{"paper_id":"2e3dff0c-bd65-453e-9d71-76a8b42d253c","body_text":"A Photolabile Backbone Amide Linker for the Solid-Phase Synthesis of Cyclic Peptides and C-Terminal Thioesters | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article A Photolabile Backbone Amide Linker for the Solid-Phase Synthesis of Cyclic Peptides and C-Terminal Thioesters Kaif R. Khan, Mary L. Niedrauer, Matthew A. Hostetler, Soo Sung Kang, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7165562/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 08 Sep, 2025 Read the published version in International Journal of Peptide Research and Therapeutics → Version 1 posted 13 You are reading this latest preprint version Abstract Introduction A new backbone amide linker has been developed for the synthesis of cyclic and C-terminally modified peptides that permits photochemical detachment of the synthesized peptide from the solid support, thus avoiding problems associated with acid deprotection conditions. Methods An initial survey of known photolabile motifs for their ability to produce a linker-bound model dipeptide in high yield and their ability to undergo efficient photochemical detachment of the model dipeptide found that the 6-nitroveratryl (Nve) motif afforded the most efficient release of the dipeptide. The problematic acylation of Nve-bound amino esters was solved through the development of the 2-hydroxy-4-carboxy-6-nitrobenzyl (Hcnb) linker, which utilizes an O-to-N transacylation to afford efficient acylation of even sterically hindered, linker-bound amino esters. Two different approaches were carried out to avoid/minimize diketopiperazine formation. This methodology was then used to synthesize cyclic peptides and peptide thioesters. Results The Hcnb linker was found to afford high yields of amino acid loading, acylation, and photolytic cleavage of model tripeptides. Attachment of the Hcnb linker to the aminomethyl TG resin permitted the solid phase synthesis of representative cyclic peptides and C-terminal thioesters in high overall yield and purity. Conclusion Hcna is a new photolabile backbone amide linker that has been used to synthesize cyclic peptides and peptide thioesters. It was found that the linker efficiently released the synthesized peptides when UV light was shed on it. This linker is also stable to acids meaning global deprotections can be done on resin and can be washed to remove by-products, releasing only the peptide upon its photocleavage from the resin. Peptide synthesis photochemistry peptide cyclization thioester formation Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction The head-to-tail cyclization of peptides (Fusetani 1993 ; Humphrey 1997 ; Katsara 2006) is a common and important strategy, employed both in nature and in synthetic applications, for improving the bioavailability and conformational rigidity of polypeptides (Kessler 1982 ; Samanen 1991; Biron 2008). The lack of either an N- or C-terminus increases proteolytic stability while internal hydrogen bonding and a more compact structure improve the membrane permeability of cyclic peptides (White 2011; Dougherty 2019). These advantages are reflected in the prominence of cyclic peptides among peptidic therapeutic agents (Veber 1981; Darkin-Rattray 1996; Coulouarn 1998; Foister 2006), such as gramicidin S (Gause 1944 ) and cyclosporine A (Shevach 1985 ). The chemical synthesis of cyclic peptides, however, has been complicated by the traditional approach to solid-phase peptide synthesis using the C-terminal carboxylate to covalently attach the growing peptide to a polymeric support. The most straightforward approach involving detachment of a fully protected linear peptide precursor from the resin followed by macrocyclization in solution is complicated by solubility problems, oligomerization and C-terminal epimerization reactions. To avoid the problems associated with head-to-tail cyclization in solution, various approaches have been devised for on-resin cyclization of peptides, including Kaiser’s Oxime resin (Mihara 1995), Richter’s thioester linker (Richter 1994), Waldmann’s hydrazide linker (Rosenbaum 2001 ), Kenner’s sulfonamide linker (Kenner 1971) and side-chain attachment of the linear peptide to the resin (Krishnamoorthy 2006). An important general strategy for the on-resin, head-to-tail cyclization of peptides was introduced by the “backbone amide linker” (BAL) strategy of Albericio and Barany (Jensen 1998). In this approach, the nitrogen atom of an internal amide bond is used to tether the growing peptide to the polymeric resin, thereby liberating both N- and C-termini for modification. It is noteworthy that this strategy also addresses the more general problem of C-terminal modification of the resin-bound peptide. Since its introduction, the BAL strategy has been employed by a number of researchers and several modifications have been developed (Bourne 1999; Bourne 2001; Boas 2002; Brask 2003; Boas 2004; Pittelkow 2005; Jessing 2006; Liley 2006; Pittelkow 2006). Additional applications of the BAL strategy involve the synthesis of C-terminal modifications, such as trithioorthoesters (Guillaumie 2000; Kappel 2005 ), aldehydes (Mori 2004; Soural 2007 ; Springer 2008), or functionalized amides. Another application of the C-terminal modification of peptides was revealed by the discovery of the native chemical ligation of peptide bonds by Kent, Muir and Dawson (Dawson 1994). Their use of peptide thioesters to couple with N-terminal cysteine residues has necessitated the development of various methods for the solid-phase synthesis of peptide thioesters (Mende 2011 ; Li 2007). Since that landmark discovery, related processes such as Staudinger ligation (Nilsson 2000) have been developed that likewise depend on the availability of peptide thioesters. In response to this need, a number of creative approaches to the solid-phase synthesis of peptide thioesters have been developed, yet the availability of C-terminal modification in the BAL strategy remains one of the most straightforward approaches to the synthesis of peptide thioesters. Although acidic deprotection conditions are widely used in peptide synthesis, there are situations involving acid-sensitive functionality that may require alternative deprotection conditions. For these cases, other methodologies have been explored and developed (Lloyd 1993; Guillier 2000; Bochet 2002; Heidler 2005 ; McAllister 2005; Scott 2006 ; Kang 2008 ). One of the most appealing ideas has been to use photochemical detachment from the resin as an extra dimension of protecting group orthogonality in solid phase peptide synthesis. Photolabile linkers (Gallop 1994; Liang 2016), in which long-wave UV radiation (300 ~ 360 nm) is used to detach an organic functional group – usually a carboxylic acid, a carbamate (a latent amine), an alcohol, or a phosphate – from a polymer or glass surface, provide an alternative to the classic acidic or basic cleavage protocols used in solid-phase organic synthesis. Ideally, photochemical deprotection should proceed with high functional group selectivity, low amounts of side reactions and involve no separation of product from reactants. Photochemical deprotection has also found favor with applications in the parallel synthesis of compound libraries (Sheehan 1973 ). Herein we describe studies directed toward the development of a photolabile BAL linker (Fig. 1 ) to address the problems associated with the BAL strategy as a general method for the synthesis of cyclic and C-terminally modified peptides. Our studies began with a survey of several known photolabile motifs for their applicability to BAL linker design (Fig. 1 ). The candidate photolabile motifs studied were the p -oxyphenacyl (Op) (Bellof 1985 ), α-methyl- p -oxyphenacyl (Mop) (Sheehan 1965; Peach 1995), p -oxybenzoin (Obz) (Bergmark 1985), α, o -dimethyl phenacyl (Dmp) (Patchornick 1970) and 6-nitroveratryl (Nve). All of these photolabile groups undergo facile n to π* or π to π* excitation at 300–360 nm and release oxygen-based substituents with few side reactions. Appropriate synthetic precursors 1 – 5 were chosen for each of the motifs, as shown in Fig. 2 . Consequently, our preliminary survey focused on the Op, Mop, Obz, Dmp, and Nve motifs, their preparation and photolysis. Of specific interest were a) the ease of anchoring amino acids to the linker, b) the rate and efficiency of photocleavage, and c) the suppression of side reactions during photolytic deprotection. To assess these questions, the dipeptide Boc-Phe-Gly-OMe ( 8 ) was chosen as a model system. After the photolabile motif was chosen, effort was directed toward its attachment to a solid support compatible with the solvents and reagents common to peptide synthesis. Finally, the problems of diketopiperazine formation, photocleavage optimization, and maximization of cyclization yields were addressed. Material and Methods Synthesis of Dmp 1-(2,5-dimethyl-4-alkylphenyl)-propanone (4b) To a mixture of propionyl chloride (0.51 mL, 5.88 mmol) and AlCl 3 (780 mg, 5.88 mmol) in CH 2 Cl 2 (25 mL) was added a solution of 4-(2,5-dimethylphenyl)butanoic acid ( 4a , 377 mg, 1.96 mmol) in CH 2 Cl 2 (5 ml) at 0°C. The reaction mixture was stirred for 1 h at the same temperature and poured into cold HCl solution (40 g ice and 10 mL of conc. HCl). The organic material was extracted by addition of CH 2 Cl 2 (25 mL) and the aqueous layer was extracted with another portion of CH 2 Cl 2 (50 mL). The combined organic layer was washed with brine (50 mL), dried with anhydrous MgSO 4 , passed through 3 cm silica gel pad, and evaporated under reduced pressure. The crude product was dissolved in oxalyl chloride (3 mL) and stirred overnight. After removal extra oxalyl chloride by evaporation under reduced pressure, the organic material was dissolved in CH 2 Cl 2 (30 mL). To this solution, benzyl amine (0.257 mL, 2.35 mmol) and DIEA (1.7 mL, 9.8 mmol) were added and stirred 3 h. The organic layer was washed with brine (30 mL), dried with anhydrous MgSO 4 , evaporated under reduced pressure and purified by flash column chromatography using 33% EtOAc in Hexanes to give 4b as a pale-yellow solid. 1-(2,5-dimethyl-4-alkylphenyl)-1-hydroxypropan-2-one oxime (4c) To a solution of 4b (0.51g, 1.50 mmol) and isoamyl nitrite (0.24 mL, 1.80 mmol) in MeOH (10 mL) at 45°C was added dropwise to concentrated HC1 (0.25 mL) and the resulting mixture was stirred for 3 h at the same temperature. After cooling to room temperature, the organic material was partitioned into EtOAc (40 mL) and H 2 O (40 mL). The organic layer was washed with brine (40 mL), dried with anhydrous MgSO 4 , passed through 3cm silica gel pad, and evaporated under reduced pressure. The residue was dissolved in MeOH (15 mL) and NaBH 4 (50 mg) was added under ice bath. After stirring for 30 min, the organic material was partitioned into EtOAc (40 mL) and HCl solution (0.1 M, 30 mL). The organic layer was washed with saturated NaHCO 3 (30 mL) and with brine (30 mL), dried over anhydrous MgSO 4 , and evaporated under reduced pressure. The residue was purified by flash column chromatography using 66% EtOAc in hexanes to give 4c as a white solid. 1-(2,5-dimethyl-4-alkylphenyl)-1-hydroxypropan-2-one (4) To a solution of 4c (0.30 g, 0.81 mmol) in DMF (2 mL) was added glyoxylic acid (60% water solution, 5 mL). After stirring for 5 h, the organic material was partitioned into EtOAc (40 mL) and H 2 O (40 mL). The organic layer was washed with brine (2 X 30 mL), dried with anhydrous MgSO 4 , evaporated under reduced pressure, and the residue was purified by flash column chromatography using 50% EtOAc in hexanes to give 4 as a pale-yellow oil. Alkylation of amino ester with photolabile linker 1–5 (6a-e) H-[Op]-Gly-OMe (6a) . To a solution of 1 , (150 mg, 0.66 mmol) in anhydrous CH 3 CN (5 mL) were added GlyOMe·HCl (248 mg, 1.98 mmol), and DIEA (0.52 mL, 2.97 mmol). The reaction mixture was stirred overnight at room temperature, evaporated under reduced pressure and partitioned into CH 2 Cl 2 (20 mL) and H 2 O (20 mL). The organic layer was washed with brine (20 mL), dried with anhydrous MgSO 4 , evaporated under reduced pressure, and purified by flash column chromatography using 33% EtOAc in hexanes to give 6a as a pale-yellow oil. H-[Mop]-Gly-OMe (6b) . To a solution of 2 , (200 mg, 0.63 mmol) in anhydrous CH 3 CN (8 mL) were added GlyOMe·HCl (237 mg, 1.89 mmol), DIEA (0.49 mL, 2.84 mmol) and tetrabutylammonium iodide (25 mg). The reaction mixture was refluxed for overnight, cooled to room temperature, evaporated under reduced pressure, and partitioned into CH 2 Cl 2 (20 mL) and H 2 O (20 mL). The organic layer was washed with brine (20 mL), dried with anhydrous MgSO 4 , evaporated under reduced pressure, and purified by flash column chromatography using 33% EtOAc in hexanes to give 6b as a colorless oil. H-[Obz]-Gly-OMe (6c) . To a solution of 3 , (123 mg, 0.39 mmol) in anhydrous MeOH (20 mL) was added GlyOMe·HCl (242 mg, 1.93 mmol). The reaction mixture was refluxed for 24 h, cooled to room temperature, evaporated under reduced pressure, and partitioned into CH 2 Cl 2 (20 mL) and H 2 O (20 mL). The organic layer was washed with brine (20 mL), dried with anhydrous MgSO 4 , evaporated under reduced pressure, and purified by flash column chromatography using 33% EtOAc in hexanes to give 6c as a pale-yellow oil. H-[Dmp]-Gly-OMe (6d) . To a solution of 4 , (200 mg, 0.57 mmol) in anhydrous toluene (20 mL) was added GlyOMe·HCl (215 mg, 1.71 mmol). The reaction mixture was refluxed for 5 h, cooled to room temperature, and partitioned into EtOAc (20 mL) and H 2 O (40 mL). The organic layer was washed with brine (40 mL), dried with anhydrous MgSO 4 , evaporated under reduced pressure, and purified by flash column chromatography using 33% EtOAc in hexanes to give 6d as a colorless oil. H-[Nve]-Gly-OMe (6e). To a solution of 5 (515 mg, 2.44 mmol) in MeOH (20 mL), were added GlyOMe·HCl (459 mg, 3.66 mmol), sodium bicarbonate (348 mg, 4.15 mmol). The reaction mixture was stirred for 4 h, cooled to 0°C. After portionwise addition of NaBH 4 (6.0 mmol, 227 mg), the mixture was stirred for 30 min and poured into HCl solution (0.1N, 50 mL). The pH of water layer was adjusted to 4 ~ 6 by dropwise addition of NaOH (1 M), and the organic material was extracted with CH 2 Cl 2 (50 mL). The aqueous layer was extracted with another portion of CH 2 Cl 2 (50 mL). The combined organic layer was dried with anhydrous MgSO 4 , evaporated under reduced pressure and purified by flash column chromatography using 50% EtOAc in hexanes to give 6e as a colorless oil. Acylation of 6a-e using symmetric anhydrides (7a-e). The symmetric anhydride of Boc-Phe was prepared in situ by mixing Boc-Phe (860 mg, 4 mmol) with EDCI (0.422 g, 2.2 mmol) in CH 2 Cl 2 (10 ml, 0.2 M) for 5 min. To the solution of symmetric anhydride was added 6a-e (1 mmol), and the reaction mixture was stirred for 24 h at room temperature. After dilution with CH 2 Cl 2 (50 ml), the organic layer was washed with brine (50 mL), dried with anhydrous MgSO 4 , and evaporated under reduced pressure. The residue was purified by flash column chromatography to obtain an Hcnb-linked dipeptide ( 7a-e ). Synthesis of Hcna Methyl ( E )-4-(2-(dimethylamino)vinyl)-3,5-dinitrobenzoate (13) . To a stirred mixture of 4-Methyl-3,5-dinitrobenzoic acid ( 12 ) (2.0 g, 8.8 mmol) in anhydrous toluene (29 mL) was added DMF dimethyl acetal (3.2 mL, 24 mmol) dropwise at ambient temperature. The resulting mixture was heated to reflux in an oil bath and became dark red in color. After 16 h, the reaction was cooled to room temperature and the solvent removed under reduced pressure by co-evaporation with MeOH. The crude residue was recrystallized from hot ethanol to afford enamine 13 as a lustrous green solid which became red upon grinding. Methyl 4-formyl-3,5-dinitrobenzoate (14). To a mixture of 13 (1.08 g, 3.66 mmol) and RuCl 3 solution (0.1 M in H 2 O, 1.2 mL, 0.13 mmol) in CH 3 CN-H 2 O (6:1, 37 mL) was added NaIO 4 (2.0 g, 9.1 mmol) in portions at rt. The color quickly dissipated, and solids formed. The reaction continued until TLC analysis showed consumption of starting material (typically 10–30 min). After stirring for 30 min, the solids were removed by vacuum filtration and washed with CH 2 Cl 2 . The filtrate was partitioned between H 2 O and CH 2 Cl 2 and the aqueous layer was extracted twice with CH 2 Cl 2 . The combined organic extracts were washed with brine and sat. aq. Na 2 S 2 O 4 , dried over Na 2 SO 4 , and concentrated under reduced pressure. The crude residue was purified by flash column chromatography (30% EA/Hex) to afford 14 as an off-white solid. Methyl 4-formyl-3-hydroxy-5-nitrobenzoate (15). A solution of 14 (678 mg, 2.67 mmol) in anhydrous DMF (5.1 mL) was added dropwise to a mixture of acetaldoxime [mixture of isomers] (325 µL, 5.33 mmol) and K 2 CO 3 (811 mg, 5.87 mmol) in anhydrous DMF (5.1 mL) dropwise at rt. The reaction mixture immediately turned bright red purple upon mixing, then brownish green after stirring overnight. After 21 h, H 2 O (10 mL) was added to the reaction and the resulting solution extracted 5x with Et 2 O. The aqueous layer was then acidified to pH ~ 1–2 with concentrated HCl and extracted 4x with CH 2 Cl 2 . The combined organic extracts were dried over Na 2 SO 4 , filtered and concentrated under reduced pressure. The crude residue was purified by flash chromatography (25% EA/Hex) to afford 15 as a bright yellow solid. 4-Formyl-3-hydroxy-5-nitrobenzoic acid (16). Solid Ba(OH) 2 ∙8 H 2 O (1.97 g, 6.24 mmol) was added in portions to a solution of 15 (468 mg, 2.08 mmol) in MeOH (23 mL) at rt. Following reaction completion (TLC), the mixture was concentrated under reduced pressure and the crude residue was partitioned between EtOAc and H 2 O. The aqueous layer was extracted twice with EtOAc, acidified to pH ~ 1–2 with concentrated HCl and extracted with EtOAc (3x). The combined organic extracts were washed with brine, dried over Na 2 SO 4 , filtered and concentrated under reduced pressure to afford 16 (379 mg, 86%) as a brown solid. General procedure for solution-phase reductive amination (17a-c). HCl-AA-OtBu (0.666 mmol) was added to a solution of 15 (200.0 mg, 0.666 mmol) and DIEA (244 µL, 1.40 mmol) in CH 3 CN (9.5 mL). The reaction was stirred at rt for 45 min. Volatiles were removed and the residue was dissolved in MeOH (3.3 m). NaBH 4 (75.6 mg, 2.00 mmol) was added, and the reaction was stirred for 15 min. Volatiles were removed and the residue was purified by flash column chromatography (20–45% EtOAc/Hex) to yield 17a-d. General procedure for solution-phase acylation (18a-c). A mixture of Fmoc-AA-OH (0.29 mmol) and DIPC (22.5 µL, 0.14 mmol) was preactivated in dry CH 2 Cl 2 : DMF (1:1) (0.6 mL) for 5 min. 17a-c (0.13 mmol) as a solution in CH 2 Cl 2 (2.1 mL) was added and the reaction was stirred for 2 h. The volatiles were removed and then dissolved in methanol (1.0 mL) and a solution of ammonium acetate (80.9mg, 1.05 mmol) in water (0.3 mL) was added and the reaction was stirred overnight. Volatiles were removed and the residues were washed with water and extracted with ethyl acetate three times, concentrated, and then purified by flash column chromatography (30–40% EA/Hex). Solid Phase Peptide Synthesis Preparation of Hcna-loaded resin (22a-b). Fmoc-Sieber Amide PS Resin (0.6 mmol/g) or TentaGel S NH 2 (0.26 mmol/g) was swelled in DMF for 15 min, drained, and treated with piperidine-DMF (1:4, 1 x 2 mL, 30 min). The mixture was drained and washed with standard wash protocol [DMF (3 x 1 mL), CH 2 Cl 2 (3 x 1 mL), MeOH (3 x 1 mL), CH 2 Cl 2 (3 x 1 mL), then DMF (3 x 1 mL)] to afford a positive Kaiser ninhydrin test. To a mixture of N -Fmoc-6-aminohexanoic acid (4 eq) and HATU (3.9 eq) in DMF (0.03 M) was added DIEA (8 eq) and this mixture was added to the resin (pre-washed with DMF) after a 5 min pre-activation period. After 2 h, the mixture was drained and washed with the standard wash protocol to afford a negative Kaiser ninhydrin test. The resin was then agitated with piperidine-DMF (1:4, 1 x 2 mL, 30 min). The mixture was drained, and the resin washed with standard wash protocol to afford a positive Kaiser ninhydrin test. To a mixture of Fmoc-Pro-OH (4 eq), HATU (3.9 eq) and HOAt (4 eq) in DMF (0.03 M) was added DIEA (8 eq) and this mixture was added to the resin (pre-washed with DMF) after a 5 min pre-activation period. After 2 h, the mixture was drained and washed with the standard wash protocol to afford a negative Kaiser ninhydrin test. The resin was then treated with piperidine-DMF (1:4, 1 x 2 mL, 30 min). The mixture was drained, and resin washed with standard wash protocol to afford a positive chloranil test. To a mixture of 16 (3 eq) and PyBOP in DMF (0.03 M) was added DIEA (6 eq) and this mixture was added to the resin (pre-washed with DMF) after a 5 min pre-activation period. After 2 h, the mixture was drained and washed with the standard wash protocol to afford a negative chloranil test. General procedure for solid-phase reductive amination. The prepared resin 22a or 22b was swelled in DMF for 15 min then treated with a mixture of H-AA-ODMA or HCl-AA-O t Bu (10 eq) and AcOH (10 eq) in CH 2 Cl 2 (0.03 M). After 3 h, the mixture was drained and washed briefly with standard wash protocol then THF-MeOH (2:1). The resin was then taken up in THF-MeOH (2:1, 0.03 M) and treated with solid NaBH 4 (5 eq). After 2 h, the mixture was drained and washed with H 2 O, standard wash protocol, then piperidine-DMF (1:4, 5 min). The resin was washed once more with standard wash protocol to afford a positive chloranil test. General procedure for loading of second amino acid residue. A flame-dried flask was charged with the 2nd Amino Acid residue (4 eq) and dry CH 2 Cl 2 (0.05 M), to which was added DIPC (2 eq) dropwise at rt. The mixture was stirred at rt for 12 min and the resulting solution was solubilized by addition of DMF (0.05 M). This solution was added directly to prepared resin 23a or 23b and the mixture was agitated for 2 h, followed by the standard wash protocol to afford a slightly positive chloranil test. This procedure was repeated once more to afford resin-bound dipeptide (and a negative chloranil test). General procedure for coupling of third amino acid residue. A solution of 1-octanthiol (10 eq), DIEA (5 eq), and DMF (0.03 M) was added to the prepared resin 24a or 24b and the mixture was agitated for 1 h, followed by the standard wash protocol. Next, a mixture of TIPS-OTf (10 eq), 2,6-Lutidine (10 eq), and DMF (0.03 M) was added to the resin and the mixture was agitated for 4 h, followed by the standard wash protocol. A flame-dried flask was charged with an Fmoc-amino acid (4 eq) and dry CH 2 Cl 2 (0.05 M), to which was added DIPC (2 eq) dropwise at rt. The mixture was stirred at rt for 12 min and the resulting mixture was diluted with DMF (0.05 M). The resin was agitated with a solution of DBU-1-Octanthiol-DMF (2:2:96, 1 x 2 mL, 3 min), washed with the standard wash protocol, and the pre-activated amino acid solution was added, taking care to minimize the time between deprotection and coupling. The mixture was agitated for 2 h, followed by standard wash protocol to afford a negative chloranil test. The resin was agitated with piperidine-DMF (1:4, 1 x 2 mL, 30 min). The mixture was drained, and the resin washed with standard wash protocol to afford a positive Kaiser ninhydrin test. Fmoc half-cycle method. A solution of 1-octanthiol (10 eq), DIEA (5 eq), and DMF (0.03 M) was added to the prepared resin 24a or 24b and the mixture was agitated for 1 h, followed by the standard wash protocol. Next, a mixture of TIPS-OTf (10 eq), 2,6-Lutidine (10 eq), and DMF (0.03 M) was added to the resin and the mixture was agitated for 4 h, followed by the standard wash protocol. The C-terminal allyl protecting group was removed using Pd(PPh 3 ) 4 (0.25 eq), PhSiH 3 (25 eq) and DCM (3 mL) and agitating the reaction mixture for 2 hours after which the following wash was done: DCM (X 3), DMF (X 3), 0.03 M sodium N,N-diethyldithiocarbamate in DMF (X 3), and DMF (X 3). H-AA n -OAllyl∙CF 3 COOH (10 eq), HATU (10 eq), HOAt (10 eq) and DIEA (20 eq) in DMF (3 mL) was mixed and added to the reaction vessel and agitated for 4 hours. The resin was washed using standard wash protocol. General procedure for peptide cyclization (condition A). After complete synthesis of the desired Fmoc or Boc-protected peptide, the C-terminal DMA protecting group was removed using Pd(PPh 3 ) 4 (1 eq) and phenylsilane (20 eq) in CH 2 Cl 2 under N 2 for 2 h, after which the resin was drained and washed with DMF (3 x 1 min), sodium N,N -diethyldithiocarbamate (0.03 M in DMF, 3 x 1 min), and CH 2 Cl 2 (3 x 1 min). The resin was agitated with piperidine-DMF (1:4, 1 x 2 mL, 30 min). The mixture was drained, and the resin washed with standard wash protocol to afford a positive Kaiser ninhydrin test. A mixture of PyAop (5 eq), HOAt (5 eq), and DIEA (10 eq) in DMF (0.03 M) was added directly to the resin. After 2 h, the mixture was drained and washed with the standard wash protocol to afford a negative Kaiser ninhydrin test. General procedure for peptide cyclization (condition B). After complete synthesis of the desired Fmoc or Boc-protected peptide, the C-terminal DMA protecting group was removed using Pd(PPh 3 ) 4 (1 eq) and phenylsilane (20 eq) in CH 2 Cl 2 under N 2 for 2 h, after which the resin was drained and washed with DMF (3 x 1 min), sodium N,N -diethyldithiocarbamate (0.03 M in DMF, 3 x 1 min), and CH 2 Cl 2 (3 x 1 min). The resin was agitated with piperidine-DMF (1:4, 1 x 2 mL, 30 min). The mixture was drained, and the resin washed with standard wash protocol to afford a positive Kaiser ninhydrin test. A mixture of PyAOP (3 eq), 2,4,6-collidine (6 eq), and DIEA (6 eq) in 9:1 CH 2 Cl 2 -DMF (0.03 M) was added directly to the resin. After 2 h, the mixture was drained and washed with the standard wash protocol to afford a negative Kaiser ninhydrin test. General procedure for thioester synthesis. After synthesis of the desired Boc-protected peptide, the C-terminal DMA protecting group was removed using Pd(PPh 3 ) 4 (1 eq) and phenylsilane (20 eq) in CH 2 Cl 2 under N 2 for 2 h, after which the resin was drained and washed with DMF (3 x 1 min), sodium N,N -diethyldithiocarbamate (0.03 M in DMF, 3 x 1 min), and CH 2 Cl 2 (3 x 1 min). A solution of 1-octanthiol (10 eq), DIEA (10 eq), and DMF (0.03 M) was added to the resin and the mixture was agitated for 1 h, followed by the standard wash protocol. Next, a mixture of TIPS-OTf (10 eq), 2,6-Lutidine (10 eq), and DMF (0.03 M) were added to the resin and the mixture was agitated for 4 h, followed by the standard wash protocol. A solution of HATU (5 eq) and DIEA (20 eq) in DMF (0.03 M) was added to the resin and agitated for a 5 min period. Without draining, thiophenol (10 eq) was added to the mixture. After 2 h, the mixture was drained and washed with the standard wash protocol to afford a negative Kaiser ninhydrin test General Procedure for Sieber Amide cleavage. An aliquot (~ 1 mg) of resin-bound peptide was treated with 200 µL TFA/H 2 O/CH 2 Cl 2 (2:1:97) and the mixture agitated for 5 min at rt. Following this, volatiles were removed by N 2 stream. The resulting residue was dissolved in 200 µL MeOH-H 2 O (1:1), filtered and analyzed by UPLC-MS. Note: Product / DKP ratio was determined by integrating 214 and 254 nm peaks and are reported as relative percentages. General Procedure for photocleavage. The resin-bound peptide was suspended in a 5 mL solution of 1:9 MeOH:CH 2 Cl 2 in a fused quartz tube and agitated under 365 nm UV light for 1 h. The solvent was filtered to remove the resin and removed under reduced pressure. The resulting residue was dissolved in MeOH-H 2 O (1:1), filtered and analyzed by UPLC-MS and HPLC. Results and Discussion Synthesis of linker-anchored dipeptides Using the precursor molecules 1–5 (Fig. 2 ), each linker was covalently attached to glycine methyl ester to afford an anchored glycine residue ( 6a - e ), which was then acylated with Boc- L -phenylalanine to afford an anchored dipeptide 7a - e (Table 1 ). Each anchored dipeptide was photolyzed at 300 nm to afford Boc-Phe-Gly-OMe ( 8 ). The linker precursors 1–5 were either commercially available or prepared using literature procedures (Stowell 1996; Vishwanathan 2006). Linkers 1 and 2 were anchored to methyl glycinate via nucleophilic displacement, whereas 3 and 4 employed the Amadori rearrangement (Amadori 1925 ; Isbell 1958 ) of an a-hydroxyketone, and 5 was attached to methyl glycinate through reductive amination. In all cases, acylation with the symmetric anhydride of Boc- L -Phe was found to afford the highest yield of anchored dipeptide. A survey of conditions for photolytic cleavage of the dipeptide found that photoexcitation at 300 nm in either methanol or chloroform, depending on the motif, afforded the fastest and cleanest reactions, but only the Dmp ( 7d ) and Nve-linked dipeptides showed acceptable photolytic reactivity. Problems arose, however, when amino acids larger than glycine were anchored to Dmp and Nve. Both the initial attachment and subsequent acylation of those anchored amino acids proved to be quite problematic and afforded anchored dipeptides in modest yields at best. In summary, Op, Mop, and Obz failed to release the dipeptide product efficiently, regardless of solvent and wavelength used, and so were discarded as linker candidates. Both the Nve and Dmp motifs displayed acceptable photolytic product release with the appropriate choices of solvent and irradiation wavelength; however, in both cases the modest yields for amino acid anchoring and acylation were insufficient for our needs, necessitating further modification to improve both steps of the reaction sequence. Development of the second-generation photolabile linker On the basis of both acylation and photocleavage yields the o -nitrobenzyl motif of the Nve linker was chosen as the basic skeleton for a second-generation linker, and the transacylation motif of the 6-hydroxy-4-methoxybenzyl (Hmb) (Sheppard 1993) and 6-hydroxynitrobenzyl (Hnb) (Smythe 1999) auxiliaries was incorporated into the linker design to improve acylation efficiency. The acid-labile Hmb auxiliary was devised by Sheppard for the protection of peptide amide backbones during the synthesis of ‘difficult’ peptide sequences. The related, photolabile Hnb auxiliary was developed by Smythe for the covalent modification of ‘difficult’ peptide sequences to facilitate their cyclization. Both the Hmb and Hnb auxiliaries employ an ortho- hydroxyl group to acylate bulky amino acids via esterification and subsequent O-to-N transacylation. It was reasoned that the addition of an o- hydroxy substituent to the o -nitrobenzyl skeleton would assist both the acylation of the first amino acid and later photolytic cleavage. Attachment of a para -carboxylate to the o -nitrobenzyl skeleton provides a tether for covalent attachment of the linker to a spacer or resin and affords additional electron withdrawal to assist both the transacylation and photolysis reactions. Thus, the 2-hydroxy-4-carboxy-6-nitrobenzyl (Hcnb) linker emerged as a second-generation candidate for a photolabile BAL linker, as exemplified by the Hcnb-glycine adduct 9 (Fig. 3 ), derived from reductive amination of the aldehyde precursor 2-hydroxy-4-carboxy-6-nitrobenzaldehyde (Hcna) with glycine methyl ester. Treatment of 9 with a symmetric anhydride (Fig. 3 ) can result in the formation of the desired dipeptide 11 either through direct N-acylation or via initial acylation of the less hindered phenol followed by subsequent O-to-N acyl transfer. This alternate pathway was envisaged as a means to overcome the limitations of the Nve motif when attached to sterically hindered amino acids. Synthesis of Hcna linker 4-formyl-3-hydroxy-5-nitrobenzoic acid ( 16 ) was prepared from commercially available 4-Methyl-3,5-dinitrobenzoic acid ( 12 ) (Fig. 4 ). A condensation reaction between 12 and DMF-DMA afforded the previously reported (Starosotnikov 2005) enamine 13 in 72% yield. Ruthenium (III) chloride-catalyzed oxidative cleavage of the enamine using sodium metaperiodate generated the aldehyde 14 in 61% yield. Unilateral nucleophilic aromatic substitution of a single nitro- group using acetaldoxime to install the ortho -phenol group afforded ester-protected linker precursor 15 in 68% yield, which was used for solution-phase studies. Finally, hydrolysis of the methyl ester group using barium hydroxide afforded the free carboxylic acid 16 in 90% yield. Solution-phase synthesis of the dipeptides using Hcnb We envisioned linking the peptide to the linker via a reductive amination between the primary amine of the first amino acid and the aldehyde precursor, in similar fashion to the loading protocol for the original BAL Linker. The resulting secondary amine can then be acylated with the next amino acid in sequence, forming a tertiary amide linkage between the peptide and linker. The presence of the ortho -phenol in Hcnb is essential for complete acylation of the secondary amine, especially when utilizing amino acids with bulky side chains. Finally, photolysis in the short-wave UV range should cause a clean release of the dipeptide from the linker. A series of dipeptides was synthesized in solution on the methyl ester-protected Hcna linker precursor 15 (Table 2 ). In solution, addition of the desired C-terminally protected amino acid to the aldehyde handle in the presence of DIEA in CH 3 CN forms the imine intermediate. Reduction of this intermediate by sodium borohydride in methanol yields the Hcnb-linked amino acids 17a-c . Acylation of the secondary amine with a pre-formed symmetric anhydride of the second amino acid resulted in acylation to give Hcnb-linked dipeptides, even in the case of bulky side-chains such as Thr. Photolysis at 365 nm in MeOH releases the desired dipeptides from the linker to give 19a-c. Due to rotomers present in peptides attached to Hcnb, yields were reported over 2 steps. Preparation of the resin-bound Hcna linker The free acid form of the Hcna linker, 16 (Fig. 4 ), was covalently anchored to aminomethyl-functionalized Tentagel resin 21a , or Sieber TG resin 21b via acylation of a resin-bound amine. A 6-aminohexanoic acid (Ahx) spacer was employed with both solid supports to improve reactivity. Additionally, a proline residue was introduced to reduce cross-linking between the amine and the aldehyde of the Hcna linker. In both cases, acylation of resin-bound amines was accomplished by coupling 16 to the free amine using either PyBOP or DCC (Fig. 5 ). Subsequent reactions of 22a were monitored by photolytic cleavage from the linker and analysis of the crude product, whereas reactions of 22b were monitored by cleavage using dilute acid and analysis of the crude product. Because of the time required for photolysis of Hcnb-linked peptides (1–2 h), reaction conditions were optimized using 22b and then applied to 22a . Loading the dipeptide by reductive amination followed by acylation The first residue was anchored to the resin-bound Hcna linker via a 2-step reductive amination under acidic conditions. (Fig. 6 ). Quantitative acylation of the resin-bound amine was accomplished using a symmetric anhydride of the second residue (4 eq, 2 x 2h) as determined by LC-MS analysis. Loading of the third amino acid One of the major challenges with the Backbone Amide Linker concept is the formation of a diketopiperazine byproduct upon deprotection of the Fmoc group at the dipeptide stage. The most common method for overcoming this problem is to employ a sterically hindered C-terminal protecting group such as t -butyl or 1,1-dimethylallyl (DMA) (Hostetler 2018 ), thereby minimizing the formation of this undesired byproduct. However, it was found that quantitative DKP formation was still obtained when using the Hcnb linker, regardless of the C-terminal protecting groups used. It was hypothesized that this results from Brønsted acid activation of the C-terminus by the nearby phenol proton, thereby catalyzing the formation of DKP. It was reasoned that this effect could be eliminated through removal of the phenol proton, thus avoiding Brønsted acid activation. The phenol group is required for efficient loading of the second amino acid residue, as well as for photocleavage of the final peptide, and so a method for temporary protection of the hydroxyl is required. Following the loading of the secondary amine, the phenol remains acylated with an excess of the second amino acid, until it is removed by the strongly nucleophilic piperidine upon deprotection of the N-terminal Fmoc group. We theorized that replacing piperidine with DBU, a relatively non-nucleophilic alternative for Fmoc deprotection, would leave the acylated phenol intact and minimize formation of the DKP byproduct. However, it was found that rapid, quantitative DKP formation was still obtained even when using DBU. Other methods were considered for blocking the phenol from interacting with the C-terminus, including silylation. However, due to the electronics of the ring, silylation of the phenol results in an exceedingly unstable silyl ether. Even attempts to observe the presence of a silyl group by LC-MS analysis were unsuccessful. The most successful protection conditions used TIPS-OTf and 2,6-Lutidine for TIPS protection of the phenol on the linker. To monitor the effectiveness of various silylation conditions, the dipeptide N-terminus was deprotected for 3 min using a cocktail of 2% v/v DBU and 2% v/v 1-Octanethiol in DMF, followed by immediate introduction of the third amino acid as a pre-formed symmetric anhydride. Various tripeptide sequences were tested to determine the efficacy of the TIPS group for reducing DKP formation (Table 3 ). In most cases, only trace amounts of DKP were observed by LC-MS, if any. The worst sequence tested was the Ala-Ala dipeptide, resulting in 12% DKP formation. To completely avoid diketopiperazine formation, we followed the method developed by the Barany group, also known as the Fmoc-half cycle. In an Fmoc-half cycle, the first amino acid used in the reductive amination step is an allyl ester. This is followed by the acylation of the second amino acid. Instead of attaching the third amino acid through the N-terminus, the allyl group of the first amino acid is deprotected and the final amino acid is attached through the C-terminus by a peptide coupling reaction. In this case, the allyl ester was deprotected with catalytic Pd(PPh 3 ) 4 and excess PhSiH 3 and then coupled with another amino acid allyl ester through the C-terminus (Fig. 7 ). Then the rest of the peptide was built through the N-terminus by Fmoc SPPS until the peptide of desired length was built. Also worth noting that we switched from t -Bu/DMA esters to allyl esters for two of the amino acids in the sequence. When the DMA group was deprotected, and the final amino acid is coupled with another DMA ester of amino acid, the 1-octanethiol (left from the Fmoc deprotection using excess diethylamine and 1-octanethiol) could also react and generate thioesters. Therefore, we switched to using allyl esters which are synthesized on Boc-protected amino acids and then esterified with allyl bromide. Before the reductive amination and Fmoc half-cycle, the Boc group is deprotected with trifluoroacetic acid and neutralized with excess DIEA for the allyl ester to participate in reaction. The choice of model cyclic peptides Two previously synthesized cyclic peptides were chosen to model the head-to-tail cyclized peptides to examine the performance of the Hcnb linker on solid support. The cyclic decapeptide c-[Arg-(D)-Phe-Pro-Glu-Asp-Asn-Tyr-Glu-Ala-Ala, 28 ] was synthesized to demonstrate the efficacy of the original Backbone Amide Linker, published by Albericio and Barany. The 14-residue cyclic peptide c-[Pro-Asp-Gly-Arg-Cys-Thr-Lys-Ser-Ile-Pro-Pro-Ile-Cys-Phe, 30 ], also known as the Sunflower Trypsin Inhibitor, is a well-known natural product. The 14-mer cyclic Sunflower Trypsin Inhibitor poses some interesting synthetic challenges due to the high prevalence of β-branched amino acids and proline residues, which make selection of a cyclization point difficult. After several attempts, it was found that reductive amination of Phe onto the linker as the first residue, followed by cyclization at Pro yielded the best results. Cyclization and subsequent photocleavage from the resin yielded the final peptide in 90% purity and 80% cleavage yield. The synthesis of the cyclic decapeptide was accomplished through reductive amination of Ala as the first residue and the subsequent linear synthesis. Following cyclization and photocleavage from the resin, the peptide was obtained in 95% purity and 90% cleavage yield. The first peptide chosen was a short-chain analogue of the natural product somatostatin, cyclo[(D)-Trp-Lys-Gly-(β)-Ala-Phe]. The synthesis was carried out according to the procedures mentioned previously. The first residue H-Phe-ODMA was loaded onto the prepared resin 22a via reductive amination. The subsequent linear synthesis was then carried out as previously discussed. The head-to-tail cyclization was accomplished on the resin using PyAOP. Photocleavage from the resin at 365 nm for 1 h yielded the final cyclic decapeptide 29 in 50% purity and 96% cleavage yield. Synthesis of C-terminal thioesters The synthesis of C-terminal thioesters is an area of interest for their use in native chemical ligation (NCL) reactions. The Hcnb linker was used to demonstrate the on-resin synthesis of thioesters in high purities. It was established by solution-phase studies that the presence of a free phenol on the linker was problematic for synthesis of C-terminal thioesters, and so the phenol was TIPS protected prior to optimizing the coupling conditions for thioester formation. Use of carbodiimides as a coupling reagent yielded low conversions (0–30%). However, switching to HATU as a coupling reagent and preactivating the carboxylic acid for 5 min prior to addition of the thiol yielded complete conversion to the desired thioester in 2 h. A series of pentapeptides were synthesized to examine the scope of C-terminal residue compatibility with thioester synthesis. It was found that β-branched amino acids yield poor conversion to the thioester. However, it was reported by Kent that these sterically hindered amino acids are not amenable to NCL due to the steric hindrance of the carbonyl required for the transthioesterification and subsequent acyl transfer. The linear sequences were synthesized according to the standard procedure outlined previously. The final amino acid in the sequence was coupled as an N-Boc-protected residue. The C-terminal DMA protecting group was removed using Pd(0) and phenylsilane. Following TIPS protection of the linker phenol, the C-terminus was pre-activated for 5 min with HATU and Hunig’s base in DMF. Addition of thiophenol resulted in 95–100% conversion of carboxylic acid to thioester in 2 h. The thioester peptides were then globally deprotected using 5% H 2 O in TFA for 1 h and cleaved from the resin using 365 nm UV irradiation for 1 h. Depending on the identity of the first amino acid residue, peptide thioesters were obtained in 75–99% purity and 66–92% cleavage yield (Table 3 ). No epimerization of the C-terminal residue was observed. Table 4 Structures and Yields of Peptide Thioesters Structure Purity, % Cleavage Yield, % H-Phe-Lys-Ala-Ala-Leu-SPh ( 31a ) 99 83 H-Ala-Glu-Phe-Leu-Phe-SPh ( 31b ) 99 66 H-Ala-Lys-Phe-Leu-Glu-SPh ( 31c ) 75 71 H-Phe-Glu-Ala-Leu-Ala-SPh ( 31d ) 99 92 Conclusions In a quest for a photolabile backbone amide linker for the solid-phase synthesis of cyclic and C-terminally modified peptides, a survey of various photolabile motifs was conducted, establishing the o -nitrobenzyl motif as the best candidate. Problems associated with many of the candidate motifs including the nitroveratryl linker led to the development of the 2-hydroxy-4-carboxy-6-nitrobenzyl (Hcnb ) linker. A convenient and scalable synthesis of a benzaldehyde precursor ( 16 ) was developed, and general conditions were found for the anchoring of various amino esters to the linker and their acylation to form anchored dipeptides in good yield. Photolysis of the various Hcnb-linked dipeptides ( 18a-c) at 365 nm cleanly deprotected them in good yield in all cases. The linker can be attached to a wide variety of amino acids, can be selectively removed in the presence of most commonly used protecting groups and tolerates treatment with acid, base and Pd 0 . It was found that the Hcnb linker could be conveniently attached to an aminoethyl TG resin using a prolyl-6-aminohexanoic acid spacer, permitting its use in solid phase peptide synthesis. The ability of the Hcnb linker to facilitate on-resin, head-to-tail cyclization of peptides was demonstrated through the synthesis of three cyclic peptides. The utility of Hcnb to synthesize C-terminal thioesters was also demonstrated. We believe that this new photolabile linker should provide a valuable tool for the synthesis of cyclic and modified peptides, depsipeptides and peptidomimetics such as oligoureas (Boeijen 1999 ; Boeijen 2001). Declarations Competing Interests. The authors declare that they have no competing interests, financial or non-financial, that are related to the work in this publication. Author Contribution K. K., M.N. and M.L. wrote the main manuscript text. SSK, M.N. and K.K. prepared the figures. All authors reviewed the manuscript. References Amadori M (1925) Atti Accad. Naz Lincei 2(6):337 Bellof D, Mutter M (1985) A New Phenacyl-Type Handle for Polymer Supported Peptide Synthesis. 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Nature 1981, 292, 55. https://doi.org/10.1016/0024-3205(84)90009-2 Viswanathan CL, Chaudhari AS (2006) Synthesis and evaluation of uterine relaxant activity for a novel series of substituted p-hydroxyphenylethanolamines. Bioorg Med Chem 14:19, 6581–6585. https://doi.org/10.1016/j.bmc.2006.06.006 White TR, Renzelman CM, Rand AC, Rezai T, McEwen CM, Gelev VM, Turner RA, Linington RG, Leung SS, Kalgutkar AS, Bauman JN, Zhang Y, Liras S, Price DA, Mathiowetz AM, Jacobson MP, Lokey RS (2011) On-resin N-methylation of cyclic peptides for discovery of orally bioavailable scaffolds. Nat Chem Biol 7:810. https://doi.org/10.1038/nchembio.664 Tables Tables 1 to 3 are available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files SupplementaryInformationAPhotolabileBackboneAmideLinkerfortheSolidPhaseSynthesisofCyclicPeptidesandCTerminalThioesters.docx Tables.docx Cite Share Download PDF Status: Published Journal Publication published 08 Sep, 2025 Read the published version in International Journal of Peptide Research and Therapeutics → Version 1 posted Editorial decision: Revision requested 04 Aug, 2025 Reviews received at journal 03 Aug, 2025 Reviews received at journal 01 Aug, 2025 Reviewers agreed at journal 28 Jul, 2025 Reviewers agreed at journal 27 Jul, 2025 Reviewers agreed at journal 27 Jul, 2025 Reviewers agreed at journal 25 Jul, 2025 Reviewers agreed at journal 25 Jul, 2025 Reviewers agreed at journal 25 Jul, 2025 Reviewers invited by journal 25 Jul, 2025 Editor assigned by journal 21 Jul, 2025 Submission checks completed at journal 21 Jul, 2025 First submitted to journal 19 Jul, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {\"props\":{\"pageProps\":{\"initialData\":{\"identity\":\"rs-7165562\",\"acceptedTermsAndConditions\":true,\"allowDirectSubmit\":false,\"archivedVersions\":[],\"articleType\":\"Research Article\",\"associatedPublications\":[],\"authors\":[{\"id\":492007854,\"identity\":\"82a51637-c748-4f3d-a91e-296c9c300e61\",\"order_by\":0,\"name\":\"Kaif R. Khan\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Purdue University West Lafayette\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Kaif\",\"middleName\":\"R.\",\"lastName\":\"Khan\",\"suffix\":\"\"},{\"id\":492007857,\"identity\":\"a62eec28-fc63-4b00-ab00-7d6b8641b08e\",\"order_by\":1,\"name\":\"Mary L. Niedrauer\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Purdue University West Lafayette\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Mary\",\"middleName\":\"L.\",\"lastName\":\"Niedrauer\",\"suffix\":\"\"},{\"id\":492007859,\"identity\":\"63e505b4-764a-4bbf-ab88-ab028df5f097\",\"order_by\":2,\"name\":\"Matthew A. Hostetler\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Purdue University West Lafayette\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Matthew\",\"middleName\":\"A.\",\"lastName\":\"Hostetler\",\"suffix\":\"\"},{\"id\":492007861,\"identity\":\"45805d5e-e5af-40b2-b350-1229764c5663\",\"order_by\":3,\"name\":\"Soo Sung Kang\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Purdue University\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Soo\",\"middleName\":\"Sung\",\"lastName\":\"Kang\",\"suffix\":\"\"},{\"id\":492007863,\"identity\":\"3480684f-64e7-4e2a-b33c-e5e25f11a567\",\"order_by\":4,\"name\":\"Mark A. Lipton\",\"email\":\"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAuElEQVRIiWNgGAWjYBACPmYwZQMiDEAEYwMhLWzMYEVppGiBKDpMihZ23uMPPu44n2dw+/A2iQ8MNrIbDhB0GF9i48wzt4sNzqWVSc5gSDMmQguPYTNv2+3EDWd4zKR5GA4nEqvlHETLH4b/RGs5ANHCwHCAGC18iTNntiUXS55hK7bsMUg2nklICz//2QMfPrbZ5fGdYd5440eFnWwfIS0MDDxgMgHCMSCoHEPLKBgFo2AUjAIsAAAucT+M51OtzgAAAABJRU5ErkJggg==\",\"orcid\":\"\",\"institution\":\"Purdue University\",\"correspondingAuthor\":true,\"prefix\":\"\",\"firstName\":\"Mark\",\"middleName\":\"A.\",\"lastName\":\"Lipton\",\"suffix\":\"\"}],\"badges\":[],\"createdAt\":\"2025-07-19 16:08:32\",\"currentVersionCode\":1,\"declarations\":\"\",\"doi\":\"10.21203/rs.3.rs-7165562/v1\",\"doiUrl\":\"https://doi.org/10.21203/rs.3.rs-7165562/v1\",\"draftVersion\":[],\"editorialEvents\":[{\"content\":\"https://doi.org/10.1007/s10989-025-10752-9\",\"type\":\"published\",\"date\":\"2025-09-08T15:57:33+00:00\"}],\"editorialNote\":\"\",\"failedWorkflow\":false,\"files\":[{\"id\":87841935,\"identity\":\"79ad1817-ce12-49c5-bb72-667b7f91fa46\",\"added_by\":\"auto\",\"created_at\":\"2025-07-29 14:19:11\",\"extension\":\"png\",\"order_by\":1,\"title\":\"Figure 1\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":22190,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eGeneral concept of amino acid anchoring and photolytic deprotection\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"1.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-7165562/v1/94fcdd9fb19a67b81a26aa1b.png\"},{\"id\":87841939,\"identity\":\"06e0f940-cc7d-4759-8b52-7c63e903df4a\",\"added_by\":\"auto\",\"created_at\":\"2025-07-29 14:19:11\",\"extension\":\"png\",\"order_by\":2,\"title\":\"Figure 2\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":34513,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eCandidate photolabile backbone amide linkers and their synthetic precursors\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"2.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-7165562/v1/62b99a13fdd8280a45fc5007.png\"},{\"id\":87841936,\"identity\":\"fe19703c-c1d8-4d24-961f-d31b3c45df81\",\"added_by\":\"auto\",\"created_at\":\"2025-07-29 14:19:11\",\"extension\":\"png\",\"order_by\":3,\"title\":\"Figure 3\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":22851,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eTransacylation motif in the Hcnb linker\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"3.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-7165562/v1/b1e13b83d449e5df1caa3b64.png\"},{\"id\":87841941,\"identity\":\"735a4775-5241-4e25-8b66-d24d5c3d9887\",\"added_by\":\"auto\",\"created_at\":\"2025-07-29 14:19:11\",\"extension\":\"png\",\"order_by\":4,\"title\":\"Figure 4\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":30868,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003ePreparation of Hnca linker \\u003cstrong\\u003e16\\u003c/strong\\u003e\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"4.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-7165562/v1/003e607b50598143cb149b90.png\"},{\"id\":87841937,\"identity\":\"2b2bac95-c917-40d5-9a29-febf099adf89\",\"added_by\":\"auto\",\"created_at\":\"2025-07-29 14:19:11\",\"extension\":\"png\",\"order_by\":5,\"title\":\"Figure 5\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":24206,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003ePreparation of resin-bound Hcna linkers \\u003cstrong\\u003e22a-b\\u003c/strong\\u003e\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"5.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-7165562/v1/9d89f2e691b68d92fe91c621.png\"},{\"id\":87843368,\"identity\":\"bcb4cd5a-f4ae-4694-a080-f6ec85a98014\",\"added_by\":\"auto\",\"created_at\":\"2025-07-29 14:35:11\",\"extension\":\"png\",\"order_by\":6,\"title\":\"Figure 6\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":28278,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eAddition of first and second amino acid residues to the Hcna linker\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"6.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-7165562/v1/37566ac1b7c6fcb3d734a1d9.png\"},{\"id\":87841947,\"identity\":\"f0e1050c-0bb9-4c89-b3fa-e116ff4f3650\",\"added_by\":\"auto\",\"created_at\":\"2025-07-29 14:19:11\",\"extension\":\"png\",\"order_by\":7,\"title\":\"Figure 7\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":25585,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eThe Fmoc-half cycle to synthesize tripeptides and avoid diketopiperazine formation\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"7.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-7165562/v1/1b479d7f5675709f567af816.png\"},{\"id\":87841957,\"identity\":\"c1f23250-4953-4177-b3e4-bf748fbd97db\",\"added_by\":\"auto\",\"created_at\":\"2025-07-29 14:19:11\",\"extension\":\"png\",\"order_by\":8,\"title\":\"Figure 8\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":47251,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eCyclization of 5-, 10-, and 14- residue cyclic peptides\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"8.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-7165562/v1/8975bf343abab791f44502e5.png\"},{\"id\":91359079,\"identity\":\"d4884417-6058-4f71-a8b8-a23e75d1c7fb\",\"added_by\":\"auto\",\"created_at\":\"2025-09-15 16:05:08\",\"extension\":\"pdf\",\"order_by\":0,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"manuscript-pdf\",\"size\":1521406,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"manuscript.pdf\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-7165562/v1/fac19e9b-0466-482f-b390-3308c3e158c4.pdf\"},{\"id\":87841973,\"identity\":\"3974f5a3-719f-4ba8-bcb5-76c4fcdcae61\",\"added_by\":\"auto\",\"created_at\":\"2025-07-29 14:19:13\",\"extension\":\"docx\",\"order_by\":1,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":53692931,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"SupplementaryInformationAPhotolabileBackboneAmideLinkerfortheSolidPhaseSynthesisofCyclicPeptidesandCTerminalThioesters.docx\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-7165562/v1/3eb9c5bdb02e80a62a8d1d93.docx\"},{\"id\":87842929,\"identity\":\"869f9b42-2efe-4e6f-9a8a-1b6eee34a975\",\"added_by\":\"auto\",\"created_at\":\"2025-07-29 14:27:11\",\"extension\":\"docx\",\"order_by\":2,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":267894,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"Tables.docx\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-7165562/v1/b131e4b8d876c515b5de750b.docx\"}],\"financialInterests\":\"No competing interests reported.\",\"formattedTitle\":\"A Photolabile Backbone Amide Linker for the Solid-Phase Synthesis of Cyclic Peptides and C-Terminal Thioesters\",\"fulltext\":[{\"header\":\"Introduction\",\"content\":\"\\u003cp\\u003eThe head-to-tail cyclization of peptides (Fusetani \\u003cspan citationid=\\\"CR17\\\" class=\\\"CitationRef\\\"\\u003e1993\\u003c/span\\u003e; Humphrey \\u003cspan citationid=\\\"CR24\\\" class=\\\"CitationRef\\\"\\u003e1997\\u003c/span\\u003e; Katsara 2006) is a common and important strategy, employed both in nature and in synthetic applications, for improving the bioavailability and conformational rigidity of polypeptides (Kessler \\u003cspan citationid=\\\"CR32\\\" class=\\\"CitationRef\\\"\\u003e1982\\u003c/span\\u003e; Samanen 1991; Biron 2008). The lack of either an N- or C-terminus increases proteolytic stability while internal hydrogen bonding and a more compact structure improve the membrane permeability of cyclic peptides (White 2011; Dougherty 2019). These advantages are reflected in the prominence of cyclic peptides among peptidic therapeutic agents (Veber 1981; Darkin-Rattray 1996; Coulouarn 1998; Foister 2006), such as gramicidin S (Gause \\u003cspan citationid=\\\"CR19\\\" class=\\\"CitationRef\\\"\\u003e1944\\u003c/span\\u003e) and cyclosporine A (Shevach \\u003cspan citationid=\\\"CR55\\\" class=\\\"CitationRef\\\"\\u003e1985\\u003c/span\\u003e). The chemical synthesis of cyclic peptides, however, has been complicated by the traditional approach to solid-phase peptide synthesis using the C-terminal carboxylate to covalently attach the growing peptide to a polymeric support. The most straightforward approach involving detachment of a fully protected linear peptide precursor from the resin followed by macrocyclization in solution is complicated by solubility problems, oligomerization and C-terminal epimerization reactions. To avoid the problems associated with head-to-tail cyclization in solution, various approaches have been devised for on-resin cyclization of peptides, including Kaiser\\u0026rsquo;s Oxime resin (Mihara 1995), Richter\\u0026rsquo;s thioester linker (Richter 1994), Waldmann\\u0026rsquo;s hydrazide linker (Rosenbaum \\u003cspan citationid=\\\"CR48\\\" class=\\\"CitationRef\\\"\\u003e2001\\u003c/span\\u003e), Kenner\\u0026rsquo;s sulfonamide linker (Kenner 1971) and side-chain attachment of the linear peptide to the resin (Krishnamoorthy 2006). An important general strategy for the on-resin, head-to-tail cyclization of peptides was introduced by the \\u0026ldquo;backbone amide linker\\u0026rdquo; (BAL) strategy of Albericio and Barany (Jensen 1998). In this approach, the nitrogen atom of an internal amide bond is used to tether the growing peptide to the polymeric resin, thereby liberating both N- and C-termini for modification. It is noteworthy that this strategy also addresses the more general problem of C-terminal modification of the resin-bound peptide. Since its introduction, the BAL strategy has been employed by a number of researchers and several modifications have been developed (Bourne 1999; Bourne 2001; Boas 2002; Brask 2003; Boas 2004; Pittelkow 2005; Jessing 2006; Liley 2006; Pittelkow 2006). Additional applications of the BAL strategy involve the synthesis of C-terminal modifications, such as trithioorthoesters (Guillaumie 2000; Kappel \\u003cspan citationid=\\\"CR29\\\" class=\\\"CitationRef\\\"\\u003e2005\\u003c/span\\u003e), aldehydes (Mori 2004; Soural \\u003cspan citationid=\\\"CR56\\\" class=\\\"CitationRef\\\"\\u003e2007\\u003c/span\\u003e; Springer 2008), or functionalized amides.\\u003c/p\\u003e\\u003cp\\u003eAnother application of the C-terminal modification of peptides was revealed by the discovery of the native chemical ligation of peptide bonds by Kent, Muir and Dawson (Dawson 1994). Their use of peptide thioesters to couple with N-terminal cysteine residues has necessitated the development of various methods for the solid-phase synthesis of peptide thioesters (Mende \\u003cspan citationid=\\\"CR39\\\" class=\\\"CitationRef\\\"\\u003e2011\\u003c/span\\u003e; Li 2007). Since that landmark discovery, related processes such as Staudinger ligation (Nilsson 2000) have been developed that likewise depend on the availability of peptide thioesters. In response to this need, a number of creative approaches to the solid-phase synthesis of peptide thioesters have been developed, yet the availability of C-terminal modification in the BAL strategy remains one of the most straightforward approaches to the synthesis of peptide thioesters.\\u003c/p\\u003e\\u003cp\\u003eAlthough acidic deprotection conditions are widely used in peptide synthesis, there are situations involving acid-sensitive functionality that may require alternative deprotection conditions. For these cases, other methodologies have been explored and developed (Lloyd 1993; Guillier 2000; Bochet 2002; Heidler \\u003cspan citationid=\\\"CR22\\\" class=\\\"CitationRef\\\"\\u003e2005\\u003c/span\\u003e; McAllister 2005; Scott \\u003cspan citationid=\\\"CR50\\\" class=\\\"CitationRef\\\"\\u003e2006\\u003c/span\\u003e; Kang \\u003cspan citationid=\\\"CR28\\\" class=\\\"CitationRef\\\"\\u003e2008\\u003c/span\\u003e). One of the most appealing ideas has been to use photochemical detachment from the resin as an extra dimension of protecting group orthogonality in solid phase peptide synthesis. Photolabile linkers (Gallop 1994; Liang 2016), in which long-wave UV radiation (300\\u0026thinsp;~\\u0026thinsp;360 nm) is used to detach an organic functional group \\u0026ndash; usually a carboxylic acid, a carbamate (a latent amine), an alcohol, or a phosphate \\u0026ndash; from a polymer or glass surface, provide an alternative to the classic acidic or basic cleavage protocols used in solid-phase organic synthesis. Ideally, photochemical deprotection should proceed with high functional group selectivity, low amounts of side reactions and involve no separation of product from reactants. Photochemical deprotection has also found favor with applications in the parallel synthesis of compound libraries (Sheehan \\u003cspan citationid=\\\"CR52\\\" class=\\\"CitationRef\\\"\\u003e1973\\u003c/span\\u003e).\\u003c/p\\u003e\\u003cp\\u003e\\u003c/p\\u003e\\u003cp\\u003eHerein we describe studies directed toward the development of a photolabile BAL linker (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003e) to address the problems associated with the BAL strategy as a general method for the synthesis of cyclic and C-terminally modified peptides. Our studies began with a survey of several known photolabile motifs for their applicability to BAL linker design (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003e). The candidate photolabile motifs studied were the \\u003cem\\u003ep\\u003c/em\\u003e-oxyphenacyl (Op) (Bellof \\u003cspan citationid=\\\"CR2\\\" class=\\\"CitationRef\\\"\\u003e1985\\u003c/span\\u003e), α-methyl-\\u003cem\\u003ep\\u003c/em\\u003e-oxyphenacyl (Mop) (Sheehan 1965; Peach 1995), \\u003cem\\u003ep\\u003c/em\\u003e-oxybenzoin (Obz) (Bergmark 1985), α,\\u003cem\\u003eo\\u003c/em\\u003e-dimethyl phenacyl (Dmp) (Patchornick 1970) and 6-nitroveratryl (Nve). All of these photolabile groups undergo facile n to π* or π to π* excitation at 300\\u0026ndash;360 nm and release oxygen-based substituents with few side reactions. Appropriate synthetic precursors \\u003cb\\u003e1\\u003c/b\\u003e\\u0026ndash;\\u003cb\\u003e5\\u003c/b\\u003e were chosen for each of the motifs, as shown in Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003e.\\u003c/p\\u003e\\u003cp\\u003e\\u003c/p\\u003e\\u003cp\\u003eConsequently, our preliminary survey focused on the Op, Mop, Obz, Dmp, and Nve motifs, their preparation and photolysis. Of specific interest were a) the ease of anchoring amino acids to the linker, b) the rate and efficiency of photocleavage, and c) the suppression of side reactions during photolytic deprotection. To assess these questions, the dipeptide Boc-Phe-Gly-OMe (\\u003cb\\u003e8\\u003c/b\\u003e) was chosen as a model system. After the photolabile motif was chosen, effort was directed toward its attachment to a solid support compatible with the solvents and reagents common to peptide synthesis. Finally, the problems of diketopiperazine formation, photocleavage optimization, and maximization of cyclization yields were addressed.\\u003c/p\\u003e\"},{\"header\":\"Material and Methods\",\"content\":\"\\u003cp\\u003e\\u003cb\\u003eSynthesis of Dmp\\u003c/b\\u003e\\u003c/p\\u003e\\u003cp\\u003e\\u003cb\\u003e1-(2,5-dimethyl-4-alkylphenyl)-propanone (4b)\\u003c/b\\u003e To a mixture of propionyl chloride (0.51 mL, 5.88 mmol) and AlCl\\u003csub\\u003e3\\u003c/sub\\u003e (780 mg, 5.88 mmol) in CH\\u003csub\\u003e2\\u003c/sub\\u003eCl\\u003csub\\u003e2\\u003c/sub\\u003e (25 mL) was added a solution of 4-(2,5-dimethylphenyl)butanoic acid (\\u003cb\\u003e4a\\u003c/b\\u003e, 377 mg, 1.96 mmol) in CH\\u003csub\\u003e2\\u003c/sub\\u003eCl\\u003csub\\u003e2\\u003c/sub\\u003e (5 ml) at 0\\u0026deg;C. The reaction mixture was stirred for 1 h at the same temperature and poured into cold HCl solution (40 g ice and 10 mL of conc. HCl). The organic material was extracted by addition of CH\\u003csub\\u003e2\\u003c/sub\\u003eCl\\u003csub\\u003e2\\u003c/sub\\u003e (25 mL) and the aqueous layer was extracted with another portion of CH\\u003csub\\u003e2\\u003c/sub\\u003eCl\\u003csub\\u003e2\\u003c/sub\\u003e (50 mL). The combined organic layer was washed with brine (50 mL), dried with anhydrous MgSO\\u003csub\\u003e4\\u003c/sub\\u003e, passed through 3 cm silica gel pad, and evaporated under reduced pressure. The crude product was dissolved in oxalyl chloride (3 mL) and stirred overnight. After removal extra oxalyl chloride by evaporation under reduced pressure, the organic material was dissolved in CH\\u003csub\\u003e2\\u003c/sub\\u003eCl\\u003csub\\u003e2\\u003c/sub\\u003e (30 mL). To this solution, benzyl amine (0.257 mL, 2.35 mmol) and DIEA (1.7 mL, 9.8 mmol) were added and stirred 3 h. The organic layer was washed with brine (30 mL), dried with anhydrous MgSO\\u003csub\\u003e4\\u003c/sub\\u003e, evaporated under reduced pressure and purified by flash column chromatography using 33% EtOAc in Hexanes to give \\u003cb\\u003e4b\\u003c/b\\u003e as a pale-yellow solid.\\u003c/p\\u003e\\u003cp\\u003e\\u003cb\\u003e1-(2,5-dimethyl-4-alkylphenyl)-1-hydroxypropan-2-one oxime (4c)\\u003c/b\\u003e To a solution of \\u003cb\\u003e4b\\u003c/b\\u003e (0.51g, 1.50 mmol) and isoamyl nitrite (0.24 mL, 1.80 mmol) in MeOH (10 mL) at 45\\u0026deg;C was added dropwise to concentrated HC1 (0.25 mL) and the resulting mixture was stirred for 3 h at the same temperature. After cooling to room temperature, the organic material was partitioned into EtOAc (40 mL) and H\\u003csub\\u003e2\\u003c/sub\\u003eO (40 mL). The organic layer was washed with brine (40 mL), dried with anhydrous MgSO\\u003csub\\u003e4\\u003c/sub\\u003e, passed through 3cm silica gel pad, and evaporated under reduced pressure. The residue was dissolved in MeOH (15 mL) and NaBH\\u003csub\\u003e4\\u003c/sub\\u003e (50 mg) was added under ice bath. After stirring for 30 min, the organic material was partitioned into EtOAc (40 mL) and HCl solution (0.1 M, 30 mL). The organic layer was washed with saturated NaHCO\\u003csub\\u003e3\\u003c/sub\\u003e (30 mL) and with brine (30 mL), dried over anhydrous MgSO\\u003csub\\u003e4\\u003c/sub\\u003e, and evaporated under reduced pressure. The residue was purified by flash column chromatography using 66% EtOAc in hexanes to give \\u003cb\\u003e4c\\u003c/b\\u003e as a white solid.\\u003c/p\\u003e\\u003cp\\u003e\\u003cb\\u003e1-(2,5-dimethyl-4-alkylphenyl)-1-hydroxypropan-2-one (4)\\u003c/b\\u003e To a solution of \\u003cb\\u003e4c\\u003c/b\\u003e (0.30 g, 0.81 mmol) in DMF (2 mL) was added glyoxylic acid (60% water solution, 5 mL). After stirring for 5 h, the organic material was partitioned into EtOAc (40 mL) and H\\u003csub\\u003e2\\u003c/sub\\u003eO (40 mL). The organic layer was washed with brine (2 X 30 mL), dried with anhydrous MgSO\\u003csub\\u003e4\\u003c/sub\\u003e, evaporated under reduced pressure, and the residue was purified by flash column chromatography using 50% EtOAc in hexanes to give \\u003cb\\u003e4\\u003c/b\\u003e as a pale-yellow oil.\\u003c/p\\u003e\\u003cp\\u003e\\u003cb\\u003eAlkylation of amino ester with photolabile linker 1\\u0026ndash;5 (6a-e)\\u003c/b\\u003e\\u003c/p\\u003e\\u003cp\\u003e\\u003cb\\u003eH-[Op]-Gly-OMe (6a)\\u003c/b\\u003e. To a solution of \\u003cb\\u003e1\\u003c/b\\u003e, (150 mg, 0.66 mmol) in anhydrous CH\\u003csub\\u003e3\\u003c/sub\\u003eCN (5 mL) were added GlyOMe\\u0026middot;HCl (248 mg, 1.98 mmol), and DIEA (0.52 mL, 2.97 mmol). The reaction mixture was stirred overnight at room temperature, evaporated under reduced pressure and partitioned into CH\\u003csub\\u003e2\\u003c/sub\\u003eCl\\u003csub\\u003e2\\u003c/sub\\u003e (20 mL) and H\\u003csub\\u003e2\\u003c/sub\\u003eO (20 mL). The organic layer was washed with brine (20 mL), dried with anhydrous MgSO\\u003csub\\u003e4\\u003c/sub\\u003e, evaporated under reduced pressure, and purified by flash column chromatography using 33% EtOAc in hexanes to give \\u003cb\\u003e6a\\u003c/b\\u003e as a pale-yellow oil.\\u003c/p\\u003e\\u003cp\\u003e\\u003cb\\u003eH-[Mop]-Gly-OMe (6b)\\u003c/b\\u003e. To a solution of \\u003cb\\u003e2\\u003c/b\\u003e, (200 mg, 0.63 mmol) in anhydrous CH\\u003csub\\u003e3\\u003c/sub\\u003eCN (8 mL) were added GlyOMe\\u0026middot;HCl (237 mg, 1.89 mmol), DIEA (0.49 mL, 2.84 mmol) and tetrabutylammonium iodide (25 mg). The reaction mixture was refluxed for overnight, cooled to room temperature, evaporated under reduced pressure, and partitioned into CH\\u003csub\\u003e2\\u003c/sub\\u003eCl\\u003csub\\u003e2\\u003c/sub\\u003e (20 mL) and H\\u003csub\\u003e2\\u003c/sub\\u003eO (20 mL). The organic layer was washed with brine (20 mL), dried with anhydrous MgSO\\u003csub\\u003e4\\u003c/sub\\u003e, evaporated under reduced pressure, and purified by flash column chromatography using 33% EtOAc in hexanes to give \\u003cb\\u003e6b\\u003c/b\\u003e as a colorless oil.\\u003c/p\\u003e\\u003cp\\u003e\\u003cb\\u003eH-[Obz]-Gly-OMe (6c)\\u003c/b\\u003e. To a solution of \\u003cb\\u003e3\\u003c/b\\u003e, (123 mg, 0.39 mmol) in anhydrous MeOH (20 mL) was added GlyOMe\\u0026middot;HCl (242 mg, 1.93 mmol). The reaction mixture was refluxed for 24 h, cooled to room temperature, evaporated under reduced pressure, and partitioned into CH\\u003csub\\u003e2\\u003c/sub\\u003eCl\\u003csub\\u003e2\\u003c/sub\\u003e (20 mL) and H\\u003csub\\u003e2\\u003c/sub\\u003eO (20 mL). The organic layer was washed with brine (20 mL), dried with anhydrous MgSO\\u003csub\\u003e4\\u003c/sub\\u003e, evaporated under reduced pressure, and purified by flash column chromatography using 33% EtOAc in hexanes to give \\u003cb\\u003e6c\\u003c/b\\u003e as a pale-yellow oil.\\u003c/p\\u003e\\u003cp\\u003e\\u003cb\\u003eH-[Dmp]-Gly-OMe (6d)\\u003c/b\\u003e. To a solution of \\u003cb\\u003e4\\u003c/b\\u003e, (200 mg, 0.57 mmol) in anhydrous toluene (20 mL) was added GlyOMe\\u0026middot;HCl (215 mg, 1.71 mmol). The reaction mixture was refluxed for 5 h, cooled to room temperature, and partitioned into EtOAc (20 mL) and H\\u003csub\\u003e2\\u003c/sub\\u003eO (40 mL). The organic layer was washed with brine (40 mL), dried with anhydrous MgSO\\u003csub\\u003e4\\u003c/sub\\u003e, evaporated under reduced pressure, and purified by flash column chromatography using 33% EtOAc in hexanes to give \\u003cb\\u003e6d\\u003c/b\\u003e as a colorless oil.\\u003c/p\\u003e\\u003cp\\u003e\\u003cb\\u003eH-[Nve]-Gly-OMe (6e).\\u003c/b\\u003e To a solution of \\u003cb\\u003e5\\u003c/b\\u003e (515 mg, 2.44 mmol) in MeOH (20 mL), were added GlyOMe\\u0026middot;HCl (459 mg, 3.66 mmol), sodium bicarbonate (348 mg, 4.15 mmol). The reaction mixture was stirred for 4 h, cooled to 0\\u0026deg;C. After portionwise addition of NaBH\\u003csub\\u003e4\\u003c/sub\\u003e (6.0 mmol, 227 mg), the mixture was stirred for 30 min and poured into HCl solution (0.1N, 50 mL). The pH of water layer was adjusted to 4\\u0026thinsp;~\\u0026thinsp;6 by dropwise addition of NaOH (1 M), and the organic material was extracted with CH\\u003csub\\u003e2\\u003c/sub\\u003eCl\\u003csub\\u003e2\\u003c/sub\\u003e (50 mL). The aqueous layer was extracted with another portion of CH\\u003csub\\u003e2\\u003c/sub\\u003eCl\\u003csub\\u003e2\\u003c/sub\\u003e (50 mL). The combined organic layer was dried with anhydrous MgSO\\u003csub\\u003e4\\u003c/sub\\u003e, evaporated under reduced pressure and purified by flash column chromatography using 50% EtOAc in hexanes to give \\u003cb\\u003e6e\\u003c/b\\u003e as a colorless oil.\\u003c/p\\u003e\\u003cp\\u003e\\u003cb\\u003eAcylation of 6a-e using symmetric anhydrides (7a-e).\\u003c/b\\u003e The symmetric anhydride of Boc-Phe was prepared \\u003cem\\u003ein situ\\u003c/em\\u003e by mixing Boc-Phe (860 mg, 4 mmol) with EDCI (0.422 g, 2.2 mmol) in CH\\u003csub\\u003e2\\u003c/sub\\u003eCl\\u003csub\\u003e2\\u003c/sub\\u003e (10 ml, 0.2 M) for 5 min. To the solution of symmetric anhydride was added \\u003cb\\u003e6a-e\\u003c/b\\u003e (1 mmol), and the reaction mixture was stirred for 24 h at room temperature. After dilution with CH\\u003csub\\u003e2\\u003c/sub\\u003eCl\\u003csub\\u003e2\\u003c/sub\\u003e (50 ml), the organic layer was washed with brine (50 mL), dried with anhydrous MgSO\\u003csub\\u003e4\\u003c/sub\\u003e, and evaporated under reduced pressure. The residue was purified by flash column chromatography to obtain an Hcnb-linked dipeptide (\\u003cb\\u003e7a-e\\u003c/b\\u003e).\\u003c/p\\u003e\\u003cp\\u003e\\u003cb\\u003eSynthesis of Hcna\\u003c/b\\u003e\\u003c/p\\u003e\\u003cp\\u003e\\u003cb\\u003eMethyl (\\u003c/b\\u003e\\u003cb\\u003eE\\u003c/b\\u003e\\u003cb\\u003e)-4-(2-(dimethylamino)vinyl)-3,5-dinitrobenzoate (13)\\u003c/b\\u003e. To a stirred mixture of 4-Methyl-3,5-dinitrobenzoic acid (\\u003cb\\u003e12\\u003c/b\\u003e) (2.0 g, 8.8 mmol) in anhydrous toluene (29 mL) was added DMF dimethyl acetal (3.2 mL, 24 mmol) dropwise at ambient temperature. The resulting mixture was heated to reflux in an oil bath and became dark red in color. After 16 h, the reaction was cooled to room temperature and the solvent removed under reduced pressure by co-evaporation with MeOH. The crude residue was recrystallized from hot ethanol to afford enamine \\u003cb\\u003e13\\u003c/b\\u003e as a lustrous green solid which became red upon grinding.\\u003c/p\\u003e\\u003cp\\u003e\\u003cb\\u003eMethyl 4-formyl-3,5-dinitrobenzoate (14).\\u003c/b\\u003e To a mixture of \\u003cb\\u003e13\\u003c/b\\u003e (1.08 g, 3.66 mmol) and RuCl\\u003csub\\u003e3\\u003c/sub\\u003e solution (0.1 M in H\\u003csub\\u003e2\\u003c/sub\\u003eO, 1.2 mL, 0.13 mmol) in CH\\u003csub\\u003e3\\u003c/sub\\u003eCN-H\\u003csub\\u003e2\\u003c/sub\\u003eO (6:1, 37 mL) was added NaIO\\u003csub\\u003e4\\u003c/sub\\u003e (2.0 g, 9.1 mmol) in portions at rt. The color quickly dissipated, and solids formed. The reaction continued until TLC analysis showed consumption of starting material (typically 10\\u0026ndash;30 min). After stirring for 30 min, the solids were removed by vacuum filtration and washed with CH\\u003csub\\u003e2\\u003c/sub\\u003eCl\\u003csub\\u003e2\\u003c/sub\\u003e. The filtrate was partitioned between H\\u003csub\\u003e2\\u003c/sub\\u003eO and CH\\u003csub\\u003e2\\u003c/sub\\u003eCl\\u003csub\\u003e2\\u003c/sub\\u003e and the aqueous layer was extracted twice with CH\\u003csub\\u003e2\\u003c/sub\\u003eCl\\u003csub\\u003e2\\u003c/sub\\u003e. The combined organic extracts were washed with brine and sat. aq. Na\\u003csub\\u003e2\\u003c/sub\\u003eS\\u003csub\\u003e2\\u003c/sub\\u003eO\\u003csub\\u003e4\\u003c/sub\\u003e, dried over Na\\u003csub\\u003e2\\u003c/sub\\u003eSO\\u003csub\\u003e4\\u003c/sub\\u003e, and concentrated under reduced pressure. The crude residue was purified by flash column chromatography (30% EA/Hex) to afford \\u003cb\\u003e14\\u003c/b\\u003e as an off-white solid.\\u003c/p\\u003e\\u003cp\\u003e\\u003cb\\u003eMethyl 4-formyl-3-hydroxy-5-nitrobenzoate (15).\\u003c/b\\u003e A solution of \\u003cb\\u003e14\\u003c/b\\u003e (678 mg, 2.67 mmol) in anhydrous DMF (5.1 mL) was added dropwise to a mixture of acetaldoxime [mixture of isomers] (325 \\u0026micro;L, 5.33 mmol) and K\\u003csub\\u003e2\\u003c/sub\\u003eCO\\u003csub\\u003e3\\u003c/sub\\u003e (811 mg, 5.87 mmol) in anhydrous DMF (5.1 mL) dropwise at rt. The reaction mixture immediately turned bright red purple upon mixing, then brownish green after stirring overnight. After 21 h, H\\u003csub\\u003e2\\u003c/sub\\u003eO (10 mL) was added to the reaction and the resulting solution extracted 5x with Et\\u003csub\\u003e2\\u003c/sub\\u003eO. The aqueous layer was then acidified to pH\\u0026thinsp;~\\u0026thinsp;1\\u0026ndash;2 with concentrated HCl and extracted 4x with CH\\u003csub\\u003e2\\u003c/sub\\u003eCl\\u003csub\\u003e2\\u003c/sub\\u003e. The combined organic extracts were dried over Na\\u003csub\\u003e2\\u003c/sub\\u003eSO\\u003csub\\u003e4\\u003c/sub\\u003e, filtered and concentrated under reduced pressure. The crude residue was purified by flash chromatography (25% EA/Hex) to afford \\u003cb\\u003e15\\u003c/b\\u003e as a bright yellow solid.\\u003c/p\\u003e\\u003cp\\u003e\\u003cb\\u003e4-Formyl-3-hydroxy-5-nitrobenzoic acid (16).\\u003c/b\\u003e Solid Ba(OH)\\u003csub\\u003e2\\u003c/sub\\u003e∙8 H\\u003csub\\u003e2\\u003c/sub\\u003eO (1.97 g, 6.24 mmol) was added in portions to a solution of \\u003cb\\u003e15\\u003c/b\\u003e (468 mg, 2.08 mmol) in MeOH (23 mL) at rt. Following reaction completion (TLC), the mixture was concentrated under reduced pressure and the crude residue was partitioned between EtOAc and H\\u003csub\\u003e2\\u003c/sub\\u003eO. The aqueous layer was extracted twice with EtOAc, acidified to pH\\u0026thinsp;~\\u0026thinsp;1\\u0026ndash;2 with concentrated HCl and extracted with EtOAc (3x). The combined organic extracts were washed with brine, dried over Na\\u003csub\\u003e2\\u003c/sub\\u003eSO\\u003csub\\u003e4\\u003c/sub\\u003e, filtered and concentrated under reduced pressure to afford \\u003cb\\u003e16\\u003c/b\\u003e (379 mg, 86%) as a brown solid.\\u003c/p\\u003e\\u003cp\\u003e\\u003cb\\u003eGeneral procedure for solution-phase reductive amination (17a-c).\\u003c/b\\u003e HCl-AA-OtBu (0.666 mmol) was added to a solution of \\u003cb\\u003e15\\u003c/b\\u003e (200.0 mg, 0.666 mmol) and DIEA (244 \\u0026micro;L, 1.40 mmol) in CH\\u003csub\\u003e3\\u003c/sub\\u003eCN (9.5 mL). The reaction was stirred at rt for 45 min. Volatiles were removed and the residue was dissolved in MeOH (3.3 m). NaBH\\u003csub\\u003e4\\u003c/sub\\u003e (75.6 mg, 2.00 mmol) was added, and the reaction was stirred for 15 min. Volatiles were removed and the residue was purified by flash column chromatography (20\\u0026ndash;45% EtOAc/Hex) to yield \\u003cb\\u003e17a-d.\\u003c/b\\u003e\\u003cdiv class=\\\"BlockQuote\\\"\\u003e\\u003cp\\u003e\\u003cb\\u003eGeneral procedure for solution-phase acylation (18a-c).\\u003c/b\\u003e A mixture of Fmoc-AA-OH (0.29 mmol) and DIPC (22.5 \\u0026micro;L, 0.14 mmol) was preactivated in dry CH\\u003csub\\u003e2\\u003c/sub\\u003eCl\\u003csub\\u003e2\\u003c/sub\\u003e: DMF (1:1) (0.6 mL) for 5 min. \\u003cb\\u003e17a-c\\u003c/b\\u003e (0.13 mmol) as a solution in CH\\u003csub\\u003e2\\u003c/sub\\u003eCl\\u003csub\\u003e2\\u003c/sub\\u003e (2.1 mL) was added and the reaction was stirred for 2 h. The volatiles were removed and then dissolved in methanol (1.0 mL) and a solution of ammonium acetate (80.9mg, 1.05 mmol) in water (0.3 mL) was added and the reaction was stirred overnight. Volatiles were removed and the residues were washed with water and extracted with ethyl acetate three times, concentrated, and then purified by flash column chromatography (30\\u0026ndash;40% EA/Hex).\\u003c/p\\u003e\\u003c/div\\u003e\\u003c/p\\u003e\\u003cp\\u003e\\u003cb\\u003eSolid Phase Peptide Synthesis\\u003c/b\\u003e\\u003c/p\\u003e\\u003cp\\u003e\\u003cb\\u003ePreparation of Hcna-loaded resin (22a-b).\\u003c/b\\u003e Fmoc-Sieber Amide PS Resin (0.6 mmol/g) or TentaGel S NH\\u003csub\\u003e2\\u003c/sub\\u003e (0.26 mmol/g) was swelled in DMF for 15 min, drained, and treated with piperidine-DMF (1:4, 1 x 2 mL, 30 min). The mixture was drained and washed with standard wash protocol [DMF (3 x 1 mL), CH\\u003csub\\u003e2\\u003c/sub\\u003eCl\\u003csub\\u003e2\\u003c/sub\\u003e (3 x 1 mL), MeOH (3 x 1 mL), CH\\u003csub\\u003e2\\u003c/sub\\u003eCl\\u003csub\\u003e2\\u003c/sub\\u003e (3 x 1 mL), then DMF (3 x 1 mL)] to afford a positive Kaiser ninhydrin test. To a mixture of \\u003cem\\u003eN\\u003c/em\\u003e-Fmoc-6-aminohexanoic acid (4 eq) and HATU (3.9 eq) in DMF (0.03 M) was added DIEA (8 eq) and this mixture was added to the resin (pre-washed with DMF) after a 5 min pre-activation period. After 2 h, the mixture was drained and washed with the standard wash protocol to afford a negative Kaiser ninhydrin test. The resin was then agitated with piperidine-DMF (1:4, 1 x 2 mL, 30 min). The mixture was drained, and the resin washed with standard wash protocol to afford a positive Kaiser ninhydrin test. To a mixture of Fmoc-Pro-OH (4 eq), HATU (3.9 eq) and HOAt (4 eq) in DMF (0.03 M) was added DIEA (8 eq) and this mixture was added to the resin (pre-washed with DMF) after a 5 min pre-activation period. After 2 h, the mixture was drained and washed with the standard wash protocol to afford a negative Kaiser ninhydrin test. The resin was then treated with piperidine-DMF (1:4, 1 x 2 mL, 30 min). The mixture was drained, and resin washed with standard wash protocol to afford a positive chloranil test. To a mixture of \\u003cb\\u003e16\\u003c/b\\u003e (3 eq) and PyBOP in DMF (0.03 M) was added DIEA (6 eq) and this mixture was added to the resin (pre-washed with DMF) after a 5 min pre-activation period. After 2 h, the mixture was drained and washed with the standard wash protocol to afford a negative chloranil test.\\u003c/p\\u003e\\u003cp\\u003e\\u003cb\\u003eGeneral procedure for solid-phase reductive amination.\\u003c/b\\u003e The prepared resin \\u003cb\\u003e22a\\u003c/b\\u003e or \\u003cb\\u003e22b\\u003c/b\\u003e was swelled in DMF for 15 min then treated with a mixture of H-AA-ODMA or HCl-AA-O\\u003cem\\u003et\\u003c/em\\u003eBu (10 eq) and AcOH (10 eq) in CH\\u003csub\\u003e2\\u003c/sub\\u003eCl\\u003csub\\u003e2\\u003c/sub\\u003e (0.03 M). After 3 h, the mixture was drained and washed briefly with standard wash protocol then THF-MeOH (2:1). The resin was then taken up in THF-MeOH (2:1, 0.03 M) and treated with solid NaBH\\u003csub\\u003e4\\u003c/sub\\u003e (5 eq). After 2 h, the mixture was drained and washed with H\\u003csub\\u003e2\\u003c/sub\\u003eO, standard wash protocol, then piperidine-DMF (1:4, 5 min). The resin was washed once more with standard wash protocol to afford a positive chloranil test.\\u003c/p\\u003e\\u003cp\\u003e\\u003cb\\u003eGeneral procedure for loading of second amino acid residue.\\u003c/b\\u003e A flame-dried flask was charged with the 2nd Amino Acid residue (4 eq) and dry CH\\u003csub\\u003e2\\u003c/sub\\u003eCl\\u003csub\\u003e2\\u003c/sub\\u003e (0.05 M), to which was added DIPC (2 eq) dropwise at rt. The mixture was stirred at rt for 12 min and the resulting solution was solubilized by addition of DMF (0.05 M). This solution was added directly to prepared resin \\u003cb\\u003e23a\\u003c/b\\u003e or \\u003cb\\u003e23b\\u003c/b\\u003e and the mixture was agitated for 2 h, followed by the standard wash protocol to afford a slightly positive chloranil test. This procedure was repeated once more to afford resin-bound dipeptide (and a negative chloranil test).\\u003c/p\\u003e\\u003cp\\u003e\\u003cb\\u003eGeneral procedure for coupling of third amino acid residue.\\u003c/b\\u003e A solution of 1-octanthiol (10 eq), DIEA (5 eq), and DMF (0.03 M) was added to the prepared resin \\u003cb\\u003e24a\\u003c/b\\u003e or \\u003cb\\u003e24b\\u003c/b\\u003e and the mixture was agitated for 1 h, followed by the standard wash protocol. Next, a mixture of TIPS-OTf (10 eq), 2,6-Lutidine (10 eq), and DMF (0.03 M) was added to the resin and the mixture was agitated for 4 h, followed by the standard wash protocol. A flame-dried flask was charged with an Fmoc-amino acid (4 eq) and dry CH\\u003csub\\u003e2\\u003c/sub\\u003eCl\\u003csub\\u003e2\\u003c/sub\\u003e (0.05 M), to which was added DIPC (2 eq) dropwise at rt. The mixture was stirred at rt for 12 min and the resulting mixture was diluted with DMF (0.05 M).\\u003c/p\\u003e\\u003cp\\u003eThe resin was agitated with a solution of DBU-1-Octanthiol-DMF (2:2:96, 1 x 2 mL, 3 min), washed with the standard wash protocol, and the pre-activated amino acid solution was added, taking care to minimize the time between deprotection and coupling. The mixture was agitated for 2 h, followed by standard wash protocol to afford a negative chloranil test. The resin was agitated with piperidine-DMF (1:4, 1 x 2 mL, 30 min). The mixture was drained, and the resin washed with standard wash protocol to afford a positive Kaiser ninhydrin test.\\u003c/p\\u003e\\u003cp\\u003e\\u003cb\\u003eFmoc half-cycle method.\\u003c/b\\u003e A solution of 1-octanthiol (10 eq), DIEA (5 eq), and DMF (0.03 M) was added to the prepared resin \\u003cb\\u003e24a\\u003c/b\\u003e or \\u003cb\\u003e24b\\u003c/b\\u003e and the mixture was agitated for 1 h, followed by the standard wash protocol. Next, a mixture of TIPS-OTf (10 eq), 2,6-Lutidine (10 eq), and DMF (0.03 M) was added to the resin and the mixture was agitated for 4 h, followed by the standard wash protocol. The C-terminal allyl protecting group was removed using Pd(PPh\\u003csub\\u003e3\\u003c/sub\\u003e)\\u003csub\\u003e4\\u003c/sub\\u003e (0.25 eq), PhSiH\\u003csub\\u003e3\\u003c/sub\\u003e (25 eq) and DCM (3 mL) and agitating the reaction mixture for 2 hours after which the following wash was done: DCM (X 3), DMF (X 3), 0.03 M sodium N,N-diethyldithiocarbamate in DMF (X 3), and DMF (X 3).\\u003c/p\\u003e\\u003cp\\u003eH-AA\\u003csub\\u003en\\u003c/sub\\u003e-OAllyl∙CF\\u003csub\\u003e3\\u003c/sub\\u003eCOOH (10 eq), HATU (10 eq), HOAt (10 eq) and DIEA (20 eq) in DMF (3 mL) was mixed and added to the reaction vessel and agitated for 4 hours. The resin was washed using standard wash protocol.\\u003c/p\\u003e\\u003cp\\u003e\\u003cb\\u003eGeneral procedure for peptide cyclization (condition A).\\u003c/b\\u003e After complete synthesis of the desired Fmoc or Boc-protected peptide, the C-terminal DMA protecting group was removed using Pd(PPh\\u003csub\\u003e3\\u003c/sub\\u003e)\\u003csub\\u003e4\\u003c/sub\\u003e (1 eq) and phenylsilane (20 eq) in CH\\u003csub\\u003e2\\u003c/sub\\u003eCl\\u003csub\\u003e2\\u003c/sub\\u003e under N\\u003csub\\u003e2\\u003c/sub\\u003e for 2 h, after which the resin was drained and washed with DMF (3 x 1 min), sodium \\u003cem\\u003eN,N\\u003c/em\\u003e-diethyldithiocarbamate (0.03 M in DMF, 3 x 1 min), and CH\\u003csub\\u003e2\\u003c/sub\\u003eCl\\u003csub\\u003e2\\u003c/sub\\u003e (3 x 1 min). The resin was agitated with piperidine-DMF (1:4, 1 x 2 mL, 30 min). The mixture was drained, and the resin washed with standard wash protocol to afford a positive Kaiser ninhydrin test. A mixture of PyAop (5 eq), HOAt (5 eq), and DIEA (10 eq) in DMF (0.03 M) was added directly to the resin. After 2 h, the mixture was drained and washed with the standard wash protocol to afford a negative Kaiser ninhydrin test.\\u003c/p\\u003e\\u003cp\\u003e\\u003cb\\u003eGeneral procedure for peptide cyclization (condition B).\\u003c/b\\u003e After complete synthesis of the desired Fmoc or Boc-protected peptide, the C-terminal DMA protecting group was removed using Pd(PPh\\u003csub\\u003e3\\u003c/sub\\u003e)\\u003csub\\u003e4\\u003c/sub\\u003e (1 eq) and phenylsilane (20 eq) in CH\\u003csub\\u003e2\\u003c/sub\\u003eCl\\u003csub\\u003e2\\u003c/sub\\u003e under N\\u003csub\\u003e2\\u003c/sub\\u003e for 2 h, after which the resin was drained and washed with DMF (3 x 1 min), sodium \\u003cem\\u003eN,N\\u003c/em\\u003e-diethyldithiocarbamate (0.03 M in DMF, 3 x 1 min), and CH\\u003csub\\u003e2\\u003c/sub\\u003eCl\\u003csub\\u003e2\\u003c/sub\\u003e (3 x 1 min). The resin was agitated with piperidine-DMF (1:4, 1 x 2 mL, 30 min). The mixture was drained, and the resin washed with standard wash protocol to afford a positive Kaiser ninhydrin test. A mixture of PyAOP (3 eq), 2,4,6-collidine (6 eq), and DIEA (6 eq) in 9:1 CH\\u003csub\\u003e2\\u003c/sub\\u003eCl\\u003csub\\u003e2\\u003c/sub\\u003e-DMF (0.03 M) was added directly to the resin. After 2 h, the mixture was drained and washed with the standard wash protocol to afford a negative Kaiser ninhydrin test.\\u003c/p\\u003e\\u003cp\\u003e\\u003cb\\u003eGeneral procedure for thioester synthesis.\\u003c/b\\u003e After synthesis of the desired Boc-protected peptide, the C-terminal DMA protecting group was removed using Pd(PPh\\u003csub\\u003e3\\u003c/sub\\u003e)\\u003csub\\u003e4\\u003c/sub\\u003e (1 eq) and phenylsilane (20 eq) in CH\\u003csub\\u003e2\\u003c/sub\\u003eCl\\u003csub\\u003e2\\u003c/sub\\u003e under N\\u003csub\\u003e2\\u003c/sub\\u003e for 2 h, after which the resin was drained and washed with DMF (3 x 1 min), sodium \\u003cem\\u003eN,N\\u003c/em\\u003e-diethyldithiocarbamate (0.03 M in DMF, 3 x 1 min), and CH\\u003csub\\u003e2\\u003c/sub\\u003eCl\\u003csub\\u003e2\\u003c/sub\\u003e (3 x 1 min). A solution of 1-octanthiol (10 eq), DIEA (10 eq), and DMF (0.03 M) was added to the resin and the mixture was agitated for 1 h, followed by the standard wash protocol. Next, a mixture of TIPS-OTf (10 eq), 2,6-Lutidine (10 eq), and DMF (0.03 M) were added to the resin and the mixture was agitated for 4 h, followed by the standard wash protocol. A solution of HATU (5 eq) and DIEA (20 eq) in DMF (0.03 M) was added to the resin and agitated for a 5 min period. Without draining, thiophenol (10 eq) was added to the mixture. After 2 h, the mixture was drained and washed with the standard wash protocol to afford a negative Kaiser ninhydrin test\\u003c/p\\u003e\\u003cp\\u003e\\u003cb\\u003eGeneral Procedure for Sieber Amide cleavage.\\u003c/b\\u003e An aliquot (~\\u0026thinsp;1 mg) of resin-bound peptide was treated with 200 \\u0026micro;L TFA/H\\u003csub\\u003e2\\u003c/sub\\u003eO/CH\\u003csub\\u003e2\\u003c/sub\\u003eCl\\u003csub\\u003e2\\u003c/sub\\u003e (2:1:97) and the mixture agitated for 5 min at rt. Following this, volatiles were removed by N\\u003csub\\u003e2\\u003c/sub\\u003e stream. The resulting residue was dissolved in 200 \\u0026micro;L MeOH-H\\u003csub\\u003e2\\u003c/sub\\u003eO (1:1), filtered and analyzed by UPLC-MS. Note: Product / DKP ratio was determined by integrating 214 and 254 nm peaks and are reported as relative percentages.\\u003c/p\\u003e\\u003cp\\u003e\\u003cb\\u003eGeneral Procedure for photocleavage.\\u003c/b\\u003e The resin-bound peptide was suspended in a 5 mL solution of 1:9 MeOH:CH\\u003csub\\u003e2\\u003c/sub\\u003eCl\\u003csub\\u003e2\\u003c/sub\\u003e in a fused quartz tube and agitated under 365 nm UV light for 1 h. The solvent was filtered to remove the resin and removed under reduced pressure. The resulting residue was dissolved in MeOH-H\\u003csub\\u003e2\\u003c/sub\\u003eO (1:1), filtered and analyzed by UPLC-MS and HPLC.\\u003c/p\\u003e\"},{\"header\":\"Results and Discussion\",\"content\":\"\\u003cp\\u003e\\u003cstrong\\u003eSynthesis of linker-anchored dipeptides\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eUsing the precursor molecules \\u003cstrong\\u003e1\\u0026ndash;5\\u003c/strong\\u003e (Fig. \\u003cspan class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003e), each linker was covalently attached to glycine methyl ester to afford an anchored glycine residue (\\u003cstrong\\u003e6a\\u003c/strong\\u003e-\\u003cstrong\\u003ee\\u003c/strong\\u003e), which was then acylated with Boc-\\u003cem\\u003eL\\u003c/em\\u003e-phenylalanine to afford an anchored dipeptide \\u003cstrong\\u003e7a\\u003c/strong\\u003e-\\u003cstrong\\u003ee\\u003c/strong\\u003e (Table \\u003cspan class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003e). Each anchored dipeptide was photolyzed at 300 nm to afford Boc-Phe-Gly-OMe (\\u003cstrong\\u003e8\\u003c/strong\\u003e).\\u003c/p\\u003e\\n\\u003cp\\u003eThe linker precursors \\u003cstrong\\u003e1\\u0026ndash;5\\u003c/strong\\u003e were either commercially available or prepared using literature procedures (Stowell 1996; Vishwanathan 2006). Linkers \\u003cstrong\\u003e1\\u003c/strong\\u003e and \\u003cstrong\\u003e2\\u003c/strong\\u003e were anchored to methyl glycinate via nucleophilic displacement, whereas \\u003cstrong\\u003e3\\u003c/strong\\u003e and \\u003cstrong\\u003e4\\u003c/strong\\u003e employed the Amadori rearrangement (Amadori \\u003cspan class=\\\"CitationRef\\\"\\u003e1925\\u003c/span\\u003e; Isbell \\u003cspan class=\\\"CitationRef\\\"\\u003e1958\\u003c/span\\u003e) of an a-hydroxyketone, and \\u003cstrong\\u003e5\\u003c/strong\\u003e was attached to methyl glycinate through reductive amination. In all cases, acylation with the symmetric anhydride of Boc-\\u003cem\\u003eL\\u003c/em\\u003e-Phe was found to afford the highest yield of anchored dipeptide. A survey of conditions for photolytic cleavage of the dipeptide found that photoexcitation at 300 nm in either methanol or chloroform, depending on the motif, afforded the fastest and cleanest reactions, but only the Dmp (\\u003cstrong\\u003e7d\\u003c/strong\\u003e) and Nve-linked dipeptides showed acceptable photolytic reactivity. Problems arose, however, when amino acids larger than glycine were anchored to Dmp and Nve. Both the initial attachment and subsequent acylation of those anchored amino acids proved to be quite problematic and afforded anchored dipeptides in modest yields at best.\\u003c/p\\u003e\\n\\u003cp\\u003eIn summary, Op, Mop, and Obz failed to release the dipeptide product efficiently, regardless of solvent and wavelength used, and so were discarded as linker candidates. Both the Nve and Dmp motifs displayed acceptable photolytic product release with the appropriate choices of solvent and irradiation wavelength; however, in both cases the modest yields for amino acid anchoring and acylation were insufficient for our needs, necessitating further modification to improve both steps of the reaction sequence.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eDevelopment of the second-generation photolabile linker\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eOn the basis of both acylation and photocleavage yields the \\u003cem\\u003eo\\u003c/em\\u003e-nitrobenzyl motif of the Nve linker was chosen as the basic skeleton for a second-generation linker, and the transacylation motif of the 6-hydroxy-4-methoxybenzyl (Hmb) (Sheppard 1993) and 6-hydroxynitrobenzyl (Hnb) (Smythe 1999) auxiliaries was incorporated into the linker design to improve acylation efficiency. The acid-labile Hmb auxiliary was devised by Sheppard for the protection of peptide amide backbones during the synthesis of \\u0026lsquo;difficult\\u0026rsquo; peptide sequences. The related, photolabile Hnb auxiliary was developed by Smythe for the covalent modification of \\u0026lsquo;difficult\\u0026rsquo; peptide sequences to facilitate their cyclization. Both the Hmb and Hnb auxiliaries employ an \\u003cem\\u003eortho-\\u003c/em\\u003ehydroxyl group to acylate bulky amino acids via esterification and subsequent O-to-N transacylation.\\u003c/p\\u003e\\n\\u003cp\\u003eIt was reasoned that the addition of an \\u003cem\\u003eo-\\u003c/em\\u003ehydroxy substituent to the \\u003cem\\u003eo\\u003c/em\\u003e-nitrobenzyl skeleton would assist both the acylation of the first amino acid and later photolytic cleavage. Attachment of a \\u003cem\\u003epara\\u003c/em\\u003e-carboxylate to the \\u003cem\\u003eo\\u003c/em\\u003e-nitrobenzyl skeleton provides a tether for covalent attachment of the linker to a spacer or resin and affords additional electron withdrawal to assist both the transacylation and photolysis reactions. Thus, the 2-hydroxy-4-carboxy-6-nitrobenzyl (Hcnb) linker emerged as a second-generation candidate for a photolabile BAL linker, as exemplified by the Hcnb-glycine adduct \\u003cstrong\\u003e9\\u003c/strong\\u003e (Fig. \\u003cspan class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003e), derived from reductive amination of the aldehyde precursor 2-hydroxy-4-carboxy-6-nitrobenzaldehyde (Hcna) with glycine methyl ester. Treatment of \\u003cstrong\\u003e9\\u003c/strong\\u003e with a symmetric anhydride (Fig. \\u003cspan class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003e) can result in the formation of the desired dipeptide \\u003cstrong\\u003e11\\u003c/strong\\u003e either through direct N-acylation or via initial acylation of the less hindered phenol followed by subsequent O-to-N acyl transfer. This alternate pathway was envisaged as a means to overcome the limitations of the Nve motif when attached to sterically hindered amino acids.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eSynthesis of Hcna linker\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003e4-formyl-3-hydroxy-5-nitrobenzoic acid (\\u003cstrong\\u003e16\\u003c/strong\\u003e) was prepared from commercially available 4-Methyl-3,5-dinitrobenzoic acid (\\u003cstrong\\u003e12\\u003c/strong\\u003e) (Fig. \\u003cspan class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003e). A condensation reaction between \\u003cstrong\\u003e12\\u003c/strong\\u003e and DMF-DMA afforded the previously reported (Starosotnikov 2005) enamine \\u003cstrong\\u003e13\\u003c/strong\\u003e in 72% yield. Ruthenium (III) chloride-catalyzed oxidative cleavage of the enamine using sodium metaperiodate generated the aldehyde \\u003cstrong\\u003e14\\u003c/strong\\u003e in 61% yield. Unilateral nucleophilic aromatic substitution of a single nitro- group using acetaldoxime to install the \\u003cem\\u003eortho\\u003c/em\\u003e-phenol group afforded ester-protected linker precursor \\u003cstrong\\u003e15\\u003c/strong\\u003e in 68% yield, which was used for solution-phase studies. Finally, hydrolysis of the methyl ester group using barium hydroxide afforded the free carboxylic acid \\u003cstrong\\u003e16\\u003c/strong\\u003e in 90% yield.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eSolution-phase synthesis of the dipeptides using Hcnb\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eWe envisioned linking the peptide to the linker via a reductive amination between the primary amine of the first amino acid and the aldehyde precursor, in similar fashion to the loading protocol for the original BAL Linker. The resulting secondary amine can then be acylated with the next amino acid in sequence, forming a tertiary amide linkage between the peptide and linker. The presence of the \\u003cem\\u003eortho\\u003c/em\\u003e-phenol in Hcnb is essential for complete acylation of the secondary amine, especially when utilizing amino acids with bulky side chains. Finally, photolysis in the short-wave UV range should cause a clean release of the dipeptide from the linker.\\u003c/p\\u003e\\n\\u003cp\\u003eA series of dipeptides was synthesized in solution on the methyl ester-protected Hcna linker precursor \\u003cstrong\\u003e15\\u003c/strong\\u003e (Table \\u003cspan class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003e). In solution, addition of the desired C-terminally protected amino acid to the aldehyde handle in the presence of DIEA in CH\\u003csub\\u003e3\\u003c/sub\\u003eCN forms the imine intermediate. Reduction of this intermediate by sodium borohydride in methanol yields the Hcnb-linked amino acids \\u003cstrong\\u003e17a-c\\u003c/strong\\u003e. Acylation of the secondary amine with a pre-formed symmetric anhydride of the second amino acid resulted in acylation to give Hcnb-linked dipeptides, even in the case of bulky side-chains such as Thr. Photolysis at 365 nm in MeOH releases the desired dipeptides from the linker to give \\u003cstrong\\u003e19a-c.\\u003c/strong\\u003e Due to rotomers present in peptides attached to Hcnb, yields were reported over 2 steps.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003ePreparation of the resin-bound Hcna linker\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe free acid form of the Hcna linker, \\u003cstrong\\u003e16\\u003c/strong\\u003e (Fig. \\u003cspan class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003e), was covalently anchored to aminomethyl-functionalized Tentagel resin \\u003cstrong\\u003e21a\\u003c/strong\\u003e, or Sieber TG resin \\u003cstrong\\u003e21b\\u003c/strong\\u003e via acylation of a resin-bound amine. A 6-aminohexanoic acid (Ahx) spacer was employed with both solid supports to improve reactivity. Additionally, a proline residue was introduced to reduce cross-linking between the amine and the aldehyde of the Hcna linker. In both cases, acylation of resin-bound amines was accomplished by coupling \\u003cstrong\\u003e16\\u003c/strong\\u003e to the free amine using either PyBOP or DCC (Fig. \\u003cspan class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003e). Subsequent reactions of \\u003cstrong\\u003e22a\\u003c/strong\\u003e were monitored by photolytic cleavage from the linker and analysis of the crude product, whereas reactions of \\u003cstrong\\u003e22b\\u003c/strong\\u003e were monitored by cleavage using dilute acid and analysis of the crude product. Because of the time required for photolysis of Hcnb-linked peptides (1\\u0026ndash;2 h), reaction conditions were optimized using \\u003cstrong\\u003e22b\\u003c/strong\\u003e and then applied to \\u003cstrong\\u003e22a\\u003c/strong\\u003e.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eLoading the dipeptide by reductive amination followed by acylation\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe first residue was anchored to the resin-bound Hcna linker via a 2-step reductive amination under acidic conditions. (Fig. \\u003cspan class=\\\"InternalRef\\\"\\u003e6\\u003c/span\\u003e). Quantitative acylation of the resin-bound amine was accomplished using a symmetric anhydride of the second residue (4 eq, 2 x 2h) as determined by LC-MS analysis.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eLoading of the third amino acid\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eOne of the major challenges with the Backbone Amide Linker concept is the formation of a diketopiperazine byproduct upon deprotection of the Fmoc group at the dipeptide stage. The most common method for overcoming this problem is to employ a sterically hindered C-terminal protecting group such as \\u003cem\\u003et\\u003c/em\\u003e-butyl or 1,1-dimethylallyl (DMA) (Hostetler \\u003cspan class=\\\"CitationRef\\\"\\u003e2018\\u003c/span\\u003e), thereby minimizing the formation of this undesired byproduct. However, it was found that quantitative DKP formation was still obtained when using the Hcnb linker, regardless of the C-terminal protecting groups used. It was hypothesized that this results from Br\\u0026oslash;nsted acid activation of the C-terminus by the nearby phenol proton, thereby catalyzing the formation of DKP. It was reasoned that this effect could be eliminated through removal of the phenol proton, thus avoiding Br\\u0026oslash;nsted acid activation. The phenol group is required for efficient loading of the second amino acid residue, as well as for photocleavage of the final peptide, and so a method for temporary protection of the hydroxyl is required. Following the loading of the secondary amine, the phenol remains acylated with an excess of the second amino acid, until it is removed by the strongly nucleophilic piperidine upon deprotection of the N-terminal Fmoc group. We theorized that replacing piperidine with DBU, a relatively non-nucleophilic alternative for Fmoc deprotection, would leave the acylated phenol intact and minimize formation of the DKP byproduct. However, it was found that rapid, quantitative DKP formation was still obtained even when using DBU. Other methods were considered for blocking the phenol from interacting with the C-terminus, including silylation. However, due to the electronics of the ring, silylation of the phenol results in an exceedingly unstable silyl ether. Even attempts to observe the presence of a silyl group by LC-MS analysis were unsuccessful. The most successful protection conditions used TIPS-OTf and 2,6-Lutidine for TIPS protection of the phenol on the linker. To monitor the effectiveness of various silylation conditions, the dipeptide N-terminus was deprotected for 3 min using a cocktail of 2% v/v DBU and 2% v/v 1-Octanethiol in DMF, followed by immediate introduction of the third amino acid as a pre-formed symmetric anhydride. Various tripeptide sequences were tested to determine the efficacy of the TIPS group for reducing DKP formation (Table \\u003cspan class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003e). In most cases, only trace amounts of DKP were observed by LC-MS, if any. The worst sequence tested was the Ala-Ala dipeptide, resulting in 12% DKP formation.\\u003c/p\\u003e\\n\\u003cp\\u003eTo completely avoid diketopiperazine formation, we followed the method developed by the Barany group, also known as the Fmoc-half cycle. In an Fmoc-half cycle, the first amino acid used in the reductive amination step is an allyl ester. This is followed by the acylation of the second amino acid. Instead of attaching the third amino acid through the N-terminus, the allyl group of the first amino acid is deprotected and the final amino acid is attached through the C-terminus by a peptide coupling reaction. In this case, the allyl ester was deprotected with catalytic Pd(PPh\\u003csub\\u003e3\\u003c/sub\\u003e)\\u003csub\\u003e4\\u003c/sub\\u003e and excess PhSiH\\u003csub\\u003e3\\u003c/sub\\u003e and then coupled with another amino acid allyl ester through the C-terminus (Fig. \\u003cspan class=\\\"InternalRef\\\"\\u003e7\\u003c/span\\u003e). Then the rest of the peptide was built through the N-terminus by Fmoc SPPS until the peptide of desired length was built.\\u003c/p\\u003e\\n\\u003cp\\u003eAlso worth noting that we switched from \\u003cem\\u003et\\u003c/em\\u003e-Bu/DMA esters to allyl esters for two of the amino acids in the sequence. When the DMA group was deprotected, and the final amino acid is coupled with another DMA ester of amino acid, the 1-octanethiol (left from the Fmoc deprotection using excess diethylamine and 1-octanethiol) could also react and generate thioesters. Therefore, we switched to using allyl esters which are synthesized on Boc-protected amino acids and then esterified with allyl bromide. Before the reductive amination and Fmoc half-cycle, the Boc group is deprotected with trifluoroacetic acid and neutralized with excess DIEA for the allyl ester to participate in reaction.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eThe choice of model cyclic peptides\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eTwo previously synthesized cyclic peptides were chosen to model the head-to-tail cyclized peptides to examine the performance of the Hcnb linker on solid support. The cyclic decapeptide c-[Arg-(D)-Phe-Pro-Glu-Asp-Asn-Tyr-Glu-Ala-Ala, \\u003cstrong\\u003e28\\u003c/strong\\u003e] was synthesized to demonstrate the efficacy of the original Backbone Amide Linker, published by Albericio and Barany. The 14-residue cyclic peptide c-[Pro-Asp-Gly-Arg-Cys-Thr-Lys-Ser-Ile-Pro-Pro-Ile-Cys-Phe, \\u003cstrong\\u003e30\\u003c/strong\\u003e], also known as the Sunflower Trypsin Inhibitor, is a well-known natural product. The 14-mer cyclic Sunflower Trypsin Inhibitor poses some interesting synthetic challenges due to the high prevalence of \\u0026beta;-branched amino acids and proline residues, which make selection of a cyclization point difficult. After several attempts, it was found that reductive amination of Phe onto the linker as the first residue, followed by cyclization at Pro yielded the best results. Cyclization and subsequent photocleavage from the resin yielded the final peptide in 90% purity and 80% cleavage yield. The synthesis of the cyclic decapeptide was accomplished through reductive amination of Ala as the first residue and the subsequent linear synthesis. Following cyclization and photocleavage from the resin, the peptide was obtained in 95% purity and 90% cleavage yield. The first peptide chosen was a short-chain analogue of the natural product somatostatin, cyclo[(D)-Trp-Lys-Gly-(\\u0026beta;)-Ala-Phe]. The synthesis was carried out according to the procedures mentioned previously. The first residue H-Phe-ODMA was loaded onto the prepared resin \\u003cstrong\\u003e22a\\u003c/strong\\u003e via reductive amination. The subsequent linear synthesis was then carried out as previously discussed. The head-to-tail cyclization was accomplished on the resin using PyAOP. Photocleavage from the resin at 365 nm for 1 h yielded the final cyclic decapeptide \\u003cstrong\\u003e29\\u003c/strong\\u003e in 50% purity and 96% cleavage yield.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eSynthesis of C-terminal thioesters\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe synthesis of C-terminal thioesters is an area of interest for their use in native chemical ligation (NCL) reactions. The Hcnb linker was used to demonstrate the on-resin synthesis of thioesters in high purities. It was established by solution-phase studies that the presence of a free phenol on the linker was problematic for synthesis of C-terminal thioesters, and so the phenol was TIPS protected prior to optimizing the coupling conditions for thioester formation. Use of carbodiimides as a coupling reagent yielded low conversions (0\\u0026ndash;30%). However, switching to HATU as a coupling reagent and preactivating the carboxylic acid for 5 min prior to addition of the thiol yielded complete conversion to the desired thioester in 2 h. A series of pentapeptides were synthesized to examine the scope of C-terminal residue compatibility with thioester synthesis. It was found that \\u0026beta;-branched amino acids yield poor conversion to the thioester. However, it was reported by Kent that these sterically hindered amino acids are not amenable to NCL due to the steric hindrance of the carbonyl required for the transthioesterification and subsequent acyl transfer. The linear sequences were synthesized according to the standard procedure outlined previously. The final amino acid in the sequence was coupled as an N-Boc-protected residue. The C-terminal DMA protecting group was removed using Pd(0) and phenylsilane. Following TIPS protection of the linker phenol, the C-terminus was pre-activated for 5 min with HATU and Hunig\\u0026rsquo;s base in DMF. Addition of thiophenol resulted in 95\\u0026ndash;100% conversion of carboxylic acid to thioester in 2 h. The thioester peptides were then globally deprotected using 5% H\\u003csub\\u003e2\\u003c/sub\\u003eO in TFA for 1 h and cleaved from the resin using 365 nm UV irradiation for 1 h. Depending on the identity of the first amino acid residue, peptide thioesters were obtained in 75\\u0026ndash;99% purity and 66\\u0026ndash;92% cleavage yield (Table \\u003cspan class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003e). No epimerization of the C-terminal residue was observed.\\u003c/p\\u003e\\n\\u003ctable id=\\\"Tab4\\\" border=\\\"1\\\" class=\\\"fr-table-selection-hover\\\"\\u003e\\n \\u003ccaption language=\\\"En\\\"\\u003e\\n \\u003cdiv class=\\\"CaptionNumber\\\"\\u003eTable 4\\u003c/div\\u003e\\n \\u003cdiv class=\\\"CaptionContent\\\"\\u003e\\n \\u003cp\\u003eStructures and Yields of Peptide Thioesters\\u003c/p\\u003e\\n \\u003c/div\\u003e\\n \\u003c/caption\\u003e\\n \\u003cthead\\u003e\\n \\u003ctr\\u003e\\n \\u003cth align=\\\"left\\\"\\u003e\\n \\u003cp\\u003eStructure\\u003c/p\\u003e\\n \\u003c/th\\u003e\\n \\u003cth align=\\\"left\\\"\\u003e\\n \\u003cp\\u003ePurity, %\\u003c/p\\u003e\\n \\u003c/th\\u003e\\n \\u003cth align=\\\"left\\\"\\u003e\\n \\u003cp\\u003eCleavage Yield, %\\u003c/p\\u003e\\n \\u003c/th\\u003e\\n \\u003c/tr\\u003e\\n \\u003c/thead\\u003e\\n \\u003ctbody\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003eH-Phe-Lys-Ala-Ala-Leu-SPh (\\u003cstrong\\u003e31a\\u003c/strong\\u003e)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e99\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e83\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003eH-Ala-Glu-Phe-Leu-Phe-SPh (\\u003cstrong\\u003e31b\\u003c/strong\\u003e)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e99\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e66\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003eH-Ala-Lys-Phe-Leu-Glu-SPh (\\u003cstrong\\u003e31c\\u003c/strong\\u003e)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e75\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e71\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003eH-Phe-Glu-Ala-Leu-Ala-SPh (\\u003cstrong\\u003e31d\\u003c/strong\\u003e)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e99\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e92\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003c/tbody\\u003e\\n\\u003c/table\\u003e\"},{\"header\":\"Conclusions\",\"content\":\"\\u003cp\\u003eIn a quest for a photolabile backbone amide linker for the solid-phase synthesis of cyclic and C-terminally modified peptides, a survey of various photolabile motifs was conducted, establishing the \\u003cem\\u003eo\\u003c/em\\u003e-nitrobenzyl motif as the best candidate. Problems associated with many of the candidate motifs including the nitroveratryl linker led to the development of the 2-hydroxy-4-carboxy-6-nitrobenzyl (Hcnb\\u003cb\\u003e)\\u003c/b\\u003e linker. A convenient and scalable synthesis of a benzaldehyde precursor (\\u003cb\\u003e16\\u003c/b\\u003e) was developed, and general conditions were found for the anchoring of various amino esters to the linker and their acylation to form anchored dipeptides in good yield. Photolysis of the various Hcnb-linked dipeptides (\\u003cb\\u003e18a-c)\\u003c/b\\u003e at 365 nm cleanly deprotected them in good yield in all cases. The linker can be attached to a wide variety of amino acids, can be selectively removed in the presence of most commonly used protecting groups and tolerates treatment with acid, base and Pd\\u003csup\\u003e0\\u003c/sup\\u003e. It was found that the Hcnb linker could be conveniently attached to an aminoethyl TG resin using a prolyl-6-aminohexanoic acid spacer, permitting its use in solid phase peptide synthesis. The ability of the Hcnb linker to facilitate on-resin, head-to-tail cyclization of peptides was demonstrated through the synthesis of three cyclic peptides. The utility of Hcnb to synthesize C-terminal thioesters was also demonstrated. We believe that this new photolabile linker should provide a valuable tool for the synthesis of cyclic and modified peptides, depsipeptides and peptidomimetics such as oligoureas (Boeijen \\u003cspan citationid=\\\"CR7\\\" class=\\\"CitationRef\\\"\\u003e1999\\u003c/span\\u003e; Boeijen 2001).\\u003c/p\\u003e\"},{\"header\":\"Declarations\",\"content\":\"\\u003ch2\\u003eCompeting Interests. \\u0026nbsp;\\u003c/h2\\u003e\\n\\u003cp\\u003eThe authors declare that they have no competing interests, financial or non-financial, that are related to the work in this publication.\\u003c/p\\u003e\\u003ch2\\u003eAuthor Contribution\\u003c/h2\\u003e\\u003cp\\u003eK. K., M.N. and M.L. wrote the main manuscript text. SSK, M.N. and K.K. prepared the figures. All authors reviewed the manuscript.\\u003c/p\\u003e\"},{\"header\":\"References\",\"content\":\"\\u003col\\u003e\\u003cli\\u003e\\u003cspan\\u003eAmadori M (1925) Atti Accad. 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Bioorg Med Chem 14:19, 6581\\u0026ndash;6585. \\u003cspan class=\\\"ExternalRef\\\"\\u003e\\u003cspan class=\\\"RefSource\\\"\\u003ehttps://doi.org/10.1016/j.bmc.2006.06.006\\u003c/span\\u003e\\u003cspan address=\\\"10.1016/j.bmc.2006.06.006\\\" targettype=\\\"DOI\\\" class=\\\"RefTarget\\\"\\u003e\\u003c/span\\u003e\\u003c/span\\u003e\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003eWhite TR, Renzelman CM, Rand AC, Rezai T, McEwen CM, Gelev VM, Turner RA, Linington RG, Leung SS, Kalgutkar AS, Bauman JN, Zhang Y, Liras S, Price DA, Mathiowetz AM, Jacobson MP, Lokey RS (2011) On-resin N-methylation of cyclic peptides for discovery of orally bioavailable scaffolds. Nat Chem Biol 7:810. \\u003cspan class=\\\"ExternalRef\\\"\\u003e\\u003cspan class=\\\"RefSource\\\"\\u003ehttps://doi.org/10.1038/nchembio.664\\u003c/span\\u003e\\u003cspan address=\\\"10.1038/nchembio.664\\\" targettype=\\\"DOI\\\" class=\\\"RefTarget\\\"\\u003e\\u003c/span\\u003e\\u003c/span\\u003e\\u003c/span\\u003e\\u003c/li\\u003e\\u003c/ol\\u003e\"},{\"header\":\"Tables\",\"content\":\"\\u003cp\\u003eTables 1 to 3 are available in the Supplementary Files section.\\u003c/p\\u003e\"}],\"fulltextSource\":\"\",\"fullText\":\"\",\"funders\":[],\"hasAdminPriorityOnWorkflow\":false,\"hasManuscriptDocX\":true,\"hasOptedInToPreprint\":true,\"hasPassedJournalQc\":\"\",\"hasAnyPriority\":false,\"hideJournal\":false,\"highlight\":\"\",\"institution\":\"\",\"isAcceptedByJournal\":true,\"isAuthorSuppliedPdf\":false,\"isDeskRejected\":\"\",\"isHiddenFromSearch\":false,\"isInQc\":false,\"isInWorkflow\":false,\"isPdf\":false,\"isPdfUpToDate\":true,\"isWithdrawnOrRetracted\":false,\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"international-journal-of-peptide-research-and-therapeutics\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":false,\"externalIdentity\":\"ijpr\",\"sideBox\":\"Learn more about [International Journal of Peptide Research and Therapeutics](http://link.springer.com/journal/10989)\",\"snPcode\":\"10989\",\"submissionUrl\":\"https://submission.nature.com/new-submission/10989/3\",\"title\":\"International Journal of Peptide Research and Therapeutics\",\"twitterHandle\":\"\",\"acdcEnabled\":true,\"dfaEnabled\":true,\"editorialSystem\":\"em\",\"reportingPortfolio\":\"Springer Hybrid\",\"inReviewEnabled\":true,\"inReviewRevisionsEnabled\":false},\"keywords\":\"Peptide synthesis, photochemistry, peptide cyclization, thioester formation\",\"lastPublishedDoi\":\"10.21203/rs.3.rs-7165562/v1\",\"lastPublishedDoiUrl\":\"https://doi.org/10.21203/rs.3.rs-7165562/v1\",\"license\":{\"name\":\"CC BY 4.0\",\"url\":\"https://creativecommons.org/licenses/by/4.0/\"},\"manuscriptAbstract\":\"\\u003ch2\\u003eIntroduction\\u003c/h2\\u003e\\u003cp\\u003eA new backbone amide linker has been developed for the synthesis of cyclic and C-terminally modified peptides that permits photochemical detachment of the synthesized peptide from the solid support, thus avoiding problems associated with acid deprotection conditions.\\u003c/p\\u003e\\u003ch2\\u003eMethods\\u003c/h2\\u003e\\u003cp\\u003eAn initial survey of known photolabile motifs for their ability to produce a linker-bound model dipeptide in high yield and their ability to undergo efficient photochemical detachment of the model dipeptide found that the 6-nitroveratryl (Nve) motif afforded the most efficient release of the dipeptide. The problematic acylation of Nve-bound amino esters was solved through the development of the 2-hydroxy-4-carboxy-6-nitrobenzyl (Hcnb) linker, which utilizes an O-to-N transacylation to afford efficient acylation of even sterically hindered, linker-bound amino esters. Two different approaches were carried out to avoid/minimize diketopiperazine formation. This methodology was then used to synthesize cyclic peptides and peptide thioesters.\\u003c/p\\u003e\\u003ch2\\u003eResults\\u003c/h2\\u003e\\u003cp\\u003eThe Hcnb linker was found to afford high yields of amino acid loading, acylation, and photolytic cleavage of model tripeptides. Attachment of the Hcnb linker to the aminomethyl TG resin permitted the solid phase synthesis of representative cyclic peptides and C-terminal thioesters in high overall yield and purity.\\u003c/p\\u003e\\u003ch2\\u003eConclusion\\u003c/h2\\u003e\\u003cp\\u003eHcna is a new photolabile backbone amide linker that has been used to synthesize cyclic peptides and peptide thioesters. It was found that the linker efficiently released the synthesized peptides when UV light was shed on it. This linker is also stable to acids meaning global deprotections can be done on resin and can be washed to remove by-products, releasing only the peptide upon its photocleavage from the resin.\\u003c/p\\u003e\",\"manuscriptTitle\":\"A Photolabile Backbone Amide Linker for the Solid-Phase Synthesis of Cyclic Peptides and C-Terminal Thioesters\",\"msid\":\"\",\"msnumber\":\"\",\"nonDraftVersions\":[{\"code\":1,\"date\":\"2025-07-29 14:19:06\",\"doi\":\"10.21203/rs.3.rs-7165562/v1\",\"editorialEvents\":[{\"type\":\"communityComments\",\"content\":0},{\"type\":\"decision\",\"content\":\"Revision requested\",\"date\":\"2025-08-04T15:20:44+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"editorInvitedReview\",\"content\":\"\",\"date\":\"2025-08-04T01:23:01+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"editorInvitedReview\",\"content\":\"\",\"date\":\"2025-08-02T00:17:40+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"reviewerAgreed\",\"content\":\"161298032965693116295026268757107377248\",\"date\":\"2025-07-28T13:07:59+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"reviewerAgreed\",\"content\":\"245571840611697797400646317627313246973\",\"date\":\"2025-07-27T23:24:28+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"reviewerAgreed\",\"content\":\"257239682984301534490561239978887348632\",\"date\":\"2025-07-27T08:39:33+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"reviewerAgreed\",\"content\":\"43040754917490440409656992499041061766\",\"date\":\"2025-07-25T21:58:45+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"reviewerAgreed\",\"content\":\"213664137239359270118469900132009387987\",\"date\":\"2025-07-25T13:59:12+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"reviewerAgreed\",\"content\":\"155410535118255107556897242650510197565\",\"date\":\"2025-07-25T08:14:51+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"reviewersInvited\",\"content\":\"\",\"date\":\"2025-07-25T08:05:14+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"editorAssigned\",\"content\":\"\",\"date\":\"2025-07-21T15:14:04+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"checksComplete\",\"content\":\"\",\"date\":\"2025-07-21T15:13:50+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"submitted\",\"content\":\"International Journal of Peptide Research and Therapeutics\",\"date\":\"2025-07-19T15:58:27+00:00\",\"index\":\"\",\"fulltext\":\"\"}],\"status\":\"published\",\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"international-journal-of-peptide-research-and-therapeutics\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":false,\"externalIdentity\":\"ijpr\",\"sideBox\":\"Learn more about [International Journal of Peptide Research and Therapeutics](http://link.springer.com/journal/10989)\",\"snPcode\":\"10989\",\"submissionUrl\":\"https://submission.nature.com/new-submission/10989/3\",\"title\":\"International Journal of Peptide Research and Therapeutics\",\"twitterHandle\":\"\",\"acdcEnabled\":true,\"dfaEnabled\":true,\"editorialSystem\":\"em\",\"reportingPortfolio\":\"Springer Hybrid\",\"inReviewEnabled\":true,\"inReviewRevisionsEnabled\":false}}],\"origin\":\"\",\"ownerIdentity\":\"1ef99044-d426-4b9d-a070-343ba23fa0da\",\"owner\":[],\"postedDate\":\"July 29th, 2025\",\"published\":true,\"recentEditorialEvents\":[],\"rejectedJournal\":[],\"revision\":\"\",\"amendment\":\"\",\"status\":\"published-in-journal\",\"subjectAreas\":[],\"tags\":[],\"updatedAt\":\"2025-09-15T16:02:20+00:00\",\"versionOfRecord\":{\"articleIdentity\":\"rs-7165562\",\"link\":\"https://doi.org/10.1007/s10989-025-10752-9\",\"journal\":{\"identity\":\"international-journal-of-peptide-research-and-therapeutics\",\"isVorOnly\":false,\"title\":\"International Journal of Peptide Research and Therapeutics\"},\"publishedOn\":\"2025-09-08 15:57:33\",\"publishedOnDateReadable\":\"September 8th, 2025\"},\"versionCreatedAt\":\"2025-07-29 14:19:06\",\"video\":\"\",\"vorDoi\":\"10.1007/s10989-025-10752-9\",\"vorDoiUrl\":\"https://doi.org/10.1007/s10989-025-10752-9\",\"workflowStages\":[]},\"version\":\"v1\",\"identity\":\"rs-7165562\",\"journalConfig\":\"researchsquare\"},\"__N_SSP\":true},\"page\":\"/article/[identity]/[[...version]]\",\"query\":{\"redirect\":\"/article/rs-7165562\",\"identity\":\"rs-7165562\",\"version\":[\"v1\"]},\"buildId\":\"XKTyCvWXoU3ODBz1xrDgd\",\"isFallback\":false,\"isExperimentalCompile\":false,\"dynamicIds\":[84888],\"gssp\":true,\"scriptLoader\":[]}","source_license":"CC-BY-4.0","license_restricted":false}