Table 1 Optimization of the Reaction Conditions^a

Entry	Endo F3 (D165A)-CPD	3-azido-propylamine (mM)	InsP₆ (μM)	Buffer	5 h		16 h
Entry	Endo F3 (D165A)-CPD	3-azido-propylamine (mM)	InsP₆ (μM)	Buffer	Ligation Yield (%)	Hydrolysis Yield (%)	Ligation Yield (%)	Hydrolysis Yield (%)
1	100 μM	50	100	PBS	100	0	100	0
2	100 μM	20	100	PBS	100	0	100	0
3	100 μM	10	100	PBS	93	7	85	15
4	100 μM	5	100	PBS	67	33	9	91
5	100 μM	2	100	PBS	6	94	0	100
6	100 μM	5	500	PBS	62	38	4	96
7	100 μM	5	200	PBS	68	32	5	95
8	100 μM	5	50	PBS	72	28	7	93
9	100 μM	5	20	PBS	66	34	36	64
10	100 μM	5	5	PBS	90	10	80	20
11	100 μM	5	1	PBS	63	25	59	41
12	100 μM	5	0.2	PBS	18	11	37	47
13	100 μM	5	5	PB pH 6.0	0	100	0	100
14	100 μM	5	5	PB pH 7.0	0	100	0	100
15	100 μM	5	5	PB pH 8.0	47	53	49	51
16	100 μM	5	5	PB pH 9.0	82	18	75	25
17	100 μM	5	5	PB	43	57	39	61
18	100 μM	5	5	HEPES	42	58	38	62
19	100 μM	5	5	Tris	62	38	57	43
20	100 μM	5	5	MOPS	45	55	40	60
21	100 μM	5	5	Tricine	45	55	41	59
22	100 μM	5	5	NaOAc	94	6	88	12
23	100 μM	2	5	NaOAc	12	88	9	91

^a The reactions were performed at 4 °C and corresponding buffer (50 mM, final pH=8.0 unless otherwise indicated). The conversion rates were measured by LC-MS.

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