Accelerated Fmoc solid-phase synthesis of peptides with aggregation-disrupting backbones

Article abstract of DOI:10.1039/c4ob02260b

In this work, we describe an accelerated solid-phase synthetic protocol for ordinary or difficult peptides involving air-bath heating and amide protection. For the Hmsb-based backbone amide protection, an optimized acyl shift condition using 1,4-dioxane w

Full text of DOI:10.1039/c4ob02260b

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Accelerated Fmoc solid-phase synthesis of
peptides with aggregation-disrupting backbones†
Cite this: DOI: 10.1039/c4ob02260b
a,b
a
b
a,c
Yi-Chao Huang, Chao-Jian Guan, Xiang-Long Tan, Chen-Chen Chen,
c
a
Qing-Xiang Guo and Yi-Ming Li*
In this work, we describe an accelerated solid-phase synthetic protocol for ordinary or difficult peptides
involving air-bath heating and amide protection. For the Hmsb-based backbone amide protection, an
optimized acyl shift condition using 1,4-dioxane was discovered. The efficiency and robustness of the
protocol was validated in the course of preparation of classical difficult peptides and ubiquitin protein
segments.
Received 23rd October 2014,
Accepted 14th November 2014
DOI: 10.1039/c4ob02260b
www.rsc.org/obc
8
tion, (4) employing more aggressive coupling reagents, such
as HATU/DIEA or COMU/DIEA single/multiple couplings, for a
Introduction
9
,10
Total synthesis of peptides and proteins provides valuable prolonged time,
and (5) applying a sophisticated microwave
1
1
molecular tools for chemical biology and medicinal chem- or conventional oil-bath heating apparatus. The second
1
istry. Despite the introduction of routine solid phase peptide group of strategy (chemical means) takes the advantage of
synthesis (SPPS) by Merrifield and the development of power- backbone amide protection to suppress the amide N–H
2
,3
12
ful native chemical ligation (NCL) by Kent,
the protein involved on-resin β-sheet formation (Fig. 1). Kiso’s group and
assembly process is often plagued by the presence of difficult Mutter’s group designed an elegant aggregation-breaking
peptide sequences. For Boc SPPS, repetitive TFA treatment of method by the incorporation of “click peptides” or “switch
1
3
resins combined with in situ neutralization coupling can mod- peptides.” This method mostly relies on the synthesis of de-
4
erate the aggregation effect. However, the toxicity of hydrogen psipeptides (O-acyl isopeptides) followed by in-solution O→N
fluoride as the cleavage reagent makes Boc SPPS less favorable intramolecular acyl transfer to restore native Xaa-Ser/Thr
to peptide chemists. While in the case of Fmoc SPPS, in which peptide bonds. In addition to this depsipeptide approach,
on-resin aggregation can easily occur through inter- or intra-
molecular chain interactions, difficult sequences pose a more
severe bottleneck toward the preparation of high-quality pep-
5
tides. To address the issue of difficult sequences, many dis-
aggregating strategies have been devised, which generally fall
into two categories.
The first group of strategy (physical means) combat difficult
sequences by adjusting the external environment during SPPS,
which includes: (1) switching typical polystyrene resins to low-
substitution high-swelling hydrophilic resins such as PEG-
6
based resins, (2) using alternative solvents or solvent mixtures
7
(
DMF, NMP, DMSO) for better swelling effects, (3) adding
chaotropic salts (LiCl, KSCN, NaClO ) into the coupling solu-
4
a
School of Medical Engineering, Hefei University of Technology, Hefei 230009,
China. E-mail: ymli@hfut.edu.cn, lym2007@mail.ustc.edu.cn
b
Department of Chemistry, Tsinghua University, Beijing, 100084, China
c
Department of Chemistry, University of Science and Technology of China, Hefei
2
†
30026, China
Fig. 1 Typical backbone protecting groups used during Fmoc SPPS.
†
Electronic supplementary information (ESI) available: General information and
These protecting groups are less sensitive to the steric hindrance of
1
13
1
2 ‡
details of synthetic procedures, H NMR, C NMR, HPLC, ESI-MS. See DOI:
0.1039/c4ob02260b
residue R and R . These protecting groups are stable under conven-
tional TFA cleavage conditions.
1
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commercially available Fmoc-protected pseudoproline dipep- acids (Ile, Val, Thr). Moreover, we successfully prepared several
tides have gained wide popularity as aggregation-breaking difficult sequences (70%–95% HPLC purities of crude pro-
1
4
building blocks 1–3. An important feature is that pseudopro- ducts) using air-bath heating batch-based SPPS in conjunction
line structures can be converted to native amides during acidic with Hmsb-based backbone amide protection. The timescale
cleavage. Unfortunately, their utility is limited to Xaa-Ser, Xaa- of a single deprotection-washing-coupling SPPS cycle can be
Thr or less frequently Xaa-Cys containing sequences. Another shortened by as much as 90 min using the new air-bath
popular method exploits the acid lability of electron-rich heating apparatus.
benzyl protected amide. Both 2,4-dimethoxybenzyl (Dmb) 4
and 2-hydroxy-4-methoxybenzyl (Hmb) 5 groups were found to
1
5,16
have considerable aggregation-disrupting effects.
These
Results and discussion
Discovery of the optimized acyl shift condition in dioxane
groups can also be cleaved with TFA to recover native amide
bonds. Importantly, Fmoc-Asp(OtBu)-(Dmb)Gly-OH and Fmoc-
Asp(OtBu)-(Hmb)Gly-OH have proven to be reliable building
blocks to completely avoid the notorious aspartimide for-
On our route to extend the applicability of Fmoc SPPS with
Hmsb-based backbone protection, we noticed Alewood group’s
report that O-acylation proceeds fast and quantitatively while
the following acyl transfer step is sluggish. According to pre-
1
7
mation. Recently, an Hmb derivative 6 was developed to
allow the incorporation of an oligo-arginine tag, which is con-
1
8
2
0a,21,25
ducive for the synthesis of transmembrane peptides, almost
exclusively difficult sequences. The major drawback of these
benzyl type protecting groups is the huge steric hindrance
vious literature,
the treatment of resins with nonpolar
solvents could facilitate on-resin intramolecular acyl transfer.
Therefore, we began our acyl shift optimization by screening
various solvents or solvent mixtures.
2
around the tertiary amide. The choice of R is usually con-
stricted to Gly and Ala. This is also the case for a photo-labile
Initially, we set two fundamental requirements for appropri-
ate solvents: good resin-swelling capability and high boiling
point. The first criterion is crucial for fast on-resin kinetics
and the second is beneficial in case of high-temperature
1
9
amide protecting group 2-nitrobenzyl (2-Nb) 7.
Interestingly, the phenol group in Hmb 5 permits fast
phenol ester formation, and then tertiary amide generation
1
6
2
6
through six-membered acyl migration. This special mechan-
SPPS. Based on this, we tested the effects of six solvents
towards the on-resin acyl shift reaction on a model pentapep-
tide, H-Tyr-Leu-(Hmsb)Leu-Ser-Lys-NH2 (Fig. 2). All deprotec-
tion and couplings were conducted under standard room
temperature Fmoc SPPS conditions except the acyl shift step.
Hmsb was synthesized using a slightly modified procedure
compared with an earlier paper (Scheme S2†). For good com-
parison, all the reactions were conducted at 50 °C for 2 h in
the presence of 5% (v/v%) DIEA (Table 2, entries 1–8). Modi-
1
2
ism slightly enhanced the residue (R and R ) tolerance of
Hmb 5 compared with Dmb 4. To speed up the rate-limiting
acyl shift step, several electron-withdrawing modifications (8
and 9) on 2-hydroxybenzyl having better acyl shift kinetics
2
0,21
were reported.
An additional advantage of these backbone
protecting groups is that they are stable to standard acidic clea-
vage cocktails. This feature is instrumental because peptides
containing tertiary amide backbones usually bear better HPLC
elution behavior (shorter retention time and sharper peak)
and greater solubility in the LC elution buffer (0.1% TFA,
acetonitrile–water = 1/1, pH 1) or the NCL buffer (6 M Gn-Cl,
2
fied cocktail K (TFA/H O/thioanisole/EDT = 87/5/5/3) was used
2
7,28
as the cleavage reagent for sulfoxide-containing peptides.
1
8,22,23
pH 4–8).
ligations of backbone-protected peptides, 2-hydroxy-6-nitro-
benzyl (Hnb) and 2-hydroxy-4-methoxy-5-methylsulfinyl-
After post-cleavage purifications and segment
8
benzyl (Hmsb) 9 were found to be cleavable by using
UV illumination and sulfoxide-reducing TFA (Fig. S1,
Scheme S1†), respectively, to generate free peptides. A similar
safety-catch
structure
2-methoxy-4-methylsulfinylbenzyl
(
Mmsb) was also very recently reported by the Albercio
2
4
group.
Previous work from Offer’s group indicates that Hmsb 9
could improve the purity and solubility of on-resin aggrega-
tion-prone peptide sequences for both manual and automated
2
1
SPPS. However, the tolerance of 9 to β-branched amino acids
was not investigated. In addition, to test the scope and feasi-
bility of this method, we thought it was necessary to prepare
longer peptides (>25 aa) with multiple Hmsb-protected
amides. Herein, we discovered a new solvent incubation proto-
col, which exhibited better acyl shift kinetics to incorporate 9.
Our results showed that the new acyl transfer condition could
Fig. 2 HPLC traces of H-Tyr-Leu-(Hmsb)Leu-Ser-Lys-NH
under different acyl shift conditions. All acyl shift reactions were carried
2
synthesized
tolerate couplings of sterically hindered β-branched amino out in 5% (v/v) DIEA/solvent at 50 °C for 2 h.
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Table 1 Optimization of the on-resin acyl shift reaction during Fmoc
SPPS to prepare H-Tyr-Leu-(Hmsb)Leu-Ser-Lys-NH
2
DIEA
[v/v %] [°C]
Temperature HPLC
Entry Solvent (mixture)
purity [%]
1
2
3
4
5
6
7
8
9
1
1
1
1
Toluene
Chloroform
THF
1,4-Dioxane
DMF
DMSO
THF–MeOH = 4/1
2
THF–H O = 4/1
1,4-Dioxane
1,4-Dioxane
DMF
1,4-Dioxane
(Acyl shift step skipped)
5
5
5
5
5
5
5
5
0
0
0
0
50
50
50
50
50
50
50
50
50
25
25
75
82
83
84
90
74
40
67
66
91
77
66
97
52
0
1
2
3
2
5
As expected, all of the nonpolar solvents afforded the
crude Hmsb-protected peptide with good HPLC purity
Table 1, entries 1–4). Interestingly, 1,4-dioxane worked better
than other less polar solvents (90% HPLC purity), possibly due (B) Ac-Ser[L/D]-(Hmsb)Ala-Lys-NH
(
Fig. 3 Racemization tests of (A) Ac-Phe[L/D]-(Hmsb)Ala-Lys-NH
2
and
con-
†
2
.
Ac-Ser[L]-(Hmsb)Ala-Lys-NH
2
‡
taining racemic sulfoxides (∼1 : 1) are separable by HPLC. Ac-Ser[D]-
Hmsb)Ala-Lys-NH containing racemic sulfoxides are separable by
HPLC.
to its excellent ability in resin-swelling and chain-solvation.
Inferior results were obtained when aprotic polar solvents
DMF and DMSO were used (Table 1, entries 5–6). In the case
of DMSO, three byproducts were detected through HPLC and
(
2
1
2
ESI-MS (Fig. 2), which corresponded to a Tyr and Leu dele-
2
2
attempted, which might afford comparable results to micro-
tion product, a Leu deletion product, and a surprising Leu
2
9
wave-based automated SPPS. Due to concerns of racemiza-
addition product. The deletion product indicated an incom-
3
0
3
tion and other side-reactions, we carried out base-free DIC/
Oxyma couplings at 75 °C (air-bath heating) for all the Fmoc
plete acyl shift reaction of Leu . The appearance of the
additional byproduct might be caused by premature Fmoc
deprotection by the N-terminal secondary amine of peptide
chain in an aprotic polar solvent in the presence of base at
higher temperature. Addition of water or methanol as a co-
solvent during acyl shift failed to produce the product with a
higher yield (Table 1, entries 7–8). Further experiments
showed that additional base was unnecessary and the acyl
shift happened more efficiently at higher temperature without
DIEA (Table 1, entries 9–12). Therefore, neat dioxane (b.p.
3
1
amino acids except Arg, Cys and His. Cysteine was coupled at
0 °C while Arg and His were coupled at room temperature.
5
Next, we synthesized Hmsb protected sequences containing
racemization-prone Phe, Ser, Cys residues (Fig. 3 and S2†).
Note that both Ac-Ser[L]-(Hmsb)Ala-Lys-NH2 and Ac-Ser[D]-
(
2
Hmsb)Ala-Lys-NH showed double peaks on HPLC (peak
volume ∼ 1 : 1) with identical molecular weights (Fig. 3B). This
was not caused by on-resin racemization of the building block
but the fact that Hmsb was synthesized as a racemic sulfoxide
compound. Interestingly, racemic Ac-Phe[L]-(Hmsb)Ala-Lys-
NH2 or Ac-Phe[D]-(Hmsb)Ala-Lys-NH2 appeared inseparable
using the same HPLC gradient. All of the LC co-injection
traces confirmed that racemization ratio was quite low (<2%)
during the manual high-temperature synthesis of Hmsb-
protected peptides (Fig. 3).
1
01 °C) was chosen as the ideal solvent in the following acyl-
shift experiments. Encouragingly, compared with Offer’s pre-
vious protocol using DMF as the sole solvent during coupling
and acyl shift, our new 1,4-dioxane involved acyl shift protocol
showed a noticeable improvement (Table 1, entries 11–13).
Scope of Hmsb protection of backbone amides
To study the reaction scope of the on-resin acyl shift reac-
To speed up Hmsb incorporation, we decided to perform both tion, we prepared another model pentapeptide H-Met(O)-Xaa-
O-acylation and acyl shift reactions at elevated temperatures. (Hmsb)Yaa-Ser-Lys-NH . The reaction efficiency was evaluated
Based on recent reports about microwave or oil-bath involved based on the HPLC purities of crude products using various
2
1
1c,d
SPPS,
we believed that manual SPPS at 50–90 °C could be Xaa/Yaa combinations (Fig. S3† and Table 2). We found that
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Table 2 Scope of the on-resin acyl shift reaction during Fmoc SPPS
using a model sequence H-Met(O)-Xaa-(Hmsb)Yaa-Ser-Lys-NH
2
Acyl shift
time [h]
HPLC
purity [%]
Entry
Xaa
Yaa
1
2
3
Leu
Asn
Phe
Arg
Pro
Trp
Ile
Leu
Asn
Phe
Arg
Gln
Tyr
Gly
Ala
Ala
Ala
Leu
Val
2
2
2
2
2
95
96
78
80
86
70
90
86
85
82
75
<5
a
4
5
6
7
8
9
1
1
1
2
12
12
12
12
24
24
Ile
Val
Thr
Val
Leu
0
1
2
Scheme 2 (A) Standard manual SPPS timeline. (B) Manual SPPS timeline
a
The acyl shift of Fmoc-Arg(Pbf)-OH was performed at room with Hmsb incorporation. (C) The SPPS protocol includes four steps,
†
temperature rather than at 75 °C.
deprotection, anchoring, coupling and acyl shift. HCTU/DIEA (RT,
5 min) or DIC/Oxyma (RT, 90 min) coupling is chosen for Fmoc-
Arg(Pbf)-OH to prevent δ-lactam formation of arginine. DIC/Oxyma (RT,
5 min) coupling is chosen for Fmoc-His(Trt)-OH to suppress racemiza-
4
4
tion. DIC/Oxyma (50 °C, 20 min) coupling is chosen for Fmoc-Cys(Trt)-
OH to suppress racemization. DIC/Oxyma (75 °C, 20 min) coupling is
‡
used for the rest of the 17 canonical amino acids. The acyl shift step
can be carried out for 2–12 h, depending on the steric bulkiness of both
the acceptor and donor amino acids of the acyl transfer. For Fmoc-
Arg(Pbf)-OH and Fmoc-His(Trt)-OH, the temperature should be set at
RT to minimize any side reactions.
duce each amino acid building block. In comparison, our new
coupling cycle (deprotection, washing and coupling) takes only
3
0 min to complete with an easily accessible air-bath shaker as
the only additional piece of equipment for manual SPPS
Scheme 2A and Fig. S4†). The actual coupling time at high
temperature is around 15 min (excluding 3–5 min for heat
(
Scheme 1 Fully optimized incorporation of Hmsb 9 consists of three
steps, (1) anchoring of salicylaldehyde onto free amino resin through exchange). Piperidine deprotection was carried out at room
reductive amination; (2) coupling of Fmoc-AA-OH to form a phenolate
under room temperature or high temperature conditions; (3) on-resin
acyl shift in 1,4-dioxane to make a protected tertiary amide bond.
temperature to suppress any base-induced side reactions.
Further tests of Fmoc SPPS assisted by air-bath heating
To evaluate the robustness of our new fast Fmoc SPPS proto-
most β-linear amino acids are compatible with the on-resin col, we selected four difficult sequences from the literature,
acyl shift (Table 2, entries 1–6). The crude peptides displayed which were tested via air-bath heating Fmoc SPPS alone and
good to excellent HPLC purity. More importantly, Xaa seems to another two sequences through high-temperature SPPS plus
tolerate all the sterically hindered β-branched amino acids Hmsb incorporation. Four difficult peptides, ACP(65–74), ABC
when Yaa = Gly or Ala (Table 2, entries 7–10). However, Val- 20mer, PnlA(A10L) and HIV-1 PR(81–99) were synthesized on a
(Hmsb)Leu and Leu-(Hmsb)Val obtained worse results indicat- 0.1 mmol scale using a 30 min coupling cycle (Scheme 2A). As
ing that the acyl shift kinetics are more sensitive to the steric shown in Fig. 4, crude peptide products were obtained at good
19,20a
bulkiness of Yaa than Xaa (Table 2, entries 11–12).
We to excellent HPLC purity (70%–95% based on peak area ratios,
believed that these experimental data would be equally helpful see Table 3). For all of the four peptides, the whole main peak
when amino acid coupling and acyl shift were encountered on analytical HPLC was collected and characterized by direct
after Hmb 5 and Hnb 8 incorporation.
injection ESI-MS.
With the scope of investigation of Hmsb protection of back-
Two additional sequences PolyL10mer and toxin protein
bone amides, we updated the procedure of incorporation of Mambalgin-1(19–40) were further synthesized using both air-
Hmsb, (1) anchoring via on-resin two-step reductive ami- bath heating SPPS and Hmsb backbone protection. PolyL10
nation; (2) coupling via O-acylation; (3) acyl shift via incubation was found insoluble in common HPLC elution buffer H O/
2
in dioxane (Scheme 1). Furthermore, we depicted a timeline ACN = 1/1 when prepared through high-temperature SPPS
5
for Fmoc SPPS, which is based on air-bath heating (50 °C and alone. However, when Hmsb was incorporated between Leu
7
6
5 °C). Traditional Fmoc SPPS requires 45 to 120 min to intro- and Leu as a backbone kink, we obtained the fully soluble
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Fig. 4 Fmoc SPPS of difficult peptides using air-bath heating. (A) ACP
(
65–74): H-VQAAIDYING-NH
HEQCNRADG-NH (C) PnlA(A10L) conotoxin: H-GCCSLPPCALNNP-
DYC-NH ; (D) HIV-1 PR(81–99): H-PVNIIGRNLLTQIGCTLNF-NH
2
; (B) ABC 20mer: H-VYWTSPFM(O)KLI-
2
;
2
2
.
Table 3 Synthesis results
HPLC
purity [%] yield [%]
Crude
b
a
Entry Peptide
Condition
Fig. 5 Fmoc SPPS of difficult peptides using air-bath heating along
1
2
3
4
5
6
7
8
9
1
1
1
1
1
ACP(65–74)
ABC20mer
PnlA(A10L) conotoxin
HIV-1PR(81–99)
PolyL10
A
A
A
A
A
B
A
B
C
D
A
D
A
D
95
92
76
70
89
N.D.
50
38
72
80
42
65
31
72
86
87
82
91
85
26
67
62
83
88
73
80
55
88
with Hmsb backbone protection. (A) PolyL10: H-LLLLL(Hmsb)
LLLLL-NH ; (B) Mambalgin-1(19–40): H-CYHNTGMPFRNLKLILQGCSSS-
2
2
NH .
†
PolyL10
nificantly improved the qualities of the crude products. Two
Hmsb deletion peaks could be identified from the mass spec-
trum of ubiquitin(1–45), which probably resulted from the
partial decomposition of tertiary amide in the ionization room
of the mass spectrometer. No significant deletion or trunca-
tion side-products could be found on the mass spectra of the
main peaks. After semi-preparative HPLC purification, ubiqui-
Mambalgin-1(19–40)
Mambalgin-1(19–40)
Mambalgin-1(19–40)
Mambalgin-1(19–40)
Ubiquitin(1–45)
Ubiquitin(1–45)
Ubiquitin(46–76)
Ubiquitin(46–76)
0
1
2
3
4
a
1
45
10
11
15
16
A = air-bath heating SPPS; B = standard room temperature SPPS; C = tin H-M(O) -F -NHNH
[G -(Hmsb)K , L -(Hmsb)E ] and
2
air-bath heating SPPS combined with single Hmsb amide protection;
D = air-bath heating SPPS combined with double Hmsb amide
protection. Crude yields were determined based on the weight of
46 76
52
53
67
68
ubiquitin H-C -G -NH
2
[D -(Hmsb)G , L -(Hmsb)H ] were
b
deprotected in a modified TFA cocktail B (2.5% H O, 2.5%
2
lyophilized crude cleavage product after ether precipitation.
4
TIPS, 2.5% EDT, 1% Bu NI) to produce the target peptides
1
45
46 76
H-M -F -NHNH2 and H-C -G -NH
2
(Fig. 6 and Scheme S1†)
in >80% HPLC yield. Compared with previously reported addi-
3
2
tives, such as NH
thioanisole/EDT,
4
I/Me
2
S, NH
4
I/TIPS and TMSBr or Bu
4
NBr/
and chemically homogeneous product (Fig. 5A and Table 3).
In the same manner, we targeted another difficult sequence
Mambalgin-1(19–40). In a control experiment, air-bath SPPS
gives the crude peptide with average purity. In contrast, both
3
3,34
in our hands, Bu
4
NI assisted sulfoxide
reduction obtained essentially equal results. However, the
Hmsb removal condition would yet need to be optimized to
give cleaner deprotection results.
3
4
35
30
one (between Leu and Gln ) or two (between Leu and
3
1
35
36
Lys; Gln and Gly ) amide-protected Mambalgin-1(19–40)
crude peptides showed better HPLC purity than the former
(
Fig. 5B and Table 3).
Finally, we applied our new protocol to two protein seg-
Conclusions
ments, ubiquitin(1–45) and ubiquitin(46–76), to verify its In summary, we successfully improved the efficiency of Hmsb-
robust performance. Both sequences were prepared with or based backbone amide protection using the key 1,4-dioxane
without Hmsb incorporation using air-bath heating SPPS. As involved acyl shift in an effort to tackle the synthesis of
shown in Fig. 6 and Table 3, backbone amide protection sig- difficult peptides. To speed up the entire synthetic scheme
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4
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(
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1 (a) H. M. Yu, S. T. Chen and K. T. Wang, J. Org. Chem.,
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1
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45
2
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46
76
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Acknowledgements
This work was supported by the National Natural Science
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