Isothiocyanyl Alanine as a Synthetic Intermediate for the Synthesis of

Oct 24, 2017 - Subhendu Sekhar Bag and Suranjan De. Bioorganic Chemistry Laboratory, Department of Chemistry, Indian Institute of Technology Guwahati ...
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Isothiocyanyl Alanine as a Synthetic Intermediate for the Synthesis of Thioureayl Alanines and Subsequent Aminotetrazolyl Alanines Subhendu Sekhar Bag* and Suranjan De Bioorganic Chemistry Laboratory, Department of Chemistry, Indian Institute of Technology Guwahati 781039, India S Supporting Information *

ABSTRACT: The synthesis of unnatural amino acids with small side-chain functionalities usable for further transformations is highly demanding for the expansion of the genetic code and other possible biotechnological applications. To this end, we wanted to report the utility of an unexplored unnatural amino acid, isothiocyanyl alanine (NCSAla = Ita), for the synthesis of another class of unnatural amino acids, thioureayl alanines (TUAla = Tua). The synthesis of a third class of unnatural amino acids, amino tetrazolyl alanines (ATzAla = Ata), in a very good yield was subsequently achieved utilizing thioureayl alanines. Thus, a variety of aliphatic- and aromatic-substituted thioureayl alanines and aromatic-substituted amino tetrazolyl alanines were successfully synthesized in good to excellent yields. The photophysical properties of three of the fluorescent unnatural amino acids from two classes were also studied and presented herein.

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INTRODUCTION In the journey of the expansion of the genetic code and other possible biotechnological applications, the synthesis of unnatural amino acids with various novel functional roles is highly demanding. In these developments, a large number of unnatural amino acids (UNAAs) have been introduced.1 Many of the reported unnatural amino acids have also been incorporated into proteins.1,2 Furthermore, several of them have been utilized as probes for investigating protein structures, functions, and dynamics and have even been applied to study interbiomolecular interactions in both in vitro and in vivo.2−6 We also have reported fluorescent triazolyl UNAAs as labels for investigating the conformation of a short peptide and addressing fundamental photophysical aspects.7 However, it has often been observed that the large sizes of labels cause problems of structural perturbation, leading these labels to be unsuitable as protein probes. Therefore, nonperturbing small functional groups as side-chain labels are highly desirable as probes over typical fluorescent probes.8 Hence, the synthesis of small side-chain-modified unnatural amino acids is highly important. As a part of our continuous research efforts in the design of unnatural amino acids via click chemistry,7 we thought that it would be worthwhile to generate an amino acid with a small and reactive functional group that can further be exploited as a precursor for other novel families of UNAAs. Thus, we became interested in exploiting the unexplored isothiocyanyl (−NCS) functional group for the generation of isothiocyanyl alanine (NCSAla = Ita) as a new family of UNAAs. The logics behind our choice are the following: (a) isothiocyanates are a very important class of chromophores long known for their analytical use, such as in the determination of the primary structures of peptides and proteins and in amino acid analysis;9 (b) recently, these chromophores have attracted much research attention because © 2017 American Chemical Society

of their stability, synthetic easiness, and wide applications in organic synthesis, such as for the synthesis of thioureas and thioamides and as precursors for various sulfur-containing biologically active heterocycles.10 For example, isothiocyanates derived from the hydrolysis of plant defense compounds, i.e., glucosinolates, have frequently been shown to act as insecticides.11 Moreover, the unique Cotton effect of chiral isothiocyanates could be utilized as an alternative for X-ray diffraction studies for stereochemical assignments.12 In peptidomimetic chemistry, the bioisosteric replacement of amide carbonyls by thiocarbonyls has been profoundly reported for the synthesis of modified peptides with new properties compared to their parent counterparts. 13 Thus, several substituted peptidyl ureas and a few peptidyl thioureas have been reported.14,15 Although there are only a few reports of isothiocyanates derived as α-isothiocyanato alkyl esters from amino acids/peptides, side-chain isothiocyanyl amino acids have not been reported in the literature.15,16 Therefore, we envisaged that the hitherto unexplored isothiocyanyl alanine (NCSAla) would be worthwhile for exploiting its synthetic applications for the generation of new classes of amino acid analogues. Furthermore, side-chain-modified NCSAla with a reactive −NCS functionality could be useful in designing side-chain fluorophoric thiouereayl amino acids/peptides and for labeling these with a fluorophore. Therefore, we wanted to report the synthesis of NCS Ala and its various synthetic applications.

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RESULTS AND DISCUSSION Synthesis of Isothiocyanyl Alanine (NCSAla = Ita). To explore our aim of the present investigation, we first synthesized Received: August 19, 2017 Published: October 24, 2017 12276

DOI: 10.1021/acs.joc.7b02103 J. Org. Chem. 2017, 82, 12276−12285

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The Journal of Organic Chemistry Scheme 1. Synthesis of Isothiocyanyl Alanine, NCSAla (1)

Scheme 2. Synthesis of Thioureayl Alanines (TUAla, 5A−R) from Isothiocyanyl Alanine (NCSAla, 1)

NCS

philic character, which allows them to readily take part in nucleophilic addition and cycloaddition reactions.10−12,17,19 Several methodologies exist for the synthesis of isothiocyanates from the corresponding amines. However, we adopted an easy and convenient literature-reported methodology, which relied on the TsCl-promoted decomposition of dithiocarbamic acid salts into isothiocyanates.20 Thus, the synthesis of NCSAla

Ala (Ita) (Scheme 1). The isothiocyanate moiety is found in many natural products,10−12,17 and some of them have been found to possess anticancer properties.18 Furthermore, isothiocyanates are also useful as reactive functional groups in chemical synthesis, such as the generation of biologically active sulfur-containing heterocycles.10−12 Isothiocyanates are wellknown synthetic intermediates owing to their strong electro12277

DOI: 10.1021/acs.joc.7b02103 J. Org. Chem. 2017, 82, 12276−12285

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The Journal of Organic Chemistry Scheme 3. Synthesis of Aminotetrazolyl Alanines (ATzAla) from Thioureayl Alanines (TUAla)

out the reaction of NCSAla with various amines in an CH3CN/ DCM (1:3) solvent mixture at 50 °C for approximately 12−24 h (Scheme 2). The reaction times were different for different amines. The aliphatic amines took less time than the aromatic amines to condense and afford the corresponding thioureayl alanines. The yields of the products also varied. All of the aromatic amines, irrespective of the nature of the substituents, except for pacetyl aniline, afforded thioureayl alanines in very good to excellent yields (81−98%). p-Acetyl aniline afforded a moderate yield of thioureayl alanine (5N, 65%), which might be because of the formation of other undetectable side products. For the aliphatic amines, the yields were good to moderate. The amine with a short carbon chain (ethanolamine, 5Q) or compact amine (cyclohexylamine, 5R) gave a very good yield (70−78%), while the long-chain amines (butyl, 5O or heptylamine, 5P) provided moderate yields (50−60%), which might be attributed to steric effects. At this stage, a correlation based on basicity/ nucleophilicity is very difficult to access. However, in summary, the novel thioureayl amino acids are the first report that can be easily utilized for catalytic transformations, which is our future research focus.24 Synthesis of Aminotetrazolyl Alanines (ATzAla = Ata) from Thioureayl Alanines (TUAla = Tua). After the successful syntheses of several thioureayl alanines (TUAla = Tua), we thought that the intermediate generated from the oxidative desulfurization of various TUAla could be trapped by external

was started from N,C-diprotected serine 2, which was converted to N,C-diprotected azido alanine 3 (DPN3Ala)7a via the reaction of the corresponding mesylate and NaN3 in DMSO in 70% yield (Scheme 1). Azido alanine 3 was then converted to diprotected amino alanine 4 (DPNH2Ala), which subsequently upon treatment with CS2 in the presence of triethylamine (Et3N) afforded the dithiocarbamic acid salt. The carbamic acid salt then reacted with TsCl to afford the desired NCSAla (1) in its diprotected form in a moderate yield of 50%.20 All of the intermediates and the final amino acid were purified by silica gel column chromatography and characterized by IR spectroscopy, NMR spectroscopy, and mass spectrometry. Synthesis of Thioureayl Alanines (TUAla = Tua). After the successful synthesis of NCSAla, we thought that it would be worthwhile to exploit the electrophilicity of the −NCS functionality first to achieve the synthesis of the hitherto unreported side-chain chiral thioureayl alanines (TUAla = Tua). Thioureas constitute an important class of compounds that possesses widespread applications in medicinal chemistry,21 as valuable building blocks for the synthesis of amides, guanidines, and a variety of heterocycles,22 for anion recognition/sensing23 and in organocatalysis.24 Thus, several methodologies have been reported for the synthesis of thioureas. Among the numerous methods,25 we utilized a mild, nontoxic and user-friendly procedure: the condensation of various aromatic, aliphatic, and alicyclic amines with isothiocyanyl alanine (NCSAla) for the synthesis of several thioureayl alanines (TUAla).25g We carried 12278

DOI: 10.1021/acs.joc.7b02103 J. Org. Chem. 2017, 82, 12276−12285

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The Journal of Organic Chemistry

Scheme 4. Plausible Mechanism for the Regiochemical Outcome for the Synthesis of Aminotetrazolyl Alanines (ATzAla) from Thioureayl Alanines (TUAla)

The regiochemistry with respect to the tetrazole moiety is a reflection of the difference in pKa’s of the amino functionalities on the thioureayl alanines.30e Thus, the less basic amine, in this case the aromatic amine, became part of the ring nitrogen, and the other more basic amine of the β-amino alanyl moiety became part of the exocyclic nitrogen with respect to the tetrazole moiety. This might be explained by considering the protonation of the more basic “N” of carbodiimide intermediate III (Scheme 4), followed by nucleophilic attack by azide to give azidoguanidyl alanine IV, which underwent electrocyclization giving products with the observed regiochemistry at the tetrazole unit. Photophysical Properties of Fluorescent Pyrenyl Amino Acids (5J, PyTUAla; 5K, MePyTUAla; and 6J, PyTzAAla). Finally, we want to highlight the preliminary photophysical properties of the fluorescent amino acids in various organic solvents. Thus, the UV−visible spectra of pyrenyl thioureayl alanine 5J (PyTUAla) showed slightly distorted absorption band shapes compared to the pyrene derivative in all solvents. The intensity of absorption followed an irregular trend with a minute shift (2−4 nm) of the absorption wavelength as the polarity of the solvent increased from toluene (329 and 346 nm) to MeOH (326 and 341 nm). However, in THF and DMF, a structureless broad absorption centered at 361 nm appeared (Figure 1a). All of these observations reflected the nominal solvatochromicity and spectral modulation by the thiourea moiety.31 Upon excitation at the long-wavelength absorption band at 345 nm, the emissions in various solvents were found to follow an irregular trend, displaying a modulated pyrene emission appearing at short wavelengths ranging from 390 to 398 nm and long-wavelength emission ranging from 410 to 425 nm. The emission was found to drastically quench in polar solvents, such as DMSO, ACN, and MeOH (Figure 1b). However, the corresponding tetrazole derivative, pyrenetetrazolyl amino alanine 6J (PyTzAAla), showed both the absorption and emission characteristics of pyrene. In the UV−visible spectra, a blueshifted solvatochromicity with an increasing absorption was observed for all three absorption bands as the solvent polarity was increased from toluene (316, 331, and 346 nm) to MeOH (311, 327, and 342 nm) (Figure 1c). Upon excitation at

nucleophiles, such as sodium azide, which would ultimately lead to the generation of another novel class of 5-aminotetrazolyl alanines ATzAla (Ata) via electrocyclization. In general, tetrazoles exhibit a wide range of biological activity.26 They can readily associate with other biological molecules via intermolecular hydrogen bonding, serve as bioisosteres of cis-amides and carboxylic acids in biologically active molecules, and are resistant to metabolic degradation.26,27 Therefore, the tetrazole class of compounds finds widespread applications for the design of commercial drug candidates and as synthetic intermediates for bioactive molecules.28 Particularly, 5-aminotetrazoles are an important class of compounds with broad applications in chemistry, biology, and materials science.29 Therefore, several synthetic methodologies have appeared in the literature for the generation of substituted tetrazoles, including 5-(substituted amino) tetrazoles.30 Recently, we also reported the regioselective and stereoselective synthesis of tetrazolyl nucleosides for the first time.26a Among the various available strategies, we adopted Cu(OAc)2-mediated desulfurization of thioureayl alanines for the formation of carbodiimide intermediates followed by their electrocyclization with azide to generate aminotetrazolyl alanines. We were the first to report this novel class of unnatural amino acids. The synthesis was carried out by stirring a solution of thioureayl alanine in DMF at 50 °C with NaN3 (3 equiv) in the presence of Cu(OAc)2/Et3N for 12 h. We synthesized various 5aminotetrazolyl alanines from the aryl thioureayl derivatives in good to excellent yields (Scheme 3). However, no tetrazole formation was observed for the alkylthiourea derivatives, which might be due to the highly basic alkyl amine moiety of alkyl thiourea inhibiting the desulfurization process in the presence of Et3N. Thus, 1-phenyltetrazolyl-5-amino alanine (6A) from phenyl thioureayl alanine (5A) was obtained in a moderate yield (Scheme 3). High yields of 1-aryl-5-aminotetrazolyl alanines were observed when the aryl group of the aryl thioureayl alanine was substituted with −Me, −Et, −nBu, dimethyl, −OH, or −Cl substituents. However, strong electron-donating (−OMe) or strong electron-withdrawing (−COMe/CN) groups reduced the yield from moderate to low (61−45%). 12279

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CONCLUSION In conclusion, we successfully synthesized and demonstrated the multifaceted use of unexplored unnatural amino acid isothiocyanyl alanine (NCSAla, Ita). A variety of aliphatic- and aromaticsubstituted thioureayl alanines from isothiocyanyl alanine and subsequently aromatic-substituted amino tetrazolyl alanines were successfully synthesized in good to excellent yields. The novel thioureayl alanine amino acids are the first report that could easily be utilized for catalytic transformations, which is our future research focus. The regiochemistry of 5-aminotetrazolyl alanines, ATzAla (Ata), the third class of novel unnatural amino acids, with respect to the tetrazole moiety was explained on the basis of the difference in the pKa’s of the amino functionalities in the thioureayl alanines. Three of the pyrene-labeled amino acids from two new classes possessed good photophysical properties, which could find applications in sensing heavy and toxic metal ions and in labeling short peptides in the future. Considering the research progress toward the expansion of the genetic code and other possible biotechnological applications, our synthesized new classes of unnatural amino acids could find widespread applications and attract a broad scientific community of organic chemists and researchers in related areas. Furthermore, the NCS Ala amino acid could be beneficial over the available nine canonical amino acids for site-specific labeling and in ligation of peptides or proteins if incorporated enzymatically or for studying peptide conformations and dynamics, which is our current research focus.

Figure 1. UV−Visible and fluorescence emission spectra of fluorescent pyrenyl thioureayl amino acid 5J (PyTUAla) (a and b) and of pyrenetetrazolyl amino acid 6J (PyTzAAla) (c and d) in various organic solvents (concentration = 10 μM).

345 nm, the three emission bands appearing at approximately 375, 395, and 415 nm experienced emission enhancements with a blueshift as the solvent polarity was increased from toluene to MeOH, except for ethyl acetate, which had a quenching effect (Figure 1d). The thiourea moiety did not show any modulation of the fluorescence of a probe probably due to the increased distance from the probe core, as was exemplified in the fluorescence of pyrenylmethyl thioureayl alanine 5K (MePyTUAla), which exhibited both the absorption and fluorescence property characteristics of pyrene and was similar to that observed for pyrenyltetrazolyl alanine. Thus, the UV−visible spectra showed characteristics of pyrenyl absorbance bands at 347, 331, and 317 nm in toluene. As the solvent polarity increased, it showed blueshifted solvatochromism along with increased absorbances at 342, 326, and 311 nm in MeOH (Figure 2a). Upon excitation

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EXPERIMENTAL SECTION

General Experimental. All of the reactions were carried out under a nitrogen atmosphere using oven-dried round-bottom flasks. Reagents were purchased at the highest commercial quality and used without further purification, unless otherwise state. Reactions were monitored by thin-layer chromatography (TLC) carried out on a 0.25 mm silica gel 60F-254 and silica gel-G (1:4) and visualized under UV light at 254 nm. Further visualization was achieved by the iodine vapor adsorbed on silica gel depending on the product type. Organic extracts were dried over anhydrous sodium sulfate. Solvents were removed in a rotary evaporator under reduced pressure. Column chromatography was performed on silica gel 60−120 mesh using a mixture of hexane and ethyl acetate as mobile solvents. The isolated compounds were characterized by 1H, 13C, and 2D NMR, IR spectroscopic techniques, and mass spectrometry. All of the NMR spectra were recorded at ambient temperature on a Bruker Ascend Aeon 600 MHz spectrometer, where the 1H frequency was 600 MHz and 13C frequency was 150 MHz. NMR spectra for all of the samples were measured in either CDCl3 or DMSO-d6. The chemical shift values were reported in ppm downfield from tetramethylsilane, using chloroform-d (δ = 7.26 for 1H NMR, δ = 77.23 for 13C NMR) or using deuterated dimethyl sulfoxide-d6 (δ = 2.50 for 1H NMR, δ = 39.50 for 13C NMR). The 1H NMR coupling constant (J) is represented in hertz (Hz). All of the NMR-FID was processed in the MestReNova v6.0.2. software. High-resolution mass spectra (HRMS) were recorded on a Water System mass spectrometer in positive mode using electrospray ionization time-of-flight (ESI-TOF) and/or atmospheric pressure chemical ionization time-of-flight (APCI-TOF) reflection experiments. IR spectra were recorded on KBr plates in a PerkinElmer spectrometer and reported in frequency of absorption (cm−1). Synthesis of N,C-Diprotected Azido Alanine (3).7 This is prepared by following our published protocol.7a 3: yield 3200 mg, 75%; IR (KBr, cm−1) υ 3422, 2105, 1714, 1666, 1499; 1H NMR (600 MHz, CDCl3) δ 5.49 (d, J = 7.8 Hz, 1H), 4.84 (d, J = 3.4 Hz, 1H), 3.76 (s, 3H), 3.55 (ddd, J = 41.1, 12.4, 4.7 Hz, 2H), 3.23 (s, 3H), 1.44 (s, 9H); 13C NMR (150 MHz, CDCl3) δ 169.6, 155.1. 80.0, 61.6, 52.3, 50.6, 32.2, 28.2, 28.2. Synthesis of N,C-Diprotected Amino Alanine (4). Azidoalanine 3 (1700 mg, 6.22 mmol) was loaded in a two-neck round-bottom flask, and dry methanol was added. Evacuation took place under a high

Figure 2. UV−Visible and fluorescence emission spectra of fluorescent pyrenylmethyl thioureayl lysine amino acid 5K (MePyTUAla) (a and b) in various organic solvents (concentration = 10 μM).

at 345 nm and increasing the solvent polarity from dioxane to MeOH, it showed emissions at 376, 396, and 420 nm with decreased intensities, which may be due to increased nonradiative decay via solvent−solute or H-bonding interactions (Figure 2b).32 12280

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The Journal of Organic Chemistry vacuum pump for 10 min, and the flask was degassed by N2 gas. Pd/C (132 mg, 1.24 mmol) was added, and a H2 atmosphere was created by a balloon. The reaction mixture was stirred at room temperature for 7 h, after which the reaction mixture was filtered through Whatmann filter paper. The solvent was evaporated under a high vacuum to afford a white semisolid product, 4, which was directly used for the next step without further purification: yield 350 mg, 88%; IR (KBr, cm−1) υ 3400, 1704, 1646, 1530; 1H NMR (600 MHz, CDCl3) δ 5.50 (d, J = 7.1 Hz, 1H), 4.69 (s, 1H), 3.76 (s, 3H), 3.20 (s, 3H), 3.04−2.98 (m, 1H), 2.85 (dd, J = 13.3, 5.8 Hz, 1H), 1.75 (s, 2H), 1.42 (s, 9H); 13C NMR (150 MHz, CDCl3) δ 171.6, 155.9, 79.9, 61.8, 53.2, 44.3, 32.2, 28.5; HRMS (ESI+) (m/z) calcd for C10H22N3O4 [M + H]+ 248.1610, found 248.1606. Synthesis of Isothiocyanyl Alanine (1, NCSAla = Ita). N,CDiprotected amino alanine (830 mg, 3.35 mmol) was loaded in a dry 50 mL round-bottom flask under a nitogen atmosphere, and dry THF was added. The reaction mixture was cooled to 0 °C, and Et3N (1.6 mL, 11.08 mmol) was added. After 5 min of stirring the solution, CS2 (0.406 mL, 6.7 mmol) was added, and the solution was stirred for 1 h at 0 °C. The tosyl chloride (704 mg, 3.68 mmol) was added, and the resulting mixture was stirred for 5 min at 0 °C, after which the reaction was completed. The reaction mixture was dried under a high vacuum, diluted with ethyl acetate, and treated with 1 N HCl, followed by washing with saturated NaHCO3 and a brine solution. The organic layer was dried over Na2SO4. After evaporation of ethyl acetate, the residue was purified by silica gel (60−120) column chromatography using hexane/ethyl acetate (Rf = 0.5, in 3:1 hexane/ethyl acetate) as an eluent to afford the desired NCSAla (1) as a light greenish solid compound: yield 800 mg, 82%; mp 80−84 °C; IR (KBr, cm−1) υ= 3343, 2223, 2120, 1697, 1658, 1526; 1H NMR (600 MHz, CDCl3) δ 5.54 (d, J = 7.5 Hz, 1H), 4.88 (d, J = 3.5 Hz, 1H), 3.84 (dd, J = 14.3, 4.4 Hz, 1H), 3.76 (s, 4H), 3.25 (s, 3H), 1.45 (s, 9H); 13C NMR (150 MHz, CDCl3) δ 168.8, 155.1, 134.1, 80.7, 62.0, 50.6, 47.1, 32.5, 28.5; HRMS (ESI+ ) (m/z) calcd for C11H19N3O4SNa [M + Na]+ 312.0989, found 312.0998. General Procedure for the Synthesis of Thioureayl Alanines (5A− Q, TUAla = tua) from Isothiocynyl Alanine (1, NCSAla). To a roundbottom flask of 5 mL of anhydrous CH2Cl2/CH3CN (3:1) was added NCS Ala (1, 200 mg, 0.69 mmol) under a nitogen atmosphere. After the flask was degassed by N2 gas for 5 min, various aliphatic/aromatic amines (0.75 mmol) were added, and the reaction mixture was heated at 50 °C for about 8−12 h. (For almost all aromatic amines, except for naphthylamine, pyrenyl amine, acetyl aniline, aminophenol, and chloroaniline, the times needed are 24, 48, 48, 18, and 18 h, respectively, and for aliphatic or cyclohexyl amines, the times needed are 5−6 h under a N2 atmosphere.) After completion of the starting material as indicated by TLC, the reaction mixture was dried under a high vacuum and directly packed into a silica gel (60−120) column. The residue was purified using hexane/ethyl acetate (1:1 to 1:4, Rf = 0.4) to afford a pure semisolid material. Phenyl Thioureayl Alanine (5A, PhTUAla). Using the general procedure and starting from 0.69 mmol of NCSAla, the desired compound 5A was obtained as a semisolid compound (Rf = 0.4 in 1:1 hexane/ethyl acetate): yield 231 mg, 87%; IR (KBr, cm−1) υ 3483, 1679, 1683, 1597, 1523, 1256; 1H NMR (600 MHz, CDCl3) δ 8.13 (d, J = 42.3 Hz, 1H), 7.41 (d, J = 6.8 Hz, 2H), 7.29−7.26 (m, 3H), 6.79 (d, J = 4.2 Hz, 1H), 5.63 (t, J = 7.5 Hz, 1H), 4.85 (d, J = 4.8 Hz, 1H), 4.07−4.06 (m, 1H), 3.85−3.70 (m, 1H), 3.77 (s, 3H), 3.19 (s, 3H), 1.37 (s, 9H); 13C NMR (150 MHz, CDCl3) δ 181.2, 170.2, 155.8, 136.1, 130.1, 127.2, 125.4, 80.3, 62.0, 50.5, 48.0, 32.5, 29.8, 28.4; HRMS (APCI+) (m/z) calcd for C17H27N4O4S [M + H]+ 383.1753, found 383.1763. Tolyl Thioureayl Alanine (5B, TolTUAla). Using the general procedure and starting from 0.36 mmol of NCSAla, the desired compound 5B was obtained as a semisolid compound (Rf = 0.4 in 1:1 hexane/ethyl acetate): yield 140 mg, 98%; IR (KBr, cm−1) υ 3440, 1707, 1693, 1648, 1535, 1252: 1H NMR (600 MHz, CDCl3) δ 8.27 (s, 1H), 7.16 (d, J = 7.8 Hz, 2H), 7.10 (d, J = 7.9 Hz, 2H), 6.67 (s, 1H), 5.63 (d, J = 12.6 Hz, 1H), 4.81 (s, 1H), 4.01 (dt, J = 13.1, 4.5 Hz, 1H), 3.73 (s, 4H), 3.14 (s, 3H), 2.30 (s, 3H), 1.34 (s, 9H); 13C NMR (150 MHz, CDCl3) δ 181.2, 170.2, 155.7, 137.1, 133.6, 130.5, 125.5, 80.1, 61.9, 50.5, 47.7, 32.4, 28.3, 21.1; HRMS (APCI+) (m/z) calcd for C18H29N4O4S [M + H]+ 397.1910, found 397.1906.

p-Ethylphenyl Thioureayl Alanine (5C, EtBTUAla). Using the general procedure and starting from 0.38 mmol of NCSAla, the desired compound 5C was obtained as a white solid compound (Rf = 0.4 in 1:1 hexane/ethyl acetate): yield 155 mg, 99%; mp 147−151 °C; IR (KBr, cm−1) υ 3442, 1701, 1651, 1533, 1253; 1H NMR (600 MHz, CDCl3) δ 7.91 (s, 1H), 7.22 (d, J = 8.2 Hz, 2H), 7.14 (d, J = 8.2 Hz, 2H), 6.66 (s, 1H), 5.56 (d, J = 5.4 Hz, 1H), 4.83 (s, 1H), 4.09−3.99 (m, 1H), 3.85−3.78 (m, 1H), 3.76 (s, 3H), 3.18 (s, 3H), 2.64 (q, J = 7.6 Hz, 2H), 1.36 (s, 9H), 1.23 (t, J = 7.6 Hz, 3H); 13C NMR (150 MHz, CDCl3) δ 181.4, 170.2, 155.8, 143.7, 133.6, 129.5, 125.7, 80.3, 62.0, 50.6, 48.1, 32.5, 28.5, 28.4, 15.5; HRMS (APCI+) (m/z) calcd for C19H31N4O4S [M + H]+ 411.2066, found 411.2073. p-Butylphenyl Thioureayl Alanine (5D, BuBTUAla). Using the general procedure and starting from 0.41 mmol of NCSAla, the desired compound 5D was obtained as a semisolid compound (Rf = 0.4 in 1:1 hexane/ethyl acetate): yield 175 mg, 97%; IR (KBr, cm−1) υ 3363, 1676, 1649, 1614, 1536, 1255. 1H NMR (600 MHz, CDCl3) δ 8.30 (s, 1H), 7.16 (d, J = 7.6 Hz, 2H), 7.12 (d, J = 8.1 Hz, 2H), 6.68 (s, 1H), 5.62 (d, J = 5.7 Hz, 1H), 4.81 (s, 1H), 4.05−3.97 (m, 1H), 3.82−3.76 (m, 1H), 3.73 (s, 3H), 3.14 (s, 3H), 2.56 (t, J = 7.7 Hz, 2H), 1.54 (dd, J = 15.1, 7.8 Hz, 2H), 1.33 (s, 12H), 0.89 (dd, J = 7.6, 7.1 Hz, 3H); 13C NMR (150 MHz, CDCl3) δ 181.1, 170.2, 155.7, 142.1, 133.7, 129.9, 125.3, 80.1, 61.9, 50.5, 47.6, 35.2, 33.5, 32.4, 28.3, 22.4, 14.0; HRMS (ESI+) (m/z) calcd for C21H35N4O4S [M + H]+ 439.2379, found 439.2378. m,p-Dimethylphenyl Thioureayl Alanine (5E, DMBTUAla). Using the general procedure and starting from 0.55 mmol of NCSAla, the desired compound 5E was obtained as a white solid compound (Rf = 0.4 in 1:1 hexane/ethyl acetate): yield 170 mg, 75%; mp 128−132 °C; IR (KBr, cm−1) υ 3415, 1706, 1653, 1538; 1H NMR (600 MHz, CDCl3) δ 8.25 (s, 1H), 7.10 (d, J = 7.5 Hz, 1H), 6.97−6.93 (m, 2H), 6.65 (s, 1H), 5.61 (d, J = 8.1 Hz, 1H), 4.81 (s, 1H), 3.99 (dd, J = 8.3, 4.8 Hz, 1H), 3.85−3.77 (m, 1H), 3.73 (s, 4H), 3.14 (s, 3H), 2.21 (s, 3H), 2.10 (s, 3H), 1.33 (s, 9H); 13 C NMR (150 MHz, CDCl3) δ 181.0, 170.3, 155.6, 138.4, 135.8, 133.7, 130.9, 126.5, 122.8, 80.0, 61.9, 50.5, 47.4, 32.4, 28.2, 19.8, 19.4; HRMS (APCI+) (m/z) calcd for C19H31N4O4S [M + H]+ 411.2066, found 411.2068. p-Hydroxyphenyl Thioureayl Alanine (5F, HBTUAla). Using the general procedure and starting from 0.39 mmol of NCSAla, the desired compound 5F was obtained as a semisolid compound (Rf = 0.4 in 1:2 hexane/ethyl acetate): yield 145 mg, 93%; IR (KBr, cm−1) υ 3471, 1690, 1637, 1538, 1249; 1H NMR (600 MHz, CDCl3) δ 8.04 (s, 1H), 7.02 (d, J = 7.6 Hz, 2H), 6.79 (d, J = 8.2 Hz, 2H), 6.53 (s, 1H), 5.78 (s, 1H), 4.87 (s, 1H), 3.97−3.86 (m, 2H), 3.74 (s, 3H), 3.16 (s, 3H), 1.39 (s, 9H); 13C NMR (150 MHz, CDCl3) δ 181.7, 170.5, 156.2, 128.0, 127.8, 116.9, 80.7, 62.0, 50.6, 47.1, 32.5, 28.5. HRMS (APCI+) (m/z) calcd for C17H27N4O5S [M + H]+ 399.1702, found 399.1715. p-Methoxyphenyl Thioureayl Alanine (5G, MOBTUAla). Using the general procedure and starting from 0.39 mmol of NCSAla, the desired compound 5G was obtained as a white solid compound (Rf = 0.4 in 1:1 hexane/ethyl acetate): yield 130 mg, 87%; mp 115−119 °C; IR (KBr, cm−1) υ 3483, 1973, 1707, 1653, 1540, 1247; 1H NMR (600 MHz, CDCl3) δ 7.71 (s, 1H), 7.17 (d, J = 8.5 Hz, 2H), 6.92 (d, J = 8.8 Hz, 2H), 6.52 (s, 1H), 5.54 (d, J = 4.4 Hz, 1H), 4.81 (s, 1H), 4.05 (dd, J = 10.2, 5.4 Hz, 1H), 3.81 (s, 3H), 3.77 (s, 4H), 3.19 (s, 3H), 1.37 (s, 9H); 13C NMR (150 MHz, CDCl3) δ 181.9, 170.2, 159.1, 155.9, 128.5, 128.0, 115.3, 80.3, 62.0, 55.6, 50.7, 48.2, 32.5, 28.4; HRMS (ESI+) (m/z) calcd for C18H29N4O5S [M + H]+ 413.1859, found 413.1843. m,m-Dimethoxyphenyl Thioureayl Alanine (5H, DMOBTUAla). Using the general procedure and starting from 0.36 mmol of NCSAla, the desired compound 5H was obtained as a white solid compound (Rf = 0.4 in 1:1 hexane/ethyl acetate): yield 135 mg, 84%; mp 139−143 °C; IR (KBr, cm−1) υ 3471, 1707, 1643, 1603, 1538, 1268; 1H NMR (600 MHz, CDCl3) δ 8.37 (dd, J = 28.0, 14.0 Hz, 1H), 6.91 (s, 1H), 6.38 (s, 2H), 6.29 (s, 1H), 5.64 (s, 1H), 4.83 (d, J = 4.4 Hz, 1H), 4.00 (dd, J = 11.9, 5.0 Hz, 1H), 3.91−3.80 (m, 1H), 3.74 (s, 9H), 3.14 (s, 3H), 1.33 (s, 9H); 13 C NMR (150 MHz, CDCl3) δ 180.7, 170.3, 161.7, 155.7, 137.9, 102.8, 99.1, 80.2, 61.9, 60.5, 55.5, 50.5, 32.4, 28.3; HRMS (APCI+) (m/z) calcd for C19H31N4O6S [M + H]+ 443.1964, found 443.1976. Naphthyl Thioureayl Alanine (5I, NapTUAla). Using the general procedure and starting from 0.41 mmol of NCSAla, the desired 12281

DOI: 10.1021/acs.joc.7b02103 J. Org. Chem. 2017, 82, 12276−12285

Article

The Journal of Organic Chemistry

compound 5O was obtained as a semisolid compound (Rf = 0.4 in 1:2 hexane/ethyl acetate): yield 75 mg, 60%; IR (KBr, cm−1) υ 3374, 1684, 1647, 1549, 1524, 1252; 1H NMR (600 MHz, CDCl3) δ 6.85 (s, 1H), 6.54 (s, 1H), 5.89 (s, 1H), 4.79 (s, 1H), 3.94 (s, 1H), 3.76 (s, 4H), 3.18 (s, 3H), 1.52 (s, 2H), 1.39 (s, 11H), 1.33 (s, 2H), 0.88 (s, 3H); 13C NMR (150 MHz, CDCl3) δ 182.0, 170.4, 156.4, 80.5, 61.9, 50.7, 43.9, 32.5, 30.9, 29.7, 28.4, 20.1, 13; HRMS (ESI+) (m/z) calcd for C15H31N4O4S [M + H]+ 363.2066, found 363.2066. n-Heptyl Thioureayl Alanine (5P, HepTUAla). Using the general procedure and starting from 0.34 mmol of NCSAla, the desired compound 5P was obtained as a white solid compound (Rf = 0.4 in 1:2 hexane/ethyl acetate): yield 80 mg; 58%; mp 112−116 °C; IR (KBr, cm−1) υ 3350, 1710, 1661, 1501, 1265; 1H NMR (600 MHz, CDCl3) δ 6.88 (s, 1H), 6.66 (s, 1H), 6.55 (s ,1H), 5.89 (s, 1H), 4.78 (s, 1H), 3.93 (s, 1H), 3.75 (s, 4H), 3.17 (s, 3H), 1.53 (s, 2H), 1.38 (s, 10H), 1.24− 1.21 (m, 9H), 0.83−0.82 (m, 3H); 13C NMR (150 MHz, CDCl3) δ 182.1, 170.4, 156.3, 80.5, 61.9, 50.7, 32.4, 31.8, 29.0, 28.9, 28.4, 28.4, 26.9, 22.6, 22.6, 14.1, 14.1; HRMS (ESI+) (m/z) calcd for C18H37N4O4S [M + H]+ 405.2537, found 405.2547. Ethanol Thioureayl Alanine (5Q, EtOHTUAla). Using the general procedure and starting from 0.29 mmol of NCSAla, the desired compound 5Q was obtained as a semisolid compound (Rf = 0.4 in 1:4, hexane/ethyl acetate): yield 71 mg, 70%; IR (KBr, cm−1) υ 3475, 1673, 1566, 1528, 1253; 1H NMR (600 MHz, CDCl3) δ 7.21 (s, 1H), 6.16 (s, 1H), 4.81 (s, 1H), 3.92 (s, 2H), 3.80 (s, 3H), 3.74 (s, 3H), 3.67− 3.39 (m, 2H), 3.20 (s, 3H), 1.41 (s, 9H); 13C NMR (150 MHz, CDCl3) δ 183.6, 171.0, 156.2, 80.6, 61.9, 61.6, 50.9, 46.8, 32.7, 29.8, 28.5; HRMS (ESI+) (m/z) calcd for C13H27N4O5S [M + H]+ 351.1702, found 351.1702. Cyclohexyl Thioureayl Alanine (5R, CyHTUAla). Using the general procedure and starting from 0.41 mmol of NCSAla, the desired compound 5R was obtained as a white solid compound (Rf = 0.4 in 1:2 hexane/ethyl acetate): yield 125 mg, 78%; mp 165−169 °C; IR (KBr, cm−1) υ 3365, 1686, 1650, 1545, 1256; 1H NMR (600 MHz, CDCl3) δ 6.87 (s, 1H), 6.35 (s, 1H), 5.82 (s, 1H), 4.79 (s, 1H), 3.95 (s, 1H), 3.76 (s, 3H), 3.62 (s, 1H), 3.19 (s, 3H), 2.02−1.94 (m, 3H), 1.71 (t, 2H), 1.60 (d, J = 12.6 Hz, 1H), 1.41 (s, 9H), 1.38−1.28 (m, 2H), 1.23−1.13 (m, 3H); 13C NMR (150 MHz, CDCl3) δ 180.9, 170.2, 156.6, 80.6, 62.0, 52.8, 50.7, 32.8, 32.5, 28.4, 25.5, 24.9, 24.8; HRMS (ESI+) (m/z) calcd for C17H33N4O4S [M + H]+ 389.2223, found 389.2229. General Procedure for the Synthesis of 5-Aminotetrazolyl Alanines (6A−G, 6I−J, 6L−N, TzAAla = Tza) from Aromatic Thioureayl Alanines (5A−G, 5I−J, 5L−N, TUAla = tua). In a flame-dried roundbottom flask various thioureayl alanines (5A−G, 5I−J, 5L−N) (0.16− 0.34 mmol) were dissolved in 5 mL of dry DMF under a N2 atmosphere. Evacuation took place under a high vacuum pump for 5 min, and the flask was degassed by N2 gas; 3 equiv of NaN3 (0.48−1.02 mmol), 0.5 equiv of Cu (OAC)2, H2O (0.08−0.17 mmol), and 3 equiv of Et3N (0.48−1.02 mmol) were added to reaction mixture while degassing was continued. Then the reaction mixture was degassed for another 2 min and stirred for 8−16 h at room temperature under a N2 atmosphere. After completion of the reaction indicated by TLC (hexane/ethyl, 1:1 to 1:2; Rf = 0.4), the residue was extracted with ethyl acetate and washed with saturated ammonium chloride and a brine solution. The organic extract was dried over Na2SO4, evaporated to dryness in a rotary evaporator, and purified on silica gel (60−120) column chromatography using hexane/ethyl (1:1 to 1:2) as an eluent to afford the desired 5aminotetrazolyl alanines (6A−G, 6I−J, 6L−N) as solid or semisolid materials with good to excellent yields (60−85%). All of the compounds were characterized by 1H and 13C NMR, IR spectroscopic techniques, and mass spectrometry. 1-Phenyltetrazolyl-5-amino Alanine (6A, PhTzAAla). Using the general procedure and starting from 0.16 mmol of PhTUAla (5A), the desired compound 6A was obtained as a white solid compound (Rf = 0.4 in 1:2 hexane/ethyl acetate): yield 40 mg, 63%; mp 130−133 °C; IR (KBr, cm−1) υ 3379, 1709, 1658, 1609, 1509, 1459, 1366, 1251; 1H NMR (600 MHz, CDCl3) δ 7.57−7.49 (m, 5H), 5.68 (d, J = 5.8 Hz, 1H), 5.38 (s, 1H), 4.90 (s, 1H), 3.92 (d, J = 10.7 Hz, 1H), 3.80 (s, 3H), 3.62 (d, J = 4.8 Hz, 1H), 3.20 (s, 3H), 1.36 (s, 9H); 13C NMR (150 MHz, CDCl3) δ 169.8, 156.3, 154.7, 133.4, 130.3, 129.8, 124.1, 80.6,

compound 5I was obtained as a semisolid compound (Rf = 0.4 in 1:1 hexane/ethyl acetate): yield 160 mg, 90%; IR (KBr, cm−1) υ 3409, 1708, 1653, 1600, 1533; 1H NMR (600 MHz, CDCl3) δ 8.15 (s, 1H), 8.03 (d, J = 8.3 Hz, 1H), 7.89 (dd, J = 11.9, 8.2 Hz, 2H), 7.58−7.20 (m, 4H), 6.51 (s, 1H), 5.40 (d, J = 7.4 Hz, 1H), 4.71−4.69 (m, 1H), 3.94 (dd, J = 11.4, 5.0 Hz, 1H), 3.80−3.76 (m, 1H), 3.65 (s, 3H), 3.06 (s, 3H), 1.26 (s, 9H); 13C NMR (150 MHz, CDCl3) δ 182.0, 170.1, 155.6, 134.8, 131.7, 130.1, 129.1, 128.6, 127.6, 127.2, 125.9, 125.6, 122.7, 80.2, 61.9, 50.3, 48.1, 32.4, 28.3; HRMS (ESI+) (m/z) calcd for C21H29N4O4S [M + H]+ 433.1910, found 433.1905. Pyrenyl Thioureayl Alanine (5J, PyTUAla). Using the general procedure and starting from 0.51 mmol of NCSAla, the desired compound 5J was obtained as a light brown solid compound (Rf = 0.4 in 1:1 hexane/ethyl acetate): yield 195 mg, 75%; mp 183−187 °C; IR (KBr, cm−1) υ 3380, 1691, 1671, 1602, 1524, 1246; 1H NMR (600 MHz, CDCl3) δ 8.49 (s, 1H), 8.21−8.13 (m, 6H), 8.10 (d, J = 8.9 Hz, 1H), 8.05−8.01 (m, 2H), 7.94 (d, J = 8.0 Hz, 1H), 6.54 (s, 1H), 5.43 (d, J = 7.1 Hz, 1H), 4.65 (d, J = 4.0 Hz, 1H), 3.96−3.79 (m, 2H), 3.56 (s, 3H), 2.98 (s, 3H), 1.01 (s, 9H); 13C NMR (150 MHz, CDCl3) δ 182.3, 170.1, 155.6, 131.3, 131.1, 131.0, 129.2, 128.7, 128.4, 127.9, 127.1, 126.7, 126.0, 126.0, 125.6, 125.5, 124.5, 121.8, 80.0, 61.8, 50.4, 48.1, 32.3, 28.0; HRMS (APCI+) (m/z) calcd for C27H31N4O4S [M + H]+ 507.2066, found 507.2063. Pyrenylmethyl Thioureayl Alanine (5K, MePyTUAla). Using the general procedure and starting from 0.69 mmol of NCSAla, the desired compound 5K was obtained as a semisolid compound (Rf = 0.4 in 2:1, hexane/ethyl acetate): yield 260 mg, 72%; mp 177−181 °C; IR (KBr, cm−1) υ 3369, 1685, 1650, 1519, 1266; 1H NMR (600 MHz, CDCl3) δ 8.12 (d, J = 2.9 Hz, 3H), 8.06−8.00 (m, 2H), 7.96 (d, J = 12.1 Hz, 3H), 7.88 (d, J = 6.7 Hz, 1H), 7.01 (s, 1H), 6.95 (s, 1H), 5.86 (s, 1H), 5.22 (m, 2H), 4.71 (s, 1H), 3.81 (s, 2H), 3.46 (s, 3H), 2.67 (s, 3H), 1.32 (s, 9H); 13 C NMR (150 MHz, CDCl3) δ 182.5, 170.5, 155.9, 131.2, 130.7, 129.13, 128.2, 127.5, 127.3, 126.1, 125.4, 124.9, 124.7, 124.6, 123.0, 80.5, 61.6, 50.5, 47.1, 32.0, 29.8, 28.3; HRMS (ESI+) (m/z) calcd for C28H33N4O4S [M + H]+ 521.2223, found 521.2232. p-Chlorophenyl Thioureayl Alanine (5L, ClBTUAla). Using the general procedure and starting from 0.34 mmol of NCSAla, the desired compound 5L was obtained as a semisolid compound (Rf = 0.4 in 1:1 hexane/ethyl acetate): yield 139 mg, 98%; mp 115−119 °C; IR (KBr, cm−1) υ 3440, 1690, 1648, 1538, 1251; 1H NMR (600 MHz, CDCl3) δ 8.75 (d, J = 11.4 Hz, 1H), 7.29 (d, J = 4.4 Hz, 2H), 7.21 (d, J = 8.3 Hz, 2H), 6.94 (s, 1H), 5.80 (s, 1H), 4.82 (s, 1H), 4.03 (s, 1H), 3.74 (s, 4H), 3.12 (s, 3H), 1.33 (s, 9H); 13C NMR (150 MHz, CDCl3) δ 181.1, 170.2, 155.9, 135.4, 132.1, 129.8, 126.4, 80.4, 61.9, 50.5, 47.6, 32.4, 28.3; HRMS (APCI+) (m/z) calcd for C17H26ClN4O4S [M + H]+ 417.1363, found 417.1371. p-Cyanophenyl Thioureayl Alanine (5M, CNBTUAla). Using the general procedure and starting from 0.36 mmol of NCSAla, the desired compound 5M was obtained as a white solid compound (Rf = 0.4 in 1:1 hexane/ethyl acetate): yield 130 mg, 88%; mp 186−190 °C; IR (KBr, cm−1) υ 3323, 2221, 1675, 1651, 1627, 1606, 1526, 1253; 1H NMR (600 MHz, CDCl3) δ 8.93 (s, 1H), 7.60 (d, J = 7.2 Hz, 2H), 7.53 (s, 2H), 7.39 (s, 1H), 5.90 (s, 1H), 4.85 (s, 1H), 4.08−3.87 (m, 2H), 3.79 (s, 3H), 3.18 (s, 3H), 1.36 (s, 9H); 13C NMR (150 MHz, CDCl3) δ 180.9, 170.3, 156.2, 141.9, 133.4, 123.4, 118.7, 108.4, 80.9, 62.0, 50.6, 47.5, 32.6, 28.4; HRMS (APCI+) (m/z) calcd for C18H26N5O4S [M + H]+ 408.1706, found 408.1716. p-Acetylphenyl Thioureayl Alanine (5N, AcBTUAla). Using the general procedure and starting from 0.36 mmol of NCSAla, the desired compound 5N was obtained as a gummy compound (Rf = 0.4 in 1:1 hexane/ethyl acetate): yield 100 mg, 65%; IR (KBr, cm−1) υ 3449, 1701, 1647, 1603, 1537. 1H NMR (600 MHz, CDCl3) δ 8.48 (s,1H), 7.97 (d, J = 7.2 Hz, 2H), 7.40 (d, J = 5.1 Hz, 2H), 7.22 (dd, J = 8.9, 4.8 Hz, 1H), 5.72 (s, 1H), 4.87 (d, J = 3.6 Hz, 1H), 4.11−4.08 (m, 1H), 3.84 (dd, J = 13.1, 6.3 Hz, 1H), 3.79 (s, 3H), 3.21 (s, 3H), 2.57 (s, 3H), 1.36 (s, 9H); 13 C NMR (150 MHz, CDCl3) δ 197.03, 180.9, 170.4, 156.0, 141.7, 134.1, 130.0, 123.0, 80.6, 62.0, 50.6, 32.5, 28.4, 26.6; HRMS (APCI+) (m/z) calcd for C19H29N4O5S [M + H]+ 425.1859, found 425.1869. n-Butyl Thioureayl Alanine (5O, BuTUAla). Using the general procedure and starting from 0.34 mmol of NCSAla, the desired 12282

DOI: 10.1021/acs.joc.7b02103 J. Org. Chem. 2017, 82, 12276−12285

Article

The Journal of Organic Chemistry 62.1, 51.0, 47.7, 32.5, 28.4; HRMS (ESI+) (m/z) calcd for C17H26N7O4 [M + H]+ 392.2046, found 392.2049. 1-Tolyltetrazolyl-5-amino Alanine (6B, TolTzAAla). Using the general procedure and starting from 0.34 mmol of TolTUAla (5B), the desired compound 6B was obtained as a solid compound (Rf = 0.4 in 1:2 hexane/ethyl acetate): yield 105 mg, 78%; mp 102−106 °C; IR (KBr, cm−1) υ 3417, 1705, 1657, 1608, 1519, 1459, 1367, 1264; 1H NMR (600 MHz, CDCl3) δ 7.36 (d, J = 8.2 Hz, 2H), 7.33 (d, J = 8.2 Hz, 2H), 5.68 (d, J = 7.6 Hz, 1H), 5.32 (s, 1H), 4.89 (d, J = 2.1 Hz, 1H), 3.88−3.86 (m, 1H), 3.78 (s, 3H), 3.60 (s, 1H), 3.17 (s, 3H), 2.41 (s, 3H), 1.35 (s, 9H); 13 C NMR (150 MHz, CDCl3) δ 169.9, 156.2, 154.7, 140.2, 130.8, 130.7, 124.0, 80.5, 62.0, 50.9, 47.5, 32.4, 28.3, 21.4; HRMS (ESI+) (m/z) calcd for C18H28N7O4 [M + H]+ 406.2203, found 406.2203. 1-(p-Ethylphenyltetrazolyl)-5-amino Alanine (6C, EtBTzAAla). Using the general procedure and starting from 0.31 mmol of EtBTUAla (5C), the desired compound 6C was obtained as a semisolid compound (Rf = 0.4 in 1:2 hexane/ethyl acetate): yield 105 mg, 80%; IR (KBr, cm−1) υ 3347, 1710, 1658, 1604, 1519, 1458, 1251; 1H NMR (600 MHz, CDCl3) δ 7.39 (d, J = 8.2 Hz, 2H), 7.35 (d, J = 8.1 Hz, 2H), 5.69 (d, J = 7.4 Hz, 1H), 5.34 (s, 1H), 4.89 (s, 1H), 3.89−3.87 (m, 1H), 3.78 (s, 3H), 3.59 (s, 1H), 3.17 (s, 3H), 2.70 (q, J = 7.6 Hz, 2H), 1.34 (s, 9H), 1.24 (dd, J = 13.8, 6.1 Hz, 3H); 13C NMR (150 MHz, CDCl3) δ 169.8, 156.2, 154.7, 146.3, 130.8, 129.6, 124.1, 80.4, 62.0, 51.0, 47.5, 32.4, 28.7, 28.3, 15.5; HRMS (ESI+) (m/z) calcd for C19H30N7O4 [M + H]+ 420.2359, found 420.2358. 1-(p-n-Butylphenyltetrazolyl)-5-amino Alanine (6D, BuBTzAAla). Using the general procedure and starting from 0.32 mmol of BuBTUAla (5D), the desired compound 6D was obtained as a semisolid compound (Rf = 0.4 in 1:2 hexane/ethyl acetate): yield 122 mg, 85%; IR (KBr, cm−1) υ 3298, 1690, 1653, 1604, 1578, 1538, 1519, 1458, 1255; 1H NMR (600 MHz, CDCl3) δ 7.36 (d, J = 7.9 Hz, 2H), 7.30 (d, J = 7.8 Hz, 2H), 5.72 (d, J = 6.7 Hz, 1H), 5.36 (s, 1H), 4.87 (s, 1H), 3.85 (s, 1H), 3.76 (s, 3H), 3.58 (s, 1H), 3.14 (s, 3H), 2.63 (t, J = 7.8 Hz, 2H), 1.58 (dt, J = 15.3, 7.7 Hz, 2H), 1.35−1.32 (m, 11H), 0.90 (t, J = 7.4 Hz, 3H); 13C NMR (150 MHz, CDCl3) δ 169.8, 156.2, 154.7, 145.0, 130.8, 130.1, 123.9, 80.3, 61.9, 50.9, 47.4, 35.3, 33.4, 32.4, 29.7, 28.3, 22.3, 14.0; HRMS (ESI+) (m/z) calcd for C21H34N7O4 [M + H]+ 448.2672, found 448.2670. 1-(m,p-Dimethyphenyltetrazolyl)-5-amino Alanine (6E, DMBTzAAla). Using the general procedure and starting from 0.28 mmol of DMBTU Ala (5E), the desired compound 6E was obtained as a semisolid compound (Rf = 0.4 in 1:1 hexane/ethyl acetate): yield 90 mg, 76%; IR (KBr, cm−1) υ 3354, 1710, 1658, 1604, 1510, 1456, 1251; 1H NMR (600 MHz, CDCl3) δ 7.26 (t, J = 8.8 Hz, 2H), 7.18 (dd, J = 7.8, 1.5 Hz, 1H), 5.68 (d, J = 7.1 Hz, 1H), 5.34 (s, 1H), 4.88−4.87 (m, 1H), 3.88−3.86 (m, 1H), 3.78 (s, 3H), 3.61−3.60 (m, 1H), 3.17 (s, 3H), 2.31 (s, 3H), 2.30 (s, 3H), 1.34 (s, 9H); 13C NMR (150 MHz, CDCl3) δ 169.9, 156.2, 154.7, 139.1, 138.8, 131.14, 130.8, 125.2, 121.2, 80.4, 62.0, 50.9, 47.4, 32.4, 28.3, 19.9, 19.7; HRMS (ESI+) (m/z) calcd for C19H30N7O4 [M + H]+ 420.2359, found 420.2360. 1-(p-Hydroxyphenyltetrazolyl)-5-amino Alanine (6F, HBTzAAla). Using the general procedure and starting from 0.34 mmol of HBTUAla (5F), the desired compound 6F was obtained as a solid compound (Rf = 0.4 in 1:2 hexane/ethyl acetate): yield 100 mg, 72%; mp 170−174 °C; IR (KBr, cm−1) υ 3353, 1698, 1674, 1659, 1617, 1517, 1458, 1279; 1H NMR (600 MHz, CDCl3 and DMSO-d6) δ 9.42 (s, 1H), 7.17 (d, J = 8.5 Hz, 2H), 6.88 (d, J = 8.5 Hz, 2H), 5.91 (d, J = 7.9 Hz, 1H), 5.55 (s, 1H), 4.78 (s, 1H), 3.71 (s, 3H), 3.62 (dd, J = 24.3, 18.1 Hz, 2H), 3.06 (s, 3H), 1.28 (s, 9H); CDCl3 and DMSO-d6) δ 170.3, 158.7, 156.1, 155.0, 126.2, 124.4, 116.7, 80.4, 61.9, 50.8, 46.2, 32.3, 28.2; HRMS (ESI+) (m/z) calcd for C17H26N7O5 [M + H]+ 408.1995, found 408.1998. 1-(p-Methoxyphenyltetrazolyl)-5-amino Alanine (6G, MOBTzAAla). Using the general procedure and starting from 0.26 mmol of MOBTUAla (5G), the desired compound 6G was obtained as a semisolid compound (Rf = 0.4 in 1:2 hexane/ethyl acetate): yield 67 mg, 61%; IR (KBr, cm−1) υ 3381, 1709, 1658, 1606, 1582, 1519, 1461, 1254; 1H NMR (600 MHz, CDCl3) δ 7.39 (d, J = 8.5 Hz, 2H), 7.02 (d, J = 8.6 Hz, 2H), 5.69 (d, J = 6.7 Hz, 1H), 5.24 (s, 1H), 4.88 (s, 1H), 3.84 (s, 4H), 3.78 (s, 3H), 3.60 (s, 1H), 3.17 (s, 3H), 1.35 (s, 9H); 13C NMR (150 MHz, CDCl3) δ 169.8, 160.6, 156.2, 154.9, 125.9, 115.3, 80.5, 62.0, 55.8, 51.0, 47.4, 32.4,

28.3; HRMS (ESI+) (m/z) calcd for C18H28N7O5 [M + H]+ 422.2153, found 422.2153. 1-Naphthyltetrazolyl-5-amino Alanine (6I, NapTzAAla). Using the general procedure and starting from 0.35 mmol of NapTUAla (5I), the desired compound 6I was obtained as a solid compound (Rf = 0.4 in 1:2 hexane/ethyl acetate): yield 93 mg, 60%; mp 190−195 °C; IR (KBr, cm−1) υ 3311, 1711, 1640, 1548, 1490, 1276; 1H NMR (600 MHz, CDCl3) δ 8.52 (d, J = 7.8 Hz, 1H), 7.85 (d, J = 8.1 Hz, 1H), 7.62 (d, J = 7.5 Hz, 1H), 7.54 (dd, J = 14.3, 7.9 Hz, 2H), 7.47 (t, J = 7.2 Hz, 1H), 6.23 (s, 1H), 6.09 (s, 1H), 4.99 (s, 1H), 3.92 (t, J = 18.8 Hz, 2H), 3.79 (s, 3H), 3.12 (s, 3H), 1.42 (s, 9H); 13C NMR (150 MHz, CDCl3) δ 170.53, 168.0, 155.9, 148.0, 132.3, 128.1, 127.0, 126.0, 125.6, 124.0, 122.1, 118.8, 80.2, 61.9, 51.2, 47.4, 32.3, 28.5; MS (APCI+) (m/z) calcd for C16H19N7O2 [(M + H) − Boc]+ 341.1600, found 341.1319. 1-Pyrenetetrazolyl-5-amino Alanine (6J, PyTzAAla). Using the general procedure and starting from 0.30 mmol of PyTUAla (5J), the desired compound 6J was obtained as a semisolid compound (Rf = 0.4 in 1:2 hexane/ethyl acetate): yield 126 mg, 81%; IR (KBr, cm−1) υ 3417, 1707, 1644, 1607, 1462, 1390; 1H NMR (600 MHz, CDCl3) δ 8.31− 8.23 (m, 3H), 8.19 (dd, J = 11.0, 4.8 Hz, 2H), 8.14−8.05 (m, 2H), 8.00 (d, J = 7.9 Hz, 1H), 7.70 (d, J = 9.1 Hz, 1H), 5.56 (d, J = 6.4 Hz, 1H), 5.00 (s, 1H), 4.83 (s, 1H), 3.82 (s, 1H), 3.75 (s, 3H), 3.71 (s, 1H), 3.13 (s, 3H), 1.15 (s, 9H); 13C NMR (150 MHz, CDCl3) δ 170.0, 156.3, 155.9, 133.1, 131.1, 130.7, 130.3, 129.6, 127.4, 127.0, 126.8, 126.6, 125.4, 125.1, 124.6, 124.2, 121.0, 80.3, 62.0, 50.9, 46.9, 32.5, 28.2; HRMS (ESI+) (m/z) calcd for C27H30N7O4 [M + H]+ 516.2359, found 516.2350. 1-(p-Chlorophenyltetrazolyl)-5-amino Alanine (6L, ClBTzAAla). Using the general procedure and starting from 0.32 mmol of ClBTUAla (5L), the desired compound 6L was obtained as a semisolid compound (Rf = 0.4 in 1:2 hexane/ethyl acetate): yield 100 mg, 74%; IR (KBr, cm−1) υ 3334, 1709, 1659, 1608, 1499, 1251; 1H NMR (600 MHz, CDCl3) δ 7.51 (d, J = 8.4 Hz, 2H), 7.47 (d, J = 8.4 Hz, 2H), 5.75 (d, J = 6.3 Hz, 1H), 5.55 (s, 1H), 4.87 (s, 1H), 3.90−3.88 (m, 1H), 3.78 (s, 3H), 3.59−3.58 (m, 1H), 3.17 (s, 3H), 1.34 (s, 9H); 13C NMR (150 MHz, CDCl3) δ 169.7, 156.4, 154.4, 135.7, 131.9, 130.4, 125.3, 80.6, 62.0, 51.0, 47.8, 32.4, 28.3; HRMS (ESI+) (m/z) calcd for C17H25ClN7O4 [M + H]+ 426.1657, found 426.1657. 1-(p-Cyanophenyltetrazolyl)-5-amino Alanine (6M, CNBTzAAla). Using the general procedure and starting from 0.21 mmol of CNBTUAla (5M), the desired compound 6M was obtained as a semisolid compound (Rf = 0.4 in 1:2 hexane/ethyl acetate): yield 54 mg, 62%; IR (KBr, cm−1) υ 3378, 2232, 1708, 1659, 1603, 1568, 1459, 1251; 1H NMR (600 MHz, CDCl3) δ 7.85 (d, J = 8.2 Hz, 2H), 7.74 (d, J = 8.2 Hz, 2H), 5.97 (s, 1H), 5.82 (s, 1H), 4.88 (s, 1H), 3.94 (s, 1H), 3.79 (s, 3H), 3.58 (s, 1H), 3.20 (s, 3H), 1.34 (s, 9H); 13C NMR (150 MHz, CDCl3) δ 169.4, 156.7, 154.3, 137.1, 134.2, 123.9, 117.6, 113.2, 80.8, 62.0, 51.0, 48.2, 32.5, 28.3; HRMS (ESI+) (m/z) calcd for C18H25N8O4 [M + H]+ 417.1999, found 417.1995. 1-(p-Acetylphenyltetrazolyl)-5-amino Alanine (6N, AcBTzAAla). Using the general procedure and starting from 0.35 mmol of AcBTUAla (5N), the desired compound 6N was obtained as a greenish white semisolid compound (Rf = 0.4 in 1:2 hexane/ethyl acetate): yield 45 mg, 45%; IR (KBr, cm−1) υ 3377, 1687, 1657, 1602, 1574, 1463, 1266; 1H NMR (600 MHz, CDCl3) δ 8.15 (d, J = 8.5 Hz, 2H), 7.68 (d, J = 8.2 Hz, 2H), 5.73 (d, J = 5.8 Hz, 1H), 5.67 (s, 1H), 4.90 (s, 1H), 3.97−3.96 (m, 1H), 3.81 (s, 3H), 3.62 (d, J = 6.3 Hz, 1H), 3.22 (s, 3H), 2.66 (s, 3H), 1.36 (s, 9H); 13C NMR (150 MHz, CDCl3) δ 196.6, 169.5, 156.6, 154.5, 130.4, 123.4, 80.8, 62.1, 51.0, 48.2, 32.5, 28.4, 26.9; HRMS (ESI+) (m/z) calcd for C19H28N7O5 [M + H]+ 434.2152, found 434.2154. UV−Visible Measurements. All of the UV−visible spectra of the amino acids (10 μM) were measured in different solvents using a UV− visible spectrophotometer with a cell of 1 cm path length. The measurements were carried out in absorbance mode. The absorbance values of the sample solutions were measured in the wavelength regime of 200−550 nm. All of the sample solutions were prepared just before doing the experiment. Fluorescence Experiments. All of the sample solutions were prepared as described in UV measurement experiments. Fluorescence spectra were obtained using a fluorescence spectrophotometer at 25 °C 12283

DOI: 10.1021/acs.joc.7b02103 J. Org. Chem. 2017, 82, 12276−12285

Article

The Journal of Organic Chemistry

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using a 1 cm path length cell. The excitation wavelengths for all of the cases were set at the excitation maxima of each sample in each solvent, and the emission spectra were measured in the wavelength regime of 300−700 nm with an integration time of 0.2 s. All of the sample solutions were prepared just before doing the experiment. A total volume of 1.0 mL of a stock solution of 2 mL of 10 μM concentration for each case was used for the fluorescence experiment in a 1 mL cell. Fluorescence emissions were collected by exciting the samples at the wavelength corresponding to their absorption maxima. Steady-state fluorescence emission spectra were recorded at room temperature as an average of five scans using an excitation slit of 3.0 nm, emission slit 3.0 nm, and scan speed of 120 nm/min.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.joc.7b02103. NMR spectra (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Fax: +91-258-2349. Tel.: +91258-2324. ORCID

Subhendu Sekhar Bag: 0000-0001-5232-4793 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors thank the Department of Biotechnology (DBT: BT/PR16620/NER/95/223/2015), New Delhi, Government of India for the generous funding. S.D. thanks the IIT Guwahati for a fellowship. The CIF, IITG is gratefully acknowledged for the 600 MHz NMR facility.

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REFERENCES

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DOI: 10.1021/acs.joc.7b02103 J. Org. Chem. 2017, 82, 12276−12285

Article

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DOI: 10.1021/acs.joc.7b02103 J. Org. Chem. 2017, 82, 12276−12285