPS), was utilized as a resin-bound
substrate for introduction of an N-formyl group. In total, seven
formylation conditions were included in this screening: (i) aliphatic
formates 1, 2,2,2-trifluoroethyl formate (4) [18], and cyanomethyl
formate (5) [19,20], (ii) aromatic reactive ester 2 [15,16] and
phenyl formate (6) [21], (iii) in situ activation of formic acid with
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J. Pept. Sci. 2017; 23: 410–415

ON-RESIN N-FORMYLATION OF PEPTIDES
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (7) [22], and (iv)
N-formylimidazole (8) [10,17]. These preliminary experiments
showed that 1 immediately could be omitted from the pool of
potential reagents because of low conversion even upon an
extended reaction time of 16 h at room temperature.
Table 2. N-formylation of 3a on different resin typesa
Formylating agent
Polystyrene (%)
PEGA (%)
ChemMatrix (%)
2
5
6
8
99.0
88.0
98.0
98.0
95.0
92.0
93.0
93.5
97.0
92.5
97.0
95.5
In Table 1, the results from formylation of model peptide 3a with
six selected reagents (in combination with solvents commonly used
in solid-phase chemistry) are given as the conversion into the
corresponding formylated peptide (3b) as determined by UV-
detected RP-HPLC analysis after reaction times of 1, 2, and 16 h
(see p. S3–S26 in Supporting Information). Both DMF and CH2Cl2
are standard solvents in SPPS, while THF is the solvent conferring
the highest swelling to traditional polystyrene-based solid
supports. After 2 h, only 2 and 8 afforded almost complete
conversion (see p. S2–S5 and S23–S26 in Supporting Information).
The high efficiency of N-formylimidazole in on-resin N-formylation
is in agreement with its reported high reactivity when employed
in formylations of primary amines under solution-phase conditions
[23,24]. However, as this reagent is not readily available through
commercial sources because of its limited stability [17], it was
considered most convenient to employ 8 as a freshly prepared
reagent in the solvent of choice. On the other hand, reagents 2, 5
and 6 are easily handled and commercially available, and they all
appeared applicable in SPPS as reported for the solution-phase
transformation [16,19,21]. In contrast, inspection of the degree of
conversion after 16 h, revealed that reagents 4 and 7 displayed
lowered reactivity when using CH2Cl2 or THF as solvent (see p.
S6–S9 and S19–S22 in Supporting Information). Hence, to retain
the possibility of adaptation to several solvents, we continued the
experiments with the set of reagents (i.e. 2, 5, 6, and 8) displaying
a final formylation degree above 85% in all solvents examined.
Compatibility of these conditions with various solid supports and
linkers was also tested, and as seen from Table 2 (see p. S4, S12,
S17, S25 and S31–S34 in Supporting Information), consistent
aReactions were performed at room temperature for 16 h using 5 equiv
of formylating agent and 10 equiv of DIPEA in DMF. The accuracy of the
measurements is 0.5% (via HPLC integration).
slightly lower formylation degrees were found for 5 (see p. S12
and S32 in Supporting Information) while all resins were found to
be equally suitable in combination with the remaining reagents
(2, 6 and 8) when an extended coupling time was employed.
However, a short reaction time comparable with that of typical
peptide couplings (0.5–2 h) would be preferable, thereby excluding
6 because it afforded a lower conversion after 2 hours (cf. Table 1;
see p. S15 and S16 in Supporting Information).
Hence, we investigated whether use of 2 or 8 might allow for
rapid formylation of an array of peptides (Table 3.; 9–13) attached
to common solid supports via different linkers. The formyl peptide
receptor ligands f-MIFL-PSM(5–12) (i.e. f-MIFLAGIIKFIK-OH, 9) [25]
and PSMα3 (i.e. f-MEFVAKLFKFFKDLLGKFLGNN-OH, 10) [26] were
synthesized on a 2-chlorotrityl functionalized polystyrene resin
(see p. S28 in Supporting Information), while PSMα2 (i.e.
f-MGIIAGIIKFIKGLIEKFTGK-OH, 11) [25] was assembled on a
NovaSyn® TentaGel™ resin pre-loaded with Fmoc-Lys(Boc)-OH
(see p. S29 in Supporting Information). In addition, N-formyl
humanin (i.e. f-MAPRGFSCLLLLTSEIDLPVKRRA-OH, 12) [27] (see p.
S29 in Supporting Information) was prepared both on a TentaGel™
resin preloaded with alanine via a Wang linker and on a
2-chlorotrityl polystyrene resin. This sequence turned out to be
surprisingly difficult, and several residues required triple-coupling
while the remaining residues were introduced via double-couplings
in order to obtain an acceptable purity prior to the formylation step
(for MW protocols see p. S41–S42 in Supporting Information). As
evident from Table 4, a satisfactory degree of formylation of longer
peptides (12–24 residues) could in most cases be accomplished
within 2 h at room temperature by using either 2 or 8. However,
for some peptides, it was beneficial to employ a repeated coupling
for an additional 1 h with fresh reagent in order to reach almost full
conversion, for example, the formylation degree for 10 when
treated with 2 in DMF could then be increased to 97%.
Finally, in the formylation step to give f-MIFL-OH (13) [28,29], it
proved feasible to reach complete conversion even with a reduced
reaction time of 20 min via formylation with 2 at an elevated
temperature by heating the mixture to 50 °C either in a
conventional heating block (20 min; using a Teflon reactor for
manual synthesis) or by MW-assisted coupling on an automated
synthesizer (MW protocol: 25 W for 2 × 15 min; see p. S43 in
Supporting Information).
In conclusion, the most versatile and convenient reagent for
on-resin N-formylation in SPPS was found to be the commercially
available activated ester 2 that displayed general applicability to
different resins, linkers, and peptides of different length prepared
manually or by automated synthesizers. High formylation degrees
were typically obtained after 1–3 h at room temperature, while
either conventional or microwave-assisted heating allowed
reduction of the formylation time to 20 min. Furthermore, the
Table 1. Effect of time and solvent on conversion of 3a in on-resin
N-formylationa
Reagent
Solvent
1 h (%)
2 h (%)
16 h (%)
2
DMF
CH2Cl2
THF
—
97.5
93.5
96.5
37.0
5.0
99.0
98.5
99.0
94.0
44.0
39.0
88.0
97.0
98.5
98.0
98.0
98.0
98.5
35.5
72.5
98.0
98.5
99.0
92.0%
96.5%
—
4
5
6
7
8
DMF
CH2Cl2
THF
2.5%
9.5%
—
17.0
25.5
11.5
43.0
73.0
74.0
91.5
46.5
35.0
68.0
91.5
97.0
97.0
DMF
CH2Cl2
THF
3.5%
24.5%
54.5%
54.0%
83.5%
—
DMF
CH2Cl2
THF
DMF
CH2Cl2
THF
34.0%
70.0%
88.0%
95.0%
—
DMF
CH2Cl2
THF
aAll reactions were performed with 5 equiv of formylating reagent and
10 equiv of DIPEA in the appropriate solvent at room temperature.
The accuracy of the measurements is 0.5% (based on HPLC
integration).
J. Pept. Sci. 2017; 23: 410–415
Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.
wileyonlinelibrary.com/journal/jpepsci

S. B. CHRISTENSEN, A. M. HANSEN AND H. FRANZYK
Table 3. Properties and characterization data of peptides
—
Sequence
Length
Overall charge
m/z
—
3a
3b
9
—
—
6
—
+3
+2
+1
+1
+2
+1
–1
Calc.
Obs.a
H-MFKFKF-NH2
282.8271
437.7353
711.4258
659.3593
769.1203
543.7110
549.2985
282.8274
437.7357
711.4261
659.3603
769.1207
543.7086
549.56b
f-MFKFKF-NH2
6
f-MIFLAGIIKFIK-OH
12
22
21
24
4
10
11
12
13
f-MEFVAKLFKFFKDLLGKFLGNN-OH
f-MGIIAGIIKFIKGLIEKFTGK-OH
f-MAPRGFSCLLLLTSEIDLPVKRRA-OH
f-MIFL-OH
aHR-MS.
bMALDI-TOF MS.
Table 4. Formylation of longer peptides assembled on various resin
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Supporting information
Additional Supporting Information may be found online in the
supporting information tab for this article.
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J. Pept. Sci. 2017; 23: 410–415
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