Late-Stage Solubilization of Poorly Soluble Peptides Using Hydrazide Chemistry

Kohei Sato,* Shoko Tanaka, Junzhen Wang, Kenya Ishikawa, Shugo Tsuda, Tetsuo Narumi,

Letter

Late-Stage Solubilization of Poorly Soluble Peptides Using

Hydrazide Chemistry

Taku Yoshiya, and Nobuyuki Mase

Cite This: Org. Lett. 2021, 23, 1653 −1658

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sı Supporting Information

ABSTRACT: A novel late-stage solubilization of peptides using

hydrazides is described. A solubilizing tag was attached through a

selective N-alkylation at a hydrazide moiety with the aid of a 2-

picoline−borane complex in 50% acetic acid−hexaﬂuoro-2-

propanol. The tag, which tolerates ligation and desulfurization

conditions, can be detached by a Cu-mediated selective oxidative

hydrolysis of the N-alkyl hydrazide. This new method was validated

through the synthesis of HIV-1 protease.

ecent developments in protein research include the use of

synthetic protein probes such as precisely labeled

We have recently developed a trityl-type tag that enables the

late-stage solubilization of poorly soluble peptides (Scheme

1C). In this method, the trityl-type tag was attached to the

proteins, proteins with novel topologies, and mirror image

proteins. A strategy for chemical protein synthesis begins with

side chain of Cys residues or other thiol groups in an on-

demand manner after preparation of the peptide by SPPS or

NCL. This strategy enables a choice of types of solubilizing

solid-phase peptide synthesis (SPPS) to prepare peptide

segments followed by chemoselective assembly of the segments

to construct a backbone of the full length of a protein. Among

the techniques to assemble peptides, native chemical ligation

tags such as poly-Arg, poly-Lys, or poly trimethyllysine, even

after the synthesis of peptides of interest, but thiol groups at

which to install the tag must be present in the peptide. This

late-stage solubilization should be useful in the challenging

syntheses of poorly soluble peptides or proteins.

(

NCL), in particular, has been widely used.

Although

modern synthetic chemistry enables the total synthesis of

proteins consisting of more than 400 residues, total chemical

We took advantage of hydrazide chemistry to achieve the

late-stage solubilization of poorly soluble peptides. Reductive

alkylation between hydrazides and aldehydes is expected to be

synthesis of proteins is challenging when dealing with poorly

soluble peptides. Consequently, a versatile method that can

enhance the solubility of peptides is needed.

potentially compatible with unprotected peptides. Seitz et al.

immobilized an unprotected peptide hydrazide on an

aldehyde-modiﬁed plate surface through hydrazone formation

followed by reduction, yielding the N-alkyl hydrazide, which is

stable under the conditions of NCL. The resulting peptides

were successfully used for on-surface NCL followed by

The temporary attachment of hydrophilic tags onto poorly

soluble peptides to improve their aqueous solubility has been

reported. In an early publication, the installation of poly-Arg

at the C-terminus of the peptide thioester was developed

(

Scheme 1A). In this method, the peptide of interest, with the

solubilizing tag on a thioester leaving group, is synthesized.

However, this solubilizing tag is eliminated during the ligation,

so the enhanced solubility cannot be retained during the

ligation and puriﬁcation steps. To overcome this problem,

many semipermanent solubilizing tags attached to side chains

desulfurization. In addition, alkyl hydrazides can be hydro-

lyzed in the presence of a Cu(II) salt to give the corresponding

carboxylic acid. On the basis of these reports, we developed

the reaction sequence in Scheme 1D for the late-stage

solubilization and removal of solubilizing tags using hydrazide

chemistry. This strategy is based on (1) installation of the

solubilizing tag, which converts the aldehyde moiety to the

or backbone amides were developed (Scheme 1B). In these

strategies, the tag can be retained during the ligation and

subsequent puriﬁcation steps, enabling the improved handling

of the peptide materials. The tags can be removed after

synthesis of the entire structure of the desired protein. Despite

these advantages, the method has a potential limitation

because the tag moiety must be introduced during SPPS,

and thus the peptide segments with the solubilizing tag must

be resynthesized if the solubility-enhancing property is

insuﬃcient.

Received: January 9, 2021

Published: February 11, 2021

2021 American Chemical Society

653

Org. Lett. 2021, 23, 1653−1658

Organic Letters

Letter

Scheme 1. Solubilization Strategies for Poorly Soluble

Peptides

Table 1. Reductive N-Alkylation of Peptide Hydrazides

HPLC purity (%)

entry

aldehyde

solvent

HFIP

50% AcOH−HFIP

Reactions were performed as follows: The peptide hydrazide 1a (1

mM) and aldehyde 2 (1.5 mM) were dissolved in the solvent and

then incubated at 37 °C for 1 h. After the addition of pic-BH (20

equiv) to the mixture, the reduction was performed at 37 °C for 1 h.

After removal of the solvent by air blowing, the crude material was

washed with Et O. The resulting precipitate was dissolved in 20%

CH CN−H O and analyzed by HPLC. Detected at 220 nm.

Reduction was performed for 24 h. Reduction was performed for 10

min.

short reduction time (10 min) resulted in the suppression of

peptide amide formation (entry 4). Using 4-formylbenzamide

(

2b) bearing an electron-withdrawing group, lower reactivity

to the reduction of the hydrazone (entry 5) was observed. In

light of these results, 4-anisaldehyde was used in the

subsequent experiments.

peptide hydrazide by a selective reductive N-alkylation, (2)

assembly of peptide segments by ligation with the tag-installed

soluble peptide, and (3) Cu(II)-mediated selective oxidative

hydrolysis of the N-alkylated hydrazide to produce the

corresponding carboxylic acid with the temporarily introduced

solubilizing tags removed. In this Letter, we used C-terminal

peptide hydrazides to evaluate the applicability of this

hydrazide chemistry to the solubilization of poorly soluble

peptides.

We then examined the Cu-mediated oxidative hydrolysis of

the N-alkyl hydrazide. The crude material prepared by

reductive alkylation of the hydrazide (1a) with 4-anisaldehyde

reacted with CuSO ·5H O (20 equiv) in 20% CH CN-

containing aqueous solution, aﬀording the corresponding

carboxylic acid (6a) in a full conversion within 1 h (Table 2,

entry 1). The hydrolysis also proceeded with decreased

amounts of CuSO (2.0 equiv) or even only catalytic amounts

In our initial attempts to complete the envisioned reactions,

of copper salt (0.1 equiv) under an oxygen atmosphere (entries

2 and 3). The benzyl alcohol (7) and 4-anisaldehyde (2a) were

observed simultaneously as coproducts during the hydrolysis.

The possible role of the Cu(II) salt would be as an oxidant to

form an acyl diazene intermediate from the N-alkyl hydrazide,

yielding a carboxylic acid and a diazene derivative (8) (Scheme

2). The diazene would aﬀord benzyl alcohol and benzaldehyde

the peptide hydrazide Ac-LYRANA-NHNH (1a) was used as

a substrate, and 1,1,1,3,3,3-hexaﬂuoro-2-propanol (HFIP)

was selected as the solvent for the installation of the

solubilizing tag. HFIP has the ability to assist in dissolving

poorly soluble peptides in other solvents, which is necessary

because this strategy is to be applied to poorly soluble

compounds. The peptide hydrazide (1a) and 4-anisaldehyde

through further oxidation or tautomerization, respectively.

(

2a), which has an electron-donating group, were reacted in

The behavior of eight C-terminal amino acids (Ala, Val, Phe,

Ser, Thr, His, Lys, and Arg) was also evaluated. As summarized

in Table 2, the desired carboxylic acids (6a−6h) were obtained

with moderate to high purity (62−91%). The epimerization of

C-terminal amino acids (Ala and Val) was suppressed (epimer

ratio >99:1, Figure S19). The peptide consisting of oxidative-

HFIP for 1 h; then, 20 equiv of 2-picoline−borane complex

(

corresponding N-alkyl hydrazide (4aa) in 42% yield (Table 1

entry 1). The increase in the reaction time to 24 h for the

reduction slightly improved the conversion, but the reaction

was incomplete (entry 2). The use of AcOH as a cosolvent

with HFIP (50% (v/v)) accelerated the reductive alkylation

pic-BH ) was added to the reaction mixture, aﬀording the

prone residues, H-WCLYRAM-NH , was stable under the Cu-

mediated oxidative hydrolysis conditions for at least 5 h while

the disulﬁde dimer was immediately formed (Figure S20).

A synthetic application of our strategy was conﬁrmed

through the synthesis of human immunodeﬁciency virus type 1

(HIV-1) protease, inhibitors of which have been used for the

(

entries 3−5). After the reduction for 1 h followed by

exposure to an air-stream and subsequent trituration with Et O

to remove the solvent and excess reagents, the N-alkylated

hydrazide was obtained in 87% yield together with a small

amount of the peptide amide (5a), which was probably

generated by reductive cleavage of the hydrazide moiety. A

treatment of acquired immunodeﬁciency syndrome. Total

chemical synthesis of this enzyme was reported by Kent et al.,

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Org. Lett. 2021, 23, 1653−1658

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Letter

Table 2. Cu-Mediated Oxidative Hydrolysis of N-Alkyl

Hydrazides and Scope of C-Terminal Amino Acids

solubilizing tag was removed by the autoprocessing property

of Gag−Pol polyprotein that occurs during maturation of the

HIV-1 virus in vivo. Being used with HIV-1 protease, which can

be matured by itself, the synthetic design does not cover other

proteins that have no such autoprocessing property.

Our synthetic plan is shown in Scheme 3. The entire

sequence of HIV-1 protease was divided into three peptide

Scheme 3. Synthetic Pathway to HIV-1 Protease

HPLC purity (%)

entry

Xaa

product

Ala

Val

Phe

Ser

Thr

His

Lys

Arg

n.d.

All reactions were performed as follows: After reductive alkylation, as

described in Table 1, the peptide was subjected to hydrolysis with

CuSO ·5H O (20 equiv) in 20% CH CN-H O at 37 °C in an air

atmosphere. After 1 h, the reaction was quenched with DTT (200

equiv) in 0.5 M HEPES buﬀer at 37 °C for 15 min. The product was

analyzed by HPLC. Detected at 220 nm. Hydrolysis was performed

with 2 equiv of CuSO ·5H O. Hydrolysis was performed with 0.1

equiv of CuSO ·5H O in an oxygen atmosphere for 19 h. n.d., not

detected.

Scheme 2. Possible Pathway for the Generation of Benzyl

Alcohol and Benzaldehyde during Cu-Mediated Oxidative

Hydrolysis

segments (9−11) with the replacement of Ala at the ligation

junctions by Cys or 1,3-thiazolidine-4-carboxo (Thz), a

protected Cys. The C-terminal segment (9) is known to

be insoluble in aqueous media, and its direct synthesis by

SPPS is diﬃcult due to the on-resin aggregation of an

elongated peptide. We therefore planned to introduce the

solubilizing tag into the insoluble peptide (9) and to apply a

pseudoproline strategy, which can suppress the on-resin

aggregation of an elongated peptide, to the synthesis of the

peptide segment.

All of the segments were synthesized as hydrazides, and the

thioester segments (10 and 11) were produced from the

corresponding hydrazides by a method developed by our

group. Using pseudoproline units at the Leu −Thr sites,

the peptide (9) was elongated with no synthetic diﬃculties.

The aldehyde-incorporated Lys 10-mer (12) as a solubilizing

tag was prepared through conventional SPPS. The reductive

N-alkylation of hydrazide 9 with the solubilizing tag 12 was

who used the solubility-enhancing properties of the C-terminal

performed in 50% AcOH−HFIP with the help of pic-BH for

Arg 10-mer tag to overcome the diﬃculty stemming from the

10 min to suppress the excess installation of 12 on the N-

insolubility of the intermediate peptide products. The

terminal Cys through the thiazolidine formation. This

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Org. Lett. 2021, 23, 1653−1658

Organic Letters

protections on the Cys residue (Figure 1 A). The resulting

Letter

successfully produced the desired segment (13) without any

oxidative hydrolysis without signiﬁcant production of side

products. Various aldehyde-containing tags can be used in this

protocol. Because the structure of a suitable solubilizing tag

(

e.g., anionic, cationic, or nonionic) depends on the peptide of

interest, such a late-stage attachment strategy is practically

preferred over trial-and-error solubilizing studies. Using this

chemistry, a His-tag or even a solid support to assist in the

puriﬁcation step could be adopted as the “tag” in the future.

Although it was demonstrated through the solubilization of C-

terminal segments, our strategy, in principle, should also be

applicable to the solubilization of N-terminal or middle

segments through the Asp/Glu side-chain hydrazides.

These applications of the developed strategy are in progress

in our laboratory.

ASSOCIATED CONTENT

sı Supporting Information

■

The Supporting Information is available free of charge at

https://pubs.acs.org/doi/10.1021/acs.orglett.1c00074.

Experimental details of syntheses, charts for the HPLC

analyses, and mass spectra of synthesized products

(PDF )

AUTHOR INFORMATION

Corresponding Author

■

Kohei Sato − Department of Applied Chemistry and

Biochemical Engineering, Faculty of Engineering, Course of

Applied Chemistry and Biochemical Engineering, Department

of Engineering, Graduate School of Integrated Science and

Technology, and Graduate School of Science and Technology,

Shizuoka University, Hamamatsu, Shizuoka 432-8561,

Japan; orcid.org/0000-0001-6877-9223;

Figure 1. Enhanced solubility of peptide 9 and HPLC analyses of

reactions for HIV-1 protease. (A) Reductive N-alkylation of 9 with 12

(t = 10 min). (B) 9 μmol of peptide 9 (left) or peptide 13 (right) in 2

mL of 6 M guanidine·HCl−0.1% TFA aq. (C) Ligation between 13

and 10 for 3 h followed by deprotection of Thz (t = 12 h). (D)

Ligation between 10 + 13 and 11 (t = 10 h). (E) Desulfurization of

Email: sato.kohei@shizuoka.ac.jp

14 (t = 13 h). (F) Cu(II)-mediated oxidative hydrolysis of 15 (t = 1

h). The calculated mass is 10703.69 Da (average isotopes).

Deconvolution of the mass spectrum yielded an observed mass of

Authors

0 704.28 Da as a proton adduct. *4-Mercaptophenylacetic acid. 10′,

Shoko Tanaka − Graduate School of Science and Technology,

Shizuoka University, Hamamatsu, Shizuoka 432-8561,

Japan

lactam form of 10 at the C-terminal Lys; 10″, hydrolyzed 10; 11′,

methoxyamide form of 11. For reaction and HPLC conditions, see the

Supporting Information .

Junzhen Wang − Department of Applied Chemistry and

Biochemical Engineering, Faculty of Engineering, Shizuoka

University, Hamamatsu, Shizuoka 432-8561, Japan

Kenya Ishikawa − Department of Applied Chemistry and

Biochemical Engineering, Faculty of Engineering, Shizuoka

University, Hamamatsu, Shizuoka 432-8561, Japan

Shugo Tsuda − Peptide Institute, Inc., Ibaraki, Osaka 567-

0085, Japan

Tetsuo Narumi − Department of Applied Chemistry and

Biochemical Engineering, Faculty of Engineering, Course of

Applied Chemistry and Biochemical Engineering, Department

of Engineering, Graduate School of Integrated Science and

Technology, Graduate School of Science and Technology, and

Research Institute of Green Science and Technology, Shizuoka

University, Hamamatsu, Shizuoka 432-8561, Japan;

orcid.org/0000-0003-2412-4035

peptide (13) showed improved solubility in an aqueous

solution compared with the C-terminal segment (9), which

lacks the solubilizing tag, enabling the puriﬁcation of the

segment (Figure 1B). Ligation between 13 and 10 followed by

ring-opening of Thz by the addition of CH ONH ·HCl and

the subsequent NCL reaction with 11 were performed in a

one-pot manner, yielding the full-length protein (14) in 33%

isolated yield (Figure 1C,D). After the desulfurization of the

resulting protein followed by HPLC puriﬁcation (70% isolated

yield, Figure 1E), the solubilizing tag was smoothly removed

with the aid of CuSO in CH CN−H O (Figure 1F). Dialysis

of the reaction mixture containing the entire HIV-1 protease

sequence (16) in the refolding buﬀer solution decreased the

Cu concentration to 1.1 ppm and aﬀorded HIV-1 protease

(

80%, determined by UV absorbance at 280 nm), which has

Taku Yoshiya − Peptide Institute, Inc., Ibaraki, Osaka 567-

0085, Japan; orcid.org/0000-0002-7276-5614

Nobuyuki Mase − Department of Applied Chemistry and

Biochemical Engineering, Faculty of Engineering, Course of

Applied Chemistry and Biochemical Engineering, Department

of Engineering, Graduate School of Integrated Science and

Technology, Graduate School of Science and Technology, and

Research Institute of Green Science and Technology, Shizuoka

the enzymatic activity to cleave its substrate peptide (Figure

S31 ).

In summary, we have developed a novel strategy that enables

late-stage solubilization of poorly soluble peptides using

hydrazide chemistry. The solubilizing tag was attached to

peptides bearing a hydrazide moiety by selective, reductive N-

alkylation and can be detached by selective Cu(II)-mediated

656

Org. Lett. 2021, 23, 1653−1658

Organic Letters

University, Hamamatsu, Shizuoka 432-8561, Japan;

Wang, Z.-P.; Xiao, L.; Zhang, L.; Tian, C.-L.; Liu, L. Expedient Total

Letter

4858−4862. (d) Zheng, J.-S.; Yu, M.; Qi, Y.-K.; Tang, S.; Shen, F.;

Synthesis of Small to Medium-Sized Membrane Proteins via Fmoc

Chemistry. J. Am. Chem. Soc. 2014, 136, 3695−3704. (e) Bello, C.;

Wang, S.; Meng, L.; Moremen, K. W.; Becker, C. F. W. A PEGylated

Photocleavable Auxiliary Mediates the Sequential Enzymatic Glyco-

sylation and Native Chemical Ligation of Peptides. Angew. Chem., Int.

Ed. 2015, 54, 7711−7715. (f) Asahina, Y.; Komiya, S.; Ohagi, A.;

Fujimoto, R.; Tamagaki, H.; Nakagawa, K.; Sato, T.; Akira, S.; Takao,

T.; Ishii, A.; Nakahara, Y.; Hojo, H. Chemical Synthesis of O-

Glycosylated Human Interleukin-2 by the Reverse Polarity Protection

Strategy. Angew. Chem., Int. Ed. 2015, 54, 8226−8230. (g) Maity, S.

K.; Mann, G.; Jbara, M.; Laps, S.; Kamnesky, G.; Brik, A. Palladium-

Assisted Removal of a Solubilizing Tag from a Cys Side Chain To

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orcid.org/0000-0001-6244-4916

Complete contact information is available at:

https://pubs.acs.org/10.1021/acs.orglett.1c00074

Notes

The authors declare no competing ﬁnancial interest.

ACKNOWLEDGMENTS

■

This research was supported in part by JSPS KAKENHI grant

number JP19K15712 and research grants from Terumo Life

Science Foundation. S.T. is grateful for the Amano Institute of

Technology scholarship. Dedicated to Professor Akira Otaka

on the occasion of his 60th birthday.

3029. (h) Zheng, J.-S.; He, Y.; Zuo, C.; Cai, X.-Y.; Tang, S.; Wang, Z.

A.; Zhang, L.-H.; Tian, C.-L.; Liu, L. Robust Chemical Synthesis of

Membrane Proteins through a General Method of Removable

Backbone Modification. J. Am. Chem. Soc. 2016, 138, 3553−3561.

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Sayers, J.; Payne, R. J. Native Chemical Ligation −Photodesulfuriza-

tion in Flow. J. Am. Chem. Soc. 2018, 140, 9020−9024. Sayers, J.;

Karpati, P. M. T.; Mitchell, N. J.; Goldys, A. M.; Kwong, S. M.; Firth,

N.; Chan, B.; Payne, R. J. Construction of Challenging Proline −

Proline Junctions via Diselenide −Selenoester Ligation Chemistry. J.

Am. Chem. Soc. 2018, 140, 13327−13334.

(15) (a) Narita, M.; Honda, S.; Umeyama, H.; Obana, S. The

Solubility of Peptide Intermediates in Organic Solvents. Solubilizing

Potential of Hexafluoro-2-propanol. Bull. Chem. Soc. Jpn. 1988, 61,

81 −284. (b) Kuroda, H.; Chen, Y.-N.; Kimura, T.; Sakakibara, S.

Powerful solvent systems useful for synthesis of sparingly-soluble

peptides in solution. Int. J. Pept. Protein Res. 1992, 40, 294−299.

(16) Sato, S.; Sakamoto, T.; Miyazawa, E.; Kikugawa, Y. One-pot

reductive amination of aldehydes and ketones with α -picoline-borane

in methanol, in water, and in neat conditions. Tetrahedron 2004, 60,

(

899−7906.

17) Acetic acid can accelerate the reductive amination. See: Abdel-

Magid, A. F.; Carson, K. G.; Harris, B. D.; Maryanoff, C. A.; Shah, R.

D. Reductive Amination of Aldehydes and Ketones with Sodium

Triacetoxyborohydride. Studies on Direct and Indirect Reductive

Amination Procedures. J. Org. Chem. 1996, 61, 3849−3862.

(18) The diﬀerence between 2a and 2b would be based on the

basicity of the nitrogen atom at a hydrazone group. That is, the

protonated hydrazonium species are more reactive to the reduction by

pic-BH₃.

(19) 4-Methoxybenzyl alcohol was generated more in the presence

of the further excess Cu(II) salt and was not converted to 4-

anisaldehyde under the hydrolytic conditions (Figures S10 and S11 ).

(20) Ghosh, A. K.; Osswald, H. L.; Prato, G. Recent Progress in the

Development of HIV-1 Protease Inhibitors for the Treatment of

HIV/AIDS. J. Med. Chem. 2016, 59, 5172−5208.

(21) Johnson, E. C. B.; Malito, E.; Shen, Y.; Rich, D.; Tang, W.-J.;

Kent, S. B. H. Modular Total Chemical Synthesis of a Human

Immunodeficiency Virus Type 1 Protease. J. Am. Chem. Soc. 2007,

(

29, 11480−11490.

22) According to the previous synthesis, Met³⁶^,⁴⁶and Cys⁶⁷were

replaced by norleucine (Nle) and 2-aminobutyric acid (Abu),

respectively. See ref 21.

23) Henkel, B.; Bayer, E. Monitoring of Solid Phase Peptide

Synthesis by FT-IR Spectroscopy. J. Pept. Sci. 1998, 4, 461−470.

24) Wohr, T.; Wahl, F.; Nefzi, A.; Rohwedder, B.; Sato, T.; Sun, X.;

(

Mutter, M. Pseudo-Prolines as a Solubilizing, Structure-Disrupting

Protection Technique in Peptide Synthesis. J. Am. Chem. Soc. 1996,

(

18, 9218−9227.

25) Sato, K.; Tanaka, S.; Yamamoto, K.; Tashiro, Y.; Narumi, T.;

Mase, N. Direct synthesis of N-terminal thiazolidine-containing

peptide thioesters from peptide hydrazides. Chem. Commun. 2018,

(

4, 9127−9130.

26) A possible explanation for this selectivity is that acetic acid can

protonate the Lys side chains and the N-terminal amino group while

the hydrazide remains unprotonated. The pK values of the conjugate

acids of the ε-amino group of Lys and glycine hydrazide in water are

reported as 10.5 for the ε-amino group, 7.69 for the α-amino group,

and 2.38 for the nitrogen of the hydrazide. See: Lindegren, C. R.;

Niemann, C. The Apparent Ionization Constants of Acethydrazide

and Glycylhydrazide. J. Am. Chem. Soc. 1949, 71, 1504.

658