The 2-(Triphenylsilyl)ethoxycarbonyl-(“Tpseoc”-) Group: A New Silicon-Based, Fluoride Cleavable Oxycarbonyl Protecting Group Highly Orthogonal to the Boc-, Fmoc- and Cbz-Groups

Starting from 2-(triphenylsilyl)ethanol a new oxycarbonyl protecting group cleavable by fluoride ion induced Peterson-elimination has been developed. Known 2-(triphenylsilyl)ethanol has been prepared from commercially available triphenylvinyl-silane by a hydroboration-oxidation sequence using the sterically hindered borane reagent 9-BBN. The silyl alcohol was subsequently transformed into its chloroformate, imidazolylcarboxylic acid ester and p-nitrophenyl carbonate and used in standard protocols for the formation of carbamates and carbonates. The Tpseoc group proved to be highly resistant against acidic conditions applied in removal of tert-butyl esters and the t-Boc-group. It also withstood catalytic hydrogenation, treatment with morpholine, methylhydrazine and Pd-reagents/allyl-scavanger combinations, conditions required to cleave Cbz-, Fmoc-, phthalimide- and Alloc-groups. The Tpseoc-group is cleaved upon treatment with TBAF/CsF at 0 °C or r.t. with cleavage times reaching from <10 min. to 24 h. Its orthogonality, ease of cleavage and UV-detectability makes the Tpseoc-group a promising alternative to other widely used silicon based amine protecting groups like the Teoc- and SES-groups.


Introduction
Masking potentially reactive sites of a polyfunctionalized organic molecule with appropriate protecting groups is a fundamental process in modern synthetic organic chemistry. Regardless of whether the target of a synthesis is a complex secondary metabolite, a protein, oligonucleotide or saccharide, they all contain functional groups like carbonyl moieties, carboxylic acid groups, alcohols, amines, among others, which might interfere with a process that converts another functional group in the desired manner. In order to navigate through the networks of reactivity in complex organic molecules a tremendous variety of protecting groups has been developed in the past and the reports of an ongoing search for more elaborated protecting strategies fill the pages of ever growing comprehensive works on the topic [1]. The field of peptide synthesis delivers illuminating insight into protecting group chemistry, as most of the functional groups mentioned above appear in this substance class. Especially protection of the amine moiety in amino acids underwent a constant evolution from simple amides to more sophisticated carbamates finally resulting in the triumvirate of amine protecting groups consisting of the Boc- [2], Cbz- [3] and Fmoc-group [4]. Those three justify their superiority by the ease of installation, mild cleavage conditions, the excellent orthogonality among each other and not the least, the vast experience gained by the community of synthetic organic chemists since their introduction in the 1930s and 70s. During our investigation of the synthesis of glycopeptide mimetics we were prospecting for an amine protecting group which should be specifically cleavable by fluoride ions under mild conditions, but at the same time resistant enough to survive the acidic conditions applied to cleave the Boc-group. Thorough examination of the literature revealed that there are only few silicon based amine protecting groups cleavable by fluoride ions with almost none of them matching our demands. By far the most popular among them is the Teoc-group [5], based on the 2-(trimethylsilyl)ethyl-(Tmse-) moiety first described in the context of a protecting-strategy for peptide synthesis by Sieber [6]. The Teoc-group is unfortunately prone to acidolysis and is not orthogonal to the Boc-group. Another frequently used amine protecting group is the sulfonamide based SES-group [7] which in turn suffers from the necessity of somewhat harsh cleavage conditions. Two at first glance very useful protecting groups appeared to be the diphenylsilyldiethylene-(DPside-) group [8] and the triisopropylsilyloxycarbonyl-(Tsoc-) group [9]. The former is introduced into molecules bearing an amine moiety via a nucleophilic substitution reaction of bis[2-(p-toluenesulfonyloxy)ethyl]diphenylsilane resulting in the formation of a 1-aza-4-silacyclohexane-derivative. Although possessing advantageous orthogonality to a variety of other amine protecting groups, the fact that it retains the basic character of the amine moiety and its limitation to sterically unhindered primary amines limit the applicability of the DPside-group. The above mentioned Tsoc-group seems so far to be the most attractive option, as it can be attached to relatively electron poor and sterically hindered primary and secondary amines. It is orthogonal to the Fmoc-, Cbz-and Boc-group and cleavage kinetics are very promising. One flaw might be that Tsoc-group can't be attached to very electron poor amines or alcohols. In addition the procedure involved in the installation of the Tsoc-group turns out to be somewhat laborious compared to the Teoc-and SES-group, where storable activated formate reagents are used in standard protocols for their introduction. In the course of our investigations we finally stumbled across 2-(triphenylsilyl)ethanol, which was used previously as a phosphate-ester protecting group in oligonucleotide synthesis [10]. The increased electronegativity of the triphenylsilyl-moiety lead to the assumption that the silicons β-effect might be sufficiently diminished to prevent the alcohol and its derivatives from undergoing acid induced Peterson-elimination [11] as observed in Teoc-derivatives. At the same time we expected superior liability to elimination induced by attack of a nucleophilic species like fluoride or hydroxyl ions at silicon due to its increased electrophilicity. Despite the estimated favorable properties of the 2-(triphenylsilyl)ethyl moiety mentioned, we found no further evidence in the literature of its use in any kind of protecting strategy, leading to the opinion that an investigation of an amine protecting group based on the oxycarbonyl derivative of 2-(triphenylsilyl)ethanol might turn out as a promising endeavor.

Results and Discussion
2-(Triphenylsilyl)ethanol (2) was previously synthesized in only moderate yields (25-30%) by hydrosilylation of vinyl acetate with triphenylsilane employing dichlorodirhodium tetracarbonyl as the catalyst [10] or, much more efficiently, by treatment of ethylene oxide with triphenylsilyl lithium [12]. Starting from commercially available triphenylvinylsilane (1) we chose instead to use a straightforward hydroboration-oxidation sequence to synthesize silyl alcohol 2 employing sterically hindered borane 9-BBN and, in regard to the expected susceptibility to elimination, the mild oxidant NaBO 3 •4H 2 O yielding alcohol 2 in an excellent yield of 88%. Borane-THF complex was also tested in the hydroboration step resulting in formation of a mixture of regioisomeric 1-(triphenylsilyl)ethanol and 2 in a ratio of 2:3 (unpublished results). This finding does not differ significantly from the regioisomer distribution observed in the hydroboration/oxidation of trimethylvinylsilane with BH 3 •THF and 9-BBN reported by Brown et al. [13]. Treatment of β-silyl alcohol 2 with p-nitrophenyl chloroformate, carbonyldiimidazole (CDI) [14] or phosgene [15], respectively resulted in a clean conversion of 2 to the corresponding p-nitrophenyl-2-(triphenylsilyl)ethyl carbonate (3), 1H-imidazole-1-carboxylic acid 2-(triphenylsilyl)ethyl ester (4) and 2-(triphenylsilyl)ethyl chloroformate (5) in yields of 88%, 88% and 94%, respectively (Scheme 1). The two former compounds 3 and 4 are crystalline, shelf stable solids and showed no sign of decomposition, as indicated by TLC, even after several weeks of storage at room temperature. Chloroformate 5 was obtained as crystalline solid by crystallization from dry n-hexane and retained its reactivity over a period of at least two months of storage in the refrigerator at −20 °C under an atmosphere of nitrogen. With the appropriate tools in hand the next step in our investigations was planned to be the Tpseoc-protection of a series of different L-leucine esters and L-proline tert-butyl ester, derivatives 11-15 (Scheme 2), and a series of variably protected 1,6-diaminohexane-derivatives, compounds 21-25 (Scheme 3). The Tpseoc-derivatives were synthesized according to general procedure A by reacting the corresponding ammonium derivatives of the amino acids 6-10 (Scheme 2) and diamines 16-20 ( The yields of Tpseoc-protected amino acid esters 11-15 and bisprotected diamines 21-24 obtained by this method were in the range of 99-87% (Table 1). Due to its base-sensitive nature, the Fmoc-protected diamine 25 was synthesized by treatment of the TFA-salt of Fmoc-1,6-diaminohexane 20 with chloroformate 5 and Hünig`s base in DCM for 3 h in a yield of 83%. On the next stage of our investigations, compounds 11-15 and 21-25 were designated to be tested for the stability of the Tpseoc-group under conditions necessary to cleave the ester function or the second amino protecting group in a competitive manner. First the Tpseoc-protected leucine and proline tert-butyl esters 11 and 12 were treated with 50% TFA in DCM, conditions usually applied to cleave tert-butyl esters [1], in the presence of 1,3-dimethoxybenzene as a cation-scavanger [16]. In both cases TLC indicated complete consumption of the starting material after 4 h at 0 °C with obviously no significant formation of side products. When we tried to obtain an analytical sample of the amorphous free acid of Tpseoc-protected L-leucine by silica gel column chromatography eluting the product with a n-hexane/ethyl acetate mixture containing 1% acetic acid or formic acid, we observed formation of a non-polar side product after passing the material through the column, as indicated by TLC. Changing the column material to acidic or neutral alumina with the same eluent or RP-8 silica gel eluting the free acid with a methanol/water mixture didn't change the outcome of the purification step. Since in all other cases of ester cleavage in 11-15 and in the competetive deprotection of the amino protecting groups in 21-25 similar problems with the purification of the free acids and amines were encountered, we chose to couple the crude products of the deprotection step in a standard peptide coupling protocol with either L-leucine tert-butyl ester for free acids or N-Boc-L-alanine for free amines. By comparing the yields of the resulting peptides 33, 34 and 35 we expected to achieve an indirect but nonetheless authentic feedback of the stability of the Tpseoc-group under the cleavage conditions examined. For the coupling step we chose to employ HBTU [17] as coupling agent, since it allows performing the coupling step in a one-pot manner and reliably leads to very high yields.

Scheme 1. Preparation of
When this strategy was applied for tert-butyl esters 11 and 12 the dipeptides 33 and 34 were obtained in yields of 71% and 69% respectively. In contrast, benzyl ester derivative 13, deprotected by hydrogenation with Pd/C (10%) in ethyl acetate, allyl ester derivative 14, cleaved by treatment with Pd(PPh 3 ) 4 /morpholine in THF [18], and methyl ester derivative 15, saponified with 1M-NaOH in THF/MeOH, yielded the same dipeptide 33 in 83%, 90% and 88% respectively. The yields obtained, together with the observation that methyl ester 15 decomposed slowly when treated with DCM/TFA 1:1 at room temperature, as indicated by TLC (half-life ca. 12 h, unpublished results), lead to the conclusion that the Tpseoc-group could very well be termed orthogonal to tert-butyl esters and the t-Boc-group, but exhibits limited stability under prolonged exposure to strong acid. Noteworthy seems the outcome of methyl ester cleavage with 1M-NaOH in ester 15 which proceeded very cleanly, as judged by TLC analysis, and lead to high yields of dipeptide 33, suggesting that the Tpseoc-group is not as prone to hydroxyl-ion induced elimination as initially expected [10]. Examination of the competitive cleavage of a second amino-protecting group in mono Tpseoc-protected diamines 21-25 (Scheme 3) drew a quite similar picture. When N-Tpseoc-N´-Boc-protected 1,6-diaminohexane 21 was treated with 20% TFA in DCM in presence of 1,3-dimethoxybenzene complete Boc-cleavage was observed after 45 min. at 0 °C. As expected, due to the shorter duration of the exposure to acid, the yield of 85% of peptide 35 obtained in the subsequent coupling step turned out to be significantly higher compared to tert-butyl ester cleavage/coupling sequence applied for 11 and 12. Cleavage of the Alloc-group in diamino-derivative 22 was achieved by treatment with tetrakis(triphenylphosphine) palladium(0) in DCM and BH 3 •Me 2 NH as allyl-scavenger [19], furnishing peptide 35 after the coupling step in 81% yield.
Cbz-derivative 23 was hydrogenated with Pd/C (10%) in dry THF as solvent instead of ethyl acetate used in deprotection of 12 (no reaction). Subsequent coupling to N-Boc-L-alanine yielded 85% of peptide 35. The best results for phthalimide-cleavage in diamino-derivative 24 were obtained by using methylhydrazine in toluene under anhydrous conditions. Scheme 4. Synthesis and testing of Tpseoc-protected alcohols, electron poor amines and amino-acids.
Heating the reaction mixture to 80 °C lead to complete cleavage of the phthalimide moiety after 36 h and subsequent peptide coupling yielded 80% of peptide 35. Treatment of Fmoc-protected diamine 25 with morpholine/DMF under anhydrous conditions followed by peptide coupling resulted in 90% recovery of peptide 35. To further explore the scope of the Tpseoc-group we desired to protect an alcohol function as the corresponding carbonate and an electron poor amine, like the indolenitrogen found in tryptophan. The capability of the Teoc-group as an alcohol protecting group was investigated earlier by Chattopadhyaya et al. and some favorable properties, especially very fast cleavage under exposure to an fluoride ion source were described [20]. In an analogical approach, prasterone 26 was transformed into its Tpseoc-carbonate with TpseocCl 5 in DCM and pyridine as the base. The reaction proceeded only slowly unless a catalytic amount of DMAP was added. After 24 h no further progression of the reaction could be observed, even upon addition of excess TpseocCl. Workup and chromatographic purification yielded moderate 66% of Tpseoc-protected prasterone 27 (Scheme 4), but the process for carbonate formation might be optimized by use of TMEDA as the base [21]. As electron poor amine N-Boc-L-tryptophan methyl ester 28 was chosen as a model compound and treatment with TpseocIm 4 in pyridine at 80 °C for 36 h resulted in formation of Tpseoc-protected tryptophan derivative 29 in satisfactory 91% yield (Scheme 4). Additionally chloroformate 5 was tested in a standard protection protocol widely used for installation of carbamate protecting groups in peptide chemistry, in which a free amino acid is treated with a chloroformate in water or water/dioxane (THF) in presence of a base like NaOH, carbonates or hydrogencarbonates [1]. To this end, L-phenylalanine 30 was reacted with TpseocCl 5 in water/dioxane and NaHCO 3 as the base. Unfortunately the obtained crude Tpseoc-L-phenylalanine 31 suffered from the same problems as Tpseoc-L-leucine described above, but coupling to leucine tert-butyl ester using the HBTU-technique resulted in formation of dipeptide 32 in an excellent yield of 98%. (Scheme 4) A so far unanswered question was the behavior of the Tpseoc-group under exposure to a fluoride ion source. Consequently the peptides 32, 33, 34 and 35 as well as the prasterone-carbonate 27 and tryptophan derivative 29 were treated with either 3 mol equivalents of TBAF•3H 2 O or a mixture of 2 mol eq. TBAF•3H 2 O/2 mol eq. CsF [8] with THF as the solvent at 0 °C or room temperature. Generally cleavage times were significantly reduced by use of TBAF•3H 2 O/CsF mixtures resulting in relatively slow Tpseoc-cleavage from the primary amine of peptide 35 (24 h, r.t.) and much faster cleavage from the primary amine of dipeptide 32 and 33 (both 6 h, r.t.) and the secondary amine in peptide 34 (90 min., r.t.). The tendency of accelerated cleavage rates going from primary amines to amines attached to sec. carbon and sec. amines might account for the release of strain induced into the molecules due to steric bulkiness of the Tpseoc-group. Both, the carbonate 27 and electron poor N-Tpseoc-derivative 29, were cleaved very fast with the reaction completed after 10 minutes at 0 °C. Accordingly the observed cleavage kinetics are significantly enhanced compared to those reported for the detachment of the Teoc-and SES-group, but at the same time much slower than those described for more fluoride sensitive silicon based amino protecting groups like the Psoc-group introduced by Wagner et al. [22]. This suggests that the Tpseoc-group could be applied orthogonally in combination with the Psoc-group or the closely related fluoride-cleavable PTMSEL-linker, which was designed for solid-phase synthesis and possesses advantageous properties with respect to its superior fluoride sensitivity [23,24]. Concerning the occurrence of racemisation in Tpseoc-protected amino acids during the deprotection step it should be mentioned that none of the Tpseoc-derivatives deprotected according to the procedure above did show any alteration of optical rotation compared to authentic samples. Also the NMR-spectra of the free amines and alcohols lacked signals caused by isomeric products, giving rise to the assumption that under given conditions no significant racemisation of the material took place. Furthermore it should be noted that generally compounds bearing the Tpseoc-moiety exhibit a very good solubility in commonly used organic solvents and almost half of the compounds were obtained as crystalline solids making them very convenient to handle.

General Procedure for the Tpseoc-protection of Aliphatic Primary and Secondary Amines A
General Procedure A: In a 25 mL round-bottom flask equipped with gas inlet and a stirring bar mixed carbonate 3 (400 mg, 0.85 mmol) was dissolved in dry DMF (5 mL) under a nitrogen atmosphere. To the solution was added the corresponding amino acid ester ammonium derivative (0.94 mmol, 1.1 eq.) and Et 3 N (356 μl, 2.56 mmol, 3 eq.) and the resulting shiny yellow solution stirred for 24 h at r.t.. After completion of the reaction (TLC) the mixture was diluted with ethyl acetate (100 mL) and transferred to a separatory funnel, washed twice each with water and 1M-NaHSO 4 -soln., three times with 5%-Na 2 CO 3 -soln. and once with brine. The organic layer was dried over Na 2 SO 4 and the solvent removed in vacuum. The residual crude Tpseoc-protected amino acid was then purified either by silica gel column chromatography or crystallization.
N-2-(Triphenylsilyl)ethoxycarbonyl-L-leucine tert-butyl ester (11). The protected leucine derivative 11 was prepared following the general procedure A from L-leucine tert-butyl ester (210 mg, 0.94 mmol, 1.1 eq.). The crude product was subjected to silica gel column chromatography with the eluent mixture n-hexane/ethyl acetate 8.   (12). The protected proline derivative 12 was prepared following general procedure A from L-proline tert-butyl ester (160 mg, 0.94 mmol, 1.1 eq.). The crude product was purified by silca gel column chromatography with the eluent mixture n-hexane/ethyl N-2-(Triphenylsilyl)ethoxycarbonyl-L-leucine benzyl ester (13). The protected leucine derivative 13 was prepared following general procedure A from L-leucine benzyl ester hydro-p-tosylate [25] (369 mg, 0.94 mmol, 1.1 eq.). The crude product was purified by silca gel column chromatography with the eluent mixture n-hexane/ethyl acetate 8  (14). The protected leucine derivative 14 was prepared following the general procedure A from L-leucine allyl ester hydro-p-tosylate [26] (25    following the deprotection protocol given below, was dissolved in dry DMF (5 mL) together with leucine tert-butyl ester hydrochloride/N-Boc-L-alanine (1.25 eq.) and HOBt (1.5 eq.) under a nitrogen atmosphere. After cooling the solution to 0 °C Hünig`s base (1.5 eq./3 eq.) and HBTU (1.5 eq.) were added, the mixture stirred for 2 h at 0 °C and for additional 14 h at r.t.. Thereafter the mixture was diluted with EtOAc, transferred to a separatory funnel, washed twice each with 1M-NaHSO 4 -soln. and sat. NaHCO 3 -soln., once with brine and dried over Na 2 SO 4 . After removal of the solvent under vacuum the residual crude Tpseoc-protected peptides were subjected to silica gel column chromatography. (33).      General procedure C for the Tpseoc-deprotection of aliphatic primary and secondary amines and alcohols [8]: In a round bottom flask equipped with gas inlet and a stirring bar the corresponding Tpseoc-protected amine or alcohol was dissolved in dry THF under an atmosphere of nitrogen. To the solution were added 2 mol eq. TBAF•3H 2 O and 2 mol eq. CsF at the temperature noted below and stirred at this temperature until completion of Tpseoc-cleavage was indicated by TLC. Then the mixture was acidified by adding 1M-NaHSO 4 soln. and stirred another 10 min. at rt. For deprotection of basic amines the solution was then diluted with additional water, transferred to a separatory funnel and extracted three times with EtOAc. The aqueous phase was then made alkaline by addition of 1N-NaOH and again extracted three times with EtOAc. Afterwards the combined organic layers were washed once each with water and brine, dried over Na 2 SO 4 and the solvent removed in vacuum, leaving the free amine in high purity. An analytical sample could be obtained by silica gel column chromatography with CHCl 3 /MeOH/Et 3 N mixtures as eluent. For non-basic amines and alcohols the mixture was neutralized by addition of sat. NaHCO 3 -soln and extracted three times with EtOAc. The combined organic layers were washed with water and brine and the residual crude product purified by silica gel column chromatography. (27). According to general procedure C Tpseoc-prasterone 27 (100 mg, 0.162 mmol) was dissolved in dry THF (2 mL) and treated with TBAF•H 2 O (102 mg, 0.323 mmol) and CsF (49 mg, 0.323 mmol) at 0 °C. TLC showed complete cleavage of the Tpseoc-group after 10 min. and chromatographic purification with the eluent mixture light petroleum ether/ethyl acetate 3:2 yielded prasterone (41 mg, 0.142 mmol, 88%) as a white solid. The material proofed to be identical to an authentic sample of prasterone as indicated by NMR-spectra and optical rotation [30]. (29). According to general procedure C Tpseoc-protected thryptophan derivative 29 (100 mg, 0.154 mmol) was dissolved in dry THF (2 mL) and treated with TBAF•3H 2 O (97 mg 0.303 mmol) and CsF (47 mg, 0.303 mmol) at 0 °C. TLC showed complete cleavage of the Tpseoc-group after 10 min. and chromatographic purification with the eluent mixture light petroleum ether/ethyl acetate 3:2 yielded N-Boc-tryptophan methyl ester (45 mg, 0.140 mmol, 91%) as colorless crystals. The material proofed to be identical to an authentic sample of N-Boc-tryptophan methyl ester as indicated by NMR-spectra and optical rotation [31]. (

Conclusions
In summary a new fluoride ion cleavable amino/alcohol protecting group based on the 2-(triphenylsilyl)ethoxycarbonyl-("Tpseoc"-) moiety was developed and installed into a series of amino acids and peptides and a steroid alcohol. The protected derivatives were synthesized via a short and efficient route starting from commercially available triphenylvinylsilane. Contrary to the Teoc-group, the Tpseoc-group proved to be highly resistant to acidic conditions necessary to cleave tert-butyl esters and the Boc-group. The Tpseoc-group was found to be compatible with a wide range of conditions, e.g., basic conditions needed to cleave methyl-esters and the Fmoc-group, catalytic hydrogenation with Pd/C in various solvents, treatment with Pd-catalysts in presence of an allyl-scavenger as applied in cleavage of allyl-based protecting groups and methylhydrazine, used in deprotection of phthaloylgroups. Cleavage of the Tpseoc-group was achieved by treatment with TBAF•3H 2 O and CsF with cleavage times ranging from 10 minutes to 24 hours. The observed cleavage kinetics are significantly enhanced compared to those reported for of Teoc-and SES-group [1]. Its general applicability as carbamate or carbonate protecting group and the fact that it can be introduced via standard methods employing a stable crystalline chloroformate reagent together with its UV-detectability make the Tpseoc-group a promising candidate for being adopted into the pool of protecting groups used more frequently in organic synthesis. Especially the very fast cleavage from electron poor amines and alcohols and its orthogonality to acid and fluoride labile linkers should make the Tpseoc-group an interesting choice as protecting group in automated solid-phase synthesis of oligonucleotides, peptides and saccharides. Adjustment of the Tpseoc-groups reactivity pattern could be established by introduction of electron releasing or electron withdrawing residues on the phenyl groups of the triphenylsilyl-moiety.