Results and Discussion
Part 1. Preparation of Peptide Thioester Building Blocks Containing Tyr(SO3H) Residue(s) by the Direct Thioesterification on Peptide Acids, Followed by Global Deprotection of the Protecting GroupsVarious methods for the preparation of peptide thioesters with Fmoc-based chemistry have been reported to date.26–31) We chose Futaki’s procedure,32) in which critical thioesterification is carried out on a fully protected peptide acid obtained with Fmoc-SPPS, for its simplicity. Before starting the peptide synthesis, we examined the direct thioesterification of Fmoc-Tyr(SO3Na)-OH as a pilot experiment (Fig. 1). In Futaki’s protocol, carbodiimide-mediated thioesterification with ethyl 3-mercaptopropionate proceeded without any notable by-products on HPLC chromatogram. The product, which was isolated by HPLC in a preparative manner, coincided with the mass number of Fmoc-Tyr(SO3Na)-S(CH2)2COOC2H5, determined by Matrix Assisted Laser Desorption/Ionization-Time of Flight (MALDI-TOF) MS analysis. In addition, 1H- and 13C-NMR spectroscopy provided data supporting this structure. This result indicates that thioesterification can be achieved on the carboxyl functional group in the presence of sulfate, stabilized by a counter-ion. Therefore, we envisioned that peptide thioesters containing Tyr(SO3H) residue(s) could be prepared by combining Futaki’s protocol for thioesterification with our original strategy for the preparation of sulfopeptides, based on the global deprotection of protecting groups with TFA at low temperatures.
In this study, we chose the N-terminal region (Ala43 to Gly74) of P-selectin glycoprotein ligand 1 (PSGL-1) as a model peptide. Three Tyr residues, namely Tyr46, Tyr48, and Tyr51, in the N-terminal of this protein are sulfated. This Tyr(SO3H) cluster is reported to participate in the ligand–receptor interactions, cooperating with the sialic acid of carbohydrate antenna on the Thr57 residue.33) We designed 13-residue peptides corresponding to PSGL-1(43–55), as peptide thioester building blocks containing Tyr(SO3H) residue(s) (Table 1), and examined their applicability in the silver-ion mediated segment condensation.34–38)
Table 1. Amino Acid Sequence of Peptide Thioester Building Blocks
[Ia–c] and Amine-Building Block
[II]| Residue | Sequence |
|---|
| 43–55 | ATEY*EYLDYDFLP [Ia] |
| ATEY*EY*LDYDFLP [Ib] |
| ATEY*EY*LDY*DFLP [Ic] |
| 56–74 | ETEPPEMLRNSTDTTPLTG [II] |
2-Chlorotrityl chloride resin (Clt-resin)39–42) was used as a solid support because a protected peptide can be quantitatively detached from this resin under mildly acidic conditions. We selected Pro55 as a C-terminal residue to avoid racemization during carbodiimide-mediated thioesterification. A sterically hindered Clt-resin is also preferable for minimizing the premature detachment of X-Pro dipeptide from the resin through diketopiperazine formation at the dipeptide step.43–46)
Protected peptides containing Tyr(SO3H) residue(s) were constructed on the Fmoc-Pro-Clt-resin according to the general procedure for Fmoc-SPPS. As the amino groups must be protected during silver-ion mediated segment condensation, the Nα-Fmoc protecting group on the N-terminal residue was retained.
The synthetic scheme for peptide thioesters containing Tyr(SO3H) residue(s), ([Ia–c]), is summarized in Fig. 2(A). Herein, we describe the preparation of monosulfated peptide thioester [Ia] in detail. After a peptide-chain was constructed using Fmoc-SPPS, the protected peptide-resin was treated with a mixture of hexafluoro-2-propanol (HFIP) and CH2Cl2 (1 : 4 v/v)47) for 40 min to detach the fully protected peptide acid from the resin. Subsequent thioesterification of the protected peptide acid was conducted using excess ethyl 3-mercaptopropionate (25 equivalents (equiv.)) and water-soluble carbodiimide (WSCDI·HCl) (15 equiv.) in the presence of N-hydroxybenzotriazole (HOBt) (15 equiv.) in N,N-dimethylformamide (DMF). After stirring the reaction mixture for 17 h at 4 °C, the starting material was nearly depleted, as evidenced by TLC. After removing the solvent by evaporation in vacuo, the oily residue was triturated several times with ether and, washed with H2O. Repeated washing with H2O was effective in removing the excess reagents used in thioesterification. After drying in vacuo, the side-chain protecting groups of the product was deprotected using 90% aqueous TFA solution (0 °C, 4 h).2,3) The crude peptide thioester containing one Tyr(SO3H) residue (Fig. 2(B)) was purified by HPLC to afford a homogeneous Fmoc-protected peptide thioester [Ia] in 13% recovery yield on HPLC-purification. The intactness of the sulfate moiety was ascertained by the negative-ion mode MALDI-TOF MS.
Peptide thioesters containing multiple Tyr(SO3H) residues ([Ib] and [Ic]) were prepared in a manner similar to that described above. HPLC chromatograms of crude [Ib] and [Ic], obtained after thioesterification followed by deprotection of the protecting groups with TFA, were shown in Fig. 2(B). Several minor peaks observed in the HPLC chromatograms coincided with the partially or fully desulfated peptides produced in the random desulfation experiments described below. Thus, the homogeneous sulfated peptide thioesters, [Ib] and [Ic], were obtained in 11 and 14% yield, respectively, on HPLC purification. A part of this relatively low recovery on HPLC-purification may be attributed to the trace contamination of thiol compound used for thioesterification in TFA-treatment. We previously pointed out that the presence of thiol compounds in TFA-deprotection system significantly accelerated the desulfation from a Tyr(SO3H) residue.3)
Information obtained by negative-ion mode MALDI-TOF MS spectrum is useful in assessing the intactness of the multiple sulfate moieties in [Ib] and [Ic]. Molecular ions [M−H]−could not be detected, however, characteristic ions with mass difference of 80 Da (SO3), [M−H−nSO3]−, were detected on the mass spectrum.48) The presence of multiple sulfate moieties was also confirmed by desulfation experiment of the purified peptides using the diluted TFA solution. In the case of the trisulfated thioester [Ic], three peaks for randomly desulfated disulfates, three peaks for monosulfates, a fully desulfated peptide, and the starting trisulfated peptide thioester were detected on HPLC chromatogram.
As the facile method for the preparation of peptide thioesters containing Tyr(SO3H) residue(s) was established, we next examined silver-ion mediated segment condensation using [Ia–c] as building blocks. Silver-ion mediated thioester segment condensation is a promising approach for the chemical synthesis of large polypeptides and proteins, in which partially protected peptide thioesters serve as building blocks. This approach has been extended to the synthesis of modified peptides and proteins, such as phosphorylated36,38) and glycosylated peptides.36,37)
Silver-ion mediated segment condensation was conducted using the protocol established by Aimoto and colleagues34–38) (Fig. 3(A)). First, AgNO3 (3 equiv.), 3,4-dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazine (HOOBt, 30 equiv.), and N,N-diisopropylethylamine (DIPEA, 20 equiv.) were mixed in dimethylsulfoxide (DMSO) and stirred for 1 h at 25 °C under light-shielding. The thioester segment [Ia] and 19-residue amine-segment [II] were dissolved in DMSO at an equivalent ratio of 1 : 1 and added to the reaction mixture. The progress of the segment condensation was monitored by HPLC. After 20 h at 25 °C, the peaks for [Ia] and [II] disappeared. Two new peaks, 1 and 2 (Fig. 3(B)), were isolated by HPLC and subjected to MALDI-TOF MS analysis. The results showed that peak 1 was an objective condensation product [Ia–II], and peak 2 was a hydrolyzed product of the thioester moiety of [Ia]. This hydrolysis of the thioester moiety, induced by the strong activation of Ag+, is reported in several cases including our previous experiments involving the synthesis of CCK-58.5)
Despite significant efforts, such as employing moisture-free drying reaction conditions, we encountered challenges in effectively reducing the hydrolysis of the thioester moiety. To improve the yield of the condensation product, we envisioned that smooth consumption of the activated thioester segment [Ia] by an excess amount of the amine-segment [II] would reduce this by-product. As shown in Fig. 3(B), the amount of hydrolyzed by-product decreased depending on the concentration of [II]. Based on the peak area calculations in the HPLC chromatogram, peak 1, corresponding to the condensation product, reached 90.6% at a five-fold excess of [II] ([Ia] : [II] = 1 : 5), whereas it remained at 64.0% at a 1 : 1 ratio of the two segments. Similarly, peptide thioester building blocks containing multiple Tyr(SO3H) residues ([Ib] and [Ic]) were subjected to silver-ion mediated segment condensation with five-fold excess of [II]. In the condensation of [Ib] and [II], the target condensation product (peak 3 in Fig. 3(B)) reached 87.7%. In addition, in the cases of [Ic] and [II], the target condensation product (peak 5 in Fig. 3(B)) reached 84.2%. In both cases, for a 1 : 1 ratio between the two segments, the target condensation products remained at 59.9 and 52.1%.
Using Nα-Fmoc-protected peptide thioesters containing Tyr(SO3H) residue(s) as building blocks, 32-residue sulfopeptides were obtained via silver-ion mediated segment condensation. The hydrolyzed product of the peptide thioester was significantly reduced using an excess amount of the amine segment [II]. Considering the time-consuming and complicated manipulation required for the preparation of peptide thioester building blocks containing Tyr(SO3H) residue(s), this excess utilization of the amine segment, which can be prepared using conventional Fmoc-SPPS without any notable attentions, seems reasonable.
Part 2. Preparation of Peptide Thioester Building Blocks Containing Tyr(SO3H) Residue(s) via Peptide AzidesAnother method for the preparation of peptide thioester building blocks containing Tyr(SO3H) residue(s) involves the azide activation of peptide hydrazide followed by thioesterification, as reported by Liu and colleagues.49–53) This method needs no special devices or auxiliaries, and this method is compatible with our strategy for the synthesis of Tyr(SO3H)-containing peptides.1–3)
First, we examined the applicability of Liu’s method for the preparation of peptide thioesters containing Tyr(SO3H) residue(s), as illustrated in Fig. 4. Two Tyr(SO3H)-containing peptides were chosen as model peptides: a 13-residue peptide of PSGL-1(43–55) [Ia] and an 11-residue peptide with two Tyr(SO3H) residues, corresponding to the N-terminal extracellular amino acid sequence (positions 9 to 19) of CC-chemokine receptor 5 (CCR5) [III].54,55)
For the synthesis of the peptide hydrazide, we prepared a NH2NH-Trt(2-Cl)-resin (NH2NH-Clt-resin) from Clt-resin according to Liu’s reports.49,52) A 13-residue peptide with one Tyr(SO3H) residue [Ia] was constructed on this hydrazine-resin using the general procedure of Fmoc-SPPS. The Nα-Fmoc protecting group on the N-terminal residue was retained for comparison with the product prepared by Futaki’s method.
The fully protected peptide-resin was treated with a mixture of TFA : triisopropylsilane (TIPS) : H2O (95 : 2.5 : 2.5 v/v) for 4 h at 0 °C to deprotect the side-chain protecting groups and detach the Fmoc-protected peptide hydrazide from the resin. After purification of the crude product using HPLC, the peptide hydrazide was subjected to an azide-conversion reaction with NaNO2 (10 equiv.) in an acidic aqueous medium (pH 3.0) containing DMF as a cosolvent at −10 °C. Complete conversion to azide was confirmed by HPLC after 30 min. Subsequently, ethyl 3-mercaptopropionate (40 equiv.) was added to the reaction mixture, and the pH of the reaction mixture was adjusted to 7.0. After the reaction mixture was stirred for 1 h at 25 °C, thioesterification was confirmed to be completed by HPLC. HPLC chromatograms of the consecutive reactions from peptide hydrazide to peptide thioester are shown in Fig. 5. The target peptide thioester [Ia] was easily obtained by HPLC-purification. A MALDI-TOF MS analysis revealed the presence of both sulfate and thioester moieties in [Ia]. In addition, the tR of the product [Ia] on HPLC coincided with tR of the peptide thioester prepared in Part 1. This peptide thioester was used as a building block for silver-ion mediated segment condensation with an excess amount of H-PSGL-1(56–74)-OH [II]. The results of the segment condensation were quite similar to those described in Part 1.
Another 11-residue peptide thioester, H-[Tyr(SO3H)10,14]-CCR5(9–19)-S-C6H4-CH2COOH [III] was similarly prepared from the corresponding peptide hydrazide constructed on the NH2NH-Clt-resin. 4-Mercaptophenylacetic acid (MPAA) was used as the thiol for thioesterification instead of ethyl 3-mercaptopropionate, considering the application in NCL. Consecutive reactions with the purified peptide hydrazide proceeded smoothly, as shown in Fig. 5. The presence of two sulfate groups in [III] was confirmed by MALDI-TOF MS. Through these two experiments, acid-labile Tyr(SO3H) residue(s) were confirmed to remain perfectly intact in each reaction step, including the oxidation with NaNO2 in an acidic aqueous medium (pH 3.0). Thus this procedure becomes an attractive alternative for the preparation of peptide thioester building blocks containing Tyr(SO3H) residue(s).
The applicability of the Tyr(SO3H)-containing peptide thioester in NCL,56–60) as a building block, was confirmed by the preparation of H-[Tyr(SO3H)10,14, Ala20]-CCR5(9–26)-OH [V], in which the original Cys20 residue is replaced with Ala20 (Fig. 6(A)). Starting with the HPLC-purified peptide hydrazide, a peptide thioester containing two Tyr(SO3H) residues [III] was prepared and, without isolation, subjected to NCL with H-CCR5(20–26)-OH [IV] containing a Cys residue at the N-terminal position. NCL was conducted at pH 8.5 and 25 °C, in the presence of tris(2-carboxyethyl)phosphine (TCEP) (10 equiv.). After 24 h, the peak corresponding to [III] disappeared, and a new peak was observed as the sole product on HPLC. The MALDI-TOF MS analysis of this product coincided with the mass number of the ligation product H-[Tyr(SO3H)10,14]-CCR5(9–26)-OH [III–IV]. The peak area of the ligation product was noticeably smaller than that of the initial peptide thioester. One of the reasons of this observation may be attributable to the detection method used in the HPLC. The thioester building block, containing an aromatic ring, shows a significantly larger peak compared to the ligation product at 235 nm.
After purification by HPLC, the ligation product was desulfurized with VA-044 (40 equiv.) in the presence of TCEP (40 equiv.) and reduced glutathione (10 equiv.) at pH 7.0 and 40 °C according to the Danishefsky’s protocol.61) After 1 h, the conversion (Cys20 to Ala20) completed, yielding H-[Tyr(SO3H)10,14, Ala20]-CCR5(9–26)-OH [V] (Fig. 6(B)). The conversion of Cys to Ala and the presence of two sulfate moieties in [V] were confirmed by a MALDI-TOF MS analysis.
Payne and colleagues developed a method for the preparation of the Tyr(SO3H)-containing peptide thioesters using a neo-pentyl (Np) protecting group12,13) for sulfate functionality, and established an NCL-desulfurization protocol for the preparation of large sulfopeptides.20–25) Interestingly, the Np-protecting group for sulfate spontaneously degrades under NCL reaction conditions; therefore, partial or full deprotection of this protecting group was observed during repetitive ligations.22,23) The results from Payne’s group and our study indicate that the Tyr(SO3H) residue(s) are fully intact under NCL-desulfurization reaction conditions.
Experimental
GeneralReagents for peptide synthesis including Fmoc-amino acid derivatives and resins [p-benzyloxybenzyl alcohol resin (Wang resin) (substituted level, 0.70 mmol/g; 100–200 mesh), and Clt-resin (substituted level, 1.57 mmol/g; 100–200 mesh)] were purchased from Watanabe Chemical Co., Ltd. (Hiroshima, Japan). Other analytical-grade chemicals were purchased from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan) and were used without further purification. DMSO (>99% dehydrated grade for organic synthesis) was purchased from FUJIFILM Wako Pure Chemical Corporation and further dehydrated on molecular sieves (4A) before use. MALDI-TOF mass spectra were obtained using a Bruker Autoflex III TOF/TOF instrument. α-Cyano-4-hydroxy cinnamic acid (CHCA), sinapinic acid (SA), and 2,4,6-trihydroxyacetophenone (THAP) were used as matrices. 1H- and 13C-NMR data were recorded at 500 and 125 MHz, respectively, using a JEOL ECA-500 instrument (JEOL) with DMSO-d6 as a deuterated solvent and an internal standard. In 13C-NMR spectrum data, the numbers of hydrogen atoms attached to carbon atoms, known from DEPT135 and HMQC spectra, are shown as follows: s, primary carbons; d, secondary carbons; t, tercially carbons; q, quaternary carbons.
General Procedure for Fmoc-SPPSFmoc-SPPS was performed manually. The side-chain protecting groups used in the synthesis were as follows: tert-butyl (tBu) for Asp, Glu, Ser, Thr, and Tyr; tert-butoxycarbonyl (Boc) for Lys; 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl (Pbf) for Arg; trityl (Trt) for Asn, Gln, and Cys. The incorporation of the Tyr(SO3H) residue was achieved using home-made Fmoc-Tyr(SO3Na)-OH.3) Nα-Fmoc protecting groups were deprotected by stirring with 20% piperidine in DMF for 20 min. After the removal of the Fmoc-group, the peptide-resin was washed with DMF (× 6), and the next amino acid residue was incorporated using a benzotriazolyoxytris(pyrrolidino)phosphonium hexafluorophosphate (PyBOP)-mediated coupling protocol [Fmoc-amino acid (3 equiv.), PyBOP reagent (3 equiv.), N-methyl morpholine (NMM) (9 equiv.)] in DMF. After agitation for 1.5 h at 20 °C and washing with DMF (× 6), a small portion of the peptide-resin was subjected to the Kaiser test.62) Upon completion of the peptide-chain assembly, the peptide-resin was successively washed with DMF (× 5), MeOH (× 5), and ether (× 5) and then dried in vacuo.
RP-HPLCRP-HPLC was performed using a SHIMADZU SCL HPLC system. For monitoring the reaction progress, small scale purification of the crude peptides, and purity assessment of the purified peptides, a Cosmosil 5C18-AR-II column (4.6 × 250 mm) was used at a flow rate of 0.8 mL/min. For preparative-scale purification of crude peptides, a Cosmosil 5C18-AR-II column (10 × 250 mm) was used at a flow rate of 2 mL/min. A solvent system consisting of solvent A (0.1 M AcONH4) and solvent B (CH3CN) was used for elution of Tyr(SO3H)-containing peptides, and a solvent system consisting of solvent C (0.1% TFA in H2O) and solvent D (0.1% TFA in CH3CN) was used for elution of other peptides. Column temperature was kept at 40 °C during HPLC. The absorbance of the eluate was measured at 235 nm or 220 nm depending on peptide.
Thioesterification of Fmoc-Tyr(SO3Na)-OHFmoc-Tyr(SO3Na)-OH (101.6 mg, 0.2 mmol) was dissolved in DMF (4 mL) and cooled to ice-cold temperatures. To this solution, ethyl 3-mercaptopropionate (126.6 µL, 1.0 mmol), WSCDI·HCl (115.1 mg, 0.6 mmol), and HOBt (81.6 mg, 0.6 mmol) were added. After the reaction mixture was stirred for 24 h at 4 °C, DMF was removed by evaporation in vacuo. The oily residue was triturated several times with ether, and dissolved in a mixture of CH3CN and H2O (1 : 2 v/v, 6 mL). After lyophilization, the obtained crude product was purified by preparative HPLC [elution, a linear gradient of B in A (40 to 70% over 40 min)] to afford Fmoc-Tyr(SO3Na)-S-(CH2)2COOC2H5 as a white fluffy powder (71.0 mg; 57%). This sample showed a single peak (tR = 36.8 min) on an analytical HPLC chromatogram [elution, a linear gradient of B in A (20 to 60% over 40 min)]. MALDI-TOF MS: m/z Calcd for C29H28NO9S2Na 621.11 (M, monoisotopic). Found 622.1 [M + H]+, 542.2 [M + H−SO3]+. Found 620.1 [M−H]−, 540.1 [M−H−SO3]−. 1H-NMR δ: 1.12 (3H, t, J = 7.2 Hz, –OCH2CH3), 2.54 (2H, t, J = 6.9 Hz, -SCH2CH2COOC2H5), 2.75 (1H, dd, J = 11.0, 13.8 Hz, Tyr-Cβ-H), 2.97 (2H, dt, J = 2.3, 6.9 Hz, -SCH2CH2COOC2H5), 3.00 (1H, dd, J = 4.1, 13.8 Hz, Tyr-Cβ-H), 4.01 (2H, q, J = 7.2 Hz, -OCH2CH3), 4.16 (1H, dd, J = 7.2, 7.2 Hz, Fmoc-9C-H), 4.20 (1H, dd, J = 7.2, 10.3 Hz, fluorenyl-CH2O-), 4.22 (1H, ddd, J = 4.1, 8.3, 11.0 Hz, Tyr-Cα-H), 4.27 (1H, dd, J = 7.2, 10.3 Hz, fluorenyl-CH2O-), 7.04 (2H, d, J = 8.5 Hz, Tyr-arom-H), 7.13 (2H, d, J = 8.5 Hz, Tyr-arom-H), 7.27 (1H, ddd, J = 0.9, 7.5, 7.5 Hz, Fmoc-arom-H), 7.30 (1H, ddd, J = 0.9, 7.5, 7.5 Hz, Fmoc-arom-H), 7.37 (1H, dd, J = 7.5, 7.5 Hz, Fmoc-arom-H), 7.38 (1H, dd, J = 7.5, 7.5 Hz, Fmoc-arom-H), 7.63 (1H, d, J = 7.5 Hz, Fmoc-arom-H), 7.66 (1H, d, J = 7.5 Hz, Fmoc-arom-H), 7.848 (1H, dd, J = 0.9, 7.5 Hz, Fmoc-arom-H), 7.850 (1H, dd, J = 0.9, 7.5 Hz, Fmoc-arom-H), 8.18 (1H, d, J = 8.3 Hz, NH). 13C-NMR δ: 15.0 (s, -OCH2CH3), 24.4 (d, -SCH2CH2COOC2H5), 34.5 (d, -SCH2CH2COOC2H5), 36.6 (d, Tyr-Cβ), 47.5 (t, Fmoc-9C), 61.2 (d, -OCH2CH3), 63.8 (t, Tyr-Cα), 66.8 (d, fluorenyl-CH2O-), 121.0 (t, 2C, Fmoc-arom-C), 121.2 (t, 2C, Tyr-arom-C), 126.2 (t, 2C, Fmoc-arom-C), 128.0 (t, 2C, Fmoc-arom-C), 128.6 (t, 2C, Fmoc-arom-C), 130.4 (t, 2C, Tyr-arom-C), 132.7 (q, Tyr-arom-C), 141.7 (q, 2C, Fmoc-arom-C), 144.6 (q, Fmoc-arom-C), 144.7 (q, Fmoc-arom-C), 153.2 (q, Tyr-arom-C), 156.8 (q, -NHCOO-), 172.1 (q, -SCH2CH2COOC2H5), 202.3 (q, Tyr-COS-).
Fmoc-[Tyr(SO3H)46]-PSGL-1(43–55)-S(CH2)2COOEt [Ia]Starting with Clt-resin (91 mg, 0.143 mmol), the peptide-chain was constructed with Fmoc-SPPS. The fully protected peptide-resin (50.1 mg) was treated with a mixture of HFIP and CH2Cl2 (1 : 4 v/v, 3 mL) at 20 °C for 40 min and filtered. The filtrate was concentrated using a N2 stream, and ice-cold ether (50 mL) was added to precipitate the fully protected peptide acid. After centrifugation, the precipitate was dried in vacuo. The obtained fully protected peptide acid (31.6 mg, 13.5 µmol) was dissolved in ice-cold DMF (2.6 mL). Then, HS-(CH2)2COOEt (42.9 µL, 0.34 mmol), WSCDI·HCl (39.0 mg, 0.20 mmol), and HOBt (27.5 mg, 0.20 mmol) were added. After the reaction mixture was stirred at 4 °C for 17 h, the solution was concentrated under reduced pressure. The resulting oily residue was triturated with ether (× 5) and H2O (× 5) to remove excess reagents and then dried in vacuo (30.8 mg, 93% from the protected peptide acid). The dried powder was then treated with ice-cold 90% aqueous TFA (3 mL) at 0 °C for 4 h. Subsequently, ether (50 mL) was added. The formed precipitate was collected by centrifugation and washed with ether (× 3). This product was dissolved in a mixture of 0.1 M AcONH4 (10 mL) and CH3CN (5 mL), and lyophilized to obtain the crude peptide thioester (24.0 mg, 86% from the protected peptide acid). A portion (10.0 mg) of the crude peptide thioester was purified by preparative HPLC, and pooled main peak fractions were lyophilized to yield the homogeneous peptide thioester [Ia] (1.32 mg, 13% in the purification step). This purified peptide thioester exhibited a sharp single peak at tR = 24.2 min on an analytical HPLC chromatogram [elution, a linear gradient of B in A (30 to 45% over 40 min)]. MALDI-TOF MS: m/z Calcd for C98H121N13O32S2 2057.2 (M, average). Found 2056.9 [M − H]−, 1977.5 [M−H−SO3]−.
Fmoc-[Tyr(SO3H)46,48]-PSGL-1(43–55)-S(CH2)2COOEt [Ib]After a peptide-chain was constructed with Fmoc-SPPS, the fully protected peptide resin (50.3 mg) was processed in a manner similar to that described for [Ia]. Using the protected peptide acid (34.4 mg, 14.6 µmol), thioesterification and subsequent deprotection with 90% TFA afforded the crude peptide thioester (26.8 mg, 86% from the protected peptide acid). A portion (10.0 mg) of the crude peptide thioester was purified by HPLC to yield the homogeneous peptide thioester [Ib] (1.13 mg, 11% in the purification step). The HPLC-purified peptide thioester exhibited a sharp single peak at tR = 19.8 min on an analytical HPLC chromatogram [elution, a linear gradient of B in A (30 to 45% over 40 min)]. MALDI-TOF MS: m/z Calcd for C98H121N13O35S3 2137.3 (M, average). Found 2056.3 [M − H−SO3]−, 1976.8 [M − H−2SO3]−.
Fmoc-[Tyr(SO3H)46,48,51]-PSGL-1(43–55)-S(CH2)2COOEt [Ic]After a peptide-chain was constructed with Fmoc-SPPS, the fully protected peptide resin (198.6 mg) was processed to afford the crude protected peptide acid (121.3 mg). Using the crude protected peptide acid (35.8 mg, 15.0 µmol), thioesterification and subsequent deprotection with 90% TFA were conducted to afford the crude peptide thioester (32.7 mg, 98% from the protected peptide acid). A portion (10.0 mg) of the crude peptide thioester was purified by HPLC to yield the homogeneous peptide thioester [Ic] (1.41 mg, 14% in the purification step). The HPLC-purified peptide thioester exhibited a sharp single peak (tR = 14.3 min) on an analytical HPLC chromatogram [elution, linear gradient of B in A (30 to 45% over 40 min)]. MALDI-TOF MS: m/z Calcd for C98H121N13O38S4 2217.3 (M, average). Found 2056.3 [M − H−2SO3]−, 1976.4 [M − H−3SO3]−.
For further confirmation of tri-sulfated peptide, the purified product (0.4 mg) was treated with 10% TFA in CH3CN/H2O (1 : 4 v/v) (0.4 mL) at 40 °C for 2 h, then the solution was lyophilized. HPLC analysis revealed three peaks corresponding to randomly disulfated peptides, three peaks corresponding to monosulfated peptides, and one peak corresponding to the non-sulfated peptide, along with a peak corresponding to the starting trisulfated peptide.
H-PSGL-1(56–74)-OH [II]The title peptide was prepared on Wang resin (250 mg, 0.175 mmol) according to a general Fmoc-SPPS protocol. After assembly of the 19-mer peptide-chain, the peptide-resin (100 mg) was treated with a deprotection reagent [TFA (2.85 mL), thioanisole (0.45 mL), trimethylsilyl bromide (0.5 mL), and m-cresol (0.1 mL)] for 2 h under ice-cooling. The obtained crude peptide was purified using HPLC in a preparative manner (21% in the purification step). MALDI-TOF MS: m/z Calcd for C86H141N23O35S 2088.0 (M, monoisotopic). Found 2089.0 [M + H]+.
Fmoc-[Tyr(SO3H)46]-PSGL-1(43–74)-OH [Ia–II]AgNO3 (0.49 mg, 2.88 µmol), HOOBt (4.75 mg, 29.1 µmol), and DIPEA (3.3 µL, 19.4 µmol) were dissolved in DMSO (110 µL) and stirred at 25 °C for 1 h. Then, a DMSO solution (200 µL) containing both the thioester segment [Ia] (2.0 mg, 0.97 µmol) and segment [II] (2.0 mg, 0.96 µmol) was added. After the mixture was stirred at 25 °C for 20 h, the formed insoluble material was removed by centrifugation. For isolation of the condensation product [Ia–II], portions of the supernatant were subjected to HPLC [column, Cosmosil 5C18-AR-II (4.6 × 250 mm); elution, a linear gradient of B in A (10 to 70% over 30 min)]. The eluate corresponding to the main peak (peak 1) (tR = 17.9 min) was pooled and lyophilized to give the objective condensation product [Ia–II] (1.37 mg, 36% isolation yield). MALDI-TOF MS: m/z Calcd for C179H252N36O65S2 4012.2 (M, average). Found 3932.1 [M − H−SO3]−. In addition, the eluate of the side peak (peak 2) (tR = 20.1 min) was found to be a hydrolyzed product of segment [Ia], Fmoc-[Tyr(SO3H)46]-PSGL-1(43–55)-OH. MALDI-TOF MS: m/z Calcd for C93H113N13O31S 1941.0 (M, average). Found 1939.4 [M−H]−, 1860.0 [M−H−SO3]−.
To improve the yield of the condensation product, the amount of [II] was increased to 2, 3, and 5 equiv. excess compared to the amount of [Ia]. The HPLC profiles of the segment condensation reactions after 20 h are shown in Fig. 3(B). Based on the peak area calculations of HPLC-chromatograms, the yield of the condensation product (peak 1) increased from 64% (1 : 1 ratio) to 91% (1 : 5 ratio).
Fmoc-[Tyr(SO3H)46,48]-PSGL-1(43–74)-OH [Ib–II]Silver-ion mediated thioester segment condensation was conducted in a manner similar to that described for [Ia–II]. The amounts of two segments were as follows: [Ib] (2.0 mg, 0.94 µmol) and [II] (9.80 mg, 4.70 µmol). Isolation of the condensation product was conducted using HPLC, as described for [Ia–II]. The eluate corresponding to the main peak (peak 3) (tR = 17.3 min) was pooled and lyophilized to give the desired condensation product [Ib–II] (3.31 mg, 86% isolation yield). MALDI-TOF MS: m/z Calcd for C179H252N36O68S3 4092.3 (M, average). Found 4090.3 [M − H]−, 4011.5 [M − H−SO3]−, 3931.5 [M − H−2SO3]−.When segment condensation was conducted using a 1 : 1 ratio of the two segments, a significant amount of the hydrolyzed product of [Ib] (peak 4) was observed. Based on the peak area calculations using HPLC-chromatograms, the yield of the condensation product increased from 60% (1 : 1 ratio) to 88% (1 : 5 ratio).
Fmoc-[Tyr(SO3H)46,48,51]-PSGL-1(43–74)-OH [Ic–II]Silver-ion mediated thioester segment condensation was conducted in a manner similar to that described for [Ia–II]. The amounts of two segments were as follows: segment [Ic] (2.0 mg, 0.90 µmol) and amine-segment [II] (9.40 mg, 4.50 µmol). The condensation product was isolated using HPLC, as described for [Ia–II]. The eluate corresponding to the main peak (peak 5) (tR = 15.9 min) was pooled and lyophilized to give the desired condensation product [Ic–II] (1.84 mg, 49% isolation yield). MALDI-TOF MS: m/z Calcd for C179H252N36O71S4 4172.4 (M, average). Found 4090.9 [M − H−SO3]−, 4010.6 [M − H−2SO3]−. When segment condensation was conducted using a 1 : 1 ratio of the two segments, a significant amount of the hydrolyzed product of [Ic] (peak 6) was observed. Based on the peak area calculations using HPLC-chromatograms, the yield of the condensation product increased from 52% (1 : 1 ratio) to 84% (1 : 5 ratio).
Reagents for the Preparation of Peptide Thioester, NCL, and DesulfurizationFor preparation of the peptide thioester from the corresponding peptide hydrazide and for NCL-desulfurization reaction, following reagent solutions were prepared: 200 mM NaNO2 solution, NaNO2 (14 mg, 0.2 mmol) in H2O (1.0 mL); 200 mM thiol solution, ethyl 3-mercaptopropionate (25.3 µL, 0.2 mmol) in a mixture of 0.2 M Na2HPO4 buffer (phosphate buffer) (pH 7.0) and DMF (4 : 1 v/v, 0.98 mL)) or MPAA (16.8 mg, 0.1 mmol) in 0.2 M phosphate buffer (pH 7.0) containing 6M guanidine hydrochloride (0.5 mL); 500 mM TCEP·HCl solution, TCEP·HCl (28.7 mg, 0.1 mmol) in H2O (0.2 mL); 40 mM reduced glutathione solution, reduced glutathione (2.46 mg, 8 µmol) in 0.2 M phosphate buffer (pH 7.0) containing 6M guanidine hydrochloride (0.2 mL); 200 mM VA-044 solution, VA-044 (12.9 mg, 40 µmol) in H2O (0.2 mL).
Fmoc-[Tyr(SO3H)46]-PSGL-1(43–55)-S-(CH2)2COOC2H5 [Ia]Starting with the Clt-resin (255 mg, 0.4 mmol), Fmoc-Pro-NHNH-Clt-resin was prepared according to the Liu’s procedure.52) Lording of the Fmoc-Pro-OH was estimated to be 0.06 mmol by quantitative measurement of the released Fmoc moiety from the resin with 20% piperidine/DMF. Using this Fmoc-Pro-NHNH-Clt-resin, a peptide-chain was constructed with Fmoc-SPPS. The dried protected peptide resin (100 mg) was treated with a mixture of TFA/triisopropylsilane (TIPS)/H2O (95 : 2.5 : 2.5 v/v, 2 mL) for 4 h at 0 °C. Then, ice-cold ether was added to obtain a precipitate. This precipitate was collected by centrifugation, and washed with ice-cold ether (× 3). The residue was dissolved in a mixture of 0.02M NH4HCO3 (4 mL) and CH3CN (2 mL), and the resin was removed by filtration. The filtrate was lyophilized to afford the crude peptide hydrazide (30.1 mg). A portion (10 mg) of this crude product was purified using HPLC [column, Cosmosil 5C18-AR-II (10 × 250 mm); elution, a linear gradient of B in A (27 to 32% over 40 min)] to give the purified Fmoc-[Tyr(SO3H)46]-PSGL-1(43–55)-NHNH2 (4.31 mg, 43% yield in the purification step). MALDI-TOF MS: m/z Calcd for C93H115N15O30S 1953.76 (M, monoisotopic). Found 1896.77 [M + Na−SO3]+, 1912.81 [M + K−SO3]+. Found 1952.76 [M − H]−, 1872.77 [M − H−SO3]−.
The purified peptide hydrazide (2.02 mg, 1.03 µmol) was dissolved in a mixture of 0.2 M phosphate buffer (pH 2.5, 0.6 mL) and DMF (0.25 mL) and, then cooled to −10 °C. A 200 mM NaNO2 solution (50 µL, 10 µmol) was added to this solution and stirred for 30 min at −10 °C. The complete conversion of peptide hydrazide (tR = 14.4 min) to peptide azide (tR = 18.9 min) was confirmed by HPLC [column, Cosmosil 5C18-AR-II (4.6 × 250 mm); elution, a linear gradient of B in A (25 to 65% over 40 min)]. Subsequently, a 200 mM HS(CH2)2COOC2H5 solution (200 µL, 40 µmol) was added dropwise, and the pH of the solution was adjusted to 7.0 with 2M NaOH. The reaction mixture was further stirred at 25 °C. After 1 h, a peak corresponding to the peptide-azide fully converted to a new peak (tR = 21.4 min) on HPLC [elution, a linear gradient of B in A (25 to 65% over 40 min)]. The crude product was directly purified using HPLC under the same HPLC conditions to afford the purified peptide thioester [Ia] (1.57 mg, 74% based on peptide hydrazide). MALDI-TOF MS: m/z Calcd for C98H121N13O32S2 2055.77 (M, monoisotopic). Found 1998.81 [M + Na−SO3]+, 2014.83 [M + K−SO3]+. Found 2054.77 [M − H]−, 1974.79 [M − H−SO3]−.
H-[Tyr(SO3H)10,14]-CCR5(9–19)-S-C6H4-CH2COOH [III]Fmoc-Pro-NHNH-Clt-resin was newly prepared from Clt-resin (255 mg, 0.4 mmol), and lording of the Fmoc-Pro-OH was estimated to be 0.14 mmol. Starting with this Fmoc-Pro-NHNH-Clt-resin, a peptide sequence was constructed with Fmoc-SPPS. The protected peptide resin (100 mg) was treated with a mixture of TFA/TIPS/H2O (95 : 2.5 : 2.5 v/v, 2 mL) for 4 h at 0 °C. The crude peptide hydrazide was obtained in a manner similar to that described for [Ia] prepared by this method: 40.9 mg. A portion (20 mg) of the crude peptide hydrazide was purified using HPLC [column, Cosmosil 5C18-AR-II (10 × 250 mm); elution, a linear gradient of B in A (14 to 19% over 40 min)] to obtain purified H-[Tyr(SO3H)10,14]-CCR5(9–19)-NHNH2 (6.53 mg, 33% in the purification step). MALDI-TOF MS: m/z Calcd for C64H90N14O27S2 1550.55 (M, monoisotopic). Found 1391.67 [M + H−2SO3]+, 1413.67 [M + Na−2SO3]+, 1429.66 [M + K−2SO3]+. Found 1469.63 [M − H−SO3]−, 1389.65 [M − H−2SO3]−.
The purified peptide hydrazide (1.58 mg, 1.0 µmol) was dissolved in 0.2 M phosphate buffer (pH 3.0) containing 6M guanidine hydrochloride (0.75 mL) and cooled to −10 °C. A 200 mM NaNO2 solution (50 µL, 10 µmol) was added to this solution and stirred for 30 min at −10 °C. Conversion of peptide hydrazide (tR = 20.7 min) to peptide azide (tR = 25.1 min) was confirmed on HPLC [elution, a linear gradient of B in A (10 to 30% over 40 min)]. Then, a 200 mM MPAA solution (200 µL, 40 µmol) was added dropwise, and the pH of the solution was adjusted to 7.0 using 2M NaOH. The reaction mixture was further stirred at 20 °C. After 1 h, the peak corresponding to the peptide-azide fully converted to a new peak (tR = 24.2 min) on HPLC [elution, a linear gradient of B in A (10 to 30% over 40 min)]. The crude product was directly purified using HPLC under the same HPLC conditions to obtain the purified peptide thioester [III] (1.28 mg, 75% based on peptide hydrazide). MALDI-TOF MS: m/z Calcd for C72H94N12O29S3 1686.54 (M, monoisotopic). Found 1549.63 [M + Na−2SO3]+. Found 1605.59 [M − H−SO3]−, 1525.60 [M − H−2SO3]−.
H-CCR5(20–26)-OH [IV]The title peptide was prepared on Fmoc-Lys(Boc)-Clt-resin (0.20 mmol) according to a general Fmoc-SPPS protocol. The fully protected peptide-resin was treated with TFA-based deprotection reagent [TFA/ethanedithiol/ H2O/TIPS (94 : 2.5 : 2.5 : 1 v/v)], and the obtained crude peptide was purified by HPLC in a preparative manner (79% in the purification step). MALDI-TOF MS: m/z Calcd for C35H65N11O10S 831.46 (M, monoisotopic). Found 832.50 [M + H]+, 854.50 [M + Na]+.
One-Pot NCL for H-[Tyr(SO3H)10,14, Cys20]-CCR5(9–26)-OH [III–IV]Starting with the purified peptide hydrazide (1.55 mg, 1.0 µmol), azide conversion and subsequent thioesterification were conducted in a manner similar to that described for [III]. Thereafter, NCL reaction was conducted without isolation of [III]. H-CCR5(20–26)-OH [IV] (1.66 mg, 2.0 µmol) dissolved in 0.2 M phosphate buffer (pH 7.0) containing 6M guanidine hydrochloride (80 µL) and 500 mM TCEP·HCl solution (20 µL, 10 µmol) were added to the reaction mixture. The pH of reaction solution was adjusted to 8.5 using 2M NaOH. Subsequently, the reaction mixture was agitated at 25 °C. After 24 h, the peak corresponding to the peptide thioester [III] (tR = 23.5 min) disappeared and a new peak (tR = 26.8 min) was detected on HPLC [elution, a linear gradient of B in A (10 to 30% over 40 min)]. This product was directly purified by HPLC under the same HPLC conditions to afford the ligation product [III–IV] (0.71 mg, 30% based on peptide hydrazide). MALDI-TOF MS: m/z Calcd for C99H151N23O37S3 2349.98 (M, monoisotopic) and 2351.59 (M, average). Found (monoisotopic) 2191.08 [M + H−2SO3]+, 2213.09 [M + Na−2SO3]+. Found (average) 2350.58 [M − H]−, 2270.52 [M − H−SO3]−, 2190.69 [M − H−2SO3]−.
H-[Tyr(SO3H)10,14, Ala20]-CCR5(9–26)-OH [V]Purified [III–IV] (0.59 mg, 0.25 µmol) was dissolved in 0.2 M phosphate buffer (pH 7.0) containing 6M guanidine hydrochloride (100 µL). Then, 500 mM TCEP·HCl (20 µL, 10 µmol), 40 mM reduced glutathione (62.5 µL, 2.5 µmol), and 200 mM VA-044 (50 µL, 10 µmol) solutions were added. The pH of this reaction solution was adjusted to 7.0 using 2M NaOH, and the reaction mixture was agitated at 40 °C. After 1 h, the peak (tR = 25.9 min) corresponding to [III–IV] disappeared and a new peak (tR = 25.1 min) was detected by HPLC [elution, a linear gradient of B in A (10 to 30% over 40 min)]. After collecting this new peak by HPLC under the same HPLC conditions, it was confirmed to be H-[Tyr(SO3H)10,14, Ala20]-CCR5(9–26)-OH [V] by MALDI-TOF MS analysis. MALDI-TOF MS: m/z Calcd for C99H151N23O37S2 2318.01 (M, monoisotopic) and 2319.52 (M, average). Found (monoisotopic) 2159.14 [M + H−2SO3]+. Found (average) 2238.66 [M − H−SO3]−, 2159.23 [M − H−2SO3]−.
