1,2-Dimethylindole-3-sulfonyl (MIS) as protecting group for the side chain of arginine

Albert Isidro a, Daniel Latassab, Matthieu Giraud b, Mercedes Álvarez*acd and Fernando Albericio*ace aInstitute for Research in Biomedicine, Barcelona Sc ien e Park, Baldiri Reixac 10, 08028 Barcelona, Spain. E-mail: albericio@irbbarcelona.org , mercedes.alvarez@irbbarcelona.org ; Fax: +3493 4037126; Tel: +3493 4037088  bLonza AG., TIDES, CH-3930, Visp, Switzerland  cCIBER-BBN, Networking Centre on Bioengineering, Bio materials and Nanomedicine, Barcelona Science Park, Baldiri Reixac 10, , 08028 Barcelona, Spain  dLaboratory of Organic Chemistry, Faculty of Pharmac y, University of Barcelona, 08028 Barcelona, Spain  eDepartment of Organic Chemistry, University of Barc elona, Martí i Franqués 1, 08028 Barcelona, Spain


Introduction
Most peptides synthesized on a solid-phase are prepared using the Fmoc/tert-butyl strategy. 1,2 Thus, -amino temporary protection is achieved with the base labile 9fluorenylmethoxycarbonyl (Fmoc) group; amino acid side chains are protected by trifluoroacetic acid (TFA)-labile protecting groups, usually t Bu derivatives; and the C-terminal amino acid is anchored to the solid support through a TFA-labile linker/handle. Nevertheless, tert-butyl-type protection of a number of amino acids is not the best option because of factors such as inefficiency in preventing side reactions or inadequate TFA lability. Among these amino acids, protection of the basic guanidinium group of Arginine (Arg) is possibly the most critical case. 3 Currently, the most frequently used TFA-labile Arg-protecting groups are based on electron-protectors. 17 Taking this into account, we chose 1,2-dimethylindole-3-sulfonyl (MIS) as a guanidinium-protecting group (Fig. 1). The extra methyl at position 2 should increase the acid lability of the protecting group and prevent electrophilic aromatic substitution. Furthermore, the 1,2-dimethylindole is commercially available. As the 1,2-dimethylindole is prone to polymerization in strong acidic conditions, sulfonation of the indole ring must be carried out in neutral or basic media. Thus, chlorosulfonic acid, which is the reagent of choice for Pmc and Pbf sulfonylation, cannot be used in the case of 1,2dimethylindole. Nevertheless, the use of sulfur trioxide pyridine complex yielded the corresponding pyridinium sulfonate in good yield. 18 Chlorination under mild conditions by treatment with oxalyl chloride yielded 1,2-dimethylindole-3-sulfonyl chloride (MIS-Cl). These conditions gave much better overall yield (80%) to those attained with Pbf and Pmc (51% and 53% respectively), 6,8 with the advantage that 1,2-dimethylindole is commercially available (Scheme 1).

Synthesis of multiple arginine-containing peptides using MIS and Pbf protection
We prepared Fmoc-Arg(MIS)-OH in a similar way to Pmc/Pbf derivatives, 6,8 using Z-Arg-OH as starting material. Z-Arg-OH was sulfonylated at the N position with MIS-Cl and the Z group was removed via catalytic hydrogenolysis. Final Fmoc protection was achieved by using Fmoc-2-mercaptobenzothiazole (Fmoc-2-MBT) because the use of other more active Fmoc derivatives leads to the formation of dipeptides or other side reactions. 19,20 As Pbf removal is more complicated in multiple Arg-containing peptides, Ac-Phe-Arg-Arg-Arg-Arg-Val-NH 2 was chosen as a model peptide to compare the acid lability of MIS and Pbf. 8,21 The corresponding Pbf-and MIS-protected peptides were prepared using standard solidphase peptide synthesis protocols on Sieber amide resin, which allows cleavage from the resin with small amounts of TFA (2%), thereby yielding the MIS-and Pbf-protected peptides respectively with excellent purity.

Optimization of the scavengers used in the removal
As MIS-OH is a polar compound, it precipitates during the ether treatment after the cleavage step. Alternative scavengers to H 2 O were tested to reduce the amounts of the strongly UV absorbant MIS-OH in order to facilitate purification. Among the scavengers tested, the optimum were 10% 3,4-dimethoxyphenol, 1,3,5-trimethoxybenzene (Tmb) or 3,5-dimethoxyphenol. The use of these scavengers reduced the amounts of MIS-OH more than 10 fold (40 times in the case of Tmb), thereby simplifying HPLC purification to yield the final product. To check the compatibility of the MIS group with Trp, 22 we first synthesized the model peptides Z-Arg(MIS)-Trp(Boc)-Ala-Gly-NH 2 and Z-Arg(Pbf)-Trp(Boc)-Ala-Gly-NH 2 on a Sieber amide resin, which were obtained with an excellent HPLC purity. Afterwards, both resins were treated with TFA-CH 2 Cl 2 -trimethoxybenzene (50 : 40 : 10) to compare the purities of Trp-containing peptides after MIS and Pbf removal. Trp alkylation or sulfonation was not detected in either case. The purity of the crude product was greater in the case of MIS and neither the MISprotected peptides nor MIS-OH were detected by LC-MS. Nevertheless, in the case of the Pbf experiment, considerable amounts of the Pbf-protected peptide were detected (34% compared to unprotected peptide, HPLC, = 220 nm).

Synthesis of Trp-containing peptides
In summary, MIS is the most acid-labile sulfonyl-type protecting group for Arg described to date. This feature makes it highly convenient for the synthesis of multiple Arg-containing peptides or peptides that contain acid-sensitive moieties. Furthermore, MIS is compatible with Trp-containing peptides.

Experimental section
Synthesis of the protecting group and Arginine protection Pyridinium 1,2-dimethylindole-3-sulfonate (1). 1,2-Dimethylindole (14.5 g, 99.8 mmol) and sulfur trioxide pyridine complex (19.1 g, 119.8 mmol) were dissolved in pyridine (70 mL) under an Ar atmosphere. The reaction mixture was refluxed for 1 h and HPLC indicated the reaction was complete (99.2% HPLC conversion, 254 nm). The reaction mixture was cooled to 60 °C and concentrated under vacuum to give a solid. The crude product was used directly for the next step. 23

1,2-Dimethylindole-3-sulfonyl chloride (MIS-Cl) (2).
All the crude product 1 obtained in the previous step was suspended in dry CH 2 Cl 2 (200 mL) under Ar atmosphere. The suspension was cooled in an ice bath and oxalyl chloride (20.0 g, 158 mmol) was slowly added. DMF (0.5 mL) was then slowly and carefully added and vigorous effervescence was observed. The reaction mixture was stirred in an ice bath for a further 30 min until the effervescence ceased and was then stirred at room temperature for 2 h. An aliquot (6 L) was then treated with MeOH for 20 min and injected into the HPLC apparatus, which showed the presence of methyl 1,2-dimethylindole-3-sulfonate and an absence of starting material. CH 2 Cl 2 (200 mL) was added to the reaction mixture and it was cooled to below 5 °C.

Z-L-Arg(MIS)-OH (3)
. Z-L-Arg-OH (2.05 g, 6.7 mmol) was suspended in 3 N aqueous NaOH (6.7 mL, 20 mmol) and acetone (13.3 mL) was added to dissolve the product. The reaction was cooled in an ice bath and 3 N aqueous NaOH (6.7 mL) and a solution of compound 2 (3.69 g, 14.7 mmol) in acetone (13.3 mL) were simultaneously added over 10 min. The reaction mixture was stirred at 0 °C for 2 h and at room temperature for a further 2 h. After that time, starting material 2 was no longer detected by TLC (hexane-EtOAc, 1 : 1). H 2 O (100 mL) was added and the suspension was washed with diethyl ether (3 × 80 mL). The aqueous phase was acidified to pH 2-3 by addition of 1 N HCl, the precipitate obtained was filtered, washed with acidic water (pH 2-3) and dried in vacuo. The crude product obtained was purified by column chromatography (CH 2 Cl 2 , MeOH, 1% HOAc). The solvent of the pure fractions was removed in vacuo to yield an oil. This process was repeated. Hexane and CH 2 Cl 2 were then sequentially added and a precipitate appeared on scratching. The solvent was decanted and the solid was washed 4 times with CH 2 Cl 2 -hexane (enough hexane to precipitate all the product) to remove HOAc and give 3 (0.70 g, 20.4% yield). 24

Fmoc-Arg(MIS)-OH (5).
H-Arg(MIS)-OH (250 mg, 0.658 mmol) was suspended in 1% aqueous Na 2 CO 3 (2 mL). 1,4-dioxane (2 mL) was added and the product was dissolved. The pH was basified to 9-10 with saturated aqueous Na 2 CO 3 (300 L in our case). Fmoc-2mercaptobenzotiazole (Fmoc-2-MBT) (256 mg, 0.658 mmol) in 1,4-dioxane (700 L) was slowly added. The pH was kept between 9 and 10 with saturated aqueous Na 2 CO 3 and the resulting suspension was stirred overnight. After 14 h of reaction, H 2 O (9 mL) was added, the pH was neutralized with 1 N HCl and the solution was washed with tert-butylmethyl ether (3 × 5 mL). The aqueous phase was acidified to pH 2-3 with 1 N HCl and extracted with EtOAc (3 × 7 mL). Note that to dissolve the precipitated product vigorous stirring is required. The organic phases were pooled, dried over dry MgSO 4 , filtered and evaporated to dryness, thereby yielding a solid. Various co-evaporations with CH 2 Cl 2 were performed to yield the desired product as a solid (207 mg, 52% yield). Mp = 137.  . Z-Arg(MIS)-OH (28.9 mg, 56 mol) was coupled using PyBOP (29.2 mg, 56 mol) HOAt (7.6 mg, 56 mol) and DIPEA (28.7 L, 168 mol) in DMF, t = 1.5 h. The resin was washed with DMF, CH 2 Cl 2 and diethyl ether, dried in vacuo and divided into 4 mg aliquots. One of them was swollen with CH 2 Cl 2 and treated with 1.5 mL of TFA-CH 2 Cl 2 -TIS-H 2 O (2 : 93 : 2.5 : 2.5) for 20 min in order to cleave the protected peptide from the resin. The resin was filtered and the collected solution was diluted with CH 2 Cl 2 and neutralised by adding DIPEA (80 L, 1.2 eq. per eq. of TFA). The solvent was then removed in vacuo, and H 2 O and AcCN were added and the solution was frozen and lyophilized. The product obtained was characterised by LC-MS (95% purity