Amide-forming chemical ligation via O-acylhydroxamic acidsDaniel L. Dunkelmanna,1, Yuki Hirataa,1, Kyle A. Totaroa, Daniel T. Cohena, Chi Zhanga, Zachary P. Gatesa,2,and Bradley L. Pentelutea,2

aDepartment of Chemistry, Massachusetts Institute of Technology, Cambridge, MA 02139

Edited by Jerrold Meinwald, Cornell University, Ithaca, NY, and approved February 28, 2018 (received for review October 20, 2017)

The facile rearrangement of “S-acyl isopeptides” to native peptidebonds via S,N-acyl shift is central to the success of native chemicalligation, the widely used approach for protein total synthesis.Proximity-driven amide bond formation via acyl transfer reactionsin other contexts has proven generally less effective. Here, weshow that under neutral aqueous conditions, “O-acyl isopeptides”derived from hydroxy-asparagine [aspartic acid-β-hydroxamic acid;Asp(β-HA)] rearrange to form native peptide bonds via an O,N-acylshift. This process constitutes a rare example of an O,N-acyl shiftthat proceeds rapidly across a medium-size ring (t1/2 ∼ 15 min),and takes place in water with minimal interference from hydroly-sis. In contrast to serine/threonine or tyrosine, which form O-acylisopeptides only by the use of highly activated acyl donors andappropriate protecting groups in organic solvent, Asp(β-HA) issufficiently reactive to form O-acyl isopeptides by treatment withan unprotected peptide-αthioester, at low mM concentration,in water. These findings were applied to an acyl transfer-basedchemical ligation strategy, in which an unprotected N-terminalAsp(β-HA)-peptide and peptide-αthioester react under aqueousconditions to give a ligation product ultimately linked by a nativepeptide bond.

chemical ligation | hydroxamic acid | O-acyl hydroxamic acid |acyl transfer | acyl shift

Contemporary protein total synthesis invariably employschemical ligation––the covalent coupling of two unprotected

peptide segments by a chemoselective reaction, in aqueous sol-vent, to form a product linked by an amide bond or analogstructure (1–3). Only a few reactions constitute the totality of thechemical ligation toolkit, highlighting the challenge of selectiveamide bond formation between unprotected peptides in water.Chemical ligation reactions that proceed in aqueous buffer in-clude native chemical ligation (4) and oxime-forming ligation (5,6); a variety of other chemistries, including Staudinger ligation(7, 8), α-ketoacid-hydroxylamine ligation (9, 10), pseudoproline-forming ligation (11), and Ser/Thr-forming ligation (12, 13), areconducted in organic or mixed aqueous/organic solvents.In native chemical ligation, an N-terminal Cys-peptide un-

dergoes thiol/thioester exchange with a peptide-αthioester togenerate a thioester intermediate, which rearranges to a peptidebond via rapid S,N-acyl shift. Key to this approach is that ami-nolysis of the thioester—which would be essentially unreactive toamines at neutral pH—is facilitated by proximity-driven, entropicactivation. The same principle was employed in two contemporarypeptide-coupling strategies based on O,N-acyl transfer (14–16).However, both strategies were ultimately limited by slow acyltransfer rates.Due to the facility of the original native chemical ligation

chemistry, many methods have been developed to extend itsapplicability beyond Xaa-Cys ligation sites, by the use of variouscysteine surrogates in place of an N-terminal cysteine (17–19). Theabilities of certain nonthiol side-chain functionalities—includingimidazoles (20) and carboxylates (21, 22)—to facilitate the aminolysisof a peptide-αthioester by N-terminal histidine or Asp/Glu-peptideshave been documented. Otherwise, extensions of native chemical

ligation have relied on the use of thiol nucleophiles to formS-acyl isopeptides, which undergo rapid S,N-acyl shifts throughsmall rings. An exception is the use of selenocysteine (23–26)and peptide-αselenoesters (27, 28), which exhibit analogous butheightened reactivity.We sought to expand the scope of nucleophiles that might be

employed in acyl transfer-based chemical ligation, and to rein-vestigate the possibility of O,N-acyl transfer across medium-sizerings. Hydroxamic acids (29) were found to be sufficiently re-active to enable the formation of an O-acyl isopeptide from apeptide-αthioester and an N-terminal Asp(β-HA)-peptide at5 mM concentrations in aqueous solvent, and without the needfor protecting groups. Unexpectedly, the resulting O-acyl iso-peptide products rearranged to native amide bonds via a facileO,N-acyl shift—apparently through a seven-member ring—withminimal interference from hydrolysis. These combined resultswere applied to the development of an amide-forming chemicalligation strategy involving reaction of an N-terminal Asp(β-HA)-peptide with a peptide-αthioester.

Results and DiscussionRearrangement of an O-Acyl Hydroxamic Acid Isopeptide to a PeptideBond. O-acyl hydroxamic acid isopeptide 1 rearranged to formnative peptide 1′ upon standing in aqueous buffer at ambienttemperature (Fig. 1A and SI Appendix, section 5). The reactionwas pH-sensitive, exhibiting a maximum between pH 5 and 6(Fig. 1B); it was substantially slower at pH 8, and did not proceedto any detectable extent at pH 2 (SI Appendix, Figs. S1 and S18).

Significance

Methods for the chemoselective formation of peptide bonds inwater generally leverage the exceptional reactivity of thiolnucleophiles. Here, we demonstrate that hydroxamic acids—incorporated into the side chain of asparagine or asparticacid—can be leveraged analogously. N-terminal hydroxy-asparagine-peptides react with peptide-αthioesters to form“O-acyl hydroxamic acid isopeptides," which rearrange to peptidebonds via a facile O,N-acyl shift. The hydroxy-asparagine residuecan be converted to either aspartic acid or asparagine, to furnishamide-linked ligation products comprising entirely canonical res-idues. Taken together, these observations form the basis of astrategy for peptide ligation that circumvents the requirementfor Xaa-Cys ligation “sites.”

Author contributions: D.L.D., Y.H., Z.P.G., and B.L.P. designed research; D.L.D., Y.H., andK.A.T. performed research; D.T.C. and C.Z. contributed new reagents/analytic tools;D.L.D., Y.H., Z.P.G., and B.L.P. analyzed data; and Z.P.G. and B.L.P. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

Published under the PNAS license.1D.L.D. and Y.H. contributed equally to this work.2To whom correspondence may be addressed. Email: zgates@mit.edu or blp@mit.edu.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1718356115/-/DCSupplemental.

Published online March 26, 2018.

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Potentially, the rate-pH maximum arises from a dependency onsimultaneous deprotonation of the Nα group, and protonation ofthe O-acyl hydroxamic acid group of isopeptide 1.To confirm the intramolecular nature of the rearrangement,

reaction time courses were carried out at two different isopep-tide concentrations (Fig. 1B and SI Appendix, section 5). Theobserved reaction half-lives did not decrease when the isopeptideconcentration was raised 10-fold, across a range of pH values.These data provide strong evidence that the reaction is first-order with respect to isopeptide, consistent with rearrangementby intramolecular acyl transfer (30).At pH 6, the half-life (t1/2) for rearrangement of 1 was on the

order of 15 min, compared with typical half-lives for O,N-acylshifts of 2–4 h in dibenzofuran frameworks (14, 31), hours todays for ester-linked thiazolidines (16), 22 h for a perylene-basedscaffold (32), and 3 h (with microwave heating) for medium tolong-range shifts in serine isopeptides (33). Thus, the rate of O-acyl hydroxamic acid isopeptide rearrangement is notable.Rearrangements of serine, threonine, and related O-acyl iso-peptides by O,N-acyl shift can also be rapid (half-lives of ∼30 s to∼10 min at neutral or mildly alkaline pH) (34–38), but theseproceed through five-member rings, which are expected to form∼104-fold faster than seven-member rings (30). Facile rear-rangement of Asp(β-HA) isopeptides is probably due in part tothe heightened reactivity of a diacyl hydroxylamine comparedwith an alkyl ester. Acyl donor strength was demonstrated re-cently to increase the rate of transfer across a salicylaldehyde-derived template, which lends support to this hypothesis (39).Several lines of evidence support the covalent structure of the

isomeric reaction product 1′ as the indicated amide. First is thechange in charge-state distribution observed upon conversion ofO-acyl hydroxamic acid 1 to product: The most abundant chargestate changes from [M+4H]4+ in 1 to [M+3H]3+ in the product1′, consistent with the loss of a basic Nα group upon conversion.Second is the susceptibility of the Asp(β-HA) residue in 1′ to

oxidative or reducing treatments to yield either Asp or Asn, re-spectively (see below). This behavior illustrates that a hydroxa-mic acid group has been revealed upon conversion of 1 to 1′.Third, and most compelling, is the observation that several am-ide products investigated were indistinguishable from authenticpeptides, upon conversion of Asp(β-HA) to Asp (SI Appendix,Figs. S65 and S66).

Synthesis of an O-Acyl Hydroxamic Acid Isopeptide from a Peptide-αThioester and Hydroxy-Asparagine-Peptide. We accessed theunique chemistry of O-acyl hydroxamic acid isopeptides by thereaction of Asp(β-HA) with thioesters (40). In a representativeexperiment, peptide-αthioester 2 (5 mM) was treated with 1.3equivalents of N-terminal Asp(β-HA)-peptide 3 (6.5 mM) in 6 Mguanidine hydrochloride, 200 mM phosphate, pH 9 buffer; after15 min, 2 had been converted toO-acyl hydroxamic acid 1 (Fig. 2).The covalent structure of 1 was confirmed by reduction of theN-O bond in acetylated analog acetyl-1, which yielded productsconsistent with the indicated O-acyl connectivity (SI Appendix,Figs. S21 and S22).The rate at which hydroxamic acids react with thioesters would

determine the practicality of chemical ligations based on thesereagents, in the same way that thiol–thioester exchange betweena Cys-peptide and a peptide-αthioester determines the rate ofnative chemical ligation (41). Accordingly, we directly comparedthe reactivity of peptide-αthioester 2 to a small panel of thiolsand hydroxamic acids (SI Appendix, section 8). At pH 7, bothacetyl hydroxamic acid and Asp(β-HA) were ∼10 or ∼30× lessreactive to 2 relative to mercaptoethanesulfonate or cysteine,respectively. Thus, reaction of a peptide-αthioester with an N-terminal Asp(β-HA)-peptide was expected to be slower than

Fig. 1. An O-acyl hydroxamic acid isopeptide derived from Asp(β-HA)rearranges to a native peptide bond in aqueous buffer. (A) Reaction scheme.(B) First-order rate constants for O-acyl isopeptide rearrangement as afunction of pH, obtained at each of two isopeptide concentrations. Rateconstants were derived from fits of LC-MS peak areas to an integrated rateexpression, as described in SI Appendix, section 5.

Fig. 2. Reaction of a peptide-αthioester with an N-terminal Asp(β-HA)-peptide yields a peptide-αO-acyl hydroxamic acid isopeptide under mildaqueous conditions. Reaction scheme and LC-MS data showing conversion ofthioester 2 to O-acyl hydroxamic acid isopeptide 1, by treatment with N-terminal Asp(β-HA)-peptide 3, at pH 9.

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reaction with an N-terminal Cys-peptide, but potentially fastenough to form the basis of a practical chemical ligation.

Chemical Ligation of Peptide-αThioesters and an Asp(β-HA)-Peptide.Our kinetic data suggested two general strategies for chemicalligation involving capture of a peptide-αthioester by Asp(β-HA),and subsequent rearrangement of the resulting O-acyl isopeptideto give a native peptide bond. In the first strategy, a peptide-αthioester would be treated with an N-terminal Asp(β-HA)-peptide at a pH intermediate between the optima for eachstep. In the second strategy, the acyl capture step would beperformed at mildly alkaline pH, where its rate is faster (pH 8–9); upon completion, the pH would be dropped to that optimalfor the isopeptide rearrangement (pH 5–6). We first exploredthe one-step strategy.Peptide-Ala-αthioester 2 (5 mM) was treated with 1.3 equiva-

lents of N-terminal Asp(β-HA)-peptide 3 (6.5 mM) in 6 Mguanidine hydrochloride, 200 mM phosphate, pH 7 buffer. Afterseveral hours, liquid chromatography-mass spectrometry (LC-

MS) analysis of reaction mixture aliquots revealed a distributionof starting materials, intermediate O-acyl hydroxamic acid iso-peptide 1, and ligation product 1′ (Fig. 3). The reaction wascomplete after 20 h, and was accompanied by formation ofpeptide-αcarboxylate derived from thioester 2 (compound 2b;∼10%). A yield of 61% was obtained for ligation product 1′ afterisolation by preparative HPLC, based on the limiting reagent 2.To test the generality of the ligation, we studied the reactions

of hydroxamic acid 3 with a range of peptide-Xaa-αthioesters (4athrough 4g) at pH 7. Table 1 shows the yields of ligation productsobtained after isolation by preparative HPLC (see SI Appendix,section 9 for LC-MS data). Reactions involving the Gly-αthio-ester, Ser-αthioester, and Tyr-αthioester were markedly fasterthan for the Ala-αthioester, and were complete within 15, 30, and60 min, respectively. Reactions involving the Lys-αthioester andTrp-αthioester were intermediate in rate, being nearly completewithin 2 h, and the Val-αthioester reacted sluggishly, as expected,requiring 48 h for complete conversion. In the case of the Lys-αthioester, lactam formation was observed to the extent of ∼10%.The tolerance of O-acyl hydroxamic acid isopeptide rearrangementto a variety of adjacent amino acid residues (Xaa-αO-acyl hydrox-yamic acid) is in contrast to the rearrangements of S-acyl iso-peptides derived from the Nα(ethanethiol) function (42), or ofStaudinger ligation intermediates (3), which are facile only forXaa-Gly and Gly-Xaa cases.A two-step ligation strategy was also explored, with the goal of

decreasing the reaction time. Peptide-αthioester 2 (5 mM) wasagain treated with 1.3 equivalents of N-terminal Asp(β-HA)-peptide 3 (6.5 mM) in 6 M guanidine hydrochloride, 200 mMphosphate buffer, but this time at pH 8. After 15 min, peptide-αthioester 2 had been converted quantitatively to intermediateO-acyl hydroxamic acid isopeptide 1, and the reaction mixturewas acidified to pH 6, to facilitate rearrangement of isopeptide 1to peptide product 1′. The rearrangement was complete within2 h (SI Appendix, Fig. S47), and ligation product 1′ was isolatedin 75% yield after preparative HPLC (compared with 61% forthe one-step approach).A two-step ligation was also carried out using peptide-Leu-

αthioester 4a, on an analytical scale. Similar to the case of Alathioester 2, two-step ligation with 4a was complete after a total of2 h 45 min (45 min at pH 8, and 2 h at pH 6; SI Appendix, Fig.S48). Similar quantities of peptide-αcarboxylate were formed byboth the one-step and two-step procedures (∼5–10%), suggest-ing that this minor side product may be a general feature of theligation (SI Appendix, Figs. S40 and S48).

Conversion of Asp(β-HA) to Asp or Asn. The utility of Asp(β-HA)-mediated chemical ligation would be extended if an Asp(β-HA)residue could be converted into either Asp or Asn, postligation.We identified conditions for these transformations, and testedthem on portions of the model ligation products 5a through 5g(Fig. 4 and SI Appendix, sections 11 and 12). Overall yields for

Fig. 3. Amide-forming chemical ligation of a peptide-αthioester and N-terminal Asp(β-HA)-peptide, at pH 7. The MS inset for the 2-h timepointcorresponds to isopeptide 1; the MS inset for the 20-h timepoint correspondsto amide product 1′. An isolated yield of 61% was obtained for 1′ afterisolation by preparative HPLC.

Table 1. Isolated yields of HPLC-purified model ligationproducts YNFRXaa-D(β-HA)FYKLS-CONH2, obtained by ligationat pH 7

Entry Xaa Ligation products Isolated yield, %

1 Ala 1′ 61 (7.8 mg)3 Leu 5a 41 (13 mg)2 Gly 5b 73 (22 mg)4 Val 5c 29 (5.7 mg)5 Ser 5d 50 (13 mg)6 Tyr 5e 41 (6.9 mg)7 Lys 5f 39 (8.8 mg)8 Trp 5g 64 (10 mg)

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ligation and subsequent conversion of Asp(β-HA) to either Aspor Asn—which include two preparative HPLC isolations—ranged from 15 to 54%, with an average of 32% (Table 2).Reduction of Asp(β-HA) to Asn was optimally achieved by

treatment with Zn, at 37 °C, in 1 M ascorbic acid (Fig. 4 and SIAppendix, section 11). A variety of conditions, including Zn inacetonitrile/water (0.1% trifluoroacetic acid), Zn in acetic acid,and Zn in 1 M HCl were attempted with generally poorer re-producibility (43, 44). The reduction required ∼20 h for com-pletion, and seemed to work better on smaller scale. Weanticipate that given sufficient impetus, milder and homoge-neous reaction conditions for the reduction of a hydroxamic acidto a carboxamide could be found.

Conversion of Asp(β-HA) to Asp proved straightforward.Nagasawa and coworkers (45) reported the liberation of benzoicacid and nitrous oxide from benzohydroxamic acid, under nitro-sation conditions. Because nitrosation of unprotected peptide-αhydrazides to yield peptide-αacyl azides (46) is enjoying widespreaduse, we decided to adapt this protocol for the oxidative hydro-lysis of hydroxamic acids. Clean conversion of Asp(β-HA) to Aspwas observed upon treatment of substrates 5a through 5g withNaNO2 in pH 1 phosphate buffer at 0 °C, for 1–2 h (Fig. 4, Table2, and SI Appendix, section 12).To confirm the covalent structure of the ligation products, we

prepared authentic peptides corresponding to products 7a and7d. These compounds were indistinguishable from 7a and 7dprepared by chemical ligation and subsequent conversion ofAsp(β-HA) to Asp (SI Appendix, Figs. S65 and S66). To investi-gate the chiral integrity of ligation products 7a and 7d, we pre-pared diastereomeric epi-7a and epi-7d, containing either a D-Leuor D-Ser residue, respectively, at the site of ligation. Diastereomerepi-7a was not detected in the crude product 7a, suggesting thatligation proceeded with negligible epimerization of the O-acylhydroxamic acid isopeptide intermediate (SI Appendix, Fig. S65).Crude product 7d, on the other hand, contained as much as 2% ofdiastereomer epi-7d (SI Appendix, Fig. S66). The propensity ofpeptide-Ser-αthioesters to epimerize has been noted (47), and maybe responsible for the comparatively worse outcome observed inthis case.

Chemical Synthesis of the Z Domain. We investigated the utility ofAsp(β-HA)-mediated chemical ligation in the synthesis of the Zdomain (48). The most successful approach involved a singlechemical ligation to form an Ala-Asp(β-HA) peptide bond (aLeu-Asn bond in the wild-type sequence). LC-MS data showingthe reaction of peptide-αthioester 8 with N-terminal Asp(β-HA)-peptide 9 in a two-step ligation are shown in Fig. 5. After 30 minat pH 8, complete conversion to O-acyl hydroxamic acid ligationproduct 10 was confirmed, and the pH was adjusted to 6. After4 h, LC-MS analysis showed conversion of 10 to an isomericspecies 10′, presumed to be the desired amide product. As forour model studies, formal hydrolysis of thioester 8 occurred tothe extent of ∼10% (product 8b). Desired product 10′ was iso-lated by preparative HPLC in 49% yield.To prepare Z domain molecules with entirely natural side

chains, the Asp(β-HA) residue of ligation product 10′ was con-verted to either Asn or Asp. Treatment of 10′ with either Zn in1 M ascorbic acid (SI Appendix, Fig. S69) or NaNO2 (SI Ap-pendix, Fig. S70) furnished the desired reaction products 10a and10b in 99% and 71% yield, respectively, corresponding tooverall yields of 49% and 35% for the ligation and Asp(β-HA)transformation sequences.

Significance. In this work, we have documented the facile rear-rangement, via O,N-acyl shift, of O-acyl isopeptides derived fromAsp(β-HA). When combined with the ability of Asp(β-HA) toform O-acyl isopeptides by reaction with peptide-αthioestersunder mild aqueous conditions, this observation forms the basisof an amide-forming chemical ligation reaction. Conversion ofthe Asp(β-HA) residue to either aspartic acid or asparaginepostligation enables the synthesis of native peptides by thisstrategy. This reaction sequence is analogous to the ligation/de-sulfurization approach to protein synthesis by native chemicalligation, and is complementary to native chemical ligation withβ-thiol-Asn (49, 50), β-thiol-Asp (51), or auxiliaries (52, 53)which enable ligation at the same Xaa-Asp and Xaa-Asn sites.Before this work, cysteine, selenocysteine, and the variety of

nonnatural amino acids functionalized with thiols and selenolswere the only amino acids known to form isopeptides with acyldonors under similarly mild conditions. A variety of isopeptidesdefined by attachment of a peptide C terminus to the side chain

Fig. 4. Conversion of an Asp(β-HA) residue to either Asn or Asp. (A) Re-action schemes. (B) LC-MS analysis of starting material and crude reactionmixtures, for a representative substrate.

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of serine, threonine, tyrosine, or tryptophan have been reported(33). But, synthesis of these compounds requires the use ofhighly activated esters and appropriate side-chain protection,rendering them of limited utility for chemical ligation.A number of considerations should be made when applying

Asp(β-HA)-mediated chemical ligations in a new context. Al-though practical reaction times have been demonstrated (hours),the reaction is ∼30-fold slower than native chemical ligation, andrequires the use of peptide-αarylthioesters. The reaction appearscompatible with a range of peptide-Xaa-αthioesters, with theexception of β-branched residues (unacceptably slow reactionrate) and Lys (lactam formation). Suitable Xaa-Asp or Xaa-Asnsites should be chosen accordingly, to maximize the chanceof success.A side reaction observed consistently in chemical ligations

involving N-terminal Asp(β-HA) and peptide-αthioesters wasapparent hydrolysis of the peptide-αthioester reaction partner, tothe extent of ∼10%. The hydrolytic stabilities of peptide-αthio-esters and peptide-αO-acyl hydroxamic acids employed in thiswork were similar at mildly alkaline pH (SI Appendix, sections17 and 18); therefore, the origin of this side product is unclear.Formation of peptide-αcarboxylate to some degree may be aninherent limitation of chemical ligations involving N-terminalAsp(β-HA), but further studies are required to resolve this issue.One other feature of ligations involving N-terminal Asp

(β-HA) should be mentioned: addition of the thiol reagent 4-mercaptophenylacetic acid (a catalyst for native chemical liga-tion), at a concentration of 50 mM, stopped productive reaction,with appearance of peptide-αcarboxylate and conversion of N-terminal Asp(β-HA) to Asn [potentially from reduction of the O-acyl hydroxamate, by a mechanism described recently (44); SIAppendix, Fig. S81]. Addition of tris(2-carboxyethyl)phosphine at20 mM was tolerated, but approximately doubled the extent ofpeptide-αcarboxylate formation (again, presumably via reductionof the O-acyl isopeptide intermediate; SI Appendix, Fig. S82).

ConclusionThe unique, mutual reactivity of peptide-αthioesters and N-ter-minal cysteine-peptides forms a basis for the total synthesis ofproteins by native chemical ligation. With continued develop-ment, we anticipate that the reaction of N-terminal Asp(β-HA)-peptides (or structural variants) with suitable acyl derivativesmay become a useful complementary method, and potentiallyform the basis of an “orthogonal” amide-forming ligation thatcan proceed in the presence of an N-terminal cysteine-peptideand/or a thioester. This would represent a powerful advance,enabling, for example, fully convergent protein synthesis withoutthe need for protecting groups or intermediate functional groupconversions (e.g., conversion of a peptide-αhydrazide ligationproduct to a peptide-αthioester) (2).

MethodsLigations. In a one-step ligation procedure, peptide-αthioester and Asp(β-HA)-peptide were dissolved in 6 M guanidine hydrochloride, 200 mM phosphate,pH 7 buffer, and combined in a plastic tube (final concentrations of 5 and 6.5 mM,respectively). The reaction mixture was allowed to stand at ambient temperature,and reaction mixture aliquots were periodically withdrawn for LC-MS analysis.Upon completion, the product was isolated by preparative HPLC, as described inSI Appendix.

Table 2. Isolated yields for the transformation of model ligation products YNFRXaa-D(β-HA)FYKLS-CONH2 to eitherYNFRXaa-DFYKLS-CONH2 or YNFRXaa-NFYKLS-CONH2

Entry Xaa Asn peptides Isolated yield, % Overall yield, % Asp peptides Isolated yield, % Overall yield, %

1 Ala 1a 71 (3.5 mg) 43 1b 79 (4.4 mg) 483 Leu 6a 69 (3.3 mg) 28 7a 62 (3.5 mg) 252 Gly 6b 56 (2.8 mg) 41 7b 46 (5.2 mg) 344 Val 6c 74 (3.2 mg) 21 7c 91 (2.3 mg) 265 Ser 6d 65 (3.3 mg) 33 7d 48 (3.0 mg) 246 Tyr 6e 82 (3.8 mg) 34 7e 80 (2.5 mg) 337 Lys 6f 40 (2.2 mg) 16 7f 38 (1.7 mg) 158 Trp 6g 84 (3.0 mg) 54 7g 45 (1.9 mg) 29

Overall yields for ligation and subsequent transformation of Asp(β-HA)—involving two HPLC purifications—are also shown.

Fig. 5. Synthesis of a 58-mer peptide by Asp(β-HA)-mediated chemical ligation.Thioester VDNKFNKEQQ10NAFYEILHLP20NA-αCO-S-C6H4-CH2-CO2H 8 and hydroxa-mic acid D(β-HA)EEQRNAF30IQSLKDDPSQ40SANILLAEAKK50LNDAQAPK58-αCONH2

9 were first combined at pH 8 for 30 min, to generate isopeptide 10. Then, thereaction mixture was acidified to pH 6; formation of amide product 10′ wasconfirmed after 4 h. TheMS insets correspond to isopeptide 10 (30-min timepoint)and amide product 10′ (4-h timepoint), respectively. Product 10′ was obtained in49% yield after isolation by preparative HPLC.

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In a two-step ligation procedure, peptide-αthioester and Asp(β-HA)-pep-tide were combined as above, but at pH 8. After 30 min, a reaction mixturealiquot was analyzed by LC-MS, to confirm complete consumption of thio-ester. Once confirmed, the reaction mixture was adjusted to pH 6, by di-lution into 10 volumes of 6 M guanidine hydrochloride, 200 mM phosphate,pH 6 buffer. Upon completion, the product was isolated by HPLC.

Conversion to Asn. AnAsp(β-HA)-containing peptidewas dissolved to 1mg/mL in1.0 M ascorbic acid, and transferred to 3-mL glass vials in 1-mL portions (thereduction was most effective when the reactionmixture was less than 1mL). Zincpowder (100 mg) and a magnetic stir bar were added to each vial. The vials weresuspended in a temperature-controlled water bath (37 °C), and the suspensionswere stirred vigorously for 20 h. Upon completion, the reaction mixtures werepooled, diluted with 95/5 water/acetonitrile, and filtered through a 0.2-μmpolytetrafluoroethylene syringe filter. The product was isolated by HPLC.

Conversion to Asp. An Asp(β-HA)-containing peptide was dissolved in 6 Mguanidine hydrochloride, 200 mM phosphate, pH 3 buffer (2 mM), in aglass vial. The solution was adjusted to pH 1, and the vial was suspended inan ice/water bath. Next, 0.2 M NaNO2 in deionized water was added, tobring the concentration to 10 mM. Upon completion, residual oxidant wasquenched by addition of 0.5 M ascorbic acid. The product was isolatedby HPLC.

ACKNOWLEDGMENTS. We thank Ethan Evans, Alex Vinogradov, and Prof.JoAnne Stubbe for insightful discussions, and Alex Mijalis for technicalassistance with automated flow-based peptide synthesis. This work wassupported by Defense Advanced Research Projects Agency Award 023504-001 (to B.L.P.), a Bristol-Myers Squibb unrestricted grant in Synthetic OrganicChemistry (B.L.P.), a Novartis Early Career Award (B.L.P.), and Novo NordiskSTAR Programme.

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