Genetically Introducing Biochemically Reactive Amino Acids Dehydroalanine and Dehydrobutyrine in Proteins

Article abstract of DOI:10.1021/jacs.9b02611

Expansion of the genetic code with unnatural amino acids (Uaas) has significantly increased the chemical space available to proteins for exploitation. Due to the inherent limitation of translational machinery and the required compatibility with biological settings, function groups introduced via Uaas to date are restricted to chemically inert, bioorthogonal, or latent bioreactive groups. To break this barrier, here we report a new strategy enabling the specific incorporation of biochemically reactive amino acids into proteins. A latent bioreactive amino acid is genetically encoded at a position proximal to the target natural amino acid; they react via proximity-enabled reactivity, selectively converting the latter into a reactive residue in situ. Using this Genetically Encoded Chemical COnversion (GECCO) strategy and harnessing the sulfur-fluoride exchange (SuFEx) reaction between fluorosulfate-l-tyrosine and serine or threonine, we site-specifically generated the reactive dehydroalanine and dehydrobutyrine into proteins. GECCO works both inter- and intramolecularly, and is compatible with various proteins. We further labeled the resultant dehydroalanine-containing protein with thiol-saccharide to generate glycoprotein mimetics. GECCO represents a new solution for selectively introducing biochemically reactive amino acids into proteins and is expected to open new avenues for exploiting chemistry in live systems for biological research and engineering.

Full text of DOI:10.1021/jacs.9b02611

Subscriber access provided by UNIV OF LOUISIANA
Communication
Genetically Introducing Biochemically Reactive Amino
Acids Dehydroalanine and Dehydrobutyrine in Proteins
Bing Yang, Nanxi Wang, Paul D. Schnier, Feng Zheng, He Zhu, Nicholas F. Polizzi, Avinash
Ittuveetil, Varma Saikam, William DeGrado, Qian Wang, Peng G. Wang, and Lei Wang
J. Am. Chem. Soc., Just Accepted Manuscript • Publication Date (Web): 30 Apr 2019
Downloaded from http://pubs.acs.org on April 30, 2019
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 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 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.
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 7
Journal of the American Chemical Society
1
2
3
4
5
6
7
8
9
Genetically Introducing Biochemically Reactive Amino Acids Dehy-
droalanine and Dehydrobutyrine in Proteins
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
Bing Yang,†,^ Nanxi Wang,†,^ Paul D. Schnier,‡,^ Feng Zheng,||,^ He Zhu,§,^ Nicholas F. Polizzi,† Avinash It-
tuveetil,§ Varma Saikam,§ William F. DeGrado,† Qian Wang,|| Peng G. Wang,§ and Lei Wang†,*
† Department of Pharmaceutical Chemistry and the Cardiovascular Research Institute, ‡Institute for Neurodegenerative Diseases, Uni-
versity of California San Francisco, San Francisco, California 94158, United States.
|| Hangzhou Research Institute of Technical Institute of Physics and Chemistry, Chinese Academy of Sciences, Hangzhou 310018,
China.
§ Department of Chemistry and Center for Therapeutics and Diagnostics, Georgia State University, Atlanta, Georgia 30302, United
States.
Supporting Information Placeholder
into proteins able to form covalent bonds with natural amino
acid residues in proximity.7-10 Nonetheless, it remains infeasi-
ble to selectively introduce biochemically reactive Uaas into
proteins in live cells, because the biochemical reactivity of the
Uaa may interfere with protein translation and other biologi-
cal processes. Nature, on the other hand, has installed the re-
active dehydroalanine (Dha) and dehydrobutyrine (Dhb)
into proteins through enzymatic posttranslational modifica-
tions, which are used to create unique intra-protein bridges in
lantipeptides and thiopeptides possessing antimicrobial and
antitumor activities.11,12 Through chemical conversion Dha
and Dhb can also be generated in vitro, and the unique struc-
ture and reactivity of a,b-unsaturated carbonyl moiety in Dha
have been harnessed for chemical mutagenesis and installa-
tion of a broad range of posttranslational modifications,
providing an invaluable route for studying proteins.13-21 How-
ever, due to the cellular incompatibility of reagents and con-
ditions used for chemical conversion, methods reported to
date cannot generate Dha or Dhb in vivo.
ABSTRACT: Expansion of the genetic code with unnatural
amino acids (Uaas) has significantly increased the chemical
space available to proteins for exploitation. Due to the inher-
ent limitation of translational machinery and the required
compatibility with biological settings, function groups intro-
duced via Uaas to date are restricted to chemically inert,
bioorthogonal, or latent bioreactive groups. To break this bar-
rier, here we report a new strategy enabling the specific incor-
poration of biochemically reactive amino acids into proteins.
A latent bioreactive amino acid is genetically encoded at a po-
sition proximal to the target natural amino acid; they react via
proximity-enabled reactivity, selectively converting the latter
into a reactive residue in situ. Using this Genetically Encoded
Chemical COnversion (GECCO) strategy and harnessing
the sulfur-fluoride exchange (SuFEx) reaction between
fluorosulfate-L-tyrosine and serine or threonine, we site-spe-
cifically generated the reactive dehydroalanine and dehydro-
butyrine into proteins. GECCO works both inter- and intra-
molecularly, and is compatible with various proteins. We fur-
ther labeled the resultant dehydroalanine-containing protein
with thiol-saccharide to generate glycoprotein mimetics.
GECCO represents a new solution for selectively introducing
biochemically reactive amino acids into proteins and is ex-
pected to open new avenues for exploiting chemistry in live
systems for biological research and engineering.
Through engineering orthogonal components for protein
translation, unnatural amino acids (Uaas) have been genet-
ically encoded in various cells and model organisms.1-5 To be
compatible with biological settings, side chains of these en-
coded Uaas are mainly chemically inert or bioorthogonal.2,3,6
A recent breakthrough is the encoding of latent bioreactive
Uaas, which are unreactive inside cells but once incorporated
Figure 1. GECCO to site-selectively introduce biochemically
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 2 of 7
reactive amino acids into proteins. The latent bioreactive Uaa
FSY reacts with a nearby Ser or Thr via proximity-enabled reac-
tivity, selectively converting the latter into Dha or Dhb.
Here we present a novel strategy to selectively introduce bi-
ochemically reactive Uaas into proteins. We genetically
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
Figure 2. Generation of Dha and Dhb on proteins via intermolecular GECCO in E. coli. (a) Structure of Afb-Z complex (PDB: 1LP1)
showing two proximal sites for placing FSY and the target Ser. (b-c) Tandem mass spectra identifying Ser and Dha at site 7 of the Afb
protein. (d-e) Tandem mass spectra identifying FSY and Tyr at site 24 of the Z protein. (f) Structure of Afb-Z complex (PDB: 1LP1)
showing two proximal sites for placing FSY and the target Thr. (g-h) Tandem mass spectra identifying Thr and Dhb at site 7 of the Afb
protein. (i-j) Tandem mass spectra identifying FSY and Tyr at site 24 of the Z protein.
encode a latent bioreactive Uaa into proteins at a site proximal
to the target position; enabled by proximity-enhanced reac-
tivity,10 the latent bioreactive Uaa then reacts with the nearby
target natural amino acid residue, selectively converting it into
a biochemically reactive Uaa (Fig. 1). Through incorporating
the latent bioreactive Uaa fluorosulfate-L-tyrosine (FSY) and
harnessing its reaction with Ser or Thr, we demonstrated the
generation of Dha and Dhb on proteins. We further showed
that the resultant Dha could be used to selectively attach sac-
charide for generating glycoprotein mimetics. We expect that
Dha and Dhb generated in situ will enable versatile chemical
means for protein research and engineering in live cells, and
that this Genetically Encoded Chemical COnversion
(GECCO) strategy will open a new avenue for selective
ACS Paragon Plus Environment

Page 3 of 7
Journal of the American Chemical Society
introduction of biochemically reactive amino acids in vivo.
5.5% and FSY to Tyr was ca. 7.2% (Fig. S2). These data sug-
1
2
3
4
5
6
7
8
We recently evolved an orthogonal tRNAPyl/FSYRS pair
that genetically incorporates Uaa FSY into proteins in E. coli
and mammalian cells.22 The incorporated FSY was found to
react with Lys, His, and Tyr in proximity through sulfur-fluo-
ride exchange (SuFEx) reaction, forming covalent protein
crosslinks in vivo. However, we did not detect any crosslink-
ing between FSY and Ser or Thr.22 In contrast, arylfluorosul-
fate installed on chemical probes was able to react with Lys,
Tyr, and Ser within a positively charged binding pocket of the
specifically bound protein, and the resultant arylfluorosulfate-
Ser adduct was found to partially hydrolyze to Dha,23-25 but
what occurred to the arylfluorosulfate warhead remains un-
characterized. Prompted by these findings, we aimed to deter-
mine whether proximal FSY and Ser incorporated into pro-
teins (instead of on small molecules) would react, whether a
positively charged microenvironment is necessary, and what
the products would be. We thus incorporated FSY and Ser at
different protein contexts without positively charged residues
nearby, and characterized their identity using high resolution
tandem MS.
gest that FSY was able to convert both Ser and Thr into the
respective Dha and Dhb, while changing itself back to the nat-
ural amino acid Tyr.
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
We first examined whether FSY would react with Ser inter-
molecularly when separately installed on two interacting pro-
teins. Specifically, we incorporated FSY at position E24 of the
Z protein, and introduced Ser at position K7 of the Z-binding
affibody (Afb) (Fig. 2a), placing them in proximity when Afb
binds with Z.26 Proteins Z(24FSY) and Afb(7Ser) were co-ex-
pressed in E. coli, allowing them to bind and react in vivo, and
then purified and characterized using MS. As expected, tan-
dem MS identified the Ser-containing peptide for the
Afb(7Ser) protein (Fig. 2b). To our delight, we also identified
this peptide with Dha at the 7Ser position (Fig. 2c). A series
of b and y ions in the tandem MS unambiguously indicate the
presence of Dha at the 7Ser position, confirming the conver-
sion of Ser to Dha. In addition, we identified the FSY-
containing peptide for the Z(24FSY) protein (Fig. 2d), indi-
cating specific incorporation of FSY. Meanwhile, the peptide
containing Tyr at the 24FSY position was also identified (Fig.
2e), suggesting the conversion of FSY to Tyr upon reacting
with Ser. Based on the peak areas of these peptides in the ex-
tracted ion chromatograms (Fig. S1), the conversion rate of
Ser to Dha was ca. 3.7% and FSY to Tyr was ca. 4.1%. These
results indicate that FSY installed proximal to Ser in proteins
could intermolecularly convert Ser into Dha.
We next examined whether FSY could convert Thr to Dhb
in proteins. Similarly, proteins Z(24FSY) and Afb(7Thr)
were co-expressed in E. coli (Fig. 2f). MS analyses of the puri-
fied proteins identified the Thr-containing peptide (Fig. 2g)
as well as the Dhb-containing peptide (Fig. 2h) of Afb(7Thr),
validating the conversion of 7Thr to Dhb. Again, for protein
Z(24FSY), the FSY-containing peptide was identified (Fig.
2i), together with this peptide wherein FSY was converted
into Tyr (Fig. 2j). Extracted ion chromatograms of these pep-
tides indicate that the conversion rate of Thr to Dhb was ca.
Figure 3. Generation of Dha on sfGFP via intramolecular
GECCO in E. coli. (a) Crystal structure of sfGFP (PDB: 2B3P)
showing site Tyr182 for FSY incorporation to target Ser intro-
duced at site Glu184 on the b-strand. (b-c) Tandem MS spectra
of sfGFP (182FSY/184Ser) expressed in E. coli identifying
182FSY/184Ser (b) and 182Tyr/184Dha (c).
We then determined whether Dha could be generated by
FSY through intramolecular conversion. We initially targeted
a Ser located on a b-strand of the super-fold green fluorescent
protein (sfGFP). FSY was incorporated at the permissive site
Tyr182 and a Ser was placed at site 184, the i+2 position so
that both side chains pointing to the same side of the b-strand
(Fig. 3a). This mutant sfGFP(182FSY/184Ser) was ex-
pressed in E. coli and then characterized by MS. The peptide
containing FSY at site 182 and Ser at site 184 was identified,
again indicating specific incorporation of FSY (Fig. 3b). As
expected, this peptide was also identified containing Tyr at
site 182 and Dha at site 184 (Fig. 3c). The conversion rate of
Ser to Dha was 1.3% (Fig. S3). We note that no peptide con-
taining FSY182/Dha184 was detected, that is, whenever Dha
was detected at site 184, site 182 was always found to be Tyr,
indicating that conversion of Ser to Dha was consequentially
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 4 of 7
associated with conversion of FSY to Tyr. The Dha conver-
sion rate was low possibly because the rigidity of the sfGFP b-
strand does not allow ample contact of FSY with Ser; compu-
tational modeling indicates that the side chains of FSY and Ser
point away from each other in their sterically allowed rota-
mers (Fig. S4).
We therefore reasoned that a good contact of FSY with the
1
2
3
4
5
6
7
8
target Ser without competition from Lys, His, and Tyr, which
are known to react with FSY,22 may promote the conversion
rate. To test this idea, we incorporated FSY into Afb at site
Asp37, which is located in a loop and has no contact with Lys,
His or Tyr, and determined how efficiently nearby Ser could
be converted into Dha. After expressing Afb(37FSY) in E. coli
followed by purification, MS characterization revealed that
Ser10 was converted to Dha in 3.9% yield and Ser-1 to Dha in
53% yield (Fig. 4a, 4b, S6). The crystal structure of Afb is
available only in complex with the Z protein, and some resi-
dues of Afb are missing in this complex structure. To under-
stand the conversion difference, we performed ab initio fold-
ing of the Afb sequence containing all residues, including ad-
ditional residues Met(-3)Thr(-2)Ser(-1) at the N-terminus
introduced by cloning. The Cb-Cb distances between site 37
and Ser-1 or Ser10 were analyzed in outputted models (Fig.
4c, 4d, S7). Many low energy/low RMSD models contain Ser-
1 with a close distance to site 37, whereas very few models
contain Ser10 with a close distance to site 37—and these few
models are very high energy (Fig. 4c-f). These data support
that better contact of FSY with Ser-1 indeed enhances the
conversion of Ser to Dha.
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
Figure 4. Generation of Dha on Afb via intramolecular GECCO
in E. coli. (a-b) Tandem mass spectra identifying Dha at Ser-1 (a)
and Ser10 (b) of the Afb protein. (c-d) Histogram of Cb-Cb dis-
tances of Ser-1 and Asp37 (c) and of Ser10 and Asp37 (d) in
4,525 low energy models of ab initio folded Afb. (e-f) Repre-
sentative models from ab initio folding of Afb showing Asp37
close to Ser-1 (e) and close to Ser10 (f). The gold structure in
(e) is the aligned Afb backbone of 1LP1.
We next tested intramolecular conversion in a less rigid pro-
tein context in ubiquitin. FSY was introduced in site 45, which
has contact with Ser65 and Lys63. After expressing ubiqui-
tin(FSY45) in E. coli followed by MS characterization, we
found that FSY predominately crosslinked with Lys63 and
Ser65 was converted to Dha in 1.7% yield (Fig. S5).
Figure 5. Labeling Dha-containing sfGFP with 1-thiol-GlcNAc.
(a) Scheme showing the structure of 1-thiol-GlcNAc and its re-
action with Dha. (b) Western blot and (c) tandem MS analysis
ACS Paragon Plus Environment

Page 5 of 7
Journal of the American Chemical Society
of reaction product confirming successful labeling of Dha with
GlcNAc.
Author Contributions
^ These authors contributed equally.
Notes
1
2
3
4
5
6
7
8
To further demonstrate the chemical identity of Dha gen-
The authors declare no competing financial interest.
erated by FSY conversion, we labeled Dha with a thiol-deri-
vatized saccharide to generate glycoprotein mimetics (Fig.
5a). Methods for preparing site-selectively glycosylated pro-
teins and mimetics are valuable for studying protein glycosyl-
ation.17,27-30 We synthesized 2-acetamido-2-deoxy-1-thio-β-
D-glucopyranose (1-thiol-GlcNAc) in 3 steps (68% overall
yield, Supplementary methods), and incubated 1-thiol-Glc-
NAc with sfGFP(182FSY/184Ser) (expressed from E. coli as
described above and containing 182Tyr/184Dha) under
ACKNOWLEDGMENT
W.F.D acknowledges NIH support (R35 GM122603). L.W.
acknowledges NIH support (R01GM118384, RF1MH114079).
9
REFERENCES
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
(1)
Genetic Code of Escherichia Coli. Science 2001, 292 (5516), 498–500.
(2) Wang, L.; Schultz, P. G. Expanding the Genetic Code. Angew.
Chem. Int. Ed. Engl. 2005, 44 (1), 34–66.
Wang, L.; Brock, A.; Herberich, B.; Schultz, P. G. Expanding the
mild conditions.
A similarly expressed and purified
sfGFP(182FSY/184Glu) protein was used as the negative
control. Western blot analysis of reaction product using an an-
tibody specific for GlcNAc showed that only the Dha-con-
taining sfGFP was labeled by 1-thiol-GlcNAc (Fig. 5b). The
reaction product was further analyzed with tandem MS,
which clearly confirmed the attachment of 1-thiol-GlcNAc
onto Dha at site 184 (Fig. 5c). These results further validate
the chemical identity of Dha generated by FSY conversion
and its value for selective protein modification.20
(3)
Liu, C. C.; Schultz, P. G. Adding New Chemistries to the Genetic
Code. Annu. Rev. Biochem. 2010, 79 (1), 413–444.
(4)
Wang, L. Engineering the Genetic Code in Cells and Animals:
Biological Considerations and Impacts. Acc. Chem. Res. 2017, 50 (11),
2767–2775.
(5)
Chen, Y.; Ma, J.; Lu, W.; Tian, M.; Thauvin, M.; Yuan, C.; Vo-
lovitch, M.; Wang, Q.; Holst, J.; Liu, M.; Vriz, S.; Ye, S.; Wang, L.; Li, D.
Heritable Expansion of the Genetic Code in Mouse and Zebrafish. Cell Res.
2017, 27 (2), 294–297.
(6)
Annu. Rev. Biophys. Biomol. Struct. 2006, 35 (1), 225–249.
(7) Xiang, Z.; Ren, H.; Hu, Y. S.; Coin, I.; Wei, J.; Cang, H.; Wang, L.
Wang, L.; Xie, J.; Schultz, P. G. Expanding the Genetic Code.
In summary, we developed a new method, GECCO, which
enables the genetically introducing of biochemically reactive
amino acids into proteins. Harnessing proximity-enabled re-
activity, a genetically incorporated latent bioreactive Uaa con-
verts a nearby target natural residue into a reactive amino acid
in situ. We demonstrated the conversion of Ser and Thr into
the reactive Dha and Dhb, respectively, and the labeling of the
Dha-containing protein with a thiol-saccharide to generate
glycoprotein mimetics. The conversion occurred both inter-
and intra-molecularly on various proteins, with the conver-
sion rate dependent on the contact between FSY and the tar-
get Ser/Thr. Dha and Dhb also represent two smallest Uaas
introduced into proteins via genetic code expansion to date.
Compared with existing methods for introducing Dha to pro-
teins via chemical transformation,20 this work presents a re-
combinant approach to produce Dha/Dhb-containing pro-
teins without extra chemical treatments. We expect that
GECCO will enable the genetically introducing of additional
biochemically reactive amino acids in proteins, thus expand-
ing new avenues for exploiting chemistry in live systems for
biological research and engineering.
Adding an Unnatural Covalent Bond to Proteins Through Proximity-En-
hanced Bioreactivity. Nat. Methods 2013, 10 (9), 885–888.
(8)
Xiang, Z.; Lacey, V. K.; Ren, H.; Xu, J.; Burban, D. J.; Jennings, P.
A.; Wang, L. Proximity-Enabled Protein Crosslinking Through Genetically
Encoding Haloalkane Unnatural Amino Acids. Angew. Chem. Int. Ed. Engl.
2014, 53, 2190–2193.
(9)
Furman, J. L.; Kang, M.; Choi, S.; Cao, Y.; Wold, E. D.; Sun, S. B.;
Smider, V. V.; Schultz, P. G.; Kim, C. H. A Genetically Encoded Aza-Michael
Acceptor for Covalent Cross-Linking of Protein-Receptor Complexes. J. Am.
Chem. Soc. 2014, 136 (23), 8411–8417.
(10) Wang, L. Genetically Encoding New Bioreactivity. N. Biotechnol.
2017, 38 (Pt A), 16–25.
(11) Li, H.; Xu, H.; Zhou, Y.; Zhang, J.; Long, C.; Li, S.; Chen, S.;
Zhou, J.-M.; Shao, F. The Phosphothreonine Lyase Activity of a Bacterial
Type III Effector Family. Science 2007, 315 (5814), 1000–1003.
(12) Repka, L. M.; Chekan, J. R.; Nair, S. K.; Van der Donk, W. A.
Mechanistic Understanding of Lanthipeptide Biosynthetic Enzymes. Chem.
Rev. 2017, 117 (8), 5457–5520.
(13) Seebeck, F. P.; Szostak, J. W. Ribosomal Synthesis of Dehydroa-
lanine-Containing Peptides. J. Am. Chem. Soc. 2006, 128 (22), 7150–7151.
(14) Wang, J.; Schiller, S. M.; Schultz, P. G. A Biosynthetic Route to
Dehydroalanine-Containing Proteins. Angew. Chem. Int. Ed. 2007, 46 (36),
6849–6851.
(15) Guo, J.; Wang, J.; Lee, J. S.; Schultz, P. G. Site-Specific Incorpo-
ration of Methyl- and Acetyl-Lysine Analogues Into Recombinant Proteins.
Angew. Chem. Int. Ed. Engl. 2008, 47 (34), 6399–6401.
(16) Wang, Z. U.; Wang, Y.-S.; Pai, P.-J.; Russell, W. K.; Russell, D. H.;
Liu, W. R. A Facile Method to Synthesize Histones with Posttranslational
Modification Mimics. Biochemistry 2012, 51 (26), 5232–5234.
(17) Wright, T. H.; Bower, B. J.; Chalker, J. M.; Bernardes, G. J. L.;
Wiewiora, R.; Ng, W. L.; Raj, R.; Faulkner, S.; Vallee, M. R. J.; Phanumartwi-
wath, A.; Coleman, O. D.; Thezenas, M. L.; Khan, M.; Galan, S. R. G.;
Lercher, L.; Schombs, M. W.; Gerstberger, S.; Palm-Espling, M. E.; Baldwin,
A. J.; Kessler, B. M.; Claridge, T. D. W.; Mohammed, S.; Davis, B. G. Post-
translational Mutagenesis: a Chemical Strategy for Exploring Protein Side-
Chain Diversity. Science 2016, 354 (6312), aag1465–aag1465.
(18) Yang, A.; Ha, S.; Ahn, J.; Kim, R.; Kim, S.; Lee, Y.; Kim, J.; Söll,
D.; Lee, H.-Y.; Park, H.-S. A Chemical Biology Route to Site-Specific Au-
thentic Protein Modifications. Science 2016, 354 (6312), 623–626.
ASSOCIATED CONTENT
Supporting Information.
The Supporting Information is available free of charge on the
ACS Publications website.
Experimental details and supporting Figures S1-7 (PDF)
AUTHOR INFORMATION
Corresponding Author
* Lei.Wang2@ucsf.edu.
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 6 of 7
(19) Freedy, A. M.; Matos, M. J.; Boutureira, O.; Corzana, F.; Guer-
S.; Wang, H.; Cravatt, B. F.; Forli, S.; Powers, E. T.; Sharpless, K. B.; Wilson,
I. A.; Kelly, J. W. “Inverse Drug Discovery” Strategy to Identify Proteins That
Are Targeted by Latent Electrophiles as Exemplified by Aryl Fluorosulfates.
J. Am. Chem. Soc. 2018, 140 (1), 200–210.
(25) Fadeyi, O. O.; Hoth, L. R.; Choi, C.; Feng, X.; Gopalsamy, A.;
Hett, E. C.; Kyne, R. E.; Robinson, R. P.; Jones, L. H. Covalent Enzyme In-
hibition Through Fluorosulfate Modification of a Noncatalytic Serine Resi-
due. ACS Chem. Biol. 2017, 12 (8), 2015–2020.
reiro, A.; Akkapeddi, P.; Somovilla, V. J.; Rodrigues, T.; Nicholls, K.; Xie, B.;
Jiménez-Osés, G.; Brindle, K. M.; Neves, A. A.; Bernardes, G. J. L. Chemose-
lective Installation of Amine Bonds on Proteins Through Aza-Michael Liga-
tion. J. Am. Chem. Soc. 2017, 139 (50), 18365–18375.
(20) Dadová, J.; Galan, S. R.; Davis, B. G. Synthesis of Modified Pro-
teins via Functionalization of Dehydroalanine. Curr. Opin. Chem. Biol. 2018,
46, 71–81.
1
2
3
4
5
6
7
8
9
(21) de Bruijn, A. D.; Roelfes, G. Catalytic Modification of Dehydro-
alanine in Peptides and Proteins by Palladium-Mediated Cross-Coupling.
Chemistry 2018, 24 (48), 12728–12733.
(26) Hogbom, M.; Eklund, M.; Nygren, P. A.; Nordlund, P. Structural
Basis for Recognition by an in Vitro Evolved Affibody. Proc. Natl. Acad. Sci.
U. S. A. 2003, 100 (6), 3191–3196.
(27) Liu, H.; Wang, L.; Brock, A.; Wong, C.-H.; Schultz, P. G. A
Method for the Generation of Glycoprotein Mimetics. J. Am. Chem. Soc.
2003, 125 (7), 1702–1703.
(28) Kiessling, L. L.; Grim, J. C. Glycopolymer Probes of Signal
Transduction. Chem. Soc. Rev. 2013, 42 (10), 4476–4491.
(29) Tiwari, V. K.; Mishra, B. B.; Mishra, K. B.; Mishra, N.; Singh, A.
S.; Chen, X. Cu-Catalyzed Click Reaction in Carbohydrate Chemistry.
Chem. Rev. 2016, 116 (5), 3086–3240.
(22) Wang, N.; Yang, B.; Fu, C.; Zhu, H.; Zheng, F.; Kobayashi, T.;
Liu, J.; Li, S.; Ma, C.; Wang, P. G.; Wang, Q.; Wang, L. Genetically Encoding
Fluorosulfate-L-Tyrosine to React with Lysine, Histidine, and Tyrosine via
SuFEx in Proteins in Vivo. J. Am. Chem. Soc. 2018, 140, 4995–4999.
(23) Chen, W.; Dong, J.; Plate, L.; Mortenson, D. E.; Brighty, G. J.; Li,
S.; Liu, Y.; Galmozzi, A.; Lee, P. S.; Hulce, J. J.; Cravatt, B. F.; Saez, E.; Pow-
ers, E. T.; Wilson, I. A.; Sharpless, K. B.; Kelly, J. W. Arylfluorosulfates Inac-
tivate Intracellular Lipid Binding Protein(S) Through Chemoselective
SuFEx Reaction with a Binding Site Tyr Residue. J. Am. Chem. Soc. 2016,
138 (23), 7353–7364.
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
(30) Li, C.; Wang, L.-X. Chemoenzymatic Methods for the Synthesis
of Glycoproteins. Chem. Rev. 2018, 118 (17), 8359–8413.
(24) Mortenson, D. E.; Brighty, G. J.; Plate, L.; Bare, G.; Chen, W.; Li,
ACS Paragon Plus Environment

Page 7 of 7
Journal of the American Chemical Society
1
2
3
Insert Table of Contents artwork here
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
7
ACS Paragon Plus Environment