Synthesis and Utility of β-Selenophenylalanine and β-Selenoleucine in Diselenide-Selenoester Ligation

DOI: 10.1021/acs.joc.9b02665

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Journal of Organic Chemistry (2020)

Update date:2022-07-30

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Authors:

Corcilius, Leo

Payne, Richard J.

Premdjee, Bhavesh

Wang, Xiaoyi

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Article abstract of DOI:10.1021/acs.joc.9b02665

The synthesis of suitably protected β-selenophenylalanine and β-selenoleucine amino acids was accomplished from Garner's aldehyde as a common starting point. These selenoamino acids were incorporated into model peptides and shown to facilitate rapid diselenide-selenoester ligation (DSL) with peptide selenoesters which, when coupled with in situ deselenization, afforded native peptide products. The utility of one-pot DSL-deselenization chemistry at phenylalanine and leucine was demonstrated through the rapid synthesis of a glycosylated interferon-γfragment and the chemokine-binding protein UL22A, respectively.

Full text of DOI:10.1021/acs.joc.9b02665

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Article

Synthesis and Utility of #-Selenophenylalanine and #-

Selenoleucine in Diselenide-Selenoester Ligation (DSL)

Xiaoyi Wang, Leo Corcilius, Bhavesh Premdjee, and Richard J Payne

J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.9b02665 • Publication Date (Web): 16 Dec 2019

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Page 1 of 13

The Journal of Organic Chemistry

Synthesis and Utility of β-Selenophenylalanine and β-Selenoleucine

in Diselenide-Selenoester Ligation (DSL).

Xiaoyi Wang, Leo Corcilius, Bhavesh Premdjee and Richard J. Payne*

School of Chemistry, The University of Sydney, NSW 2006, Australia

richard.payne@sydney.edu.au

Supporting Information Placeholder

ABSTRACT: The synthesis of suitably protected β-selenophenylalanine and β-selenoleucine amino acids was accomplished from Gar-

ner’s aldehyde as a common starting point. These selenoamino acids were incorporated into model peptides and shown to facilitate rapid

diselenide-selenoester ligation (DSL) with peptide selenoesters which, when coupled with in situ deselenization, afforded native peptide

products. The utility of one-pot DSL-deselenization chemistry at phenylalanine and leucine was demonstrated through the rapid synthesis of

a glycosylated interferon- fragment and the chemokine-binding protein UL22A, respectively.

namely γ-Se-Pro²⁹and β-Se-Phe³⁰, thus enabling access to Xaa-

Pro and Xaa-Phe junctions following chemoselective deseleni-

INTRODUCTION

The field of protein science has greatly benefited from the ad-

vent of peptide ligation chemistry^1-3that allows proteins to be

chemically synthesized by convergent assembly of smaller pol-

ypeptide fragments. The most widely used methodology is na-

tive chemical ligation (NCL)⁴which involves the chemoselec-

tive reaction between a peptide with an N-terminal cysteine

residue and another fragment functionalized at the C-terminus

as a thioester. These two species react through a reversible and

rate-limiting transthioesterification step, followed by a rapid

intramolecular S-to-N acyl shift to afford a native amide bond

(Scheme 1A). The method has been significantly expanded by

the use of desulfurization chemistry to convert Cys to native

Ala residues^5-7and through the use of synthetic thiol-derived

amino acids which can also be desulfurized to native amino ac-

ids following the ligation event^8-24(Scheme 1B). The slow ki-

netics of NCL reactions at sterically hindered ligation junctions,

coupled with the lack of chemoselectivity of desulfurization

(that results in the removal of all thiol groups, including those

of native Cys residues) has prompted exploration of ligation

chemistry at the 21^stamino acid selenocysteine (Sec)^25-27. The

weaker C-Se bond strength in Sec, when compared to the C-S

bond in Cys residues, enables the chemoselective deseleniza-

tion of Sec to Ala in the presence of unprotected Cys, a finding

first reported by Metanis and Dawson.²⁸This chemistry has

provided the impetus to perform NCL at selenoamino acids,

zation. Despite the increased nucleophilicity of Sec, which

should lead to a significant rate enhancement in NCL, reaction

kinetics with peptide thioesters are slow due to the low reduc-

tion potential of the diselenide (-381 mV), which limits the

steady state concentration of the reactive selenolate under

standard NCL conditions that employ thiol reductants.

We recently reported a novel peptide ligation reaction between

peptide dimers bearing an N-terminal selenocystine (the oxi-

dized form of Sec) and peptide selenoesters - dubbed the

diselenide-selenoester ligation [DSL, (Scheme 1C)].^31-32The re-

action proceeds in aqueous denaturing ligation buffer in the ab-

sence of a reductant with reactions proceeding cleanly and to

completion within 10 min, even at sterically-encumbered liga-

tion junctions such as Val and Ile (cf. NCL where these reactions

take 48 h or longer³³). Importantly, DSL reactions can also be

coupled with in situ deselenization to afford alanine at the liga-

tion junction (or serine through an oxidative deselenization re-

action^34-35). The concept of one-pot DSL-deselenization has re-

cently been extended to amino acids other than the 21^stamino

acid Sec, e.g. -Se-Pro³⁶, β-Se-Asp and γ-Se-Glu³⁷. Together,

these selenoamino acids have been used to rapidly access na-

tive selenoproteins as well as synthetic libraries of small post-

translationally modified proteins³⁸through one-pot DSL-

deselenization. However, the DSL reaction manifold is still

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Page 2 of 13

limited to reactions at a select number of proteinogenic amino

acids.

ether was cleaved using TBAF to yield primary alcohol 10,

which was subjected to TEMPO/BAIB oxidation to yield the

carboxylic acid 11. Reduction of the selenocyanate with NaBH₄

in the presence of PMBCl then afforded the Boc-PMB-(β-Se)Leu

building block 5 with an overall yield of 12% over 8 steps.

Herein, we report the development of one-pot DSL-

deselenization chemistry at β-Se-Phe and β-Se-Leu to expand

the scope of the DSL methodology (Scheme 1D). As part of this

work, we also intended to establish a general synthetic route to

β-Se-Phe and β-Se-Leu from a common precursor. The power

of the DSL-deselenization chemistry at β-Se-Phe and β-Se-Leu

is highlighted in the synthesis of a fragment of human inter-

feron-γ and a virus-derived chemokine-binding protein, UL22A,

respectively.

Scheme 2. Synthesis of Boc‐PMB‐(β‐Se)Phe (2) from

Garner’s aldehyde.

Scheme 1. The evolution of chemical ligation reactions

from NCL to DSL

RESULTS AND DISCUSSION

The synthesis of both target selenoamino acids was proposed

from Garner’s aldehyde 1 as a common starting point. The tar-

get p-methoxybenzyl (PMB) protected β-Se-Phe [Boc-PMB-(β-

Se)Phe] building block 2 was first synthesized over 9 steps

from Garner’s aldehyde 1 through slight modifications to our

previously reported method³⁰(Scheme 2). Specifically, to avoid

the tedious work-up associated with the pyridinium dichro-

mate oxidation, the final oxidation of primary alcohol 3 was in-

stead carried out using 2,2,6,6-tetramethyl-1-piperidinyloxy

(TEMPO) and (diacetoxyiodo)benzene (BAIB). The resulting

selenocyanated amino acid 4 was reductively alkylated with

PMB-Cl to afford the desired Boc-PMB-(β-Se)Phe building

block 2. When preparing the corresponding Boc-PMB-(β-

Se)Leu building block 5 (Scheme 3), it was noted that the initial

Grignard addition of iPrMgBr to Garner’s aldehyde 1 delivered

the alcohol intermediate 6 as only the syn-diastereomer (vs. 2:3

syn/anti for PhMgBr), thereby alleviating the need for a lengthy

oxidation and reduction sequence to obtain diastereomeri-

cally-pure material. However, attempts to displace the corre-

sponding syn-mesylate with KSeCN were unsuccessful due to

the unfavourable conformational preference imposed by the

oxazolidine (the syn-mesylate was also observed to be unreac-

tive during the original Boc-PMB-(β-Se)Phe synthesis³⁰).

Therefore, the isopropylidene moiety was first removed to

yield diol 7. The primary alcohol was then protected as the

TBDMS ether 8, which underwent smooth mesylation and

KSeCN displacement at the secondary alcohol to afford the anti-

selenocyanate 9 in 60% yield over 2 steps. Next, the TBDMS

Scheme 3. Synthesis of Boc‐PMB‐(β‐Se)Leu (5) from

Garner’s aldehyde.

In order to evaluate the utility of selenoamino acid building

blocks 2 and 5 in DSL chemistry, each was coupled to the N-

terminus of the Rink amide resin-bound model pentapeptide

SPGYS, which was assembled through Fmoc-strategy solid-

phase peptide synthesis (Fmoc-SPPS, Scheme 4). After global

cleavage of the acid-labile side chain protecting groups, and

subsequent PMB deprotection, the two model peptides were

afforded as their corresponding diselenide dimers 12 and 13

in 38% and 36%, respectively, after HPLC purification. The two

selenopeptide fragments were next evaluated in the additive-

free DSL reaction with a wide range of peptidyl selenoesters

LYRANX-SePh (14a‐f) bearing different C-terminal residues

(Tables 1 and 2). The additive-free DSL reactions were con-

ducted by simply dissolving both the peptide diselenide dimer

12 or 13 and selenoester 14a‐f in 6 M Gn·HCl, 0.1 M Na₂HPO₄

aqueous buffer (final pH 6.1-6.4 without adjustment)³². In each

case, three intermediates were observed in the ligation mixture

corresponding to I) ligated product bearing an additional

equivalent of the C-terminal fragment as a side chain peptidyl

selenoester, II) the symmetric diselenide of the ligated product,

and III) the asymmetric diselenide encompassing the ligated

product with a side chain phenylselenyl group (III was only ob-

served for ligations with β-Se-Phe peptide 12). It was notewor-

thy that all ligations proceeded on a dramatically reduced time-

scale compared with the thiolated counterparts, β-SH-Phe¹¹or

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The Journal of Organic Chemistry

β-SH-Leu^12-13, under NCL conditions. Remarkably, the ligations

between diselenide dimer 12 and 13 and the selenoester 14d

and 14e, containing sterically hindered C-terminal residues Ile

and Val, reached completion in 1 h for Phe (Table 2, entries 4

and 5) and 3 h for Leu (Table 1, entries 4 and 5). This compares

favorably to ligations of the thiol counterparts, β-SH-Phe (24 h)

and β-SH-Leu (8 h), under NCL conditions. Given the similar

rates of ligation for the β-Se-Leu and β-Se-Phe peptides (bear-

ing opposite stereochemistry at the β position), it can be in-

ferred that the configuration of the selenium auxiliary does not

dramatically affect the rate of ligation. This is in contrast to the

corresponding β-SH-Leu amino acid, for which the S to N acyl

shift becomes rate-limiting when the thiol is anti-configured.¹³

En-

try

Selenoester

(14)

Ligation

Time

Desel. Time* Yield of

a X=Ala (A)

b X=Ser (S)

c X=Leu (L)

d X=Ile (I)

e X=Val (V)

10 min

1 h

16 h

58%

69%

82%

55%

64%

Without purification, the DSL mixtures were degassed with ar-

gon and then subjected to in situ deselenization with tris(2-car-

boxyethyl)phosphine hydrochloride (TCEP) and DL-

dithiothreitol (DTT) to convert I‐III into the native peptide

products. The deselenization of β-Se-Leu peptides proceeded

cleanly in 16 h to yield Xaa-Leu peptides 15a‐e in 55-82% yield

over two steps after HPLC purification (Table 1). In contrast,

the deselenization of β-Se-Phe, was initially accompanied by

two diastereomeric, β-hydroxylated peptide byproducts de-

spite extensive degassing (see Figure S3 in Supporting Infor-

mation). These β-hydroxylated byproducts were also observed

during NCL-deselenization reactions between thioesters and

the selenopeptide 12 in previous work³⁰. In order to suppress

the hydroxylated byproducts, we developed a competing oxy-

gen scavenger p-carboxybenzyl diselenide 16 which was syn-

thesized in two steps from 4-bromomethylbenzoic acid

(Scheme 5). Diselenide 16 was introduced into the reaction

mixture prior to deselenization with the hope that the benzylic

radical generated upon deselenization would out-compete that

of the Phe side chain for oxygen. The radical initiator VA-044

was also added to the deselenization mixture to accelerate the

reaction, which was slowed down by the addition of 16. Thank-

fully, under the modified conditions, the unwanted β-hydrox-

ylation side-reaction was suppressed from ~ 35% to < 2% as

judged by analytical UPLC-MS, and the reactions were complete

within 5 minutes when the reactions were performed at 37 °C.

Following HPLC purification native peptide products 17a‐f

were afforded in 51-74% over the two steps (Table 2). It should

be noted that deselenization of the β-Se-Leu peptides can also

pushed to completion within 5 min (instead of 16 h) through

addition of VA-044 and incubation at 37 °C. However, these

conditions are not selective, and will also lead to desulfuriza-

tion of Cys residues.

3 h

*Deselenization was conducted using TCEP (50 equiv.) and DTT (50 equiv.)

at rt.

Table 2. Reaction time and yield for one-pot DSL-deselenization at β-Se-Phe.

Entry Selenoester

Ligation

Time

Desel. Time* Yield of

(14)

a X=Ala (A)

b X=Ser (S)

c X=Leu (L)

d X=Ile (I)

e X=Val (V)

f X=Thr (T)

5 min

1 h

5 min

68%

66%

74%

62%

51%

65%

1 h

*Deselenization was conducted using TCEP (50 equiv.) and DTT (200 equiv.)

in the presence of 16 (4.4 equiv) and VA-044 (1.7 equiv.) at 37°C.

Scheme 4. Synthesis of model selenopeptides contain‐

ing β‐selenol amino acid building blocks (A: β‐Se‐Phe; B:

β‐Se‐Leu) at N‐termini.

Scheme 5. Synthesis of oxygen scavenger p‐carboxyben‐

zyl diselenide 16.

In order to evaluate the robustness of Boc-PMB-(β-Se)Phe

building block 2 in ligation towards more complex targets, fo-

cus was directed towards the synthesis of a truncated version

of human interferon-γ (IFN-γ 75-131, 18), a cysteine-free 57-

mer glycopeptide bearing an N-linked -GlcNAc moiety at

Table 1. Reaction time and yield for one-pot ligation-deselenization at β-Se-Leu.

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Page 4 of 13

the ligation to proceed to completion within 30 min (as judged by

Asn120 (Scheme 6). IFN-γ is a cytokine secreted by T lympho-

cytes and natural killing cells and imparts crucial antimicrobial

and antiviral functions in human innate and acquired immunity.

The target IFN-γ 75-131 peptide 18 was disconnected almost

evenly into two fragments, selenoester IFN-γ 75-104 19, pos-

sessing a C-terminal phenylalanine residue, and diselenide di-

mer IFN-γ 105-131 20, containing an N-terminal β-Se-Phe.

Both IFN-γ fragments were assembled on 2-chlorotrityl chlo-

ride resin via Fmoc-SPPS, using a per-O-acetylated Fmoc-

Asn[GlcNAc(OAc)₃]-OH building block³⁹to install the glycan in

diselenide dimer IFN-γ 105-131 20. The DSL ligation of se-

lenoester IFN-γ 75-104 19 and diselenide dimer IFN-γ 105-131

20 was performed by dissolving peptides 19 and 20 in 6 M

Gn·HCl, 0.1 M Na₂HPO₄aqueous buffer and the final pH ad-

justed to 6.1-6.4. The DSL reaction proceeded smoothly and to

completion within 5 min, as evidenced by the generation of a yel-

low precipitate of (PhSe)₂, and confirmed by UPLC-MS analysis.

Upon hexane extraction of (PhSe)₂, an aqueous solution of oxygen

scavenger 16 was added to the ligation reaction, and the result-

ing mixture was degassed for 10 minutes under a stream of ar-

gon before the addition of radical initiator VA-044. Following

treatment with TCEP (50 eq.) and DTT (200 eq.), the deseleni-

zation mixture was sealed and incubated at 37 °C, resulting in

clean conversion to IFN-γ 75-131 18 within 5 min without any

observed hydroxylated byproducts. The crude reaction mix-

ture was purified by reverse-phase HPLC to afford the final gly-

copeptide IFN-γ 75-131 18 in 51% yield over two steps.

UPLC-MS analysis); the superior kinetics of DSL meant that no

ligation to the thioester at Gly-76 was oberved, despite the steric

accessibility of this site. After hexane extraction of (PhSe)₂, and

deselenization, the UL22A 77-103 fragment 24 was added to the

same Eppendorf tube together with TCEP (25 mM) and trifluoro-

ethanethiol (TFET, 2 vol.%) as a thiol additive⁴². Finally, a stand-

ard radical desulfurization was performed to convert β-SH-Asp

into native Asp, which provided UL22A 21-103 (21) in 40%

yield over 4 steps after HPLC purification.

Scheme 7. One‐pot three‐component kinetically‐con‐

trolled assembly of UL22A 21‐103 (21) via DSL at β‐Se‐

Leu followed by NCL at β‐thiol‐Asp.

In order to improve the efficiency and yield for the assembly of

UL22A, the target protein was subsequently re-disconnected

into two fragments, selenoester UL22A 21-71 25 and diselenide

dimer UL22A 72-103 26, which we hoped would enable the syn-

thesis of UL22A via a single DSL reaction (Scheme 8). Unfortu-

nately, on this occasion the two fragments did not ligate under

standard DSL conditions, which we attributed to steric hinderance

at the Val-Leu junction i.e. a combination of a large diselenide

dimer fragment and a bulky valine selenoester. In order to test this

hypothesis, we performed an ‘additive’ DSL reaction⁴¹. Specifi-

cally, a combination of (PhSe)₂and TCEP was included in the

ligation mixture to reduce diselenide 26. It is noteworthy that

under these conditions, 5-8% of diselenide dimer 26 was dese-

lenized due to the presence of TCEP. However, the ligation pro-

ceeded smoothly, providing the target UL22A protein in 58%

yield following hexane extraction, deselenization and HPLC pu-

rification. The need for additives in this case (and not in the

model Val-Leu ligation) highlights the fact that protein ligation

reactions are strongly dependent on the specific nature of the

fragments, not only on the sterics of the reactive termini.

Scheme 6. Assembly of IFN‐γ 75‐131 (18) via one‐pot

DSL‐deselenization chemistry using β‐Se‐Phe.

Next, we investigated the utility of the β-Se-Leu building block

5 in the synthesis of a larger target, namely the chemokine-

binding protein from human cytomegalovirus called UL22A (21)

(Scheme 7). UL22A has been shown to bind to RANTES with

high affinity⁴⁰and we have recently shown that sulfation of two

tyrosine residues leads to significant improvements in affin-

ity⁴¹. We first attempted to perform a one-pot three-fragment

kinetically-controlled ligation starting with DSL (using β-Se-

Leu) followed by NCL (β-SH-Asp ligation⁹). Specifically, the full-

length protein was disconnected into N-terminal fragment

UL22A 21-51 as a C-terminal phenylselenoester 22, bifunc-

tional middle fragment UL22A 52-76 as diselenide thioester di-

mer 23 and C-terminal fragment UL22A 77-103 as thiolated pep-

tide bearing an N-terminal β-SH-Asp 24. UL22A 21-51 selenoes-

ter 22 was first ligated to UL22A 52-76 diselenide dimer 23 un-

der DSL conditions (6 M Gn·HCl, 0.1 M Na₂HPO₄aqueous buffer

at a final pH of 6.1-6.4). Incubation of the reaction at 37 °C allowed

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The Journal of Organic Chemistry

UPLC-MS was performed on a Shimadzu LC-MS 2020 system

equipped with a Nexera X2 LC-30AD pump and a Nexera X2

SPD-M30A diode array detector coupled to a Shimadzu 2020

mass spectrometer (ESI) operating in positive mode. Peptides

were analyzed using an Acquity UPLC BEH 1.7 µm (C18) 2.1 x

50 mm column at a flow rate of 0.6 mL min^-1using a mobile

phase of 0.1% formic acid (FA) in water (Solvent A) and 0.1%

FA in acetonitrile (Solvent B) and a linear gradient of 0-30% B

over 8 min or 0-50% B over 8 min or 0-70% B over 8 min.

Analytical reverse-phase UPLC was performed on a Waters Ac-

quity UPLC system equipped with a PDA eλ detector (λ = 210 –

400 nm), Sample Manager FAN and Quaternary Solvent Man-

ager (H-class) modules at 30 °C. Peptides were analyzed using

a Waters Acquity UPLC BEH 1.7 µm 2.1 x 50 mm column (C18)

at a flow rate of 0.6 mL min^-1using a mobile phase composed of

0.1% trifluoroacetic acid (TFA) in H₂O (Solvent A) and 0.1% tri-

fluoroacetic acid (TFA) in acetonitrile (Solvent B) and a linear

gradient as specified. Chromatograms were analyzed using Wa-

ters Empower software.

Scheme 8. Alternative one‐pot assembly of UL22A 21‐

103 (21) through a single DSL reaction at β‐Se‐Leu.

CONCLUSION

Preparative reverse-phase HPLC was performed using a Wa-

ters 600 Multisolvent Delivery System and Waters 500 pump

with 2996 photodiode array detector or Waters 490E Program-

mable wavelength detector operating at 214, 230 and 280 nm.

Model peptides, UL22A fragments and Interferon-γ fragments

were purified on a Waters Sunfire 5 µm (C18) 19 x 150 mm

preparative column operating at a flow rate of 7 mL min^-1.

UL22A proteins and Interferon-γ proteins were purified on a

XBridge Peptide BEH 5 µm (C18) 300 Å 10 × 250 mm semi-pre-

parative column operating at a flow rate of 4 mL min^-1. Prepar-

ative HPLC used a mobile phase of 0.1% trifluoroacetic acid in

water (Solvent A) and 0.1% trifluoroacetic acid in acetonitrile

(Solvent B) or 0.1% formic acid (FA) in water (Solvent A) and

0.1% FA in acetonitrile (Solvent B) and a linear gradient as

specified. Ligation yields were adjusted to account for the re-

moval of aliquots for reaction monitoring (e.g. UPLC-MS, pH

measurement).

In summary, we have demonstrated the feasibility of a general

synthetic route towards two suitably protected β-selenoamino

acids, β-Se-Phe and β-Se-Leu, from a single commercially avail-

able precursor, Garner’s aldehyde. We have demonstrated the

utility of β-Se-Phe and β-Se-Leu in rapid DSL with a range of

peptidyl aryl selenoesters. Notably, the β-hydroxylation by-

products previously observed during the deselenization of β-

Se-Phe were circumvented by the introduction of a novel radi-

cal scavenger p-carboxybenzyl diselenide. Furthermore, β-Se-

Phe and β-Se-Leu were successfully applied to the one-pot as-

sembly of a fragment of human interferon-γ and the chemokine-

binding protein, UL22A. Future work in our group will aim to build

on the toolbox of selenoamino acids that can be used in DSL to

further expand the scope of this methodology as well as the appli-

cation of these DSL with these selenoamino acids to larger protein

targets.

Materials

EXPERIMENTAL SECTION

General Procedures

Commercially available materials were purchased from Merck,

Sigma Aldrich, AK Scientific Inc or Mimotopes and used without

further purification. Amino acids, coupling reagents and resins

were obtained from PCAS Biomatrix Inc, Novabiochem or GL

Biochem. N,N-dimethylformamide (DMF) was obtained as pep-

tide synthesis grade from Merck or Labscan. Dichloromethane

was purchased from Merck. Thin layer chromatography was

performed on Merck TLC silica gel 60 0.25 nm F524 silica plates

and visualized by UV light at 254 nm or using vanillin or ninhy-

drin stain. Flash column chromatography was performed on

230-400 mesh silica gel from Grace. Solid phase peptide syn-

thesis was conducted in polypropylene syringes from Torviq

containing Teflon frits. A stirring hotplate with round bottom

flask adapter was used for large-scale reactions that required

heating. An Eppendorf tube heating block (Benchmark) was

used for ligation reactions that required heating.

¹H NMR and ¹³C{¹H} NMR spectra were recorded at 300 K using

a Bruker Avance DPX 400 spectrometer. Chemical shifts are re-

ported in parts per million (ppm) and are referenced to solvent

residual signals: CDCl₃(δ 7.26 [¹H], 77.2 [¹³C]) and DMSO-d₆(δ

2.50 [¹H], 39.52 [¹³C]). ¹H NMR data is reported as chemical

shift (δ), multiplicity (s = singlet, d = doublet, t = triplet, q =

quartet, dd = doublet of doublets, ddd = doublet of doublet of

doublets), relative integral, coupling constant (J Hz) and assign-

ment where possible. ¹³C{¹H} NMR data is reported as chemical

shift (δ). ¹H NMR assignments were made on the basis of COSY

spectra where applicable. Low-resolution mass spectra were

recorded on a Shimadzu 2020 mass spectrometer (ESI) operat-

ing in positive mode. High resolution ESI mass spectra were

measured on a BrukerDaltonics Apex Ultra 7.0 T fourier trans-

form mass spectrometer (FTICR). MALDI-TOF mass spectra

were measured on a Bruker autoflex speed MALDI-TOF instru-

ment using a matrix of sinapinic acid in water/acetonitrile (7:3

v/v) containing 0.1 vol% TFA in linear mode. Infrared (IR) ab-

sorption spectra were recorded on a Bruker ALPHA Spectrom-

eter with Attenuated Total Reflection (ATR) capability, using

OPUS 6.5 software. Optical rotations of enantioenriched com-

pounds were recorded on a Perkin–Elmer 341 polarimeter at

589 nm (sodium D line) with a cell path length of 1 dm. Concen-

trations are reported as g/100 mL.

Synthesis of β‐Selenoamino acid Building Blocks 2 and 5:

(2R,3S)‐2‐((tert‐butoxycarbonyl)amino)‐3‐phenyl‐3‐selenocya‐

natopropanoic acid (4). To a solution of 3³⁰(1.88 g, 5.29 mmol)

in a mixed solvent of MeCN/H₂O 1:1 (v/v, 40 mL) was added

(diacetoxyiodo)benzene (8.52 g, 26.45 mmol) and TEMPO

(0.41 g, 2.7 mmol) in one portion. The orange solution was

stirred at 25 °C for 3 h and then concentrated under a stream

of N₂followed by lyophilization. The crude product was puri-

fied

flash

column

chromatography

(96:2:2

CH₂Cl₂:MeOH:AcOH) to afford carboxylic acid 4 (1.39 g, 71%)

as a yellow oil. ¹H NMR (CDCl₃, 300 MHz) δ 7.35 (m, 5H, Ar-H),

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5.27 (m, 1H, NH), 5.20 (m, 1H, benzylic CH), 4.97-4.85 (m, 1H,

CH_α), 1.45 (s, 9H, Boc). The characterization data is in agree-

2961, 2927, 2874, 1685, 1503, 1392, 1366, 1258, 1166, 1064,

1012, 803; H NMR (CDCl₃, 400 MHz) δ 5.28 (br s, 1H, NH),

3.77-3.72 (m, 3H, CH₂, CHNH), 3.48-3.46 (m, 1H, CHOH), 3.12

(br s, 2H, OH), 1.77-1.68 (m, 1H, CH(CH₃)₂), 1.43 (s, 9H,

C(CH₃)₃), 0.99 (d, 3H, J = 6.6 Hz, CH(CH₃)_2a), 0.90 (d, 3H, J = 6.7

Hz, CH(CH₃)_2b); ¹³C{¹H} NMR (CDCl₃, 100MHz) δ 156.6, 79.8,

78.0, 65.4, 52.4, 31.1, 28.5, 19.1, 18.9; HRMS (ESI⁺): m/z calcd.

for C₁₁H₂₃NO₄Na [M+Na]⁺256.1523, found 256.1524.

ment with previously reported literature data by Malins et al.

(2R,3S)‐2‐((tert‐butoxycarbonyl)amino)‐3‐((4‐methoxyben‐

zyl)selanyl)‐3‐phenylpropanoic acid (2). To a solution of com-

pound 4 (1.38 g, 3.73 mmol) in THF (26 mL) at 0 °C was added

NaBH₄(0.28 g, 7.5 mmol) in 95% EtOH (3.7 mL). The reaction

was stirred for 1 h at 0 °C before the addition of PMB-Cl (2.02

mL, 14.92 mmol) and degassed 2 M NaOH solution (10 mL). The

reaction mixture was warmed to room temperature overnight

and poured into 1 M citric acid (200 mL). The aqueous layer

was extracted with EtOAc (3 × 200 mL), and the combined or-

ganic layers were washed with water (3 × 200 mL) and brine

(300 mL), dried over Na₂SO₄, filtered, and concentrated in

vacuo. The crude product was purified via flash column chro-

matography (3:10 to 100:0 EtOAc/hexane v/v + 0.1% AcOH),

affording the final building block 2 (1.25 g, 72%) as a pale-yel-

tert‐Butyl‐((2R,3R)‐1‐((tert‐butyldimethylsilyl)oxy)‐3‐hydroxy‐

4‐methylpentan‐2‐yl)carbamate (8). syn-Diol 7 (2.08 g, 8.95

mmol) and imidazole (1.22 g, 17.9 mmol) were dissolved in dry

CH₂Cl₂(8.5 mL) followed by addition of tert-butyldimethylsilyl

chloride (1.62 g, 10.7 mmol) in one portion at 0 °C. The mixture

was warmed to room temperature and the reaction stirred for

12 h. After complete consumption of the starting material, the

reaction was quenched with saturated aq. NH₄Cl solution (20

mL) and extracted with CH₂Cl₂(60 mL × 3). The combined or-

ganic layers were dried over MgSO₄, filtered and concentrated

in vacuo. The crude product was purified by flash column chro-

matography (5:95, EtOAc/Hexane, R_f= 0.28) to give TBS-

protected syn diol 8 as a yellow oil (3.00 g, 8.66 mmol,

97%). ꢀꢄ^ꢂ_ꢁ^ꢃ= -26.4° (c 0.54, CH₂Cl₂); IR (ν/cm^-1, film) 3444,

2956, 2929, 2858, 1716, 1692, 1498, 1472, 1390, 1366, 1254,

1168, 1097, 1017, 835, 776; ¹H NMR (CDCl₃, 400 MHz) δ 5.19

(d, 1H, J = 8.5 Hz, NH), 3.97 (dd, 1H, J = 3.4 Hz, 10.2 Hz, CH_2a),

3.78 (dd, 1H, J = 2.3 Hz, 10.2 Hz, CH_2b), 3.70 (m, 1H, CHNH), 3.51

(m, CHOH), 1.74-1.69 (m, 1H, CH(CH₃)₂), 1.43 (s, 9H, C(CH₃)₃),

0.99 (d, 3H, J = 6.6 Hz, CH(CH₃)_2a), 0.91-0.87 (m, 12H, SiC(CH₃)₃,

CH(CH₃)_2b), 0.07 (s, 6H, Si(CH₃)₂); ¹³C{¹H} NMR (CDCl₃, 100MHz)

δ 156.0, 79.3, 67.1, 51.3, 30.9, 28.5, 26.0, 19.1, 19.0, 18.3, -5.5;

HRMS (ESI⁺): m/z calcd. for C₁₇H₃₇NO₄SiNa [M+Na]⁺370.2384,

found 370.2386.

low oil. H NMR (CDCl₃, 300 MHz) δ 7.28-7.26 (m, 5H, Ar-H

(phenyl)), 7.11 (d, 2H, J = 9.0 Hz, Ar-H (PMB)), 6.78 (d, 2H, J =

6.0 Hz, Ar-H (PMB)), 5.01-4.98 (m, 1H, NH), 4.83 (m, 1H, CH_α),

4.33 (m, 1H, benzylic CH), 3.78 (s, 3H, OCH₃), 3.73-3.54 (m, 2H,

PMB benzylic CH₂), 1.41 (s, 9H, Boc). The characterization data

is in agreement with previously reported literature data by Ma-

lins et al.³⁰

tert‐Butyl

(R)‐4‐((R)‐1‐hydroxy‐2‐methylpropyl)‐2,2‐dime‐

thyloxazolidine‐3‐carboxylate (6). Magnesium turnings (0.27 g,

11 mmol) were dried in a 3-necked round bottom flask in vacuo

with heat and stirring for 0.5 h before the addition of dry Et₂O

(2 mL). A spatula tip of iodine was quickly added to the above

mixture and the resulting brown suspension was stirred vigor-

ously at 25 ^oC for 30 min. Isopropyl bromide (0.52 mL, 5.5

mmol) in Et₂O (3 mL) was added to the mixture dropwise at

0 °C, and the mixture was subsequently stirred at 40 °C for 1 h.

A solution of Garner’s aldehyde 1 (970 mg, 4.25 mmol) in Et₂O

(5 mL) was added dropwise to the Grignard reagent at 0 °C. The

reaction mixture was continually stirred at 0 °C and allowed to

warm to 25 °C over 5 h. The reaction was quenched with satu-

rated aq. NH₄Cl solution (8 mL) at 0 °C before extraction with

EtOAc (20 mL × 3). The combined organic layers were dried

over MgSO₄, filtered and concentrated in vacuo. The crude

product was purified by flash column chromatography (15:85,

EtOAc/Hexane, R_f= 0.32) to afford syn-alcohol 6 as a white

solid (630 mg, 2.29 mmol, 54%). ꢀꢄ^ꢂ_ꢁ^ꢃ= +54.5° (c 0.33, CH₂Cl₂);

m.p. 82-83 °C; IR (ν/cm^-1, film) 3399, 2963, 2935, 2875, 1657,

1400, 1366, 1248, 1172, 1108, 1058, 1018, 865; ¹H NMR (CDCl₃,

400 MHz) δ 4.04 (br s, 1H, CHNH), 3.94-3.91 (m, 1H, CH_2a), 3.76-

3.74 (m, 1H, CH_2b), 3.50-3.48 (m, 1H, CHOH), 1.70-1.64 (m, 1H,

CH(CH₃)₂), 1.59-1.44 (m, 15H, CH(CH₃)₂, C(CH₃)₃), 1.02 (d, 3H, J

= 6.8 Hz, CH(CH₃)_2a), 0.89 (d, 3H, J = 6.7 Hz, CH(CH₃)_2b); ¹³C{¹H}

NMR (CDCl₃, 75 MHz) δ 155.7, 99.1, 79.4, 77.3, 65.8, 45.4, 29.9,

29.3, 28.5, 19.1, 18.7, 17.6; HRMS (ESI⁺): m/z calcd. For

tert‐Butyl‐((2R,3S)‐1‐((tert‐butyldimethylsilyl)oxy)‐4‐methyl‐3‐

selenocyanatopentan‐2‐yl)carbamate (9). Triethylamine (1.80

mL, 13.0 mmol) and mesyl chloride (1.04 mL, 10.4 mmol) were

slowly added into a solution of TBS-protected syn diol 8 (3.00

g, 8.66 mmol) in dry CH₂Cl₂(30 mL) at 0 °C. The reaction mix-

ture was stirred at 0 °C for 40 min before being quenched with

saturated aq. NH₄Cl solution (40 mL) followed by extraction

with CH₂Cl₂(60 mL × 3). The combined organic layers were

dried over MgSO₄, filtered and concentrated in vacuo to yield

the crude mesylate as a pale yellow solid. The crude mesylate

and potassium selenocyanate (18.71 g, 129.9 mmol) were dis-

solved in dry MeCN (40 mL) and stirred at 65 °C for 24 h. The

reaction mixture was concentrated in vacuo, diluted with

CH₂Cl₂(50 mL) and poured into water (70 mL). The organic

layer was separated and the aqueous layer was extracted with

CH₂Cl₂(70 mL × 2). The combined organic layers were dried

over MgSO₄, filtered and concentrated in vacuo. The crude

product was purified by flash column chromatography (4:96,

EtOAc/Hexane, R_f= 0.31) to yield selenocyanate 9 as a yellow

oil (2.26 g, 5.19 mmol, 60%). ꢀꢄ^ꢂ_ꢁ^ꢃ= -52.3° (c 0.34, CH₂Cl₂); IR

(ν/cm^-1, film) 2959, 2930, 2885, 2858, 2149 (SeCN), 1715,

1490, 1391, 1366, 1255, 1170, 1103, 1046, 836, 779; ¹H NMR

(CDCl₃, 400 MHz) δ 5.27 (d, 1H, J = 8.6 Hz, NH), 4.08-4.07 (m,

1H, CHNH), 3.87 (dd, 1H, J = 2.2 Hz, 10.7 Hz, CH_2a), 3.73 (dd, 1H,

J = 1.92 Hz, 10.6 Hz, CH_2b), 3.51-3.47 (m, 1H, CHSeCN), 2.11-

2.02 (m, 1H, CH(CH₃)₂), 1.47 (s, 9H, C(CH₃)₃), 1.30-1.28 (m, 3H,

CH(CH₃)_2a), 1.13-1.12 (d, 3H, J = 6.7 Hz, CH(CH₃)_2b) 0.91 (s, 9H,

SiC(CH₃)₃), 0.10 (s, 3H, Si(CH₃)_2a), 0.09 (s, 3H, Si(CH₃)_2b); ¹³C{¹H}

NMR (CDCl₃, 100 MHz) δ 155.1, 106.3(SeCN), 80.4, 64.1, 62.7,

51.2, 33.2, 29.8, 28.5, 26.1, 21.3, 18.6, -5.2, -5.3; HRMS (ESI⁺):

m/z calcd. for C₁₈H₃₆N₂O₃SeSiNa [M+Na]⁺459.1552, found

459.1554.

₁₄H₂₇NO₄Na [M+Na]⁺296.1832, found 296.1833.

tert‐Butyl ((2R,3R)‐1,3‐dihydroxy‐4‐methylpentan‐2‐yl)carba‐

mate (7). 0.5 M aq. HCl (20 mL) was added slowly to a solution

of syn alcohol 6 (2.54 g, 9.29 mmol) in THF (380 mL). The mix-

ture was stirred at 25 °C for 24 h before being quenched by the

addition of NaHCO₃(1.7 g, 20 mmol), followed by co-evapora-

tion with toluene to dryness. The crude material was diluted

with EtOAc (100 mL), filtered through cotton wool and concen-

trated in vacuo. The crude product was purified by flash col-

umn chromatography (1:1, v/v, EtOAc/Hexane, R_f= 0.34) to

obtain syn diol 7 as a yellow oil (2.09 g, 8.95 mmol,

96%). ꢀꢄ^ꢂ_ꢁ^ꢃ= -15.1° (c 0.37, CH₂Cl₂); IR (ν/cm^-1, film) 3377,

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The Journal of Organic Chemistry

tert‐Butyl ((2R,3S)‐1‐hydroxy‐4‐methyl‐3‐selenocyanatopentan‐

Synthesis of 4,4'‐(diselanediylbis(methylene))dibenzoic acid (16).

4-(Bromomethyl)benzoic acid (0.510 g, 2.37 mmol) was dis-

solved in dry THF (6 mL) and the solution was cooled to 0 °C.

The resulting solution was treated with potassium selenocya-

nate (0.510 g, 3.55 mmol) in one portion at 0 °C and stirred at

25 °C for 0.5 h. The reaction mixture was diluted with 100 mL

of a mixed solvent (40:60:2, v/v/v, ethyl acetate:hexane:AcOH)

and filtered through celite. The filtrate was concentrated, re-

dissolved in 95% EtOH (2 mL) and cooled to 0 °C before the

addition of a solution of NaBH₄(0.13 g, 3.6 mmol) in 95% EtOH

(2 mL) at 0 °C. The reaction was then stirred at 0 °C under an

argon atmosphere for 2 h before being quenched by 1 M HCl

until pH 1-2. The yellow precipitate was filtered out, washed

with H₂O (3 mL × 2) and then dried in vacuo overnight to afford

16 as a pale yellow solid (0.260 g, 1.21 mmol, 51%). ¹H NMR

(DMSO-d₆, 400 MHz) δ 12.88 (br s, 2H, COOH), 7.88 (d, 4H, J =

8.4 Hz, Ar-H), 7.33 (d, 4H, J = 8.4 Hz, Ar-H), 4.01 (s, 4H, CH₂);

¹³C{¹H} NMR (DMSO-d₆, 125 MHz) δ 167.1, 144.4, 129.4, 129.3,

129.1, 30.9; LRMS (ESI⁺): m/z calcd. for C₁₆H₁₄NaO₄Se₂[M+Na]⁺

452.9, found 453.1. The ¹H NMR data is in agreement with that

2‐yl)carbamate (10). A solution of tetrabutylammonium fluo-

ride (5.02 mL, 5.02 mmol, 1 M in THF) was added dropwise to

a solution of TBDMS ether 9 (1.82 g, 4.18 mmol) in dry THF (32

mL) and the reaction mixture was stirred at 25 °C for 30 min.

CaCO₃(1.06 g), DOWEX ^®50W X8 (200-400 mesh, 3.11 g) and

MeOH (7.53 mL) were added to the reaction flask and the reac-

tion was stirred for 1 h. The resulting suspension was filtered

through celite, washed with MeOH and concentrated in vacuo.

The crude product was purified by flash column chromatog-

raphy (3:7, EtOAc/Hexane, R_f= 0.32) to afford 10 as a yellow

oil (0.97 g, 3.03 mmol, 72%). ꢀꢄ^ꢂ_ꢁ^ꢃ= -34.4° (c 0.28, CH₂Cl₂); IR

(ν/cm^-1, film) 3421, 2967, 2932, 2155 (SeCN), 1687, 1505,

1392, 1368, 1250, 1168, 1052, 803; ¹H NMR (CDCl₃, 400 MHz)

δ 5.33 (d, 1H, J = 7.8 Hz, NH), 4.07 (m, 1H, CHNH), 3.97 (dd, 1H,

J = 3.3 Hz, 11.2 Hz, CH_2a), 3.83 (dd, 1H, J = 2.4 Hz, 11.3 Hz, CH_2b),

3.55-3.52 (m, 1H, CHSeCN), 2.35 (br s, 1H, OH), 2.18-2.11 (m,

1H, CH(CH₃)₂), 1.46 (s, 9H, C(CH₃)₃), 1.21 (d, 3H, J = 6.6 Hz,

CH(CH₃)_2a), 1.14 (d, 3H, J = 6.6 Hz, CH(CH₃)_2b); ¹³C{¹H} NMR

(CDCl₃, 100 MHz) δ 155.4, 105.2 (SeCN), 80.5, 63.2, 62.2, 52.2,

31.9, 28.5, 21.6, 21.5; HRMS (ESI⁺): m/z calcd. for

C₁₂H₂₂N₂O₃SeNa [M+Na]⁺345.0688, found 345.0688.

reported by Lin et al.⁴³

(2R,3S)‐2‐((tert‐butoxycarbonyl)amino)‐4‐methyl‐3‐selenocya‐

natopentanoic acid (11). Primary alcohol 10 (158 mg, 0.490

mmol) was dissolved in a mixed solvent of MeCN/H₂O 1:1 (v/v,

4 mL) before the addition of (diacetoxyiodo)benzene (0.79 g,

2.5 mmol) and TEMPO (38 mg, 0.25 mmol) in one portion. The

orange solution was stirred at 25 °C for 3 h and then concen-

trated under a stream of N₂followed by lyophilization. The

crude product was purified by flash column chromatography

(96:2:2, CH₂Cl₂:MeOH:AcOH, R_f= 0.33) to provide 11 as a yel-

low oil (149 mg, 0.44 mmol, 90%). ꢀꢄ^ꢂ_ꢁ^ꢃ= -42.7° (c 0.33,

CH₂Cl₂); IR (ν/cm^-1, film) 2965, 2923, 2852, 2153 (SeCN), 1705,

1503, 1457, 1393, 1369, 1253, 1160, 1057, 1019, 856, 801; ¹H

NMR (CDCl₃, 400 MHz) δ 5.52 (d, 1H, J = 5.5 Hz, NH), 4.87-4.74

(m, 1H, CHNH), 3.49-3.47 (m, 1H, CHSeCN), 2.30-2.22 (m, 1H,

CH(CH₃)₂), 1.47 (s, 9H, C(CH₃)₃), 1.21 (d, 6H, J = 5.9 Hz,

CH(CH₃)₂); ¹³C{¹H} NMR (CDCl₃, 100 MHz) δ 172.5 (COOH),

155.4, 102.8 (SeCN), 81.7, 60.9, 55.2, 29.8, 28.4, 21.2; HRMS

(ESI⁺): m/z calcd. For C₁₂H₂₀N₂O₄SeNa [M+Na]⁺359.0481,

found 359.0481.

General procedures for solid phase peptide synthesis

(SPPS)

Preloading of 2‐chlorotrityl‐chloride resin: 2-chlorotrityl-chlo-

ride resin (1.5 mmol/g loading capacity) was swollen in dry

CH₂Cl₂for 30 min and then washed thoroughly (CH₂Cl₂× 5, DMF

× 5, CH₂Cl₂× 5 and DMF × 5, ~ 3 mL per wash). A solution of

Fmoc-AA-OH (2 equiv.) and iPr₂NEt (4 equiv.) in dry DMF (final

concentration 0.2 M) was added and the resin was agitated for

3-16 h at 25 °C (or 16 h for β-branched amino acids). Following

a thorough wash (DMF × 5, CH₂Cl₂× 5, DMF × 5 and CH₂Cl₂× 5),

the resin was treated with a capping solution (17:2:1, v/v/v,

CH₂Cl₂/MeOH/iPr₂NEt, 3 mL) for 30 min at 25 °C. The resin was

washed again (CH₂Cl₂× 5, DMF × 5, CH₂Cl₂× 5 and DMF × 5) and

subjected to iterative peptide assembly (Fmoc-SPPS).

Preloading of Chemmatrix® Trityl‐OH resin: Chemmatrix®

Trityl-OH resin (0.3 mmol/g loading capacity) was swollen in

dry CH₂Cl₂for 30 min and then washed thoroughly (CH₂Cl₂× 5,

DMF × 5 and CH₂Cl₂× 5). An activating solution (10 vol% SOCl₂

in dry CH₂Cl₂, 4 mL) was then added and the resin was shaken

for 2 h at 25 °C. After filtration, the resin was washed with dry

CH₂Cl₂(10 × 3 mL) and 10 vol% iPr₂NEt in dry CH₂Cl₂(10 mL).

Then a solution of Fmoc-AA-OH (4 equiv.) and iPr₂NEt (8 equiv.)

in dry DMF (final concentration 0.2 M) was added to the resin

and the mixture was agitated for 3 h (16 h for β-branched

amino acids). The resin was washed again (DMF × 5, CH₂Cl₂× 5,

DMF × 5 and CH₂Cl₂× 5), and treated with a solution of

CH₂Cl₂/MeOH/iPr₂Net (17:2:1 v/v/v, 3 mL) for 30 min at 25 ^oC.

The resin was finally washed (CH₂Cl₂× 5, DMF × 5, CH₂Cl₂× 5

and DMF × 5) and subjected to iterative peptide assembly

(Fmoc-SPPS).

(2R,3S)‐2‐((tert‐Butoxycarbonyl)amino)‐3‐((4‐methoxyben‐

zyl)selanyl)‐4‐methylpentanoic acid (5). To a solution of 11

(209 mg, 0.870 mmol) in THF (6 mL) was added a solution of

NaBH₄(65 mg, 1.73 mmol) in 95% EtOH (0.86 mL) at 0 °C un-

der an argon atmosphere. After stirring the reaction mixture at

0 ^oC for 1 h, p-methoxybenzyl chloride (0.542 g, 3.46 mmol) and

aq. 2 M NaOH (degassed, 2.31 mL) were added while maintain-

ing the reaction temperature at 0 °C. The mixture was allowed

to warm up to 25 °C and stirred for a further 16 hours. The re-

action was quenched with AcOH (2 mL) and co-evaporated

with toluene to dryness. The crude product was purified by

flash column chromatography (97:1:2, CH₂Cl₂:MeOH:AcOH, R_f=

0.35) to provide β-selenoleucine building block 5 as a yellow

oil (224 mg, 0.520 mmol, 60%). ꢀꢄ^ꢂ_ꢁ^ꢃ= -72.7° (c 0.29, CH₂Cl₂);

IR (ν/cm^-1, film) 2960, 2926, 2853, 1715, 1610, 1511, 1458,

1393, 1368, 1248, 1171, 1035, 831; ¹H NMR (CDCl₃, 400 MHz)

δ 7.19 (d, 2H, J = 8.5 Hz, Ar-H), 6.80 (d, 2H, J = 8.0 Hz, Ar-H), 5.19

(d, 1H, J = 7.1 Hz, NH), 4.66 (s, 1H, CHNH), 3.77 (s, 3H, OCH₃),

3.75-3.74 (m, 2H, CH₂), 2.61 (m, 1H, CHSe), 2.03-2.01 (m, 1H,

CH(CH₃)₂), 1.45 (s, 9H, C(CH₃)₃), 0.97 (d, 6H, CH(CH₃)₂); ¹³C{¹H}

NMR (CDCl₃, 100 MHz) δ 175.2 (COOH), 158.7, 155.5, 130.3,

114.1, 80.6, 56.2, 55.4, 51.6, 29.8, 28.9, 28.4, 21.4, 21.0; HRMS

(ESI⁺): m/z calcd. for C₁₉H₂₉NO₅SeNa [M+Na]⁺454.1103, found

454.1101.

Preloading of Rink amide resin: Fmoc-Rink amide resin (0.8

mmol/g loading capacity) was swollen in dry CH₂Cl₂for 30 min

and then washed thoroughly (CH₂Cl₂× 5, DMF × 5 and CH₂Cl₂×

5). The N-terminal Fmoc group was removed by piperidine (20

vol%) in DMF (3 mL, 2 x 5 min). The resin was washed thor-

oughly (DMF × 5, CH₂Cl₂× 5, DMF × 5) before a solution of

Fmoc-AA(PG)-OH (4 equiv.), N,N'-Diisopropylcarbodiimide

(DIC, 4 equiv.), and Oxyma (4 equiv.) in DMF (final concentra-

tion 0.3 mmol/mL) was added and the mixture was agitated for

3 h (16 h for β-branched amino acids). The loaded resin was

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The Journal of Organic Chemistry

Page 8 of 13

washed again (CH₂Cl₂× 5, DMF × 5, CH₂Cl₂× 5 and DMF × 5) and

subjected to iterative peptide assembly (Fmoc-SPPS).

Following filtration, the resin was washed thoroughly (DMF ×

5, CH₂Cl₂× 5, DMF × 5).

Estimation of amino acid loading: The resin was treated with 20%

piperidine/DMF (2 × 3 mL, 5 min) and 50 μL of the combined

deprotection solution was diluted to 10 mL using 20% piperi-

dine/DMF in a 10 mL volumetric flask. The UV absorbance of

the resulting piperidine-fulvene adduct was measured (λ = 301

nm, ε = 7800 M^-1cm^-1) to estimate the amount of amino acid

loaded onto the resin.

Synthesis of H‐(β‐Se)FSPGYS‐NH₂dimer (12). A solution of Boc-

β-Se(PMB)-Phe-OH 2 (28 mg, 0.060 mmol, 1.2 equiv.), HOAt (8

mg, 0.06 mmol, 1.2 equiv.) and DIC (9 μL, 0.06 mmol, 1.2 equiv.)

in DMF (0.6 mL, 0.1 M amino acid concentration) was added to

H-SPGYS (0.185 g, 0.05 mmol, 1.0 equiv., side-chain protected)

on Rink Amide resin (0.3 mmol/g loading) and the reaction ves-

sel was shaken for 16 h at 25 °C. The PMB-protected selenopep-

tide was cleaved by treatment with a mixture of TFA, triiso-

propylsilane (TIS) and water (95:5:5 v/v/v) for 2 h at 25 °C.

The resulting peptide solution was concentrated under a

stream of N₂, and the peptide was precipitated from cold Et₂O

and separated via centrifugation. The crude peptide was dis-

solved in TFA (10 mL) and added dropwise into a mixture of

DMSO/TFA (1:4 v/v, 10 mL) at 0 °C and the PMB group was

cleaved off after 16 h. The crude diselenide peptide solution

was concentrated (~ 4 mL), resuspended in cold diethyl ether

(0 °C) and centrifuged. The pellet was purified by preparative

reverse phase HPLC (10-20% MeCN in H₂O over 40 min, 0.1%

TFA, Sunfire prep C18 OBD 5 µm 19 × 150 mm column) and

lyophilized to give the desired peptide dimer 12 (13.9 mg, 19

µmol, 38%).

Iterative peptide assembly (Fmoc‐SPPS): Deprotection: The

resin was treated with 20% piperidine/DMF (2 × 3 mL, 5 min)

and washed with DMF (5 × 3 mL), CH₂Cl₂(5 × 3 mL) and DMF

(5 × 3 mL). General amino acid coupling: A solution of protected

amino acid (4 equiv.), benzotriazol-1-yl-oxytripyrrolidino-

phosphonium hexafluorophosphate (PyBOP, 4 equiv.) and 4-

methylmorpholine (NMM, 8 equiv.) in DMF (final concentra-

tion 0.1 M) was added to the resin. After 1 h, the resin was

washed with DMF (5 × 3 mL), CH₂Cl₂(5 × 3 mL) and DMF (5 ×

3 mL). Capping: Acetic anhydride/pyridine (1:9 v/v) was

added to the resin (3 mL). After 3 min the resin was washed

with DMF (5 × 3 mL), CH₂Cl₂(5 × 3 mL) and DMF (5 × 3 mL).

Cleavage: A mixture of TFA, triisopropylsilane (TIS) and water

(95:5:5 v/v/v) was added to the resin. After 3 h, the resin was

washed with TFA (3 × 2 mL). Work‐up: The combined cleavage

and TFA wash solutions were concentrated under a stream of

nitrogen to less than 5 mL. Cold diethyl ether (30 mL) was

added to precipitate the peptide, the suspension cooled at -

20 °C and centrifuged at 4000 rpm for 5 min. The pellet was

then dissolved in water containing 0.1% TFA or FA, filtered, pu-

rified by preparative HPLC and analyzed by UPLC-MS and ESI

mass spectrometry.

Synthesis of H‐(β‐Se)LSPGYS‐NH₂dimer (13). A solution of Boc-

β-Se(PMB)-Leu-OH 5 (26 mg, 0.06 mmol, 1.2 equiv.), HOAt (8

mg, 0.06 mmol, 1.2 equiv.) and DIC (9 μL, 0.06 mmol, 1.2 equiv.)

in DMF (0.6 mL, 0.1 M amino acid concentration) was added to

H-SPGYS (0.185 g, 0.05 mmol, 1.0 equiv., side-chain protected

and resin-bound) on Rink Amide resin (0.3 mmol/g loading)

and the reaction was shaken for 16 h at 25 °C. The PMB-

selenopeptide was cleaved by treatment with a mixture of TFA,

triisopropylsilane (TIS) and water (95:5:5 v/v/v) for 2 h at

25 °C. The cleaved peptide solution was concentrated under a

stream of N₂, and the peptide was precipitated from cold Et₂O

and separated via centrifugation. The crude peptide was dis-

solved in TFA (10 mL) and added dropwise into a mixture of

DMSO/TFA (v/v, 1:4, 10 mL) at 0 °C and the PMB group was

cleaved off after 16 h. The crude diselenide peptide in solution

was concentrated (~ 4 mL), precipitated with cold Et₂O and

separated by centrifugation. The resulting pellet was purified

by preparative reverse phase HPLC (10-20% B over 40 min, 0.1%

TFA, Sunfire prep C18 OBD 5 µm 19 × 150 mm column) and

lyophilized to give the desired peptide dimer 13 (12.7 mg, 18

µmol, 36%).

Automated solid‐phase peptide synthesis

Automated Fmoc-SPPS was carried out on a Biotage Initiator⁺

Alstra microwave peptide synthesizer equipped with an inert

gas manifold. General synthetic protocols for Fmoc-deprotec-

tion and capping were carried out in accordance with the man-

ufacturer’s specifications. Standard amino acid couplings were

performed for 20 min at 50 °C under microwave irradiation in

the presence of amino acid (0.3 M in DMF), Oxyma (0.5 M in

DMF) and DIC (0.5 M in DMF). Peptide cleavage and work-up

were carried out as described above for manual SPPS.

Coupling conditions for synthetic amino acids:

Coupling of Boc-PMB-(β-Se)Phe (2): After the removal of the N-

terminal Fmoc group, a solution of PMB-β-Se-Phe 2 (1.2 equiv.),

1-Hydroxy-7-azabenzotriazole (HOAt, 1.2 equiv.) and DIC (1.2

equiv.) in DMF (0.07 M amino acid concentration) was added

to the elongated peptide on resin and the reaction was shaken

for 16 h at 25 °C. Following filtration, the resin was washed

thoroughly (DMF × 5, CH₂Cl₂× 5, DMF × 5).

General procedure for one‐pot DSL‐deselenization of model pep‐

tide 12 with an N‐terminal β‐Se‐Phe with 14a‐f: The diselenide

peptide H-(β-Se)FSPGYS-NH₂12 (2.00 mg, 1.36 µmol, 0.5

equiv.) and selenoester Ac-LYRANX-SePh 14a‐f (X= Ala, Ser ,

Leu, Ile, Val, Thr, 3.54 µmol, 1.3 equiv.) were dissolved in liga-

tion buffer (545 µL, 6 M Gn·HCl, 0.1 M Na₂HPO₄, pH= 7.2) at a

concentration of 5 mM with respect to the selenopeptide mon-

omer (6.5 mM with respect to the selenoester) and to a final pH

of 6.1-6.4 (without adjustment). The reaction was agitated and

allowed to stand at 25 °C. 1 µL aliquots were taken from the

reaction mixture at various time points and diluted with 40 µL

of Milli-Q H₂O for analytical UPLC-MS and HPLC (Sunfire C18 5

µm 2.1 × 150 mm column) [or UPLC (ACQUITY UPLC BEH C18

1.7μm 2.1 × 50 mm column)] analysis until the diselenide pep-

tide was totally consumed and ligation products were gener-

ated. The ligation mixture was washed with hexane (10 × 600

µL) to remove (PhSe)₂, and treated with a solution of 4,4'-(dise-

lanediylbis(methylene))-dibenzoic acid 16 (60 µL, 200 mM in

6 M Gn·HCl, 0.1 M Na₂HPO₄ligation buffer, pH= 6.5). The

Coupling of Boc-PMB-(β-Se)Leu (5). A solution of Boc-PMB-(β-

Se)Leu 5 (26 mg, 0.06 mmol, 1.2equiv.), HOAt (8 mg, 0.06 mmol,

1.2 equiv.), DIC (9 μL, 0.06 mmol, 1.2 equiv.) in DMF (0.6 mL,

0.1 M amino acid concentration) was added to the resin and the

reaction was shaken for 16 h at 25 °C. Following filtration, the

resin was washed with DMF (5 × 3 mL), CH₂Cl₂(5 × 3 mL) and

DMF (5 × 3 mL).

Coupling of Fmoc‐Asn[GlcNAc(OAc)₃]‐OH. After the removal of

the N-terminal Fmoc group, a solution of peracetylated Fmoc-

Asn[GlcNAc(OAc)₃]-OH³⁹(2 equiv.), (7-Azabenzotriazol-1-

yloxy)tripyrrolidinophosphonium

hexafluorophosphate

(PyAOP, 2 equiv.) and NMM (4 equiv.) in DMF (0.08 M amino

acid concentration) was added to the elongated resin-bound

peptide and the reaction was shaken for 16 h at 25 °C.

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resulting yellow mixture was degassed with argon for 10 min.

LYRANTFSPGYS-NH₂17f as a white solid (2.54 mg, 1.79 µmol,

65% over 2 steps).

Meanwhile, a solution of TCEP (250 mM, 72 mg, 50 equiv.) and

DTT (100 mM, 144 mg, 200 equiv.) was prepared by dissolving

the two components in ligation buffer (1 mL, 6 M Gn·HCl, 0.1 M

Na₂HPO₄, pH= 7.2) and adjusting to pH 6-6.8 with 2 M aq. NaOH.

The TCEP/DTT solution was degassed with argon for 10 min.

Then, radical initiator 2,2'-Azobis[2-(2-imidazolin-2-yl)pro-

pane]dihydrochloride (VA-044, 1.5 mg, 4.6 µmol) was added as

a solid into the ligation mixture under argon before the addi-

tion of the degassed TCEP/DTT solution (605 µL) in one por-

tion. The deselenization reaction was sealed, agitated, and in-

cubated at 37 °C for 5 min. 1 µL of aliquots were taken for ana-

lytical UPLC-MS and HPLC or UPLC analysis once the deseleni-

zation was completed. The crude one-pot ligation-deseleniza-

tion product was purified by reverse phase preparative HPLC

(15-45% MeCN in H₂O over 40 min, 0.1% TFA, Sunfire prep C18

OBD 5 µm 19 × 150 mm column) and lyophilized to afford the

native peptide as a white solid.

General procedure for one‐pot DSL‐deselenization of model pep‐

tide 13 with an N‐terminal β‐Se‐Leu with 14a‐e: The diselenide

peptide H-(β-Se)LSPGYS-NH₂13 (2.00 mg, 1.43 µmol, 0.5

equiv.) and selenoester Ac-LYRANX-SePh 14a‐e (X= Ala, Ser ,

Leu, Ile, Val, 3.72 µmol, 1.3 equiv.) were dissolved in ligation

buffer (570 µL, 6 M Gn·HCl, 0.1 M Na₂HPO₄, pH= 7.2) to the con-

centration of 5 mM with respect to the selenopeptide monomer

(6.5 mM with respect to the selenoester) and to a final pH of

6.1-6.4 (without adjustment). The reaction was agitated and al-

lowed to stand at 25 °C. 1 µL aliquots were taken from the re-

action mixture at various time points and diluted with 40 µL of

Milli-Q H₂O for analytical UPLC-MS and HPLC (Sunfire C18 5 µm

2.1 × 150 mm column) [or UPLC (ACQUITY UPLC BEH C18

1.7μm 2.1 × 50 mm column)] analysis until the diselenide pep-

tide was totally consumed and ligation products were gener-

ated. The ligation mixture was washed with hexane (10 × 600

µL) to remove (PhSe)₂and degassed with argon for 10 min.

Meanwhile, a solution of TCEP (250 mM, 72 mg, 50 equiv.) and

DTT (250 mM, 36 mg, 50 equiv.) was prepared by dissolving

the two components in ligation buffer (1 mL, 6 M Gn·HCl, 0.1 M

Na₂HPO₄, pH= 7.2) and adjusting to pH 5-5.5 with 2 M aq. NaOH.

The TCEP/DTT solution was degassed with argon for 10 min

before adding (570 µL) into the ligation mixture in one portion.

The deselenization reaction was sealed, agitated, and kept on

bench at room temperature for 16 h. 1 µL of aliquots were

taken for analytical UPLC-MS and HPLC or UPLC analysis until

the completion of deselenization. The crude one-pot ligation-

deselenization product was purified by reverse phase prepara-

tive HPLC (15-45% B over 40 min, 0.1% TFA, Sunfire prep C18

OBD 5 µm 19 × 150 mm column) and lyophilized to afford the

native peptide product as a white solid.

Ac‐LYRANAFSPGYS‐NH₂(17a). The title compound was pro-

duced via the one-pot ligation-deselenization method outlined

above using diselenide peptide H-(β-Se)FSPGYS-NH₂12 (2.12

mg, 1.44 µmol, 0.5 equiv.) and selenoester Ac-LYRANA-SePh

14a (3.32 mg, 3.74 µmol, 1.3 equiv.). The crude ligation- dese-

lenization product was purified and lyophilized to obtain Ac-

LYRANAFSPGYS-NH₂17a as a white solid (2.72 mg, 1.96 µmol,

68% over 2 steps).

Ac‐LYRANSFSPGYS‐NH₂(17b). The title compound was pro-

duced via the one-pot ligation-deselenization method outlined

above using diselenide peptide H-(β-Se)FSPGYS-NH₂12 (2.08

mg, 1.42 µmol, 0.5 equiv.) and selenoester Ac-LYRANS-SePh

14b (3.33 mg, 3.69 µmol, 1.3 equiv.). The crude ligation-dese-

lenization product was purified and lyophilized to obtain Ac-

LYRANSFSPGYS-NH₂17b as a white solid (2.62 mg, 1.87 µmol,

66% over 2 steps).

Ac‐LYRANALSPGYS‐NH₂(15a). The title compound was pro-

duced via the one-pot ligation-deselenization method outlined

above using diselenide peptide H-(β-Se)LSPGYS-NH₂13 (2.20

mg, 1.57 µmol, 0.5 equiv.) and selenoester Ac-LYRANA-SePh

14a (3.63 mg, 4.09 µmol, 1.3 equiv.). The crude ligation-dese-

lenization product was purified and lyophilized to obtain Ac-

LYRANALSPGYS-NH₂15a as a white solid (2.47 mg, 1.83 µmol,

58% over 2 steps).

Ac‐LYRANLFSPGYS‐NH₂(17c). The title compound was pro-

duced via the one-pot ligation-deselenization method outlined

above using diselenide peptide H-(β-Se)FSPGYS-NH₂12 (2.26

mg, 1.54 µmol, 0.5 equiv.) and selenoester Ac-LYRANL-SePh

14c (3.72 mg, 4.00 µmol, 1.3 equiv.). The crude ligation- dese-

lenization product was purified and lyophilized to obtain Ac-

LYRANLFSPGYS-NH₂17c as a white solid (3.26 mg, 2.28 µmol,

74% over 2 steps).

Ac‐LYRANSLSPGYS‐NH₂(15b). The title compound was pro-

duced via the one-pot ligation-deselenization method outlined

above using diselenide peptide H-(β-Se)LSPGYS-NH₂13 (2.10

mg, 1.50 µmol, 0.5 equiv.) and selenoester Ac-LYRANS-SePh

14b (3.64 mg, 4.03 µmol, 1.3 equiv.). The crude ligation-dese-

lenization product was purified and lyophilized to obtain Ac-

LYRANSLSPGYS-NH₂15b as a white solid (2.82 mg, 2.06 µmol,

69% over 2 steps).

Ac‐LYRANIFSPGYS‐NH₂(17d). The title compound was pro-

duced via the one-pot ligation-deselenization method outlined

above using diselenide peptide H-(β-Se)FSPGYS-NH₂12 (1.92

mg, 1.31 µmol, 0.5 equiv.) and selenoester Ac-LYRANI-SePh

14d (3.16 mg, 3.40 µmol, 1.3 equiv.). The crude ligation- dese-

lenization product was purified and lyophilized to obtain Ac-

LYRANIFSPGYS-NH₂17d as a white solid (2.32 mg, 1.62 µmol,

62% over 2 steps).

Ac‐LYRANLLSPGYS‐NH₂(15c). The title compound was pro-

duced via the one-pot ligation-deselenization method outlined

above using diselenide peptide H-(β-Se)LSPGYS-NH₂13 (2.03

mg, 1.45 µmol, 0.5 equiv.) and selenoester Ac-LYRANL-SePh

14c (3.51 mg, 3.77 µmol, 1.3 equiv.). The crude ligation-dese-

lenization product was purified and lyophilized to obtain Ac-

LYRANLLSPGYS-NH₂15c as a white solid (3.30 mg, 2.34 µmol,

82% over 2 steps).

Ac‐LYRANVFSPGYS‐NH₂(17e). The title compound was pro-

duced via the one-pot ligation-deselenization method outlined

above using diselenide peptide H-(β-Se)FSPGYS-NH₂12 (2.10

mg, 1.43 µmol, 0.5 equiv.) and selenoester Ac-LYRANV-SePh

14e (3.41 mg, 3.72 µmol, 1.3 equiv.). The crude ligation- dese-

lenization product was purified and lyophilized to obtain Ac-

LYRANVFSPGYS-NH₂17e as a white solid (2.07 mg, 1.46 µmol,

51% over 2 steps).

Ac‐LYRANILSPGYS‐NH₂(15d). The title compound was pro-

duced via the one-pot ligation-deselenization method outlined

above using diselenide peptide H-(β-Se)LSPGYS-NH₂13 (1.96

mg, 1.40 µmol, 0.5 equiv.) and selenoester Ac-LYRANI-SePh

14d (3.39 mg, 3.64 µmol, 1.3 equiv.). The crude ligation-dese-

lenization product was purified and lyophilized to obtain Ac-

Ac‐LYRANTFSPGYS‐NH₂(17f). The title compound was pro-

duced via the one-pot ligation-deselenization method outlined

above using diselenide peptide H-(β-Se)FSPGYS-NH₂12 (2.02

mg, 1.38 µmol, 0.5 equiv.) and selenoester Ac-LYRANT-SePh

14f (3.29 mg, 3.58 µmol, 1.3 equiv.). The crude ligation-dese-

lenization product was purified and lyophilized to obtain Ac-

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Page 10 of 13

LYRANILSPGYS-NH₂15d as a white solid (2.13 mg, 1.53 µmol,

55% over 2 steps).

Synthesis of IFN‐γ (75‐131) (18). The ligation of IFN-γ (75-104)

selenoester 19 (2.44 mg, 0.63 µmol, 1.1 equiv.) and IFN-γ (105-

131) diselenide dimer 20 (2.03 mg, 0.57 µmol, 1 equiv. with re-

spect to selenopeptide monomer) was performed by dissolving

both solids in ligation buffer (100 µL, 6 M Gn·HCl, 0.1 M

Na₂HPO₄, pH= 7.2, 5 mM with respect to selenopeptide mono-

mer) and the final pH adjusted to 6.1-6.4 by 0.5 M aq. NaOH.

The resulting solution was sealed, agitated and incubated at

25 °C for 5 min, after which time UPLC-MS analysis indicated

complete conversion into the ligated peptide. The ligation mix-

ture was extracted with hexane (10 × 200 µL) to remove

(PhSe)₂, an then treated with a solution of 4,4'-(diselan-

ediylbis(methylene))dibenzoic acid 16 (30 µL, 200 mM in 6 M

Gn·HCl, 0.1 M Na₂HPO₄ligation buffer, pH= 6.5 ). The resulting

yellow mixture was degassed with argon for 10 min. Mean-

while, a solution of TCEP (250 mM, 72 mg, 50 equiv.) and DTT

(100 mM, 144 mg, 200 equiv.) was prepared by dissolving the

two components in ligation buffer (1 mL, 6 M Gn·HCl, 0.1 M

Na₂HPO₄, pH= 7.2) and adjusting to pH 6-6.8 with 2 M aq. NaOH.

The TCEP/DTT solution was degassed with argon for 10 min.

Then, radical initiator 2,2'-Azobis[2-(2-imidazolin-2-yl)pro-

pane]dihydrochloride (VA-044, 1.5 mg, 4.64 µmol) was added

as a solid into the ligation mixture under argon before the ad-

dition of the degassed TCEP/DTT solution (130 µL) in one por-

tion. The deselenization reaction was sealed, agitated, and in-

cubated at 37 °C for 5 min, at which point the in situ deseleni-

zation reaction was shown to have reached completion by

UPLC-MS. IFN-γ 75-131 was purified by preparative reverse

phase HPLC (20-80% MeCN in H₂O over 60 min, 0.1% TFA,

XBridge Peptide BEH Prep C18 300 Å 5 µm 10 × 250 mm col-

umn) and lyophilized to give IFN-γ (75-131) 18 (2.13 mg, 0.29

µmol, 51% over 2 steps) as a white solid. Note: Reported iso-

lated yield takes into account the aliquots removed for analyti-

cal UPLC monitoring at each step.

Ac‐LYRANVLSPGYS‐NH₂(15e). The title compound was pro-

duced via the one-pot ligation-deselenization method outlined

above using diselenide peptide H-(β-Se)LSPGYS-NH₂13 (2.00

mg, 1.43 µmol, 0.5 equiv.) and selenoester Ac-LYRANV-SePh

14e (3.40 mg, 3.72 µmol, 1.3 equiv.). The crude ligation-dese-

lenization product was purified and lyophilized to obtain Ac-

LYRANVLSPGYS-NH₂15e as a white solid (2.53 mg, 1.83 µmol,

64% over 2 steps).

Synthesis of IFN-γ (75-131) glycopeptide (18)

N‐terminal fragment IFN‐γ (75‐104) (19). The N-terminal frag-

ment IFN-γ (75-104) (19) (30 µmol) was prepared by loading

Fmoc-Phe-OH on 2-chlorotrityl chloride resin and elongating to

the target peptide sequence using automated Fmoc-SPPS as de-

scribed in the general procedures. The side-chain protected

peptide was acetylated at its N-terminus and cleaved from

resin via treatment with HFIP/CH₂Cl₂(3:7, v/v, 4 mL × 2, 1.5 h

each time) for 3 h and concentrated in vacuo. The cleaved side-

chain protected peptide and (PhSe)₂(0.31 g, 1 mmol, 50 equiv.)

were dissolved in anhydrous DMF (0.5 mL) under argon at 0 °C.

Bu₃P (0.24 mL, 1 mmol, 50 equiv.) was added dropwise and the

mixture was stirred for 3 h at 0 °C before the removal of solvent

under a stream of N₂. The crude peptide was treated with a TFA

cleavage cocktail (90:5:5, v/v/v, TFA:TIS:H₂O, 10 mL) at 0 °C

and stirred for 3 h at room temperature. The cleaved peptide

solution was concentrated under a stream of N₂, and the pep-

tide was precipitated from cold Et₂O and separated via centrif-

ugation. The crude peptide selenoester was purified by prepar-

ative reverse phase HPLC (30-80% MeCN in H₂O over 40 min,

0.1% TFA, Sunfire Prep C18 5 µm 19 × 150 mm column) and

lyophilized to give the desired peptide selenoester IFN-γ (75-

104) 19 (32.5 mg, 8.33 µmol, 28%).

C‐terminal fragment IFN‐γ (105‐131) (20). The C-terminal frag-

ment IFN-γ (105-131) 20 (30 µmol) was prepared by loading

Fmoc-Lys(Boc)-OH on to 2-chlorotrityl chloride resin and elon-

gating to the desired peptide sequence using automated Fmoc-

SPPS as described in the general procedures. Then a deacetyla-

tion solution (10% NH₂NH₂in DMF, 4 mL) was added to the

elongated peptide on resin after its N-terminal Fmoc group was

deprotected, and the reaction was shaken for 16 h at 25 °C. Af-

ter thoroughly washing the resin, a solution of β-Se(PMB)-Phe-

OH 2 (20 mg, 0.042 mmol, 1.2 equiv.), HOAt (6 mg, 0.042 mmol,

1.2 equiv.) and DIC (7 μL, 0.042 mmol, 1.2 equiv.) in DMF (0.6

mL, 0.07 M amino acid concentration) was added to the elon-

gated peptide on resin and the reaction was shaken for 16 h at

25 °C. The fully assembled peptide was globally cleaved using a

TFA cleavage cocktail (90:5:5, v/v/v, TFA:TIS:H₂O) for 3 h at

25 °C. The cleaved peptide solution was concentrated under a

stream of N₂, and the peptide was precipitated from cold Et₂O

and separated via centrifugation. The crude PMB-protected

peptide was purified by preparative reverse phase HPLC (20-

50% B over 50 min, 0.1% TFA, Sunfire Prep C18 5 µm 19×150

mm column) and lyophilized to yield the PMB-protected se-

lenopeptide (34 mg). The PMB group of the purified peptide

was removed after the peptide was agitated in a mixed solution

of DMSO/ligation buffer (6 M Gn·HCl, 0.1 M Na₂HPO₄, pH=

7.2)/TFA (1:1:3, v/v/v, 3 mL, 16 mg/mL) for 0.5 h at 25 °C. The

resulting solution was diluted with 0.1% TFA/H₂O and purified

by preparative reverse phase HPLC (20-50% MeCN in H₂O over

50 min, 0.1% TFA, Sunfire Prep C18 5 µm 19 × 150 mm column)

to give the desired diselenide peptide 20 (28.1 mg, 3.93 µmol

as dimer, 26% from resin loading) after lyophilization.

Synthesis of UL22A (21).

Synthesis of N‐terminal fragment UL22A (21‐51) (22). The N-

terminal fragment UL22A 21-50 (50 µmol) (22) was prepared

by loading Fmoc-Thr(tBu)-OH on 2-chlorotrityl chloride resin

and elongating to the desired peptide sequence using auto-

mated Fmoc-SPPS as described in the general procedures. The

N-terminal alanine residue at position 21 was incorporated as

Boc-Ala-OH. The side-chain protected peptide was cleaved

from resin via treatment with HFIP/CH₂Cl₂(3:7, v/v, 4 mL, 2 x

1.5 h) and the combined filtrates were concentrated in vacuo.

The cleaved side-chain protected peptide and (PhSe)₂(0.78 g,

2.5 mmol, 50 equiv.) were dissolved in anhydrous DMF (2.5 mL)

under argon at 0 °C. The reaction mixture was treated with

Bu₃P (0.62 mL, 2.5 mmol, 50 equiv.) dropwise and stirred for 3

h at 0 °C before the removal of solvent under a stream of N₂.

The crude peptide was treated with a TFA cleavage cocktail

(90:5:5, v/v/v, TFA:TIS:H₂O, 10 mL) at 0 °C and stirred for 3 h

at room temperature. The peptide solution was concentrated

under a stream of N₂, and the peptide was precipitated from

cold Et₂O and separated via centrifugation. The crude peptide

selenoester was purified by preparative reverse phase HPLC

(10-30% MeCN in H₂O over 40 min, 0.1% TFA, XBridge BEH300

Prep C18 5 µm 19 × 150 mm column) and lyophilized to give

the desired peptide selenoester 22 (30.8 mg, 9.25 µmol, 19%).

Synthesis of middle bifunctional fragment of UL22A (52‐76) (23).

The middle fragment of UL22A (52-76) (23) (50 µmol) was

prepared by loading Fmoc-Gly-OH on preactivated trityl-OH

ChemMatrix® resin and elongating to the desired peptide se-

quence using automated Fmoc-SPPS as described in the gen-

eral procedures. A solution of Boc-β-Se(PMB)-Leu-OH 5 (26 mg,

0.06 mmol, 1.2 equiv.), HOAt (8 mg, 0.06 mmol, 1.2 equiv.) and

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DIC (9 μL, 0.06 mmol, 1.2 equiv.) in DMF (0.6 mL, 0.1 M amino

to pH 5.2 with 2 M aq. NaOH and degassing with Argon for 10

min before the addition (125 µL) into the ligation mixture in

one portion (1.7 mM respect to diselenide thioester dimer).

The deselenization reaction was sealed, agitated, and allowed

to stand at room temperature. After 6 h, the in situ deseleniza-

tion reaction was complete according to UPLC-MS.

acid concentration) was added to the elongated peptide on

resin and the reaction was shaken for 16 h at 25 °C. The side-

chain protected peptide was cleaved from resin via treatment

with HFIP/CH₂Cl₂(3:7, v/v, 4 mL, 2 x 1.5 h) and the combined

filtrates were concentrated in vacuo. The cleaved side-chain

protected peptide was dissolved in anhydrous DMF (2 mL) un-

der argon at -30 °C followed by the addition of ethyl-mercapto-

propionate (0.19 mL, 1.5 mmol, 30 equiv.), iPr₂NEt (0.043 mL,

0.25 mmol, 5 equiv.) and PyBOP (0.13 g, 0.25 mmol, 5 equiv.).

The solution was stirred for 5 h at -30 °C before the removal of

solvent under a stream of N₂. The crude peptide was treated

with a TFA cleavage cocktail (90:5:5, v/v/v, TFA:TIS:H₂O, 10

mL) at 0 °C and stirred for 3 h at room temperature. The

cleaved peptide solution was concentrated under a stream of

N₂, and the peptide was precipitated from cold Et₂O and sepa-

rated via centrifugation. The crude PMB-protected peptide was

purified by preparative reverse phase HPLC (0-50% MeCN in

H₂O over 30 min, 0.1% TFA, XBridge BEH300 Prep C18 5 µm 19

× 150 mm column) and lyophilised to yield the pure PMB-

protected peptide thioester intermediate (12.57 mg, 4.32 µmol,

9 %). The PMB group of the purified peptide thioester (12.57

mg, 4.32 µmol) was removed after being agitated in a mixed so-

lution of DMSO/ ligation buffer (6 M Gn·HCl, 0.1 M Na₂HPO₄,

pH= 7.2)/TFA (1:1:3, v/v/v, 2 mL, 6 mg/mL) for 3 h at room

temperature. The resulting solution was diluted with 0.1%

TFA/H₂O and purified by preparative reverse phase HPLC (0-

50% MeCN in H₂O over 30 min, 0.1% TFA, XBridge BEH300

Prep C18 5 µm 19 × 150 mm column) to give the desired

diselenide peptide thioester 23 (4.32 mg, 1.55 µmol, 3% from

resin loading) after lyophilization.

To the deselenized mixture was added UL22A (77-103) 24

(3.95 mg, 1.22 µmol, 2 equiv.) and TCEP solution (20 µL, 250

mM solution in 6 M Gn·HCl/0.1 M Na₂HPO₄ligation buffer, pH

7.1, to achieve a final TCEP concentration of 25 mM) and then

adjusted to pH 7.2 (1.5 mM respect to diselenide thioester di-

mer). TFET (4 µL, 2 vol.%) was added and the reaction was in-

cubated at 37 °C. UPLC-MS analysis indicated complete con-

sumption of thioester UL22A (21-76) after 6 h.

A solution of TCEP (150 mM, 21 mg, 50 equiv.) and reduced

glutathione (200 mM, 30 mg, 67 equiv.) was prepared by dis-

solving the two components in ligation buffer (0.5 mL, 6 M

Gn·HCl, 0.1 M Na₂HPO₄, pH = 7.2) and adjusting to pH 6.8 with

2 M aq. NaOH. 210 µL of this solution was subsequently added

into the ligation mixture (0.75 mM respect to diselenide thioe-

ster dimer). The resulting mixture was degassed with argon for

10 min followed by the addition of VA-044 (4.5 mM, 0.61 mg, 3

equiv.). The reaction was sealed, agitated and incubated at

37 °C for 16 h. The crude peptide was purified by preparative

reverse phase HPLC (0-40% MeCN in H₂O over 45 min, 0.1%

TFA, XBridge BEH300 Prep C18 5 µm 10 × 150 mm column)

and lyophilized to give native UL22A (21-103) 21 (2.22 mg,

0.25 µmol, 40% over 4 steps) as a white solid. Note: The re-

ported isolated yield takes into account the aliquots removed

for analytical UPLC monitoring in each step.

Synthesis of N‐terminal Fragment UL22A (21‐71) (25). The N-

terminal fragment UL22A (21-71) (25) (20 µmol) was pre-

pared by loading Fmoc-Val-OH on 2-chlorotrityl chloride resin

and elongating to the target peptide sequence using automated

Fmoc-SPPS as described in the general procedures. The side-

chain protected peptide was cleaved from resin via treatment

with HFIP/CH₂Cl₂(3:7, v/v, 4 mL, 2 x 1.5 h) and the combined

filtrates were concentrated in vacuo. The cleaved side-chain

protected peptide and (PhSe)₂(0.31 g, 1 mmol, 50 equiv.) were

dissolved in anhydrous DMF (0.5 mL) under argon at 0 °C. Bu₃P

(0.24 mL, 1 mmol, 50 equiv.) was added dropwise and the mix-

ture was stirred for 3 h at 0 °C before the removal of solvent

under a stream of N₂. The crude peptide was treated with a TFA

cleavage cocktail (90:5:5, v/v/v, TFA:TIS:H₂O, 10 mL) at 0 °C

and stirred for 3 h at room temperature. The cleaved peptide

solution was concentrated under a stream of N₂, and the pep-

tide was precipitated from cold Et₂O and separated via centrif-

ugation. The crude peptide selenoester was purified by prepar-

ative reverse phase HPLC (15-45% MeCN in H₂O over 30 min,

0.1% TFA, Sunfire Prep C18 5 µm 19 × 150 mm column) and

lyophilized to give the desired peptide selenoester UL22A (21-

71) (25) (27.7 mg, 4.63 µmol, 23%).

Synthesis of C‐terminal fragment UL22A (77‐103) (24). The C-

terminal fragment UL22A (77-103) (24) (50 µmol) was pre-

pared by loading Fmoc-Gln(Trt)-OH on 2-chlorotrityl chloride

resin and elongating to the desired peptide sequence using au-

tomated Fmoc-SPPS as described in the general procedures. A

solution of syn-Boc-Asp(tBu, STmob)-OH⁹(30 mg, 0.06 mmol,

1.2 equiv.), HOAt (8 mg, 0.06 mmol, 1.2 equiv.) and DIC (9 μL,

0.06 mmol, 1.2 equiv.) in DMF (0.6 mL, 0.1 M amino acid con-

centration) was added to the elongated peptide on resin and

the reaction was shaken for 16 h at 25 °C. The fully assembled

peptide was globally cleaved by treating with a TFA cleavage

cocktail (90:5:5, v/v/v, TFA:TIS:H₂O) for 3 h at 25 °C. The

cleaved peptide solution was concentrated under a stream of

N₂, and the peptide was precipitated from cold Et₂O and sepa-

rated via centrifugation. The crude β-thio-Asp containing pep-

tide was purified by preparative reverse phase HPLC (0-20%

MeCN in H₂O over 30 min, 0.1% TFA, XBridge BEH300 Prep C18

5 µm 19 × 150 mm column) and lyophilised to afford the de-

sired peptide 24 (33.20 mg, 10.26 µmol, 21%).

Synthesis of UL22A protein (21) via the 3-component strategy:

The ligation of UL22A (21-51) selenoester 22 (4 mg, 1.22 µmol,

2 equiv.) and diselenide dimer UL22A (52-76) 23 (1.7 mg, 0.31

µmol, 0.5 equiv.) was performed by dissolving both solids in li-

gation buffer (62 µL, 6 M Gn·HCl, 0.1 M Na₂HPO₄, pH = 7.2, 5

mM respect to diselenide thioester dimer) together and the fi-

nal pH was adjusted to 6.2 with 1 M aq. NaOH. The resulting

solution was agitated and incubated at 37 °C for 0.5 h, after

which time UPLC-MS analysis indicated full conversion into the

corresponding ligation products. The ligation mixture was

washed with hexane (10 × 200 µL) to remove (PhSe)₂and de-

gassed with argon for 10 min. Meanwhile, a solution of TCEP

(250 mM, 72 mg, 50 equiv.) and DTT (250 mM, 36 mg, 50 equiv.)

was prepared by dissolving the two components in ligation

buffer (1 mL, 6 M Gn·HCl, 0.1 M Na₂HPO₄, pH = 7.2), adjusting

Synthesis of C‐terminal fragment UL22A (72‐103) (26). The C-

terminal fragment UL22A (72-103) 26 (30 µmol) was prepared

by loading Fmoc-Gln(Trt)-OH on 2-chlorotrityl chloride resin

and elongating to the desired peptide sequence using auto-

mated Fmoc-SPPS as described in the general procedures. A so-

lution of Boc-β-Se(PMB)-Leu-OH 5 (18 mg, 0.042 mmol,

1.2equiv.), HOAt (6 mg, 0.042 mmol, 1.2 equiv.) and DIC (7 μL,

0.042 mmol, 1.2 equiv.) in DMF (0.6 mL, 0.07 M amino acid con-

centration) was added to the elongated peptide on resin and

the reaction was shaken for 16 h at 25 °C. The fully assembled

peptide was globally cleaved using a TFA cleavage cocktail

(90:5:5, v/v/v, TFA:TIS:H₂O) for 3 h at 25 °C. The cleaved

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The Journal of Organic Chemistry

Page 12 of 13

peptide solution was concentrated under a stream of N₂, and

the peptide was precipitated from cold Et₂O and separated via

centrifugation. The crude PMB-protected peptide was purified

by preparative reverse phase HPLC (0-20% B over 30 min, 0.1%

TFA, Sunfire Prep C18 5 µm 19 × 150 mm column) and lyophi-

lized to yield PMB-protected selenopeptide (48 mg). The PMB

group of the purified peptide was removed after being agitated

in a mixed solution of DMSO/ligation buffer (6 M Gn·HCl, 0.1 M

Na₂HPO₄, pH = 7.2)/TFA (1:1:3, v/v/v, 3 mL, 16 mg/mL) for 0.5

h at 25 °C. The resulting solution was diluted with 0.1%

TFA/H₂O and purified by preparative reverse phase HPLC (0-

30% MeCN in H₂O over 40 min, 0.1% TFA, Sunfire Prep C18 5

µm 19 × 150 mm column) to give the desired diselenide peptide

26 (41 mg, 4.32 µmol as dimer, 29% from resin loading) after

lyophilization.

Author Contributions

The manuscript was written through contributions of all au-

thors. All authors have given approval to the final version of

the manuscript.

ACKNOWLEDGMENT

We would like to acknowledge grant support from an ARC Dis-

covery Project (DP190101526). We would also like to thank

the John A. Lamberton Research Scholarship for PhD funding

(to X. W.).

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Synthesis of UL22A protein (21) via one‐pot DSL‐deselenization

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Supporting Information

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Synthesis and Utility of β-Selenophenylalanine and β-Selenoleucine in Diselenide-Selenoester Ligation

DOI: 10.1021/acs.joc.9b02665

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