Synthesis of Four-Disulfide Insulin Analogs via ... - ACS Publications

Mar 20, 2017 - peptide synthesizer were obtained from Midwest Biotech. Fmoc-. Cys(StBu)-OH .... using Origin software (OriginLab, Northampton, MA)...
0 downloads 0 Views 575KB Size
Subscriber access provided by University of Newcastle, Australia

Article

Synthesis of Four-Disulfide Insulin Analogs via Sequential Disulfide Bond Formation Fangzhou Wu, John P. Mayer, Vasily G. Gelfanov, Fa Liu, and Richard D. DiMarchi J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.6b03078 • Publication Date (Web): 20 Mar 2017 Downloaded from http://pubs.acs.org on March 21, 2017

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

The Journal of Organic Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 15

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

The Journal of Organic Chemistry

Synthesis of Four-Disulfide Insulin Analogs via Sequential Disulfide Bond Formation Fangzhou Wu1, John P. Mayer2, Vasily G. Gelfanov2, Fa Liu2* and Richard D. DiMarchi1,2* 1 Department of Chemistry, Indiana University, Bloomington, Indiana, 47405. 2 Novo Nordisk Research Center Indianapolis, Indianapolis, Indiana, 46241. Correspondence should be directed to Dr. Fa Liu: [email protected] or Prof. Richard DiMarchi: [email protected].

1 ACS Paragon Plus Environment

The Journal of Organic Chemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 15

Table of Contents Graphic OH StBu H

G I V EQCC

H2N

O

Acm O N H

H

tBu

Acm

Trt OH

tBu

O

H

Mmt

CC S L YQ L E N Y CN O

Trt

F V NCH L CGS H L V E A L Y L V CGE RG F F

H

H N O

P K T H2N

OH

O

HO

Sequential Disulfide Bond Formation 1. Thio-nitropyridine directed on-resin thiolysis 2. Thio-nitropyridine directed solution thiolysis 3. Acm/I2 oxidation 4. O-to-N acyl transfer 5. TFA/DMSO oxidation Step 1

G I V E Q C C T S C C S L Y Q L E N Y C N

H

Step 5

OH

Step 2

Step 3 H

F V NCH L CGS H L V E A L Y L V CGE RG F F Y T P K T

OH

Four-disulfide Bond Insulin Analog

2 ACS Paragon Plus Environment

Page 3 of 15

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

The Journal of Organic Chemistry

Abstract Naturally-occurring, multiple cysteine-containing peptides are a structurally-unique class of compounds with a wide range of therapeutic and diagnostic applications. The development of reliable, precise chemical methods for their preparation is of paramount importance to facilitate exploration of their utility. We report here a straightforward and effective approach based on stepwise, sequentially-directed disulfide bond formation, exemplified by the synthesis of four-disulfide bond-containing insulin analogs. Cysteine protection consisted of tert-butylthiol (StBu), thiol-trimethoxyphenyl (STmp), trityl (Trt), 4methoxytrityl (Mmt), S-acetamidomethyl (Acm) and tert-butyl (tBu). This report describes chemistry that is broadly applicable to cysteine-rich peptides and the influence of a fourth disulfide bond on insulin bioactivity.

3 ACS Paragon Plus Environment

The Journal of Organic Chemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 4 of 15

Introduction Disulfide-rich, bioactive macromolecules are widely represented in nature. Disulfide bonds stabilize higher-order structures that confer much enhanced chemical, biophysical and metabolic stability1-4. These higher-order structures are also integral to high potency and specificity in biological action, with conotoxins, epidermal growth factors and the insulin superfamily peptides representing prominent examples5-9. Synthetic access to these peptides is essential to enable investigation of their structure-activity relationships and subsequent evaluation as drug candidates10-12. Despite the availability of a number of established cysteine protection and activation strategies, applying these in orthogonal fashion to multiple disulfide-containing peptides is rarely straightforward. Conventional approaches utilize single step, air-mediated oxidation of native cysteines to form thermodynamically favored, multiple disulfides13-16. Similarly, redox buffer systems are often also used to fold linear intermediates into native disulfide-linked structures7,13. An alternative strategy involves sequential disulfide formation directed by orthogonal thiolprotection17-21. This method is not dependent on native structure and as such can facilitate access to a broader set of structures which are otherwise not synthetically accessible10. The application of a stepwise disulfide bond formation strategy has largely been restricted to peptides with three or fewer disulfide bonds10,12 with four disulfide bond syntheses only rarely reported22-24. Accordingly, we set out to exemplify a synthetic route to peptides possessing four disulfide bonds via a sequential disulfide bond assembly process. We report the synthesis and biochemical assessment of five peptides analogous to human insulin where each of the four disulfides formed sequentially through the use of six different cysteine protecting groups.

H

G I V EQCC T S CC S L YQ L E N Y CN

H

F V NCH L CGS H L V E A L Y L V CGE RG F F Y T P K T

OH

OH

A10-B4 Four-disulfide Insulin 1

H

G I V E QCC T S CC S L YQ L E NY CN

H

F V NQC L CGS H L V E A L Y L V CGE RG F F Y T P K T

OH

OH

A10-B5 Four-disulfide Insulin 2

H

G I V EQCC T S CC S L YQ L E N Y CN

H

F V NQHCCGS H L V E A L Y L V CGE RG F F Y T P K T

OH

OH

A10-B6 Four-disulfide Insulin 3

H

H

T GY KG I A C E CCQH Y C T DQE F I N Y C P P V T E S S S S S S S A A

H E H T CQ L DD P A H PQG K CGS D L V N Y HE E K C E E E E A

OH

OH

Snail Insulin Con-Ins G2 4

H

H

G I V CQCC T S I C S L YQ L E N Y CN

OH

H E H T CQ L DD P F V NQH L CGS H L V E A L Y L V CGE RG F F Y T P K T

OH

Human/Snail Insulin Hybrid 5

Figure 1. Structure of four-disulfide insulin analogs (The 4th disulfide bond is highlighted in red.)

Results and Discussions 4 ACS Paragon Plus Environment

Page 5 of 15

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

The Journal of Organic Chemistry

Since its discovery nearly a century ago insulin has served an indispensable role in the treatment of diabetes. Native insulin consists of two individual chains (A and B-chain) which are connected by three disulfide bonds. Disruption of any of the three bonds causes a dramatic loss in structural stability and biological activity28. Conversely, the site-specific introduction of a fourth disulfide bond was observed to modestly enhance chemical and biophysical stability, as well as in vivo activity. The work was enabled by biosynthesis and native folding, with the fourth disulfide optimally positioned at A10-B4 125,26. An initial survey was conducted to determine the optimal residue in the B1-B6 region to crosslink with residue A1027. Yeast expression provided four of the six targeted insulin analogs with formation of the B5 2 and B6 3 analogs impaired by poor folding efficiency, purportedly resulting from non-native structures. Our synthetic strategy based on stepwise, directed disulfide bond formation enabled the preparation of the B5 2 and B6 3 insulins as well as its extension to other four-disulfide insulin analogs with less homology to human insulin (Figure 1).

Scheme 1. Application of the “O-to-N” acyl shift in peptide synthesis Insulin has historically proved a challenging synthetic target, in part due to the biophysical properties of the highly hydrophobic A-chain28,29. Recently, Liu et al. noted that incorporation of isoacyl dipeptides significantly improved solubility and chromatographic handling of the A-chain and A-B heterodimers to provide a highly effective synthetic route to human insulin. The isoacyl dipeptide strategy is based upon the well-established O-N acyl transfer at serine or threonine ester to improve the synthesis and chromatography properties of hydrophobic peptides30,31(Scheme 1). Consequently, we employ the isoacyl approach in the present work. To assemble the fourth disulfide bond in a regio-specific manner, the two corresponding Cys residues were protected as tert-Butyl (tBu) (residues A10 with B4, B5 or B6). The three native disulfides (A6-A11, A7-B7 and A20-B19) were initially constructed through the selective use of StBu, Mmt, Trt and Acm-based cysteine protection22,32 (Schemes 2 and 3). The resulting Cys protection scheme enabled a sequential synthetic route to fourdisulfide insulin analogs with the first two disulfide bonds formed through thio-nitropyridine (SNPy) activation, the third by iodine oxidation of two Cys(Acm) residues and the fourth by DMSO/TFA oxidation of two Cys(tBu) residues33. Human insulin A-chain, with Cys residues protected as A6-StBu, A7-Acm, A10-tBu, A11-Mmt and A20-Trt containing an isoacyl dipeptide ThrA8-SerA9 was assembled by using Fmoc-based solid-phase peptide synthesis on Rink-amide ChemMatrix® resin (Scheme 2)30,34,35. The C-terminal residue was anchored to the solid support through the β-carboxyl of Fmoc-Asp-OtBu, intended to convert to native Asn through cleavage from the resin. Upon completion of peptide assembly, the peptidyl-resin 6 was treated with 20% 2-mercaptoethanol in DMF to remove the StBu group at CysA6, and the liberated thiol activated as CysA6-SNPy by treatment with 10 equiv 2,2’-dithiobis(5-nitropyridine) (DTNP) in DCM. The resulting resin 7 was subsequently treated with 1% trifluoroacetic acid (TFA), 5% triisopropylsilane (TIS) in DCM to selectively remove the Mmt group at CysA11 with the unmasked free thiol immediately undergoing SNPy-directed thiolysis to form the intra-A chain disulfide bond at CysA6-CysA11 (Scheme 2). Following acid treatment of resin 8 and RP-HPLC purification of the crude peptide, A-chain 9 was obtained in 16% yield based upon the initial resin substitution.

5 ACS Paragon Plus Environment

The Journal of Organic Chemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 6 of 15

Rink-amide ChemMatrix resin

S

Mmt

NH

G I V EQCC T S CC S L YQ L E N Y CD

H

Acm

O

Trt

tBu Peptidyl-resin 6

1. 2-mercaptoethanol in DMF, 60 min 2. 10 equiv DTNP in DCM, 60 min NO 2 N S

Mmt

NH

G I V E QCC T S CC S L YQ L E N Y CD

H

Acm

O

Trt

tBu Peptidyl-resin 7 1% TFA, 5% TIS in DCM

NH

G I V EQCC T S CC S L YQ L E N Y CD

H

Acm

O

Trt

tBu Peptidyl-resin 8

90% TFA, 5% TIS, 5% H 2O, 2 h

H

G I V E QCC T S CC S L YQ L E N Y CN Acm

tBu

OH

H

A-chain 9

Scheme 2. Synthesis of human insulin A-chain 9 (The isoacyl dipeptide is highlighted in red).

Human insulin B-chain, with the Cys residues protected as B4-tBu (or B5-tBu, or B6tBu), B7-Acm and B19-Trt and containing the isoacyl dipeptide at TyrB26-ThrB27, was assembled on a Thr pre-loaded HMPB ChemMatrix® resin (Figure S1, Supporting Information). Cleavage of B-chain resin 10 was initially attempted in TFA with 15 equiv DTNP. According to previous reports this approach should simultaneously cleave the peptideresin bond and activate CysB19 to the SNPy derivative29, 30. However, LC-MS analysis revealed that the tBu group protecting the CysB4 was simultaneously cleaved and subsequently modified by DTNP to yield the undesired (CysB4-SNPy, CysB7-Acm, CysB19SNPy)-B-chain. This premature deprotection of Cys-tBu was consistent with earlier reports of employing DTNP in TFA36, 37. The desired product was obtained through a 2-step procedure with the resin cleavage conducted in a conventional TFA cocktail, followed by DTNP treatment (Figure 2a). DTNP activation of crude B-chain 11 in a mixed solvent of acetic acid and DMF was incomplete with ~60% completion after 8 h. Presuming the slow reaction to be a function of poor B-chain solubility, a switch to DMSO resulted in complete B-chain activation within 10 min38, with the CysB5-tBu group remaining intact (Figure 2b). Application of this method provided the B-chain (12, 13 and 14) used in preparation of insulin analogs 1, 2 and 3 in 18% yield after RP-HPLC purification, based upon the initial resin substitution.

6 ACS Paragon Plus Environment

Page 7 of 15

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

The Journal of Organic Chemistry

a tBu H

Acm

HMPB ChemMatrix Resin

Trt

F V N Q C L C G S H L V E A L Y L V C G E R G F F Y T P K T Peptidyl-resin 10 90% TFA, 5% TIS, 5% H2O, 2 h

tBu H

Acm

H

F V N Q C L C G S H L V E A L Y L V C G E R G F F Y T P K T

OH

B-chain 11 10% AcOH in DMSO, 1.5 equiv DTNP, 10 min

N tBu H

Acm

NO2

S

F V N Q C L C G S H L V E A L Y L V C G E R G F F Y T P K T

OH

B-chain 12

b

Figure 2. Synthesis of human insulin B-chain 12. a): Synthetic scheme for the preparation of B-chain 12; b): Analytical HPLC traces of converting B-chain 11 to 12 by DTNP in DMSO ( λ = 214 nm, and the isoacyl dipeptide is highlighted in red).

Equimolar amounts of A-chain 9 and its B-chain counterpart 13 were mixed in an aqueous acetate buffer (6 M urea, pH 4.5) to form the A20-B19 disulfide bond. The reaction was complete within 90 min and the isoacyl motifs were found to be stable under these conditions30 (Scheme 3, Figure 3). The resulting mixture was diluted with acetic acid prior to treatment with 25 equiv of iodine (I2) which formed the third disulfide from the A7-B7 CysAcm protected residues. An excess amount of I2 was quenched by the addition of ascorbic acid, and the resulting A-B heterodimer 15 was purified by RP-HPLC. The lyophilized peptide was solubilized in ammonium bicarbonate buffer (50 mM, pH 8.0) for 10 min undergoing O-to-N acyl shifts to afford the amide backbone A-B heterodimer 1630. The fourth disulfide bond A10-B4 (or B5, B6) was formed by treatment of heterodimer 16 with 5% DMSO/TFA at room temperature which was completed within 2 h at 80% yield, as determined by LC-MS (Figure 3). The crude peptide was precipitated by the addition of diethyl ether and purified by RP-HPLC to provide the insulin A10-B4 analog 1 in 16% yield, as calculated from purified A-chain 9. The synthesis of the A10-B5 analog 2 and A10-B6 analog 3 employed the same method as established for analog 1. The yields of final, purified four-disulfide insulin analogs were 8% and 18% respectively (Figure S2 and S3, Supporting Information). While analog 1 is structurally identical to that previously prepared by yeast-based biosynthesis, the other two analogs have not been previously reported. The relatively lower yield of the A10-B5 analog 2 was attributed to unexpected disulfide bond exchange during two of the chemical reactions, these being the isoacyl shift and TFA/DMSO treatment. This side-reaction resulted in a contaminating product which closely eluted with analog 2 (Figure S3, Supporting Information).

7 ACS Paragon Plus Environment

The Journal of Organic Chemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

G I V E Q C C T S C C S L Y Q L E N Y C N

H

Acm

N

H

OH

H

tBu A-chain 9 NO 2

S

Acm

tBu

Page 8 of 15

F V NC H L CG S H L V E A L Y L V CGE RGF F Y T P K T

OH

B-chain 13 1. Mixed in 100 mM NH 4OAc, 8 M urea (pH 4.5), 90 min 2. 25 equiv I 2 in AcOH/H2O, 20 min

G I V E Q C C T S C C S L Y Q L E N Y C N

H

OH

tBu tBu H

F V NCH L CG S H L V E A L Y L V CGE RGF F Y T P K T

OH

A-B heterodimer 15 1. 25 mM NH 4HCO 3, 20% ACN (pH 8), 10 min 2. Lyophilization

G I V E Q C C T S C C S L Y Q L E N Y C N

H

OH

tBu tBu H

F V NCH L CGS H L V E A L Y L V CGERG F F Y T P K T

OH

A-B heterodimer 16 5% DMSO in TFA, RT, 2 h

A10-B4 Four-disulfide Insulin 1

Scheme 3. Synthesis of A10-B4 four-disulfide insulin analog 1 (The isoacyl dipeptide is highlighted in red).

Figure 3. Analytical HPLC traces for the preparation of A10-B4 four-disulfide insulin 1 ( λ = 214 nm)

We applied this newly-established synthetic route to other insulin-like peptides to assess its versatility when applied to peptides with less homology to native human insulin39,40. The recent identification of snail-derived insulin Con-Ins G2 4 from the venom gland transcriptome of C.geographus drew our interest given its unique fourth native disulfide bond, and a sizably different sequence from human insulin (Figure 1)41. The chemical synthesis of 4 has not previously been reported. We also designed a hybrid of peptide 4 and human insulin, which constitutes human/snail insulin hybrid 5 with a fourth disulfide bond added to human insulin (Figure 1). Synthesis of snail insulin 4 A-chain 17 was initiated by coupling Fmoc-Ala-OH to NovaSyn TGA resin via the symmetrical anhydride method. The peptide was assembled by standard Fmoc-based SPPS with on-resin construction of its intra-A-chain disulfide bond. We employed cysteine-thiol protection of A8-tBu, A10-STmp, A11-Acm, A15-Mmt and A24-Trt (Scheme S1, Supporting Information). The S-Tmp group was selected for the snail insulin Achain synthesis to facilitate a more efficient on-resin deprotection by reducing agents than achievable with StBu thiol protection42. As both snail insulin A- and B-chains possessed sufficient solubility in aqueous buffers, no isoacyl-dipeptide was required. Snail insulin Achain 17 containing the desired intra-chain disulfide bond (CysA10-CysA15) was obtained in 20% yield after resin cleavage and RP-HPLC purification (Scheme S1, Supporting Information).

8 ACS Paragon Plus Environment

Page 9 of 15

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

The Journal of Organic Chemistry

Snail insulin B-chain 18 (CysB4-tBu, CysB16-Acm, CysB28-Trt) was synthesized by conventional Fmoc-based SPPS and activated with DTNP in DMSO, following the same procedure developed for the human insulin B-chain synthesis (Scheme 4). Ligation of snail insulin A-chain 17 and B-chain 18 in 6 M urea, ammonium acetate buffer as presented previously for the synthesis of 1-3, gave the snail insulin heterodimer (Scheme 4, Figure 4). The second inter-chain disulfide was subsequently installed by iodine-mediated oxidative deprotection of the Acm group to yield A-B heterodimer 19 (Scheme 4, Figure 4). After purification by RP-HPLC and lyophilization, snail insulin A-B heterodimer 19 was treated with 5% DMSO in TFA to generate the last disulfide bond to afford snail insulin 4 (Scheme 4, Figure 4). The overall synthetic yield for snail insulin 4 was 8% as calculated from purified A-chain 17. The slightly lower yield of snail insulin 4 as compared with 1-3 was attributed to the unexpected low quality iodine oxidation step following SNPy-directed chain combination (Figure 4). H

T GY K G I A C E C CQH Y C T DQE F I N Y C P P V T E S S S S S S S A A tBu

Acm

A-chain 17

NO 2

N Acm

tBu H

OH

H

S

H E H T CQ L DD P A H P QGK CGS D L V N Y H E E K C E E E E A

OH

B-chain 18 1. Mixed in 100 mM NH 4OAC, 6 M Urea (pH 6.8), 10 min 2. 25 equiv I 2 in AcOH/H2O, 20 min tBu H

T GY K G I A C E C CQH Y C T DQE F I N Y C P P V T E S S S S S S S A A

OH

tBu H

H E H T CQ L DD P A H P QGK CGS D L V N Y H E E K C E E E E A

OH

A-B heterodimer 19 5% DMSO in TFA, RT, 2 h

Snail Insulin 4

Scheme 4. Synthesis of snail insulin 4

Figure 4. Analytical HPLC of synthetic process for snail insulin 4 ( λ = 214 nm)

Since the design of human/snail insulin hybrid 5 was largely based upon the human insulin sequence, the isoacyl-dipeptides were required in its synthesis to enhance peptide solubility. The remaining approach was identical to that of peptide 4. The overall yield for 5 was 18% based on A-chain 20 (Scheme S2, Figure S4 in the Supporting Information).

9 ACS Paragon Plus Environment

The Journal of Organic Chemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 10 of 15

The biological activity of the four-disulfide insulin analogs was assessed in an engineered-cell line using an insulin receptor phosphorylation assay for both receptor isoforms (type A and type B). Relative potency was calculated by comparing the EC50 value of the analog versus authentic human insulin standard. The synthetic A10-B4 analog 1 functioned as a full agonist at both receptor isoforms with slightly enhanced potency, which was consistent with the original report25 (Table 1 and Figure S5 in the Supporting Information). The high in vitro potency of analog 1 confirmed that all four disulfide bonds were paired correctly, as mis-paired disulfide bonded peptides are well-known to exhibit much lower activity2840. The bioactivity of A10-B5 analog 2 and A10-B6 analog 3 are reported here for the first time. A10-B5 analog 2 showed full maximal agonism at both receptor isoforms but with significantly reduced potency of 25% at receptor A and 12% at receptor B (Table 1, Figure S5 in the Supporting Information). In stark contrast, the A10-B6 analog 3 was completely inactive at each receptor isoform (Table 1, Figure S5 in the Supporting Information). To explore the possibility that 3 may function as an antagonist, a fixed insulin concentration of 6 nM was used to stimulate the receptor in the presence of various concentrations of 3. We observed no appreciable changes in insulin receptor activation at any concentration of 3, confirming that this peptide lacks antagonist activity. The loss of function of this analog is most likely caused by the alteration of protein conformation by the additional disulfide linkage or the LeuB6 to Cys mutation. (Figure S5, Supporting Information). The four-disulfide snail insulin 4 proved to be devoid of bioactivity at human insulin receptors (Table 1) We postulated that the comparative low potency of human/snail insulin hybrid 5 indicated that the absence of activity in 4 is a combination of the structural constraint imposed by the additional disulfide, as witnessed in analog 3 and the sizable differences in its primary sequence (Table 1). Analog 5 exhibited an intermediate potency between 2 and 3 suggesting that the unique location of this fourth disulfide bond might be unfavorable and likely a result of its position within the A-chain N-terminus, as extensions to the B-chain are typically well tolerated. Table 1. In vitro activities of four-disulfide insulin analogs and snail insulin

Analogs A10-B4 analog 1 A10-B5 analog 2 A10-B6 analog 3 Snail insulin 4 Human/Snail insulin hybrid 5

Relative Potency at IR-A (% of native insulin) 156% 25% Inactive Inactive

Relative Potency at IR-B (% of native insulin) 125% 12% Inactive Inactive

5%

3%

Conclusions The results demonstrate a straightforward and effective synthetic methodology used in preparing this set of five insulin-related analogs, containing four-disulfide bonds. While at least two of the peptides could not be prepared through a conventional biosynthetic route, they were readily accessible by our method. Peptide 1 exhibits bioactivity that is consistent with that previously reported following its preparation through native oxidative folding by yeast biosynthesis. Peptides 2 and 3 broaden the structure-activity relationship by providing access to two additional insulin analogs that were not obtainable through biosynthesis. This underscores the unique virtue of stepwise, disulfide bond formation employing orthogonal

10 ACS Paragon Plus Environment

Page 11 of 15

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

The Journal of Organic Chemistry

cysteine protection to achieve a fuller interrogation of the relationship between structure and function. This report embellishes the chemical synthesis of multiple-disulfide containing peptides through a sequentially-directed disulfide bond strategy. The synthetic method was validated using the four-disulfide based insulin analogs, and a recently-reported snail insulin that contains an unprecedented fourth disulfide bond. Each synthesis utilized standard, automated Fmoc-based solid phase assembly of the A- and B-chains, followed by a stepwise, regio-selective formation of each distinct disulfide bond, as enabled by site-specific orthogonal cysteine protection. We believe that this general approach is applicable to other cysteine-rich peptides without methionine or tryptophan residues and worthy of investigation as the examples provided within this study demonstrate how a single additional bond can impact the bioactivity of a macromolecules.

Material and Experimental Sections General information All reagents and solvents were purchased and used directly unless otherwise noted, including N,N’-dimethylforamide (DMF), methanol, dichloromethane (DCM), acetonitrile (ACN), diethyl ether, trifluoroacetic acid (TFA), 2-mercaptoethanol, piperidine, 6-chloride-hydroxybenzotriazole (6-Cl-HOBt), N,N’-diisopropylcarbodiimide (DIC), N,N’-diisopropylethylamine (DIEA), anisole, triisopropylsilane (TIS), 2,2’dithiobis(5-nitropyridine) (DTNP), dimethyl sulfoxide, acetic acid, iodide, ammonium acetate, ascorbic acid and urea. Amino acid cartridges for the automated peptide synthesizer were obtained from Midwest Biotech. Fmoc-Cys(StBu)-OH, Fmoc-Cys(Mmt)-OH and FmocCys(tBu)-OH were products of Chemimpex international. Fmoc-Cys(Acm)-OH, BocThr[Fmoc-Tyr(tBu)]-OH and Boc-Ser[Fmoc-Thr(tBu)]-OH were obtained from AAPPTec. H-Rink Amide ChemMatrix resin was product from PCAS biomatrix Inc. Water (H2O) was produced from a Thermo Scientific Genpure water purification system. Analytical LC-MS was performed on an Agilent 1260 infinity LC-MS system coupled with Agilent 6120 quadrupole mass spectrometer and a Phenomenex Kinetex C8 2.6 μ 100Å (75 × 60 mm) column. The analysis was conducted at 1 mL/min flow rate using a linear gradient of 10% to 80% B over 10 min (Buffer A: 0.05% TFA-containing H2O; Buffer B: 0.05% TFA-containing 90% aqueous acetonitrile (ACN). LC traces were recorded by measuring UV absorbance at a wavelength of 214 nm. Preparative HPLC was performed using a Waters HPLC instrument including a Waters controller model 600, Waters dual wavelength detector 2487 connected with a Prostar model 701 fractions collector and a Luna 10μ C8 100Å AXIA packed (25 × 21.2 mm) reversed phase column. Flow rate varied from 10-15 mL/min with a linear gradient of 15% to 50% B over 80 min (Buffer A: 0.1% TFA-containing 10% aqueous ACN; Buffer B: 0.1% TFA-containing ACN). Peptide synthesis Peptides were assembled on an Applied Biosystem 433A automated peptide synthesizer. The synthesizer was programmed to operate customary Fmoc-based synthetic protocol on 0.1 mmol scale and utilized 20% piperidine in NMP for N-terminal Fmoc deprotection with DIC/6-Cl-HOBt employed for amino acid coupling. Coupling and deprotection times employed by the automated peptide synthesizer were 37 and 11 mins, respectively. Isoacyl dipeptides Boc-Ser[Fmoc-Thr(tBu)]-OH and Boc-Ser[Fmoc-Tyr(tBu)]OH were filled into regular amino acid cartridges and coupled by the synthesizer using standard instrument procedures during the corresponding A- and B-chain synthesis.

11 ACS Paragon Plus Environment

The Journal of Organic Chemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 12 of 15

Synthesis of human insulin A-chain 9 A-chain 9 was assembled on Rink amide ChemMatrix resin (0.1 mmol). The first residue was introduced by coupled Fmoc-Asp-OtBu to the Rink amide resin using the automated peptide synthesizer. Upon completion of peptide chain assembly, the resin was treated with 20% 2-mercaptoethanol in DMF (8 mL) in the presence of DIEA (1 mmol, 174 µL) for 1 h, subsequently washed with DMF (6 × 10 mL), DCM (6 × 10 mL) and DMF (6 × 10 mL). Next, the resin was mixed with DCM (10 mL) containing DTNP (1.29 mmol, 400 mg). The mixture was gently agitated for 1 h. The solution was removed by suction and resin was washed with DMF (6 × 10 mL), DCM (6 × 10 mL) then treated with 1% TFA, 5% TIS in DCM (6 × 3 min, 10 mL each). Peptidyl-resin was further agitated in DCM (10 mL) for 1 hour at room temperature. In the final step, the resin was washed with DCM (6 × 10 mL), briefly dried in vacuum and the crude peptide was cleaved from solid support by treatment with TFA/TIS/H2O (90:5:5, 15 mL total) for 2 h. The resin was filtered off, and peptide was precipitated by chilled diethyl ether. The pellet was collected, washed with ether, and solubilized in 20% aqueous acetonitrile containing 0.1% TFA for preparative RP-HPLC purification. 40 mg of A-chain 9 was obtained after lyophilization of the pooled fractions with a yield of 16% based upon resin substitution. Synthesis of human insulin B-chain Each of insulin B-chains (12, 13 and 14) was assembled on a Thr or Ala-preloaded HMPB ChemMatrix resin (0.1 mmol) utilizing the automated peptide synthesizer. Upon completion of peptide chain assembly, the resin was treated with TFA cleavage cocktail (15 mL) containing 5% TIS, 5% H2O at rt for 2 h. Crude peptides were subsequently recovered after diethyl ether precipitation and washings, and dissolved in 0.1% TFA-containing 20% aqueous acetonitrile for lyophilization. Crude, lyophilized B-chain (100~150 mg) was solubilized in DMSO (9 mL) containing glacial acetic acid (1 mL) with the addition of DTNP (1.5 equiv, 25~30 mg depend on the actual B-chain amount). The resulting solution was stirred at rt for 10 min before it was diluted with H2O and purified by preparative RP-HPLC. Following lyophilization of the pooled fractions, pure B-chains were obtained with a yield of around 25% based upon initial resin substitution. Construction of human insulin A-B heterodimer with CysA20-CysB19, CysA7-CysB7 disulfide bonds A-chain 9 (1 equiv, 20 mg) and its paired B-chain partners (1 equiv, 30 mg) were mixed and solubilized in 0.1 M ammonium acetate, 6 M urea (5 mL, pH 4.5). The resulting solution was stirred at rt for 2 h and the progress of the thiolysis chain combination was monitored by LC-MS analysis. Upon completion, the mixture was directly diluted with acetic acid (20 mL) and H2O (10 mL). I2 (25 equiv, 25~35 mg) solubilized in methanol (0.8 mL) was subsequently added and the solution was further stirred for 20 min before the reaction was terminated by addition of ascorbic acid (70 mM in H2O) to yield a colourless solution. The resulting A-B heterodimer was obtained following preparative HPLC purification and lyophilisation of selected fractions. Synthesis of four-disulfide human insulin analogs by TFA/DMSO oxidation A-B heterodimer with two Cys(tBu) residues (~10 mg) was dissolved in anhydrous TFA (1.9 mL) with the addition of DMSO (0.1 mL). The mixture was stirred at rt for 2 h. Upon completion, crude four-disulfide insulin analogs were recovered by diethyl ether precipitation and subsequently solubilized in 0.1% TFA containing aqueous acetonitrile for preparative HPLC purification. Four-disulfide insulin analogs were typically isolated with a 30-40% yield in this step.

In vitro biochemical characterizations of four-disulfide insulin analogs To measure receptor phosphorylation insulin-A or B receptor transfected HEK293 cells were plated in poly-lysine-coated 96 well tissue culture plates (Costar #3596, Cambridge, MA) and cultured

12 ACS Paragon Plus Environment

Page 13 of 15

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

The Journal of Organic Chemistry

in Dulbecco’s modified Eagle medium (DMEM) supplemented with antibiotics, 10 mM 4-(2hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) and 0.25% bovine growth serum (HyClone SH30541, Logan, UT) for 16-20 h at 37oC, 5% CO2 and 90% humidity. Serial dilutions of insulin or test analogs were prepared in DMEM supplemented with 0.5% bovine serum albumin (Roche Applied Science #100350, Indianapolis, IN) and added to the wells with adhered cells. After 15 min incubation at 37oC in humidified atmosphere with 5% CO2 the cells were fixed with 5% paraformaldehyde for 20 min at room temperature, washed twice with phosphate buffered saline (PBS) pH 7.4 and blocked with 2% bovine serum albumin in PBS for 1 h. The plate was then washed three times and filled with horseradish peroxidaseconjugated antibody against phosphotyrosine (Upstate biotechnology #16-105, Temecula, CA) reconstituted in PBS with 2% bovine serum albumin per manufacturer’s recommendation. After 3 h incubation at room temperature the plate was washed 4 times and 0.1 mL of 3,3’,5,5,-tetramethylbenzidine (TMB) single solution substrate (Invitrogen, #00-2023, Carlsbad, CA) was added to each well. Color development was stopped 5 min later by adding 0.05 mL 1N HCl. Absorbance at 450 nm was measured on Titertek Multiscan MCC340 (ThermoFisher, Pittsburgh, PA). Absorbance vs peptide concentration dose response curves were plotted and EC50 values were determined by using Origin software (OriginLab, Northampton, MA)

Supporting Information Available Synthetic scheme for the preparation of snail insulin 4 and human/snail insulin hybrid 5, and additional analytical HPLC and mass spec. data of synthetic four-disulfide human insulin analogs.This material is available free of charge via the Internet at http://pubs.acs.org

13 ACS Paragon Plus Environment

The Journal of Organic Chemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 14 of 15

Reference (1) Swaisgood, H. E. Biotechnol. Adv. 2005, 23, 71-73. (2) Colgrave, M. L.; Craik, D. J. Biochemistry. 2004, 43, 5965-5975. (3) Postma, T. M.; Albericio, F. Eur. J. Org. Chem. 2014, 2014, 3519-3530. (4) Creighton, T. E. BioEssays. 1988, 8, 57-63. (5) Vetter, I.; Lewis, R. J. Curr. Top. Med. Chem. 2012, 12, 1546-1552. (6) Shabanpoor, F.; Separovic, F.; Wade, J. D. Vitam. Horm. 2009, 80, 1-31. (7) Liu, F.; Zaykov, A. N.; Levy, J. J.; DiMarchi, R. D.; Mayer, J. P. J. Pept. Sci. 2016, 22, 260-270. (8) Lewis, R. J.; Garcia, M. L. Nat. Rev. Drug. Discovery. 2003, 2, 790-802. (9) Akondi, K. B.; Muttenthaler, M.; Dutertre, S.; Kaas, Q.; Craik, D. J.; Lewis, R. J.; Alewood, P. F. Chem. Rev. 2014, 114, 5815-5847. (10) Boulegue, C.; Musiol, H. J.; Prasad, V.; Moroder, L. Chem. Today. 2006, 24, 24-36. (11) King, G. F. Expert. Opin. Biol. Ther. 2011, 11, 1469-1484. (12) Andreu, D.; Albericio, F.; Sole, N. A.; Munson, M. C.; Ferrer, M.; Barany, G. Humana Press, Inc. 1994, 45, 91. (13) Tofteng, A. P.; Jensen, K. J.; Schaffer, L.; Hoeg-Jensen, T. Chembiochem. 2008, 9, 2989-2996. (14) Mamathambika, B. S.; Bardwell, J. C. Annu. Rev. Cell. Dev. Bol. 2008, 24, 211-235. (15) Avital-Shmilovici, M.; Mandal, K.; Gates, Z. P.; Phillips, N. B.; Weiss, M. A.; Kent, S. B. J. Am. Chem. Soc. 2013, 135, 3173-3185. (16) Beld, J.; Woycechowsky, K. J.; Hilvert, D. Biochemistry. 2007, 46, 5382-5390. (17) Shabanpoor, F.; Hossain, M. A.; Lin, F.; Wade, J. D. Methods. Mol. Biol. 2013, 1047, 81. (18) (a) Akaji, K.; Fujino, K.; Tatsumi, T.; Kiso, Y. J. Am. Chem. Soc. 1993, 115, 1138411392. (b) Barany, G.; Merrifield, R. B. J. Am. Chem. Soc. 1977, 99, 7363-7365. (19) Liobet, A. I.; Alvaez, M.; Albericio, F. Chem. Rev. 2009, 109, 2455-2504. (20) Okumura, M.; Shimamoto, S.; Hidaka, Y. The FEBS journal. 2012, 279, 2283-2295. (21) Gongora-Benitez, M.; Tulla-Puche, J.; Paradis-Bas, M.; Werbitzky, O.; Giraud, M.; Albericio, F. Biopolymers. 2011, 96, 69. (22) Cuthbertson, A.; Indrevoll, B. Org. Lett. 2003, 5, 2955-2957. (23) Mochizuki, M.; Tsuda, S.; Tanimura, K.; Nishiuchi, Y. Org. Lett. 2015, 17, 2202-2205. (24) Dekan, Z.; Mobli, M.; Pennington, M. W.; Fung, E.; Nemeth, E.; Alewood, P. F. Angew. Chem., Int. Ed. 2014, 53, 2931-2934. (25) Vinther, T. N.; Norrman, M.; Ribel, U.; Huus, K.; Schlein, M.; Steensgaard, D. B.; Pedersen, T. A.; Pettersson, I.; Ludvigsen, S.; Kjeldsen, T.; Jensen, K. J.; Hubalek, F. Prot. Sci. 2013, 22, 296-305. (26) Vinther, T. N.; Kjeldsen, T. B.; Jensen, K. J.; Hubalek, F. J. Pept. Sci. 2015, 21, 797-806. (27) Vinther, T. N.; Pettersson, I.; Huus, K.; Schlein, M.; Steensgaard, D. B.; Sorensen, A.; Jensen, K. J.; Kjeldsen, T.; Hubalek, F. Prot. Sci. 2015, 24, 779-788. (28) Mayer, J. P.; Zhang, F.; DiMarchi, R. D. Biopolymers. 2007, 88, 687-713. (29) Liu, F.; Luo, E. Y.; Flora, D. B.; Mayer, J. P. Org. Lett. 2013, 15, 960-963. (30) Liu, F.; Luo, E. Y.; Flora, D. B.; Mezo, A. R. Angew Chem., Int. Ed. 2014, 53, 39833987. (31) Taniguchi, A.; Sohma, Y.; Kimura, M.; Okada, T.; Ikeda, K.; Hayashi, Y.; Kimura, T.; Hirota, S.; Matsuzaki, K.; Kiso, Y. J. Am. Chem. Soc. 2006, 128, 696-697. (32) Cuthbertson, A.; Indrevoll, B. Tetrahedron. Lett. 2000, 41, 3661-3663. (33) Koide, T.; Otaka, A.; Fujii, N. Chem. Pharm. Bull. 1993, 41, 1030-1034. (34) Galande, A. K.; Weissleder, R.; Tung, C.-H. J. Comb. Chem. 2005, 7, 174-177. (35) Garcia-Ramos, Y.; Paradis-Bas, M.; Tulla-Puche, J.; Albericio, F. J. Pept. Sci. 2010, 16, 675-678. (36) Harris, K. M.; Flemer, S., Jr.; Hondal, R. J. J. Pept. Sci. 2007, 13, 81-93. (37) A. L. Schroll, R. J. Hondal, S. Flemer, Jr. J. Pept. Sci. 2012, 18, 155-162 (38) J. Han, S. Cheng, R. DiMarchi. Biopolymers. 2009, 92, 371-371 (39) Zaykov, A. N.; Mayer, J. P.; Gelfanov, V. G.; DiMarchi, R. D. ACS. Chem. Biol. 2014, 9, 683-691.

14 ACS Paragon Plus Environment

Page 15 of 15

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

The Journal of Organic Chemistry

(40) Narhi, L. O.; Hua, Q. X.; Arakawa, T.; Fox, G. M.; Tsai, L.; Rosenfeld, R.; Holst, P.; Miller, J. A.; Weiss, M. A. Biochemistry. 1993, 32, 5214-5221. (41) S-Hemami, H.; Gajewiak, J.; Karanth, S.; Robinson, S. D.; Ueberheide, B.; Douglass, A. D.; Schlegel, A.; Imperial, J. S.; Watkins, M.; Bandyopadhyay, P. K.; Yandell, M.; Li, Q.; Purcell, A. W.; Norton, R. S.; Ellgaard, L.; Olivera, B. M. Proc. Natl. Acad. Sci. U S A. 2015, 112, 1743-1748. (42) Postma, T. M.; Giraud, M.; Albericio, F. Org. Lett. 2012, 14, 5468-5471.

15 ACS Paragon Plus Environment