47

Supplementary data

Table SI: Inter- and intra-interactions of SARS 3CLpro dimers

Atom pairs in the interacting amino acid residues with minimal contact distances (< 3.5 Å)

are listed.

Residue 1 Atom 1 Residue 2 Atom 2 Distance (Å) Interactions of N-terminus from protomer A

A Ser1 Og B Glu166 OE2 2.56 A Ser1 N B Glu166 OE1 2.48 A Ser1 N B His172 N 2.83 A Gly2 O B Ser139 Og 2.90 A Lys5 N AGlu290 OE1 2.53 A Ala7 N B Val125 O 3.01 A Ala7 O B Val125 N 2.74

Interactions of N-terminus from protomer B B Ser1 N AGlu166 OE1 2.95 B Ser1 N A His172 N 3.16 B Ala7 N A Val125 O 3.04 B Ala7 O A Val125 N 2.80

Interactions of C-terminus from protomer A A Ser301 N A Val297 N 2.62 A Val303 O B Ser123 Og 3.53 A Gln306 OE1 A Lys12 NZ 3.19

48

Scheme S1. Synthesis of AG7088 (Compound 1)

HO2C CO2H

NH2

L-Glutamic acid

MeO2C CO2Me

NHBocNC

NO

H

BocHN CO2Me

i, ii iii

BocHN

NO

H

CO2Et

iv-vi

HONH2

O

OH

L-Serine

O

OMeO

FOTf

O

OMe

COOHNH

NH O O

Ot-Bu NO

F

O

OMe

viii x xi, xii

xiii, xiv

10 11

Cbz-L-Valine

xv (11), xvi

HR

1213 R = Cbz

14 R = Bocxvii

1.

2.

3.

6 7 8H3N CO2Et

NHO

NH

O N

O

O

F

O

OH

9X

O

PhCH2O PhCH2O

O

NO

F

O

OH

BocO N

O

Cl

1a (AG7088)

NH

O N

O

ONH

F

O

CO2Et

NHO

15 17

16

vii

xviii, xix

xx, xxi

(9)

xxii

Reagents and conditions: (i) Me3SiCl, MeOH, 0 oC, 18 h; then Boc2O, Et3N, 0 25 oC, 4 h; 96%. (ii)

LiN(SiMe3)2, THF, 78 oC, 3 h; then BrCH2CN, 3.5 h; 82%. (iii) H2, cat. PtO2, MeOH, CHCl3, 25 oC, 12 h;

then NaOAc, reflux, 12 h; 81%. (iv) NaBH4, LiCl, THF, EtOH, 25 oC, 18 h; 89%. (v) pyridine-SO3, Me2SO,

CH2Cl2, (i-Pr)2NEt, 10 oC, 3 h. (vi) [EtO2CCHPO(OEt)2] Na+, THF, 78 oC, 1 h; 75% yield for two steps.

(vii) HCl, 1,4-dioxane, rt, 2 h. (viii) HBr, NaNO2, KBr, H2O, 10 oC, 12 h. (ix) KOH, EtOH, 0 oC, 12 h. (x)

Me2SO4, CH2Cl2, cat. (PhCH2)Et3N+ Cl , rt, 24 h; 70% for three steps. (xi) 4-FC6H4MgBr, CuBr-Me2S,

THF, 35 oC, 1 h; 86%. (xii) (CF3SO2)2O, 2,6-lutidine, CH2Cl2, 0 oC, 40 min. (xiii) 1,1'-

carbonyldiimidazole, THF, rt, 1 h. (xiv) CH3CO2t-Bu, LiN(i-Pr)2, THF, 78 oC, 1 h; 65%. (xv) NaH, THF, 0

oC, 30 min; then triflate 11, THF, 0 oC to rt, 24 h. (xvi) CF3CO2H, CH2Cl2, rt, 24 h; 71% for two steps. (xvii)

H2, Pd/C, Boc2O, MeOH, rt, 10 h; 83%. (xviii) LiOH (1.1 equiv), H2O, 1 h, 0 oC; 90%. (xix) allyl iodide,

Cs2CO3, DMF, 45 oC, 5 h; 85%. (xx) N-methylmorpholine (NMM), CH2Cl2, 0 25 oC, 2 h; 88%. (xxi)

Pd(PPh3)4, morphloine, THF, 25 oC, 3 h; 84%. (xxii) HOBt, EDCI, (i-Pr)2NEt, CH2Cl2, 0 25 oC, 20 h; 70%.

49

Scheme S2. Combinatorial synthesis of peptide conjugated esters 2a d and 4a d.

BocHN CO2HBocHN

HN

RO

O

OMeH3N

ROMe

O

NH

HN

RO

O

OMe

O

R'Cl+

i ii R'COCl

iii

iv

NH

HN

RO

O

OH

O

R' +v

Boc-L-Val 18

2

R = PhCH2, 4-FC6H4CH2

R' = PhCH2O, 5-Me-1,2-oxazole

NH

NO

H

CO2EtNH

HN

RO

OO

R'

BocHN CO2Et

NH

HN

RO

O

NH

O

R' CO2EtNH

HN

RO

O

OH

O

R'+

BocHN CO2Me

Boc-Phe-OMe 22

1.

2.

19 20

21

23

v

H3N CO2Et

NHO

9

X

4

Reagents and conditions: (i) DCC, HOBt, Et3N, DMF, CH2Cl2, 0 25 oC, 12 24 h; 81 89%. (ii) CF3CO2H, CH2Cl2, 25 oC, 3 h. (iii) NMM, CH2Cl2, 0 25 oC, 4 h. (iv) LiOH, MeOH, 0 oC, 2 h. (v) HOBt, EDCI, (i-Pr)2NEt, CH2Cl2, 0 25 oC, 20 h.