Synthesis of Functional Amino Acids Bearing 1%2C3-Dithiane Modification

Journal of Chinese Pharmaceutical Sciences http://www.jcps.ac.cn 195

Synthesis of functional amino acids bearing 1,3dithiane modification Ying Yang, Chao Wang *

Department of Medicinal Chemistry, School of Pharmaceutical Sciences, Peking University Health Science Center, Beijing 100191, China

Abstract: Two protected single amino acid chelates, N α FmocN ε , N ε di((2,2dimethyl1, 3dithian5yl)methyl)Llysine (7) and N α FmocN ε (2,2dimethyl1,3dithian5yl)methyl, N ε BocLlysine (9), were synthesized by modifying the side chain of lysine with 1,3dithiane through direct reductive Nalkylation protocol. These amino acids have potential uses in peptide chemistry. Keywords: Amino acid chelate; 1,3Dithiane; Reductive Nalkylation

CLC number: R916 Document code: A Article ID: 1003–1057(2011)2–195–04

Received date: 20101107. * Corresponding author. Tel.: 861082805049; Email: [email protected]

doi:10.5246/jcps.2011.02.025

1. Introduction

Radiolabeled peptides can be used to target a variety of disease tissues through interaction with specific cell receptors [1] . Introduction of an entity that allows facile labeling with medically useful radionuclides such as 99m Tc for diagnosis and 186/188 Re for targeted therapy is desirable. The general approaches for the linking of metal cations to a readily prepared peptide are through the use of bifunctional chelates to form metalchelating agent peptide complex. However, the limitations of this strategy for peptide labeling are its poor residue selectivity and potentially significant alteration of the peptide’s structure and receptor binding affinity. In view of the need for better labeling strategy and

the importance of the thiolate ligands in coordina tion with heavy metals to form stable complex, we designed a novel amino acid chelate that was prepared from modified amino acid analogues. The protected thiols in a 1,3dithiane structure as the potential chelating functionality were introduced to the side chain of lysine for effective coordination with 99m Tc (Fig. 1). We anticipated such amino acid chelate can be used in peptide synthesis and this specific residue can be incorporated into any position along a peptide sequence.

In our earlier experiments, two protected amino acid chelators, NFmocO((S,S′diacetamidomethyl) 3,3′dithioether)isobutylLtyrosine and NFmoc O((S,S ′isopropylidene)3,3′dithioether)isobutyl Ltyrosine were synthesized from NFmoctyrosine methyl ester with the hydroxyl components by Mitsunobu reaction [2] . Those amino acids have been used in peptide synthesis by solution method or by solid phase method. The protection of the sulfhydryl group could be removed by deprotecting procedures commonly used in peptide chemistry, and the 1,3 dithiols units can be easily regenerated via the form of 1,2dithiolanes.

Figure 1. Schematic representation of labeling peptide with 99m Tc: (A) The sequence of a bioactive peptide; (B) An example of a bifunctional chelate linking radioactive metal cation to the peptide; (C) Amino acid chelate modified peptide for labeling with 99m Tc. AAC = amino acid chelate.

(A)

(B)

(C)

H-AA 1 -AA 2 -AA 3 ........ AA n-1 -AA n -OH

H-AAC-AA 2 -AA 3 ........ AA n-1 -AA n -OH

H-AA 1 -AAC-AA 3 ........ AA n-1 -AA n -OH

H-AA 1 -AA 2 -AAC ........ AA n-1 -AA n -OH

H-AA 1 -AA 2 -AA 3 ........ AAC-AA n -OH

N N

S S

O

(CH 2 ) n C O

AA 1 -AA 2 -AA 3 ........ AA n-1 -AA n -OH

O

Tc O

H-AA 1 -AA 2 -AA 3 ........ AA n-1 -AAC-OH N H O

(CH 2 ) 4

N

HS SH SH HS

AAC =

Note

Copyright © 2011 Journal of Chinese Pharmaceutical Sciences, School of Pharmaceutical Sciences, Peking University http://www.jcps.ac.cn

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Y. Yang et al. / Journal of Chinese Pharmaceutical Sciences 20 (2011) 195–198 196

The purpose of this study was to develop a new single amino acid chelate that has multithiolate moiety in one amino acid residue. The reactions used in the preparation of such specific functional amino acids are shown in Scheme 1.

2. Results and discussion

Aldehydes are often prepared by oxidation of alcohols. It also can be synthesized from carboxylic acids via Weinreb amides under mild conditions [3] . In our experiment, we attempted to prepare aldehyde (4) with these procedures and the results showed that the reductive method using Weinreb amide afforded corresponding aldehyde satisfactorily. Reductive amination of aldehydes is a very useful

reaction in organic preparation. In the case of amino acids, the reductive alkylation with aldehydes is generally performed after protecting the carboxyl group as an ester. The reports that detail the reductive alkylation of amino acids with a free acid functionality are rare. In our experiments, the required dialkylated product (7) and the monoalkylated product (8) were obtained in one pot under mild condition. Compounds 7 and 8 were isolated in 20% and 46% yield, respectively, by silica column chromatography. The εimino group of monoalkyl derivative (8) was protected by the Boc group, which can be used for peptide synthesis by the Fmoc strategy. AbdelMagid et al [4] have recently demonstrated

that NaBH(OAc)3 can be used effectively as a mild reagent in the reductive amination of aldehydes and ketones with shorter reaction time and excellent yields. This method was confirmed in our experiment, because the efficiency of NaBH(OAc)3 was much higher than that of NaBH3CN or NaBH4. The final aim of our researches is to synthesize a

series of bioactive peptides that contain the key structural feature of multithiols in one amino acid residue, and that residue can be incorporated into any position of a peptide sequence for 99m Tc labeling [5] . The amino acid chelates synthesized in the present report could have potential uses in peptide chemistry.

3. Experimental

Melting points were measured using a microscope hotstage apparatus and are uncorrected. NMR and MS spectroscopic data were obtained on the Bruker500 and ZQ2000 instruments, respectively. Elemental analyses were performed on a PE2400 elemental analyzer. All reactions were monitored by TLC using precoated plates of silica gel 60 F254 (Merck).

3.1. 3Mercapto2(mercaptomethyl)propoinic acid (1)

It was prepared according to the literature proce dure [6] .

3.2. 2,2Dimethyl1,3dithiane5carboxylic acid (2)

To a well stirred solution of 1 (1.9 g, 12.5 mmol) in CHCl3 (15 mL), acetone (1.45 g, 25 mmol) and BF3/ethyl ether (0.76 mL) were added at 0 ºC . The reaction mixture was stirred at room temperature overnight. It was washed with saturated aqueous

Scheme 1. Reagents and conditions: (a) CH3COCH3, BF3/Et2O, CHCl3, 0 ºC, 50%; (b) CH3NHOCH3, TBTU, DMF, 56%; (c) LiAlH4, THF, 0 ºC, 100%; (d) DMP, CH2Cl2, 50%; (e) NaBH(OAc)3, THF, 20% (7), 46% (8); (f) NaHCO3/H2O, Boc2O, 70%. TBTU = 2 (1HBenzotriazole1yl)1,1,3,3tetramethyluronium tetrafluoroborate; DMP = DessMartin periodinane; Fmoc = 9Fluorenylmethoxycarbonyl; Boc = tbutyloxycarbonyl.

S S

OH O

SH SH

OH O

S S

N O

S S

H O

S S

OH

O

4

N H

NH 2

Fmoc OH

O N H

(CH 2 ) 4

Fmoc OH

O

N

S S

N H

(CH 2 ) 4

Fmoc OH

O

N

S S S S

R

R = H R = Boc

8 6 7

9

1 2 3

5

a b

c

d

4 e

f


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NaHCO3 (3×3 mL) and water (3×3 mL), dried over anhydrous Na2SO4 and concentrated in vacuo to give a crude product, which was recrystallized from ethyl ether–petroleum ether to afford a white needle crystal (0.92 g, 50%). Mp 131–132 ºC; ESIMS: m/z 193 (M+H) + , 215 (M+Na) + ; 1 H NMR (500 MHz, CDCl3) δ: 1.67 (s, 3H), 1.81 (s, 3H), 2.90 (m, 1H), 3.06 (dd, 2H, J1 3.2 Hz, J2 14.8 Hz), 3.20 (dd, 2H, J1 10.1 Hz, J2 14.8 Hz).

3.3. 2,2Dimethyl1,3dithiane5carboxylic acid methoxymethylamide (3)

To a well stirred solution of 2 (1.5 g, 7.8 mmol) in DMF (20 mL), N, Odimethylhydroxylamine hydrochloride (0.92 g, 9.36 mmol) and N, N diisopropyl–ethylamine (2.57 mL, 1.94 g, 1.5 mmol) were added. The reaction mixture was stirred for 30 min at room temperature, then TBTU (3.37 g, 10.4 mmol) was added. The reaction mixture was stirred for 3 h, TLC showed the reaction was completed. The reaction mixture was poured into water and NaCl was added, and the mixture was extracted using EtOAc (3×10 mL). The organic layer was washed with water (3×3 mL), dried over anhydrous Na2SO4 and concentrated in vacuo to give a white solid, which was purified by crystallization from ethyl ether to afford a white crystal (1.0 g, 56%). Mp 64–65 ºC; ESIMS: m/z 236 (M+H) + , 258 (M+Na) + , 274 (M+K) + ; 1 H NMR (500 MHz, CDCl3) δ: 1.62 (s, 3H), 1.89 (s, 3H), 2.76 (dd, 2H, J1 2.5 Hz, J2 14.5 Hz), 3.16 (m, 1H), 3.22 (s, 3H), 3.28 (dd, 2H, J1 11.8 Hz, J2 13.4 Hz), 3.76 (s, 3H).

3.4. 2,2Dimethyl1,3dithiane5carbaldehyde (4)

Method A: To a well stirred solution of 3 (0.45 g, 1.9 mmol) in THF (10 mL), LiAlH4 (0.095 g, 2.47 mmol) was added at 0 ºC. The reaction was quenched by 10% KHSO4 after 1.5 h stirring, then the mixture was filtered. The filtrate was extracted using CH2Cl2 (3×10 mL). The organic layer was washed with water (3×3 mL), dried over anhydrous Na2SO4 and concentrated in vacuo to give a yellow

oil (0.41 g, >100%), which was directly used for next stage preparation without further purification. 1 H NMR (500 MHz, CDCl3) δ: 1.72 (s, 3H), 1.73 (s, 3H), 2.73 (m, 1H), 3.08 (dd, 2H, J1 8.6 Hz, J2 14.6 Hz), 3.15 (dd, 2H, J1 3.3 Hz, J2 14.6 Hz), 9.79 (s, 1H). Method B: To a well stirred solution of 5 (0.04 g,

0.225 mmol) in CH2Cl2, NaHCO3 (0.113 g, 1.35 mmol) and DMP (0.143 g, 0.338 mmol) were added at room temperature. The mixture was stirred for another 2 h until 5 was disappeared. Then CH2Cl2 (15 mL) was added, and the reaction was quenched with Na2S2O3

(0.235 g, 1.35 mmol). The mixture was washed with 5% NaHCO3 (2×5 mL) and water (3×3 mL), dried over anhydrous Na2SO4 and concentrated in vacuo to give a yellow oil, which is used directly without further purification.

3.5. 2,2Dimethyl1,3dithiane5methanol (5)

It was obtained as a a white crystal according to the previous report [2] .

3.6. N α FmocLlysine (6)

It was obtained as a white crystal in 50% yield according to the literature method [7,8] .

3.7. N α FmocN ε , N ε di((2,2dimethyl1,3dithian 5yl)methyl)Llysine (7) and N α FmocN ε (2,2 dimethyl1,3dithian5yl)methylLlysine (8)

To a well stirred solution of 4 (0.41 g, ≤1.9 mmol) in THF (7 mL), compound 6 (0.458, 0.95 mmol) was added at room temperature. It was stirred for 30 min, and NaBH(OAc)3 (0.5 g, 2.38 mmol) was added. The reaction mixture was stirred for 3 h at room temperature until the reagent disappeared (the reaction was monitored by TLC). The reaction mixture was poured into water and stirred. The mixture was filtrated, and the precipitate was purified by column chromatography (CHCl3–MeOH–HOAc– EtOAc–petroleum ether, 20:1:0.4:2:2) to get the product 7 as a white solid (0.09 g, 20%) and product 8 (0.2 g, 46%) as a white solid.


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Compound 7, mp 106–107 ºC. +1.43 (MeOH; c 1.0); ESIMS:m/z 688 (M+H) + ; 1 H NMR (500 MHz, CDCl3) δ: 1.29 (m, 2H), 1.42 (m, 2H), 1.54 (m, 1H), 1.67 (s, 6H), 1.68 (s, 6H), 1.97 (m, 1H), 2.15 (m, 2H), 2.63–2.77 (m, 8H), 2.84 (m, 2H), 3.02 (m, 4H), 3.04 (m, 1H), 4.25 (m, 2H), 4.48 (m, 1H), 7.33 (t, 2H, J 6.5 Hz), 7.41 (t, 2H, J 6.5 Hz), 7.65 (m, 2H), 7.78 (d, 2H, J 7 Hz). Anal. calcd. for C35H48N2O4S2∙H2O: C, 59.46; H, 7.13; N, 3.96. Found: C, 59.65; H, 7.24; N, 3.58. Compound 8, mp 192–193 ºC; ESIMS: m/z 529

(M+H) + . It is used directly in the next preparation.

3.8. N α FmocN ε (2,2dimethyl1,3dithian5yl) methyl, N ε BocLlysine (9)

To a flask, compound 8 (0.084 g, 0.159 mmol), water (5 mL), NaHCO3 (0.38 g, 0.454 mmol) and Boc2O (0.643 g, 0.295 mmol) were added in sequence. The mixture was stirred at room temperature and became clear 20 min later. The mixture was stirred for another 1 h at room temperature. Then the pH of the mixture was adjusted to 2 with 5% KHSO4. The mixture was extracted with EtOAc (3×10 mL). The organic layer was washed with water (3×3 mL), dried over anhydrous Na2SO4 and concentrated in vacuo to give a yellow oil, which was purified by column chromatography (EtOAc–petroleum ether– HOAc, 5:1:0.4) to get the desired product as a

white solid (0.07 g, 70%). Mp 77–78 ºC; –0.70 (MeOH; c 1.0); HRESIMS: m/z 629.26945 (M+H) + , calcd. 629.27136, 651.25303 (M+Na) + ; found 651.25330; 1 H NMR (500 MHz, CDCl3) δ: 1.29 (m, 2H), 1.41 (m, 2H), 1.48 (s, 9H), 1.60 (m, 1H), 1.68 (s, 3H), 1.75 (s, 3H), 1.96 (m, 1H), 2.13 (m, 1H), 3.22 (m, 2H), 3.32 (m, 2H), 4.26 (m, 1H), 4.43 (m, 3H), 7.34 (t, 2H, J 7.5 Hz), 7.42 (t, 2H, J 7.5 Hz), 7.63 (m, 2H), 7.78 (d, 2H, J 8 Hz).

References

[1] Fischman, A.J.; Babich, J.W.; Strauss, H.W. J. Nucl. Med.

1993, 34, 2253–2260.

[2] Sun, Y.; Li, X.Q.; Meng, Y.P.; Wang, C. Syn. Commun.

2008, 38, 3303–3310.

[3] Marielle, P.; Catherine, P.; Laurent, G.; Annie, H.;

Martinez, J.; Fehrentz J.A. React. Funct. Polym. 1999, 41,

255–261.

[4] AbdelMagid, A.F.; Carson, K.G.; Harris, B.C.; Maryanoff,

C.A.; Shah, R.D. J. Org. Chem. 1996, 61, 3849–3862.

[5] Liu, S.; Edwards, D.S. Chem. Rev. 1999, 99, 2235–2268.

[6] Singh, R.; Whitesides, G.M. J. Am. Chem. Soc. 1990,

112, 1190–1197.

[7] Leman, L.J.; Weinberger, D.A.; Huang, Z,Z.; Wilcoxen,

K.M.; Ghadiri, M.R. J. Am. Chem. Soc. 2007, 129, 2959–

2966.

[8] Haldar, D. Tetrahedron. 2008, 64, 186–190.

20D [ ] α 20

D [ ] α

多巯基功能性保护氨基酸的合成杨颖, 王超 *

北京大学医学部药学院药物化学系,北京 100191

摘要: 本文通过直接还原N烷基化的反应将1,3二噻烷衍生物导入赖氨酸侧链, 合成了两个保护的氨基酸络合剂N α

FmocN ε , N ε di((2,2dimethyl1,3dithian5yl)methyl)Llysine (7) 及 N α FmocN ε ((2,2dimethyl1,3dithian5yl)methyl, N ε

BocLlysine (9)。此保护氨基酸将在多肽化学应用中发挥作用。

关键词:氨基酸络合剂; 1,3二噻烷衍生物;还原N烷基化反应


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Synthesis of Functional Amino Acids Bearing 1%2C3-Dithiane Modification